Influenc Of Female Mice Exposed To PFASs On Reproductive Endocrine And Thyroid Hormones Leading To Relevant Outcomes

BackgroundPerfluorooctanyl sulfonate(PFOS),a degradation product of sulfonyl-based fluorochemicals,is used extensively in industrial and household applications,such as stain-resistant coatings for fabrics and carpets,oil-resistant coatings for paper products approved for food contact,fire-fighting foams,mining and oil well surfactants,floor polishes,and insecticide formulations.PFOS is 8 carbon and 17 fluorine ion compound.Some reaserchs report that high serum level of PFOS in women associate with a longer waiting time to pregnancy and later age of menarche.The 1-to 2-week exposure to PFOS(10 mg/kg)has been reported to cause a persistent diestrus in rats.Average level of serum PFOS in human has been reported to range from 145 to 3490 ng/ml.The exposure of rats to 0.1 mg/kg of PFOS during gavage day 2-21 produces a serum level of 370 in rats.PFOS is well absorbed but poorly metabolized and cleared,and geometric mean half-life of serum elimination of PFOS in humans has been estimated to be approximately 4.8 years.However,no studies to date have assessed the influence of long-term exposure to an environmental-dose of PFOS on female reproductive endocrine and function.Hypothalamic-pituitary-gonadal(HPG)hormones are very important for female reproductive health including follicular development,maturation and ovulation.Gonadotropin-releasing hormone(GnRH)stimulates the secretion of luteinizing hormone(LH)and follicle stimulating hormone(FSH).The gonadotropins enhance biosynthesis and secretion of estrogen(E2).Steroidogenesis is primarily regulated by the steroidogenic acute regulatory(StAR)protein and cytochrome P450-mediated cholestrol side-chain cleavage enzyme CYP11A1(P450scc).Researchs in recent years indicated that estrogen receptor a(ERa)did not express in GnRH neurons.E2 regulates positivey the AVPV-kisspeptin neurons to induce the production of LH surge and ovulation.Perfluorobutanesulfonyl fluoride(PBSF)is excreted rapidly in male and female urine.One study indicates that parental exposure to PFBS can cause a slight delay of preputial separation in male rat offspring.PFOS has been reported to reduce the levels of maternal thyroid hormones leading to the fetal hypothyroxinemia.Several lines of evidence suggest that the thyroid hormones are critical for growth development and reproductive tract development in rodent models.Objective1.To determine the influence of chronic exposure to low dose of PFOS in female mice on reproductive endocrine and ovary function.2.To examine the effects of prenatal exposure to PFBS on perinatal thyroid function and growth-development in female miceThe First Part Influence of chronic exposure to low dose of PFOS in female mice on reproductive endocrine and ovary functionMaterials and Methods1.Two-month-old female mice were given the oral treatment with PFOS(O.lmg/kg/day)for consecutive six months.The estrous cycle was examined by vaginal smears daily.2.Follicles and corpora lutea were counted in the sections of ovary stained with hematoxylin and eosin(HE).3.Kisspeptin neurons were examined by immunohistochemistry.4.The serum E2,P4,LH,FSH and GnRH were measured by radioimmunoassay.5.Levels of Kissl,GnRH,StAR,P450scc,3?-HSD and P450arom mRNA were measured by RT-PCR6.Using Chromatin immunoprecipitation assay(CHIP),the acetylation of StAR or P450scc was examined.Results1.In comparison with control mice,the mice treated with PFOS(PFOS mice)exhibited the prolongation of estrous cycle starting in the third month,mainly increased the duration of diestrus.2.At proestrus,the levels of E2,FSH,LH and GnRH in PFOS mice were reduced compared to control mice.At diestrus,the level of E2was lower in PFOS-mice than those in control mice.In comparison with control mice,the level of StAR mRNA and StAR histone H3K14 acetylation were significantly reduced at proestrus or diestrus in PFOS-mice.3.PFOS mice showed a significant decrease in the number of antral and preovulatory follicles,the number of corpora luteum and level of P4.The LH-surge failed to be observed in PFOS-mice.4.At proestrus,the number of kisspeptin immunoreactive cell bodies(kisspeptin+cells)in AVPV and the level of Kissl mRNA in AVPV were reduced in PFOS mice compared to controls.In PFOS-mice were treated with the same high-dose of E2,the levels of AVPV-kissl mRNA and serum P4,and the LH-surge could be recovered.Conclusion(1)The chronic exposure to low-dose PFOS can suppress histone acetylation of StAR leading to reduction of E2 biosynthesis.(2)The reduction of E2 inhibits the activation of AVPV-kisspeptin neurons at proestrus and the production of LH-surge;the decline of E2 reduces follicle's development,maturation and ovulation.—The Second Part Effects of prenatal exposure to PFBS on perinatal thyroid function and growth-development in female miceMaterials and Methods1.The pregnant mice received the treatment with PFBS(50,200 or 500 mg/kg/day)from GD1 to GD19 by gavage.2.The time of eye opening,vaginal opening and first estrus were examined.3.Estrous cycle was examined by vaginal cytology.4.The number of follicles and corpora lutea and thickness of uterine tissue were observed in the sections of ovaries and uterus stained with hematoxylin and eosin(HE).5.The serum E2?P4?LH?T3?T4?TSH and GnRH were measured by enzyme-linked immunosorbent assay(ELISA)kits.Level of TRH mRNA was examined by RT-PCR.Results1.The female pups exposed prenatally to PFBS(200 and 500 mg/kg/day)(PFBS-mice)showed a significant decline of body weights in neonatal mice,pubertal and adult periods.There was a slight but significant delay of eyes opening PFBS-mice compared to controls.2.In comparison with control mice,PFBS-mice showed a delayed vaginal opening and first estrus.3.PFBS-mice(postnatal days 35-60)showed a prolongation of diestrus,decrease of ovaries and uterus sizes and weight with decline in numbers of follicles and corpora luteum.4.The level of serum E2 was reduced in puberty PFBS-mice,but not in neonatal and adult PFBS-mice,which was associated with a slight increase in the level of LH.The levels of serum T3 and T4 were permanently reduced in neonatal,puberty or adult PFBS-mice without the changes in levels of serum TSH and TRH mRNA.5.The dams exposed to PFBS(200 and 500 mg/kg/day)showed a significant decline in the levels of T4 and T3 rather than TSH and TRH.ConclusionThe prenatal exposure of female mice to PFBS(200/500 mg/kg)leads to a permanent hypothyroidism,which impairs the perinatal growth and development,and reproductive function.