Rathi R, Colenbrander B, Stout T A E, Bevers M M, Gadella B M
Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Mol Reprod Dev. 2003 Jan;64(1):120-8. doi: 10.1002/mrd.10216.
Progesterone (P(4)) is a physiological inducer of the acrosome reaction (AR) in stallion spermatozoa. However, the capacitation-dependent changes that enable progesterone binding, and the nature of the signaling cascade that is triggered by progesterone and results in induction of the AR, are poorly understood. The aim of the current study was, therefore, to investigate the protein kinase dependent signaling cascades involved in progesterone-mediated induction of the AR in stallion spermatozoa. In addition, we aimed to determine whether bicarbonate, an inducer of sperm capacitation, acted via the same pathway as P(4) or whether it otherwise synergized P(4)-mediated induction of the AR. We examined the effect on AR progression of specific inhibitors and stimulators of protein kinase A (PKA), protein kinase C (PKC), protein kinase G (PKG), and protein tyrosine kinase (PTK), in the presence or absence of 15 mM bicarbonate and/or 1 microg/ml progesterone. Progression of the AR was assessed using the Pisum sativum agglutinin conjugated to fluorescein iso thiocyanate (PSA-FITC) staining technique. Bicarbonate specifically activated a PKA-dependent signaling pathway, whereas the effect of P(4) was independent of PKA. Conversely, while P(4)-mediated AR induction was dependent on PTK, the effects of bicarbonate were PTK-independent. Finally, although the AR inducing effects of both P(4) and bicarbonate were sensitive to staurosporin, a potent blocker of PKC activity at moderate (50 nM) concentrations, the effect of P(4) was completely blocked at 50 nM staurosporin, whereas that of bicarbonate was only completely inhibited by much higher concentrations (2 microM) where staurosporin also inhibits PKA activity. In conclusion, P(4)-mediated activation of the AR is dependent on a pathway that includes both PTK and PKC. While the effects of bicarbonate on the AR are mediated via a separate PKA-dependent signaling pathway, P(4) and bicarbonate have synergistic effects on the AR.
孕酮（P(4)）是种马精子顶体反应（AR）的生理诱导剂。然而，使孕酮结合的获能依赖性变化以及由孕酮触发并导致顶体反应诱导的信号级联的性质，目前还知之甚少。因此，本研究的目的是调查参与孕酮介导的种马精子顶体反应诱导的蛋白激酶依赖性信号级联。此外，我们旨在确定精子获能诱导剂碳酸氢盐是否通过与P(4)相同的途径起作用，或者它是否以其他方式协同P(4)介导的顶体反应诱导。我们研究了在存在或不存在15 mM碳酸氢盐和/或1微克/毫升孕酮的情况下，蛋白激酶A（PKA）、蛋白激酶C（PKC）、蛋白激酶G（PKG）和蛋白酪氨酸激酶（PTK）的特异性抑制剂和刺激剂对顶体反应进程的影响。使用与异硫氰酸荧光素偶联的豌豆凝集素（PSA-FITC）染色技术评估顶体反应的进程。碳酸氢盐特异性激活了一条依赖PKA的信号通路，而P(4)的作用不依赖PKA。相反，虽然P(4)介导的顶体反应诱导依赖于PTK，但碳酸氢盐的作用不依赖PTK。最后，尽管P(4)和碳酸氢盐的顶体反应诱导作用对星形孢菌素敏感，星形孢菌素在中等（50 nM）浓度下是PKC活性的有效阻断剂，但在50 nM星形孢菌素时P(4)的作用被完全阻断，而碳酸氢盐的作用仅在更高浓度（2 microM）时被完全抑制，此时星形孢菌素也抑制PKA活性。总之，P(4)介导的顶体反应激活依赖于一条包括PTK和PKC的信号通路。虽然碳酸氢盐对顶体反应的作用是通过一条独立的依赖PKA的信号通路介导的，但P(4)和碳酸氢盐对顶体反应有协同作用。
