Maternal High Fat Diet Combined With Bisphenol A Exposure Cause Multigenerational Malfunction Of Femal Reproductive Systems

Objective:The environmental pollutant Bisphenol A（BPA）is one of the common endocrine disruptors（EDCs）,which is easily exposed to the population and disrupts reproductive function via affecting the hypothalamus-pituitary gonadal axis（HPG）.Previous studies have demonstrated that overnutrition affects fertility.While the effects of BPA exposure or nutritional imbalance on reproductive ability have been tested extensively,studies on the long-term consequences of simultaneous exposure to both BPA and overnutrition induced obesity for the reproductive health remains to be determined.In fact,the effects of maternal diet-induced obesity combined with environmental pollutant exposure on the reproductive system are of greater concern,especially on the first-and second-generation female offspring in adulthood.Both maternal obesity and maternal exposure to adverse environmental factors are the important factors influencing the growth of offspring.Herein we aim to determine the effects of maternal exposure to BPA in combination with high fat feeding on the reproduction and key molecular mechanism in subsequent generations of female mice.In this study,ICR female mice（F0）were exposed to dietary fat intake（Low-fat diet,LFD or High-fat diet,HFD）with or without BPA given by gavage.F0 mice were mated with untreated males to generate the first generation（F1）,and then adult F1female mice were mated with normal males to create the second generation（F2）.F1 and F2 female offspring received normal drinking water and normal diet chow.To observe the effects of maternal high-fat diet feeding induced obesity with BPA exposure on reproductive characteristic and function in the next generations.Besides,to investigate the key molecules involved in the reproductive dysfunction of female offspring caused by the intergeneration of multiple adverse factors exposure in the mother,so as to further understand the complex influence pathways.Methods:Six-week-old ICR female（F0）mice were purchased and allowed to acclimatize to the facility for one week before transfer to Specific Pathogen Free（SPF）animal room.F0 were randomly received four different treatments before and during pregnancy until weaning（10weeks+3 weeks+3 weeks）:LFD（10%of fat,LFD+vehicle）;BPA（LFD+500μg/kg/day BPA）;HFD（60%of fat,HFD+vehicle）;HFD+BPA（HFD+500μg/kg/day BPA）.BPA was dissolved in 0.01%ethanol in PBS and freshly prepared every day for gavage.The F0 female mice were mated with age-matched healthy males to generate F1 female offspring.The F1females were mated with untreated males at the age of 12w to create F2female offspring.A vaginal plug or vaginal fluid smear with sperm indicated successful conception（0.5day）.The first generation（F1）of female offspring were labeled as O₁-LFD,O₁-BPA,O₁-HFD,O₁-HFD+BPA,the second generation（F2）of female offspring were labeled O₂-LFD,O₂-BPA,O₂-HFD,O₂-HFD+BPA.F1 and F2 female offspring were received normal drinking water and normal diet chow.Results:LC-MS/MS result showed directly exposure to BPA led to increased urine BPA concentrations compared to those mice without BPA exposure in the F0 generation.It found that exposure to HFD+BPA had higher urine BPA levels（2.493±1.151μg/ml）than single BPA（1.923±0.569μg/ml）and the similar urinary BPA levels in the LFD（0.474±0.147μg/ml）and HFD（0.544±0.133μg/ml）mice;F1 and F2 female mice from maternal BPA plus HFD exposure showed higher urine BPA levels than those from maternal BPA exposure solely:O₁-HFD+BPA（39.888±20.236ng/ml）>O₁-HFD（20.183±6.683ng/ml）>O₁-BPA（1.799±3.659ng/ml）>O₁-LFD（0.047±0 ng/ml）;O₂-HFD+BPA（59.328±15.921ng/ml）>O₂-HFD（23.215±21.714ng/ml）>O₂-BPA（9.328±11.733ng/ml）>O₂-LFD（1.949±2.536 ng/ml）.The combination of maternal HFD and BPA additively prolonged F0 pregnancy length,increased the possibility of dystocia and increased the birth weight in the F1,and BPA exposure alone showed reduce the number of newborns in the F1 generation;In the O₁-HFD+BPA、O₁-HFD group of the female F1generation,the mice also showed dystocia when they were mated,but there was no difference in the birth weight and the number of births in the F2generation of newborns.Maternal exposure to BPA combined with HFD feeding advanced the vaginal opening day which means early puberty;altered estrous cyclicity,especially lengthened diestrus;decreased developmental follicle but increased corpus luteum numbers;decreased serum FSH level.Moreover,the combination increased ovarian Esr1 in the F1 generation and elevated Esr2 expression in the F2 generation;decreased hypothalamic Kiss1 expression in the F1 and raised hypothalamic Kiss1expression in the F2 female mice,Kiss1 promoter specifically showed hypomethylation in the F1 generation while hypermethylation in the F2generation.Conclusions:This study indicated that maternal exposure to BPA in combination with HFD exerted a multigenerational effect on female reproduction,and that HFD can accentuate BPA concentration in female offspring of mothers with higher levels of BPA.Further experiments are needed to explore the mechanisms underlying this intergenerational effect.