Study Of MAPK-AEBPβ Pathway On Cell Apoptosis, Cell Cycle And Hormone Secretion In Porcine Granulosa Cell

Mammalian folliculogenesis is involved in follicular growth and astresia. The follicular growth included the regulation of proliferation, cell cycle control and differentiation of GCs. However, only a few of follicles undergo ovulation and the majority of follicles undergo atresia before ovulation. This degenerative process is initiated or caused by granulosa cell apoptosis and eventually lead to follicle atresia. GCs produce steroids, sense follicle stimulating hormone (FSH) and LH in ovarian micro-environment and promote growth of delicate oocyte. Therefore, cultured ovarian GCs are ideal models to study molecular mechanisms of gene regulation during folliculogenesis. However, the mechanisms of granulosa cells apoptosis are complex progresses and not clear. Therefore, cultured the granulosa cells in vitro, addition the inhibitors, RNAi, transfection, western blot, real time PCR, flow cytometry, ELISA were performed to study the role of the pathway MAPK-CEBPβ in the porcine granulosa cells through detecting key genes expression, hormone secretion (P4, E2), cell apoptosis, cell cycle. The study investigated the affect and mechanisms of MAPK-CEBPP on granulosa cells in order to get the mechanisms of the MAPK-CEBP(3regulated ovulation, luteinization in pig and improve porcine reproduction. This study contains the following two parts:Experiment1:study the role of the U0126and RU486which are the inhibitors of MAPK3/1(ERK1/2) and progesterone receptor in the porcine GCs.In this part study, the results showed that luteinizing hormone (LH) and exogenous human chorionic gonadotropins (hCG) significantly increased the expression of CEBP(3in porcine GCs, and the protein expression of p-ERKl/2, CEBPβ and p-p38MAPK were decreased after treatment with the inhibitors of ERK1/2(U0126) and PGR (RU486). Furthermore, FBS has no affect on the role of U0126and RU486in the porcine GCs, and the GCs in S and G2/M phase of cell cycle was decreased and GO/Gl phase was increased after U0126treatment, but the GCs in S phase of cell cycle was decreased and had no affect on the GO/G1and G2/M phase after RU486treatment. Further research found that U0126could survival cell apoptosis, but RU486could promote cell apoptosis. More importantly, it markedly down regulated the concentration of estradiol (E2) and progesterone (P4) in the culture medium after U0126and RU486treatment. To uncover the regulatory mechanism of ERK1/2and PGR on porcine GCs, we detected the genes related with cell cycle, cell apoptosis, steroids synthesis, ovulation and luteinization by QPCR, we found that the mRNA expression of bcl-2and bax has no affect, p53and cyclinAl was up-regulated, while genes related to pro-apoptosis (Caspase-3), hormonal synthesis (CYP11A1and Runx2), cell cycle (cyclinB1), ovulation (IGFBP4) and luteinization (PTGFR) were down-regulated after U0126treatment. However, we also found that the mRNA expression of bcl-2, bax, p53, caspase-3, Runx2, cyclinA1, cyclinB1and PTGFR were all up-regulated; while genes related to hormonal synthesis (CYP11A1) and ovulation (IGFBP4) was down-regulated after RU486treatment. In conclusion, this part study reveals that ERK1/2and PGR are key regulators of porcine GCs through modulation of cell cycle, apoptosis, steroid synthesis, and other regulators of folliculogenesis.Experiment2:study the role of the gene CEBPp in the porcine GCs by RNAi.In this part, we constructed the CEBPP RNA vectors successfully, and the best vector pshRNA-2was chosen, which can knock down the expression of CEBPβ gene successfully, confirmed by mRNA and protein level analyzed by real time PCR and western blot, respectively. We also found that knockdown of CEBPβ significantly increased the expression of p-ERK1/2by WB. Furthermore, CEBPp knockdown arrested the GCs at S phase of cell cycle, but had no effects on cell apoptosis. More importantly, it markedly down regulated the concentration of estradiol (E2) and progesterone (P4) in the culture medium. To uncover the regulatory mechanism of CEBPβ knockdown on cell cycle, apoptosis and steroids synthesis, we found that the mRNA expression of bcl-2(anti-apoptosis), StAR and Runx2(steroid hormone synthesis) was up-regulated, while genes related to apoptosis (Caspase-3and p53), hormonal synthesis (CYP11A1) and cell cycle (cyclinAl, cyclinB1, cyclinD1) were down-regulated, suggesting that knockdown of CEBPP may inhibit apoptosis, regulate cell cycle and hormone secretions at the transcriptional level in porcine GCs. Furthermore, knockdown of CEBPβ significantly increased the expression of PTGS2and decreased the expression of IGFBP4, Has2and PTGFR which are important for folliculogenesis in porcine GCs. In conclusion, this part study reveals that CEBPβ is a key regulator of porcine GCs through modulation of cell cycle, apoptosis, steroid synthesis, and other regulators of folliculogenesis.The results of this study indicated that MAPK-CEBPβ pathway not only regulated the porcine granulosa cells apoptosis, cell cycle and hormone secretion, but also involved in many genes’expression in the GCs, such as genes related to cell apoptosis, cell cycle and hormone secretion as well as genes related to oocyte growth, ovulation and luteinization. In the conclusion, the MAPK-CEBPβ pathway play important role in the porcine granulosa cells, and might be involved in the regulation of folliculogenesis, ovulation, and luteinization through regulation of many genes expression in porcine GCs and GCs growth. These results can provide reference for the study of porcine folliculogenesis and ovulation, as well as lay foundation for improving pig fertility.