Effects Of Prenatal DEHP Exposure On Expression Of Three Proteins Related To Reproductive Regulation In Hypothalamus Of Adult Offspring Rat

Objective:To investigate the potential effects of prenatal DEHP exposure on reproductive regulation of hypothalamic neuroendocrine system,the protein expressions of estrogen receptor alpha(ER?),Kisspeptin and aromatase(CYP19)in the anteroventral periventricular nucleus(AVPV)and medial preoptic nucleus(MPN)of adult offspring rats were detected.Methods:32 pregnant rats were randomly divided into four groups(n = 8 per group)and treated respectivly with corn oil(vehicle control),2 mg/kg,10 mg/kg or 50 mg/kg DEHP by gavage from gestational day 14(G14)to G19.The dosing volume was 1m L/100 g.Postnatal day 0(PND 0)was determined by the birth of pup.From PND21,the pups were weaned and were housed four same-sex littermates per cage.On PND70,one male and one female rat per litter(n = 8 per sex per group)were randomly chosen to be deeply anesthetized with chloral hydrate and were checked for lack of reflexes.Then the rats were perfused by 4% paraformaldehyde(PFA)through the transcardial pathway.After fixation,brain tissues were harvested and cut into coronal sections of 40-?m thickness using a vibrating blade microtome.Based on The Rat Brain in Stereotaxic Coordinates(6th),the AVPV and MPN were identified.One section per nuclei was chosen from the same plane of each animal.Immunohistochemistry was performed for ER?,Kisspeptin and CYP19 in the AVPV and MPN.Confocal microscopy was used to compare the density of ER?,Kisspeptin and CYP19.From the surface of each section,a stack of 10 images(with an interval of 1 ?m)was captured using a confocal microscope.Using NIH Image J 1.46 r software,the images were processed and analyzed for immunoreactive areas.The optical density of each protein in each group was quantified and then compared by one-way ANOVA and LSD test using SPSS.Results:(1)No significant difference was observed for ER? expression in male AVPV.2mg/kg DEHP decreased ER? expression in female AVPV(P = 0.023).A decreasing tendency of ER? expression was observed in female AVPV of 10 mg/kg DEHP group(P = 0.052).(2)No significant difference was observed for Kisspeptin expression in male AVPV.A decreasing tendency of Kisspeptin expression was observed in female AVPV of 2 mg/kg DEHP group(P = 0.060).10 mg/kg and 50 mg/kg DEHP decreased Kisspeptin expression in female AVPV(P = 0.012 and P = 0.040).(3)No significant differences were observed for CYP19 expression in male and female AVPV.(4)No significant differences were observed for ER? expression in male and female MPN.(5)No significant differences were observed for CYP19 expression in male and female MPN.Conclusions:(1)Prenatal DEHP exposure could decrease ER? and Kisspeptin expression in female AVPV while no effect was observed in male AVPV.(2)Based on our experiment condition,no effect of prenatal DEHP exprosure on CYP19 expression were observed in the AVPV of adult rats.(3)Based on our experiment condition,no effect of prenatal DEHP exposure on ER? and CYP19 expression were observed in the MPN of adult rats.(4)Prenatal DEHP exposure disrupted ER? and Kisspeptin expression in adult AVPV in a sex-specific manner and its impact on ER? expression showed low-dose effects.