小鼠支持细胞中血睾屏障基因缺失导致精子发生微环境变化的研究

Blood-testis barrier (BTB) is a special structure comprising of several kinds of cell junctions (including adherens junction with CDH2 as one of its basic components) between Sertoli cells in seminiferous tubules, which prevents the exchange of macromolecules between blood and seminiferous tubules to provide a specific microenvironment required of for spermatogenesis. However, it remains unclear about the components of this microenvironment, the impact on spermatogenesis brought by changes on those components and whether these changes can be detected in serum. In this project, we will use mice with Cdh2 conditionally knocked out in Sertoli cells as an animal model to study the influence of BTB damage to spermatogenetic microenvironment and spermatogenesis itself. On the basis of findings in the Cdh2 knockout mice, we will detect proteins whose expression levels changed with the same tendency in mice lacking Cdh2,Cx43 or Claudin 11 in Sertoli cells. Through functional experiments, we will finally combine proteomics with bioinformatics techniques to find proteins differentially expressed in the testis of the knockout mice. We will try to sort out several proteins associated with spermatogenesis, which might be the components of the microenvironment for spermatogenesis, than identify the proteins in spermatogenesis microenvironment by functional analysis of the selected differentially expressed proteins. For the proteins specially expressed in testis (of Wild type mice) whose expression level decreases significantly in knockout mice, we will examine their levels in the serum of mice and patients with oligozoospermia, with the hope to find serum molecular markers for BTB damage and changes on spermatogenetic microenvironment. The results will further our understanding of mechanisms underlying abnormalities in spermatogenesis and by which microenvironment affects spermatogenesis, and provide candidates for the molecular diagnosis of oligozoospermia patients.

血睾屏障（blood-testis barrier，BTB）是在生精小管的支持细胞间由多种细胞连接（包括以CDH2为主要成分的粘附连接）形成的特殊结构，阻止血液和生精小管内分子的交流等，从而保证精子发生所需的特殊微环境。但这种微环境含何种成分，其改变如何影响精子发生及会不会反映在血清中，尚不清楚。本项目以睾丸支持细胞Cdh2特异敲除小鼠为模型，探讨Cdh2敲除导致精子发生异常的机制。在此基础上，发现在支持细胞Cdh2、Cx43和Claudin 11敲除鼠睾丸中丰度有类似改变的蛋白，通过功能鉴定，筛选其中影响精子发生的分子，以期发现精子发生微环境组成分子；选择只在睾丸（正常小鼠）中表达、但在敲除鼠睾丸中丰度显著降低的蛋白，测定血清含量，以期发现血睾屏障和精子发生微环境的血清标记物。为揭示精子发生微环境对精子发生的作用及机制、阐明精子发生异常的发病机理、实现相关不育症的分子诊断提供候选分析。

血睾屏障（blood-testis barrier，BTB）是在生精小管的支持细胞间由多种细胞连接形成的特殊结构，其保证了精子发生所需的特殊微环境。为了研究血睾屏障的功能，鉴别血睾屏障微环境组分，我们以条件性敲除小鼠为模型，综合利用细胞遗传学、分子生物学等方法开展了系统的研究。. 在研究过程中，我们发现CDH2蛋白的表达和定位随小鼠睾丸的发育过程发生了明显变化；支持细胞中Cdh2敲除会导致小鼠生殖力降低，睾丸血睾屏障功能受损，生殖细胞从精原细胞中期到减数分裂II中期的进程异常，并且减数分裂阶段细胞难以继续在曲细精管中完成精子发生过程，会提前脱落至附睾。我们还利用多种血睾屏障受损模型，研究了支持细胞Cdh2、Cx43单独敲除和Cdh2与Cx43双敲除后，睾丸中支持细胞和生殖细胞发育情况等，发现小鼠睾丸支持细胞Cdh2和Cx43双敲除小鼠精子发生异常的表型较Cx43或Cdh2单独敲除不完全一致，表明血睾屏障不同组分（如缝隙连接和锚定连接）是以独立的方式调节精子发生，并且这种调节可能与支持细胞中β-catenin的表达变化有关。此外，我们还发现支持细胞中的Rac1参与了血睾屏障对精子发生的调节，其在血睾屏障受损后（Cdh2敲除）小鼠睾丸中表达量下降，进一步研究发现支持细胞中Rac1敲除小鼠精子发生也存在异常，并且Rac1对精子发生调节可能是通过微环境分子Nociceptin而介导的。. 在项目资助下，我们发表SCI论文15篇；通过项目的实施，培养了博士后3人，博士生5人和硕士生1人。项目所取得的相关研究成果为阐明少精子症、无精子症等人类精子发生异常的发病机理提供了新的理论基础，并有望为诊断、治疗人类不育提供了新的思路。