Adverse Effects And Mechanism Of Bisphenol A Exposure On Mitochondria And Endoplasmic Reticulum Of Male Germ Cells

As an environmental endocrine disrupter,bisphenol A?bisphenol,A,BPA?can enter the living things through diet,respiration and other means.Consistent with its wide distribution in the natural environment,BPA was found in animals'and human body fluids.A large number of studies have shown that BPA has adverse effects on the immune system,respiratory system,and so on.In the male reproductive system,BPA exposure is related to genital dysplasia and decreased semen quality.However,the mechanism underlying BPA-induced male reproductive toxicity remains unclear.Mitochondria and endoplasmic reticulum play a vital role in maintaining normal function of cells.Previous studies have found that environmental pollutants can cause endoplasmic reticulum and mitochondrial damage,suggesting that mitochondria and endoplasmic reticulum might play an important role in BPA induced toxicity in male reproductive system.We established in vitro model of BPA exposed mouse spermatocyte-derived GC-2 cells and in vivo model of BPA exposed Kunming mouse to evaluate the male reproductive toxic effects of BPA exposure and the adverse effect on the structure and function of mitochondria,and further to explore its mechanism.Contents1.Established in vivo model of BPA induced male reproductive toxicity.After 5 weeks exposure of BPA,we evaluated the effects of BPA exposure on semen quality and testicular morphology of mice.2.Established in vitro model of BPA exposed mouse spermatocyte-derived GC-2 cells to evaluate the toxicity of BPA exposure in GC-2 cells.We observed the morphology of GC-2cells,used MST and EdU to analyze GC-2 cell viability and proliferation,detected cell cycle and apoptosis by flow cytometry,used DCFH-DA probe to analyze intracellular ROS,evaluated mitochondrial structure by JC-1 detection kit,detected the level of Ca2+in cells by Fluo4 AM staining method,analyzed morphological changes of endoplasmic reticulum by endoplasmic reticulum staining kit and electron microscopy.3.Based on the established animal and cell models,we analyzed the effects of BPA exposure on the expression of the mitochondrial pathway and endoplasmic reticulum pathway related molecules.Results1.After 5 weeks exposure of BPA,the body weight and epididymis index of mice had no obvious change,the testis index of mice exposed to 300mg/kg/d BPA was decreased;the sperm with rapid motility of mice in 30mg/kg/d and 300mg/kg/d exposure group was significantly lower than that in control group,the sperm with slow motion in BPA exposed group was significantly higher than that in normal control group.The results showed that BPA exposure reduced the semen quality in mice.2.The morphological changes of testis in mice exposed to BPA for 5 weeks were observed.The results showed that the testis in BPA exposed mice showed different degrees of pathological changes,spermatogenic cells in the seminiferous tubules were disordered,and some of the spermatogenic cells fell off to the lumen,the extent of the lesion in the seminiferous tubules increased with increasing doses,the seminiferous tubules exhibited substantial shrinkage andvacuolation,and most spermatogenic cells were missing.TUNEL staining confirmed that the apoptosis rate in the testes increased with increasing BPA dose.3.The intracellular ROS was increased in GC-2 cells exposed to BPA,and pretreatment of antioxidants-NAC could partially reverse BPA induced inhibition of cell proliferation.These results suggested that ROS might be involved in the regulation of BPA triggered inhibiting of GC-2 cell viability.4.BPA exposure decreased the mitochondrial membrane potential and trigged abnormal expression of mitochondrial pathway related molecules,such as caspase3,caspase9,bcl2,Bax and bad.Mitochondria related molecular cyt-c released from mitochondria to cytoplasm,and nuclear transcription factor AIF transfered from mitochondria to nuclear in BPA exposed GC-2 cells.The mitochondrial pathway related molecules were also activated in the testis of BPA exposed mice.These results showed that BPA exposure had adverse effect on mitochondria,and mitochondrial pathway was activated.The mitochondrial pathway might play a role in BPA induced male reproductive toxicity.5.BPA exposure triggered abnormality of numbers and morphology in endoplasmic reticulum,and decreased intracellular Ca²⁺level.The markers of endoplasmic reticulum stress,GRP78 and caspase12 were activated,and endoplasmic reticulum pathway related molecules,such as PERK,p-PERK,EIF2 and p-EIF2 alpha,chop and ATF6 are activated in GC-2 cells exposed to BPA.The vivo model also confirmed this result.Pretreatment of antioxidant could partially reverse the activation of endoplasmic reticulum pathway induced by BPA exposure in GC-2 cells.These results suggested that endoplasmic reticulum was another target organelle of BPA exposure induced male reproductive injury,BPA exposure might activate endoplasmic reticulum pathway and endoplasmic reticulum pathway played a vital role in BPA triggered male reproductive toxicity.ConclusionBPA exposure induced spermatogenic cell toxicity in vitro and induced male reproductive toxicity in vivo,resulting in apoptosis of spermatogenic cells and reduction of sperm motility.Endoplasmic reticulum and mitochondria are target organelles of BPA exposure trigged male reproductive damage.Further studies showed BPA exposure activated mitochondrial pathway and endoplasmic reticulum pathway.ROS regulated mitochondrial pathway and endoplasmic reticulum pathway regulated might play an important regulatory role in male reproductive toxicity induced by BPA.