本文選題：人精子 + 17β-雌二醇��； 參考：《四川大學》2007年碩士論文

【摘要】： 受精是生物完成繁衍后代使命必須歷經的重要過程，受精受到多方面因素的制約和影響。精子進入雌性生殖道后，經過重重淘汰篩選，到達輸卵管的壺腹部，此過程中精子發(fā)生了結構和功能的變化即精子激活，最終才能穿過透明帶，實現(xiàn)與卵的接觸、融合并完成受精過程。精子激活的主要表現(xiàn)為細胞內鈣離子濃度的升高，頂體反應，超激活運動和受精能力的獲得。精子的激活受到許多生物因素的調節(jié)，，目前有關精子激活的分子機制及其影響因素尚未完全闡明。有研究表明，女性生殖道中各種激素的水平尤其是雌激素濃度遠遠高于血液中的基礎水平，雌激素可能影響人和一些動物精子功能及受精能力；如雌激素可以提高精子胞內鈣離子濃度，增強精子運動能力及相關蛋白的表達。有實驗證明，雌激素可能通過與精子膜上的受體或結合位點相結合，可能是通過非基因組效應實現(xiàn)其對精子功能的快速調節(jié)效應。但是，目前國內外的研究都尚未闡明雌激素非基因組效應的具體作用機制及參與的信號轉導通路，也尚未成功分離出雌激素的膜受體，其化學結構、生物學功能特點、以及基因定位等都有待進一步研究。 為此進行了以下研究：第一，用不同濃度偶聯(lián)牛血清白蛋白的17β—雌二醇(E_2—BSA)短時間處理人精子，運用計算機輔助精子分析系統(tǒng)檢測精子活動力，以人精子穿透去透明帶金黃地鼠卵異種體外受精實驗檢測精子受精率，觀察雌激素對精子運動能力和受精能力的影響。第二，用不同跨膜信號轉導通路特異性抑制劑阻斷相應跨膜信號轉導后，再用E_2—BSA處理精子，采用流式細胞術檢測精子胞內鈣離子濃度變化，以證明參與E_2—BSA對精子非基因組效應的信號轉導通路。第三，用E_2—BSA處理精子，提取精子總蛋白，采用Western blot檢測信號蛋白的激活情況，以進一步確定參與非基因組效應的信號轉導下游路徑，探討雌激素非基因組效應的機制。 實驗結果如下： 一、0.5μM E_2—BSA作用后，A級精子百分比、平均曲線運動速度(VCL)、平均直線運動速度(VSL)、平均路徑速度(VAP)均有提高，但與作用前相比差異不具有顯著性(P＞0.05)；1μM E_2—BSA作用后，A級精子百分比、VCL、VSL、VAP均明顯提高(P＜0.05)；5μM E_2—BSA作用后，A級精子百分比、VCL、VAP明顯提高(P＜0.05)，但對VSL作用無顯著差異(P＞0.05)。 二、自身對照受精率偏低(fertilization rate FR＜30％)的精子，經0.5μM E_2—BSA作用后其受精率改變并無明顯差異(P＞0.05)；經1μM E_2—BSA、5μM E_2—BSA分別作用后受精率明顯提高(P＜0.05)。自身受精率較高(FR＞30％)的精子，經0.5μM E_2—BSA、1μM E_2—BSA作用后其受精率并無明顯改變，P＞0.05；5μM E_2—BSA作用后精子受精率降低(P＜0.05)。 三、腺苷酸環(huán)化酶特異性抑制劑SQ22536、磷脂酶C特異性抑制劑U73122、酪氨酸蛋白激酶特異性抑制劑Genistein處理精子后均明顯抑制由E_2—BSA引起的精子胞內鈣離子濃度的上升(P＜0.05)。 四、用Western blot方法檢測到精子經E_2—BSA處理后，磷脂酶C(PLC)和蛋白激酶C(PKC)的激活，并且傳統(tǒng)雌激素受體阻斷劑tamoxifen不能抑制該效應，進一步證明PLC-PKC信號途徑參與E_2—BSA對人精子的非基因組效應。 結論：E_2—BSA對人精子的運動能力、受精能力具有快速調節(jié)效應，參與E_2—BSA對精子功能的非基因組快速調節(jié)效應的信號轉導通路包括：腺苷酸環(huán)化酶途徑、磷脂酶C途徑和酪氨酸蛋白激酶途徑。
[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.
【學位授予單位】：四川大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R321

【參考文獻】

相關期刊論文 前2條

1 何彥芳,岳利民,何亞平,張金虎,鄭潔,高小平;17β-雌二醇誘導人精子頂體反應及胞內鈣離子增加的研究[J];四川大學學報(醫(yī)學版);2005年04期

2 鄭潔,何亞平,張金虎,何彥芳,高小平,岳利民;人精子中芳香化酶表達與精子功能的關系[J];生殖與避孕;2005年05期

本文編號：2001938