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）：哺乳动物的精子在射精后无法立即施肥。他们在居住在女性区域中的有限时间段内后获得施肥能力。在女性生殖道中发生的生理变化，使精子能够受精的精子构成了“精子能力”的现象（Austin，1952； Chang，1951）。使用小鼠作为实验模型，我们已经证明了电容与蛋白质子集的酪氨酸（Tyr）磷酸化的增加有关（Visconti等，1995a）。我们还证明，蛋白质Tyr磷酸化以及电容的增加受到cAMP依赖性途径的调节（Visconti等，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）。尽管在理解调节磷酸化过程中调节磷酸化的机制方面取得了进展，但对这种磷酸化级联反应的蛋白质靶标的身份以及参与精子功能调节的激酶和磷酸酶的认同知之甚少。最近，我们使用了2维（2D）聚丙烯酰胺凝胶电泳（PAGE）方法，结合了串联质谱法（MS/MS）分析（Ficarro等，2003）来识别在人类精子中能力期间经历py磷酸化的蛋白质。在同一项工作中，我们分析了来自电容的人类精子群中MS/MS的精确磷酸化序列，并鉴定出在Tyr和Tyr和丝氨酸（Ser）和苏氨酸（THR）中磷酸化的序列（Ficarro等，2003）。在蛋白质靶标中，含瓣膜蛋白的蛋白质（VCP），一种来自同一家族的ATPase在同一家族中，发现在电容期间被发现在电容过程中被磷酸化。另外，VCP的免疫定位显示出伴随精子电容的荧光模式变化。该提案的基本假设结合了上述发现，并假定在电容过程中特异性磷酸化以及负责其磷酸化的激酶的蛋白质是调节哺乳动物精子能力所必需的。该提案的目的是进一步阐明控制蛋白质磷酸化精子中蛋白质磷酸化级联反应的序列。磷酸化蛋白底物的表征和所涉及的精子功能调节的激酶将为受精过程的药理控制提供新的靶标。