线粒体融合蛋白MFN2在精子发生过程中的作用及机制研究

Spermatogenesis is a complicated biological process in mammals, which involved in a large number of testis-specific and/or preferential gene expression regulation. Although lots of genes were identified by many of investigators are believed to control or regulate spermatogenesis, the genetic mechanism of gene regulation during spermatogenesis is largely unknown. We previously found that the transcript of Mitofusin 2 (Mfn2) was expressed in mouse testes preferentially and highly expressed in pachytene spermatocytes and round spermatids. Based on these expression profiles of Mfn2, we hypothesized that Mfn2 is essential for regulation of spermatogenesis. In this project, we are going to utilize the Cre-Loxp knock-out strategy to obtain two types of Mfn2 conditional knock-out mouse models to inactivate Mfn2 from different stage of male germ cells. One is Stra8-Cre mediate Mfn2 knock-out mouse model, another is Ddx4-Cre mediate Mfn2 knock-out mouse model. We will characterize the phenotype differences of these two types of Mfn2 conditional knock-out male mice to identify the roles of Mfn2 during spermatogenesis, and further reveal the functions and mechanism of Mfn2 in spermatogenesis. It is hope that a better understanding on how can Mfn2 genetically control spermatogenesis and what is the mechanism of Mfn2 regulation, and will be helpful to provide a new evidence and theory for gene target therapy on aberrant spermatogenesis caused by gene mutation.

哺乳动物精子发生是个极其复杂的生物学过程，涉及了很多睾丸特异或优势表达基因对其进行调控。尽管国内外学者已发现了一些基因参与调控精子发生，但其具体的调控作用机理仍然不十分清楚。申请人在前期的工作中发现线粒体融合蛋白基因Mfn2在小鼠睾丸中优势表达，并在精母细胞和圆形精子中高度表达，因此推测Mfn2基因参与调控小鼠精子发生过程。本课题拟利用Cre-Loxp敲除策略构建两种不同的生殖细胞条件性敲除Mfn2小鼠模型：1）Stra8-Cre介导的Mfn2敲除小鼠；2）Ddx4-Cre介导的Mfn2敲除小鼠，通过观察比较这两种条件性敲除小鼠表型的异同，确定Mfn2在精子发生过程中的作用，并进一步探寻与其相互作用的因子，以揭示Mfn2在调控精子发生过程中的具体机制。通过本课题的研究，可望阐明线粒体融合蛋白MFN2在精子发生过程中的遗传作用机制，为基因突变导致的精子发生障碍的靶向治疗提供新的理论基础。

男性不育症已成为影响男性生殖健康的主要因素之一，有关男性不育与精子发生相关的研究越来越受到重视。精子发生是个极其复杂的生物学过程，涉及了很多睾丸特异或优势表达的基因对其进行调控。尽管国内外学者已发现了一些基因参与调控精子发生，但精子发生过程中的一些基因调控的遗传作用机理仍然不十分清楚。MFN2是第一个被发现可以调节线粒体外膜融合的基因之一，与多种神经性疾病的发生发展有关，但在精子发生过程中的作用仍未尽可知。我们在本研究中利用Cre/Loxp敲除策略构建了在雄性生殖细胞中条件性敲除Mfn2的小鼠模型（Mfn2-cKO），发现雄性敲除小鼠不育，附睾中精子数量显著减少，精子形态异常。进一步对敲除小鼠生殖细胞发育过程进行分析发现，Mfn2-cKO小鼠生殖细胞（包括粗线期精母细胞、圆形精子细胞）中的线粒体、内质网等超微结构出现异常。在分子机制上，本研究发现MFN2与很多piRNA通路相关蛋白互作，比如MIWI、DDX4、GASZ、TDRKH、MSY2等，且MFN2参与调控生殖细胞中相关基因的选择性剪接。此外，本研究还意外发现了表观遗传调控因子UHRF1有可能也参与调控精子发生过程，基于此，我们构建了Uhrf1生殖细胞条件性敲除小鼠模型（Uhrf1-cKO），发现Uhrf1-cKO小鼠出现不育的表型，精子发生阻滞在粗线期精母细胞阶段。更为有趣的是，敲除小鼠精母细胞中的逆转录转座子出现异常激活现象。总之，本研究结果将有助于为由基因突变导致的精子发生障碍的靶向治疗提供新的理论基础和实验依据。