支持细胞因子调控精原干细胞更新与分化和精母细胞减数分裂机制的研究

Our previous studies have demonstrated that local temporal "heating" testis could induce a revisable functional differentiation change in Sertoli cells and followed by a revisable failure of spermatogenesis at all stages via Sertoli cell transcription factors which seem to be a particularly important component of spermatogonial stem cells (SSCs) niche and blood-testis barrier (BTB). Sertoli cells are the only somatic cells in the seminiferous tubule and closely interact with SSCs and primary/secondary spermatocytes. On the basis of our previous NSFC project, we have established an efficient in vitro co-culture system of mouse "SSCs/testicular somatic cells" isolated from 2.5-day-old mice to produce haploid sperms and obtain health offsprings following micro-insemination. Utill now, the molecular mechanism of Sertoli cell transcription factor function in the "SSC self-renewal/ differentiation" and "meiosis" is not clear. In the present project, Gata4-knockout mice (Gata4flox/flox, AMH-Cre) and Ar-mutant mice (Ar-/Y) will be employed to explore the molecular mechanisms of the transcription factor GATA4 and AR, as well as their signaling transduction in the early spermatogenesis in vivo. On the basis of the observed phenotypes, furthermore, we could develop an in vitro system based on the established mouse SSCs/testis somatic coculture by adding the deficient paracrine factor(s) or activate/suppress the paracrine signaling pathways to rescue spermatogenesis. Our findings would be not only expand the known paracrine role of Sertoli cells in regulation of the early spermatogenesis, but might be also to provide a method for obtaining their own children in the non-obstructive azoospermia populations.

在上一个主持的自然科学基金（已结题）中，我们揭示了"热"通过调控支持细胞可逆性去分化影响精子发生的各个环节，并发现许多支持细胞转录因子在精原干细胞（SSCs）微环境中发挥重要作用。在此基础上，我们建立了小鼠"SSCs/睾丸体细胞"体外培养模型，能高效获得功能性单倍体精细胞。支持细胞是曲细精管内与精细胞接触的唯一体细胞，探究其表达的转录因子在SSCs自我更新与分化和减数分裂中的功能及作用机制是目前研究的热点。本课题拟利用支持细胞Gata4特异性敲除小鼠和Ar缺失突变小鼠"在体"研究支持细胞转录因子GATA4和AR及其介导的旁分泌信号途径对SSCs和减数分裂的作用；在表型及机制研究基础上，拟在敲除/突变小鼠"SSCs/睾丸体细胞"培养体系中添加缺乏的生长因子或激活/抑制异常的信号通路，以达到"挽救"敲除/突变小鼠精子发生缺陷的目的。本课题将揭示支持细胞对早期精子发生的转录与旁分泌调控机制。

减数分裂是在由Sertoli细胞间形成的BTB紧密联接中完成的。Sertoli细胞作为曲细精管内与精母细胞接触的唯一体细胞，发挥结构支持、旁分泌调控精母细胞减数分裂的关键功能。前期相关研究中，我们发掘了许多调控精子发生的Sertoli细胞转录因子,其中AR、GATA4和WT-1是最为重要的转录因子。探究它们对SSCs自我更新与分化和精母细胞减数分裂的影响及其调控早期精子发生的旁分泌信号通路是本课题提出的最主要的科学问题。