REGULATION OF TESTICULAR GONOCYTE DEVELOPMENT

本申请的目的是确定调节新生儿睾丸生殖细胞增殖的机制。这一过程的异常可能危及整个精子发生过程。我们已经表明，新生儿生殖细胞特异性表达蛋白C ζ（ZetaPKC）和磷脂酰肌醇3-激酶（PI 3-激酶），这可能在PDGF激活的细胞增殖中发挥作用。因此，我们研究了PDGF对3日龄大鼠分离的生殖细胞的影响。由于新生儿支持细胞分泌17 β-雌二醇，我们也测试了它的作用。发现这两种药物均刺激生殖细胞增殖。这些结果构成了我们假设的基础，即PDGF和雌二醇激活的信号转导通路之间的串扰驱动生殖细胞有丝分裂，发育和分化为精原细胞。我们的第一个目标是确定生殖细胞中PDGF转导途径的组成部分。zetaPKC和PI 3-激酶在这一途径中的作用将通过研究每种酶的表达、激酶活性和底物来进行研究。还将继续确定级联反应的两个关键要素，MEK和MAP激酶。这两个实验应该揭示了生殖细胞中PDGF通路的重要组成部分，并确认zetaPKC和PI 3-激酶是否是该级联反应的一部分。第二个目的是确定在生殖细胞中PDGF和雌二醇通路之间是否存在串扰。这将通过研究1）雌激素拮抗剂对PDGF依赖性细胞增殖、激酶活性和蛋白磷酸化模式的影响和2）PDGF和PDGF激活的激酶抑制剂对雌二醇依赖性增殖的影响来检查。第三个目的是检查围产期暴露于外源性雌激素是否对生殖细胞功能有不利影响。用雌激素处理孕鼠，检测其子代生殖细胞的功能，包括对PDGF和雌二醇的反应以及凋亡细胞的百分比。这些实验应该揭示，如果雌激素在体内的生殖细胞成熟中发挥作用，并可能提供关于异种雌激素对男性生殖功能的影响的澄清。

The objectives of this application are to determine the mechanisms regulating neonatal testicular gonocyte proliferation. Anomalies of this process could jeopardize the entire spermatogenic process. We have shown that neonatal gonocytes specifically expressed the protein C zeta (ZetaPKC), and the phosphatidylinositol 3-kinase (PI 3-kinase), which may play a role in PDGF-activated cell proliferation. Thus, we examined the effect of PDGF on isolated gonocytes from 3-day-old rats. Since neonatal Sertoli cells secrete 17beta-estradiol, we also tested its effect. Both agents were found to stimulate gonocyte proliferation. These results constitute the basis of our hypothesis that the cross talk between the PDGF- and estradiol-activated signal transduction pathways drives gonocyte mitosis, development and differentiation into spermatogonia. Our first aim is to determine the components of the PDGF transduction pathway in gonocytes. The role of zetaPKC and PI 3-kinase in this pathway will be examined, by studying the expression, kinase activity, and substrates of each enzyme. The identification of two key elements of the cascade, the MEK and MAP-kinase, will also be pursued. These two experiments should reveal important components of PDGF pathway in gonocytes and confirm if zetaPKC and PI 3-kinase are part of this cascade. The second aim is to determine if there is a cross talk between PDGF and estradiol pathways in gonocytes. This will be examined by studying 1) the effects of estrogen antagonists of PDGF-dependent cell proliferation, kinase activities and protein phosphorylation patterns and 2) the effects of PDGF and PDGF-activated kinase inhibitors on estradiol-dependent proliferation. The third aim is to examine if perinatal exposure to xenoestrogens has detrimental effects on gonocyte functions. Pregnant rats will be treated with estrogens, and their functions of the gonocytes derived from their offspring will be examined, including the responses to PDGF and estradiol and percent of apoptotic cells. These experiments should reveal if estrogens play role in vivo in gonocyte maturation, and may provide clarifications regarding the effects of xenoestrogens on male reproductive functions.