NHE1介导猪植入前胚胎母源-合子转换期发育阻滞的分子机理研究

The sodium-hydrogen exchanger isoform-1 (NHE1) is a transmembrane protein that regulates intracellular pH independently and adjusts cell volume homeostasis when co-activated with a bicarbonate-chloride exchanger. The aberrant activation of NHE1 gives rise to the developmental arrests at the maternal-zygote transition in mammalian preimplantation embryos when organic osmolytes are absent. However, the molecular mechanism underlying the NHE1 activation causes the developmental block in early embryos is elusive. To answer these questions, methods such as intracellular pH measurements in a single cell, live-cell imaging, RNA-Seq high-throughput sequencing, gene knockdown by morpholino, gene overexpression, co-immunoprecipitation protein-protein and so on will be employed. We will characterize how NHE1 is activated via a signaling mechanism involving FAK, JAK2 and other tyrosine kinase(s), which need(s) to be identified. Furthermore, we will determine whether the activation of G2/M phase checkpoint, different expression profile of maternal effect gene(s) and small RNA(s) or epigenetic modification of DNA and histone causes the developmental block at the 4-cell stage in preimplantation porcine embryos mediated by NHE1. Finally, we will evaluate the possibility that improving the efficiency of both culture and cryopreservation for porcine oocytes and preimplantation embryos through optimizing the cell volume regulation by regulating the activities of NHE1and glycine transporter I (GLYT1), which serves as a unique organic osmolyte transporter in oocytes and preimplantation embryos. This study will help to better understand the mechanism of early embryo development, and potentially improve the application and safety of assisted reproductive technology in animals.

跨膜蛋白Na+/H+泵（NHE1）单独激活可以调控细胞内pH值，与HCO3-/Cl-泵偶联激活可以调控细胞体积大小。在缺乏有机渗透压调节物质时，NHE1异常激活导致哺乳动物胚胎在母源-合子转换期发育阻滞，但其分子机制还不清楚。本研究利用单细胞pH测定、活细胞成像、RNA-Seq高通量测序以及基因Morpholino敲低或过表达、免疫共沉淀等方法研究包括JAK2和FAK在内的酪氨酸激酶调控猪植入前胚胎NHE1的信号通路以及G2/M期检验点、差异表达的母源基因和small RNAs以及基因组/组蛋白表观修饰在NHE1介导的猪4-细胞胚胎发育阻断中的作用；检测能否通过调控NHE1和甘氨酸转运蛋白GLYTI（卵母细胞和胚胎特有的有机渗透压调控通道）的活性来优化细胞体积调控提高猪卵母细胞和植入前胚胎体外培养和冷冻保存效率。该工作对提高动物胚胎发育机制的认识和动物辅助生殖技术的应用与安全具有重要意义。

哺乳动物植入前胚胎细胞内无机离子和有机渗透压调节物质的比例失衡导致物种特异的母源-合子转换期胚胎发育阻断，但其具体的调控机制还不清楚。当高渗溶液使胚胎体积缩小后，胚胎激活NHE1使无机离子导入细胞内恢复其体积，但高浓度离子影响细胞功能，胚胎再导入不带电荷的有机渗透压物质置换部分离子而维持正常的体积和功能。因此细胞体积的正确调控是胚胎体外发育的先决条件。本研究利用单细胞转录组技术筛选了在缺乏有机渗透压调节物质的情况下，高渗溶液诱导的发育阻断的猪4-细胞胚胎与正常胚胎差异表达的基因。利用单细胞pH测定、活细胞成像、免疫荧光和免疫印迹技术揭示了酪氨酸激酶JAK2，而非FAK，可能参与了高渗诱导的NHE1的激活。细胞周期检测发现阻断的胚胎停滞在细胞周期的S期。生物信息学分析和RT-qPCR验证实验揭示发育阻断的胚胎已启动了合子基因组激活，但合子基因组激活不完全。另外，调控RNA剪接、DNA甲基化和组蛋白H3K4me3和H3K27me3表观遗传修饰相关的酶基因表达异常。通过优化细胞体积调控向猪卵丘-卵母细胞复合体（COCs）玻璃化冷冻液、解冻液和体外成熟培养液中添加有机渗透压调节物质甘氨酸可以降低冷冻或解冻诱导的渗透压损失而提高猪卵母细胞玻璃化冷冻保存效率。最后，通过调整猪卵母细胞体外成熟（IVM）液的渗透压和甘氨酸的浓度筛选了比现在常规的猪IVM液更加高效的成熟培养液。该工作对提高动物胚胎发育机制的认识和动物辅助生殖技术的应用与效率具有重要意义。