| With the intensification of the process of industrialization, a large number of environmental estrogens(EEs) released which caused spermatogenesis dysfunction. There is no effective prevention method and has aroused great attention of domestic and foreign ecologists and toxicologists. Bisphenol-A(BPA) is a kind of typical EEs that harmful to male reproduction.Since BPA and the kidney deficiency infertility not only cause male reproduct ive disorders and abnormal of sperm count and quality, but also appeared related behavioral changes, at the same time, this abnormal could be antagonistic by reinforcing kidney drugs. Our studies shows that kidney-yang deficiency rats occurs not only reduced sperm count and quality, but also weight loss, unresponsive, arched curled, chills, dry hair thinning and other behavioral changes However, those performances have improved after gave jingui shenqi pills. Therefore, according to the th-eory of “kidney controlling reproduction” and “Prescription and Syndrome correspo-nding”, we hypothesized that male reproduction disorders caused by BPA may belong to the category of kidney deficiency infertility.The observation of the changes on animal's behavioral such as elevated plus maze, open field test, exhaustive swimming time may reflect its activity, the stateof panic and endurance. What's more, the sperm count and quality, the number of fertility of male animals is the intuitive indicator of fertility. In addi-tion, the a bnormal of testosterone(T), estradiol(E2) and T/E2 levels were closed with male i nferility. G protein coupled estrogen receptor(GPER) could regulate germ cell pro liferation and differentiation through non-genomic pathways.Therefore, we will Looking for the correlation of male reproduction disorders caused by BPA and kidney deficiency infertility through compare the behaviors, hormone levels and the expression of GPER, In this way, we will evaluvate whether the reproduction disorders caused by BPA belong to kidney infertility areas, which will provide new ideas and strategies for the prevention and treatment of male reproduction disorders caused by BPA.Part one: The behavioral changes of reproduction disorders with BPA and deficiencyof the kidney mouseObjective:To explore the correlation of BPA reproductive disorders and deficiency of kidney infertility, through observing the changes of behavior of mouse models.Methods:1) Putting mouses in metabolic cages, and monitor the change of body weight dailywater intake and urine output. 2) Using elevated plus maze apparatus to detect the percentage of mouses into openarms and the percentage of open arms residengce time to evaluate the degree ofpanic and anxiety in mouse by computer digital sampling and analysis system. 3) Using open field test apparatus to detecte the number of entering in central region ofmouse and the percentage of central region residence time, evaluate the activity andanxiety by data automatic analysis system.4) Putting mouse in a water pool that length×width of 65cm×50cm, and depth of50cm.using loaned-swimming test, lead wire was fixed on tail root 3 cm that it wasequivalent to 5% of body weight of mouse. When the mouse submerged into water5 s for three consecutive times as the exhaustive swimming time, detailed record theexhausting time, and evaluate the endurance of mouse.Results: 1) Compared with the normal group and negative control group, the mouses body massof BPA group was significantly reduced(P <0.05), testicular weight also significantlyreduced(P <0.01). Compared with BPA group, the body mass of kidney-yangdeficiency group were no significant changed(P> 0.05), and testis weight increasedsignificantly(P <0.05); and the body mass of kidney-yin deficiency group of mousewas significantly reduced(P <0.01), the testis weight did not changed significantly(P> 0.05). 2) Compared with the normal group and negative control group, the water intake ofBPA group and kidney-yang deficiency group mouse were decreased from the fifthday, but the difference was not statistically significant(P>0.05), the urine of twogroups increased significantly from the sixth day(P<0.05), but no significantdifference between two groups(P>0.05). Compared with the BPA group,the waterintake of kidney-yin deficiency mouse increased significantly from modeling thirdday(P<0.05), but no significant differences in urine volume(P>0.05). 3) Compared with the normal group and negative control group, the percentage of BPAmouses open arms and the retention time significantly reduced(P<0.01); comparedwith the BPA group, the percentage into the open arms and retention time ofkidney-yang deficiency mouses was no significant changed(P>0.05), thepercentage of open arms of kidney-yin deficiency mouse was increased significantly(P<0.01), while the percentage of retention time was no significant changes(P<0.05). 4) Compared with the normal group and negative control group, the number ofentering the central area and the percentage of residence time in BPA mouse weresignificantly reduced(P <0.01). Compared with the BPA group, the number ofentering central area of kidney-yang deficiency mouses were significantly reduced(P<0.05), but the percentage of residence time were no significant change(P>0.05);kidney-yin deficiency mouses were significantly reduced(P<0.01), the percentageof the central zone residence time was significantly increased(P<0.05). 5) Compared with the normal group and negative control group, the exhaustiveswimming time of BPA mouse were significantly shorter(P <0.01). Compared withthe BPA group, the exhaustive swimming time of kidney-yang deficiency mousewere not change significantly(P>0.05); and kidney-yin deficiency mouse weresignificantly increased(P<0.05). 6) Compared with the normal group, BPA group and kidney-yang deficiency mouse areall have appeared more pronounced chills, curled hair dry, dull, and the emergenceof the phenomenon of falling sparse.When remove from the water to the hair iscompletely dry time is significantly extended, and kidney-yin deficiency groupshow to be dry hair, dull, and the emergence of sparse off, but no chills, curledperformance.Conclusion:The body weight, testis weight, water intake of BPA and kidney-yang deficiency mouse have similarwith each other, but the body weight of kidney-yin deficiency mouse decreased more significantly, water intake also increased significantly, but the urine was less than the other two groups.The BPA group and kidney-yang deficiency mouse were panic state, reduced activity and decreased endurance, and with dry hair, dull, and the emergence of sparse shedding similar behavioral manifestations; The kidney-yin deficiency mouse also appeared panic state, increase activities, stamina declined. However, BPA group and kidney-yang deficiency group were increased, accompanied by dry hair, dull, but there was no fear cold, huddled performance. In a conclusion, the model of infertility caused by BPA are more similar with kidney-yang model.Part two: The change of semen quality and hormone levels of BPA and kidney-yangdeficiency groupObjective:By detecting the number of sperm in mouse, sperm motility and percentage of abnormal sperm and serum T, E2 and T/E2, in order to explore the correlation.of BPA and kidney deficiency about spermatogenesis and hormone levelsMethods: 1) Detecting mouse serum T, E2, and T/E2 with Roche chemiluminescence. 2) 3) Supplant semen from vas deferens and epididymis, adding 3 ml of saline to preparesperm suspension at 37?. Water bath to fully liquefaction. Detect sperm count, thepercentage of abnormal sperm with automatic sperm analyzer(SQAV).Male and female mice were caged with male and female ratio of 3: 1 was observed inmale infertility litter.Results: 1) Compared with the normal group and negative control group, The serum T and T/E2of BPA mouse was significantly reduced(P<0.05), but E2 did not changesignificantly, the difference was not statistically significant(P>0.05). Compared withthe BPA group, the T level did not change significantly of kidney-yang deficiencymouse(P>0.05), E2 was slightly decreased, but the difference was not statisticallysignificant(P> 0.05), T/E2 was increased(P<0.05); the serum T was significantlyincreased of kidney-yin deficiency(P <0.01), but E2 and T/E2 were significantlyincreased(P <0.05). 2) Compared with the normal group and negative control group, the sperm count, thepercentage of motile sperm decreased significantly, while the percentage of abnormalsperm was increased(P<0.01). Compared with the BPA group, the sperm count andsperm abnormality rate of kidney-yang deficiency mouse did not change significantly(P>0.05), but the percentage of motile sperm increased(P<0.05); the number ofsperm increase significantly(P<0.01), the percentage of motile sperm and spermabnormality rate also increased significantly(P<0.05).3) Compared with the normal group and negative control group, the rearing number ofBPA is 3.8 males, of which four non-pregnant females(P <0.05). Compared with theBPA group, the average rearing number of kidney-yang deficiency was 4.33, but therewere four non-pregnant females, the difference was not statistically significant(P>0.05); the average rearing number of kidney-yin deficiency was 6.46, including fournon-pregnant females(P <0.05).Conclusion:The serum T and T/E2 of BPA and kidney-yang deficiency mouse were significantly reduced, but E2 did not change significantly, compared with BPA group, the T, E2 and T/E2 of kidney-yin deficiency group were increased. The sperm counts and the percentage of abnormal sperm of BPA group and kidney-yang deficiency mouse were increased, but the percentage of motile sperm of BPA significantly reduced, kidney-yang deficiency mouse was increased; the number of sperm, the percentage of motile sperm and sperm deformity rate were increased. BPA group was similar with kidney-yang deficiency mouse at rearing number; but kidney-yin deficiency was higher than BPA group.In conclusion, it was correlated there are many similaries about T, E2, T / E2, the percentage of abnormal sperm as well as sperm countsbetween BPA and kidney-yang deficiency mousePart three: The expression of GPER in BPA and kidney deficiency groupObjective:By observing the expression of GPER in order to investigate the relationship between BPA and kidney deficiency model.Methods: 1) Observe the testis structure of mouse by HE dyeing. 2) Observe the expression of testicular GPER located by Immunohistochemical. 3) Observe the expression of testicular GPER by Immunofluorescenc.Results: 1) Compared with the normal group and negative control group, mouse testisseminiferous tubules of BPA group, kidney-yang deficiency and kidney-yindeficiency showed different degrees of official parietal damaged, thinning, thecontinuity of the basement membrane damage, and germ cells were arrangeddisorderly, uneven distribution of leydig cells, the seminiferous epithelium layer andthe cavity sperm count were reduced. 2) GPER were expressed in cytoplasm and membrane of mouse testis leydig cellsbetween the experimental groups by immunoreactivity and the nucleus werenegative. There were no expression in Sertoli cells and spermatogenic cells ofseminiferous tubules. 3) The GPER expressed in testis leydig cells of normal group and control group, therewas weak fluorescence,but could be clearly visible(+); The GPER expressedfluorescent bright of BPA group and clearly visible(+++), it were significantlydifference when compared with normal control group(P<0.05). the fluorescent wasbright of kidney-yang deficiency group, it clearly visible(+++), and there was nosignificant difference when compared with the BPA group(P>0.05); the positiveexpression of fluorescent kidney-yin deficiency group, but clearly visible(+), andBPA group significantly decreased(P<0.05).Conclusion: Both BPA group, kidney-yang deficiency group and kidney-yin deficiency mouse testicular tissue structure are destroyed. and the uneven distribution of seminiferous tubule. The continuity of basement membrane was damaged, and the lumen wall thinning, the germ cells were disorderly arranged on the wall, the lumen cavity, the number of sperm within the lumen was reduced. GPER expressed in membrane and cytoplasm of Leydig cell, The expression of GPER was bright fluorescence in kidney-yang deficiency group mouse testis, it was visible andare closed with BPA group, the expression GPER fluorescence of kidney-yin deficiency group mouse was positive but with a little decreased. W e may draw a conclusion that both BPA group and kidney-yang deficiency mouse have higher expression of GPER, disordered serum hormone levels, lower sperm quality. |
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