SEMA3C在卵母细胞发育中的作用机制研究

Oocyte maturation is a prerequisite for normal fertilization. The oocyte degeneration will result in total fertilization failure, thus causing infertility. The etiology and pathogenesis of oocyte degeneration is still rarely reported. The applicant has previously collected a large consanguineous family with repeated oocyte degeneration during in-vitro fertilization (IVF) cycles. Using whole exome sequencing and genome-wide linkage analysis, the heterozygous missense mutation c.506 A>G (p. N169S) of SEMA3C was identified as the casual mutation. It was reported that SEMA3C was highly expressed in human oocytes, but the detailed functions were unclear. In this project, cell lines from ovary, oocytes cultured in vitro and oocyte specific knockout of sema3c mouse model will be used to clarify the function and molecular mechanism of SEMA3C during follicular development and oocyte maturation. Cell biology and molecular biology methods will be integrated to demonstrate the associated molecular mechanism of oocyte degeneration caused by heterozygous missense mutation c.506 A>G (p. N169S). The obtained research results would provide novel theoretical basis and potential target for the diagnosis and treatment of oocyte degeneration, and shed new light on successful associated reproduction technology.

成熟的卵母细胞是正常受精的先决条件。若卵母细胞发生退变则无法正常受精，从而导致不孕。卵母细胞退变的病因和发病机制目前还鲜有报道。申请者前期针对一个卵母细胞退变的不孕症大家系，通过全外显子组测序和全基因组连锁分析进行突变检测，将致病位点定位于SEMA3C基因的杂合错义突变c.506 A>G (p. N169S)。SEMA3C在人卵母细胞中高表达，但功能尚不清楚。本项目拟利用卵巢来源的细胞系，体外培养的卵母细胞以及SEMA3C的卵母细胞特异性基因敲除小鼠模型，1).解析SEMA3C在卵泡发育和卵母细胞成熟过程中的具体功能；2）.综合运用细胞生物学及分子生物学的方法阐明其作用机制；3）.揭示杂合错义突变c.506 A>G (p. N169S) 导致卵母细胞退变的分子机理。本研究有望为IVF-ET周期中卵母细胞退变的诊断、治疗提供理论依据和潜在靶点，为成功实施人类辅助生殖技术开辟新的思路。

空卵泡综合征指促排卵时，卵巢中有多个卵泡发育，超声检测及血雌二醇水平正常，但取卵手术时经反复冲洗及抽吸，获得的卵母细胞数明显低于超声下大卵泡数，部分患者甚至无法获得卵母细胞。还有一种比较少见且更为严重的特殊类型空卵泡综合征，表现为取卵时可偶尔见到已经退变、裂解成碎片的卵母细胞。关于空卵泡综合征的遗传学发病机制，既往只报道了LCHGR突变。而对于空卵泡综合征卵母细胞退变，目前尚无研究报道其原因。本团队收集了几十例空卵泡综合征卵母细胞退变的患者及家系，通过全外显子组测序的方法发现ZP3 p.A134T突变或ZP1双等位基因突变可分别导致空卵泡综合征卵母细胞退变。ZP3和ZP1编码ZP3和ZP1糖蛋白，是卵母细胞透明带的重要组份。其中，ZP3 p.A134T突变影响ZP3和ZP2糖蛋白的结合，发挥显性负效应作用；而ZP1双等位基因突变则可使ZP1糖蛋白在胞质内滞留，影响ZP1向胞外分泌。两类突变最终分别影响透明带形成，导致卵母细胞退变及空卵泡综合征。本研究为空卵泡综合征卵母细胞退变提供了遗传学病因，临床上可在卵子异常或退变的患者中开展透明带基因筛查，以明确病因，及时改变助孕策略，可避免对患者反复促排卵造成的经济损失和精神打击。