Phosphorylation Events During Sperm Capacitation

描述(申请人提供)：哺乳动物的精子不能在射精后立即使卵子受精。它们在雌性体内居住了一段有限的时间后就获得了受精能力。女性生殖道中发生的使精子能够受精的生理变化构成了“精子获能”现象(Austin，1952；Chang，1951)。使用小鼠作为实验模型，我们已经证明了获能与蛋白质子集的酪氨酸(Tyr)磷酸化增加有关(Visconi等人，1995A)。我们还证明了蛋白质Tyr磷酸化和获能的增加是由cAMP依赖的途径调节的(Visconi等人，1995b)。随后在包括人类在内的其他物种的精子中证实了这种调控途径的存在(Leclerc等人，1996；Osheroff等人，1999)。尽管在获能过程中调节磷酸化的机制方面取得了这些进展，但对于这一磷酸化级联反应的蛋白靶标以及参与调节精子功能的激酶和磷酸酶的同一性知之甚少。最近，我们使用二维(2D)聚丙烯酰胺凝胶电泳法(PAGE)结合串联质谱仪(MS/MS)分析(Ficarro等人，2003)来鉴定在人类精子获能过程中经历Tyr磷酸化的蛋白质。在同一项工作中，我们用MS/MS分析了一组获能人类精子中蛋白质的准确磷酸化序列，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年)。在蛋白质靶标中，含有Valosin的蛋白(VCP)被发现在获能过程中被酪氨酸磷酸化。VCP是SNARE相互作用蛋白N-乙基马来酰亚胺可溶性因子(NSF)的同一家族的ATPase。此外，VCP的免疫定位显示，随着精子获能，荧光模式发生了变化。支持这一建议的假设结合了上述一组发现和假设，即在获能过程中特定磷酸化的蛋白质以及负责其磷酸化的蛋白是调节哺乳动物精子获能所必需的。这项建议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。对参与精子功能调节的磷酸化蛋白底物和激酶(S)的表征将为受精过程的药物控制提供新的靶点。

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.