ROLE OF PREGNANCY-SPECIFIC B1 GLYCOPROTEIN IN THE TESTIS

该提案的长期目标是描绘蜂窝 影响睾丸发育和精子发生的因素。 的 目前的目标是使用大鼠作为动物模型， 妊娠特异性β 1-糖蛋白（PSG）在妊娠中的生物学作用 睾丸 人胎盘中存在多种PSG。 最近 研究表明，PSG也以高丰度存在于睾丸中。 已显示来自人胎盘的PSG具有免疫抑制作用， 促进增长的活动。 睾丸中PSG的存在提高了 关于其在睾丸代谢中的功能作用的问题。 为了解决这个 问题，第一步是建立PSG在大鼠睾丸中的存在。 这将通过大鼠睾丸PSG cDNA的分子克隆来实现。 PSG cDNA的可用性将允许产生基因特异性 用于进一步研究的寡核苷酸探针以及制备 通过克隆cDNA的体外表达纯化蛋白质。 分子 通过筛选大鼠睾丸cDNA，克隆PSG cDNA 以人PSG cDNA为探针，构建了人PSG cDNA文库。 克隆的核苷酸结构 cDNA将通过桑格双脱氧链终止法测定。 由这些cDNA编码的蛋白质将通过使用哺乳动物细胞来生产， 体外表达系统 纯化的重组蛋白将用于 为后续研究提供单克隆抗体。 收购双方 cDNA和抗体将允许PSG的精确细胞定位 在大鼠睾丸中。 cDNA将用于原位杂交研究， 抗体将用于免疫组织化学研究，以确定细胞 其中PSG mRNA和蛋白质存在的类型。 PSG的存在， 不同发育阶段的精子细胞亦会作进一步研究。 如果大鼠睾丸中存在多种PSG， 将用基因特异性探针检查不同的PSG。 表达 测定大鼠睾丸发育不同阶段的PSG 在mRNA和蛋白质水平上存在不同年龄的差异。 初始功能 PSG的分析将包括细胞结合靶点的鉴定， 检查PSG对生殖细胞和其他睾丸细胞的影响， 文化 细胞结合实验将用标记的蛋白质进行， Scatchard分析 PSG对这些细胞生长和形态的影响 细胞将被研究。 拟议的项目将阐明 PSG在睾丸发育中的生理功能， 大鼠精子发生，并应提供一个模型系统， 这些蛋白质在人体内的研究。

The long term objective of this proposal is the delineation of cellular factors affecting testicular development and spermatogenesis. The immediate goal is to use the rat as an animal model for examining the biological role of the pregnancy-specific beta1-glycoprotein (PSG) in the testis. Multiple species of PSG are present in human placenta. Recent studies showed that PSG is also present in high abundance in the testis. PSG derived from human placenta has been shown to possess immunosuppressive and growth promoting activities. the presence of PSG in testis raises the question about its functional role in testis metabolism. To address this question, the first step is to establish the presence of PSG in rat testis. This will be achieved by molecular cloning of rat testicular PSG cDNA. Availability of PSG cDNAs will allow generation of gene-specific oligonucleotide probes for further investigation as well as preparation of pure proteins by in vitro expression of the cloned cDNAs. Molecular cloning of PSG cDNA will be achieved by screening of rat testicular cDNA library using human PSG cDNA as probe. Nucleotide structure of cloned cDNAs will be determined by Sanger's dideoxy chain termination methods. Proteins encoded by these cDNAs will be produced by using mammalian cell in vitro expression systems. Purified recombinant proteins will be used to raise monoclonal antibodies for subsequent studies. Acquisition of both cDNAs and antibodies will permit the precise cellular localization of PSG in rat testis. cDNAs will be used for in situ hybridization studies while antibodies will be used for immunohistochemical studies to define the cell types in which PSG mRNA and protein are present. The presence of PSG in spermatids at different stages of development will also be further studied. If multiple species of PSG are present in rat testis, the occurrence of the different PSG will be examined with gene-specific probes. Expression of PSG at different stage of testicular development will be determined in rats of different ages at both the mRNA and protein levels. Initial functional analysis of PSG will include identification of cellular binding target and examination of effects of PSG on germ cells and other testicular cells in culture. Cell binding experiments will be done with labeled proteins and Scatchard analysis. Effects of PSG on growth and morphology of these cells will be studied. The proposed project will shed light on the physiological functions of the PSG in testicular development and spermatogenesis in the rat and should provide a model system for further studies of these proteins in the human.