Gene Expression In Spermatogenic Cells

这些研究将确定和描述内在遗传程序的关键组成部分，该程序控制男性生殖细胞的发育和功能，并最终确定导致男性生育的条件。目前正在应用的方法是鉴定在雄性生殖细胞中特异表达的基因，使用基因敲除方法来确定它们编码的蛋白质的作用，使用酵母双杂交和缺失突变来鉴定对雄配子发育至关重要的蛋白质-蛋白质相互作用，以及制备抗血清来确定特定基因产物的时空分布。许多基因只在雄性生殖细胞中表达，人们正在研究一些编码蛋白质的基因，这些蛋白质的功能对于配子发育和/或功能的新方面是必不可少的。(1)雄性生殖细胞减数分裂的开始和进展：维甲酸调节进入减数分裂，但进入的时间在雄性和雌性生殖细胞之间不同。我们已经鉴定了一个新的同源异型盒转录因子RHOX13，并假设维甲酸信号涉及其基因的激活。Rhox13基因从胚胎E13.5天开始在雄性和雌性生殖细胞中转录。RHOX13的翻译发生在E13.5卵巢，与雌性生殖细胞进入减数分裂相一致。RHOX13的翻译在出生后第4天开始，并使雄性生殖细胞进入减数分裂。我们假设RNA结合蛋白NANOS2与Rhox13转录本的3‘UTR结合，以抑制其翻译。目前的研究正在检验这一假设。(2)精子发生过程中减数分裂进程的调控：精子发生过程中从减数分裂前期I到中期I的调控机制尚不清楚。以前的研究表明，CDK1和Cyclin B1以外的蛋白质可能在精子发生过程中调节这一过程。条件性基因打靶方法正被用来确定这些蛋白质是否对雄性生殖细胞减数分裂的进程是必不可少的。(3)新型支架蛋白和糖酵解酶在精子运动中的作用：鞭毛是最复杂的细胞器之一，它的许多蛋白质是由减数分裂后单倍体精子发生阶段表达的基因编码的。我们使用了各种方法来鉴定纤维鞘的成分，这是精子鞭毛的关键细胞骨架成分。这些包括结构蛋白、信号转导锚定蛋白和糖酵解酶，其中大多数是由只在雄性生殖细胞中表达的基因编码的。这导致我们假设哺乳动物的精子需要糖酵解产生的ATP来运动，而纤维鞘是糖酵解酶的重要支架。这一假说已经通过基因打靶得到证实，目前的研究正在确定不同的糖酵解酶和纤维鞘的结构成分是如何组装的，以及它们的功能是如何调节的。

These studies will identify and characterize key components of the intrinsic genetic program that controls development and function of male germ cells and that ultimately define the conditions responsible for male fertility. The approaches currently being applied are to identify genes expressed specifically in male germ cells, use the gene knockout approach to define the roles of the proteins they encode, employ yeast two-hybrid assays and deletion mutagenesis to identify protein-protein interactions essential for development of the male gamete, and prepare antisera to determine the temporal-spatial distribution of specific gene products. Many genes are expressed only in male germ cells and selected genes are being studied that encode proteins whose functions are essential for novel aspects of gamete development and/or function. (1) Initiation and progression of meiosis in male germ cells: Retinoic acid regulates entry into meiosis, but the timing of entry differs between male and female germ cells. We have identified a novel homeobox transcription factor RHOX13 and hypothesize that retinoic acid signaling involves activation of its gene. The Rhox13 gene is transcribed in male and female germ cells beginning on embryonic day E13.5. Translation of RHOX13 occurs in the E13.5 ovary and coincides with the entry of female germ cells into meiosis. Translation of RHOX13 begins on postnatal day P4 and proceeds male germ cells entry into meiosis. We hypothesize that the RNA-binding protein NANOS2 binds to the 3' UTR of the Rhox13 transcript to suppress its translation. Current studies are testing this hypothesis. (2) Regulation of meiotic progression in spermatogenesis: The mechanisms regulating transition from prophase I to metaphase I of meiosis during spermatogenesis are uncertain. Previous studies have suggested that proteins other than or in addition to CDK1 and Cyclin B1 may regulate this process during spermatogenesis. Conditional gene targeting approaches are being used to determine if these proteins are essential for the progression of meiosis in male germ cells. (3) The role of novel scaffold proteins and glycolytic enzymes in sperm motility: The flagellum is one of the most complex of all cell organelles and many of its proteins are encoded by genes expressed during post-meiotic haploid phase of spermatogenesis. We have used a variety of approaches to identify components of the fibrous sheath, a key cytoskeletal component of the sperm flagellum. These included structural proteins, signal transduction anchoring proteins, and glycolytic enzymes, most of which are encoded by genes only expressed in male germ cells. This led us to hypothesize thatmammalian sperm require ATP produced by glycolysis for motility and the fibrous sheath is an essential scaffold for the glycolytic enzymes. This hypothesis has been confirmed using gene targeting and current studies are determining how the different glycolytic enzymes and structural components of the fibrous sheath are assembled and how their functions are regulated.