Epididymal PMCA4 Expression Functional Impact and Mechanism of Sperm Uptake

附睾 PMCA4 表达的功能影响和精子摄取机制

基本信息

批准号：
8675894
负责人：
PATRICIA ANASTASIA DELEON
金额：
$ 7.58万
依托单位：
UNIVERSITY OF DELAWARE
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8675894
关键词：
Accounting Assisted Reproductive Technology Binding Sites Biological Assay C-terminal Ca(2+)-Transporting ATPase Catalytic Domain Cell membrane Cells Confocal Microscopy Cytoplasm Data Detection Duct (organ) structure Dyes Enzymes Epididymis Extracellular Space Face Family Female Fertility Flow Cytometry Funding Genes Glycosylphosphatidylinositols Homeostasis Human Hydrophobic Interactions Image Immunofluorescence Immunologic Immunohistochemistry In Situ In Vitro Incubated Infertility Integral Membrane Protein Investigation Knock-out Label Lead Ligand Binding Link Lipid Bilayers Liquid substance Male Infertility Mammalian Cell Mediating Membrane Membrane Fusion Messenger RNA Mus Nature Outer Leaflet of the Lipid Bilayer Pattern Process Protein Isoforms Protein-Serine-Threonine Kinases Proteins RNA Splicing Rattus Reporting Research Research Project Grants Rest Reverse Transcriptase Polymerase Chain Reaction Role Signal Transduction Sperm Maturation Sperm Motility Surface Testis Therapeutic Time Tissues Transmembrane Domain Transmission Electron Microscopy Ultracentrifugation Variant Vesicle Western Blotting Wild Type Mouse artificial vesicle asthenospermia cell motility efflux pump human PHEMX protein improved insight link protein mRNA Expression male member mouse model novel therapeutics public health relevance research study sperm cell sperm function sperm protein uptake

项目摘要

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.

描述（由申请人提供）：精子几乎没有细胞质，其绝大多数蛋白质存在于质膜（PM）上。其中许多蛋白质具有生育调节作用，在缺乏合成机制的情况下，精子在睾丸后成熟过程中从附睾获取它们。迄今为止，大多数已鉴定的附睾获得性精子蛋白都是与附睾脂质双层外叶相连的糖基磷脂酰肌醇 (GPI)。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，已知膜囊泡可将 GPI 连接蛋白转移至精子。 DeLeon 实验室过去的研究有助于理解精子摄取 GPI 相关附睾蛋白的机制，我们现在准备启动对 TM 蛋白的研究，由于其折叠性质，精子不太可能获得 TM 蛋白。 GPI 连接蛋白所采用的机制。在本应用中，我们重点关注 PMCA4（质膜 Ca2+-ATP 酶 4），这是一种 10 TM 蛋白，其 4b 亚型是小鼠精子中主要的 Ca2+ 外排泵，其缺失会导致活力丧失和不育。令人兴奋的初步结果表明，小鼠附睾表达 4b 和 4a 亚型的 mRNA，在清除 Ca2+ 和维持体内平衡方面更有效。我们还表明，PMCA4a 存在于尾部附睾腔液 (ELF) 中，有趣的是，尾部精子中的含量比带头精子中的含量更高。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，在精子表面获得，以促进其成熟和发挥功能。使用 Pmca4 缺失和野生型 (WT) 小鼠，我们在此 R03 应用中的具体目标是： 1) 通过分析组织中的 mRNA 和蛋白质亚型来研究 PMCA4 在小鼠附睾所有三个区域中的表达模式和分泌：以及 ELF 和精子中的情况，以及 2) 确定 PMCA4/a 是否以及如何在 WT 和 Pmca4 无效尾部精子表面体外获得，以及获得是否增加PMCA4 酶活性、Ca2+ 清除率和运动性。我们将重点关注膜囊泡或附睾体在将 PMCA4/a 递送至精子表面方面的作用，并在 CD9 四跨膜蛋白的存在的帮助下，通过膜融合介导细胞间的蛋白质转移。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，以开始了解 PMCA4 传递到 PM 的过程。总之，这些实验将提供有关 TM 蛋白摄取机制以及附睾 PMCA4a 在精子成熟和功能中的作用的新信息。该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，以完全机械地阐明囊泡在 PMCA4a 和其他 TM 蛋白递送中的作用，并确定在 ART 中使用人工囊泡的可能性（辅助生殖技术）通过体外受精（IVF）将蛋白质输送给有缺陷的精子，以提高其受精能力。