Effects And Bad Outcomes Of Exposure To Environmental Estrogens On Female Reproductive Endocrine Fuction

BackgroundEnvironmental estrogens are natural or synthetic compounds in the environment with estrogenic activity.Since environmental estrogens can disrupt the actions of endogenous hormones and the regulation of endocrine system,they are also called environmental endocrine disruptors(EEDs).In recent years,since exposure to triclosan(TCS)from commodity and personal care,perfluorooctane sulfonate(PFOS)from coating of non-stick pans and plasticizer bisphenol A(BPA)become more extensive,the accumulation of these chemicals in body is increasing gradually.The reproductive system is one of the target organs highly sensitive to environmental estrogens.The epidemic data showed that environmental estrogens are in relation with the infertility,abortion,menstrual cycle disorder and so on,but the mechanisms remain largely unknown.Female reproductive function is controlled by hypothalamic-pituitary-ovary(HPO)axis.Hypothalamic gonadotropin-releasing hormone(GnRH)neurons release GnRH,stimulating the secretion of luteinizing hormone(LH)and follicle stimulating hormone(FSH)from anterior pituitary,thus increasing the synthesis and release of estrogen(E2)in ovaries,whereas E2 can negatively regulate the GnRH neurons.Recent researches have proved that the GnRH neurons don't express estrogen receptor(ER),whereas kisspeptin neurons located in the anteroventral periventricular nucleus(AVPV)express ER.The activated ER stimulates the AVPV-kisspeptin neurons to release kisspeptin,then activate the kisspeptin receptor in GnRH neurons,increase the release of GnRH and LH,induce the production of preovulatory LH surge(LH-surge)and increase the level of E2.However,it remains unclear whether environmental estrogens influence the E2-induced positive feedback in AVPV-kisspeptin neurons,leading to reproductive endocrine disorders.In addition,the metabolism of E2 plays an important part in regulating reproductive endocrine.Estrogen sulfotransferase(EST)is the main enzyme to catalyze the sulfoconjugation of E2.The reduction of EST expression or activity can increase the proportion of activated E2,leading to many symptoms of high levels of E2.It's reported that TCS could uncompetitively combine with EST,inhibiting the activity of EST.Whether the inhibition of EST by TCS affects the pregnance remains unknown so far.Research content and Objective1.The results of in vitro experiments showed that environmental estrogen TCS could uncompetitively combine with EST,inhibiting the activity of EST.The EST knockoff pregnant mice showed overhigh level of activated E2,leading to fetal death.The first research content is to discussion whether exposure to TCS disorders the metabolism of E2,leading to spontaneous abortion.2.Previous researches have proved that environmental level of BPA showed estrogenic activity,disordered the estrous cycle and induced precocious puberty,whereas low dose of BPA didn't directly influence the biosynthesis of E2 in granulosa cells.Since low dose of BPA can cross the blood-brain barrier,the second research content is to test whether exposure to low dose of BPA influence E2-induced positive feedback in AVPV-kisspetin in neurons,leading to hypothalamic-pituitary-ovary reproductive endocrine axis disorders.3.Previous research found that exposure to high dose of PFOS caused cell toxicity and prolonged estrous cycle.We have reported that low dose of PFOS could reduce the biosynthesis of E2 through epigenetic mechanism in granulosa.The third research content is to discuss the central regulatory mechanism of PFOS disorder the hypothalamic-pituitary-ovary reproductive endocrine axis.Experimental methods and ResultsMaterials and Methods1.Samples collection and preparation:(1)The study included 113 eligible cases with medically unexplained spontaneous abortion and 339 eligible controls.Their urine,serum and plasma samples were frozen at-20?.(2)Three-month-old female ICR mice were given oral gavage with TCS(1,10,100 mg/kg/day)from day 5.5 of gestation(GD5.5)to GD15.5(TCS-mice).2.The urinary concentration of TCS was measured with ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)3.The levels of SULT1E1 mRNA in placenta,EST protein and the activity of EST in placenta and plasma were examined by RT-qPCR,western-blot and liquid scintillation counting,respectively.4.Histology of placenta was examined by hematoxylin and eosin(HE).5.The levels of serum E2,P4,?-HCG,T3 and T4 were measured by radioimmunoassay(RIA).6.The levels of serum free E2,S-E2 and plasm fibrin degradation product(FDP)were examined by enzyme-linked immunoassay(ELISA).7.The platelet aggregation,prothrombin time(PT)and activated partial thromboplastin time(APTT)were measured using platelet aggregation analyzer and automatic coagulometer,respectively.Results1.Compared to normal pregnant women,the urinary concentration of TCS in spontaneous abortion patients was significantly increased.2.According to the level of TCS in spontaneous abortion patients,the female mice were given oral gavage with TCS(1-100 mg/kg/day)from GD5.5 to GD15.5.The urine results showed that 10-100 mg/kg was equivalent to that in abortion patients with a high exposure to TCS.3.80%TCS-mice showed a progressive fetal death(about 28.4%)and live fetal body weight was significantly reduced from GD15.5,which were dose-dependent on TCS.It indicated that the increasingly exposure to TCS was in relation with spontaneous abortion.4.TCS-mice showed placental thrombus and hemorrhage with tissue necrosis.It indicated that TCS enhanced placental thrombosis,leading to abortion.5.The level of plasm EST protein in spontaneous abortion patients and TCS-mice did not differ significantly from controls,whereas,the activity of EST was significantly reduced.Though the level of serum total E2 did not changed,the level of free E2(activated E2)was significantly increased,the level of S-E2 was reduced.It indicated that TCS could increase the level of activated E2 in gestation.6.The levels of FDP and ADP-induced platelet aggregation were significantly increased in TCS-mice,whereas PT and APTT were declined.It indicated that TCS enhanced coagulation function in gestation.7.The administration of ER antagonist ICR182,780 prevented the placental thrombosis and necrosis,and partially reduced the fetal death.It indicated that the activated E2 could enhance the coagulation function,inducing placental thrombosis.SummaryEnvironmental estrogen TCS?inhibited the activity of EST in placenta-*increased the level of activated E2?+activated blood coagulation factor?induced placental thrombosis and necrosis?leading to spontaneous abortion.Materials and Methods1.Three-month-old female ICR mice were used to prepare BPA models.The estrous cycle was examined by vaginal smears daily.Oral administration(p.o.)of BPA:the mice were given single oral treatment with BPA(20 ?g/kg/day)at proestrus,estrus and diestrus(BPA-mice),respectively.Intracerebroventricular(i.c.v.)injection of BPA:BPA(2.0 nmol/3 ?l/mouse)was infused(i.c.v.)at proestrus,estrus and diestrus,respectively.In addition,the mice were given BPA at doses of 0.02,0.2,2.0,20.0,200 nmol/3?l/mouse at proestrus.2.Drugs treatment:E2 was subcutaneous(s.c.)injected;The ERa antagonist MPP,the ER? antagonist PHTPP or the GPR54 antagonist P234 was injected into lateral ventricle.3.The number of AVPV-kisspeptin positive(kisspeptin+)cells was examined by immunohistochemistry.4.The levels of AVPV/ARC Kiss1,ER? and POA-GnRH mRNA were measured by RT-qPCR.5.The levels of serum E2,LH,FSH,P4 and LH-surge were measured by radioimmunoassay(RIA).Results1.At proestrus,the levels of serum E2,LH and FSH and hypothalamic GnRH mRNA in BPA-mice were significantly higher than controls,whereas,the levels of serum LH and FSH and hypothalamic GnRH mRNA did not differ differently from controls at diestrus and estrus.It indicated that BPA could enhance the activity of HPG axis at proestrus.2.Compared to controls,the levels of AVPV-kiss1 mRNA and AVPV-kisspeptin protein were significantly increased at proestrus,but not at diestrus and estrus.It indicated that BPA could stimulate the expression of AVPV-kisspeptin in high level of E2.3.At proestrus,at 6 h after application of BPA(i.c.v.),the level of AVPV-kiss1 mRNA was increased dose-dependently.By contrast,in diestrus,estrus or ovariectomized(OVX)mice,the application of BPA had no effect on AVPV-kiss1 mRNA.However,in E2-treated OVX female mice,the application of BPA significantly increased the level of AVPV-kiss1 mRNA.It indicated that BPA could directly enhance E2-induced positive feedback in AVPV-kisspetin neurons.4.At proestrus,at 6 h after application of BPA(i.c.v.),the levels of GnRH mRNA,LH and E2 were increased dose-dependently compared to control mice,which were blocked by the GPR54 blocker P234.It indicated that BPA enhanced the activity of HPG axis through stimulating the expression of AVPV-kisspeptin.5.In OVX mice,the application of E2 increased dose-dependently the level of AVPV-kiss1 mRNA(EC50=45.20 ?g/kg).The administration of BPA could shift the curve of E2-increased AVPV-kiss1 mRNA leftward(EC50=15.25 ?g/kg).It indicated that BPA could enhance E2-induced AVPV-kisspeptin expression.6.The effects of BPA on AVPV-kisspeptin expression,GnRH,LH and E2 could be blocked by the ERa antagonist MPP,but not the ER? antagonist PHTPP.It indicated that ERa was the molecular target of BPA-induced AVPV-kisspeptin expression.SummaryAdult exposure to BPA?enhanced ERa-induced AVPV-kisspeptin expression?disordered the function of HPG axis.Materials and Methods1.Two-month-old female ICR mice were used to prepare PFOS models.Oral administration(p.o.)of PFOS:the mice were given oral gavage of PFOS(10 mg/kg/day)once or for consecutive 30 days(PFOS-mice).Intracerebroventricular(i.c.v.)injection of PFOS:PFOS(1.5 mM/3 ?l/mouse)was infused(i.c.v.)singly at proestrus or diestrus.2.Drugs treatment:E2 was subcutaneous(s.c.)injected;The ERa antagonist MPP,the ER? antagonist PHTPP,the GPR54 antagonist P234 or kisspeptin-10 was injected into lateral ventricle.3.The expression of AVPV-kisspeptin was examined as same as the second part.4.The histology of ovary and the number of corpus luteum were examined by hematoxylin and eosin(HE)5.The levels of serum reproductive hormones were measured as same as the second part.6.The level of hypothalamic GnRH was measured by ELISA.Results1.The mice treated with PFOS(PFOS-mice)for 30 days showed an obvious prolongation of diestrus2.Compared with control mice,the number of corpora lutea was significantly reduced in PFOS-mice,meanwhile the LH-surge could not be observed.It indicated that PFOS could inhibit ovulation in mice.3.Compared with control mice,the level of E2 was reduced at proestrus and diestrus.In addition,the levels of LH and GnRH were significantly reduced at proestrus.It indicated that PFOS could inhibit the activity of HPG axis.4.At proestrus,the levels of AVPV-kisspeptin+cells,Kiss1 mRNA and kisspeptin protein were significantly lower than control mice.It indicated that PFOS could inhibit the expression of AVPV-kisspeptin.5.At proestrus,8 h after administration(p.o.)of PFOS(0.015-150 mM/3?l)or injection(i.c.v.)of PFOS(0.01-100 mg/kg),the level of AVPV-Kiss1 mRNA was decreased dose-dependently,meanwhile the levels of GnRH,LH and E2 were reduced too.It indicated that PFOS could reduce directly the expression of AVPV-kisspeptin,suppressing the activity of HPG axis.6.In OVX mice,the application of E2 increased dose-dependently the level of AVPV-kiss1 mRNA(EC50=40.10 ?g).The administration of PFOS could suppress the E2-induced AVPV-kisspeptin expression,leading to the curve of E2-increased AVPV-kiss1 mRNA shifted leftward(EC50=79.13 ?g).In addition,PFOS could inhibit the ER? agonist PPT-induced AVPV-kisspeptin expression.Similar to the effect of ERa antagonist MPP,PFOS could suppress the E2-induced AVPV-kisspeptin expression too.It indicated that ERa was the molecular target of PFOS-inhibited AVPV-kisspeptin expression.7.Injection(i.c.v.)of kisspeptin-10 could attenuate the inhibition of PFOS on reproductive hormones and LH-surge.It indicated that PFOS reduced the reproductive hormones through inhibiting AVPV-kisspeptin expression.SummaryEnvironmental estrogen PFOS?inhibited ER?-induced AVPV-kisspeptin expression?suppressed the activity of HPG axis?damaged the generation of LH-surge?inhibited ovulation and prolonged diestrus.ConclusionIn non-gestational females,environmental estrogens could increase or reduce the expression of AVPV-kisspeptin through ER?,disorder the function of HPG axis,damage the generation of LH-surge,and then inhibit ovulation;Whereas in gestation,environmental estrogens could increase the level of activated E2 through inhibiting the activity of EST,enhance the coagulation function,and then induce placental thrombosis,leading to spontaneous abortion.