Ashizawa K, Wishart G J, Katayama S, Takano D, Ranasinghe A R A H, Narumi K, Tsuzuki Y
Laboratory of Animal Reproduction, Faculty of Agriculture, University of Miyazaki, Japan.
Reproduction. 2006 Jun;131(6):1017-24. doi: 10.1530/rep.1.01069.
The signal transduction pathways involved in the regulation of the acrosome reaction and motility of fowl spermatozoa were investigated. The motility and acrosomal integrity of fowl spermatozoa in TES/NaCl buffer, with or without homogenised inner perivitelline layers (IPVL), prepared from laid fowl eggs, was almost negligible at 40 degrees C. In the presence of 2 mmol CaCl(2)/l at 40 degrees C, motility became vigorous and the acrosome reaction was stimulated when IPVL was added. In the absence of Ca(2+), motility was stimulated by the addition of calyculin A and okadaic acid, both specific inhibitors of protein phosphatase-type 1 (PP1) and -type 2A (PP2A), but Okadaic acid, which is a weaker inhibitor of PP1, did not completely restore motility at 40 degrees C. However, the acrosome reaction was significantly and equally stimulated in a dose-dependent manner by both inhibitors in the range of 10-1000 nmol/l, when spermatozoa were incubated with IPVL but without Ca(2+). These inhibitors did not stimulate the acrosome reaction in the absence of IPVL. The vigorous motility of spermatozoa, stimulated by the addition of Ca(2+), was reduced gradually as the concentrations of SC-9, a selective activator of protein kinase C (PKC), were increased and a similar SC-9-induced inhibition was observed in the acrosome reaction in the presence of Ca(2+) and IPVL. These results confirm that IPVL is necessary for the activation of the acrosome reaction in fowl spermatozoa and that Ca(2+) plays an important role in the stimulation of motility and acrosomal exocytosis. Furthermore, it appears that the intracellular molecular mechanisms for the regulation of acrosome reaction of fowl spermatozoa are different from those for the restoration of motility, i.e., protein dephosporylation involving PP1 and/or PP2A in the former, and PP1 alone in the latter case. In addition, the activation of PKC may contribute to a decrease in the flagellar movement and acrosome reaction of fowl spermatozoa.
研究了参与调控家禽精子顶体反应和运动性的信号转导途径。在40℃时，置于含有或不含有从产蛋家禽卵制备的匀浆内卵黄膜层（IPVL）的TES/NaCl缓冲液中的家禽精子，其运动性和顶体完整性几乎可以忽略不计。在40℃且存在2 mmol CaCl₂/L的情况下，当添加IPVL时，精子运动变得活跃，顶体反应受到刺激。在无Ca²⁺时，添加特异性蛋白磷酸酶1型（PP1）和2A型（PP2A）抑制剂花萼海绵诱癌素A和冈田酸可刺激精子运动，但冈田酸作为PP1的较弱抑制剂，在40℃时不能完全恢复精子运动。然而，当精子与IPVL一起孵育但无Ca²⁺时，在10 - 1000 nmol/L范围内，两种抑制剂均以剂量依赖性方式显著且同等程度地刺激顶体反应。在无IPVL时，这些抑制剂不刺激顶体反应。添加Ca²⁺所刺激的精子活跃运动，随着蛋白激酶C（PKC）的选择性激活剂SC - 9浓度的增加而逐渐降低，并且在存在Ca²⁺和IPVL的情况下，在顶体反应中也观察到类似的SC - 9诱导的抑制作用。这些结果证实，IPVL是激活家禽精子顶体反应所必需的，并且Ca²⁺在刺激精子运动和顶体胞吐作用中起重要作用。此外，似乎家禽精子顶体反应调控的细胞内分子机制与运动恢复的机制不同，即前者涉及PP1和/或PP2A的蛋白去磷酸化，而后者仅涉及PP1。此外，PKC的激活可能导致家禽精子鞭毛运动和顶体反应的减少。
