ORCID Profile
0000-0002-6006-8475
Current Organisation
UNSW Sydney
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Publisher: Oxford University Press (OUP)
Date: 17-02-2023
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.
Publisher: Oxford University Press (OUP)
Date: 02-08-2019
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.
Publisher: Oxford University Press (OUP)
Date: 22-01-2020
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.
Publisher: Oxford University Press (OUP)
Date: 02-02-2017
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.
Publisher: Impact Journals, LLC
Date: 24-05-2019
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.
Publisher: Wiley
Date: 10-2023
DOI: 10.1002/MRD.23709
Publisher: Ubiquity Press, Ltd.
Date: 16-12-2016
DOI: 10.5334/IJIC.2654
Publisher: Public Library of Science (PLoS)
Date: 13-08-2015
Publisher: Elsevier BV
Date: 12-2018
Publisher: Springer Science and Business Media LLC
Date: 08-11-2022
DOI: 10.1038/S41467-022-34294-6
Abstract: Human beings are made of ~50 trillion cells which arise from serial mitotic isions of a single cell - the fertilised egg. Remarkably, the early human embryo is often chromosomally abnormal, and many are mosaic, with the karyotype differing from one cell to another. Mosaicism presumably arises from chromosome segregation errors during the early mitotic isions, although these events have never been visualised in living human embryos. Here, we establish live cell imaging of chromosome segregation using normally fertilised embryos from an egg-share-to-research programme, as well as embryos deselected during fertility treatment. We reveal that the first mitotic ision has an extended prometaphase/metaphase and exhibits phenotypes that can cause nondisjunction. These included multipolar chromosome segregations and lagging chromosomes that lead to formation of micronuclei. Analysis of nuclear number and size provides evidence of equivalent phenotypes in 2-cell human embryos that gave rise to live births. Together this shows that errors in the first mitotic ision can be tolerated in human embryos and uncovers cell biological events that contribute to preimplantation mosaicism.
Publisher: Oxford University Press (OUP)
Date: 24-09-2018
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.
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.
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.
Publisher: Mary Ann Liebert Inc
Date: 10-03-2019
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.
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.
Publisher: JMIR Publications Inc.
Date: 03-2017
DOI: 10.2196/RESPROT.6498
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.
Publisher: Wiley
Date: 17-02-2020
Publisher: Cold Spring Harbor Laboratory
Date: 14-01-2023
DOI: 10.1101/2023.01.13.523952
Abstract: Aneuploid human eggs (oocytes) are a major cause of infertility, miscarriage and chromosomal disorders. Such aneuploidies increase greatly as women age, originating from defective linkages between sister-chromatids (cohesion) in meiosis. We found evidence that loss of a specific pool of the cohesin protector protein, shugoshin 2 (Sgo2) contributes to this phenomenon. Our data indicate that Sgo2 preserves sister chromatid cohesion in meiosis by protecting a ‘cohesin bridge’ between sister chromatids. In human oocytes, Sgo2 localizes to both sub-centromere cups and the pericentromeric bridge which spans the sister chromatid junction. Sgo2 normally colocalizes with cohesin, however, in oocytes from older women, Sgo2 is frequently lost specifically from the pericentromeric bridge and sister chromatid cohesion is weakened. Mps1 and Bub1 kinase activities maintain Sgo2 at sub-centromeres and the pericentromeric bridge. Removal of Sgo2 throughout meiosis I by Mps1 inhibition reduces cohesion protection, increasing the incidence of single chromatids at meiosis II. Therefore, Sgo2 deficiency in human oocytes can exacerbate the effects of maternal age by rendering residual cohesin at pericentromeres vulnerable to loss in anaphase I. Our data show that maternal age-dependent loss of Sgo2 at the pericentromere bridge in human oocytes impairs cohesion integrity and contributes to the increased incidence of aneuploidy observed in human oocytes with advanced maternal age.
Publisher: Elsevier BV
Date: 12-2015
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Bettina Mihalas.