Capacitation IVM improves cumulus function and oocyte quality in minimally stimulated mice.

PURPOSE: Oocyte in vitro maturation (IVM) is a patient-friendly reproductive technology but lower success rates than IVF have limited its uptake. Capacitation-IVM (CAPA-IVM) is an innovative new IVM system currently undergoing clinical evaluation. This study aimed to determine temporal effects of the pre-IVM phase of CAPA-IVM on cumulus function and oocyte developmental competence in mildly-stimulated mice. METHODS: Immature cumulus oocyte complexes (COCs) derived from mildly stimulated (23 h PMSG) 28-day-old mice underwent pre-IVM for 0-24 h in medium containing c-type natriuretic peptide (CNP), E2, FSH and insulin, prior to IVM (CAPA-IVM). The effect of pre-IVM duration on cumulus cell function and embryo development post-CAPA-IVM/IVF was assessed. RESULTS: Day 6 blastocyst rate increased incrementally with increasing pre-IVM duration: 40.6 ± 2.0%, 45.8 ± 1.2%, 52.2 ± 3.5%, 53.3 ± 5.9%, and 59.9 ± 2.5% for 0, 2, 6, 12, and 24 h pre-IVM, respectively (P < 0.01). DNA content/COC, a measure of cumulus cell proliferation, was significantly higher with 24 h pre-IVM group compared to 0, 2, or 6 h pre-IVM (P < 0.001). Pre-IVM for 24 h significantly increased cumulus expansion and mRNA expression of matrix genes Has2 and Tnfaip6 and Areg relative to no pre-IVM control (P < 0.01). Cumulus-oocyte gap-junctional communication (GJC) was maintained throughout 24 h pre-IVM (P < 0.0001), and GJC loss was slowed during the subsequent IVM phase, whilst meiotic resumption was accelerated (P < 0.05). Pre-IVM increased COC ATP and ADP content (P < 0.05), but not AMP, ATP/ADP, and energy charge. CONCLUSION: The pre-IVM phase of CAPA-IVM improves the quality of IVM oocytes in a temporally dependent manner and significantly influences cumulus cell function including increased cell proliferation, cumulus expansion, and prolonged cumulus-oocyte GJC.

In vitro survival of follicles in prepubertal ewe ovarian cortex cryopreserved by slow freezing or non-equilibrium vitrification.

PURPOSE: Vitrification is a well-accepted fertility preservation procedure for cryopreservation of oocytes and embryos but little is known regarding ovarian tissue, for which slow freezing is the current convention. The aim of the present study was to assess the efficiency of non-equilibrium vitrification compared to conventional slow freezing for ovarian cortex cryopreservation. METHODS: Using prepubertal sheep ovaries, the capacity of the tissue to sustain folliculogenesis following cryopreservation and in vitro culture was evaluated. Ovarian cortex fragments were cultured in wells for 9 days, immediately or after cryopreservation by conventional slow freezing or non-equilibrium vitrification in straws. During culture, follicular populations within cortex were evaluated by histology and immunohistochemistry for PCNA and TUNEL. Steroidogenic activity of the tissue was monitored by assay for progesterone and estradiol in spent media. RESULTS: No significant differences in follicle morphology, PCNA, or TUNEL labeling were observed between cryopreservation methods at the initiation of culture. Similar decreases in the proportion of primordial follicle population, and increases in the proportion of growing follicles, were observed following culture of fresh or cryopreserved ovarian tissue regardless of cryopreservation method. At the end of culture, PCNA and TUNEL-positive follicles were not statistically altered by slow freezing or vitrification in comparison to fresh cultured fragments. CONCLUSIONS: Overall, for both cryopreservation methods, the cryopreserved tissue showed equal capacity to fresh tissue for supporting basal folliculogenesis in vitro. Taken together, these data confirm that both non-equilibrium vitrification and slow-freezing methods are both efficient for the cryopreservation of sheep ovarian cortex fragments.

Non-canonical cyclic AMP SMAD1/5/8 signalling in human granulosa cells.

Development of mammalian ovarian follicles is promoted by the combined action of endocrine cues and paracrine factors. Follicle stimulating hormone (FSH), through the action of cAMP drives follicular growth and development. The oocyte secretes powerful growth factors such as bone morphogenetic protein 15 (BMP15) to regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation through the activation of SMAD1/5/8. This study investigated the role of the cAMP signalling pathway on SMAD1/5/8 action in human granulosa cells. Cyclic AMP enhanced BMP15-induction of a SMAD1/5/8-specific BRE reporter. Moreover, in the absence of BMP ligand, cAMP also activated SMAD1/5/8-induced BRE activity. Cyclic AMP increased canonical downstream targets of BMP signalling such as inhibitor of differentiation (ID) mRNA expression. The observed effects were not mediated by secretion of BMPs as cAMP did not promote BMP ligand mRNA expression and a BMP extracellular antagonist, the BMP type II receptor ectodomain, did not affect cAMP-induced ID mRNA expression. Finally, the ERK1/2 pathway was shown to be required for the maintenance of cAMP-induced SMAD1/5/8 activity. Together our results suggest a novel and non-canonical pathway for cAMP signalling in human granulosa cells. Cyclic AMP appears to promote SMAD1/5/8 pathway activity intracellularly and has the ability to activate canonical SMAD1/5/8 downstream targets. Our results add another layer of complexity to the interactions between endocrine signalling and oocyte-secreted BMP ligands during folliculogenesis. Given the importance of both cAMP and SMAD1/5/8 pathways in follicular development, these interactions are likely required for the fine-tuning of oocyte paracrine signalling by endocrine stimuli.

Evidence from animal models on the pathogenesis of PCOS.

Polycystic ovarian syndrome (PCOS) is the most common endocrine condition in women, and is characterized by reproductive, endocrine and metabolic features. However, there is no simple unequivocal diagnostic test for PCOS, its etiology remains unknown and there is no cure. Hence, the management of PCOS is suboptimal as it relies on the ad hoc empirical management of its symptoms only. Decisive studies are required to unravel the origins of PCOS, but due to ethical and logistical reasons these are not possible in humans. Experimental animal models for PCOS have been established which have enhanced our understanding of the mechanisms underlying PCOS and propose novel mechanism-based therapies to treat the condition. This review examines the findings from various animal models to reveal the current knowledge of the mechanisms underpinning the development of PCOS, and also provides insights into the implications from these studies for improved clinical management of this disorder.

Recent and emerging reproductive biology research in Australia and New Zealand: highlights from the Society for Reproductive Biology Annual Meeting, 2017.

Research in reproductive science is essential to promote new developments in reproductive health and medicine, agriculture and conservation. The Society for Reproductive Biology (SRB) 2017 conference held in Perth (WA, Australia) provided a valuable update on current research programs in Australia and New Zealand. This conference review delivers a dedicated summary of significant questions, emerging concepts and innovative technologies presented in the symposia. This research demonstrates significant advances in the identification of precursors for a healthy pregnancy, birth and child, and discusses how these factors can influence disease risk. A key theme included preconception parental health and its effect on gametogenesis, embryo and fetal development and placental function. In addition, the perturbation of key developmental checkpoints was shown to contribute to a variety of pathological states that have the capacity to affect health and fertility. Importantly, the symposia discussed in this review emphasised the role of reproductive biology as a conduit for understanding the transmission of non-communicable diseases, such as metabolic disorders and cancers. The research presented at SRB 2017 has revealed key findings that have the prospect to change not only the fertility of the present generation, but also the health and reproductive capacity of future generations.

Multigenerational obesity-induced perturbations in oocyte-secreted factor signalling can be ameliorated by exercise and nicotinamide mononucleotide.

STUDY QUESTION: Can maternal and offspring high-fat diet (HFD)-induced changes in mRNA expression levels in mice be ameliorated by interventions in female offspring? SUMMARY ANSWER: Our results indicate that exercise and nicotinamide mononucleotide (NMN) can ameliorate the negative effects of maternal and post-weaning HFD in female offspring. WHAT IS KNOWN ALREADY: Maternal and post-weaning HFD can perturb offspring developmental trajectories. As rates of maternal obesity are rising globally, there is a need for effective treatments in offspring to ameliorate the negative effects from a maternal obesogenic environment. Modulation of the nicotinamide adenine dinucleotide (NAD+) pathway by exercise and the NAD+ precursor NMN has previously been shown to reduce the effects of obesity. STUDY DESIGN SIZE DURATION: This study consisted of a multigenerational study using C57Bl6 mice. Mice were fed a control (chow) or HFD ad libitum throughout mating, pregnancy and lactation (n = 13-25). Female offspring (n = 72) were then also supplied either a chow or HFD post-weaning. At 9 weeks of age offspring from HFD dams were subjected to exercise on a treadmill for 9 weeks or at 16 weeks of age administered NMN (i.p.) for 2.5 weeks. At 18.5 weeks mice were euthanized and ovaries and cumulus-oocyte complexes (COC) were collected to examine the possibility of ameliorating the negative effects of maternal and post-weaning HFD. PARTICIPANTS/MATERIALS SETTING METHODS: Ovary and COC mRNA expression was analysed using RT-qPCR. An initial screen of candidate genes was developed to test which molecular pathways may be involved in generating adverse reproductive system effects. For histological analysis, ovarian tissue was fixed in paraformaldehyde and embedded in paraffin and stained with haematoxylin and eosin. The numbers of primordial, primary, secondary and antral follicles were counted. MAIN RESULTS AND THE ROLE OF CHANCE: In the offspring's COC, maternal obesity increased both growth differentiation factor 9 (Gdf9: 2-fold; P < 0.05, HFD versus chow) and bone morphogenetic protein 15 (Bmp15: 4-fold; P < 0.05, HFD versus chow) mRNA expression levels while exercise and NMN interventions did not regulate Gdf9 and Bmp15 in the same manner. In whole ovary, maternal diet programmed a 25-50% reduction in FSH receptor and sirtuin-3 mRNA expression levels in daughter ovaries (P < 0.05, HFD versus chow). There was a significant interaction between HFD and intervention on the proportion of large preantral and preovulatory follicles (P < 0.05). However, the increase in preovulatory follicles did not translate to increased oocyte yield. NMN administration resulted in reduced body weight in HFD-fed individuals. LIMITATIONS REASONS FOR CAUTION: It is unclear if the changes in oocyte mRNA expression levels reported here will impact oocyte quality and fertility in offspring. Offspring ovulation rate or fecundity could not be studied here and fertility trials are required to determine if the changes in gene expression do reduce fertility. WIDER IMPLICATIONS OF THE FINDINGS: Our results demonstrate that maternal and offspring HFD perturbs key signalling pathways that are known to regulate fertility in mice, highlighting the importance of interventions in helping to prevent the declining rates of fertility in the context of the current obesity epidemic. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants and fellowships from the National Health and Medical Research Council to R.B.G. (APP1023210, APP1062762, APP1117538) and to M.J.M. and D.A.S. (APP1044295). DAS is a consultant to and inventor on patents licenced to Ovascience, Metrobiotech and GlaxoSmithKline. The other authors declare that there is no conflict of interest.

The current state of reproductive biology research in Australia and New Zealand: core themes from the Society for Reproductive Biology Annual Meeting, 2016.

Because reproduction is essential for all life, it is central to our understanding of all aspects of biology. The Society for Reproductive Biology (SRB) 2016 conference held on the Gold Coast (Qld, Australia) displayed the current breadth of reproductive research in Australia and New Zealand, with additional insights from world leaders in the field. This conference review provides a focused summary of the key questions, emerging ideas and novel technologies that were presented in the symposia. Presented research demonstrated key advances in how stem cell biology may allow us to better understand pluripotency, as well as how environmental and lifestyle factors, such as circadian disruption, smoking, alcohol and diet, affect gametogenesis, embryo implantation, placental function and reproductive capacity. Sessions also highlighted the role of reproductive biology in providing insight into the mechanisms and processes governing a wide range of biological science disciplines, including cancer research and therapies, oncofertility, conservation of native species and chronic non-communicable diseases. Recurring themes included the importance of male and female gamete quality for reproductive potential and the critical and varied roles of the placenta in the maintenance of a healthy pregnancy. Dysregulation of reproductive processes can contribute to a variety of pathological states that affect future health, fertility and fecundity. Research being conducted by the SRB has the potential to shape not only the fertility of the current generation, but also the health and reproductive viability of future generations.

Relative mRNA expression and immunolocalization for transforming growth factor-beta (TGF-ß) and their effect on in vitro development of caprine preantral follicles.

This study aimed to evaluate the immunolocalization and messenger RNA (mRNA) expression for transforming growth factor-beta (TGF-ß) and its receptors (TGF-ßRI and RII), as well as mRNA expression for P450 aromatase and FSH receptor in caprine preantral follicles. The effects of TGF-ß, FSH alone, or in association on the in vitro follicular development were also assessed. Immunohistochemical analyses showed the expression of TGF-ß and its receptors in oocytes of all follicle stages and granulosa cells of primary and secondary follicles. mRNA for TGF-ß receptors and for FSH receptor (FSHR) was present in preantral follicles as well as in oocytes and granulosa cells of antral follicles. Isolated secondary follicles were cultured in α-minimum essential medium (MEM) alone or supplemented with either FSH (100 ng/ml), TGF-ß (10 ng/ml), or TGF-ß + FSH for 18 d. TGF-ß increased significantly oocyte diameter when compared to FSH alone and control. After 18 d of culture, all groups showed a significant reduction in P450 aromatase and FSHR mRNA levels in comparison to fresh control. In contrast, treatment with FSH significantly increased the mRNA expression for TGF-ß in comparison to fresh control and other treatments. In conclusion, the findings showed that TGF-ß and its receptors are present in caprine ovarian follicles. Furthermore, they showed a positive effect on oocyte growth in vitro.

[Regulating pre-antral follicle development: a brake on depletion of the ovarian reserve]. / Régulation de la croissance des follicules pré-antraux, un frein à l'épuisement de la réserve ovarienne.

The process of folliculogenesis is a complex and dynamic process that is regulated by positive and negative factors. During folliculogenesis in the mammalian ovary, the primordial follicle pool is gradually reduced through successive waves of follicle growth. It is important that this pool is carefully regulated, otherwise premature ovarian failure will occur. This process results in the ovulation of an ovocyte or more commonly, the loss of ovocytes through atresia. The majority of research has concentrated on the positive regulators of follicle growth with very little attention on the negative regulators. Nonetheless, there are several factors that have been observed to inhibit follicle activation and development. In this review, we summarize those here.

Seasonal variation in the ovarian function of sows.

The modern domestic sow exhibits a period of impaired reproductive performance known as seasonal infertility during the late summer and early autumn months. A reduction in farrowing rate due to pregnancy loss is the most economically significant manifestation of this phenomenon. Presently, little is known of the aetiology of seasonal pregnancy loss in the pig. Recent findings represent a major advancement in the understanding of sow reproductive physiology and implicate poor oocyte developmental competence as a contributing factor to pregnancy loss during the seasonal infertility period. It has also been demonstrated that ovarian activity is depressed during the seasonal infertility period. The reduction in oocyte quality is associated with decreased levels of progesterone in follicular fluid during final oocyte maturation in vivo. The recent identification of sow-specific risk factors, such as parity for late pregnancy loss, should improve breeding herd efficiency by allowing producers to tailor management interventions and/or culling protocols that target animals identified as having a greater risk of late pregnancy loss during the seasonal infertility period.

Seasonal effects on oocyte developmental competence in sows experiencing pregnancy loss.

Recently, oocyte quality in sows culled for reasons unrelated to fertility was found to decline during the period of seasonal infertility. Wean-to-service interval (WSI) has also been associated with pregnancy loss in sows mated during the period of seasonal infertility. The aims of this study were to determine whether WSI and season are associated with changes in oocyte developmental competence in sows experiencing early (before Day 35 of gestation) and late (after Day 35 of gestation) pregnancy loss. Ovaries were collected in pairs from sows sourced from commercial piggeries that were culled for reasons related to infertility after being mated in summer and winter/spring. Sows were grouped according to their pregnancy loss type, their previous WSI and the presence or absence of corpora lutea (CL) on their ovaries. Oocyte developmental competence was assessed following in vitro maturation, artificial activation and parthenote development in vitro. In sows culled for early-pregnancy loss, there was a greater number of CL present on ovaries collected in spring compared to those collected in summer (11.57±3.3 vs. 9.26±0.99; P<0.05). Also, the proportion of oocytes developing to the blastocyst stage was greater in summer than in spring (55.9±5.2% vs. 31.2±6.4%; P<0.05). In sows culled for late-pregnancy loss, a greater proportion of oocytes developed to the blastocyst stage in winter compared with late-spring (64.3±7.0% vs. 34.1±6.6%; P<0.05). In addition, the blastocyst formation rate of oocytes was lower in sows that displayed a WSI≤6 days than in sows that displayed a WSI>6 days (37.8±7.3% vs. 62.2±6.9%; P<0.05). The results of the present study indicate that sows culled for pregnancy loss exhibit seasonal changes in oocyte developmental competence. The mechanism which causes WSI to be prolonged does not appear to result in reduced oocyte developmental competence. While poor oocyte quality and the mechanism that increases WSI may contribute to pregnancy loss during the seasonal infertility period, the findings suggest that these factors are not the main drivers of early and late pregnancy loss throughout the year.