The Protective Effects Of Cyanidin-3-O-glucoside On Heat Stress-induced Male Reproductive Damage

Testis is the main site of spermatogenesis.It is necessary to ensure that the testicular temperature must be 2?8? below the body core temperature to maintain the best microenvironment for production of motile and fertile sperm.Heat stress can increase testicular temperature and cause spermatogenesis disturbance,thereby leading to the decrease of male reproductive ability and even infertility.Cyanidin-3-O-glucoside(C3G)is classical type of flavonoids abundant in fruit and vegetables,and is considered as a potential functional ingredient to protect male reproductive damage due to the remarkable antioxidant and other health-promoting benefits.To explore the protective mechanism of C3 G and its metabolite(PCA)on male reproductive damage induced by heat stress,we established the model of heat stress and gave C3 G and PCA intervention by intragastric administration.The main outcomes of this study are as follows:1.To explore the mechanism of heat stress-induced testicular injury,male mice(7?8 weeks)were used to establish male reproductive damage model induced by single heat stress in hot water bath(43?,30min).Otherwise,the sperm quality was determined by the computer-aided semen analysis system(CASA).And,the damage of testicular tissue of male mice were evaluated by hematoxylin-eosin staining,the immunofluorescence(IF)and western blotting(WB).According to results,heat stress can decrease the weight of testis,sperm counts,sperm motility,sperm vitality,increase sperm morphology,and induce the phosphorylation of e IF2? and the formation of stress granules(SGs).2.In order to investigate the protective effects of C3 G and PCA on heat stress-induced testicular injury,we gave the pretreatment with C3 G,PCA and ascorbic acid at the dose of 100mg/kg· bw by gavage for 7 days before heat stress.Then,C3 G,PCA and ascorbic acid were given once a day until the end of experiment.The hematoxylin-eosin staining was applied to observe histopathological changes of testis and epididymis,and the external diameter and thickness of seminiferous tubules were evaluated.At the same time,the sperm counts,sperm motility,sperm vitality and sperm morphology were measured by CASA.The results indicated that heat stress is considered to cause testicular damage,thereby leading to atrophy,vacuolation and disintegration of seminiferous tubules,and reducing the external diameter and the thickness of seminiferous tubules.C3 G and PCA can effectively improve testicular damage.3.To evaluate the effects of C3 G and PCA on the formation of SGs and the autophagy of spermatogenic cells were evaluated,The WB was applied to analyze the expression level of related proteins about SGs and autophagy in testis.The IF was used to measure the expression of SGs in testis.The results showed that heat stress(0 day)can induce the phosphorylation of e IF2? and the formation of stress granules,which can initiate the protective mechanism of autophagy and reduce the apoptosis of spermatogenic cells in testis.C3 G and PCA can reduce external stress of testis via health-promoting benefits,thereby reducing the phosphorylation level of e IF2? and suppressing the formation of stress granules,enhancing heat stress tolerance of testicular and maintaining normal spermatogenic cells.4.In order to explore the protective effects of C3 G and PCA on antioxidant system of testicular,the lipid peroxidation products and related antioxidant enzymes in testicular tissue were determined by kit.Real-time quantitative PCR(q PCR)was used to detect the expression of apoptosis-related genes induced by endoplasmic reticulum stress.TUNEL fluorescence was used to measure apoptosis.The results suggested that heat stress can easily lead to oxidative stress and endoplasmic reticulum stress,and then lead to apoptosis of spermatogenic cell.C3 G and PCA can alleviate oxidative stress and endoplasmic reticulum stress induced by heat stress through improving antioxidant system and regulating IRE1?-XBP1 pathway,to ensure the orderly progress of spermatogenesis.In conclusion,C3 G and PCA can increase heat stress tolerance of testicular by reducing the level of e IF2? phosphorylation and inhibiting the formation of stress granules.Meanwhile,C3 G and PCA effectively improve antioxidant system of testicular and regulate IRE1?-XBP1 pathway to alleviate apoptosis induced by oxidative stress and endoplasmic reticulum stress,so as to decrease spermatogenesis disturbance and testicular injury.The results of this study can provide a reliable theoretical basis for C3 G and PCA to improve male reproductive damage induced by heat stress,such as unhealthy living habits,occupational factors and pathological causes.