Mechanisms Of Gonadotropin On CNP/NPR2expression And Regulation In Mouse Ovary

Mammalian oocytes arrest at prophase of meiosis I at around birth and they remain arrested at this stage until puberty when the preovulatory surge of luteinizing hormone(LH)causes oocyte meiotic resumption. When immature oocytes are isolated from follicles and cultured in vitro, the oocytes spontaneously resume meiosis because of their removal from the inhibitive environment of follicles. So, some researchers think that a certain substance existing in the follicle that inhibits oocyte maturation. A recent study showed that C type natriuretic peptide(CNP) and its cognate receptor, the natriuretic peptide receptor 2(NPR2)have been shown to maintain oocytes arrest. The binding of CNP(mainly expressed in mural granulosa cells) and NPR2(a guanylyl cyclase, mainly expressed in cumulus cells) promotes meiotic arrest by increasing cyclic guanosine monophosphate(cGMP) production. And then the cGMP diffuses to oocytes via gap junctions, and maintains oocyte meiotic arrest. The nature of CNP/NPR2 signals inhibiting oocyte maturation is now better understood. However,the role gonadotrophin in regulation of CNP/NPR2 is not clearly. In this study, we use mouse before sexual maturity, mouse granulosa cells, mouse cumulus oocyte complex(COCs),mouse preovulatory follicles as models to study the molecular regulatory mechanisms behind CNP/NPR2 and gonadotrophin. And the related methods used in this study were real-time quantitative PCR, protein immunoblot, adddtion of small molecule compounds and oocyte morphology analysis. Our research will provide more evidence for the mechanisms of mammalian oocytes arrest. The results are as follow:1. The effects of gonadotrophin on EGFR, MAPK3/1, CNP and NPR2 expression and activation in mouse ovarian. We used mouse before sexual maturity as model to study the activation of EGFR and MAPK3/1 and to study the expression CNP and NPR2 of mouse ovarian after stimulation with pregnant mare serum gonadotropin(PMSG) at 0 h, 6 h, 12 h, 18 h, 24 h, 36 h and 48 h and sequently with human chorionic gonadotropin(hCG) at 1 h, 2 h, 4h, 8 h and 16 h. The results showed that PMSG simulates the CNP and NPR2 expression and the highest expression was observed at 48 h, and then decrease at 2 h after stimulation with hCG. The activation pattern of EGFR and MAPK3/1 is similar. The phosphorylation levels of EGFR and MAPK3/1 was clearly detected in COCs at 6 h, but did not at 48 h, and thegreatest activation was observed from 18 h to 30 h after stimulation with PMSG. After stimulation with hCG, the phosphorylation of EGFR and MAPK3/1 was observed again at 1h and reached peak levels by 2-4 h.2. The effects of hCG on CNP expression in vitro cultured mouse granlulosa cells. We used cultured mouse granlulosa cells as model to study the relationships of EGFR, MAPK3/1,LH and CNP. Results showed that under in vitro cultured mouse granlulosa cells, the hCG decreases the CNP mRNA expression. Further research found that that hCG-induced CNP downregulation is depend on the EGFR and MAPK3/1 signaling pathways.3. The role of NPR2 in FSH-mediated mouse oocyte meiotic resumption.We used cultured mouse COCs as model to study the relationships of EGFR, MAPK3/1, FSH, GVBD and NPR2. Results showed that FSH stimulated the expression of EGF-like factors, the activation of MAPK3/1, a decrease in NPR2 m RNA and oocyte meiotic resumption.Moreover, the FSH-induced decrease in NPR2 and oocyte meiotic resumption occurred via the MAPK3/1 singling pathway, which was activated by the EGFR singling pathway.4. The effects of LH on NPR2 expression and activity in vitro cultured mouse preovulatory follicles. We used cultured mouse preovulatory follicles as model to study the effects of LH on NPR2 expression and activity. Results showed that LH-induced the decrease NPR2 activity and NPR2 mRNA expression may be different. One hand, LH binding to LH receptor rapidly stimulated release of EGF-like factors of the cell surface via PKA signaling,and then the EGF-like factors binding and activation of the EGFR to decrease NPR2 activity of follicle. On the other hand, LH binding to LH receptor steadily promoted EGF-like factors transcription and translation via PKA signaling, and then the EGF-like factors binding and activation of the EGFR decreased NPR2 mRNA expression.As the results showed above, the evidence from in vivo and in vitro experiments showed gonadotropin are releated with the CNP/NPR2 expression and regulation and this process may need the activation of EGFR and MAPK3/1 signaling pathway.