| Objective: This study established an animal model of obese rats to investigate the expression and localization of zinc-related proteins(PRDM16,ZBTB32 and ZAG)in testicular and epididymal adipose in order to investigate the value of zinc-related proteins in the reproduction of obese rats.Methods: Twenty clean male SD rats were purchased from the Animal Experimental Center of Hebei Medical University with an average body weight of(103±11)g.After one week of adaptive feeding,20 SD rats were randomly divided into 6 normal rats and 14 obese rats;normal rats were fed with normal diet,and obese rats were fed with high fat diet.The body weight of the rats were tested every week.At the end of the 12 th week,the obesity model was successfully constructed with the body mass of the obese rats being 20% higher than the average body mass of the normal rats.Finally,7models of obese rats were successfully established.The bilateral testis and epididymal adipose of rats were weighed.Meanwhile,the caudal epididymis tissue was cut up in saline solution.The sperm concentration and viability of rats in different groups were recorded by adding 10 ?L to sperm counting board.HE and immunohistochemistry were used to observe the morphological changes of testis in obese rats and the localization of PRDM16,ZBTB32 and ZAG in testicular tissue.Western Blot was used to detect the relative expression of PRDM16,ZBTB32 and ZAG in different groups of rats.The expression of PRDM16,ZBTB32 and ZAG in mRNA of different groups were detected by quantitative real-time PCR.Results:1.Analysis of semen parameters in normal and obese ratsBy analyzing the semen of two groups of rats,it was found that the sperm concentration and motility of obese rats were significantly decreased,and the difference was statistically significant(P<0.05).2.Morphological changes of testis in normal and obese ratsThe results of HE staining showed that the testicular seminiferous tubules were composed of multiple layers of spermatogenic cells at different developmental stages.In the normal group,the testicular seminiferous tubules were arranged neatly,and the spermatogenic cells in the lumen were normal in structure,with clear layers and a large number of spermatozoa.However in the obese group,the seminiferous tubules were loosely arranged,the interstitial space was widened,the testicular spermatogenic epithelial structure was incomplete,the number of spermatogenic cells was reduced and vacuolar-like changes occurred,and the number of spermatozoa in the lumen was reduced.3.Location of PRDM16,ZBTB32 and ZAG in testicular tissueImmunohistochemical results showed that PRDM16,ZBTB32 and ZAG were mainly expressed in spermatogenic cytoplasm of rat testis,and the expression of PRDM16 in testicular tissue of obese rats was significantly lower than that of normal group,while the expression of ZBTB32 and ZAG were not significantly different from that of normal rats(P>0.05).4.Expression of PRDM16,ZBTB32 and ZAG mRNA in the testicular and epididymal adipose of two groups of ratsThe results of quantitative real-time PCR showed that the expression of PRDM16 mRNA in the testis and epididymal adipose tissue of the obese rats was significantly decreased,and the difference was statistically significant(P<0.05).The expression of ZAG mRNA was significantly decreased in the epididymal adipose tissue of obese rats(P<0.05),but there was no significant difference in the expression of ZAG mRNA between normal and obese groups in rat testis(P>0.05).There was no significant difference in the expression of ZBTB32 mRNA between testis and epididymal adipose in normal and obese rats(P>0.05).5.Expression of PRDM16,ZBTB32 and ZAG proteins in testis and testicular adipose tissue of two groups of ratsWestern Blot results showed that compared with the normal group,the expression of PRDM16 protein in the testis and epididymal adipose of the obese group was significantly lower,the difference was statistically significant(P > 0.05).Compared with the normal group,the expression of ZAG protein in the testis of obese rats had no significant difference(P>0.05),but the expression of ZAG protein in epididymal adipose was significantly lower than that of the normal group,the difference was statistically significant(P<0.05).There was no significant difference in the expression of ZBTB32 protein between the two groups of testicular and epididymal adipose in rats(P>0.05).Conclusion:1.Compared with the normal group,the testicular tissue morphology of the obese group was significantly impaired,sperm concentration and sperm motility were significantly reduced,suggesting that obesity can affect male sexual reproduction by affecting semen quality.2.Zinc-related proteins PRDM16,ZBTB32 and ZAG were mainly expressed in spermatogenic cytoplasm of rats;the expression of PRDM16 in testis and epididymal adipose of obese rats was significantly lower than that of normal rats,and the expression of ZAG in epididymal adipose was significantly lower than that of normal rats,suggesting that obesity may lead to abnormal expression of zinc-related proteins and affect reproduction of male rats. |
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