精子成熟新机制- - 附睾小体递入mRNAs及蛋白翻译的研究

Sperm obtain many proteins during maturation in epididymis. To identify these proteins and elucidate the underlying mechanisms is not only important for sperm maturation, but also is a rational basis for the research of therapies for male infertility, and for the development of future contraception.We found that most cell-free mRNA in human semen is contained inside seminal microvesicles, and most cell-free mRNA in mouse epididymis is contained inside the epididymosome. In somatic cells, microvesicles has been found to deliver mRNAs, which can then be translatedinto proteins. In our experiments,human seminal microvesicles or mouse epididymosome can also deliver mRNAs into sperm. And the ability of protein translation in mammal spermwas confirmed recently by at least two groups and our lab. Our overall working hypothesis for the present proposal is that proteins translated from mRNAs delivered by epididymosome is a new mechanism for sperm maturation. That is, during sperm transit in epididymis, epididymosome can deliver mRNAs into sperm and then these mRNAs can be translated into proteins. In that way, sperm obtain many epididymal proteins. These proteins contribute to sperm maturation and sperm functions. In this project, we will obtain the specific mRNAs profile in epididymosome of mouse cauda epididymis by microarray, and observe the delivering of some specific mRNAs into sperm and the subsequent protein translation, as well as the subcellular localization of these proteins. By using a specific inhibitor for sperm protein translation, we will study the protein synthesis of sperm in cauda epididymis in vivo by a 2D-PAGE, and identify some proteins subsequently by a mass spectrometer, as well as the effects of the translation inhibition on sperm functions. Our overall goal of this project is to reveal a new mechanism for the change of protein composition and sperm maturation in epididymis. These specific mRNAs in epididymosome of cauda epididymis, and proteins synthesized in sperm of cauda epididymis will be new targets for the research of sperm maturation and sperm functions.

附睾是精子成熟的主要场所，探索精子成熟过程中基因与蛋白分子调控的新机制具有很好的理论和应用价值。我们前期研究发现人精浆游离mRNA主要存在于精浆的小体中，小鼠附睾液的游离mRNA也主要存在于附睾小体中。已知小体可在体细胞间传递mRNA并翻译蛋白，我们的实验已证明附睾小体可以与精子融合并递入mRNAs；而精子有合成蛋白的能力。因此，我们提出假说：精子在附睾移行过程中，附睾小体与之融合并递入mRNAs，被精子用于合成蛋白，这些蛋白在精子成熟和精子功能方面发挥重要作用。本课题将筛出小鼠附睾尾部小体特异性mRNAs表达谱，研究特异性mRNAs进入小鼠附睾精子中后蛋白翻译情况及部位；进一步研究附睾尾部精子蛋白合成的图谱和对精子功能的影响。目的是探索一种精子成熟和获得附睾蛋白的新分子机制，附睾尾部小体特异性mRNAs及精子蛋白合成图谱也可为研究精子成熟与功能的提供新靶点。

精子功能成熟主要发生在附睾，精子在附睾移行过程中，获得附睾表达的一些蛋白，研究获得这些蛋白的分子机制和种类，是精子成熟的重要内容，也是男性不育诊疗和研发男性避孕途径的理论基础。本课题首先验证了精子仍具有合成蛋白的能力，发现附睾小体含有丰富的mRNAs且能被用于翻译蛋白；进一步鉴定了附睾小体被细胞摄取后附睾小体内mRNAs被用于翻译蛋白的种类，也表明附睾小体可以被精子摄取并利用其mRNAs翻译蛋白，这可能是附睾精子成熟获得蛋白的一种新分子机制；通过体内外实验，鉴定了小鼠附睾尾部精子蛋白合成图谱（56种蛋白），以及抑制其蛋白合成后对精子功能的影响，这些蛋白为进一步研究精子成熟与功能提供了新靶点。课题还进行了紧密相关内容的细化和拓展，主要包括小鼠附睾小体亚型的分析、基于存在形式（包括小体）的RNA提取、精液中小体传递物质对于精子功能的可能影响等。