KCTD19在精子发生减数分裂过程中的作用及机制

Spermatogenesis is the process of producing mature sperm by germ cell proliferation and differentiation. Abnormal development would lead to male infertility. Our preliminary data demonstrated that potassium channel tetramerization domain protein KCTD19 is highly expressed in the spermatocytes and round spermatids. KCTD19 knockout mice were sterile owing to the arrest of spermatogenesis meiosis at the metaphase step. However, it is unclear regarding KCTD19 functions and the related mechanisms in spermatogenesis and meiosis. To address key scientific problems, based on the frontier progress and our previous research findings, our aim is to clarify the function of KCTD19 and the mechanism between KCTD19 and HDAC1 in the testicular spermatogenesis meiosis. We hypothesize that the formation of KCTD19-HDAC1 complex would lead to histone deacetylation modification during spermatogenesis meiosis, then to screen and test the downstream signaling moleculars. The related experiments will be performed, including CRISPR/Cas9 KCTD19 knockout mouse and a series of technology, such as spermatocyte chromosome spread, spermatocyte separation using STA-PUT velocity sedimentation, RNA-seq, ChIP-Seq. The results are crucial for understanding the regulating mechanisms spermatogenesis meiosis, and provide an important experimental basis for the treatment and drug research of germ cell-related diseases in animals and humans.

精子发生是生殖细胞增殖和分化产生成熟精子的过程，发育异常会导致雄性不育。申请人前期的研究结果发现，钾离子通道四聚化结构域蛋白KCTD19在睾丸精母细胞和圆形精子细胞中高表达，敲除KCTD19导致雄性小鼠不育，其生殖细胞发育阻滞在减数分裂中期，但KCTD19在精子发生减数分裂过程中的功能和相关的机理尚不清楚。本项目正是针对这一关键性科学问题，拟采用CRISPR／Cas9技术构建KCTD19敲除小鼠为模型，利用精母细胞染色体铺片、STA-PUT沉降法分选精母细胞、RNA-Seq和ChIP-Seq等技术，深入研究KCTD19在精子发生减数分裂过程中的功能，阐明KCTD19与HDAC1复合体引起组蛋白去乙酰化修饰的作用机制，筛选并验证参与KCTD19调控的下游信号分子。研究结果为调控精子发生减数分裂过程的机制提供新的理论依据，为动物和人类生殖细胞相关疾病的治疗和药物研究提供重要的实验依据。

精子发生是一个错综复杂而有规律的细胞分化过程，经历精原细胞有丝分裂、精母细胞减数分裂和精子形成三个阶段。减数分裂是生殖细胞独有的细胞分裂方式，也是形成单倍体精子细胞的关键环节，被分成细线期、偶线期、粗线期、双线期和终变期。这些阶段经历DNA双链断裂与修复，同源染色体的联会与重组、性染色体沉默等一系列关键步骤，任何一步出错都可能引起减数分裂阻滞，从而导致不育。申请人前期的研究结果发现，钾离子通道四聚化结构域蛋白KCTD19在睾丸精母细胞和圆形精子细胞中高表达，敲除Kctd19导致雄性小鼠不育，其生殖细胞发育阻滞在减数分裂中期。. 通过免疫共沉淀-质谱和体外Co-IP发现KCTD19与锌指蛋白ZFP541、去乙酰化酶HDAC1、去乙酰化酶HDAC2和末端脱氧核苷酸转移酶相互作用蛋白DNTTIP1形成一个复合体。ZFP541是睾丸中特异性表达蛋白，通过定量PCR、免疫印迹、免疫组化和免疫荧光方法证明ZFP541从细线期精母细胞中开始表达持续到圆形精子，而KCTD19从粗线期精母细胞开始表达持续到圆形精子。通过CRISPR/Cas9方法建立Zfp541敲除小鼠模型，敲除Zfp541雄性小鼠不育，生殖细胞发育阻滞在双线期，而敲除Kctd19或者Zfp541都会导致早期粗线期向中后期粗线期的发育出现延迟。此外，精母细胞染色体铺片免疫荧光结果显示敲除Zfp541影响DNA双链的修复、同源染色体的交叉互换和XY染色体的联会。RNA-seq、CUT&Tag和双荧光素酶活性实验分析结果显示ZFP541直接结合并激活减数分裂和减数分裂后发育过程中关键基因的表达，包括Kctd19；反之KCTD19也能增强ZFP541的转录活性。综上所述，我们的研究结果显示ZFP541与KCTD19形成一个转录激活复合体调控雄性小鼠精母细胞中减数分裂和减数分裂后精子形成关键基因的表达，为调控精子发生减数分裂的机制提供新的理论依据，为动物和人类生殖细胞相关疾病的治疗和药物研究提供重要的实验依据。