ESTROGEN RECEPTORS IN MALE REPRODUCTION

男性生殖中的雌激素受体

• 批准号: 6432400
• 负责人: EDWARD MITCHELL EDDY
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432400
• 关键词: estrogen receptors; fertility; follicle stimulating hormone; gene mutation; genetically modified animals; hormone regulation /control mechanism; laboratory mouse; luteinizing hormone; male; male reproductive system; reproductive development; testosterone

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.

雌激素受体α基因突变的纯合雄性小鼠（ERKO小鼠）是不育的，而雌激素受体β基因突变的纯合雄性小鼠是可育的。该项目的目的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，黄体酮可以与人类精子的膜受体结合，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，膜相关孕激素受体（MAPR）调节男性生殖过程。目的是确定MAPR mRNA和蛋白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小鼠不育是由于生精细胞内雌激素调节过程的破坏，还是相关体细胞内雌激素调节过程的破坏，或者两者兼而有之。我们假设雌激素受体不直接调节生殖细胞的发育，但睾丸和/或附睾的体细胞需要雌激素受体。我们通过将来自ERKO+/-或ERKO-/-供体小鼠睾丸的生精细胞移植到经布硫芬处理以消除大多数内源性生精细胞的等基因野生型小鼠的睾丸中来解决这个问题。在允许精子再生后，接受者与野生型雌性交配。ERKO供鼠为C57Bl/6J（黑色），雄性受体为白化型C57Bl/6J，野生型雌性为白化型CD-1。这允许使用毛色标记来识别从移植的生殖细胞（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-/-受体生殖细胞的两窝小鼠繁育成功，其中一窝有两只黑色幼鼠和两只白色幼鼠，另一窝有10只黑色幼鼠。这些结果支持了男性生殖细胞不直接需要雌激素受体来发育的假设，并表明ERKO男性不育是因为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对此目的没有给出有用的结果。然后采用cDNA抑制减法杂交的方法，生成ERKO和野生型小鼠睾丸中差异表达基因的cDNA文库。差异表达的克隆目前正在被表征，而金属蛋白酶-2 （TIMP-2）的组织抑制剂是进一步研究的早期候选者。当它们可用时，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。其他研究人员假设膜相关孕激素受体（MAPR）调节男性生殖过程。从猪肝膜中纯化的黄体酮结合复合物中确定了丰富的蛋白质的肽序列。这些序列用于设计PCR引物和制备猪血管平滑肌cDNA文库筛选探针。分离到与25-Dx序列同源性显著（76%）的cDNA。该cDNA是从NIEHS实验室Douglas Bell博士的大鼠肝脏文库中分离出来的，并建议编码二恶英诱导蛋白。我们使用大鼠25-Dx cDNA克隆小鼠同源基因，并进行了Northern blot， Western blot和免疫组织化学研究，重点研究雄性小鼠生殖系统，因为报道了黄体酮在顶体反应中的作用。由于假定的MAPR缺乏黄体酮受体应有的特征（类固醇结合结构域、钙结合结构域或激酶结构域），我们假设黄体酮结合蛋白复合体中的其他蛋白质也具有这些特征。对小鼠肝脏和睾丸文库进行酵母双杂交筛选，以鉴定与假定的MAPR相关的其他蛋白质。使用了几种不同的诱饵结构，进行了一系列广泛的筛选，并对100多个克隆进行了测序。然而，没有发现重叠克隆的多个拷贝，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在孕激素结合蛋白复合体中存在的特征。由于缺乏25-Dx编码的蛋白质与其他可能构成黄体酮受体的蛋白质之间存在关联的证据，MAPR项目目前已被搁置。