Na+ and K+ role in capacitation-associated hyperpolariz*

描述（由申请人提供） PI（Visconti）和外国合作者（FC; Darszon）研究计划的长期目标是了解精子受精能力的分子基础。哺乳动物的精子在射精后不能立即使卵子受精。它们在雌性生殖道中停留一段时间后获得受精能力。在雌性生殖道中发生的使精子能够受精的生理变化构成了“精子获能”现象。PI的工作集中在控制这个知之甚少的过程的信号事件上。FC的工作重点是研究离子通道和哺乳动物和无脊椎动物精子中离子事件的调节。最近，他们描述了一种内向整流钾离子通道，有助于与获能相关的超极化。FC和Pi共同证明了Na+/HCO 3-协同转运蛋白存在于小鼠精子中，并在小鼠精子获能中发挥作用。本FIRCA申请基于这些研究期间的观察结果。PI和FC的结果表明，小鼠精子静息膜电位（Era）对Na ~+和K ~+离子的渗透性有重要贡献。在第一个目标中，将进行实验，以确定外部Na+如何影响精子Em之前和之后的获能和Na+转运蛋白参与。在第二个目标中，将通过膜片钳技术测量小鼠生精细胞中的Na+通道，并使用平面双层测量未获能和获能精子中的Na+通道。将探索生精细胞和精子中阿米洛利敏感性Na+通道和超极化及环核苷酸门控（HCN）通道的存在。最后，在第三个目标中，将进行实验以研究存在于小鼠生精细胞中的参与精子获能的内向整流性K+（Kir）通道的调节。RT-PCR和免疫学方法将用于建立这些Kir通道的分子身份。Darszon博士是本申请中提出的所有技术的国际公认的专家。对获能的分子机制和离子在获能中的作用的清楚理解与基于精子的非激素避孕方法的新靶点的研究以及对改善不育夫妇的受精率的调查有关。

DESCRIPTION (provided by applicant) The long-term goals of the research programs of both the PI (Visconti) and Foreign Collaborator (FC; Darszon) are to understand the molecular basis of the sperm ability to fertilize. Mammalian sperm are not able to fertilize eggs immediately after ejaculation. They acquire the capacity to fertilize after residing in the female tract for a finite period of time. The physiological changes occurring in the female reproductive tract, that render the sperm able to fertilize, constitute the phenomenon of "sperm capacitation". Work by the PI has focused on the signaling events controlling this poorly understood process. Work of the FC has focused on the study of ion channels and on the regulation of ionic events in mammalian and invertebrate sperm. Recently they described an inward rectifier K+ channel that contributes to the hyperpolarization associated to capacitation. Together the FC and Pi have demonstrated that a Na+/HCO3- cotransporter is present in mouse sperm and has a role in mouse sperm capacitation. This FIRCA application is based on observations made during these studies. The findings of the PI and FC indicate that the mouse sperm resting membrane potential (Era) has an important contribution of permeability towards Na+ and K+ ions. In the first aim, experiments will be carried out to determine how external Na+ influences sperm Em before and after capacitation and which Na+ transporters are involved. In the second aim Na+ channels will be measured in mouse spermatogenic cells by patch clamp techniques and in uncapacitated and capacitated sperm using planar bilayers. The presence of amiloride sensitive Na+ channels and hyperpolarization and cyclic nucleotide gated (HCN) channels in spermatogenic cells and sperm will be explored. Finally, in the third aim, experiments will be conducted to study the regulation of the inward rectifier K+ (Kir) channel present in mouse spermatogenic cells that participates in sperm capacitation. RT-PCR and immunological approaches will be used to establish the molecular identity of these Kir channels. Dr. Darszon is an internationally recognized expert in all the techniques proposed in the present application. A clear understanding of the molecular mechanisms of capacitation and the role of ions in capacitation is relevant to research in sperm based new targets for non hormonal contraceptive approaches as well as to investigation on improvements of the fertilization rates in infertile couples.