| Bisphenols（BPs）,the chemicals that widely used in the manufacture of industrial and daily consumer products,can interfere with the synthesis and metabolism of endogenous hormones,causing metabolic disruptions.Prenatal and postnatal exposure to BPA can lead to insulin resistance and metabolic dysfunction.Moreover,the presence of BPs in cord blood and milk can bring potential longterm effects on offspring.Besides,the skeletal muscle development initials during the embryonic stage and most of the myofibers formation during the fetal stage.As the largest insulin target tissue,skeletal muscle plays an essential role in keeping glucose homeostasis and energy balance.Only a few studies have focused on the effects of bisphenols on skeletal muscle tissue.In the present study,pregnant ewes were treated with bisphenols to evaluate the effects of bisphenols exposure on fetal skeletal muscle development through in vivo and in vitro.Pregnant sheep（N = 7 8 / group）were treated with BPA or BPS（0.5 mg/kg/day）from gestational day（GD）30 to 100.After 20 days washout period,at GD120,fetal hindlimb skeletal muscle was harvested.The cross-sectional area of myofibers was tested using HE staining.The expression of genes related to the different fiber types,myogenic regulatory factors,fiber size were detected using q RT-PCR.The expression of proteins associated with different fiber types was evaluated using Western blotting in skeletal muscle tissues.Fetal primary myoblasts were isolated and purified to investigate proliferation and differentiation into myotubes.Moreover,mouse C2C12 and rat L6 myoblast cell lines were used as the test model,the cytotoxicity and proliferation of C2C12 myoblasts were detected using MTT and Ed U assays,separately.The area and diameter of differentiated myotubes were using immunofluorescence staining.The protein expression of insulin signaling molecules in C2C12 myoblasts and differentiated myotubes were conducted using Western blotting.Results showed as following:1.In vivo results of animal experiments:（1）The mean area of myofibers was found to be significantly larger in maternal BPA and BPS treatment groups for both female and male fetuses when compared to their control counterparts.（2）In maternal BPA-treated group,female fetuses had higher MYH1,and in maternal BPS-treated group,female fetuses had more MYH2 and MYH7 m RNA expression compared to their control counterparts（P < 0.05）,while there were no differences were observed in male fetuses（P > 0.05）.（3）In maternal BPS-treated group,female fetuses had more fiber hypertrophy promoter（JUNB）,myoblasts fusion promoter（My D88）and more myogenic regulatory factors（Myf5,Myog,Myo D and MRF4）m RNA expression compared to their control counterparts（P < 0.05）.No differences were observed in males（P > 0.05）.（4）Comparisons of skeletal muscle tissue between sex and within group,revealed female fetuses had higher fast MHC expression in all the control,BPA-and BPS-treated groups,and slow MHC expression only in female fetuses in the maternal BPS-treated group（P < 0.05）.2.In vitro results of animal experiments:（1）Increased the area and diameter of differentiated myotubes independent of sex upon differentiation were shown upon differentiation（P < 0.05）after gestational BPA or BPS treatment.Females had larger myofiber and differentiated myotubes than males in all treatment groups after induced differentiation（P < 0.05）.（2）Myoblast proliferation and insulin responsiveness were not significantly altered after gestational BPA or BPS exposure（P > 0.05）.3.Results of myoblast cell lines in vitro:（1）C2C12 myoblasts were more susceptible to bisphenols compared to L6 myoblasts.The cytotoxicity order of the three chemicals was BPA > BPF > BPS.（2）C2C12 myoblast proliferation was promoted only in 10-4 M BPF exposure group and the fusion index was increased after exposure to either BPS or BPF at doses of 10-4 M,10-6 M,10-8 M and 10-10 M.（3）C2C12 and L6 myoblasts were treated with non-cytotoxic dose（10-5 M）of BPA,BPS or BPF for 5 d,BPS and BPF reduced baseline expression of p-AKT（Thr）and p-GSK-3β,but not decreased downstream effectors such as mTOR and GLUT4.In conclusion,gestational sheep exposure to BPA or BPS from gestational day（GD）30 to 100 leads to increasing myofiber area and larger myotubes,inducing fetal fiber hypertrophy in skeletal muscle independent of sex.Mouse C2C12 myoblasts is a more sensitive model in vitro for testing the effect of bisphenols exposure on myogenesis.Exposure to BPS and BPF could reduce baseline expression of pAKT（Thr）and p-GSK-3β,but not influence downstream effectors such as mTOR and GLUT4.BPS or BPF exposure evaluated here does not result in impaired insulin responsiveness.However,partially modulate early effectors of the insulin receptor signaling pathway were down-regulated.The safety of BPA substates applied in industrial production needs to be further explored. |
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