Melatonin Relieves Busulfan-induced Spermatogonial Stem Cell Apoptosis Of Mouse Testis By Inhibiting Endoplasmic Reticulum Stress

Studies show that the male infertility is related with genetic defects,environmental toxicants and cancer treatment with reproductive toxicity.The main reason of male infertility is the abnormal spermatogenesis caused by the impaired spermatagonial stem cells.Chronic myeloid leukemia is a myeloproliferative tumor characterized by unrestricted expansion of pluripotent bone marrow stem cells.Busulfan is widely used for the pretreatment of the hematopoietic stem cell transplantation in patients with chronic myeloid leukemia,although it can improve the survival rate of patients,but it causes the male infertility by damaging testis and reducing sperm count.Therefore,the underlying mechanism should be explored,and new agents should be developed to protect the male reproductive system from busulfan-induced damage.Endoplasmic reticulum stress?ERS?is triggered by a disturbance in endoplasmic reticulum homeostasis as a result of changes in redox state,nutrition and Ca²⁺level and protein synthesis rate and the occurrence of pathogens or inflammatory stimulation.ERS is a protective mechanism in physiological and pathological conditions,ERS is the key to the maintenance of cell homeostasis,however,continued ERS caused a variety of diseases,including cancer,inflammation,neurodegenerative diseases,metabolic diseases and immune diseases.The important role of ERS in development,metabolism and immunity has been confirmed in various cell types.Stem cells with the special ability to self-renew and differentiate into various somatic cells,which are involved in development,tissue renewal and certain disease processes in various tissues.Although several studies linking ERS to toxicants induced apoptosis,studies have yet to determine whether ERS is a contributing factor to the apoptosis of spermatogenic stem cells and busulfan-induced testicular damage.Melatonin is a kind of indole hormone synthesized by pineal gland.It is a well-known broad-spectrum antioxidant,anti-inflammatory and antitumour agent.Melatonin binds to the specific receptor or cytoplasmic protein and activates a series of signaling pathways.Studies have shown that melatonin can regulate the apoptosis process of normal cells and cancer cells,but the effects of melatonin on busulfan-induced ERS in mouse testes damage and spermatogenic stem cells apoptosis are less documented.In this experiment,we evaluated ERS activity by the immunofluorescence staining and Western blotting,and found that ERS signal strongly activated in busulfan treated mouse testis?in vivo?and spermatogonial stem cells?in vitro?.The expression of ERS related apoptosis proteins such as Caspase12,CHOP and Caspase3 was enhanced,the expression of P53 and PUMA apoptosis protein increases,thus,busulfan mainly induced testicular injury and germ cell apoptosis by activating ERS.In this study,the effects of melatonin were measured by the morphological analysis,histological analysis,immunofluorescence staining,flow cytometry and Western blotting.We found that melatonin can inhibit the ERS marker expression of GRP78,ATF6,pIRE1 XBP1 in mouse testis?in vivo?and spermatogonial stem cells?in vitro?.In addition,melatonin can inhibit the expression of apoptotic protein Caspase12,CHOP,Caspase3,P53 and PUMA.This study shows that ERS is an important mediator for busulfan-induced apoptosis.The attenuation of ERS by melatonin can prevent busulfan-treated SSCs apoptosis and protect busulfan-treated testes from damage.Thus,this study suggests that melatonin may alleviate the side effects of busulfan for male patients during clinical treatment.