Epididymal PMCA4 Expression Functional Impact and Mechanism of Sperm Uptake

描述（由申请方提供）：精子几乎没有细胞质，其绝大多数蛋白质存在于质膜（PM）上。这些蛋白质中的许多是生育调节蛋白质，并且在缺乏合成机制的情况下，精子在睾丸后成熟期间从附睾获得它们。迄今为止，大多数附睾获得的精子蛋白是糖基磷脂酰肌醇（GPI）-连接到PM的脂质双层的外小叶。然而，最近有报道称，跨膜（TM）蛋白也在附睾表达，并存在于膜囊泡上，已知膜囊泡将GPI连接的蛋白转移到精子。DeLeon实验室过去的研究有助于了解精子摄取GPI连接的附睾蛋白的机制，我们现在准备对TM蛋白进行研究，由于其折叠性质，不太可能通过GPI连接蛋白所采用的机制获得。在本申请中，我们专注于PMCA 4（质膜Ca 2 +-ATPase 4），一种10 TM蛋白，其4 b同种型是小鼠精子中主要的Ca 2+外排泵，其缺失导致运动力丧失和不育。令人兴奋的初步结果表明，小鼠附睾表达的mRNA的4 b以及4a亚型，这是更有效地清除Ca 2+和维持稳态。我们还表明，PMCA 4a是存在于尾部附睾腔液（ELF）和有趣的是，更丰富的尾部比头部精子。我们的中心假设是PMCA 4在附睾中表达，并在附睾腔液（ELF）中分泌，在那里它在精子表面获得，用于其成熟和功能。使用Pmca 4 null和野生型（WT）小鼠，我们在R 03应用中的具体目标是：1）通过分析组织以及ELF和精子中的mRNA和蛋白质同种型来研究PMCA 4在小鼠附睾的所有三个区域中的表达模式和分泌，以及2）为了确定是否以及如何在体外在WT和Pmca 4无效尾部精子的表面上获得PMCA 4/a，以及是否获得增加PMCA 4酶活性、Ca 2+清除和运动性。我们将集中在膜囊泡或附睾体的作用，在提供PMCA 4/a的精子表面，辅助的存在下的CD 9 tetraspanin介导细胞间的蛋白质转移通过膜融合。精子PM -囊泡相互作用将使用免疫金标记在超微结构图像中可视化，以开始理解PMCA 4递送至PM的过程。总之，这些实验将提供新的信息，TM蛋白的摄取机制和附睾PMCA 4a在精子成熟和功能中的作用。这个探索性项目的试点数据将使我们能够申请（通过R 01机制）资金，以完全机械地阐明囊泡在PMCA 4a和其他TM蛋白质递送中的作用，并确定在ART（辅助生殖技术）中使用人工囊泡将蛋白质递送给缺陷精子以改善其精子能力的可能性，通过IVF。

DESCRIPTION (provided by applicant): Almost devoid of cytoplasm, sperm have the vast majority of their proteins present on the plasma membrane (PM). Many of these proteins are fertility-modulating and in the absence of synthetic machinery sperm acquire them from the epididymis during their post-testicular maturation. To date, the majority of epididymally-acquired sperm proteins identified are glycosyl phosphatidylinositol-(GPI)-linked to the outer leaflet of th PM's lipid bilayer. However, it has recently been reported that transmembrane (TM) proteins are also epididymally-expressed and present on membranous vesicles that are known to transfer GPI-linked proteins to sperm. Past research in the DeLeon Lab has contributed to an understanding of the mechanisms of sperm uptake of GPI-linked epididymal proteins, and we are now poised to launch an investigation for TM proteins which, due to their folded nature, are unlikely to be acquired by the mechanisms employed by GPI- linked proteins. In this application we focus on PMCA4 (Plasma Membrane Ca2+-ATPase 4), a 10 TM protein, whose 4b isoform is the major Ca2+ efflux pump in murine sperm where its absence leads to loss of motility and to infertility. Exciting Preliminary results indicate murine epididymal expression of the mRNA for the 4b as well as the 4a isoform which is more efficient in clearing Ca2+ and maintaining homeostasis. We also show that PMCA4a is present in caudal epididymal luminal fluid (ELF) and interestingly, more abundant in caudal than caput sperm. Our central hypothesis is that PMCA4 is expressed in the epididymis and is secreted in the epididymal luminal fluid (ELF) where it is acquired on the sperm surface for their maturation and function. Using Pmca4 null and wild-type (WT) mice, our Specific Aims in this R03 application are: 1) To investigate the expression pattern and secretion of PMCA4 in all three regions of the murine epididymis by analyzing the mRNA and protein isoforms in tissues as well as in ELF and sperm, and 2) To determine if, and how, PMCA4/a is acquired in vitro on the surface of WT and Pmca4 null caudal sperm, and if acquisition increases PMCA4 enzymatic activity, Ca2+ clearance, and motility. We will focus on the role of membranous vesicles or epididymosomes, in delivering PMCA4/a to the sperm surface, assisted by the presence of CD9 tetraspanin which mediates cell-to-cell protein transfer via membrane fusion. Sperm PM - vesicle interaction will be visualized in ultrastructural images using immunogold labeling to begin to understand the process of delivery of PMCA4 to the PM. Together, these experiments will provide new information on the mechanism of uptake of TM proteins and the role of epididymal PMCA4a in sperm maturation and function. Pilot data from this exploratory project will allow us to apply (through the R01 mechanism) for funding to fully mechanistically elucidate the role of vesicles in the delivery of PMCA4a and other TM proteins, and to determine the possibility of using artificial vesicles in ART (assisted reproductive technology) to deliver the proteins to deficient sperm to improve their fertilizing capabilities, via IVF.