BMP4和RA在诱导性多能干细胞向雄性配子分化中的作用及其信号转导机制

Bone morphogenetic protein 4 (BMP4) and retinoic acid (RA) play crucial roles during the derivation of male gametes from the induced pluripotent stem cells (iPS cells). However, it remains unknown about the roles and molecular mechanisms of BMP4 and RA on this process. We have established the system for the derivation of male gametes from mouse iPS cells using in vitro RA induction and in vivo ectopic transplantation. In this project, iPS cells was induced with BMP4 to differentiate into primordial germ cells (PGCs). After selection of PGCs by fluorescence-activated cell sorting, RA was applied to induce PGCs differentiating into male gametes..RT-PCR, immunocytochemistry, Western blots, and small interference RNAs (siRNAs) will be utilized to determine the effects and Smad 1/5/8 signaling pathways of BMP4 on differentiation of iPS cells to PGCs, as well as the roles and RA/RAR signaling pathways of RA on differentiation of PGCs to male gametes. We will unveil the roles and signaling transduction pathways of BMP4 and RA on the differenatiation of the iPS cells into male gametes, which would provide a novel insight into the molecular mechanism of spermatogenesis. This project would offer new approaches for treating male infertility and developing new male contraception.

骨形成蛋白4（BMP4）和维甲酸（RA）在诱导性多能干细胞(iPS)向雄性配子分化中发挥重要作用。然而，BMP4和RA在此过程中的作用及其分子机制尚未完全阐明。我们前期建立了体外RA诱导联合体内异位移植诱导小鼠iPS细胞向雄性配子分化的体系。本项目是在此基础上，首先采用BMP4诱导iPS细胞向原始生殖细胞（PGCs）定向分化，再通过流式细胞分选PGCs，然后经RA诱导其向雄性配子分化。利用RT-PCR、免疫细胞化学、Western blots、小分子RNA干扰等技术研究BMP4对iPS细胞向PGCs分化的影响及其Smad 1/5/8信号转导，同时研究RA在PGCs向雄性配子分化的作用及RA/RAR信号通路调控。本项目将研究BMP4和RA在 iPS细胞向雄性配子分化的作用及其调控机理，为阐明精子发生提供新的机制，为男性不育治疗和男性避孕提供新的途径。

骨形成蛋白 4（BMP4）和维甲酸（RA）在诱导性多能干细胞(iPS)向雄性配子分化中发挥重要作用。我们利用Real-time PCR、免疫细胞化学、流式细胞术和Western blots等方法研究BMP4 和RA 在iPS 细胞向雄性配子分化的作用及其调控机理，发现：(1) BMP4可诱导iPS细胞向雄性配子分化，其中100 ng/ml BMP4效果最为显著；(2) Noggin可部分抑制iPS细胞向雄性配子分化，其中100 ng/ml Noggin效果最显著；(3)在iPS细胞向雄性配子分化过程中，BMP4通过结合受体BMPR1a和BMPR1b和BMPR2，进一步激活Smad1/5，转录因子gata4表达以及靶基因Id1和Id2，最终参与雄性生殖细胞分化过程；(4) RA能促进小鼠iPS细胞表达PGC相关基因、生精细胞相关基因和减数分裂相关基因，同时诱导其分化为PGC细胞；(5) RA通过激活转录因子sohlh1，下调细胞周期蛋白Cyclin A、Cylcin E 和Cdk2 ，抑制iPS细胞增殖，诱导其向雄性配子分化。以上结果揭示了BMP4 和 RA 在 iPS 细胞向雄性配子分化的作用及其信号转导机制，为男性不育治疗提供了新的靶点。