Protein Kinase C (PKC)-Mitogen Activated Protein Kinase (Mapk) Signaling Pathway And Cholesterol Production In Granulosa Cell-Oocyte Co-Culture System In Mice

There is an interaction between granulosa cells and denuded oocytes during meiotic resumption in granulosa cells-oocytes co-culture system. Granulosa cells plays important roles by secreting substances into the culture medium needed by oocytes. The culture medium can facilitate the substances transduction from granulosa cells to the denuded oocytes (DOs). These secreted factors are important for the maturation of incompetent denuded oocytes. The secreted factors from granulosa cells can be increased by coculture granulosa cells with denuded oocyte (mean %GVBD up to 64.1±1.8%) compare with DOs alone (mean %GVBD 20.4±4.8%). It suggested that DOs alone can not absorb nutrition directly from the culture medium, hence granulosa cells can serve as a mediator to mature the denuded oocytes. It can be explained because there are signaling pathways in the granulosa cells. GVBD percentage of DOs can be elevated by addition of Follicle Stimulating Hormone (FSH). FSH can act its role directly and indirectly on DOs since this experiment demonstrated that there was no significantly different of GVBD percentage between FSH cultured with DOs alone (71.9±0.1%) and FSH cultured with GCs+DOs(81.0±0.6%).The secreted factors have been released by granulosa cells cocultured with DOs after 12 h incubation time. This culture time is recommended because the obtained supernatant can induce GVBD percentage of new DOs up to 71.7±3% (in the absence of FSH) and 81.1±1.4%(in the presence of FSH). There is no significantly different on GVBD percentage between 12 h and 24 h incubation time for GCs+DOs coculture group. It suggested that 12 h is enough to culture the incompetent DOs. In the presence of FSH, GVBD percentage of DOs cultured in the supernatant collected from GCs (59.9±2.8%) is lower than that of GCs+DOs group (81.1±1.4%). Granulosa cells can act its role in inducing GVBD event in a DOs dependent manner. It suggested that there is bidirectional communication between granulosa cells and denuded oocytes.Mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) have been investigated being two of many substances secreted by granulosa cells during meiotic maturation since FSH failed to induce GVBD event in the presence of 10 uM MEK inhibitor (U0126) or that of Staurosporine. U0126 inhibit MAPK activation and decrease GVBD percentage of DOs and GCs+DOs group after 12 h incubation time (24.12% and 25.86%, respectively). The supernatant collected from these treatment fail to support further 24 h culture time. MAPK signaling pathway has important role in granulosa cells and denuded oocytes for oocyte maturation. MAPK activation can be detected by immunohistochemistry analysis, GCs and DO express phosphorylated MAPK during maturation process. As U0126, Staurosporine (PKC inhibitor) makes FSH fail to induce MAPK phosphorylation (in western blot result) and decrease GVBD percentage (8.8±1.5% for DOs alone and 19.8±5.2% for GCs+DOs). PKC signaling pathway apparently has a pivotal role in granulosa cells and denuded oocyte. In addition of PMA (total PKC activator), PKC can be stimulated in both cells. It suggested PMA can replace the role of FSH. In granulosa cells, PKC αβγ (cPKC),δε (nPKC) and ξ (aPKC) are activated during oocyte maturation. Meanwhile, in denuded oocyte there are more than 6 PKC isoforms are participated in inducing GVBD event because GVBD percentage of DOs alone in Go 6983 (28.2±5.1%) and Bisindolylmaleimide (31.4±2.5%) is higher (P<0.05) than that of Staurosporine (8.8±1.5%). In western blot analysis, Staurosporine blocks MAPK phosphorylation in granulosa cells. MAPK is at the downstream of PKC pathway.Signaling pathways and metabolic activity are part of bidirectional communication between granulosa cells and denuded oocytes. Protein synthesis and phosphorylation are involved in FSH-induced meiotic resumption. Protein synthesis inhibitor Cycloheximide (CHX) has been reported affect depletion of GVBD percentage in DOs alone (30.0%) or GCs+DOs group (48.9%) cultured with FSH for 12 h. CHX makes FSH fail to induce protein synthesis in granulosa cells and denuded oocytes. The obtained supernatant from CHX treatment can not supply enough nutrient for new DOs, furthermore decreased GVBD percentage after 24 h incubation time in the supernatant (33.9%,29.9% and 46.6% for DOs alone, GCs alone and GCs+DOs, respectively). In the western blot analysis, CHX can not inhibit MAPK phosphorylation, but decreased oocyte GVBD. It suggested that MAPK and protein synthesis participate in inducing meiotic resumption via different signaling pathways, or MAPK is at the upstream of protein synthesis and oocyte GVBD. In commassive brilliant blue staining, phosphorylated proteins of FSH treated granulosa cells are more than that of U0126, CHX and Staurosporine treated granulosa cells. Protein phosphorylation has been investigated participates in oocyte maturation. Besides protein, cholesterol is also involved in FSH-induced meiotic resumption. Cholesterol production increase after addition of FSH (0.24±0.49 umol/1mg protein), and this effect can be inhibited by Staurosporine (0.02±0.003 umol/lmg protein), U0126 (0.12±0.016 umol/lmg protein) and CHX (0.04±0.001 umol/lmg protein). It demonstrated that PKC, MAPK and protein synthesis participate in stimulating cholesterol production.In summary, this experiment result suggested that granulosa cells-oocyte co-culture sytem greatly stimulates secreted factors needed by oocyte in 12 h. PKC, MAPK, protein and cholesterol have been investigated become the secreted factors released by granulosa cells. MAPK is at the downstream of PKC signaling pathway. Otherwise, MAPK and protein synthesis are stimulated in different signaling pathway. In granulosa cells, PKC α,β,γ,δ,ε,ξ induce meiotic maturation but in denuded oocytes there are more than 6 PKC isoforms involved in meiotic maturation. FSH stimulates cholesterol production mediated by PKC, MAPK and protein synthesis.