基于转录组学和脂质组学研究α-亚麻酸影响绵羊睾丸间质细胞睾酮合成的机制

Alpha-linolenic acid (ALA) is a kind of omega-3 polyunsaturated fatty acids (ω-3 PUFAs). Our preliminary results illustrated that ALA contained in diet affected fatty acids composition and cholesterol accumulation, stimulated PUFAs metabolism and steroidogenesis related genes (FADS1, ELOVL2, StAR and P450scc) expression in testis tissues, and increased plasma estradiol and testosterone concentration in Hu sheep. Additionally, ALA also stimulated mice Leydig cells proliferation and StAR gene expression in vitro. Built on the preliminary data, we hypothesize that ALA may regulate testosterone biosynthesis via PUFAs metabolism pathways. The project will firstly explore how ALA regulates cell proliferation and testosterone biosynthesis in Leydig cells using techniques of ELISA, Western blot, TUNEL, flow cytometry, etc. To understand this regulation mechanism, RNA-seq and LC-MS/MS techniques will be used to comprehensive analyze the transcriptomics and lipidomics of Leydig cells treated with ALA or not. Finally, the hypothesis mechanism will be testified from different aspects with techniques of RNA interference and over-expression. In conclusion, our finding will provide new theoretical evidence for improving sheep fertility by nutrition regulation.

α-亚麻酸（ALA）是重要的ω-3多不饱和脂肪酸（ω-3 PUFAs），项目组在前期的研究中发现，富含ALA的日粮能够改变湖羊睾丸组织中脂肪酸组成，增加胆固醇积累，促进PUFAs代谢和睾酮合成关键基因(FADS1、ELOVL2、StAR和P450scc)的表达以及类固醇激素生成。在体外，ALA也可促进小鼠间质细胞增殖和StAR基因表达。因此，推测ALA可能通过调节PUFAs代谢途径调控间质细胞睾酮合成。本项目在利用ELISA、Western blot、TUNEL及流式细胞技术研究ALA调节绵羊睾丸间质细胞增殖及睾酮合成基础上，进一步利用RNA-seq和LC-MS/MS检测平台，结合转录组学和脂质组学等方法从PUFAs代谢通路角度解析其可能的作用机制，并利用RNA干扰或过表达技术对可能机制加以验证。所得结果将为通过营养调控来提高绵羊繁殖力提供理论依据。

Alpha-亚麻酸（α-linolenci acid, ALA）在动物繁殖活动中具有重要的生理作用，ALA及其代谢产物可能影响睾酮的合成分泌。间质细胞主要利用脂类物质合成、分泌睾酮，促进睾丸发育和精子发生。因此，本项目以公羊睾丸间质细胞为研究对象，联合分子生物学技术、转录组学和代谢组学方法，对ALA调控间质细胞增殖和睾酮分泌分子机制进行解析，结果显示添加α亚麻酸后，细胞内ALA、EPA、AA、ETA等ω-3、ω-6 PUFAs代谢物升高，刺激Phospholipase A2相关基因表达，细胞内磷脂代谢活动加强，PGG2和Glycerol生成增加，同时引起Unspecific monooxygenase酶相关基因CYP2C/D/E等以及11-beta-hydroxysteroid dehydrogenase酶相关基因HSD11B表达，引起Testosterone合成增加，而利用RNA干扰技术沉默PLA2基因表达则抑制ALA对睾酮合成的刺激作用。在此基础之上，利用亚麻籽作为ALA的来源，发现初情期前公羊日粮中添加富含ALA的亚麻籽（油）可刺激睾丸发育、睾酮合成、PUFAs代谢以及精子发生。本课题的顺利实施，为睾酮的合成机理补充新的理论依据，也为生产上指导早期培育优秀种公羊提供了指导方法。