Temporal expression pattern of steroid-metabolizing enzymes in bovine COC during in vitro maturation employing different gonadotropin concentrations.

Steroid hormones are regulators in the fine-tuned process of follicular development. During final maturation in vivo a switch from oestradiol (E2) to progesterone (P4) dominance within the follicle is well-described. This change is accompanied by the resumption of meiosis and results in the maturation of the oocyte. It also suggests the important role of these hormones. However, present in vitro maturation (IVM) systems do not completely mimic the in vivo situation, resulting in oocytes of reduced quality. Aim of the study was to determine the temporal pattern of steroid hormone concentrations in the IVM medium of bovine cumulus-oocyte-complexes (COC) at defined time points. The influence of different gonadotropin supplementations during IVM on oocyte maturation, as well as the molecular quality of the oocytes and their corresponding cumulus cells was investigated. COCs were obtained from abattoir-derived ovaries and matured in medium added with different compounds of gonadotropins (eCG/hCG; FSH/LH, each at 0.05 IU or 0.01 IU; only FSH; without gonadotropins) employing a standard protocol without oil overlay. In experiment 1, medium, oocytes and cumulus cells were collected at different time points (0 h [control], 4 h, 8 h, 12 h, 16 h, 20 h, 24 h) after IVM in just eCG/hCG-supplemented medium. In experiment 2, medium, oocytes and cumulus cells were collected at 0 h (control) and after 24 h of IVM with all above-named supplements. The E2 concentration remained similar during IVM whereas P4 concentration increased during experiment 1. No significant changes could be determined after the addition of different gonadotropins (experiment 2). These results suggest that during IVM the temporal pattern of E2 and P4 did not correspond with the pattern during final maturation in vivo. RT-qPCR was used to assess the relative abundance of developmentally important genes in oocytes (BMP15; GDF9; ZAR1; PGR; PGRMC1/2; G6PD; StAR; ESR1/2; SULT1E1; STS; SOAT) and cumulus cells (ESR1/2; FSHR; LHCGR; CYP19A1; HSD3B1; PGR; PGRMC1/2; SULT1E1; STS; SOAT) at all collection points in both experiments. Most transcripts follow a time-regulated mRNA expression pattern during the entire in vitro maturation period. In addition, the expression of the analyzed transcripts was not influenced by the different gonadotropin supplementations during the IVM period. In all, this underlines that present conditions of IVM do not reflect the in vivo situation and require further optimisation.

The effects of kinase modulation on in vitro maturation according to different cumulus-oocyte complex morphologies.

Successful production of transgenic pigs requires oocytes with a high developmental competence. However, cumulus-oocyte complexes (COCs) obtained from antral follicles have a heterogeneous morphology. COCs can be classified into one of two classes: class I, with five or more layers of cumulus cells; and class II, with one or two layers of cumulus cells. Activator [e.g., epidermal growth factor (EGF)] or inhibitors (e.g., wortmannin and U0126) are added to modulate kinases in oocytes during meiosis. In the present study, we investigated the effects of kinase modulation on nuclear and cytoplasmic maturation in COCs. Class I COCs showed a significantly higher developmental competence than class II COCs. Moreover, the expression of two kinases, AKT and ERK, differed between class I and class II COCs during in vitro maturation (IVM). Initially, inhibition of the PI3K/AKT signaling pathway in class I COCs during early IVM (0-22 h) decreased developmental parameters, such as blastocyst formation rate, blastomere number, and cell survival. Conversely, EGF-mediated AKT activation in class II COCs enhanced developmental capacity. Regarding the MAPK signaling pathway, inhibition of ERK by U0126 in class II COCs during early IVM impaired developmental competence. However, transient treatment with U0126 in class II COCs increased oocyte maturation and AKT activity, improving embryonic development. Additionally, western blotting showed that inhibition of ERK activity negatively regulated the AKT signaling pathway, indicative of a relationship between AKT and MAPK signaling in the process underlying meiotic progression in pigs. These findings may help increase the developmental competence and utilization rate of pig COCs with regard to the production of transgenic pigs and improve our understanding of kinase-associated meiosis events.

Primary Culture of Human Cumulus Cells Requires Stearoyl-Coenzyme A Desaturase 1 Activity for Steroidogenesis and Enhancing Oocyte In Vitro Maturation.

Stearoyl-coenzyme A desaturase 1 (SCD1) is a key enzyme in lipid metabolism and is expressed in cumulus cells. The objective of the present study was to evaluate the effect of SCD1 inhibition in human cumulus cells on triglyceride content, steroidogenesis, and oocyte in vitro maturation. Human cumulus cells were exposed to SCD1 inhibitor CAY10566 (SCDinhib) alone or in combination with oleic acid in primary culture. The SCDinhib markedly suppressed triglyceride accumulation (-47%, P = .01), aromatase gene expression (-36%, P = .02), and estradiol production (-49%, P = .01) even at a dose not affecting cell viability and apoptosis. Human immature oocytes at the germinal vesicle (GV) stage were cocultured with pretreated cumulus cells. The rate of oocytes reaching the metaphase II stage was significantly lower in coculture with SCDinhib-treated cumulus cells than with control cumulus cells (-18%, P < .01), which recovered by oleic acid supplementation. This finding on in vitro maturation rate was also reproducible with mouse GV oocytes. The results suggest that SCD1 activity is required for cumulus cell lipid storage and steroidogenesis. In addition, oocyte maturation is negatively affected by SCD1 inhibition in cumulus cells, possibly due to a deficient lipid-mediated paracrine support.

Overexpression of hyaluronan synthase 2 and gonadotropin receptors in cumulus cells of goats subjected to one-shot eCG/FSH hormonal treatment for ovarian stimulation.

Hormonal ovarian stimulation may affect transcripts in somatic cells of cumulus-oocyte complexes (COCs) and affect the resulting oocyte quality. Here, in parallel with morphological classification and in vitro maturation (IVM) rate analysis, we investigated the expression of hyaluronan synthase 2 (HAS2), gonadotropic receptors (FSHR and LHR) and connexin 43 (GJA1) in cumulus cells (CCs) from goat COCs after multi-dose FSH (MD) or one-shot FSH/eCG (OS) treatments, using bovine COCs as control groups. The MD treatment produced more large follicles, and the resulting COCs had a better morphology and IVM rate than were obtained with OS. The OS treatment produced COCs with increased HAS2, FSHR, LHR and GJA1 expression. This gene expression pattern was also observed in the CCs of COCs that showed poor morphological characteristics. On the other hand, the mRNA levels were more similar between groups after IVM; FSHR and LHR were the main genes that showed decreased expression. Some events that occurred in bovine CCs during IVM, such as cell expansion, increased HAS2 expression and decreased GJA1 expression, were less evident or did not occur in goat COCs. In conclusion, increasing HAS2, FSHR, LHR and GJA1 expression in goat COCs does not confer greater meiotic competence to oocytes. Instead, it may result from poor regulation of gene expression in CCs by lower quality oocytes. Finally, cumulus expansion, together with HAS2 upregulation and GJA1 downregulation, seems to be more important for bovine COCs than for goat COCs. Additional studies are needed to investigate the importance of other HAS isoforms and connexins in goat COCs.

Down-regulation of the CYP19A1 gene in cumulus cells of infertile women with endometriosis.

Aromatase plays a fundamental role in the establishment of oocyte quality, which might be compromised in infertile women with endometriosis. The expression of the CYP19A1 gene (that encodes aromatase) was compared in cumulus cells and oestradiol concentrations in the follicular fluid of infertile women with and without endometriosis submitted to ovarian stimulation for intracytoplasmic sperm injection. Cumulus cells were isolated and the expression of the CYP19A1 was quantitated through real-time polymerase chain reaction. Oestradiol concentrations in follicular fluid were measured by chemiluminescence immunoassay. A lower expression of the CYP19A1 in the cumulus cells of infertile women with endometriosis was observed compared with controls (0.17 ± 0.13 and 0.56 ± 0.12, respectively), and no significant difference in the follicular fluid oestradiol concentrations was observed between groups. Our results show reduced expression of the CYP19A1 in cumulus cells of infertile women with endometriosis, which may play a role in the pathogenesis of endometriosis-related infertility.

Alteration of energy metabolism gene expression in cumulus cells affects oocyte maturation via MOS-mitogen-activated protein kinase pathway in dairy cows with an unfavorable "Fertil-" haplotype of one female fertility quantitative trait locus.

Prim'Holstein heifers selected for the "Fertil-" homozygous haplotype of QTL-Female-Fert ility-BTA3 showed a greater rate of early pregnancy failure and slower embryo development after IVM suggesting lower oocyte quality than those selected for "Fertile+". We aimed to ascertain intrafollicular factors related to lower oocyte quality in "Fertil-" cows. Analysis of individual oocytes showed meiotic progression delay in "Fertil-" compared with "Fertil+" dairy cows after in vivo maturation and IVM (P < 0.05). Expression of several genes localized to QTL-F-Fert-BTA3 or related to meiosis and mitogen-activated protein kinase pathway was analyzed in individual metaphase-II oocytes using reverse transcription- real-time polymerase chain reaction. Energy metabolism, apoptosis, extracellular matrix, and QTL-F-Fert-BTA3 genes were analyzed in surrounding cumulus cells (CC). In vivo, a significant decrease in prostaglandin synthase PTGES1 and PTGS2 expression coupled with lower PTGS2 protein abundance in CC and reduced expression of MOS in enclosed metaphase-II oocytes from "Fertil-" cows was observed. IVM strongly deregulated gene expression in CC and in oocytes compared with in vivo; nevertheless, differential expression of several genes including PEX19, NAMPT and MOS was observed between the two haplotypes. During IVM, PTGS2 activity inhibitor NS398 (50 µM) led to lower expression of fatty acid synthase (FASN) in CC and of MOS in treated metaphase-II oocytes. Using immunofluorescence, MOS protein was localized to a midbody-like contractile ring separating the polar body from the ooplasm, suggesting a role in the terminal stage of oocyte maturation. Our results suggest that factors involved in prostaglandin synthesis and lipid metabolism in CC could impair oocyte maturation, and might be involved in the reduced fertility of "Fertil-" cows.

Intracellular glutathione content, developmental competence and expression of apoptosis-related genes associated with G6PDH-activity in goat oocyte.

PURPOSE: To associate glucose-6-phosphate dehydrogenase (G6PDH) activity in goat oocytes with intracellular glutathione (GSH) content, meiotic competence, developmental potential, and relative abundance of Bax and Bcl-2 genes transcripts. METHODS: Goat oocytes were exposed to brilliant cresyl blue (BCB) staining test and categorized into BCB(+) (blue-cytoplasm), and BCB(-) (colorless-cytoplasm) groups. A group of oocytes were not exposed to BCB test and was considered as a control group. After maturation in vitro, a group of oocytes were used for determination of nuclear status and intracellular GSH content while another group was subjected to parthenogenetic activation followed by in vitro embryo culture. RESULTS: We found that BCB(+) oocytes not only yielded higher rate of maturation, but also showed an increased level of intracellular GSH content than BCB(-) and control oocytes. Furthermore, BCB(+) oocytes produced more blastocysts than BCB(-) and control oocytes. Our data revealed that the expression of anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) genes were interacted with G6PDH-activity in mature oocyte, their surrounding cumulus cells, and blastocyst-stage embryos. CONCLUSIONS: The results of this study demonstrate that selection of goat oocytes based on G6PDH-activity through the BCB test improves their developmental competence, increases intracellular GSH content, and affects the expression of the apoptosis-related genes.

Heat stress effects on the cumulus cells surrounding the bovine oocyte during maturation: altered matrix metallopeptidase 9 and progesterone production.

When the effects of heat stress are detrimental during maturation, cumulus cells are intimately associated with the oocyte. To determine the extent to which heat stress affects these cells, in this study, transcriptome profiles of the cumulus that surrounded control and heat-stressed oocytes (41 °C during the first 12 h only and then shifted back to 38.5 °C) during in vitro maturation (IVM) were compared using Affymetrix bovine microarrays. The comparison of cumulus-derived profiles revealed a number of transcripts whose levels were increased (n=11) or decreased (n=13) ≥ twofold after heat stress exposure (P<0.01), sufficient to reduce the development of blastocysts by 46.4%. In a separate study, quantitative PCR (qPCR) was used to confirm heat-induced differences in the relative abundances of the transcripts of five different genes (caveolin 1, matrix metallopeptidase 9, FSH receptor, Indian hedgehog homolog, and inducible nitric oxide synthase). Heat stress exposure resulted in >1.7-fold decrease in the protein levels of latent matrix metallopeptidase 9 (proMMP9). Heat-induced reductions in transcript levels were noted at 6 h IVM with reductions in proMMP9 protein levels at 18 h IVM (P=0.0002). Independent of temperature, proMMP9 levels at 24 h IVM were positively correlated with the development rate of blastocysts (R²=0.36; P=0.002). The production of progesterone increased during maturation; heat-induced increases were evident by 12 h IVM (P=0.002). Both MMP9 and progesterone are associated with the developmental competence of the oocyte; thus, it seems plausible for some of the negative consequences of heat stress on the cumulus-oocyte complex to be mediated through heat-induced perturbations occurring in the surrounding cumulus.

Steroidogenesis and the influence of MAPK activity during in vitro maturation of porcine cumulus oocyte complexes.

The aim of this study was to investigate steroidogenesis within porcine cumulus oocyte complexes during in vitro maturation and to examine the possible influence of the mitogen-activated protein kinase (MAPK). Porcine cumulus oocyte complexes were matured in vitro with and without the MAPK kinase inhibitor U0126 for 0, 5, 26 and 46 h. The 17ß-estradiol and progesterone concentration in the culture medium were then determined. In addition, the mRNA levels of StAR, Cyp11A1, 3ß-HSD and Cyp19A1 in cumulus cells were analysed by RT-PCR. Using an immunoblot, the MAPK phosphorylation in cumulus cells and oocytes was examined. During the first 26 h of in vitro maturation, 17ß-estradiol secretion was predominant, whereas, after a culture period of 46 h, the progesterone secretion decreased conspicuously. Under the influence of U0126, the secretion of 17ß-estradiol increased progressively during the complete maturation period, while progesterone secretion was completely inhibited. The mRNA levels of StAR and Cyp11A1 were not altered by U0126; however, corresponding to the hormone secretion, the gene expression of Cyp19A1 was up-regulated and the expression of 3ß-HSD down-regulated. The results suggested an influence of the MAPK on steroidogenesis in cumulus cells comparable to a luteinization factor. Hormone synthesis in cumulus cells during oocyte maturation seems to be regulated by altering expression of Cyp19A1 and 3ß-HSD.

LH-induced neuregulin 1 (NRG1) type III transcripts control granulosa cell differentiation and oocyte maturation.

Epidermal growth factor (EGF)-like factors [amphiregulin (AREG), betacellulin, and epiregulin] are induced by LH and activate the EGF receptor (ERBB1)/ERK1/2 pathway in granulosa cells and cumulus cells of preovulatory follicles to impact ovulation. However, the expression and roles of other ERBB family members and their ligands have not been explored in detail. Herein, we document that two transcripts of the neuregulin (Nrg1) gene are expressed in granulosa cells, and that the type III Nrg1 is induced during ovulation in an ERK1/2 and C/EBPß-dependent manner. Western blotting shows that intact (75 kDa) and secreted (45 kDa) forms of neuregulin 1 (NRG1) are present in the ovary. NRG1 likely binds to ERBB3/ERBB2 complexes that are expressed in granulosa cells and cumulus cells. In cultured granulosa cells, NRG1 selectively stimulates the phosphorylation of AKT/PKB compared to ERK1/2. However, when granulosa cells were cultured with NRG1 and AREG, the phosphorylation of ERK1/2 was markedly enhanced as compared with that by AREG alone. Cotreatment with NRG1 and AREG also increased progesterone production. When cumulus-oocyte complexes (COCs) were cultured with both NRG1 and AREG, the matured oocytes exhibited significantly higher developmental competence as compared with that of oocytes cultured with AREG alone. Collectively, these results document that the expression of type III NRG1 is induced in granulosa cells during ovulation and that NRG1 enhances AREG-induced ERK1/2 phosphorylation in both granulosa cells and cumulus cells. The NRG1 pathway has two roles: one is to enhance AREG-induced progesterone production in granulosa cells, and the other is to regulate oocyte maturation by a cumulus cell-dependent mechanism.

Growth differentiation factor 9 signaling requires ERK1/2 activity in mouse granulosa and cumulus cells.

Ovarian folliculogenesis is driven by the combined action of endocrine cues and paracrine factors. The oocyte secretes powerful mitogens, such as growth differentiation factor 9 (GDF9), that regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation. This study investigated the role of the epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinase 1 and 2 (ERK1/2; also known as MAPK3/1) signaling pathway on GDF9 action on granulosa cells. Results show that mitogenic action of the oocyte is prevented by pharmacological inhibition of the EGFR-ERK1/2 pathway. Importantly, EGFR-ERK1/2 activity as well as rous sarcoma oncogene family kinases (SFK) are required for signaling through SMADs, mediating GDF9, activin A and TGFbeta1 mitogenic action in granulosa cells. GDF9 could not activate ERK1/2 or affect EGF-stimulated ERK1/2 in granulosa cells. However, induction of the SMAD3-specific CAGA reporter by GDF9 in granulosa cells required active EGFR, SFKs and ERK1/2 as did GDF9-responsive gene expression. Finally, the EGFR-SFKs-ERK1/2 pathway was shown to be required for the maintenance of phosphorylation of the SMAD3 linker region. Together our results suggest that receptivity of granulosa cells to oocyte-secreted factors, including GDF9, is regulated by the level of activation of the EGFR and resulting ERK1/2 activity, through the requisite permissive phosphorylation of SMAD3 in the linker region. Our results indicate that oocyte-secreted TGFbeta-like ligands and EGFR-ERK1/2 signaling are cooperatively required for the unique granulosa cell response to the signal from oocytes mediating granulosa cell survival and proliferation and hence the promotion of follicle growth and ovulation.

Targeted disruption of Mapk14 (p38MAPKalpha) in granulosa cells and cumulus cells causes cell-specific changes in gene expression profiles that rescue COC expansion and maintain fertility.

MAPK14 (p38MAPKalpha) is critical for FSH and prostaglandin E (PGE)2 signaling cascades in granulosa cells (GCs) and cumulus cell-oocyte complexes (COCs) in culture, indicating that this kinase might impact follicular development and COC expansion in vivo. Because Mapk14 knockout mice are embryonic lethal, we generated GC specific Mapk14 knockout mice (Mapk14gc(-/-)) by mating Mapk14(fl/fl) and Cyp19-Cre mice. Unexpectedly, the Mapk14gc(-/-) female mice were fertile. Analyses of gene expression patterns showed that amphiregulin (Areg) and epiregulin (Ereg), two key regulators of ovulation and COC expansion, were up-regulated in the GCs but down-regulated in cumulus cells of the mutant mice in vivo. COCs from the mutant mice expanded and expressed matrix-related genes, if cultured with AREG, but not when cultured with forskolin or PGE2, the latter being a key factor regulating MAPK14 activity in cumulus cells. Conversely, when GCs from the Mapk14gc(-/-) mice were cultured with forskolin, they produced more Areg and Ereg mRNA than did wild-type GCs. These results indicate that disruption of Mapk14 selectively alters the expression of Areg and other genes in each cell type. Greater AREG and EREG produced by the GCs appears to by-pass and compensate for the critical need for MAPK14 signaling and induction of Areg/Ereg (and hence matrix genes) by PGE2 in cumulus cells of the mutant mice. In conclusion, although MAPK14 is not overtly essential for preovulatory follicle development or events associated with ovulation and luteinization in vivo, it does impact gene expression profiles.

Cumulus cell-enclosed oocytes acquire a capacity to synthesize GSH by FSH stimulation during in vitro maturation in pigs.

We investigated (i) follicle stimulating hormone (FSH)-modulated changes in the expression of glutathione (GSH) and its rate-limiting enzyme, glutamate cysteine ligase (GCL), in porcine oocytes and cumulus cells, and (ii) the contribution of gap-junctional communications (GJCs) in cumulus-oocyte complexes (COCs) to intraoocyte GSH accumulation. In experiment (i), COCs were cultured for 48 h with (+FSH group) or without FSH (-FSH group). The GSH content of oocytes increased with cultivation time in the +FSH group, but decreased in the -FSH group. The GSH content of cumulus cells at 48 h was also higher in the +FSH group than that in the -FSH group. Expression of GCL subunit mRNAs in oocytes and cumulus cells was increased by FSH stimulation until 12 h, and then fell to the baseline level. On the other hand, the amount of GCL subunit proteins in oocytes and cumulus cells increased gradually throughout the period of culture with FSH. In experiment (ii), blocking of GJCs in COCs during 0-24 h of culture led to a decrease in the GSH content of oocytes at 24 h of culture, whereas the GSH content at 48 h of culture did not differ even after blocking of the GJCs during 24-48 h of culture. These findings indicate that FSH initiates GSH synthesis in cumulus cells and oocytes by modulating the expression of GCL, and that porcine oocytes are able to synthesize GSH without GJC-mediated support from cumulus cells, at least in the later half of maturation culture.

[Realization pathways of prolactin modulating influence on the cAMP-dependent mechanism of meiosis regulation in bovine oocytes].

Cyclic AMP is one of the key regulators of mammalian meiosis. In the present work, realization pathways of the previously revealed modulating influence of prolactin (PRL) on the cAMP-dependent mechanisms of meiosis regulation in bovine oocytes were studied. A comparative investigation of individual and combined effects of PRL (50 ng/ml) and an activator of adenylate cyclase forskolin (FK. 20 microM) on the meiotic reinitiation and nuclear maturation completion in cumulus-enclosed and cumulus-free oocytes was performed. It has been shown that the pattern of PRL influence on the meiotic resumption in oocytes devoid of cumulus cells depends on the presence of FK in the culture medium. Furthermore, realization of this influence is not associated with the activation of cytoplasmic isoforms of protein kinase C. It has been also found that PRL inhibits the retarding action of FK on the completion of oocyte nuclear maturation in both the presence and absence of cumulus cells. The findings suggest that PRL may modulate the functioning of the cAMP-dependent mechanism of meiosis regulation by the direct action on bovine oocytes, with realization of the action being independent of the metabolic coupling of oocytes with cumulus cells.

Mobilisation of Ca2+ stores and flagellar regulation in human sperm by S-nitrosylation: a role for NO synthesised in the female reproductive tract.

Generation of NO by nitric oxide synthase (NOS) is implicated in gamete interaction and fertilisation. Exposure of human spermatozoa to NO donors caused mobilisation of stored Ca(2+) by a mechanism that did not require activation of guanylate cyclase but was mimicked by S-nitroso-glutathione (GSNO; an S-nitrosylating agent). Application of dithiothreitol, to reduce protein -SNO groups, rapidly reversed the actions of NO and GSNO on [Ca(2+)](i). The effects of NO, GSNO and dithiothreitol on sperm protein S-nitrosylation, assessed using the biotin switch method, closely paralleled their actions on [Ca(2+)](i). Immunofluorescent staining revealed constitutive and inducible NOS in human oviduct and cumulus (the cellular layer investing the oocyte). 4,5-diaminofluorescein (DAF) staining demonstrated production of NO by these tissues. Incubation of human sperm with oviduct explants induced sperm protein S-nitrosylation resembling that induced by NO donors and GSNO. Progesterone (a product of cumulus cells) also mobilises stored Ca(2+) in human sperm. Pre-treatment of sperm with NO greatly enhanced the effect of progesterone on [Ca(2+)](i), resulting in a prolonged increase in flagellar excursion. We conclude that NO regulates mobilisation of stored Ca(2+) in human sperm by protein S-nitrosylation, that this action is synergistic with that of progesterone and that this synergism is potentially highly significant in gamete interactions leading to fertilisation.

Coculture with cumulus cells improves maturation of mouse oocytes denuded of the cumulus oophorus: observations of nuclear and cytoplasmic events.

OBJECTIVE: To study the mechanisms by which cumulus cells (CCs) promote oocyte maturation by observing the effect of removing the cumulus oophorus on nuclear and cytoplasmic events during in vitro maturation. DESIGN: Experimental animal study. SETTING: Academic institution. ANIMAL(S): Mice of the Kun-ming breed. INTERVENTION(S): Cumulus-free oocytes were cultured alone (DOs) or with a CC monolayer (coDOs), and the nuclear and cytoplasmic events were compared with those of oocytes matured in vivo or in vitro with the cumulus intact (COCs). MAIN OUTCOME MEASURE(S): Nuclear progression, spindle assembly, behavior of cortical granules (CGs) and mitochondria, levels of glutathione (GSH), and dynamics of maturation-promoting factor (MPF) activity during oocyte maturation under different conditions. RESULT(S): Cumulus removal increased MPF activity and accelerated the transition from the G2 to the M phase and the redistribution of CGs. Spindle assembly and mitochondrial congregation were impaired. In addition, removal of the cumulus caused a precocious exocytosis of CGs, leading to zona hardening and reduced penetrability of oocytes by sperm. After DOs were matured on the CC monolayer, however, these parameters were much improved, and the DOs acquired characteristics closer to those of cumulus-invested oocytes matured in vivo or in vitro. The level of intracellular GSH of DOs, on the other hand, did not differ from that of oocytes matured as COCs, suggesting that the mouse DOs can synthesize GSH on their own. CONCLUSION(S): While denuding mouse oocytes of CCs impaired in vitro cytoplasmic maturation, coculture with CCs promoted maturation, possibly through the regulation of MPF activity and meiotic progression.

PI3-kinase and mitogen-activated protein kinase in cumulus cells mediate EGF-induced meiotic resumption of porcine oocyte.

Previous studies have shown that epidermal growth factor (EGF) has the ability to promote in vitro cultured porcine oocyte maturation. However, little is known about the detailed downstream events in EGF-induced meiotic resumption. We designed this study to determine the relationship of EGF, EGFR, phosphatidylinositol 3-kinase (PI3-kinase), MAPK, and germinal vesicle breakdown (GVBD) during oocyte maturation. Our results showed that GVBD in cumulus-enclosed oocytes (CEOs) but not in denuded oocytes (DOs) was induced by EGF in a dose-dependent manner, which indicated that cumulus cells but not oocyte itself were the main target for EGF-induced meiotic resumption. Furthermore, we found that MAPK in cumulus cells rather than in oocyte was activated immediately after EGF administration. To explore whether EGF exerts its functions through MAPK pathway, the activities of EGF receptor (EGFR) and MAPK were inhibited by employing AG1478 and U0126, respectively. Inhibition of MAPK blocked EGF-induced GVBD, whereas inhibition of EGFR prevented MAPK activation. Both AG1478 and U0126 could lead to the failure of EGF-induced GVBD singly. Notably, we found that LY294002, a specific inhibitor of PI3-kinase, effectively inhibited EGF-induced MAPK activation as well as subsequent oocyte meiotic resumption and this inhibition could not be reversed by adding additional EGF. Thus, PI3-kinase-induced MAPK activation in cumulus cells mediated EGF-induced meiotic resumption in porcine CEOs. Together, this study provides evidences demonstrating a linear relationship of EGF/EGFR, PI3-kinase, MAPK and GVBD and presents a relatively definitive mechanism of EGF-induced meiotic resumption of porcine oocyte.