附睾小体内Hnrnph1在精子成熟中的作用及机制研究

Accumulating evidence indicates epididymosomes carry RNAs and protein and can deliver it to immature sperm, which play essential roles in sperm maturation. However, the molecular mechanism is still unclear. In our previous study, using microarray analysis to detect and quantify differences in mRNA expression between epididymosomes from caput epididymis and cauda epididymis, we identified Hnrnph1 is high expressed in caput epididymosomes. We further provided evidences showing that epididymosomes contain both Hnrnph1 mRNA and protein. Furthermore, we found that epididymosomal Hnrnph1 can deliver to sperm and had a potential to be translated into proteins. Our data also showed that Hnrnph1 interacts with Hnrnpa2b1 (a known protein plays key role in sorting of RNA into exosomes). So we build hypothesis that epididymosomal Hnrnph1 contribute to sperm maturation. In this project, we plan to systematically study the molecular mechanism of the interaction between Hnrnph1 and Hnrnpa2b1 in sperm maturation, enrich the molecular biology theory, and which will benefit the diagnosis and treatment of male infertility.

附睾小体传递的RNA和蛋白在精子成熟中有重要作用，然而其分子机制还知之甚少。我们前期通过基因芯片筛选了附睾头部来源和尾部来源的附睾小体中差异表达的基因，鉴定了Hnrnph1在头部附睾小体中高表达。通过定量PCR和western blot进一步证实了在附睾小体内含有Hnrnph1的mRNA及蛋白质。附睾小体内Hnrnph1的mRNA和蛋白可被递入到精子中，且mRNA可被翻译出蛋白。此外，我们还发现Hnrnph1蛋白能与Hnrnpa2b1蛋白（在选择性包裹RNA进入外泌体发挥重要功能）存在相互作用。因此，我们提出假说： Hnrnpa2b1可通过调控Hnrnph1分选进入附睾小体从而参与精子成熟的调控过程。本项目拟明确Hnrnph1/Hnrnpa2b1相互作用在精子成熟中的分子机制，丰富相关分子生物学理论，为并为男性不育的诊断、治疗及避孕药物的开发提供新的理论依据和靶点。

附睾小体传递的RNA、蛋白在精子成熟中起着重要作用，然而其具体的分子机制还知之甚少。我们前期研究表明Hnrnph1在头部附睾小体中高表达，且Hnrnph1和Hnrnpa2b1存在相互作用。本课题在附睾上皮细胞系（DC2细胞）中证实Hnrnph1可与Hnrnpa2b1相互作用，调控mRNA、miRNA、lncRNA等分选进入附睾小体。在该调控作用下，Hnrnph1可被附睾上皮细胞特异性分选并富集于头部的附睾小体中。下调DC2细胞中Hnrnph1，精子前向运动、非前向运动、总活力、超活化运动显著下降。测序结果表明下调Hnrnph1后，附睾小体中CXCL1、IL6、SOCS3、NTF3、FGF9等调节因子的表达量显著改变。过表达DC2细胞中FGF9后，精子前向运动、非前向运动、总活力显著下降。上述研究结果表明Hnrnph1与Hnrnpa2b1相互作用，调控Hnrnph1分选进入附睾小体，进而调节FGF9的表达，影响精子活力。本课题初步阐明了Hnrnph1对精子活力的调控作用，丰富了精子成熟的分子机制，同时也为男性不育的诊断、治疗及避孕药物的开发提供新的理论依据和靶点。