Pem Homeobox Gene Function During Spermatogenesis

描述（由申请人提供）：缺乏关于决定男性生育力的分子机制的知识。因此，男性非甾体避孕药和不孕症治疗的发展受到限制。睾丸支持细胞与生殖细胞之间的通讯是精子发生的必要条件。睾酮通过与睾丸支持细胞雄激素受体结合，调节基因转录，从而调节精子发生。目前已知的唯一一个睾丸激素调控的支持细胞特异性基因是Pem同源框基因。转录因子如同源异型盒基因的适当调节和表达是正常发育所必需的。因此，Pem的表达可能是精子发生的调节因子。我们是第一个表明，支持细胞表达Pem的睾丸激素依赖性曲细精管阶段VI-VIII。出生后，Pem首先表达，正好在第一次生殖细胞减数分裂之前，同时在支持细胞中诱导雄激素受体。在睾酮耗竭的小鼠中，我们的工作表明，单独的睾酮足以诱导秘鲁表达。目前，这种严格控制的Pem表达的功能尚不清楚。秘鲁基因功能的研究是重要的，因为秘鲁可能是一个转录因子，调节基因参与雄激素的反应性和凋亡。本项目的主要目标是确定秘鲁表达的功能。所检验的假设是Pem表达调节关键的支持细胞与生殖细胞的通讯。检验这一假设的第一个目的是确定支持细胞中Pem表达上调或下调的基因。这一目的将通过用来自用Pem表达载体转染的Sertoli细胞的cDNA或用来自用空载体转染的Sertoli细胞的cDNA筛选小鼠微阵列来实现。未代表的基因或新的PEM调节基因将通过PI先前使用的基于PCR的消减杂交方法分离。第二个目标将决定Pem所结合的识别元件。为此，将同位素标记的随机寡核苷酸与单独的Pem蛋白、Pem加Sertoli细胞核提取物或对照混合，免疫沉淀，PCR扩增，通过迁移率变动测定确认，克隆和测序。该提案是一项重点计划，将导致分子靶点的发展，以进一步了解精子发生和男性生育力。

DESCRIPTION (provided by applicant): Knowledge of molecular mechanisms determining male fertility is lacking. As a result, development of male nonsteroidal contraceptives and treatments for infertility has been limited. Communication between Sertoli cells of the testis and germ cells is essential for spermatogenesis. Testosterone regulates spermatogenesis through binding Sertoli cell androgen receptors and resultant gene transcription. The only testosterone-regulated, Sertoli cell-specific gene known to date is the Pem homeobox gene. Appropriate regulation and expression of transcription factors such as homeobox genes are required for normal developmental events. Pem expression, therefore, may serve as a regulator of spermatogenesis. We were the first to show that Sertoli cells express Pem specifically during the testosterone-dependant seminiferous tubule stages VI-VIII. Postnatally, Pem is first expressed just prior to the first germ cell meiosis concurrent with the induction of androgen receptor in Sertoli cells. In hormone-depleted mice, our work demonstrated that testosterone alone is sufficient to induce Peru expression. Currently, the function of this tightly controlled expression of Pem is unknown. The study of Peru gene function is important because Peru may be a transcription factor that regulates genes involved in androgen responsiveness and apoptosis. The primary goal of this project is to determine the function of Peru expression. The hypothesis tested is that Pem expression regulates critical Sertoli cell to germ cell communication. The first aim to test this hypothesis will be to determine genes that are upregulated or downregulated by Pem expression in Sertoli cells. This aim will be achieved by screening a mouse microarray with cDNA from Sertoli cells transfected with a Pem expression vector or with cDNA from Sertoli cells transfected with an empty vector. Unrepresented genes or novel Pem-regulated genes will be isolated by a PCR-based subtraction hybridization method the PI has used previously. The second aim will determine the recognition element to which Pem binds. For this aim, isotope-labeled random oligonucleotides will be mixed with Pem protein alone, Pem plus Sertoli cell nuclear extract or control, immunoprecipitated, PCR amplified, confirmed by mobility shift assay, cloned and sequenced. This proposal is a focused plan that will lead to the development of molecular targets to further the understanding of spermatogenesis and male fertility.