摘要
目的
探讨血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)对不育症精子获能、运动参数和胞内Ca~(2+)的影响及其可能的作用方式。
材料和方法
由32例精液常规正常、血清抗精子抗体阴性的不育症患者通过手淫获得精液样本并经上游优化法制备精子悬液。
本试验共分为三部分:
第一部分AngⅡ对不育症精子获能的影响
不同浓度的AngⅡ在体外与经上游优化处理的不育症精子悬液共同孵育,以孕酮诱发顶体反应,利用络合异硫氰酸荧光素的豌豆凝集素(FITC-PSA)标记精子顶体,该物质在488nm波长的激发光照射下,在510～550nm处发绿色荧光,通过流式细胞仪检测荧光强度,间接反映AngⅡ对精子获能的影响;
第二部分AngⅡ对不育症精子运动参数的影响
不同浓度的AngⅡ在体外与经上游优化处理的不育症精子悬液共同孵育,分别于20、40min利用计算机辅助精子分析(computer-assisted sperm analysis,CASA)检测人精子运动参数,评价不同时间段精子运动参数的变化;
第三部分AngⅡ对不育症精子胞内Ca~(2+)的影响及作用机制
不同浓度的AngⅡ在体外与经上游优化处理的不育症精子悬液共同孵育,采用Fura-2/AM作为Ca~(2+)探针,以350nm～380nm为激发波长,465nm为发射波长,经荧光分光光度计检测精子内游离Ca~(2+)荧光强度的变化以及Losartan(AngⅡ受体AT_1拮抗剂)、EGTA[ethyleneglycol bis(2-aminoethyl ether)tetraacetic acid,Ca~(2+)螯合剂]对其的抑制作用来评价。
结果
1、在未获能的精子悬液中AngⅡ(10nmol/L和100nmol/L)均可明显加速精子获能和增强孕酮诱发的顶体反应(p＜0.05);
2、AngⅡ(10nmol/L和100nmol/L)均能显著提高不育症患者精子活率及运动参数(p＜0.05);
3、AngⅡ(10nmol/L和100nmol/L)能与AT_1受体结合激发胞内Ca~(2+)浓度升高(p＜0.001),Losartan、EGTA可以明显阻断和抑制这些过程。
结论
AngⅡ对不育症精子能够显著促进获能和明显提高运动参数;其作用机制可能是通过AT_1受体介导,经改变精子细胞内Ca~(2+)的浓度来实现。本研究结果提示,AngⅡ对不育症精子功能有极为重要的影响,可在辅助生殖技术中被有效地使用,来为临床和科研工作服务。
Objective
To investigate the effect of angiotensinⅡ(AngⅡ)on the sperm motility and capacitation and intracellular calcium in male infertility in vitro and to explore its mechanism.
Material and Methods
Sperm were aseptically obtained by masturbation and prepared by swim-up technique from 32 infertile men.For the initial investigations,male infertility,all found to have normal semen parameters and negative antisperm antibody of blood serum.
The present study is composed of the following three sub-article:
Part one:Effect of AngiotensⅡon the capacitation in infertile men.
Spermatozoa suspensions was incubated with the different concentrations of AngⅡat 37℃in vitro and acrosome reaction induced by progesterone were determined by means of a flow cytometer.The capacitation effect were assessed using FITC-PSA analysis,which can radiate green fluorescence at 510～550nm wavelength as excited by 488nm wavelength laser.
Part two:Influence of angiotensinⅡon sperm motility in infertile men.
Spermatozoa suspensions was incubated with the different concentrations of AngⅡat 37℃in vitro.Measurements were carried out at 20,40min for patients in all specimens and motive parameters were measured by computer-aided sperm analysis(CASA).
Part three:Effect of AngiotensⅡon spermatozoa intracellular calcium in infertile men and to explore its mechanism.
Spermatozoa suspensions was incubated with the different concentrations of AngⅡat 37℃in vitro.Fluorescence intensity of spermatozoa intracellular Ca~(2+)were measured useing fluoreacent probe fura-2/AM by fluorospectrophotometer.Furthermore,the inhibiting effect of Losartana(a AngⅡblocker)and EGTA[ethyleneglycol bis(2-aminoethyl ether)tetraacetic acid] was also observed.
Results
1.In uncapacitated suspensions,AngⅡ(10nmol/L or 100nmol/L)significantly increased the acrosome reaction induced by progesterone and accelerated capacitation in uncapacitated sperm of infertile men(p＜0.05).
2.The results showed that AngⅡ(10nmol/L or 100nmol/L)can significantly affect infertile men spermatozoa motility incubated(p＜0.05).
3.In addition,same concentration of AngⅡstimulated increase in the intracellular Ca~(2+) concentration(p＜0.001).However,these effects of AngⅡwere obviously inhibited by Losartan (a AngⅡblocker)and prevented by EGTA.
Conclusion
These results indicat that AngⅡ(10nmol/L or 100nmol/L)can significantly accelerate capacitation in uncapacitated sperm and increase the sperm mobility of infertile men. Mechanisms of action of AngⅡmay be mediated by AT_1 receptor and accompanied by altering the intracellular Ca~(2+)concentration.The responses obtained in vitro suggest that AngⅡcould have significant effects on sperm function of infertile men in vivo,it could be used effectively in assisted reproductive technology and could also be served in infertility clinics and scientific research.
引文
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