Jeremy Thompson

The effect of glucosamine concentration on the development and sex ratio of bovine embryos

Publisher: Elsevier BV

Date: 2008

DOI: 10.1016/J.ANIREPROSCI.2006.12.014

Abstract: Glucosamine is a component of hyaluronic acid and an alternative substrate to glucose for the extracellular matrix synthesis of COCs. Its addition to an IVM medium reduces the glucose consumption of bovine COCs. Glucosamine is also metabolized to UDP-N-acetyl glucosamine (UDP-GlcNAc) via the hexosamine biosynthesis pathway and is utilized for O-linked glycosylation by the X-linked enzyme, O-linked GlcNAc transferase (OGT). Moreover, the inactivation of the second X chromosome in female embryos is influential in producing the sex ratio bias observed in vitro when embryos are cultured in the presence of glucose above 2.5mM. Accordingly, the aim of this study is to examine whether the presence of glucosamine during maturation or embryo culture causes a sex ratio bias in bovine blastocysts. Glucosamine was added to the medium in three different embryo developmental periods: in vitro maturation, the one-cell to eight-cell stage (before the maternal-zygotic transition, MZT), and the eight-cell to blastocyst stage (after MZT). When glucosamine was added during in vitro maturation, the developmental competence of oocytes was severely compromised. However, the sex ratio of embryos was not influenced. When glucosamine was added to embryo culture medium during development from one-cell to eight-cell stage (before MZT), it affected neither the development nor the sex ratio of bovine embryos. Finally, when glucosamine was added after MZT, the development rate of embryos was severely decreased, and the sex ratio was skewed toward males. Moreover, an inhibitor of OGT, benzyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside (BADGP), negated the effect of glucosamine on the sex ratio when it was added to embryo culture medium from the eight-cell to blastocyst stage (after MZT). These results suggest that, like glucose, the supplementation of glucosamine into the medium skewed the sex ratio to males and that OGT, an X-linked enzyme, was involved in this phenomenon. Moreover, this effect of glucosamine was limited only to when it was present in the embryo culture medium after MZT.

Dysregulation of bisphosphoglycerate mutase during in vitro maturation of oocytes

Publisher: Springer Science and Business Media LLC

Date: 30-05-2021

DOI: 10.1007/S10815-021-02230-0

Differential expression of oxygen-regulated genes in bovine blastocysts

Publisher: Wiley

Date: 2007

DOI: 10.1002/MRD.20617

Abstract: Low oxygen conditions (2%) during post-compaction culture of bovine blastocysts improve embryo quality, which is associated with a small yet significant increase in the expression of glucose transporter 1 (GLUT-1), suggesting a role of oxygen in embryo development mediated through oxygen-sensitive gene expression. However, bovine embryos to at least the blastocyst stage lack a key regulator of oxygen-sensitive gene expression, hypoxia-inducible factor 1alpha (HIF1alpha). A second, less well-characterized protein (HIF2alpha) is, however, detectable from the 8-cell stage of development. Here we use differential display to determine additional gene targets in bovine embryos in response to low oxygen conditions. While development to the blastocyst stage was unaffected by the oxygen concentration used during post-compaction culture, differential display identified oxygen-regulation of myotrophin and anaphase promoting complex 1 expression, with significantly lower levels observed following culture under 20% oxygen than 2% oxygen. These results further support the hypothesis that the level of gene expression of specific transcripts by bovine embryos alters in response to changes in the oxygen environment post-compaction. Specifically, we have identified two oxygen-sensitive genes that are potentially regulated by HIF2 in the bovine blastocyst.

Foreword

Publisher: Elsevier BV

Date: 2007

DOI: 10.1016/J.THERIOGENOLOGY.2006.09.008

Periconception onset diabetes is associated with embryopathy and fetal growth retardation, reproductive tract hyperglycosylation and impaired immune adaptation to pregnancy

Publisher: Springer Science and Business Media LLC

Date: 02-2018

DOI: 10.1038/S41598-018-19263-8

Abstract: Diabetes has been linked with impaired fertility but the underlying mechanisms are not well defined. Here we use a streptozotocin-induced diabetes mouse model to investigate the cellular and biochemical changes in conceptus and maternal tissues that accompany hyperglycaemia. We report that streptozotocin treatment before conception induces profound intra-cellular protein β- O -glycosylation ( O -GlcNAc) in the oviduct and uterine epithelium, prominent in early pregnancy. Diabetic mice have impaired blastocyst development and reduced embryo implantation rates, and delayed mid-gestation growth and development. Peri-conception changes are accompanied by increased expression of pro-inflammatory cytokine Trail , and a trend towards increased Il1a , Tnf and Ifng in the uterus, and changes in local T-cell dynamics that skew the adaptive immune response to pregnancy, resulting in 60% fewer anti-inflammatory regulatory T-cells within the uterus-draining lymph nodes. Activation of the heat shock chaperones, a mechanism for stress deflection, was evident in the reproductive tract. Additionally, we show that the embryo exhibits elevated hyper- O -GlcNAcylation of both cytoplasmic and nuclear proteins, associated with activation of DNA damage (ɣH2AX) pathways. These results advance understanding of the impact of peri-conception diabetes, and provide a foundation for designing interventions to support healthy conception without propagation of disease legacy to offspring.

Plasma progesterone and LH concentrations during oestrous synchronization in female fallow deer (Dama dama)

Publisher: Elsevier BV

Date: 05-1989

DOI: 10.1016/0378-4320(89)90054-7

Superovulation of farmed red deer (Cervus elaphus) and fallow deer (Dama dama) : Incidence of ovulation and changes in plasma hormone concentrations during the pre-ovulatory period in relation to ova

Publisher: Elsevier BV

Date: 03-1995

DOI: 10.1016/0378-4320(94)01351-L

REDOX regulation of early embryo development

Publisher: Bioscientifica

Date: 04-2002

DOI: 10.1530/REP.0.1230479

Abstract: Preimplantation embryonic development is associated with a change in preference in energy metabolism pathways. Although oxidative phosphorylation is obligatory in most species throughout preimplantation development, an increasing role for energy derived from glycolysis is associated with compaction and blastulation. Such a shift in metabolic pathway preference is desirable as the embryo faces an increasingly hypoxic environment in utero. We hypothesize that this shift in metabolic preference is associated with a change in the reduction-oxidation (REDOX) state within the embryo, affecting not only the energy production required for development, but also the activity of REDOX-sensitive transcription factors, which may alter gene expression patterns. Shifts in intracellular REDOX state may also contribute to spatial differences in cell activity, especially after compaction, and perhaps even major embryonic events such as fertilization, genome activation and cellular differentiation.

Redox and anti-oxidant state within cattle oocytes following in vitro maturation with bone morphogenetic protein 15 and follicle stimulating hormone

Publisher: Wiley

Date: 26-02-2015

DOI: 10.1002/MRD.22470

Abstract: The developmental competence of cumulus oocyte complexes (COCs) can be increased during in vitro oocyte maturation with the addition of exogenous oocyte-secreted factors, such as bone morphogenetic protein 15 (BMP15), in combination with hormones. FSH and BMP15, for ex le, induce different metabolic profiles within COCs-namely, FSH increases glycolysis while BMP15 stimulates FAD and NAD(P)H accumulation within oocytes, without changing the redox ratio. The aim of this study was to investigate if this BMP15-induced NAD(P)H increase was due to de novo NADPH production. Cattle COCs were cultured with FSH and/or recombinant human BMP15, resulting in a significant decrease in glucose-6-phosphate dehydrogenase activity (P < 0.05). Inhibition of isocitrate dehydrogenase (IDH) during this process decreased NAD(P)H intensity threefold in BMP15-treated oocytes, suggesting that BMP15 stimulates IDH and NADPH production via the tricarboxylic acid cycle. As NADPH is a reducing agent, reduced glutathione (GSH), H2O2, and mitochondrial activity were also measured to assess the general redox status of the oocyte. FSH alone decreased GSH levels whereas the combination of BMP15 and FSH sustained higher levels. Expression of genes encoding glutathione-reducing enzymes were also lower in oocytes cultured in the presence of FSH alone. BMP15 supplementation further promoted mitochondrial localization patterns that are consistent with enhanced developmental competence. Metabolomics revealed significant consumption of glutamine and production of alanine by COCs matured with both FSH and BMP15 compared to the control (P < 0.05). Hence, BMP15 supplementation differentially modulates reductive metabolism and mitochondrial localization within the oocyte. In comparison, FSH-stimulation alone decreases the oocytes' ability to regulate cellular stress, and therefore utilizes other mechanisms to improve developmental competence.

The effects of 2,4-dinitrophenol and d-glucose concentration on the development, sex ratio, and interferon-tau (IFNT) production of bovine blastocysts

Publisher: Wiley

Date: 21-10-2016

DOI: 10.1002/MRD.22590

Effect of the oxidative phosphorylation uncoupler 2,4-dinitrophenol on hypoxia-inducible factor-regulated gene expression in bovine blastocysts

Publisher: CSIRO Publishing

Date: 2004

DOI: 10.1071/RD04027

Abstract: In cattle embryos, development to the blastocyst stage is improved in the presence of 10 μm 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, coincident with an increase in glycolytic activity following embryonic genome activation. The present study examined redox-sensitive gene expression and embryo development in response to the addition of DNP post-compaction. 2,4-Dinitrophenol increased the expression of hypoxia-inducible factor 1α and 2α (HIF1α, HIF2α) mRNA. Although HIF1α protein remained undetectable in bovine blastocysts, HIF2α protein was localised within the nucleus of trophectoderm and inner cell mass (ICM) cells of blastocysts cultured in the presence or absence of DNP, with a slight increase in staining evident within the ICM in blastocysts cultured in the presence of DNP. However, the expression of GLUT1 and VEGF mRNA, genes known to be regulated by HIFs, was unaffected by the addition of DNP to the culture. Although the development of Grade 1 and 2 blastocysts was unaltered by the addition of DNP post compaction in the present study, a significant increase in the proportion of ICM cells was observed. Results indicate that 10 μm DNP improves the quality of bovine embryos, coincident with increased HIF2α protein localisation within ICM cells and increased HIFα mRNA levels. Therefore, the results demonstrate redox-regulated expression of HIF2.

A study relating the composition of follicular fluid and blood plasma from individual Holstein dairy cows to the in vitro developmental competence of pooled abattoir-derived oocytes

Publisher: Elsevier BV

Date: 07-2014

DOI: 10.1016/J.THERIOGENOLOGY.2014.03.011

Abstract: The fertility of high-performance (high milk yield) dairy breeds such as the Holstein within the Australian dairy herd has been on the decline for the past two decades. The 12-month calving interval for pasture-based farming practices results in oocyte maturation coinciding with peak lactation, periods of negative energy balance, and energy partitioning for lactation, causing energy deficiency in some organ systems, including the reproductive system. Oocyte developmental competence (the ability to undergo successful fertilization, embryo development, and establishment of pregnancy) is intrinsically linked with the composition of follicular fluid (FF). The aim of this study was to determine if there was a relationship between the fat and carbohydrate levels in plasma and FF and the ability to support in vitro oocyte maturation (IVM). Plasma and FF were collected in vivo from eight Holstein cows between 52 and 151 days post-partum. Plasma glucose trended (P = 0.072) higher and triglyceride levels were significantly higher than in FF (P < 0.05), but there were no relationships between FF and plasma composition. Glucose FF concentration was negatively related to follicular lactate and nonesterified fatty acid (NEFA) levels and days post-partum. Conversely, FF triglyceride concentrations were positively related to FF NEFA levels and negatively related to milk fat and protein composition. Abattoir-derived cumulus-oocyte complexes were cultured in either 50% FF (FF-IVM) or 50% plasma (plasma-IVM), with on-time embryo development then assessed. Although there were no differences between animals, the blastocyst rates after FF-IVM were negatively related to plasma glucose and days post-partum and positively related to body condition score and plasma NEFA levels. In comparison to the previous studies, total NEFA levels in FF were not related to animal parameters and did not influence oocyte developmental competence in vitro. Results from this study suggest that days post-partum and body condition score influence carbohydrate metabolism within the follicular environment, and this may be attributed to the pasture-based feed system applied in the Australian dairy industry.

Hemoglobin: a Gas Transport Molecule That Is Hormonally Regulated in the Ovarian Follicle in Mice and Humans1

Publisher: Oxford University Press (OUP)

Date: 2015

DOI: 10.1095/BIOLREPROD.114.124594

Abstract: An increasing number of nonerythroid tissues are found to express hemoglobin mRNA and protein. Hemoglobin is a well-described gas transport molecule, especially for O2, but also for NO, CO2, and CO, and also acts as a reactive oxygen species scavenger. We previously found Hba-a1 and Hbb mRNA and protein at high levels within mouse periovulatory cumulus cells, but not in cumulus following in vitro maturation. This led us to investigate the temporal and spatial regulation in follicular cells during the periovulatory period. Cumulus-oocyte complexes were collected from equine chorionic gonadotropin/human chorionic gonadotropin-treated peripubertal SV129 female mice and collected and analyzed for gene expression and protein localization at a variety of time points over the periovulatory period. A further cohort matured in vitro with different forms of hemoglobin (ferro- and ferrihemoglobin) under different O2 atmospheric conditions (2%, 5%, and 20% O2) were subsequently fertilized in vitro and cultured to the blastocyst stage. Murine mRNA transcripts for hemoglobin were regulated by stimulation of the ovulatory cascade, in both granulosa and cumulus cells, and expression of HBA1 and HBB was highly significant in human granulosa and cumulus, but erythrocyte cell marker genes were not. Several other genes involved in hemoglobin function were similarly luteinizing hormone-regulated, including genes for heme biosynthesis. Immunohistochemistry revealed a changing localization pattern of HBA-A1 protein in murine cumulus cells and oocytes following the ovulatory signal. Significantly, no positive staining for HBA-A1 protein was observed within in vitro-matured oocytes, but, if coincubated with ferro- or ferrihemoglobin, cytoplasmic HBA-A1 was observed, similar to in vivo-derived oocytes. Addition of ferro-, but not ferrihemoglobin, had a small, positive effect on blastocyst yield, but only under either 2% or 20% O2 gas atmosphere. The identification of hemoglobin within granulosa and cumulus cells poses many questions as to its function in these cells. There are several possible roles, the most likely of which is either an O2 or NO sequestering molecule perhaps both roles are engaged. The strong endocrine regulation during the periovulatory period suggests to us that one potential function of hemoglobin is to provide a short-lived hypoxic environment by binding very tightly any available O2. This, in turn, facilitates the differentiation of the follicle towards corpus luteum formation by enabling the stabilization of a key transcription factor known to initiate such differentiation: hypoxia inducible factor.

Improving oocytematuration in vitro

Publisher: Cambridge University Press

Date: 24-10-2012

DOI: 10.1017/CBO9781139135030.019

The pivotal role of glucose metabolism in determining oocyte developmental competence

Publisher: Bioscientifica

Date: 04-2010

DOI: 10.1530/REP-09-0345

Abstract: The environment that the cumulus oocyte complex (COC) is exposed to during either in vivo or in vitro maturation (IVM) can have profound effects on the success of fertilisation and subsequent embryo development. Glucose is a pivotal metabolite for the COC and is metabolised by glycolysis, the pentose phosphate pathway (PPP), the hexosamine biosynthesis pathway (HBP) and the polyol pathway. Over the course of oocyte maturation, a large proportion of total glucose is metabolised via the glycolytic pathway to provide substrates such as pyruvate for energy production. Glucose is also the substrate for many cellular functions during oocyte maturation, including regulation of nuclear maturation and redox state via the PPP and for the synthesis of substrates of extracellular matrices (cumulus expansion) and O -linked glycosylation (cell signalling) via the HBP. However, the oocyte is susceptible to glucose concentration-dependent perturbations in nuclear and cytoplasmic maturation, leading to poor embryonic development post-fertilisation. For ex le, glucose concentrations either too high or too low result in precocious resumption of nuclear maturation. This review will discuss the relevant pathways of glucose metabolism by COCs during in vivo maturation and IVM, including the relative contribution of the somatic and gamete compartments of the COC to glucose metabolism. The consequences of exposing COCs to abnormal glucose concentrations will also be examined, either during IVM or by altered maternal environments, such as during hyperglycaemia induced by diabetes and obesity.

Chromosome constitution of human embryos generated after in vitro maturation including 3-isobutyl-1-methylxanthine in the oocyte collection medium

Publisher: Oxford University Press (OUP)

Date: 04-12-2014

DOI: 10.1093/HUMREP/DEU329

Abstract: Do cleavage-stage embryos obtained from oocytes matured in vitro after pre-incubation with a phosphodiesterase inhibitor (IBMX) carry more chromosomal abnormalities than those generated from oocytes matured in vivo? The rate and type of chromosomal abnormalities in normally developing cleavage-stage embryos generated with an in vitro maturation (IVM) system including pre-incubation with IBMX are not different from those observed in supernumerary embryos obtained from oocytes matured in vivo. Very limited information is available about the chromosomal constitution of IVM embryos. Previous studies were carried out using FISH on single biopsied blastomeres or arrested whole embryos and only provided fragmentary information on chromosomal abnormalities in IVM embryos. There is no systematic study of chromosomal abnormalities in all blastomeres of human Day 3 embryos with good morphology. Between July 2012 and December 2012, 16 young (age <35 years old) egg donors underwent 18 IVM cycles for the generation of research embryos. Eighteen embryos developed to Day 3 and were analysed using array comparative genomic hybridization (aCGH). Immature oocytes were retrieved from 2 to 10 mm follicles after mild ovarian stimulation with gonadotrophins but without hCG ovulation trigger. At collection, oocytes were pre-incubated with 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor and matured in vitro. After IVM culture, mature oocytes were microinjected with sperm from a single donor. Embryos were cultured to Day 3 after ICSI and all blastomeres of 18 good-morphology embryos were collected in idually for aCGH. Oocyte maturation rate in vitro was 50.2% (120/239). The mean fertilization rate was 68.3% (82/120) and 30.5% (25/82) of fertilized oocytes developed into a morphologically good quality embryo on Day 3 after ICSI. Of these, 18 embryos that developed well up to Day 3 were analysed using aCGH. Eighty of the 123 blastomeres analysed showed at least one chromosomal abnormality. Three out of eighteen embryos had completely normal cells. A single embryo carried a meiotic abnormality, 11 embryos were mosaic and three were chaotic. Although the aneuploidy data of this study are too limited to allow statistical analysis, these data are comparable to our own published data on the chromosome constitution of whole day 3 and day 4 embryos after conventional ART. Array CGH technology determines relative quantification of chromosomal domains but does not allow for the visualization of chromosomal rearrangements, assessment of ploidy or detection of uniparental isodisomy. Conclusions drawn on segmental abnormalities should be treated with caution. Although the limited number of embryos analysed here precludes firm conclusions, they provide valuable data on possible causes of the reduced potential of IVM embryos. This is the first study to describe the complete chromosome complement of all single blastomeres of good-morphology day 3 embryos obtained with IVM (including the presence of IBMX in a pre-incubation medium). The results demonstrate that a high proportion of good-morphology embryos are aneuploid and that there is no obvious increase in aneuploidies as a result of IVM which seems to suggest that the reduced efficiency of IVM technology compared with standard IVF may be accounted for by factors other than aneuploidy, such as cytoplasmic defects or reduced endometrial receptivity. This study was funded by the TBM (Applied Biomedical Research with Societal Finality) programme of the IWT (Agency for Innovation through Science and Technology - Flanders, 110680) and by a Methusalem grant of the Vrije Universiteit Brussel. C.S. is a post-doctoral fellow of the Fund for Scientific Research Flanders (FWO - Vlaanderen). K.J. is a PhD student funded by the FWO. The University of Adelaide owns a patent family associated with IVM technologies that is licensed to Cook Medical. R.B.G. and J.G.T. are inventors. The remaining authors have no conflict of interest to declare.

Development of in vitro embryo production systems for red deer (Cervus elaphus)

Publisher: Elsevier BV

Date: 03-2002

DOI: 10.1016/S0378-4320(01)00201-9

Abstract: The following experiments investigated the use of sheep serum (SS) as a capacitating agent for red deer (Cervus elaphus) sperm during in vitro fertilisation. Red deer oocytes were collected at slaughter and matured in vitro for 24h in TCM-199 supplemented with 10% foetal calf serum, 10 microg ml(-1) FSH and LH, and 1microg ml(-1) of oestradiol. Fertilisation medium was IVF-SOF modified to contain 5mM Ca(2+) and no glucose. Experiment 1 investigated the addition of heparin, BSA (8 mg ml(-1)) or 20% SS. All oocytes were penetrated when IVF-SOF was supplemented with SS compared to 10 and 0% penetration when either heparin or BSA was present (P<0.01). However, 43.8% of these oocytes were polyspermic when the medium contained SS. In Experiment 2, the effect of sperm concentration on penetration rates during in vitro fertilisation was investigated. Total sperm penetration and monospermic penetration rates increased with increased sperm concentrations in a log linear manner (P<0.001) and both approached an asymptote at 0.4 x 10(6) sperm ml(-1) with 93.6 and 77% for total and monospermic penetration, respectively. Polyspermic fertilisation also increased with increasing sperm concentrations (P<0.05) but was variable (range 3.5+/-4.2 to 42.3+/-10.6%), especially at the lower sperm concentrations. Experiment 3 investigated the viability of these oocytes after transfer into red deer recipients. Fifteen 2- and 4-cell embryos were transferred into the oviducts of synchronized recipients 28 h post in vitro insemination. An additional fourteen embryos (8-10 cell) were transferred into synchronised recipients after 48 h of in vitro culture in either SOFaaBSA (n=10) or on red deer epithelial oviduct monolayers (n=4). Five (33% 5/15) of the recipients that received 2- and 4-cell embryos were pregnant at Day 45 (verified by ultrasonography) and four recipients subsequently calved. One recipient receiving an embryo cultured in SOFaaBSA was pregnant at Day 45 and subsequently calved. The birth of five normal calves indicate that full developmental competence of red deer oocytes matured and fertilised in vitro can be achieved by the techniques described.

Prematuration with cyclic adenosine monophosphate modulators alters cumulus cell and oocyte metabolism and enhances developmental competence of in vitro- matured mouse oocytes

Publisher: Oxford University Press (OUP)

Date: 08-2014

DOI: 10.1095/BIOLREPROD.114.118471

Abstract: Oocyte in vitro maturation (IVM) is an important assisted reproductive technology and research tool. The adoption of IVM into routine clinical practice has been hindered by its significantly lower success rates compared to conventional in vitro fertilization. Cyclic AMP (cAMP) modulation and follicle-stimulating hormone (FSH), independently, have long been known to improve IVM oocyte developmental competence. This study comprehensively examined the effects of FSH and cAMP/cGMP modulation, alone and in combination, on IVM oocyte metabolism and developmental outcomes. Mouse cumulus-oocyte complexes (COCs) were subjected to a 1 h prematuration phase ± the cAMP modulator forskolin and cAMP/cGMP modulator 3-isobutyl-1-methylxanthine followed by IVM ± FSH. Prematuration with these cyclic nucleotide modulators or IVM with FSH significantly improved oocyte developmental competence and reduced spindle abnormalities compared to spontaneous IVM (no treatment) however, these two treatments in combination endowed even greater developmental competence (improved subsequent blastocyst rates and quality P < 0.05), albeit blastocyst yield and quality remained significantly lower than that of oocytes matured in vivo. A significant additive effect of combined IVM treatments was evident as increased COC lactate production and oxygen consumption and enhanced oocyte oxidative metabolism, ATP production, ATP:ADP ratio, and glutathione levels (P < 0.05). Nevertheless, IVM increased reactive oxygen species production, particularly as a consequence of FSH addition, relative to in vivo matured oocytes. In conclusion, improvements in the embryo yield following IVM is associated with increased COC oxygen consumption and oocyte oxidative metabolism, but these remain metabolically and developmentally less competent relative to in vivo derived oocytes.

Oocyte-Secreted Factors in Domestic Animals

Publisher: Wiley

Date: 13-03-2013

DOI: 10.1002/9781118538074.CH4

Beta-Oxidation Is Essential for Mouse Oocyte Developmental Competence and Early Embryo Development1

Publisher: Oxford University Press (OUP)

Date: 12-2010

DOI: 10.1095/BIOLREPROD.110.084145

Abstract: Oocyte and embryo metabolism are closely linked with their subsequent developmental capacity. Lipids are a potent source of cellular energy, yet little is known about lipid metabolism during oocyte maturation and early embryo development. Generation of ATP from lipids occurs within mitochondria via beta-oxidation of fatty acids, with the rate-limiting step catalyzed by carnitine palmitoyl transferase I (CPT1B), a process also requiring carnitine. We sought to investigate the regulation and role of beta-oxidation during oocyte maturation and preimplantation development. Expression of Cpt1b mRNA, assessed by real-time RT-PCR in murine cumulus-oocyte complexes (COCs), increased following hormonal induction of oocyte maturation and ovulation in vivo with human chorionic gonadotropin (5 IU) and in embryos reaching the blastocyst stage. Beta-oxidation, measured by the production of (3)H(2)O from [(3)H]palmitic acid, was significantly increased over that in immature COCs following induction of maturation in vitro with epidermal growth factor (3 ng/ml) and follicle-stimulating hormone (50 mIU/ml). The importance of lipid metabolism for oocyte developmental competence and early embryo development was demonstrated by assessing the rate of embryo development following inhibition or upregulation of beta-oxidation with etomoxir (an inhibitor of CPT1B) or L-carnitine, respectively. Inhibition of beta-oxidation during oocyte maturation or zygote cleavage impaired subsequent blastocyst development. In contrast, L-carnitine supplementation during oocyte maturation significantly increased beta-oxidation, improved developmental competence, and in the absence of a carbohydrate energy supply, significantly increased 2-cell cleavage. Thus, carnitine is an important cofactor for developing oocytes, and fatty acids are an important energy source for oocyte and embryo development.

Effects of high concentrations of hyaluronan in culture medium on development and survival rates of fresh and frozen-thawed bovine embryos produced in vitro

Publisher: Bioscientifica

Date: 07-2002

DOI: 10.1530/REP.0.1240141

Abstract: Hyaluronic acid (HA) is the main glycosaminoglycan present in follicular, oviductal and uterine fluids. The main functions of HA include dynamic processes that are mediated through interaction with extracellular matrix components, regulation of gene expression, cell proliferation and cell differentiation. HA increases the viscosity of solutions and also has several physiological functions, including regulation of water distribution and water-binding capacity. The addition of 6 mg HA ml(-1) to synthetic oviduct fluid (SOF SOF-HA) culture medium on day 5 (IVF = day 0) significantly (P 0.001) increased the viscosity of the medium in comparison with SOF culture medium containing BSA (SOF-BSA). On day 8, rate of blastocyst development in SOF-HA culture medium was significantly (P 0.05) higher than in SOF-BSA culture medium (38.2 versus 29.3%). The number of trophectoderm cells and the total number of cells of expanded blastocysts cultured in the presence of HA were significantly (P 0.01) higher in comparison with expanded blastocysts cultured in the presence of BSA (88.9 +/- 7.3 versus 67.6 +/- 3.0 and 130.1 +/- 10.9 versus 104.8 +/- 2.5, respectively). After freezing and thawing, the percentage of day 8 embryos that re-expanded and hatched when cultured with SOF-HA was greater than that of embryos cultured with SOF-BSA (11.3 and 10.5% versus 75.5 and 36.8%, respectively). After thawing, the ATP contents of in vivo-derived, SOF-HA and SOF-BSA expanded blastocysts were similar. The embryos cultured with HA showed less ultrastructural deviation and de-differentiation after freezing and thawing than the embryos cultured with BSA. This study demonstrates that HA improves the developmental capacity of bovine embryos under in vitro conditions and is warranted as a supplement for in vitro production of bovine embryos, particularly if they are to be cryopreserved.

Metabolism of pyruvate by pre-elongation sheep embryos and effect of pyruvate and lactate concentrations during culture in vitro

Publisher: CSIRO Publishing

Date: 1993

DOI: 10.1071/RD9930417

Abstract: In the first of two experiments, utilization of [1-14C]pyruvate by 8-cell and blastocyst-stage embryos derived in vivo was examined during a 3-h incubation in HEPES-buffered synthetic oviduct fluid (SOF) medium in the presence or absence of other substrates. In the second, a factorial design examined the effect of pyruvate (0, 0.33, 1.0 and 3.3 mM) and lactate (3.3, 10 and 33 mM) on development of 1- and 2-cell sheep embryos cultured in vitro in a modified SOF medium (containing glucose, glutamine and modified Eagle's medium non-essential amino acids). Peak utilization of [1-14C]pyruvate was unaffected by the presence or absence of other energy substrates. In contrast, rate of utilization was affected by the addition of other energy substrates, with half maximal utilization occurring at either 0.4 +/- 0.2 mM or 1.2 +/- 0.2 mM for 8-cells and either 0.2 +/- 0.2 mM or 1.3 +/- 0.3 mM for blastocysts when incubated in the absence or presence of other energy substrates respectively. In the second experiment the proportion of embryos developing to blastocysts was inhibited by high lactate levels (P 0.001), but was generally not affected by pyruvate concentration. However, there was a significant interaction (P 0.001) between pyruvate and lactate when both were present in the medium. At 0.33 mM pyruvate, 3.3 mM lactate supported good development (83 +/- 8% blastocysts) whereas 10 mM lactate supported less development (50 +/- 11%). However, at the higher levels of pyruvate this effect was lost.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonesterified Fatty Acid-Induced Endoplasmic Reticulum Stress in Cattle Cumulus Oocyte Complexes Alters Cell Metabolism and Developmental Competence1

Publisher: Oxford University Press (OUP)

Date: 2016

DOI: 10.1095/BIOLREPROD.115.131862

Abstract: Reduced oocyte quality has been associated with poor fertility of high-performance dairy cows during peak lactation, due to negative energy balance. We examined the role of nonesterified fatty acids (NEFAs), known to accumulate within follicular fluid during under- and overnutrition scenarios, in causing endoplasmic reticulum (ER) stress of in vitro maturated cattle cumulus-oocyte complexes (COCs). NEFA concentrations were: palmitic acid (150 μM), oleic acid (200 μM), and steric acid (75 μM). Abattoir-derived COCs were randomly matured for 24 h in the presence of NEFAs and/or an ER stress inhibitor, salubrinal. Total and hatched blastocyst yields were negatively impacted by NEFA treatment compared with controls, but this was reversed by salubrinal. ER stress markers, activating transcription factor 4 (Atf4) and heat shock protein 5 (Hspa5), but not Atf6, were significantly up-regulated by NEFA treatment within whole COCs but reversed by coincubation with salubrinal. Likewise, glucose uptake and lactate production, measured in spent medium s les, showed a similar pattern, suggesting that cumulus cell metabolism is sensitive to NEFAs via an ER stress-mediated process. In contrast, while mitochondrial DNA copy number was recovered in NEFA-treated oocytes, oocyte autofluorescence of the respiratory chain cofactor, FAD, was lower following NEFA treatment of COCs, and this was not reversed by salubrinal, suggesting the negative impact was via reduced mitochondrial function. These results reveal the significance of NEFA-induced ER stress on bovine COC developmental competence, revealing a potential therapeutic target for improving oocyte quality during peak lactation.

The effects of 2,4-dinitrophenol and D-glucose concentration on the quality, development, sex ratio, and IFN-tau production of bovine blastocysts

Publisher: Oxford University Press (OUP)

Date: 05-2008

DOI: 10.1093/BIOLREPROD/78.S1.207A

Correction to: Vitrification within a nanoliter volume: oocyte and embryo cryopreservation within a 3D photopolymerized device

Publisher: Springer Science and Business Media LLC

Date: 07-09-2022

DOI: 10.1007/S10815-022-02610-0

1D-2 Environmental influences during preimplantation embryo development: the role of oxygen

Publisher: Elsevier BV

Date: 09-2007

DOI: 10.1016/S0378-3782(07)70008-7

Bone morphogenetic protein 15 and fibroblast growth factor 10 enhance cumulus expansion, glucose uptake, and expression of genes in the ovulatory cascade during in vitro maturation of bovine cumulus–o

Publisher: Bioscientifica

Date: 07-2013

DOI: 10.1530/REP-13-0079

Abstract: Oocyte-secreted factors (OSFs) regulate differentiation of cumulus cells and are of pivotal relevance for fertility. Bone morphogenetic protein 15 (BMP15) and fibroblast growth factor 10 (FGF10) are OSFs and enhance oocyte competence by unknown mechanisms. We tested the hypothesis that BMP15 and FGF10, alone or combined in the maturation medium, enhance cumulus expansion and expression of genes in the preovulatory cascade and regulate glucose metabolism favouring hyaluronic acid production in bovine cumulus–oocyte complexes (COCs). BMP15 or FGF10 increased the percentage of fully expanded COCs, but the combination did not further stimulate it. BMP15 increased cumulus cell levels of mRNA encoding a disintegrin and metalloprotease 10 ( ADAM10 ), ADAM17 , hiregulin ( AREG ), and epiregulin ( EREG ) at 12 h of culture and of prostaglandin (PG)-endoperoxide synthase 2 ( PTGS2 ), pentraxin 3 ( PTX3 ) and tumor necrosis factor alpha-induced protein 6 ( TNFAIP6 ( TSG6 )) at 22 h of culture. FGF10 did not alter the expression of epidermal growth factor-like factors but enhanced the mRNA expression of PTGS2 at 4 h, PTX3 at 12 h, and TNFAIP6 at 22 h. FGF10 and BMP15 stimulated glucose consumption by cumulus cells but did not affect lactate production or levels of mRNA encoding glycolytic enzymes phosphofructokinase and lactate dehydrogenase A. Each growth factor increased mRNA encoding glucosamine:fructose-6-PO 4 transaminases, key enzymes in the hexosamine pathway leading to hyaluronic acid production, and BMP15 also stimulated hyaluronan synthase 2 ( HAS2 ) mRNA expression. This study provides evidence that BMP15 and FGF10 stimulate expansion of in vitro -matured bovine COCs by driving glucose metabolism toward hyaluronic acid production and controlling the expression of genes in the ovulatory cascade, the first acting upon ADAM10 , ADAM17 , AREG , and EREG and the second on downstream genes, particularly PTGS2 .

Development of bright and biocompatible nanoruby and its application to background-free time-gated imaging of G-protein-coupled receptors

Publisher: American Chemical Society (ACS)

Date: 02-11-2017

DOI: 10.1021/ACSAMI.7B12665

Abstract: At the forefront of developing fluorescent probes for biological imaging applications are enhancements aimed at increasing their brightness, contrast, and photostability, especially toward demanding applications of single-molecule detection. In comparison with existing probes, nanorubies exhibit unlimited photostability and a long emission lifetime (∼4 ms), which enable continuous imaging at single-particle sensitivity in highly scattering and fluorescent biological specimens. However, their wide application as fluorescence probes has so far been hindered by the absence of facile methods for scaled-up high-volume production and molecularly specific targeting. The present work encompasses the large-scale production of colloidally stable nanoruby particles, the demonstration of their biofunctionality and negligible cytotoxicity, as well as the validation of its use for targeted biomolecular imaging. In addition, optical characteristics of nanorubies are found to be comparable or superior to those of state-of-the-art quantum dots. Protocols of reproducible and robust coupling of functional proteins to the nanoruby surface are also presented. As an ex le, NeutrAvidin-coupled nanoruby show excellent affinity and specificity to μ-opioid receptors in fixed and live cells, allowing wide-field imaging of G-protein coupled receptors with single-particle sensitivity.

Cumulin and FSH Cooperate to Regulate Inhibin B and Activin B Production by Human Granulosa-Lutein Cells In Vitro

Publisher: The Endocrine Society

Date: 08-02-2019

DOI: 10.1210/EN.2018-01026

Abstract: The oocyte-secreted factors bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) interact functionally, and it is hypothesized that this interaction may be mediated by formation of a GDF9:BMP15 heterodimer termed cumulin. GDF9 and BMP15 regulate folliculogenesis and ovulation rate and have been shown to regulate inhibin and activin, local regulators of folliculogenesis. The objective of this study was to determine whether cumulin regulates granulosa cell inhibin and activin production and whether this requires cooperation with FSH. Human granulosa-lutein (hGL) cells collected from patients undergoing in vitro fertilization were cultured with or without FSH with various forms of recombinant cumulin (native and cysteine mutants, with or without the prodomains), and cysteine mutant GDF9 or BMP15. Messenger RNA expression of the subunits of inhibins/activins (INHA, INHBA, INHBB) and secretion of inhibin A, inhibin B, and activin B were measured. Mature forms and proforms of cumulin stimulated comparable INHBB mRNA expression and secretion of inhibin B and activin B, whereas GDF9 or BMP15 exhibited no effect. Cumulin, but not GDF9 or BMP15, interacted synergistically with FSH to increase INHBB mRNA and inhibin B expression. FSH markedly stimulated INHA, which encodes the α subunit of inhibin A/B, and suppressed activin B. Cumulin with or without FSH did not significantly alter inhibin A. Together these data demonstrate that cumulin, but not GDF9 or BMP15, exerts paracrine control of FSH-induced regulation of inhibin B and activin B. The prodomains of cumulin may have a minimal role in its actions on granulosa cells.

Bidirectional communication between cumulus cells and the oocyte: Old hands and new players?

Publisher: Elsevier BV

Date: 07-2016

DOI: 10.1016/J.THERIOGENOLOGY.2016.04.019

Abstract: Cumulus cell-oocyte communication is an essential feature of mammalian reproduction. Established mechanisms involve the bidirectional transfer of ions and small molecules through gap junctions that fundamentally regulate the process of oocyte maturation. Also, well established is the paracrine signaling from the oocyte to the cumulus, which regulates much of the flow of ions and molecules to the oocyte and orchestrates many of the associated local signaling events around ovulation, which is the key to establishing oocyte competence to sustain early embryo development. Less well-characterized and new potential players include exosomal transfer of noncoding RNAs from cumulus to oocytes and the recent observations of the presence of hemoglobin in oocytes and cumulus cells. The impact of these new communication pathways is either poorly defined or even unknown. Finally, signaling between the two cell types most likely continues after ovulation and even fertilization however, this too is largely undefined but may play roles in substrate transport, sperm chemotaxis and "trapping", and potential signaling to the rest of the reproductive tract.

Effect of specific phosphodiesterase isoenzyme inhibitors during in vitro maturation of bovine oocytes on meiotic and developmental capacity

Publisher: Oxford University Press (OUP)

Date: 10-2004

DOI: 10.1095/BIOLREPROD.103.024828

In vivo survival of transferred sheep embryos following puncture of the zona pellucida and in vitro culture

Publisher: Elsevier BV

Date: 03-1994

DOI: 10.1016/0378-4320(94)90008-6

Maternal factors and the risk of birth defects after IVF and ICSI: A whole of population cohort study

Publisher: Wiley

Date: 17-10-2016

DOI: 10.1111/1471-0528.14365

Abstract: To assess the contribution of maternal factors to major birth defects after in vitro fertilisation (IVF), intracytoplasmic sperm injection (ICSI), and natural conception. Retrospective cohort study in South Australia for the period January 1986 to December 2002. A whole of population study. A census of all IVF and ICSI linked to registries for births, pregnancy terminations, and birth defects (diagnosed before a child's fifth birthday). Odds ratios (ORs) for birth defects were calculated among IVF, ICSI, and natural conceptions for maternal age, parity, pre-pregnancy BMI, smoking, pre-existing diseases, and conditions in pregnancy, with adjustment for confounding factors. Birth defects classified by International Classification of Diseases (ninth revision) and British Paediatric Association (ICD9-BPA) codes. There were 2211 IVF, 1399 ICSI, and 301 060 naturally conceived births. The unadjusted prevalence of any birth defect was 7.1, 9.9, and 5.7% in the IVF, ICSI, and natural conception groups, respectively. As expected, the risk of birth defects increased with maternal age among the natural conceptions. In contrast, for IVF and ICSI combined, relative to natural conceptions, births to women aged ≤29 years had a higher risk (adjusted odds ratio, aOR 1.42 95% confidence interval, 95% CI 1.04-1.94), births to women aged 35-39 years had no difference in risk (aOR 1.01 95% CI 0.74-1.37), and births to women aged ≥40 years had a lower risk of defects (aOR 0.45 95% CI 0.22-0.92). Defects were also elevated for nulliparity, anaemia, and urinary tract infection in births after ICSI, but not after IVF. The usual age-birth defect relationship is reversed in births after IVF and ICSI, and the associations for other maternal factors and defects vary between IVF and ICSI. Risk of birth defects in women over 40 years is lower after infertility treatment than for natural conceptions.

Dioxin Affects Glucose Transport via the Arylhydrocarbon Receptor Signal Cascade in Pluripotent Embryonic Carcinoma Cells

Publisher: The Endocrine Society

Date: 12-2007

DOI: 10.1210/EN.2007-0254

Abstract: Intoxication by dioxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads, among other damages, to early embryo loss, fetal malformations, and cardiovascular toxicity. Apart from binding to the arylhydrocarbon receptor (AhR), the mechanism of TCDD-mediated embryo toxicity is still unclear. We investigated possible modes of a TCDD-mediated toxicity, particularly in glucose metabolism, in pluripotent P19 mouse embryonic carcinoma cells. Undifferentiated P19 cells were exposed to 1–100 nm TCDD and characterized for AhR signaling. For studying cell differentiation, P19 cells were exposed to 10 nm TCDD at stage of embryoid body formation, and analyzed on glucose metabolism and cardiac differentiation during the next 3 wk. TCDD treatment activated the AhR-signaling cascade within 1 h, confirmed by AhR translocation, induction of cytochrome P450 1A1 expression, and activation of the xenobiotic response element. Although cell viability and transcription of the cardiac marker protein α-myosin heavy chain were affected, TCDD did not inhibit the differentiation of P19 cells to pulsating cardiomyocytes. TCDD significantly down-regulated the expression levels of the glucose transporter (GLUT) isoforms 1 and 3. After 24-h TCDD treatment, GLUT1 was no longer localized in the plasma membrane of P19 cells. The impaired GLUT expression correlated with a lower glucose uptake in 5-d-old embryoid bodies. The TCDD effects were mediated by AhR, as shown by preculture with the AhR antagonist α-naphthoflavone. Our data demonstrate that an AhR-mediated disturbance in GLUT expression and insufficient glucose uptake may be major mechanisms in TCDD embryo toxicity.

Development of in vitro embryo production systems for red deer (Cervus elaphus)

Publisher: Elsevier BV

Date: 03-2002

DOI: 10.1016/S0378-4320(01)00199-3

Abstract: In vitro fertilisation (IVF) protocols for red deer have yielded low fertilisation rates, with no embryo development beyond the eight-cell stage when heparin was used as the in vitro capacitation agent. As this low fertilisation rate may result from reduced motility, the present study investigated the use of red deer oviduct epithelial cell monolayers (COEM) and conditioned medium (Cm) from the monolayers to maintain red deer sperm motility in vitro. A second experiment compared the fertilisability of red deer sperm pre-incubated for 4-12h on COEM or for 4h in TALP medium supplemented with 20 microg of heparin.COEM was superior in maintaining red deer sperm motility compared with either Sp-TALP alone or Cm (P<0.05). COEM sustained sperm motility at levels comparable to the initial motility over the 24h period. The motility of sperm incubated in Sp-TALP and Cm was similar and had declined to less than 10% by 4h and no motile sperm were observed by 8h. Overall, the penetration rates of in vitro red deer oocytes were low (5-28%) regardless of sperm treatment. Sperm pre-incubated on COEM penetrated more oocytes than sperm incubated with heparin (P 0.50). Penetration rates were greater across all treatments when both sperm and oocytes were co-incubated for 24h compared to 12h (P<0.001). There were no differences in penetration rates among the four donor stags used in the study. It was concluded that COEM sustains red deer sperm motility in vitro during the 24h observation period. Pre-incubating sperm on COEM does increase sperm penetration rates compared with heparin alone, but at a rate too low and variable to be used on a routine basis. Overall, the penetration rates were comparable to those previously reported for red deer even though differences in heparin concentration, fertilisation systems and stags were used.

Gray level Co-occurrence Matrices (GLCM) to assess microstructural and textural changes in pre-implantation embryos.

Publisher: Wiley

Date: 08-2016

DOI: 10.1002/MRD.22680

Abstract: The preimplantation embryo is extraordinarily sensitive to environmental signals and events such that perturbations can alter embryo metabolism and program an altered developmental trajectory, ultimately affecting the phenotype of the adult in idual indeed, the physical environment associated with in vitro embryo culture can attenuate development. Defining the underlying metabolic changes and mechanisms, however, has been limited by the imaging technology used to evaluate metabolites and structural features in the embryo. Here, we assessed the impact of in vitro fertilization and culture on mouse embryos using three metabolic markers: peroxyfluor 1 (a reporter of hydrogen peroxide), monochlorobimane (a reporter of glutathione), and Mitotracker Deep Red (a marker of mitochondria). We also evaluated the distribution pattern of histone 2AX gamma (γH2AX) in the nuclei of 2- and 8-cell embryos and blastocysts to investigate the degree of DNA damage caused by in vitro embryo culture. In vitro-fertilized embryos, in vivo-developed embryos, and in vivo-fertilized embryos recovered and cultured in vitro were compared at the 2-, 8-cell, and blastocyst stages. In addition to assessments based on fluorescence intensity, textural analysis using Gray Level Co-occurrence Matrix (GLCM), a statistical approach that assesses texture within an image, was used to evaluate peroxyfluor 1, monochlorobimane, and Mitotracker Deep Red staining in an effort to develop a robust metric of embryo quality. Our data provide strong evidence of modified metabolic parameters identifiable as altered fluorescence texture in embryos developed in vitro. Thus, texture-analysis approach may provide a means of gaining additional insight into embryo programming beyond conventional measurements of staining intensity for metabolic markers. Mol. Reprod. Dev. 83: 701-713, 2016 © 2016 Wiley Periodicals, Inc.

THE PEAK OF OXYGEN CONSUMPTION AT THE TIME OF SPERM PENETRATION IS ASSOCIATED WITH AN INCREASE IN REACTIVE OXYGEN SPECIES PRODUCTION IN BOVINE ZYGOTES

Publisher: CSIRO Publishing

Date: 2009

DOI: 10.1071/RDV21N1AB118

Abstract: Oxygen is essential for the generation of cellular energy (ATP) via oxidative phosphorylation, and thus oxygen consumption is a key indicator of metabolic activity within cells. Increased oxidative activity at the time of fertilization has been described for marine invertebrate oocytes. The objective of the present study was to determine if changes in oxygen consumption would occur at the time of fertilization and cell cleavage in bovine zygotes. The oxygen consumption of presumptive zygotes (n = 57) was measured in idually and continuously from 6 until 30 h after IVF using the Embryoscope™ (Unisense Fertilitech A/S). A control group of in vitro matured oocytes (n = 9) that had not been fertilized was also measured continuously from 6 h after transfer to IVF medium. Time-lapse images were acquired during the measurements at intervals of approximately 36 min and used for evaluation of the developmental progress. Oocytes/zygotes were subsequently stained with Hoechst 33342 for more accurate classification by fluorescence microscopy. Cohorts of oocytes/zygotes (n = 55) were in idually stained with REDOX-Sensor Red CC-1 and Hoechst 33342 at 0, 7, 12, 17 and 24 h after IVF and imaged by confocal microscopy to determine the REDOX potential and the distribution pattern as well as nuclear stage. Data was analyzed statistically using Proc Mixed, SAS. The mean oxygen consumption of the developing zygotes peaked markedly at 9 h after fertilization (0.552 nL h–1 value 14% over and above baseline oxygen consumption) and fell abruptly until 20 h after IVF (0.482 nL h–1). Following this, an oxygen peak of lower magnitude (0.487 nL h–1 value 1% over and above baseline oxygen consumption) was observed at the time of the first cleavage (detected between 21 to 32 h). The curve pattern for the control group was statistically different: the mean oxygen consumption was at its greatest from 8 to 12 h after IVF and then fell gradually until 30 h after IVF with no visible small increase in oxygen consumption at the supposed time of first cleavage. Mean respiration rates of the oocytes in the control group were significantly greater than those of the developing zygotes at all time points during the measurements. The mean pixel intensity of the REDOX-Sensor Red CC-1 staining was significantly greater at 7 h and at 24 h after IVF, when compared with the other group. The two distinct peaks of oxygen consumption were coincident with the REDOX pulses occurring at the time of sperm penetration and cell cleavage. However, due to the considerably higher magnitude of the oxygen peak occurring around the time of sperm penetration (signaling fertilization) we were led to speculate that only this peak was associated with an increased reactive oxygen species production. The REDOX pulse observed at the time of cleavage did not seem to be an oxygen-derived REDOX pulse because the oxygen consumption by the bovine zygotes was only changed marginally. ASL is supported by FCT, Portugal. The authors thank Unisense-Fertilitech for their collaboration.

Foreword

Publisher: CSIRO Publishing

Date: 1997

DOI: 10.1071/RDV17N3_FO

Successful in vitro culture of early cleavage stage embryos recovered from superovulated red deer ()

Publisher: Elsevier BV

Date: 07-1995

DOI: 10.1016/0093-691X(95)00174-7

Abstract: Three separate embryo culture systems were evaluated for their ability to support development of early cleavage stage red deer (Cervus elaphus ) embryos: ligated sheep oviducts (Treatment A) cervine oviduct epithelial monolayer in TCM 199 + 10% deer serum (Treatment B) synthetic oviduct fluid + 20% human serum at 7% O(2) atmosphere (Treatment Q. In addition, 2 superovulation protocols were compared for their efficacy in producing early cleavage stage embryos. Twenty red deer (2 to 7 yr old) were synchronized in April with intravaginal CIDR devices for 12 d. All animals received a total of 0.4 units of ovine FSH administered in 8 equal doses, 12 h apart, beginning 72 h before removal of CIDR devices. The deer additionally received 200 IU PMSG, either with the first FSH injection (Group 1, n = 10) or with the last FSH injection (Group 2, n = 10). Hinds were placed with fertile stags following withdrawal of CIDR devices. Ova were collected by surgical recovery 63 h post CIDR removal. At the time of collection, animals in Group 2 had a significantly greater mean (+/- SEM) ovulation rate (11.2 +/- 2.4 vs 5.3 +/- 2.4), with more animals responding to treatment (>1 ovulation), than the animals in Group 1 (10/10 vs 4/10). Late in the breeding season (June), 10 additional red deer (Group 3, Experiment 2) were superovulated using the same protocol as for the deer in Group 2, with ova collection advanced by 24 h. Mean (+/- SEM) ovulation rate was 6.4 +/- 1.2 with 9 10 animals responding. Ova recovery did not differ among the groups (range 73 to 87%). Superovulation treatment did not affect cultured embryo development to the morula/blastocyst stage. Furthermore, there was no difference among the 3 culture systems in their support of development either to the morula (range 50 to 58%) or to the blastocyst (range 22 to 26%) stage. After laparoscopic transfer of 4 morula/blastocyst embryos to recipient red deer (2 from Treatment B and 2 from Treatment C) 2 live calves were born from embryos cultured in Treatment B.

Super-multiplexed fluorescence microscopy via photostability contrast

Publisher: The Optical Society

Date: 06-06-2018

DOI: 10.1364/BOE.9.002943

Implantation and Early Development

Publisher: Informa UK Limited

Date: 2006

DOI: 10.1080/14647270600636509

Glucose deprivation, oxidative stress and peroxisome proliferator-activated receptor-α (PPARA) cause peroxisome proliferation in preimplantation mouse embryos

Publisher: Bioscientifica

Date: 09-2009

DOI: 10.1530/REP-09-0038

Abstract: Ex vivo two-cell mouse embryos deprived of glucose in vitro can develop to blastocysts by increasing their pyruvate consumption however, zygotes when glucose-deprived cannot adapt this metabolic profile and degenerate as morulae. Prior to their death, these glucose-deprived morulae exhibit upregulation of the H + -monocarboxylate co-transporter SLC16A7 and catalase, which partly co-localize in peroxisomes. SLC16A7 has been linked to redox shuttling for peroxisomal β-oxidation. Peroxisomal function is unclear during preimplantation development, but as a peroxisomal transporter in embryos, SLC16A7 may be involved and influenced by peroxisome proliferators such as peroxisome proliferator-activated receptor-α (PPARA). PCR confirmed Ppara mRNA expression in mouse embryos. Zygotes were cultured with or without glucose and with the PPARA-selective agonist WY14643 and the developing embryos assessed for expression of PPARA and phospho-PPARA in relation to the upregulation of SLC16A7 and catalase driven by glucose deprivation, indicative of peroxisomal proliferation. Reactive oxygen species (ROS) production and relationship to PPARA expression were also analysed. In glucose-deprived zygotes, ROS was elevated within 2 h, as were PPARA expression within 8 h and catalase and SLC16A7 after 12–24 h compared with glucose-supplied embryos. Inhibition of ROS production prevented this induction of PPARA and SLC16A7. Selective PPARA agonism with WY14643 also induced SLC16A7 and catalase expression in the presence of glucose. These data suggest that glucose-deprived cleavage stage embryos, although supplied with sufficient monocarboxylate-derived energy, undergo oxidative stress and exhibit elevated ROS, which in turn upregulates PPARA, catalase and SLC16A7 in a classical peroxisomal proliferation response.

Maternal supply of omega-3 polyunsaturated fatty acids alter mechanisms involved in oocyte and early embryo development in the mouse

Publisher: American Physiological Society

Date: 02-2008

DOI: 10.1152/AJPENDO.00409.2007

Abstract: Despite the well-known benefits of omega-3 ( n-3) polyunsaturated fatty acid (PUFA) supplementation on human health, relatively little is known about the effect of n-3 PUFA intake on fertility. More specifically, the aim of this study was to determine how oocyte and preimplantation embryo development might be influenced by n-3 PUFA supply and to understand the possible mechanisms underlying these effects. Adult female mice were fed a control diet or a diet relatively high in the long-chain n-3 PUFAs for 4 wk, and ovulated oocytes or zygotes were collected after gonadotropin stimulation. Oocytes were examined for mitochondrial parameters (active mitochondrial distribution, mitochondrial calcium and membrane potential) and oxidative stress, and embryo developmental ability was assessed at the blastocyst stage following 1) in vitro fertilization (IVF) or 2) culture of in vivo-derived zygotes. This study demonstrated that exposure of the oocyte during maturation in the ovary to an environment high in n-3 PUFA resulted in altered mitochondrial distribution and calcium levels and increased production of reactive oxygen species. Despite normal fertilization and development in vitro following IVF, the exposure of oocytes to an environment high in n-3 PUFA during in vivo fertilization adversely affected the morphological appearance of the embryo and decreased developmental ability to the blastocyst stage. This study suggests that high maternal dietary n-3 PUFA exposure periconception reduces normal embryo development in the mouse and is associated with perturbed mitochondrial metabolism, raising questions regarding supplementation with n-3 PUFAs during this period of time.

Biosensors for detecting stress in developing embryos

Publisher: SPIE

Date: 09-12-2016

DOI: 10.1117/12.2244645

Oocyte-secreted factors directly affect oocyte developmental competence during in vitro maturation of the bovine cumulus-oocyte complex

Publisher: CSIRO Publishing

Date: 2006

DOI: 10.1071/RDV18N2AB327

Abstract: Paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) regulate a broad range of cumulus cell functions including proliferation, differentiation, and apoptosis. The capacity of oocytes to regulate their own microenvironment by OSFs may in turn contribute to oocyte developmental competence. The aim of this study was to determine if OSFs have a direct influence on bovine oocyte developmental competence during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were obtained by aspiration of -mm follicles from abattoir-derived ovaries. IVM was conducted in Bovine VitroMat (Cook Australia, Eight Mile Plains, Brisbane, Australia) supplemented with 0.1 IU/mL rhFSH for 24 h under 6% CO2 in air at 38.5�C. In the first experiment, COCs were co-cultured with denuded oocytes (DOs, 5/COC in 10 �L) beginning at either 0 or 9-h of IVM. To generate the 9-h DO group, COCs were first cultured intact for 9-h and then denuded. In the second experiment, specific OSFs, recombinant bone morphogenetic protein-15 (BMP-15) and growth differentiation factor 9 (GDF-9), were prepared as partially purified supernatants of transfected 293H cells, and used as 10% v/v supplements in Bovine VitroMat. Treatments were: (1) control (no supplement), (2) BMP-15, (3) GDF-9, (4) BMP-15 and GDF-9, and (5) untransfected 293H control. Following maturation, in vitro production of embryos was performed using the Bovine Vitro system (Cook Australia) and blastocysts were examined on Day 8 for development. Developmental data were arcsine-transformed and analyzed by ANOVA, followed by Tukey's test. Cell numbers were analyzed by ANOVA. Co-culturing intact COCs with DOs from 0 or 9 h did not affect cleavage rate, but increased (P 0.001) the proportion of cleaved embryos that reached the blastocyst stage post-insemination (50.6 � 1.9 and 61.3 � 1.9%, respectively), compared to COCs cultured alone (40.7 � 1.4%). Therefore, paracrine factors secreted by DOs increased the developmental competence of oocytes matured as COCs. OSFs also improved embryo quality, as co-culture of COCs with DOs (0 or 9 h) significantly increased total cell (156.1 � 1.3 and 159.1 � 1.3, respectively) and trophectoderm (105.7 � 1.3 and 109.8 � 0.4, respectively) numbers, compared to control COCs (total = 148 � 1.2, trophectoderm = 98.2 � 0.8, P 0.001). BMP-15 alone or with GDF-9 also significantly (P 0.001) increased the proportion of oocytes that reached the blastocyst stage post insemination (57.5 � 2.4% and 55.1 � 4.5%, respectively), compared to control (41.0 � 0.9%) and 293H-treated (27.1 � 3.1%) COCs. GDF-9 also increased blastocyst yield (49.5 � 3.9%) but not significantly. These results are the first to demonstrate that OSFs, and particularly BMP-15 and GDF-9, directly affect bovine oocyte developmental competence. These results have far-reaching implications for improving the efficiency of IVM in domestic species and human infertility treatment, and support the role of OSF production by oocytes as a diagnostic marker for developmental competence.

The application of progesterone-containing CIRD™ devices to superovulated ewes

Publisher: Elsevier BV

Date: 06-1990

DOI: 10.1016/0093-691X(90)90048-X

Effect of in-vivo and in-vitro environments on the metabolism of the cumulus-oocyte complex and its influence on oocyte developmental capacity

Publisher: Oxford University Press (OUP)

Date: 2003

DOI: 10.1093/HUMUPD/DMG009

Abstract: There has been an improvement in the blastocyst rates achieved following in-vitro embryo production that can largely be attributed to improved embryo culture conditions based on an increased knowledge of the in-vivo environment, as well as the metabolic needs of the embryo. Despite this, in-vitro oocyte maturation (IVM) conditions have remained largely unchanged. Within the antral follicle, numerous events affect oocyte maturation and the acquisition of developmental competency, including: interactions between somatic cells of the follicle (in particular cumulus cells) and the oocyte the composition of follicular fluid and the temperature and vascularity of the follicular environment. Many of these factors change with follicle size and oocyte growth. In contrast, culture conditions for IVM are based on somatic cells that often do not reflect the follicular environment, and/or have complex compositions or additives such as macromolecule supplements that are undefined in nature. Metabolites included in media such as glucose, pyruvate, oxygen and amino acids have been shown to have differential influences on oocyte maturation and competency. Manipulation of these factors and application of gained knowledge of the in-vivo environment may result in improved in-vitro oocyte maturation and overall in-vitro embryo production.

Hemoglobin: potential roles in the oocyte and early embryo†

Publisher: Oxford University Press (OUP)

Date: 06-05-2019

DOI: 10.1093/BIOLRE/IOZ078

Abstract: Hemoglobin (Hb) is commonly known for its capacity to bind and transport oxygen and carbon dioxide in erythroid cells. However, it plays additional roles in cellular function and health due to its capacity to bind other gases including nitric oxide. Further, Hb acts as a potent antioxidant, quenching reactive oxygen species. Despite its potential roles in cellular function, the preponderance of Hb research remains focused on its role in oxygen regulation. There is increasing evidence that Hb expression is more ubiquitous than previously thought, with Hb and its variants found in a myriad of cell types ranging from macrophages to spermatozoa. The majority of nonerythroid cell types that express Hb are situated within hypoxic environments, suggesting Hb may play a role in hypoxia-inducible factor-regulated gene expression by controlling the level of oxygen available or as an adaptation to low oxygen providing a mechanism to store oxygen. Oocyte maturation and preimplantation embryo development occur within the low oxygen environments of the antral follicle and oviduct/uterus, respectively. Interestingly, Hb was recently found in human cumulus and granulosa cells and murine cumulus–oocyte complexes and preimplantation embryos. Here, we consolidate and analyze the research generated todate on Hb expression in nonerythroid cells with a particular focus on reproductive cell types. We outline future directions of this research to elucidate the role of Hb during oocyte maturation and preimplantation embryo development and finally, we explore the potential clinical applications and benefits of Hb supplementation during the in vitro culture of gametes and embryos.

Mode of oocyte maturation affects EGF-like peptide function and oocyte competence.

Publisher: Oxford University Press (OUP)

Date: 16-04-2013

DOI: 10.1093/MOLEHR/GAT028

Abstract: The function and impact of epidermal growth factor (EGF)-like peptide signalling during ovulation and in vivo oocyte maturation (IVV) has been recently characterized, however, little is currently known about the effect of oocyte in vitro maturation (IVM) on this pathway. The aim of this study was to examine expression and functional aspects of three EGF-like peptides ( hiregulin, epiregulin and betacellulin) and their common receptor (EGFR) in cumulus cells during mouse oocyte IVM compared with IVV. Cumulus-oocyte complexes (COCs) were collected from prepubertal mice either 46 h post-eCG (IVM) or 46 h post-eCG plus 0.5-12 h post-hCG (IVV). Time course experiments showed mRNA expression of all three EGF-like peptides and hiregulin protein in IVM media were significantly lower for the majority of FSH-supplemented IVM compared with IVV. The supplementation of EGF during IVM yielded EGF-like peptide expression levels comparable with IVV and hiregulin/epiregulin supplemented IVM. However, despite this, EGF activation of the COC EGFR remained significantly lower at 3 and 6 h of IVM than in vivo, and levels were similar to those observed during FSH-supplemented IVM. The addition of exogenous epiregulin during IVM significantly increased blastocyst rates, and epiregulin and hiregulin improved blastocyst quality, compared with FSH or EGF. In conclusion, findings from this study suggest that the widely used IVM additives, FSH and EGF, are inadequate propagators of the essential EGF-like peptide signalling cascade. In contrast, the use of epiregulin and/or hiregulin during IVM leads to improved oocyte developmental competence and therefore may be preferable IVM additives than FSH or EGF.

Partitioning of glucose carbon in post-compaction ovine embryos

Publisher: Elsevier BV

Date: 03-1995

DOI: 10.1016/0378-4320(94)01350-U

Requirement for glucose during in vitro culture of sheep preimplantation embryos

Publisher: Wiley

Date: 04-1992

DOI: 10.1002/MRD.1080310405

Abstract: Glucose utilization by sheep embryos was examined in 8-cell (N = 36) and blastocyst (N = 36) stages, by measuring conversion of [5-3H]glucose to 3H2O. Fifty percent glucose utilization occurred at 0.79 +/- 0.69 mM for 8-cell embryos and -0.06 +/- 0.15 mM for blastocysts. Development of 1- and 2-cell sheep embryos (N = 264) was examined under different glucose concentrations (0, 1.5, 3, or 6 mM) and in the presence or absence of 0.33 mM pyruvate and 3.3 mM lactate (PL). Overall, the presence of glucose was detrimental (P less than 0.001) to embryonic development. By contrast, the presence of pyruvate and lactate was beneficial (P less than 0.001) to development. An interaction was observed between the concentration of glucose and presence or absence of PL (P less than 0.05). An optimum level of glucose occurs at 0-3 mM in the presence of PL (P less than 0.1). Development to the blastocyst stage was observed in medium when supplemented with amino acids and albumin alone. Thus, glucose metabolism is not critical for embryonic development, but beneficial at low concentrations. High concentrations can inhibit development, possibly by inhibiting the tricarboxylic acid (TCA) cycle. Sheep embryos may also be using amino acids as an energy source for development.

Nano-liter perfusion microfluidic device made entirely by two-photon polymerization for dynamic cell culture with easy cell recovery

Publisher: Springer Science and Business Media LLC

Date: 11-01-2023

DOI: 10.1038/S41598-023-27660-X

Abstract: Polydimethylsiloxane (PDMS) has been the material of choice for microfluidic applications in cell biology for many years, with recent advances encompassing nano-scaffolds and surface modifications to enhance cell-surface interactions at nano-scale. However, PDMS has not previously been amenable to applications which require complex geometries in three dimensions for cell culture device fabrication in the absence of additional components. Further, PDMS microfluidic devices have limited capacity for cell retrieval following culture without severely compromising cell health. This study presents a designed and entirely 3D-printed microfluidic chip (8.8 mm × 8.2 mm × 3.6 mm) using two-photon polymerization (2PP). The ‘nest’ chip is composed of ten channels that deliver sub-microliter volume flowrates (to ~ 600 nL/min per channel) to 10 in idual retrievable cell s le ‘cradles’ that interlock with the nest to create the microfluidic device. Computational fluid dynamics modelling predicted medium flow in the device, which was accurately validated by real-time microbead tracking. Functional capability of the device was assessed, and demonstrated the capability to deliver culture medium, dyes, and biological molecules to support cell growth, staining and cell phenotype changes, respectively. Therefore, 2PP 3D-printing provides the precision needed for nanoliter fluidic devices constructed from multiple interlocking parts for cell culture application.

Fibroblast growth factor 17 and bone morphogenetic protein 15 enhance cumulus expansion and improve quality of in vitro-produced embryos in cattle

Publisher: Elsevier BV

Date: 08-2015

DOI: 10.1016/J.THERIOGENOLOGY.2015.03.031

Abstract: Bone morphogenetic protein 15 (BMP15) and members of the fibroblast growth factor (FGF) family are expressed by the oocyte and are involved in the control of cumulus cell function. We tested the hypothesis that FGF17, alone or combined with BMP15 in the maturation medium, enhances cumulus expansion, meiosis progression, embryonic development, and expression of mRNA encoding key genes regulating expansion (prostaglandin-endoperoxide synthase 2 [PTGS2], hyaluronan synthase 2 [HAS2], tumor necrosis factor-stimulated gene 6 [TNFAIP6], and pentraxin 3 [PTX3]) and markers of oocyte developmental competence (phosphofructokinase [PFKP], gremlin [GREM1], versican [VCAN], and the genomic progesterone receptor [nPR]) in cumulus cells. Fibroblast growth factor 17 and BMP15 increased the percentage of fully expanded cumulus-oocyte complexes (COCs), but there was no additive effect when both were combined. Neither FGF17 nor BMP15 altered the percentage of oocytes reaching meiosis II at the end of COC culture or cleavage and blastocyst rates after IVF. However, embryo quality, as assessed by the number of cells in the inner cell mass, was improved by the combination of FGF17 with BMP15. Fibroblast growth factor 17 alone did not alter gene expression in cumulus cells at the end of IVM, whereas BMP15 increased PTGS2 and PTX3 mRNA levels. The combination of FGF17 and BMP15 increased nPR mRNA abundance in cumulus cells but did not change the expression of other markers of developmental competence. This study provides novel evidence that FGF17 enhances cumulus expansion in bovine COCs submitted to IVM and that the supplementation of the IVM medium with FGF17 and BMP15 may improve embryo quality.

Reevaluation and evolution of the simulated physiological oocyte maturation system

Publisher: Elsevier BV

Date: 09-2015

DOI: 10.1016/J.THERIOGENOLOGY.2015.03.032

Mathematical modelling of oxygen concentration in bovine and murine cumulus-oocyte complexes.

Publisher: Bioscientifica

Date: 06-2006

DOI: 10.1530/REP.1.00974

Abstract: Immature oocytes benefit from nutrient modification of the follicular environment by the surrounding cumulus mass. However, the oxygen concentration that the oocyte may be exposed to could be lower than the antral follicular concentration due to the metabolism of surrounding cumulus cells. Using metabolic data previously determined, we have developed a mathematical model of O 2 diffusion across the bovine and murine cumulus–oocyte complex. From this we have determined that across a physiological range of external pO 2 , less than 0.25% and 0.5% O 2 is removed by cumulus cells within the bovine and murine cumulus–oocyte complex respectively. Our model differs from others as it: incorporates a term that allows for nonlinear variation of the oxygen consumption rate with oxygen concentration considers two regions (oocyte and cumulus) sharing a common boundary, both of which consume oxygen at different non linear rates. Cumulus cells therefore remove little O 2 , thus sparing this essential gas for the oocyte, which is dependent on ATP generation via oxidative phosphorylation.

Some effects of chondroitin sulphate on the in-vitro acrosome reaction in ram spermatozoa

Publisher: Elsevier BV

Date: 10-1986

DOI: 10.1016/0378-4320(86)90055-2

Addition of superoxide dismutase and catalase does not necessarily overcome developmental retardation of one-cell mouse embryos during in-vitro culture

Publisher: CSIRO Publishing

Date: 1992

DOI: 10.1071/RD9920167

Abstract: There is evidence that developmental blocks observed in mouse embryos during culture in vitro may be the result of free radical-induced cellular dysfunction. We have further investigated this possibility by examining the effects of 5% O2 and the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), on mouse 1-cell embryo development. The results demonstrate that development of CF-1 strain embryos is enhanced by incubating in 5% CO2, 5% O2, 90% N2 (5/5/90) compared with 5% CO2 in air (5/air) (P 0.01) and by culturing in the presence of other embryos. Superoxide dismutase significantly improved embryonic development (35 +/- 5% v. 17 +/- 4% morulae/blastocysts, P 0.001) at a concentration of 100 units mL-1 when embryos were gassed under 5/5/90. At a concentration of 1000 units mL-1, SOD was detrimental to development (P 0.001). Injection of SOD and/or CAT into embryos had not effect on development. Development has also been examined in four strains of mice (CF-1, Quackenbush (random-bred strains), Balb-C (inbred) and the F1 hybrid of CBA x C57B1) in the presence or absence of 100 units SOD mL-1 and 1000 units CAT mL-1. Embryonic development was markedly different among the four strains examined, with F1 hybrid Balb-C (P 0.001), Balb-C CF-1 (P 0.05) and Quackenbush embryos performing very poorly compared with the embryos of the other three strains (P 0.001).

Regulation of gene expression in bovine blastocysts in response to oxygen and the iron chelator desferoxamine

Publisher: Oxford University Press (OUP)

Date: 07-2007

DOI: 10.1095/BIOLREPROD.106.058826

Abstract: Low (2%) oxygen conditions during postcompaction culture of bovine blastocysts improve embryo quality and are associated with small increases in the expression of glucose transporter 1 (SLC2A1), anaphase promoting complex (ANAPC1), and myotrophin (MTPN), suggesting a role for oxygen in the regulation of embryo development, mediated through oxygen-sensitive gene expression. However, bovine embryos, to at least the blastocyst stage, lack detectable levels of the key regulator of oxygen-sensitive gene expression, hypoxia-inducible 1 alpha (HIF1A), while the less well-characterized HIF2 alpha protein is readily detectable. Here we report that other key HIF1 regulated genes are not significantly altered in their expression pattern in bovine blastocysts in response to reduced oxygen concentrations postcompaction-with the exception of lactate dehydrogenase A (LDHA), which was significantly increased following 2% oxygen culture. Antioxidant enzymes have been suggested as potential HIF2 target genes, but their expression was not altered following low-oxygen culture in the bovine blastocyst. The addition of desferrioxamine (an iron chelator and inducer of HIF-regulated gene expression) during postcompaction stages significantly increased SLC2A1, LDHA, inducible nitric oxide synthase (NOS2A), and MTPN gene expression in bovine blastocysts, although development to the blastocyst stage was not significantly affected. These results further suggest that expression of genes, known to be regulated by oxygen via HIF-1 in somatic cells, is not influenced by oxygen during preimplantation postcompaction bovine embryo development. Oxygen-regulated expression of LDHA and SLC2A1 in bovine blastocysts suggests that regulation of these genes may be mediated by HIF2. Furthermore, the effect of a reduced-oxygen environment on gene expression can be mimicked in vitro through the use of desferrioxamine. These results further support our data that the bovine blastocyst stage embryo is unique in its responsiveness to oxygen compared with somatic cells, in that the lack of HIF1-mediated gene expression reduces the overall response to low (physiological) oxygen environments, which appear to favor development.

Biphasic in vitro maturation with C-type natriuretic peptide enhances the developmental competence of juvenile-goat oocytes

Publisher: Public Library of Science (PLoS)

Date: 23-08-2019

DOI: 10.1371/JOURNAL.PONE.0221663

Utilization of endogenous fatty acid stores for energy production in bovine preimplantation embryos

Publisher: Elsevier BV

Date: 05-2012

DOI: 10.1016/J.THERIOGENOLOGY.2011.12.008

Abstract: Although current embryo culture media are based on carbohydrate metabolism of embryos, little is known about metabolism of endogenous lipids. L-carnitine is a β-oxidation cofactor absent in most culture media. The objective was to investigate the influence of L-carnitine supplementation on bovine embryo development. Abattoir-derived bovine cumulus oocyte complexes were cultured and fertilized. Post-fertilization, presumptive zygotes were transferred into a basic cleavage medium ± carbohydrates (glucose, lactate and pyruvate) ± 5 mm L-carnitine and cultured for 4 days in vitro. In the absence of carbohydrates during culture, embryos arrested at the 2- and 4-cell stages. Remarkably, +L-carnitine increased development to the morula stage compared to +carbohydrates alone (P < 0.001). The beneficial effects of L-carnitine were further demonstrated by inclusion of carbohydrates, with 14-fold more embryos reaching the morula stage after culture in the +carbohydrates +L-carnitine group compared to the +carbohydrates group (P < 0.05). Whereas there was a trend for +L-carnitine to increase ATP (P = 0.09), ADP levels were higher and ATP: ADP ratio were 1.9-fold lower (main effect, P < 0.05) compared to embryos cultured in -L-carnitine. Therefore, we inferred that +L-carnitine embryos were more metabolically active, with higher rates of ATP-ADP conversion. In conclusion, L-carnitine supplementation supported precompaction embryo development and there was an additive effect of +L-carnitine +carbohydrates on early embryo development, most likely through increased β-oxidation within embryos.

Urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) expression and activity during early embryo development in the cow.

Publisher: Springer Science and Business Media LLC

Date: 12-2001

DOI: 10.1007/S429-001-8004-7

Abstract: Several extracellular matrix (ECM)-degrading proteinases are hypothesised to play important roles during early mammalian development. In particular, urokinase-type plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP-9) are expressed in peri-implantation mouse, sheep, and pig embryos and are implicated in the implantation process. These proteinases are not expressed in early (pre-blastocyst) mouse, sheep or pig embryos. The aim of this study was to establish the gene expression and proteolytic activity of uPA and MMP-9 in in vitro-produced (IVP) cow embryos. Using RT-PCR, mRNA transcripts for uPA and MMP-9 were detected during the first 7 days of development. To investigate the activity of these proteinases, conditioned media from various stages of development (days 2, 3, 4, 5 and 7) were assayed for uPA activity by chromogenic assay and MMP-9 activity by gelatin zymography. Both uPA and MMP-9 activities were detected in the media s les indicating the production and secretion of these proteinases. This pattern of proteinase expression is novel in comparison to the mouse where uPA and MMP-9 are only expressed from the blastocyst stage onwards. The results of this study suggest that these ECM proteinases have a role prior to implantation in the cow, in contrast to that exhibited by mouse, sheep and pig embryos.

Pre‐maturation with cAMP modulators in conjunction with EGF‐like peptides during in vitro maturation enhances mouse oocyte developmental competence

Publisher: Wiley

Date: 22-02-2014

DOI: 10.1002/MRD.22307

Abstract: Recent studies have independently shown that cyclic adenosine 3'5'-monophosphate (cAMP) modulation prior to in vitro maturation (IVM) and epidermal growth factor (EGF)-like peptide supplementation during IVM improve subsequent oocyte developmental outcomes. This study investigated the effects of an IVM system that incorporates these two concepts. Cumulus-oocyte complexes (COCs) were collected from pre-pubertal mice either 46 hr post-equine chorionic gonadotropin (eCG) (IVM) or post-eCG + post-human chorionic gonadotropin (hCG) stimulation (in vivo maturation IVV). IVM COCs were treated with the cAMP modulators forskolin and IBMX for 1, 2, or 4 hr (pre-IVM phase) prior to IVM. COCs then underwent IVM with the EGF-like peptides hiregulin or epiregulin, or with the common IVM stimulants follicle-stimulating hormone (FSH) or EGF. A pre-IVM phase increased the size of the subsequent blastocysts' inner-cell-mass compared to standard IVM, regardless of IVM treatment (P < 0.05). Unlike FSH or EGF, hiregulin or epiregulin significantly increased blastocyst quality (trophectoderm and total cell numbers) and/or yield (P < 0.01) compared to standard IVM, and were the only treatments that produced blastocysts comparable to IVV-derived blastocysts. Forskolin acutely up-regulated EGF-like peptide mRNA expression after a 2-hr pre-IVM phase (P < 0.001), although EGF receptor and ERK1/2 activities were not significantly different than control. IVV-like levels of EGF-like peptide mRNA expression during IVM were maintained only by supplementing with EGF-like peptides and EGF, since expression levels induced by FSH were significantly lower in vitro than during IVV. However, EGF receptor and ERK1/2 phosphorylation levels were not significantly different across treatment groups. In conclusion, a pre-IVM phase in conjunction with IVM in the presence of EGF-like peptides endows high oocyte developmental competence, as evidenced by increased embryo yield and/or quality relative to FSH and EGF.

Haemoglobin expression in in vivo murine preimplantation embryos suggests a role in oxygen-regulated gene expression

Publisher: CSIRO Publishing

Date: 2019

DOI: 10.1071/RD17321

Abstract: Haemoglobin expression is not restricted to erythroid cells. We investigated the gene expression of the haemoglobin subunits haemoglobin, alpha adult chain 1 (Hba-a1) and haemoglobin, beta (Hbb), 2,3-bisphosphoglycerate mutase (Bpgm) and the oxygen-regulated genes BCL2/adenovirus E1B interacting protein 3 (Bnip3), solute carrier family 2 (facilitated glucose transporter), member 1 (Slc2a1) and N-myc downstream regulated gene 1 (Ndrg1) in the murine preimplantation embryo, comparing invivo to invitro gene expression. Relatively high levels of Hba-a1 and Hbb were expressed invivo from the 2-cell to blastocyst stage in contrast, little or no expression occurred invitro. We hypothesised that the presence of haemoglobin invivo creates a low oxygen environment to induce oxygen-regulated gene expression, supported by high expression of Slc2a1 and Ndrg1 in invivo relative to invitro embryos. In addition, analysis of an invitro-derived human embryo gene expression public dataset revealed low expression of haemoglobin subunit alpha (HBA) and HBB, and high expression of BPGM. To explore whether there was a developmental stage-specific effect of haemoglobin, we added exogenous haemoglobin either up to the 4-cell stage or throughout development to the blastocyst stage, but observed no difference in blastocyst rate or the inner cell mass to trophectoderm cell ratio. We conclude that haemoglobin in the invivo preimplantation embryo raises an interesting premise of potential mechanisms for oxygen regulation, which may influence oxygen-regulated gene expression.

Effect of oxygen concentration on in-vitro development of preimplantation sheep and cattle embryos

Publisher: Bioscientifica

Date: 07-1990

DOI: 10.1530/JRF.0.0890573

Abstract: Two-cell sheep embryos and 2-4-cell and 8-cell cow embryos were cultured for 5 days in stoppered test-tubes in Synthetic Oviduct Fluid supplemented with 32 mg BSA/ml. The medium had been previously equilibrated with one of the following O2 concentrations (sheep: 0, 2, 4, 6, 8, 10, 12, 17, 20% cow: 0, 4, 8, 12, 17, 20%). At the end of culture embryos were examined for morphology and stained to assess numbers of nuclei. Mean (+/- s.e.m.) nuclei/embryo was highest at 8% O2 for sheep embryos (23.6 +/- 3.1), 4% for 2-4-cell cow embryos (23.2 +/- 6.1) and 8% for 8-cell cow embryos (29.6 +/- 5.2). The minimum number of nuclei/embryo occurred at 20% O2 in each case (10.3 +/- 0.9, 10.3 +/- 2.7, 14.5 +/- 2.4, respectively) with similar values also recorded at 0% O2 (10.8 +/- 1.9, 16.5 +/- 6.0, 14.6 +/- 2.4, respectively). Analysis of the proportion of embryos reaching at least the morula stage demonstrated a significant quadratic component for the different oxygen concentrations for sheep (P less than 0.01) and cow (P less than 0.05) embryos. A number of sheep and cow embryos showed abnormalities, suggesting that the culture conditions require further refinement. The results confirm that, under lowered oxygen levels, development of sheep and cattle embryos can occur through the 8- to 16-cell block in a simple defined medium without somatic cell support.

Effect of hexoses and gonadotrophin supplementation on bovine oocyte nuclear maturation during in vitro maturation in a synthetic follicle fluid medium

Publisher: CSIRO Publishing

Date: 2005

DOI: 10.1071/RD04135

Abstract: In vitro oocyte maturation (IVM) culture conditions have been relatively unchanged over the past few decades and remain suboptimal. In contrast, studies of the in vivo environment have led to significant improvements to in vitro embryo culture technologies. The aim of the present study was to determine the effect of maturing bovine cumulus–oocyte complexes (COCs) in medium based on the composition of bovine follicular fluid (Bovine VitroMat Cook Australia, Eight Mile Plain, Qld, Australia). In particular, the effect of different glucose concentrations and glucosamine supplementation on meiotic maturation was determined. Culturing COCs in the presence of gonadotrophins in Bovine VitroMat, containing either physiological glucose concentrations (2.3 mm) or 5.6 mm (equivalent to levels in Tissue Culture Medium 199 (TCM199)) supplemented with glucosamine resulted in comparable cumulus expansion to COCs cultured in TCM199 plus gonadotrophins. However, nuclear maturation was 1.3-fold lower in Bovine VitroMat cultures containing 2.3 mm glucose compared with 5.6 mm glucose and this effect was independent of glucosamine supplementation. Investigations into the effects of different glucose concentrations and gonadotrophin supplementation during the initial 6 h of maturation demonstrated that COCs cultured in Bovine VitroMat with 5.6 mm glucose without gonadotrophins had a twofold acceleration of the rate of meiotic resumption, yet the rate of polar body formation was decreased by approximately 20% compared with cultures in 2.3 mm glucose and TCM199. However, this effect was not seen when COCs were cultured for the initial 16 h in Bovine VitroMat + 5.6 mm minus gonadotrophins or in Bovine VitroMat + 2.3 mm glucose ± gonadotrophins. These data demonstrate that glucose concentrations and the timing of the introduction of gonadotrophin during IVM have variable effects on nuclear maturation. Manipulation of glucose concentrations may be a mechanism to influence oocyte meiotic progression and may lead to the development of improved IVM systems, allowing for an increased developmental capacity of bovine oocytes.

Improvement in sperm DNA quality using an oral antioxidant therapy

Publisher: Elsevier BV

Date: 2009

DOI: 10.1016/S1472-6483(10)60024-7

Abstract: Oxidative stress is now recognized as a common pathology that affects up to half of all infertile men. One of the principal mechanisms by which oxidative stress produces infertility is by damage to sperm DNA, either through direct oxidation of the DNA by reactive oxygen species (ROS) or by the initiation of apoptosis. The objective of this study was to determine if an oral antioxidant/mineral supplement could improve sperm DNA integrity in men with known oxidative stress. A total of 50 infertile men identified as exhibiting oxidative stress were administered oral antioxidant therapy for a period of 3 months. All participants were assessed at entry and exit for sperm DNA integrity with terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling, apoptosis with annexin V, protamination with chromomycin A(3) and ROS production with nitro blue tetrazolium assay. Sperm concentration, motility and morphology, together with assessment of serum male reproductive hormones (LH, FSH, testosterone, anti-Mullerian hormone), were also monitored. The principal finding that emerged from this study was that antioxidant therapy resulted in significant improvements in sperm DNA integrity (P = 0.002) and protamine packaging (P < 0.001), accompanied by a reduction in seminal ROS production (P = 0.027) and apoptosis (P = 0.004). No significant changes in routine sperm parameters (concentration, motility, morphology) or male reproductive hormones were observed.

Birthweight and the effects of culture media.

Publisher: Oxford University Press (OUP)

Date: 29-12-2017

DOI: 10.1093/HUMREP/DEW337

Glucose utilization by sheep embryos derived in vivo and in vitro

Publisher: CSIRO Publishing

Date: 1991

DOI: 10.1071/RD9910571

Abstract: Embryos were collected from superovulated donors at various intervals from onset of oestrus, ranging from Day 1.5 to Day 6. In addition, blastocysts obtained from the culture of 1-cell embryos collected in vivo or of oocytes matured and fertilized in vitro were used to assess the effects of in vitro manipulation and culture on glucose utilization. Glycolytic activity was determined by the conversion of [5-3H]glucose to 3H2O, and oxidation of glucose was determined by the conversion of [U-14C]glucose to 14CO2. Glucose utilization increases significantly from the 8-cell stage and during compaction and blastulation. Glucose oxidation was at a relatively low level (5-12% of total utilization) compared with glycolysis. No difference was observed between the glycolytic activity of blastocysts derived from in vivo or in vitro sources. However, glucose oxidation was lower (P less than 0.05) in blastocysts derived from the culture of 1-cell embryos or from oocytes matured and fertilized in vitro. Exogenous tricarboxylic acid cycle substrates (i.e. pyruvate and lactate supplied in the medium) affected the level of glucose oxidation.

Altered pregnancy outcomes in mice following treatment with the hyperglycaemia mimetic, glucosamine, during the periconception period

Publisher: CSIRO Publishing

Date: 2013

DOI: 10.1071/RD11313

Abstract: Exposure of cumulus–oocyte complexes to the hyperglycaemia mimetic, glucosamine, during in vitro maturation impairs embryo development, potentially through upregulation of the hexosamine biosynthesis pathway. This study examined the effects of in vivo periconception glucosamine exposure on reproductive outcomes in young healthy mice, and further assessed the effects in overweight mice fed a high-fat diet. Eight-week-old mice received daily glucosamine injections (20 or 400 mg kg–1) for 3–6 days before and 1 day after mating (periconception). Outcomes were assessed at Day 18 of gestation. Glucosamine treatment reduced litter size independent of dose. A high-fat diet (21% fat) for 11 weeks before and during pregnancy reduced fetal size. No additional effects of periconception glucosamine (20 mg kg–1) on pregnancy outcomes were observed in fat-fed mice. In 16-week-old mice fed the control diet, glucosamine treatment reduced fetal weight and increased congenital abnormalities, but did not alter litter size. As differing effects of glucosamine were observed in 8-week-old and 16-week-old mice, maternal age effects were assessed. Periconception glucosamine at 8 weeks reduced litter size, whereas glucosamine at 16 weeks reduced fetal size. Thus, in vivo periconception glucosamine exposure perturbs reproductive outcomes in mice, with the nature of the outcomes dependent upon maternal age.

Effects of recombinant human follicle-stimulating hormone on embryo development in mice

Publisher: American Physiological Society

Date: 05-2005

DOI: 10.1152/AJPENDO.00398.2004

Abstract: We have developed a protocol using recombinant human follicle-stimulating hormone (rhFSH) to induce ovarian stimulation in the mouse to investigate its impact on preimplantation embryo development. Embryos were collected from adult female C57Bl/6 × CBA F 1 mice treated with rhFSH (0, 2.5, 5.0, 10.0, or 20.0 IU) or 5 IU equine chorionic gonadotropin (eCG). Embryos were also recovered from nontreated control mice. Embryos were cultured in vitro for 88 h, and the stage of development was morphologically assessed. The allocation of cells to the inner cell mass or trophectoderm of blastocysts was determined by differential nuclear staining. The expression of insulin-like growth factor 2 (IGF-II), the insulin-like growth factor receptor (IGF-II receptor), and vascular endothelial growth factor (VEGF) in blastocysts was measured by real-time RT-PCR. Blastocyst development was reduced in the 10 (72.3 ± 5.1%) and 20 (77.3 ± 5.6%) IU rhFSH groups compared with control embryos (96.7 ± 1.0%). The number of inner cell mass cells was reduced ( P 0.001) in the 5, 10, and 20 IU rhFSH groups and the eCG group compared with control embryos. We did not find any effect of rhFSH treatment on IGF-II, IGF-II receptor, or VEGF expression in blastocysts compared with the control group. eCG treatment, however, significantly increased the expression of IGF-II in blastocysts. These results indicate that ovarian stimulation with rhFSH impairs the in vitro development of preimplantation mouse embryos, and these results may have potential implications for clinical ovarian stimulation during infertility treatment and subsequent embryo quality.

Promotion of EGF receptor signaling improves the quality of low developmental competence oocytes.

Publisher: Elsevier BV

Date: 07-2015

DOI: 10.1016/J.YDBIO.2015.05.008

Abstract: Oocytes acquire developmental competence with progressive folliculogenesis. Cumulus oocyte complexes (COCs) from small antral follicles have inherent low competence and are poorly responsive to hiregulin (AREG) which normally mediates oocyte maturation and ovulation. Using low competence porcine COCs, in an in vitro AREG-induced oocyte maturation system, the combined exposure to N(6),2'-O-dibutyryladenosine 3':5' cyclic monophosphate (cAMP) and bone morphogenetic protein 15 (B15) and growth differentiation factor 9 (G9) was necessary to enhance the rate of oocyte meiotic maturation and blastocyst formation. Furthermore, the combination of cAMP+B15+G9 enabled AREG-stimulated cumulus expansion and increased expression of the matrix-related genes HAS2, TNFIPA6 and PTGS2. Additionally, the combination enhanced p-ERK1/2 which is downstream of the EGF receptor. The enhanced nuclear maturation and blastocyst formation rates with the combinational treatment were ablated by an EGF receptor phosphorylation inhibitor. These results indicate that cAMP and oocyte-secreted factors cooperate to promote EGF receptor functionality in developing COCs, representing a key component of the acquisition of oocyte developmental competence.

Alterations in mouse embryo intracellular pH by DMO during culture impair implantation and fetal growth

Publisher: Elsevier BV

Date: 08-2010

DOI: 10.1016/J.RBMO.2010.05.001

Abstract: The preimplantation embryo is highly susceptible to in-vitro stress, and although this does not necessarily perturb blastocyst development, it can significantly affect embryo physiology and the ability to form a viable pregnancy. This study determined that the preimplantation mouse embryo is highly sensitive to a small decrease in intracellular pH (<0.2 pH units). Embryos cultured in media containing a weak acid (5,5-dimethyl-2,4-oxazolidinedione DMO) formed blastocysts with decreased cell number and inner cell mass number, as well as increased apoptosis, even though blastocyst development and morphology were unchanged. Interestingly, the effects were similar regardless of whether the pH stress was present for a short-term 'acute' exposure (during the zygote to 2-cell, or 2-cell to 8-cell ision) or an extended 'chronic' period of time (continually from the zygote to the blastocyst stage). Exposure to DMO during the first cleavage ision did not alter implantation however, fetal weight and crown-rump length were significantly decreased (P<0.05). In contrast, continuous exposure to DMO throughout preimplantation development reduced not only implantation but also fetal weight and crown-rump length. This study highlights the importance of correct intracellular pH and demonstrates that slight deviations can significantly impact embryo development and viability.

Hormonally regulated follicle differentiation and luteinization in the mouse is associated with hypoxia inducible factor activity

Publisher: Elsevier BV

Date: 07-10-2010

DOI: 10.1016/J.MCE.2010.06.008

Abstract: Hypoxia inducible factors (HIFs) are transcription factors that mediate physiological responses to hypoxia. Hypoxia is established as the major inducer of HIFs, but stimuli such as transition metals and hormones also induce HIF target genes. Whilst the ovarian granulosa cell layer is known to be avascular and the follicle is vascularised via the thecal cell layer, little is known about the role of hypoxia or HIFs in regulating ovarian function. In this study, we hypothesized that hypoxia as well as non-hypoxic stimuli cooperate in promoting follicle differentiation and luteinization via HIF activity and resultant gene regulation. We quantitatively measured the HIF1alpha protein response to hCG in ovarian granulosa cell cultures and in vivo and developed a transgenic (HRE(4)-SV40-EGFP) HIF reporter mouse line. We observed a time-dependent increase of HIF1alpha protein levels in granulosa cells post-hCG in vivo, maximal around time of ovulation. hCG alone was unable to promote HIF1alpha protein accumulation in cultured granulosa cells, but increased protein abundance was observed when combined with a hypoxic stimulus. HRE-EGFP ovaries showed no follicular EGFP in stages prior to antrum formation. However, HIF regulated EGFP was maximally induced in granulosa cells around the time of ovulation and readily observed in corpora lutea. There was also an increase in HIF regulated EGFP activity in the corpora lutea from functional to regressing stages. Taken together, these observations establish the notion that HIFs play a role during follicular differentiation and luteinization.

Oxygen consumption and energy metabolism of the early mouse embryo

Publisher: Wiley

Date: 08-1996

DOI: 10.1002/(SICI)1098-2795(199608)44:4<476::AID-MRD7>3.0.CO;2-I

Abstract: The effect of early enteral nutrition (EN) in patients with acute pancreatitis (AP) has been confirmed. In recent years, some researchers provided new strategy that immediate EN was offered after admission. The effect and safety of immediate EN was unclear because of the different results among studies. The study aimed to implement the meta analysis of randomized controlled trials (RCT) to confirm the effect and safety between the immediate EN group and the early refeeding group. Four electronic databases including PubMed, EMBASE, the Cochrane Library and China National Knowledge Internet (CNKI) were searched from inception to July 2021. Endnote X7.0 software was used to manage all the relevant citations. Then data extraction and evaluation of risk of bias for included studies were performed after initial selection and full-text selection. All statistical analyses were performed by Review Manager 5.3 version software. 5 randomized controlled trials (RCT) involving 372 patients were included in the present study. The meta analysis revealed that immediate EN after admission in patients with AP could significantly decrease the length of hospital stay (LOHS) (Mean difference [MD] = 2.57, 95% confidence interval [CI] = 0.41-4.72) and the intolerance of feeding (risk ratio [RR] = 0.78, 95%CI = 0.63-0.95), compared with early refeeding. But immediate EN couldn't significantly decrease the incidence of readmission after discharging (RR = 0.51, 95%CI = 0.12-2.27), the incidence of progression to severe pancreatitis (RR = 0.76, 95%CI = 0.15-3.76), the incidence of complications (RR = 1.12, 95%CI = 0.50-2.49) and the values of C-reactive protein (CRP) and leukocyte counts (MD = 1.05, 95%CI = 0.15-2.26 and MD = 0.11, 95%CI = 0.59-0.80), compared with early refeeding. Compared with early refeeding, immediate EN after admission could safely reduce LOHS and intolerance of feeding in patients with AP.

Reactive oxygen species production and redox state in parthenogenetic and sperm-mediated bovine oocyte activation

Publisher: Bioscientifica

Date: 05-2013

DOI: 10.1530/REP-13-0017

Abstract: The knowledge concerning redox and reactive oxygen species (ROS)-mediated regulation of early embryo development is scarce and remains controversial. The aim of this work was to determine ROS production and redox state during early in vitro embryo development in sperm-mediated and parthenogenetic activation of bovine oocytes. Sperm-mediated oocyte activation was carried out in IVF-modified synthetic oviductal fluid (mSOF) with frozen–thawed semen. Parthenogenetic activation was performed in TALP plus ionomycin and then in IVF-mSOF with 6-dimethylaminopurine plus cytochalasin B. Embryos were cultured in IVF-mSOF. ROS and redox state were determined at each 2-h interval (7–24 h from activation) by 2′,7′-dichlorodihydrofluorescein diacetate and RedoxSensor Red CC-1 fluorochromes respectively. ROS levels and redox state differed between activated and non-activated oocytes ( P .05 by ANOVA). In sperm-activated oocytes, an increase was observed between 15 and 19 h ( P .05). Conversely, in parthenogenetically activated oocytes, we observed a decrease at 9 h ( P .05). In sperm-activated oocytes, ROS fluctuated throughout the 24 h, presenting peaks around 7, 19, and 24 h ( P .05), while in parthenogenetic activation, peaks were detected at 7, 11, and 17 h ( P .05). In the present work, we found clear distinctive metabolic patterns between normal and parthenogenetic zygotes. Oxidative activity and ROS production are an integral part of bovine zygote behavior, and defining a temporal pattern of change may be linked with developmental competence.

PVA and BSA have different effects on development and metabolism of bovine embryos produced in vitro

Publisher: Elsevier BV

Date: 1998

DOI: 10.1016/S0093-691X(98)90584-2

Development of in vitro embryo production systems for red deer (Cervus elaphus)

Publisher: Elsevier BV

Date: 03-2002

DOI: 10.1016/S0378-4320(01)00200-7

Abstract: The time course of in vitro red deer nuclear oocyte maturation was determined. Ovaries were obtained at slaughter and oocytes were aspirated from follicles greater than 2mm in diameter. Oocytes with compact cumulus cells were matured in 50 microl microdrops (10 per drop) under mineral oil containing TCM 199 supplemented with 0.33 mM pyruvate, 10 microg LH and FSH, 1 microg oestradiol and 10% foetal bovine serum. Oocytes were matured at 39 degrees C and 5% CO(2) in air. At 3h intervals (0-27 h) oocytes were removed from incubation, cumulus expansion scored and removed, and fixed oocytes in ethanol:acetic acid (3:1) for 48 h. Oocytes were stained with lacmoid (1%) and nuclear maturation assessed. Oocytes were arrested in the germinal vesicle (GV) stage at aspiration and up to 6h of incubation. The nuclear membrane began to disperse after 6h and by 10.6+/-0.6h of incubation 75% of the oocytes exhibited germinal vesicle breakdown (GVBD). The mean time for 50% of the oocytes to reach metaphase one (MI) and metaphase two (MII) was 11.7+/-0.4 and 24.8+/-0.9h, respectively. Cumulus oophorus were tightly compacted at aspiration and did not begin expansion until 12h of culture. Full expansion was complete by 18 h of culture. Corona radiata cells did not begin expansion until 15 h and were fully expanded by 24h. Results indicate that in vitro red deer oocyte maturation follows a similar time course of nuclear maturation as reported for bovine and ovine oocytes.

The definition of IVM is clear—variations need defining

Publisher: Oxford University Press (OUP)

Date: 22-09-2016

DOI: 10.1093/HUMREP/DEW208

Abstract: Oocyte in vitro maturation (IVM) is currently defined as the maturation in vitro of immature cumulus-oocyte complexes collected from antral follicles. This is the original definition as first described by Pincus and Enzmann and then by Edwards many decades ago, and this clear and unambiguous definition has served us well ever since. In an attempt to clarify apparent differences among clinicians, the following revised definition of IVM was recently proposed: 'The retrieval of oocytes from small and intermediate sized follicles in an ovary before the largest follicle has surpassed 13 mm in mean diameter'. As such, this proposed definition should encompass the use of hCG triggering. To change the clear and long-serving definition of IVM to fit varying clinical practices requires a compelling justification based on solid scientific and clinical grounds. We are of the opinion that the proposed revised definition of IVM is counterintuitive as it includes protocols that are intended to mature oocytes in vivo The proposed definitions are cumbersome and indeed further complicate the situation. It is not scientifically rational to base the definition on follicular size, and the definition ignores the vast corporate knowledge acquired from the many decades and >6000 publications in animal research that universally practices IVM as per the existing definition. Furthermore, such a definition can lead to false results in interpreting the follow-up of children conceived using IVM. Hence, we see no rationale to change the existing definition of IVM. However, we agree that variations on IVM require alternative nomenclature-these definitions need to be intuitive and need to clearly distinguish themselves from the existing long-standing definition of IVM. This would help to clarify the recent confusion within the clinical ART community as to what is and what is not, IVM.

Oxygen consumption and ROS production are increased at the time of fertilization and cell cleavage in bovine zygotes

Publisher: Oxford University Press (OUP)

Date: 07-09-2010

DOI: 10.1093/HUMREP/DEQ221

Abstract: Oxygen consumption is a key indicator of metabolic activity within embryos. Increased oxidative activity and REDOX changes at the time of fertilization have been suggested to signal Ca(2+) oscillations after sperm penetration. The objective of the present study was to determine the oxygen consumption and the REDOX status of zygotes and early embryos at the time of sperm penetration and cell cleavage and to investigate how metabolism relates to key temporal events and developmental competence. In idual oxygen-consumption rates of bovine in vitro matured oocytes and presumptive zygotes (n = 101) were measured using the Nanorespirometer at 0, 7, 12, 17 and 24 h after IVF. Using the Embryoscope, oxygen-consumption profiles of in idual oocytes and embryos (n = 75) were recorded repeatedly from 6 h until 30 h after IVF and time-lapse images were acquired, at intervals of ∼36 min. Oocytes and embryos were stained with Hoechst 33342 and visualization of nuclear stage was performed by fluorescence microscopy. To determine the REDOX status, cohorts of oocytes and zygotes (n = 55) were in idually stained with REDOX-Sensor Red CC-1 and Hoechst 33342 at 0, 7, 12, 17 and 24 h after IVF and subsequently imaged by confocal microscopy. A peak of oxygen consumption was observed at the time of fertilization and a smaller rise and fall in oxygen consumption could be detected prior to the first cell cleavage. Increased reactive oxygen species production was also observed at 7 h and then at 24 h after IVF, just preceding the first embryonic cleavage. There are specific events during embryo development that appear to be associated with a change in oxygen consumption and REDOX state, indicating that both have a role in sperm-mediated oocyte activation and cell cleavage in bovine embryos.

Effects of hyaluronic acid supplementation on in vitro development of bovine embryos in a two-step culture system

Publisher: Elsevier BV

Date: 1999

DOI: 10.1016/S0093-691X(99)91813-7

Beyond oxygen: complex regulation and activity of hypoxia inducible factors in pregnancy

Publisher: Oxford University Press (OUP)

Date: 19-11-2009

DOI: 10.1093/HUMUPD/DMP046

Rationally Designed Probe for Reversible Sensing of Zinc and Application in Cells

Publisher: American Chemical Society (ACS)

Date: 27-09-2017

DOI: 10.1021/ACSOMEGA.7B00923

A randomised control trial examining the effect of an antioxidant (Menevit) on pregnancy outcome during IVF-ICSI treatment

Publisher: Wiley

Date: 06-2007

DOI: 10.1111/J.1479-828X.2007.00723.X

The effect of streptozotocin-induced hyperglycemia on N-and O-linked protein glycosylation in mouse ovary

Publisher: Oxford University Press (OUP)

Date: 28-08-2018

DOI: 10.1093/GLYCOB/CWY075

Abstract: Post-translational modification of proteins namely glycosylation influences cellular behavior, structural properties and interactions including during ovarian follicle development and atresia. However, little is known about protein glycosylation changes occurring in diabetes mellitus in ovarian tissues despite the well-known influence of diabetes on the outcome of successful embryo implantation. In our study, the use of PGC chromatography-ESI mass spectrometry in negative ion mode enabled the identification of 138 N-glycans and 6 O-glycans on the proteins of Streptozotocin-induced (STZ) diabetic mouse ovarian tissues (n = 3). Diabetic mouse ovaries exhibited a relative decrease in sialylation, fucosylation and, to a lesser extent, branched N-linked glycan structures, as well as an increase in oligomannose structures on their proteins, compared with nondiabetic mouse ovaries. Changes in N-glycans occurred in the diabetic liver tissue but were more evident in diabetic ovarian tissue of the same mouse, suggesting an organ-specific effect of diabetes mellitus on protein glycosylation. Although at a very low amount, O-GalNAc glycans of mice ovaries were present as core type 1 and core type 2 glycans with a relative increase in the NeuGc:NeuAc ratio as the most significant difference between control and diabetic ovarian tissues. STZ-treated mice also showed a trend towards an increase in TNF-α and IL1-B inflammatory cytokines, which have previously been shown to influence protein glycosylation.

Failure to launch: aberrant cumulus gene expression during oocyte in vitro maturation

Publisher: Bioscientifica

Date: 03-2017

DOI: 10.1530/REP-16-0426

Abstract: In vitro maturation (IVM) offers significant benefits for human infertility treatment and animal breeding, but this potential is yet to be fully realised due to reduced oocyte developmental competence in comparison with in vivo matured oocytes. Cumulus cells occupy an essential position in determining oocyte developmental competence. Here we have examined the areas of deficient gene expression, as determined within microarrays primarily from cumulus cells of mouse COCs, but also other species, between in vivo matured and in vitro matured oocytes. By retrospectively analysing the literature, directed by focussing on downregulated genes, we provide an insight as to why the in vitro cumulus cells fail to support full oocyte potential and dissect molecular pathways that have important roles in oocyte competence. We conclude that the roles of epidermal growth factor signalling, the expanded extracellular matrix, cumulus cell metabolism and the immune system are critical deficiencies in cumulus cells of IVM COCs.

A dual-mode mobile phone microscope using the onboard camera flash and ambient light

Publisher: Springer Science and Business Media LLC

Date: 19-02-2018

DOI: 10.1038/S41598-018-21543-2

Abstract: Mobile phone microscopes are a natural platform for point-of-care imaging, but current solutions require an externally powered illumination source, thereby adding bulk and cost. We present a mobile phone microscope that uses the internal flash or sunlight as the illumination source, thereby reducing complexity whilst maintaining functionality and performance. The microscope is capable of both brightfield and darkfield imaging modes, enabling microscopic visualisation of s les ranging from plant to mammalian cells. We describe the microscope design principles, assembly process, and demonstrate its imaging capabilities through the visualisation of unlabelled cell nuclei to observing the motility of cattle sperm and zooplankton.

Regulation of sheep oocyte maturation using cAMP modulators

Publisher: Elsevier BV

Date: 2013

DOI: 10.1016/J.THERIOGENOLOGY.2012.09.020

Abstract: Physical removal of mammalian cumulus-oocyte complexes (COCs) from ovarian follicles results in spontaneous resumption of meiosis, largely because of a decrease in cAMP concentrations, causing asynchrony between cytoplasmic and nuclear maturation and decreased oocyte developmental competence. The aim of this study was to modulate cAMP concentrations within ovine COCs to delay spontaneous nuclear maturation and improve developmental competence. Abattoir-derived sheep COCs were cultured for 2 hours (pre-IVM) in 100 μM forskolin (FSK) plus 500 μM 3-isobutyl-1-methylxanthine (IBMX). Pre-IVM (100 μM FSK and 500 μM IBMX) culture increased COC cAMP concentrations 10-fold compared with controls (P < 0.05). With regard to nuclear maturation, with FSK and IBMX and/or with FSH and cilostamide delayed completion of meiosis (metaphase II) by 3 to 4 hours compared with standard IVM (FSH-stimulated induction of meiosis). In this study, pre-IVM (with FSK and IBMX) followed by IVM (with FSH and cilostamide), increased ovine COC cAMP concentrations and delayed, but did not inhibit, completion of nuclear maturation. This did not affect embryo development rates, but increased total cell number of blastocysts compared with IVM with FSH alone (103 ± 6 vs. 66 ± 4 cells, respectively mean ± SEM P < 0.05). We inferred that regulation of ovine oocyte cAMP concentrations during IVM improved embryo quality compared with embryos produced by standard IVM methods.

Heparin and cAMP modulators interact during pre-in vitro maturation to affect mouse and human oocyte meiosis and developmental competence.

Publisher: Oxford University Press (OUP)

Date: 04-04-2013

DOI: 10.1093/HUMREP/DET086

Abstract: Does heparin ablate the advantageous effects of cyclic adenosine mono-phosphate (cAMP) modulators during pre-in vitro maturation (IVM) and have a deleterious effect in standard oocyte IVM? Heparin interrupts energy metabolism and meiotic progression and adversely affects subsequent development of oocytes under conditions of elevated cAMP levels in cumulus-oocyte complexes (COCs) after pre-IVM treatment with forskolin. In animal IVM studies, artificial regulation of meiotic resumption by cAMP-elevating agents improves subsequent oocyte developmental competence. Heparin has no effect on spontaneous, FSH- or epidermal growth factor (EGF)-stimulated meiotic maturation. An in vitro cross-sectional study was conducted using immature mouse and human COCs. Depending on in idual experimental design, COCs were treated during pre-IVM with or without heparin, in the presence or absence of forskolin and/or 3-isobutyl-1-methylxanthine (IBMX), and then COC function was assessed by various means. Forty-two women with polycystic ovaries (PCOs) or polycystic ovarian syndrome (PCOS) donated COCs after oocyte retrieval in a non-hCG-triggered IVM cycle. COCs were collected in pre-IVM treatments and then cultured for 40 h and meiotic progression was assessed. COCs from 21- to 24-day-old female CBA F1 mice were collected 46 h after stimulation with equine chorionic gonadotrophin. Following treatments, COCs were checked for meiotic progression. Effects on mouse oocyte metabolism were measured by assessing oocyte mitochondrial membrane potential using JC-1 staining and oocyte ATP content. Post-IVM mouse oocyte developmental competence was assessed by in vitro fertilization and embryo production. Blastocyst quality was evaluated by differential staining of inner cell mass (ICM) and trophectoderm (TE) layers. In the absence of heparin in pre-IVM culture, the addition of cAMP modulators did not affect human oocyte MII competence after 40 h. In standard IVM, heparin supplementation in pre-IVM did not affect MII competence however, when heparin was combined with cAMP modulators, MII competence was significantly reduced from 65 to 15% (P < 0.05). In mouse experiments, heparin alone in pre-IVM significantly delayed germinal vesicle breakdown (GVBD) so that fewer GVBDs were observed at 0 and 1 h of IVM (P < 0.05), but not by 2 or 3 h of IVM. Combined treatment with IBMX and forskolin in the pre-IVM medium produced a large delay in GVBD such that no COCs exhibited GVBD in the first 1 h of IVM, and the addition of heparin in pre-IVM further significantly delayed the progression of GVBD (P < 0.05), in a dose-dependent manner (P < 0.01). Combined IBMX and forskolin treatment of mouse COCs during pre-IVM significantly increased mitochondrial membrane potential and ATP production in the oocyte at the end of pre-IVM (P < 0.05), and significantly improved fertilization, embryo development and quality (P < 0.05). However, heparin abolished the IBMX + forskolin-stimulated increase in mitochondrial membrane potential and ATP production (P < 0.05), and adversely affected embryonic cleavage, development rates and embryo quality (P < 0.05). This latter adverse combinational effect was negated when mouse COCs were collected in heparin and IBMX for 15 min, washed and then cultured for 45 min in IBMX and forskolin without heparin. Experiments in mice found that heparin ablation of the advantageous effects of cAMP modulators during pre-IVM was associated with altered oocyte metabolism, but the mechanism by which heparin affects metabolism remains unclear. This study has revealed a novel and unexpected interaction between heparin and cAMP modulators in pre-IVM in immature mouse and human oocytes, and established a means to collect oocytes using heparin while modulating oocyte cAMP to improve developmental potential.

Development of the NBT assay as a marker of sperm oxidative stress.

Publisher: Wiley

Date: 08-01-2010

DOI: 10.1111/J.1365-2605.2008.00941.X

Abstract: Oxidative stress is a well-established cause of male infertility, with reactive oxygen species (ROS) causing infertility principally by impairing sperm motility and DNA integrity. Currently, most clinics do not test their infertile patients for the presence of oxidative stress because the available tests are expensive or difficult to perform. As antioxidant therapy may improve sperm DNA integrity and pregnancy outcomes, it has become apparent that there is an unmet clinical need for an inexpensive and easy-to-perform assay to identify sperm oxidative stress. The aim of this study was to develop a standardized protocol for performing a photometric nitro blue tetrazolium (NBT) assay for the measurement of seminal ROS production via production of coloured formazan, whilst correlating these results with impaired sperm function (motility and DNA integrity). Semen s les from 21 fertile and 36 male aetiology infertile men were assessed for ROS production (NBT assay), sperm DNA integrity (TUNEL), apoptosis (Annexin V) and sperm motility. Infertile men's semen contained on average fourfold higher levels of ROS than fertile men. The production of ROS by sperm was positively correlated with sperm DNA fragmentation and apoptosis, whilst being negatively correlated with sperm motility. Receiver-operating characteristic plot analysis established a cut-off point of 24 microg formazan/10(7) sperm having a sensitivity of 91.7% and a specificity of 81% for determining the fertility status of an in idual. This study has been successful in establishing a standardized protocol for performing a photometric seminal NBT assay that has significant clinical utility in identifying men with impaired fertility because of oxidative stress.

Bovine embryo culture in vitro: New developments and post-transfer consequences

Publisher: Oxford University Press (OUP)

Date: 12-2000

DOI: 10.1093/HUMREP/15.SUPPL_5.59

Abstract: The past decade has seen a significant shift away from co-culture systems for cattle blastocyst production. In particular, recent adoption of sequential media systems has increased performance. However, wholly defined systems, such as the replacement of albumin with nonbiological macromolecules, fail to reproduce the nutritive role that this molecule has during development. Cattle blastocysts developed in protein-free medium are metabolically compromised. A further new concept is the use of metabolic inhibitors to stimulate embryo development in vitro. Non-toxic levels of NaN3, 2,4-dinitrophenol or very low oxygen atmospheres (approximately 2%) significantly increase both the yield (by approximately 10-20%) and the quality of blastocysts when these treatments are applied during the peri-compaction period in vitro. Nevertheless, there are also negative consequences of cattle embryo culture, such as fetal oversize and/or significant post-day 35 fetal loss. We have recently found that much of this loss is due to failure of normal allantoic development within the conceptus. Early fetal development is supported by vascularization within the yolk sac, but from day 35 to day 110, loss occurs through poor nutrient supply and an inability to remove nitrogenous wastes, leading to fetal death around day 35. The cause of disrupted allantois development has not been identified as yet, but may share a common 'cause-effect' mechanism with the fetal oversize syndrome.

Oxygen concentration during mouse oocyte in vitro maturation affects embryo and fetal development.

Publisher: Oxford University Press (OUP)

Date: 25-07-2007

DOI: 10.1093/HUMREP/DEM203

Abstract: Little is known of how the oxygen environment in the ovarian follicle affects oocyte and embryo development, but this has an important impact on the conditions used for in vitro maturation (IVM) of oocytes. We investigated the effect of varying oxygen concentrations during IVM on subsequent pre and post-implantation development. IVM of mouse cumulus-oocyte complexes (COCs) was performed under 2, 5, 10 or 20% O(2) (6% CO(2), balance N(2)). In vivo-matured COCs were collected post ovulation. Embryos were generated by IVF and culture. Blastocyst development, cell number and apoptosis were assessed, and fetal and placental outcomes analysed following embryo transfer at day 18 of pregnancy. Oxygen concentration during IVM did not affect oocyte maturation or subsequent fertilization, cleavage and blastocyst development rates. Maturation of oocytes under 2% O(2) increased blastocyst trophectoderm cell number compared with all groups and numbers at 5% were higher than 20% (both P < 0.05). Percentage of apoptotic cells was increased in blastocysts developed from 2% O(2)-matured oocytes, compared with maturation at 5% O(2) or in vivo (P < 0.05). Rates of embryo implantation and development into a viable fetus were not altered by IVM oxygen. However, fetal weight was reduced following oocyte maturation at 5% O(2) compared wiht 20% O(2) and maturation at 5% O(2) also reduced placental weight, when compared with in vivo-matured oocytes (both P < 0.05). Level of O(2) exposure during oocyte maturation can alter the cellular composition of blastocysts, but these changes in cell number do not correlate with the altered fetal and placental outcomes after transfer.

A mixed bag: A perspective on the regulation of IVF in Australia

Publisher: Informa UK Limited

Date: 06-2005

DOI: 10.1080/14647270500030621

Abstract: Regulation of ART in Australia consists of a mixed bag of Federal and State legislation and a national regulatory authority involved in accreditation. Access to IVF technology is variable between states and much ethical debate surrounds issues such as "social sexing" and other access differences, as well as the fate of surplus embryos. Little public debate on restriction of multiple embryo transfer practices has occurred, most likely due to public ignorance of the associated health risks.

Development during mid to late pregnancy following either in vitro embryo transfer or artificial insemination in cattle

Publisher: Elsevier BV

Date: 1999

DOI: 10.1016/S0093-691X(99)91787-9

Perturbations in mouse embryo development and viability caused by ammonium are more severe after exposure at the cleavage stages

Publisher: Oxford University Press (OUP)

Date: 02-2006

DOI: 10.1095/BIOLREPROD.105.046235

Abstract: The presence of ammonium in culture medium has a detrimental effect on embryo physiology and biochemistry however, the stage at which the embryo is most sensitive to this effect is unknown. The aim of this study was to determine the exact stage at which the embryo is most vulnerable to ammonium by exposing the preimplantation embryo to 300 muM ammonium either at the precompaction stage (between the zygote and two-cell or the two-cell to eight-cell) or at the postcompaction stage (between the eight-cell and blastocyst). This study determined that exposure of embryos to ammonium at the precompaction stage from either the zygote to two-cell stage or from the two-cell to the eight-cell stage did not affect the rate of development to the blastocyst stage however, the resultant blastocysts had decreased cell numbers and inner cell mass cells. Furthermore, these blastocysts had increased levels of cellular apoptosis and perturbed levels of Slc2a3 expression and glucose uptake. Transfer of these blastocysts revealed that, while implantation was not affected, the number of fetuses was reduced by culture with ammonium at the precompaction stage and fetal development was delayed, as observed by reduced crown-rump length and maturity. In contrast, the later stage embryo was more resistant to the negative effects of ammonium, with only Slc2a3 expression and fetal maturity affected. This raises the possibility that the later stage embryo is more able to protect itself from in vitro-derived stress and that the majority of in vitro-induced damage to mouse embryos is inflicted at the early stages of development.

Oocytes prevent cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins

Publisher: The Company of Biologists

Date: 15-11-2005

DOI: 10.1242/JCS.02644

Abstract: Paracrine factors secreted by the oocyte regulate a broad range of cumulus cell functions. Characteristically, cumulus cells have a low incidence of apoptosis and we proposed that this is due to oocyte-secreted factors acting in an anti-apoptotic manner. Bovine cumulus-oocyte complexes (COC) were aspirated from abattoir-derived ovaries and oocytectomized (OOX) by microsurgical removal of the oocyte. OOX were treated with doses of either denuded oocytes (DO) or various growth factors for 24 hours (± rFSH 0.1 IU/ml). Proportions of apoptotic cumulus cells were assessed using TUNEL and laser confocal scanning microscopy followed by image analysis. Quantification of Bcl-2 and Bax proteins in OOX was undertaken by western analysis. Oocyte removal led to a significant increase in cumulus cell apoptosis compared with COC controls (35% versus 9% TUNEL positive, respectively P& .001). Levels of OOX apoptosis were significantly reversed (P& .001) in a dose-dependent manner when co-cultured with oocytes. Furthermore, the anti-apoptotic effect of oocyte-secreted factors followed a gradient from the site of the oocyte(s). Growth differentiation factor 9 (GDF9) had no significant effect on cumulus cell apoptosis. By contrast, cumulus cell apoptosis was significantly (P& .001) reduced by bone morphogenetic proteins (BMP) 15, 6 or 7. Accordingly, levels of anti-apoptotic Bcl-2 were high in OOX+DO and OOX+BMP15 and low with OOX+GDF9 or OOX alone, whereas the reverse was observed for pro-apoptotic Bax. DO, BMP15 and BMP6 were also able to protect cumulus cells from undergoing apoptosis induced by staurosporine. FSH partially prevented apoptosis in all treatment groups (P& .001). Follistatin and a BMP6 neutralizing antibody, which antagonized the anti-apoptotic effects of BMP15 and BMP6, respectively, whether alone or combined, blocked ∼50% of the anti-apoptotic actions of oocytes. These results are the first to demonstrate that oocyte-secreted factors, and particularly BMP15 and BMP6, maintain the low incidence of cumulus cell apoptosis by establishing a localized gradient of bone morphogenetic proteins.

The effects of washing and protein supplementation on the acrosome reaction of ram spermatozoa in vitro

Publisher: Elsevier BV

Date: 07-1985

DOI: 10.1016/0378-4320(85)90043-0

Temporal effects of exogenous oocyte-secreted factors on bovine oocyte developmental competence during IVM

Publisher: CSIRO Publishing

Date: 2011

DOI: 10.1071/RD10323

Abstract: We investigated whether paracrine signalling between the bovine oocyte and cumulus cells is altered during the course of in vitro maturation (IVM). Bovine COCs were cocultured with denuded oocytes or treated with specific oocyte-secreted factors, namely recombinant bone morphogenetic protein (BMP)-15 or growth differentiation factor (GDF)-9, beginning from 0 or 9 h IVM. To generate a 9-h denuded oocyte (DO) group, COCs were cultured intact for the first 9 h of IVM and then denuded. Coculturing intact COCs with DOs denuded immediately after collection or following 9 h of maturation did not affect cleavage rate, but improved blastocyst yield (P 0.05) on Day 8 (51 and 61%, respectively P 0.05) and cell number compared with COCs cultured alone (41%). Significantly, we observed higher levels of endogenous GDF-9 and BMP-15 protein in oocytes of COCs matured for 9 h compared with no incubation. The addition of 175 ng mL–1 GDF-9 or 10% v/v BMP-15 from partially purified transfected 293H cell supernatant for 24 h IVM significantly enhanced development to the blastocyst stage from 40% (control) to 51 and 47%, respectively (P 0.05). However, treatment of COCs with GDF-9 or BMP-15 between 9 and 24 h of IVM did not increase blastocyst yield. These results provide evidence of quantitative and possibly qualitative temporal changes in oocyte paracrine factor production during IVM.

The Promise of in Vitro Maturation in Assisted Reproduction and Fertility Preservation

Publisher: Georg Thieme Verlag KG

Date: 2011

DOI: 10.1055/S-0030-1268701

Abstract: An innovative approach to in vitro maturation (IVM) for application in infertility treatment and fertility preservation is required to bring this patient-friendly treatment into routine practice. Current approaches to IVM never report more than a 10 to 15% implantation rate per embryo transferred, which is two to three times lower and early pregnancy losses are higher than in conventional in vitro fertilization/intracytoplasmic sperm injection. The cornerstone of such an innovative culture technique is the use of pharmacological compounds that allow synchronization of nuclear and cytoplasmic maturation processes within the oocyte. The rationale of a prolonged oocyte maturation period is to promote a longer interaction between the immature oocyte with adequately conditioned cumulus cells. Successful introduction of a new approach to IVM will reduce the requirement of fertility hormones and will be less invasive to the patient's daily life by reducing the need for monitoring of serum hormone levels and intravaginal ultrasound. The new IVM conditions will reduce a whole range of minor and major complications in assisted reproductive technology and finally will also reduce the total cost for treatment. The minimal invasiveness of this procedure will benefit cancer patients who want to store gonadal tissue before undergoing therapy that devastates subsequent germ-cell competence.

Recombinant human follicle-stimulating hormone alters maternal ovarian hormone concentrations and the uterus and perturbs fetal development in mice.

Publisher: American Physiological Society

Date: 10-2006

DOI: 10.1152/AJPENDO.00079.2006

Abstract: Gonadotropins are routinely administered to produce multiple oocytes for clinical in vitro fertilization (IVF) treatment, laboratory research, and livestock industries. Studies in mice have shown gonadotropin stimulation using equine chorionic gonadotropin (eCG) affects the endometrium, implantation, and fetal development. Evidence from clinical studies also indicates that stimulation with recombinant human follicle-stimulating hormone (rhFSH) may be detrimental to the endometrium and implantation rates. We investigated the effect of rhFSH in mice on maternal plasma hormone concentrations and uterine gene and protein expression and the effect of a stimulated maternal environment on pregnancy. Adult females were stimulated with rhFSH or eCG, followed by human chorionic gonadotropin (hCG). On day 4 of pseudopregnancy, mice either had embryos transferred to the uterus or were killed, and blood and uterine s les were collected. Pregnancy outcomes were examined on day 15. Gonadotropin stimulation increased plasma progesterone concentrations on day 4 compared with controls, whereas estradiol concentrations were unaffected. Stimulation also reduced uterine leukemia inhibitory factor ( Lif) mRNA, but the expression of estrogen and progesterone receptors ( Esr1 and Pgr), homeobox gene Hoxa10, and Vegf mRNA were unchanged. Furthermore, distribution of uterine PGR protein expression was altered by stimulation, but LIF protein was unchanged. Stimulated embryo transfer recipients had lower pregnancy rates than controls, and fetuses from the rhFSH group had reduced weight, length, and maturity. These results demonstrate that gonadotropin stimulation with rhFSH or eCG alters the preimplantation maternal environment, which results in reduced pregnancy rates and fetal development in the mouse.

Antiretroviral treatment change among HIV, hepatitis B virus and hepatitis C virus co-infected patients in the Australian HIV Observational Database

Publisher: Wiley

Date: 05-2005

DOI: 10.1111/J.1468-1293.2005.00280.X

Abstract: To assess the impact of highly active antiretroviral therapy (HAART) on rates of change of antiretroviral treatment among patients co-infected with hepatitis B virus (HBV) and/or hepatitis C virus (HCV) in the Australian HIV Observational Database (AHOD). Analysis was based on 805 of the 2218 patients recruited to the AHOD by March 2003, who had commenced HAART after 1 January 1997, who had recorded test results for HBV surface antigen and anti-HCV antibody, and who had follow-up of more than 3 months. The effect of hepatitis co-infection on the rate of antiretroviral treatment change after commencing HAART was assessed using a random-effect Poisson regression model. Among those included in the analyses, the prevalences of HBV and HCV were 4.8% and 12.8%, respectively. The overall rate of combination antiretroviral treatment change was 0.74 combinations per year. Factors independently associated with an increased rate of change of combination antiretroviral treatment were: prior AIDS-defining illness prior exposure to double combination antiretroviral therapy and antiretroviral treatment class. Co-infection with HBV and/or HCV was not found to be significantly associated with the rate of combination antiretroviral treatment change. While both HBV and HCV co-infections are relatively common in the AHOD, they do not appear to be serious impediments to the treatment of HIV-infected patients.

Bleaching-assisted multichannel microscopy

Publisher: OSA

Date: 2017

DOI: 10.1364/OMP.2017.OMW2D.3

Sperm DNA damage is associated with assisted reproductive technology pregnancy

Publisher: Wiley

Date: 08-09-2008

DOI: 10.1111/J.1365-2605.2007.00803.X

Abstract: The literature suggests an association between sperm DNA damage and assisted reproductive technology (ART) outcomes. However, previous studies involved the transfer of multiple embryos, which has complicated the interpretation of the results. The aim of this study was to determine the relationship between the levels of sperm DNA damage and fertilization rate, embryo development as well as pregnancy outcome, following single embryo transfer. Patients (n = 113) undergoing in vitro fertilization (IVF) (n = 45) and intra-cytoplasmic sperm injection (ICSI) (n = 68) were assessed for their levels of sperm DNA damage in the s le used for insemination. DNA damage was determined using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling (TUNEL). The relationship between DNA damage and outcomes were assessed using regression analysis. Overall data showed no association between sperm DNA damage and fertilization rate, or embryo development in vitro. However, when IVF was the insemination method, there was a significant negative correlation between fertilization rates and sperm DNA damage (p < 0.05). When ICSI was the insemination technique, low sperm DNA damage was associated with successful pregnancy (37.8 +/- 5.7% DNA damaged sperm) compared with failed implantation (52.9 +/- 3.9% DNA damaged sperm, p < 0.05). Our results suggest that sperm DNA damage as measured by the TUNEL assay may provide an indicator for patients with poor fertilization rates and/or those unable to achieve pregnancy following ART treatment.

A New Window into Ovarian Follicle Development

Publisher: Oxford University Press (OUP)

Date: 09-11-2016

DOI: 10.1095/BIOLREPROD.116.146647

A NEW APPROACH TO IN VITRO MATURATION (IVM) AND EMBRYO IN VITRO PRODUCTION: INDUCED IVM SUBSTANTIALLY IMPROVES EMBRYO YIELD AND PREGNANCY OUTCOMES

Publisher: CSIRO Publishing

Date: 2010

DOI: 10.1071/RDV22N1AB272

Abstract: Oocyte in vitro maturation (IVM) is the rate-limiting step in the in vitro production (IVP) of embryos. Oocyte maturation in vivo is a highly orchestrated, induced process, whereby cAMP-mediated meiotic arrest is overridden by the gonadotrophin surge prior to ovulation. However, aspirated oocytes resume maturation spontaneously compromising developmental competence. Hence, we hypothesized that establishing an induced system in vitro would synchronize oocyte-somatic cell communication leading to improved oocyte quality. Abattoir-collected bovine or 129/Sv mouse oocytes were treated for the first 1 to 2 h in vitro (pre-IVM) with the adenylate cyclase activator forskolin (100 μM, 50 μM, respectively) and a nonspecific phosphodiesterase (PDE) inhibitor, IBMX (500 μM, 50 μM), which substantially increased cumulus-oocyte complex (COC) cAMP (bovine, 180 v. 2 fmol/COC, treated v. control P 0.001). To maintain oocyte cAMP levels and prevent precocious oocyte maturation, IVM media (VitroMat + BSA) contained an oocyte-specific (type 3) PDE inhibitor, cilostamide (20 μM, 0.1 μM), plus FSH to induce maturation. The net effect of this system (induced-IVM) was to increase oocyte-cumulus cell gap-junctional communication (bovine: 1000 ± 148 v. 340 ± 73 unit, treated v. control P 0.05) and to slow meiotic progression through prophase I to metaphase II, extending the normal IVM interval (bovine: 30 v. 24 h, mouse: 22 v. 18 h treated v. control). FSH was required to complete maturation and FSH-induced maturation was prevented by an epidermal growth factor receptor inhibitor, AG1478 (2.5 μM), demonstrating induced oocyte maturation functions via secondary autocrine signaling within the cumulus cell compartment. These effects on COC functions had profound consequences for oocyte developmental potential. In completely serum-free bovine IVP, induced-IVM more than doubled blastocyst yield (69 v. 27%, treated v. control P 0.05) and improved blastocyst quality (186 v. 132 blastomeres). To achieve these rates, the pre-IVM phase, the modified IVM conditions, and delayed IVF were all required. Adapting the system to the mouse, induced-IVM increased blastocyst rate (86 v. 55%, treated v. control P 0.05), implantation rate (51 v. 25% P 0.01), fetal survival rate (29 v. 5% P 0.01) and fetal weight (0.9 v. 0.5 g P 0.01). All these embryonic and fetal outcomes in mice were equivalent (P 0.05) using induced-IVM to levels obtained from in vivo-matured control oocytes (conventional IVF). Data were analyzed by ANOVA. In conclusion, induced-IVM mimics some of the characteristics of oocyte maturation in vivo and substantially improves oocyte developmental outcomes in 2 disparate mammalian species. Adaption of this new approach to clinical/field conditions should lead to new opportunities for a wide range of reproductive biotechnologies. Such a notable increase in IVM efficiency could see IVP as the preferred embryo production technology in future livestock artificial breeding programs. Funded by an Australian Research Council Linkage Grant and Cook Australia. Thanks to M. Sasseville, M. Lane, and D. T. Armstrong.

Implications of glycolytic and pentose phosphate pathways on the oxidative status and active mitochondria of the porcine oocyte during IVM

Publisher: Elsevier BV

Date: 12-2016

DOI: 10.1016/J.THERIOGENOLOGY.2015.11.008

Abstract: Glycolysis and the pentose phosphate pathway (PPP) were modulated in porcine cumulus-oocyte complexes during IVM by the addition of inhibitors and stimulators of key enzymes of the pathways to analyze their influence on the oxidative status, active mitochondria, and maturation of the oocyte. The influence of pharmacologic and physiological inhibitors of glycolysis (Sodium fluoride and ATP) and PPP (6-Aminonicotinamide and nicotinamide adenine dinucleotide phosphate) was validated by assessing glucose and lactate turnover and brilliant cresyl blue staining in oocytes. Inhibitors of glycolysis and PPP activity significantly perturbed nuclear maturation, oxidative metabolism (Redox Sensor Red CC-1), and active mitochondria (Mitotracker Green FM) within oocytes (P < 0.05). In comparison, physiological stimulators of glycolysis (adenosine monophosphate) and PPP (nicotinamide adenine dinucleotide phosphate) did not affect any of evaluated parameter. In the absence of modulators, fluctuations in the oocyte oxidative activity and active mitochondria were observed during porcine IVM. The inhibition of glycolysis and PPP modified the pattern of oxidation and mitochondrial fluctuation, resulting in impaired meiotic progression. We demonstrated the relationship between carbohydrate metabolism in COC and oocyte redox status necessary for porcine oocyte IVM.

Peri-conceptual cytokines - Setting the trajectory for embryo implantation, pregnancy and Beyond

Publisher: Wiley

Date: 07-2011

DOI: 10.1111/J.1600-0897.2011.01039.X

Abstract: The peri-conceptual environment influences the early embryo to impart long-term consequences for the fetus and neonate however, the underlying mechanisms are not well defined. We argue that the cytokine network acting in the female reproductive tract during the pre- and peri-implantation period integrates environmental information to program the embryo and fine-tune the maternal immune response and endometrial remodelling to determine implantation success. As well as sex steroid hormones and male seminal fluid factors, female tract cytokines are influenced by agents signalling via the Toll-like receptors including the microbiome and a plethora of metabolic, chemical and other stressors. In mouse models, an altered peri-conceptual cytokine environment induced by cytokine deficiency, inflammatory insults or dysregulated seminal fluid signalling is associated with adverse effects on embryo development, pregnancy viability and reproductive outcome. The cytokine network provides a pivotal mechanism through which environmental factors influence both embryo development and receptivity of the uterus.

In vitro maturation of Mammalian oocytes: outcomes and consequences.

Publisher: Georg Thieme Verlag KG

Date: 03-2008

DOI: 10.1055/S-2008-1042955

Abstract: The application of in vitro maturation (IVM) of oocytes as a technology to assist animal production and clinical infertility treatment remains poor because of the reduced developmental competence of oocytes after IVM, despite several decades of research. Reduced meiotic maturation rates, fertilization rates, and blastocyst production reveal short-term developmental insufficiency of oocytes when compared with in vivo-matured counterparts. However, there is an increasing body of evidence that demonstrates the capacity of IVM efficiency to be improved, some of which is reviewed here. Of more concern is the role that IVM of oocytes may play in causing or accentuating long-term development and health of fetuses and neonates after in vitro production of embryos and embryo transfer. This is a largely unexplored area, yet the application of such techniques, especially the safety of clinical IVM, is significant and requires monitoring before acceptance as a routine procedure.

Simulated physiological oocyte maturation (SPOM): A novel in vitro maturation system that substantially improves embryo yield and pregnancy outcomes

Publisher: Oxford University Press (OUP)

Date: 24-09-2010

DOI: 10.1093/HUMREP/DEQ246

Abstract: Oocyte in vitro maturation (IVM) reduces the need for gonadotrophin-induced ovarian hyperstimulation and its associated health risks but the unacceptably low conception regnancy rates have limited its clinical uptake. We report the development of a novel in vitro simulated physiological oocyte maturation (SPOM) system. Bovine or mouse cumulus-oocyte complexes (COCs) were treated with cAMP modulators for the first 1-2 h in vitro (pre-IVM), increasing COC cAMP levels ∼100-fold. To maintain oocyte cAMP levels and prevent precocious oocyte maturation, COCs were treated during IVM with an oocyte-specific phosphodiesterase inhibitor and simultaneously induced to mature with FSH. Using SPOM, the pre-IVM and IVM treatments synergized to increase bovine COC gap-junctional communication and slow meiotic progression (both P < 0.05 versus control), extending the normal IVM interval by 6 h in bovine and 4 h in mouse. FSH was required to complete maturation and this required epidermal growth factor signalling. These effects on COC had profound consequences for oocyte developmental potential. In serum-free conditions, SPOM increased bovine blastocyst yield (69 versus 27%) and improved blastocyst quality (184 versus 132 blastomeres both P < 0.05 versus standard IVM). In mice, SPOM increased (all P < 0.05) blastocyst rate (86 versus 55% SPOM versus control), implantation rate (53 versus 28%), fetal yield (26 versus 8%) and fetal weight (0.9 versus 0.5 g) to levels matching those of in vivo matured oocytes (conventional IVF). SPOM is a new approach to IVM, mimicing some characteristics of oocyte maturation in vivo and substantially improving oocyte developmental outcomes. Adaption of SPOM for clinical application should have significant implications for infertility management and bring important benefits to patients.

The difference in pregnancy rates between elective single embryo transfer (SET) compared to double embryo transfer is dependent on the implantation rates of embryos being transferred. Using mathematical modeling to determine when SET becomes a viable option.

Publisher: Oxford University Press (OUP)

Date: 08-2006

DOI: 10.1093/HUMREP/DEL241

Mathematical modeling of glucose supply toward successful in vitro maturation of Mammalian oocytes.

Publisher: Mary Ann Liebert Inc

Date: 09-2008

DOI: 10.1089/TEN.TEA.2008.0036

Abstract: In vitro maturation-whereby an oocyte is harvested from an ovary just before full maturation, matured in the laboratory, fertilized, and then transplanted back to the uterus-has important benefits over, but is significantly less successful than, traditional in vitro fertilization. Inadequate in vitro nutrient environments are believed to be a prime reason for the low success, but understanding of the in vivo environment, which needs to be better replicated in the laboratory, is still lacking. We here consider mathematical modeling as an aid to increasing that understanding. A general mathematical model suitable for examining the in vivo concentrations of a nutrient in the cumulus-oocyte complex (COC) is presented. We then tailor the model to consider glucose concentration. Experimental data are used to obtain information on glucose uptake in the COC for use in the model. Finally, we solve the model to estimate glucose concentration in the COC. With the information currently available, the model indicates a significant reduction in glucose concentration from the follicular fluid across the cumulus matrix to the oocyte.

Microarray analysis of mRNA from cumulus cells following in vivo or in vitro maturation of mouse cumulus-oocyte complexes.

Publisher: CSIRO Publishing

Date: 2013

DOI: 10.1071/RD11305

Abstract: The IVM of mammalian cumulus–oocyte complexes (COCs) yields reduced oocyte developmental competence compared with oocytes matured in vivo. Altered cumulus cell function during IVM is implicated as one cause for this difference. We have conducted a microarray analysis of cumulus cell mRNA following IVM or in vivo maturation (IVV). Mouse COCs were sourced from ovaries of 21-day-old CBAB6F1 mice 46 h after equine chorionic gonadotrophin (5 IU, i.p.) or from oviducts following treatment with 5 IU eCG (61 h) and 5 IU human chorionic gonadotrophin (13 h). IVM was performed in α-Minimal Essential Medium with 50 mIU FSH for 17 h. Three independent RNA s les were assessed using the Affymetrix Gene Chip Mouse Genome 430 2.0 array (Affymetrix, Santa Clara, CA, USA). In total, 1593 genes were differentially expressed, with 811 genes upregulated and 782 genes downregulated in IVM compared with IVV cumulus cells selected genes were validated by real-time reverse transcription–polymerase chain reaction (RT-PCR). Surprisingly, haemoglobin α (Hba-a1) was highly expressed in IVV relative to IVM cumulus cells, which was verified by both RT-PCR and western blot analysis. Because haemoglobin regulates O2 and/or nitric oxide availability, we postulate that it may contribute to regulation of these gases during the ovulatory period in vivo. These data will provide a useful resource to determine differences in cumulus cell function that are possibly linked to oocyte competence.

Effect of delayed supplementation of fetal calf serum to culture medium on bovine embryo development in vitro and following transfer

Publisher: Elsevier BV

Date: 04-1998

DOI: 10.1016/S0093-691X(98)00071-5

Abstract: Supplementation of synthetic oviduct fluid (SOF) medium plus amino acids and bovine serum albumin (BSA) with either fetal calf serum (FCS) or charcoal-treated FCS (CT-FCS) from Day 5 of development was investigated to determine if either in vitro or post-transfer development was altered. Development to the compact morula stage or beyond was similar for all 3 treatments. However, blastocyst development at Day 7 was accelerated when serum was added to the medium (21.6, 40.1 and 39.4% blastocysts from cleaved embryos for BSA, FCS and CT-FCS, respectively P < 0.01), but cell number of the resulting embryos was unaffected. Furthermore, addition of CT-FCS decreased the between replicate variation in embryo development and produced more Grade 1 and 2 quality embryos (25.8%) than BSA supplementation (18.1% P < 0.05). The transfer of Grade 1 and 2 embryos at Day 7 following culture resulted in similar pregnancy and embryo survival rates for the 3 treatments, with a tendency for lower embryo survival of embryos cultured in FCS (embryo survival at Day 50 = 37.7% vs 53.3% and 57.6% for FCS, BSA and CT-FCS, respectively P = 0.1). Significant fetal loss from Day 50 to term occurred within all 3 treatments. There were no birth weight differences for calves amongst the 3 culture treatments however, one of the sires produced calves that were significantly heavier than expected, suggesting a possible sire-by-embryo interaction. These results demonstrate that addition of FCS may promote blastocyst development however, there was also a tendency for lower embryo survival. Thus charcoal treatment of FCS is recommended, because it decreases variability in embryo development between runs and results in embryo survival rates to term similar to that BSA-supplemented media.

Disruption of mitochondrial malate-aspartate shuttle activity in mouse blastocysts impairs viability and fetal growth

Publisher: Oxford University Press (OUP)

Date: 02-2009

DOI: 10.1095/BIOLREPROD.108.069864

Post hCG follicle differentiation in the mouse is associated with an increase in hypoxia inducible factor activity

Publisher: Oxford University Press (OUP)

Date: 05-2008

DOI: 10.1093/BIOLREPROD/78.S1.127C

A biophotonic approach to measure pH in small volumes in vitro: Quantifiable differences in metabolic flux around the cumulus‐oocyte‐complex (COC)

Publisher: Wiley

Date: 04-12-2019

DOI: 10.1002/JBIO.201960038

Abstract: Unfertilised eggs (oocytes) release chemical biomarkers into the medium surrounding them. This provides an opportunity to monitor cell health and development during assisted reproductive processes if detected in a non-invasive manner. Here we report the measurement of pH using an optical fibre probe, OFP1, in 5 μL drops of culture medium containing single mouse cumulus oocyte complexes (COCs). This allowed for the detection of statistically significant differences in pH between COCs in culture medium with no additives and those incubated with either a chemical (cobalt chloride) or hormonal treatment (follicle stimulating hormone) both of which serve to induce the release of lactic acid into the medium immediately surrounding the COC. Importantly, OFP1 was shown to be cell-safe with no inherent cell toxicity or light-induced phototoxicity indicated by negative DNA damage staining. Pre-measurement photobleaching of the probe reduced fluorescence signal variability, providing improved measurement precision (0.01-0.05 pH units) compared to previous studies. This optical technology presents a promising platform for the measurement of pH and the detection of other extracellular biomarkers to assess cell health during assisted reproduction.

Measuring embryo metabolism to predict embryo quality

Publisher: CSIRO Publishing

Date: 2016

DOI: 10.1071/RD15340

Abstract: Measuring the metabolism of early embryos has the potential to be used as a prospective marker for post-transfer development, either alone or in conjunction with other embryo quality assessment tools. This is necessary to maximise the opportunity of couples to have a healthy child from assisted reproduction technology (ART) and for livestock breeders to efficiently improve the genetics of their animals. Nevertheless, although many promising candidate substrates (e.g. glucose uptake) and methods (e.g. metabolomics using different spectroscopic techniques) have been promoted as viability markers, none has yet been widely used clinically or in livestock production. Herein we review the major techniques that have been reported these are ided into indirect techniques, where measurements are made from the embryo’s immediate microenvironment, or direct techniques that measure intracellular metabolic activity. Both have strengths and weaknesses, the latter ruling out some from contention for use in human ART, but not necessarily for use in livestock embryo assessment. We also introduce a new method, namely multi- (or hyper-) spectral analysis, which measures naturally occurring autofluorescence. Several metabolically important molecules have fluorescent properties, which we are pursuing in conjunction with improved image analysis as a viable embryo quality assessment methodology.

Glycolytic pathway activity: Effect on IVM and oxidative metabolism of bovine oocytes

Publisher: CSIRO Publishing

Date: 2013

DOI: 10.1071/RD12193

Abstract: The aim of the present study was to determine the effect of altering glycolytic pathway activity during bovine IVM on the meiotic maturation rate, oxidative activity, mitochondrial activity and the mitochondrial distribution within oocytes. Glycolytic activity was manipulated using two inhibitors (ATP, NaF) and a stimulator (AMP) of key enzymes of the pathway. Inhibition of glucose uptake, lactate production and meiotic maturation rates was observed when media were supplemented with ATP or NaF. The addition of AMP to the maturation medium had no effect on glucose uptake, lactate production or meiotic maturation. In the absence of gonadotrophin supplementation, AMP stimulated both glucose uptake and lactate production. However, AMP also decreased cytoplasmic maturation, as determined by early cleavage. During IVM, oocyte oxidative and mitochondrial activity was observed to increase at 15 and 22 h maturation. Inhibiting glycolysis with ATP or NaF led to a reduced oxidative and mitochondrial pattern compared with the respective control groups. Stimulation of the pathway with AMP increased oxidative and mitochondrial activity. A progressive mitochondrial migration to the central area was observed during maturation oocytes treated with ATP, NaF or AMP showed limited migration. The present study reveals the effects of altering glycolytic pathway activity in cumulus–oocyte complexes, revealing the link between glycolysis of the cumulus–oocyte complex and the oxidative and mitochondrial activity of the oocyte.

Oxygen-regulated gene expression in bovine blastocysts

Publisher: Oxford University Press (OUP)

Date: 10-2004

DOI: 10.1095/BIOLREPROD.104.028639

Sperm DNA damage: is altered carbohydrate metabolism to blame?

Publisher: Elsevier BV

Date: 09-2007

DOI: 10.1016/J.FERTNSTERT.2007.07.170

A Dual Sensor for pH and hydrogen peroxide using polymer-coated optical fibre tips

Publisher: MDPI AG

Date: 17-12-2015

DOI: 10.3390/S151229893

Effect of 2,4-dinitrophenol on the energy metabolism of cattle embryos produced by in vitro fertilization and culture

Publisher: CSIRO Publishing

Date: 2002

DOI: 10.1071/RD02038

Abstract: In cattle embryos, the proportion of ATP produced by glycolysis increases following the major activation of the embryonic genome, and development to the blastocyst stage is improved in the presence of 10 µM 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, from Day 5 to Day 7 of culture. In Experiment 1 of the present study, culture of cattle embryos in the presence of 10 µM DNP from Day 5 to Day 7 stimulated development to the blastocyst stage, but had no significant effects on oxygen, pyruvate or glucose uptake, or on lactate production. In Experiment 2, culture of cattle embryos in the presence of 10 µM DNP from Day 5 to Day 7, stimulated the metabolism of [2-14C]pyruvate (a measure of Krebs cycle activity) on all of Days 5, 6 and 7, and stimulated metabolism of [5-3H]glucose (a measure of glycolysis) on Day 7 only. The results show that 10 µM DNP stimulates oxidative and glycolytic metabolism in Day-5 to Day-7 cattle embryos, but this does not fully explain the observed increase in developmental competence. We propose that partial inhibition or uncoupling of oxidative phosphorylation may reduce the level of intracellular reactive oxygen species production, thereby facilitating development.

Boronate probes for the detection of hydrogen peroxide release from human spermatozoa

Publisher: Elsevier BV

Date: 04-2015

DOI: 10.1016/J.FREERADBIOMED.2015.01.015

Abstract: Human spermatozoa are compromised by production of reactive oxygen species (ROS), and detection of ROS in spermatozoa is important for the diagnosis of male infertility. The probes 2',7'-dichlorohydrofluorescein diacetate (DCFH), dihydroethidium (DHE), and MitoSOX red (MSR) are commonly used for detecting ROS by flow cytometry however, these probes lack sensitivity to hydrogen peroxide (H2O2), which is particularly damaging to mammalian sperm cells. This study reports the synthesis and use of three aryl boronate probes, peroxyfluor-1 (PF1), carboxyperoxyfluor-1, and a novel probe, 2-(2-ethoxyethoxy)ethoxyperoxyfluor-1 (EEPF1), in human spermatozoa. PF1 and EEPF1 were effective at detecting H2O2 and peroxynitrite (ONOO(-)) produced by spermatozoa when stimulated with menadione or 4-hydroxynonenal. EEPF1 was more effective at detection of ROS in spermatozoa than DCFH, DHE, or MSR furthermore it distinguished poorly motile sperm as shown by greater ROS production. EEPF1 should therefore have a significant role in the diagnosis of oxidative stress in male infertility, cryopreservation, age, lifestyle, and exposure to environmental toxicants.

Lamb Birth Weight is Affected by Culture System Utilized during in Vitro Pre-Elongation Development of Ovine Embryos

Publisher: Oxford University Press (OUP)

Date: 12-1995

DOI: 10.1095/BIOLREPROD53.6.1385

Abstract: It has previously been reported that ovine embryos cultured in Synthetic Oviduct Fluid medium supplemented with 20% human serum (SOF+HS) develop into lambs with a high birth weight. We have investigated this phenomenon by culturing ovine zygotes in SOF+HS or a serum-free version of Synthetic Oviduct Fluid with BSA and amino acids (SOFaaBSA) in place of serum. Zygotes were either obtained from superovulated and naturally mated ewes or produced in vitro. Embryos were subsequently transferred to synchronized recipient ewes (n = 63). An additional group of ewes (n = 16) served as flock fertility and lambing controls. Development of zygotes to stages suitable for transfer (i.e., good to excellent compact morulae or blastocysts) was not affected by medium (SOFaaBSA = 53 +/- 5% vs. SOF+HS = 59 +/- 5%) but was affected by source (in vivo-derived = 74 +/- 5% vs. in vitro-derived = 35 +/- 5%, p < 0.001). Embryos incubated in SOF+HS were morphologically different from those incubated in SOFaaBSA, having abundant lipid droplets. Pregnancy rate (65%) and embryo survival (48%) of recipients determined by ultrasonography on approximately Day 60 of pregnancy did not differ between medium treatments or source of embryo. Mean weight of lambs from embryos cultured in SOF+HS (4.2 +/- 0.2 kg) was significantly heavier than that of controls (3.4 +/- 0.2 kg, p < 0.01) or of lambs from embryos cultured in SOFaaBSA (3.5 +/- 0.2 kg, p < 0.05). Furthermore, mean gestation length was longer in recipients receiving embryos incubated in SOF+HS (147 +/- 1 days) than in SOFaaBSA (145 +/- 1 day, p < 0.05). Reasons for this birth weight and gestation length difference are unclear, but our data suggest that different culture conditions can produce embryos with differing morphology, apparent chemical composition, and rate of development, resulting in lambs with differing gestation length and birth weight.

Donor and recipient ewe factors affecting in vitro development and post-transfer survival of cultured sheep embryos

Publisher: Elsevier BV

Date: 12-1995

DOI: 10.1016/0378-4320(95)01432-2

Human assisted conception: A cautionary tale. Lessons from domestic animals

Publisher: Oxford University Press (OUP)

Date: 12-1998

DOI: 10.1093/HUMREP/13.SUPPL_4.184

Abstract: A variety of embryo-based technologies used in farm animal reproduction, including embryo culture, nuclear transfer, embryo-somatic cell co-culture and asynchronous embryo transfer can lead to the production of large offspring the so-called large calf/lamb syndrome. In some cases, abnormalities in the fetus and newborn are apparent. The nature of these associations is explored with emphasis on the biological differences between in-vivo- and in-vitro-produced embryos. A unifying framework and research programme aimed at explaining anomalies in early embryo development is then proposed in terms of the response of somatic cells and embryos to cellular stress. The review concludes with a caution against developments in assisted conception technologies, in man and domestic animals, being determined too much by the needs of commerce at the expense of research on the molecular, biochemical and physiological basis of early mammalian development.

Glucosamine supplementation during in vitro maturation inhibits subsequent embryo development: Possible role of the hexosamine pathway as a regulator of developmental competence

Publisher: Oxford University Press (OUP)

Date: 05-2006

DOI: 10.1095/BIOLREPROD.105.048553

Abstract: Glucose concentration during cumulus-oocyte complex (COC) maturation influences several functions, including progression of oocyte meiosis, oocyte developmental competence, and cumulus mucification. Glucosamine (GlcN) is an alternative hexose substrate, specifically metabolized through the hexosamine biosynthesis pathway, which provides the intermediates for extracellular matrix formation during cumulus cell mucification. The aim of this study was to determine the influence of GlcN on meiotic progression and oocyte developmental competence following in vitro maturation (IVM). The presence of GlcN during bovine IVM did not affect the completion of nuclear maturation and early cleavage, but severely perturbed blastocyst development. This effect was subsequently shown to be dose-dependent and was also observed for porcine oocytes matured in vitro. Hexosamine biosynthesis upregulation using GlcN supplementation is well known to increase O-linked glycosylation of many intracellular signaling molecules, the best-characterized being the phosphoinositol-3-kinase (PI3K) signaling pathway. We observed extensive O-linked glycosylation in bovine cumulus cells, but not oocytes, following IVM in either the presence or the absence of GlcN. Inhibition of O-linked glycosylation significantly reversed the effect of GlcN-induced reduction in developmental competence, but inhibition of PI3K signaling had no effect. Our data are the first to link hexosamine biosynthesis, involved in cumulus cell mucification, to oocyte developmental competence during in vitro maturation.

Glucosamine supplementation during in vitro maturation leads to perturbed developmental capacity of bovine cumulus oocyte complexes

Publisher: CSIRO Publishing

Date: 2005

DOI: 10.1071/RDV17N2AB300

Abstract: Glucose is a primary energy substrate required for successful in vitro oocyte maturation (IVM). However, most maturation media contain more glucose than that seen in follicular fluid (2.3 mM vs. 5.6 mM in TCM199). Glucosamine (Glc) as an alternative substrate for extracellular matrix during cumulus expansion reduced glucose uptake by bovine cumulus oocyte complexes (COCs, Sutton-McDowall et al. 2004 Reproduction 128, 313–319). As this could enable a reduction in glucose concentrations to physiological levels in IVM medium of COCs, the aim of this study was to investigate the influence of Glc supplementation on oocyte developmental capacity. Bovine COCs were matured in synthetic follicular fluid medium (SFFM, a defined medium based on the composition of follicular fluid, plus 5.6 mM glucose, FSH, hCG and BSA, Sutton-McDowall et al. 2004 Reprod. Fertil. Dev. 16 sup, 204) ± 5 mM Glc. After 24 h, either nuclear maturation (rep = 8, n = 160) or blastocyst development 8 days post-fertilization (rep = 5, n = 400) was determined. Data was arcsine transformed and analyzed by ANOVA, followed by Tukey's test. While the presence of Glc did not affect the completion of nuclear maturation and early cleavage, +Glc led to severely perturbed blastocyst development (−Glc, 32.5 ± 1.9% vs. +Glc, 4.7 ± 3.9%, P 0.001). Glc supplementation in somatic cells is well-known to down-regulate the phosphatidylinositol-3-kinase (PI3K) signalling pathway, reducing protein synthesis and other cell survival mechanisms. Therefore, oocyte protein synthesis (measured by [2,3,4,5,6-3H] phenylalanine incorporation, rep = 5, n = 200) and embryo development (rep = 6, n = 720) following IVM in SFFM ± Glc ± EGF (a PI3K pathway stimulator) was determined. Glc supplementation led to a 40% decrease in protein synthesis compared to −Glc, while the combination of +Glc + EGF significantly increased protein synthesis by 60%. However, IVM + EGF + Glc did not improve blastocyst rates (main effect: −Glc 41.6 ± 6.6% vs. +Glc, 6.6 ± 1.7%, P 0.001). Additionally, COCs were also cultured in SFFM ± 50 μM LY294002 (a specific PI3K inhibitor) and nuclear maturation (rep = 5, n = 200) or blastocyst development 8 days post-fertilization (rep = 4, n = 200) was determined. Despite the presence of LY294002 leading to 43% less COCs completing nuclear maturation (P 0.001), blastocyst development was not affected (mean = 38.8 ± 3.2%). These results demonstrate that Glc supplementation during IVM has no effect on nuclear maturation or early development but is detrimental to oocyte developmental capacity by severely perturbing blastocyst development. However, the diminished developmental capacity appears to be independent of the well-characterized Glc down-regulation of the PI3K signalling pathway. This work was supported by the Australian Research Council (SPIRT, C00107702) and Cook Australia Pty Ltd.

Localised hydrogen peroxide sensing for reproductive health

Publisher: SPIE

Date: 05-05-2015

DOI: 10.1117/12.2178680

Effect of inhibitors and uncouplers of oxidative phosphorylation during compaction and blastulation of bovine embryos cultured in vitro

Publisher: Bioscientifica

Date: 2000

DOI: 10.1530/REPROD/118.1.47

Abstract: The effect of inhibiting ATP production via oxidative phosphorylation during pericompaction of in vitro produced bovine embryos was investigated. This was achieved by: (i) varying the atmospheric O2 concentration (0, 1, 2, 4 and 7%) (ii) addition of oxidative phosphorylation inhibitors, NaN3 and antimycin A and (iii) addition of 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation from electron transport. The development of embryos under various O2 concentrations from day 5 to day 7 of development indicated that an optimal concentration occurred at about 2%. Addition of NaN3 revealed that doses above 100 mumol l-1 were toxic to embryo development, but that concentrations of 5-10 mumol l-1 stimulated embryo development by 10-25%. A similar result was observed after addition of 2,4-dinitrophenol, whereas antimycin A was inhibitory at doses as low as 1 mumol l-1. At concentrations of NaN3 or 2,4-dinitrophenol that stimulated embryo development, the number of cells of the resulting blastocysts was also significantly increased. Addition of NaN3 from day 1 of development inhibited subsequent development. Metabolic data of NaN3-treated embryos revealed that O2 uptake was significantly lower at inhibitory doses (100 mumol l-1). A significant (P 0.05) log linear increase in glucose uptake was measured between the three concentrations of NaN3 (0, 10 and 100 mumol l-1). These results demonstrate that ATP production via oxidative phosphorylation is essential for bovine embryo development in vitro. However, transient (subacute) inhibition appears to be beneficial to embryo development and the number of cells, perhaps by creating a more favourable intracellular environment.

A novel embryo culture media supplement that improves pregnancy rates in mice

Publisher: Bioscientifica

Date: 03-2017

DOI: 10.1530/REP-16-0517

Abstract: The preimplantation embryo in vivo is exposed to numerous growth factors in the female reproductive tract, which are not recapitulated in embryo culture media in vitro . The IGF2 and plasminogen activator systems facilitate blastocyst development. We hypothesized that the addition of IGF2 in combination with urokinase plasminogen activator (uPA) and plasminogen could improve rates of blastocyst hatching and implantation in mice. B6BcF1 and CBAB6F2 mouse embryos were ided into one of four supplemented culture media treatment groups: (1) control (media only) (2) 12.5 nM IGF2 (3) 10 µg/mL uPA and 5 µg/mL plasminogen or (4) a combination of IGF2, uPA and plasminogen treatments. Embryo development to blastocyst stage and hatching were assessed before transfer to pseudopregnant recipient females and implantation, pregnancy rates and postnatal growth were assessed. After 90.5 h of culture, IGF2 + U + P treatment increased the percentage of B6BcF1 embryos that were hatching/hatched and percentage developing to blastocyst stage compared with controls ( P 0.02). Following B6BcF1 embryo transfer, IGF2 + U + P treatment increased implantation sites at day 8 of pregnancy compared with controls ( P 0.05). Replication in the CBAB6F2 mouse strain showed significant improvements in pregnancy rates at days 8 and 18 but not in blastocyst development. No adverse effects were seen on gestational age, litter size or birthweight, or the reproductive capacity of offspring of IGF2 + U + P treated embryos. For embryos susceptible to detrimental effects of in vitro culture, IGF2, uPA and plasminogen supplementation of culture media can improve pregnancy success, but the effect of treatment is dependent on the mouse strain.

Bone morphogenetic protein 15 in the pro-mature complex form enhances bovine oocyte developmental competence.

Publisher: Public Library of Science (PLoS)

Date: 24-07-2014

DOI: 10.1371/JOURNAL.PONE.0103563

Growth factors and cytokines in embryo development

Publisher: Cambridge University Press

Date: 27-03-2014

DOI: 10.1017/CBO9781139059053.010

The effect of peri-conception hyperglycaemia and the involvement of the hexosamine biosynthesis pathway in mediating oocyte and embryo developmental competence

Publisher: Wiley

Date: 27-01-2014

DOI: 10.1002/MRD.22299

Abstract: The environment that the oocyte is exposed to during the peri-conception period can have a significant impact on oocyte developmental competence (the ability of the oocyte to support fertilisation and subsequent embryo development) and the long-term health of the resulting offspring. This is particularly true for maternal hyperglycaemia. While maternal hyperglycaemia during early pregnancy through term development has been extensively studied, the effects on the oocyte itself, and the underlying mechanisms, remain largely unknown. There is increasing evidence, however, for the role of the fuel-sensing hexosamine biosynthesis pathway in mediating the effects of hyperglycaemia in many different cell types. In this review, we will focus on the reproductive consequences of maternal hyperglycaemia during the peri-conceptual period and the role of the hexosamine pathway in mediating these processes.

[5-3H]-glucose and [1-14C]-pyruvate utilization by fresh and frozen-thawed day 7 IVP bovine blastocysts cultured in PVA- or BSA-supplemented medium

Publisher: Elsevier BV

Date: 1998

DOI: 10.1016/S0093-691X(98)90586-6

In vitro culture and embryo metabolism of cattle and sheep embryos — a decade of achievement

Publisher: Elsevier BV

Date: 07-2000

DOI: 10.1016/S0378-4320(00)00096-8

Abstract: At the beginning of the 1990s, co-culture of cattle and sheep embryos was the most favoured method to support embryo development, but the use of this system has h ered progress in raising the efficiency of embryo production. Furthermore, little was known of the requirements of embryos and the biochemistry of early embryo development. As the decade progressed, energy metabolism studies improved our understanding of the energy substrate requirements for embryo development. Furthermore, an appreciation of the reproductive tract environment increased. This resulted in more "defined" systems, which have evolved further in the development of "sequential" media systems, where components change in accordance to the needs of the embryo. Nevertheless, wholly defined systems, such as the replacement of albumin with PVA, are less able to support similar levels of development as protein-containing medium, and the resulting embryos are metabolically compromised. This highlights the nutritive role of albumin. One area in which much work has been conducted, but yet no unifying theory has emerged, is that of the interactive roles of growth factors (including autocrine aracrine), cytokines and extra-cellular matrix molecules in the development of a viable embryo. A new concept is that of regulation of energy metabolism. Compounds such as ethylenediamine tetraacetic acid (EDTA), NaN(3) and 2,4-dinitrophenol have been shown to increase embryo development and quality of resulting embryos. This demonstrates that the process of ATP production is a key regulator of in vitro embryo development.

Non-invasive detection and monitoring of biochemistry in cells and tissues by decomposing autofluorescence

Publisher: OSA

Date: 2014

DOI: 10.1364/ACPC.2016.AS3I.1

Oocyte-secreted factors enhance oocyte developmental competence

Publisher: Elsevier BV

Date: 08-2006

DOI: 10.1016/J.YDBIO.2006.06.026

Abstract: The capacity of fully grown oocytes to regulate their own microenvironment by paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) may in turn contribute to oocyte developmental competence. Here, we investigated if OSFs have a direct influence on oocyte developmental competence during in vitro maturation (IVM). Bovine cumulus-oocyte complexes (COCs) were aspirated from abattoir-derived ovaries and matured in serum-free medium. COCs were either co-cultured with denuded oocytes (DOs) or treated with specific OSFs: recombinant bone morphogenetic protein 15 (BMP15) and/or growth differentiation factor 9 (GDF9). Following maturation, embryos were fertilized and cultured in vitro and blastocyst development and cell number were assessed on day 8. Co-culturing intact COCs with DOs did not affect cleavage rate, but increased (P<0.001) the proportion of cleaved embryos that reached the blastocyst stage post-insemination from 39% to 51%. OSFs also altered blastocyst cell allocation as co-culture of COCs with DOs significantly increased total and trophectoderm cell numbers, compared to control COCs. BMP15 alone, GDF9 alone or the two combined all (P<0.05) increased the proportion of oocytes that reached the blastocyst stage post-insemination from 41% (controls) to 58%, 50% and 55%, respectively. These results were further verified in neutralization experiments of the exogenous growth factors and of the native OSFs. Follistatin and the kinase inhibitor SB-431542, which antagonize BMP15 and GDF9, respectively, neutralized the stimulatory effects of the exogenous growth factors and impaired the developmental competence of control COCs. These results demonstrate that OSFs, and particularly BMP15 and GDF9, enhance oocyte developmental competence and provide evidence that OSF regulation of the COC microenvironment is an important determinant of oocyte developmental programming.

Hyperglycaemic conditions perturb mouse oocyte in vitro developmental competence via beta-O-linked glycosylation of Heat shock protein 90

Publisher: Oxford University Press (OUP)

Date: 07-04-2014

DOI: 10.1093/HUMREP/DEU066

Abstract: What is the effect of beta-O-linked glycosylation (O-GlcNAcylation) on specific proteins in the cumulus-oocyte complex (COC) under hyperglycaemic conditions? Heat shock protein 90 (HSP90) was identified and confirmed as being O-GlcNAcylated in mouse COCs under hyperglycaemic conditions (modelled using glucosamine), causing detrimental outcomes for embryo development. O-GlcNAcylation of proteins occurs as a result of increased activity of the hexosamine biosynthesis pathway, which provides substrates for cumulus matrix production during COC maturation, and also for O-GlcNAcylation. COCs matured under hyperglycaemic conditions have decreased developmental competence, mediated at least in part through the mechanism of increased O-GlcNAcylation. This study was designed to examine the effect of hyperglycaemic conditions (using the hyperglycaemic mimetic, glucosamine) on O-GlcNAc levels in the mouse COC, and furthermore to identify potential candidate proteins which are targets of this modification, and their roles in oocyte maturation. COCs from 21-day-old superovulated CBA × C57BL6 F1 hybrid female mice were matured in vitro (IVM). Levels of O-GlcNAcylated proteins, HSP90 and O-GlcNAc transferase (OGT, the enzyme responsible for O-GlcNAcylation) in COCs were measured using western blot, and localization observed using immunocytochemistry. For glycosylated HSP90 levels, and to test OGT-HSP90 interaction, immunoprecipitation was performed prior to western blotting. Embryo development was assessed using in vitro fertilization and embryo culture post-maturation. Addition of the hyperglycaemic mimetic glucosamine to IVM medium for mouse COCs increased detectable O-GlcNAcylated protein levels (by western blot and immunocytochemistry), and this effect was reversed using an OGT inhibitor (P < 0.05). HSP90 was identified as a target of O-GlcNAcylation in the COC, and inhibition of HSP90 during IVM reversed glucosamine-induced decreases in oocyte developmental competence (P < 0.05). We also demonstrated the novel finding of an association between HSP90 and OGT in COCs, suggesting a possible client-chaperone relationship. In vitro maturation of COCs was used so that treatment time could be limited to the 17 h of maturation prior to ovulation. Additionally, glucosamine, a hyperglycaemic mimetic, was used because it specifically activates the hexosamine pathway which provides the O-GlcNAc moieties. The results in this study should be confirmed using in vivo models of hyperglycaemia and different HSP90 inhibitors. This study leads to a new understanding of how diabetes influences oocyte competence and provides insight into possible therapeutic interventions based on inhibiting HSP90 to improve oocyte quality. This work was supported by a programme grant from the National Health and Medical Research Council, Australia, ID 453556. J.G.T. is a recipient of funding from and a consultant to Cook Medical Pty Ltd. The other authors have no conflicts of interest to declare.

Amphiregulin co-operates with bone morphogenetic protein 15 to increase bovine oocyte developmental competence: effects on gap junction-mediated metabolite supply.

Publisher: Oxford University Press (OUP)

Date: 19-02-2014

DOI: 10.1093/MOLEHR/GAU013

Abstract: This study assessed the participation of hiregulin (AREG) and bone morphogenetic protein 15 (BMP15) during maturation of bovine cumulus-oocyte complexes (COCs) on cumulus cell function and their impact on subsequent embryo development. AREG treatment of COCs enhanced blastocyst formation and quality only when in the presence of BMP15. Expression of hyaluronan synthase 2 was enhanced by follicle-stimulating hormone (FSH) but not by AREG, which was reflected in the level of cumulus expansion. Although both FSH and AREG stimulated glycolysis, AREG-treated COCs had higher glucose consumption, lactate production and ratio of lactate production to glucose uptake. Autofluorescence levels in oocytes, indicative of NAD(P)H and FAD(++), were increased with combined AREG and BMP15 treatment of COCs. In contrast, these treatments did not alter autofluorescence levels when cumulus cells were removed from oocytes, even in the presence of other COCs, suggesting that oocyte-cumulus gap-junctional communication (GJC) is required. FSH contributed to maintaining GJC for an extended period of time. Remarkably, BMP15 was equally effective at maintaining GJC even in the presence of AREG. Hence, AREG stimulation of COC glycolysis and BMP15 preservation of GJC may facilitate efficient transfer of metabolites from cumulus cells to the oocyte thereby enhancing oocyte developmental competence. These results have implications for improving in vitro oocyte maturation systems.

Oxygen concentration during in vitro maturation of murine oocytes affects blastocyst cell lineage

Publisher: CSIRO Publishing

Date: 2005

DOI: 10.1071/SRB05ABS233

Abstract: Follicular antral oxygen tension is thought to influence subsequent oocyte developmental competence. Despite this, in vitro maturation (IVM) is routinely performed in either 5 or 20% O2 and while low O2 has been shown to be beneficial to embryo development in many species, the effect of altering O2 concentration during IVM has not been adequately investigated. Here we investigated the effects of a range of O2 concentrations during IVM on meiotic maturation and subsequent embryo development after IVF. Ovaries from eCG-stimulated CBA F1 female mice (21 days) were collected and intact cumulus oocyte complexes (COCs) cultured for 17–18 h under 2, 5, 10 or 20% O2 (6% CO2 and balance of N2). Matured COCs were denuded of cumulus cells, fixed and stained (1% aceto-orcein) for visualisation of maturation status. No significant difference in maturation rates between treatment groups was observed. Following IVF (performed under 5% O2, 6% CO2 and balance of N2), no difference in fertilisation rates between treatment groups was observed in a randomly selected cohort 7 h post-fertilisation. There was also no significant difference in cleavage rates after 24 h or ability to reach blastocyst stage after 96 h, with a tendency (P = 0.079) for more blastocysts in 2% O2. However there was a significant increase in the number of trophectoderm cells present in the resulting blastocysts (P 0.05) in the 2% O2 group (35 ± 2.1) compared to 20% O2 (25 ± 2.8). Our data suggests that O2 concentration during IVM does not influence nuclear maturation or subsequent fertilisation, cleavage and blastocyst development rates. However, maturation in 2% O2 significantly alters subsequent cell lineage within blastocysts to favour trophectoderm development. Such skewed trophectoderm cell number may influence embryo viability. Funded by NHMRC and NIH.

Phenotypes of the ovarian follicular basal lamina predict developmental competence of oocytes

Publisher: Oxford University Press (OUP)

Date: 26-12-2008

DOI: 10.1093/HUMREP/DEN447

Oocytes prevent bovine cumulus cell apoptosis by maintaining a morphogenic paracrine gradient of bone morphogenetic proteins

Publisher: CSIRO Publishing

Date: 2005

DOI: 10.1071/SRB05ABS232

Abstract: Paracrine factors secreted by the oocyte regulate a broad range of cumulus cell (CC) functions. Previously we have shown that the low incidence of apoptosis in CCs is due to unidentified oocyte-secreted factors (OSF) acting in an anti-apoptotic manner. Here we examine the nature of the paracrine network of oocyte BMP growth factors and their binding proteins regulating CC apoptosis. Bovine cumulus–oocyte complexes (COC) were aspirated from abattoir-derived ovaries and oocytes microsurgically removed to create oocytectomized (OOX) complexes. OOX were treated with denuded oocytes (DO) or various growth factors for 24 h, then CC apoptosis was assessed using TUNEL together with confocal microscopy plus image analysis and by Western blotting for Bcl-2 and Bax. CC apoptosis was significantly (P 0.001) reduced by DO, bone morphogenetic protein 15 (BMP15), BMP6 or BMP7 as assessed by TUNEL. Accordingly, expression of anti-apoptotic Bcl-2 was high in OOX+DO and OOX+BMP15, and low with OOX+GDF9 and OOX alone, whereas the reverse was observed for pro-apoptotic Bax. Combined treatment of OOXs with BMP6 and BMP15 did not further decrease apoptosis levels beyond that of BMP15 alone (P 0.05), suggesting no additive effect of these two BMPs. Follistatin (FS) effectively antagonized BMP15 anti-apoptotic effects, and likewise, a BMP6 neutralizing antibody (NAb) antagonized the inhibitory effect of BMP6. Gremlin blocked BMP7 anti-apoptotic effects on CCs, but had no significant effect on BMP15. FS or BMP6. NAb antagonized ~50% of the anti-apoptotic activity of oocytes however, these effects were not additive suggesting the additional involvement of other OSF. These results indicate for the first time that OSF (BMP15 and BMP6 in particular) maintains the low incidence of CC apoptosis by establishing a localized morphogenic gradient of bone morphogenetic proteins.

Observations on the loss of the cellular vestment surrounding superovulated sheep oocytes

Publisher: Elsevier BV

Date: 12-1987

DOI: 10.1016/0378-4320(87)90040-6

Oocyte-secreted factor(s) regulate apoptosis of bovine cumulus cells

Publisher: CSIRO Publishing

Date: 2004

DOI: 10.1071/SRB04ABS252

Abstract: Paracrine factors secreted by the oocyte affect cumulus cell proliferation and differentiation. These factors may also act in an anti-apoptotic manner, maintaining the low incidence of cellular apoptosis within cumulus cells. The purpose of this study was to determine whether the incidence of apoptosis within cumulus cells is regulated by oocyte-secreted factors (OSF). Bovine cumulus-oocyte complexes (COC) aspirated from abattoir-derived ovaries were randomly allocated to 3 treatments: (1) groups of 5 intact COC (2) groups of 5 oocytectomised complexes (OOX), where oocytes were removed microsurgically and (3) groups of 5 OOX co-cultured with 25 denuded oocytes (DO). To examine a dose effect of OSF, OOX were also cultured with increasing numbers of DO (OOX+5DO, OOX+25DO, OOX+50DO). In both experiments, complexes were cultured in their respective treatments for 24 h in 50 �l of oocyte maturation medium (+/– rFSH 0.1 IU/mL). Apoptosis was assessed using TUNEL, with all nuclei counterstained with propidum iodide (PI). Nikon TE2000 laser confocal scanning microscopy was used to visualise and quantify the incidence of apoptosis (TUNEL/PI). The proportion of apoptotic cells was determined by image analysis. Oocyte removal lead to a significant increase in cumulus cell apoptosis (OOX, 67% apoptotic COC, 15% P� �0.001). However, the incidence of apoptosis in OOX was restored to COC levels when co-cultured with DO (18% P� �0.05, COC v. OOX+DO). FSH prevented apoptosis in all treatment groups (P� �0.05) decreasing the incidence by 27% in OOX and by 12% in COC. Cumulus cell apoptosis within OOX (+/– FSH) was reduced in a dose dependent manner by treating with increasing numbers of DO. These results indicate for the first time that oocyte-secreted factor(s) regulate the incidence of apoptosis within cumulus cells.

Vitrification within a nanoliter volume: oocyte and embryo cryopreservation within a 3D photopolymerized device

Publisher: Springer Science and Business Media LLC

Date: 11-08-2022

DOI: 10.1007/S10815-022-02589-8

Abstract: Vitrification permits long-term banking of oocytes and embryos. It is a technically challenging procedure requiring direct handling and movement of cells between potentially cytotoxic cryoprotectant solutions. Variation in adherence to timing, and ability to trace cells during the procedure, affects survival post-warming. We hypothesized that minimizing direct handling will simplify the procedure and improve traceability. To address this, we present a novel photopolymerized device that houses the s le during vitrification. The fabricated device consisted of two components: the Pod and Garage . Single mouse oocytes or embryos were housed in a Pod, with multiple Pods docked into a Garage. The suitability of the device for cryogenic application was assessed by repeated vitrification and warming cycles. Oocytes or early blastocyst-stage embryos were vitrified either using standard practice or within Pods and a Garage and compared to non-vitrified control groups. Post-warming, we assessed survival rate, oocyte developmental potential (fertilization and subsequent development) and metabolism (autofluorescence). Vitrification within the device occurred within ~ 3 nL of cryoprotectant: this volume being ~ 1000-fold lower than standard vitrification. Compared to standard practice, vitrification and warming within our device showed no differences in viability, developmental competency, or metabolism for oocytes and embryos. The device housed the s le during processing, which improved traceability and minimized handling. Interestingly, vitrification-warming itself, altered oocyte and embryo metabolism. The Pod and Garage system minimized the volume of cryoprotectant at vitrification—by ~ 1000-fold—improved traceability and reduced direct handling of the s le. This is a major step in simplifying the procedure.

Artificial blastocyst collapse prior to vitrification significantly improves Na+/K+-ATPase-dependent post-warming blastocoel re-expansion kinetics without inducing endoplasmic reticulum stress gene ex

Publisher: CSIRO Publishing

Date: 2019

DOI: 10.1071/RD17500

Abstract: Blastocoel expansion during embryo development is known to be reliant on the Na+/K+-ATPase pump, but little is known about the relative contribution of active (Na+/K+-ATPase pump) and facilitated diffusion (aquaporins) water transport during blastocoel re-expansion after vitrification. The aims of this study were to examine potential effects of artificial blastocoel collapse (ABC) on markers of embryo stress and the contribution of active and facilitated diffusion water transport mechanisms to blastocoel re-expansion. Day 5 mouse embryos were vitrified using either a standard protocol, laser pulse ABC, a hyperosmotic sucrose ABC protocol or both laser pulse and sucrose. Using real-time polymerase chain reaction, no differences were found in the gene expression of the endoplasmic reticulum (ER) stress markers activating transcription factor 4 (Atf4) or heat shock protein 90-alpha (Hsp90α) 2h after warming. Similarly, expression of the Na+/K+-ATPase pump gene, ATPase, Na+/K+ transporting, beta 1 polypeptide (Atp1b1) and protein did not differ between groups. Aquaporin 8 (Aqp8) gene expression was significantly lower in the laser+sucrose ABC group than in fresh controls, and aquaporin 3 (Aqp3) expression significantly higher in standard vitrified embryos compared with all other groups. Ouabain, a potent and specific Na+/K+-ATPase pump inhibitor, inhibited blastocoel re-expansion in both standard protocol- and laser ABC-vitrified embryos, reducing both groups to the same rate of re-expansion 3h after warming. These results demonstrate that ABC before vitrification does not alter mRNA or protein expression of Na+/K+-ATPase, or mRNA levels of ER stress genes Atf4 and Hsp90α. Activity of the pump may be increased in ABC embryos, with potential compensation by AQP3 when it is compromised.

Exogenous protein affects developmental competence and metabolic activity of bovine pre-implantation embryos in vitro

Publisher: CSIRO Publishing

Date: 1998

DOI: 10.1071/R98077

Abstract: The role of exogenous protein during bovine pre-implantation embryo development in two in vitro production systems was investigated. Morphological development, survival after vitrification and metabolic activity before and after vitrification were recorded in blastocysts generated in vitro in synthetic oviduct fluid (SOF) medium in the presence of either bovine serum albumin (BSA) or polyvinyl-alcohol (PVA). Metabolic activity was determined by measuring oxygen consumption, glucose and pyruvate uptake as well as lactate production. Development to blastocysts and survival after vitrification were reduced significantly in medium lacking protein. Of the metabolic parameters measured, only pyruvate uptake was increased significantly in embryos cultured in medium supplemented with PVA. Whereas in BSA-supplemented medium pyruvate uptake was correlated with lactate production, in PVA-supplemented medium glucose uptake was correlated with lactate production. Lactate production increased significantly after vitrification as compared with fresh embryos. Thus, exogenously added protein significantly alters oxidative metabolism. In medium lacking protein, the additional pyruvate may be used for the maintenance of intracellular amino acid pools. Vitrification appears to alter glycolytic metabolic profiles indicating a stress-response. In conclusion, the perturbed metabolism corresponding to reduced developmental capacity of embryos produced under protein-free conditions emphasizes the ambiguity between maximum develop-ment, technical and hygienic requirements and physiological demands of the early bovine embryo in vitro. The use of well-defined recombinant proteins might assist in closing this gap.

Epigenetic risks related to assisted reproductive technologies: Short- and long-term consequences for the health of children conceived through assisted reproduction technology: more reason for caution

Publisher: Oxford University Press (OUP)

Date: 11-2002

DOI: 10.1093/HUMREP/17.11.2783

Abstract: Does the manipulation of gametes and embryos as practised in human IVF invoke perturbations in fetal and neonatal phenotype? There is increasing evidence that the answer is 'yes', although the degree of perturbation may be less acute than observed in other species. However, the long-term consequences are not known, and may prove to be considerable. There is now a substantial body of evidence from animal models suggesting that assisted reproductive technologies (ART) are associated with altered outcomes in fetal and neonatal development. Epigenetic modification of gene expression is an attractive hypothesis that accounts for these differences and is one of a number of causal pathways that may be activated by cellular stress invoked during manipulation. Here we widen the debate to propose that environment-induced cellular stress also acts to modify fetal and placental gene expression, potentially also contributing to phenotype skewing after ART.

Effect of epidermal growth factor-like peptides on the metabolism of in vitro-matured mouse oocytes and cumulus cells

Publisher: Oxford University Press (OUP)

Date: 03-2014

DOI: 10.1095/BIOLREPROD.113.115311

Abstract: Oocyte in vitro maturation (IVM) is an assisted reproductive technology that involves the maturation of cumulus-oocyte complexes (COCs) that are then capable of normal development. We have shown that epidermal growth factor (EGF)-like peptide signaling is perturbed in mouse COCs undergoing IVM when matured with follicle-stimulating hormone (FSH) and/or EGF, but supplementation of IVM with EGF-like peptides hiregulin or epiregulin improves oocyte developmental competence. Here we aimed to determine whether EGF-like peptides regulate COC metabolism. Immature 129/Sv mouse COCs underwent IVM with FSH, EGF, hiregulin, epiregulin, betacellulin, or no treatment (control). Epiregulin significantly increased intraoocyte flavin adenine dinucleotide (FAD) and REDOX (reduction and oxidation) ratio compared to FSH and control. Amphiregulin and epiregulin significantly increased the proportion of J aggregates (from JC-1) in oocyte mitochondria compared to control, FSH, or EGF, and this coupled with FAD and REDOX measures indicates greater mitochondrial activity. There were no differences in glucose consumption, lactate production, or glycolysis between COCs matured with FSH, EGF, and EGF-like peptides. COCs matured with EGF or EGF-like peptides exhibited significantly higher mRNA expression of the hexosamine biosynthesis pathway (HBP) rate-limiting enzyme gene Gfpt2, Has2 expression, and global beta-O-linked glycosylation of proteins, compared to control or FSH, suggesting greater HBP activity. Our findings suggest that 1) EGF-like peptides, particularly epiregulin, induce more oocyte mitochondrial activity than EGF or FSH and 2) EGF-like peptides and EGF induce greater HBP activity, enabling more hyaluronic acid synthesis and protein beta-O-linked glycosylation. These metabolic alterations may be a mechanism by which EGF-like peptides increase oocyte developmental competence.

Current status and future trends of the clinical practice of human oocyte in vitro maturation

Publisher: Cambridge University Press

Date: 31-03-2011

DOI: 10.1017/CBO9780511734755.018

The temporal relationship between oocyte maturation and early fertilisation events in relation to the pre-ovulatory LH peak and preimplantation embryo development in red deer (Cervus elaphus)

Publisher: Elsevier BV

Date: 05-2008

DOI: 10.1016/J.ANIREPROSCI.2007.03.013

Abstract: The temporal relationships among oocyte maturation, gamete transport and fertilisation following the pre-ovulatory luteinsing hormone surge in red deer were established and secondly, early preimplantation development to the blastocyst stage in relation to the onset of oestrus was determined for red deer. In the first series of observations, oestrus was synchronised in April (N=22), for the fixed time recovery of gametes from 0 to 36 h after the estimated pre-ovulatory LH peak. Matings were observed and the time of the LH peak was determined from the retrospective analysis of blood plasma collected at 3h intervals. Gametes were recovered surgically and the meiotic status of follicular and ovulated oocytes assessed. Spermatozoa were recovered from the oviduct and their motility analysed by videomicroscopy. Nineteen of 22 hinds exhibited a pre-ovulatory LH surge and were observed to mate. Oocyte metaphase I occurred between 11 and 18 h, and metaphase II was completed within the follicle between 20 and 25 h following the pre-ovulatory LH peak. Fertilised ova were recovered from 30 to 36 h in both the ulla and isthmic portions of the oviduct. Motile spermatozoa were first recovered from the isthmus and the ulla at 13 and 21 h, respectively, after the LH peak. Hyperactive spermatozoa were observed in both the isthmus and the ulla flushings but only from the eight hinds that had ovulated. In the second series of observations, 16 mature hinds were synchronised and allocated to groups for embryo collection on days 3, 5 and 7 after oestrus. Eight embryos were recovered an 8-cell at 90 h, 3 morulae at 137, 138 and 186 h, and 4 blastocysts at 180, 182 and 190 h post-mating. Blastocysts were only recovered from the uterine horns and the mean+/-S.E.M. number of nuclei per blastocyst was 93.5+/-10.0 with a range of 66-114 cells. The results of this study will improve the application of assisted reproductive technologies to red deer as they indicate that oocyte maturation, fertilisation and early embryonic development of the red deer is similar to other domestic ruminants with the exception that the red deer embryo enters the uterus at the blastocyst stage.

Cumulus expansion and glucose utilisation by bovine cumulus-oocyte complexes during in vitro maturation: The influence of glucosamine and follicle-stimulating hormone

Publisher: Bioscientifica

Date: 09-2004

DOI: 10.1530/REP.1.00225

Abstract: Glucose is an important metabolite and its presence during in vitro oocyte maturation (IVM) can have profound effects on the oocyte’s developmental capacity. We have demonstrated that glucose uptake increases over a 24 h IVM period, with most accounted for as l -lactate production. However, as maturation proceeds, l -lactate production remains constant, suggesting an alternative role for glucose metabolism. We hypothesised that in the latter stages of oocyte maturation, glucose not accounted for by l -lactate production is utilised for FSH-stimulated extracellular matrix (ECM) synthesis. To examine precursor utilisation for synthesis of ECM, bovine cumulus–oocyte complexes (COCs) were matured in ± FSH and/or glucosamine (an alternative substrate of matrix components). Measurements included COC diameters, glucose consumption and l -lactate production in spent media and [U- 14 C]glucose incorporation into ECM. FSH significantly stimulated both diameter and glucose consumption during 20–24 h maturation compared with unstimulated complexes, although co-incubation with glucosamine and FSH decreased total glucose consumption 1.7-fold compared with FSH alone ( P 0.05). Furthermore, there was a linear relationship between glucose and l -lactate metabolism in the presence of glucosamine, suggesting that the majority of glucose was being utilised for l -lactate production via glycolysis. In the presence of glucosamine, twofold less [U- 14 C]glucose was incorporated into matrix compared with COCs cultured without glucosamine. These results support the hypothesis that there is a link between glucose and glucosamine uptake in FSH-stimulated ECM synthesis. Furthermore, glucose has multiple fates within the COC during maturation and levels of utilisation are dependent on the composition of the maturation environment.

Inhibitors of mitochondrial ATP production at the time of compaction improve development of in vitro produced porcine embryos

Publisher: Wiley

Date: 04-12-2000

DOI: 10.1002/1098-2795(200101)58:1<39::AID-MRD6>3.0.CO;2-B

Female tract cytokines and developmental programming in embryos

Publisher: Springer New York

Date: 2015

DOI: 10.1007/978-1-4939-2480-6_7

Abstract: In the physiological situation, cytokines are pivotal mediators of communication between the maternal tract and the embryo. Compelling evidence shows that cytokines emanating from the oviduct and uterus confer a sophisticated mechanism for 'fine-tuning' of embryo development, influencing a range of cellular events from cell survival and metabolism, through ision and differentiation, and potentially exerting long-term impact through epigenetic remodelling. The balance between survival agents, including GM-CSF, CSF1, LIF, HB-EGF and IGFII, against apoptosis-inducing factors such as TNFα, TRAIL and IFNg, influence the course of preimplantation development, causing embryos to develop normally, adapt to varying maternal environments, or in some cases to arrest and undergo demise. Maternal cytokine-mediated pathways help mediate the biological effects of embryo programming, embryo plasticity and adaptation, and maternal tract quality control. Thus maternal cytokines exert influence not only on fertility and pregnancy progression but on the developmental trajectory and health of offspring. Defining a clear understanding of the biology of cytokine networks influencing the embryo is essential to support optimal outcomes in natural and assisted conception.

The effects of glucose and glucosamine on cumulus expansion and nuclear maturation of bovine cumulus-oocyte complexes

Publisher: CSIRO Publishing

Date: 2004

DOI: 10.1071/RDV16N1AB327

Abstract: Glucose is the primary energy substrate consumed by bovine COCs during in vitro maturation (IVM), with most accounted for by glycolysis (L-lactate production). However, antral follicular fluid (FF) contains less than half the glucose of standard IVM media (TCM199=5.6mM, FF=2.3mM). We have previously demonstrated that from 20 to 24h of IVM, a significant proportion of the glucose utilized is directed into pathways other than lactate production (Sutton et al., 2003 Reproduction 126, 27–34). We hypothesize that glucose is utilized for cumulus matrix synthesis. The aim of this study was to determine the influence of glucosamine (an intermediate for matrix components) on FSH-stimulated glucose uptake and cumulus expansion. The influence of different glucose concentrations and glucosamine on nuclear maturation was also investigated. Bovine COCs were collected from abattoir-derived ovaries. In Exp. 1, in idual COCs (n=60, 3 replicates) were cultured in 10-μL drops of TCM199 (plus pyruvate, hCG and BSA, containing 5.6mM glucose), ±FSH (0.1IUmL−1) and ±glucosamine (5mM). After 20h, COCs were transferred to fresh media and cultured a further 4h. Cumulus expansion and glucose/L-lactate levels in spent medium from 0–4-h and 20–24-h culture periods were measured. In Experiment 2, COCs (n=300, 6 replicates) were cultured in groups of 10 in 100μL of Bovine FF medium (a defined medium based on the composition of bovine antral FF, also containing amino acids, FSH, hCG and BSA) ±glucosamine (5mM) in 2.3 or 5.6mM glucose, or in conventional TCM199 IVM media (as above). Nuclear maturation was assessed at 24 and 30h using orcein staining. Treatment differences were determined using two-way ANOVA. The influence of FSH and glucosamine (Exp. 1) on the measured parameters was evident at 20–24h, with FSH increasing diameter, glucose uptake and L-lactate production (P& .05). Although glucosamine alone did not influence diameter or glucose/L-lactate concentrations, glucosamine plus FSH led to a decrease in glucose uptake compared to FSH-stimulation alone (P& .05). The proportion of oocytes at MII (Exp. 2) was significantly lower when COCs were cultured in low glucose (main effect, 24h: 2.3mM=38% v. 5.6mM=64% P& .005). The presence of glucosamine tended to stimulate meiotic maturation (main effect, 24h: 0mM=45% v. 5mM=59% P=0.1). MII frequency in TCM199 controls at 24h was 68%. These experiments support the hypothesis that synthesis of cumulus matrix is a major pathway for glucose metabolism, especially in the absence of glucosamine. Furthermore, oocytes matured in media based on a physiological concentration of glucose (2.3mM), have delayed meiosis compared to oocytes cultured in higher glucose (5.6mM). Thus, glucose has multiple functions, involving matrix formation and meiosis regulation during bovine IVM. Supplementation of medium with glucosamine appears to partly reduce the dependency of COCs on glucose. Supported by Australian Research Council and COOK Australia.

Molecular Filtration Properties of the Mouse Expanded Cumulus Matrix: Controlled Supply of Metabolites and Extracellular Signals to Cumulus Cells and the Oocyte1

Publisher: Oxford University Press (OUP)

Date: 10-2012

DOI: 10.1095/BIOLREPROD.111.096271

Abstract: While formation of the expanded cumulus matrix and its importance for oocyte maturation and ovulation are well described, its function in these processes remains unknown. The degree of expansion and expression of cumulus matrix genes are positively correlated with oocyte quality, suggesting that this matrix plays a key role in oocyte maturation. Based on recognized filtration properties of analogous matrices, we investigated whether the cumulus matrix acts as a molecular filter by assessing diffusion of fluorescently labeled dextrans (neutral and negatively charged) and hydrophilic (glucose) and hydrophobic (cholesterol) metabolites in cumulus oocyte complexes (COCs). Expanded in vivo-matured COCs resisted absorption of glucose and cholesterol compared to unexpanded COCs. In vitro-matured (IVM) COCs have a pronounced deficiency in cumulus matrix proteins and have poor oocyte quality. Here we demonstrate that IVM cumulus matrix has deficient filtration properties, with dextran and glucose and cholesterol molecules diffusing more readily into IVM than in vivo-matured COCs. Taking the inverse approach, we found that prostaglandin E2 (PGE2), synthesized by cumulus cells, is retained within the matrix of in vivo-matured COCs but IVM COCs have reduced capacity to retain PGE2, secreting significantly more into the medium. This is the first demonstration of a biophysical property of the cumulus matrix. The ability to regulate metabolite supply from the surrounding environment while sequestering vital signaling factors, such as PGE2, is likely to impact oocyte maturation. Thus, IVM may reduce oocyte quality due to dysregulated control of metabolites and signaling molecules.

Extending prematuration with cAMP modulators enhances the cumulus contribution to oocyte antioxidant defence and oocyte quality via gap junctions

Publisher: Oxford University Press (OUP)

Date: 22-02-2016

DOI: 10.1093/HUMREP/DEW020

Abstract: Can bovine oocyte antioxidant defence and oocyte quality be improved by extending the duration of pre-in vitro maturation (IVM) with cyclic adenosine mono-phosphate (cAMP) modulators? Lengthening the duration of cAMP-modulated pre-IVM elevates intra-oocyte reduced glutathione (GSH) content and reduces hydrogen peroxide (H2O2) via increased cumulus cell-oocyte gap-junctional communication (GJC), associated with an improvement in subsequent embryo development and quality. Oocytes are susceptible to oxidative stress and the oocyte's most important antioxidant glutathione is supplied, at least in part, by cumulus cells. A temporary inhibition of spontaneous meiotic resumption in oocytes can be achieved by preventing a fall in cAMP, and cyclic AMP-modulated pre-IVM maintains cumulus-oocyte GJC and improves subsequent embryo development. This study consisted of a series of 10 experiments using bovine oocytes in vitro, each with multiple replicates. A range of pre-IVM durations were examined as the key study treatments which were compared with a control. The study was designed to examine if one of the oocyte's major antioxidant defences can be enhanced by pre-IVM with cAMP modulators, and to examine the contribution of cumulus-oocyte GJC on these processes. Immature bovine cumulus-oocyte complexes were treated in vitro without (control) or with the cAMP modulators 100 µM forskolin (FSK) and 500 µM 3-isobutyl-1-methyxanthine (IBMX), for 0, 2, 4 or 6 h (pre-IVM phase) prior to IVM. Oocyte developmental competence was assessed by embryo development and quality post-IVM/IVF. Cumulus-oocyte GJC, intra-oocyte GSH and H2O2 were quantified at various time points during pre-IVM and IVM, in the presence and the absence of functional inhibitors: carbenoxolone (CBX) to block GJC and buthionine sulfoximide (BSO) to inhibit glutathione synthesis. Pre-IVM with FSK + IBMX increased subsequent blastocyst formation rate and quality compared with standard IVM (P < 0.05), regardless of pre-IVM duration. The final blastocyst yields (proportion of blastocysts/immature oocyte) were 26.3% for the control, compared with 39.2, 35.2 and 34.2%, for the 2, 4 and 6 h pre-IVM FSK + IBMX treatments, respectively. In contrast to standard IVM (control), pre-IVM with cAMP modulators maintained open gap junctions between cumulus cells and oocytes for the duration (6 h) of pre-IVM examined, and persisted for a further 8 h in the IVM phase. Cyclic AMP-modulated pre-IVM increased intra-oocyte GSH levels at the completion of both pre-IVM and IVM, in a pre-IVM duration-dependent manner (P < 0.05), which was ablated when GJC was blocked using CBX (P < 0.05). By 4 h of pre-IVM treatment with cAMP modulators, oocyte H2O2 levels were reduced compared the control (P < 0.05), although this beneficial effect was lost when oocytes were co-treated with BSO. Inhibiting glutathione synthesis with BSO during pre-IVM ablated any positive benefits of cAMP-mediated pre-IVM on oocyte developmental competence (P < 0.01). It is unclear if the improvement in oocyte antioxidant defence and developmental competence reported here is due to direct transfer of total and/or reduced glutathione from cumulus cells to the oocyte via gap junctions, or whether a GSH synthesis signal and/or amino acid substrates are supplied to the oocyte via gap junctions. Embryo transfer experiments are required to determine if the cAMP-mediated improvement in blastocyst rates leads to improved live birth rates. IVM offers significant benefits to infertile and cancer patients and has the potential to significantly alter ART practice, if IVM efficiency in embryo production could be improved closer to that of conventional IVF (using ovarian hyperstimulation). Pre-IVM with cAMP modulators is a simple and reliable means to improve IVM outcomes. This work was supported by grants and fellowships from the National Health and Medical Research Council of Australia (1007551, 627007, 1008137, 1023210) and by scholarships from the Chinese Scholarship Council (CSC) awarded to H.J.L. and the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad awarded to S.S. The Fluoview FV10i confocal microscope was purchased as part of the Sensing Technologies for Advanced Reproductive Research (STARR) facility, funded by the South Australian Premier's Science and Research Fund. We acknowledge partial support from the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). We declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

In vitro development of early sheep embryos is superior in medium supplemented with human serum compared with sheep serum or human serum albumin

Publisher: Elsevier BV

Date: 09-1992

DOI: 10.1016/0378-4320(92)90020-E

Hyperglycaemia: A new player in oocyte epigenetics

Publisher: Elsevier BV

Date: 09-2014

DOI: 10.1016/J.ANIREPROSCI.2014.06.017

Microstructured Optical Fibers and Live Cells: A Water-Soluble, Photochromic Zinc Sensor

Publisher: American Chemical Society (ACS)

Date: 13-09-2013

DOI: 10.1021/BM401040V

Abstract: A new biologically compatible Zn(II) sensor was fabricated by embedding a Zn(II) sensing spiropyran within the surface of a liposome derived from Escherichia coli lipids (LSP2). Solution-based experiments with increasing Zn(II) concentrations show improved aqueous solubility and sensitivity compared to the isolated spiropyran molecule (SP2). LSP2 is capable of sensing Zn(II) efflux from dying cells with preliminary data indicating that sensing is localized near the surface membrane of HEK 293 cells. Finally, LSP2 is suitable for development into a nanoliter-scale dip-sensor for Zn(II) using microstructured optical fiber as the sensing platform to detect Zn(II) in the range of 100 ρM with minimal photobleaching. Existing spiropyran based sensing molecules can thus be made biologically compatible, with an ability to operate with improved sensitivity using nanoscale liquid s le volumes. This work represents the first instance where photochromic spiropyran molecules and liposomes are combined to create a new and multifunctional sensing entity for Zn(II).

The effect of discrete wavelengths of visible light on the developing murine embryo

Publisher: Springer Science and Business Media LLC

Date: 23-06-2022

DOI: 10.1007/S10815-022-02555-4

Abstract: A current focus of the IVF field is non-invasive imaging of the embryo to quantify developmental potential. Such approaches use varying wavelengths to gain maximum biological information. The impact of irradiating the developing embryo with discrete wavelengths of light is not fully understood. Here, we assess the impact of a range of wavelengths on the developing embryo. Murine preimplantation embryos were exposed daily to wavelengths within the blue, green, yellow, and red spectral bands and compared to an unexposed control group. Development to blastocyst, DNA damage, and cell number/allocation to blastocyst cell lineages were assessed. For the longer wavelengths (yellow and red), pregnancy/fetal outcomes and the abundance of intracellular lipid were investigated. Significantly fewer embryos developed to the blastocyst stage when exposed to the yellow wavelength. Elevated DNA damage was observed within embryos exposed to blue, green, or red wavelengths. There was no effect on blastocyst cell number/lineage allocation for all wavelengths except red, where there was a significant decrease in total cell number. Pregnancy rate was significantly reduced when embryos were irradiated with the red wavelength. Weight at weaning was significantly higher when embryos were exposed to yellow or red wavelengths. Lipid abundance was significantly elevated following exposure to the yellow wavelength. Our results demonstrate that the impact of light is wavelength-specific, with longer wavelengths also impacting the embryo. We also show that effects are energy-dependent. This data shows that damage is multifaceted and developmental rate alone may not fully reflect the impact of light exposure.

Influence of oocyte-secreted factors and culture duration on the metabolic activity of bovine cumulus cell complexes

Publisher: Bioscientifica

Date: 07-2003

DOI: 10.1530/REP.0.1260027

Abstract: Intracellular communication between the cumulus cell complex and the oocyte is essential for numerous processes during oocyte maturation. The aim of this study was to determine the interaction between oocyte-secreted factors and the metabolic activity of bovine cumulus cell complexes during in vitro maturation (IVM). Cumulus-oocyte complexes (COCs) were aspirated from ovaries derived from an abattoir and ided into four treatment groups: (i) intact COCs, (ii) oocytectomized complexes (OOX), in which the ooplasm was microsurgically removed, (iii) OOX co-cultured with denuded oocytes (OOX+DO) and (iv) DO. The complexes were cultured in idually in IVM media. After 0-4, 10-14 and 20-24 h of culture, the utilization of oxygen, glucose, pyruvate and L-lactate by the complexes was measured. The metabolic activity of the DO was undetectable. There were no significant differences in metabolic measurement among any of the treatment groups, indicating that the metabolism of the cumulus complex is not affected by the presence of the oocyte. When metabolic activity for the complexes was analysed relative to time in culture, there was an approximate twofold increase in the consumption of oxygen, glucose and pyruvate over the 24 h period (P .05), although production of L-lactate remained constant. The relationship between total glucose uptake and L-lactate production indicated that the majority of glucose consumed at the start of culture was being utilized via glycolysis, but by the cessation of the maturation period, there was significant utilization of glucose elsewhere, possibly for the formation of cumulus extracellular matrix. These results indicate that metabolism of COC does not reflect biochemical activity of the oocyte. Nevertheless, the metabolic requirements of the COC increase throughout maturation.

Metabolic differences in bovine cumulus-oocyte complexes matured in vitro in the presence or absence of follicle-stimulating hormone and bone morphogenetic protein 15

Publisher: Oxford University Press (OUP)

Date: 10-2012

DOI: 10.1095/BIOLREPROD.112.102061

Abstract: Bidirectional communication between cumulus cells and the oocyte is necessary to achieve oocyte developmental competence. The aim of the present study was to examine the effects of recombinant human bone morphogenetic protein 15 (rhBMP15) and follicle-stimulating hormone (FSH) supplementation on bovine cumulus-oocyte complex (COC) metabolism during maturation. Bovine COCs were matured in the presence of absence of FSH, rhBMP15, or both for 23 h. The addition of FSH and rhBMP15 increased blastocyst development (without rhBMP15 and FSH, 28.4% ± 7.4% with FSH and rhBMP15, 51.5% ± 5.4% P < 0.05). Glucose uptake and lactate production was significantly increased by greater than 2-fold with FSH (P < 0.05), whereas rhBM15 supplementation did not increase these levels. rhBMP15 supplementation (regardless of FSH) significantly decreased ADP levels in COCs, leading to an increase in ATP:ADP ratios (P < 0.05). Indicators of mitochondrial activity and cellular REDOX, oxidized flavin adenine dinucleotide (FAD(++)) and reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), levels within the oocyte of COCs were significantly higher with rhBMP15 alone, whereas the presence of FSH diminished the rhBMP15 effect. Regardless of treatment, no changes in REDOX state (FAD(++):NAD(P)H). The significant increase in FAD(++) and NAD(P)H in COCs with rhBMP15 was mediated via cumulus cells, because no differences were found in denuded oocytes cultured in the presence or absence of FSH, rhBMP15, or both. The present study demonstrates that a principal metabolic consequence of FSH supplementation of COCs is to alter the glycolytic rate of cumulus cells, whereas that of rhBMP15 is to regulate oxidative phosphorylation in the oocyte, even though it acts via cumulus cells. These effects are tempered when FSH and rhBMP15 are present together but, nonetheless, yield the best oocyte developmental competence.

Oxygen-regulated gene expression in murine cumulus cells.

Publisher: CSIRO Publishing

Date: 2015

DOI: 10.1071/RD13249

Abstract: Oxygen is an important component of the environment of the cumulus–oocyte complex (COC), both in vivo within the ovarian follicle and during in vitro oocyte maturation (IVM). Cumulus cells have a key role in supporting oocyte development, and cumulus cell function and gene expression are known to be altered when the environment of the COC is perturbed. Oxygen-regulated gene expression is mediated through the actions of the transcription factors, the hypoxia-inducible factors (HIFs). In the present study, the effect of oxygen on cumulus cell gene expression was examined following in vitro maturation of the murine COC at 2%, 5% or 20% oxygen. Increased expression of HIF-responsive genes, including glucose transporter-1, lactate dehydrogenase A and BCL2/adenovirus E1B interacting protein 3, was observed in cumulus cells matured at 2% or 5%, compared with 20% oxygen. Stabilisation of HIF1α protein in cumulus cells exposed to low oxygen was confirmed by western blot and HIF-mediated transcriptional activity was demonstrated using a transgenic mouse expressing green fluorescent protein under the control of a promoter containing hypoxia response elements. These results indicate that oxygen concentration influences cumulus cell gene expression and support a role for HIF1α in mediating the cumulus cell response to varying oxygen.

Comparison between in vivo-derived and in vitro-produced pre-elongation embryos from domestic ruminants

Publisher: CSIRO Publishing

Date: 1997

DOI: 10.1071/R96079

Abstract: In vitro production of ruminant embryos has become routine and is increasingly available as a commercial service to dairy, meat and wool producers. However, the efficiency of producing viable embryos and the development of such embryos after transfer to recipients are perceived to be inferior to that which occurs in vivo. The present review outlines the biochemical and morphological similarities and differences between embryos produced In vitro and those produced in vivo. Some measures of metabolism are not markedly different between In vitro- and in vivo-derived blastocysts. However, at a cellular and subcellular level, differences in metabolism, morphology and ultrastructure have been described, as has susceptibility to manipulation and cryopreservation. Most importantly are the differences in lambing and calving rates and the reports of abnormal fetal development from embryos produced In vitro. These latter observations are of major concern, as they suggest that the In vitro environment may affect subsequent developmental physiology. At the extreme, these effects may not be expressed until adult life. Further efforts to improve the efficiency of In vitro embryo production must be accompanied by a commitment to assess the long-term consequences of these procedures. Extra keyword: development.

Effect of oxygen and glucose availability during in vitro maturation of bovine oocytes on development and gene expression

Publisher: Springer Science and Business Media LLC

Date: 24-05-2021

DOI: 10.1007/S10815-021-02218-W

Metabolic co-dependence of the oocyte and cumulus cells: essential role in determining oocyte developmental competence

Publisher: Oxford University Press (OUP)

Date: 06-10-2020

DOI: 10.1093/HUMUPD/DMAA043

Abstract: Within the antral follicle, the oocyte is reliant on metabolic support from its surrounding somatic cells. Metabolism plays a critical role in oocyte developmental competence (oocyte quality). In the last decade, there has been significant progress in understanding the metabolism of the cumulus–oocyte complex (COC) during its final stages of growth and maturation in the follicle. Certain metabolic conditions (e.g. obesity) or ART (e.g. IVM) perturb COC metabolism, providing insights into metabolic regulation of oocyte quality. This review provides an update on the progress made in our understanding of COC metabolism, and the metabolic conditions that influence both meiotic and developmental competence of the oocyte. The PubMed database was used to search for peer-reviewed original and review articles. Searches were performed adopting the main terms ‘oocyte metabolism’, ‘cumulus cell metabolism’, ‘oocyte maturation’, ‘oocyte mitochondria’, ‘oocyte metabolism’, ‘oocyte developmental competence’ and ‘oocyte IVM’. Metabolism is a major determinant of oocyte quality. Glucose is an essential requirement for both meiotic and cytoplasmic maturation of the COC. Glucose is the driver of cumulus cell metabolism and is essential for energy production, extracellular matrix formation and supply of pyruvate to the oocyte for ATP production. Mitochondria are the primary source of ATP production within the oocyte. Recent advances in real-time live cell imaging reveal dynamic fluctuations in ATP demand throughout oocyte maturation. Cumulus cells have been shown to play a central role in maintaining adequate oocyte ATP levels by providing metabolic support through gap junctional communication. New insights have highlighted the importance of oocyte lipid metabolism for oocyte oxidative phosphorylation for ATP production, meiotic progression and developmental competence. Within the last decade, several new strategies for improving the developmental competence of oocytes undergoing IVM have emerged, including modulation of cyclic nucleotides, the addition of precursors for the antioxidant glutathione or endogenous maturation mediators such as epidermal growth factor-like peptides and growth differentiation factor 9/bone morphogenetic protein 15. These IVM additives positively alter COC metabolic endpoints commonly associated with oocyte competence. There remain significant challenges in the study of COC metabolism. Owing to the paucity in non-invasive or in situ techniques to assess metabolism, most work to date has used in vitro or ex vivo models. Additionally, the difficulty of measuring oocyte and cumulus cell metabolism separately while still in a complex has led to the frequent use of denuded oocytes, the results from which should be interpreted with caution since the oocyte and cumulus cell compartments are metabolically interdependent, and oocytes do not naturally exist in a naked state until after fertilization. There are emerging tools, including live fluorescence imaging and photonics probes, which may provide ways to measure the dynamic nature of metabolism in a single oocyte, potentially while in situ. There is an association between oocyte metabolism and oocyte developmental competence. Advancing our understanding of basic cellular and biochemical mechanisms regulating oocyte metabolism may identify new avenues to augment oocyte quality and assess developmental potential in assisted reproduction.

MODULATION OF PORCINE NUCLEAR MATURATION STATUS USING A SPECIFIC PHOSPHODIESTERASE INHIBITOR

Publisher: CSIRO Publishing

Date: 2010

DOI: 10.1071/RDV22N1AB356

Abstract: Compared to other livestock species, IVM porcine COCs have poor developmental competence.Thisismost likely due to poor cytoplasmic maturation and asynchronous nuclear maturation. The resumption of nuclear maturation is largely regulated by cyclic adenosine monophosphate(cAMP), with increasing intra-oocyte levels prolonging cumulus-oocyte gap junction communication and delaying meiotic resumption. Modulation of cAMP levels using phosphodiesterase (PDE) inhibitors during bovine IVM significantly improves developmental competence (Thomas et al. 2004 Biol. Reprod. 71, 1142-1149). Hence, the aim of this study was to determine the effect of a type 3 PDE inhibitor (cilostamide, CIL) supplementation during porcine IVM on nuclear maturation, using a defined culture system. COCs derived from follicles of prepubertal gilts were cultured in groups of 10 in 100μL IVM medium (VitroMat, IVF Vet Solutions, Adelaide, Australia) +100 m IU mL-1 rhFSH +4 mg mL-1 BSA and nuclear maturation was assessed using orcein dye. Exp. 1: IVM medium was supplemented with 0, 5, 10, and 20 μM CIL or 0, 0.01, 0.1, 1, and 10 μM CIL and nuclear maturation was determined at 24 h and 44 h. Exp. 2: COCs were cultured in ± 0.1 μM CIL and nuclear maturation was assessed at 24 h, 44 h, 48 h, 52 h, and 56 h. Proportions of COCs at each stage of nuclear maturation were arcsine transformed and differences determined using a general linear model and Bonferroni post hoc test. Four replicates per experiment were performed with 20 COCs used for each treatment and/or time point. Results for Exp. 1 revealed no differences in the rate of nuclear maturation after 24 h of culture. After 44 h, 77% of COCs incubated in the absence of CIL were at metaphase II (MII) compared to 35-45% MII when cultured in the presence of 1, 5, 10, or 20 μM CIL (P 0.001). Furthermore, CIL supplementation resulted in approximately half the COCs arresting at germinal vesicle stage (GV) after 44 h of culture. While there were no significant differences in the MII rates of COCs cultured in 0, 0.01, and 0.1 μM CIL, significantly more COCs were at metaphase I (MI) at 44 h compared to control COCs (0 μM CIL, P 0.05). The time course experiment (Exp 2) demonstrated that nuclear maturation was delayed by 12 h with 0.1 μM CIL, compared to the absence of CIL, with comparable MII rates being achieved at 56 h for 0.1 μM CIL (72%) and 44 h for controls (0 μM CIL = 73%). These results demonstrate that porcine oocyte maturation can be induced in vitro by FSH in the presence of a low dose of type 3 PDE inhibitor resulting in a delay in the resumption and completion of nuclear maturation. Further investigations are underway to determine if CIL treatment prolongs gap junction communication and improves the developmental competence of porcine oocytes. This work was supported by the National Institutes of Health (USA) and National Health and Medical Research Council (Australia).

Effect of varying glucose and glucosamine concentration in vitro on mouse oocyte maturation and developmental competence

Publisher: CSIRO Publishing

Date: 2013

DOI: 10.1071/RD12275

Abstract: The effects of hyper- and hypo-glycaemic conditions during the in vitro maturation of mouse cumulus–oocyte complexes on developmental competence were examined, with an emphasis on the role of the hexosamine biosynthesis pathway. A low (1 mM) glucose concentration achieved optimal oocyte competence (3-fold higher blastocyst development rate compared with high (30 mM) glucose, P 0.05). In addition, glucose supplementation during only the first hour after release from the follicle was necessary and sufficient to support oocyte maturation and embryo development to the blastocyst stage. Glucosamine (a known hyperglycaemic mimetic and specific activator of the hexosamine pathway) was able to substitute for glucose during this first hour, indicating that flux through the hexosamine pathway is essential for oocyte competence. In the absence of glucose throughout the maturation period, glucosamine was not able to increase developmental competence, and at higher concentrations (2.5 and 5 mM) had a detrimental effect on MII and blastocyst development rates, compared with controls (P 0.05). These experiments underscore the importance of glucose metabolic pathways during in vitro maturation and support the concept that excess flux through the hexosamine pathway has detrimental consequences.

Pentose phosphate pathway activity: Effect on in vitro maturation and oxidative status of bovine oocytes

Publisher: CSIRO Publishing

Date: 2014

DOI: 10.1071/RD12397

Abstract: The relationship between pentose phosphate pathway (PPP) activity in cumulus–oocyte complexes (COCs) and oxidative and mitochondrial activity in bovine oocytes was evaluated with the aim of analysing the impact of two inhibitors (NADPH and 6-aminonicotinamide (6-AN)) and a stimulator (NADP) of the key enzymes of the PPP on the maturation rate, oxidative and mitochondrial activity and the mitochondrial distribution in oocytes. The proportion of COCs with measurable PPP activity (assessed using brilliant cresyl blue staining), glucose uptake, lactate production and meiotic maturation rate diminished when 6-AN (0.1, 1, 5 and 10 mM for 22 h) was added to the maturation medium (P 0.05). The addition of NADPH did not modify glucose uptake or lactate production, but reduced PPP activity in COCs and meiotic maturation rates (P 0.05). The presence of NADP (0.0125, 0.125, 1.25 and 12.5 mM for 22 h of culture) in the maturation medium had no effect on PPP activity in COCs, glucose uptake, lactate production and meiotic maturation rate. However, in the absence of gonadotropin supplementation, NADP stimulated both glucose uptake and lactate production at 12.5 mM (the highest concentration tested P 0.05). NADP did not modify cleavage rate, but decreased blastocyst production (P 0.05). During IVM, oocyte oxidative and mitochondrial activity was observed to increase at 15 and 22 h maturation, which was also related to progressive mitochondrial migration. Inhibiting the PPP with 6-AN or NADPH led to reduced oxidative and mitochondrial activity compared with the respective control groups and inhibition of mitochondrial migration (P 0.05). Stimulation of the PPP with NADP increased oxidative and mitochondrial activity at 9 h maturation (P 0.05) and delayed mitochondrial migration. The present study shows the significance of altering PPP activity during bovine oocyte IVM, revealing that there is a link between the activity of the PPP and the oxidative status of the oocyte.

Complex Interactions Between Hypoxia Inducible Factors, Insulin-Like Growth Factor-II and Oxygen in Early Murine Trophoblasts

Publisher: Elsevier BV

Date: 11-2007

DOI: 10.1016/J.PLACENTA.2007.05.009

Abstract: The human first trimester placenta experiences a low oxygen environment. The hypoxia inducible factors (HIFs) mediate the response to low oxygen, inducing genes such as insulin-like growth factor (IGF)-II. Interestingly, IGF-II has been shown to promote placental growth and function. Currently, the interaction between oxygen, IGF-II and HIFs in the regulation of trophoblast behaviour are unclear. Murine implantation sites from days 5.5-10.5 were collected for immunohistochemical analyses. Use of the hypoxia marker pimonidazole indicated that the early mouse implantation site is exposed to low oxygen levels similar to those seen in the early human placenta. HIF-1alpha protein immunostaining was also observed in the implantation site. Culturing murine ectoplacental cones in decreasing oxygen concentrations (20%, 5% and 1% O(2)), either with or without the addition of IGF-II, induced complex responses by trophoblasts in terms of their migration and differentiation. Following 3 days exposure to low oxygen there was reduced EPC outgrowth, reduced Igf2 and increased Tpbp mRNA levels, suggesting commitment to the spongiotrophoblast lineage. In addition, Hif-1alpha mRNA levels were decreased, whilst Hif-2alpha mRNA was unchanged. This decrease in Hif-1alpha may be due to the observed increase in antisense (as) Hif-1alpha mRNA levels in 1% cultures. Furthermore, expression of Hif-2alpha and the HIF target genes: asHif-1alpha, Vegf and Slc2a1 were reduced under low oxygen with the addition of IGF-II. In conclusion, Hif-1alpha and Hif-2alpha are differentially regulated by oxygen and IGF-II in cultured trophoblast cells and asHif-1alpha may mediate the response to prolonged hypoxia in murine trophoblasts.

Protein synthesis and uptake by bovine day 7 embryos produced under different incubation systems

Publisher: Elsevier BV

Date: 1998

DOI: 10.1016/S0093-691X(98)90588-X

Programming embryo development with a pre-implantation inflammatory insult

Publisher: Elsevier BV

Date: 08-2010

DOI: 10.1016/J.JRI.2010.06.075

Oocyte maturation: Emerging concepts and technologies to improve developmental potential in vitro

Publisher: Elsevier BV

Date: 2007

DOI: 10.1016/J.THERIOGENOLOGY.2006.09.027

Abstract: Oocyte in vitro maturation (IVM) is an important reproductive technology that generates mature oocytes that are capable of supporting preimplantation embryo development and full development to term. There is great clinical and commercial incentive to improve the efficiency of the technology, however, progress has been slow over the past decade. A critical challenge is to understand what constitutes oocyte developmental competence and the mechanisms governing it. We have taken the approach of studying in detail oocyte-somatic cell interactions including, oocyte-cumulus cell (CC) gap-junctional communication, and bidirectional paracrine signalling between the two cell types. It is becoming clear that, compared to oocytes matured in vivo, IVM oocytes undergo maturation prematurely as they are still in the process of acquiring developmental competence in vivo, and the molecular cascade reinitiating meiosis differs entirely to that in vivo. Attempts to enhance oocyte developmental competence by attenuating the spontaneous meiotic resumption of oocytes in vitro have been met with mixed success. Kinase inhibitors that prevent maturation-promoting factor activity have, in general, been ineffectual on promoting oocyte developmental potential post-IVM. In contrast, agents that modulate oocyte cAMP during IVM show greater potential, possibly as these compounds extend oocyte-CC gap-junctional communication. An important concept that is now emerging is that the oocyte secretes potent growth factors that regulate fundamental aspects of CC function and thereby determine the distinctive phenotype of the cumulus-oocyte complex. The capacity of an oocyte to regulate its own microenvironment by oocyte-secreted factors (OSFs) may constitute an important component of oocyte developmental competence. In support of this notion, we have recently demonstrated that supplementing IVM media with exogenous OSFs improves oocyte developmental potential, as evidenced by enhanced pre- and post-implantation embryo development.

Hyperspectral microscopy can detect metabolic heterogeneity within bovine post-compaction embryos incubated under two oxygen concentrations (7% versus 20%)

Publisher: Oxford University Press (OUP)

Date: 28-08-2017

DOI: 10.1093/HUMREP/DEX261

Abstract: Can we separate embryos cultured under either 7% or 20% oxygen atmospheres by measuring their metabolic heterogeneity? Metabolic heterogeneity and changes in metabolic profiles in morula exposed to two different oxygen concentrations were not detectable using traditional fluorophore and two-channel autofluorescence but were detectable using hyperspectral microscopy. Increased genetic and morphological blastomere heterogeneity is associated with compromised developmental competence of embryos and currently forms the basis for embryo scoring within the clinic. However, there remains uncertainty over the accuracy of current techniques, such as PGS and time-lapse microscopy, to predict subsequent pregnancy establishment. The impact of two oxygen concentrations (7% = optimal and 20% = stressed) during post-fertilisation embryo culture was assessed. Cattle embryos were exposed to the different oxygen concentrations for 8 days (D8 embryo developmental competence) or 5 days (D5 metabolism measurements). Between 3 and 4 experimental replicates were performed, with 40-50 embryos per replicate used for the developmental competency experiment, 10-20 embryos per replicate for the fluorophore and two-channel autofluorescence experiments and a total of 21-22 embryos used for the hyperspectral microscopy study. In-vitro produced (IVP) cattle embryos were utilised for this study. Post-fertilisation, embryos were exposed to 7% or 20% oxygen. To determine impact of oxygen concentrations on embryo viability, blastocyst development was assessed on D8. On D5, metabolic heterogeneity was assessed in morula (on-time) embryos using fluorophores probes (active mitochondria, hydrogen peroxide and reduced glutathione), two-channel autofluorescence (FAD and NAD(P)H) and 18-channel hyperspectral microscopy. Exposure to 20% oxygen following fertilisation significantly reduced total blastocyst, expanded and hatched blastocyst rates by 1.4-, 1.9- and 2.8-fold, respectively, compared to 7% oxygen (P 0.05). While there were no significant differences in two-channel autofluorescent profiles of morula exposed to 7% and 20% oxygen (main effect, P > 0.05), morula that subsequently progressed to the blastocyst stage had significantly higher levels of FAD and NAD(P)H fluorescence compared to arrested morula (P < 0.05), with no change in the redox ratio. Hyperspectral autofluorescence imaging (in 18-spectral channels) of the D5 morula revealed highly significant differences in four features of the metabolic profiles of morula exposed to the two different oxygen concentrations (P < 0.001). These four features were weighted and their linear combination revealed clear discrimination between the two treatment groups. Metabolic profiles were assessed at a single time point (morula), and as such further investigation is required to determine if differences in hyperspectral signatures can be detected in pre-compaction embryos and oocytes, using both cattle and subsequently human models. Furthermore, embryo transfers should be performed to determine the relationship between metabolic profiles and pregnancy success. Advanced autofluorescence imaging techniques, such as hyperspectral microscopy, may provide clinics with additional tools to improve the assessment of embryos prior to transfer. This study was funded by the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003). The Fluoview FV10i confocal microscope was purchased as part of the Sensing Technologies for Advanced Reproductive Research (STARR) facility, funded by the South Australian Premier's Science and Research Fund. The authors declare there are no conflict of interest.

Oocyte-secreted factors: Regulators of cumulus cell function and oocyte quality

Publisher: Oxford University Press (OUP)

Date: 05-01-2008

DOI: 10.1093/HUMUPD/DMM040

Abstract: Oocyte quality is a key limiting factor in female fertility, yet we have a poor understanding of what constitutes oocyte quality or the mechanisms governing it. The ovarian follicular microenvironment and maternal signals, mediated primarily through granulosa cells (GCs) and cumulus cells (CCs), are responsible for nurturing oocyte growth, development and the gradual acquisition of oocyte developmental competence. However, oocyte-GC/CC communication is bidirectional with the oocyte secreting potent growth factors that act locally to direct the differentiation and function of CCs. Two important oocyte-secreted factors (OSFs) are growth-differentiation factor 9 and bone morphogenetic protein 15, which activate signaling pathways in CCs to regulate key genes and cellular processes required for CC differentiation and for CCs to maintain their distinctive phenotype. Hence, oocytes appear to tightly control their neighboring somatic cells, directing them to perform functions required for appropriate development of the oocyte. This oocyte-CC regulatory loop and the capacity of oocytes to regulate their own microenvironment by OSFs may constitute important components of oocyte quality. In support of this notion, it has recently been demonstrated that supplementing oocyte in vitro maturation (IVM) media with exogenous OSFs improves oocyte developmental potential, as evidenced by enhanced pre- and post-implantation embryo development. This new perspective on oocyte-CC interactions is improving our knowledge of the processes regulating oocyte quality, which is likely to have a number of applications, including improving the efficiency of clinical IVM and thereby providing new options for the treatment of infertility.

Mechanisms contributing to the reduced developmental competence of glucosamine-exposed mouse oocytes

Publisher: CSIRO Publishing

Date: 2010

DOI: 10.1071/RD09193

Abstract: Glucosamine (GlcN) is a widely used hyperglycaemia mimetic because of its ability to upregulate the ‘energy-sensing’ hexosamine biosynthesis pathway in a dose-dependent manner. A previous study demonstrated that addition of GlcN (2.5–5 mM) during IVM of cattle and pig cumulus–oocyte complexes (COC) inhibited development following fertilisation and early cleavage. In the present study, we demonstrate that the addition of 2.5 mM GlcN during IVM of mouse COCs similarly inhibits embryo development, with the degree of inhibition dependent upon the availability of glucose in the maturation medium. Furthermore, we determined that the effect of GlcN is likely mediated by the cumulus cell vestment, because we failed to observe inhibitory effects of GlcN following maturation of denuded (and therefore already developmentally compromised) oocytes. As with cattle oocytes, inhibition of O-linked glycosylation of unknown proteins within mouse cumulus cells significantly reversed the effects of GlcN. Finally, we also provide preliminary evidence that GlcN may inhibit the pentose phosphate metabolic pathway within the oocyte, an effect possibly mediated by cumulus cells in intact COCs. Collectively, our results demonstrate that GlcN inhibits the developmental competence of IVM mouse oocytes and suggest that this occurs via cumulus cell-mediated mechanisms. Therefore, the in vitro addition of GlcN is a useful experimental tool to determine the mechanisms of hyperglycaemic responses within COCs.

Pioneering contributions by Robert Edwards to oocyte in vitro maturation (IVM)

Publisher: Oxford University Press (OUP)

Date: 15-11-2013

DOI: 10.1093/MOLEHR/GAT075

Abstract: The history of in vitro maturation (IVM) of mammalian oocytes, especially of human oocytes, holds a special place for Robert Edwards. He was the first to comprehensively examine and demonstrate maturation of human oocytes in vitro and in so doing he changed the course of medicine by fertilizing them in vitro. In reviewing his contribution, we have examined the state of the field at the time and discuss his pioneering insights into mammalian oocyte biology. We will also discuss how some of the major concepts and challenges identified by Edwards 50 years ago remain among the major challenges facing IVM today.

Murine HIF-1a localisation by immunohistochemistry in a mouse reproductive tissue

Publisher: CSIRO Publishing

Date: 2005

DOI: 10.1071/SRB05ABS304

Abstract: Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors consisting of an α and β subunit. The level of O2 within cells regulates the stability of HIF-1α and HIF-1 is considered the primary mediator of cellular responses to hypoxia, helping restore O2 homeostasis by promoting the expression of hypoxia-sensitive genes involved in cell survival, angiogenesis, cell proliferation and metabolism. There are few published studies investigating the role of HIF-1 protein in mouse tissues using immunohistochemistry, due to the lack of a reliable protocol and the inability of many commercially available antibodies to detect murine HIF-1α protein. We have developed a protocol that has allowed us to analyse the presence and location of HIF-1α protein in the mouse epididymis and found that it was predominantly located in the nucleus of discrete principal cells in the epididymis of mice housed under normoxic conditions and sacrificed by cervical dislocation. Interestingly, a 2.5× increase (P 0.05) of HIF-1α protein staining intensity in both the nucleus and cytoplasm of principal cells in the epididymis was detected in mice housed under normoxic conditions but sacrificed with CO2 gas, compared to mice sacrificed by cervical dislocation. HIF-1α protein detection was 3-fold increased in the nucleus and cytoplasm of principal cells when mice were exposed to hypoxic conditions (6% O2 for 1 h). Our results demonstrate that murine HIF-1α can be detected in discrete cells under normoxic conditions, suggesting local differences in O2. Acute hypoxic responses, via deliberate exposure or even CO2 euthanasia can significantly upregulate HIF-1α protein levels. Further studies will investigate the role of HIFs and hypoxia in male and female reproductive function.

Development of sheep preimplantation embryos in media supplemented with glucose and acetate

Publisher: Elsevier BV

Date: 08-1989

DOI: 10.1016/0093-691X(89)90322-1

Abstract: Two experiments were conducted to examine the effect of supplemental glucose (G 1.5 mM) and/or acetate (A 0.5 mM) on the development of early sheep embryos to blastocysts when cultured in vitro in glucose-free synthetic oviductal fluid (SOF)+sheep serum or bovine serum albumin (BSA). In Experiment 1, 2- to 4-cell, 8- to 16-cell and >16-cell embryos were cultured in SOF, SOF+G, SOF+A or SOF+G+A. All media were supplemented with 10% sheep serum. In addition, embryos were cultured in either microdrops under polysiloxane oil or in multiwell dishes. Overall, development to the blastocyst stage was 3%, 30% and 68% for 2- to 4-cell, 8- to 16-cell and >16-cell stages, respectively, suggesting that an 8-cell developmental block existed under our culture conditions. Glucose supplementation had little effect on embryo development, and no overall effect was observed from the addition of acetate. In Experiment 2, 8- to 16-cell embryos were cultured in SOF or SOF+G, both supplemented with BSA. Development to the blastocyst stage was 25% and 18%, respectively. The results show that the presence of glucose or acetate did little to enhance embryonic development in our incubation systems. Further work is required to evaluate fully the energy requirements for development of the early sheep embryo.

Oxygen consumption by Day 7 bovine blastocysts: determination of ATP production

Publisher: Elsevier BV

Date: 07-1996

DOI: 10.1016/0378-4320(96)01477-7

Exogenous growth differentiation factor 9 in oocyte maturation media enhances subsequent embryo development and fetal viability in mice

Publisher: Oxford University Press (OUP)

Date: 16-11-2008

DOI: 10.1093/HUMREP/DEM140

Abstract: Successful oocyte in vitro maturation (IVM) would eliminate the need for hormonal stimulation used in assisted reproduction techniques. Unfortunately, oocytes matured in vitro have compromised developmental competence possibly due to disrupted oocyte-cumulus communication resulting from inappropriate levels of oocyte-secreted factors such as growth differentiation factor 9 (GDF9). Hence, the aim of this study was to investigate the effects of exogenous GDF9 during IVM of mouse oocytes on development and subsequent fetal viability. Cumulus-oocyte complexes from pregnant mare's serum gonadotrophin primed mice were cultured with or without 200 ng/ml exogenous recombinant GDF9, 50 mIU/ml FSH and 10 ng/ml epidermal growth factor (EGF). After 18 h, cumulus expansion was scored and oocytes were fertilized in vitro. Cleavage, blastocyst development, blastocyst total, inner cell mass (ICM) and trophectoderm cell numbers were assessed. Viability of embryos was assessed by transfer to recipient females and pregnancy outcome determined at day 15. Oocytes matured with exogenous GDF9 in the presence of FSH and EGF had higher rates of development, percentage of hatching blastocyst and blastocyst total and ICM cell numbers (all P < 0.05). Although implantation rate and fetal and placental weights were not affected, the number of viable fetuses at day 15 was increased with exogenous GDF9. Exogenous GDF9 during IVM improved embryo development and fetal viability and provides a promising approach for human IVM.

Estimation of glucose uptake by ovarian follicular cells.

Publisher: Springer Science and Business Media LLC

Date: 16-07-2011

DOI: 10.1007/S10439-011-0353-Y

Abstract: In vitro maturation (IVM) of mammalian oocytes provides an alternative to traditional in vitro fertilization techniques for clinical treatment of infertility or animal breeding. IVM involves the collection of oocytes from the ovary prior to ovulation, with maturation occurring in a laboratory environment. The success of IVM is highly sensitive to the in vitro nutrient environment. The nurse cells surrounding the oocyte, known as cumulus cells, regulate this environment and removal of these cells reduces the ability of the oocyte to develop following insemination. Determining the nature of the interaction between the oocyte and cumulus cells, collectively called the cumulus-oocyte complex (COC), is a difficult task experimentally. Here we use a combination of experimental and mathematical techniques to investigate glucose transport within bovine COCs and find quantitative estimates of the glucose uptake rates of the oocyte and cumulus cells. Surprisingly, our modeling shows the rate of uptake of glucose by the oocyte to increase and then decrease with concentration, a result that needs further experimental investigation but which supports the expectation that high and low glucose concentrations are detrimental to oocyte development. The methodology described is suitable for use across species and for investigating the transport of other important nutrients within the COC.

Stress response genes are suppressed in mouse preimplantation embryos by granulocyte-macrophage colony-stimulating factor (GM-CSF)

Publisher: Oxford University Press (OUP)

Date: 08-09-2009

DOI: 10.1093/HUMREP/DEP307

Abstract: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to promote the development and survival of human and mouse preimplantation embryos however, the mechanism of action of GM-CSF in embryos is not defined. Mouse blastocysts were cultured from zygote stage in vitro with and without recombinant mouse GM-CSF (rmGM-CSF), and in vivo developed blastocysts were flushed from Csf2 null mutant and wild-type mice. The effect of GM-CSF on blastocyst expression of stress response and apoptosis genes was evaluated by microarray, qPCR and immunochemistry. Microarray analysis of the gene transcription profile showed suppression of stress response and apoptosis gene pathways in blastocysts exposed to rmGM-CSF in vitro. qPCR analysis confirmed that rmGM-CSF inhibited expression of heat shock protein (HSP) and apoptosis pathway genes Cbl, Hspa5, Hsp90aa1, Hsp90ab1 and Gas5 in in vitro blastocysts. Immunocytochemical analysis of HSP 1 (HSPA1A/1B HSP70), BAX, BCL2 and TRP53 (p53) in in vitro blastocysts showed that HSPA1A/1B and BCL2 proteins were less abundant when embryos were cultured with rmGM-CSF. BAX and TRP53 were unchanged at the protein level, but Bax mRNA expression was reduced after GM-CSF treatment. In in vivo developed blastocysts, Csf2 null mutation caused elevated expression of Hsph1 but not other stress response genes. We conclude that GM-CSF inhibits the cellular stress response and apoptosis pathways to facilitate embryo growth and survival, and the protective effects of GM-CSF are particularly evident in in vitro culture media, whereas in vivo other cytokines can partly compensate for absence of GM-CSF.

The impact of nutrition of the cumulus oocyte complex and embryo on subsequent development in ruminants

Publisher: Japanese Society of Animal Reproduction

Date: 2006

DOI: 10.1262/JRD.17089

Abstract: Cumulus-oocyte complexes (COCs) and early embryos rely on a histotrophic nutrition source for energy production and the synthesis of macromolecules. There is accumulating evidence suggesting that the balance of supply and demand for energy and other anabolic substrates during oocyte maturation and very early stages of development programmes subsequent developmental potential, and this may include subsequent fetal growth trajectory. One ex le is the role of glucose (Glc) during cumulus-oocyte complex maturation. Glucose is an essential nutrient for maturation, especially its role during cumulus expansion. Our laboratory has shown that during in vitro culture, too little glucose during cumulus-oocyte complex maturation affects meiotic competence. We have focussed on glucose (Glc) metabolism through the hexosamine biosynthesis pathway (HBP) during COC maturation in vitro. The HBP in somatic cells is regarded as a "fuel-sensing" pathway and its interaction with cell signalling systems and transcriptional regulation is increasingly apparent. Up-regulation of the HBP during oocyte maturation in vitro has negative consequences for subsequent development. Another ex le is the role of hypoxia (low O2) during peri-compaction development. My laboratory believes that ruminant embryos during compaction, blastulation and subsequent development in the uterine cavity lack a key hypoxia responsive element. Because of this, hypoxia is important for normal development in ruminants but perturbs further development in rodents. The implication of these ex les to the fundamental concept of peri-conception nutritional programming of development are discussed.

Embryo culture and long-term consequences

Publisher: CSIRO Publishing

Date: 2007

DOI: 10.1071/RD06129

Abstract: The development of pre-elongation (ruminants) and preimplantation (e.g. mouse and humans) embryos ex vivo has evolved over the past four decades into a reliable technology that is used as a research tool in developmental biology, as well as other embryo technologies, for application in infertility treatment, species conservation and selective breeding. It is clear from a variety of embryo culture studies that adaptive responses by embryos during culture can lead to significant alterations in subsequent developmental profiles, the mechanisms of which are not entirely clear but are unlikely to be limited to a single mechanism because this does not account for the variability seen in responses and the emerging list of specific cellular stressors that cause long-term deviations in fetal development. Epigenetic mechanisms, especially deviation of methylation patterns, and adaptation via causal pathways linking gene expression signalling with critical developmental time points, especially of placental development, are two candidates. Observational studies on post-transfer consequences must now be designed so that specific candidate pathways are followed to elucidate their role in perturbed development following transfer.

Effect of glutathione synthesis stimulation during in vitro maturation of ovine oocytes on embryo development and intracellular peroxide content

Publisher: Elsevier BV

Date: 03-2002

DOI: 10.1016/S0093-691X(02)00643-X

Abstract: Cysteamine and beta-mercaptoethanol supplementation of in vitro maturation (IVM) medium has been found to increase intracellular glutathione (GSH) content in oocytes and to improve embryo development and quality in several species. The objective of this experiment was to study the effect of cysteamine and beta-mercaptoethanol added during IVM of sheep oocytes on GSH synthesis and embryo development. Furthermore, we examined if cysteamine addition (hence GSH production) had an effect on the reduction of the intracellular peroxide content. We matured oocytes obtained from ovaries collected at a slaughterhouse in vitro in the presence of 0, 50, 100, and 200 microM cysteamine (Experiment 1) or with 0, 50, 100, and 200 microM beta-mercaptoethanol (Experiment 2). Following fertilization and embryo development, there was a increasing level of morula and blastocyst development in the presence of cysteamine, reaching significance in the presence of 200 microM (P < 0.05). However, beta-mercaptoethanol did not influence on the rate of embryo development. GSH levels were measured in oocytes matured in the presence or absence of 200 microM cysteamine (Experiment 3) or 50 microM beta-mercaptoethanol (Experiment 4), with or without buthionine sulfoximide (BSO), an inhibitor of GSH synthesis. Results demonstrated that for both cysteamine and beta-mercaptoethanol, intracellular GSH levels increased against control values (P < 0.01), which was abolished in the presence of BSO. Finally, we reduced intracellular peroxide levels, as measured by the relative fluorescence of the intracellular peroxide probe, carboxy-H2DCFDA, in the presence of either 200 microM cysteamine or 50 microM beta-mercaptoethanol (Experiment 5). These results demonstrate that cysteamine, but not beta-mercaptoethanol, when present during IVM, stimulates sheep embryo development both cysteamine and beta-mercaptoethanol stimulate GSH synthesis the increase in intracellular GSH is associated with a decrease in peroxide levels within oocytes.

Total protein content and protein synthesis within pre-elongation stage bovine embryos

Publisher: Wiley

Date: 06-1998

DOI: 10.1002/(SICI)1098-2795(199806)50:2<139::AID-MRD3>3.0.CO;2-L

Abstract: Wheat production requires at least ~ 2.4% increase per year rate by 2050 globally to meet food demands. However, heat stress results in serious yield loss of wheat worldwide. Correspondingly, wheat has evolved genetic basis and molecular mechanisms to protect themselves from heat-induced damage. Thus, it is very urgent to understand the underlying genetic basis and molecular mechanisms responsive to elevated temperatures to provide important strategies for heat-tolerant varieties breeding. In this review, we focused on the impact of heat stress on morphology variation at adult stage in wheat breeding programs. We also summarize the recent studies of genetic and molecular factors regulating heat tolerance, including identification of heat stress tolerance related QTLs/genes, and the regulation pathway in response to heat stress. In addition, we discuss the potential ways to improve heat tolerance by developing new technologies such as genome editing. This review of wheat responses to heat stress may shed light on the understanding heat-responsive mechanisms, although the regulatory network of heat tolerance is still ambiguous in wheat. The online version contains supplementary material available at 10.1007/s42994-021-00064-z.

Deconstructing autofluorescence: non-invasive detection and monitoring of biochemistry in cells and tissues (Conference Presentation)

Publisher: SPIE

Date: 17-05-2016

DOI: 10.1117/12.2212443

Fabrication on the microscale: a two-photon polymerized device for oocyte microinjection

Publisher: Springer Science and Business Media LLC

Date: 12-05-2022

DOI: 10.1007/S10815-022-02485-1

Abstract: Intracytoplasmic sperm injection (ICSI) addresses male sub-fertility by injecting a spermatozoon into the oocyte. This challenging procedure requires the use of dual micromanipulators, with success influenced by inter-operator expertise. We hypothesized that minimizing oocyte handling during ICSI will simplify the procedure. To address this, we designed and fabricated a micrometer scale device that houses the oocyte and requires only one micromanipulator for microinjection. The device consisted of 2 components, each of sub-cubic millimeter volume: a Pod and a Garage. These were fabricated using 2-photon polymerization. Toxicity was evaluated by culturing single-mouse presumptive zygotes (PZs) to the blastocyst stage within a Pod, with several Pods (and embryos) docked in a Garage. The development was compared to standard culture. The level of DNA damage/repair in resultant blastocysts was quantified (γH2A.X immunohistochemistry). To demonstrate the capability to carry out ICSI within the device, PZs were microinjected with 4-μm fluorescent microspheres and cultured to the blastocyst stage. Finally, the device was assessed for oocyte traceability and high-throughput microinjection capabilities and compared to standard microinjection practice using key parameters (pipette setup, holding then injecting oocytes). Compared to standard culture, embryo culture within Pods and a Garage showed no differences in development to the blastocyst stage or levels of DNA damage in resultant blastocysts. Furthermore, microinjection within our device removes the need for a holding pipette, improves traceability, and facilitates high-throughput microinjection. This novel device could improve embryo production following ICSI by simplifying the procedure and thus decreasing inter-operator variability.

Culture without the petri-dish

Publisher: Elsevier BV

Date: 2007

DOI: 10.1016/J.THERIOGENOLOGY.2006.09.016

Abstract: Automation of oocyte maturation and embryo production techniques is a new and exciting development in the field of reproductive technologies. There are two areas where increased automation is having an impact: in the area of embryo diagnostics and in the process of embryo production itself. Benefits include decreased staffing and skill requirements for production and assessment of embryos, as well as increasing quality management systems by removing the "human" factor. However, the uptake of new technologies is likely to be slow, as costs and the conservative nature of the Assisted Reproduction Technology industry to adopt new techniques.

Regulation of bovine oocyte meiotic and developmental capacity by glucose and glucosamine

Publisher: CSIRO Publishing

Date: 2004

DOI: 10.1071/SRB04ABS204

Abstract: Glucose is an important substrate for in vitro oocyte maturation (IVM) and is metabolised by cumulus oocyte complexes (COCs) via glycolysis or is used for extracellular matrix (ECM) synthesis. Follicular glucose concentration is significantly lower than commonly used IVM media (2.3�mM v. 5.6�mM in TCM199). Glucosamine is an alternative substrate for ECM and supplementation to IVM media reduces glucose uptake by COCs. The aim of this study was to determine the effect of glucose and glucosamine supplementation during IVM on bovine oocytes. First, bovine COCs (n�=�400) were matured in TCM199 (containing pyruvate, BSA, hCG and FSH), or synthetic follicular fluid medium (SFFM a defined medium based on bovine follicular fluid composition) with 2.3�mM or 5.6�mM glucose���5�mM glucosamine and nuclear maturation was assessed after 24 and 30�h. Significantly less COCs matured in 2.3�mM glucose completed nuclear maturation compared to COCs matured in 5.6�mM glucose (P� �0.05), whereas glucosamine had no effect on meiotic maturation. We then compared oocyte developmental capacity following IVM (n�=�600) in TCM199 or SFFM�+�5.6�mM glucose���5mM glucosamine. Blastocyst production was severely perturbed when COCs were matured in the presence of glucosamine (–glucosamine 32% v. +glucosamine 4% P� �0.001). To determine the cause of this reduction in oocyte developmental competence, we investigated oocyte protein synthesis by maturing COCs (n�=�100) in SFFM�+�5.6�mM glucose���5mM glucosamine�+�1�mM L-[2,3,4,5,6–3H] phenylalanine. In the presence of glucosamine, oocyte protein synthesis was reduced 40% compared to oocytes matured in control medium (P� �0.05). These results demonstrate that while glucosamine supplementation has no effect on oocyte nuclear maturation, cytoplasmic maturation is compromised, as demonstrated by perturbed oocyte protein synthesis and embryo development. In contrast, glucose concentration has a significant influence on meiotic progression. This provides a useful model to investigate the mechanisms of establishment of developmental competence in oocytes following maturation.

236 effect of cyclic adenosine monophosphate modulator regulators in association with bmp15 on bovine embryo development in vitro.

Publisher: CSIRO Publishing

Date: 2014

DOI: 10.1071/RDV27N1AB236

Abstract: BMP15 is a promising peptide to improve oocyte competence also, addition of cyclic adenosine monophosphate modulator (cAMP) regulators prevents spontaneous maturation in vitro and promotes embryo development. We aimed to assess embryo development after prematuration [pre-in vitro maturation (IVM)] with IBMX and Forskolin (FSK) and maturation in the presence or absence of a purified pro mature region of BMP15. Immature cumulus-oocyte complexes (COC) were cultured in vitroMat (IVF Vet Solutions, Adelaide, Australia) plus 4 mg mL–1 fatty acid free-BSA and rhFSH (0.1 IU mL–1), then ided into the following treatment groups: 1) spontaneous IVM: 24 h of IVM 2) spontaneous IVM + BMP15: 24 h of IVM in the presence of BMP15 (100 ng mL–1) 3) Pre 2 h: pretreatment with IBMX (500 µM Sigma-Aldrich) and FSK (100 µM Sigma-Aldrich) for 2 h following 24 h maturation and 4) Pre 2 h + BMP15: pretreatment with IBMX and FSK for 2 h following 24 h maturation in the presence of BMP15 (100 ng mL–1). After maturation, oocytes were inseminated and zygotes were cultured for 5 days in VitroCleave (IVF Vet Solutions, Adelaide, Australia) and transferred into VitroBlast (IVF Vet Solutions, Adelaide, Australia) until blastocyst assessment (Days 7 and 8). Zona-intact embryos were retrieved to assess differential staining of trophectoderm and inner cell mass. Data were transformed into a logarithm and analysed by 1-way ANOVA and post hoc least significant difference using SigmaStat software (SPSS Inc., San Jose, CA, USA P 0.05). There was no difference among groups on cleavage rates or blastocyst rates at Day 7 however, both Pre 2 h treatments increase hatched blastocyst rates at Day 8 of embryo development (Table 1). Supplementation with BMP15 increased total blastocyst rates at Day 8, regardless of pretreatment with IBMX+FSK (Table 1). Our data demonstrate that embryos from oocytes matured in the presence of BMP15 or pretreated with IBMX+FSK increase trophectoderm and total cell numbers however, no differences were observed for inner cell mass. We conclude that Pre 2 h treatment or BMP15 increase embryo development however, no effect of cAMP regulators in association with BMP15 on embryo development was observed. Table 1.Embryo development Supported by FAPESP (project numbers: 2012/1073-8 2013/12960-9 2013/05083-1 2012/50533-2).

Biological hydrogen peroxide detection with aryl boronate and benzil BODIPY-based fluorescent probes

Publisher: Elsevier BV

Date: 06-2018

DOI: 10.1016/J.SNB.2018.01.198

Defining the requirements for bovine embryo culture

Publisher: Elsevier BV

Date: 1996

DOI: 10.1016/0093-691X(95)00352-9

Glutathione synthesis during in vitro maturation of ovine oocytes: Effect of cysteamine and β-mercaptoethanol

Publisher: Elsevier BV

Date: 1999

DOI: 10.1016/S0093-691X(99)91927-1

The Ovarian Antral Follicle: Living on the Edge of Hypoxia or Not?1

Publisher: Oxford University Press (OUP)

Date: 06-2015

DOI: 10.1095/BIOLREPROD.115.128660

Abstract: Oocytes within antral follicles are thought to have restricted access to O2, as follicle vascularity is not adjacent and both granulosa and cumulus cells are metabolically active. Indeed, measured follicular antrum partial pressure (pO2) is regarded as low, but accurate and direct measurement represents a technical challenge that has yet to be overcome. The oocyte itself is highly dependent on oxidative phosphorylation for survival and competence for further development following fertilization, and it has been suggested that follicular pO2 levels are correlated with this capacity for further development. It is clear that gonadotropins are involved in regulating antrum formation, follicle vascularization, cellular differentiation, and the hypoxia-inducible factors (HIF), which are mainly regulated by dissolved O2 concentration. A newly discovered player in this story is the intracellular production of hemoglobin by both granulosa and cumulus cells, as well as the oocyte. Furthermore, cellular hemoglobin levels are dynamic, responding to the ovulatory luteinizing hormone (LH) surge. We hypothesize that this gas transport and antioxidant molecule is involved in the prevention of hypoxic response signaling by HIFs within the preovulatory antral follicle and the transition of granulosa cells to luteal tissue by facilitating the stabilization of HIFs, enabling induction of luteinization signaling. Another possible role is by sequestering nitric oxide (NO) during the ovulatory period, which may facilitate the resumption of meiosis in the oocyte. Testing these hypotheses will be challenging but important if the regulation of ovarian function is to be fully understood.

Oocyte maturation and quality: role of cyclic nucleotides

Publisher: Bioscientifica

Date: 11-2016

DOI: 10.1530/REP-15-0606

Abstract: The cyclic nucleotides, cAMP and cGMP, are the key molecules controlling mammalian oocyte meiosis. Their roles in oocyte biology have been at the forefront of oocyte research for decades, and many of the long-standing controversies in relation to the regulation of oocyte meiotic maturation are now resolved. It is now clear that the follicle prevents meiotic resumption through the actions of natriuretic peptides and cGMP – inhibiting the hydrolysis of intra-oocyte cAMP – and that the pre-ovulatory gonadotrophin surge reverses these processes. The gonadotrophin surge also leads to a transient spike in cAMP in the somatic compartment of the follicle. Research over the past two decades has conclusively demonstrated that this surge in cAMP is important for the subsequent developmental capacity of the oocyte. This is important, as oocyte in vitro maturation (IVM) systems practised clinically do not recapitulate this cAMP surge in vitro , possibly accounting for the lower efficiency of IVM compared with clinical IVF. This review particularly focuses on this latter aspect – the role of cAMP/cGMP in the regulation of oocyte quality. We conclude that clinical practice of IVM should reflect this new understanding of the role of cyclic nucleotides, thereby creating a new generation of ART and fertility treatment options.

Developmental ability of in vitro matured sheep oocytes collected during the nonbreeding season and fertilized in vitro with frozen ram semen

Publisher: Elsevier BV

Date: 11-1991

DOI: 10.1016/0093-691X(91)90342-B

Abstract: During the nonbreeding season, oocytes recovered from ovaries of FSH-primed or nonprimed ewes were matured in the presence or absence of granulosa cells collected from ovaries of primed or nonprimed ewes prior to in vitro fertilization with either fresh or frozen-thawed sperm. Following fertilization, ova were cultured for 24 h in synthetic oviduct fluid medium (SOF) supplemented with 20% human serum at 39 degrees C under humidified 5% CO(2), 5% O(2), 90% N(2) and then assessed for cleavage. Overall, 52% of ova cleaved. Cleavage was not affected by the source of sperm. Significantly more oocytes from primed follicles cleaved after 24 hours than those from nonprimed follicles (P<0.001). Maturation of oocytes in the presence of granulosa cells from nonprimed ewes resulted in a lower cleavage rate (44%, P<0.05) than in the presence of granulosa from primed ewes (59%) or no granulosa cells (50%). Oocytes (n = 508) from primed ewes were matured in the presence of granulosa cells (also from primed ewes) and fertilized in vitro with frozen-thawed sperm. Following in vitro culture for 24 hours, 68 of the 270 (53%) cleaved embryos were transferred to 17 recipient ewes, 15 of which remained pregnant to term, producing 24 lambs. The remaining 202 cleaved embryos were cultured for a further 5 days, of which 73 appeared to reach the morula/blastocyst stage and 61 were transferred to 16 recipients. Two ewes remained pregnant to term producing two lambs. These results demonstrate that production of sheep embryos using in vitro maturation and fertilization techniques is possible in the nonbreeding season. However, the poor viability of embryos obtained following extended culture needs to be resolved before such techniques can be usefully applied.

Influence of hyaluronic acid synthesis and cumulus mucification on bovine oocyte in vitro maturation, fertilisation and embryo development

Publisher: CSIRO Publishing

Date: 2007

DOI: 10.1071/RD06134

Abstract: During cumulus–oocyte complex (COC) maturation, cumulus expansion involves the deposition of mucoelastic compounds, especially hyaluronic acid, synthesised from glucose via the hexosamine biosynthesis pathway. The aim of the present study was to determine the effects of uridine monophosphate (UMP) and 6-diazo-5-oxo-l-norleucine (DON), inhibitors of hyaluronic acid synthesis, during bovine oocyte in vitro maturation (IVM) on cumulus expansion, glucose uptake, protein synthesis, cumulus cell number, meiotic maturation, cleavage rate and subsequent embryo development. A further aim of the study was to examine the effect of hyaluronic acid on sperm capacitation and acrosome reaction in relation to the capacity of COCs to be fertilised in vitro. A low correlation between glucose uptake and degree of cumulus expansion was observed. Total and partial inhibition of cumulus expansion was observed with DON and UMP, respectively, and was accompanied by a decrease in glucose uptake with DON. Total protein content and cumulus cell number per COC increased during IVM, but was unaffected by the presence of DON or UMP, as was oocyte meiotic maturation. Rates of cleavage and blastocyst development decreased in oocytes matured with DON and UMP, although this inhibition was reversed when the in vitro fertilisation (IVF) medium contained heparin. Hyaluronic acid induced capacitation and the acrosome reaction, and in IVF medium prevented the inhibition of cleavage and blastocyst development by DON in a similar fashion to heparin. Hyaluronic acid synthesis during cumulus mucification contributes to the penetration and fertilisation of bovine oocytes, most likely by facilitating the processes of capacitation and acrosome reaction. Mucification during IVM is independent of cumulus cell proliferation, COC protein content, oocyte meiotic maturation and subsequent developmental competence once fertilised.

Oxygen-regulated expression of GLUT-1, GLUT-3, and VEGF in the mouse blastocyst

Publisher: Wiley

Date: 2005

DOI: 10.1002/MRD.20183

Abstract: The oxygen concentration used in the incubation atmosphere during embryo culture influences embryo development rates and embryo quality. In somatic cells, oxygen levels can influence the expression of a range of genes, including glucose transporters, glycolytic enzymes, and angiogenic growth factors. Many of these oxygen-regulated genes have important roles in embryonic development and metabolism. The aim of this study was to determine whether oxygen regulates gene expression in the preimplantation mouse blastocyst. Mouse embryos were cultured from the 1-cell to morula stage under 7% oxygen, followed by culture under 20, 7, or 2% oxygen to the blastocyst stage. Expression of glucose transporter (GLUT)-1, GLUT-3, and vascular endothelial growth factor (VEGF) in blastocysts was measured by real-time reverse transcription PCR. Development from morula to blastocyst was not altered by culture under different oxygen conditions. Expression of GLUT-1, GLUT-3, and vascular endothelial growth (VEGF) was increased by 2- to 4-fold in embryos cultured under 2% oxygen, when compared to embryos cultured under 20 or 7% oxygen, and when compared to embryos developed in vivo (all P < 0.001). These results suggest that the preimplantation mouse embryo has the capacity to detect and respond to low oxygen availability with changes in expression of oxygen-regulated genes.

Effect of culturing mouse embryos under different oxygen concentrations on subsequent fetal and placental development.

Publisher: Wiley

Date: 28-03-2006

DOI: 10.1113/JPHYSIOL.2005.102681

339 OXYGEN CONCENTRATION DURING IN VITRO MATURATION OF MURINE OOCYTES INFLUENCES SUBSEQUENT FETAL AND PLACENTAL OUTCOMES

Publisher: CSIRO Publishing

Date: 2007

DOI: 10.1071/RDV19N1AB339

Abstract: Although the oxygen environment of the ovarian follicle is thought to influence oocyte developmental competence, little is known of the optimal oxygen environment for oocyte in vitro maturation (IVM). Previously, we found that oxygen concentration (either 2, 5, 10, or 20% O2 6% CO2 and balance of N2) during IVM of murine oocytes had no effect on maturation rate or subsequent fertilization, cleavage, and blastocyst development rates. However, 2% O2 results in blastocysts with a higher (P & 0.05) trophectoderm cell number (mean � SEM, 35.1 � 2.3) when compared to 20% (19.4 � 1.7), with 5 and 10% O2 yielding similar but intermediate cell numbers. When examined for cell apoptosis by TUNEL labelling, the 2% O2 IVM-derived embryos were also found to have a significantly higher percentage of cells undergoing apoptosis compared to the 5% O2 IVM-derived embryos and embryos derived from in vivo matured oocytes (unpublished data). Although the blastocyst development rate is not affected by varying oxygen environment during oocyte maturation, the resultant blastocysts exhibited signs of differing quality. The aim of this study was to investigate the effect of varying oxygen during IVM on post-transfer outcomes. Immature cumulus-oocyte complexes (COCs) were collected from the ovaries of eCG-stimulated CBAB6F1 females (21 days) and cultured for 17–18 h under 2, 5, or 20% O2, whereas in vivo-matured COCs were also collected post-hCG. After IVF/C (both under 5% O2), 6 blastocysts were transferred to each uterine horn of pseudopregnant Swiss recipients. Fetal and placental parameters were measured on Day 18 of pregnancy. The ability of the embryos to implant or develop was not altered by IVM oxygen concentration. However, the average weight of fetuses derived from 5% O2 matured oocytes was reduced (823.3 � 28.1 mg, P & 0.05) compared to those in the 20% O2 group (928.5 � 26.1 mg). The average weight of the placenta in the 5% O2 group was also reduced (87.4 � 4.0 mg) compared to those derived from in vivo-matured oocytes (104.5 � 5.4 mg). In contrast, the fetal:placental weight ratio was unchanged in the 5% O2 treatment, suggesting these placentae, although small, are still efficient. This is the first evidence that programming of fetal lacental growth occurs from treatments applied during oocyte maturation.

Quantitative non-invasive cell characterisation and discrimination based on multispectral autofluorescence features

Publisher: Springer Science and Business Media LLC

Date: 31-03-2016

DOI: 10.1038/SREP23453

Abstract: Automated and unbiased methods of non-invasive cell monitoring able to deal with complex biological heterogeneity are fundamentally important for biology and medicine. Label-free cell imaging provides information about endogenous autofluorescent metabolites, enzymes and cofactors in cells. However extracting high content information from autofluorescence imaging has been hitherto impossible. Here, we quantitatively characterise cell populations in different tissue types, live or fixed, by using novel image processing and a simple multispectral upgrade of a wide-field fluorescence microscope. Our optimal discrimination approach enables statistical hypothesis testing and intuitive visualisations where previously undetectable differences become clearly apparent. Label-free classifications are validated by the analysis of Classification Determinant (CD) antigen expression. The versatility of our method is illustrated by detecting genetic mutations in cancer, non-invasive monitoring of CD90 expression, label-free tracking of stem cell differentiation, identifying stem cell subpopulations with varying functional characteristics, tissue diagnostics in diabetes and assessing the condition of preimplantation embryos.

Effects of differing oocyte-secreted factors during mouse in vitro maturation on subsequent embryo and fetal development.

Publisher: Springer Science and Business Media LLC

Date: 11-01-2014

DOI: 10.1007/S10815-013-0152-5

IVM media are designed specifically to support immature cumulus-oocyte complexes not denuded oocytes that have failed to respond to hyperstimulation

Publisher: Elsevier BV

Date: 08-2011

DOI: 10.1016/J.FERTNSTERT.2011.06.012

Oocyte-secreted factor activation of SMAD 2/3 signaling enables initiation of mouse cumulus cell expansion.

Publisher: Oxford University Press (OUP)

Date: 05-2007

DOI: 10.1095/BIOLREPROD.106.057471

Abstract: Expansion of the mouse cumulus-oocyte complex (COC) is dependent on oocyte-secreted paracrine factors. Transforming growth factor beta (TGFB) superfamily molecules are prime candidates for the cumulus expansion-enabling factors (CEEFs), and we have recently determined that growth differentiation factor 9 (GDF9) alone is not the CEEF. The aim of this study was to examine oocyte paracrine factors and their signaling pathways that regulate mouse cumulus expansion. Using RT-PCR, oocytes were found to express the two activin subunits, Inhba and Inhbb, and activin A and activin B both enabled FSH-induced cumulus expansion of oocytectomized (OOX) complexes. Follistatin, an activin-binding protein, neutralized activin-induced expansion but had no effect on oocyte-induced expansion. The type I receptors for GDF9 and activin are activin receptor-like kinase 5 (ALK5) and ALK4, respectively, both of which activate the same SMAD 2/3 signaling pathway. We examined the requirement for this signaling system using an ALK 4/5/7 inhibitor, SB-431542. SB-431542 completely ablated FSH-stimulated GDF9-, activin A-, activin B-, and oocyte-induced cumulus expansion. Moreover, SB-431542 also antagonized epidermal growth factor-stimulated, oocyte-induced cumulus expansion. Using real-time RT-PCR, SB-431542 also attenuated GDF9-, activin A-, and oocyte-induced OOX expression of hyaluronan synthase 2, tumor necrosis factor alpha-induced protein 6, prostaglandin synthase 2, and pentraxin 3. This study provides evidence that the CEEF is composed of TGFB superfamily molecules that signal through SMAD 2/3 to enable the initiation of mouse cumulus expansion.

Effect of pre-maturation with C-type natriuretic peptide and 3-isobutyl-1-methylxanthine on cumulus-oocyte communication and oocyte developmental competence in cattle

Publisher: Elsevier BV

Date: 03-2019

DOI: 10.1016/J.ANIREPROSCI.2019.01.007

Abstract: In vitro embryo production depends on oocyte competence, which is acquired during folliculogenesis, involving cytoplasmic and nuclear processes. In vitro maturation (IVM) induces spontaneous resumption of meiosis, preventing full competence acquisition. The incorporation of a pre-IVM phase with supplementation with C-type natriuretic peptide (CNP) and 3-Isobutyl-1-methylxanthine (IBMX) was used with the aim of improving developmental competence of cattle oocytes. In a preliminary experiment, COCs were cultured with increasing CNP concentrations and nuclear stage assessment was performed. Supplementation with both 100 and 200 nM CNP resulted in more germinal vesicle (GV) arrest at 6 h of culture than those in the control group (79.3%, 76.4% and 59.2%, respectively). In a second experiment, use of 100 nM CNP plus 500 μM IBMX resulted in retention of more oocytes in the GV stage (92.0%) at 6 h of culture compared to supplementation with either CNP or IBMX alone (74.8% and 86.7%, respectively). A subsequent assessment of the effect of the pre-IVM system (6-h of culture with CNP plus IBMX), followed by 20-h of IVM, with comparison to the control at 24-h of IVM was performed. Blastocyst development rate was greater after the pre-IVM phase (45.1% compared with 34.5%). The inclusion of the pre-IVM phase also resulted in an enhanced mitochondrial activity in matured oocytes and sustained integrity of transzonal projections for longer after IVM. In conclusion, CNP and IBMX function synergistically to arrest meiosis in cattle oocytes during a pre-IVM phase, which improves cumulus-oocyte communication and embryo development.

University Of Adelaide

Location: Australia

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Murdoch University

Location: Australia

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Fertilis

Location: Australia

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AgResearch

Location: New Zealand

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ARC Centre Of Excellence For Nanoscale BioPhotonics

Start Date: 2014

End Date: 2020

Funder: Australian Research Council

Inflammatory Cytokines As Causal Agents In Peri-conception Programming Of Offspring Health

Start Date: 2015

End Date: 2017

Funder: National Health and Medical Research Council

Technology For Hormone-free Infertility Treatment

Start Date: 2014

End Date: 2017

Funder: National Health and Medical Research Council

EGF Peptide Signalling Improves Oocyte Maturation And Quality

Start Date: 2014

End Date: 2017

Funder: National Health and Medical Research Council

A Media Formulation To Improve Implantation Rates And Pregnancy Outcome Following ART

Start Date: 2008

End Date: 2010

Funder: National Health and Medical Research Council

Improving The Efficiency Of Bovine Oocyte Maturation In Vitro

Start Date: 2005

End Date: 2008

Funder: Australian Research Council

Nanosampling Sensors For Real-time Embryo Monitoring

Start Date: 2011

End Date: 2014

Funder: Australian Research Council

Development Of Engineered Novel Growth Factors For Infertility Treatment

Start Date: 2011

End Date: 2014

Funder: National Health and Medical Research Council

Reproductive Foundations For A Healthy Start To Life.

Start Date: 2007

End Date: 2011

Funder: National Health and Medical Research Council

ARC/NHMRC Research Network In Genes And Environment In Development

Start Date: 2004

End Date: 2009

Funder: Australian Research Council

Linkage Projects - Grant ID: LP110200736

Start Date: 04-2012

End Date: 12-2015

Amount: $420,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0562536

Start Date: 12-2005

End Date: 12-2009

Amount: $117,444.00

Funder: Australian Research Council

Special Research Initiatives - Grant ID: SR0354622

Start Date: 11-2003

End Date: 03-2005

Amount: $20,000.00

Funder: Australian Research Council

Research Networks - Grant ID: RN0457079

Start Date: 09-2004

End Date: 08-2009

Amount: $1,500,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE170100241

Start Date: 07-2017

End Date: 06-2018

Amount: $480,000.00

Funder: Australian Research Council