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。女性生殖道发生的使精子能够受精的生理变化构成了“精子获能”现象。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磷酸化增加有关(Visconi等人，1995A；Visconi等人，2002年)。我们还证明了蛋白酪氨酸磷酸化和获能的增加是由涉及蛋白激酶A(PKA)的cAMP依赖的途径调节的(Visconi等人，1997；Visconti等人，1995 b)。随后，在牛(Galantino-Hmer等人，1997)、人(Leclerc等人，1996；Osheroff等人，1999)、猪(Kalab等人，1998)和仓鼠(Visconi等人，1999)等物种的精子中证明了这一调控途径的存在。尽管在理解获能过程中的磷酸化调控机制方面取得了这些进展，但对于这种磷酸化级联反应的蛋白靶标以及参与获能调控的激酶和磷酸酶的同一性还知之甚少。这项建议的目的是确定在获能过程中受磷酸化调控的蛋白质。此外，我们还将努力确定参与调节人和小鼠精子获能的激酶(S)，并进一步阐明控制蛋白质Tyr磷酸化和获能的反应序列。支持这一假设的假设是，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蛋白)参与了哺乳动物精子获能状态的调节。对参与获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。为此，我们提出了以下目标：1)鉴定精子获能过程中经历磷酸化的蛋白质；2)鉴定和表征精子中存在的与获能有关的蛋白。3)探讨获能过程中精子激酶和磷酸化蛋白的调节及功能。

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". Using the mouse as an experimental model, we have demonstrated that capacitation is associated with an increase in the tyrosine phosphorylation of a subset of proteins (Visconti et al., 1995a; Visconti et al., 2002). We have also demonstrated that the increase in protein tyrosine phosphorylation as well as capacitation were regulated by a cAMP-dependent pathway involving protein kinase A (PKA) (Visconti et al., 1997; Visconti et al., 1995b). The presence of this regulatory pathway has subsequently been demonstrated in sperm from other species such as bovine (Galantino-Homer et al., 1997), human (Leclerc et al., 1996; Osheroff et al., 1999), boar (Kalab et al., 1998) and hamster (Visconti et al., 1999c). 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 capacitation. The objective of this proposal is to identify the proteins regulated by phosphorylation during capacitation. In addition, our efforts will be conducted to identify the kinase(s) involved in the regulation of capacitation of human and mouse sperm and to further elucidate the sequence of reactions that control protein tyr phosphorylation and capacitation. The hypotheses underlying this proposal postulate that proteins specifically phosphorylated during capacitation (as well as the kinases responsible for their phosphorylation) are involved in the regulation of the capacitation state of mammalian sperm. Characterization of the phosphorylated protein substrates and the kinase(s) involved in the regulation of capacitation will provide novel targets for pharmacological control of the fertilization process. To these ends, we propose the following aims: 1) To identify proteins which undergoes phosphorylation during sperm capacitation, 2) To identify and characterize kinases present in sperm playing a role in capacitation. 3) To investigate the regulation and function of sperm kinases and of proteins undergoing phosphorylation during capacitation.