树鼩精子特异性钙通道CatSper生化特性及功能研究

Biochemical Characteristics and Functional Studies of the Sperm-specific Calcium Channel CatSper in Tupaia belangeri

【目的】探究树鼩精子中的特异性钙通道CatSper生化特性及其在树鼩精子中的亚细胞定位,并阐明CatSper对树鼩精子运动的调控作用。【方法】选择性成熟、1~2岁龄雄性树鼩,用CO_(2)处死后采集树鼩精液,统计精子活力并提取蛋白质。利用Western blotting和免疫组化检测精子中CatSper家族存在情况及其亚细胞定位。通过显微镜观察不同钙离子通道抑制剂(CatSper抑制剂HC-056456,瞬时受体电位香草酸亚型1(TRPV1)抑制剂Capsazepine)与孕酮(P_(4))共孵育后树鼩精子的运动度。使用钙离子探针法检测不同抑制剂和P_4共孵育对树鼩精子钙离子浓度的影响。【结果】树鼩精子中存在CatSper及TRPV1通道蛋白,CatSper定位于精子头部及鞭毛。与对照组相比,当HC-056456浓度为100、200、400、1 000 nmol/L时,树鼩精子运动度和钙离子浓度均显著降低(P<0.05),Capsazepine浓度为10、20、40μmol/L时,树鼩精子运动度和钙离子浓度均显著降低(P<0.05)。与Capsazepine组相比,HC-056456与P_4共孵育时,树鼩精子钙离子浓度明显升高,且当P_4浓度为10、20、30μmol/L时两组间差异显著(P<0.05)。Capsazepine、HC-056456与P_(4)共孵育时,两组间树鼩精子运动度无显著差异(P>0.05)。【结论】CatSper通道对树鼩精子运动和钙离子转运调控至关重要,通过P_4激活从而提高精子内钙离子浓度,本研究结果对于深入研究精子功能及精子受精能力调控机制具有重要意义。

【Objective】The purpose of this experiment was to explore the biochemical characteristics of specific calcium channel CatSper and its subcellular location in Tupaia belangeri sperm,and to clarify the regulatory role of CatSper on the motility of Tupaia belangeri sperm.【Method】Sexually mature male Tupaia belangeri aged 1 to 2 years were selected,semen was collected after being killed with CO_(2),sperm motility was observed and protein was extracted.Western blotting and immunohistochemistry were used to detect the presence and subcellular localization of CatSper family in sperm.Different calcium channel inhibitors(CatSper inhibitor HC-056456,transient receptor potential vanillate subtype 1(TRPV1)inhibitor Capsazepine)co-incubated with progesterone(P_(4))were used to observe the motility of Tupaia belangeri sperm.Calcium probe method was used to detect the effects of different inhibitors and P_(4) co-incubation on calcium ion concentration of Tupaia belangeri sperm.【Result】CatSper and TRPV1 channel protein were present in Tupaia belangeri sperm,and CatSper was located in the sperm head and flagella.Compared with control group,sperm motility and calcium ion concentration were significantly decreased when HC-056456 concentrations were 100,200,400 and 1000 nmol/L(P<0.05),and Capsazepine concentrations were 10,20 and 40μmol/L,sperm motility and calcium ion concentration were significantly decreased(P<0.05).Compared with Capsazepine group,when HC-056456 was co-incubated with P_(4),the calcium ion concentration of Tupaia belangeri sperm was significantly increased,and there were significant differences between the two groups when P_(4) concentrations were 10,20 and 30μmol/L(P<0.05).When Capsazepine and HC-056456 were co-incubated with P_(4),there was no significant difference in sperm motility between the two groups(P>0.05).【Conclusion】CatSper channel played an important role in the regulation of sperm motility and calcium ion transport in Tupaia belangeri,and the calcium ion concentration in sperm was increased through P_(4) activation.The results were of great significance for further research on the regulatory mechanism of sperm function and sperm fertilization ability.