The Protective Effects Of Cyanidin-3-O-glucoside On Cadmium-induced Spermatogenic Dysfunction In Male Pubertal Mice

Objective:Cadmium(Cd),a widespread environmental contaminant,has been generally recognized as a primary cause of male infertility due to an attenuated effect on semen quality through oxidative damage,disturbance of endocrine system and others negative responses.Comparing to sexual maturity,the male reproductive system in puberty is more sensitive to the toxicity of Cd.Cyanidin-3-O-glucoside(C3G)is a classical type of flavonoid accompanied by remarkable anti-oxidative ability and constitutes a large proportion of phytonutrient intake in the average daily dietary intake,which has been considered as the underlying ingredient against Cd-induced reproductive toxicity.This study sought to demonstrate the protective effect of C3 G against Cd-induced spermatogenic dysfunction and also the corresponding mechanism in vivo by treating the male pubertal mice exposed to Cd with C3 G as a dietary supplement.Methods:Mice at age of 25 days were given cadmium chloride at the dose of 5.0 mg/kg·bw by gavage once a day,meanwhile,500 mg/kg·diet C3 G was added into the feeds for free-feeding.After treating for 30 days until sexual maturity,the mice were sacrificed,subsequently,the serum,testis,epididymis,sperms,hypothalamus,and pituitary were removed for the measurement of following parameters.(1)We first estimated the protective effect of C3 G against Cd-induced testis damage and decreased sperm quality.The hematoxylin-eosin staining was used for the histological evaluation of testis and epididymis,and spermatogenic epithelial score.The computer-aided semen analysis system was used for the determination of sperm counts,abnormal rate,motility,vitality,and other motion parameters.In addition,the lactate dehydrogenase and its isozyme in testis were indicated by the commercial kit.(2)The impact of exposure to Cd and intervention of C3 G on blood-testis barrier was considered by analyzing the corresponding proteins with the techniques of immunohistochemistry(IHC),immunofluorescence(IF),and western blot(WB).(3)The possible effects of C3 G against on spermatogenesis was discussed by evaluating spermatogenic epithelial cycle via Periodic Acid-Schiff staining.After that,the IHC,IF,WB,real-time quantitative PCR(qPCR)and enzyme-linked immune sorbent assay(ELISA)were used to present the alteration of histone-protamine exchange in the period of spermiogenesis and histone modification.(4)The anti-oxidative ability of C3 G in testis was evaluated by conducting the content and activity of anti-oxidative enzymes and advanced lipoxidation end products in testis.The DNA damage marker of sperm was identified by IF,the cell apoptosis was performed by Terminal deoxynucleotidyl transferase dUTP nick end labeling,and the expression of proteins associated with apoptosis signaling pathway was quantified by WB,for exploring the anti-apoptosis mechanism of C3 G on oxidative damage mediated-sperm cell apoptosis caused by Cd in testis.(5)The sex hormone concentration in serum was conducted by ELISA kit,the gene expression related to synthesis of gonadotropin-releasing hormone in hypothalamus and the gene expression of hormone receptor in pituitary and testis was quantified by qPCR,and the protein expression involved in synthesis and secretion of testosterone in testis was performed by WB,for discussing the regulation of C3 G on Cd-induced dysfunction of hypothalamus-pituitary-gonad axis.Results:(1)C3G significantly increased the total number,motility,and vitality of sperms compared to the Cd injury group,which means C3 G can improve the quality of sperm.(2)After exposure to Cd,the distribution and expression of proteins related to blood-testis barrier junction were consistent to the untreated mice,which means Cd does not show toxicity to blood-testis barrier under this exposed condition in puberty.(3)Exposure to Cd in male puberty can retard the exchange of histone to protamine via the abnormal modification of histone in sperm nucleus,which disrupts the spermatogenesis at the period of spermiogenesis.Nevertheless,C3 G treatment can maintain a regular histone modification,and then normalize the exchange of histone to protamine,subsequently protect the spermiogenesis from Cd damage.(4)The failure of spermiogenesis results in a vulnerable sperm nucleus that easily to be attacked by Cd-induced oxidative stress,which can cause sperm DNA damage in Cd injury group testis accompanied by cell apoptosis.The C3 G treatment can predominantly restore the antioxidative system and protect the sperm from the Cd-induced oxidative damage.(5)Exposure to Cd in male puberty can disturb the function of the hypothalamus-pituitarygonad axis and cause an abnormal corresponding hormone secretion,especially a higher testosterone concentration.C3 G treatment can ameliorate the gonadotropin-releasing hormone synthesis in the hypothalamus,the expression of hormone receptor in pituitary and testis,and testosterone synthesis in testis,which reestablished the sex hormone secretion that contributes to normal spermatogenesis.Conclusion:C3G effectively protects the spermatogenesis in male pubertal mice against the damage from Cd via normalizing histone modification for releasing the retardation of histone to protamine exchanges in spermiogenesis,improving the anti-oxidative system of testis associated with its ability to anti-apoptosis,and restoring the function of hypothalamus-pituitary-gonad axis for maintaining a steady sex hormone synthesis secretion.The results of this study suggest that consumption of anthocyanins can be protective against Cd-induced male pubertal reproductive dysfunction.