Varano Gabriele, Lombardi Adriana, Cantini Giulia, Forti Gianni, Baldi Elisabetta, Luconi Michaela
Department of Clinical Physiopathology, Endocrinology Unit, DENOTHE Center for Research, Transfer and High Education, University of Florence, Florence, Italy.
Hum Reprod. 2008 Dec;23(12):2652-62. doi: 10.1093/humrep/den314. Epub 2008 Aug 26.
Protein tyrosine phosphorylation is one of the main processes associated with sperm activation. Although this process and its targets have been well characterized, only few tyrosine kinases have been identified so far and their roles in spermatozoa are still largely unknown. In this study, we report the presence and localization of Src kinase in ejaculated human spermatozoa and investigate its role in regulating the processes underlying sperm activation.
Specific anti-Src antibodies, against different epitopes of the protein, identified a single band of approximately 70 kDa relating to a protein which is mainly localized in the post-acrosomal region of the head, neck and midpiece. By immunoprecipitation and immunofluorescence techniques performed with antibodies against Src phosphorylated at Tyr416, which identifies the active kinase, we showed an increased phosphorylation during sperm capacitation. Blocking Src activity with SU6656 resulted in a significant reduction in the protein tyrosine phosphorylation. Moreover, this inhibitor also blocked the progesterone-induced acrosome reaction and interfered with the calcium response to progesterone evaluated in fura-2-loaded spermatozoa. No effect on sperm motility and hyperactivation resulted from incubation with SU6656.
We identified a novel Src isoform in human spermatozoa, which appears to be involved in regulating sperm capacitation, calcium fluxes, tyrosine phosphorylation and acrosome reaction.
蛋白质酪氨酸磷酸化是与精子激活相关的主要过程之一。尽管这一过程及其靶点已得到充分表征，但迄今为止仅鉴定出少数酪氨酸激酶，它们在精子中的作用仍大多未知。在本研究中，我们报告了Src激酶在射出的人类精子中的存在和定位，并研究了其在调节精子激活相关过程中的作用。
针对该蛋白不同表位的特异性抗Src抗体识别出一条约70 kDa的单带，该蛋白主要定位于头部、颈部和中段的顶体后区域。通过使用针对在Tyr416位点磷酸化的Src的抗体进行免疫沉淀和免疫荧光技术（该位点可识别活性激酶），我们发现精子获能过程中磷酸化增加。用SU6656阻断Src活性导致蛋白酪氨酸磷酸化显著降低。此外，该抑制剂还阻断了孕酮诱导的顶体反应，并干扰了用fura - 2负载的精子中对孕酮的钙反应。用SU6656孵育对精子活力和超激活没有影响。
我们在人类精子中鉴定出一种新的Src异构体，它似乎参与调节精子获能、钙通量、酪氨酸磷酸化和顶体反应。
