Effect Of Letrozole On Spermatogenesis In Cadmium-exposed Mice And Its Molecular Mechanism

The heavy metal cadmium is proposed to be one of the environmental endocrine disruptors of spermatogenesis.Cadmium-induced inhibition of spermatogenesis is associated with a hormone secretion disorder.Letrozole is an aromatase inhibitor that increases peripheral androgen levels and stimulate spermatogenesis.However,the potential protective effects of letrozole on cadmium-induced reproductive toxicity remain to be elucidated.In this study,male mice were administered Cd Cl₂（4 mg/kg BW）by oral gavage alone or in combination with letrozole（0.25 mg/kg BW）for 30 days.Cd exposure caused a significant decrease in body weight,sperm count,motility,vitality and plasma testosterone levels.Histopathological changes revealed extensive vacuolization and decreased spermatozoa in the lumen.However,in the Cd+letrozole group,letrozole treatment compensated for deficits in sperm parameters（count,motility,and vitality）induced by Cd.In addition,the results of the letrozole-treated group revealed a systematic array of all germ cells,a preserved basement membrane and relatively less vacuolization.Letrozole treatment significantly increased serum testosterone levels,which were reduced by Cd.For a mechanistic examination,RNA-seq was used to profile alterations in gene expression in response to letrozole.Compared with the Cd-treated group,RNA-Seq analysis showed that 214 genes were differentially expressed in animals treated with letrozole.Gene ontology（GO）enrichment analysis and KEGG signaling pathway analysis showed that steroid biosynthetic processes were the processes most affected by letrozole treatment.Furthermore,expression of the testosterone synthesis-related genes LHCGR（luteinizing hormone/choriogonadotropin receptor）,Cyp11a1（cytochrome P450,family 11,subfamily a,polypeptide 1）,Cyp17a1（Cytochrome P450,family 17,subfamily a,polypeptide 1）and Hsd3b6（3 beta-and steroid delta-isomerase 6）was significantly downregulated in Cd-treated testes,but these genes maintained similar expression levels in letrozole-treated testes as those in the control group.However,the transcript levels of inflammatory cytokines,such as IL-1βand IL-6,and oxidative stress-related genes（Nrf2,Nqo1,and Ho-1）showed no changes.In addition,we performed real-time quantitative polymerase chain reaction and protein immunoblotting experiments on testosterone synthesis-related genes to further validate the transcriptome results,and these genes were consistently expressed at the m RNA level and protein level.The present study suggests that the potential protective effect of letrozole on Cd-induced reproductive toxicity might be mediated by the upregulation of the expression levels of genes related to the steroid hormone synthesis pathway,which would beneficially increase testosterone synthesis to achieve optimum protection of sperm quality and spermatogenesis.