Study On The Effects And Mechanism Of Non-genomic Effects Of 17-Estradiol On Human Spermatozoa

Fertilization is an important process of reproduction, and it is affected bymany biological factors. After spermatozoa entering into the female genitaltract, only one spermatozoon can complete the fertilization at the ampulla offallopian tube. The level of various hormones in the female genital tract,especially, the level of estrogen concentration is far more than the basic levelin the blood, estrogen can influence the motility and fertiliablility ofspermatozoa.Some studies show that estrogen can obviously influence the motility andfertilization of spermatozoa in human and animals; the estrogen can increasethe Ca²⁺ concentration and activity of protein in spermatozoa. The researchresults show that estrogen can influence the spermatozoa function by rapideffects in which the estrogen may bind with the receptor or binding site on themembrane, and the results we obtained before have been proved this. Up tonow, the mechanism of estrogen non-genomic effects and the transmembranesignal transduction haven't been demonstrated. The membrane receptor has notbeen separated and the chemical frame, biological function and gene structure of receptor remain to be clarified.In present study, the sperm formulary assay, sperm penetration ofzona-free hamster egg assay, flow cytometry (FCM) and Western blotting areused to investigate the effects of estrogen on spermatozoa and the possiblemechanism.1) To evaluate the role of estrogen non-genomic effects on spermatozoafunction, spermatozoa were treated with the membrane impermeable17β-estradiol which conjugated to bovine serum albumin (E₂-BSA) atdifferent concentrations, then to detect the changes of motility andfertilization.2) To study the mechanism of estrogen non-genomic effects,spermatozoa were treated with three inhibitor of transmembrane signaltransduction and E₂-BSA, to compared the changes of [Ca²⁺]_i between theihibitor and control by flow cytometry. Western blotting was used to detectthe activation of the signal proteins after the spermatozoa were treated withE₂-BSA and tamoxifen of estrogen receptor inhibitor.The results were as follows:1. 0.5μM E₂—BSA had no effects on the percentage of A grade, thevelocity of curve locomotion(VCL), the velocity of straight locomotion(VSL)and the velocity of average path(VAP), P＞0.05; but 1μM E₂—BSAsignificantly increased the percentage of A grade, VCL, VSL and VAP,P＜0.05; 5μM E₂—BSA can increased the percentage of A grade, VCL andVAP, P＜0.05, but had no significantly effects on VSL, P＞0.05. 2. 0.5μM E₂—BSA had no effects on the rate of fertilization of thespermatozoa for control FR＜30ï¼…, P＞0.05; and 1μM E₂—BSA and 5μM E₂—BSA can significantly increased the rate of fertilization of the spermatozoa(control FR＜30ï¼…), P＜0.05. But, 0.5μM E₂—BSA and 1μM E₂—BSA hadno effects on the rate of fertilization of the spermatozoa for control FR＞30ï¼…,P＞0.05; and 5μM E₂—BSA can significantly reduced the rate of fertilizationof the spermatozoa (control FR＞30ï¼…),P＜0.053. Adenylyl cyclase(AC) inhibitor SQ22536, phospholipase C(PLC)inhibitor U73122 and protein tyrosine kinase(PTK) inhibitor Genistein all canreduce the increase of [Ca²⁺]_i which raised by 1μM E₂—BSA.4. The PLC protein and PKC protein were significantly enhanced by 1μME₂—BSA and the activation of PLC can't inhibited by tamoxifen.In conclusion, E₂—BSA can significantly increase the motility andfertilization of human spermatozoa by estrogen non-genomic effects and thisrole is related to AC,PLC and PTK transmembran signal transduction.