维甲酸调控精原细胞分化的途径及其分子机制研究

Spermatogonial differentiation, the transition of A aligned (Aal) spermatogonia into A1 spermatogonia, is an initial step of mammalian spermatogenesis, which is irreversibly committed toward gamete production. Retinoic acid (RA), the active metabolite of vitamin A is essential for the spermatogonial differentiation; however, it remains unclear whether RA signaling controls spermatogonial differentiation directly through spermatogonia or Sertoli cells or both. The molecular mechanisms by which RA signaling controls the spermatogonial differentiation remain largely unaddressed.Our long-term goal is to identify the essential networks involved in spermatogonial differentiation. The goal of this proposal is to test the hypothesis that RA signaling via its target genes directly controls spermatogonial differentiation. Our hypothesis has been formulated on the basis of supportive preliminary data. In this application, we propose to determine whether and how RA signaling controls spermatogonial differentiation. Specifically, we first hypothesize that RA signaling directly drives spermatogonial differentiation and we propose to determine whether RA signaling cell-autonomously controls spermatogonial differentiation. We then hypothesize that RA signaling controls spermatogonial differentiation through its target genes andwe propose to determine the molecular mechanisms by which RA signaling controls the spermatogonial differentiation.Successful completion of the proposed research will help identify regulatory networks that control spermatogonial differentiation. A better understanding of spermatogonial differentiation has important implications for male reproduction and contraception.

精原细胞分化是精子发生的关键步骤，也是精子发生不可逆转的起始阶段。维甲酸信号系统已被证明为精原细胞分化所必需；然而，国内外对维甲酸信号系统能否直接通过精原细胞自身调控精原细胞分化，以及其调控精原细胞分化的分子机制仍知之甚少。申请人的长期目标是剖析并阐明调控精原细胞分化的分子机理。基于申请人的预实验结果，本项目假设维甲酸信号系统通过其靶基因直接调控精原细胞分化。为了验证此假设，本课题主要研究内容包括：（1）维甲酸信号系统是否通过精原细胞自身直接调控精原细胞分化？或者是通过支持细胞间接调控精原细胞分化？还是通过精原细胞和支持细胞共同调控精原细胞分化？（2）维甲酸信号系统调控精原细胞分化的分子机制是什么？完成该项目将为以精原细胞分化为靶标的男性不育症的诊治和抗生育方法的开发奠定技术和理论基础。

哺乳动物精子发生是一个高度复杂和特化的细胞发育过程。其中，精原细胞分化是精子发生的关键步骤，也是精子发生不可逆转的开始。维甲酸作为维生素A在体内的活性形式，在精原细胞分化过程中发挥着重要的调控作用。然而，维甲酸通过何种途径以及调控精原细胞分化的具体分子机制，学术界知之甚少。应用特异性失活生殖细胞或支持细胞内维甲酸信号系统的小鼠模型，我们揭示了维甲酸信号系统通过精原细胞直接调控精原细胞分化，其作用并不依赖于支持细胞；进一步证实了维甲酸主要通过调控精原细胞内Hist1 cluster以及其它细胞周期相关基因的表达，控制精原细胞G1/S期的转换，从而调控精原细胞分化。. 哺乳动物精子发生是一个连续而非同步化的过程。精子发生的非同步化使分离获取高纯度和均一的生精细胞变得极其困难；同时，缺少高效率和成熟的哺乳动物精子发生体外研究模型，这些均阻碍了哺乳动物精子发生调控机制的研究。基于上述维甲酸调控精原细胞分化的分子机理，结合精子发生同步化方法与生殖细胞特异性荧光标记小鼠模型，我们成功地建立了能够获取高纯度、处于任意发育阶段生精细胞的实验体系。应用单细胞转录组测序的方法，绘制了小鼠精子发生过程的转录组精细图谱。. RNA代谢在精子发生中高度复杂，而其中的调控机制尚未完全阐明。m6A RNA修饰是一种广泛而保守的表观遗传修饰，参与多种RNA的代谢调控。已有的体外研究表明m6A RNA修饰参与mRNA的降解、储存、可变剪接等多种转录后调控以及翻译调控。然而，对m6A RNA修饰是否以及如何在生理条件下调控哺乳动物器官发生和发育包括精子发生所知甚少。应用m6A RNA甲基转移酶Mettl3和／或Mettl14的条件性基因敲除小鼠模型，绘制了小鼠不同发育阶段生精细胞的m6A RNA修饰图谱，揭示了m6A RNA修饰通过调控精子发生过程中关键基因的转录后翻译，从而控制精子发生的分子机制。. 为最终阐明哺乳动物精子发生包括精原细胞分化、减数分裂启动和发展以及精子形成等关键生物学事件的分子机制立下了坚实的基础。