Epididymal PMCA4 Expression Functional Impact and Mechanism of Sperm Uptake

描述（由申请人提供）：精子几乎没有细胞质，绝大多数蛋白质存在于质膜（PM）上。这些蛋白质中有许多是调节生育能力的，在没有合成机制的情况下，精子在睾丸后成熟过程中从附睾获得这些蛋白质。迄今为止，鉴定的大多数附睾获得性精子蛋白是糖基磷脂酰肌醇-(GPI)-与PM脂质双分子层的外小叶相连。然而，最近有报道称，跨膜（TM）蛋白也在附睾中表达，并存在于已知将gpi相关蛋白转移到精子的膜泡上。DeLeon实验室过去的研究有助于理解精子摄取GPI连接的附睾蛋白的机制，我们现在准备启动TM蛋白的研究，由于它们的折叠性质，不太可能被GPI连接蛋白所采用的机制获得。在本应用中，我们关注PMCA4（质膜Ca2+- atp酶4），一种10 TM蛋白，其4b异构体是小鼠精子中主要的Ca2+外排泵，其缺失导致运动性丧失和不育。令人兴奋的初步结果表明，小鼠附睾中4b和4a亚型的mRNA表达更有效地清除Ca2+和维持体内平衡。我们还发现PMCA4a存在于尾侧附睾腔液（ELF）中，有趣的是，在尾侧精子中比在头侧精子中更丰富。我们的中心假设是PMCA4在附睾中表达，并在附睾腔液（ELF）中分泌，并在精子表面获得成熟和功能。使用Pmca4 null和野生型小鼠(WT),我们的具体目标是在这个R03应用程序是:1)调查的表达模式和分泌Pmca4在所有三个地区的鼠附睾通过分析mRNA和蛋白亚型组织以及精灵和精子,和2)来确定,以及如何,Pmca4 /获得体外表面WT Pmca4零尾精子,如果收购增加Pmca4酶活性、Ca2 +间隙和能动性。我们将重点关注膜泡或附睾在将PMCA4/a运送到精子表面的作用，在CD9四联蛋白的帮助下，CD9四联蛋白通过膜融合介导细胞间蛋白转移。精子PM -囊泡相互作用将通过免疫金标记在超微结构图像中可视化，以开始了解PMCA4传递到PM的过程。总之，这些实验将提供关于TM蛋白摄取机制和附睾PMCA4a在精子成熟和功能中的作用的新信息。这个探索性项目的试点数据将使我们能够（通过R01机制）申请资金，以充分阐明囊泡在PMCA4a和其他TM蛋白的输送中的作用，并确定在ART（辅助生殖技术）中使用人工囊泡的可能性，通过体外受精将蛋白质输送到缺陷精子中，以提高其受精能力。

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.