NEUROENDOCRINE CONTROL OF SPERMATOGENESIS

糖蛋白激素包括一个结构上相关的蛋白质家族，它们产生于脑下垂体(TSH、FSH、LH)和胎盘(CG)。这些激素是异二聚体，每个都包含一个共同的α-亚基和不同的β-亚基，它们赋予激素不同的生物学活性。这个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。在该项目的早期阶段，我们发现cAMP反应转录因子cAMP反应元件调节器(CREM)在睾丸中高水平表达，在大鼠12天的生精周期中，这些因子的编码基因的表达是自动调节的，替代外显子剪接、内部翻译和替代启动子的使用将转录激活因子循环地转化为抑制因子。特别是，我们发现在精子发生的II-VIII阶段，外显子W与CREB mRNA的剪接提前终止翻译，并激活两个隐蔽的内部翻译位点，导致合成抑制CREB亚型(I-CREB)。我们还假设，垂体促性腺激素FSH作用于支持细胞的受体，在生精小管内产生cAMP的周期性波动，从而调节CREB和CREM的活性以及其他生殖细胞发育所必需的靶基因。然而，最近相当明确的证据质疑了FSH对男性生育能力的基本要求。这些发现导致我们提出，在没有FSH信号的情况下，在睾丸和圆形精子细胞中局部产生的激素PACAP(垂体腺苷环化酶激活肽)作用于支持细胞中的它的受体，产生cAMP信号的周期波。其目的是：(1)制备靶向缺失外显子W的小鼠，以测试所提出的I-Crebs在精子发生过程中的周期性表达在体内的功能重要性；(2)制备睾丸特异性PACAP启动子的靶向干扰小鼠，建立睾丸特异性PACAP启动子控制下表达LacZ转录报告基因的转基因小鼠，建立睾丸特异性PACAP启动子控制下表达LacZ转录报告基因的转基因小鼠，检测SRY相关同源框因子Sox5在睾丸特异性PACAP启动子调控中的作用，以及SRY相关同源框因子Sox5在睾丸特异性PACAP启动子调控中的作用；(3)研究在Sertoli细胞上表达的PACAP 1型受体胞外区新的选择性剪接(插入)外显子在PACAP异肽结合亲和力和受体偶联信号转导通路中的作用。这些研究对于理解控制精子发生和生育的分子机制具有潜在的相关性。

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.