Secretory Vesicle Function During Spermatogenesis & Fertilization in C. elegans

精子发生过程中的分泌囊泡功能

• 批准号: 7495134
• 负责人: STEVEN W. L'HERNAULT
• 金额: $ 29.07万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-10 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7495134
• 关键词: Accounting; Acrosome; Address; Affect; Animals; Binding; Biogenesis; Biology; Bone Diseases; Caenorhabditis elegans; Cell membrane; Cell surface; Cells; Cellular biology; Child; Couples; Data; Defect; Development; Disease; Ensure; Event; Fanconi Syndrome; Fertilization; Genes; Germ Cells; Goals; Human; Immune System Diseases; Infertility; Kidney; Ligands; Malignant Neoplasms; Mammals; Membrane Protein Traffic; Men' s Role; Molecular; Mutation; Nature; Nerve Degeneration; Neurodegenerative Disorders; Organelles; Osteogenesis; Osteoporosis; Pharmacology; Phenotype; Physiological; Play; Preparation; Process; Proteins; Proton Pump; Public Health; Regulation; Renal function; Role; Secretory Vesicles; Sperm Motility; Spermatids; Spermatogenesis; System; Testing; Ubiquitin; Ubiquitination; Work; base; desire; developmental genetics; egg; falls; human disease; infancy; innovation; loss of function; men; mutant; receptor function; sperm cell; sperm function; ubiquitin-protein ligase; vacuolar H+-ATPase; zygote

DESCRIPTION (provided by applicant): The long-term goal of this work is to determine the molecular and cellular mechanisms required for sperm-egg fusion during fertilization. This proposal addresses how a sperm secretory vesicle (acrosome) forms properly so that it can fuse with and create a fertilization-competent cell surface on the spermatozoon. Sperm secretory vesicles acquire an acidic internal pH during their maturation due to vacuolar (V-) ATPase activity, and this occurs in many animals, including humans. However, the mechanisms that regulate V-ATPase activity in sperm secretory vesicles are not understood, mostly because a spermatogenesis system suitable for its analysis had not been identified. Our preliminary data show that Caenorhabditis elegans spermatogenesis is ideally suited for analyzing the V-ATPase role and regulation during biogenesis and function of sperm secretory vesicles. In particular, mutations in the spe-5 gene alter a V-ATPase subunit that is spermatogenesis-specific, so acidification of sperm secretory vesicles can be studied in the absence of somatic V-ATPase defects, which are usually lethal. The spe-16 gene encodes a spermatogenesis-specific ubiquitin E3 ligase and how it participates in developmentally controlled acidification of sperm secretory vesicles will be determined. The focused objective of this proposal is to determine how the spe-5 and spe-16 encoded proteins cooperate to regulate internal pH in sperm secretory vesicles and what happens when this regulation is disrupted. We use three specific aims to test the overall hypothesis that the internal pH of sperm secretory vesicles must fall in order for this organelle to fuse with the plasma membrane. Specific aim #1 is to identify the role of the V-ATPase during sperm secretory vesicle maturation. Specific aim #2 is to identify how absence of V-ATPase function affects sperm motility and function during fertilization. Specific aim #3 is to identify why secretory vesicles fail to acidify in spe-16 loss of function mutant sperm. The unusual nature of the spe-16 mutant phenotype ensures it utilizes a previously undescribed way of regulating an animal V-ATPase. Ubiquitin is conjugated to proteins in normal human sperm, including those in the acrosome, and ubiquitination defects occur in infertile men; the role of ubiquitination is not understood in either case. Defects in ubiquitin biology are associated with various cancers, neurodegenerative diseases and many other disease processes. In addition to its role during spermatogenesis, V-ATPase function is defective in diseases that include renal Fanconi syndrome and the bone diseases, infantile ostepetrosis and osteoporosis. This proposal is focused on how sperm develop the ability to fertilize an egg. This is a problem of significant public health impact because ~15% of couples desiring children are infertile and many cases are due to defective sperm. The processes we study, in addition to functioning in sperm, also play a role in proper kidney function and bone formation.

描述（由申请人提供）：这项工作的长期目标是确定受精过程中精子-卵子融合所需的分子和细胞机制。这一建议解决了精子分泌囊泡（顶体）如何正确形成，使其能够与精子融合并在精子上产生受精能力细胞表面。由于液泡（V-） atp酶的活性，精子分泌囊泡在成熟过程中获得酸性内部pH值，这种情况发生在包括人类在内的许多动物中。然而，调节精子分泌囊中v - atp酶活性的机制尚不清楚，主要是因为适合其分析的精子发生系统尚未确定。我们的初步数据表明，秀丽隐杆线虫的精子发生非常适合分析v - atp酶在生物发生过程中的作用和调控以及精子分泌囊的功能。特别是，spe-5基因的突变改变了精子发生特异性的v - atp酶亚基，因此可以在没有体细胞v - atp酶缺陷的情况下研究精子分泌囊泡的酸化，而体细胞v - atp酶缺陷通常是致命的。spe-16基因编码精子发生特异性泛素E3连接酶，以及它如何参与发育控制的精子分泌囊泡酸化将被确定。本研究的主要目的是确定spe-5和spe-16编码蛋白如何协同调节精子分泌囊泡的内部pH值，以及当这种调节被破坏时会发生什么。我们使用三个特定的目标来测试精子分泌囊泡的内部pH值必须下降以使细胞器与质膜融合的总体假设。具体目的#1是确定v - atp酶在精子分泌囊泡成熟过程中的作用。具体目标#2是确定v - atp酶功能的缺失如何影响受精过程中精子的活力和功能。具体目标#3是确定为什么在spe16功能丧失突变精子中分泌囊泡不能酸化。spe-16突变表型的不寻常性质确保它利用先前描述的调节动物v - atp酶的方式。泛素与正常人类精子中的蛋白质结合，包括顶体中的蛋白质，而泛素化缺陷发生在不育男性中；在这两种情况下，泛素化的作用都不清楚。泛素生物学缺陷与各种癌症、神经退行性疾病和许多其他疾病过程有关。除了在精子发生过程中发挥作用外，v - atp酶功能在包括肾范可尼综合征和骨病、婴儿骨质疏松症和骨质疏松症在内的疾病中也存在缺陷。