Impact Of Neonatal Exposure To Different Doses Of Bisphenol-A On The Puberty Of Female Rats

Objective:To observe vaginal opening day（VOD），hypothalamic kiss-1gene anduterus and ovarian estrogen receptors (Estrogen Receptor, ER) gene expression levelchanges and their protein level changes in neonatal rats exposure to different doses ofbisphenol-A（BPA）.Methods: Neonatal female SD rats were randomly divided into six groups: controlgroup, vehicle group,17β-estradiol group(17β-estradiol, E2,10μg d-1), low-dose BPAgroup (25μg kg-1d-1), medium-dose BPA group (50μg kg-1d-1) and high-dose BPA group(250μg kg-1d-1). The rats got7subcutaneous injections over postnatal day(PND)0-6andwere sacrificed on the VOD and weighed. The VOD was recorded. The hypothalamus,uterus and ovary were removed, weighed and then calculated the organ/body weight ratio.Immunohistochemistry was used to observe the localization of kisspeptin inhypothalamus. Hematoxylin-eosin staining (HE staining) was used to observe the changesof the morphologies of uterus and ovary. Real-time PCR was used to observe the mRNAlevel changes of hypothalamic kiss-1gene and ER gene in uterus and ovary. Western-blotwas used to observe the protein level changes of hypothalamic kisspeptin and ER in uterusand ovary.Results:1. The impact on weight：after the seven subcutaneous injections from PND0to PND6，there was no significant difference between the control group and other sixdrug groups on the weight of rats.（P>0.05）2. The VODs were advanced by E2,50μg kg-1d-1and250μg kg-1d-1BPA exposure（P<0.05）,not the vehicle and25μg kg-1d-1BPA exposure.（P>0.05）3. From the results of immunohistochemistry, the kisspeptinexpression was almost on nerve cells in the hypothalamus.4. The HE staining shows thatmyometrium and endometrium thickness of uterus in control，vehicle，25μg kg-1d-1,50μg kg-1d-1and250μg kg-1d-1BPA groups were significantly thicker than the E2group； the development of ovarian follicular was much lower than other five groups.5. On theresults of the organ/body weight ratio,the hypothalamus/body weight ratio was notdifferent between the treatment groups and the control group （P>0.05）. Theuterus/body weight ratio and the ovary/body weight ratio in E2group were much lowerthan that in control group （P<0.05）,while there was no significant difference in theuterus/body weight ratio and the ovary/body weight ratio compared3BPA groups tocontrol group （P>0.05）.6. From the results of RT-PCR, in the groups ofvehicle,25μg kg-1d-1,50μg kg-1d-1and250μg kg-1d-1BPA, there were no significantdifference in the mRNA expressions of hypothalamic kiss-1and ER in uterus and ovarycompared with control group on the VOD（P>0.05）.The hypothalamic kiss-1mRNAexpression,ERα mRNA expression in uterus and ERβ mRNA expression in ovary werelower in E2group than the control group（P<0.05）, while the ERβ mRNA expression inuterus and ERα mRNA expression in ovary in E2group were not significantly differentfrom these of control group.6. From the results of Western-Blot, in the groups ofvehicle,25μg kg-1d-1,50μg kg-1d-1and250μg kg-1d-1BPA, there were no significantdifference in the protein expressions of hypothalamic kisspeptin and ER protein expressionin uterus and ovary compared with control group on the VOD （P>0.05）. Thehypothalamic kisspeptin expression and protein level expression of ERα in uterus and ERβin ovary were lower in E2group than the control group（P<0.05）,while the protein level ofERβ in uterus and ERα in ovary were no significant difference between E2group andcontrol group（P>0.05）。Conclusions:①Daily subcutaneous injection had no great effection on body weightsof rats during the period of treatment.②There was kisspeptin expression on nerve cellsin the hypothalamus.③Neonatal50μg kg-1d-1and250μg kg-1d-1BPA exposure inducedpremature puberty of rats.④The premature puberty above mentioned was not caused bythe changes of the hypothalamic kiss-1mRNA and kisspeptin expression, in addition therewere no great changes on the hypothalamus/body weight ratio, uterus/body weight ratio,ovary/body weight ratio, the morphology of uterus and ovary or the expression of mRNAand protein level changes in ER on uterus and ovary.⑤Neonatal vehicle and25μg kg-1d-1BPA exposure did not induce premature puberty of rat.⑥Neonatal10μg d-1E2exposuremight not only lead to the premature puberty of rat, but also caused the reduction of the hypothalamic kiss-1mRNA expression, uterus ERα mRNA and ovary ERβ mRNAexpressions and their protein level expression compared with control group,as well as thedevelopment of uterus and ovary on VOD were much lower than these in control group.