LH-EGFR信号升高卵丘细胞中钙离子水平的分子机理

Our previous results indicate that natriuretic peptide type C (NPPC, also commonly known as C-type natriuretic peptide, CNP) produced by follicular mural granulosa cells stimulates the generation of cyclic guanosine 3’,5’-monophosphate (cGMP) by its cognate natriuretic peptide receptor NPR2 in cumulus cells, maintains meiotic arrest of oocyte within antral follicle. Further studies suggest that Luteinizing hormone (LH) participates in oocyte meiosis by the inactivity of NPR2. LH induces the expression of epidermal growth factor (EGF)-like proteins in mural granulosa cells that act on cumulus cells.The activation of EGF receptor (EGFR) could decrease NPR2 activity by elevating calcium in cumulus cells, resulting oocyte meiotic resumption.Calcium is also required for cumulus expansion.However, it is not clear how LH-EGFR signal elevates calcium in cumulus cells. Sphingosine Kinase (SphK) inhibitor SKI-II could completely inhibit LH-EGFR signal-elevated calcium in cumulus cells. The objective of the present project is to study the relationship of LH-EGFR signaling and SphK activity;the mechanism that S1P mobilizates calcium from endoplasmic reticulum calcium stores; the testification with conditional knock out mice. These results will further understand the mechanism of LH-EGFR signal on oocyte meiotic resumption, which will be benefit for improving the quality of oocyte in vitro culture, and providing foundation for clinic application.

我们前期结果证实颗粒细胞分泌的C-型钠肽及其受体NPR2维持了卵母细胞减数分裂阻滞；而促黄体素（LH）通过转激活卵丘细胞上的表皮生长因子受体（EGFR）来升高钙离子水平，导致NPR2活性下降、卵母细胞减数分裂恢复。钙离子也参与了LH-EGFR信号诱导的卵丘扩展。但是LH-EGFR信号引起钙离子升高的分子机理不清楚。初步实验表明，LH-EGFR信号能升高1-磷酸鞘氨醇(S1P)水平；鞘氨醇激酶(SphK)的抑制剂可以完全抑制LH-EGFR信号引起的钙离子水平增加。本项目将利用体内和体外卵泡培养模型研究LH-EGFR信号是否通过激活SphK产生S1P，导致了钙离子水平的增加；S1P动员内质网中钙离子释放的分子机理；同时利用基因敲除小鼠对上述分子进行功能验证。最终确定LH-EGFR信号引起卵丘细胞钙离子升高的分子机理，为完善卵母细胞成熟机制、提高卵母细胞体外成熟质量以及临床研究和应用奠定基础。

前期研究表明，由颗粒细胞分泌的C-型钠肽NPPC与卵丘细胞中的受体NPR2结合，维持了卵母细胞减数分裂阻滞。而LH-EGFR信号通过钙信号下降NPR2的亲和性，导致了减数分裂的恢复。我们目前的研究表明TGF-beta的配体和受体主要表达在卵泡颗粒细胞中。FSH可通过TGF-beta信号上调NPPC的表达，维持了卵母细胞成熟阻滞；LH-EGFR信号通过激活SphK，导致颗粒细胞中的S1P水平升高，进而引起钙离子水平的升高，NPR2失活，减数分裂恢复。发现S1P可通过PLN抑制钙-ATPase的活性，从而引起颗粒细胞内钙离子水平的升高；同时，S1P可以通过TPX2以及DAZL维持纺锤体正常组装，促进胚胎的正常发育。在小腔卵泡中敲除Sgpl1导致了S1P水平升高，通过钙信号使NPR2失活，会导致小腔卵泡发育停滞。此外，颗粒细胞中糖酵解增强通过 mTOR信号促进原始卵泡的激活。研究结果阐明了LH-EGFR信号，通过激活SphK，升高颗粒细胞中的S1P水平，下降NPR2活性，促进卵母细胞成熟。S1P也能够促进卵母细胞成熟过程中纺锤体的正常组装。这些结果进一步补充和完善了卵泡发育和卵母细胞理论，为临床诊断和治疗不孕症奠定了理论基础。相关研究结果发表在Cell Death Dis、Histochem Cell Biol等杂志上。