Isolation and proteomic analysis of the sperm annulus

精子环的分离和蛋白质组学分析

• 批准号: 7707328
• 负责人: PATRICIA L. MORRIS
• 金额: $ 8.04万
• 依托单位: POPULATION COUNCIL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-15 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7707328
• 关键词: Acrosome; Antibodies; Binding; Biology; Chemicals; Comprehension; Contraceptive Agents; Data; Electrons; Event; Fertility; Fertilization; Germ Cells; Goals; Immunofluorescence Immunologic; Infertility; Japan; Knockout Mice; Knowledge; Laboratories; Lead; Male Contraceptions; Male Contraceptive Agents; Mass Spectrum Analysis; Mechanics; Methods; Mind; Mus; Organelles; Planets; Play; Population; Positioning Attribute; Proteins; Proteome; Proteomics; Reproductive Biology; Role; Sperm Count Procedure; Sperm Tail; Staging; Structure; Swimming; System; Testing; Tyrosine; Tyrosine Phosphorylation; Work; cell motility; contraceptive target; design; improved; information gathering; innovation; male; men; novel; novel therapeutics; public health relevance; research study; scaffold; sperm analysis; sperm cell; sperm function

DESCRIPTION (provided by applicant): With world population expanding at a logarithmic rate, the planet will reach approximately 10 billion people in about 40 years. The demand for more contraceptive options, especially targeting the male, thus is also expected to rise. To identify new targets for male contraception, studies must be undertaken to understand critical events that sperm must undergo to become functional gametes. With this goal in mind, we propose to study the annulus, a macromolecular septin structure located on the sperm tail. Sperm that are produced without an annulus are otherwise normal structurally, but they fail to swim and cannot undergo capacitation. This condition has been shown in the laboratory with septin 4 knockout mice as well as clinically in a population of infertile men. Together, these data establish that the annulus plays a vital role in sperm function, but its mechanisms of action are unknown. We propose to isolate the annulus and determine its proteome using mass spectrometry. Both low and high stringency conditions will be used to isolate the annulus to identify as many annulus-associating proteins as possible. We will isolate the annulus at four different stages of sperm function (immotile, motile, hypermotile, and fertilization competent). Identified annulus and annulus-associated proteins will be confirmed by immunofluorescence. The identification of proteins that alter their protein tyrosine phosphorylation will also be done. From the information gathered, we will develop hypotheses about how the annulus is functioning and test them in wild-type sperm by inhibiting the proposed function and observing the effect, as well as rescue experiments in sept4-/- sperm to see if function can be restored. Understanding the vital role the annulus plays in sperm function will advance our comprehension of how these mechanisms work together to make a sperm cell become a functional gamete. This information may lead to new therapeutics for infertile men as well as novel post- testicular male contraceptives. PUBLIC HEALTH RELEVANCE: The goal of this project is to isolate the sperm annulus and determine its proteome. This data will advance the fields of septin, contraceptive, and reproductive biology. Putative new therapies for infertility as well as novel male contraceptive targets may arise from this work.

描述（由申请人提供）：随着世界人口以对数率的扩大，地球将在大约40年内达到约100亿人。因此，人们对更多避孕措施，尤其是针对男性的需求也有望上升。为了确定男性避孕的新目标，必须进行研究以了解精子必须经历的关键事件才能成为功能配子。考虑到这个目标，我们建议研究精子尾部上的大分子septin结构。没有任何环的生产的精子在结构上是正常的，但它们无法游泳，无法承受电容。在实验室中，已有SEPTIN 4基因敲除小鼠以及临床上的不育男性在实验室中显示了这种情况。总之，这些数据表明，环在精子功能中起着至关重要的作用，但其作用机理尚不清楚。我们建议使用质谱分离环并确定其蛋白质组。低严格条件和高严格条件都将用于隔离环，以识别尽可能多的环形缔解蛋白。我们将在四个不同阶段的精子功能（Immotile，Motile，Hypermotile和Fustrization）处分离环。鉴定的环和与环相关的蛋白质将通过免疫荧光确认。还将完成改变其蛋白质酪氨酸磷酸化的蛋白质的鉴定。从收集的信息中，我们将通过抑制拟议的功能并观察效果以及在sept4 - / - 精子中观察效果，​​以查看功能是否可以恢复功能，从而在野生型精子中建立有关如何在野生型精子中进行测试的假设。了解环中精子功能中的重要作用将提高我们对这些机制如何共同起作用以使精子细胞成为功能配子的理解。这些信息可能会导致不育男性以及新型的睾丸后男性避孕药的新疗法。 公共卫生相关性：该项目的目标是隔离精子环并确定其蛋白质组。这些数据将推进Septin，避孕和生殖生物学的领域。这项工作可能会引起假定的不孕症以及新型男性避孕靶标的新疗法。