孕酮对精子获能及顶体反应影响的研究

发布时间：2018-12-26 15:58

【摘要】： 本研究主要探讨的是近年来发展日益迅猛的人类辅助生殖领域(ART)中孕酮对精子获能及顶体反应所产生的影响。受精率的高低是影响ART成功率最为关键的一步,而国内国际对孕酮在此方面作用机理的相关研究并不多,故选择本课题深入探讨。目的:用不同浓度孕酮处理人精子,即在精子获能培养液中添加不同浓度孕酮,通过观察应用孕酮与否对精子透明带结合试验结果的影响,以人卵透明带结合实验来观察精子获能和顶体反应的发生情况,为临床及实验室提供此方面的研究信息。方法:通过上游法和密度梯度离心法处理正常标准的精液后,取精子悬液6组分别组成终浓度分别为0.1μmol/L、0.5μmol/L、1μmol/L、5μmol/L、20μmol/L、500μmol/L的孕酮溶液,培养箱中培养3.5小时获能,再与常规体外受精-胚胎移植或卵胞浆内单精子注射周期中受精失败的取卵后7天内的Ⅱ、Ⅲ级卵子体外共培养2h后,轻柔吹打下黏附于透明带上的精子。先将卵子移至新培养液中,以内径260μm吸管轻柔吹打下卵子透明带上松散结合的精子。将卵子移至小培养皿中油下制备的4μL液滴中,内径120μm的吸管吹打下卵子透明带上紧密结合的精子,然后计数卵子透明带上结合精子的总数目。采用SPSS11.5进行t检验及方差分析,对结果进行统计学处理。结果:孕酮处理精子3.5小时,20μmol/L、5μmol/L的浓度与空白组比较对精子结合透明带的比例有显著提高作用,用500μmol/L孕酮处理,精子结合透明带的比例反而下降,在3.5小时处理时间中甚至是极其显著的下降,用浓度在1μmol/L,0.5μmol/L,0.1μmol/L的孕酮组与空白组比较P＞0.05,统计学上无差异。结论:孕酮浓度在20μmol/L、5μmol/L时,可在一定程度上增强精子获能,从而提高精卵结合的能力,浓度在500μmol/L时,过高的孕酮浓度显著降低人精子顶体膜和质膜的流动性,并引起膜分子的聚集,反而加强了膜的稳定性,从而降低了顶体反应的发生率,导致精子获能减弱,降低了精卵结合力。浓度在1μmol/L,0.5μmol/L,0.1μmol/L等低水平的孕酮组与空白组比较P＞0.05,统计学上无差异,说明在低浓度的孕酮下,孕酮对精子获能及项体反应没有影响。这些说明孕酮只有在一定浓度范围内才会发挥其促进精子获能及顶体反应的发生。孕酮作为雌性动物生殖道内主要的激素之一,对精子获能及顶体反应的发生是至关重要的,研究并弄清这些激素的代表物-孕酮的作用机理,有利于对其它相关因素作用机理的了解,可以为人类体外受精等辅助生殖技术的发展提供理论依据,也有利于提高体外受精率和保证胚胎移植后的胎儿发育质量。
[Abstract]:The purpose of this study was to investigate the effects of progesterone on sperm capacitation and acrosome reaction in the field of human assisted reproduction (ART), which has been developing rapidly in recent years. Fertilization rate is the most important step to affect the success rate of ART. However, there are few domestic and international studies on the mechanism of progesterone in this respect. Objective: to treat human sperm with different concentrations of progesterone, that is, to add different concentrations of progesterone to sperm capacitation medium, and to observe the effect of progesterone application on the results of zona-binding test. To observe the occurrence of sperm capacitation and acrosome reaction by the combination of human pellucida and to provide information for clinical and laboratory research. Methods: after the normal semen was treated with upstream method and density gradient centrifugation method, the progesterone solution with final concentration of 0.1 渭 mol/L,0.5 渭 mol/L,1 渭 mol/L,5 渭 mol/L,20 渭 mol/L,500 渭 mol/L was obtained from sperm suspension of 6 groups. The incubator was incubated for 3.5 hours and co-cultured with conventional IVF-embryo transfer or intracytoplasmic sperm injection cycle (ICSI) within 7 days after fertilization failed. Gently blow down the sperm attached to the pellucida. The egg was first transferred to the new culture medium, and the loose bound sperm of the clear band of the egg was laid down by the inner diameter of 260 渭 m straw. The egg was transferred to the 4 渭 L liquid droplets prepared under the oil in the petri dish, and the 120 渭 m inner diameter straw was used to blow down the tightly bound sperm in the clear band of the egg, and then counted the total number of the sperm bound in the clear band of the egg. T-test and ANOVA were performed by SPSS11.5, and the results were analyzed statistically. Results: the concentration of 20 渭 mol/L,5 渭 mol/L significantly increased the ratio of spermatozoa binding pellucida band when treated with progesterone for 3.5h, but the proportion of spermatozoa binding pellucida band decreased when treated with 500 渭 mol/L progesterone. There was no statistical difference between progesterone group (1 渭 mol/L,0.5 渭 mol/L,0.1 渭 mol/L) and control group (P > 0.05). Conclusion: when the concentration of progesterone is 20 渭 mol/L,5 渭 mol/L, sperm capacitation can be enhanced to a certain extent, and the ability of sperm-egg binding can be improved. When the concentration of progesterone is 渭 mol/L, the fluidity of human acrosome membrane and plasma membrane can be significantly reduced by the high concentration of progesterone. The aggregation of membrane molecules enhances the stability of the membrane and reduces the incidence of acrosome reaction, resulting in the reduction of sperm capacitation and the decrease of sperm-egg binding power. There was no statistical difference between progesterone group and blank group (P > 0.05), which indicated that progesterone had no effect on spermatozoa capacitation and thighbody reaction at low concentration of progesterone (1 渭 mol/L,0.5 渭 mol/L,0.1 渭 mol/L). These results suggest that progesterone can promote sperm capacitation and acrosome reaction only within a certain concentration range. As one of the main hormones in the reproductive tract of female animals, progesterone is very important to the development of sperm capacitation and acrosome reaction. It is helpful to understand the mechanism of other related factors, to provide theoretical basis for the development of assisted reproductive technology such as human in vitro fertilization, to improve in vitro fertilization rate and to ensure the quality of fetal development after embryo transfer.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R321

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