The Mechanism Of Luteinizing Hormone-induced EGF-like Factors Expression And Mouse Oocyte Meiotic Resumption

Elevated cGMP which is produced by NPR2 (natriuretic peptide receptor 2, also known as NPRB)binding to NPPC (natriuretic peptide C, also known as CNP) in mural granulosa cells, maintains the meiotic blockade of oocytes within antral follicles. A surge of LH (luteinizing hormone) from the pituitary gland induces the expression of EGF (epidermal growth factor)-like factors, which triggers oocyte maturation and ovulation. We investigated the mechanism of LH-induced EGF-like factors expression, and the function of NPR2 that was inhibited by EGFR signaling in oocyte maturation.In the present study, there was a rapid increase of phosphorylated CREB (cAMP response element binding protein) after the activation of LHR (LH receptor) by hCG (human chorionic gonadotropin).Large antral follicles from eCG (pregnant mare serum gonadotropin, also known as PMSG)-primed mice were cultured in the medium with LH to stimulate the expression of EGF-like factors. In this system, the phosphorylation of CREB was increased in granulosa cells, and the CREB functional inhibitor KG-501 significantly reduced LH-induced EGF-like factors expression, oocyte meiotic resumption and cumulus cells expansion. In cultured granulosa cells, the reduction of CREB expression by Creb siRNA also repressed LH-induced gene expression of EGF-like factors. Furthermore, the inactivation of MAPK3/1 (mitogen-activated protein kinase 3/1) by U0126 had an inhibitory effect on LH-induced CREB phosphorylation and EGF-like factors gene expression. Meanwhile, the activation of LHR increased the phosphorylation levels of Akt (protein kinase B, also known as PKB), which was involved in LH-induced CREB phosphorylation and EGF-like factors gene expression.Next, we also studied the decreasing effects of EGFR signaling on NPR2 function, which involed in EGF-induced oocyte maturation. COCs (cumulus-oocyte complexs) are cultured in the medium with 30 nM NPPC to prevent oocyte spontaneous maturation. In this system, EGF could stimulate oocyte meiotic resumption after 4 hours of incubation. Further study showed that EGF elevated intracellular calcium concentrations of cumulus cells and decreased cGMP (cyclic guanosine monophosphate) levels in cumulus cells and oocytes, and calcium-elevating reagents SIP (sphingosine-1-phosphate) mimicked the effects of EGF on oocyte maturation and cGMP levels. EGF-mediated cGMP levels and meiotic resumption could be reversed by EGF receptor inhibitor AG 1478 and the calcium chelator BAPTA/AM.EGF also decreased the expression of Npr2 mRNA in cumulus cells, which may not be involved in meiotic resumption, because the block of NPR2 protein de novo synthesis by cyclohgeximide had no effect on NPPC and EGF-mediated oocyte maturation. However, EGF had no effect on oocyte maturation when meiotic arrest was maintained in the present of cGMP analog 8-Br-cGMP(8-bromoadenosine-cGMP). These data show that elevating calcium concentrations induced by EGFR signaling decrease NPR2 guanylyl cyclase activity, and trigger the resumption of oocyte meiotic.Additionally, EGFR signaling also involve in LH-induced follicular volume increasing and TGF-?(transforming growth factor-(3) signal may involve in this process, which is crucial for ovulation.Thus, LH induces MAPK3/1 and Akt activation, both of which are required for CREB activation that promotes the expression of EGF-like factors in granulosa cells. Activated EGFR signaling induces meiotic resumption by elevating calcium concentrations of cumulus cells to decrease NPR2 guanylyl cyclase activity.