线粒体融合在雄性生殖细胞发育中的作用及机制研究

The morphology of mitochondria is dynamically regulated through multiple proteins including GTPases, Mfn1 and Mfn2. Specifically, clustered mitochondria can be found in characteristic cytoplasmic structure, named nuage, in germline cells. It has been reported that a number of germ cell-specific proteins such as GASZ are localized at nuage. GASZ null mutation led to male infertility in mice, accompanied by decreased expression of dislocalized nuage proteins. Our previous study demonstrated that ectopic GASZ enhanced germ cell derivation from both human and mouse embryonic stem cells. In addition, our preliminary data showed that GASZ promoted mitochondria fusion dependent on the existence of Mfn1 and Mfn2. Further, we found that dislocalization of GASZ from mitochondria led to abnormal development of testis in mice, suggesting that GASZ and Mfn1/2-mediated mitochondrial fusion may play an important role in germ cell maturation. To determine if Mfn1/2 proteins are indeed required for germ cell formation at different developmental stages and to understand the underlying molecular mechanism of mitochondrial fusion in germ cells, in this grant appication, we propose to examine the changes in morphology, DNA copies, and metabolism of mitochondria, in localization and expression of nuage proteins, as well the germ cell development and maturation upon deletion of mitochondrial signal of GASZ and germ cell-specific Mfn1/2 knockout in mice, spermatogonia, and in embryonic stem cells. This study will contribute to our understanding of the molecular regulation of germ cell development, but also to the pathology and drug discovery for human infertility.

线粒体受GTP水解酶Mfn1/2等的调控可发生融合，是生殖细胞中特殊结构nuage的主要组成部分。GASZ等多种生殖特异性基因定位于nuage，缺失时引起其他nuage蛋白的定位改变、表达下降，生殖细胞发育阻滞。我们的前期研究发现GASZ依赖于Mfn1/2促进线粒体的融合，其过表达利于胚胎干细胞向生殖细胞的分化。GASZ线粒体定位信号缺失会导致其促生殖细胞形成的功能丧失，雄性小鼠不育。为了阐明GASZ及Mfn1/2所介导的线粒体融合影响生殖细胞成熟的作用机理，我们拟利用GASZ及Mfn1/2的基因敲除小鼠模型，结合胚胎干细胞分化体系及体外分子试验，确定GASZ线粒体信号缺失对线粒体功能及小鼠生殖发育的影响；明确Mfn1/2在小鼠生殖发育不同阶段的独特功能; 验证GASZ与Mfn1/2的相互作用对生殖细胞成熟及对胚胎干细胞向生殖细胞分化的影响，为不孕不育的基因治疗及药物开发提供新的思路。

在精子形成过程中，生殖细胞的形态，功能，代谢需求及环境发生了巨大的变化，这需要线粒体的功能亦随之变化以适应发育及代谢需求。我们发现生殖细胞特异性蛋白GASZ的C端存在着线粒体定位信号，使之定位于线粒体的外膜，并可相互作用，促进线粒体的融合，而这种融合依赖于GASZ同线粒体融合的关键GTPase Mfn1/2之间的相互作用。当Mfn1/2缺失时，GASZ促进线粒体聚集及融合的现象消失。而GASZ促进生殖细胞发育的能力与其线粒体定位亦密切相关，基于GASZ敲除小鼠模型的实验表明，GASZ线粒体定位信号缺失会导致生殖细胞发育受阻，nuage的结构丧失，piRNA形成减少，线粒体代谢异常，睾丸变小，雄性小鼠不育。这一系列的研究结果提示GASZ及Mfn1/2所介导的线粒体融合在生殖细胞的发育中可能起着关键性的作用。同时，通过利用MFN1, MFN2, 及MFN1/2在生殖细胞中条件敲除的小鼠模型，我们发现MFN1敲除引起与GASZ MLS缺失类似的结果，即生殖细胞形成缺陷，睾丸变小，线粒体代谢异常，雄性小鼠不育。但MFN1与MFN2敲除对雄性及雌性生殖发育的影响不同，其作用机制亦有差异。通过MFN1与MFN2在Sertoli细胞中的条件敲除发现其对生殖支持细胞Sertoli Cells的功能亦有影响。