Phosphorylation Events During Sperm Capacitation

精子获能期间的磷酸化事件

• 批准号: 7232402
• 负责人: Pablo E. Visconti
• 金额: $ 24.35万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7232402
• 关键词: ATP phosphohydrolase; Accounting; Acrosome Reaction; Amino Acids; Cyclic AMP; Ejaculation; Event; Experimental Models; Family; Female; Fertilization; Human; Label; Lead; Link; Maleimides; Methods; Molecular; Mus; Pathway interactions; Pattern; Peptides; Phosphopeptides; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Play; Polyacrylamide Gel Electrophoresis; Population; Process; Protein Tyrosine Kinase; Proteins; Reaction; Recombinants; Regulation; Regulatory Pathway; Role; Serine; Site; Specificity; Sperm Capacitation; Techniques; Threonine; Time; Two-Dimensional Gel Electrophoresis; Tyrosine; Tyrosine Phosphorylation; Tyrosine Phosphorylation Site; Work; austin; design; novel; poly(tyrosyl-glutamic acid); protein aminoacid sequence; protein function; reproductive; sperm cell; sperm function; synthetic peptide; tandem mass spectrometry; two-dimensional; valosin-containing protein; zygote

DESCRIPTION (provided by applicant): Mammalian sperm are not able to fertilize eggs immediately after ejaculation. They acquire fertilization capacity after residing in the female tract for a finite period of time. The physiological changes occurring in the female reproductive tract rendering the sperm able to fertilize constitute the phenomenon of "sperm capacitation" (Austin, 1952; Chang, 1951). Using the mouse as an experimental model, we have demonstrated that capacitation is associated with an increase in the tyrosine (tyr) phosphorylation of a subset of proteins (Visconti et al., 1995a). We have also demonstrated that the increase in protein tyr phosphorylation as well as capacitation are regulated by a cAMP-dependent pathway (Visconti et al., 1995b). The presence of this regulatory pathway has subsequently been demonstrated in sperm from other species including human (Leclerc et al., 1996; Osheroff et al., 1999). Despite these advances in understanding the mechanisms regulating phosphorylation during capacitation, little is known about the identity of the protein targets of this phosphorylation cascade and of the kinases and phosphatases involved in the regulation of sperm function. Recently, we have used a 2 dimensional (2D) polyacrylamide gel electrophoresis (PAGE) approach combined with tandem mass spectrometry (MS/MS) analysis (Ficarro et al., 2003) to identify proteins that undergo tyr phosphorylation during capacitation of human sperm. In the same work, we have analyzed the exact phosphorylated sequence by MS/MS in proteins from a population of capacitated human sperm and identified sequences phosphorylated in tyr as well as in serine (ser) and threonine (thr) (Ficarro et al., 2003). Among the protein targets, valosin-containing protein (VCP), an ATPase from the same family of the SNARE-interacting protein N-ethyl maleimide soluble factor (NSF) was found to be tyr phosphorylated during capacitation. In addition, immunolocalization of VCP showed a change in fluorescent pattern accompanying sperm capacitation. The hypotheses underlying this proposal combine the above set of findings and postulate that proteins specifically phosphorylated during capacitation as well as the kinases responsible for their phosphorylation are required for the regulation of mammalian sperm capacitation. The objective of this proposal is to further elucidate the sequence of reactions that control protein phosphorylation cascades in mammalian sperm. Characterization of the phosphorylated protein substrates and the kinase(s) involved in the regulation of sperm function will provide novel targets for pharmacological control of the fertilization process.

描述（由申请人提供）：哺乳动物精子在射精后不能立即使卵子受精。它们在雌性生殖道中停留一段有限的时间后获得受精能力。女性生殖道中发生的使精子能够受精的生理变化构成“精子获能”现象（Austin，1952；Chang，1951）。使用小鼠作为实验模型，我们证明获能与蛋白质子集酪氨酸 (tyr) 磷酸化的增加有关（Visconti 等人，1995a）。我们还证明了蛋白质 tyr 磷酸化的增加以及获能是由 cAMP 依赖性途径调节的（Visconti 等人，1995b）。随后在包括人类在内的其他物种的精子中证明了这种调节途径的存在（Leclerc 等，1996；Osheroff 等，1999）。尽管在了解获能期间调节磷酸化的机制方面取得了这些进展，但人们对这种磷酸化级联的蛋白质靶标以及参与精子功能调节的激酶和磷酸酶的身份知之甚少。最近，我们使用二维 (2D) 聚丙烯酰胺凝胶电泳 (PAGE) 方法结合串联质谱 (MS/MS) 分析（Ficarro 等，2003）来鉴定在人类精子获能过程中经历酪氨酸磷酸化的蛋白质。在同一工作中，我们通过 MS/MS 分析了来自获能人类精子群体的蛋白质中的精确磷酸化序列，并鉴定了 tyr 以及丝氨酸 (ser) 和苏氨酸 (thr) 中的磷酸化序列（Ficarro 等人， 2003）。在蛋白质靶标中，含缬洛辛的蛋白质 (VCP) 是一种来自 SNARE 相互作用蛋白 N-乙基马来酰亚胺可溶性因子 (NSF) 同一家族的 ATP 酶，被发现在获能过程中被酪氨酸磷酸化。此外，VCP 的免疫定位显示伴随精子获能的荧光模式发生变化。该提议的假设结合了上述一组发现，并假设在获能过程中特异性磷酸化的蛋白质以及负责其磷酸化的激酶是哺乳动物精子获能调节所必需的。该提案的目的是进一步阐明控制哺乳动物精子中蛋白质磷酸化级联的反应序列。参与精子功能调节的磷酸化蛋白底物和激酶的表征将为受精过程的药理学控制提供新的靶点。