Cumulus cell contact during oocyte maturation in mice regulates meiotic spindle positioning and enhances developmental competence

In this study, we collected follicular fluid, granulosa cells, and cumulus cells from antral follicles at specific time intervals following equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) treatment of gilts. The treatment with eCG increased the production of estrogen coordinately with up-regulated proliferation of granulosa and cumulus cells. eCG also induced the expression ofLHCGRandPGRin cumulus cells and progesterone accumulation was detected in follicular fluid prior to the LH/hCG surge. Moreover, progesterone and progesterone receptor (PGR) were critical for FSH-inducedLHCGRexpression in cumulus cells in culture. The expression ofLHCGRmRNA in cumulus cells was associated with the ability of LH to induce prostaglandin production, release of epidermal growth factor (EGF)-like factors, and a disintegrin and metalloprotease with thrombospondin-like repeats 1 expression, promoting cumulus cell oocyte complexes (COCs) expansion and oocyte maturation. Based on the unique expression and regulation ofPGRandLHCGRin cumulus cells, we designed a novel porcine COCs culture system in which hormones were added sequentially to mimic changes observedin vivo. Specifically, COCs from small antral follicles were pre-cultured with FSH and estradiol for 10 h at which time progesterone was added for another 10 h. After 20 h, COCs were moved to fresh medium containing LH, EGF, and progesterone. The oocytes matured in this revised COC culture system exhibited greater developmental competence to blastocyst stage. From these results, we conclude that to achieve optimal COC expansion and oocyte maturation in culture the unique gene expression patterns in cumulus cells of each species need to be characterized and used to increase the effectiveness of hormone stimulation.

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Effect of epidermal growth factor-like peptides on pig cumulus cell expansion, oocyte maturation, and acquisition of developmental competence in vitro: comparison with gonadotropins

Reproduction ◽

10.1530/rep-10-0418 ◽

2011 ◽

Vol 141 (4) ◽

pp. 425-435 ◽

Cited By ~ 48

Author(s):

Radek Procházka ◽

Michal Petlach ◽

Eva Nagyová ◽

Lucie Němcová

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Oocyte Maturation ◽

Cumulus Cell ◽

Cumulus Cells ◽

Blastocyst Stage ◽

Human Chorionic Gonadotrophin ◽

Developmental Competence ◽

Epidermal Growth

The aim of this work was to assess the FSH-stimulated expression of epidermal growth factor (EGF)-like peptides in cultured cumulus–oocyte complexes (COCs) and to find out the effect of the peptides on cumulus expansion, oocyte maturation, and acquisition of developmental competencein vitro. FSH promptly stimulated expression of amphiregulin (AREG) and epiregulin (EREG), but not betacellulin (BTC) in the cultured COCs. Expression ofAREGandEREGreached maximum at 2 or 4 h after FSH addition respectively. FSH also significantly stimulated expression of expansion-related genes (PTGS2,TNFAIP6, andHAS2) in the COCs at 4 and 8 h of culture, with a significant decrease at 20 h of culture. Both AREG and EREG also increased expression of the expansion-related genes; however, the relative abundance of mRNA for each gene was much lower than in the FSH-stimulated COCs. In contrast to FSH, AREG and EREG neither stimulated expression ofCYP11A1in the COCs nor an increase in progesterone production by cumulus cells. AREG and EREG stimulated maturation of oocytes and expansion of cumulus cells, although the percentage of oocytes that had reached metaphase II was significantly lower when compared to FSH-induced maturation. Nevertheless, significantly more oocytes stimulated with AREG and/or EREG developed to blastocyst stage after parthenogenetic activation when compared to oocytes stimulated with FSH alone or combinations of FSH/LH or pregnant mares serum gonadotrophin/human chorionic gonadotrophin. We conclude that EGF-like peptides do not mimic all effects of FSH on the cultured COCs; nevertheless, they yield oocytes with superior developmental competence.

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Protein deubiquitination during oocyte maturation influences sperm function during fertilisation, antipolyspermy defense and embryo development

Reproduction Fertility and Development ◽

10.1071/rd14012 ◽

2015 ◽

Vol 27 (8) ◽

pp. 1154 ◽

Cited By ~ 9

Author(s):

Young-Joo Yi ◽

Miriam Sutovsky ◽

Won-Hee Song ◽

Peter Sutovsky

Keyword(s):

Embryo Development ◽

Oocyte Maturation ◽

26S Proteasome ◽

Developmental Competence ◽

Meiotic Spindle ◽

Cortical Granule ◽

Sperm Function ◽

Post Translational Modification ◽

Preimplantation Embryo Development ◽

Porcine Oocyte

Ubiquitination is a covalent post-translational modification of proteins by the chaperone protein ubiquitin. Upon docking to the 26S proteasome, ubiquitin is released from the substrate protein by deubiquitinating enzymes (DUBs). We hypothesised that specific inhibitors of two closely related oocyte DUBs, namely inhibitors of the ubiquitin C-terminal hydrolases (UCH) UCHL1 (L1 inhibitor) and UCHL3 (L3 inhibitor), would alter porcine oocyte maturation and influence sperm function and embryo development. Aberrant cortical granule (CG) migration and meiotic spindle defects were observed in oocytes matured with the L1 or L3 inhibitor. Embryo development was delayed or blocked in oocytes matured with the general DUB inhibitor PR-619. Aggresomes, the cellular stress-inducible aggregates of ubiquitinated proteins, formed in oocytes matured with L1 inhibitor or PR-619, a likely consequence of impaired protein turnover. Proteomic analysis identified the major vault protein (MVP) as the most prominent protein accumulated in oocytes matured with PR-619, suggesting that the inhibition of deubiquitination altered the turnover of MVP. The mitophagy/autophagy of sperm-contributed mitochondria inside the fertilised oocytes was hindered by DUB inhibitors. It is concluded that DUB inhibitors alter porcine oocyte maturation, fertilisation and preimplantation embryo development. By regulating the turnover of oocyte proteins and mono-ubiquitin regeneration, the DUBs may promote the acquisition of developmental competence during oocyte maturation.

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Oocyte Induction of EGF Responsiveness in Somatic Cells Is Associated With the Acquisition of Porcine Oocyte Developmental Competence

Endocrinology ◽

10.1210/en.2014-1884 ◽

2015 ◽

Vol 156 (6) ◽

pp. 2299-2312 ◽

Cited By ~ 27

Author(s):

Lesley J. Ritter ◽

Satoshi Sugimura ◽

Robert B. Gilchrist

Keyword(s):

Somatic Cell ◽

Oocyte Maturation ◽

Egf Receptor ◽

Somatic Cells ◽

Cumulus Cell ◽

Developmental Competence ◽

Cumulus Expansion ◽

Cell Functions ◽

Oocyte Developmental Competence

Abstract Oocytes progressively acquire the competence to support embryo development as oogenesis proceeds with ovarian folliculogenesis. The objectives of this study were to investigate oocyte-secreted factor (OSF) participation in the development of somatic cell epidermal growth factor (EGF) responsiveness associated with oocyte developmental competence. A well-established porcine model was employed using oocytes from small (<4 mm) vs medium sized (>4 mm) antral follicles, representing low vs moderate developmental competence, respectively. Cumulus-oocyte complexes (COCs) were treated in vitro with inducers of oocyte maturation, and cumulus cell functions and oocyte developmental competence were assessed. COCs from small follicles responded to FSH but, unlike COCs from larger follicles, were incapable of responding to EGF family growth factors known to mediate oocyte maturation in vivo, exhibiting perturbed cumulus expansion and expression of associated transcripts (HAS2 and TNFAIP6). Low and moderate competence COCs expressed equivalent levels of EGF receptor (EGFR) mRNA; however, the former had less total EGFR protein leading to failed activation of phospho-EGFR and phospho-ERK1/2, despite equivalent total ERK1/2 protein levels. Native OSFs from moderate, but not from low, competence oocytes established EGF responsiveness in low competence COCs. Four candidate recombinant OSFs failed to mimic the actions of native OSFs in regulating cumulus expansion. Treatment with OSFs and EGF enhanced oocyte competence but only of the low competence COCs. These data suggest that developmental acquisition by the oocyte of capacity to regulate EGF responsiveness in the oocyte's somatic cells is a major milestone in the oocyte's developmental program and contributes to coordinated oocyte and somatic cell development.

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

Human Reproduction Update ◽

10.1093/humupd/dmaa043 ◽

2020 ◽

Vol 27 (1) ◽

pp. 27-47

Author(s):

Dulama Richani ◽

Kylie R Dunning ◽

Jeremy G Thompson ◽

Robert B Gilchrist

Keyword(s):

Oocyte Maturation ◽

Cell Metabolism ◽

Cumulus Cell ◽

Cumulus Cells ◽

Oocyte Quality ◽

Developmental Competence ◽

Atp Production ◽

Oocyte Developmental Competence ◽

Metabolic Conditions

Abstract 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.

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Role for cumulus cell-produced EGF-like ligands during primate oocyte maturation in vitro

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.90930.2008 ◽

2009 ◽

Vol 296 (5) ◽

pp. E1049-E1058 ◽

Cited By ~ 19

Author(s):

Jenna K. Nyholt de Prada ◽

Young S. Lee ◽

Keith E. Latham ◽

Charles L. Chaffin ◽

Catherine A. VandeVoort

Keyword(s):

Rhesus Macaque ◽

Oocyte Maturation ◽

Egf Receptor ◽

Cumulus Cell ◽

Cumulus Cells ◽

Developmental Competence ◽

Lower Percentage ◽

Egfr Activation

The developmental competence of in vitro-matured (IVM) rhesus macaque cumulus oocyte complexes (COCs) is deficient compared with in vivo-matured (IVM) oocytes. To improve oocyte quality and subsequent embryo development following IVM, culture conditions must be optimized. A series of experiments was undertaken to determine the role of epidermal growth factor (EGF) during IVM of rhesus macaque COCs. The addition of Tyrphostin AG-1478 (a selective inhibitor of the EGF receptor EGFR) to the IVM medium yielded fewer oocytes maturing to metaphase II of meiosis II (MII), decreased cumulus expansion, and a lower percentage of embryos that developed to the blastocyst stage compared with untreated IVM controls, indicating that EGFR activation is important for IVM maturation in the rhesus macaque. However, the addition of recombinant human EGF (r-hEGF) to the IVM medium did not enhance outcome. The expression of mRNAs encoding the EGF-like factors amphiregulin, epiregulin, and betacellulin in cumulus cells indicates that these factors produced by cumulus cells may be responsible for maximal EGFR activation during oocyte maturation, precluding any further effect of exogenous r-hEGF. Additionally, these results illustrate the potential futility of exogenous supplementation of IVM medium without prior knowledge of pathway activity.

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Recent insights into oocyte - follicle cell interactions provide opportunities for the development of new approaches to in vitro maturation

Reproduction Fertility and Development ◽

10.1071/rd10225 ◽

2011 ◽

Vol 23 (1) ◽

pp. 23 ◽

Cited By ~ 141

Author(s):

Robert B. Gilchrist

Keyword(s):

Oocyte Maturation ◽

In Vitro Maturation ◽

Egf Receptor ◽

Cumulus Cell ◽

Follicle Cell ◽

Cell Interactions ◽

Developmental Competence ◽

New Approaches

The last 5–10 years of research in ovarian and oocyte biology has delivered some major new advances in knowledge of the molecular and cellular processes regulating oocyte maturation and oocyte developmental competence. These new insights include, among others: (1) the knowledge that oocytes regulate granulosa and cumulus cell differentiation, ovulation rate and fertility via the secretion of soluble paracrine growth factors; (2) new perspectives on the participation of cyclic nucleotides, phosphodiesterases and gap junctions in the regulation of oocyte meiotic arrest and resumption; and (3) the new appreciation of the mechanisms of LH-induced oocyte maturation and ovulation mediated by the follicular cascade of epidermal growth factor (EGF)-like peptides, the EGF receptor and their intracellular second messengers. These recent insights into oocyte–follicle cell interactions provide opportunities for the development of new approaches to oocyte in vitro maturation (IVM). Laboratory IVM methodologies have changed little over the past 20–30 years and IVM remains notably less efficient than hormone-stimulated IVF, limiting its wider application in reproductive medicine and animal breeding. The challenge for oocyte biologists and clinicians practicing IVM is to modernise clinical IVM systems to benefit from these new insights into oocyte–follicle cell interactions in vivo.

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LIMK1/2 inhibitor LIMKi 3 suppresses porcine oocyte maturation

PeerJ ◽

10.7717/peerj.2553 ◽

2016 ◽

Vol 4 ◽

pp. e2553 ◽

Cited By ~ 4

Author(s):

Ru-Xia Jia ◽

Xing Duan ◽

Si-Jing Song ◽

Shao-Chen Sun

Keyword(s):

Oocyte Maturation ◽

Polar Body ◽

Meiotic Spindle ◽

Actin Dynamics ◽

Immunofluorescent Staining ◽

Meiotic Maturation ◽

Spindle Positioning ◽

Oocyte Meiosis ◽

Porcine Oocyte ◽

Oocyte Meiotic Maturation

LIMKi 3 is a specific selective LIMK inhibitor against LIMK1 and LIMK2, while LIMK1 and LIMK2 are the main regulators of actin cytoskeleton to participate in many cell activities. However, the effect of LIMKi 3 in porcine oocyte meiosis is still unclear. The present study was designed to investigate the effects of LIMKi 3 and potential regulatory role of LIMK1/2 on porcine oocyte meiotic maturation. Immunofluorescent staining of p-LIMK1/2 antibody showed that LIMK1/2 was localized mainly to the cortex of porcine oocyte, which co-localized with actin. After LIMKi 3 treatment, the diffusion of COCs became weak and the rate of polar body extrusion was decreased. This could be rescued by moving oocytes to fresh medium. After prolonging the culture time of oocytes, the maturation rate of porcine oocyte increased in LIMKi 3 groups, indicating that LIMKi 3 may suppress the cell cycle during porcine oocyte maturation. We also found that after LIMKi 3 treatment actin distribution was significantly disturbed at porcine oocyte membranes and cytoplasm, indicating the conserved roles of LIMK1/2 on actin dynamics. Next we examined the meiotic spindle positioning in porcine oocyte, and the results showed that a majority of spindles were not attached to the cortex of porcine oocyte, indicating that LIMKi 3 may affect actin-mediated spindle positioning. Taken together, these results showed that LIMK1/2 inhibitor LIMKi 3 had a repressive role on porcine oocyte meiotic maturation.

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Bovine oocyte maturation: acquisition of developmental competence

Reproduction Fertility and Development ◽

10.1071/rd19255 ◽

2020 ◽

Vol 32 (2) ◽

pp. 98

Author(s):

Bernard A. J. Roelen

Keyword(s):

Oocyte Maturation ◽

Developmental Competence ◽

Cell Contact ◽

Intimate Contact ◽

Homologous Chromosomes ◽

Paracrine Signalling ◽

Sister Chromatids ◽

Direct Cell

Although millions of oocytes are formed during embryo and fetal development in the cow, only a small fraction of these will form a developmentally competent oocyte and be fertilised. Development to competence relies on an intimate contact between the oocyte and the surrounding somatic cells in ovarian follicles, via both direct cell–cell contact and paracrine signalling. An important aspect of oocyte maturation is the segregation of homologous chromosomes and subsequently sister chromatids to form a haploid oocyte. Furthermore, the cytoplasm needs to be prepared for the formation of pronuclei and nuclear reprogramming to form a totipotent zygote. Conditions such as high levels of fatty acids or oxidative stress constrain the developmental competence of oocytes, and a better insight into these processes may help improve in vitro and in vivo oocyte maturation success. In addition, identification of the developmentally competent oocyte is useful for the efficiency of (artificial) reproduction.

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Characterisation of the cellular and molecular responses of ovine oocytes and their supporting somatic cells to pre-ovulatory levels of LH and FSH during in vitro maturation

Reproduction ◽

10.1530/rep-12-0031 ◽

2012 ◽

Vol 144 (2) ◽

pp. 195-207 ◽

Cited By ~ 5

Author(s):

Matthew Cotterill ◽

Sally L Catt ◽

Helen M Picton

Keyword(s):

Oocyte Maturation ◽

Granulosa Cells ◽

Cumulus Cell ◽

Developmental Competence ◽

Cumulus Expansion ◽

Serum Free ◽

Blastocyst Rate ◽

The Impact

The response of Graafian follicles to pre-ovulatory surge levels of FSH and LH in vivo triggers the terminal differentiation of granulosa cells and oocyte maturation. In polyovular species, the LH-driven signalling uses the epidermal growth factor (EGF)-like ligands AREG, EREG and BTC to promote oocyte maturation and cumulus expansion. This experimental series used a physiologically relevant ovine in vitro maturation (IVM) system to evaluate the impact of exposure to pre-ovulatory levels (100 ng/ml) of LH and FSH on ovine cumulus cell expression of EGF-like ligands in vitro. The serum-free sheep IVM system supported high levels (91.4%) of gonadotrophin-induced maturation of cumulus-enclosed oocytes and embryo development to the blastocyst stage (34.5%). Results were equivalent to a serum-based IVM system (85.1% IVM, 25.8% blastocyst rate; P>0.05) but were significantly different (P<0.05) to serum-free medium without gonadotrophins (69.5% IVM; 8.0% blastocyst rate). Ovine BTC was cloned and sequenced. Gonadotrophin-induced AREG, EREG, BTC and EGFR expressions were quantified in cumulus and mural granulosa cells during IVM. A rapid induction of AREG expression was apparent in both cell types within 30 min of gonadotrophin exposure in vitro. LHCGR (LHR) was detected in mural cells and FSHR in both cumulus and mural granulosa cells. The data confirm the involvement of AREG and EGFR during gonadotrophin-induced cumulus expansion, oocyte maturation and the acquisition of developmental competence by sheep oocytes matured in vitro.

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