CYCLIC NUCLEOTIDES AND GERM CELLS

有充分的证据表明，坎普是另一个参与 成熟精子功能的调节。两者都在发展中 生殖细胞和精子缺乏已知的受体和Gs蛋白。 在体细胞中用于激活腺苷环化酶，但表达 高水平的磷酸二酯酶(PDE)。因此，我们建议测试 假设PDE及其调控在 雄配子中cAMP水平的控制。理解这些问题的本质 配子的PDE也很重要，因为药物抑制 这些酶是控制cAMP水平的有效方法 不活动精子的治疗。我们的初步数据表明 编码cAMP特异性PDE(cAMP-PDE)和钙调蛋白的两个基因- 调控的PDE(CaM-PDE)在精子发生过程中被激活，PDE 这些基因的产物存在于精子中。属性和 两个PDE的分布将由西方分析和 免疫细胞化学。由于这些形式可溶于精子细胞，但 在精子中的颗粒部分中恢复，蛋白质 将对导致这种划分的修改进行调查。在……里面 认为精子在附睾处获得运动能力是 与cAMP增加和PDE活性降低有关， 导致抑制PDE活性的机制将是 调查过了。不同性别精液中PDE蛋白水平的比较 附睾的一部分将通过蛋白质分析和 与酶的活性有关。磷酸化的状态和 头部和尾部对这些酶活性的影响 下定决心。因为咖啡因，一种低浓度的非选择性PDE抑制剂 不育症患者的精子质量已被用于提高精子质量。 患者，一种使用形式选择性的新的药理学方法 将开发用于操纵人类精子PDE的抑制剂 活动。PDE亚型存在于射出的人类精子和 它们的定位将用生化和 可用的免疫学工具。新型PDE特异性抑制剂 因为在精子中表达的形式将被用于研究 PDE抑制对运动和顶体反应的影响。如果 在人类中发现了PDE亚型分布的差异，亚型- 选择性抑制剂将被用来操纵头部或大脑中的cAMP水平 在精子的鞭毛中，从而选择性地影响运动性 或者顶体反应。此外，我们建议建立和测试一个 转基因小鼠模型研究cAMP在成熟过程中的作用 精子。通过对发育中cAMP水平的遗传操作 精子细胞和精子，这个第二信使在 运动性的获得将在现场确定。这些研究将 提高我们对精子成熟控制机制的理解 将为药理改进提供重要信息 精子质量。

There is ample evidence that cAMP is a second messenger involved in regulation of the function of the maturing spermatozoon. Both developing germ cells and spermatozoa are devoid of known receptors and Gs protein that in somatic cells serves to activate adenylate cyclase but express high levels of phosphodiesterases (PDEs). We therefore propose to test the hypothesis that PDEs and their regulation play a crucial role in the control of cAMP levels in the male gamete. Understanding the nature of the PDEs of the gamete is also important because pharmacological inhibition of these enzymes is an effective approach to manipulate cAMP levels for treatment of immotile spermatozoa. Our preliminary data demonstrate that two genes encoding a cAMP-specific PDE (cAMP-PDE) and a calmodulin- regulated PDE (CaM-PDE) are activated during spertmatogenesis and the PDE products of these genes are present in spermatozoa. The properties and distribution of the two PDEs will be followed by western analysis and immunocytochemistry. Since these forms are soluble in spermatids but recovered in the particulate fraction in spermatozoa, the protein modification causing this compartmentalization will be investigated. In view of the fact that acquisition of sperm motility in the epididymis is associated with an increase in cAMP and a reduction of the PDE activity, the mechanism leading to inhibition of the activity of the PDEs will be investigated. The PDE protein levels in spermatozoa from different portions of the epididymis will be quantitated by western analysis and related to the enzyme activity. The state of phosphorylation and the impact on activity of these enzymes from caput and cauda will be determined. Because caffeine, a non selective PDE inhibitor with low potency has been used to improve human sperm quality in infertile patients, a novel, pharmacological approach using form-selective inhibitors will be developed to manipulate human spermatozoon PDE activity. The PDE isoforms present in ejaculated human spermatozoa and their localization will be characterized using biochemical and immunological tools available. Novel classes of PDE inhibitors specific for the forms expressed in the spermatozoon will be used to study the effects of PDE inhibition on motility and acrosomal reaction. If the difference in PDE isoform distribution is found in human, isoform- selective inhibitors will be used to manipulate cAMP levels in the head or in the flagellum of the spermatozoon, thus selectively affecting motility or acrosomal reaction. In addition, we propose to establish and test a transgenic mouse model to study the role of cAMP in the maturing spermatozoa. By genetic manipulation of cAMP levels in developing spermatids and spermatozoa, the role of this second messenger in the acquisition of motility will be determined in situ. These studies will improve our understanding of the mechanisms controlling sperm maturation and will provide important information for a pharmacological improvement of sperm quality.