Research On Networks Regulatinglh-induced Meiosis Resumption In Mouse Oocyte

Oocytes meiotic progression begins in mammalian ovaries during fetal life but becomes arrested for prolonged periods at the diplotenestage of meiotic prophase until the LH surge. High levels of c GMP regulated by CNP, secreted by granulosa cells, inhibit the activation process required for oocyte reentry into the cell cycle. EGF/EGFR signaling is activated after LH surge, which induces the decrease of c GMP in the oocytes, following with oocyte maturation and ovulation. It indicates that the regulation of c GMP levels via CNP/NPR2 and EGF/EGFR signaling pathways is crucial for oocyte maturation. However, the regulating mechanisms of FSH and LH on CNP/NPR2 and EGF/EGFR signaling pathways and the function of these two pathways on LH-induced decrease of c GMP are unclear.To delineate the related signaling pathways involved in the regulation of c GMP levels and their relationships during oocyte maturation, we carried out HE staining, Immunohistochemistry, Immunofluorescence, Quantitative Real-time PCR, Enzyme immunometric assay, Radioimmunoassay, Western Blot et al on the levels of ovarian follicle, cumulus oocyte complex and oocyte of different EGFR knockout mouse models. The main specific results are as follows:1. C57BL/6J×129Sv type mice used in this study resposed well to Gn RH with noemal development of follicles and oocytes. After induction of 5 IU PMSG for 44-48 h, there were many preovulatory follicles on the surface of C57BL/6J×129Sv ovaries and the expression and distribution of EGFR in the follicle was normal. And then inject mice with 5 IU h CG, there were a number of MII-stage oocytes in the oviducts. The fertilization rate was low after IVF, while the development and morphology of early embryonic after fertilization was good. The ratio of spontaneous maturation of oocytes underwent IVM was high, and the morphological observation of chromosomes showed good condition.2. Oocyte maturation was inhibited significantly in Egfrdelta/flCyp19Cre/+ mice. As the Western blot shown, EGFR protein levels in Egfrfl/flCyp19Cre/+ and Egfrdelta/flCyp19Cre/+ mice decreased significantly to 35% and 15% of control, respectively. After superovulation through PMSG and h CG, cumulus cells closely surrounded the oocytes in the ovarian follicles, and it is difficult to strip the oocytes from cumulus cells by hyaluronidase. Oocytes from Egfrfl/flCyp19Cre/+ mice went through GVBD at this time, while there were still 60% oocytes from Egfrdelta/flCyp19Cre/+ mice were maintain at GV stage. Meanwhile, the oocyte number collected from oviducts significantly decreased in the Egfrfl/flCyp19Cre/+ and Egfrdelta/flCyp19Cre/+ mice, especially in Egfrdelta/flCyp19Cre/+ mice, it was only about 18% as control group. Among those successful ovulated oocytes, some were still in GV stage, and even more than 50% oocytes were in GV in Egfrdelta/flCyp19Cre/+ mice.3. Production of CNP in granulosa cells was unregulated by FSH and downregulated by LH. A genome-wide and genetic investigation of the gonadotropin control of NPPC were performed, which showed a positive regulation by FSH and a negative regulation by LH and confirmed that this mechanism functions regulating c GMP production both in mural granulosa cells and cumulus cells, while no function on the regulation of NPPA and NPPB. Receptor NPR2 expressed both on surface of mural granulosa cells and cumulus cells with a higher level on cumuls cells. FSH/PMSG increased NPR2 expression gradually, and decresed significantly after 44 h. Three hours treatment of LH/h CG inhibited its expression. There was no effect of CNP on LH-induced oocyte GVBD in in vitro cultured preovulatory follicles, while CNP increased the c GMP levels in cultured mural granulosa cells and inhibited oocyte GVBD in cultured cumulus oocyte complexes. In the cultured mural granulosa cells, LH/h CG inhibited the gather of c GMP by CNP, which suggested a decrease of sensitivity of mural granulosa cells to CNP stimulation. Besides, the expression of NPPC in Egfrdelta/flCyp19Cre/+ mice still decresed significantly responsing to the regulation of LH as wild type mice. It indicated that the delete of EGFR didn’t affect the fuction of LH on NPPC.4. EGF/EGFR network regulated LH-induced c GMP decrease, while was not necessary for LH-dependent NPPC m RNA decrease. LH/h CG treatment causes a major decrease in both c GMP and NPPC m RNA accumulation in vivo and in vitro. However, the c GMP downregulation was very fast, preceding the decrease in NPPC m RNA by more than 1 h. Areg, an EGF-like factor, suppresses NPPC m RNA levels in cultured follicles to the same extent as LH, and this effect is completely prevented by EGFR kinase inhibitor AG1478. However, the LH-dependent suppression of NPPC is insensitive to AG1478. Similarly, NPPC suppression by LH occurs in follicles from 4 kinds of EGFR null mice. These findings document that EGFR signaling is sufficient to downregulate CNP, but is not necessary for LH action. When c GMP concentration in the follicle is measured, the short-term, but not long-term, LH effects on c GMP are prevented by AG1478, suggesting that ligand availability may be responsible for the late response. Further study showed that EGFR phosphorylation happened at 15-30 min, which indicated the activation of EGF/EGFR signaling. These findings demonstrate that EGFR-dependent events are involved in the short-term regulation of c GMP, whereas the long-term effects may involve regulation of the CNP.To sum up, we verified the crucial regulation functions of CNP/NPR2 and EGF/EGFR signaling pathways during oocyte development and maturation in the cell, tissue and in vivo levels by using ovarian granulosa cell specific knockout mouse models in this study. These findings demonstrate that redundant pathways are involved in the regulation of c GMP during this process. The early and fast decrease of c GMP mediated by EGF/EGFR network is essential for oocyte maturation. This study provides important experimental evidences for the understanding of the oocyte maturation and new research ideas for the prevention and treatment of oocytes related infertility in clinical research.