The Effects Of G9a-Mediated Histone Methylation On Cadmium-Induced Male Reproductive System Damage

BackgroundCadmium(Cd)is a well-known toxic heavy metal,arising primarily from industrial contamination,food,drinking water and cigarette smoke.Male reproductive system is an important target in Cd exposure.A vast number of studies have shown that cadmium is associated with male infertility by causing oxidative stress.Certain studies have reported that epigenetic modification especially histone methylation occurs during spermatogenesis.G9 a plays a vital role in performing the monomethylation and dimethylation of H3K9(marked by H3K9me1 and H3K9me2,respectively),which plays a key regulatory role in the male reproductive system.However,little is known about the effect of histone modification on male fertility damage,and evidence is lacking regarding to the mechanisms involved.Methods(1)3-week-old male C57BL/6J mice were exposed to Cd by intraperitoneal injection with 2 mg/kg of body weight(bw)for 1,3 and 5 days,once a day.After exposure,hematoxylin and eosin(H&E)staining was applied to evaluate the cauda epididymal ducts and seminiferous epithelium histological examination,terminal dUTP nick-end labeling(TUNEL)assay and western blot for cleaved caspase 3,PARP,Bax and Bcl-2 was used to evaluate testicular cell apoptosis.Moreover,after treatment,male mice were kept until 8 weeks.Sperm counts were analyzed by computer-assisted sperm analysis(CASA),male fertility assay was evaluated by percent of impregnated females and litter size.(2)To address whether Cd-induced testicular cell apoptosis and male fertility damage was involved in G9a-mediated histone methylation,we conducted a 2 mg/kg of body weight(bw)exposure procedure for for 1,3 and 5 days by intraperitoneal injection and then evaluated the expressional levels of G9 a,H3K9me1 and H3K9me2 by western-blot analysis and immunofluorescence assay.Furthermore,we examined whether BIX-01294 can abolish H3K9me1 and H3K9me2 modifications after Cd exposure.BIX-01294 pretreatment attenuated the increased expression levels of histone modification marks and testicular cell apoptosis were evaluated by western-blot and immunofluorescence.Terminal d UTP nick-end labeling(TUNEL)assay was also applied to evaluate testicular apoptosis.Subsequently,male fertility,sperm counts and testes histological examinations were also assessed when mice were kept until 8-week-old.Results(1)We found that Cd exposure for 3 days and 5 days administration,the percentage of impregnated females and the litter size of the impregnated females significantly decreased in all of Cd-treated group.Following Cd treatment,the testis exhibited widespread structural abnormalities,which is concomitant with a mark increase in the seminiferous tubule score on day 5 of Cd exposure.Testicular cell apoptosis as evaluated by terminal d UTP nick-end labeling(TUNEL)assay and immunoblotting with increased levels of cleaved caspase 3,PARP and Bax and a decreased level of Bcl-2.(2)The result showed that the levels of G9 a,H3K9me1 and H3K9me2 were significantly increased following cadmium treatment as evaluated by western-blot and immunofluorescence analysis.Moreover,BIX-01294 pretreatment drastically prevented Cd-induced the activation of histone modification marks and testicular cell apoptosis.Furthermore,BIX-01294 pretreatment can increase sperm quality and attenuated Cd-induced structural changes.In addition,the percentage of impregnated females and the litter size of the impregnated females were significantly increased in the BIX-01294 preatment group.ConclusionIn summary,our results demonstrated that pubertal Cd exposure induced activation of G9 a followed by increased levels of H3K9me1 and H3K9me2,in addition to male fertility damage with a reduced pregnancy rate and litter size of pregnant females,which is partially due to decreased sperm counts,epididymal weight and relative epididymal weight.Furthermore,Cd exposure caused testicular cell apoptosis.Pretreatment with pharmacological inhibition of G9 a activity prevented Cd-induced male fertility damage and testicular cell apoptosis.These rodent data revealed that G9a-mediated histone methylation plays a key regulatory role in Cd-induced testicular cell apoptosis and male fertility damage.BIX-01294 has an anti-apoptotic and protective role against Cd toxicity in male fertility,which indicates its usefulness as a promising pharmacological candidate for preventing the reproductive toxicity of Cd.