The Role Of Autophagy And Apoptosis In The Effect Of Gallic Acid On The Reproductive Performance Of Male Brandt’s Vole (Lasiopodomys Brandtii)

Brandt’s vole(Lasiopodomys brandtii)is a kind of small herbivorous rodent in the steppe.Tannic acid(TA)is one of the tannins known to be found in plants that Brandt’s voles feed on.Our previous studies have proved that tannic acid could affect the reproduction of Brandt’s vole,but its mechanism remains unknown.Tannic acid was reported to be decomposed into gallic acid(GA)and glucose in the gastrointestinal tract of mammals.GA is one kind of phenolic acid,which is also plant secondary metabolite widely found in nature.It contains polyhydroxyl groups in its structure and has a strong antioxidant capacity.Reports indicated that phenolic compounds could affect autophagy,apoptosis and animal reproduction.Based on the above reasons,we speculated that the effect of tannic acid on reproductive performance of Brandt’s voles might be related to its decomposition product GA.Therefore,this study would investigate whether GA affect the reproductive ability of male Brandt’s voles.Adolescent and adult male Brandt’s voles were divided into control(0.5%CMC-Na),low dose GA(100 mg·kg-1·day-1)and high doses GA(200mg·kg-1·day-1)group.GA was administered orally by gavage,from postnatal day 40 and 90,respectively,for two consecutive weeks.During the experiment,food intake was recorded daily,and body weight was recorded every three days.The sperm quality(sperm concentration,ratio of normal sperm,abnormal sperm and dead sperm)of Brandt’s vole was detected by trypan blue staining.Enzyme linked immunosorbent assay(ELISA)was used to detect the levels of Follicle Stimulating Hormone(FSH),Luteinizing Hormone(LH),Testosterone(T)in Brandt’s voles serum and testicular tissues besides content of catalase(CAT),superoxide dismutase(SOD)in testicular tissue.The testicular tissue sections were prepared and the testicular morphological changes were detected by Hematoxylin-eosin staining(HE).Autophagy in testicular tissues was detected by transmission electron microscopy(TEM),and apoptosis in testicular tissues was detected by TdT-mediated dUTP Nick-End Labeling(TUNEL).Real-time quantitative PCR(RT-qPCR)was used to detect the expression levels of autophagy and apoptosis genes in the gonads of male Brandt’s vole.Western blot(WB)was used to detect the expression of autophagy,apoptosis and related proteins in the gonads of male Brandt’s vole.This study would be benefit to explain the reasons and mechanism of TA on the reproduction of Brandt’s voles,enrich the theory of co-evolution of animals and plants,and provide new ideas for the population control of Brandt’s vole.The results were as follows:1.There was no significant difference in body weight and food intake when adolescent and adult male Brandt’s voles were exposed to different doses of GA.2.Compared with the control group,the gonadal index and sperm concentration of Brandt’s voles in low-dose group were significantly increased,and the rate of abnormal sperm was significantly reduced.Rates of abnormal and dead sperm increased significantly in the high-dose group.The results of HE staining showed that low-dose GA could promote the development of testis in adolescent male Brandt’s vole,while the testis in high-dose GA group was damaged.GA increased the inner diameter of the seminiferous tubule of adult male Brandt’s voles,but there was no significant change in their morphological structure.ELISA results showed that low dose GA significantly increased the levels of FSH and LH in male Brandt voles,while high dose GA decreased the level of T.3.The antioxidant level of adolescent voles increased in the low-dose GA group and decreased in the high-dose GA group.The antioxidant level of adult Brandt’s voles increased in both treatment groups,but the high-dose GA group was not as effective as the low-dose group.4.TEM results showed that GA treatment could enhance the autophagy activity in the testis tissue of adolescent male Brandt’s vole,while the adult group was inhibited.TUNEL results showed that the number of apoptotic cells in adolescent male Brandt’s voles decreased significantly in the low-dose group and significantly increased in the high-dose group,while there was no change in adult voles.5.RT-qPCR results showed that the LC3 gene expression of adolescent Brandt’s vole was significantly increased in both treatment groups,and the expression of Caspase-3 gene was significantly decreased in the low-dose group.Western Blot results showed that the protein expressions of p-Akt increased and cleaved Caspase-3 decreased in adolescent low-dose GA group,while the protein expressions of p-mTOR reduced and LC3 increased in high-dose GA group.In the adult high-dose GA group,p-AKT,p-mTOR levels were increased,and LC3 level decreased.These results indicated that AKT/mTOR pathway was involved in the regulation of GA on autophagy and apoptosis of testicular tissues in Brandt voles.These results indicated that GA has a dose-response on the reproductive ability of male Brandt’s vole.Lower doses of GA promoted the development of male Brandt’s voles by improving sperm quality,antioxidant capacity,and promoting the synthesis of reproductive hormones,while higher doses of GA caused reproductive function damage.The effect of GA on reproductive system may be based on the regulation of Akt/mTOR pathway on autophagy and apoptosis.