Study On The Adverse Effects And Mechanism Induced By Glyphosate In Male Mouse Germ Cells

Objective:To study the effects and molecular mechanism of Glyphosate induced reproductive toxicity effects in male mice.Methods:In vivo study1.8-week-old male mice were oral administered with different concentrations of glyphosate for 35 d to establish in vivo model.2.Organ index of testes and epididymis were obtained after glyphosate exposure.3.Mice Semen quality including Sperm Motility and Sperm Morphology were determined by Sperm Class Analyzer.4.Microscopic changes of testes tissue samples were observed under light microscope.5.Apoptotic cells of testes tissue were detected by terminal deoxynucleotidyl transferase d UTP nick end labeling method.In vitro study1.Morphology of the GC-2 cells after glyphosate treatment was observed by optical microscope and Transmission Electron Microscopy.2.Cell cycle arrest and apoptosis were detected by Flow cytometry.Mechanism research1.Western Blot was used to detect cell cycle arrest and apoptosis related proteins.2.The mRNA and protein level of XAF1 in GC-2 cells were determined by qRT-PCR,Western blot and immunofluorescence method.3.The protein levels of XAF1 in testes were determined by Western blot and immunofluorescence method.4.siRNA were used to investigate the related molecular mechanism of glyphosate induced reproductive toxicity effects.Results:In vivo study1.The general condition of glyphosate treated mice had no obvious changes compared with the control group after oral administration of glyphosate for 35 d.The body weight,testis index and epididymis index in glyphosate treated mice had no significant differences compared with the control group.2.The fast moving sperm count was significantly decreased after treated with Glyphosate.Meanwhile,the numbers of motionless sperm in glyphosate treated groups were obviously increased.3.The sperm density in the high dose of glyphosate treated decreased significantly compared with the control group.The sperm malformation rate increased significantly in 180mg/kg Gly and 540mg/kg Gly group increased obviously.4.After glyphosate exposure for 35 d,there were different pathological changes of testes in glyphosate exposure groups including a large number of vacuoles,disorder of spermatogenic cells and decreased number of sperm in seminiferous tubule.Besides,the injury of testes showed dose-dependent manner.5.The apoptotic cells of testes increased in glyphosate exposure groups determined by TUNNEL method.In vitro study1.The GC-2 cell morphology under the optical microscope showed significant changes after treated with glyphosate for 48 h.The cell volume became smaller,the shape became round,the cell gap increased and the death cell increased.2.Typical apoptotic characteristics,mitochondrial swelling and endoparasitic reticulum injury were observed under transmission electron microscopy(TEM)in GC-2 cells treated with glyphosate for 48 h.3.Glyphosate can inhibit the growth and result in G1 phase arrest and apoptosis in GC-2 cells after exposure for 48 h.Mechanism research1.The expressions of apoptosis and cell cycle related proteins,such as p21,CyclinD1,Cyclin E and caspase3,changed obviously after glyphosate treatment.2.After glyphosate treated for 48 h,the mRNA expression level of XAF1 increased.The 25mg/L Gly dose group increased significantly compared with the normal control group.Western blot and immunofluorescence results showed that Gly treatment can promote the protein expression of XAF1 in GC-2 cells;3.The expression of XAF1 in the testis of mice was up-regulated,especially in the 180mg/kg Gly and 540mg/kg Gly dose groups.4.After Gly(25mg/L)treated for 48 h,the death GC-2 cell of si XAF1 in group was less than the NC group.The apoptosis rate was significantly lower compared with NC group.Besides,the activation of pro-apoptotic proteins was attenuated after XAF1 interference.Conclusion:1.Glyphosate exposure for 35 d can lead to obvious male reproductive toxicity in mice such as pathological changes of testis tissue and decreased semen quality.2.Glyphosate can result in G1 phase arrest and apoptosis in CC-2 cells.In vivo experiments also confirmed that Gly exposure could lead to cells apoptosis in mice testes;3.XAF1 participated in Gly induced apoptosis in GC-2 cells.