17β-雌二醇对人精子功能的非基因组调节效应及其机制的研究

发布时间：2018-06-10 04:14

本文选题：人精子 + 17β-雌二醇　；参考：《四川大学》2007年硕士论文

【摘要】： 受精是生物完成繁衍后代使命必须历经的重要过程，受精受到多方面因素的制约和影响。精子进入雌性生殖道后，经过重重淘汰筛选，到达输卵管的壶腹部，此过程中精子发生了结构和功能的变化即精子激活，最终才能穿过透明带，实现与卵的接触、融合并完成受精过程。精子激活的主要表现为细胞内钙离子浓度的升高，顶体反应，超激活运动和受精能力的获得。精子的激活受到许多生物因素的调节，，目前有关精子激活的分子机制及其影响因素尚未完全阐明。有研究表明，女性生殖道中各种激素的水平尤其是雌激素浓度远远高于血液中的基础水平，雌激素可能影响人和一些动物精子功能及受精能力；如雌激素可以提高精子胞内钙离子浓度，增强精子运动能力及相关蛋白的表达。有实验证明，雌激素可能通过与精子膜上的受体或结合位点相结合，可能是通过非基因组效应实现其对精子功能的快速调节效应。但是，目前国内外的研究都尚未阐明雌激素非基因组效应的具体作用机制及参与的信号转导通路，也尚未成功分离出雌激素的膜受体，其化学结构、生物学功能特点、以及基因定位等都有待进一步研究。为此进行了以下研究：第一，用不同浓度偶联牛血清白蛋白的17β—雌二醇(E_2—BSA)短时间处理人精子，运用计算机辅助精子分析系统检测精子活动力，以人精子穿透去透明带金黄地鼠卵异种体外受精实验检测精子受精率，观察雌激素对精子运动能力和受精能力的影响。第二，用不同跨膜信号转导通路特异性抑制剂阻断相应跨膜信号转导后，再用E_2—BSA处理精子，采用流式细胞术检测精子胞内钙离子浓度变化，以证明参与E_2—BSA对精子非基因组效应的信号转导通路。第三，用E_2—BSA处理精子，提取精子总蛋白，采用Western blot检测信号蛋白的激活情况，以进一步确定参与非基因组效应的信号转导下游路径，探讨雌激素非基因组效应的机制。实验结果如下：一、0.5μM E_2—BSA作用后，A级精子百分比、平均曲线运动速度(VCL)、平均直线运动速度(VSL)、平均路径速度(VAP)均有提高，但与作用前相比差异不具有显著性(P＞0.05)；1μM E_2—BSA作用后，A级精子百分比、VCL、VSL、VAP均明显提高(P＜0.05)；5μM E_2—BSA作用后，A级精子百分比、VCL、VAP明显提高(P＜0.05)，但对VSL作用无显著差异(P＞0.05)。二、自身对照受精率偏低(fertilization rate FR＜30％)的精子，经0.5μM E_2—BSA作用后其受精率改变并无明显差异(P＞0.05)；经1μM E_2—BSA、5μM E_2—BSA分别作用后受精率明显提高(P＜0.05)。自身受精率较高(FR＞30％)的精子，经0.5μM E_2—BSA、1μM E_2—BSA作用后其受精率并无明显改变，P＞0.05；5μM E_2—BSA作用后精子受精率降低(P＜0.05)。三、腺苷酸环化酶特异性抑制剂SQ22536、磷脂酶C特异性抑制剂U73122、酪氨酸蛋白激酶特异性抑制剂Genistein处理精子后均明显抑制由E_2—BSA引起的精子胞内钙离子浓度的上升(P＜0.05)。四、用Western blot方法检测到精子经E_2—BSA处理后，磷脂酶C(PLC)和蛋白激酶C(PKC)的激活，并且传统雌激素受体阻断剂tamoxifen不能抑制该效应，进一步证明PLC-PKC信号途径参与E_2—BSA对人精子的非基因组效应。结论：E_2—BSA对人精子的运动能力、受精能力具有快速调节效应，参与E_2—BSA对精子功能的非基因组快速调节效应的信号转导通路包括：腺苷酸环化酶途径、磷脂酶C途径和酪氨酸蛋白激酶途径。
[Abstract]:Fertilization is an important process that must be passed through the mission of the biological reproduction of the offspring. Fertilization is restricted and influenced by many factors. After the sperm enters the female reproductive tract, it passes through a lot of elimination and screening to reach the ampulla of the fallopian tube. In this process, sperm has changed the structure and function, that is, sperm activation can eventually be passed through the zona pellucida. The activation of sperm is mainly manifested in the increase of intracellular calcium concentration, acrosome reaction, hyperactivation and fertilization. The activation of sperm is regulated by many biological factors. The molecular mechanism of sperm activation and its influencing factors have not yet been fully elucidated. The level of various hormones in the female reproductive tract, especially the estrogen concentration, is far higher than the basic level in the blood. Estrogen may affect the sperm function and fertilization ability of human and some animals, such as estrogen can increase the intracellular calcium concentration, enhance the ability of sperm motility and the expression of related proteins. By combining with the receptor or binding site on the sperm membrane, it may be possible to realize its rapid regulation effect on sperm function through non genomic effect. However, the specific mechanisms of the non genomic effect of estrogen and the signal transduction pathway involved in the non genomic effect of estrogen have not been elucidated at present, and the estrin has not been successfully separated. The membrane receptors, their chemical structure, biological function and gene location need to be further studied.
The following studies have been done: first, using 17 beta estradiol (E_2 - BSA) with different concentrations of bovine serum albumin (BSA) for short time treatment of human sperm, the sperm motility was detected by a computer assisted sperm analysis system, and the sperm fertilization rate was detected by human sperm penetration to the oocytes of the oocytes in the zona pellucida, and the estrogen was observed. Second. Second, after blocking the corresponding transmembrane signal transduction by the specific inhibitors of different transmembrane signal transduction pathways, the sperm was treated with E_2 BSA, and the intracellular calcium concentration changes were detected by flow cytometry to demonstrate the signal transduction pathway involved in the non genomic effect of E_2 - BSA. Third, the sperm was treated with E_2 - BSA, the total sperm protein was extracted, and the activation of the signal protein was detected by Western blot, so as to further determine the downstream pathway of signal transduction involved in the non genome effect and explore the mechanism of the non genomic effect of estrogen.
The experimental results are as follows:
First, after the action of 0.5 M E_2 - BSA, the percentage of A-level sperm, the average curve velocity (VCL), the mean linear velocity (VSL) and the average path velocity (VAP) were improved, but the difference was not significant (P > 0.05) compared with that before the action (P > 0.05). After the action of 1 u M E_2 BSA, the percentage of a spermatozoon, VCL, VSL, were obviously improved (5 mu). After SA treatment, the percentages of A-level sperm, VCL and VAP increased significantly (P < 0.05), but there was no significant difference in the effect of VSL (P > 0.05).
Two, the fertilization rate of sperm with low fertilization rate (fertilization rate FR < 30%) was not significantly different after the effect of 0.5 mu M E_2 BSA (P > 0.05), and the fertilization rate was significantly increased after 1 u M E_2 BSA, 5 mu M E_2 - 0.05. The fertilization rate did not change significantly after BSA treatment, P > 0.05; sperm fertilization rate decreased after 5 M E_2 - BSA (P < 0.05).
Three, adenylate cyclase specific inhibitor SQ22536, phospholipase C specific inhibitor U73122, and Genistein, a tyrosine protein kinase specific inhibitor, significantly inhibited the increase of intracellular calcium concentration caused by E_2 - BSA (P < 0.05).
Four, the Western blot method was used to detect the activation of phospholipase C (PLC) and protein kinase C (PKC) after the sperm was treated with E_2 BSA, and the traditional estrogen receptor blocker tamoxifen could not inhibit the effect, and further demonstrated that the PLC-PKC signaling pathway was involved in the non genomic effect of E_2 BSA on human sperm.
Conclusion: E_2 - BSA has a rapid regulation effect on the ability of human sperm movement and fertilization. The signal transduction pathways involved in the non genomic rapid regulation effect of E_2 - BSA on sperm function include adenylate cyclase pathway, phospholipase C pathway and tyrosine protein kinase pathway.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R321

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