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，每一种含有一个共同的α亚基和不同的β亚基。这赋予激素不同的生物活性。该项目的总体目标是了解控制糖蛋白激素的表达和作用的机制。在项目的早期阶段，我们发现camp反应转录因子，camp反应元件调节剂（CREM），在睾丸中高水平表达，编码这些因子的基因的表达在大鼠12天的生精周期中被自动调节，并且外显子剪接、内部翻译和启动子的交替使用周期性地将转录激活因子相互转化为抑制因子。特别是，我们发现，在精子发生的II-VIII阶段，外显子W剪接到CREB mRNA中，会过早地终止翻译并激活两个隐式内部翻译位点，导致CREB抑制剂异构体（i -CREB）的合成。我们还假设垂体促性腺激素FSH作用于支持细胞中的受体，产生精小管中cAMP的周期性波动，从而调节生殖细胞发育所必需的CREB和CREM等靶基因的活性。然而，最近相当明确的证据质疑卵泡刺激素对男性生育能力的基本要求。这些发现使我们提出，在缺乏FSH信号的情况下，睾丸和圆形精子中局部产生的激素PACAP（垂体腺苷酸环化酶激活肽）作用于其在支持细胞中的受体，产生cAMP信号的周期性波。目的是：(1)制备具有W外显子靶向缺失的小鼠，以测试在精子发生过程中提出的i - creb周期性表达在体内的功能重要性；(2)制备靶向破坏睾丸特异性PACAP启动子的小鼠，构建睾丸特异性PACAP启动子控制下表达LacZ转录报告子的转基因小鼠，构建睾丸特异性PACAP启动子控制下表达LacZ转录报告子的转基因小鼠，并检测sry相关同源盒因子Sox5在睾丸启动子调控中的作用。并研究sry相关同源盒因子Sox5在睾丸特异性PACAP启动子调控中的作用；(3)研究在支持细胞上表达的PACAP 1型受体胞外结构域的新选择性剪接（插入）外显子在PACAP异肽结合亲和力和受体偶联信号转导途径中的作用。这些研究对理解控制精子发生和生育的分子机制具有潜在的相关性。