Sox30在罗非鱼精子发生中的功能及作用机制

The regulation mechanism of animal spermatogenesis has been broadly investigating. Previous report showed that Sox30 is expressed in mammalian germ cells. Our previous studies indicate that Sox30 is also expressed in the germ cells of the Nile tilapia testis. The promoter of Sox30 gene has potential binding sites for Dmrt1 and Sf-1 that are highly expressed in testis. The onset of meiosis was delayed and some sperms have short tail or two tails in the male heterozygous mutants of the Sox30 gene. These results suggested that Sox30 may play important roles in spermatogonial differentiation and spermatogenesis. In this study, by using CRISPR/Cas9 knock-out technology, immunohistochemical staining, ChIP-seq, luciferase assays, EMSA and ChIP-PCR, we are planning to survey the function of Sox30 in spermatogenesis of the Nile tilapia, and to decipher the response of the Sox30 gene to the transcription factors (such as Dmrt1 and Sf-1) and the regulation of the Sox30 protein on the transcription of the spermatogenesis related genes (such as Sycp3 and Spo11). This study will not only elucidate the function and signaling pathway of the Sox30 gene during spermatogenesis in the Nile tilapia, but also provide new insight into the regulatory mechanism of male sterility in animals.

动物精子发生的调控机制一直是学术界的研究热点之一。已有研究报道，转录因子Sox30在哺乳动物精巢中高表达；我们的前期研究显示，尼罗罗非鱼Sox30基因主要在精巢的生殖细胞中表达，其启动子含Dmrt1和Sf-1等精巢高表达转录因子的潜在结合位点，其杂合突变导致精巢减数分裂延迟及精子鞭毛变短。本项目拟以尼罗罗非鱼Sox30基因为靶标，综合利用CRISPR/Cas9基因敲除、免疫组化、转录组测序、ChIP-seq、荧光素酶报告基因实验、EMSA和ChIP-PCR等方法，系统分析Sox30在尼罗罗非鱼精子发生中的功能，并探究Sox30对Dmrt1和Sf-1等精巢高表达转录因子的应答机制及其对Sycp3和Spo11等精子发生相关基因转录的调控作用。相关研究结果不仅有助于丰富和完善罗非鱼精子发生的分子调控机制，还可为动物雄性不育的机理解析提供新的思路。

动物精子发生是一个复杂的由精原干细胞分化发育为成熟精子的过程。我们在罗非鱼中的前期研究发现转录因子Sox30在精巢中高表达，但其是否参与调控精子发生过程尚不清楚。本研究围绕Sox30在罗非鱼精子发生过程中的功能及分子作用机制研究，取得如下发现：（1）免疫荧光实验表明，Sox30在罗非鱼精巢中的表达主要定位于精母细胞、精子细胞和成熟精子。（2）基于CRISPR/Cas9系统对罗非鱼Sox30基因进行敲除突变，结果使精子尾部变短或卷曲、精子活力下降，最终导致雄性亚不育，表明Sox30在罗非鱼精子发生中具有重要调控作用。（3）基于RNA-seq技术分析发现，罗非鱼Sox30突变导致精巢中出现2024个差异表达基因，其中ift140、ptprb、tssk6、igf3和cyp11c1等精子发生相关基因的表达显著下调。（4）利用染色质免疫共沉淀测序（ChIP-Seq）检测精巢基因组中与Sox30结合的DNA区段，发现ift140和ptprb的启动子区均含有Sox30的结合峰；基于荧光素酶报告基因实验证实，Sox30对ift140和ptprb启动子的活性具有正调控作用。综合分析表明，Sox30通过直接调控ift140和ptprb等精子发生相关基因的转录来参与精子发生过程。（5）综合应用荧光素酶报告基因实验、凝胶迁移分析（EMSA）、ChIP-PCR和RT-qPCR实验证实，精巢高表达转录因子Dmrt1通过结合Sox30基因启动子上的特异基序来直接正调控其转录。上述系列创新性发现系统解析了Sox30在罗非鱼精子发生中的关键作用与分子机制，完善了动物精子发生的分子调控网络。