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: 7349913
• 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/7349913
• 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蛋白融合所需的分子和细胞机制。该提案介绍了精子分泌囊泡（Acrosom）如何正确形成，以便它可以与精子上的精子融合并创建具有施肥能力的细胞表面。精子分泌囊泡由于液泡（V-）ATPase活性在成熟期间获得酸性内部pH，并且在包括人类在内的许多动物中都会发生这种情况。然而，尚不清楚调节精子分泌囊泡中V-ATPase活性的机制，主要是因为尚未鉴定出适合其分析的精子发生系统。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精子分泌囊泡生物发生和功能期间V-ATPase的作用和调节。特别是，SPE-5基因中的突变改变了精子发生特异性的V-ATPase亚基，因此在没有体细胞V-ATPase缺陷的情况下，可以研究精子分泌囊泡的酸化，通常是致命的。 SPE-16基因编码精子发生特异性的泛素E3连接酶，以及它如何参与精子分泌囊泡的发育控制酸化。该提案的重点目标是确定SPE-5和SPE-16编码蛋白如何配合以调节精子分泌囊泡中的内部pH，以及该调节中断时会发生什么。我们使用三个特定的目标来检验总体假设，即精子分泌囊泡的内部pH值必须降低，以使该细胞器与质膜融合。具体目的1是确定V-ATPase在精子分泌囊泡成熟过程中的作用。具体目的＃2是确定缺乏V-ATPase功能如何影响受精过程中的精子运动和功能。特定目的＃3是确定为什么分泌囊泡在SPE-16功能突变体精子丧失中无法酸化的原因。 SPE-16突变表型的异常性质确保了它利用以前未描述的调节动物V-ATPase的方式。泛素与正常人精子中的蛋白质相结合，包括基体中的蛋白质，泛素化缺陷发生在不育男性中。无论哪种情况，泛素化的作用均不理解。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的作用外，V-ATPase功能在包括肾脏Fanconi综合征和骨骼疾病，婴儿骨质疾病和骨质疏松症的疾病中有缺陷。 该提议的重点是精子如何发展卵子的能力。这是一个重大公共卫生影响的问题，因为约有15％的夫妻渴望儿童不育，许多病例是由于精子缺陷所致。除精子中功能外，我们研究的过程在适当的肾功能和骨形成中也起作用。