Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for appropriate expression of steroidogenic and ovulation-related genes that impact oocyte maturation in vivo and in vitro

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.

Download Full-text

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.

Download Full-text

188 IMPROVEMENT OF IN VITRO CANINE OOCYTE MATURATION BY OVIDUCTAL SECRETOME

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab188 ◽

2017 ◽

Vol 29 (1) ◽

pp. 202 ◽

Cited By ~ 1

Author(s):

A. Lange-Consiglio ◽

C. Perrini ◽

P. Esposti ◽

F. Cremonesi

Keyword(s):

Oocyte Maturation ◽

In Vitro Maturation ◽

Cumulus Cell ◽

Follicular Development ◽

Cumulus Cells ◽

Chi Square ◽

Cell Layers ◽

Hoechst Staining

The in vitro maturation of canine oocyte is problematic because it is difficult to reproduce the oviducal microenvironment where the in vivo maturation occurs. Because cells are able to communicate with each other by paracrine action, oviducal cells could be in vitro cultivated to obtain the conditioned medium (CM) consisting of soluble factors and microvesicles (MV), which represent a carrier for nonsoluble molecules including microRNA. The aim of the present work was to investigate the effect of the addition of CM or MV, secreted by oviducal cells, to the canine in vitro maturation medium. To generate CM, cells from oviducts of 3 animals in late oestrus were cultured for 5 days at 38.5°C in a humidified atmosphere of 5% CO2. Supernatants were collected, pooled, centrifuged at 2500 × g, and stored at −80°C. Microvesicles were obtained by ultracentrifugation of CM at 100,000 × g for 1 h at 4°C and measured for concentration and size by a Nanosight instrument. Ovaries were obtained from 50 healthy domestic bitches (1–4 years old) of different breeds that underwent ovariectomy regardless of the oestrous cycle. Cumulus-oocyte complexes were released by slicing the ovarian cortex with a scalpel blade, and only Grade 1 cumulus-oocyte complexes (darkly granulated cytoplasm and surrounded by 3 or more compact cumulus cell layers) 110 to 120 µm in diameter were selected for culture. Maturation was performed at 38.5°C in a humidified atmosphere of 5% CO2 and 5% of O2 in bi-phasic systems: 24 h in SOF with 5.0 μg mL−1 of LH followed by 48 h in SOF supplemented with 10% of oestrous bitch serum and 10% CM or 50, 75, 100, or 150 × 106 MV mL−1 labelled with PKH-26. Control was the same medium without CM or MV. Oocytes were observed under a fluorescent microscope to detect metaphase II (MII), by Hoechst staining, and the incorporation of MV. Statistical analysis was performed by chi-square test. Results show that canine oviducal cells secreted MV of 234 ± 23 nm in size, underling that these MV fall within the shedding vesicles category. The incorporation of labelled MV occurred at first in cumulus cells, at 48 h of maturation, and then, at 72 h, in oocyte cytoplasm. These MV had a positive effect on maturation rate (MII) at the concentration of 75 and 100 × 106 MV mL−1 compared with CM and control (20.34 and 21.82 v. 9.09 and 3.95%, respectively). The concentration of 150 × 106 MV mL−1 provided only 9.26% of MII. To understand the role of MV, we assessed the expression of 3 microRNA (miRNA-30b, miR-375, and miR-503) that are involved in some key pathways (WNT, MAPK, ERbB, and TGFβ) regulating follicular development and meiotic resumption. The lower rate of MII with the higher concentration of MV is possibly due to the high level of miR-375, which recent literature shows to suppress the TGFβ pathway, leading to impaired oocyte maturation. In conclusion, the oviducal MV, or specific microRNA, are involved in cellular trafficking during oocyte maturation, and their possible use in vitro could facilitate the exploitation of canine reproductive biotechnologies.

Download Full-text

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.

Download Full-text

Changes of follicular fluid composition during estrous cycle: The effects on oocyte maturation and embryo development in vitro

Indian Journal of Animal Research ◽

10.18805/ijar.b-1030 ◽

2019 ◽

Author(s):

A.A. Mohammed ◽

T. Al-Shaheen ◽

S. Al-Suwaiegh

Keyword(s):

Embryo Development ◽

Oocyte Maturation ◽

Follicular Fluid ◽

Extracellular Fluid ◽

Cumulus Cells ◽

Fluid Composition ◽

Lh Surge ◽

Antral Follicles ◽

Positive Effects

Oocytes are bathed in extracellular fluid of the antral follicles, which is termed follicular fluid (FF). Follicular fluid is synthesized from secretions of theca, granulosa, and cumulus cells and from a transudate of blood plasma. Oocytes persist in meiotic arrest in antral follicles until luteinizing hormone (LH) surge or removal the oocytes from the ovarian follicles. This suggests that FF before LH surge might contain meiosis inhibiting factor(s). The microvasculatory bed of the follicular wall and the composition of FF undergo changes during follicular growth and development, which is important for oocyte maturation and subsequent embryo development. Therefore, it is expected that FF composition and components might change according to timing of FF aspiration from follicles. Hence, negative or positive effects could be expected when FF supplemented during oocyte maturation in vitro. Nutrition effects on microvasculatory bed of follicles and their sizes. Thus, the nutritional status of animals is a factor affected on oocyte maturation and embryo development. The present article reviews and discusses these effects.

Download Full-text

EGF-Like Factors Induce Expansion of the Cumulus Cell-Oocyte Complexes by Activating Calpain-Mediated Cell Movement

Endocrinology ◽

10.1210/en.2012-1059 ◽

2012 ◽

Vol 153 (8) ◽

pp. 3949-3959 ◽

Cited By ~ 27

Author(s):

Ikko Kawashima ◽

Zhilin Liu ◽

Lisa K. Mullany ◽

Toshihiro Mihara ◽

Joanne S. Richards ◽

...

Keyword(s):

Chorionic Gonadotropin ◽

Human Chorionic Gonadotropin ◽

Egf Receptor ◽

Cumulus Cell ◽

Cumulus Cells ◽

Receptor Activation ◽

Calpain Inhibitor ◽

Cell Detachment ◽

Calpain Activity

Cumulus cell-oocyte complex (COC) expansion is obligatory for LH-induced ovulation and is initiated by LH induction of the epidermal growth factor (EGF)-like factors that mediate the synthesis of the hyaluronan-rich matrix and hyaluronan-stabilizing factors. COC expansion also involves the movement of cumulus cells within the matrix by mechanisms that have not been characterized. We document herein that two proteases, calpain 2 and to a lesser extent calpain 1, are expressed in cumulus cells and that the proteolytic activity of these enzymes is rapidly and significantly increased in COC isolated from human chorionic gonadotropin-induced ovulatory follicles in vivo. Stimulation of calpain activity was associated with proteolytic degradation of paxillin and talin (two components of focal adhesion complexes), cell detachment, and the formation of cell surface bleb-like protrusions. Injection of a calpain inhibitor in vivo reduced 1) human chorionic gonadotropin-stimulated calpain enzyme activity, 2) cell detachment, 3) membrane protrusion formation, and 4) COC expansion by mechanisms that did not alter Has2 expression. During EGF-like factor induction of COC expansion in culture, calpain activity was increased by ERK1/2 and intracellular Ca2+ signaling pathways. Inhibition of calpain activity in cultured COC blocked cumulus cell detachment, protrusion formation, and the vigorous movement of cumulus cells. As a consequence, COC expansion was impaired. Collectively, these results show that two highly coordinated processes control COC expansion. One process involves the synthesis of the hyaluronan matrix, and the other mediates cumulus cell detachment and movement. The latter are controlled by calpain activation downstream of the EGF receptor activation of the Ca2+ pathway and ERK1/2 pathways.

Download Full-text

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.

Download Full-text

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

Reproduction ◽

10.1530/rep-16-0426 ◽

2017 ◽

Vol 153 (3) ◽

pp. R109-R120 ◽

Cited By ~ 16

Author(s):

Hannah M Brown ◽

Kylie R Dunning ◽

Melanie Sutton-McDowall ◽

Robert B Gilchrist ◽

Jeremy G Thompson ◽

...

Keyword(s):

Gene Expression ◽

Cell Metabolism ◽

Cumulus Cell ◽

Cumulus Cells ◽

Developmental Competence ◽

Oocyte Competence ◽

Oocyte Developmental Competence ◽

Growth Factor Signalling

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.

Download Full-text

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

Reproduction Fertility and Development ◽

10.1071/rd11305 ◽

2013 ◽

Vol 25 (2) ◽

pp. 426 ◽

Cited By ~ 23

Author(s):

Karen L. Kind ◽

Kelly M. Banwell ◽

Kathryn M. Gebhardt ◽

Anne Macpherson ◽

Ashley Gauld ◽

...

Keyword(s):

Microarray Analysis ◽

Cell Function ◽

Cumulus Cell ◽

Cumulus Cells ◽

Chorionic Gonadotrophin ◽

Developmental Competence ◽

Rt Pcr ◽

Oocyte Competence

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 samples 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.

Download Full-text

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.

Download Full-text

The beneficial effects of cumulus cells and oocyte-cumulus cell gap junctions depends on oocyte maturation and fertilization methods in mice

PeerJ ◽

10.7717/peerj.1761 ◽

2016 ◽

Vol 4 ◽

pp. e1761 ◽

Cited By ~ 15

Author(s):

Cheng-Jie Zhou ◽

Sha-Na Wu ◽

Jiang-Peng Shen ◽

Dong-Hui Wang ◽

Xiang-Wei Kong ◽

...

Keyword(s):

Gap Junctions ◽

Gap Junction ◽

Oocyte Maturation ◽

Cumulus Cell ◽

Cumulus Cells ◽

Early Embryo ◽

Blastocyst Formation ◽

Beneficial Effects

Cumulus cells are a group of closely associated granulosa cells that surround and nourish oocytes. Previous studies have shown that cumulus cells contribute to oocyte maturation and fertilization through gap junction communication. However, it is not known how this gap junction signaling affectsin vivoversusin vitromaturation of oocytes, and their subsequent fertilization and embryonic development following insemination. Therefore, in our study, we performed mouse oocyte maturation and insemination usingin vivo- orin vitro-matured oocyte-cumulus complexes (OCCs, which retain gap junctions between the cumulus cells and the oocytes),in vitro-matured, denuded oocytes co-cultured with cumulus cells (DCs, which lack gap junctions between the cumulus cells and the oocytes), andin vitro-matured, denuded oocytes without cumulus cells (DOs). Using these models, we were able to analyze the effects of gap junction signaling on oocyte maturation, fertilization, and early embryo development. We found that gap junctions were necessary for bothin vivoandin vitrooocyte maturation. In addition, for oocytes maturedin vivo, the presence of cumulus cells during insemination improved fertilization and blastocyst formation, and this improvement was strengthened by gap junctions. Moreover, for oocytes maturedin vitro, the presence of cumulus cells during insemination improved fertilization, but not blastocyst formation, and this improvement was independent of gap junctions. Our results demonstrate, for the first time, that the beneficial effect of gap junction signaling from cumulus cells depends on oocyte maturation and fertilization methods.

Download Full-text