Eileen McLaughlin

Small molecule Hedgehog pathway antagonists

Publisher: Royal Society of Chemistry (RSC)

Date: 2017

DOI: 10.1039/C6OB01959E

Abstract: Leveraging our quinolone-1-(2 H )-one based Hedgehog signalling pathway (HSP) inhibitors we have developed two new classes of HSP inhibitors based on: l -tryptophan and benzo[1,3]dioxol-5-ylmethyl-[2-(1 H -indol-3-yl)-ethyl]-amine.

Modeling Secondary Electron Imaging at Atomic Resolution Using a Focused Coherent Electron Probe

Publisher: Oxford University Press (OUP)

Date: 08-2014

DOI: 10.1017/S143192761400213X

Advances in human primordial follicle activation and premature ovarian insufficiency

Publisher: Bioscientifica

Date: 2020

DOI: 10.1530/REP-19-0201

Abstract: In women, the non-growing population of follicles that comprise the ovarian reserve is determined at birth and serves as the reservoir for future fertility. This reserve of dormant, primordial follicles and the mechanisms controlling their selective activation which constitute the committing step into folliculogenesis are essential for determining fertility outcomes in women. Much of the available data on the mechanisms responsible for primordial follicle activation focuses on a selection of key molecular pathways, studied primarily in animal models, with findings often not synonymous in humans. The excessive induction of primordial follicle activation may cause the development of premature ovarian insufficiency (POI), a condition characterised by menopause before age 40 years. POI affects 1–2% of all women and is accompanied by additional health risks. Therefore, it is critical to further our understanding of primordial follicle activation in order to diagnose, treat and prevent premature infertility. Research in primordial follicle activation has focused on connecting new molecules to already established key signalling pathways, such as phosphatidylinositol 3-Kinase (PI3K) and mammalian target of rapamycin (mTOR). Additionally, other aspects of the ovarian environment, such as the function of the extracellular matrix, in contributing to primordial follicle activation have gained traction. Clinical applications are examining replication of this extracellular environment through the construction of biological matrices mimicking the 3D ovary, to support follicular growth through to ovulation. This review outlines the importance of the events leading to the establishment of the ovarian reserve and highlights the fundamental factors known to influence primordial follicle activation in humans presenting new horizons for female infertility treatment.

The small non-coding RNA profile of mouse oocytes is modified during aging

Publisher: Impact Journals, LLC

Date: 24-05-2019

DOI: 10.18632/AGING.101947

The atomic absorption determination of antimony,arsenic, bismuth, cadmium, lead, and tin in iron, copper,and zinc alloys with the graphite furnace

Publisher: Elsevier BV

Date: 12-1975

DOI: 10.1016/S0003-2670(01)85082-0

Effects of cryopreservation on the intracellular calcium concentration of human spermatozoa and its response to progesterone

Publisher: Wiley

Date: 02-1994

DOI: 10.1002/MRD.1080370216

Abstract: The intracellular free calcium concentration [Ca2+]i of sperm from 23 ejaculates was measured before and after cryopreservation using the fluorescent probe Fura-2. Spermatozoa were treated with 3.18 microM progesterone so that the regulation of [Ca2+]i in a dynamic situation could be studied. [Ca2+]i (nM) was 290 +/- 13 in fresh spermatozoa vs. 550 +/- 26 in cryopreserved s les (mean +/- S.E.M. P 1 microM after 10-20 seconds. [Ca2+]i then declined to a slightly raised basal level over the next 30-40 seconds. This phenomenon occurred in all the fresh s les, but about half the frozen thawed s les failed to respond. The peak [Ca2+] attained by frozen s les which did respond after the addition of progesterone was similar to that observed with fresh sperm. The calcium channel blocker verapamil (200 microM) completely inhibited the transient rise in [Ca2+]i produced by progesterone, but 100 microM verapamil had only a partial effect. We conclude that (1) cryopreservation causes a substantial elevation of the [Ca2+]i in human spermatozoa and (2) damage to the plasma membrane during cryopreservation may result in the loss of the progesterone receptor. Both factors may contribute to the loss of fertility after cryopreservation.

Heat Shock Protein member A2 forms a stable complex with angiotensin converting enzyme and protein disulfide isomerase A6 in human spermatozoa

Publisher: Oxford University Press (OUP)

Date: 16-12-2016

DOI: 10.1093/MOLEHR/GAV073

Abstract: Given the importance of the chaperone Heat Shock Protein A2 (HSPA2) in the regulation of male fertility, this study aimed to identify and characterize additional proteins that may rely on the activity of this chaperone in human spermatozoa. In view of the findings in this study we propose that angiotensin converting enzyme (ACE) and protein disulfide isomerase A6 (PDIA6) are novel interacting proteins of HSPA2 and that this multimeric complex may participate in key elements of the fertilization cascade. The molecular chaperone HSPA2 plays a pivotal role in the remodelling of the sperm surface during capacitation. Indeed, human spermatozoa that are deficient in HSPA2 protein expression lack the ability to recognize human oocytes, resulting in repeated IVF failure in a clinical setting. Moreover, our recent work has shown that defective HSPA2 function induced by oxidative stress leads to the aberrant surface expression of one of its interacting proteins, arylsulfatase A, and thus contributes to a loss of sperm-zona pellucida adhesion. Human spermatozoa were collected from fertile donors, capacitated and prepared for Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) analysis. Protein complexes resolved via BN-PAGE were excised and their constituents were identified using mass spectrometry. The interactions between ACE, PDIA6 and HSPA2 were then confirmed using immunoprecipitation and proximity ligation assays and the localization of these proteins was assessed in isolated spermatozoa and commercially available human testis tissue sections. Finally, pharmacological inhibition of ACE was performed to assess the role of ACE in human sperm capacitation. Herein we have identified ACE and PDIA6 as potential HSPA2-interacting proteins and shown that this assemblage resides in membrane raft microdomains located in the peri-acrosomal region of the sperm head. Additionally, the surface expression of PDIA6, but not ACE, was shown to be dynamically regulated during sperm capacitation and, like that of previously characterized HSPA2-interacting proteins, this surface expression proved vulnerable to oxidative stress. In terms of the functional significance of this protein complex, pharmacological inhibition of ACE significantly reduced the ability of human spermatozoa to undergo an agonist induced acrosome reaction (P < 0.01). While these results provide a descriptive analysis of the PDIA6/ACE/HSPA2 complex, this study provides the impetus for further investigation into the role of PDIA6 and ACE in human sperm function. As our research group, and others, have shown that HSPA2 is compromised in the spermatozoa of men with oocyte recognition defects, the characterization of these HSPA2-interacting proteins provides important insight into the complexity of the cellular pathways that may be affected in the spermatozoa of infertile in iduals. Large scale proteomics data can be accessed through the Proteomics Identifications Database (PRIDE). This work was supported by the National Health and Medical Research Council. Grant # APP1046346. The authors have no competing interests to declare.

Atomic Resolution Imaging of Light Elements in a Crystalline Environment using Dynamic Hollow-Cone Illumination Transmission Electron Microscopy

Publisher: Oxford University Press (OUP)

Date: 10-06-2020

DOI: 10.1017/S1431927620001658

Abstract: Multiple electron scattering and the nonintuitive nature of image formation with coherent radiation complicate the interpretation of conventional transmission electron microscopy images. Precession of the illuminating beam in transmission electron microscopy (TEM) can lead to more robust and interpretable images with some penalty to image contrast, a technique known as dynamic hollow-cone illumination TEM. We demonstrate direct and robust imaging of light and heavy atoms in a crystalline environment with this technique. This method is similar to the annular bright-field technique in scanning transmission electron microscopy, via the principle of reciprocity. Dynamic hollow-cone illumination TEM is challenging in practice due to sensitivity to the misalignment of the precession axis, microscope objective aperture, and crystal zone axis.

The Musashi Family of RNA Binding Proteins: Master Regulators of Multiple Stem Cell Populations

Publisher: Springer Netherlands

Date: 2013

DOI: 10.1007/978-94-007-6621-1_13

Abstract: In order to maintain their unlimited capacity to ide, stem cells require controlled temporal and spatial protein expression. The Musashi family of RNA-binding proteins have been shown to exhibit this necessary translational control through both repression and activation in order to regulate multiple stem cell populations. This chapter looks in depth at the initial discovery and characterisation of Musashi in the model organism Drosophila, and its subsequent emergence as a master regulator in a number of stem cell populations. Furthermore the unique roles for mammalian Musashi-1 and Musashi-2 in different stem cell types are correlated with the perceived diagnostic power of Musashi expression in specific stem cell derived oncologies. In particular the potential role for Musashi in the identification and treatment of human cancer is considered, with a focus on the role of Musashi-2 in leukaemia. Finally, the manipulation of Musashi expression is proposed as a potential avenue towards the targeted treatment of specific aggressive stem cell cancers.

Jumping the gun: Smoking constituent BaP causes premature primordial follicle activation and impairs oocyte fusibility through oxidative stress

Publisher: Elsevier BV

Date: 04-2012

DOI: 10.1016/J.TAAP.2012.01.028

Abstract: Benzo(a)pyrene (BaP) is an ovotoxic constituent of cigarette smoke associated with pre-mature ovarian failure and decreased rates of conception in IVF patients. Although the overall effect of BaP on female fertility has been documented, the exact molecular mechanisms behind its ovotoxicity remain elusive. In this study we examined the effects of BaP exposure on the ovarian transcriptome, and observed the effects of in vivo exposure on oocyte dysfunction. Microarray analysis of BaP cultured neonatal ovaries revealed a complex mechanism of ovotoxicity involving a small cohort of genes associated with follicular growth, cell cycle progression, and cell death. Histomorphological and immunohistochemical analysis supported these results, with BaP exposure causing increased primordial follicle activation and developing follicle atresia in vitro and in vivo. Functional analysis of oocytes obtained from adult Swiss mice treated neonatally revealed significantly increased levels of mitochondrial ROS/lipid peroxidation, and severely reduced sperm-egg binding and fusion in both low (1.5mg/kg/daily) and high (3mg/kg/daily) dose treatments. Our results reveal a complex mechanism of BaP induced ovotoxicity involving developing follicle atresia and accelerated primordial follicle activation, and suggest short term neonatal BaP exposure causes mitochondrial leakage resulting in reduced oolemma fluidity and impaired fertilisation in adulthood. This study highlights BaP as a key compound which may be partially responsible for the documented effects of cigarette smoke on follicular development and sub-fertility.

Higher gonadotrophin surge-attenuating factor bioactivity is found in small follicles from superovulated women

Publisher: Bioscientifica

Date: 10-1994

DOI: 10.1677/JOE.0.1430033

Abstract: Ovine and rat pituitary bioassays for gonadotrophin surgeattenuating factor (GnSAF) were utilized to determine whether the level of GnSAF bioactivity in pooled human follicular fluid (hFF) from superovulated women varied according to follicle diameter (≤11 mm, 12–15 mm and 16–21 mm follicles examined using the ovine bioassay, or ≤10 mm, 11–13 mm, 14–17 mm, 18–20 mm, 21–24 mm and ≥ 25 mm follicles examined using the rat bioassay). When tested using dispersed ovine pituitary cells, GnSAF bioactivity, expressed in terms of the reduction in gonadotrophin-releasing hormone (GnRH)-induced LH secretion, was inversely related to follicle diameter ( P ·01). In response to 5 μl hFF/well from follicles of ≤ 11, 12–15 and 16–21 mm diameter, GnRH-induced LH secretion was reduced to 40·5±6·6.9%, 65·2±6·6% and 83·7±7·9% of control respectively. A similar inverse relationship was observed when a second batch of hFF s les from different sized follicles was tested using rat pituitary cell monolayers. Expressing GnSAF bioactivity in terms of the dose required to suppress GnRH-induced LH secretion by rat pituitary cells to 50% of the maximal suppression observed (ED 50 ), the three smallest follicle size pools contained the most GnSAF (ED 50 values of 0·13, 2·79 and 5·36 μl hFF/well from follicles of ≤ 10, 11–13 and 14–17 mm respectively). The ED 50 values for follicles of 18–20, 21–24 and ≥25 mm were 8·81, 27·1 and 60·0 μl hFF/well respectively. Thus hFF from follicles ≤ 11 mm was over 450 times more potent than hFF from follicles ≥ 25 mm in suppressing GnRH-induced LH release. The ED 50 values for inhibin bioactivity (measured as the suppression of basal FSH secretion from rat pituitary monolayers) were much less variable than those for GnSAF bioactivity (between 0·85 and 0·13 μl hFF/well). Inhibin immunoreactivity, measured by a two-site immunoradiometric assay, followed the same pattern as inhibin bioactivity with lowest concentrations in the smallest follicles (41·96 ng/ml) and highest concentrations in the three largest follicle size groups (56·48–64·48 ng/ml). The specific effects of inhibin on GnRH-induced LH and basal FSH release in these pituitary bioassays were determined by incubating culture dishes with pure recombinant human inhibin at doses of 0·025–25 ng/well. In both the sheep and rat pituitary monolayers, basal FSH was suppressed (ED 50 =0·02 and 0·16 ng/well respectively). However, while inhibin markedly stimulated GnRH-induced LH secretion from ovine pituitary monolayers (ED 50 =0·04 ng/well), it suppressed GnRH-induced LH secretion from rat pituitary monolayers (ED 50 =0·31 ng/well) by 13%. The ergent effects of inhibin on GnRH-induced LH secretion in the two culture systems, and the relative insensitivity of GnRH-induced LH secretion to recombinant human inhibin in the rat system, indicates that the inverse relationship between GnSAF concentrations and follicular diameter cannot be an artefact of inhibin bioactivity. In addition, when hFF was fractionated by hydrophobic interaction chromatography using phenyl Sepharose, fractions which contained the greatest amounts of GnSAF bioactivity differed from those which contained peak levels of bioactive or immunoreactive inhibin. These results support in vivo observations that small follicles are important regulators of gonadotrophin secretion in superovulated women. Concentrations of GnSAF fall as the follicles approach an ovulatory size which enables positive steroid feedback on pituitary responses to hypothalamic GnRH, leading to the preovulatory LH surge. Journal of Endocrinology (1994) 143 , 33–44

Neonatal immune activation depletes the ovarian follicle reserve and alters ovarian acute inflammatory mediators in neonatal rats†

Publisher: Oxford University Press (OUP)

Date: 10-10-2017

DOI: 10.1093/BIOLRE/IOX123

Abstract: Normal ovarian development is crucial for female reproductive success and longevity. Interruptions to the delicate process of initial folliculogenesis may lead to ovarian dysfunction. We have previously demonstrated that an early life immune challenge in the rat, induced by administration of lipopolysaccharide (LPS) on postnatal day (PND) 3 and 5, depletes ovarian follicle reserve long term. Here, we hypothesized that this neonatal immune challenge leads to an increase in peripheral and ovarian inflammatory signaling, contributing to an acute depletion of ovarian follicles. Morphological analysis of neonatal ovaries indicated that LPS administration significantly depleted PND 5 primordial follicle populations and accelerated follicle maturation. LPS exposure upregulated circulating interleukin 6, tumor necrosis factor alpha (TNFa), and C-reactive protein on PND 5, and upregulated ovarian mRNA expression of Tnfa, mitogen-activated protein kinase 8 (Mapk8/Jnk1), and growth differentiation factor 9 (Gdf9) (P < 0.05). Mass spectrometry and cell signaling pathway analysis indicated upregulation of cellular pathways associated with acute phase signaling, and cellular survival and assembly. Apoptosis assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling indicated significantly increased positive staining in the ovaries of LPS-treated neonates. These findings suggest that increased proinflammatory signaling within the neonatal ovary may be responsible for the LPS-induced depletion of the primordial follicle pool. These findings also have implications for female reproductive health, as the ovarian reserve is a major determinate of female reproductive longevity.

Signal transducer and activator of transcription (STAT) 1 and STAT3 are expressed in the human ovary and have Janus kinase 1-independent functions in the COV434 human granulosa cell line

Publisher: CSIRO Publishing

Date: 2020

DOI: 10.1071/RD20098

Abstract: Ovarian granulosa cells are fundamental for oocyte maintenance and maturation. Recent studies have demonstrated the importance of members of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathway in the granulosa cell population of mouse and horse ovaries, with perturbation of JAK1 signalling in the mouse shown to impair oocyte maintenance and accelerate primordial follicle activation. The presence and role of the JAK/STAT pathway in human granulosa cells has yet to be elucidated. In this study, expression of JAK1, STAT1 and STAT3 was detected in oocytes and granulosa cells of human ovarian sections from fetal (40 weeks gestation) and premenopausal ovaries (34–41 years of age n=3). To determine the effects of JAK1 signalling in granulosa cells, the human granulosa-like cell line COV434 was used, with JAK1 inhibition using ruxolitinib. Chemical inhibition of JAK1 in COV434 cells with 100nM ruxolitinib for 72h resulted in significant increases in STAT3 mRNA (P=0.034) and p-Y701-STAT1 protein (P=0.0117), demonstrating a role for JAK1 in modulating STAT in granulosa cells. This study implicates a conserved role for JAK/STAT signalling in human ovary development, warranting further investigation of this pathway in human granulosa cell function.

Localization and significance of molecular chaperones, heat shock protein 1, and tumor rejection antigen gp96 in the male reproductive tract and during capacitation and acrosome reaction

Publisher: Oxford University Press (OUP)

Date: 02-2005

DOI: 10.1095/BIOLREPROD.104.034470

The rise of testicular germ cell tumours: the search for causes, risk factors and novel therapeutic targets

Publisher: F1000 Research Ltd

Date: 19-02-2013

DOI: 10.12688/F1000RESEARCH.2-55.V1

Abstract: Since the beginning of the 20th century there has been a decline in the reproductive vitality of men within the Western world. The declining sperm quantity and quality has been associated with increased overt disorders of sexual development including hypospadias, undescended testes and type II testicular germ cell tumours (TGCTs). The increase in TGCTs cannot be accounted for by genetic changes in the population. Therefore exposure to environmental toxicants appears to be a major contributor to the aetiology of TGCTs and men with a genetic predisposition are particularly vulnerable. In particular, Type II TGCTs have been identified to arise from a precursor lesion Carcinoma in situ (CIS), identified as a dysfunctional gonocyte however, the exact triggers for CIS development are currently unknown. Therefore the transition from gonocytes into spermatogonia is key to those studying TGCTs. Recently we have identified seven miRNA molecules (including members of the miR-290 family and miR-136, 463* and 743a) to be significantly changed over this transition period. These miRNA molecules are predicted to have targets within the CXCR4, PTEN, DHH, RAC and PDGF pathways, all of which have important roles in germ cell migration, proliferation and homing to the spermatogonial stem cell niche. Given the plethora of potential targets affected by each miRNA molecule, subtle changes in miRNA expression could have significant consequences e.g. tumourigenesis. The role of non-traditional oncogenes and tumour suppressors such as miRNA in TGCT is highlighted by the fact that the majority of these tumours express wild type p53, a pivotal tumour suppressor usually inactivated in cancer. While treatment of TGCTs is highly successful, the impact of these treatments on fertility means that identification of exact triggers, earlier diagnosis and alternate treatments are essential. This review examines the genetic factors and possible triggers of type II TGCT to highlight target areas for potential new treatments.

The chemokine CXCL12 and its receptor CXCR4 are implicated in human seminoma metastasis

Publisher: Wiley

Date: 15-03-2013

DOI: 10.1111/J.2047-2927.2013.00081.X

Abstract: Seminoma and non-seminoma tumours increasingly occur within the western population. These tumours originate from carcinoma in situ (CIS) cells, which arise from dysfunctional gonocytes. CXCL12 and its receptors, CXCR4 and CXCR7, have been implicated in migration, proliferation and survival of gonocytes and their precursors and progeny, primordial germ cells and spermatogonial stem cells respectively. We previously found evidence that several miRNA molecules predicted to modulate CXCR4 signalling are differentially expressed during the differentiation of gonocytes into spermatogonia in mice. Bioinformatic analysis predicted these miRNA to modulate CXCR4 signalling, leading us to hypothesize that CXCL12-mediated CXCR4 signalling is involved in the disrupted differentiation of gonocytes that underpins CIS formation. Indeed, we detected CXCL12 in Sertoli cells of normal human testis, and relatively high expression in tumour stroma with concomitant weak staining in dispersed tumour cells. In contrast, CXCR4 was expressed in spermatogonial and meiotic germ cells of normal testis and in the majority of tumour cells. Quantitative RT-PCR identified elevated CXCR4 transcript levels in seminoma compared with normal testis and to non-seminoma, potentially reflecting the higher proportion of dysfunctional germ cells within seminomas. In the normal testis, expression of CXCR4 downstream signalling molecules phospho-MEK1/2 and phospho-ERK1/2 correlated with CXCR4/CXCL12 expression. Strikingly, this correlation was absent in seminoma and non-seminoma s les, suggesting that CXCL12 signalling is disrupted. Proliferation rate and cell survival were not altered by CXCL12 in either seminoma (TCam-2) or non-seminoma (833ke) cell lines. However, CXCL12 exposure induced TCam-2 cell invasion though simulated basement membrane, while in contrast, we provide the novel evidence that CXCR4-expressing non-seminoma cell lines 833ke and NTera2/D1 do not invade in response to CXCL12. These findings indicate that CXCL12 expression in the human testis may selectively influence seminoma migration and metastasis, correlating with its importance in gonocyte and spermatogonial stem cell biology.

Inhibition of arachidonate 15-lipoxygenase (ALOX15) prevents 4HNE-induced protein damage in male germ cells

Publisher: Oxford University Press (OUP)

Date: 02-02-2017

DOI: 10.1093/BIOLRE/IOX005

Abstract: Lipid peroxidation products, such as 4-hydroxynonenal (4HNE), are causative agents responsible for extensive protein damage within the male and female germlines. Recently, we have demonstrated that 4HNE production can initiate the proteolytic degradation of the molecular chaperone Heat Shock Protein A2 (HSPA2) in male germ cells. These events may be partially responsible for HSPA2 deficiency in the spermatozoa of patients that repeatedly fail in vitro fertilization. Given this, mechanisms that limit the production of 4HNE will be highly advantageous for the preservation of male fertility. The propagation of 4HNE in somatic cells has been linked to the enzymatic actions of arachidonate 15-lipoxygenase (ALOX15), a member of the lipoxygenase family of proteins. In view of this association, this study sought to explore ALOX15 as a physiological target to manipulate the levels of 4HNE produced in the male germline. Herein, we have demonstrated that ALOX15 is markedly upregulated in response to oxidative stress in round spermatids and the GC-2 cell line. Pharmacological inhibition of ALOX15 in GC-2 cells resulted in a significant reduction in both mitochondrial and cytoplasmic reactive oxygen species, as well as a dramatic reduction in 4HNE. Importantly, the reduced bioavailability of this aldehyde appears to confer positive downstream effects to its target proteins such that HSPA2 could be protected from damage by 4HNE. Taken together, these results suggest that the actions of ALOX15 are intimately tied to the production of 4HNE. Thus, the ALOX15 protein may be a promising new target for the mitigation of germline oxidative stress.

Investigation of the expression and functional significance of the novel mouse sperm protein, a disintegrin and metalloprotease with thrombospondin type 1 motifs number 10 (ADAMTS10)

Publisher: Wiley

Date: 28-03-2012

DOI: 10.1111/J.1365-2605.2011.01235.X

Abstract: Fertilization represents the culmination of a series of complex interactions between male and female gametes. Despite advances in our understanding, the precise molecular mechanisms underlying these fundamental interactions remain largely uncharacterized. There is however growing recognition that this process requires the concerted action of multiple sperm receptors that possess affinity for complementary zona pellucida ligands and those that reside on the surface of the oolemma. Among the candidate sperm proteins that have been implicated in fertilization, those belonging to the ADAM (a disintegrin and metalloprotease) family of proteases have received considerable attention. The focus of the studies described herein has been the characterization of a closely related member of this protease family, ADAMTS10 (a disintegrin and metalloprotease with thrombospondin type 1 motifs number 10). We have demonstrated that ADAMTS10 is expressed during the later stages of mouse spermatogenesis and incorporated into the acrosomal domain of developing spermatids. During sperm maturation, the protein appears to be processed before being expressed on the surface of the peri-acrosomal region of the head. Our collective data suggest that, from this position, ADAMTS10 participates in sperm adhesion to the zona pellucida. Indeed, pre-incubation of capacitated spermatozoa with either galardin, a broad spectrum inhibitor of metalloprotease activity, or anti-ADAMTS10 antisera elicited a significant reduction in their ability to engage in zona adhesion. Overall, these studies support the notion that sperm-oocyte interactions involve considerable functional redundancy and identify ADAMTS10 as a novel candidate in the mediation of these fundamentally important events.

Diabetic ketoacidosis in patients with SARS-CoV-2: a systematic review and meta-analysis

Publisher: Springer Science and Business Media LLC

Date: 26-10-2021

DOI: 10.1186/S13098-021-00740-6

Abstract: One possible reason for increased mortality due to SARS-CoV-2 in patients with diabetes is from the complication of diabetic ketoacidosis (DKA). To re-evaluate the association of SARS-CoV-2 and development of DKA and analyse the demographic and biochemical parameters and the clinical outcomes in COVID-19 patients with DKA. A systematic review and meta-analysis. Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement was followed. Electronic databases (Proquest, Medline, Embase, Pubmed, CINAHL, Wiley online library, Scopus and Nature) were searched from 1 December 2019 to 30 June 2021 in the English language using the following keywords alone or in combination: COVID-19 OR SARS-CoV-2 AND diabetic ketoacidosis OR DKA OR ketosis OR ketonemia OR hyperglycaemic emergency OR hyperglycaemic crisis . We included studies in adults and children of all ages in all healthcare settings. Binary logistic regression model was used to explore the effect of various demographic and biochemical parameters variables on patient’s final treatment outcome (survival or death). Of the 484 papers that were identified, 68 articles were included in the systematic review and meta-analysis (54 case report, 10 case series, and 4 cohort studies). Studies involving 639 DKA patients with confirmed SARS-CoV-2 [46 (7.2%) were children and 334 (52.3%) were adults] were analyzed. The median or mean patient age ranged from 1 years to 66 years across studies. Most of the patients (n = 309, 48.3%) had pre-existing type 2 diabetes mellitus. The majority of the patients were male (n = 373, 58.4%) and belonged to Hispanic (n = 156, 24.4%) and black (n = 98, 15.3%) ethnicity. The median random blood glucose level, HbA1c, pH, bicarbonate, and anion gap in all included patients at presentation were 507 mg/dl [IQR 399–638 mg/dl], 11.4% [IQR 9.9–13.5%], 7.16 [IQR 7.00–7.22], 10 mmol/l [IQR 6.9–13 mmol/l], and 24.5 mEq/l [18–29.2 mEq/l] respectively. Mortality rate was [63/243, 25.9%], with a majority of death in patients of Hispanic ethnicity (n = 17, 27% p = 0.001). The odd ratios of death were significantly high in patients with pre-existing diabetes mellitus type 2 [OR 5.24, 95% CI 2.07–15.19 p = 0.001], old age (≥ 60 years) [OR 3.29, 95% CI 1.38–7.91 p = 0.007], and male gender [OR 2.61, 95% CI 1.37–5.17 p = 0.004] compared to those who survived. DKA is not uncommon in SARS-CoV-2 patients with diabetes mellitus and results in a mortality rate of 25.9%. Mortality key determinants in DKA patients with SARS-CoV-2 infection are in iduals with pre-existing diabetes mellitus type 2, older age [≥ 60 years old], male gender, BMI ≥ 30, blood glucose level 1000 mg/dl, and anion gap ≥ 30 mEq/l.

ENDOMETRIOSIS AND IVF: EFFECT OF PRIOR THERAPY

Publisher: Elsevier BV

Date: 02-1986

DOI: 10.1016/S0140-6736(86)90815-9

Abstract: Gene duplications alter the genetic constitution of organisms and can be a driving force of molecular evolution in humans and the great apes. In this context, the study of genomic disorders has uncovered the essential role played by the genomic architecture, especially low copy repeats (LCRs) or segmental duplications (SDs). In fact, regardless of the mechanism, LCRs can mediate or stimulate rearrangements, inciting genomic instability and generating dynamic and unstable regions prone to rapid molecular evolution. In humans, copy-number variation (CNV) has been implicated in common traits such as neuropathy, hypertension, color blindness, infertility, and behavioral traits including autism and schizophrenia, as well as disease susceptibility to HIV, lupus nephritis, and psoriasis among many other clinical phenotypes. The same mechanisms implicated in the origin of genomic disorders may also play a role in the emergence of segmental duplications and the evolution of new genes by means of genomic and gene duplication and triplication, exon shuffling, exon accretion, and fusion/fission events.

Immunology: The indirect immunobead test for seminal antisperm antibodies and fertilization rates at in-vitro fertifization

Publisher: Oxford University Press (OUP)

Date: 07-1996

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A019411

Abstract: A series of 183 patients with positive indirect immunobead tests on semen was studied to determine the correlation in semen between specific antibody types, binding sites, antibody concentration, and fertilizing ability. IgM was present in only 44 ejaculates and was present in sufficient quantity to cause significant binding to immunobeads (i.e. >20% of motile donor spermatozoa) in only three of them. There was no correlation between the percentages of motile donor spermatozoa that bound IgA and IgG immunobeads but the two classes of beads generally bound to the same region of the spermatozoa. A total of 63 couples went on to attempt in-vitro fertilization (IVF) treatment, all with mature eggs recovered. Of these mature eggs, 44% were fertilized and cleaved normally in comparison to 68% in a group of patients with tubal disease. Fertilization rates in in iduals followed a bimodal distribution with a substantial number of couples experiencing zero or very poor rates (0-20%), the mode for the remainder lying between 60 and 80%. The fertilization rate tended to decrease as the amount of antibody increased. The percentage of donor spermatozoa that bound to immunobeads, taken as the greater of IgA and IgG, was selected by logistic regression as a significant predictor of poor fertilization (rate <=25%). The predictive power of the equation was improved by including the motile normal sperm concentration but the equation could only account for a small proportion of the total variation in fertilization rate. The presence of antibodies to the sperm head was highly correlated with the antibody concentration but was not selected as a predictor of fertilization. We conclude that the nature of the antigen against which the seminal antisperm antibody is directed may be as important as the antibody concentration in affecting sperm function. There seems to be little practical value in measuring IgM in seminal plasma.

Ovarian ageing: Where are we now? And where to next?

Publisher: Elsevier BV

Date: 06-2021

DOI: 10.1016/J.COEMR.2021.01.005

The association between preoperative anemia, blood transfusion need, and postoperative complications in adult cardiac surgery, a single center contemporary experience

Publisher: Springer Science and Business Media LLC

Date: 07-01-2023

DOI: 10.1186/S13019-023-02132-5

Differential Roles of HOW in Male and Female Drosophila Germline Differentiation

Publisher: Public Library of Science (PLoS)

Date: 06-12-2011

DOI: 10.1371/JOURNAL.PONE.0028508

miRNA and mammalian male germ cells

Publisher: Oxford University Press (OUP)

Date: 11-10-2012

DOI: 10.1093/HUMUPD/DMR041

Abstract: BACKGROUND Achieving the correct spatial and temporal expression of germ-cell-specific genes is fundamental to the production of viable healthy spermatozoa. Notably, post-transcriptional gene regulation resulting in the repression of protein translation is central to many embryonic processes, and is particularly active during spermatogenesis. In this review, we discuss microRNA (miRNA) regulation of target gene expression in relation to mammalian spermatogenesis, the establishment of testicular germ cell tumours (TGCT) and the potential use of miRNA manipulation for cancer therapy and fertility regulation. METHODS Journal databases such as PubMed were searched using key words, including miRNA, testis, spermatogenesis, germ cell, testicular cancer and cancer. RESULTS In the past decade, the deployment of small non-coding RNA molecules, including miRNA, by the cell, has been recognized as among the most important mechanisms of fine-tuning translational regulation in differentiating cell types. For key regulators of male gametogenesis, high levels of gene expression do not always correspond to elevated levels of protein expression. Cumulatively this indicates that enhancement and repression of post-transcriptional regulatory mechanisms are essential to the success of spermatogenesis. There is also growing evidence that this form of regulation contributes to the aetiology of both TGCT and spermatocytic tumours. CONCLUSIONS miRNA plays an essential role in regulation of genes during the process of spermatogenesis. Disruption of this regulation has the ability to contribute to the neoplastic development of germ cell tumours. However, targeted knockdown of specific miRNA molecules has the potential to form both anti-oncogenic reagents and underpin the basis for novel contraceptive technologies.

The in vivo expression of dipeptidyl peptidases 8 and 9

Publisher: SAGE Publications

Date: 06-07-2009

DOI: 10.1369/JHC.2009.953760

Abstract: The dipeptidyl peptidase IV (DPIV) enzyme family contains both potential and proven therapeutic targets. Recent reports indicate the presence of DP8 and DP9 in peripheral blood lymphocytes, testis, lung, and brain. For a more comprehensive understanding of DP8 and DP9 tissue and cellular expression, mRNA and enzyme activity were examined. Many organs from C57BL/6 wild-type and DPIV gene-knockout mice were examined DP8/9 enzyme activity was detected in the immune system, brain, testis, muscle, and epithelia. In situ hybridization localized DP8 and DP9 mRNA to lymphocytes and epithelial cells in liver, gastrointestinal tract, lymph node, spleen, and lung. DP8 and DP9 mRNA was detected in baboon and mouse testis, and DP9 expression was elevated in human testicular cancers. DP8 and DP9 mRNA were ubiquitous in day 17 mouse embryo, with greatest expression in epithelium (skin and gastrointestinal tract) and brain. Thus, DP8 and DP9 are widely expressed enzymes. Their expression in lymphocytes and epithelia indicates potential for roles in the digestive and immune systems. This manuscript contains online supplemental material at www.jhc.org . Please visit this article online to view these materials.

Adenosine triphosphate and motility characteristics of fresh and cryopreserved human spermatozoa

Publisher: Wiley

Date: 02-1994

DOI: 10.1111/J.1365-2605.1994.TB01203.X

Abstract: The concentration of ATP and the motility of human spermatozoa was measured in fresh and cryopreserved cells from the same 15 ejaculates. No coherent picture of the relationship between motility and ATP concentration emerged in whole semen or in spermatozoa washed by repeated centrifugation and resuspension in Biggers Whitten and Whittingham medium. This may have been due to the presence of dead spermatozoa and contaminating cells. After preparation on a Percoll gradient, the ATP concentration in fresh and cryopreserved spermatozoa was the same (6 +/- 0.7 nmol/10(8) spermatozoa) but 85 +/- 2.5% of the fresh spermatozoa were progressively motile with an average path velocity of 55 +/- 3.5 microns/s compared to corresponding values of 33 +/- 5.3% and 44 +/- 3.4 microns/s in frozen/thawed spermatozoa. This suggests that the poor motility of cryopreserved spermatozoa does not result from deficient ATP production. No relationship was found between ATP concentration and the ability of motile spermatozoa in the ejaculate to survive freezing.

Biochemical Characterization of Caenorhabditis elegans Ferritins

Publisher: American Chemical Society (ACS)

Date: 04-04-2023

DOI: 10.1021/ACS.BIOCHEM.3C00005

The RNA-binding protein Musashi-2 (MSI2) controls mRNA processing and translational regulation via interactions with SFPQ and PIWIL1 during mammalian spermatogenesis

Publisher: Bioscientifica

Date: 23-09-2014

DOI: 10.1530/REPABS.1.P222

Identification and characterization of a novel Mt-retrotransposon highly represented in the female mouse germline

Publisher: Elsevier BV

Date: 04-2006

DOI: 10.1016/J.YGENO.2005.08.015

Abstract: The control of primordial follicle recruitment into the growing follicle population is a major limiting process in female reproduction. In order to gain insight into the molecular processes occurring at the time of primordial follicle activation, a subtractive hybridization analysis was performed between cDNAs prepared from temporally distinct mouse neonatal ovarian tissues that differed according to the state of primordial follicle activation. One highly represented clone associated with activation was an Mt retrotransposon-like sequence designated Mtfull, which was subsequently cloned and determined to be novel and restricted in expression to the ovary. The polyadenylated 1684-bp sequence has long terminal repeats, is predicted to be noncoding, and is the predominant Mti-related sequence present in the mouse ovary. In situ hybridization further localized Mtfull expression to the oocyte and confirmed that expression is concomitant with follicle activation. Together with in silico data, we predict Mtfull plays an essential role in folliculogenesis through regulation of gene expression.

Is there a role for immunocontraception?

Publisher: Elsevier BV

Date: 03-2011

DOI: 10.1016/J.MCE.2010.04.004

Abstract: The world's population is continuing to grow at an alarming rate and yet no novel methods of contraception have been introduced since 1960s. The paucity of our current contraceptive armoury is indicated by the 46 million abortions that are performed each year, largely in developing countries where population growth is greatest. Thus, whatever new forms of fertility control we develop for the next millennium, the particular needs of developing countries should be borne in mind. Contraceptive vaccines have the potential to provide safe, effective, prolonged, reversible protection against pregnancy in a form that can be easily administered in the Third World. In this review we consider the contraceptive targets that might be pursued, how vaccines might be engineered and the problems generated by inter-in idual variations in antibody titre. We conclude that the specifications for a safe, effective, reversible vaccine are more likely to be met in animals than man.

Hidden gems in the niche: a new approach to the study of spermatogonial stem cells

Publisher: Medknow

Date: 14-01-2013

DOI: 10.1038/AJA.2012.150

Damaging legacy: maternal cigarette smoking has long-term consequences for male offspring fertility

Publisher: Oxford University Press (OUP)

Date: 30-09-2014

DOI: 10.1093/HUMREP/DEU235

Abstract: What are the effects on fertility of cigarette smoke-induced toxicity on male offspring exposed during the gestational/weaning period? Maternal cigarette smoke exposure during the gestational/weaning period causes long-term defects in male offspring fertility. Cigarette smoke is a well-known reproductive toxicant which is particularly harmful to both fetal and neonatal germ cells. However, recent studies suggest a significant portion of young mothers in the developed world still smoke during pregnancy. In the context of male reproductive health, our understanding of the effects of in utero exposure on offspring fertility is limited. In this study, 27 C57BL/6 5-week-old female mice were exposed via the nose-only to cigarette smoke (treatment) or 27 were exposed to room air (control) for 6 weeks before being housed with stud males to produce litters. In the treatment group, smoke exposure continued throughout mating, pregnancy and lactation until weaning of pups at 21 days post birth. Male offspring were examined at post-natal days 3, 6, 12, 21 and 98 (adult). Approximately 108 maternal smoke-exposed C57BL/6 offspring and controls were examined. Spermatogenesis was examined using testicular histology and apoptosis/DNA damage was assessed using caspase immunohistochemistry and TUNEL. Sertoli cell morphology and fluctuations in the spermatogonial stem cell population were also examined using immunohistochemistry. Microarray and QPCR analysis were performed on adult testes to examine specific long-term transcriptomic alteration as a consequence of maternal smoke exposure. Sperm counts and motility, zona/oolemma binding assays, COMET analysis and mitochondrial genomic sequencing were also performed on spermatozoa obtained from adult treated and control mice. Fertility trials using exposed adult male offspring were also performed. Maternal cigarette smoke exposure caused increased gonocyte and meiotic spermatocyte apoptosis (P < 0.01) as well as germ cell depletion in the seminiferous tubules of neonatal and juvenile offspring. Aberrant testicular development characterized by abnormal Sertoli and germ cell organization, a depleted spermatogonial stem cell population (P < 0.01), atrophic seminiferous tubules and increased germ cell DNA damage (P < 0.01) persisted in adult offspring 11 weeks after exposure. Microarray analysis of adult offspring testes associated these defects with meiotic germ cell development, sex hormone metabolism, oxidative stress and Sertoli cell signalling. Next generation sequencing also revealed a high mitochondrial DNA mutational load in the testes of adult offspring (P < 0.01). Adult maternal smoke-exposed offspring also had reduced sperm counts with spermatozoa exhibiting morphological abnormalities (P < 0.01), affecting motility and fertilization potential. Odf2, a spermatozoa flagellum component required for coordinated ciliary beating, was also significantly down-regulated (P < 0.01) in maternal smoke-exposed adult offspring, with aberrant localization along the spermatozoa flagellum. Adult maternal smoke-exposed offspring took significantly longer to impregnate control females and had a slight but significant (P < 0.01) reduction in litter size. This study examined only one species (mouse) using a smoking model which only simulates human cigarette smoke exposure. This study represents the first comprehensive animal model of maternal smoking on male offspring reproductive function, suggesting that exposure during the gestational/weaning period causes long-term defects in male offspring fertility. This is due to a compromised spermatogonial stem cell population resulting from gonocyte apoptosis and impaired spermatogenic development. This results in significant germ cell damage and Sertoli cell dysfunction, impacting germ cell number, tubule organization, DNA damage and spermatozoa in adult offspring. This study strengthens the current literature suggesting that maternal exposure impairs male offspring fertility, which is currently debated due to conflicting studies. This study was funded by the Australian Research Council, Hunter Medical Research Institute, National Health and Medical Research Council of Australia and the Newcastle Permanent Building Society Charitable Trust. The authors declare no conflict of interest.

Accreditation standards and guidelines for IVF laboratories

Publisher: Informa UK Limited

Date: 2000

DOI: 10.1080/1464727002000198941

INTRAUTERINE INSEMINATION

Publisher: Elsevier BV

Date: 1987

DOI: 10.1016/S0140-6736(87)90083-3

Abstract: Dental healthcare workers (DHCWs) are at high risk of occupational exposure to droplets and aerosol particles emitted from patients' mouths during treatment. We evaluated the effectiveness of an air cleaner in reducing droplet and aerosol contamination by positioning the device in four different locations in an actual dental clinic. We applied computational fluid dynamics (CFD) methods to solve the governing equations of airflow, energy and dispersion of different-sized airborne droplets/aerosol particles. In a dental clinic, we measured the supply air velocity and temperature of the ventilation system, the airflow rate and the particle removal efficiency of the air cleaner to determine the boundary conditions for the CFD simulations. Our results indicate that use of an air cleaner in a dental clinic may be an effective method for reducing DHCWs' exposure to airborne droplets and aerosol particles. Further, we found that the probability of droplet/aerosol particle removal and the direction of airflow from the cleaner are both important control measures for droplet and aerosol contamination in a dental clinic. Thus, the distance between the air cleaner and droplet/aerosol particle source as well as the relative location of the air cleaner to both the source and the DHCW are important considerations for reducing DHCWs' exposure to droplets/aerosol particles emitted from the patient's mouth during treatments.

130. CONDITIONAL TARGETED DELETIONS OF STAT3 TO IDENTIFY ITS ROLE IN OOCYTESAND GRANULOSA CELLS

Publisher: CSIRO Publishing

Date: 2010

DOI: 10.1071/SRB10ABS130

Abstract: Signal transducers and activators of transcription-3 (STAT3) is a transcription factor activated by JAK kinases after cytokine-receptor binding. We have identified active phospho-STAT3 in granulosa cells of ovarian follicles from the very early activated stage. Abundant STAT3 has also been identified by us and others in oocytes, cumulus and granulosa cells of mature preovulatory follicles. Further, we have found the STAT responsive gene product SOCS4 is present in granulosa cells of early activated and growing follicles (1). To determine the role of STAT3 in follicle growth and ovulation we have generated three unique lines of conditional STAT3 null mice with STAT3 deletion in granulosa cells or oocytes. Mice with STAT3 gene sequenced flanked by LoxP elements (STAT3fl/fl) were crossed with lines expressing Cre-recombinase in granulosa cells (AmhR2-Cre, FSHR-Cre) or oocytes (ZP3-Cre). Fertility analysis of STAT3fl/fl Zp3-Cre females crossed with wildtype males showed no effect on fertility from STAT3 deletion in the oocyte. In contrast, both FSHR-Cre and AmhR2-Cre mediated granulosa deficient STAT3 lines had significantly reduced litter sizes 1.4-fold and 1.9-fold lower respectively compared to control littermates (STAT3fl/+ Cre+ or STAT3fl/fl Cre-). Furthermore AmhR2-Cre mediated STAT3 deletion resulted in a significant 1.9-fold reduction in litters per month and an increased time to first litter. Surprisingly we did not find any difference in ovulation rate after eCG+hCG stimulated superovulation, nor in naturally cylcing mice. Zygotes flushed the morning after mating and placed into culture showed no deficit in cleavage of 2-cell embryos or development to blastocyst. A potential uterine deficiency was investigated and gross morphological observations indicate a defect in the uteri which is consistent with the recent report of Cre-recombinase expression in uterine cells derived from the Mullerian duct mesenchyme (2). Thus STAT3 deficiency in oocytes does not lead to infertility while AmhR2-Cre and FSHR-Cre mediated STAT3 deficiency leads to a sub-fertile phenotype. (1) Keightley RA, et al. 2009 Reprod, Fertil Dev 21(Suppl.): 509.(2) Gonzalez G, Behringer RR. 2009 Mol Reprod Dev 76: 678–688.

Expression of rabbit IL-4 by recombinant myxoma viruses enhances virulence and overcomes genetic resistance to myxomatosis.

Publisher: Elsevier BV

Date: 06-2004

DOI: 10.1016/J.VIROL.2004.02.031

Dynamin 2 is essential for mammalian spermatogenesis

Publisher: Springer Science and Business Media LLC

Date: 11-10-2016

DOI: 10.1038/SREP35084

Abstract: The dynamin family of proteins play important regulatory roles in membrane remodelling and endocytosis, especially within brain and neuronal tissues. In the context of reproduction, dynamin 1 (DNM1) and dynamin 2 (DNM2) have recently been shown to act as key mediators of sperm acrosome formation and function. However, little is known about the roles that these proteins play in the developing testicular germ cells. In this study, we employed a DNM2 germ cell-specific knockout model to investigate the role of DNM2 in spermatogenesis. We demonstrate that ablation of DNM2 in early spermatogenesis results in germ cell arrest during prophase I of meiosis, subsequent loss of all post-meiotic germ cells and concomitant sterility. These effects become exacerbated with age, and ultimately result in the demise of the spermatogonial stem cells and a Sertoli cell only phenotype. We also demonstrate that DNM2 activity may be temporally regulated by phosphorylation of DNM2 via the kinase CDK1 in spermatogonia, and dephosphorylation by phosphatase PPP3CA during meiotic and post-meiotic spermatogenesis.

Letters to the Editor

Publisher: Oxford University Press (OUP)

Date: 10-2000

DOI: 10.1093/HUMREP/15.10.2248

Staying Alive: PI3K Pathway Promotes Primordial Follicle Activation and Survival in Response to 3MC-Induced Ovotoxicity

Publisher: Oxford University Press (OUP)

Date: 12-04-2012

DOI: 10.1093/TOXSCI/KFS137

Abstract: 3-Methylcholanthrene (3MC) is a potent ovotoxicant capable of causing premature ovarian failure through primordial follicle depletion. Despite 3MCs ovotoxicity having been established for 30 years, relatively little information exists on the mechanisms. In this study, we examined the effects of 3MC exposure on the immature ovarian follicle population. Microarray analysis revealed a complex mechanism of 3MC-induced ovotoxicity involving a number of cellular processes associated with xenobiotic metabolism, ovarian cancer, cell cycle progression, and cell death. 3MC exposure was also found to induce developing follicle atresia and aberrant primordial follicle activation via the stimulation of PI3K/Akt and mammalian target of rapamycin (mTOR) signaling pathways. Inhibition of PI3K/Akt signaling resulted in the severe depletion of the primordial follicle pool, with further analysis identifying increased Akt1-stimulated Bad phosphoinhibition in 3MC-treated primordial follicles. Our results suggest that the primordial follicle pool enters a "prosurvival" state upon 3MC exposure and that its depletion is due to a vicious cycle of primordial follicle activation in an attempt to replace developing follicles undergoing follicular atresia.

Proteomic and functional analysis of human sperm detergent resistant membranes

Publisher: Wiley

Date: 22-07-2007

DOI: 10.1002/JCP.22615

Abstract: Mammalian spermatozoa attain the ability to fertilize an oocyte as they negotiate the female reproductive tract. This acquisition of functional competence is preceded by an intricate cascade of biochemical and functional changes collectively known as "capacitation." Among the universal correlates of the capacitation process is a remarkable remodeling of the lipid and protein architecture of the sperm plasma membrane. While the mechanisms that underpin this dynamic reorganization remain enigmatic, emerging evidence has raised the prospect that it may be coordinated, in part, by specialized membrane microdomains, or rafts. In the present study we have demonstrated that human spermatozoa express recognized markers of membrane rafts. Further, upon depletion of membrane cholesterol through either physiological (capacitation) or pharmacological (methyl-β-cyclodextrin) intervention, these membrane rafts appear to undergo a polarized redistribution to the peri-acrosomal region of the sperm head. This finding encourages speculation that membrane rafts represent platforms for the organization of proteins involved in sperm-oocyte interactions. Support for this notion rests with the demonstration that membrane rafts isolated on the basis of their biochemical composition in the form of detergent resistant membranes (DRMs), possess the ability to adhere to homologous zona pellucidae. Furthermore a comprehensive proteomic analysis of the DRMs identified a number of proteins known for their affinity for the zona pellucida in addition to other candidates putatively involved in the mediation of downstream binding and/or fusion with the oolemma. Collectively these data afford novel insights into the subcellular localization and potential functions of membrane rafts in human spermatozoa.

Surface determination through atomically resolved secondary-electron imaging

Publisher: Springer Science and Business Media LLC

Date: 17-06-2015

DOI: 10.1038/NCOMMS8358

Abstract: Unique determination of the atomic structure of technologically relevant surfaces is often limited by both a need for homogeneous crystals and ambiguity of registration between the surface and bulk. Atomically resolved secondary-electron imaging is extremely sensitive to this registration and is compatible with faceted nanomaterials, but has not been previously utilized for surface structure determination. Here we report a detailed experimental atomic-resolution secondary-electron microscopy analysis of the c(6 × 2) reconstruction on strontium titanate (001) coupled with careful simulation of secondary-electron images, density functional theory calculations and surface monolayer-sensitive aberration-corrected plan-view high-resolution transmission electron microscopy. Our work reveals several unexpected findings, including an amended registry of the surface on the bulk and strontium atoms with unusual seven-fold coordination within a typically high surface coverage of square pyramidal TiO 5 units. Dielectric screening is found to play a critical role in attenuating secondary-electron generation processes from valence orbitals.

Expression of extracellular superoxide dismutase in the human male reproductive tract, detected using antisera raised against a recombinant protein

Publisher: Oxford University Press (OUP)

Date: 03-1998

DOI: 10.1093/MOLEHR/4.3.235

Abstract: Mammalian spermatozoa are particularly susceptible to the deleterious effects of reactive oxygen species and lipid peroxidation, which ultimately lead to impaired fertility. A number of enzymes are present in the male reproductive tract which may play a role in preventing oxidative damage in particular, the epididymis is the site of synthesis and secretion of large amounts of extracellular superoxide dismutase (eSOD). In order to study the distribution of eSOD in the male reproductive tract, and distinguish it from other related superoxide dismutase isoenzymes (e.g. cytosolic SOD), polyclonal antisera have been raised against a recombinant human eSOD fusion protein, expressed in bacterial cells. This protein was expressed from a synthetic gene fragment, using preferred Escherichia coli codons, designed to overcome the problems associated with the high guanine+cytosine content of the natural human eSOD transcript. Using this antiserum, eSOD can be readily detected in a range of human reproductive tissues as well as in human seminal plasma. However, the presence of similar levels of eSOD in the seminal plasma of vasectomized men (probably of prostatic origin) precludes its use as a simple diagnostic indicator of eSOD activity levels in the epididymis.

Motoring through: the role of kinesin superfamily proteins in female meiosis

Publisher: Oxford University Press (OUP)

Date: 18-04-2017

DOI: 10.1093/HUMUPD/DMX010

Abstract: The kinesin motor protein family consists of 14 distinct subclasses and 45 kinesin proteins in humans. A large number of these proteins, or their orthologues, have been shown to possess essential function(s) in both the mitotic and the meiotic cell cycle. Kinesins have important roles in chromosome separation, microtubule dynamics, spindle formation, cytokinesis and cell cycle progression. This article contains a review of the literature with respect to the role of kinesin motor proteins in female meiosis in model species. Throughout, we discuss the function of each class of kinesin proteins during oocyte meiosis, and where such data are not available their role in mitosis is considered. Finally, the review highlights the potential clinical importance of this family of proteins for human oocyte quality. To examine the role of kinesin motor proteins in oocyte meiosis. A search was performed on the Pubmed database for journal articles published between January 1970 and February 2017. Search terms included 'oocyte kinesin' and 'meiosis kinesin' in addition to in idual kinesin names with the terms oocyte or meiosis. Within human cells 45 kinesin motor proteins have been discovered, with the role of only 13 of these proteins, or their orthologues, investigated in female meiosis. Furthermore, of these kinesins only half have been examined in mammalian oocytes, despite alterations occurring in gene transcripts or protein expression with maternal ageing, cryopreservation or behavioral conditions, such as binge drinking, for many of them. Kinesin motor proteins have distinct and important roles throughout oocyte meiosis in many non-mammalian model species. However, the functions these proteins have in mammalian meiosis, particularly in humans, are less clear owing to lack of research. This review brings to light the need for more experimental investigation of kinesin motor proteins, particularly those associated with maternal ageing, cryopreservation or exposure to environmental toxicants.

119. IDENTIFICATION AND CHARACTERISATION OF SURFACE PROTEIN COMPLEXES IN HUMAN SPERMATOZOA

Publisher: CSIRO Publishing

Date: 2010

DOI: 10.1071/SRB10ABS119

Abstract: A unique characteristic of mammalian spermatozoa is that upon ejaculation, they are unable to recognise and bind to an ovulated oocyte. These functional attributes are only realised following the sperms ascent of the female reproductive tract whereupon they undergo a myriad of biochemical and biophysical changes collectively referred to as ‘capacitation’. Since spermatozoa are both transcriptionally and translationally quiescent cells, this functional transformation must be engineered by a combination of post-translational modification and spatial reorganisation of existing sperm proteins. Indeed, evidence from our laboratory suggests that a key attribute of capacitation is the remodeling of the sperm surface architecture leading to the assembly and / or presentation of multimeric sperm-oocyte receptor complex(es). Through the novel application of Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE), we have secured the first direct evidence that human spermatozoa express a number of these protein complexes on their surface. Furthermore, we have demonstrated that a subset of these complexes harbour putative zona adhesion proteins and display strong affinity for solubilised zona pellucidae. In this study, we have extended our findings through the characterisation of one such complex containing arylsulfatase A (ASA), a protein with recognised affinity for sulfated ligands present within the zona pellucida. Through the application of immunohistochemistry and flow cytometry we revealed that ASA undergoes a capacitation-associated translocation to become expressed on the apical region of the human sperm head, a location compatible with a role in the mediation of sperm-zona pellucida interactions. This dramatic relocation was completely abolished by incubation of capacitating spermatozoa in exogenous cholesterol, suggesting that it may be driven in part by alteration in the membrane fluidity characteristics. Our current research is focused on confirming the role of ASA in human sperm-zona pellucida adhesion and elucidating the precise cellular mechanisms that underpin the proteins translocation to the cell surface.

A New Understanding, Guided by Single-Cell Sequencing, of the Establishment and Maintenance of the Ovarian Reserve in Mammals

Publisher: S. Karger AG

Date: 19-09-2023

DOI: 10.1159/000526426

Abstract: b i Background: /i /b Oocytes are a finite and non-renewable resource that are maintained in primordial follicle structures. The ovarian reserve is the totality of primordial follicles, present from birth, within the ovary and its establishment, size, and maintenance dictates the duration of the female reproductive lifespan. Understanding the cellular and molecular dynamics relevant to the establishment and maintenance of the reserve provides the first steps necessary for modulating both in idual human and animal reproductive health as well as population dynamics. b i Summary: /i /b This review details the key stages of establishment and maintenance of the ovarian reserve, encompassing germ cell nest formation, germ cell nest breakdown, and primordial follicle formation and activation. Furthermore, we spotlight several formative single-cell sequencing studies that have significantly advanced our knowledge of novel molecular regulators of the ovarian reserve, which may improve our ability to modulate female reproductive lifespans. b i Key Messages: /i /b The application of single-cell sequencing to studies of ovarian development in mammals, especially when leveraging genetic and environmental models, offers significant insights into fertility and its regulation. Moreover, comparative studies looking at key stages in the development of the ovarian reserve across species has the potential to impact not just human fertility, but also conservation biology, invasive species management, and agriculture.

Mechanisms of tethering and cargo transfer during epididymosome-sperm interactions

Publisher: Springer Science and Business Media LLC

Date: 18-04-2019

DOI: 10.1186/S12915-019-0653-5

126. JAK/STAT SIGNALLING IN FOLLICULOGENESIS

Publisher: CSIRO Publishing

Date: 2010

DOI: 10.1071/SRB10ABS126

Abstract: Primordial follicle activation marks the first stage of pre-pubertal ovarian folliculogenesis, and is therefore fundamental to female fertility. Entry into development is initiated by a group of pleiotropic cytokines and growth factors, originating in and acting upon both the oocyte and granulosa support cells of the ovarian follicle through the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signalling pathway. Pivotal to this process is the transcriptional regulation of target genes via STAT complexes and negative regulation by the Suppressors of Cytokine Signalling (SOCS) family of proteins. Preliminary evidence indicates that STAT3 facilitates the activation of primordial follicles, while SOCS4 counterbalances the activity of STAT3, mediating the controlled release of primordial follicles into the growing pool throughout reproductive life. Leukemia Inhibitory Factor (LIF) has been previously demonstrated as a key granulosa cell derived cytokine involved in inducing primordial follicle activation. Through both quantitative gene expression (qPCR) and immunoblotting we have demonstrated that LIF can significantly upregulate STAT3 mRNA production (~2-fold) as well as increase STAT3 protein phosphorylation within neonatal mouse ovarian explants culture. Furthermore, through the generation of a recombinant SOCS4 protein construct, and its use in subsequent protein-protein pull-downs, we were able to multiple targets involved in oocyte maturation, STAT3 interactions, and JAK/STAT signaling. These targets were also found to be significantly upregulated via qPCR analysis in neonatal mouse ovaries treated with LIF. These results support our current model for the involvement of STAT3 and SOCS4 in a basic negative feedback loop within the JAK/STAT signalling pathway that results in the regulation of primordial follicle activation and development.

121. SHORT TERM XENOBIOTIC EXPOSURE COMPROMISES LONG TERM OOCYTE VIABILITY

Publisher: CSIRO Publishing

Date: 2010

DOI: 10.1071/SRB10ABS121

Abstract: Mammalian females are born with a finite number of non-renewing primordial follicles, the majority of which remain in a quiescent state for many years. These follicles serve as the primary source of all developing oocytes in the ovary, and cannot be regenerated post fetal development. Due to their non-renewing nature, these “resting” oocytes are particularly vulnerable to environmental and toxic insults, especially to those which are capable of inducing oxidative stress. Recent evidence suggests that certain synthetic chemical compounds, known as xenobiotics, have the potential to generate oxidative stress through the production of free oxygen radicals (ROS) as a byproduct of the cell’s detoxification process. Given the redox sensitive nature of the mammalian oocyte, we hypothesise that xenobiotic exposure may have adverse effects on long term oocyte viability. In this study, we attempted to identify the effects of short term xenobiotic exposure on long term oocyte viability. Female Swiss neonatal mice (day 4) were administered 7 daily consecutive doses of 4-Vinylcyclohexene diepoxide (40mg/kg/daily 80mg/kg/daily) Methoxychlor (50mg/kg/daily 100mg/kg/daily) or Menadione (7.5mg/kg/daily 15mg/kg/daily). Mice were then superovulated at 6wks and their oocytes collected for analysis. Sperm-egg fusion assays revealed a significant decrease (P 0.01) in sperm egg binding (1.4–7 fold) and fusion (4–20 fold) in a dose dependent manner for all three xenobiotic treatments in vivo, signifying a decrease in oocyte membrane fluidity. Follow-up lipid peroxidation analysis on xenobiotic cultured oocytes also showed a significant (P 0.01) dose dependent increase (1.3–2.5 fold) in membrane lipid peroxidation for each xenobiotic compared to the control. These results provide some of the first evidence of short term xenobiotic exposure causing long term oocyte dysfunction, possibly interfering with the fluidity and/or elasticity of the oocyte plasma membrane through xenobiotic ROS induced lipid peroxidation.

The lipid peroxidation product 4-hydroxynonenal contributes to oxidative stress-mediated deterioration of the ageing oocyte

Publisher: Springer Science and Business Media LLC

Date: 24-07-2017

DOI: 10.1038/S41598-017-06372-Z

Abstract: An increase in intraovarian reactive oxygen species (ROS) has long been implicated in the decline in oocyte quality associated with maternal ageing. Oxidative stress (OS)-induced lipid peroxidation and the consequent generation of highly electrophilic aldehydes, such as 4-hydroxynonenal (4-HNE), represents a potential mechanism by which ROS can inflict damage in the ageing oocyte. In this study, we have established that aged oocytes are vulnerable to damage by 4-HNE resulting from increased cytosolic ROS production within the oocyte itself. Further, we demonstrated that the age-related induction of OS can be recapitulated by exposure of germinal vesicle (GV) oocytes to exogenous H 2 O 2 . Such treatments stimulated an increase in 4-HNE generation, which remained elevated during in vitro oocyte maturation to metaphase II. Additionally, exposure of GV oocytes to either H 2 O 2 or 4-HNE resulted in decreased meiotic completion, increased spindle abnormalities, chromosome misalignments and aneuploidy. In seeking to account for these data, we revealed that proteins essential for oocyte health and meiotic development, namely α-, β-, and γ-tubulin are vulnerable to adduction via 4-HNE. Importantly, 4-HNE-tubulin adduction, as well as increased aneuploidy rates, were resolved by co-treatment with the antioxidant penicillamine, demonstrating a possible therapeutic mechanism to improve oocyte quality in older females.

Structure Retrieval at Atomic Resolution in the Presence of Multiple Scattering of the Electron Probe

Publisher: American Physical Society (APS)

Date: 26-12-2018

DOI: 10.1103/PHYSREVLETT.121.266102

Addressing preservation of elastic contrast in energy-filtered transmission electron microscopy

Publisher: Elsevier BV

Date: 03-2016

DOI: 10.1016/J.ULTRAMIC.2015.10.001

Assessment of microRNA expression in mouse epididymal epithelial cells and spermatozoa by next generation sequencing

Publisher: Elsevier BV

Date: 12-2015

DOI: 10.1016/J.GDATA.2015.09.012

Drosophila Rbp6 Is an Orthologue of Vertebrate Msi-1 and Msi-2, but Does Not Function Redundantly with dMsi to Regulate Germline Stem Cell Behaviour

Publisher: Public Library of Science (PLoS)

Date: 27-11-2012

DOI: 10.1371/JOURNAL.PONE.0049810

Redox activity associated with the maturation and capacitation of mammalian spermatozoa

Publisher: Elsevier BV

Date: 04-2004

DOI: 10.1016/J.FREERADBIOMED.2004.01.017

The APC/C activator FZR1 is essential for meiotic prophase I in mice

Publisher: The Company of Biologists

Date: 15-03-2014

DOI: 10.1242/DEV.104828

Abstract: Fizzy-related 1 (FZR1) is an activator of the Anaphase promoting complex/cyclosome (APC/C) and an important regulator of the mitotic cell ision cycle. Using a germ-cell-specific conditional knockout model we examined its role in entry into meiosis and early meiotic events in both sexes. Loss of APC/CFZR1 activity in the male germline led to both a mitotic and a meiotic testicular defect resulting in infertility due to the absence of mature spermatozoa. Spermatogonia in the prepubertal testes of such mice had abnormal proliferation and delayed entry into meiosis. Although early recombination events were initiated, male germ cells failed to progress beyond zygotene and underwent apoptosis. Loss of APC/CFZR1 activity was associated with raised cyclin B1 levels, suggesting that CDK1 may trigger apoptosis. By contrast, female FZR1Δ mice were subfertile, with premature onset of ovarian failure by 5 months of age. Germ cell loss occurred embryonically in the ovary, around the time of the zygotene-pachytene transition, similar to that observed in males. In addition, the transition of primordial follicles into the growing follicle pool in the neonatal ovary was abnormal, such that the primordial follicles were prematurely depleted. We conclude that APC/CFZR1 is an essential regulator of spermatogonial proliferation and early meiotic prophase I in both male and female germ cells and is therefore important in establishing the reproductive health of adult male and female mammals.

The RNA-binding protein Musashi is required intrinsically to maintain stem cell identity

Publisher: Proceedings of the National Academy of Sciences

Date: 30-05-2006

DOI: 10.1073/PNAS.0600906103

Abstract: A key goal of regenerative medicine is an understanding of the genetic factors that define the properties of stem cells. However, stem cell research in mammalian tissue has been h ered by a paucity of stem cell-specific markers. Although increasing evidence suggests that members of the Musashi (Msi) family of RNA-binding proteins play important functions in progenitor cells, it remains unclear whether there is a stem cell-autonomous requirement for Msi because of an inability to distinguish stem cells from early-lineage cells in mammalian tissues. Here, using the Drosophila testis as a model system for the study of stem cell regulation, we show specific evidence for a cell-autonomous requirement for Msi family proteins in regulating stem cell differentiation, leading to the identification of an RNA-binding protein required for spermatogonial stem cell maintenance. We found that loss of Msi function disrupts the balance between germ-line stem cell renewal and differentiation, resulting in the premature differentiation of germ-line stem cells. Moreover, we found that, although Msi is expressed in both somatic and germ cells, Msi function is required intrinsically in stem cells for maintenance of stem cell identity. We also discovered a requirement for Msi function in male meiosis, revealing that Msi has distinct roles at different stages of germ cell differentiation. We describe the complementary expression patterns of the murine Msi paralogues Msi1 and Msi2 during spermatogenesis, which support the idea of distinct, evolutionarily conserved roles of Msi.

Exploiting Machine Learning Algorithms and Methods for the Prediction of Agitated Delirium After Cardiac Surgery: Models Development and Validation Study

Publisher: JMIR Publications Inc.

Date: 23-10-2019

DOI: 10.2196/14993

Abstract: Delirium is a temporary mental disorder that occasionally affects patients undergoing surgery, especially cardiac surgery. It is strongly associated with major adverse events, which in turn leads to increased cost and poor outcomes (eg, need for nursing home due to cognitive impairment, stroke, and death). The ability to foresee patients at risk of delirium will guide the timely initiation of multimodal preventive interventions, which will aid in reducing the burden and negative consequences associated with delirium. Several studies have focused on the prediction of delirium. However, the number of studies in cardiac surgical patients that have used machine learning methods is very limited. This study aimed to explore the application of several machine learning predictive models that can pre-emptively predict delirium in patients undergoing cardiac surgery and compare their performance. We investigated a number of machine learning methods to develop models that can predict delirium after cardiac surgery. A clinical dataset comprising over 5000 actual patients who underwent cardiac surgery in a single center was used to develop the models using logistic regression, artificial neural networks (ANN), support vector machines (SVM), Bayesian belief networks (BBN), naïve Bayesian, random forest, and decision trees. Only 507 out of 5584 patients (11.4%) developed delirium. We addressed the underlying class imbalance, using random unders ling, in the training dataset. The final prediction performance was validated on a separate test dataset. Owing to the target class imbalance, several measures were used to evaluate algorithm’s performance for the delirium class on the test dataset. Out of the selected algorithms, the SVM algorithm had the best F1 score for positive cases, kappa, and positive predictive value (40.2%, 29.3%, and 29.7%, respectively) with a P=.01, .03, .02, respectively. The ANN had the best receiver-operator area-under the curve (78.2% P=.03). The BBN had the best precision-recall area-under the curve for detecting positive cases (30.4% P=.03). Although delirium is inherently complex, preventive measures to mitigate its negative effect can be applied proactively if patients at risk are prospectively identified. Our results highlight 2 important points: (1) addressing class imbalance on the training dataset will augment machine learning model’s performance in identifying patients likely to develop postoperative delirium, and (2) as the prediction of postoperative delirium is difficult because it is multifactorial and has complex pathophysiology, applying machine learning methods (complex or simple) may improve the prediction by revealing hidden patterns, which will lead to cost reduction by prevention of complications and will optimize patients’ outcomes.

Randomized controlled trial of cervical cap with intracervical reservoir versus standard intracervical injection to inseminate cryopreserved donor semen

Publisher: Oxford University Press (OUP)

Date: 1996

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A019044

Abstract: A prospective controlled study of donor insemination without sperm preparation or ovarian stimulation was performed to compare the use of a cervical cap incorporating an intracervical reservoir with a standard intracervical injection technique to inseminate 0.5 ml cryopreserved semen. Treatments were alternated in successive cycles in each patient after initial randomized selection. A total of 198 patients had 635 treatment cycles (median 3, range 1-7), 309 with reservoir and 326 by standard injection. A total of 56 women became pregnant, 24 (7.8% per cycle) with the reservoir and 32 (9.8% per cycle) by injection. There were no significant differences between the pregnancy rates per cycle overall or cycle-specific cumulative rates calculated using the life-table method. There were no significant differences in age, parity, baseline gonadotrophin measurements, mid-luteal serum progesterone concentrations, frequency of adverse fertility factors in the woman or her partner's cause of infertility between women who conceived and those who failed to conceive. We conclude that use of a cervical reservoir and cap for donor insemination does not offer any advantage over standard intracervical insemination.

Reduced Chromosome Cohesion Measured by Interkinetochore Distance Is Associated with Aneuploidy Even in Oocytes from Young Mice1

Publisher: Oxford University Press (OUP)

Date: 02-2013

DOI: 10.1095/BIOLREPROD.112.104786

Abstract: It is becoming clear that reduced chromosome cohesion is an important factor in the rise of maternal age-related aneuploidy. This reduction in cohesion has been observed both in human and mouse oocytes, and it can be measured directly by an increase with respect to maternal age in interkinetochore (iKT) distance between a sister chromatid pair. We have observed variations in iKT distance even in oocytes from young mice and wondered if such differences may predispose those oocytes displaying the greatest iKT distances to be becoming aneuploid. Therefore, we used two methods, one pharmacological (Aurora kinase inhibitor) and one genetic (Fzr1 knockout), to raise aneuploidy rates in oocytes from young mice (age, 1-3 mo) and to examine if those oocytes that were aneuploid had greater iKT distances. We observed that for both Aurora kinase inhibition and Fzr1 knockout, iKT distances were significantly greater in those oocytes that became aneuploid compared to those that remained euploid. Based on these results, we propose that in idual oocytes undergo loss in chromosomal cohesion at different rates and that the greater this loss, the greater the risk for becoming aneuploid.

The role of the molecular chaperone heat shock protein A2 (HSPA2) in regulating human sperm-egg recognition

Publisher: Medknow

Date: 2015

DOI: 10.4103/1008-682X.151395

RNA binding proteins in spermatogenesis: an in depth focus on the Musashi family

Publisher: Medknow

Date: 2015

DOI: 10.4103/1008-682X.151397

Scrambled and fried: Cigarette smoke exposure causes antral follicle destruction and oocyte dysfunction through oxidative stress

Publisher: Elsevier BV

Date: 09-2013

DOI: 10.1016/J.TAAP.2013.05.009

Abstract: Cigarette smoke is a reproductive hazard associated with pre-mature reproductive senescence and reduced clinical pregnancy rates in female smokers. Despite an increased awareness of the adverse effects of cigarette smoke exposure on systemic health, many women remain unaware of the adverse effects of cigarette smoke on female fertility. This issue is compounded by our limited understanding of the molecular mechanisms behind cigarette smoke induced infertility. In this study we used a direct nasal exposure mouse model of cigarette smoke-induced chronic obstructive pulmonary disease to characterise mechanisms of cigarette-smoke induced ovotoxicity. Cigarette smoke exposure caused increased levels of primordial follicle depletion, antral follicle oocyte apoptosis and oxidative stress in exposed ovaries, resulting in fewer follicles available for ovulation. Evidence of oxidative stress also persisted in ovulated oocytes which escaped destruction, with increased levels of mitochondrial ROS and lipid peroxidation resulting in reduced fertilisation potential. Microarray analysis of ovarian tissue correlated these insults with a complex mechanism of ovotoxicity involving genes associated with detoxification, inflammation, follicular activation, immune cell mediated apoptosis and membrane organisation. In particular, the phase I detoxifying enzyme cyp2e1 was found to be significantly up-regulated in developing oocytes an enzyme known to cause molecular bioactivation resulting in oxidative stress. Our results provide a preliminary model of cigarette smoke induced sub-fertility through cyp2e1 bioactivation and oxidative stress, resulting in developing follicle depletion and oocyte dysfunction.

Abstract # 1761 Neonatal immune activation with lipopolysaccharide and a ‘second hit’ of adulthood stress alters ovarian inflammatory mediators: Implications for female subfertility

Publisher: Elsevier BV

Date: 10-2016

DOI: 10.1016/J.BBI.2016.07.054

Prophylactic and therapeutic vaccination protects sperm health from Chlamydia muridarum-induced abnormalities

Publisher: Oxford University Press (OUP)

Date: 17-02-2023

DOI: 10.1093/BIOLRE/IOAD021

Abstract: Chlamydia is the most common bacterial sexually transmitted infection worldwide and it is widely acknowledged that controlling the r ant community transmission of this infection requires vaccine development. In this study, for the first time, we elucidate the long-term response to male mouse chlamydial vaccination with chlamydial major outer membrane protein (MOMP) and ISCOMATRIX (IMX) both prophylactically and in a novel therapeutic setting. Vaccination significantly reduced and, in some cases, cleared chlamydial burden from the prostates, epididymides, and testes, which correlates with high IgG and IgA tires in tissues and serum. Important markers of sperm health and fertility were protected including sperm motility and proteins associated with fertility in men. Within splenocytes, expression of IFNγ, TNFα, IL17, IL13, IL10, and TGFβ were changed by both infection and vaccination within CD4 and CD8 T cells and regulatory T cells. Within the testicular tissue, phenotypic and concentration changes were observed in macrophages and T cells (resident and transitory). This revealed some pathogenic phenotypes associated with infection and critically that vaccination allows maintenance of testicular homeostasis, likely by preventing significant influx of CD4 T cells and promoting IL10 production. Finally, we demonstrated the testes contained immature (B220+) B cells and mature (CD138+) Chlamydia-specific plasma cells. Thus, through vaccination, we can maintain the healthy function of the testes, which is vital to protection of male fertility.

Suppressor of cytokine signaling 4 (SOCS4): Moderator of ovarian primordial follicle activation

Publisher: Wiley

Date: 22-12-2011

DOI: 10.1002/JCP.22837

Abstract: Mammalian ovarian primordial follicle activation and regulation is considered as one of the most important stages of folliculogenesis and as such requires exquisite control. Selection of quiescent follicles to enter the growing pool determines the rate of supply of maturing follicles over the female reproductive lifespan. To coordinate this process a range of positive and negative input signals contribute to determine follicle fate. This study demonstrates that the cytokine Leukemia Inhibitory Factor (LIF) activates the Janus Kinase 1/Signal Transducers and Activators of Transcription 3 (JAK1/STAT3) signaling pathway in pre-granulosa cells and positively regulates primordial follicle activation. Negative regulation of the JAK/STAT pathway is controlled by the suppressor of cytokine signaling 4 (SOCS4) protein, which target members of negative feedback loops, Cardiotrophin like Cytokine (CLC), Poly (rC) Binding Protein 1 (PCBP1), and Cytosolic Malate Dehydrogenase (MDH1) to suppress follicle growth and development.

Grandmaternal smoke exposure reduces female fertility in a murine model, with great-grandmaternal smoke exposure unlikely to have an effect

Publisher: Oxford University Press (OUP)

Date: 10-04-2017

DOI: 10.1093/HUMREP/DEX073

Abstract: What effect does multigenerational (F2) and transgenerational (F3) cigarette smoke exposure have on female fertility in mice? Cigarette smoking has a multigenerational effect on female fertility. It has been well established that cigarette smoking decreases female fertility. Furthermore, a growing body of evidence suggests that smoking during pregnancy decreases the fertility of daughters and increases cancer and asthma incidence in grandchildren and great-grandchildren. Six-week-old C57BL/6 female mice were exposed nasally to cigarette smoke or room air (controls) for 5 weeks prior to being housed with males. Females continued to be exposed to smoke throughout pregnancy and lactation until pups were weaned. A subset of F1 female pups born to these smoke and non-smoke exposed females were bred to create the F2 grandmaternal exposed generation (multigenerational). Finally, a subset of F2 females were bred to create the F3 great-grandmaternal exposed generation (transgenerational). The reproductive health of F2 and F3 females was examined at 8 weeks and 9 months. Ovarian and oocyte quality was examined in smoke exposed and control animals. A small-scale fertility trial was performed before ovarian changes were examined using ovarian histology and immunofluorescence and/or immunoblotting analysis of markers of apoptosis (TUNEL) and proliferation (proliferating cell nuclear antigen (PCNA) and anti-Mullerian hormone (AMH)). Oocyte quality was examined using immunocytochemistry to analyze the metaphase II spindle and ploidy status. Parthenogenetic activation of oocytes was used to investigate meiosis II timing and preimplantation embryo development. Finally, diestrus hormone serum levels (FSH and LH) were quantified. F2 smoke exposed females had no detectable change in ovarian follicle quality at 8 weeks, although by 9 months ovarian somatic cell proliferation was reduced (P = 0.0197) compared with non-smoke exposed control. Further investigation revealed changes between control and smoke exposed F2 oocyte quality, including altered meiosis II timing at 8 weeks (P = 0.0337) and decreased spindle pole to pole length at 9 months (P = 0.0109). However, no change in preimplantation embryo development was observed following parthenogenetic activation. The most noticeable effect of cigarette smoke exposure was related to the subfertility of F2 females F2 smoke exposed females displayed significantly increased time to conception (P = 0.0042) and significantly increased lag time between pregnancies (P = 0.0274) compared with non-smoke exposed F2 females. Conversely, F3 smoke exposed females displayed negligible oocyte and follicle changes up to 9 months of age, and normal preimplantation embryo development. None. This study focused solely on a mouse model of cigarette smoke exposure to simulate human exposure. Our results demonstrate that grandmaternal cigarette smoke exposure reduces female fertility in mice, highlighting the clinical need to promote cessation of cigarette smoking in pregnant women. This study was funded by the Australian Research Council, National Health and Medical Research Council, Hunter Medical Research Institute, Newcastle Permanent Building Society Charitable Trust, and the University of Newcastle Priory Research Centers in Chemical Biology, Healthy Lungs and Grow Up Well. The authors declare no conflict of interest.

Intracellular calcium measurements in individual human sperm demonstrate that the majority can respond to progesterone

Publisher: Elsevier BV

Date: 12-1995

DOI: 10.1016/S0015-0282(16)57989-8

Abstract: To determine if human spermatozoa could be immobilized and intracellular calcium measurements made on in idual cells to measure what proportion can respond to P. Spermatozoa were loaded with Fura 2 (Sigma Chemical Co., Poole, Dorest, United Kingdom) and suspended in 10% gelatin at 37 degrees C. A thin layer of the suspension was cooled to room temperature (20 degrees C to 25 degrees C) and [Ca2+]i was measured with a fluorescence microscope equipped with dual wavelength excitation and an image analysis system. University-based laboratory. Semen was obtained from four fertile donors to a donor insemination program. None. [Ca2+]i was calculated from the ratio of Fura 2 fluorescence excited at 340 nm and that excited at 366 nm. One hundred six of 114 sperm examined (93%) demonstrated a significant response to P but the size and duration of the response was variable. These data demonstrate that most sperm can respond to P.

Changing expression and subcellular distribution of karyopherins during murine oogenesis

Publisher: Bioscientifica

Date: 12-2015

DOI: 10.1530/REP-14-0585

Abstract: Mammalian oocyte growth and development is driven by a strict program of gene expression that relies on the timely presence of transcriptional regulators via nuclear pores. By targeting specific cargos for nucleo-cytoplasmic transport, karyopherin (KPN) proteins are key to the relocation of essential transcription factors and chromatin-remodelling factors into and out of the nucleus. Using multiple complementary techniques, here we establish that KPNA genes and proteins are dynamically expressed and relocalised throughout mouse oogenesis and folliculogenesis. Of the KPNAs examined ( Kpna1 , Kpna2 , Kpna3 , Kpna4 , Kpna6 , Kpna7 , Kpnb1 , Ipo5 and Xpo1 ), all were expressed in the embryonic ovary with up-regulation of protein levels concomitant with meiotic entry for KPNA2, accompanied by the redistribution of the cellular localisation of KPNA2 and XPO1. In contrast, postnatal folliculogenesis revealed significant up-regulation of Kpna1 , Kpna2 , Kpna4 , Kpna6 and Ipo5 and down-regulation of Kpnb1 , Kpna7 and Xpo1 at the primordial to primary follicle transition. KPNAs exhibited different localisation patterns in both oocytes and granulosa cells during folliculogenesis, with three KPNAs – KPNA1, KPNA2 and IPO5 – displaying marked enrichment in the nucleus by antral follicle stage. Remarkably, varied subcellular expression profiles were also identified in isolated pre-ovulatory oocytes with KPNAs KPNA2, KPNB1 and IPO5 detected in the cytoplasm and at the nuclear rim and XPO1 in cytoplasmic aggregates. Intriguingly, meiotic spindle staining was also observed for KPNB1 and XPO1 in meiosis II eggs, implying roles for KPNAs outside of nucleo-cytoplasmic transport. Thus, we propose that KPNAs, by targeting specific cargoes, are likely to be key regulators of oocyte development.

The impact of oxidative stress on chaperone-mediated human sperm-egg interaction

Publisher: Oxford University Press (OUP)

Date: 07-09-2015

DOI: 10.1093/HUMREP/DEV214

Abstract: How does oxidative stress impact upon human sperm-egg interaction and in particular the formation of zona pellucida-receptor complexes on the sperm surface? Oxidative stress during human sperm capacitation resulted in the chemical alkylation of the molecular chaperone heat shock protein A2 (HSPA2), a concomitant reduction in surface expression of the zona pellucida-receptor arylsulphatase A (ARSA) and a severe loss of zona pellucida binding ability. An inability to bind to the zona pellucida is commonly encountered in the defective spermatozoa generated by male infertility patients however, the underlying mechanisms remain unresolved. Recent studies have revealed that zona pellucida binding is mediated by molecular chaperones, particularly HSPA2, that facilitate the formation of multimeric zona pellucida-receptor complexes on the surface of mammalian spermatozoa during capacitation. Spermatozoa were collected from healthy normozoospermic donors (n = 15). Low levels of oxidative stress were induced in populations of non-capacitated spermatozoa by a 1 h treatment with 4-hydroxynonenal (4HNE) or hydrogen peroxide (H2O2) and then these insults were removed and cells were capacitated for 3 h. Motility, membrane fluidity, protein tyrosine phosphorylation and lipid raft distribution were evaluated after sperm capacitation to determine the impact of oxidative stress on this process. The surface expression of ARSA and sperm adhesion molecule 1 (SPAM1) was observed using fluorescence microscopy, and the ability of treated cells to interact with homologous human zonae pellucidae was assessed through gamete co-incubation. Proximity ligation was used to evaluate the state of the HSPA2-laden zona pellucida-receptor complex and an immunoprecipitation approach was taken to establish the chemical alkylation of HSPA2 by the cytotoxic lipid aldehyde 4HNE. The validity of these findings was then tested through treatment of oxidatively stressed cells with the nucleophile penicillamine in order to scavenge lipid aldehydes and limit their ability to interact with HSPA2. All experiments were performed on s les pooled from two or more donors per replicate, with a minimum of three replicates. The oxidative treatments employed in this study did not influence sperm motility or capacitation-associated changes in membrane fluidity, tyrosine phosphorylation and lipid raft redistribution. However, they did significantly impair zona pellucida binding compared with the capacitated control (P < 0.01). The reduction in zona pellucida binding was associated with the impaired surface expression (P < 0.02) of a zona pellucida-receptor complex comprising HSPA2, SPAM1 and ARSA. Proximity ligation and immunoprecipitation assays demonstrated that impaired zona pellucida binding was, in turn, associated with the chemical alkylation of HSPA2 with 4HNE and the concomitant disruption of this zona pellucida-receptor complex. The use of penicillamine enabled a partial recovery of ARSA surface expression and zona pellucida adherence in H2O2-treated cells. These data suggest that the ability of low levels of oxidative stress to disrupt sperm function is mediated by the production of lipid aldehydes as a consequence of lipid peroxidation and their adduction to the molecular chaperone HSPA2 that is responsible for co-ordinating the assembly of functional zona pellucida-receptor complexes during sperm capacitation. While these results extend only to one particular zona pellucida-receptor complex, we postulate that oxidative stress may more broadly impact upon sperm surface architecture. In this light, further study is required to assess the impact of oxidative stress on additional HSPA2-laden protein complexes. These findings link low levels of oxidative stress to a severe loss of sperm function. In doing so, this work suggests a potential cause of male infertility pertaining to a loss of zona pellucida recognition ability and will contribute to the more accurate diagnosis and treatment of such conditions.

Dynamic Hedgehog signalling pathway activity in germline stem cells

Publisher: Wiley

Date: 24-02-2014

DOI: 10.1111/J.2047-2927.2014.00187.X

The Composition and Significance of Lipid Rafts in Mouse Spermatozoa.

Publisher: Oxford University Press (OUP)

Date: 05-2008

DOI: 10.1093/BIOLREPROD/78.S1.187D

Activin and BMP Signalling in Human Testicular Cancer Cell Lines, and a Role for the Nucleocytoplasmic Transport Protein Importin-5 in Their Crosstalk

Publisher: MDPI AG

Date: 24-03-2023

DOI: 10.3390/CELLS12071000

Abstract: Testicular germ cell tumours (TGCTs) are the most common malignancy in young men. Originating from foetal testicular germ cells that fail to differentiate correctly, TGCTs appear after puberty as germ cell neoplasia in situ cells that transform through unknown mechanisms into distinct seminoma and non-seminoma tumour types. A balance between activin and BMP signalling may influence TGCT emergence and progression, and we investigated this using human cell line models of seminoma (TCam-2) and non-seminoma (NT2/D1). Activin A- and BMP4-regulated transcripts measured at 6 h post-treatment by RNA-sequencing revealed fewer altered transcripts in TCam-2 cells but a greater responsiveness to activin A, while BMP4 altered more transcripts in NT2/D1 cells. Activin significantly elevated transcripts linked to pluripotency, cancer, TGF-β, Notch, p53, and Hippo signalling in both lines, whereas BMP4 altered TGF-β, pluripotency, Hippo and Wnt signalling components. Dose-dependent antagonism of BMP4 signalling by activin A in TCam-2 cells demonstrated signalling crosstalk between these two TGF-β superfamily arms. Levels of the nuclear transport protein, IPO5, implicated in BMP4 and WNT signalling, are highly regulated in the foetal mouse germline. IPO5 knockdown in TCam-2 cells using siRNA blunted BMP4-induced transcript changes, indicating that IPO5 levels could determine TGF-β signalling pathway outcomes in TGCTs.

The yield, motility and performance in the hamster egg test of human spermatozoa prepared from cryopreserved semen by four different methods

Publisher: Oxford University Press (OUP)

Date: 05-1992

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A137714

Abstract: Different procedures were investigated for the dilution of human cryopreserved semen and the preparation of an enriched population of motile spermatozoa for assisted reproduction. The dilution of a 0.25 ml straw of cryopreserved human semen by addition of 2.0 ml Ham's F-10 buffer in one step caused a large decrease in the proportion of motile spermatozoa. This was due to osmotic stress because many of the diluted spermatozoa exhibited swollen tails. To a large extent the damage could be avoided by adding the buffer in 0.10-ml aliquots at 30-s intervals. Spermatozoa obtained after such dilution of cryopreserved human semen were subjected to the swim-up procedure, to centrifugation on two-step gradients of Nycodenz or Percoll, or to filtration through glass fibre paper and compared with respect to yield, motility parameters and penetrating ability in the hamster egg test. The swim-up procedure yielded spermatozoa with excellent motility but only 12% of the available motile spermatozoa were recovered. On both Nycodenz and Percoll gradients, greater than 40% of the available motile spermatozoa were recovered and the average velocity of the spermatozoa was not significantly less than for the swim-up technique. When A23187 was used to promote acrosome reactions in the hamster egg test, Percoll-prepared spermatozoa achieved an average of 8.6 decondensed sperm heads/egg compared to 1.9 for Nycodenz and 1.3 for the swim-up procedure. The yield from glass fibre paper filtration was only 12% and the velocity of the spermatozoa and their performance in the hamster egg test was significantly poorer than in all the other methods.(ABSTRACT TRUNCATED AT 250 WORDS)

149. TRANSLATIONAL CONTROL IN FOLLICULOGENESIS AND OOCYTE DEVELOPMENT: A ROLE FOR RNA-BINDING PROTEIN MUSASHI-1

Publisher: CSIRO Publishing

Date: 2010

DOI: 10.1071/SRB10ABS149

Abstract: Control of the maternal mRNA pool during oocyte maturation is crucial to the correct temporal and spatial expression of proteins, particularly during oocyte transcriptional quiescence. We have identified Musashi-1 as being present within the oocyte/ovary, where this RNA-binding protein is believed to act as a translational repressor of target mRNAs. Recent studies in mammalian neural and intestinal systems have identified a number of cell cycle regulators as potential targets of Msi-1. Using Msi-1 protein-RNA immunoprecipitation, we have also identified musashi-2 (msi-2) and c-mos as putative targets in the mouse oocyte. To further study these targets, a transgenic mouse was produced to overexpress Msi-1 exclusively in the oocyte. QPCR analysis, performed on intact ovaries of wild type (WT) and Tg mice, confirmed a 1.5-fold increase in msi-1 expression in tgMsi-1/+ ovaries in excess of WT ovary expression. QPCR analysis of Msi-1 target expression, performed on intact WT and Tg ovaries, in conjunction with transcript obtained from the Msi-1 protein-RNA immunoprecipitation, revealed an overall increase in expression in the tgMsi-1/+ and Msi-1 IP s les, respectively, of p21WAF-1 (~2.5-fold undetected), cdkn2a (~2-fold undetected), notch1 (~3-fold undetected), c-mos (no difference ~41-fold) and msi-2 (~7-fold ~10-fold). Immunohistochemical analysis of Msi-2 protein expression in transgenic juvenile mouse ovaries,demonstrated a decrease in expression of Msi-2 in tgMsi-1/+ ovaries, when compared to WT ovary expression, suggesting that Msi-2 mRNA is translationally repressed by Msi-1. Therefore, preliminary analysis suggests that Msi-1 may play a role inregulating transcripts of genes necessary for processes characteristic of meiotic progression and oocyte development.

Use of home insemination in programmes of artificial insemination with donor semen.

Publisher: BMJ

Date: 15-10-1983

DOI: 10.1136/BMJ.287.6399.1110

Abstract: Ewing sarcoma is rarely shown to develop this intravascular extension so the decision of the initial treatment is more difficult. We report a 7-year-old boy of this sarcoma with extension into superior vena cava (SVC) and right atrium (RA), who was successfully treated with initial surgery. Intravascular extension was observed from the azygous vein to SVC and finally RA. The removal of the intravascular extension was done, 7 days before chemotherapy was started. The initial surgery for the intravascular extension may have decreased a risk of pulmonary tumor embolism and this made the chemotherapy done safe in this patient.

142. microRNA AND EARLY MALE GERM CELLS

Publisher: CSIRO Publishing

Date: 2010

DOI: 10.1071/SRB10ABS142

Abstract: MicroRNAs are short regulatory noncoding RNA molecules that bind to the 3′ untranslated region of mRNA targets to control translation therefore influencing the abundance of many different protein molecules. Aberrant expression of miRNA is linked to many diseases and developmental abnormalities. Testicular Germ Cell Tumours (TGCT) develop from Carcinoma in situ cells which have been identified as dysfunctional gonocytes. Due to the continual rise in the rate of testicular cancer in the developed world, the molecular mechanisms underlying the failure of gonocytes to differentiate into spermatogonia is of great interest. Gonocytes from post natal day 1 testes and spermatogonia from day 7–9 mice were enriched by 2–4% BSA gradient sedimentation. Total RNA including microRNA was extracted and analysed in by Illumina miRNA microarray. Total RNA was also reverse transcribed using specific primers and analysed by qPCR. Three biological replicates were performed in both the microarray and qPCR experiments. Bioinformatic analysis with SAM (Significance Analysis of Microarrays) identified seven significantly different miRNA molecules between spermatogonia and gonocytes. qPCR analysis confirmed two miRNAs were significantly upregulated in spermatogonia (743a, 463*) and three miRNAs were significantly down regulated in spermatogonia (293, 290-5p, 291a-5p). Several miRNA molecules were selected for further study (293, 290-5p, 136, and 146a) and overexpression assays first in the P19 cell line, then in isolated spermatogonia will help determine their function. In the future the role of these molecules in human seminoma will be analysed using overexpression within a seminoma cell line. It is hypothesised that these miRNA molecules control genes involved in male development and differentiation, such as stella, nanog and oct3/4, and may also play a role in tumour development. In conclusion miRNA expression is significantly different between gonocytes and spermatogonia and we propose that this results in the initiation of differentiation and commencement of spermatogenesis.

The Role of the RNA-Binding Protein, Musashi-1, in Murine Spermatogonial Stem Cell Maintenance.

Publisher: Oxford University Press (OUP)

Date: 05-2008

DOI: 10.1093/BIOLREPROD/78.S1.228C

Attitudes towards donor insemination—a post-Warnock survey

Publisher: Oxford University Press (OUP)

Date: 11-1987

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A136625

Abstract: In a multi-centred study, a total of 799 patients, donors and health-care professionals concerned with artificial insemination with donor semen (AID) responded to a questionnaire regarding their attitudes towards current provision of AID services and proposed legislation. There was little support for any fundamental change in the way in which AID is practised, at least in those centres. The anonymous status of the donor met with universal agreement. Although there was some support for the communication of non-identifying details to the recipient couple, where they wanted them, there was no support for any legislation which might give the AID child a right of access to details of the donor. The greatest ergence of opinion was over the question of who should have access to AID treatment and whether or not screening procedures should be applied to prospective parents. Most respondents felt that the closed and confidential relationship between the clinic and the other parties involved should not automatically be extended to general practitioners or any national bodies. In respect of specific recommendations of the Warnock Committee, there was support for changes which might legitimize or assist the present system, but not for any which might be restrictive.

268. Suppressors of cytokine signalling (SOCS): roles in ovarian follicle activation

Publisher: CSIRO Publishing

Date: 2008

DOI: 10.1071/SRB08ABS268

Abstract: Oocytes are sequestered in primordial follicles before birth and remain quiescent in the ovary for decades, until recruited into the growing pool throughout the reproductive years. Therefore activation of follicle growth is a major biological checkpoint that controls female reproductive potential. However we are only just beginning to elucidate the cellular mechanisms required, for either maintenance of the quiescent primordial pool, or initiation of follicle growth. Analysis of microarray data derived from neonatal mouse ovaries indicated that members of the Suppressors of Cytokine Signalling SOCS family of proteins may play pivotal roles in folliculogenesis. We undertook a detailed analysis of gene and protein expression patterns of the eight members of the SOCS family, namely CIS and SOCS1–7, within adult and neonatal mouse ovaries. Quantitative real time PCR and immunohistochemistry was performed to determine mRNA levels and cellular localisation in the ovaries of cycling and new born animals. SOCS proteins were expressed largely within the oocytes of developing follicles and in the granulosa cells of the larger preovulatory follicles. Expression of SOCS4 in the granulosa cells and SOCS5 within the oocyte was coincident with the activation of oocyte growth and the differentiation of squamous pregranulosa to cuboidal granulosa cells. Our investigation has identified a role for the SOCS family proteins within the ovary and SOCS4 and SOCS5 as major regulators of cytokine signalling pathways in follicle activation and development.

Parental Intention to Support Video Game Play by Children With Autism Spectrum Disorder: An Application of the Theory of Planned Behavior

Publisher: American Speech Language Hearing Association

Date: 04-2015

DOI: 10.1044/2015_LSHSS-13-0080

Abstract: The purpose of this study was to determine parental attitudes regarding engagement with video games by their children with autism spectrum disorder (ASD) and whether attitudes vary based on ASD symptom severity. Online survey methodology was used to gather information from parents of children with ASD between the ages of 8 and 12 years. The finalized data set included 152 cases. Descriptive statistics and frequency analyses were used to examine participant demographics and video game play. Descriptive and inferential statistics were used to evaluate questions on the theory of planned behavior. Regression analyses determined the predictive ability of the theory of planned behavior constructs, and t tests provided additional descriptive information about between-group differences. Children with ASD play video games. There are no significant differences in the time, intensity, or types of games played based on severity of ASD symptoms (mild vs. moderate). Parents of children with ASD had positive attitudes about video game play. Parents of children with ASD appear to support video game play. On average, parents indicated video game play was positive for their children with ASD, particularly if they believed the games were having a positive impact on their child's development.

Identification of Cytochrome P450-Reductase as the Enzyme Responsible for NADPH-Dependent Lucigenin and Tetrazolium Salt Reduction in Rat Epididymal Sperm Preparations1

Publisher: Oxford University Press (OUP)

Date: 07-2004

DOI: 10.1095/BIOLREPROD.104.027748

Developmental Expression of Musashi-1 and Musashi-2 RNA-Binding Proteins During Spermatogenesis: Analysis of the Deleterious Effects of Dysregulated Expression1

Publisher: Oxford University Press (OUP)

Date: 05-2014

DOI: 10.1095/BIOLREPROD.113.115261

Abstract: Spermatogenesis is a complex developmental process whereby diploid spermatogenic stem cells become haploid and undergo a series of morphological changes to produce physically mature spermatozoa. Crucial to this process are a number of RNA-binding proteins, responsible for the posttranscriptional control of essential mRNAs and particularly pertinent to the two periods of inactive transcription that occur in spermatogenesis. One such group of RNA-binding proteins is the Musashi family, specifically Musashi-1 (MSI1) and Musashi-2 (MSI2), which act as key translational regulators in various stem cell populations and have been linked with the induction of tumorigenesis. In the present study, we examined the differential expression of mammalian MSI1 and MSI2 during germ cell development in the mouse testis. MSI1 was found to be predominately localized in mitotic gonocytes and spermatogonia, whereas MSI2 was detected in meiotic spermatocytes and differentiating spermatids. Extensive examination of the function of Musashi in spermatogenesis was achieved through the use of two transgenic mouse models with germ cell-specific overexpression of full-length isoforms of Msi1 or Msi2. These models demonstrated that aberrant expression of either Msi1 or Msi2 has deleterious effects on normal spermatogenesis, with Msi2 overexpression resulting in male sterility. Studies undertaken on human testicular seminoma tumors provide further insights into the relevance of MSI1 and MSI2 overexpression as diagnostic markers to human stem cell cancers. Overall this study provides further evidence for the unique functions that RNA-binding protein isoforms occupy within spermatogenesis, and introduces the potential manipulation of the Musashi family proteins to elucidate the mechanisms of posttranscriptional gene expression during germ cell development.

ENDOMETRIOSIS AND OVULATORY DISORDER: REDUCED FERTILISATION IN VITRO COMPARED WITH TUBAL AND UNEXPLAINED INFERTILITY

Publisher: Elsevier BV

Date: 08-1985

DOI: 10.1016/S0140-6736(85)90289-2

Abstract: In-vitro fertilisation (IVF) was carried out once for each of 104 couples who had a single cause of infertility. The group with tubal damage was used as the reference for normal fertilising capacity of both oocytes and sperms: the IVF rates were 68% (71/105) per mature oocyte and 88% (37/42) for couples from whom mature oocytes were recovered. Couples with poor sperm/mucus penetration had reduced IVF rates: 32% (12/38) per oocyte and 60% (9/15) per couple. Sperm function, which was judged normal by means of standard seminal analysis and mucus penetration, was confirmed by normal IVF in unexplained infertility: 63% (37/59) per oocyte and 90% (18/20) per couple. Despite favourable sperm function in their partners, women with endometriosis (without tubal damage) had reduced IVF rates: 33% (19/58) per oocyte and 60% (9/15) per couple. These findings indicate that ovulatory disorder is present in endometriosis and suggest that it causes the associated infertility.

Analysis of the small non-protein-coding RNA profile of mouse spermatozoa reveals specific enrichment of piRNAs within mature spermatozoa

Publisher: Informa UK Limited

Date: 21-09-2017

DOI: 10.1080/15476286.2017.1356569

65. Localisation of tyrosine phosphorylated proteins on mouse spermatozoa during zona pellucida interaction and characterisation of sperm surface phosphoproteins

Publisher: CSIRO Publishing

Date: 2003

DOI: 10.1071/SRB03AB65

Detection of chlamydia infection within human testicular biopsies

Publisher: Oxford University Press (OUP)

Date: 10-2019

DOI: 10.1093/HUMREP/DEZ169

Abstract: Can Chlamydia be found in the testes of infertile men? Chlamydia can be found in 16.7% of fresh testicular biopsies and 45.3% of fixed testicular biopsies taken from a selection of infertile men. Male chlamydial infection has been understudied despite male and female infections occurring at similar rates. This is particularly true of asymptomatic infections, which occur in 50% of cases. Chlamydial infection has also been associated with increased sperm DNA damage and reduced male fertility. We collected diagnostic (fixed, n = 100) and therapeutic (fresh, n = 18) human testicular biopsies during sperm recovery procedures from moderately to severely infertile men in a cross-sectional approach to s ling. The diagnostic and therapeutic biopsies were tested for Chlamydia-specific DNA and protein, using real-time PCR and immunohistochemical approaches, respectively. Serum s les matched to the fresh biopsies were also assayed for the presence of Chlamydia-specific antibodies using immunoblotting techniques. Chlamydial major outer membrane protein was detected in fixed biopsies at a rate of 45.3%. This was confirmed by detection of chlamydial DNA and TC0500 protein (replication marker). C. trachomatis DNA was detected in fresh biopsies at a rate of 16.7%, and the sera from each of these three positive patients contained C. trachomatis-specific antibodies. Overall, C. trachomatis-specific antibodies were detected in 72.2% of the serum s les from the patients providing fresh biopsies, although none of the patients were symptomatic nor had they reported a previous sexually transmitted infection diagnosis including Chlamydia. No reproductively healthy male testicular biopsies were tested for the presence of Chlamydia DNA or proteins or Chlamydia-specific antibodies due to the unavailability of these s les. Application of Chlamydia-specific PCR and immunohistochemistry in this human male infertility context of testicular biopsies reveals evidence of a high prevalence of previously unrecognised infection, which may potentially have a pathogenic role in spermatogenic failure. Funding for this project was provided by the Australian NHMRC under project grant number APP1062198. We also acknowledge assistance from the Monash IVF Group and Queensland Fertility Group in the collection of fresh biopsies, and the Monash Health and co-author McLachlan (declared equity interest) in retrieval and sectioning of fixed biopsies. E.M. declares an equity interest in the study due to financing of fixed biopsy sectioning. All other authors declare no conflicts of interest. N/A

Identification of Sites of STAT3 Action in the Female Reproductive Tract through Conditional Gene Deletion

Publisher: Public Library of Science (PLoS)

Date: 07-2014

DOI: 10.1371/JOURNAL.PONE.0101182

Neonatal lipopolysaccharide exposure impairs sexual development and reproductive success in the Wistar rat

Publisher: Elsevier BV

Date: 05-2011

DOI: 10.1016/J.BBI.2011.01.004

Expression of c-Kit receptor mRNA and protein in the developing, adult and irradiated rodent testis

Publisher: Bioscientifica

Date: 03-2006

DOI: 10.1530/REP.1.00968

Abstract: Germ cell proliferation, migration and survival during all stages of spermatogenesis are affected by stem cell factor signalling through the c-Kit receptor, the expression and function of which are vital for normal male reproductive function. The present study comprehensively describes the c-Kit mRNA and protein cellular expression profiles in germ cells of the postnatal and adult rodent testis, revealing their significant elevation in synthesis at the onset of spermatogenesis. Real-time PCR analysis for both mice and rats matched the cellular mRNA expression profile where examined. Localization studies in normal mouse testes indicated that both c-Kit mRNA and protein are first detectable in differentiating spermatogonia. In addition, all spermatogonia isolated from 8-day-old mice displayed detectable c-Kit mRNA, but 30–50% of these lacked protein expression. The c-Kit mRNA and protein profile in normal rat testes indicated expression in gonocytes, in addition to differentiating spermatogonia. However, in the irradiated adult rat testes, in which undifferentiated spermatogonia are the only germ cell type, mRNA was also detected in the absence of protein. This persisted at 3 days and 1 and 2 weeks following treatment with gonadotrophin-releasing hormone (GnRH) antagonist to stimulate spermatogenesis recovery. By 4 weeks of GnRH antagonist treatment, accompanying the emergence of differentiating spermatogonia, both mRNA and protein were detected. Based on these observations, we propose that c-Kit mRNA and protein synthesis are regulated separately, possibly by influences linked to testis maturation and circulating hormone levels.

Extracorporeal Membrane Oxygenation in the Middle East and India During the COVID-19 Pandemic

Publisher: Elsevier BV

Date: 2021

DOI: 10.2139/SSRN.3790467

RBM5 Is a Male Germ Cell Splicing Factor and Is Required for Spermatid Differentiation and Male Fertility

Publisher: Public Library of Science (PLoS)

Date: 25-07-2013

DOI: 10.1371/JOURNAL.PGEN.1003628

93. Acute neonatal immune challenge: Implications for the programming of later life female sub-fertility

Publisher: Elsevier BV

Date: 09-2014

DOI: 10.1016/J.BBI.2014.06.113

Patients With Systemic Right Ventricle Are at Higher Risk of Chylothorax After Cavopulmonary Connections

Publisher: Elsevier BV

Date: 11-2018

DOI: 10.1016/J.ATHORACSUR.2018.06.077

Abstract: Chylothorax is a rare but severe complication after pediatric cardiac surgical procedures and is related to significant morbidity and mortality. It is suspected to be more frequent after single-ventricle staged palliation procedures, but focused studies on chylothorax in patients with univentricular heart physiology are scarce. From January 2008 to December 2016, a total of 289 patients underwent 376 cavopulmonary connection (CPC) procedures over 9 years (superior cavopulmonary connection [SCPC], 199 Fontan completion, 177). Patients were classified according to whether they had a chylothorax (group 1) or not (group 2). Chylothorax was confirmed on a pleural fluid test. The rate of chylothorax after a CPC procedure was 19.7% (74 of 376): 15.6% after SCPC and 24.3% after Fontan completion. Mean follow-up was 4.3 ± 0.1 years. Systemic right ventricle was more frequent in group 1 than in group 2 (64.9% vs 46%, respectively p = 0.003). Chylothorax was associated with a higher rate of early reoperation (p = 0.001) and late failure of the CPC (p < 0.001). Late mortality was also more frequent in group 1 than in group 2 (17.6% vs 4.3% p < 0.001). By multivariate analysis, having a systemic right ventricle was the only identified predictor for the development of chylothorax (odds ratio, 2.49 95% confidence interval, 1.4 to 4.7 p = 0.004). The incidence of chylothorax in patients undergoing the univentricular pathway procedure is higher than previously suggested. Having a systemic right ventricle is a significant risk factor for developing a chylothorax after a CPC.

Characterization of a novel role for the dynamin mechanoenzymes in the regulation of human sperm acrosomal exocytosis

Publisher: Oxford University Press (OUP)

Date: 30-08-2017

DOI: 10.1093/MOLEHR/GAX044

Abstract: Does dynamin regulate human sperm acrosomal exocytosis? Our studies of dynamin localization and function have implicated this family of mechanoenzymes in the regulation of progesterone-induced acrosomal exocytosis in human spermatozoa. Completion of an acrosome reaction is a prerequisite for successful fertilization in all studied mammalian species. It follows that failure to complete this unique exocytotic event represents a common aetiology in the defective spermatozoa of male infertility patients that have failed IVF in a clinical setting. Recent studies have implicated the dynamin family of mechanoenzymes as important regulators of the acrosome reaction in murine spermatozoa. The biological basis of this activity appears to rest with the ability of dynamin to polymerize around newly formed membrane vesicles and subsequently regulate the rate of fusion pore expansion. To date, however, the dynamin family of GTPases have not been studied in the spermatozoa of non-rodent species. Here, we have sought to examine the presence and functional significance of dynamin in human spermatozoa. Dynamin expression was characterized in the testis and spermatozoa of several healthy normozoospermic in iduals. In addition, we assessed the influence of selective dynamin inhibition on the competence of human spermatozoa to undergo a progesterone-induced acrosome reaction. A minimum of five biological and technical replicates were performed to investigate both inter- and intra-donor variability in dynamin expression and establish statistical significance in terms of the impact of dynamin inhibition. The expression and the localization of dynamin in the human testis, epididymis and mature spermatozoa were determined through the application of immunofluorescence, immunoblotting and/or electron microscopy. Human semen s les were fractionated via density gradient centrifugation and the resultant populations of good and poor quality spermatozoa were induced to capacitate and acrosome react in the presence or absence of selective dynamin inhibitors. The acrosome integrity of live spermatozoa was subsequently assessed via the use of fluorescently conjugated Arachis hypogea lectin (PNA). The influence of dynamin phosphorylation and the regulatory kinase(s) responsible for this modification in human spermatozoa were also assessed via the use of in situ proximity ligation assays and pharmacological inhibition. In all experiments, ≥100 spermatozoa were assessed/treatment group and all graphical data are presented as the mean values ± SEM, with statistical significance being determined by ANOVA. Dynamin 1 (DNM1) and DNM2, but not DNM3, were specifically localized to the acrosomal region of the head of human spermatozoa, an ideal position from which to regulate acrosomal exocytosis. In keeping with this notion, pharmacological inhibition of DNM1 and DNM2 was able to significantly suppress the rates of acrosomal exocytosis stimulated by progesterone. Furthermore, our comparison of dynamin expression in good and poor quality spermatozoa recovered from the same ejaculate, revealed a significant reduction in the amount of DNM2 in the latter subpopulation of cells. In contrast, DNM1 was detected at equivalent levels in both subpopulations of spermatozoa. Such findings are of potential significance given that the poor quality spermatozoa proved refractory to the induction of a progesterone stimulated acrosome reaction. In seeking to identify the regulatory influence of progesterone on DNM2 function, we were able to establish that the protein is a substrate for CDK1-dependent phosphorylation. The functional significance of DNM2 phosphorylation was illustrated by the fact that pharmacological inhibition of CDK1 elicited a concomitant suppression of both DNM2-Ser764 phosphorylation and the overall rates of progesterone-induced acrosomal exocytosis. N/A. This was an in vitro study performed mainly on ejaculated human spermatozoa. This experimental paradigm necessarily eliminates the physiological contributions of the female reproductive tract that would normally support capacitation and acrosomal responsiveness. This study identifies a novel causative link between dynamin activity and the ability of human spermatozoa to complete a progesterone-induced acrosome reaction. Such findings encourage a more detailed analysis of the contribution of dynamin dysregulation as an underlying aetiology in infertile males whose spermatozoa are unable to penetrate the zona pellucida. This research was supported by a National Health and Medical Research Council of Australia Project Grant (APP1103176) awarded to B.N. and E.A.M. The authors report no conflict of interest.

py4DSTEM: A Software Package for Four-Dimensional Scanning Transmission Electron Microscopy Data Analysis

Publisher: Oxford University Press (OUP)

Date: 21-05-2021

DOI: 10.1017/S1431927621000477

Abstract: Scanning transmission electron microscopy (STEM) allows for imaging, diffraction, and spectroscopy of materials on length scales ranging from microns to atoms. By using a high-speed, direct electron detector, it is now possible to record a full two-dimensional (2D) image of the diffracted electron beam at each probe position, typically a 2D grid of probe positions. These 4D-STEM datasets are rich in information, including signatures of the local structure, orientation, deformation, electromagnetic fields, and other s le-dependent properties. However, extracting this information requires complex analysis pipelines that include data wrangling, calibration, analysis, and visualization, all while maintaining robustness against imaging distortions and artifacts. In this paper, we present py4DSTEM, an analysis toolkit for measuring material properties from 4D-STEM datasets, written in the Python language and released with an open-source license. We describe the algorithmic steps for dataset calibration and various 4D-STEM property measurements in detail and present results from several experimental datasets. We also implement a simple and universal file format appropriate for electron microscopy data in py4DSTEM, which uses the open-source HDF5 standard. We hope this tool will benefit the research community and help improve the standards for data and computational methods in electron microscopy, and we invite the community to contribute to this ongoing project.

Making it through meiosis: APC/C FZR1 has an essential role in meiotic prophase I in germ cell development

Publisher: Bioscientifica

Date: 23-09-2014

DOI: 10.1530/REPABS.1.S028

Spermatids do it differently! Paip2a—the essential regulator of spermiogenesis?

Publisher: Medknow

Date: 11-2010

DOI: 10.1038/AJA.2010.142

Controlling the swimming speed of human sperm by varying the incubation temperature and its effect on cervical mucus penetration

Publisher: Wiley

Date: 04-1992

DOI: 10.1111/J.1365-2605.1992.TB01121.X

Abstract: The objective of the present experiments was to study the effect of sperm velocity as a single variable on the ability of sperm to penetrate cervical mucus in a modified Kremer test. Sperm incubated at 13, 22 and 37 degrees C exhibited progressive velocities of 25 +/- 1.7, 40 +/- 2.1 and 56 +/- 2.1 microns sec-1 (mean +/- SEM, n = 6) respectively, but the percentage of progressively motile sperm, their lateral head displacement and the viscoelastic properties of cervical mucus remained comparatively unchanged over this temperature range. The number of sperm which penetrated the mucus and the percentage of successful collisions were correlated strongly with the average velocity of the sperm population (r = 0.82 and r = 0.72 respectively). It is concluded that sperm velocity has an important influence on the penetration of cervical mucus because it governs the frequency of collisions with the mucus interface and is determined by the thrust generated by the flagellum which also determines the ability of the sperm to traverse the mucus interface.

The effect of A23187 concentration and exposure time on the outcome of the hamster egg penetration test

Publisher: Wiley

Date: 04-1991

DOI: 10.1111/J.1365-2605.1991.TB01074.X

Abstract: We investigated the conditions required to enhance the performance of human sperm in the hamster egg penetration test with the free acid form of A23187. The best performance was observed after stimulation with 2 microM A23187 for 1 h when the median penetration rate with sperm from fertile donors was 100% of eggs with 5.8 decondensed sperm heads/egg. Extending the stimulation period with 2 microM A23187 to 2 or 3 h, resulted in a progressive decrease in the penetration rate. In the absence of A23187, the penetration rate was lower (0.7 decondensed sperm heads/egg after 1 h) but increased with stimulation time. A similar picture was observed with sperm from patients taken for an IVF programme. For a pool of cryopreserved semen, the coefficient of variation of the penetration rate after stimulation with 2 microM A23187 for 1 h, expressed as decondensed sperm heads/egg, was 11% within and 20% between assays. There was no correlation between the outcome of the hamster egg penetration test and the percentage motility, velocity or lateral head displacement of the sperm measured after the same stimulation regime. However, in IVF patients the initial velocity and lateral head displacement of the sperm (zero time) were correlated with the best result from the hamster egg penetration test (r = 0.62 and 0.57 respectively). No motility changes characteristic of capacitation were detected. We conclude that stimulation with 2 microM A23187 (free acid) for 1 h prior to the addition of the zona free hamster eggs can produce a high penetration rate with fertile s les and provides a convenient and robust protocol for the assay. However, when carried out in this way the test does not assess the ability of the sperm to capacitate.

Adding Insult to Injury: Effects of Xenobiotic-Induced Preantral Ovotoxicity on Ovarian Development and Oocyte Fusibility

Publisher: Oxford University Press (OUP)

Date: 09-09-2010

DOI: 10.1093/TOXSCI/KFQ272

Abstract: Mammalian females are born with a finite number of nonrenewing primordial follicles, the majority of which remain in a quiescent state for many years. Because of their nonrenewing nature, these "resting" oocytes are particularly vulnerable to xenobiotic insult, resulting in premature ovarian senescence and the formation of dysfunctional oocytes. In this study, we characterized the mechanisms of ovotoxicity for three ovotoxic agents, 4-vinylcyclohexene diepoxide (VCD), methoxychlor (MXC), and menadione (MEN), all of which target immature follicles. Microarray analysis of neonatal mouse ovaries exposed to these xenobiotics in vitro revealed a more than twofold significant difference in transcript expression (p < 0.05) for a number of genes associated with apoptotic cell death and primordial follicle activation. Histomorphological and immunohistological analysis supported the microarray data, showing signs of primordial follicle activation and preantral follicle atresia both in vitro and in vivo. Sperm-oocyte fusion assays on oocytes obtained from adult Swiss mice treated neonatally revealed severely reduced sperm-egg binding and fusion in a dose-dependent manner for all the xenobiotic treatments. Additionally, lipid peroxidation analysis on xenobiotic-cultured oocytes indicated a dose-dependent increase in oocyte lipid peroxidation for all three xenobiotics in vitro. Our results reveal a novel mechanism of preantral ovotoxicity involving the homeostatic recruitment of primordial follicles to maintain the pool of developing follicles destroyed by xenobiotic exposure and to our knowledge provide the first documented evidence of short-term, low- and high-dose (VCD 40-80 mg/kg/day, MXC 50-100 mg/kg/day, MEN 7.5-15 mg/kg/day) neonatal exposure to xenobiotics causing long-term reactive oxygen species-induced oocyte dysfunction.

Beyond Frontiers: Feasibility and Outcomes of Prolonged Veno-Venous Extracorporeal Membrane Oxygenation in Severe Acute Respiratory Distress Syndrome

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 22-01-2021

DOI: 10.1097/MAT.0000000000001367

Abstract: Extracorporeal membrane oxygenation (ECMO) use in acute respiratory failure is increasing. We aim to compare characteristics and outcomes of patients with prolonged (≥21 days) veno-venous (VV) ECMO runs (pECMO), to patients with short ( days) VV ECMO runs (sECMO). The observational retrospective single-center study compared patients who received VV ECMO from January 2018 to June 2019 at Prince Mohamed Bin Abdulaziz Center in Riyadh, Saudi Arabia. Forty-three patients were supported with VV ECMO during the study period, of whom 37 are included as six patients were still receiving ECMO at time of data collection: 24 sECMO and 13 pECMO patients. Baseline characteristics and comorbidities were similar except pECMO patients were older and had a lower P/F ratio (61 [58–68] vs . 71[58–85.5], p = 0.05). Survival to hospital discharge (69% vs. 83%, p = 0.32 pECMO vs. sECMO) and 90 day survival (62% vs. 75%, p = 0.413 pECMO vs. sECMO) were similar among groups. At 1 year follow-up, all patients were still alive and independently functioning except for one patient in the pECMO group who required a walking aid related to trauma. In this single-center study, patients requiring pECMO had similar short- and long-term survival to those requiring sECMO duration.

Definitive Evidence for the Nonmitochondrial Production of Superoxide Anion by Human Spermatozoa

Publisher: The Endocrine Society

Date: 05-2006

DOI: 10.1210/JC.2005-2711

Abstract: Oxidative stress in the male germ line has been associated with poor fertility, impaired embryonic development, miscarriage, and childhood disease. Such stress is known to be associated with the peroxidation of unsaturated fatty acids in the sperm plasma membrane and oxidative DNA damage to both the nuclear and mitochondrial genomes. However, the source of the free radicals responsible for such damage is still unresolved. The objective of this study was to chemically validate the use of dihydroethidium (DHE) as a probe for detecting the generation of superoxide anion by human spermatozoa and to examine the relationship between this activity and defective sperm function. DHE and SYTOX green were used in conjunction with flow cytometry and HPLC to investigate superoxide generation by human spermatozoa. Cause and effect relationships were established using menadione to artificially drive superoxide production by these cells. HPLC, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and spectrofluorometry were used to demonstrate that human spermatozoa generate the superoxide-specific product, 2-hydroxyethidium, from DHE. Spontaneous superoxide production by human spermatozoa was found to originate from a nonmitochondrial source and was inversely correlated with sperm motility. A causative relationship between superoxide generation and sperm function was demonstrated when the pharmacological stimulation of this activity with menadione was shown to result in both severe motility loss and DNA damage. These studies validate a methodology for investigating the origins of oxidative stress in the male germ line and demonstrate, for the first time, the significance of superoxide generation by human spermatozoa in the etiology of this condition.

Proteolytic degradation of heat shock protein A2 occurs in response to oxidative stress in male germ cells of the mouse

Publisher: Oxford University Press (OUP)

Date: 08-12-2017

DOI: 10.1093/MOLEHR/GAW074

Abstract: Does oxidative stress compromise the protein expression of heat shock protein A2 (HSPA2) in the developing germ cells of the mouse testis? Oxidative stress leads to the modification of HSPA2 by the lipid aldehyde 4-hydroxynonenal (4HNE) and initiates its degradation via the ubiquitin-proteasome system. Previous work has revealed a deficiency in HSPA2 protein expression within the spermatozoa of infertile men that have failed fertilization in a clinical setting. While the biological basis of this reduction in HSPA2 remains to be established, we have recently shown that the HSPA2 expressed in the spermatozoa of normozoospermic in iduals is highly susceptible to adduction, a form of post-translational modification, by the lipid aldehyde 4HNE that has been causally linked to the degradation of its substrates. This modification of HSPA2 by 4HNE adduction dramatically reduced human sperm-egg interaction in vitro. Moreover, studies in a mouse model offer compelling evidence that the co-chaperone BCL2-associated athanogene 6 (BAG6) plays a key role in regulating the stability of HSPA2 in the testis, by preventing its ubiquitination and subsequent proteolytic degradation. Dose-dependent studies were used to establish a 4HNE-treatment regime for primary culture(s) of male mouse germ cells. The influence of 4HNE on HSPA2 protein stability was subsequently assessed in treated germ cells. Additionally, sperm lysates from infertile patients with established zona pellucida recognition defects were examined for the presence of 4HNE and ubiquitin adducts. A minimum of three biological replicates were performed to test statistical significance. Oxidative stress was induced in pachytene spermatocytes and round spermatids isolated from the mouse testis, as well as a GC-2 cell line, using 50-200 µM 4HNE or hydrogen peroxide (H2O2), and the expression of HSPA2 was monitored via immunocytochemistry and immunoblotting approaches. Using the GC-2 cell line as a model, the ubiquitination and degradation of HSPA2 was assessed using immunoprecipitation techniques and pharmacological inhibition of proteasomal and lysosomal degradation pathways. Finally, the interaction between BAG6 and HSPA2 was examined in response to 4HNE exposure via proximity ligation assays. HSPA2 protein levels were significantly reduced compared with controls after 4HNE treatment of round spermatids (P < 0.01) and GC-2 cells (P < 0.001) but not pachytene spermatocytes. Using GC-2 cells as a model, HSPA2 was shown to be both adducted by 4HNE and targeted for ubiquitination in response to cellular oxidative stress. Inhibition of the proteasome with MG132 prevented HSPA2 degradation after 4HNE treatment indicating that the degradation of HSPA2 is likely to occur via a proteasomal pathway. Moreover, our assessment of proteasome activity provided evidence that 4HNE treatment can significantly increase the proteasome activity of GC-2 cells (P < 0.05 versus control). Finally, 4HNE exposure to GC-2 cells resulted in the dissociation of HSPA2 from its regulatory co-chaperone BAG6, a key mediator of HSPA2 stability in male germ cells. While these experiments were performed using a mouse germ cell-model system, our analyses of patient sperm lysate imply that these mechanisms are conserved between mouse and human germ cells. This study suggests a causative link between non-enzymatic post-translational modifications and the relative levels of HSPA2 in the spermatozoa of a specific sub-class of infertile males. In doing so, this work enhances our understanding of failed sperm-egg recognition and may assist in the development of targeted antioxidant-based approaches for ameliorating the production of cytotoxic lipid aldehydes in the testis in an attempt to prevent this form of infertility. Not applicable. This work was supported by the National Health and Medical Research Council of Australia (APP1101953). The authors have no competing interests to declare.

The use of C57Bl/6 × CBA F1 hybrid cross as a model for human age‐related oocyte aneuploidy

Publisher: Wiley

Date: 2017

DOI: 10.1002/MRD.22766

Dextrocardia, aortopulmonary window with transposition of the great arteries, case report

Publisher: Saudi Heart Association

Date: 04-2010

DOI: 10.1016/J.JSHA.2010.02.007

Quinolone-1-(2H)-ones as hedgehog signalling pathway inhibitors

Publisher: Royal Society of Chemistry (RSC)

Date: 2016

DOI: 10.1039/C6OB00606J

Abstract: A series of quinolone-2-(1 H )-ones derived from a Ugi-Knoevenagel three- and four-component reaction were prepared exhibiting low micromolar cytotoxicity against a panel of eight human cancer cell lines known to possess the Hedgehog Signalling Pathway (HSP) components, as well as the seminoma TCAM-2 cell line.

Psychosocial and Economic Differences Between Donors and Recipients.

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 07-2014

DOI: 10.1097/00007890-201407151-02799

Secondary electron imaging at atomic resolution using a focused coherent electron probe

Publisher: American Physical Society (APS)

Date: 05-02-2013

DOI: 10.1103/PHYSREVB.87.054102

167. MUSASHI FAMILY OF RNA BINDING PROTEINS: CELL CYCLE REGULATORS IN SPERMATOGENESIS

Publisher: CSIRO Publishing

Date: 2010

DOI: 10.1071/SRB10ABS167

Abstract: Mammalian meiosis is a tightly regulated process involving specialized cell cycle progression and morphogenetic changes. We have demonstrated that the Musashi family of RNA binding proteins is implicated in the regulation of spermatogonial stem self renewal and germ cell differentiation. Here we describe the novel mechanism by which the Musashi family proteins, Msi1 and Msi2, act to control exit from spermatogonial mitotic lification and normal entry into meiosis. Gene and protein analysis indicated overlapping Msi1 and Msi2 profiles in enriched populations of isolated germ cells and reciprocal subcellular expression patterns in spermatogonia and pachytene spermatocytes/ round spermatids in testes sections. Recombinant Msi1 protein-RNA pulldown and microarray analysis coupled with in vitro shRNA knockdown studies in spermatogonial culture and subsequent immunoprecipitation and qPCR established that Msi1 targeted Msi2 mRNA for post transcriptional translational repression. Immunoprecipitation of Msi2 target mRNA and subsequent qPCR together with in vitro shRNA knockdown studies inround spermatidculture identified a cell cycle inhibitor protein CDKN1C (p57kip2) as the principal target of Msi2 translational inhibition. Immunolocalisation of CDKN1C protein indicated that expression of this cell cycle regulator coincided with the nuclear import of Msi1 and the appearance of cytoplasmic Msi2 expression in early pachytene spermatocytes. Using a transgenic Msi1 overexpression mouse model in conjunction with quantitative gene and protein expression, we confirmed Msi1 targeting of Msi2 and subsequent Msi2 targeting of CDKN1C for translational repression in vivo. Ectopic overexpression of Msi1 in germ cellsinduces substantial Msi2 downregulation and aberrant CDKN1C expression, resulting in abnormal spermatogenic differentiation, germ cell apoptosis/arrest and sterility. In conclusion, our results indicate a sophisticated molecular switch encompassing cell cycle protein regulation by Musashi family proteins, is required for normal exit from mitotic ision, entry into meiosis and post meiotic germ cell differentiation.

Prediction of mechanical properties for ferritic ODS steel using the nanoindentation test

Publisher: AIP Publishing

Date: 2021

DOI: 10.1063/5.0068160

Large angle illumination enabling accurate structure reconstruction from thick samples in scanning transmission electron microscopy

Publisher: Elsevier BV

Date: 02-2019

DOI: 10.1016/J.ULTRAMIC.2018.12.010

Abstract: Most reconstructions of the electrostatic potential of a specimen at atomic resolution assume a thin and weakly scattering s le, restricting accurate quantification to specimens only tens of Ångströms thick. We demonstrate that using large-angle-illumination scanning transmission electron microscopy (STEM)-a probe forming aperture with convergence angle larger than about 50 mrad-allows us to better meet the weak phase object approximation and thereby accurately reconstruct the electrostatic potential in s les thicker than the order of 100 Å.

The Impact of Aging on Macroautophagy in the Pre-ovulatory Mouse Oocyte

Publisher: Frontiers Media SA

Date: 29-06-2021

DOI: 10.3389/FCELL.2021.691826

Abstract: Accompanying the precipitous age-related decline in human female fertility is an increase in the proportion of poor-quality oocytes within the ovary. The macroautophagy pathway, an essential protein degradation mechanism responsible for maintaining cell health, has not yet been thoroughly investigated in this phenomenon. The aim of this study was to characterize the macroautophagy pathway in an established mouse model of oocyte aging using in-depth image analysis-based methods and to determine mechanisms that account for the observed changes. Three autophagy pathway markers were selected for assessment of gene and protein expression in this model: Beclin 1 an initiator of autophagosome formation, Microtubule-associated protein 1 light chain 3B a constituent of the autophagosome membrane, and lysosomal-associated membrane protein 1 a constituent of the lysosome membrane. Through quantitative image analysis of immunolabeled oocytes, this study revealed impairment of the macroautophagy pathway in the aged oocyte with an attenuation of both autophagosome and lysosome number. Additionally, an accumulation of hisomes greater than 10 μm 2 in area were observed in aging oocytes, and this accumulation was mimicked in oocytes treated with lysosomal inhibitor chloroquine. Overall, these findings implicate lysosomal dysfunction as a prominent mechanism by which these age-related changes may occur and highlight the importance of macroautophagy in maintaining mouse pre-ovulatory oocyte quality. This provides a basis for further investigation of dysfunctional autophagy in poor oocyte quality and for the development of therapeutic or preventative strategies to aid in the maintenance of pre-ovulatory oocyte health.

Encyclopedia of Gerontology and Population Aging

Publisher: Springer International Publishing

Date: 2020

DOI: 10.1007/978-3-319-69892-2

Alternative splicing: a mechanism for spermatogonia differentiation, meiosis progression and spermatid maturation

Publisher: Bioscientifica

Date: 23-09-2014

DOI: 10.1530/REPABS.1.P201

MeasureIce: accessible on-the-fly measurement of ice thickness in cryo-electron microscopy

Publisher: Springer Science and Business Media LLC

Date: 15-08-2022

DOI: 10.1038/S42003-022-03698-X

Abstract: Ice thickness is arguably one of the most important factors limiting the resolution of protein structures determined by cryo-electron microscopy (cryo-EM). The amorphous atomic structure of the ice that stabilizes and protects biological s les in cryo-EM grids also imprints some additional noise in cryo-EM images. Ice that is too thick jeopardizes the success of particle picking and reconstruction of the biomolecule in the worst case and, at best, deteriorates eventual map resolution. Minimizing the thickness of the ice layer and thus the magnitude of its noise contribution is thus imperative in cryo-EM grid preparation. In this paper we introduce MeasureIce, a simple, easy to use ice thickness measurement tool for screening and selecting acquisition areas of cryo-EM grids. We show that it is possible to simulate thickness-image intensity look-up tables, also usable in SerialEM and Leginon, using elementary scattering physics and thereby adapt the tool to any microscope without time consuming experimental calibration. We benchmark our approach using two alternative techniques: the “ice channel” technique and tilt-series tomography. We also demonstrate the utility of ice thickness measurement for selecting holes in gold grids containing an Equine apoferritin s le, achieving a 1.88 Ångstrom resolution in subsequent refinement of the atomic map.

The contribution of subtle oocyte or sperm dysfunction affecting fertilization in endometriosis-associated or unexplained infertility: a controlled comparison with tubal infertility and use of donor s

Publisher: Oxford University Press (OUP)

Date: 07-1998

DOI: 10.1093/HUMREP/13.7.1825

Abstract: This study aims to determine the relative contribution of oocyte and/or sperm dysfunction to the reduction of fertilization rates in vitro in cases of minor endometriosis and prolonged unexplained infertility. The results of in-vitro fertilization (IVF) treatment with ovarian stimulation have been compared between couples with the above conditions and women with tubal infertility (as control for oocyte function) and the use of donor spermatozoa (as control for sperm function). Fertilization and cleavage rates using husband's spermatozoa were significantly reduced in endometriosis couples (56%, n = 194, P < 0.001) and further significantly reduced in couples with unexplained infertility (52%, n = 327, P < 0.001) compared with tubal infertility (60%, n = 509). Using donor spermatozoa the rates were the same as using husband's spermatozoa in tubal infertility (61%, n = 27) or endometriosis (55%, n = 21) but significantly though only partly improved with unexplained infertility (57%, n = 60, P < 0.02). In unexplained infertility, a significantly increased proportion of couples experienced complete failure of fertilization and cleavage in a cycle (5-6% versus 2-3%). However, complete failure was not usually repetitive, and the affected couples did not account for the overall reduction in fertilization and cleavage rates, which remained significantly lower in the rest of the unexplained and endometriosis groups. Implantation and pregnancy rates appeared similar in all groups. The benefit of IVF treatment in cases of minor endometriosis and prolonged unexplained infertility is due to superabundance of oocytes obtained by stimulation. The reduction in natural fertility associated with endometriosis appears to be at least partly due to a reduced fertilizing ability of the oocyte. In unexplained infertility, there is distinct impairment due to otherwise unsuspected sperm dysfunction but probably also oocyte dysfunction.

Reduced ability to recover from spindle disruption and loss of kinetochore spindle assembly checkpoint proteins in oocytes from aged mice.

Publisher: Informa UK Limited

Date: 23-04-2014

DOI: 10.4161/CC.28897

MiMIC analysis reveals an isoform specific role for Drosophila Musashi in follicle stem cell maintenance and escort cell function

Publisher: Springer Science and Business Media LLC

Date: 12-11-2022

DOI: 10.1038/S41420-022-01245-5

Abstract: The Drosophila ovary is regenerated from germline and somatic stem cell populations that have provided fundamental conceptual understanding on how adult stem cells are regulated within their niches. Recent ovarian transcriptomic studies have failed to identify mRNAs that are specific to follicle stem cells (FSCs), suggesting that their fate may be regulated post-transcriptionally. We have identified that the RNA-binding protein, Musashi (Msi) is required for maintaining the stem cell state of FSCs. Loss of msi function results in stem cell loss, due to a change in differentiation state, indicated by upregulation of Lamin C in the stem cell population. In msi mutant ovaries, Lamin C upregulation was also observed in posterior escort cells that interact with newly formed germ cell cysts. Mutant somatic cells within this region were dysfunctional, as evidenced by the presence of germline cyst collisions, fused egg chambers and an increase in germ cell cyst apoptosis. The msi locus produces two classes of mRNAs (long and short). We show that FSC maintenance and escort cell function specifically requires the long transcripts, thus providing the first evidence of isoform-specific regulation in a population of Drosophila epithelial cells. We further demonstrate that although male germline stem cells have previously been shown to require Msi function to prevent differentiation this is not the case for female germline stem cells, indicating that these similar stem cell types have different requirements for Msi, in addition to the differential use of Msi isoforms between soma and germline. In summary, we show that different isoforms of the Msi RNA-binding protein are expressed in specific cell populations of the ovarian stem cell niche where Msi regulates stem cell differentiation, niche cell function and subsequent germ cell survival and differentiation.

An evaluation of the role of relaxin in the penetration of cervical mucus by spermatozoa

Publisher: Oxford University Press (OUP)

Date: 10-1988

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A136797

Abstract: Relaxin-like immunoreactivity was measured in seminal plasma from men who were separated into two groups, on the basis of a previous positive or negative result in a postcoital cervical mucus penetration test. There was no difference in the relaxin concentration between the groups. The effect of exogenous porcine relaxin (0, 10 or 100 ng/ml) on human cervical mucus penetration in vitro by washed human spermatozoa was studied using a capillary tube preparation. In the positive postcoital test group the highest relaxin concentration (100 ng/ml) tended to inhibit cervical mucus penetration, although this effect was only significant for one of the parameters measured (number of spermatozoa penetrating to the 10-mm mark). The same trend was apparent for the negative postcoital test group, but no differences were significant. The results are in direct contrast to previous reports that relaxin can stimulate human spermatozoa motility and cervical mucus penetration.

Antibiotic treatment based on seminal cultures from asymptomatic male partners in in-vitro fertilization is unnecessary and may be detrimental

Publisher: Oxford University Press (OUP)

Date: 06-1996

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A019361

Abstract: We questioned the policy of routine microbiological culture of semen prior to in-vitro fertilization (IVF) with a view to prescribing antibiotics to reduce the risk of introducing seminal infection into the embryo culture system. An initial retrospective study examined serum microbiology reports of 449 couples undergoing IVF or gamete intra-Fallopian transfer (GIFT). In semen s les taking >/=1 days to reach the microbiology laboratory compared with same-day delivery there was increased frequency of significant culture of enterococci (27 versus 15%, P /=2 days there was increased frequency of significant culture of Gram-negative bacilli (31 versus 12%, P < 0.01) and of overall culture of other potentially pathogenic organisms (26 versus 14%, P < 0.01). We questioned diagnostic accuracy and relevance. Therefore, in a prospective study, semen and high vaginal swabs obtained on the day of oocyte collection were cultured from 100 couples having IVF or GIFT, of whom 52 male partners had been treated with antibiotics following positive pre-IVF semen culture. The presence of bacteria in semen s les used only for IVF (n = 90) did not reduce fertilization rates nor lead to infection of the embryo culture system. However, there was an increased incidence of significant culture of vaginal Gram-negative bacilli in patients with treated partners compared with untreated partners [15/52 (29%) versus 5/48 (10%), P < 0.05]. Thus antibiotic therapy in the male partner may increase the likelihood of inoculation of antibiotic-resistant pathogenic bacteria from the vagina into the embryo culture system during vaginal oocyte collection. In asymptomatic patients, microbiological screening of semen s les prior to IVF treatment and subsequent treatment with antibiotic therapy in those with positive cultures appears to be unnecessary and may be detrimental to IVF outcome.

British Andrology Society guidelines for the assessment of post vasectomy semen samples (2002)

Publisher: BMJ

Date: 11-2002

DOI: 10.1136/JCP.55.11.812

Abstract: The British Andrology Society guidelines for the assessment of post vasectomy semen s les recommend that initial assessment is undertaken 16 weeks post vasectomy and after the patient has produced at least 24 ejaculates. The laboratory should examine a freshly produced seminal fluid specimen by direct microscopy and if no sperm are seen the centrifugate should be examined for the presence of motile and non-motile spermatozoa. It is recommended that the clinician should give clearance after the production of two consecutive sperm free ejaculates. In cases of persistent identification of non-motile spermatozoa the referring clinician should advise the patient regarding the cessation of other contraceptive precautions. Surgeons are responsible both preoperatively and postoperatively for the counselling of couples regarding complications and the possibility of late recanalisation after clearance.

MiMIC analysis reveals an isoform specific role for Drosophila Musashi in follicle stem cell maintenance and escort cell function

Publisher: Cold Spring Harbor Laboratory

Date: 26-08-2020

DOI: 10.1101/2020.08.25.265769

Abstract: The Drosophila ovary is regenerated from germline and somatic stem cell populations that have provided fundamental conceptual understanding on how adult stem cells are regulated within their niches. Recent ovarian transcriptomic studies have failed to identify mRNAs that are specific to follicle stem cells (FSCs), suggesting that their fate may be regulated post-transcriptionally. We have identified that the RNA-binding protein, Musashi (Msi) is required for maintaining the stem cell state of FSCs. Loss of msi function results in stem cell loss, not due to cell death, but mutant FSCs upregulate Lamin C, indicating a change in differentiation state. In msi mutant ovaries, Lamin C upregulation was also observed in posterior escort cells and mutant somatic cells within regions 2/3 were dysfunctional, as evidenced by the presence of germline cyst collisions and fused egg chambers. The msi locus produces two classes of mRNAs (long and short). We show that FSC maintenance and escort cell function specifically requires the long transcripts, thus providing the first evidence of isoform-specific regulation in a population of Drosophila epithelial cells. We further demonstrate that although male germline stem cells have previously been shown to require Msi function to prevent differentiation this is not the case for female germline stem cells, indicating that these similar stem cell types have different requirements for Msi, in addition to the differential use of Msi isoforms between soma and germline.

Awakening the oocyte: controlling primordial follicle development

Publisher: Bioscientifica

Date: 2009

DOI: 10.1530/REP-08-0118

Abstract: Oocytes are sequestered in primordial follicles before birth and remain quiescent in the ovary, often for decades, until recruited into the growing pool throughout the reproductive years. Therefore, activation of follicle growth is a major biological checkpoint that controls female reproductive potential. However, we are only just beginning to elucidate the cellular mechanisms required for either maintenance of the quiescent primordial follicle pool or initiation of follicle growth. Understanding the intracellular signalling systems that control oocyte maintenance and activation has significant implications for improving female reproductive productivity and longevity in mammals, and has application in domestic animal husbandry, feral animal population control and infertility in women.

A scoping review of the information provided by fertility smartphone applications

Publisher: Informa UK Limited

Date: 30-03-2021

DOI: 10.1080/14647273.2021.1871784

Abstract: The growth of smartphone application use across areas of female reproductive health has led to increased interest into their functions and benefits. This scoping review aims to determine the nature and extent of the peer-reviewed literature presented on fertility-based apps, to identify the reliability of the information within the apps, and to determine the ability of this information to educate users. A systematic search of six databases was conducted in April 2020, returning a total of 21,158 records. After duplicate removal, title and abstract screening exclusionary steps, 27 records were reviewed and charted. Records covered a variety of reproductive health themes including contraception, sexual health, and family planning, and used a range of methodologies. The accuracy of fertility information within the apps reported in these studies was variable, but overall there was a lack of depth in the coverage of content in apps. It was common for studies in this review to base fertile window algorithms on stringent cycle length and variability requirements, limiting the applicability of information delivered to users. Furthermore, studies from app affiliates often lacked collaborations with researchers, minimising the potential for fertility knowledge improvements integrated across the suite of female reproductive health apps.

Resolution of Virtual Depth Sectioning from Four-Dimensional Scanning Transmission Electron Microscopy

Publisher: Oxford University Press (OUP)

Date: 12-07-2023

DOI: 10.1093/MICMIC/OZAD068

Abstract: One approach to three-dimensional structure determination using the wealth of scattering data in four-dimensional (4D) scanning transmission electron microscopy (STEM) is the parallax method proposed by Ophus et al. (2019. Advanced phase reconstruction methods enabled by 4D scanning transmission electron microscopy, Microsc Microanal25, 10–11), which determines the scattering matrix and uses it to synthesize a virtual depth-sectioning reconstruction of the s le structure. Drawing on an equivalence with a hypothetical confocal imaging mode, we derive contrast transfer and point spread functions for this parallax method applied to weakly scattering objects, showing them identical to earlier depth-sectioning STEM modes when only bright field signal is used, but that improved depth resolution is possible if dark field signal can be used. Through a simulation-based study of doped Si, we show that this depth resolution is preserved for thicker s les, explore the impact of shot noise on the parallax reconstructions, discuss challenges to making use of dark field signal, and identify cases where the interpretation of the parallax reconstruction breaks down.

Pharmacological inhibition of arachidonate 15-lipoxygenase protects human spermatozoa against oxidative stress.

Publisher: Oxford University Press (OUP)

Date: 13-03-2018

DOI: 10.1093/BIOLRE/IOY058

Abstract: One of the leading causes of male infertility is defective sperm function, a pathology that commonly arises from oxidative stress in the germline. Lipid peroxidation events in the sperm plasma membrane result in the generation of cytotoxic aldehydes such as 4-hydroxynonenal (4HNE), which accentuate the production of reactive oxygen species (ROS) and cause cellular damage. One of the key enzymes involved in the metabolism of polyunsaturated fatty acids to 4HNE in somatic cells is arachidonate 15-lipoxygenase (ALOX15). Although ALOX15 has yet to be characterized in human spermatozoa, our previous studies have revealed a strong link between ALOX15 activity and the levels of oxidative stress and 4HNE in mouse germ cell models. In view of these data, we sought to assess the function of ALOX15 in mature human spermatozoa and determine whether the pharmacological inhibition of this enzyme could influence the level of oxidative stress experienced by these cells. By driving oxidative stress in vitro with exogenous H2O2, our data reveal that 6,11-dihydro[1]benzothiopyrano[4,3-b]indole (PD146176 a selective ALOX15 inhibitor) was able to significantly reduce several deleterious, oxidative insults in spermatozoa. Indeed, PD146176 attenuated the production of ROS, as well as membrane lipid peroxidation and 4HNE production in human spermatozoa. Accordingly, ALOX15 inhibition also protected the functional competence of these cells to acrosome react and bind homologous human zonae pellucidae. Together, these results implicate ALOX15 in the propagation of oxidative stress cascades within human spermatozoa and offer insight into potential therapeutic avenues to address male in fertility that arises from oxidative stress.

168. IDENTIFICATION AND CHARACTERISATION OF SURFACE PROTEIN COMPLEXES IN HUMAN SPERMATOZOA

Publisher: CSIRO Publishing

Date: 2009

DOI: 10.1071/SRB09ABS168

Abstract: Upon leaving the testis mammalian spermatozoa are functionally incompetent and are thus unable to fertilize an oocyte. As the spermatozoa ascend the female reproductive tract, functional maturity is achieved through a complex cascade of biophysical and biochemical changes known as capacitation. An important aspect of this final maturation phase is the remodelling of the sperm surface architecture to enable it to interact with the zona pellucida, a glycoprotein matrix that surrounds the oocyte, and initiate fertilisation. While originally thought to be underpinned by a simple lock and key mechanism, emerging evidence has suggested that this interaction may instead be mediated by a multimeric recognition complex that is formed on the sperm surface during capacitation. However, to date the presence and composition of such a complex has yet to be described. Through the application of Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE), we have provided evidence that human spermatozoa express a number of high molecular weight protein complexes on their surface. Furthermore, the affinity of these surface expressed complexes for the zona pellucida was assessed utilising solubilised human zona pellucida and the technique of Far Western Blotting. Among the complexes that showed affinity for the zona pellucida we identified one comprising 14 subunits of the 20S proteasome. Interestingly, the 20S proteasome has previously been implicated in various aspects of mammalian fertilisation, including zona pellucida penetration and the acrosome reaction, although its precise role in these events has yet to be elucidated. Collectively, these results demonstrate the presence of multimeric protein complexes on the surface of human spermatozoa, and support their putative role in the initial interaction between the sperm and the zona pellucida. Our current research is focused on elucidation of the role of the 20S proteasome in human sperm-zona binding and further investigation of surface expressed protein complexes.

Hematogenous dissemination of Chlamydia muridarum from the urethra in macrophages causes testicular infection and sperm DNA damage†

Publisher: Oxford University Press (OUP)

Date: 02-08-2019

DOI: 10.1093/BIOLRE/IOZ146

Abstract: The incidence of Chlamydia infection, in both females and males, is increasing worldwide. Male infections have been associated clinically with urethritis, epididymitis, and orchitis, believed to be caused by ascending infection, although the impact of infection on male fertility remains controversial. Using a mouse model of male chlamydial infection, we show that all the major testicular cell populations, germ cells, Sertoli cells, Leydig cells, and testicular macrophages can be productively infected. Furthermore, sperm isolated from vas deferens of infected mice also had increased levels of DNA damage as early as 4 weeks post-infection. Bilateral vasectomy, prior to infection, did not affect the chlamydial load recovered from testes at 2, 4, and 8 weeks post-infection, and Chlamydia-infected macrophages were detectable in blood and the testes as soon as 3 days post-infection. Partial depletion of macrophages with clodronate liposomes significantly reduced the testicular chlamydial burden, consistent with a hematogenous route of infection, with Chlamydia transported to the testes in infected macrophages. These data suggest that macrophages serve as Trojan horses, transporting Chlamydia from the penile urethra to the testes within 3 days of infection, bypassing the entire male reproductive tract. In the testes, infected macrophages likely transfer infection to Leydig, Sertoli, and germ cells, causing sperm DNA damage and impaired spermatogenesis.

Glycogen synthase kinase 3 regulates acrosomal exocytosis in mouse spermatozoa via dynamin phosphorylation

Publisher: Wiley

Date: 25-03-2015

DOI: 10.1096/FJ.14-265553

Abstract: The dynamin family of GTPases has been implicated as novel regulators of the acrosome reaction, a unique exocytotic event that is essential for fertilization. Dynamin activity during the acrosome reaction is accompanied by phosphorylation of key serine residues. We now tested the hypothesis that glycogen synthase kinase 3 (GSK3) is the protein kinase responsible for dynamin phosphorylation at these phosphosites in mouse spermatozoa. Pharmacologic inhibition of GSK3 in mature mouse spermatozoa (CHIR99021: IC50 = 6.7 nM) led to a significant reduction in dynamin phosphorylation (10.3% vs. 27.3% P < 0.001), acrosomal exocytosis (9.7% vs. 25.7% P < 0.01), and in vitro fertilization (53% vs. 100% P < 0.01). GSK3 was shown to be present in developing germ cells where it colocalized with dynamin in the peri-acrosomal domain. However, additional GSK3 was acquired by maturing mouse spermatozoa within the male reproductive tract, via a novel mechanism involving direct interaction of sperm heads with extracellular structures known as epididymal dense bodies. These data reveal a novel mode for the cellular acquisition of a protein kinase and identify a key role for GSK3 in the regulation of sperm maturation and acrosomal exocytosis.

Interlaboratory Reproducibility of a Single-Locus Sequence-Based Method for Strain Typing of Aspergillus fumigatus

Publisher: American Society for Microbiology

Date: 05-2009

DOI: 10.1128/JCM.00124-09

Abstract: Seven international laboratories tested the recently proposed single-locus typing strategy for Aspergillus fumigatus subtyping for interlaboratory reproducibility. Comparative sequence analyses of portions of the locus AFUA_3G08990, encoding a putative cell surface protein (denoted CSP), was performed with a panel of Aspergillus isolates. Each laboratory followed very different protocols for extraction of DNA, PCR, and sequencing. Results revealed that the CSP typing method was a reproducible and portable strain typing method.

Contraceptive vaccines

Publisher: Informa Healthcare

Date: 08-2003

DOI: 10.1517/14712598.3.5.829

Abstract: The control of human fertility would be revolutionised by the development of a safe, effective, long-acting contraceptive vaccine. The pursuit of this objective has involved the selection of appropriate targets within the reproductive process that are amenable to interference with antibodies. To date, three major targets have been researched. The zona pellucida (ZP) plays key roles in folliculogenesis, fertilisation and early development, and is comprised of powerful cell-specific antigens. The induction of infertility requires high ZP antibody titres that are difficult to maintain without inducing ovarian pathology characterised by a premature loss of primordial follicles. As a premature menopause would be a high price to pay for long-term contraception, this approach to a vaccine cannot progress until the cause of the ovarian pathology has been resolved. Sperm surface antigens represent another promising approach to contraceptive vaccine development. While there is some clinical data to support the likely efficacy of this strategy, none of the gamete-specific molecules characterised to date have fulfilled this promise. Anti-human chorionic gonadotropin (hCG) vaccines terminate pregnancy by preventing the maternal recognition of pregnancy. This vaccine has reached the stage of clinical trials, and preliminary indications are that the approach is safe and potentially effective. However, reliability may be an issue, given the observed inter-in idual variability in antibody generation. The future of contraceptive vaccine development will clearly involve a continuation of the intense search for suitable targets and the development of improved immunisation procedures that exploit the latest innovations in vaccine technology.

Effects of cryopreservation on the human sperm acrosome and its response to A23187

Publisher: Bioscientifica

Date: 09-1993

DOI: 10.1530/JRF.0.0990071

Abstract: The proportion of human spermatozoa from 28 ejaculates to lose their acrosomes during cryopreservation was measured and correlated with the number that became immotile or lost the integrity of their plasma membrane. The ability of washed spermatozoa to acrosome react in response to A23187 before and after cryopreservation was compared. Motility was assessed by time-lapse photography intact acrosomes were stained with fluorescein conjugated Pisum sativum agglutinin and dead spermatozoa were stained with bisbenzimide (H33258). Twenty-four per cent of spermatozoa lost their acrosomes during freezing and thawing, but the number that did so was not correlated with the number that became immotile or non-viable. Frozen spermatozoa exhibited fewer spontaneous acrosome reactions than did fresh spermatozoa (5 versus 13% after 4 h), but they responded to A23187 in a similar way. Although frozen spermatozoa were significantly more likely to die during the incubation, the data do not suggest that degenerative acrosome loss had a major influence on the results. In the hamster egg test frozen-thawed spermatozoa achieved more penetrations than did fresh spermatozoa when stimulated with 0 or 1 mumol A23187 l-1 but considerably fewer when stimulated with 4 mumol A23187 l-1. The following conclusions were made. First, cryopreservation damage to the acrosome, the plasma membrane and the flagellum can occur independently. Second, acrosome function is maintained after cryopreservation as long as the organelle remains mechanically intact. Third, some spermatozoa that lose their acrosomes during cryopreservation remain viable and can fuse with zona-free hamster eggs.

Developmental expression of the dynamin family of mechanoenzymes in the mouse epididymis

Publisher: Oxford University Press (OUP)

Date: 20-12-2016

DOI: 10.1095/BIOLREPROD.116.145433

Abstract: The mammalian epididymis is an exceptionally long ductal system tasked with the provision of one of the most complex intraluminal fluids found in any exocrine gland. This specialized milieu is continuously modified by the combined secretory and absorptive of the surrounding epithelium and thus finely tuned for its essential roles in promoting sperm maturation and storage. While considerable effort has been focused on defining the composition of the epididymal fluid, relatively less is known about the intracellular trafficking machinery that regulates this luminal environment. Here, we characterize the ontogeny of expression of a master regulator of this machinery, the dynamin family of mechanoenzymes. Our data show that canonical dynamin isoforms were abundantly expressed in the juvenile mouse epididymis. However, in peripubertal and adult animals dynamin takes on a heterogeneous pattern of expression such that the different isoforms displayed both cell- and segment-specific localization. Thus, dynamin 1 and 3 were predominately localized in the distal epididymal segments (corpus and cauda), where they were found within clear and principal cells, respectively. In contrast, dynamin 2 was expressed throughout the epididymis, but localized to the Golgi apparatus of the principal cells in the proximal (caput) segment and the luminal border of these cells in more distal segments. These dynamin isoforms are therefore ideally positioned to play complementary, nonredundant roles in the regulation of the epididymal milieu. In support of this hypothesis, selective inhibition of dynamin altered the profile of proteins secreted from an immortalized caput epididymal cell line.

Exploiting Machine Learning Algorithms and Methods for the Prediction of Agitated Delirium After Cardiac Surgery: Models Development and Validation Study (Preprint)

Publisher: JMIR Publications Inc.

Date: 11-06-2019

DOI: 10.2196/PREPRINTS.14993

Abstract: elirium is a temporary mental disorder that occasionally affects patients undergoing surgery, especially cardiac surgery. It is strongly associated with major adverse events, which in turn leads to increased cost and poor outcomes (eg, need for nursing home due to cognitive impairment, stroke, and death). The ability to foresee patients at risk of delirium will guide the timely initiation of multimodal preventive interventions, which will aid in reducing the burden and negative consequences associated with delirium. Several studies have focused on the prediction of delirium. However, the number of studies in cardiac surgical patients that have used machine learning methods is very limited. his study aimed to explore the application of several machine learning predictive models that can pre-emptively predict delirium in patients undergoing cardiac surgery and compare their performance. e investigated a number of machine learning methods to develop models that can predict delirium after cardiac surgery. A clinical dataset comprising over 5000 actual patients who underwent cardiac surgery in a single center was used to develop the models using logistic regression, artificial neural networks (ANN), support vector machines (SVM), Bayesian belief networks (BBN), naïve Bayesian, random forest, and decision trees. nly 507 out of 5584 patients (11.4%) developed delirium. We addressed the underlying class imbalance, using random unders ling, in the training dataset. The final prediction performance was validated on a separate test dataset. Owing to the target class imbalance, several measures were used to evaluate algorithm’s performance for the delirium class on the test dataset. Out of the selected algorithms, the SVM algorithm had the best F1 score for positive cases, kappa, and positive predictive value (40.2%, 29.3%, and 29.7%, respectively) with a italic P /italic =.01, .03, .02, respectively. The ANN had the best receiver-operator area-under the curve (78.2% italic P /italic =.03). The BBN had the best precision-recall area-under the curve for detecting positive cases (30.4% italic P /italic =.03). lthough delirium is inherently complex, preventive measures to mitigate its negative effect can be applied proactively if patients at risk are prospectively identified. Our results highlight 2 important points: (1) addressing class imbalance on the training dataset will augment machine learning model’s performance in identifying patients likely to develop postoperative delirium, and (2) as the prediction of postoperative delirium is difficult because it is multifactorial and has complex pathophysiology, applying machine learning methods (complex or simple) may improve the prediction by revealing hidden patterns, which will lead to cost reduction by prevention of complications and will optimize patients’ outcomes.

Next Generation Sequencing Analysis Reveals Segmental Patterns of microRNA Expression in Mouse Epididymal Epithelial Cells

Publisher: Public Library of Science (PLoS)

Date: 13-08-2015

DOI: 10.1371/JOURNAL.PONE.0135605

Letters to the Editor

Publisher: Oxford University Press (OUP)

Date: 10-2000

DOI: 10.1093/HUMREP/15.10.2247-A

Non-coding RNA in spermatogenesis and epididymal maturation

Publisher: Springer Netherlands

Date: 12-12-2016

DOI: 10.1007/978-94-017-7417-8_6

Abstract: Testicular germ and somatic cells express many classes of small ncRNAs, including Dicer-independent PIWI-interacting RNAs, Dicer-dependent miRNAs, and endogenous small interfering RNA. Several studies have identified ncRNAs that are highly, exclusively, or preferentially expressed in the testis and epididymis in specific germ and somatic cell types. Temporal and spatial expression of proteins is a key requirement of successful spermatogenesis and large-scale gene transcription occurs in two key stages, just prior to transcriptional quiescence in meiosis and then during spermiogenesis just prior to nuclear silencing in elongating spermatids. More than 60 % of these transcripts are then stockpiled for subsequent translation. In this capacity ncRNAs may act to interpret and transduce cellular signals to either maintain the undifferentiated stem cell population and/or drive cell differentiation during spermatogenesis and epididymal maturation. The assignation of specific roles to the majority of ncRNA species implicated as having a role in spermatogenesis and epididymal function will underpin fundamental understanding of normal and disease states in humans such as infertility and the development of germ cell tumours.

Molecular Mechanisms Responsible for Increased Vulnerability of the Ageing Oocyte to Oxidative Damage

Publisher: Hindawi Limited

Date: 2017

DOI: 10.1155/2017/4015874

Abstract: In their midthirties, women experience a decline in fertility, coupled to a pronounced increase in the risk of aneuploidy, miscarriage, and birth defects. Although the aetiology of such pathologies are complex, a causative relationship between the age-related decline in oocyte quality and oxidative stress (OS) is now well established. What remains less certain are the molecular mechanisms governing the increased vulnerability of the aged oocyte to oxidative damage. In this review, we explore the reduced capacity of the ageing oocyte to mitigate macromolecular damage arising from oxidative insults and highlight the dramatic consequences for oocyte quality and female fertility. Indeed, while oocytes are typically endowed with a comprehensive suite of molecular mechanisms to moderate oxidative damage and thus ensure the fidelity of the germline, there is increasing recognition that the efficacy of such protective mechanisms undergoes an age-related decline. For instance, impaired reactive oxygen species metabolism, decreased DNA repair, reduced sensitivity of the spindle assembly checkpoint, and decreased capacity for protein repair and degradation collectively render the aged oocyte acutely vulnerable to OS and limits their capacity to recover from exposure to such insults. We also highlight the inadequacies of our current armoury of assisted reproductive technologies to combat age-related female infertility, emphasising the need for further research into mechanisms underpinning the functional deterioration of the ageing oocyte.

DNA damage contributes to transcriptional and immunological dysregulation of testicular cells during Chlamydia infection

Publisher: Wiley

Date: 20-02-2021

DOI: 10.1111/AJI.13400

Abstract: Chlamydia is the most commonly reported sexually transmitted bacterial infection, with 127 million notifications worldwide each year. Both males and females are susceptible to the pathological impacts on fertility that Chlamydia infections can induce. However, male chlamydial infections, particularly within the upper reproductive tract, including the testis, are not well characterized. In this study, using mouse testicular cell lines, we examined the impact of infection on testicular cell lineage transcriptomes and potential mechanisms for this impact. The somatic cell lineages exhibited significantly fragmented genomes during infection. Likely resulting from this, each of the Leydig, Sertoli and germ cell lineages experienced extensive transcriptional dysregulation, leading to significant changes in cellular biological pathways, including interferon and germ‐Sertoli cell signalling. The cell lineages, as well as isolated spermatozoa from infected mice, also contained globally hypomethylated DNA. Cumulatively, the DNA damage and epigenetic‐mediated transcriptional dysregulation observed within testicular cells during chlamydial infection could result in the production of spermatozoa with abnormal epigenomes, resulting in previously observed subfertility in infected animals and congenital defects in their offspring.

P-157. The contribution of subtle oocyte or sperm dysfunction to endometriosis-associated and unexplained infertility

Publisher: Oxford University Press (OUP)

Date: 06-1997

DOI: 10.1093/HUMREP/12.SUPPL_2.194-A

Intraoperative ventilation: improving physiology, or preventing harm?

Publisher: Elsevier BV

Date: 03-2016

DOI: 10.1093/BJA/AEW013

10. Modulation of inflammatory signalling pathways in the ovary by immune challenge in early life

Publisher: Elsevier BV

Date: 09-2013

DOI: 10.1016/J.BBI.2013.07.022

Letters to the Editor

Publisher: Oxford University Press (OUP)

Date: 10-2000

DOI: 10.1093/HUMREP/15.10.2247-B

RNA binding protein Musashi‐2 regulates PIWIL1 and TBX1 in mouse spermatogenesis

Publisher: Wiley

Date: 04-10-2017

DOI: 10.1002/JCP.26168

Abstract: RNA-binding proteins (RBP) are important facilitators of post-transcriptional gene regulation. We have previously established that nuclear overexpression of the RBP Musashi-2 (MSI2) during male germ cell maturation is detrimental to sperm cell development and fertility. Herein we determine the genes and pathways impacted by the upregulation of Msi2. Microarray analysis and qPCR confirmed differential gene expression in factors fundamental to the cell cycle, cellular proliferation, and cell death. Similarly, comparative protein expression analysis via iTRAQ, immunoblot, and immunolocalization, identified differential expression and localization of important regulators of transcription, translation, RNA processing, and spermatogenesis. Specifically, the testis-expressed transcription factor, Tbx1, and the piRNA regulator of gamete development, Piwil1, were both found to be targeted for translational repression by MSI2. This study provides key evidence to support a fundamental role for MSI2 in post-transcriptional regulation during male gamete development.

Chlamydia infection in males; an underappreciated problem

Publisher: Elsevier BV

Date: 06-2016

DOI: 10.1016/J.JRI.2016.04.160

Intracellular signalling during female gametogenesis

Publisher: Oxford University Press (OUP)

Date: 17-12-2012

DOI: 10.1093/MOLEHR/GAS065

Abstract: Female reproductive potential is dictated by the size of the primordial follicle pool and the correct regulation of oocyte maturation and activation--events essential for production of viable offspring. Although a substantial body of work underpins our understanding of these processes, the molecular mechanisms of follicular and oocyte development are not fully understood. This review summarizes recent findings which have improved our conception of how folliculogenesis and oocyte competence are regulated, and discusses their implications for assisted reproductive techniques. We highlight evidence provided by genetically modified mouse models and in vitro studies which have refined our understanding of Pi3k/Akt and mTOR signalling in the oocyte and have discovered a role for Jak/Stat/Socs signalling in granulosa cells during primordial follicle activation. We also appraise a novel role for the metal ion zinc in the regulation of meiosis I and meiosis II progression through early meiosis inhibitor (Emi2) and Mos-Mapk signalling, and examine studies which expand our understanding of intracellular calcium signalling and extrinsic Plcζ in stimulating oocyte activation.

153. XENOBIOTICS; INFLUENCE ON OVARIAN FOLLICULAR DEVELOPMENT

Publisher: CSIRO Publishing

Date: 2009

DOI: 10.1071/SRB09ABS153

Abstract: The mammalian female reproductive lifespan is largely defined by a finite pool of ovarian follicles established around the time of birth. It is now understood that certain synthetic chemical compounds, known as xenobiotics, can cause premature ovarian senescence through the destruction of small ovarian follicles. Although the ovotoxic effects of these chemicals are well documented, the exact molecular mechanisms behind their action are only just becoming understood. Recent evidence suggests that bioactivation of xenobiotics by Phase I detoxifying enzymes may lead to the generation of free oxygen radicals (ROS), which we suspect may perturb intracellular signalling pathways in primordial follicles. In this study we attempted to identify ovarian follicle signalling pathways activated by xenobiotic exposure using ovotoxic agents which target immature follicles. Neonatal ovaries obtained from 3/4-day old Swiss mice were exposed to either 4-Vinylcyclohexene (25µM), Methoxychlor (25µM) or Menadione (5µM) for 96hrs using our in vitro culture system. Total RNA was then collected and analysed using Affymetrix Mouse Genome 430 2.0 Arrays. Bioinformatic analysis identified between ~500–1000 genes with a two-fold significant difference in gene expression (p .05) for each xenobiotic compared to the control. Differentially expressed genes were analysed for pathways and molecular functions using Ingenuity Pathways Analysis (Ingenuity Systems). In agreement with the current literature, many of the genes belonged to toxic response pathways, such as Xenobiotic metabolism (10) p53 (15) and Apoptosis (11) signalling. However, the vast majority of the differentially expressed genes belonged to canonical pathways implicated in follicular development, such as PI3K/AKT (18), Wnt/ b -catenin (21), and JAK/Stat (8) signalling. Further qPCR analysis has confirmed a substantial increase in the transcription factor Sox4 and cell cycle inhibitor Cdkn2a in 4-Vinylcyclohexene and Menadione treated ovaries respectively. These results suggest that xenobiotics which target primordial follicles may exert part of their ovotoxic effects by perturbing signalling pathways involved in follicular activation and development.

Recruitment of gamete donors and payment of expenses

Publisher: Oxford University Press (OUP)

Date: 05-1998

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A019705

Abstract: Experience of early-life socioeconomic deprivation (ELSD) may increase the risk of mental disorders in young adulthood. This association may be mediated by structural and functional alterations of the hippoc us. We conducted a prospective cohort study on 122 participants of the European Longitudinal Study of Pregnancy and Childhood. Information about ELSD was collected via questionnaire from mothers during the first 18 months of participants' lives. At age 23-24, participants underwent examination by structural magnetic resonance imaging, resting-state functional connectivity and assessment of depressive symptoms (Mood and Feelings Questionnaire) and anxiety (Spielberger State-Trait Anxiety Inventory). The association of ELSD with brain outcomes in young adulthood was assessed with correlations, linear regression (adjusting for sex, socioeconomic position and mother's mental health) and moderated mediation analysis. Higher ELSD was associated with greater depressive symptoms (B = 0.22 p = 0.001), trait anxiety (B = 0.07 p = 0.02) and lower global connectivity of the right hippoc us (B = -0.01 p = 0.02). These associations persisted when adjusted for covariates. In women, lower global connectivity of the right hippoc us was associated with stronger trait anxiety (B = -4.14 p = 0.01). Global connectivity of the right hippoc us as well as connectivity between the right hippoc us and the left middle temporal gyrus mediated the association between ELSD and trait anxiety in women. Higher ELSD correlated with a lower volume of the right hippoc us in men, but the volume of the right hippoc us was not related to mental health. Early preventive strategies targeted at children from socioeconomically deprived families may yield long-lasting benefits for the mental health of the population.

Fitting a square beam in a square camera: Novel condenser apertures for low-dose transmission electron microscopy

Publisher: Research Square Platform LLC

Date: 15-09-2023

DOI: 10.21203/RS.3.RS-3316272/V1

Effect of aging on superovulation efficiency, aneuploidy rates, and sister chromatid cohesion in mice aged up to 15 months

Publisher: Oxford University Press (OUP)

Date: 02-2012

DOI: 10.1095/BIOLREPROD.111.095711

Abstract: Human eggs are highly aneuploid, with female age being the only known risk factor. Here this aging phenomenon was further studied in Swiss CD1 mice aged between 1 and 15 mo. The mean number of eggs ± SEM recovered from mice following superovulation peaked at 22.5 ± 3.8 eggs/oviduct in 3-mo-old females, decreasing markedly between 6 and 9 mo old, and was only 2.1 ± 0.2 eggs/oviduct by 15 mo. Measurement of aneuploidy in these eggs revealed a low rate, ∼3-4%, in mice aged 1 and 3 mo, rising to 12.5% by 9 mo old and to 37.5% at 12 mo. Fifteen-month-old mice had the highest rate of aneuploidy, peaking at 60%. The in situ chromosome counting technique used here allowed us to measure with accuracy the distance between the kinetochores in the sister chromatids of the eggs analyzed for aneuploidy. We observed that this distance increased in eggs from older females, from 0.38 ± 0.01 μm at 1 mo old to 0.82 ± 0.03 μm by 15 mo. Furthermore, in 3- to 12-mo-old females, aneuploid eggs had significantly larger interkinetochore distances than euploid eggs from the same age, and measurements were similar to eggs from the oldest mice. However, the association between aneuploidy and interkinetochore distance was not observed at the oldest, 15-mo age, despite such measurements being maximal. We conclude that in aging CD1 mice, a reduction in the ovulated egg number precedes a rise in aneuploidy and, furthermore, except at very advanced ages, increased interkinetochore distance is associated with aneuploidy.

In vitro fertilization: Fertilization failure due to toxic catheters

Publisher: Springer Science and Business Media LLC

Date: 02-1987

DOI: 10.1007/BF01555438

Abstract: Antibodies that neutralize (nAbs) genetically erse HIV-1 strains have been recovered from a subset of HIV-1 infected subjects during chronic infection. Exact mechanisms that expand the otherwise narrow neutralization capacity observed during early infection are, however, currently undefined. Here we characterized the earliest nAb responses in a subtype A HIV-1 infected Rwandan seroconverter who later developed moderate cross-clade nAb breadth, using (i) envelope (Env) glycoproteins from the transmitted/founder virus and twenty longitudinal nAb escape variants, (ii) longitudinal autologous plasma, and (iii) autologous monoclonal antibodies (mAbs). Initially, nAbs targeted a single region of gp120, which flanked the V3 domain and involved the alpha2 helix. A single amino acid change at one of three positions in this region conferred early escape. One immunoglobulin heavy chain and two light chains recovered from autologous B cells comprised two mAbs, 19.3H-L1 and 19.3H-L3, which neutralized the founder Env along with one or three of the early escape variants carrying these mutations, respectively. Neither mAb neutralized later nAb escape or heterologous Envs. Crystal structures of the antigen-binding fragments (Fabs) revealed flat epitope contact surfaces, where minimal light chain mutation in 19.3H-L3 allowed for additional antigenic interactions. Resistance to mAb neutralization arose in later Envs through alteration of two glycans spatially adjacent to the initial escape signatures. The cross-neutralizing nAbs that ultimately developed failed to target any of the defined V3-proximal changes generated during the first year of infection in this subject. Our data demonstrate that this subject's first recognized nAb epitope elicited strain-specific mAbs, which incrementally acquired autologous breadth, and directed later B cell responses to target distinct portions of Env. This immune re-focusing could have triggered the evolution of cross-clade antibodies and suggests that exposure to a specific sequence of immune escape variants might promote broad humoral responses during HIV-1 infection.

Contraception Targets in Mammalian Ovarian Development

Publisher: Springer Berlin Heidelberg

Date: 2010

DOI: 10.1007/978-3-642-02062-9_4

Abstract: In the human ovary, early in pre-natal life, oocytes are surrounded by pre-granulosa follicular cells to form primordial follicles. These primordial oocytes remain dormant, often for decades, until recruited into the growing pool throughout a woman's adult reproductive years. Activation of follicle growth and subsequent development of growing oocytes in pre-antral follicles are major biological checkpoints that determine an in idual females reproductive potential. In the past decade, great strides have been made in the elucidation of the molecular and cellular mechanisms underpinning maintenance of the quiescent primordial follicle pool and initiation and development of follicle growth. Gaining an in-depth knowledge of the intracellular signalling systems that control oocyte preservation and follicle activation has significant implications for improving female reproductive productivity and alleviating infertility. It also has application in domestic animal husbandry, feral animal population control and contraception in women.

A Unique Combination of Male Germ Cell miRNAs Coordinates Gonocyte Differentiation

Publisher: Public Library of Science (PLoS)

Date: 20-04-2012

DOI: 10.1371/JOURNAL.PONE.0035553

Tyrosine phosphorylation activates surface chaperones facilitating sperm-zona recognition

Publisher: The Company of Biologists

Date: 15-07-2004

DOI: 10.1242/JCS.01214

Abstract: Mammalian spermatozoa undergo a series of molecular and biochemical changes collectively termed capacitation prior to acquiring the ability to fertilise the oocyte. Although phosphorylation of sperm proteins on tyrosine residues has been recognised as an important component of this process, the precise relationship between the phosphorylation status of mammalian spermatozoa and their capacity for fertilisation has remained unclear. In this study we demonstrate a causal relationship between tyrosine phosphorylation in spermatozoa and sperm-zona interaction. The phosphotyrosine expression associated with sperm capacitation localised to internal flagellar structures in permeabilised cells but could also be detected on the exterior surface of the sperm head in live cells. Importantly, almost all spermatozoa bound to the zona pellucida demonstrated this pattern of phosphoprotein localisation, compared to fewer than 15% of the free-swimming population. These data suggest that tyrosine phosphorylation plays a significant role in remodelling the sperm surface, so that these cells are able to recognise the zona pellucida. Phosphoproteome analysis yielded the first evidence of molecular chaperones, endoplasmin (erp99) and heat shock protein 60 (hsp60), as targets for phosphorylation on the surface of mouse spermatozoa, whereas immunofluorescence localised these proteins to the precise region of the sperm head that participates in zona recognition. Based on these results, we propose a novel mechanism for mammalian gamete interaction whereby the activation of sperm-surface chaperones by tyrosine phosphorylation during capacitation may trigger conformational changes facilitating the formation of a functional zona pellucida receptor complex on the surface of mammalian spermatozoa.

Translational control in germ cell development: A role for the RNA-binding proteins Musashi-1 and Musashi-2

Publisher: Wiley

Date: 2011

DOI: 10.1002/IUB.499

Abstract: Mammalian gametogenesis is a complex process involving specialised cell cycle progression and differentiation. As part of their differentiation, germ cells experience periods of transcriptional inactivation and chromatin inaccessibility whilst continuing to coordinate the correct temporal and spatial expression of genes required for continued development. To overcome these obstacles, mammalian germ cells express a wide variety of sequence-specific RNA-binding proteins, which assist in the translational control of many mRNA transcripts which are produced and stored during periods of high mRNA synthesis. In this review we focus on the Musashi family of RNA-binding proteins, a highly conserved family of translational regulatory proteins whose recent identification in germ cells of Drosophila and Xenopus, as well as their well described role in processes such as cell cycle progression and stem cell identity, has led us to investigate the role of these proteins in mammalian germ cell development.

Studies of Fertility Smartphone Apps Reveal a Lack of Insight and Scientific Information

Publisher: World Scientific Pub Co Pte Ltd

Date: 09-2022

DOI: 10.1142/S2661318222740942

Abstract: Background: Female reproductive health and fertility smartphone applications have grown rapidly into a 400 million dollar “femtech” industry. As such, there has been increased interest in their functions and benefits. Claims of app participation in research is a marketing tool for consumers, however there is little known about the current scope of reproductive health app research and specifically how it is performed and reported. Aim: This study aimed to determine the nature and extent of the peer-reviewed literature presented on fertility-based apps, to identify the reliability of the information within the apps, and to determine the ability of this information to educate users. Method: A systematic search of six databases was conducted in April 2020, returning a total of 21,158 records. After duplicate removal, title and abstract screening exclusionary steps, 27 records were reviewed and charted. Results: Fertility smartphone apps covered a variety of reproductive health themes including contraception, sexual health, and family planning, and used a range of research methodologies including prospective, retrospective and observational. A majority (63%) of the studies on apps specified ovulation windows as the sole information on fertility given to users. Around 52% of studies in the review based fertile window algorithms on stringent menstrual cycle length and variability requirements (typically 20-40 day length day variability). Furthermore, a third of studies in the scoping review contained authors that were affiliates or developers from an app company, and of these studies, 15% did not have co-authors independent to the app advisory board or employees. Conclusion: Female reproductive health apps have the potential to fulfil a range of reproductive health needs. However, they currently do so with severe limitations when it comes to the menstrual cycle variability, and they do not adequately succeed in multi-functional capabilities. Furthermore, the scope of information available in apps via the user interface is a major barrier to independent review for accuracy. Our findings support the recommendation for open source sharing of app contents. This would ensure developers are accountable to consumers, healthcare professionals and researchers. This study highlights lack of research evaluating the effectiveness of an app’s contents to translate to knowledge or behaviour change in participants.

144. ROLE OF RNA-BINDING PROTEIN, MUSASHI-1 (Msi-1), IN MURINE FOLLICULOGENESIS AND OOCYTE DEVELOPMENT

Publisher: CSIRO Publishing

Date: 2009

DOI: 10.1071/SRB09ABS144

Abstract: Follicular development and oocyte maturation in mammals requires the temporal and spatial control of protein production. Consequently, it is hypothesised that the preovulatory follicle represses mRNA translation until specific proteins are required during oocyte maturation. Increasingly RNA-binding proteins are being recognised as important contributors to germ cell development, particularly during oocyte transcriptional quiescence. We have identified the presence of RNA-binding protein musashi-1 (Msi-1) mRNA within the mouse ovary and mature mouse oocyte, where the protein is believed to act as a translational repressor by binding to specific sequences within the 3' UTR of target mRNA molecules. Recent studies in various mammalian systems have identified p21 WAF1, cdkn2a, notch and m-numb as potential targets of Msi-1. We have also identified morf4l1 as a potential target through preliminary pulldown and microarray analysis using a GST tagged Msi-1 recombinant protein. To further study these potential targets, a transgenic Msi-1 mouse was produced to overexpress the RNA-binding protein in the developing oocyte. Real time PCR, performed on intact ovaries of WT and Tg mice, has so far demonstrated a 1.5-fold increase in Msi-1 expression in tgMsi-1/+ ovaries, above WT ovary expression. Real time PCR analysis of Msi-1 target mRNA expression has also shown an overall increase in expression in the tgMsi-1/+ ovaries of p21 WAF1 (~2.5-fold), cdkn2a (~2-fold), and notch (~3-fold). However m-numb and morf4l1 do not appear to be targets of Msi-1 in the oocyte, with no significant difference in expression between the WT and tgMsi-1/+ ovaries analysed. Functional quantification of oocyte development reveals a significantly less oocytes produced from superovulated juvenile mice compared with wild type litter mates. Therefore, preliminary analysis suggests that Msi-1 may play a role in binding the transcripts of genes necessary for cell cycle regulation and chromatin remodelling, characteristic of meiotic progression and oocyte development.

Structure retrieval with fast electrons using segmented detectors

Publisher: American Physical Society (APS)

Date: 27-04-2016

DOI: 10.1103/PHYSREVB.93.134116

Implementation of new ECMO centers during the COVID-19 pandemic: experience and results from the Middle East and India

Publisher: Springer Science and Business Media LLC

Date: 22-06-2021

DOI: 10.1007/S00134-021-06451-W

Biosynthesis of the Canine Zona Pellucida Requires the Integrated Participation of Both Oocytes and Granulosa Cells1

Publisher: Oxford University Press (OUP)

Date: 08-2004

DOI: 10.1095/BIOLREPROD.104.028779

Janus kinase JAK1 maintains the ovarian reserve of primordial follicles in the mouse ovary

Publisher: Oxford University Press (OUP)

Date: 24-09-2018

DOI: 10.1093/MOLEHR/GAY041

Abstract: Is the Janus kinase and signal transducer and activator of transcription (JAK-STAT) signalling pathway involved in ovarian follicle development and primordial follicle activation? JAK1 is a key factor involved in the regulation of primordial follicle activation and maintenance of the ovarian reserve. A series of integrated, intrinsic signalling pathways (including PI3K/AKT, mTOR and KITL) are responsible for regulating the ovarian reserve of non-growing primordial follicles and ultimately female fertility. The JAK-STAT signal transduction pathway is highly conserved with established roles in cell ision and differentiation. Key pathway members (specifically JAK1, STAT3 and SOCS4) have been previously implicated in early follicle development. A laboratory animal study was undertaken using the C57Bl/6 inbred mouse strain as a model for human ovarian follicle development. To determine which Jak genes were most abundantly expressed during primordial follicle activation, mRNA expression was analysed across a developmental time-course, with ovaries collected from female mice at post-natal days 1 (PND1), 4 (PND4), 8 (PND8), as well as at 6 weeks (6WK) and 7 months (7MTH) (n ≥ 4). Functional analysis of JAK1 was performed on PND2 mouse ovaries subjected to in vitro explant culture treated with 12.5 μM Ruxolitinib (JAK inhibitor) or vehicle control (DMSO) for 48 h prior to histological assessment (n ≥ 4). The expression and localization of the JAK family during ovarian follicle development in the C57Bl/6 inbred mouse strain were evaluated using quantitative PCR, immunoblotting and immunolocalisation. Functional studies were undertaken using the JAK inhibitor Ruxolitinib to investigate the underpinning cellular mechanisms via biochemical in vitro inhibition and histological assessment of intact neonate ovaries. All experiments were replicated at least three times using tissue from different mice unless otherwise stated. Jak1 is the predominant Jak mRNA expressed in the C57Bl/6 mouse ovary across all developmental time-points assessed (P ≤ 0.05). Forty-eight hour inhibition of JAK1 with Ruxolitinib of PND2 ovaries in vitro demonstrated concomitant acceleration of primordial follicle activation and apoptosis (P ≤ 0.001) and upregulation of downstream JAK-STAT pathway members STAT3 and suppressors of cytokine signalling 4 (SOCS4). N/A. Results are shown in one species, the C57Bl/6 mouse strain as an established model of human ovary development. Ruxolitinib also inhibits JAK2, with decreased efficacy. However, Jak2 mRNA had limited expression in the mouse ovary, particularly at the neonatal stages of follicle development, thus any effect of Ruxolitinib on primordial follicle activation was unlikely to be mediated via this isoform. This study supports a key role for JAK1 in the maintenance and activation of primordial follicles, with potential for targeting the JAK-STAT pathway as a method of regulating the ovarian reserve and female fertility. This project has been funded by the Australian National Health and Medical Research Council (G1600095) and The Hunter Medical Research Institute Bob and Terry Kennedy Children's Research Project Grant in Pregnancy & Reproduction (G1501433). All authors declare no conflict of interests.

The MicroRNA Signature of Mouse Spermatozoa Is Substantially Modified During Epididymal Maturation1

Publisher: Oxford University Press (OUP)

Date: 10-2015

DOI: 10.1095/BIOLREPROD.115.132209

Abstract: In recent years considerable effort has been devoted to understanding the epigenetic control of sperm development, leading to an increased appreciation of the importance of RNA interference pathways, and in particular miRNAs, as key regulators of spermatogenesis and epididymal maturation. It has also been shown that sperm are endowed with an impressive array of miRNA that have been implicated in various aspects of fertilization and embryo development. However, to date there have been no reports on whether the sperm miRNA signature is static or whether it is influenced by their prolonged maturation within the male reproductive tract. To investigate this phenomenon, we employed next-generation sequencing to systematically profile the miRNA signature of maturing mouse spermatozoa. In so doing we have provided the first evidence for the posttesticular modification of the sperm miRNA profile under normal physiological conditions. Such modifications include the apparent loss and acquisition of an impressive cohort of some 113 and 115 miRNAs, respectively, between the proximal and distal epididymal segments. Interestingly, the majority of these changes occur late in maturation and include the uptake of novel miRNA species in addition to a significant increase in many miRNAs natively expressed in immature sperm. Because sperm are not capable of de novo transcription, these findings identify the epididymis as an important site in establishing the sperm epigenome with the potential to influence the peri-conceptual environment of the female reproductive tract, contribute to the inheritance of acquired characteristics, and/or alter the developmental trajectory of the resulting offspring.

Characterisation of mouse epididymosomes reveals a complex profile of microRNAs and a potential mechanism for modification of the sperm epigenome

Publisher: Springer Science and Business Media LLC

Date: 23-08-2016

DOI: 10.1038/SREP31794

Abstract: Recent evidence has shown that the sperm epigenome is vulnerable to dynamic modifications arising from a variety of paternal environment exposures and that this legacy can serve as an important determinant of intergenerational inheritance. It has been postulated that such exchange is communicated to maturing spermatozoa via the transfer of small non-protein-coding RNAs (sRNAs) in a mechanism mediated by epididymosomes small membrane bound vesicles released by the soma of the male reproductive tract (epididymis). Here we confirm that mouse epididymosomes encapsulate an impressive cargo of microRNAs (miRNAs), a developmentally important sRNA class, the majority (~60%) of which are also represented by the miRNA signature of spermatozoa. This includes miRNAs that were found exclusively in epididymal sperm and epididymosomes, but not in the surrounding soma. We also documented substantial changes in the epididymosome miRNA cargo, including significant fold changes in almost half of the miRNAs along the length of the epididymis. Finally, we provide the first direct evidence for the transfer of several prominent miRNA species between mouse epididymosomes and spermatozoa to afford novel insight into a mechanism of intercellular communication by which the sRNA payload of sperm can be selectively modified during their post-testicular maturation.

Value of the hamster oocyte test and computerised measurements of sperm motility in predicting if four or more viable embryos will be obtained in an IVF cycle

Publisher: Wiley

Date: 27-04-2001

DOI: 10.1046/J.1365-2605.2001.00284.X

Abstract: The experimental group consisted of men from 81 couples waiting for in vitro fertilization (IVF), about half of whom had sperm dysfunction defined by a negative post-coital test. A diagnostic semen s le was subjected to a hamster oocyte penetration test (HOPT) after stimulation of the acrosome reaction with A23187 +/- pentoxifylline and to computerized sperm motility measurements (CASA) as well as conventional semen analysis according to the WHO protocol. Logistic regression was used to identify parameters that predicted the probability of achieving four or more viable embryos at IVF among the 65 couples from whom four or more oocytes were collected. The number of oocytes available and whether the woman had previously been pregnant (ever pregnant) were important factors but once these had been taken into account a number of sperm parameters had additional predictive power. The most useful of these were the percentage sperm static (CASA) or the percent sperm progressively motile (conventional semen analysis) in the Percoll preparation. A model incorporating the number of oocytes collected, ever pregnant and percentage sperm static achieved 85% correct prediction of outcome in the experimental dataset but only 62% correct prediction in an independent set of 280 IVF cycles. The percentage of hamster oocytes penetrated was a significant predictor but had no advantage over simple motility measurements. The results illustrate the difficulty of basing a prognosis for achieving satisfactory fertilization in IVF on the properties of spermatozoa.

Differential cell death decisions in the testis: evidence for an exclusive window of ferroptosis in round spermatids.

Publisher: Oxford University Press (OUP)

Date: 13-03-2019

DOI: 10.1093/MOLEHR/GAZ015

Abstract: Oxidative stress is a major aetiology in many pathologies, including that of male infertility. Recent evidence in somatic cells has linked oxidative stress to the induction of a novel cell death modality termed ferroptosis. However, the induction of this iron-regulated, caspase-independent cell death pathway has never been explored outside of the soma. Ferroptosis is initiated through the inactivation of the lipid repair enzyme glutathione peroxidase 4 (GPX4) and is exacerbated by the activity of arachidonate 15-lipoxygenase (ALOX15), a lipoxygenase enzyme that facilitates lipid degradation. Here, we demonstrate that male germ cells of the mouse exhibit hallmarks of ferroptosis including a caspase-independent decline in viability following exposure to oxidative stress conditions induced by the electrophile 4-hydroxynonenal or the ferroptosis activators (erastin and RSL3), as well as a reciprocal upregulation of ALOX15 and down regulation of GPX4 protein expression. Moreover, the round spermatid developmental stage may be sensitized to ferroptosis via the action of acyl-CoA synthetase long-chain family member 4 (ACSL4), which modifies membrane lipid composition in a manner favourable to lipid peroxidation. This work provides a clear impetus to explore the contribution of ferroptosis to the demise of germline cells during periods of acute stress in in vivo models.

Two alternative methods for the retrieval of somatic cell populations from the mouse ovary

Publisher: Oxford University Press (OUP)

Date: 10-05-2021

DOI: 10.1093/MOLEHR/GAAB033

Abstract: Many modern techniques employed to uncover the molecular fundamentals underlying biological processes require dissociated cells as their starting point/substrate. Investigations into ovarian endocrinology or folliculogenesis, therefore, necessitate robust protocols for dissociating the ovary into its constituent cell populations. While in the mouse, methods to obtain in idual, mature follicles are well-established, the separation and isolation of single cells of all types from early mouse follicles, including somatic cells, has been more challenging. Herein we present two methods for the isolation of somatic cells in the ovary. These methods are suitable for a range of applications relating to the study of folliculogenesis and mouse ovarian development. First, an enzymatic dissociation utilising collagenase and a temporary, primary cell culture step using neonatal mouse ovaries which yields large quantities of granulosa cells from primordial, activating, and primary follicles. Second, a rapid papain dissociation resulting in a high viability single cell suspension of ovarian somatic cells in less than an hour, which can be applied from embryonic to adult ovarian s les. Collectively these protocols can be applied to a broad array of investigations with unique advantages and benefits pertaining to both.

A Three-Dimensional Reconstruction Algorithm for Scanning Transmission Electron Microscopy Data from a Single Sample Orientation

Publisher: Oxford University Press (OUP)

Date: 10-2022

DOI: 10.1017/S1431927622012090

Abstract: Increasing interest in three-dimensional nanostructures adds impetus to electron microscopy techniques capable of imaging at or below the nanoscale in three dimensions. We present a reconstruction algorithm that takes as input a focal series of four-dimensional scanning transmission electron microscopy (4D-STEM) data. We apply the approach to a lead iridate, PbIrO, and yttrium-stabilized zirconia, YZrO, heterostructure from data acquired with the specimen in a single plan-view orientation, with the epitaxial layers stacked along the beam direction. We demonstrate that Pb–Ir atomic columns are visible in the uppermost layers of the reconstructed volume. We compare this approach to the alternative techniques of depth sectioning using differential phase contrast scanning transmission electron microscopy (DPC-STEM) and multislice ptychographic reconstruction.

Knockout of RNA Binding Protein MSI2 Impairs Follicle Development in the Mouse Ovary: Characterization of MSI1 and MSI2 during Folliculogenesis

Publisher: MDPI AG

Date: 26-06-2015

DOI: 10.3390/BIOM5031228

Through the smoke: Use of in vivo and in vitro cigarette smoking models to elucidate its effect on female fertility

Publisher: Elsevier BV

Date: 12-2014

DOI: 10.1016/J.TAAP.2014.10.010

Abstract: A finite number of oocytes are established within the mammalian ovary prior to birth to form a precious ovarian reserve. Damage to this limited pool of gametes by environmental factors such as cigarette smoke and its constituents therefore represents a significant risk to a woman's reproductive capacity. Although evidence from human studies to date implicates a detrimental effect of cigarette smoking on female fertility, these retrospective studies are limited and present conflicting results. In an effort to more clearly understand the effect of cigarette smoke, and its chemical constituents, on female fertility, a variety of in vivo and in vitro animal models have been developed. This article represents a systematic review of the literature regarding four of experimental model types: 1) direct exposure of ovarian cells and follicles to smoking constituents' in vitro, 2) direct exposure of whole ovarian tissue with smoking constituents in vitro, 3) whole body exposure of animals to smoking constituents and 4) whole body exposure of animals to cigarette smoke. We summarise key findings and highlight the strengths and weaknesses of each model system, and link these to the molecular mechanisms identified in smoke-induced fertility changes.

All Your Eggs in One Basket: Mechanisms of Xenobiotic Induced Female Reproductive Senescence

Publisher: InTech

Date: 29-02-2012

DOI: 10.5772/33022

89. The effect of kit ligand on follicle growth initiation in cultured rabbit and mouse ovaries

Publisher: CSIRO Publishing

Date: 2003

DOI: 10.1071/SRB03AB89

A comparison of the freezing of human semen in the uncirculated vapour above liquid nitrogen and in a commercial semi-programmable freezer

Publisher: Oxford University Press (OUP)

Date: 08-1990

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A137176

Abstract: The cooling rates inside 0.25-ml semen straws filled with glycerol, egg yolk, citrate buffer were compared between a standard vapour freezing procedure and freezing in a Nicool LM-10 semi-programmable freezer. During vapour freezing, the cooling rate at the bottom of the straw was much faster than at the top and there was considerable variation between replicates. By contrast, position within the straw did not affect the cooling rate in the Nicool LM-10 procedure and replicates were more consistent. More motile spermatozoa survived the Nicool freezing procedure and their lateral head displacement was greater than vapour frozen spermatozoa. However the percentage of intact spermatozoa and their velocity was very similar after the two procedures. We conclude that the freezing procedure using the Nicool LM-10 provides a comparatively economical way to achieve consistent semen freezing for research studies.

A novel germ cell protein, SPIF (sperm PKA interacting factor), is essential for the formation of a PKA/TCP11 complex that undergoes conformational and phosphorylation changes upon capacitation

Publisher: Wiley

Date: 22-04-2016

DOI: 10.1096/FJ.201500136R

Characterization of structure and expression of the Dzip1 gene in the rat and mouse

Publisher: Elsevier BV

Date: 02-2006

DOI: 10.1016/J.YGENO.2005.10.007

Abstract: A transcript encoding a rat homologue of DZIP1 (DAZ-interacting protein) was isolated from testis RNA. Like human DZIP1, it contains a C(2)H(2) zinc finger domain. A predicted mouse homologue of DZIP1 was found in the GenBank database. Genome analysis indicated that while DZIP1 and mouse Dzip1 contain 22 and 20 exons, respectively, the rat sequence was intronless, confirmed by PCR on genomic DNA. This rat Dzip1 sequence is homologous to mouse Dzip1 exons 1-6 and DZIP1 exons 5-9. As this rat sequence was shorter than DZIP1 it was designated rat Dzip1S. The rat genome also contained a further predicted homologue of DZIP1 displaying conserved linkage homology with mouse Dzip1 and DZIP1. This sequence, if expressed, is the true rat homologue of DZIP1, designated rat Dzip1. Rat Dzip1S mRNA was present in all tissues examined by qualitative RT-RCR, and in situ hybridization of rat testis confirmed that expression of rat Dzip1S mRNA was confined to the spermatogenic lineage, specifically premeiotic spermatogonia.

An essential role for Polycomb Repressive Complex 2 in the mouse ovary

Publisher: Bioscientifica

Date: 03-2022

DOI: 10.1530/REP-21-0361

Abstract: Polycomb repressive complex 2 (PRC2) catalyses the repressive epigenetic modification of histone 3 lysine 27 tri-methylation (H3K27me3) and functions as a key epigenetic regulator during embryonic development. PRC2 is known to regulate the development of a range of tissues by transcriptional silencing of genes that control cell differentiation, but its roles in female germline and ovarian development remain unknown. Using a mouse model with hypomorphic embryonic ectoderm development (EED) function that reduced H3K27me3 in somatic and germ cells, we found that PRC2 was required for survival, with more than 95% of female animals dying before birth. Although surviving adult EED hypomorphic females appeared morphologically similar to controls and were fertile, Eed hypo/hypo adult ovaries were abnormal, with altered morphology characterised by abnormal follicles. Early Eed hypo/hypo and control fetal ovaries were morphologically similar, and germ cells entered meiosis normally. Immunofluorescent analyses of somatic and germline markers indicated that ovarian development in Eed hypo/hypo ovaries was similar to heterozygous and WT controls. However, TUNEL analyses revealed higher rates of apoptosis in the ovarian surface epithelium, and transcriptional analyses revealed changes in genes regulating epithelial and steroidogenic cell differentiation, possibly foreshadowing the defects observed in adult ovaries of hypomorphic females. While it was possible to analyse early-mid fetal ovarian development, postnatal stages were inaccessible due to the high level of lethality during late fetal stages. Despite this limitation, the data we were able to obtain reveal a novel role for EED in the ovary that is likely to alter ovarian development and ovarian function in adult animals.

The Saudi Critical Care Society extracorporeal life support chapter guidance on utilization of veno-venous extracorporeal membrane oxygenation in adults with acute respiratory distress syndrome and sp

Publisher: Saudi Medical Journal

Date: 06-2021

DOI: 10.15537/SMJ.2021.42.6.20200520

Baseball pitching biomechanics in relation to pain, injury, and surgery: A systematic review

Publisher: Elsevier BV

Date: 2021

DOI: 10.1016/J.JSAMS.2020.06.015

Synthesis and Characterization of 17 Cr Ferritic ODS Steel Developed Through Vacuum Hot Pressing

Publisher: Springer Singapore

Date: 27-09-2020

DOI: 10.1007/978-981-15-6619-6_30

Differential expression profiles of conserved Snail transcription factors in the mouse testis

Publisher: Wiley

Date: 30-01-2018

DOI: 10.1111/ANDR.12465

Abstract: Snail transcription factors are key regulators of cellular transitions during embryonic development and tumorigenesis. The closely related SNAI1 and SNAI2 proteins induce epithelial-mesenchymal transitions (EMTs), acting predominantly as transcriptional repressors, while the functions of SNAI3 are unknown. An initial examination of Snai2-deficient mice provided evidence of deficient spermatogenesis. To address the hypothesis that Snail proteins are important for male fertility, this study provides the first comprehensive cellular expression profiles of all three mammalian Snail genes in the post-natal mouse testis. To evaluate Snail transcript expression profiles, droplet digital (dd) PCR and in situ hybridization were employed. Snai1, 2 and 3 transcripts are readily detected at 7, 14, 28 days post-partum (dpp) and 7 weeks (adult). Unique cellular expression was demonstrated for each by in situ hybridization and immunohistochemistry using Western blot-validated antibodies. SNAI1 and SNAI2 are in the nucleus of the most mature germ cell types at post-natal ages 10, 15 and 26. SNAI3 is only detected from 15 dpp onwards and is localized in the Sertoli cell cytoplasm. In the adult testis, Snai1 and Snai2 transcripts are detected in spermatogonia and spermatocytes, while Snai3 is in both germ and Sertoli cells. SNAI1 protein is evident in nuclei of spermatogonia, spermatocytes, round spermatids and elongated spermatids (Stages IX-XII). SNAI2 is present in the nuclei of spermatogonia and spermatocytes, with a faint signal detected in round spermatids. SNAI3 was detected only in Sertoli cell cytoplasm, as in juvenile testes. Additionally, colocalization of SNAI1 and SNAI2 with previously identified key binding partners, LSD1 and PRC2 complex components, provides strong evidence that these important functional interactions are conserved during spermatogenesis to control gene activity. These distinct expression profiles suggest that each Snail family member has unique functions during spermatogenesis.

Chemokines: Role in Germ Cell Migration and Survival.

Publisher: Oxford University Press (OUP)

Date: 05-2008

DOI: 10.1093/BIOLREPROD/78.S1.53C

Cryopreservation and storage of spermatozoa

Publisher: CRC Press

Date: 11-11-2008

DOI: 10.3109/9780203092934-24

MeasureIce: Accessible on-the-fly measurement of ice thickness in cryo-electron microscopy

Publisher: Cold Spring Harbor Laboratory

Date: 09-12-2021

DOI: 10.1101/2021.12.08.471523

Abstract: Ice thickness is arguably one of the most important factors limiting the resolution of protein structures determined by cryo electron microscopy. The amorphous atomic structure of the ice that stabilizes and protects biological s les in cryo-EM grids also imprints some addition noise in the TEM images. Ice that is too thick jeopardizes the success of particle picking and reconstruction of the biomolecule in the worst case and, at best, deteriorates eventual map resolution. Minimizing the thickness of the ice layer and thus the magnitude of its noise contribution is thus imperative in cryo-EM grid preparation. In this paper we introduce MeasureIce, a simple, easy to use ice thickness measurement tool for screening and selecting acquisition areas of cryo-EM grids. We show that it is possible to simulate thickness-image intensity look-up tables using elementary scattering physics and thereby adapt the tool to any microscope without time consuming experimental calibration. We benchmark our approach using two alternative techniques: the “ice-channel” technique and tilt-series tomography. We also demonstrate the utility of ice thickness measurement for selecting holes in gold grids containing an Equine apoferritin s le, achieving a 1.88 Ångstrom resolution in subsequent refinement of the atomic map.

Gonadotrophin surge-attenuating factor bioactivity is present in follicular fluid from naturally cycling women

Publisher: Oxford University Press (OUP)

Date: 1995

DOI: 10.1093/HUMREP/10.1.68

Abstract: Rat pituitary monolayer bioassays were used to compare gonadotrophin surge-attenuating factor (GnSAF) bioactivity in follicular fluid from 12 follicles in 10 spontaneously cycling women with that in pooled follicular fluid from women undergoing ovulation induction. Expressed as ED50S (microliter follicular fluid/well producing 50% of maximal effect), GnSAF bioactivity was detectable in all spontaneous follicular fluid s les (1.4-33.3 microliters/well) and in follicular fluid from women undergoing ovulation induction (6.8 microliters/well). This GnSAF bioactivity was unaffected by pre-incubation with an inhibin antibody. When the data were grouped according to whether the recovered oocytes fertilized in vitro or not, the fertilized group contained significantly greater GnSAF bioactivity than the unfertilized group (5.3 +/- 1.1 and 14.1 +/- 2.6 microliters/well respectively, P < 0.05). While both inhibin bioactivity (9.7 +/- 1.4 and 28.9 +/- 12.1 microliters/well) and immunoreactivity (36.8 +/- 2.2 and 21.0 +/- 3.0 and ng/ml) were also greater (P < 0.01) in the fertilized compared with the unfertilized groups respectively, there were no other significant differences between the two groups. We conclude that GnSAF is found in follicular fluid from spontaneously cycling women, supporting in-vivo evidence for the involvement of GnSAF in feedback control of the ovary-pituitary axis.

Acute neonatal immune challenge: Lipopolysaccharide exposure alters neonatal ovarian inflammatory pathways and mediators

Publisher: Elsevier BV

Date: 10-2015

DOI: 10.1016/J.BBI.2015.06.136

Maternal Smoke Exposure Impairs the Long-Term Fertility of Female Offspring in a Murine Model1

Publisher: Oxford University Press (OUP)

Date: 02-2016

DOI: 10.1095/BIOLREPROD.115.135848

Abstract: The theory of fetal origins of adult disease was first proposed in 1989, and in the decades since, a wide range of other diseases from obesity to asthma have been found to originate in early development. Because mammalian oocyte development begins in fetal life it has been suggested that environmental and lifestyle factors of the mother could directly impact the fertility of subsequent generations. Cigarette smoke is a known ovotoxicant in active smokers, yet disturbingly 13% of Australian and 12% of US women continue to smoke throughout pregnancy. The focus of our investigation was to characterize the adverse effects of smoking on ovary and oocyte quality in female offspring exposed in utero. Pregnant mice were nasally exposed to cigarette smoke for 12 wk throughout pregnancy/lactation, and ovary and oocyte quality of the F1 (maternal smoke exposed) generation was examined. Neonatal ovaries displayed abnormal somatic cell proliferation and increased apoptosis, leading to a reduction in follicle numbers. Further investigation found that altered somatic cell proliferation and reduced follicle number continued into adulthood however, apoptosis did not. This reduction in follicles resulted in decreased oocyte numbers, with these oocytes found to have elevated levels of oxidative stress, altered metaphase II spindle, and reduced sperm-egg interaction. These ovarian and oocyte changes ultimately lead to subfertility, with maternal smoke-exposed animals having smaller litters and also taking longer to conceive. In conclusion, our results demonstrate that in utero and lactational exposure to cigarette smoke can have long-lasting effects on the fertility of the next generation of females.

Do fertilin beta and cyritestin play a major role in mammalian sperm-oolemma interactions? A critical re-evaluation of the use of peptide mimics in identifying specific oocyte recognition proteins

Publisher: Oxford University Press (OUP)

Date: 04-2001

DOI: 10.1093/MOLEHR/7.4.313

Abstract: Integrins have been proposed to play a role in mammalian sperm-oocyte interactions for many years. To a large extent this hypothesis stems from the ability of short synthetic peptides, based on the disintegrin-like domains of two sperm surface integral membrane proteins, fertilin beta and cyritestin, to inhibit sperm--oocyte binding and fusion in vitro. Here we argue that such peptide mimics lack specificity in these simple IVF assay systems. Hence, whilst not precluding a role for fertilin beta and cyritestin in sperm-oolemma interactions, this lack of specificity indicates the need for considerable caution when interpreting results obtained using this approach.

Immune regulation of ovarian development: programming by neonatal immune challenge

Publisher: Frontiers Media SA

Date: 2013

DOI: 10.3389/FNINS.2013.00100

33. Programming of reproductive development by neonatal immunological challenge: Evidence for transgenerational inheritance

Publisher: Elsevier BV

Date: 09-2012

DOI: 10.1016/J.BBI.2012.07.057

Perioperative prediction of agitated (hyperactive) delirium after cardiac surgery in adults – The development of a practical scorecard

Publisher: Elsevier BV

Date: 12-2017

DOI: 10.1016/J.JCRC.2017.07.045

Abstract: Delirium is a temporary mental disorder that occurs frequently among hospitalized patients. In this study we sought to develop a user-friendly scorecard based on perioperative features to identify patients at risk of developing agitated delirium after cardiac surgery. Retrospective analysis was performed on adult patients undergoing cardiac surgery in a single center. A parsimonious predictive model was created, with subsequent internal validation. Then a simple scorecard was developed that can be used to predict the probability of agitated delirium. Among the 5584 patients who met the study criteria, 614 (11.4%) developed postoperative agitated delirium. Independent predictors of postoperative agitated delirium were age, male gender, history of cerebrovascular disease, procedure other than isolated Coronary Arteries Bypass Surgery, transfusion of blood products within the first 48h, mechanical ventilation for >24h, length of stay in the Intensive Care Unit. The scorecard stratified patients into 4 categories at risk of postoperative agitated delirium ranging from 30%. Using a large cohort of adult patient's undergoing cardiac surgery, a user-friendly scorecard was developed and validated, which will facilitate the implementation of timely interventions to mitigate adverse effects of agitated delirium in this high risk population.

Cis-Unsaturated Fatty Acids Stimulate Reactive Oxygen Species Generation and Lipid Peroxidation in Human Spermatozoa

Publisher: The Endocrine Society

Date: 10-2006

DOI: 10.1210/JC.2006-1309

Abstract: Defective sperm function is the largest defined cause of human infertility however, the etiology of this condition is poorly understood. Although oxidative stress is acknowledged as a key contributor to this pathology, there are also data indicating that defective human spermatozoa contain abnormally high amounts of cis-unsaturated fatty acids. This study investigated whether a causative relationship exists between these two attributes of impaired semen quality. The objective of this study was to determine whether polyunsaturated fatty acids can induce oxidative stress in human spermatozoa. Dihydroethidium and SYTOX Green were used in conjunction with flow cytometry and HPLC to investigate reactive oxygen species (ROS) generation by human spermatozoa after fatty acid exposure. Arachidonic acid (AA) induced a time- and dose-dependent increase in ROS generation by human spermatozoa that led to the promotion of peroxidative damage and a loss of sperm motility. This effect could not be blocked with inhibitors of the cyclooxygenase or lipoxygenase pathways of AA metabolism, rotenone, protein kinase C antagonists, or known inhibitors of plasma membrane redox systems. However, ROS generation could be triggered with other cis-unsaturated fatty acids including linoleic and docosahexaenoic acids. Saturated fatty acids, methyl esters of unsaturated fatty acids, or other hiphiles were all ineffective. However in a cell-free system, AA could trigger a redox signal via mechanisms that were profoundly disrupted by diphenylene iodonium, a flavoprotein inhibitor. The presence of excess unsaturated fatty acids in defective human spermatozoa may precipitate the oxidative stress encountered in male infertility.

HOW Is Required for Stem Cell Maintenance in the Drosophila Testis and for the Onset of Transit-Amplifying Divisions

Publisher: Elsevier BV

Date: 04-2010

DOI: 10.1016/J.STEM.2010.02.016

Abstract: The mechanisms by which germline stem cells (GSCs) in the Drosophila testis undergo asymmetric ision to regenerate a stem cell as well as a daughter (gonialblast) that will only undergo a further four mitotic isions prior to entering premeiotic S phase and differentiating into a cyst of spermatocytes are not fully resolved. Here we demonstrate that the HOW RNA-binding protein is required for maintenance of CycB and therefore mitotic progression in GSCs and gonialblasts as well as determining the timing of the spermatogonial isions. HOW is normally expressed in a complementary pattern to Bam in the germline and bam mRNA is bound by HOW in vivo. Ectopic expression of the HOW(L) isoform is associated with a delay in accumulation of Bam to the level required for differentiation, resulting in extra mitotic isions. Spatiotemporal regulation of HOW expression is therefore required to specify the four spermatogonial transit- lifying isions.

The contribution of the toxicity of a glycerol-egg yolk-citrate cryopreservative to the decline in human sperm motility during cryopreservation

Publisher: Wiley

Date: 05-1993

DOI: 10.1016/0020-7292(93)90712-6

Chronic testicular Chlamydia muridarum infection impairs mouse fertility and offspring development†.

Publisher: Oxford University Press (OUP)

Date: 22-01-2020

DOI: 10.1093/BIOLRE/IOZ229

Abstract: With approximately 131 million new genital tract infections occurring each year, Chlamydia is the most common sexually transmitted bacterial pathogen worldwide. Male and female infections occur at similar rates and both cause serious pathological sequelae. Despite this, the impact of chlamydial infection on male fertility has long been debated, and the effects of paternal chlamydial infection on offspring development are unknown. Using a male mouse chronic infection model, we show that chlamydial infection persists in the testes, adversely affecting the testicular environment. Infection increased leukocyte infiltration, disrupted the blood:testis barrier and reduced spermiogenic cell numbers and seminiferous tubule volume. Sperm from infected mice had decreased motility, increased abnormal morphology, decreased zona-binding capacity, and increased DNA damage. Serum anti-sperm antibodies were also increased. When both acutely and chronically infected male mice were bred with healthy female mice, 16.7% of pups displayed developmental abnormalities. Female offspring of chronically infected sires had smaller reproductive tracts than offspring of noninfected sires. The male pups of infected sires displayed delayed testicular development, with abnormalities in sperm vitality, motility, and sperm-oocyte binding evident at sexual maturity. These data suggest that chronic testicular Chlamydia infection can contribute to male infertility, which may have an intergenerational impact on sperm quality.

Fitting a square beam in a square camera: novel condenser apertures for low-dose transmission electron microscopy

Publisher: Cold Spring Harbor Laboratory

Date: 15-08-2023

DOI: 10.1101/2023.08.13.553155

Abstract: In transmission electron microscopy (TEM) cameras are square or rectangular but beams are round. With a beam size chosen to fill the camera at a given image magnification, the circular lobes of the beam will extend beyond the camera’s field of view and irradiate areas that are not acquired on the camera, damaging and precluding them from future acquisitions if the s le is beam sensitive. In this paper we present development of condenser aperture plates for TEM that have square and rectangular apertures which improve the efficiency of s le area usage by 44% or greater in low dose TEM applications. We demonstrate that the use of these apertures is compatible with high-resolution cryogenic (cryo) TEM by reconstructing sub 2 Å apo-ferritin models from a datasets recorded with both square and rectangular apertures. Moreover the design of our aperture plates should improve the flexibility of 2 condenser systems for cryo-TEM acquisitions with multiple shots per hole by tailored matching of beam sizes to camera sizes at each magnification.

Safe cryopreservation of sperm and embryos

Publisher: Informa UK Limited

Date: 1998

DOI: 10.1080/1464727982000198181

216. c-kit Expression study: timing of onset in rodent testis and irradiated rat testis model

Publisher: CSIRO Publishing

Date: 2005

DOI: 10.1071/SRB05ABS216

Abstract: Primordial germ cell and spermatogonial cell function is essential for normal male fertility. These cells require Sertoli–germ cell interactions, specifically somatic cell-derived stem cell factor (SCF) that acts through the c-kit receptor to govern primordial germ cell migration in the foetus, spermatogonial differentiation during puberty and adulthood, and Leydig cell steroidogenesis. We performed a comprehensive study of the c-kit mRNA expression profile in the pre- and post-pubertal mouse and rat testes by in situ hybridisation. Expression of c-kit mRNA was first visualised in germ cells after birth, with the levels concordant with the number and appearance of the differentiated spermatogonial subtypes in both the rat and the mouse. We also studied c-kit expression in the irradiated adult rat testis, in which only undifferentiated spermatogonia are present. After treatment with Cetrorelix, GnRH antagonist (3 days, 1, 2 and 4 weeks) germ cell maturation is re-initiated. Expression of c-kit messenger RNA was observed in the undifferentiated spermatogonia in both untreated and treated testes sections. In contrast, c-kit protein expression was undetectable until 4 weeks of hormone treatment. This suggests that c-kit mRNA and protein expression are differentially regulated and that protein expression relates to somatic cell function.

Prismatic 2.0 – Simulation software for scanning and high resolution transmission electron microscopy (STEM and HRTEM)

Publisher: Elsevier BV

Date: 12-2021

DOI: 10.1016/J.MICRON.2021.103141

Germ cell specific overactivation of WNT/βcatenin signalling has no effect on folliculogenesis but causes fertility defects due to abnormal foetal development

Publisher: Springer Science and Business Media LLC

Date: 06-06-2016

DOI: 10.1038/SREP27273

Abstract: All the major components of the WNT signalling pathway are expressed in female germ cells and embryos. However, their functional relevance in oocyte biology is currently unclear. We examined ovaries collected from TCFGFP mice, a well-known Wnt reporter mouse model, and found dynamic changes in the Wnt/βcatenin signalling activity during different stages of oocyte development and maturation. To understand the functional importance of Wnt signalling in oocytes, we developed a mouse model with the germ cell-specific constitutive activation of βcatenin using cre recombinase driven by the DEAD (Asp-Glu-Ala-Asp) box protein 4 ( Ddx4 ) gene promoter. Histopathological and functional analysis of ovaries from these mutant mice (Ctnnb1 ex3 cko) showed no defects in ovarian functions, oocytes, ovulation and early embryonic development. However, breeding of the Ctnnb1 ex3 cko female mice with males of known fertility never resulted in birth of mutant pups. Examination of uteri from time pregnant mutant females revealed defects in ectoderm differentiation leading to abnormal foetal development and premature death. Collectively, our work has established the role of active WNT/βcatenin signalling in oocyte biology and foetal development, and provides novel insights into the possible mechanisms of complications in human pregnancy such as repeated spontaneous abortion, sudden intrauterine unexpected foetal death syndrome and stillbirth.

Kif4 Is Essential for Mouse Oocyte Meiosis

Publisher: Public Library of Science (PLoS)

Date: 26-01-2017

DOI: 10.1371/JOURNAL.PONE.0170650

Linking stress and infertility: A novel role for Ghrelin

Publisher: The Endocrine Society

Date: 27-07-2017

DOI: 10.1210/ER.2016-1133

Abstract: Infertility affects a remarkable one in four couples in developing countries. Psychological stress is a ubiquitous facet of life, and although stress affects us all at some point, prolonged or unmanageable stress may become harmful for some in iduals, negatively impacting on their health, including fertility. For instance, women who struggle to conceive are twice as likely to suffer from emotional distress than fertile women. Assisted reproductive technology treatments place an additional physical, emotional, and financial burden of stress, particularly on women, who are often exposed to invasive techniques associated with treatment. Stress-reduction interventions can reduce negative affect and in some cases to improve in vitro fertilization outcomes. Although it has been well-established that stress negatively affects fertility in animal models, human research remains inconsistent due to in idual differences and methodological flaws. Attempts to isolate single causal links between stress and infertility have not yet been successful due to their multifaceted etiologies. In this review, we will discuss the current literature in the field of stress-induced reproductive dysfunction based on animal and human models, and introduce a recently unexplored link between stress and infertility, the gut-derived hormone, ghrelin. We also present evidence from recent seminal studies demonstrating that ghrelin has a principal role in the stress response and reward processing, as well as in regulating reproductive function, and that these roles are tightly interlinked. Collectively, these data support the hypothesis that stress may negatively impact upon fertility at least in part by stimulating a dysregulation in ghrelin signaling.

Motility characteristics and membrane integrity of cryopreserved human spermatozoa

Publisher: Bioscientifica

Date: 07-1992

DOI: 10.1530/JRF.0.0950527

Abstract: The motility characteristics of washed spermatozoa from 50 normal ejaculates were measured by time-lapse photography, before and after cryopreservation. Plasma membrane integrity was assessed by the hypo-osmotic swelling test and with the supravital fluorescent dye bisbenzimide (H33258). There was a marked decline in the percentage of progressively motile spermatozoa after cryopreservation, the extent varying widely among donors. Results were, however, consistent between different ejaculates from the same in idual. The ability of spermatozoa to survive cryopreservation could not be predicted from the properties of the semen beforehand. The mean velocity of the spermatozoa was significantly reduced after freezing, but the lateral head displacement was unaltered. There was a significant reduction in the proportion of spermatozoa with intact plasma membranes after cryopreservation and the results of the hypo-osmotic swelling test and H33258 tests correlated closely. There was no correlation between the declines in the percentage of motile spermatozoa, or intact spermatozoa and the sperm velocity. We conclude that membrane rupture is not the sole cause of loss of motile spermatozoa during freezing and that the decrease in the proportion of motile spermatozoa is caused, at least in part, by a separate process from that responsible for the decrease in the average swimming speed of spermatozoa.

Immunocontraceptive effects on female rabbits infected with recombinant myxoma virus expressing rabbit ZP2 or ZP3.

Publisher: Oxford University Press (OUP)

Date: 03-2005

DOI: 10.1095/BIOLREPROD.105.046268

Abstract: Recombinant myxoma viruses expressing rabbit zona pellucida 2 (rZP2) or rabbit zona pellucida 3 (rZP3) glycoproteins were constructed and tested in domestic rabbits to assess their potential to induce autoimmune infertility. The recombinant virus expressing rZP2 had no effect on fertility or ovarian histology, despite all animals developing antibodies against the rZP2 antigen. However, recombinant viruses expressing rZP3 induced infertility in 70% of animals at the first breeding. Serum antibodies were relatively short-lived, but antibody was bound to zona pellucida of all rabbits from Day 10 onward. There was no obvious correlation between infertility and rZP3 antibody titer. There was a transient inflammatory response in the ovaries of rZP3-immunized rabbits at Day 15 but no T-cell response to rZP3 could be detected at any time. Dysfunctional follicular formation was present in ovaries from rabbits infected with rZP3-expressing viruses 15-40 days postinfection but this had disappeared at later time points. A recombinant myxoma virus expressing a modified rZP3 antigen with the C-terminal hydrophobic putative anchor sequence deleted was also tested. This virus did not induce either infertility or an antibody response against the zona pellucida. Thus, the context of antigen presentation was crucial for an autoimmune response.

The association between reproductive health smartphone applications and fertility knowledge of Australian women

Publisher: Springer Science and Business Media LLC

Date: 04-03-2020

DOI: 10.1186/S12905-020-00912-Y

Abstract: Previous studies have identified that women living in developed countries have insufficient knowledge of factors which may be contributing to the increasingly high global infertility rates such as maternal age and assisted reproductive technologies. There is a large market of reproductive health smartphone applications, yet little is known about the advantages these apps may confer to users in regards to reproductive health knowledge. An anonymous, online survey of women living in Australia aged 18 and above was open March–June 2018, until ≥200 responses were acquired for statistical power. Respondents answered questions regarding knowledge about general fertility and related factors (age, cyclic fertility, smoking, obesity, miscarriage rate, and success of assisted reproductive technologies). Fertility knowledge was compared in respondents who did or did not use apps relating to female reproductive health. Additionally the functions preferred in reproductive health apps was described by app using respondents. Sociodemographic information was also collected, and relevant data within the dataset was subject to multivariable modelling for the outcome of the fertility knowledge questions. Of the 673 respondents that completed the survey, 43.09% reported using mobile phone applications relating to female reproductive health. On average, respondents answered only three of the six fertility knowledge questions correctly. App using respondents were more likely to score better on one question, related to fertility during the menstrual cycle ( p 0.001). App users most commonly reported using the menstrual tracking function in apps (82.4%), which may account for the increased knowledge of cyclic fertility. This data provides preliminary evidence toward the usefulness of smartphone applications as a medium for providing information about fertility to women. A limited understanding of one’s own fertility was demonstrated despite being essential for the decision-making of women throughout their reproductive years.

Identification of relaxin immunoreactivity in human follicular fluid

Publisher: Oxford University Press (OUP)

Date: 12-1986

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A136465

Abstract: Relaxin immunoreactivity has been found in s les of human follicular fluid collected from artificially stimulated pre-ovulatory follicles. The crude extract caused a reduction in the height of the contractions in a rat uterine strip bioassay. The reactive material eluted from Sephadex G50 in two major peaks. The first contained approximately 60% of the immunoreactivity and had an elution position corresponding to that of porcine relaxin, indicating a mol. wt of approximately 6000 daltons. The second peak was of a lower mol. wt, but its exact size and significance are unknown. A possible role for relaxin in the process of follicular rupture is suggested.

Dynamin 2‐dependent endocytosis is essential for mouse oocyte development and fertility

Publisher: Wiley

Date: 17-02-2020

DOI: 10.1096/FJ.201902184R

Pentoxifylline acts synergistically with A23187 to increase the penetration of zona-free hamster oocytes by cryopreserved human spermatozoa

Publisher: Wiley

Date: 08-1994

DOI: 10.1111/J.1365-2605.1994.TB01243.X

Abstract: The number of cryopreserved human spermatozoa which penetrated zona-free hamster oocytes after stimulation with 2 mumol A23187 per litre was increased by the further addition of 0.6 or 3.6 mmol pentoxifylline per litre. With spermatozoa prepared by washing by repeated centrifugation, the median numbers of sperm heads/egg were 1.9, 7.9 and 10.8 in the presence of 0, 0.6 or 3.6 mmol pentoxifylline per litre, respectively. A similar effect was observed with spermatozoa prepared on a Percoll gradient. As A23187 inhibited sperm motility, and this was exacerbated by pentoxifylline, the increased penetration rate of hamster oocytes cannot be explained by improved sperm motility. The number of spermatozoa stimulated to acrosome react by 2 mumol A23187 per litre was increased 3-fold by 3.6 mmol pentoxifylline per litre and 4-fold by 5 mmol caffeine per litre. These data suggest that cAMP may act synergistically with Ca2+ to stimulate the acrosome reaction. Pentoxifylline may improve the fertility of poor-quality human spermatozoa by enhancing their ability to respond to the Ca2+ signal produced by binding to the zona pellucida.

217.Expression of the chemokine CXCL12 and its receptor CXCR4 in the activating mammalian ovarian follicle

Publisher: CSIRO Publishing

Date: 2004

DOI: 10.1071/SRB04ABS217

Abstract: The mammalian ovary contains a finite number of oocytes that is determined during oogenesis in fetal life. As most of these oocytes are destined to undergo apoptosis, the initial primordial follicle population represents a valuable resource for the clinical manipulation of the female germ cell pool. However the events underlying the activation of the resting primordial follicle remain relatively poorly understood. A comparison was undertaken between whole 2-day and 7-day neonate mouse ovaries which represents ovaries with primordial follicles and primordial follicles / newly activated follicles respectively. The comparison took place at the level of gene expression utilizing cDNA microarray analysis. The mRNAs for the chemokine CXCL12 and its receptor CXCR4, were consistently shown to be up-regulated approximately 2-fold in 7-day tissue compared with 2-day tissue. Microarray results were confirmed for CXCL12 by real-time PCR analysis. CXCL12 and CXCR4 have been identified as essential for development of the haemopoietic, nervous and cardiovascular systems and have also been shown to be involved in the foetal migration of primordial germ cells to the gonads. These genes were selected for further analysis due to the known importance of other cytokine family members in primordial follicle activation. In situ hybridisation studies of CXCL12 revealed mRNA expression in oocytes of all stages, including primordial follicles, as well as epithelial, corpora luteal and stromal cells. In contrast CXCR4 mRNA expression appears to be restricted primarily to oocytes of all stages. This co-expression of ligand and receptor within the oocyte suggests an autocrine signaling mechanism. The age-dependent increase in CXCL12 and CXCR4 gene expression is likely to be a result of an increased oocyte cytoplasmic volume and/or the proportion of stromal cells present as follicles begin to activate in the neonate ovary. Examination of the timing of protein expression is currently underway to identify the role this chemokine signaling pathway plays in the initial activation of the mammalian primordial follicle.

Hedgehog signalling pathway inhibitors as cancer suppressing agents

Publisher: Royal Society of Chemistry (RSC)

Date: 2014

DOI: 10.1039/C3MD00334E

Significance of Mitochondrial Reactive Oxygen Species in the Generation of Oxidative Stress in Spermatozoa

Publisher: The Endocrine Society

Date: 08-2008

DOI: 10.1210/JC.2007-2616

Abstract: Context: Male infertility has been linked with the excessive generation of reactive oxygen species (ROS) by defective spermatozoa. However, the subcellular origins of this activity are unclear. Objective: The objective of this study was to determine the importance of sperm mitochondria in creating the oxidative stress associated with defective sperm function. Method: Intracellular measurement of mitochondrial ROS generation and lipid peroxidation was performed using the fluorescent probes MitoSOX red and BODIPY C11 in conjunction with flow cytometry. Effects on sperm movement were measured by computer-assisted sperm analysis. Results: Disruption of mitochondrial electron transport flow in human spermatozoa resulted in generation of ROS from complex I (rotenone sensitive) or III (myxothiazol, antimycin A sensitive) via mechanisms that were independent of mitochondrial membrane potential. Activation of ROS generation at complex III led to the rapid release of hydrogen peroxide into the extracellular space, but no detectable peroxidative damage. Conversely, the induction of ROS on the matrix side of the inner mitochondrial membrane at complex I resulted in peroxidative damage to the midpiece and a loss of sperm movement that could be prevented by the concomitant presence of α-tocopherol. Defective human spermatozoa spontaneously generated mitochondrial ROS in a manner that was negatively correlated with motility. Simultaneous measurement of general cellular ROS generation with dihydroethidium indicated that 68% of the variability in such measurements could be explained by differences in mitochondrial ROS production. Conclusion: We conclude that the sperm mitochondria make a significant contribution to the oxidative stress experienced by defective human spermatozoa.

Understanding the Villain: DMBA-Induced Preantral Ovotoxicity Involves Selective Follicular Destruction and Primordial Follicle Activation through PI3K/Akt and mTOR Signaling

Publisher: Oxford University Press (OUP)

Date: 23-07-2011

DOI: 10.1093/TOXSCI/KFR195

Abstract: 7,12-Dimethylbenz-[a]anthracene (DMBA) is an environmental carcinogen which has a potent ovotoxic affect on rat and mouse ovaries, causing complete follicular depletion resulting in premature ovarian failure. Although the overall effects of DMBA on ovarian folliculogenesis are well known, little is known about the exact molecular mechanisms behind its ovotoxicity. In this study, we characterized the mechanisms behind DMBA-induced ovotoxicity in immature follicles. Microarray analysis of neonatal mouse ovaries exposed to DMBA in vitro revealed a multilayered mechanism of DMBA-induced neonatal ovotoxicity involving a distinct cohort of genes and ovarian signaling pathways primarily associated with follicular atresia, tumorigenesis, and follicular growth. Histomorphological and immunohistological analysis supported the microarray data, showing evidence of primordial follicle activation and preantral follicle atresia both in vitro and in vivo. Further immunohistological analysis identified increased Akt1 phosphorylation, mTOR activation, and decreased FOXO3a expression in DMBA-treated primordial oocytes. Our results reveal a novel mechanism of DMBA-induced preantral ovotoxicity involving selective immature follicle destruction and primordial follicle activation involving downstream members of the PI3K/Akt and mTOR signaling pathways.

326. SPIF - A NOVEL TESTIS-SPECIFIC GENE AND ITS INTERACTION WITH PKA

Publisher: CSIRO Publishing

Date: 2010

DOI: 10.1071/SRB10ABS326

Abstract: The activation of protein kinase A (PKA) is strongly implicated in capacitation and sperm motility. However, the full pathway is yet to be elucidated. To identify potential PKA binding partners in sperm, a yeast two-hybrid assay was performed using the testis specific catalytic subunit (Cs) of PKA as the ‘bait’ to screen a mouse testis cDNA library. A novel cDNA clone termed Sperm PKA Interacting Factor (SPIF) was identified from the screen on three separate occasions. The interaction was confirmed by a protein pull-down using a C-terminal recombinant protein to SPIF and a PKACs antibody. During cloning and sequence analysis, SPIF was found to contain two isoforms a full length (4770 bp) and a truncated form (2784 bp) with alternate start sites and an identical 3′ end, with only the full length isoform containing the PKA binding motif. SPIF was found to be testis specific using PCR and Northern Blotting with high expression levels in round spermatids and adult testis. The interaction between SPIF and PKA was further demonstrated with protein co-localisation in round spermatids and in the midpiece and flagellum of mouse sperm. In summary, we have identified a novel testis specific gene that in concert with PKA could prove to be an essential link in the incomplete capacitation pathway

On Regenerating the Ovary and Generating Controversy

Publisher: Elsevier BV

Date: 09-2005

DOI: 10.1016/J.CELL.2005.09.004

Oxidative damage in naturally aged mouse oocytes is exacerbated by dysregulation of proteasomal activity.

Publisher: Elsevier BV

Date: 12-2018

DOI: 10.1074/JBC.RA118.005751

New insights into the molecular mechanisms of sperm-egg interaction

Publisher: Springer Science and Business Media LLC

Date: 20-04-2007

DOI: 10.1007/S00018-007-6552-X

Abstract: At the moment of insemination millions of mammalian sperm cells are released into the female reproductive tract in order to find a single cell - the oocyte. The spermatozoa subsequently ignore the thousands of cells they make contact with during their journey to the site of fertilisation, until they reach the surface of the oocyte. At this point, they bind tenaciously to the acellular coat, known as the zona pellucida, that surrounds the oocyte and initiate the chain of cellular interactions that will culminate in fertilization. These exquisitely cell- and species-specific recognition events are among the most strategically important cellular interactions in biology. Understanding the cellular and molecular mechanisms that underpin them has implications for diagnosis of the aetiology of human infertility and the development of novel targets for fertility regulation. Herein, we describe two models indicating the plethora of highly orchestrated molecular interactions underlying successful sperm zona binding and sperm oocyte fusion.

Cryopreservation, screening and storage of sperm — The challenges for the twenty-first century

Publisher: Informa UK Limited

Date: 2002

DOI: 10.1080/1464727022000199941

Abstract: The advent of HIV and the serious nature of the sequelae resulted in a major reassessment of artificial insemination practices in the UK. The development of human semen cryopreservation had enormous impact on reproductive medicine and the availability of cryopreserved quarantined donor semen became a mainstay for the treatment of male infertility in the UK. The regulation and accreditation of assisted reproductive technologies and the introduction of peer-reviewed guidelines have largely standardized clinical and laboratory practice. The introduction of assisted fertilization techniques such as intracytoplasmic sperm injection, testicular sperm retrieval and improved oncology treatments have placed pressure on reproductive biologists and cryobiologists to design and use cryopreservation protocols for the optimum survival of sperm.

Optimisation of handling, activation and assessment procedures for Bufo marinus spermatozoa

Publisher: CSIRO Publishing

Date: 2007

DOI: 10.1071/RD06124

Abstract: In the present study, we investigated handling, activation and assessment procedures for cane toad (Bufo marinus) spermatozoa. Optimisation of these techniques will facilitate the maintenance of sperm viability during cryopreservation and during in vitro fertilisation (IVF) techniques in reproduction technologies for endangered species. Spermatozoa were taken from testicular macerates and assessed using plasma membrane integrity assays (live/dead stains) and quantitative scores of motility parameters. In the assessment of sperm viability using live/dead stains, there were small but significant differences in the percentage of sperm from cryopreserved s les staining positive with propidium iodide, Hoechst H33258 and Trypan blue these differences were not large and all stains performed acceptably. Spermatozoa were activated by dilution of testicular macerates in water at one of two dilution ratios (1 : 6 or 1 : 20) with or without 0.1–5.0 mm theophylline. Sperm plasma membrane integrity (unstained spermatozoa) was unaffected by either dilution ratio (osmolarity) or theophylline concentration. However, sperm motility was significantly affected by osmolarity and theophylline concentration. The stimulation of sperm motility increased with higher theophylline concentrations and these strongly interacted with lower osmolarities through a higher dilution ratio of sperm macerates with water. Spermatozoa were exposed to increasing centrifugation forces to determine tolerance to physical stresses encountered during washing procedures. Forces between 50 and 800g were associated with a significant reduction in motility (mean 56 ± 3% decreasing to 27 ± 3%), but did not affect staining. In conclusion, centrifugation should be minimised in anuran sperm washing procedures osmotic shock associated with higher dilution ratios reduces the capacity of anuran sperm to achieve high percentages of motile sperm, leading to a likely trade-off between dilution required for activation and sperm motility to optimise IVF fertilisation rates and optimal conditions for sperm motility after activation occur at lower dilutions of suspensions with 5.0 mm theophylline. The present study has improved protocols for the handling of anuran sperm during pre- and post-cryopreservation procedures.

Machine learning decision tree algorithm role for predicting mortality in critically ill adult COVID-19 patients admitted to the ICU

Publisher: Elsevier BV

Date: 07-2022

DOI: 10.1016/J.JIPH.2022.06.008

Arrhythmogenic right ventricular cardiomyopathy: use of a left ventricular assist device as a bridge to transplantation?

Publisher: Springer Science and Business Media LLC

Date: 13-06-2013

DOI: 10.1007/S10047-013-0716-2

Abstract: The principal characteristic of arrhythmogenic right ventricular cardiomyopathy (ARVC) is the tendency for ventricular arrhythmia and sudden death to occur without overt ventricular dysfunction. Current recommendations for management of patients with ARVC include insertion of an automated implantable cardioverter-defibrillator (AICD) to prevent sudden cardiac death. However, despite the use of AICD and/or anti-arrhythmic drugs some patients suffer recurrent ventricular arrhythmias unresponsive to optimum medical management. We present two cases of ARVC with refractory recurrent ventricular arrhythmias that were successfully managed by left ventricular assist device (LVAD) implantation, as a bridge to transplant (BTT). These two cases are unconventional ex les of use of LVAD, given the predominant right ventricular pathology of ARVC and the arrhythmogenic nature of their presentation. The novelty of these cases should be taken in the context of increasing pressure to standardize indications for use of mechanical circulatory support.

Reactive oxygen species (ROS) production and the outcome of diagnostic tests of sperm function

Publisher: Wiley

Date: 08-1999

DOI: 10.1046/J.1365-2605.1999.00174.X

Abstract: Washed sperm suspensions from 64 out of 89 (72%) randomly selected infertility patients produced detectable reactive oxygen species (ROS) compared to 17 out of 67 (25%) prospective semen donors (p < 0.01, Chi-square test). Among patients, the median sperm concentration in ejaculates which yielded sperm suspensions that generated detectable levels of ROS was lower than in those which did not: 36.2 (15.63-57.64) vs. 71.5 (22-108) x 10(6)/mL, respectively (median (interquartile range), p < 0.05, Kruskal-Wallis test). In s les that produced ROS, the basal rate of production and the rates after stimulation with 50 mumol N-formyl met leu phe (N-FMLP) l-1 or with 100 nmol phorbol 12-myristate 13-acetate (PMA) l-1 were significantly and inversely correlated with sperm concentration in the ejaculate (r = -0.43, -0.41 and -0.35, respectively, p < 0.01 Spearman's rank correlation). The rate of ROS production showed no relationship to the motility of spermatozoa in semen, whether evaluated visually or via computer assisted semen analysis. However, there was a significant negative correlation (r = -0.370) between the motile, normal sperm concentration (MNSC) and basal ROS production, and when stimulated with N-FMLP (r = -0.311) or with PMA (r = -0.249) (all p < 0.05). In patient s les that generated detectable ROS, the ability of the spermatozoa to retain motility for 24 h after preparation on a 40/80% Percoll gradient was negatively correlated with basal ROS production (r = -0.310, p < 0.05). ROS production was also related to the outcome of in vitro sperm mucus penetration tests. Unstimulated levels of ROS production showed a significant (p < 0.05), negative correlation with the number of progressively motile spermatozoa present in mucus after 15 (r = -0.379) and 60 (r = -0.362) min. These results suggest that sperm s les with increased ROS tend to have poor semen quality and reduced performance in a number of routine, diagnostic sperm function tests.

Norethisterone treatment to control timing of the IVF cycle

Publisher: Oxford University Press (OUP)

Date: 11-1986

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A136454

Abstract: The use of norethisterone to control the timing of the preceding menstrual cycle and in consequence the timing of the in-vitro fertilization (IVF) cycle has been evaluated in a therapeutic IVF programme in which oocyte recovery was limited to 2 days each week. A consecutive series of 181 cycles after norethisterone and 29 untreated controls were compared. Menstruation occurred 2-3 days after norethisterone as planned in 82% of patients overall and in 87% of patients whose menstrual cycle length varied by no more than 2 days about the median. Norethisterone treatment did not significantly affect the outcome of IVF treatment compared with the controls in respect to cycles abandoned (12 versus 0%, respectively), peak follicular diameter (mean 18.1 mm versus 18.3 mm 48 h before laparoscopy), oocyte recovery rate (4.6 versus 4.5 per patient), oocyte morphology (63% versus 52% mature), or fertilization rate (72 versus 65% of mature oocytes). Clinical pregnancies were too few for comparison (rates 27 versus 9% per laparoscopy) but the overall rate (23%) indicated effectiveness of the methods. Prior norethisterone treatment appears to be an effective and useful means of controlling the timing of the oocyte recovery in IVF treatment.

The BRCA1-binding protein BRAP2 can act as a cytoplasmic retention factor for nuclear and nuclear envelope-localizing testicular proteins

Publisher: Elsevier BV

Date: 12-2013

DOI: 10.1016/J.BBAMCR.2013.05.015

Abstract: Regulation of nuclear protein import is central to many cellular processes such as development, with a key mechanism being factors that retain cargoes in the cytoplasm that normally localize in the nucleus. The breast cancer antigen BRCA1-binding protein BRAP2 has been reported as a novel negative regulator of nuclear import of various nuclear localization signal (NLS)-containing viral and cellular proteins, but although implicated in differentiation pathways and highly expressed in tissues including testis, the gamut of targets for BRAP2 action in a developmental context is unknown. As a first step towards defining the BRAP2 interactome, we performed a yeast-2-hybrid screen to identify binding partners of BRAP2 in human testis. Here we report characterization for the first time of three of these: the high mobility group (HMG)-box-domain-containing chromatin component HMG20A, nuclear mitotic apparatus protein NuMA1 and synaptic nuclear envelope protein SYNE2. Co-immunoprecipitation experiments indicate association of BRAP2 with HMG20A, NuMA1, and SYNE2 in testis, underlining the physiological relevance of the interactions, with immunohistochemistry showing that where BRAP2 is co-expressed with HMG20A and NuMA1, both are present in the cytoplasm, in contrast to their nuclear localization in other testicular cell types. Importantly, quantitative confocal microscopic analysis of cultured cells indicates that ectopic expression of BRAP2 inhibits nuclear localization of HMG20A and NuMA1, and prevents nuclear envelope accumulation of SYNE2, the first report of BRAP2 altering localization of a non-nuclear protein. These results imply for the first time that BRAP2 may have an important role in modulating subcellular localization during testicular development.

207.Oolemmal proteomics: identification of oocyte cell surface protein complexes involved in sperm - egg interaction

Publisher: CSIRO Publishing

Date: 2004

DOI: 10.1071/SRB04ABS207

Abstract: While the molecular basis of sperm-oolemma interaction is of considerable biological significance, the protein(s) involved in this process are yet to be identified. In particular, the point at which developing mammalian oocytes acquire the capacity to bind to, and fuse with, capacitated, acrosome-reacted spermatozoa is yet to be clearly defined. Previous reports suggested that mature metaphase II (MII) mouse oocytes possessed the capacity to bind and fuse with spermatozoa while, in contrast, immature germinal vesicle (GV) phase oocytes did not. In this study oocytes were cultured in α-MEM containing 5% fetal calf serum. For GV oocytes, α-MEM was also supplemented with 1 μM Milrinone to prevent GV breakdown. Standard murine IVF was performed in 100 μL α-MEM media droplets containing 10 to 15 oocytes and 2.5×104 capacitated spermatozoa. These studies found that no significant difference existed in the levels of sperm-oocyte binding or fusion between freshly isolated GV oocytes (14 sperm/egg bound, 7 sperm/egg fused), cultured arrested GV oocytes (16 sperm/egg bound, 7 sperm/egg fused) or cultured MII phase oocytes (17 sperm/egg bound, 8 sperm/egg fused). Interestingly, upon fusion MII oocytes commenced sperm nuclear decondensation whereas arrested GV oocytes did not. Comparison of biotinylated oolemmal surface proteins revealed markedly different protein profiles between the GV oolemma and the MII oolemma. The MII oolemma revealed a multitude of proteins ranging in size from approximately 20 to 200 kDa, whilst the GV oolemma revealed only six middle range molecular weight proteins ranging from approximately 50 to 90 kDa. The protein(s) implicated in sperm-egg interaction must be one or more of those proteins found in both oocyte populations. Comparison of these two profiles has greatly reduced the number of possible candidates, allowing possible identification of the proposed GPI-linked sperm receptor.

Cloning and sequence analysis of rat fertilin alpha and beta-- developmental expression, processing and immunolocalization

Publisher: Oxford University Press (OUP)

Date: 09-1997

DOI: 10.1093/MOLEHR/3.9.801

Abstract: Fertilin alpha and beta are members of the MDC (metalloproteinase-like, disintegrin-like, cysteine-rich) protein family and are expressed on the sperm surface where they have been proposed to play a role in mammalian fertilization. Inhibition of sperm-oocyte binding and sperm-oocyte fusion make fertilin an attractive target for the development of an immunocontraceptive vaccine. Full-length cDNAs encoding alpha and beta fertilin subunits were isolated from a rat testis cDNA library and sequenced. Using reverse transcription-polymerase chain reaction (RT-PCR), the developmental expression of fertilin alpha and beta was determined in pre-pubertal and mature rat testes. Fertilin alpha mRNA was present at all stages of development, suggesting that it is not exclusively expressed in post-meiotic germ cells. In contrast, fertilin beta mRNA was first identified in day 19 testes, coincident with the presence of pachytene spermatocytes. Polyclonal antisera raised against a 28-residue peptide (corresponding to part of the disintegrin domain) and two recombinant fusion proteins identified a 90 kDa protein in testicular sperm extracts and a 60 kDa protein in caput and cauda epididymidal sperm extracts, the predicted sizes for rat fertilin beta precursor and mature protein respectively. Indirect immunofluorescence using the anti-peptide antisera stained the acrosomal cap of permeabilized testicular, caput and caudal spermatozoa and elongating spermatids in testicular sections.

Anaphylactic and nonanaphylactic reactions to SARS-CoV-2 vaccines: a systematic review and meta-analysis

Publisher: Springer Science and Business Media LLC

Date: 16-10-2021

DOI: 10.1186/S13223-021-00613-7

Abstract: Currently there is no systematic review and meta-analysis of the global incidence rates of anaphylactic and nonanaphylactic reactions to SARS-CoV-2 vaccines in the general adult population. To estimate the incidence rates of anaphylactic and nonanaphylactic reactions after COVID-19 vaccines and describe the demographic and clinical characteristics, triggers, presenting signs and symptoms, treatment and clinical course of confirmed cases. A systematic review and meta-analysis. Preferred Reporting Items for Systematic Reviews and Meta-Analyses [PRISMA] statement was followed. Electronic databases (Proquest, Medline, Embase, Pubmed, CINAHL, Wiley online library, and Nature) were searched from 1 December 2020 to 31 May 2021 in the English language using the following keywords alone or in combination: anaphylaxis , non-anaphylaxis , anaphylactic reaction , nonanaphylactic reaction , anaphylactic/anaphylactoid shock , hypersensitivity , allergy reaction , allergic reaction , immunology reaction , immunologic reaction , angioedema , loss of consciousness , generalized erythema , urticaria , urticarial rash , cyanosis , grunting , stridor , tachypnoea , wheezing , tachycardia , abdominal pain , diarrhea , nausea , vomiting and tryptase . We included studies in adults of all ages in all healthcare settings. Effect sizes of prevalence were pooled with 95% confidence intervals (CIs). To minimize heterogeneity, we performed sub-group analyses. Of the 1,734 papers that were identified, 26 articles were included in the systematic review (8 case report, 5 cohort, 4 case series, 2 randomized controlled trial and 1 randomized cross-sectional studies) and 14 articles (1 cohort, 2 case series, 1 randomized controlled trial and 1 randomized cross-sectional studies) were included in meta-analysis. Studies involving 26,337,421 vaccine recipients [Pfizer-BioNTech (n = 14,505,399) and Moderna (n = 11,831,488)] were analyzed. The overall pooled prevalence estimate of anaphylaxis to both vaccines was 5.0 (95% CI 2.9 to 7.2, I 2 = 81%, p = 0.0001), while the overall pooled prevalence estimate of nonanaphylactic reactions to both vaccines was 53.9 (95% CI 0.0 to 116.1, I 2 = 99%, p = 0.0001). Vaccination with Pfizer-BioNTech resulted in higher anaphylactic reactions compared to Moderna (8.0, 95% CI 0.0 to 11.3, I 2 = 85% versus 2.8, 95% CI 0.0 to 5.7, I 2 = 59%). However, lower incidence of nonanaphylactic reactions was associated with Pfizer-BioNTech compared to Moderna (43.9, 95% CI 0.0 to 131.9, I 2 = 99% versus 63.8, 95% CI 0.0 to 151.8, I 2 = 98%). The funnel plots for possible publication bias for the pooled effect sizes to determine the incidence of anaphylaxis and nonanaphylactic reactions associated with mRNA COVID-19 immunization based on mRNA vaccine type appeared asymmetrical on visual inspection, and Egger’s tests confirmed asymmetry by producing p values 0.05. Across the included studies, the most commonly identified risk factors for anaphylactic and nonanaphylactic reactions to SARS-CoV-2 vaccines were female sex and personal history of atopy. The key triggers to anaphylactic and nonanaphylactic reactions identified in these studies included foods, medications, stinging insects or jellyfish, contrast media, cosmetics and detergents, household products, and latex. Previous history of anaphylaxis and comorbidities such as asthma, allergic rhinitis, atopic and contact eczema/dermatitis and psoriasis and cholinergic urticaria were also found to be important. The prevalence of COVID-19 mRNA vaccine-associated anaphylaxis is very low and nonanaphylactic reactions occur at higher rate, however, cutaneous reactions are largely self-limited. Both anaphylactic and nonanaphylactic reactions should not discourage vaccination.

Identification of RARhoGAP, a novel putative RhoGAP gene expressed in male germ cells☆

Publisher: Elsevier BV

Date: 08-2004

DOI: 10.1016/J.YGENO.2004.03.004

Drosophila Musashi is required in the ovary to regulate follicle stem cell behaviour and maintain niche homeostasis

Publisher: Elsevier BV

Date: 07-2017

DOI: 10.1016/J.MOD.2017.04.496

Analysis of lipid peroxidation in human spermatozoa using BODIPY C11

Publisher: Oxford University Press (OUP)

Date: 27-02-2007

DOI: 10.1093/MOLEHR/GAL119

Abstract: Lipid peroxidation is known to be a major factor in the aetiology of defective sperm function. Although biochemical assays for this process exist, they are relatively insensitive and require large numbers of spermatozoa a condition that cannot be met with many infertility specimens. Recently, a new approach for monitoring peroxidative damage has been introduced, involving the probe BODIPY (581/591) C(11), which readily incorporates into cells and undergoes a spectral emission shift when attacked by reactive oxygen metabolites. We have examined the applicability of this probe as an indicator of oxidative stress in human sperm populations using flow cytometry as an end point. The measurement of peroxidation with BODIPY C(11) demonstrated significant dependence on the presence of a ferrous ion promoter (P < 0.001), which was significantly enhanced in sperm recovered from low-density Percoll fractions (P < 0.05) and was particularly damaging to the sperm midpiece. Iron-induced radical formation was suppressed by ascorbate in a dose-dependent manner (P < 0.001) and could only be promoted by Fe(II) and Cu(II) nickel, zinc and Fe(III) were ineffective. The Fe(II)-promoted BODIPY C(11) signal was significantly correlated with the measurement of reactive oxygen species generation with dihydroethidium. We conclude that BODIPY C(11) is an extremely useful probe for indexing peroxidative damage in human spermatozoa.

CXCR4/SDF1 interaction inhibits the primordial to primary follicle transition in the neonatal mouse ovary

Publisher: Elsevier BV

Date: 05-2006

DOI: 10.1016/J.YDBIO.2006.02.012

Abstract: The molecular mechanisms behind the entry of the primordial follicle into the growing follicle pool remain poorly understood. To investigate this process further, a microarray-based comparison was undertaken between 2-day postpartum mouse ovaries consisting of primordial follicles/naked oocytes only and those with both primordial follicles and newly activated follicles (7-day postpartum). Gene candidates identified included the chemoattractive cytokine stromal derived factor-1 (SDF1) and its receptor CXCR4. SDF1 and CXCR4 have been implicated in a variety of physiological processes including the migration of embryonic germ cells to the gonads. SDF1-alpha expression increased with the developmental stage of the follicle. Embryonic expression was found to be dichotomous post-germ cell migration, with low expression in the female. Immunohistochemical studies nonetheless indicate that the autocrine pattern of expression ligand and receptor begins during embryonic life. Addition of recombinant SDF1-alpha to neonatal mouse ovaries in vitro resulted in significantly higher follicle densities than for control ovaries. TUNEL analysis indicated no detectable difference in populations of apoptotic cells of treated or control ovaries. Treated ovaries also contained a significantly lower percentage of activated follicles as determined by measurement of oocyte diameter and morphological analysis. Treatment of cultured ovaries with an inhibitor of SDF1-alpha, AMD3100, ablated the effect of SDF1-alpha. By retaining follicles in an unactivated state, SDF1/CXCR4 signaling may play an important role in maintaining the size and longevity of the primordial follicle pool.

322. PROTEOMIC AND FUNCTIONAL ANALYSIS OF HUMAN SPERM DETERGENT RESISTANT MEMBRANES

Publisher: CSIRO Publishing

Date: 2010

DOI: 10.1071/SRB10ABS322

Abstract: Mammalian spermatozoa attain the ability to fertilize an oocyte as they negotiate the female reproductive tract. This acquisition of functional competence is preceded by an intricate cascade of biochemical and functional changes collectively known as ‘capacitation’. Among the universal correlates of the capacitation process is a remarkable remodeling of the lipid and protein architecture of the sperm plasma membrane. While the fundamental mechanisms that underpin this dynamic reorganization remain enigmatic, emerging evidence has raised the prospect that it may be coordinated, at least in part, by specialized membrane microdomains, or rafts. In the studies described herein we have demonstrated that human spermatozoa express recognized markers of membrane rafts. Further, upon depletion of cellular cholesterol through either physiological (capacitation) or pharmacological (methyl-β-cyclodextrin) intervention, these membrane rafts appear to undergo a polarized redistribution to the peri-acrosomal region sperm head. The polarized targeting of membrane rafts to the sperm head encourages speculation that they represent platforms for the organization of proteins involved in sperm-oocyte interactions. Support for this notion rests with the demonstration that membrane rafts isolated on the basis of their biochemical composition in the form of detergent resistant membranes (DRMs), possess the ability to adhere to homologous zona pellucida. Furthermore a comprehensive proteomic analysis of the DRMs identified a number of proteins known for their affinity for the zona pellucida in addition to other candidates putatively involved in the mediation of downstream binding and/or fusion with the oolemma. Collectively these data afford novel insights into the sub-cellular localization and potential functions of membrane rafts in human spermatozoa.

324. SLX4, A KEY REGULATOR OF MEIOSIS AND DNA REPAIR IN THE MALE GERMLINE

Publisher: CSIRO Publishing

Date: 2010

DOI: 10.1071/SRB10ABS324

Abstract: Genome integrity relies on the ability of DNA repair proteins to open, cleave and recombine DNA. Slx4 (synthetic lethal of unknown function) or BTBd12 (BTBD12 domain-containing protein 12) the human homologue is a DNA repair protein which has multiple independent cellular roles. Importantly, it is a component in Holliday junction homologous recombination (HR) where it interacts with other proteins to repair double strand breaks (DSB). SLX4 forms a heterodimer complex with SLX1, an endonuclease, in response to DNA damage. This complex works by recruiting several associated proteins including XPF- ERCC1 a 3′ flap endonuclease, to DSB allowing proper single strand annealing repair of the DNA. An essential part of meiosis in the male and female germ cell formation is DNA recombination which is initiated by DSB. Meiotic error at this stage can cause aneuploidy and infertility. As SLX4 acts as a key regulator of recombination we proposed that loss of this protein would cause dysfunction in germ cell development. Breeding pairs of SLX4+/– heterozygous mice were obtained from EUCOMM and germline transmission of the null allele confirmed. Adult mice were normal and healthy but analysis of the male reproductive tract confirmed abnormally small testes (wt+/– vs testes). Further investigation demonstrated hypospermatogenesis with major loss of meiotic spermatocytes and post meiotic and spermatids. Epididymal sperm retrieval confirmed that the SLX4–/– male does produce poorly motile sperm with significant acrosomal abnormalities and associated failure of sperm-zona pellucida binding (2.5 ± 1.6 vs 13.6 ± 3.6 sperm bound) when compared to heterozygote or wild type littermates. Interestingly the female SLX4 null mouse also has small ovaries compared with het and wt littermates (wt vs null). Histomorphological analysis reveals a significant reduction in the ovarian follicle pool compared to wild type littermates with concomitant increased levels of apoptosis. Early breeding trials confirm significant loss of fertility in null males and females probably due to deficient sperm development and oocyte dysfunction. Our study now aims to further examine the role of SLX4 in germ cell development in the testes and ovary and its impact on infertility.

The effect of halving the standard dose of cryopreserved semen for donor insemination: a controlled study of conception rates

Publisher: Oxford University Press (OUP)

Date: 02-1994

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A138502

Abstract: Employing a common standard technique of intra-cervical insemination from straws of cryopreserved donor semen, a volume of 0.25 ml of 0.5 ml was inseminated in alternate cycles to determine if the lower volume could be used without a decrease in the conception rate. A total of 177 women were recruited and received a median of four cycles of treatment. Of these, 90 women became pregnant, 47 with 0.5 ml and 43 with 0.25 ml inseminations. The conception rates were identical for both volumes in the first nine cycles of treatment and the cumulative rates were 57.7 and 59.3%, respectively. Subsequently more pregnancies were achieved with 0.5 ml than 0.25 ml semen (nine pregnancies in 73 further cycles versus three pregnancies in 68 cycles, respectively), although the difference was not statistically significant. There were no significant differences in the women's ages, luteinizing hormone, follicle stimulating hormone, progesterone, mucus quality, mucus pH, parity or partner's diagnosis between those women who became pregnant and those who failed to conceive with either insemination dose. We conclude that the volume of semen inseminated into the cervical canal without a cervical cap can be decreased to 0.25 ml without an adverse effect on the conception rate at least in the first 9 months of treatment. This will allow more effective use to be made of valuable screened and quarantined cryopreserved semen.

509. REGULATING MURINE FOLLICLE DEVELOPMENT BY STIMULATION OF THE JAK2/STAT3 SIGNAL TRANSDUCTION PATHWAY

Publisher: CSIRO Publishing

Date: 2009

DOI: 10.1071/SRB09ABS509

Abstract: Follicular development requires the recruitment of primordial follicles into the growing follicle pool following initiation of multiple cytokine signalling pathways. Suppression of follicular development is thought to be key to maintaining the population of primordial follicles and allowing for controlled release of these follicles throughout the reproductive lifespan of the female. However, little is known of the processes and signalling molecules that suppress primordial follicle activation and early follicle growth. Our group has identified significant upregulation of the Janus Kinase 2 (JAK2)/ Signal Transducer and Activator of Transcription 3 (STAT3) signalling pathway inhibitor the Suppressor of Cytokine Signalling 4 (SOCS4) that coincides with the initial wave of follicular activation in theneonatal mouse ovary. Further studies by our group have localised the SOCS4 protein to the granulosa cells of activating and growing follicles, suggesting SOCS4 expression may be linked to follicular activation. We have focused on examining protein localisation and gene expression patterns of the eight SOCS family members CIS and SOCS1-7. We have recently demonstrated that co-culture of neonatal ovaries with Kit Ligand (KL) for 2 days increases the mRNA levels of all SOCS genes. We also demonstrated the co-localisation of SOCS2 proteins with the KL receptor c-kit in the mural granulosa cells of antral, and large pre-antral follicles suggesting a significant role for SOCS2 in the later stages of follicular development. We have also shown that culturing ovaries with the potent JAK2 inhibitor AG490 substantially reduces mRNA levels of all SOCS and STAT genes that we have so far measured. We hypothesise a significant role for JAK2/STAT3 signalling in promoting the activation and early growth of ovarian follicles. Our investigations have identified significant roles for JAK2/STAT3 and the SOCS family in the regulation of ovarian follicle development.

320. THE ROLE OF FIZZY RELATED 1 IN MALE MEIOSIS

Publisher: CSIRO Publishing

Date: 2010

DOI: 10.1071/SRB10ABS320

Abstract: Accurate chromosome segregation during mitosis and meiosis is facilitated by a regulatory complex known as the Anaphase Promoting Cyclosome (APC), an ubiquitin ligase complex that tags proteins with ubiquitin. Subsequently targeted proteins are recognised by the 26S proteosome and degraded. In mammalian cells, two temporally regulated co-activators are required for the APC to function fizzy and fzr1. In studies of female oocyte development fzr1 has been demonstrated to play an important role in maintaining G2 arrest during meiosis by controlling spatial levels of the cell cycle protein Cyclin B1 but the role of Fzr1 in spermatogenesis remains unknown. Germ cell specific conditional knockout fzr1mice were generated using the DDX4-Cre and flox/flox fzr1 mouse lines and initial gross morphological analysis indicated that at 7 weeks of age null mice possessed significantly smaller testes (21.81mg ± 0.23mg) when compared to heterozygote (99.86mg ± 1.58mg) and wildtype littermates (93.06mg ± 1.16mg) n = 3 P 0.0001. Quantitative gene expression analysis confirmed almost complete absence of fzr1 transcript in testes (20-fold decrease) in comparison to wild-type. Immunoblotting and immunohistochemistry revealed no expression of Fzr1 protein in meiotic and post meiotic germ cells when compared to heterozygote and wild type littermates. Histomorphological analysis of testes tissue sections revealed Fzr1 null males exhibited spermatogenic arrest and a complete absence of round spermatids with concomitant apoptosis in the residual spermatocytes. Epididymal examination confirmed a complete lack of mature spermatozoa in the cauda epididymis of null males. In contrast, both wild type and heterozygote mice displayed normal spermatogenesis and epididymal sperm analysis indicated no distinguishable differences in seminal characteristics with normal motility, morphology and sperm-zona binding capacity. Based on these observations we hypothesise that Fzr1 plays a significant role in the establishment and maintenance of meiosis possibly through regulation of key cell cycle proteins.

The contribution of the toxicity of a glycerol-egg yolk-citrate cryopreservative to the decline in human sperm motility during cryopreservation

Publisher: Bioscientifica

Date: 08-1992

DOI: 10.1530/JRF.0.0950749

Abstract: The contribution of the toxicity of glycerol-egg yolk-citrate (GEYC) cryopreservative medium to the loss of function of human spermatozoa during cryopreservation was determined by investigating the effect of mixing semen with the medium on sperm motility. The percentage of progressively motile spermatozoa, velocity (micron s-1) and lateral head displacement (micron) (mean +/- SEM, n = 28) were 55 +/- 4.1, 47 +/- 2.7, 4.4 +/- 0.2 and 32 +/- 3.8, 40 +/- 2.5, 3.6 +/- 0.25 and 15 +/- 2.5, 28 +/- 1.1, 2.8 +/- 0.15 in suspensions of washed spermatozoa prepared from fresh, GEYC-treated and frozen-thawed semen, respectively. The variables changed only slightly after incubation for 3 h. The toxicity of GEYC did not vary significantly between s les which survived the complete freeze-thaw cycle well or very poorly. The toxicity of GEYC is responsible for about 50% of the loss of progressively motile spermatozoa during the complete cryopreservation process, but has little effect on the quality of motility. Susceptibility to GEYC does not explain observed differences in the ability of semen s les to survive freezing.

Composition and significance of detergent resistant membranes in mouse spermatozoa

Publisher: Wiley

Date: 2009

DOI: 10.1002/JCP.21575

Abstract: Mammalian spermatozoa acquire the ability to fertilize an oocyte as they ascend the female reproductive tract. This process is characterized by a complex cascade of biophysical and biochemical changes collectively know as "capacitation." The attainment of a capacitated state is accompanied by a dramatic reorganization of the surface architecture to render spermatozoa competent to recognize the oocyte and initiate fertilization. Emerging evidence indicates that this process is facilitated by molecular chaperone-mediated assembly of a multimeric receptor complex on the sperm surface. However, the mechanisms responsible for gathering key recognition molecules within this putative complex have yet to be defined. In this study, we provide the first evidence that chaperones partition into detergent resistant membrane fractions (DRMs) within capacitated mouse spermatozoa and co-localize in membrane microdomains enriched with the lipid raft marker, G(M1) ganglioside. During capacitation, these microdomains coalesce within the apical region of the sperm head, a location compatible with a role in sperm-zona pellucida interaction. Significantly, DRMs isolated from spermatozoa possessed the ability to selectively bind to the zona pellucida of unfertilized, but not fertilized, mouse oocytes. A comprehensive proteomic analysis of the DRM fractions identified a total of 100 proteins, a number of which have previously been implicated in sperm-oocyte interaction. Collectively, these data provide compelling evidence that mouse spermatozoa possess membrane microdomains that provide a platform for the assembly of key recognition molecules on the sperm surface and thus present an important mechanistic insight into the fundamental cell biological process of sperm-oocyte interaction.

Phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) follicular signalling is conserved in the mare ovary

Publisher: CSIRO Publishing

Date: 2018

DOI: 10.1071/RD17024

Abstract: The mare ovary is unique in its anatomical structure however, the signalling pathways responsible for physiological processes, such as follicular activation, remain uncharacterised. This provided us with the impetus to explore whether signalling molecules from important folliculogenesis pathways, phosphoinositide 3-kinase rotein kinase B (PI3K/AKT) and Janus kinase/signal transducer and activator of transcription (JAK/STAT), are conserved in the mare ovary. Messenger RNA expression of six genes important in follicle development was measured using quantitative polymerase chain reaction and protein localisation of key pathway members (PI3K, AKT1, phosphatase and tensin homologue (PTEN), JAK1, STAT3 and suppressor of cytokine signalling 4 (SOCS4)) was compared in tissue from fetal and adult mare ovaries. Tissue from adult ovaries exhibited significantly increased levels of mRNA expression of PI3K, AKT1, PTEN, JAK1, STAT3 and SOCS4 compared with tissue from fetal ovaries. PI3K, AKT1, JAK1 and STAT3 demonstrated redistributed localisation, from pregranulosa cells in fetal development, to both the oocyte and granulosa cells of follicles in the adult ovary, whilst negative feedback molecules PTEN and SOCS4 were only localised to the granulosa cells in the adult ovary. These findings suggest that the PI3K/AKT and JAK/STAT signalling pathways are utilised during folliculogenesis in the mare, similarly to previously studied mammalian species, and may serve as useful biomarkers for assessment of ovary development in the horse.

The University Of Canberra

Location: Australia

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University Of Wollongong

Location: Australia

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Western Sydney University

Location: Australia

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Location: Australia

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Chlamydia Infection In Males

Start Date: 2014

End Date: 2017

Funder: National Health and Medical Research Council

Male Chlamydia Infections: The Key Role Of Macrophages In Testicular Dissemination And Disrupted Spermatogenesis

Start Date: 2018

End Date: 2022

Funder: National Health and Medical Research Council

Investigation Of The Mechanisms Underpinning HSPA2 Dysfunction In The Spermatozoa Of Infertile Patients

Start Date: 2016

End Date: 2018

Funder: National Health and Medical Research Council

Towards A Chlamydia Vaccine For Males: The Key Role Of Mucosal IgA

Start Date: 2009

End Date: 2011

Funder: National Health and Medical Research Council

An Advanced Mass Spectrometry Facility For Applications In Proteomics And Organic Chemistry

Start Date: 2009

End Date: 2009

Funder: Australian Research Council

High Throughput Proteomics - Thermo Finnigan ProteomeX LCQ Integrated Proteomics Workstation

Start Date: 2005

End Date: 2005

Funder: Australian Research Council

An Integrated Liquid Chromatography Mass Spectrometry Nuclear Magnetic Resonance (LC-MS-NMR) Facility For Applications In Proteomics And Organic Chemistry

Start Date: 2010

End Date: 2010

Funder: Australian Research Council

Role Of Musashi In The Regulation Of Cell Cycle Proteins

Start Date: 2012

End Date: 2014

Funder: Australian Research Council

The Causes Of Aneuploidy In Oocytes

Start Date: 2009

End Date: 2011

Funder: National Health and Medical Research Council

ARC Centre Of Excellence In Biotechnology And Development

Start Date: 2003

End Date: 2010

Funder: Australian Research Council

Confocal Laser Scanning Microscopy For Live Cell Imaging

Start Date: 2009

End Date: 2009

Funder: Australian Research Council

Establishment Of A Comprehensive Regional Biophysical Analysis Facility

Start Date: 2011

End Date: 2011

Funder: Australian Research Council

(Laser Microdissection Microscopy System For Cell And Development Biology)

Start Date: 2010

End Date: 2010

Funder: Australian Research Council

Biotechnology Applications In The Veterinary Pharmaceutical Industry: Pharmacological Manipulation Of Ovarian Function In Domestic Animals

Start Date: 2004

End Date: 2006

Funder: Australian Research Council

Multifunctional Confocal Laser Scanning Microscope With Time Resolved And Two Photon Imaging And Fluorescence Correlation Capabilities

Start Date: 2004

End Date: 2004

Funder: Australian Research Council

Cellular Signals Controlling Oocyte Activation

Start Date: 2008

End Date: 2010

Funder: Australian Research Council

(Gamete-specific Knockout Of Fizzy-Related To Examine Its Meiotic Role In Oocytes And Sperm)

Start Date: 2011

End Date: 2014

Funder: Australian Research Council

High-Speed Confocal Microscope Live Cell Recording System

Start Date: 2004

End Date: 2004

Funder: Australian Research Council

Discovery Projects - Grant ID: DP110100418

Start Date: 06-2011

End Date: 04-2016

Amount: $600,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0878355

Start Date: 03-2008

End Date: 12-2012

Amount: $270,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100008

Start Date: 2010

End Date: 12-2010

Amount: $350,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100142

Start Date: 2010

End Date: 07-2011

Amount: $500,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0453683

Start Date: 2004

End Date: 12-2010

Amount: $465,000.00

Funder: Australian Research Council

ARC Centres Of Excellence - Grant ID: CE0348239

Start Date: 06-2003

End Date: 12-2010

Amount: $15,878,900.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0561173

Start Date: 2005

End Date: 12-2005

Amount: $207,189.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989084

Start Date: 2009

End Date: 12-2009

Amount: $275,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP120100224

Start Date: 06-2012

End Date: 12-2015

Amount: $280,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989105

Start Date: 2009

End Date: 11-2009

Amount: $495,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP200103463

Start Date: 01-2020

End Date: 01-2023

Amount: $510,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE110100078

Start Date: 02-2011

End Date: 12-2011

Amount: $500,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0453630

Start Date: 2004

End Date: 12-2004

Amount: $274,692.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0453608

Start Date: 2004

End Date: 12-2004

Amount: $579,230.00

Funder: Australian Research Council