| Objective:To research the effects of the growth and development of mouse, testi-cular weight and sperma to genic function by different feeding alcohol. To understand the preparation of testis paraffin sections and HE staining. And to master terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) of detecting the changes of the apoptosis of spermatogenic cells and the immunohistochemistry dyeing technology to measure specimens in each group and to explore the expression of Caspase-3 protein and Survivin protein in various experimental groups, and to test the relevance of them and germ cell apoptosis induced by alcohol.Methods:The experiments were conducted respectively with distilled water, red wine, low and high dose of white alcohol, which given to mouse by 0.01ml/g.We measured mouse body weight and growth and changes of state per week during the experiment. After 3 spermatogenic cycle, we successfully established the mouse models with alcohol toxicity. We measured immediately the testes weight of mice in each group after their death. Mice's testis were prepared to paraffin sections,we observed testis seminiferous tubules and germ cell's morphological changes by using HE staining,we applied TUNEL to detect the germ cell apoptotic index (AI).Then SP immunohistochemical method were tested for the expression of Caspase-3 and Survivin protein in each experimental group, and the immunore activity score(IRS) is used for semiquantitative analysis, that is IRS:0:negative (-),1～4:weak positive (+), 5-8:moderately positive (++),9-12:strong positive (+++).SPSS17.0 statistical software are used for statistical processing of experimental data, that is:quantitative variables are analyzed by one-way ANOVA,ordinal variables by non-parametric Kruskal-Wallis test, and the relationship between two variables by Spearman rank correlation.Results:We successfully established mouse model of alcohol-based. It showed normal appetite, good spirits in control and the wine group, but the mouse appeared drunk in alcohol group.The mouse increased the average weight was the most in red wine group, while high-dose alcohol group was increased by at least, and it was significantly less than the low dose group (P=0.003), with increasing doses of liquor, the less weight gained in mouse.At the same time, the relative weight of testis (testes relative to their body weight before the experiment) and seminiferous tubule diameter changes in HE stained were also showed the basic consistency of the results.We expioted TUNEL to detect the AI of spermatogenic cells, and we found the average of germ cells'AI in distilled water group was 0.1394±0.0104,which was higher than the red wine group (0.1016±0.0124, P=0.000), but lower than the alcohol group (0.2213±0.0149,0.2582±0.0177, respectivelyP=0.000, P=0.000), and the germ cells' AI in red wine group was lowest, while high-dose alcohol toxicity was the highest, and with increasing the dose of liquor,the more AI of spermatogenic cells.We found the expression of Caspase-3 protein in alcohol toxicity group was higher than in control and red wine group,and with the increase of alcohol concentration, the stronger the expression of Caspase-3 protein; but Survivn protein showed opposite. While the mouse'testis Caspase-3 protein in wine group was lower than other groups, while Survivin protein expression was the most. With the degree of apoptosis of spermatogenic cells increasing, the expression of Caspase-3 was stronger, and was positive correlation (r=0.717, P<0.01); but the expression of Survivn was weaker and negative correlation (r=-0.631, P<0.01).Conclusion:Moderate feeding red wine is beneficial of the growth and development of mammals, testis and spermatogenic function, while consumption of liquor is harmful, and with the increase of the concentration of liquor, the more worse.This experiment confirmed the expression of between Survivn and Caspase-3 protein induced by drinking in germ cells'apoptosis were closely related. However, the mechanism maybe related to some elements in the wine or alcohol, but it can not be determined and need to further confirmed. In this study, it may change people's recognition of drinking, as well as provide new ideas to clinical treatment of male infertility. |
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