Li Xinhong, Wang Lirui, Li Yuhua, Zhao Na, Zhen Linqing, Fu Jieli, Yang Qiangzhen
Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
Shanghai Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China.
Anim Reprod Sci. 2016 Sep;172:39-51. doi: 10.1016/j.anireprosci.2016.07.001. Epub 2016 Jul 2.
Considering the importance of calcium (Ca(2+)) in regulating sperm capacitation, hyperactivation and acrosome reaction, little is known about the molecular mechanism of action of this ion in this process. In the present study, assessment of the molecular mechanism from the perspective of energy metabolism occurred. Sperm motility variables were determined using computer-assisted sperm analysis (CASA) and the phosphorylation of PKA substrates, tyrosine residues and AMP-activated protein kinase (AMPK) were analyzed by Western blot. Moreover, intracellular sperm-specific glyceraldehyde 3-phosphatedehydrogenase (GAPDH) activity, 3'-5'-cyclic adenosine monophosphate (cAMP) and adenosine 5'-triphosphate (ATP) concentrations were assessed in boar sperm treated with Ca(2+). Results of the present study indicated that, under greater extracellular Ca(2+)concentrations (≥3.0mM), sperm motility and protein phosphorylation were inhibited. Interestingly, these changes were correlated with that of GAPDH activity, AMPK phosphorylation, cAMP and ATP concentrations. The negative effects of Ca(2+) on these intracellular processes were attenuated by addition of the calmodulin (CaM) inhibitor W7 and the inhibitor of calmodulin-dependent protein kinase (CaMK), KN-93. In the presence of greater extracellular Ca(2+), however, the phosphorylation pathway was suppressed by H-89. Taken together, these results suggested that Ca(2+) had a dual role in regulating boar sperm motility and protein phosphorylation due to the changes of cAMP and ATP concentrations, in response to cAMP-mediated signal transduction and the Ca(2+) signaling cascade. The present study provided some novel insights into the molecular mechanism underlying the effects of Ca(2+) on boar sperm as well as the involvement of energy metabolism in this mechanism.
鉴于钙（Ca(2+)）在调节精子获能、超激活和顶体反应中的重要性，关于该离子在此过程中的分子作用机制却知之甚少。在本研究中，从能量代谢的角度对分子机制进行了评估。使用计算机辅助精子分析（CASA）测定精子活力变量，并通过蛋白质免疫印迹分析蛋白激酶A（PKA）底物、酪氨酸残基和AMP激活的蛋白激酶（AMPK）的磷酸化情况。此外，还评估了用Ca(2+)处理的公猪精子中细胞内精子特异性甘油醛-3-磷酸脱氢酶（GAPDH）活性、3',5'-环磷酸腺苷（cAMP）和三磷酸腺苷（ATP）浓度。本研究结果表明，在较高的细胞外Ca(2+)浓度（≥3.0 mM）下，精子活力和蛋白质磷酸化受到抑制。有趣的是，这些变化与GAPDH活性、AMPK磷酸化、cAMP和ATP浓度的变化相关。添加钙调蛋白（CaM）抑制剂W7和钙调蛋白依赖性蛋白激酶（CaMK）抑制剂KN-93可减弱Ca(2+)对这些细胞内过程的负面影响。然而，在存在较高细胞外Ca(2+)的情况下，H-89可抑制磷酸化途径。综上所述，这些结果表明，由于cAMP和ATP浓度的变化，Ca(2+)在调节公猪精子活力和蛋白质磷酸化方面具有双重作用，这与cAMP介导的信号转导和Ca(2+)信号级联反应有关。本研究为Ca(2+)对公猪精子影响的分子机制以及能量代谢在该机制中的作用提供了一些新的见解。
