The Effect Of Tannic Acid On Autophagy In Gonad Of Brandt's Voles (Lasiopodomys Brandtii)

It has been proven that antioxidants can affect the expression of autophagy related proteins in animal cells and variations of autophagy in germ cells can affect the reproductive performance of animals.A large number of studies have shown that TA(tannic acid,one kind of hydrolyzed tannins)has antioxidant function.However,it is less reported that whether TA could affect the reproduction of animals by regulating the autophagy in germ cells.In this study,we chose male Brandt's voles as research object.When Brandt's voles were 28-day-old after birth,they were treated with TA in their drinking water with different concentrations(0 mg mL-1,3 mg mL-1 and 6 mg mL-1,as control,low dose and high dose group,respectively)to the age of 8 or 13 weeks.The food intake was recorded every other day,and body weight was measured weekly.The ELISA was used to assay the activity of Superoxide dismutase(SOD),Catalase(CAT),Glutathione peroxidase(GSH-Px)and the content of Malondialdehyde(MDA),Testosterone(T),Follicle-stimulating hormone(FSH),Luteinizing hormone(LH)in the serum.Under the light microscope,testicular tissue structures were observed,and the sperm concentration,mortality rate and deformity rate in epididymis were calculated.Real-time quantitative PCR(RT-qPCR)was used to detect the mRNA expression level of autophagy related protein genes LC3,Beclinl and p62.and immunocytochemistry(IHC)was used to detect the protein expression level of autophagy related protein LC3,Beclinl and p62(play important roles in the initiation,extension and degradation of autophagy,respectively)in the testicular tissues of Brandt's voles.Autophagy in testicular tissue was also observed under transmission electron microscopy(TEM).The aim of this study was to investigate the effects of TA on antioxidant,gonadal autophagy and gonadal function in male Brandt's voles.The results would enrich the theory of coevolution between plants and animals.Our studies were as follows.1.The food intake and body weight of male Brandt's voles treated with TA to 13-week-age increased significantly in the high-dose TA group,compared with the control group,but no significant difference was observed between other groups.2.Compared with the control group,the enzyme activity of SOD,GSH-Px and CAT in the serum of male Brandt's voles treated with TA to the age of 8 and 13 weeks were significantly increased in the low-dose TA group.The enzyme activity of serum SOD,GSH-Px and CAT in the male Brandt's voles treated with TA to the 8-week-age significantly decreased in the high-dose TA group,but the activity of three kinds of antioxidant enzymes in the male Brandt's voles treated with TA to the the 13-week-age significantly increased in the high-dose TA group;The serum MDA of TA treatment group showed a decreasing trend compared with that of control group and there was statistically significant difference between the high-dose group and the control group.Our results suggest that TA can be used as an antioxidant to improve the antioxidative capacity of male Brandt's voles.3.It was observed by TEM that high-dose of TA caused obvious sperm damage in 8-week-old and increased sperm deformity rate in 13-week-old of male Brandt's voles.The degree of autophagy was further measured at the molecular level.The results of RT-qPCR showed that low-dose TA reduced the expression of autophagy-related genes in the initiation and extension stages in the 8-week-old,but increased the gene expression at the initiation stage in the 13-week-old of male Brandt's voles;The expression of all 3 genes increased in the high-dose group compared with the low-dose group,but the differences were not significant;The expression of autophagy gene in degradation stage was not abnormal.Immunohistochemistry staining results also showed that low-dose TA decreased,while high-dose TA increased the expression of autophagy-related proteins at the initiation stage of autophagy;There was no significant difference in the main proteins at the extension stages,but the expression of autophagy related proteins possessed the trend of increasing in the high-dose group and decreasing in the low-dose group;There was no significant accumulation of protein at degradation stage,indicating that TA did not cause autophagy dysfunction of gonad in male Brandt's voles,but had a certain regulatory effect.4.Our results showed that TA would increase the testis and epididymis index of male Brandt's voles,and it was found that with TA dose increasing,gonadal index increased in the male Brandt^s voles treated with TA to the age of 13 weeks.HE staining results showed that high-dose of TA could damage the testicular tissues structure of 8-week-old male Brandt's voles.Low-dose TA increased the sperm concentration and reduced the mortality and deformity rate,while the high-dose group significantly increased the sperm mortality and deformity rate in 8-week-old of male Brandt's voles.Low-dose of TA increased the serum levels of reproductive related hormones in male Brandt's voles.In summary,in both 8-week-old and 13-week-old male Brandt's voles,low-dose TA could increase the content of reproductive related hormones,the gonad index,the number of epididymal sperm and reduce the rate of sperm deformity and mortality.Meanwhile,low-dose TA enhanced their antioxidative ability and alleviate the autophagy in their gonads.So,our study suggested that TA,to some extent,can improve the gonadal function of Brandt's voles by mediating the antioxidation and autophagy in their gonads.