Effects Of Maternal Exposure To Bisphenol A During Pregnancy And Lactation On Endochondral Ossification In Offspring And The Regulatory Role Of Wnt/β-catenin Signaling Pathway

Objective: Bisphenol A(BPA)is a known environmental endocrine disrupting chemical,which was used to synthesize polycarbonate plastics and epoxy resins.Previous studies have indicated that prenatal BPA exposure decreased femur and tail length,impaired growth plate and mineralization in offspring.However,the effect of prenatal BPA exposure on endochondral ossification and underlying mechanisms are unclear at present.In order to explore the effect of prenatal BPA exposure on endochondral ossification and Wnt/β-catenin signaling pathway,we treated prenatal Wistar rats with or without BPA to observe the effects of prenatal BPA exposure on endochondral ossification,including chondrocyte proliferation,differentiation,terminal differentiation,apotosis,osteoclastogenesis and other processes of endochondral ossification.At the same time,we explored the effect of prenatal BPA exposure on wnt/β-catenin signaling pathway.Our goal was to provide further data support for the study of the relationship between prenatal BPA exposure,endochondral ossification and Wnt/β-catenin signaling pathway.Method: Eighteen female and 18 male Wistar rats at age of 8-week were obtained from Experimental Animal Center of China Medical University.After a week of adaptation,male and female rats were 1:1 matched.The day of detection of the vaginal plug was designated the first day of gestational day(GD 0).Pregnant rats were randomly divided into control group,0.01 mg/L BPA group and 0.1 mg/L BPA group,containing 6 animals each.BPA was given via drinking water from GD 0 to PND 21.After weaning,the offspring were fed with distilled water and anesthetized at age of 4-week and 8-week.The length from mouse to anus,tail length and body length were measured.The left femur and tibia were dissected out,and then measured with vernier caliper.The left tibias were fixed in 4% phosphate-buffered formalin for48 h,followed by decalcificated in 15% EDTA and then embedded in paraffin.Five-μm-thick sections were prepared from these paraffin blocks.For quantitative histology of the growth plate,sections were stained with hematoxylin-eosin,observed under 10× microscope,photographed,and analyzed by Image J software.The growth plate height,resting zone height,proliferative zone height and hypertrophic zone height was measured in four equal parts of each image.Furthermore,to observe the effect of prenatal BPA exposure on the formation of secondary ossification center and trabecular in offspring,we performed Alcian blue/Van Gieson double staining on tibia sections and observed under microscope.Immunohistochemical staining was used to detect the expression of cartilage proliferation markers(PCNA,Col2a1),differentiation markers(Col10a1),terminal differentiation markers(MMP13,OPN),TUNEL staining was used to detect chondrocyte apoptosis,TRAP immunohistochemical staining was used to detect osteoclastogenesis,RANKL,OPG immunohistochemical staining was used to detect the effect of prenatal BPA exposure on RANKL/OPG signaling pathway.To investigate whether Wnt/β-catenin signaling pathway was involved in prenatal BPA exposure,we detected the expressionof Wnt/β-catenin downstream effector protein β-catenin and the negative feedback regulator CXXC5 by immunohistochemistry.Results: 1.Effect of prenatal BPA exposure on body weight and body length: The length of tibia,femur and tail were significantly decreased in prenatal BPA exposure offspring(P<0.05).These changes were more obvious at age of 4-week.2.The effect of prenatal BPA exposure on growth plate morphological: Compared with controls,the height of total growth plate,proliferation zone,hypertrophy zone were significantly decreased in prenatal BPA exposure offspring(P<0.05),furthermore,the growth plate chondrocytes were disordered,and the distance between chondrocyte columns was increased in prenatal BPA exposure offspring.For male rats,only total growth plate height was significantly decreased in prenatal BPA exposure offspring at age of 4-week(P<0.05),and there were no significant changes among three groups at age of 8-week.The secondary ossification center of prenatal BPA exposure offspring was significantly smaller and the trabecular was thinner and shorter than controls,these changes were more obvious in 4-week offspring.3.The effect of prenatal BPA exposure on chondrocyte proliferation and differentiation: Compared with controls,prenatal BPA exposure significantly decreased the expression of chondrocyte proliferation markers(PCNA,Col2a1)and differentiation markers(Col10a1)in offspring rats(P<0.05).4.The effect of prenatal BPA exposure on chondrocyte terminal differentiation: Compared with controls,prenatal BPA exposure significantly increased the expression of terminal differentiation markers(MMP13,OPN)in offspring rats(P<0.05).5.The effect of prenatal exposure on chondrocyte apoptosis:The rate of chondrocyte apotosis were significantly increased in Prenatal BPA exposure groups compared with controls(P<0.05).6.The effect of prenatal BPA exposure on osteoclastogenesis: Compared with controls,the number of osteoclasts in prenatal BPA exposure groups were reduced(P<0.05).There were no significant difference among three groups in RANKL expression level while the expression of OPG were significantly increased in prenatal BPA exposure offspring,thus the RANKL/OPG ratio were decreased in prenatal BPA exposure groups compred with controls(P<0.05).7.The effect of prenatal BPA exposure on Wnt/β-catenin signaling pathway: Prenatal BPA exposure decreased the expression of β-catenin while stimulated the expression of CXXC5(P<0.05).Conclusions: 1.Prenatal BPA exposure decreased total growth plate height and each zone height in female offspring.2.Prenatal BPA exposure inhibited chondrocyte proliferation and differentiation,accelerated terminal differentiation and apoptosis,and inhibited osteoclastogensis by suppressing RANKL/OPG signaling pathway in offspring,thus regulated endochondral ossification.3.Prenatal BPA exposure may inhibited endochondral ossification by downregulating Wnt/β-catenin signaling pathway via stimulating the expression of CXXC5.