NEUROENDOCRINE CONTROL OF SPERMATOGENESIS

精子发生的神经内分泌控制

• 批准号: 6590022
• 负责人: JOEL F HABENER
• 金额: $ 24.33万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6590022
• 关键词: RNA splicing; biological signal transduction; cAMP response element binding protein; cooperative study; cyclic AMP; genetically modified animals; hormone receptor; hormone regulation /control mechanism; laboratory mouse; male reproductive system; neuroendocrine system; neuropeptides; receptor expression; reproductive development; spermatogenesis

The glycoprotein hormones comprise a structurally related family of proteins produced in the pituitary gland (TSH, FSH, LH) and in the placenta (CG). The hormones are the heterodimers, each containing a common alpha-subunit and different beta-subunits., which confer distinct biological activities to the hormones. The overall goals of this project have been to understand the mechanisms that control the expression and actions of the glycoprotein hormones. During earlier phases of the project we found that the cAMP-response transcription factors, cAMP-response element modulator (CREM), are expressed at high levels in the testis, that the expression of the genes encoding these factors is autoregulated during the 12-day spermatogenic cycles in the rat, and that alternative exon splicing, internal translation, and alternative promoter usage cyclically interconverts transcriptional activators to repressors. In particular, we have discovered that the splicing of exon W into CREB mRNA during stages II-VIII of spermatogenesis prematurely terminates translation and activates two cryptic internal translation sites, resulting in the synthesis of inhibitor CREB isoforms (I-CREBs). We also have hypothesized that the pituitary gonadotropin FSH acts on receptors in Sertoli cells to generate cyclical fluctuations of cAMP in the seminiferous tubule, resulting in the regulation of the activities of CREB and CREM and other target genes essential for the development of germ cells. However, recent rather definitive evidence questions the essential requirement of FSH for male fertility. These findings lead us to propose that in the absence of FSH signaling, the hormone PACAP (pituitary adenylyl cyclase activating peptide), produced locally in the testis and round spermatids, acts on its receptors in Sertoli cells to produce the cyclical waves of cAMP signaling. The Aims are to: (1) Prepare mice with a targeted deletion of exon W to test the functional importance in vivo of the proposed cyclical expression of I-CREBs during spermatogenesis; (2) Prepare mice with a targeted disruption of the testis-specific PACAP promoter, create transgenic mice expressing the LacZ transcriptional reporter under control of the testis-specific PACAP promoter, create transgenic mice expressing the LacZ transcriptional reporter under control of the testis-specific PACAP promoter, and to examine the role of the SRY-related homeobox factor Sox5 in the regulation of the testis-promoter, and to examine the role of the SRY-related homeobox factor Sox5 in the regulation of the testis-specific PACAP promoter; (3) To examine the role of the novel alternatively spliced (inserted) exon in the extracellular domain of the PACAP type 1 receptor, expressed on Sertoli cells, on the binding affinities of PACAP isopeptides and receptor coupling to signal transduction pathways. These studies have potential relevance to understanding the molecular mechanisms controlling spermatogenesis and fertility.

糖蛋白激素包括在垂体（TSH，FSH，LH）和胎盘（CG）中产生的结构相关的蛋白质家族。激素是异二聚体，每种二聚体都包含一个常见的α-亚基和不同的β-亚基。该项目的总体目标是了解控制糖蛋白激素表达和作用的机制。在该项目的较早阶段，我们发现在睾丸中，CAMP响应转录因子（CREM）在高水平中表达，即编码这些因子的基因的表达在12天内进行自动调节。大鼠中的精子循环，以及替代的外显子剪接，内部翻译和替代启动子用法可周期性地将转录激活因子与阻遏物相互互连。特别是，我们发现，在精子发生的II-VIII期间，外显子W片段过早终止翻译并激活两个隐性内部翻译位点，从而导致抑制剂CREB同工型（I-Crebs）合成。我们还假设，垂体性促性腺激素FSH作用于Sertoli细胞中的受体，以产生cAMP的周期性波动，从而调节CREB和CREM和CREM的活性以及其他靶基因对生殖细胞发育必不可少的。但是，最近相当确定的证据质疑FSH对男性生育的基本要求。这些发现使我们提出，在没有FSH信号传导的情况下，激素PACAP（垂体腺苷酸环化酶激活肽）在睾丸和圆形的精子中局部产生，作用于其在Sertoli细胞中的受体上，以产生cAMP信号传导的周期性波。目的是：（1）准备具有外显子W的靶向缺失的小鼠，以测试精子发生过程中I-CREB的拟议周期性表达在体内的功能重要性； （2）准备小鼠对睾丸特异性PACAP启动子的有针对性破坏，创建在控制睾丸特异性PACAP启动子的控制下表达LACZ转录记者的转基因小鼠，创建表达LACZ特异性转录报告基因控制的转基因小鼠PACAP启动子，并检查与SRY相关的同型副盒因子SOX5在调节睾丸启动器中的作用，并检查与SRY相关的同源性同源型因子SOX5在调节睾丸特异性PACAP启动子调节中的作用； （3）在检查新颖的（插入）外显子中，在Sertoli细胞上表达的PACAP 1型受体的细胞外域（插入）外显子对PACAP Isophtides和受体偶联到信号转导途径的结合亲和力。这些研究与理解控制精子发生和生育能力的分子机制具有潜在的相关性。