ROLE OF CYCLIC NUCLEOTIDES IN SPERMATID DIFFERENTIATION

精子细胞分化为精子涉及到独特基因的转录激活和配子特异性细胞器（包括顶体和鞭毛）的组装。本提案的目的是确定cAMP依赖性途径在雄配子分化过程中的作用。我们已经取得了重大进展，在圆形和伸长精子细胞的cAMP合成和降解的酶的特点。使用我们已经开发的工具和模型，我们现在建议研究这些酶的调节机制和cAMP信号在精子细胞特异性基因表达中的作用。本提案中所述的实验是按照沿着两个具体目标组织的。在第一个具体目标中，我们将讨论腺苷酸环化酶和磷酸二酯酶在控制精子细胞分化过程中cAMP水平方面的作用。这些酶的表达和亚细胞定位以及它们在成熟精子中的保留将在体内精子细胞发育过程中进行研究。这些酶的调节机制将在重组系统和完整的圆形和伸长精子细胞中进行研究。第二个具体目标将致力于了解cAMP信号通路是如何参与基因表达的调控。体外研究将研究cAMP浓度的变化如何影响cAMP调节的转录因子的磷酸化和精子细胞特异性基因的表达。转基因过表达或同源重组的遗传方法将用于在体内操纵cAMP依赖性途径。这些体内模型的分析将阐明cAMP信号如何控制这些生殖细胞的基因表达和终末分化。这些研究将进一步加深我们对精子发生和精子功能调控的认识。此外，它们将有助于确定配子产生和生育力的药理学操纵的新靶点。

Differentiation of spermatids into spermatozoa involves activation of transcription of unique genes and assembly of gamete-specific organelles including the acrosome and the flagellum. The objective of this proposal is to determine the role of the cAMP-dependent pathway during this differentiative process of the male gamete. We have made major progress in characterizing the enzymes involved in cAMP synthesis and degradation in round and elongating spermatids. Using the tools and models that we have developed, we now propose to study the mechanisms of regulation of these enzymes and the role of cAMP signaling in the spermatid-specific gene expression. The experiments described in this proposal are organized along two Specific Aims. With the first Specific Aim, we will address the role of adenylyl cyclases and phosphodiesterases in the control of cAMP levels in differentiating spermatids. The expression and subcellular localization of these enzymes as well as their retention in mature spermatozoa will be studied during spermatid development in vivo. The mechanisms of regulation of these enzymes will be investigated in a reconstitution system and in intact round and elongating spermatids. The second Specific Aim will be devoted to understanding how the cAMP signaling pathway is involved in the regulation of gene expression. In vitro studies will investigate how changes in cAMP concentration impact phosphorylation of cAMP-regulated transcription factors and expression of spermatid-specific genes. A genetic approach of transgenic overexpression or homologous recombination will be used to manipulate the cAMP- dependent pathway in vivo. Analysis of these in vivo models will elucidate how cAMP signaling controls gene expression and terminal differentiation of these germ cells. The proposed studies will further our understanding of regulation of spermatogenesis and sperm function. Moreover, they will help to identify novel targets for pharmacological manipulation of gamete production and fertility.