小鼠精子体外培养过程中Ca2+对微管蛋白乙酰化的影响
Ca2+ Regulates the Acetylation of Tubulin in Mouse Sperm in Vitro

微管蛋白的乙酰化作用对细胞代谢过程,维持细胞的稳定性及运动性具有特殊作用,而有关哺乳动物精子蛋白乙酰化修饰的研究报道较少,且成熟精子蛋白乙酰化作用机制尚未明确。本研究利用精子活力参数计算机辅助检测(CASA)技术、蛋白免疫印迹(WB)以及免疫荧光等实验技术,分析测定小鼠精子体外培养过程中,不同浓度Ca2+以及HCO-3、环腺苷酸(dbcAMP)等精子获能调节因子对微管蛋白乙酰化的影响。结果表明,小鼠精子中分子量约为55、37、26 kDa微管蛋白发生乙酰化修饰,获能培养后微管蛋白乙酰化程度明显高于未获能培养;钙离子对精子运动能力以及微管蛋白乙酰化修饰起双重调节作用,低浓度Ca2+(≤ 2.0 mmol/L)促进精子活力及微管蛋白乙酰化(P<0.05),而高浓度Ca2+ (4.0 mmol/L)明显抑制精子活力MOT (48.88%)、曲线运动速度VCL (99.46 μm/s)及微管蛋白乙酰化(P<0.05);加入钙离子级联信号调节因子钙调蛋白抑制剂(W7)和钙调蛋白激酶抑制剂(KN-93),能有效缓解高浓度Ca2+ (4.0 mmol/L)对微管蛋白乙酰化的抑制作用,而钙离子载体(A23187)能进一步增强高浓度Ca2+对小鼠精子微管蛋白乙酰化的抑制作用;dbcAMP、异丁基黄嘌呤(IBMX)及PKA抑制剂(H-89)等cAMP-PKA磷酸化信号因子影响微管蛋白乙酰化,但不符合cAMP-PKA磷酸化信号通路调节规律。免疫荧光结果显示乙酰化微管蛋白主要分布在精子尾部的中段和主段,且主段最为明显。本研究提示Ca2+可能通过调节微管蛋白的乙酰化,从而影响精子的活力及受精能力。
Acetylation of tubulin has a special role in regulating cell metabolism and maintaining cell stability and motility.However,protein acetylation,one of the post-translational modifications (PTMs),has not been well studied in mammalian sperm.The influence mechanism of protein acetylation on mature sperm is still unclear.The objective of the present work was to investigate the impact of Ca2+ concentrations and signal factors such as HCO-3 and 3’-5’-cyclic adenosine (cAMP) analog dibutytyl-cAMP (dbcAMP) on tubulin acetylation of mouse sperm in vitro.Computer-assisted sperm analysis (CASA),Western blot (WB) and immunofluorescence were used in the present experiment.Our results showed that,acetylation of α-tubulin on 55,7,26 kDa were detected,the acetylation level in capacitated sperm was higher than that in un-capacitated sperm.Ca2+ played a biphasic role in regulating tubulin acetylation and sperm motility.Low concentration of Ca2+ (≤2.0 mmol/L) promoted sperm motility and tubulin acetylation (P<0.05).However,under high extracellular Ca2+concentration (4.0 mmol/L),sperm motility (MOT) (48.88%) and curvilinear velocity (VCL) (99.46 μm/s) were distinctly decreased and tubulin acetylation was inhibited compared with non-capacitation treatment (P<0.05).Interestingly,the negative effect of high concentration of Ca2+ (4.0 mmol/L) could be relieved by addition of calmodulin inhibitors W7 and calmodulin kinase inhibitor KN-93.However,the inhibition of Ca2+ (4.0 mmol/L) on acetylation could be further enhanced with the treatment of calcium ionophore A23187.In addition,the cAMP-PKA signal factors such as dbcAMP,phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and N-[2-(p-Bromocinnamylamino) ethyl]-5-isoquinolinesufonamide·2HCL (H-89) promoted the acetylation of α-tubulin,but the promotion mechanism did not conform to the cAMP-PKA signaling