ROLE OF PREGNANCY-SPECIFIC B1 GLYCOPROTEIN IN THE TESTIS

这项提案的长期目标是划定细胞 影响睾丸发育和精子发生的因素。这个 近期的目标是用大鼠作为动物模型来研究 妊娠特异的β1-糖蛋白(PSG)在胚胎发育中的生物学作用 睾丸。人体胎盘中存在多种PSG。近期 研究表明，PSG在睾丸中的含量也很高。 从人胎盘中提取的PSG已被证明具有免疫抑制作用 和促进成长活动。睾丸中PSG的存在提高了 关于它在睾丸新陈代谢中的功能作用的问题。要解决这个问题 问题，第一步是确定PSG在大鼠睾丸中的存在。 这将通过分子克隆大鼠睾丸PSG基因来实现。 PSG cDNA的可获得性将允许产生基因特异性的 用于进一步研究的寡核苷酸探针以及制备 通过体外表达纯化蛋白所克隆的cDNAs。分子 通过筛选大鼠睾丸基因实现PSG基因的克隆 以人PSG基因为探针构建文库。克隆的核糖核酸结构 CDNA将通过桑格的双脱氧链终止法来确定。 这些cDNA编码的蛋白质将通过使用哺乳动物细胞在 体外表达系统。纯化的重组蛋白将用于 制备单抗，为后续研究做准备。收购这两家公司 CDNA和抗体将使PSG的精确细胞定位成为可能 在老鼠的睾丸里。CDNA将用于原位杂交研究，而 抗体将用于免疫组织化学研究，以确定细胞 PSG mRNA和蛋白存在的类型。PSG在中国的存在 处于不同发育阶段的精子细胞也将得到进一步研究。 如果大鼠睾丸中存在多种PSG，则 不同的PSG将用基因特异性探针进行检测。的表达 测定大鼠睾丸发育不同阶段的PSG 在信使核糖核酸和蛋白质水平上的不同年龄。初始泛函 PSG的分析将包括鉴定细胞结合靶标和 三七总皂苷对小鼠生殖细胞及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和 Scatchard分析。三七总皂苷对其生长和形态的影响 将对细胞进行研究。拟议中的项目将揭示 三七总皂甙在睾丸发育和发育中的生理作用 大鼠精子发生，应为进一步研究提供一个模型系统 对人体内这些蛋白质的研究。

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.