ESTROGEN RECEPTORS IN MALE REPRODUCTION

雌激素受体α基因突变纯合的雄性小鼠（ERKO小鼠）是不育的，而雌激素受体β基因突变纯合的雄性小鼠是可育的。该项目的目标是确定雌激素在雄性生殖细胞发育中的作用，雄性ERKO小鼠不育的原因，以及睾丸和附睾中雌激素调节基因的身份。此外，据报道孕酮与人精子上的膜受体结合，导致信号转导途径的快速激活，而不会对基因转录产生影响。据推测，膜相关孕酮受体（MAPR）调节男性生殖过程。目的是确定MAPR mRNA和蛋白质在男性生殖系统中的位置和时间，并确定MAPR在精子发生，精子成熟和受精过程中的功能。目前尚不清楚雄性ERKO小鼠是否由于生精细胞内、相关体细胞内或两者内雌激素调节过程的破坏而不育。我们假设雌激素受体不直接调节生殖细胞的发育，但需要在睾丸和/或附睾的体细胞。我们通过将来自ERKO+/-或ERKO-/-供体小鼠睾丸的生精细胞移植到已用白消安处理以消除大多数内源性生精细胞的同基因野生型小鼠的睾丸中来解决这一问题。在允许精子发生再生后，受体与野生型雌性交配。ERKO供体小鼠是C57 Bl/6 J（黑色），雄性受体是白化病C57 BL/6 J，野生型雌性是白化病CD-1。这允许使用毛色标记来识别由移植的生殖细胞（黑色）和再生的内源性生精细胞（白色）产生的后代。最近，接受ERKO-/-供体生殖细胞的受体繁殖了两窝小鼠，一窝有两只黑色幼崽和两只白色幼崽，另一窝有10只黑色幼崽。这些结果支持了雄性生殖细胞发育不直接需要雌激素受体的假设，并表明ERKO雄性不育是由于睾丸或附睾体细胞的功能缺陷。其他研究试图确定睾丸和附睾体细胞中受雌激素调节并导致ERKO男性不育的基因。尝试使用差异显示RT-PCR用于此目的没有得到有用的结果。cDNA抑制消减杂交方法，然后被用来产生一个cDNA文库，代表基因差异表达的ERKO和野生型小鼠的睾丸。差异表达克隆目前正在进行表征，金属蛋白酶组织抑制剂-2（TIMP-2）是进一步研究的早期候选者。当它们变得可用时，我们希望使用小鼠基因微阵列来鉴定相对于野生型小鼠，ERKO小鼠睾丸和附睾中直接或间接诱导或下调的基因。我们将确定ERKO和野生型小鼠基因表达特征的差异是否能识别导致ERKO雄性不育的功能变化。其他研究者假设膜相关孕酮受体（MAPR）调节男性生殖过程。确定了从猪肝膜纯化的胆甾酮结合复合物中存在的丰富蛋白质的肽序列。利用这些序列设计PCR引物，制备探针，筛选猪血管平滑肌cDNA文库。分离出与25-Dx cDNA具有显著序列同源性（76%）的cDNA。该cDNA是从NIEHS的道格拉斯贝尔博士实验室的大鼠肝脏文库中分离的，并被认为编码二恶英诱导蛋白。我们使用大鼠25-Dx cDNA克隆小鼠同源物，并进行了北方印迹，Western印迹和免疫组织化学研究，重点是雄性小鼠生殖系统，因为报告的孕酮在顶体反应中的作用。由于假定的MAPR缺乏孕酮受体的部分特征（类固醇结合结构域、钙结合结构域或激酶结构域），我们推测孕酮结合蛋白复合物中的其他蛋白质将具有这些特征。进行小鼠肝脏和睾丸文库的酵母双杂交筛选以鉴定与推定的MAPR相关的其他蛋白质。使用了几种不同的诱饵构建体，进行了广泛的一系列筛选，并对超过100个克隆进行了测序。然而，重叠克隆的多个拷贝未被鉴定，这通常发生在特异性相互作用的蛋白质中，并且分离的克隆不具有预测存在于胆固醇结合蛋白复合物中的特征。由于缺乏25-Dx编码的蛋白质与可能构成孕酮受体的其他蛋白质之间存在关联的证据，MAPR项目目前已被搁置。

Male mice homozygous for a mutation in the estrogen receptor alpha gene (ERKO mice) are infertile, while those homozygous for a mutation in the estrogen receptor beta gene are fertile. The goals of this project are to determine the role of estrogen in male germ cell development, the causes of infertility in male ERKO mice, and the identity of estrogen-regulated genes in the testis and epididymis. In addition, progesterone is reported to bind to membrane receptors on human sperm to cause rapid activation of signal transduction pathways without causing effects on gene transcription. It has been hypothesized that a membrane-associated progesterone receptor (MAPR) regulates male reproductive processes. The goals are to determine where and when MAPR mRNA and protein are present in the male reproductive system, and to define the function of MAPR during spermatogenesis, sperm maturation and fertilization. It is unknown if male ERKO mice are infertile due to disruption of estrogen-regulated processes within spermatogenic cells, within associated somatic cells, or both. We hypothesized that estrogen receptors do not directly regulate germ cell development, but are required by somatic cells in the testis and/or epididymis. We addressed this by transplanting spermatogenic cells from the testes of ERKO+/- or ERKO-/- donor mice to those of isogenic wild-type mice that had been treated with busulphan to eliminate most endogenous spermatogenic cells. After allowing regeneration of spermatogenesis, recipients were mated with wild-type females. The ERKO donor mice were C57Bl/6J (black), the male recipients were albino C57BL/6J, and the wild-type females were albino CD-1. This allowed the use of coat color markers to identify offspring produced from transplanted germ cells (black) and from regenerating endogenous spermatogenic cells (white). Two litters of mice were sired recently by recipients that received germ cells from ERKO-/- donors, one with two black pups and two white pups, and one with 10 black pups. These results support the hypothesis that male germ cells do not directly require the estrogen receptor for development, and suggest that ERKO males are infertile because of functional deficiencies in somatic cells of the testis or epididymis. Other studies seek to identify genes in somatic cells of the testis and epididymis that are regulated by estrogen and lead to infertility in ERKO males. Attempts to use differential display RT-PCR for this purpose did not give useful results. A cDNA suppression subtractive hybridization method then was used to generate a cDNA library representing genes differentially expressed in the testis of ERKO and wild-type mice. Differentially expressed clones currently are being characterized, and a tissue inhibitor of metalloproteinase-2 (TIMP-2) is an early candidate for further study. When they become available, we hope to use mouse gene microarrays to identify genes that are directly or indirectly induced or down-regulated in the testis and epididymis of ERKO mice relative to wild-type mice. We will determine if differences in gene expression signatures in ERKO and wild-type mice identify the functional changes that lead to infertility in ERKO males. Other investigators hypothesized that a membrane-associated progesterone receptor (MAPR) regulates male reproductive processes. Peptide sequences were determined for an abundant protein present in a progesterone-binding complex purified from porcine liver membranes. Those sequences were used to design PCR primers and to generate a probe for screening a porcine vascular smooth muscle cDNA library. A cDNA was isolated with significant sequence homology (76%) to the 25-Dx cDNA. This cDNA was isolated from a rat liver library in the laboratory Dr. Douglas Bell at NIEHS and suggested to encode a dioxin-inducible protein. We used the rat 25-Dx cDNA to clone the mouse homolog and carried out Northern blot, Western blot, and immunohistochemical studies, focusing on the male mouse reproductive system because of the reported role of progesterone in the acrosome reaction. Because the putative MAPR lacks the features that should be part of a progesterone receptor (steroid-binding domain, calcium-binding domain, or kinase domain), we postulated that other proteins in the progesterone-binding protein complex would have these features. Yeast two-hybrid screens of mouse liver and testis libraries were carried out to identify other proteins that associate with the putative MAPR. Several different bait constructs were used, an extensive series of screens performed, and over one hundred clones sequenced. However, multiple copies of overlapping clones were not identified, as usually occurs for proteins that interact specifically, and the clones isolated did not have the features that would be predicted to be present in a progesterone-binding protein complex. The MAPR project has been shelved for the present time because of the lack of evidence of an association between the protein encoded by 25-Dx and other proteins likely to constitute a progesterone receptor.