电压依赖性钙通道在人类弱精子症精子中的改变
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摘要
目的:探讨电压依赖性钙通道病变导致人类弱精子症的发生作用及其机制。
方法:将不同浓度的特异性L-型钙通道拮抗剂硝苯地平在体外与生育男性经上游优化处理的精液分别共同孵育10、20、30、60 min ,采用计算机辅助的精子分析系统分别检测精子的运动参数,包括精子活率、精子活力分级、畸形率、平均路径速度、精子侧摆幅度和摆动幅度;然后应用半定量反转录酶-聚合酶链式反应(RT-PCR)技术分别检测正常人类运动精子和弱精子症精子中各类型电压依赖性钙通道的功能性亚单位α1 mRNA相对表达量的变化;最后应用实时荧光定量逆转录-多聚酶链反应(real-time qPCR)、免疫印迹(Western blotting)和荧光免疫细胞化学技术分别检测L型VDCC功能性α1C亚单位在正常人类运动精子和弱精子症精子中的变化。
结果:硝苯地平在体外既对人精子形态有明显性改变,也对精子活率、精子活力分级(a+b级百分率)、精子侧摆幅度和摆动幅度等指标的影响差异有非常显著性(p<0.001);与正常运动精子相比,弱精子症精子中电压依赖性钙通道的α1B、α1C、α1E、α1G、α1H mRNA表达的差异具有极显著意义(p<0.01),其中弱精子症精子中的L型VDCCα1C分别在分子水平、蛋白水平和细胞水平上的差异都存在着显著性变化。
结论: L-型钙通道拮抗剂在体外可直接导致可逆性的精子运动能力下降,L型和/或非L型电压依赖性钙通道都存在于精子细胞膜上,是调控正常人类精子运动的主要电压依赖性钙通道,并且进一步证明L型VDCCα1C功能性亚单位基因的改变显著影响着正常人类精子的运动,导致男性不育,并将成为研究人类弱精子症病因和治疗人类弱精子症的新靶点。
Purpose : To explore the relationship between the changes of the voltage-dependent calcium channels functional in the asthenospermia sperm and asthenospermia
Methods: Sperms were aseptically obtained by masturbation and prepared by swim-up technique to produce a spermatozoa solution of high motility from 20 healthy fertile men. The spermatozoa solution was incubated with the different types CCBs at 37℃in vitro. Motive parameters were measured by computer-aided sperm analysis (CASA). Measurements were carried out at 10,20,30 and 60 minutes in all specimens included live rate, progressive motility, malformed rate, VAP, ALH and WOB. then use reverse transcription polymerase chain reaction to examine and quantitate the multitype VDCCsα1 mRNA expressions between the normal and the asthenospermia. The last to use Real-time qPCR, Western blotting and Immunocytochemical technique to examine the changes of L type VDCCα1C between the normal and the asthenospermia.
Results: The results showed that the nifedipine can not only significantly affect human sperm’s shape but also spermatozoa motility incubated in vitro (p<0.001). Comparing with the normal sperms, there are the markedly differences in the expressions of the asthenospermia’sα1B、α1C、α1E、α1G andα1H mRNA messages(p<0.01). and there are the markedly differences of the asthenospermia’s L type VDCCα1C at molecular level, protein level and cellular level.
Conclusions:L-type CCB can directly cause reversible male infertility in vitro. Based on VDCC mRNAs relative expression levels, we propose that both L-type and non-L-type VDCCs all may be gates for the external calcium influx, required for the motility. Changes of the L type VDCCα1C gene influenced motility of normal sperm significantly, and it would be a new target of the study and treatment in asthenospermia.
方法:将不同浓度的特异性L-型钙通道拮抗剂硝苯地平在体外与生育男性经上游优化处理的精液分别共同孵育10、20、30、60 min ,采用计算机辅助的精子分析系统分别检测精子的运动参数,包括精子活率、精子活力分级、畸形率、平均路径速度、精子侧摆幅度和摆动幅度;然后应用半定量反转录酶-聚合酶链式反应(RT-PCR)技术分别检测正常人类运动精子和弱精子症精子中各类型电压依赖性钙通道的功能性亚单位α1 mRNA相对表达量的变化;最后应用实时荧光定量逆转录-多聚酶链反应(real-time qPCR)、免疫印迹(Western blotting)和荧光免疫细胞化学技术分别检测L型VDCC功能性α1C亚单位在正常人类运动精子和弱精子症精子中的变化。
结果:硝苯地平在体外既对人精子形态有明显性改变,也对精子活率、精子活力分级(a+b级百分率)、精子侧摆幅度和摆动幅度等指标的影响差异有非常显著性(p<0.001);与正常运动精子相比,弱精子症精子中电压依赖性钙通道的α1B、α1C、α1E、α1G、α1H mRNA表达的差异具有极显著意义(p<0.01),其中弱精子症精子中的L型VDCCα1C分别在分子水平、蛋白水平和细胞水平上的差异都存在着显著性变化。
结论: L-型钙通道拮抗剂在体外可直接导致可逆性的精子运动能力下降,L型和/或非L型电压依赖性钙通道都存在于精子细胞膜上,是调控正常人类精子运动的主要电压依赖性钙通道,并且进一步证明L型VDCCα1C功能性亚单位基因的改变显著影响着正常人类精子的运动,导致男性不育,并将成为研究人类弱精子症病因和治疗人类弱精子症的新靶点。
Purpose : To explore the relationship between the changes of the voltage-dependent calcium channels functional in the asthenospermia sperm and asthenospermia
Methods: Sperms were aseptically obtained by masturbation and prepared by swim-up technique to produce a spermatozoa solution of high motility from 20 healthy fertile men. The spermatozoa solution was incubated with the different types CCBs at 37℃in vitro. Motive parameters were measured by computer-aided sperm analysis (CASA). Measurements were carried out at 10,20,30 and 60 minutes in all specimens included live rate, progressive motility, malformed rate, VAP, ALH and WOB. then use reverse transcription polymerase chain reaction to examine and quantitate the multitype VDCCsα1 mRNA expressions between the normal and the asthenospermia. The last to use Real-time qPCR, Western blotting and Immunocytochemical technique to examine the changes of L type VDCCα1C between the normal and the asthenospermia.
Results: The results showed that the nifedipine can not only significantly affect human sperm’s shape but also spermatozoa motility incubated in vitro (p<0.001). Comparing with the normal sperms, there are the markedly differences in the expressions of the asthenospermia’sα1B、α1C、α1E、α1G andα1H mRNA messages(p<0.01). and there are the markedly differences of the asthenospermia’s L type VDCCα1C at molecular level, protein level and cellular level.
Conclusions:L-type CCB can directly cause reversible male infertility in vitro. Based on VDCC mRNAs relative expression levels, we propose that both L-type and non-L-type VDCCs all may be gates for the external calcium influx, required for the motility. Changes of the L type VDCCα1C gene influenced motility of normal sperm significantly, and it would be a new target of the study and treatment in asthenospermia.
引文
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8. Publicover SJ, Barratt CL. Voltage-operated Ca2+ channels and the acrosome reaction: which channels are present and what do they do? [J].Hum Reprod, 1999,14(4):873-879.
9. Goodwin LO, Karabinus DS, Pergolizzi RG, Benoff S. L-type voltage-dependent calcium channel alpha-1C subunit mRNA is present in ejaculated human spermatozoa[J]. Mol Hum Reprod, 2000,6(2):127-136.
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2. World Health Organization. The global burden of reproductive health[J]. Progress in human reproduction research, 1997, 42:2-3.
3. Niederberger C. The clinical conundrum of complete asthenospermia[J]. Androl,2004,25(5):679-680.
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6. Jagannathan S, Punt EL, Gu Y, Arnoult C, Sakkas D, Barratt CL, Publicover SJ. Identification and localization of T-type voltage-operated calcium channel subunits in human male germ cells. Expression of multiple isoforms[J]. Biol Chem, 2002,277(10):8449-8456.
7. Publicover SJ, Barratt CL. Voltage-operated Ca2+ channels and the acrosome reaction: which channels are present and what do they do? [J].Hum Reprod, 1999,14(4):873-879.
8. Goodwin LO, Karabinus DS, Pergolizzi RG, Benoff S. L-type voltage-dependent calcium channel alpha-1C subunit mRNA is present in ejaculated human spermatozoa[J]. Mol Hum Reprod, 2000,6(2):127-136.
9. Park JY, Ahn HJ, Gu JG, et al. Molecular identification of Ca2+ channels in human sperm [J]. Exp Mol Med,2003,35(4):285-292.
10. Sakata Y, Saegusa H, Zong S, et al. Ca(v)2.3 (alpha1E) Ca2+ channelparticipates in the control of sperm function[J]. FEBS Lett,2002,516(1-3):229-233.
11. Sakata Y, Saegusa H, Zong S, et al. Analysis of Ca(2+) currents in spermatocytes from mice lacking Ca(v)2.3 (alpha(1E)) Ca(2+) channel[J]. Biochem Biophys Res Commun,2001,288(4):1032-1036.
12. Trevino CL, Felix R, Castellano LE et al. Expression and differential cell distribution of low-threshold Ca(2+) channels in mammalian male germ cells and sperm[J]. FEBS Lett,2004,563(1-3):87-92.
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13 Bains R, Miles DM, Carson RJ, Adeghe J. Hyaluronic acid increases motility/intracellular CA2+ concentration in human sperm in vitro. Arch Androl, 2001; 47(2): 119-125.
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16 Jagannathan S, Punt EL, Gu Y, Arnoult C, Sakkas D, Barratt CL, Publicover SJ. Identification and localization of T-type voltage-operated calcium channel subunits in human male germ cells. Expression of multiple isoforms. Biol Chem, 2002; 277(10): 8449-8456.
17 Goodwin LO, Karabinus DS, Pergolizzi RG, Benoff S. L-type voltage-dependent calcium channel alpha-1C subunit mRNA is present in ejaculated human spermatozoa. Mol Hum Reprod, 2000; 6(2): 127-136.
18 Park JY, Ahn HJ, Gu JG, et al. Molecular identification of Ca2+ channels in human sperm [J]. Exp Mol Med. 2003; 35(4): 285-92.
19 Trevino CL, Felix R, Castellano LE, et.al. Expression and differential cell distribution of low-threshold Ca(2+) channels in mammalian male germ cells and sperm [J]. FEBS Lett. 2004; 563(1-3): 87-92.
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