Na+ /K+ role /capacitation-associated hyperpolarization

描述(由申请人提供) PI(Visconi)和国外合作者(FC；Darszon)的研究计划的长期目标都是了解精子受精能力的分子基础。哺乳动物的精子不能在射精后立即使卵子受精。它们在雌性体内居住了一段有限的时间后，就获得了受精的能力。女性生殖道发生的生理变化，使精子能够受精，构成了“精子获能”现象。PI的工作重点是控制这一鲜为人知的过程的信号事件。FC的工作重点是研究哺乳动物和无脊椎动物精子中的离子通道和离子事件的调节。最近，他们描述了一种内向整流钾通道，它促成了与获能相关的超极化。Fc和PI一起证明了Na/HCO3-共转运体存在于小鼠精子中，并在小鼠精子获能过程中发挥了作用。这项FIRCA申请是基于在这些研究期间所做的观察。PI和FC的结果表明，小鼠精子静息膜电位(ERA)对Na、K离子的通透性有重要贡献。在第一个目标中，将进行实验，以确定外部钠在获能前后如何影响精子Em，以及哪些钠转运体参与其中。在第二个目标中，我们将使用膜片钳技术测量小鼠生精细胞中的Na通道，并使用平面双层技术测量未获能精子和获能精子中的Na通道。阿米洛利敏感钠通道和超极化和环核苷酸门控(HCN)通道的存在将被探索在生精细胞和精子中。最后，在第三个目标中，将进行实验来研究小鼠生精细胞中存在的内向整流钾(KIR)通道的调节，该通道参与精子获能。将使用RT-PCR和免疫学方法来确定这些KIR通道的分子特性。Darszon博士在本申请中提出的所有技术方面是国际公认的专家。明确获能的分子机制和离子在获能中的作用，对于研究以精子为基础的非激素避孕方法的新靶点以及提高不孕夫妇的受精率具有重要意义。

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.