Cumulus Cell Function Research Articles

332 GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF) ENHANCES CUMULUS CELLS EXPANSION OF IN VITRO-MATURED BOVINE CUMULUS - OOCYTE COMPLEXES IN A CHEMICALLY DEFINED MEDIUM

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine that stimulates proliferation, differentiation and function in different cells types. We have previously demonstrated (Bücher DD et al. 2008 Reprod. Dom. Anim. 43 (Suppl. 3), 146 abst.) that both subunits of GM-CSF receptor are expressed in granulosa cells from antral follicles in bovine ovaries. Also, we determined that the cytokine enhances glucose uptake through facilitative hexose transporters in granulosa cells in primary culture. The goals of the present study were to characterize the expression of GM-CSF receptor in cumulus cells and oocytes from bovine antral follicles and to determine its effects on in vitro-matured bovine COCs in a chemically defined medium. To determine the presence of a and |5 subunits of GM-CSF receptor, COCs were aspirated from follicles <8 mm in diameter, fixed, and submitted to immunocytochemistry. To study the effect of GM-CSF on in vitro maturation of oocytes, COCs (n =481) were cultured using serum-free medium (SOF) containing 0, 1, 10, and 100 ng mL-1 of human recombinant GM-CSF (R&D Systems, Inc., Minneapolis, MN, USA) for 22 h at 39°C, 5% CO2 in humidified air. Nuclear stage, cumulus expansion, cumulus cell number, and viability were analyzed after in vitro maturation. Cumulus expansion was assessed using the cumulus expansion index (CEI) (Fagbohun C and Down S 1990 Biol. Reprod. 42, 413-423). Nuclear stage was evaluated using aceto-orcein stain. To determine cumulus cell viability and number, COCs (n = 10-12 per group) were transferred into an Eppendorf tube and cumulus cells were removed by vortexing for 3 min, stained with trypan blue and counted with a hemocytometer. The study was conducted in 6 replicates. Data from cumulus expansion and cell number were analyzed by Kruskal-Wallis analysis. Data for nuclear stage and cell viability were analyzed by chi-square analysis and one way ANOVA, respectively. Both receptor subunits were present in cumulus cells and oocytes from COCs. COCs cultured in 10 and 100 ng mL-1 GM-CSF had CEI scores (0.8 and 1.22, respectively) greater (P < 0.01) than controls (0.2), but the proportion of COCs displaying second metaphase did not differ (P = 0.5) among treatment groups. GM-CSF at a concentration of 100 ng mL-1 increased (P < 0.01) cumulus cell viability by more than 20% compared to the control group. Similarly, GM-CSF at concentrations of 10 and 100 ng mL-1 increased (P < 0.05) cumulus cell number by more than 20% and 45%, respectively, from the control group. The use of a specific inhibitor of PI3 kinase (Ly294002; 10 and 100 μM) blocked the stimulatory effect of GM-CSF on cumulus expansion, cell viability, and cell number. In conclusion, the results of the study suggest a plausible modulator role of GM-CSF in the metabolism and function of cumulus cells and oocytes during in vitro maturation. Funding from Faculty of Veterinary Sciences, Universidad Austral de Chile, MECESUP AUS-0005, AUS-0601, and DID D-2006-24 and from Universidad Católica de Temuco, research grant 2007 DGI-CDA-04.

Cumulus gene expression as a predictor of human oocyte fertilisation, embryo development and competence to establish a pregnancy

The close relationship between cumulus cell function and oocyte developmental competence indicates that analysis of cumulus gene expression is a potential non-invasive method to aid embryo selection and IVF outcome. Cumulus was isolated from 674 oocytes from 75 women undergoing ICSI and gene expression analysed by quantitative RT-PCR. Cumulus expression of cyclooxygenase 2 (PTGS2) was higher with mature oocytes, whereas brain-derived neurotrophic factor (BDNF) was lower when fertilisation was normal. Expression levels of gremlin (GREM1) and BDNF were weak positive and negative predictors of embryo quality respectively. Ranking of GREM1 expression within cohorts of oocytes showed that oocytes associated with the highest GREM1 expression were more likely to be transferred or cryopreserved than discarded (49 vs 33%, P<0.02), although the clinical pregnancy rate was not significantly different. This study demonstrates both the feasibility and difficulties of this method of analysis in the largest such group studied thus far. Novel relationships between BDNF expression and fertilisation were identified, and the potential value of GREM1 expression as a marker of embryo quality supports the further assessment of GREM1 analysis in the context of embryo selection.

The Cumulus Cell-Secreted Factor Neuregulin 1 Maintains the Cumulus Cell Function in Ovulated COCs To Enhance Fertilization and Developmental Competence in Porcine Oocytes.

It has been known that cumulus cells support oocyte meiotic and cytoplasmic maturation. Our previous study showed that the EGF like factors (amphiregulin, epiregulin and betacellulin) are expressed in granulosa cells and cumulus cells and act on cumulus cells to induce cumulus cell-oocyte complex (COC) expansion and oocyte maturation in mice and in pigs. Additionally, when ovulated mouse COCs were cultured with amphiregulin, this factor activated/phosphorylated the EGF receptor (ErbB1) and maintained cumulus expansion and the expression of specific genes when expression of endogenous amphiregulin is decreased markedly. Moreover, we have shown recently that the maintenance of the COC matrix and the secretion of specific chemokines from cumulus cells enhance successful fertilization via supporting sperm motility and induction of sperm capacitation. These data suggested that additional ligand members for the ErbB family are required to act to maintain cumulus cell function after ovulation. However, there is little information about what mechanisms regulate cumulus cells in oviductal COCs. Therefore, this study was designed to determine the kinetic expression patterns of ErbB family and the ligand in cumulus cells in vivo and in vitro, and then activate the ErbB signaling pathway to stimulate oocyte maturation in COCs in culture. For this, intact follicles and cumulus cells were collected at early, antral and ovulatory stages of follicle, or from COCs in the oviduct at specific time intervals following eCG and hCG treatment of gilts. FSH/eCG increased levels of EGF receptor and ErbB3 protein in cumulus cells, whereas ErbB2 remained constant and ErbB4 was down regulated. Immuno-histochemistry localized ErbB3 to cumulus cells after eCG and hCG stimulation. Because we found high expression of neuregulin 1 (Nrg1) mRNA in cumulus cells from our rat COC micro-array database, and because RT-PCR analyses showed that Nrg1 expression in cumulus cells of porcine COCs was increased after hCG stimulation and reached maximum levels after ovulation, we sought to determine if NGR1 might regulate cumulus cell functions via ErbB3. When porcine COCs were cultured in vitro with FSH alone, the expression of Nrg1 in cumulus cells did not increase. Therefore, to determine if NRG1 might enhance the actions of FSH, NRG1 was added to FSH-containing medium at 24 hr of culture and COC expansion and oocyte maturation were analyzed after an additional 20-hr culture (total 44 hr culture period). The addition of NRG1 strongly induced the phosphorylation of ErbB3 but not ErbB1, which resulted in the activation of both ERK1/2 and PI 3-kinase-PKB pathways in cumulus cells. The COCs exhibited improved cumulus cell survival (no apoptosis) and sustained expansion. The developmental competence of fertilized oocytes to the blastocyst stage was also enhanced by NGR1. From these results, we show for the first time that NRG1 is a novel COC secreted EGF-like factor that acts via autocrine mechanisms to activate ErbB3 expressed on cumulus cells within COCs after ovulation, and thereby helps maintain cumulus cell function to improve oocyte developmental competence, maybe due to suppress oocyte aging. Supported in part by JSPS-18688016 (MS). (poster)

Fibroblast growth factors and epidermal growth factor cooperate with oocyte-derived members of the TGFbeta superfamily to regulate Spry2 mRNA levels in mouse cumulus cells.

Mouse oocytes produce members of the transforming growth factor beta (TGFbeta) superfamily, including bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), as well as fibroblast growth factors (FGFs). These growth factors cooperate to regulate cumulus cell function. To identify potential mechanisms involved in these interactions, the ability of fully grown oocytes to regulate expression of BMP or FGF antagonists in cumulus cells was examined. Oocytes promoted cumulus cell expression of transcripts encoding antagonists to TGFbeta superfamily members, including Grem2, Htra1, Htra3, and Nog mRNAs. In contrast, oocytes suppressed cumulus cell expression of Spry2 mRNA, which encodes a regulator of receptor tyrosine kinase signals, such as FGF and epidermal growth factor (EGF) receptor signals. The regulation of Spry2 mRNA levels in cumulus cells was studied further as a model for analysis of potential mechanisms for cooperativity of FGF/EGF signaling with oocyte-derived members of the TGFbeta superfamily. Oocytes suppressed basal and FGF-stimulated Spry2 mRNA levels in cumulus cells but promoted EGF-stimulated levels. Furthermore, recombinant TGFbeta superfamily proteins, including BMP15 and GDF9, mimicked these effects of oocytes. Elevated expression of Spry2 mRNA in cumulus and mural granulosa cells correlated with human chorionic gonadotropin-induced expression of mRNAs encoding EGF-like peptides. Therefore, oocyte-derived members of the TGFbeta superfamily suppress FGF-stimulated Spry2 mRNA levels before the luteinizing hormone surge but promote Spry2 mRNA levels stimulated by EGF receptor-mediated signals after the surge.

Interleukin-6: An Autocrine Regulator of the Mouse Cumulus Cell-Oocyte Complex Expansion Process

Ovulation has long been regarded as a process resembling an inflammatory response. Recent studies indicate that genes associated with innate immune responses were also expressed during the ovulation process. Because the innate immune genes are induced in cumulus cell oocyte complexes (COCs) later than the inflammation-associated genes, we hypothesize that COC expansion is dependent on specific sequential changes in cumulus cells. Because IL-6 is a potent mediator of immune responses, we sought to determine what factors regulate the induction of Il6 mRNA in COCs and what impact IL-6 alone would have on COC expansion. We found that the levels of Il6 mRNA increased dramatically during COC expansion, both in vivo and in vitro. Moreover, IL-6, together with its soluble receptor (IL-6SR), could bypass the need for either amphiregulin and/or prostaglandin E2 to induce the expansion of COCs. This ability of IL-6/IL-6SR to induce COC expansion was blocked by the inhibitors to p38MAPK, MAPK kinase 1/2, and Janus kinase. More importantly, when COCs were in vitro maturated in the presence of IL-6, they had a significantly higher embryo transfer rate than the ones without IL-6 and comparable with in vivo matured oocytes. IL-6/IL-6SR activated multiple signaling pathways (Janus kinase/signal transducer and activator of transcription, ERK1/2, p38MAPK, and AKT) and progressively induced genes known to impact COC expansion, genes related to inflammation and immune responses, and some transcription factors. Collectively, these data indicate that IL-6 alone can act as a potent autocrine regulator of ovarian cumulus cell function, COC expansion, and oocyte competence.

The Effect of Several ?-Tocopherol Concentrations on Swine Oocyte Maturation and Embryo Culture

Free radicals play significant roles in the physiological processes happening in the ovary such as steroidogenesis, folliculogenesis, corpus luteum progesterone release and degeneration, and follicle rupture during ovulation. As defence, living systems employ antioxidants that they either produce or take up from the environment. Among these ?-topherol has been shown to improve swine oocyte maturation and the development of bovine and swine embryos. The goal of this study was to establish the influence of several ?-tocopherol concentrations on swine oocyte maturation, viability, the function of cumulus cells and in vitro embryo development in order to improve culture media. Pig oocytes were cultured for 45 hours at 38°C in 5% CO2 atmosphere; in M199 containing several ?-tocopherol (5, 10, 20, 40 and 80 µM) concentrations and cumulus expansion was assessed. Then they were fertilized in TALP medium using spermatozoa capacitated by centrifugation and incubated for 16-18 hours Afterwards the presumed zygotes were cultured in NCSU-23 droplets supplemented with ?-tocopherol (5, 10, 20, 40 and 80 µM) and covered with mineral oil for 92 hours at 38°C in 5% CO2 atmosphere. At the end of this period the number of embryos that had developed to the 2 cells, 4-8 cells and morula stages was counted and compared to the control, and the differences analyzed using ANOVA and interpreted using the Student test. The addition of 20 µM ?-tocopherol to the maturation medium lead to a significant (p<0.05) increase in the number of matured oocytes. Culturing swine embryos in media supplemented with 5 ?M, 10 ?M and 40 ?M ?-tocopherol also had a beneficial effect in their development to the morula stage resulting in higher number of viable swine embryos

Identification of Potential Markers of Oocyte Competence Expressed in Bovine Cumulus Cells Matured with Follicle-Stimulating Hormone and/or Phorbol Myristate Acetate In Vitro

Oocyte competence is the ability of the oocyte to complete maturation, undergo successful fertilization, and reach the blastocyst stage. Cumulus cells are indispensable for this process. Their removal significantly affects the blastocyst rates. Moreover, the properties and functions of cumulus cells are regulated by the oocyte. They also reflect the oocyte's degree of maturation. Our study was aimed at identifying markers of oocyte competence that are expressed in bovine cumulus cells. In a previous study in our laboratory, the blastocyst yield following FSH or phorbol myristate acetate (PMA) treatment was 45%%. Therefore, we tested four sets of conditions during the first 6 h of in vitro maturation (IVM): FSH (0.1 microg/ml), PMA (0.1 microM), FSH ++ PMA, and negative control. Extracts from each IVM treatment were hybridized against the same negative control on a microarray containing a partial library of differentially expressed transcripts in the cumulus of competent oocytes collected at 6 h after LH in vivo. Common positive clones between diffrentially treated cells were selected, and 15 candidates were validated by real-time PCR. Based on this, the main candidates expressed in cumulus cells and that could be valuable and indirect markers of oocyte competence are hyaluronan synthase 2 (HAS2), inhibin betaA (INHBA), epidermal growth factor receptor (EGFR), gremlin 1 (GREM1), betacellulin (BTC), CD44, tumor necrosis factor-induced protein 6 (TNFAIP6), and prostaglandin-endoperoxide synthase 2 (PTGS2). These biomarkers could be potential candidates to predict oocyte competence and to select higher-quality embryos for transfer. Additionally, these indirect predictors of oocyte competence and follicular health could improve our knowledge of gene expression patterns in the cumulus and yield insights into the molecular pathways controlling oocyte competence.

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

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.

260. Synergistic effects of cAMP modulating agents in pre-IVM and in IVM on bovine cumulus and oocyte functions

Cyclic AMP plays a crucial role in oocyte maturation. It has been shown that prolonging spontaneous maturation by modulating oocyte cAMP levels during in vitro maturation (IVM) improves developmental outcomes. This study sought to assess the effect of inclusion of cAMP modulators during a pre-IVM phase and during IVM on cumulus cell (CC) and oocyte function. Bovine cumulus-oocyte complexes (COCs) were precultured for 2 h in medium supplemented with/without cAMP modulators: 0.5 mM IBMX and 0.1 mM forskolin (FK). Cyclic AMP in control COCs dropped significantly from 15 ± 4 to 2 ± 1 fmol/COC within 30 min, whereas FK+IBMX during pre-IVM increased levels to 180 ± 19 fmol/COC, which had notable persistent effects on several COC functions throughout IVM. Cyclic AMP modulators delayed oocyte GV progression; after 2 h of pre-IVM, 67 ± 5% were at GVII stage compared (P &lt; 0.0001) to 12 ± 3% in controls, 66 ± 5% of which had progressed to GVIII. This was associated with a significantly higher level of oocyte-CC gap junction mediated communication (1000 ± 148 v. 340 ± 73 units). We next assessed the interaction of cAMP modulators in pre-IVM and IVM (20uM cilostamide) phases. After 9 h of IVM, significantly more oocytes were arrested at GV stage when modulators were present in both pre-IVM and IVM phases, compared with in IVM alone (96 ± 1 v. 74 ± 2%, P &lt; 0.05). Moreover, progression to MII was delayed by modulators in pre-IVM and IVM (24 ± 1, 75 ± 2, 92 ± 1% MII at 20, 24 and 28 h, respectively), compared with in IVM alone (78 ± 1, 95 ± 1, 98 ± 2% MII). After 24 h of IVM, intra-oocyte cAMP levels remained 15-fold higher (P &lt; 0.05) with modulators in both phases compared with in IVM alone. These results show that the efficacy of cAMP modulators during IVM is substantially improved by management of COC cAMP levels during the pre-IVM phase. Such an approach has profound effects on CC and oocyte function, including increasing oocyte-CC communication and delaying meiotic resumption.

Hormone-Induced Expression of Tumor Necrosis Factor α-Converting Enzyme/A Disintegrin and Metalloprotease-17 Impacts Porcine Cumulus Cell Oocyte Complex Expansion and Meiotic Maturation via Ligand Activation of the Epidermal Growth Factor Receptor

The epidermal growth factor (EGF)-like growth factors, amphiregulin (AREG) and epiregulin (EREG), are expressed in murine cumulus oocyte complexes (COCs) where they impact the function of cumulus cells and oocyte maturation during LH-mediated ovulation. Because TNFalpha-converting enzyme (TACE)/a disintegrin and metalloprotease-17 (ADAM17) is essential for ectodomain shedding of AREG and EREG from the surface of other cell types, the expression and function of TACE/ADAM17 was analyzed in a porcine COC culture system in which FSH- and LH-mediated expansion and oocyte meiotic maturation have been well characterized and shown to occur between 20 and 40 h. In this model, Areg, Ereg, and Tace/Adam17 mRNAs increased significantly with maximal levels observed between 5 and 20 h of culture with FSH plus LH. TACE/ADAM17 protein and protease activity were up-regulated markedly at 10 h and maintained to 40 h. Treatment of COCs with the TACE/ADAM17-selective inhibitor TNFalpha-processing inhibitor-2 (TAPI-2) significantly suppressed in a time-dependent manner downstream targets of EGF receptor activation such as ERK1/2 phosphorylation, Ptgs2, Has2, and Tnfaip6 mRNA expression, hormone-induced COC expansion, and meiotic maturation of the oocytes. Addition of EGF to COCs cultured in the presence of FSH/LH reversed the inhibitory effects of TAPI-2 on these ovulation-related processes. Gonadotropin-induced phosphorylation of ERK1/2 was also inhibited in rat granulosa cells treated with TAPI-2 or after transfection with Tace/Adam17 small interfering RNA. Induced expression of Tnfaip6 mRNA was also reduced by Tace/Adam17 small interfering RNA. Thus, TACE/ADAM17 is induced and the activity is involved in porcine COC expansion as well as oocyte meiotic maturation through the activation of EGF receptor in cumulus cells.

MOUSE OOCYTES PROMOTE STEROL BIOSYNTHESIS IN CUMULUS CELLS: MORE EVIDENCE FOR OOCYTEREGULATION OF METABOLIC COOPERATIVITY BETWEEN GRANULOSA CELLS AND OOCYTES

To evaluate the function(s) of oocyte-derived BMP15 and GDF9 on the development and function of cumulus cells before LH-stimulation of cumulus expansion, we previously compared the transcriptomes of cumulus cells from eCG-primed wildtype (WT), Bmp15−/−, and Bmp15−/−Gdf9+/− double mutant (DM) mice by micro-array analysis, and found that transcripts encoding enzymes of metabolic pathways in cumulus cells, particularly glycolysis, sterol biosynthesis, inositol metabolism, and pentose phosphate, were significantly affected. Here we have focused on the sterol biosynthesis pathway and studied its regulation in cumulus cells by oocytes. Oocytectomy (OOX) of WT cumulus cell-oocyte complexes reduced the expression in cumulus cells of 15 of 17 mRNAs encoding enzymes for sterol biosynthesis. This reduction was prevented when OOX cumulus cells were co-cultured with fully-grown oocytes (FGOs). Associated with the down-regulation of these transcripts' expression, the rate of de novo cholesterol biosynthesis was reduced by more than 90% in OOX cumulus cells. The reduction in cholesterol biosynthesis was recovered to only 50% of the control level by co-culture with FGOs. This suggests that contact between oocytes and cumulus cells is necessary for cumulus cell cholesterol biosynthesis. The expression of 6 mRNAs encoding enzymes in this pathway, Mvd, Fdps, Sqle, Lss, and Tm7sf2, was also significantly lower in cumulus cells of Bmp15−/− and DM, compared to that in WT mice, and these low mRNA levels were reflected in reduced levels of cholesterol synthesis (55% and 25% of WT level in Bmp15−/− and DM cumulus cells, respectively). These results suggest that the oocyte-derived paracrine factors, including GDF9 and BMP15, enhance sterol biosynthesis in cumulus cells by promoting the expression of mRNAs encoding the key enzymes in this pathway. Comparison of the expression of all the transcripts encoding enzymes in this pathway in cumulus cells and oocytes revealed that 7 mRNAs, Mvk, Pmvk, Fdps, Sqle, Cyp51, Sc4mol, and Ebp, were robustly expressed in cumulus cells, but barely detectable in oocytes. Moreover, the level of de novo synthesized cholesterol in cumulus cell-enclosed oocytes was about 5-fold higher than that in denuded oocytes. We conclude that oocytes are deficient in synthesizing sterols and require cumulus cells to provide them with cholesterol and the other intermediates of the sterol biosynthetic pathway. Moreover, mouse oocyte-derived BMP15 and GDF9 profoundly affect the metabolic functions of cumulus cells before the LH surge. Supported by HD23839 (Y-QS, KS, JJE), HD21970 (KS, KW, JJE), HD33438 (SAP, MMM) and 5F32HD46335 (SAP). (platform)

卵丘細胞において発現するADAM17は，EGF-like factorを活性化させ，卵子成熟と卵丘細胞の膨潤を誘起する

卵丘細胞において発現するADAM17は，EGF-like factorを活性化させ，卵子成熟と卵丘細胞の膨潤を誘起する

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

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<0.001). Levels of OOX apoptosis were significantly reversed (P<0.001) 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<0.001) 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<0.001). Follistatin and a BMP6 neutralizing antibody, which antagonized the anti-apoptotic effects of BMP15 and BMP6, respectively, whether alone or combined, blocked approximately 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.

Presence of cumulus cells during in vitro fertilization protects the bovine oocyte against oxidative stress and improves first cleavage but does not affect further development

The present study was conducted to evaluate the function of cumulus cells during bovine IVF Oocytes within cumulus-oocyte complexes (COCs) or denuded oocytes (DOs) were inseminated in control medium, or DOs were inseminated in cumulus cell conditioned medium (CCCM). DOs exhibited reduced cleavage and blastocyst formation rates when compared with intact COCs. The reduced blastocyst formation rate of DOs resulted from reduced first cleavage but subsequent embryo development was not changed. Live-dead staining and staining for apoptotic cells revealed no differences in blastocysts from oocytes fertilized as COC or DO. Fertilization of DOs in CCCM partially restored the cleavage rate, suggesting that factors secreted by cumulus cells are important for fertilization but that physical contact between oocytes and cumulus cells is required for optimal fertilization and first cleavage. Exposure of COCs to hydrogen peroxide shortly before fertilization reduced the cleavage rate, but did not lead to enhanced death of cumulus cells or oocyte death. Exposure of DOs to hydrogen peroxide, however, resulted in oocyte death and a complete block of first cleavage, suggesting that cumulus cells protect the oocyte against oxidative stress during fertilization.

Nitric oxide influences the meiotic maturation of porcine oocytes cultured in hypoxanthine‐supplemented medium

Nitric oxide (NO) plays an important role in many pathological processes. The present investigation was undertaken to ascertain the effects of NO at high level on porcine oocyte meiotic development. Cumulus-enclosed oocytes (CEOs) and denuded oocytes (DOs) were obtained from the follicles (2-6 mm diameter). Exogenous NO was provided with sodium nitroprusside (SNP). Control (0 mM), 0.1 mM, 1 mM or 10 mM SNP was introduced to the medium. Meanwhile, 0.1 mM, 1 mM and 10 mM preincubated SNP was also added to the medium respectively. The cumulus expansion, cumulus cells DNA fragmentation, oocyte meiotic maturation and degeneration were determined 44 h after incubation. SNP inhibited cumulus expansion and cumulus cells DNA fragmentation in a dose-dependent manner. Significantly fewer CEOs treated by any level of SNP resumed meiosis than control (p < 0.05) and more CEOs exposed to 0.1 mM or 1 mM SNP were arrested at GV stage. Moreover, SNP increased the percentage of CEOs at metaphase I (MI) stage but decreased the percentage of CEOs at metaphase II (MII) stage. 0.1 mM or 1 mM SNP showed no influence on the proportion of germinal vesicle breakdown of DOs and only 10 mM SNP reduced the proportion. SNP accelerated degeneration of both CEOs and DOs, especially at higher concentrations. DOs were more sensitive to SNP toxicity. Preincubated SNP showed no effect on cumulus expansion, cumulus cells DNA fragmentation, oocyte meiotic maturation or degeneration at lower concentration, but higher concentrations had effect, especially on the viability of porcine oocytes. Taken together, high level of NO inhibits porcine oocyte meiotic maturation, especially the transition from MI to MII, by mediating the functions of cumulus cells.

Role of cumulus cells during maturation of porcine oocytes in the rise in intracellular Ca 2+ induced by inositol 1,4,5-trisphosphate

At the time of fertilization, release of inositol 1,4,5-trisphosphate (IP3) into the cytoplasm of oocytes is said to be induced by hydrolysis of phosphatidylinositol bis phosphate (PI2) via activation of phospholipase C and is responsible for the Ca 2+ oscillation in oocytes immediately after sperm penetration. On the other hand, cumulus cells have been reported to play an important role in cytoplasmic maturation of mammalian oocytes and to affect embryonic development after fertilization. To obtain more information on the role of cumulus cells in cytoplasmic maturation of oocytes, the effects of cumulus cells on the rise in [Ca 2+]i and the rates of activation and development of porcine mature oocytes induced by IP3 injection were investigated. Mature porcine oocytes that had been denuded of their cumulus cells in the early stage of the maturation period had a depressed rise in [Ca 2+]i (4.0–6.0) and reduced rates of activation (31.4–36.8%) and development (10.0–24.4%) induced by IP3 injection compared with those of their cumulus-enclosed counterparts (7.3, 69.1% and 43.8%; P < 0.05). The [Ca 2+]i rise and the rates of activation and development depressed by the removal of cumulus cells were restored by adding pyruvate to the maturation medium. Furthermore, the IP3 injection-induced depression of [Ca 2+]i rise in mature oocytes derived from cumulus-denuded oocytes (DOs) was restored when they were cultured in a medium with pyruvate (3.9–6.3, P < 0.05). Also, mature oocytes from cumulus–oocyte complexes (COCs) cultured in a medium without glucose had a lower rise in [Ca 2+]i than that in mature oocytes from COCs cultured with glucose (7.4–6.0, P < 0.05). Cumulus cells supported porcine oocytes during maturation in the rise in [Ca 2+]i induced by IP3 and the following activation and development of porcine oocytes after injection of IP3. Moreover, we inferred that a function of cumulus cells is to produce pyruvate by metabolizing glucose and to provide oocytes with pyruvate during maturation, thereby promoting oocyte sensitivity to IP3.

276 THE EFFECT OF CUMULUS CELLS DURING MATURATION ON THE RISE IN THE CONCENTRATION OF INTRACELLULAR Ca2+ ([Ca2+]i) OF PORCINE OOCYTES INDUCEDBY INOSITOL 1,4,5-TRISPHOSPHATE

Increase of inositol 1,4,5-triphosphate (IP3) in the cytoplasm of mammalian oocytes is said to be responsible for [Ca2+]i oscillation observed in the oocytes immediately after sperm penetration, and the [Ca2+]i oscillation is known to be essential for the development of embryos. On the other hand, cumulus cells have been reported to play an important role in cytoplasmic maturation of oocytes and affecting the embryonic development. To obtain more information about the role of cumulus cells in cytoplasmic maturation, the effects of cumulus cells during maturation on the rise in [Ca2+]i and on the rate of activation of porcine mature oocytes induced by IP3 injection were investigated. The immature porcine oocytes were divided into three groups: COCs (intact cumulus-oocyte complexes), DOs (oocytes denuded of their cumulus cells), Co-culture (DOs attached to separated cumulus cells). These groups of immature oocytes were cultured in NCSU23 46 h for maturation. To examine the function of cumulus cells, two groups of immature oocytes were also prepared: DOs + pyruvate (DOs put into NCSU23 with pyruvate) and COCs-glucose free (COCs put into NCSU23 without glucose). The mature oocytes from each group were loaded with Ca2+ indicator fluorescent dye Fura2-AM, and then were irradiated by 340 nm and 360 nm of ultraviolet immediately after the injection of IP3. The intensities of emission light caused by the irradiation of 340 nm and 360 nm ultraviolet were recorded as E340 and E360. Since coupling of Ca2+ and the dye intensifies E340, but does not change E360, the level of [Ca2+]i was shown as R (ratio = E340/E360) in this study. Activation rate was calculated by counting the number of the oocytes that formed pronuclei by injection of IP3. ANOVA and Student's t-test were used in this study. Transient rise in [Ca2+]i was observed in the mature oocytes from every group. The peak R of the rise in [Ca2+]i of the mature oocytes derived from COCs, Dos, and Co-culture and induced by IP3 were 7.2, 4.0, and 6.9, respectively. The R of DOs was significantly lower than those of the others (P &lt; 0.05). Also, the activation rate of the mature oocytes from DOs was significantly lower than those from COCs and Co-culture (31, 66, and 66%). The mature oocytes from DOs + pyruvate showed the same level of peak R compared with those from COCs (7.4 and 6.3), but COCs-glucose free showed a slight but significantly lower peak R compared with the mature oocytes from COCs (6.0 and 7.4, P &lt; 0.05). In conclusion, cumulus cells appeared to support the rise in [Ca2+]i of porcine oocytes induced by IP3 during maturation and the following activation. Moreover, a function of cumulus cells supposedly produces pyruvate by metabolizing glucose and provides it to oocytes during maturation for promoting the cytoplasmic maturation. A part of this study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan MEXT, and by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST.

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

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 &lt; 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 &gt; 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.

327 OOCYTE-SECRETED FACTORS DIRECTLY AFFECT OOCYTE DEVELOPMENTAL COMPETENCE DURING IN VITRO MATURATION OF THE BOVINE CUMULUS - OOCYTE COMPLEX

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 &gt;3-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 &lt; 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 &lt; 0.001). BMP-15 alone or with GDF-9 also significantly (P &lt; 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.

Effects of adding luteinizing hormone to a medium containing follicle stimulating hormone on progesterone‐induced differentiation of cumulus cells during meiotic resumption of porcine oocytes

ABSTRACTIn the present study, we investigated the effects of adding luteinizing hormone (LH) to a medium containing follicle stimulating hormone (FSH) on the shift in expression of progesterone receptor (PR) isoforms (PR‐A and PR‐B) and the roles in function of cumulus cells of cumulus‐oocyte complexes (COC). The level of PR‐B mRNA in cumulus cells was up‐regulated by FSH during the first 16‐h cultivation but the level was significantly decreased at 20 h. The decrease of PR‐B mRNA was accelerated when COC were cultured with FSH and LH. Still, a high level of total PR mRNA was maintained in cumulus cells cultured with or without the addition of LH up to 20 h, suggesting that the expression of PR isoforms was shifted from PR‐B to PR‐A in cumulus cells. The reduction of PR‐B was also induced by addition of progesterone to FSH‐containing medium. The addition of LH or progesterone to FSH‐containing medium stimulated cumulus expansion of COC as compared with that of COC cultured with FSH. In the expanded COC, ADAMTS‐1 which is expressed in granulosa cells and cumulus cells in rodent follicles through LH‐induced progesterone‐ and PR‐dependent pathway, was more accumulated within the COC matrix. These results suggest that the addition of LH or progesterone to FSH‐containing medium is required for the differentiation of cumulus cells, such as cumulus expansion, mediated by the shift from PR‐B to PR‐A in them.