Oocyte maturation and quality: role of cyclic nucleotides

Advance Publication first posted online on 15 July 2016 - Accepted manuscriptThe 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 preovulatory 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 2 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 practiced clinically do not recapitulate this cAMP surge in vitro, possibly accounting for the lower efficiency of IVM compared to clinical IVF. This review focuses in particular 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.Gilchrist RB, Luciano AM, Richani D, Zeng HT, Wang X, De Vos M, Sugimura S, Smitz J, Richard FJ and Thompson J

Participation of the adenosine salvage pathway and cyclic AMP modulation in oocyte energy metabolism

A follicular spike in cyclic AMP (cAMP) and its subsequent degradation to AMP promotes oocyte maturation and ovulation. In vitro matured (IVM) oocytes do not receive the cAMP increase that occurs in vivo, and artificial elevation of cAMP in IVM cumulus-oocyte complexes improves oocyte developmental potential. This study examined whether mouse oocytes can use the cAMP degradation product AMP to generate ATP via the adenosine salvage pathway, and examined whether pharmacological elevation of cAMP in IVM cumulus-oocyte complexes alters ATP levels. Oocytes cultured with isotopic 13C5-AMP dose-dependently produced 13C5-ATP, however total cellular ATP remained constant. Pharmacological elevation of cAMP using forskolin and IBMX prior to IVM decreased oocyte ATP and ATP:ADP ratio, and promoted activity of the energy regulator AMPK. Conversely, cumulus cells exhibited higher ATP and no change in AMPK. Culture of oocytes without their cumulus cells or inhibition of their gap-junctional communication yielded lower oocyte 13C5-ATP, indicating that cumulus cells facilitate ATP production via the adenosine salvage pathway. In conclusion, this study demonstrates that mouse oocytes can generate ATP from AMP via the adenosine salvage pathway, and cAMP elevation alters adenine nucleotide metabolism and may provide AMP for energy production via the adenosine salvage pathway during the energetically demanding process of meiotic maturation.Dulama Richani, Cathy F. Lavea, Raji Kanakkaparambil, Angelique H. Riepsamen, Michael J. Bertoldo, Sonia Bustamante and Robert B. Gilchris

Cumulin, an Oocyte-secreted Heterodimer of the Transforming Growth Factor-β Family, Is a Potent Activator of Granulosa Cells and Improves Oocyte Quality

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-specific growth factors with central roles in mammalian reproduction, regulating species-specific fecundity, ovarian follicular somatic cell differentiation, and oocyte quality. In the human, GDF9 is produced in a latent form, the mechanism of activation being an open question. Here, we produced a range of recombinant GDF9 and BMP15 variants, examined their in silico and physical interactions and their effects on ovarian granulosa cells (GC) and oocytes. We found that the potent synergistic actions of GDF9 and BMP15 on GC can be attributed to the formation of a heterodimer, which we have termed cumulin. Structural modeling of cumulin revealed a dimerization interface identical to homodimeric GDF9 and BMP15, indicating likely formation of a stable complex. This was confirmed by generation of recombinant heterodimeric complexes of pro/mature domains (pro-cumulin) and covalent mature domains (cumulin). Both pro-cumulin and cumulin exhibited highly potent bioactivity on GC, activating both SMAD2/3 and SMAD1/5/8 signaling pathways and promoting proliferation and expression of a set of genes associated with oocyte-regulated GC differentiation. Cumulin was more potent than pro-cumulin, pro-GDF9, pro-BMP15, or the two combined on GC. However, on cumulus-oocyte complexes, pro-cumulin was more effective than all other growth factors at notably improving oocyte quality as assessed by subsequent day 7 embryo development. Our results support a model of activation for human GDF9 dependent on cumulin formation through heterodimerization with BMP15. Oocyte-secreted cumulin is likely to be a central regulator of fertility in mono-ovular mammals

Cumulin and FSH cooperate to regulate inhibin B and activin B production by human granulosa-lutein cells in vitro

Meeting abstract, from the 35th Annual Meeting of the European Society of Human Reproduction and Embryology

Niclosamide reduces glucagon sensitivity via hepatic PKA inhibition in obese mice: implications for glucose metabolism improvements in type 2 diabetes

Type 2 diabetes (T2D) is a global pandemic. Currently, the drugs used to treat T2D improve hyperglycemic symptom of the disease but the underlying mechanism causing the high blood glucose levels have not been fully resolved. Recently published data showed that salt form of niclosamide improved glucose metabolism in high fat fed mice via mitochondrial uncoupling. However, based on our previous work we hypothesised that niclosamide might also improve glucose metabolism via inhibition of the glucagon signalling in liver in vivo. In this study, mice were fed either a chow or high fat diet containing two different formulations of niclosamide (niclosamide ethanolamine salt - NENS or niclosamide - Nic) for 10 weeks. We identified both forms of niclosamide significantly improved whole body glucose metabolism without altering total body weight or body composition, energy expenditure or insulin secretion or sensitivity. Our study provides evidence that inhibition of the glucagon signalling pathway contributes to the beneficial effects of niclosamide (NENS or Nic) on whole body glucose metabolism. In conclusion, our results suggest that the niclosamide could be a useful adjunctive therapeutic strategy to treat T2D, as hepatic glucose output is elevated in people with T2D and current drugs do not redress this adequately.Md. Kamrul Hasan Chowdhury, Nigel Turner, Nicholas L. Bentley, Abhirup Das, Lindsay E. Wu, Dulama Richani, Sonia Bustamante, Robert B. Gilchrist, Margaret J. Morris, Peter R. Shepherd, Greg C. Smit

The epidermal growth factor network: role in oocyte growth, maturation and developmental competence

Advanced Access publication on September 20, 2017BACKGROUND: The LH surge induces great physiological changes within the preovulatory follicle, which culminate in the ovulation of a mature oocyte that is capable of supporting embryo and foetal development. However, unlike mural granulosa cells, the oocyte and its surrounding cumulus cells are not directly responsive to LH, indicating that the LH signal is mediated by secondary factors produced by the granulosa cells. The mechanisms by which the oocyte senses the ovulatory LH signal and hence prepares for ovulation has been a subject of considerable controversy for the past four decades. Within the last 15 years several significant insights have been made into the molecular mechanisms orchestrating oocyte development, maturation and ovulation. These findings centre on the epidermal growth factor (EGF) pathway and the role it plays in the complex signalling network that finely regulates oocyte maturation and ovulation. OBJECTIVE AND RATIONALE: This review outlines the role of the EGF network during oocyte development and regulation of the ovulatory cascade, and in particular focuses on the effect of the EGF network on oocyte developmental competence. Application of this new knowledge to advances in ART is examined. SEARCH METHODS: The PubMed database was used to search for peer-reviewed original and review articles concerning the EGF network. Publications offering a comprehensive description of the role of the EGF network in follicle and oocyte development were used. OUTCOMES: It is now clear that acute upregulation of the EGF network is an essential component of the ovulatory cascade as it transmits the LH signal from the periphery of the follicle to the cumulus-oocyte complex (COC). More recent findings have elucidated new roles for the EGF network in the regulation of oocyte development. EGF signalling downregulates the somatic signal 3′5′-cyclic guanine monophosphate that suppresses oocyte meiotic maturation and simultaneously provides meiotic inducing signals. The EGF network also controls translation of maternal transcripts in the quiescent oocyte, a process that is integral to oocyte competence. As a means of restricting the ovulatory signal to the Graffian follicle, most COCs in the ovary are unresponsive to EGF-ligands. Recent studies have revealed that development of a functional EGF signalling network in cumulus cells requires dual endocrine (FSH) and oocyte paracrine cues (growth differentiation factor 9 and bone morphogenetic protein 15), and this occurs progressively in COCs during the last stages of folliculogenesis. Hence, a new concept to emerge is that cumulus cell acquisition of EGF receptor responsiveness represents a developmental hallmark in folliculogenesis, analogous to FSH-induction of LH receptor signalling in mural granulosa cells. Likewise, this event represents a major milestone in the oocyte’s developmental progression and acquisition of developmental competence. It is now clear that EGF signalling is perturbed in COCs matured in vitro. This has inspired novel concepts in IVM systems to ameliorate this perturbation, resulting in improved oocyte developmental competence. WIDER IMPLICATIONS: An oocyte of high quality is imperative for fertility. Elucidating the fundamental molecular and cellular mechanims by which the EGF network regulates oocyte maturation and ovulation can be expected to open new opportunities in ART. This knowledge has already led to advances in oocyte IVM in animal models. Translation of such advances into a clinical setting should increase the efficacy of IVM, making it a viable treatment option for a wide range of patients, thereby simplifying fertility treatment and bringing substantial cost and health benefits.Dulama Richani and Robert B. Gilchris

Somatic guidance for the oocyte

Abstract not availableRobert B. Gilchrist and Dulama Richan

A sensitive method for the separation and quantification of low-level adenine nucleotides using porous graphitic carbon-based liquid chromatography and tandem mass spectrometry

Abstract not availableSonia Bustamante, Robert B. Gilchrist, Dulama Richan

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

Background: 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. Objective and Rationale: 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. Search Methods: 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'. Outcomes: 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. Wider Implications: 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.Dulama Richani, Kylie R. Dunning, Jeremy G. Thompson, and Robert B. Gilchris