孕酮對精子獲能及頂體反應(yīng)影響的研究
發(fā)布時間：2018-12-26 15:58
【摘要】： 本研究主要探討的是近年來發(fā)展日益迅猛的人類輔助生殖領(lǐng)域(ART)中孕酮對精子獲能及頂體反應(yīng)所產(chǎn)生的影響。受精率的高低是影響ART成功率最為關(guān)鍵的一步,而國內(nèi)國際對孕酮在此方面作用機(jī)理的相關(guān)研究并不多,故選擇本課題深入探討。 目的:用不同濃度孕酮處理人精子,即在精子獲能培養(yǎng)液中添加不同濃度孕酮,通過觀察應(yīng)用孕酮與否對精子透明帶結(jié)合試驗(yàn)結(jié)果的影響,以人卵透明帶結(jié)合實(shí)驗(yàn)來觀察精子獲能和頂體反應(yīng)的發(fā)生情況,為臨床及實(shí)驗(yàn)室提供此方面的研究信息。方法:通過上游法和密度梯度離心法處理正常標(biāo)準(zhǔn)的精液后,取精子懸液6組分別組成終濃度分別為0.1μmol/L、0.5μmol/L、1μmol/L、5μmol/L、20μmol/L、500μmol/L的孕酮溶液,培養(yǎng)箱中培養(yǎng)3.5小時獲能,再與常規(guī)體外受精-胚胎移植或卵胞漿內(nèi)單精子注射周期中受精失敗的取卵后7天內(nèi)的Ⅱ、Ⅲ級卵子體外共培養(yǎng)2h后,輕柔吹打下黏附于透明帶上的精子。先將卵子移至新培養(yǎng)液中,以內(nèi)徑260μm吸管輕柔吹打下卵子透明帶上松散結(jié)合的精子。將卵子移至小培養(yǎng)皿中油下制備的4μL液滴中,內(nèi)徑120μm的吸管吹打下卵子透明帶上緊密結(jié)合的精子,然后計(jì)數(shù)卵子透明帶上結(jié)合精子的總數(shù)目。采用SPSS11.5進(jìn)行t檢驗(yàn)及方差分析,對結(jié)果進(jìn)行統(tǒng)計(jì)學(xué)處理。結(jié)果:孕酮處理精子3.5小時,20μmol/L、5μmol/L的濃度與空白組比較對精子結(jié)合透明帶的比例有顯著提高作用,用500μmol/L孕酮處理,精子結(jié)合透明帶的比例反而下降,在3.5小時處理時間中甚至是極其顯著的下降,用濃度在1μmol/L,0.5μmol/L,0.1μmol/L的孕酮組與空白組比較P＞0.05,統(tǒng)計(jì)學(xué)上無差異。結(jié)論:孕酮濃度在20μmol/L、5μmol/L時,可在一定程度上增強(qiáng)精子獲能,從而提高精卵結(jié)合的能力,濃度在500μmol/L時,過高的孕酮濃度顯著降低人精子頂體膜和質(zhì)膜的流動性,并引起膜分子的聚集,反而加強(qiáng)了膜的穩(wěn)定性,從而降低了頂體反應(yīng)的發(fā)生率,導(dǎo)致精子獲能減弱,降低了精卵結(jié)合力。濃度在1μmol/L,0.5μmol/L,0.1μmol/L等低水平的孕酮組與空白組比較P＞0.05,統(tǒng)計(jì)學(xué)上無差異,說明在低濃度的孕酮下,孕酮對精子獲能及項(xiàng)體反應(yīng)沒有影響。這些說明孕酮只有在一定濃度范圍內(nèi)才會發(fā)揮其促進(jìn)精子獲能及頂體反應(yīng)的發(fā)生。孕酮作為雌性動物生殖道內(nèi)主要的激素之一,對精子獲能及頂體反應(yīng)的發(fā)生是至關(guān)重要的,研究并弄清這些激素的代表物-孕酮的作用機(jī)理,有利于對其它相關(guān)因素作用機(jī)理的了解,可以為人類體外受精等輔助生殖技術(shù)的發(fā)展提供理論依據(jù),也有利于提高體外受精率和保證胚胎移植后的胎兒發(fā)育質(zhì)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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R321
本文編號：2392353
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R321
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