The Alleviation Effect Of Melatonin On Hexavalent Chromium-induced Male Germ Cells Damage And Its Mechanism

The health of breeding sires is vital to the development of the livestock industry.Spermatogenesis is a complex and highly organized process with high sensitivity to external stimuli.Environmental pollution has emerged as one of the major factors affecting male fertility.Of these,hexavalent chromium（Cr（VI））is among the top 10 environmental pollutants,recognized as an endocrine disruptor and a reproductive toxicant.Due to the rapid industrialization,the concentration of Cr（VI）in the environment has risen sharply.Chromium-contaminated wastewater and chromium residue stacking could pollute groundwater,rivers and lakes,and then endanger farmland,aquatic products,livestock and human health.Research have revealed that excessive exposure to Cr（VI）poses a serious threat to male reproductive health.Cr（VI）exposure causes serious effects such as testicular atrophy,spermatogenic arrest and poor semen quality,and even male infertility.However,the specific damage process of Cr（VI）-induced male reproductive disorders and its network regulatory mechanisms have not been systematically explored.As an endogenous antioxidant,melatonin（Me）holds potent antioxidative and anti-inflammatory properties,becoming a potential candidate for treatment of a variety of diseases.Nevertheless,the mitigating effect of melatonin pretreatment on Cr（VI）-induced spermatogenic damage and its epigenetic regulatory mechanism are unclear.In this study,we used intraperitoneal injection of Cr（VI）in mice as an experimental animal model to investigate the occurrence and development of male reproductive system injury during one spermatogenic cycle after Cr（VI）exposure using histological morphological,immunofluorescence and CASA analyses.The pre-protective effects of melatonin against Cr（VI）-induced reproductive injury were also evaluated.In combination with in vitro,we deeply explored the regulatory network of Cr（VI）exposure on germ cell damage,as well as the pre-protective mechanism of melatonin against Cr（VI）-induced germ cell toxicity from the perspective of epigenetic modification.The main findings are as follows:（1）The testicular damage caused by Cr（VI）was persistent until day 21 after administration and then started to alleviate,with clear alleviation on day 35.Cr（VI）exposure resulted in massive loss of various types of spermatogenic cells,vacuolation,shrinking and even the empty tubules containing only Sertoli cells.Cr（VI）treatment swiftly triggered apoptosis in differentiating spermatogonia and spermatocytes.Sertoli cells were the most resistant in the seminiferous tubules,with no effect in number but disruption of the intercellular blood-testis barrier.The cauda epididymis sperm quality dropped abruptly after Cr（VI）exposure,persisting until day 21 after administration,followed by slow recovery.（2）Pretreatment with melatonin evidently relieved Cr（VI）-induced testicular damage and accelerated spermatogenic restoration,generating almost normal phenotype on day 35.For one thing,melatonin was found to exert a robust role in promoting undifferentiated spermatogonial self-renewal in response to Cr（VI）-induced cell elimination.For another,melatonin effectively inhibited Cr（VI）-induced germ cell apoptosis.Meanwhile,melatonin contributed to maintain the integrity of the blood-testis barrier and accelerated its reconstruction.In cauda epididymis sperm,pretreatment with melatonin significantly inhibited the Cr（VI）-induced decrease in sperm viability and density as well as the increase in sperm malformation,improving sperm quality.The addition of melatonin protected the fertility of male mice.（3）Cr（VI）exposure induced ROS accumulation,causing abnormal expression of mitochondrial fusion and fission genes,which resulted in disturbed mitochondrial dynamics in mouse undifferentiated spermatogonia.Meanwhile,Cr（VI）decreased the global m6A level by downregulating METTL3,which reduced the m6A enrichment level on mitochondrial fusion genes Mfn2 and Opa1 as well as mitochondrial autophagy genes Bnip3and Nix,inducing mitophagy.（4）Cr（VI）induced ROS accumulation also activated the HIF1α-BNIP3 pathway to regulate mitophagy in mouse undifferentiated spermatogonia.In addition to decreasing the expression of METTL3,Cr（VI）also downregulated the expression of m6A recognition protein YTHDF2.YTHDF2 directly interacted with Hif1αand Bnip3 m RNAs,mediating their decay in an m6A-dependent manner and participating in Cr（VI）-induced mitophagy.（5）Melatonin pretreatment inhibited Cr（VI）-induced decrease in cell viability and ROS accumulation in mouse undifferentiated spermatogonia.The protective roles of melatonin against Cr（VI）-induced mitophagy were exerted by restoration of METTL3-mediated RNA m6A modification and activation of mitochondrial fusion proteins MFN2 and OPA1,as well as inhibition of the mitophagy BNIP3/NIX receptor pathway.（6）Cr（VI）induced DNA damage,cycle arrest and apoptosis in mouse differentiating spermatogonia.Melatonin could protect spermatogonia from Cr（VI）-triggered damage via elimination of reactive oxygen species（ROS）as well as via suppression of ATM-p53phosphorylation and the mitogen-activated protein kinase（MAPK）pathway.Prior administration of melatonin also prevented the Cr（VI）-caused enrichment of H3K9me3 in the Mad1,Mad2 and Bcl2 gene promoter regions,precluding the G₂/M arrest and apoptosis in spermatogonia.（7）Cr（VI）exposure triggered mitophagy,ferroptosis and apoptosis in porcine undifferentiated spermatogonia.Cr（VI）activated mitophagy and ferroptosis pathways,respectively,which together promote apoptosis in porcine undifferentiated spermatogonia.Melatonin pretreatment effectively mitigated Cr（VI）-induced damage in porcine undifferentiated spermatogonia.In conclusion,this study systematically evaluated the damage of Cr（VI）on the male reproductive system and analyzed the mechanisms of Cr（VI）-induced spermatogonia damage.Melatonin demonstrated potent preventive effects against Cr（VI）-induced reproductive damage at both in vivo and in vitro levels.This study not only contributes to the understanding of fertility disorders in the context of environmental pollution,but also offers theoretical references for treating livestock reproductive diseases,improving livestock fertility,and promoting healthy livestock breeding.Furthermore,it provides a foundation and direction for the clinical prevention and treatment of male infertility caused by environmental pollution.