Prp3在果蝇睾丸生殖干细胞自我更新和分化过程中的作用及机制研究

The homeostasis of self-renewal and differentiation in germline stem cells (GSCs) plays an important role in non-obstructive azoospermia (NOA) and testicular germ cell tumor (TGCT). Prp3, as an U4/U6 related small nuclear ribonucleoprotein particle (snRNP), is a key component of the precursor mRNA splicing, but its biological function is still unknown. This project intends to investigate the function and mechanism of Prp3 in the Drosophila testis. We have demonstrated previously that Prp3 deficiency reduced the male fertility in Drosophila. Knockdown of Prp3 in early germ cells resulted in reduced testicular size and loss of germ cells, while knockdown of Prp3 in spermatogonia and cyst cell blocked germ cell differentiation and finally led to tumor formation. Based on these results, we propose a hypothesis that Prp3 may regulate the self-renewal and differentiation of GSCs in Drosophila testes. In the present study, we intend to understand the function and mechanism of Prp3 in the GSC self-renewal and differentiation process of the Drosophila testis by in vivo and in vitro methods. This finding will reveal the significant role of pre-mRNA splicing factors and provide new insights for understanding the pathogenic mechanism of human NOA and TGCT.

生殖干细胞自我更新和分化过程的稳态平衡在非梗阻性无精子症和睾丸生殖细胞肿瘤中发挥了重要作用。Prp3作为U4/U6相关的snRNP，是前体mRNA剪接体的关键元件，但其生物学功能仍然未知。本项目拟运用模式动物果蝇来研究Prp3在睾丸中的功能及机制。申请者在前期研究中发现，Prp3缺陷可降低雄性果蝇的生育能力。在早期生殖细胞中敲减Prp3可导致睾丸体积变小和生殖细胞缺失的表型，而在精原细胞和包囊细胞中敲减Prp3可出现生殖细胞分化阻滞，并最终导致肿瘤的形成。基于此提出假说：Prp3可能调控果蝇睾丸生殖干细胞的自我更新和分化作用。本项目拟在前期的研究基础上，从分子、细胞和整体水平上理解Prp3在果蝇睾丸生殖干细胞自我更新和分化过程中的作用及机制，为理解前体mRNA剪接因子在人非梗阻性无精子症和睾丸生殖细胞肿瘤中的致病机理提供新视野。

生殖干细胞的自我更新和分化过程受到生殖干细胞微环境的严格调控，然而其分子机理尚未完全阐明。前期，本课题组在果蝇睾丸中解析了生殖干细胞的调控网络，并鉴定到了Prp3。在国家自然科学基金的支持下，我们以Prp3为切入点，深入理解Prp3及其相关因子在果蝇睾丸生殖干细胞自我更新和分化过程中的作用。研究发现，Prp3通过生殖干细胞来影响其自我更新，也通过分化因子Bam在精原细胞中调控分化过程。有趣的是，Prp3还可以利用包囊细胞通过非自主性细胞效应来促进生殖细胞的分化过程。本项目进一步对前体mRNA剪接相关因子进行了拓展性研究，阐明了SmD3调控生殖干细胞自我更新和分化过程的作用机理。本项目还利用体外实验证实了Prp3及其相关因子介导细胞的增殖和凋亡过程。本项目初步探索了前体mRNA剪接体在果蝇睾丸生殖干细胞微环境中的调控机理，通过解析前体mRNA剪接体相关亚基的结合蛋白调控网络，我们发现核糖体也参与调控果蝇睾丸的生殖干细胞微环境。本项目研究为人非梗阻性无精子症和睾丸生殖细胞肿瘤的临床诊治提供了潜在的分子靶点，为深入理解其致病机理提供了理论基础。