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）：几乎没有细胞质，精子中具有绝大多数蛋白质存在于质膜上（PM）。这些蛋白质中的许多是生育能力调节的，在没有合成机械的情况下，精子在它们的死后成熟过程中从附睾中获取它们。迄今为止，确定的大多数附睾所获得的精子蛋白是糖基磷脂酰肌醇 - （GPI） - 与PM脂质双层的外部小叶连接在一起。然而，最近据报道，跨膜（TM）蛋白也具有附睾表达，并存在于已知将GPI连接蛋白转移到精子上的膜囊泡上。过去在DELEON实验室的研究有助于理解GPI连接的附子蛋白的精子吸收机制，现在我们准备对TM蛋白进行研究，由于其折叠性质，该蛋白不可能由GPI-GPI-连接蛋白所采用的机制来获取。在此应用中，我们专注于10 TM蛋白的PMCA4（质膜Ca2+ -ATPase 4），其4B同工型是鼠精子中主要的Ca2+外排泵，其缺失导致运动能力丧失和不孕症。令人兴奋的初步结果表明4B的mRNA的鼠附子表达以及4A同工型，在清除Ca2+和维持稳态方面更有效。我们还表明，PMCA4A存在于尾骨腔液体（ELF）中，有趣的是，尾部比Caput精子更丰富。我们的中心假设是，PMCA4在附睾中表达，并在附睾流体（ELF）中分泌，在该腔体液体（ELF）中，在精子表面获得其成熟和功能。使用PMCA4 NULL和野生型（WT）小鼠，我们在此R03应用中的具体目的是：1）调查在鼠附子症的所有三个区域中PMCA4的表达模式和分泌的表达方式和分泌，通过分析在ELF和PMERS中的组织以及PMCA 4的效果和pMCA 4的效果，通过分析mRNA和蛋白质同工型，以确定在ELF和PMCA中的效果。无尾精子，如果采集会增加PMCA4酶活性，Ca2+清除率和运动性。我们将重点关注膜囊泡或附子体在将PMCA4/A传递到精子表面的作用，这是在CD9四跨地下蛋白通过膜融合中介导细胞到细胞蛋白转移的辅助。精子PM-囊泡相互作用将在超微结构图像中使用免疫质量标记来可视化，以开始了解PMCA4向PM的传递过程。总之，这些实验将提供有关TM蛋白摄取机理的新信息，以及附睾PMCA4A在精子成熟和功能中的作用。该探索性项目的试验数据将使我们能够（通过R01机制）申请资金，以完全机械机械地阐明囊泡在PMCA4A和其他TM蛋白的传递中的作用，并确定在艺术中使用人工囊泡的可能性（辅助生殖技术）以使蛋白质提供蛋白质，以使蛋白质能够为超级效率提高蛋白质，以提高其肥料，以提高其肥料Cababf。