Bisphenol A enhances kisspeptin neurons in anteroventral. periventricular nucleus of female mice

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1 Page of 0 Accepted Preprint first posted on February 0 as Manuscript JOE--0 JOURNAL OF ENDOCRINOLOGY 0 Bisphenol A enhances kisspeptin neurons in anteroventral periventricular nucleus of female mice Short title: BPA-stimulated kisspeptin neurons in AVPV Xiaoli Wang,, Fei Chang, Yinyang Bai, Fang Chen, Jun Zhang,Ling Chen, State Key Lab of Reproductive Medicine, Department of Physiology, Nanjing Medical University, Nanjing 00, China, MOE-Shanghai Key Laboratory of Children s Environmental Health, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 000, China 0 Correspondence author: Ling Chen, Ph.D. & MD Address: Department of Physiology, Nanjing Medical University, Hanzhong Road 0, Nanjing, China. Tel: +--, Fax: lingchen@njmu.edu.cn Key words: Bisphenol-A (BPA); kisspeptin neurons; estrogen (E); estrogen receptor (ER); hypothalamic-pituitary-gonadal (HPG) axis Word count: Copyright 0 by the Society for Endocrinology.

2 Page of Abstract Bisphenol-A (BPA), an environmental estrogen, adversely affects female reproductive health. However, the underlying mechanisms remain largely unknown. We found that oral administration (p.o.) of BPA (0 µg/kg) to adult female mice at proestrus, but not at estrus or diestrus, significantly increased levels of plasma E, LH and FSH, and GnRH mrna within h. The BPA-application at proestrus, but not at diestrus, could elevate the levels of Kiss mrna and kisspeptin protein in anteroventral periventricular nucleus (AVPV) within h. By contrast, the level of Kiss mrna in arcuate nucleus (ARC) was hardly altered by the BPA-application. In addition, at proestrus a single injection (i.c.v.) of BPA dose-dependently enhanced the AVPV-kisspeptin expression within h, which was sensitive to E-depletion by ovariectomy (OVX) and the ERα antagonist. Similarly, the BPA-injection (i.c.v.) at proestrus could elevate the levels of plasma E, LH and GnRH mrna within h in a dose-dependent manner, which was blocked by the antagonists of GPR or ERα. The BPA-injection (i.c.v.) at proestrus failed to alter the timing and peak concentration of LH-surge generation. In OVX mice, the application of E induced a dose-dependent increase in AVPV-Kiss mrna level, indicative of E-induced positive feedback, which was enhanced by BPA-injection (i.c.v.). The levels of ERα and ERβ mrna in AVPV and ARC did not differ significantly between vehicle and BPA-treated groups. The present study provides in vivo evidence that exposure of adult female mice to a low-dose BPA disrupts hypothalamic-pituitary-gonadal reproductive endocrine through enhancing AVPV-kisspeptin expression and release. Introduction

3 Page of Environmental estrogenic chemical bisphenol-a (BPA) is an industrial monomer used in production of polycarbonates and epoxy resins. There is considerable potential for human exposure to this compound from the lining of food cans, plastic ware and dental sealants. Studies in humans have also determined that BPA can be measured in serum, urine, amniotic fluid, follicular fluid, placental tissue and umbilical cord blood (Ikezuki, et al. 00). Estimates of oral exposure to BPA are 0-0 mg during the first hour following administration of dental sealant (Markey, et al. 00) and up to. mg per day from food cans (Brotons, et al. ). Although many women are exposed to BPA worldwide, the reproductive health risks and complications of BPA exposure during adulthood have not been evaluated. Recently, the increased incidence of BPA exposure in humans is associated with reproductive diseases including recurrent miscarriage (Sugiura-Ogasawara, et al. 00), premature delivery (Cantonwine, et al. 00) and polycystic ovary syndrome (Kandaraki, et al. 0). In animal experiments, BPA exposure has been shown to alter estrous cyclicity (Lee, et al. 0; Rubin, et al. 00), impair reproductive capacity (Cabaton, et al. 0) and disturb hormonal levels (Fernandez, et al. 00; Rubin et al. 00). However, the underlying mechanisms remain largely unknown. In the female, the regular estrous cycle is controlled by pulse mode and surge mode of gonadotrophin-releasing hormone (GnRH)/luteinizing hormone (LH) release, which is regulated by feedback action of estrogen (E) secreted from the ovarian mature follicles (Adachi, et al. 00). However, GnRH neurons do not express estrogen receptor α (ERα), they accept E signaling largely from ERα-expressing neurons elsewhere within the hypothalamus, such as the kisspeptin neurons in the arcuate nucleus (ARC) and anteroventral

4 Page of periventricular nucleus (AVPV) (Kinoshita, et al. 00). The kisspeptin neurons are known to send projections to preoptic area (POA) in close proximity to GnRH neurons. Approximately 0% of GnRH neurons express G protein-coupled receptor (GPR) that are intensely activated by kisspeptin (Pinilla, et al. 0). The kisspeptin binding to GPR enhances the release of GnRH, presumably through Ca + -mediated process (Stathatos, et al. 00; Strock and Diverse-Pierluissi 00). The GnRH antagonist acyline blocks the ability of kisspeptin to stimulate gonadotrophin release in the rat, mouse, and primate (Gottsch, et al. 00). The response of the Kiss gene to E is opposite between these two nuclei ARC and AVPV. E decreases Kiss mrna in ARC, termed E-induced negative feedback, whereas increases Kiss mrna in AVPV, termed E-induced positive feedback (Dungan, et al. 00). Many pieces of evidence suggest that kisspeptin neuron in AVPV, as a target of E positive feedback, is considered to regulate GnRH/LH-surge generation (Ohkura, et al. 00). The ARC kisspeptin neuronal population, as a target of E negative feedback, is considered to be involved in the regulation of GnRH pulse generation (Ohkura et al. 00). Administration of exogenous kisspeptin to the brain is able to enhance GnRH expression and release, while the GPR antagonist blocks the response of GnRH neurons to exogenous kisspeptin in female rats. Given estrogenicity of BPA, it can presumably disturb or mimic E-action to affect the E-induced feedback regulation in kisspeptin neurons, which leads to abnormal estrous cycle and reproductive endocrine. BPA mimics endogenous E-actions by binding to membranous or nucleus ERs (Washington, et al. 00). By its competitive action to endogenous E (Fang, et al. 000), BPA has been reported to disturb the hypothalamic-pituitary-gonadal (HPG) endocrine

5 Page of system, resulting in the impairment of reproductive function (Markey et al. 00). However, BPA is considered to be a very weak environmental E, because its affinity for ERs is 0,000 00,000-fold weaker than that of estradiol (Okada, et al. 00). Results from recent studies have revealed that BPA can stimulate cellular responses at very low concentrations via rapid induction of calcium uptake and nongenomic cell signaling (Quesada, et al. 00; Wozniak, et al. 00) involving membrane-associated forms of the ERs (Wetherill, et al. 00), in addition to the effects initiated by BPA binding to the classical nuclear or genomic ERs (Welshons, et al. 00). The objectives of this study were to dissect the influence of BPA on kisspeptin-gpr and hypothalamic-pituitary-gonadal reproductive endocrine axis that is well known to be critical for control of ovulation and estrus cycle. Human exposures are most likely through the oral route, thus adult female mice were treated with a single oral administration (p.o.) of BPA (0 µg/kg) at proestrus, estrus or diestrus, respectively. To determine whether BPA acts directly in AVPV- and ARC-kisspeptin neurons, we performed a single intracerebroventricular (i.c.v.) injection of BPA in proestrus or diestrus female mice. AVPVand ARC-kisspeptin expression, GPR-mediated GnRH expression, secretion of LH, FSH and E, generation of LH-surge were examined within h after the BPA-application (p.o.) or (i.c.v.) to evaluate the acute effects of BPA on kisspeptin neurons and hypothalamic-pituitary-gonadal reproductive endocrine. Finally, we examined the influence of BPA on E-enhanced AVPV-kisspeptin expression and involvement of ERα and ERβ to explore the possible targets and molecular mechanisms underlying BPA-actions in kisspeptin-gpr mediated HPG axis. Our results indicate that the exposure of adult female

6 Page of 0 mice to a low-dose of BPA stimulates AVPV-kisspeptin neurons to disrupt hypothalamic-pituitary-gonadal reproductive endocrine axis. 0 0 Materials and methods Animals Studies were performed according to the protocols for animal use (Institute of Laboratory Animal Resources ). The use of animals was approved by Animal Care and Ethical Committee of Nanjing Medical University. All efforts were made to minimize animal suffering and to reduce the number of animals used. Twelve-week-old female ICR mice (Oriental Bio Service Inc., Nanjing), weighing approximately 0± g, were used at the beginning of this study. The animals were housed in stainless steel cages with wood bedding to minimize additional exposure to endocrine disrupting chemicals (temperature ± C, humidity ±%, and : h light/dark cycle) in Animal Research Center of Nanjing University. They had free access to food and water before and after all procedures. Examination of estrous cycles Estrous cycle was daily examined by vaginal lavage with 0.% saline at h. The fluid was spotted thinly on a microscope slide, and the dried slides were stained with toluidine blue. The estrous cycle stage was determined by microscopic examination. We examined vaginal cytology for a total of days. According to the types of vaginal epithelium cells (leukocyte cells, nucleated cells and cornified cells), diestrus (D), estrus (E) and proestrus (P) were determined in all mice, as described by (Goldman, et al. 00). Exposure to BPA

7 Page of Oral administration (p.o.) of BPA: After - regular estrous cycles were determined, the mice were treated with BPA at 0 µg/kg body weight/day. BPA (> % purity; Sigma-Aldrich Inc., St. Louis, MO, USA) was dissolved in 00 µl corn oil. Control mice were treated with corn oil at the same volume. The dose was chosen according to a recent report (Lee, et al. 0) that the oral administration of BPA at or 00 µg/kg observably affects the duration of estrus phase and the levels of reproductive hormones in adult female Sprague-Dawley rats. The lowest observed adverse effect level (LOAEL) for BPA established by the U.S. Environmental Protection Agency (EPA) is 0 mg/kg body weight/day, and the U.S. EPA reference dose (and the U.S. Food and Drug Administration acceptable daily intake) is 0 µg/kg body weight/day (U.S. EPA ). Intracerebroventricular (i.c.v.) injection of BPA: BPA was dissolved in DMSO and then in 0.% saline to a final concentration of 0.% DMSO. To determine the direct action of BPA in kisspeptin neurons, BPA (0, 0.0, 0.,.0, 0.0 and 00.0 nm/ µl) was injected into right lateral ventricle (anteroposterior +0. mm, lateral +0. mm, dorsoventral. mm) using a stepper-motorized micro-syringe (Stoelting, Wood Dale, IL, USA) with a rate of µl/min. These doses were chosen because the treatment with BPA (00 pm-00 nm) can decrease viability of mouse granule cells (Xu, et al. 00). The mice infused with the same volume vehicle served as the control group. Ovariectomy (OVX) Ovaries were removed from mice anesthetized by the intraperitoneal injection (i.p.) of chloralhydrate (00 mg/kg). Briefly, the anesthetized animal s ventral surface was shaved and cleaned and the ovaries dissected out through midline incisions in the skin and abdominal

8 Page of musculature. After ovary removal, the muscle was sutured and the skin closed with sterile wound clips. Treatment with drugs Treatment with E: () To prepare the E-treated OVX-mice, mice were subcutaneously implanted at the time of OVX with a silastic brand (Dow Corning, Midland, MI) capsule containing 0. µg E (in sesame oil, Sigma-Aldrich Corp. St., Louis, MO) as described by Christian, et al. (Christian, et al. 00). This E-treatment paradigm results in constant physiological levels of E in the circulation beginning on day. () In th day after OVX, the mice were given the subcutaneous injection (s.c.) of E dissolved in sesame oil at dose of 0, 0.,, 0, 00, 00 and 00 µg/kg for consecutive days to examine the E-induced AVPV-kisspeptin expression. Treatment with receptor antagonists: The ERα antagonist,-bis (-hydroxyphenyl)--methyl--[-(-piperidinylethoxy)phenol] -H-pyrazole dihydrochloride (MPP) and the ERβ antagonist -[-phenyl-,-bis(trifluoromrthyl)pyrazolo(,-a)pyrimidin--yl]phenol (PHTPP, Tocris Cookson Ltd., Avonmouth, UK) and the GPR antagonist peptide (p, Sigma-Aldrich Corp., St. Louis, MO) were dissolved in ethanol, and then in 0.% saline (final ethanol concentration was less than 0.%). MPP (0 nmol /mouse; Harrington, et al. 00), PHTPP (0 nmol/mouse; (Compton, et al. 00; Harrington, et al. 00) and p ( nmol /mouse; Roseweir, et al. 00) were injected (i.c.v., µl/mouse) at 0 min prior to BPA-injection using a stepper-motorized micro-syringe at a rate of 0. µl/min. The drugs were prepared freshly on the day of experiment. Control mice were given an equal volume of vehicle. Measurement of plasma hormones

9 Page of Blood samples were taken by jugular venipuncture under anesthetized conditions with chloralhydrate (00 mg/kg, i.p.) in 0-00 h at diestrus (D), proestrus (P) and estrus (E), respectively. Plasma (total 00 µl per mouse) was separated by centrifugation at C and stored at -0 C until assay. Hormonal levels were measured using a radioimmunoassay (RIA) kit provided by the National Hormone and Peptide Programme (Baltimore, MD). The intraand inter-assay coefficients of variation are.% and.% for LH,.% and 0.% for FSH, % and.% for E, respectively. The lowest detectable levels were 0. ng/ml for LH, 0. ng/ml for FSH and pg/ml for E, respectively. Observation of LH-surge At proestrus mice were anesthetized with pentobarbital ( mg/00 µl, i.p.) (Clarkson, et al. 00) and blood (00 µl per time) was collected at h by an automatic blood sampling system (DraQ, EICOM, Kyoto, Japan) as described previously (Ishii, et al. 0). An equivalent volume (00 µl) of heparinised saline ( U/ml normal saline; CP Pharmaceuticals Ltd, Wrexham, UK) was replaced through the atria cannula after each blood collection. Plasma was separated by centrifugation and stored at -0 C until assay. Reverse transcription, quantitative polymerase chain reaction (RT-qPCR) The regions of AVPV, ARC and POA at proestrus or diestrus at h after BPA-application (p.o.) or (i.c.v.) were collected from the frozen slices (-mm thick) and stored at -0 C until assay. The primer sequences of Kiss, GnRH, ERα and GAPDH mrna were designed and RT-qPCR was performed according to the publication (Xi, et al. 0). Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Purified RNA with an A0/A0 ratio of.-.0 was used.

10 Page 0 of RNA ( µg) was reversed transcribed using high-capacity cdna of the reverse transcription kit RT (TaKaRa Biotechnology CO., Ltd) in accordance with the instructions. PCR reactions were conducted using a Light Cycler Fast Start DNA Master SYBR Green I kit and an ABI Prism 00 Sequence Detection System (Applied Biosystems, Foster City, California, USA). The copy number of transcripts was calculated in reference to the parallel amplifications of known concentrations of the respective cloned PCR fragments. Standard curves were constructed and amplification efficiencies were between 0. and 0.. The relative expression of genes was determined using the - ct method with normalization to GAPDH expression. Based on melting curve analyses there were no primer-dimers or secondary products formed. There was only one PCR product amplified for each set of primers. Immunohistochemistry of kisspeptin Mice at 0-00 h of proestrus were anaesthetized with ketamine/xylazine and perfused intraventricularly with % paraformaldehyde. Brains were post-fixed overnight in % paraformaldehyde, and then were transferred gradually into % and 0% sucrose until they settled. Sections (0 µm thick) through AVPV (0. mm anterior to bregma and 0. mm posterior to bregma)(mayer and Boehm 0) were cut using a cryostat. Free-floating sections were incubated in 0.% sodium metaperiodate for 0 min and then in % sodium borohydride for 0 min. They were pre-incubated with % normal fetal goat serum (blocking solution) for 0 min, and then incubated in rabbit anti-kisspeptin polyclonal antibody (:000, Millipore, Billerica, MA) for h at C as described previously (Penatti, et al. 0; Quennell, et al. 0). Sections were treated with biotin-conjugated goat anti-rabbit IgG (:00; vector laboratories, Burlingame, CA, USA) for h at C. The immunoreactivity 0

11 Page of was visualized with the standard avidin-biotin complex reaction with Ni-, -diaminobenzidine (DAB, Vector Laboratories). Sections were mounted on Super-Frost Plus slides (VWR Scientific, West Chester, Pa., USA), air-dried, and dehydrated in graded ethanol, and cleared with Citrosolv, after which cover slips were applied. In every experiment, incubation of sections without the primary antibodies served as negative controls for immunohistochemistry. Kisspeptin immunoreactive (kisspeptin + ) cells in AVPV were counted using a conventional light microscope (Olympus PD0) with a 0 objective. The kisspeptin + cells were counted in sections of AVPV per brain. The values obtained from mice were averaged to provide each group mean value. Western blotting analysis The regions of AVPV at 0-00 h of proestrus and the regions of ARC at 0-00 h of diestrus were collected from the frozen slices ( mm thick) of brain using -gauge stainless steel tubing, and then stored at -0 C until assay. The amount of protein was determined using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). Protein (0 µg) was separated by SDS-PAGE and transferred to membranes. The membranes were incubated with rabbit anti-kisspeptin polyclonal antibody (:000, Millipore, Billerica, MA) at C overnight. After several PBS rinses, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (:000, Millipore) for h, and then were developed using an enhanced chemilumiescence detection Kit (Millipore). Western blotting bands were scanned and analyzed with image analysis software (Image J, NIH). Data analysis/statistics Data were retrieved and processed with the software Micro cal Origin.. The group data

12 Page of 0 were expressed as the mean ± standard error (SE). The experiment data were evaluated by Bartlett s test to examine the equality of variances. After the homogeneity of variance was determined, statistical differences among values for individual groups were determined by t-test or analysis of one- or two-way ANOVA, followed by the Bonferroni post hoc test. Statistical analysis was performed using Stata software (STATA Corporation, USA). Differences at P<0.0 were considered statistically significant. 0 0 Results Influence of BPA-application (p.o.) on activation of HPG axis and kisspeptin neurons The oral administration (p.o.) of BPA (0 µg/kg) were given at diestrus, proestrus and estrus, respectively (see the time chart of experimental procedure; Figure A). At h after the BPA-application, the levels of reproductive hormones and the GnRH mrna, and the kisspeptin expression were examined. Two-way ANOVA analysis showed significant effects of estrous cycle (EC), BPA-application (BPA) and their interaction (EC BPA) on the levels of plasma E (EC: F (, ) =., P<0.0; BPA: F (, ) =., P<0.0; EC BPA: F (, )=00., P<0.0; Figure B), LH (EC: F (, ) =., P<0.0; BPA: F (, ) =., P<0.0; EC BPA: F (, ) =., P<0.0; Figure C), FSH (EC: F (, ) =., P<0.0; BPA: F (, ) =., P<0.0; EC BPA: F (, ) =.0, P<0.0; Figure D). In control mice, the level of plasma E was higher at proestrus (P<0.0; Figure B) or estrus (P<0.0) than that in diestrus. The BPA-application further increased the levels of plasma E at proestrus (P<0.0) and estrus (P<0.0), but not at diestrus (P>0.0), compared to controls. By contrast, the elevation of plasma LH (Figure C) or FSH (Figure D) was observed at proestrus of control

13 Page of mice, compared to estrus and diestrus (P<0.0). At proestrus, the BPA-application could elevate the levels of LH (P<0.0) and FSH (P<0.0), but at estrus or diestrus it could not (P>0.0). Similarly, the expression of GnRH was affected by estrous cycle (F (, ) =., P<0.0; Figure E), BPA-application (F (, ) =., P<0.0) and their interaction (F (, )=., P<0.0). In comparison with control mice, the level of GnRH mrna in POA was largely elevated by the BPA-application at proestrus (P<0.0; Figure E), but not at estrus and diestrus (P>0.0). The levels of Kiss mrna in AVPV and ARC were altered during estrous cycle (AVPV: F (, ) =., P<0.0; Figure F, ARC: F (, ) =., P<0.0; Figure G, respectively). Two-way ANOVA analysis showed the influence of BPA-application and interaction of EC BPA on AVPV-Kiss mrna (BPA: F (, ) =., P<0.0; EC BPA: F (, ) =.0, P<0.0), but not on ARC-Kiss mrna (BPA: F (, ) =0., P>0.0; EC BPA: F (, ) =., P>0.0). The level of AVPV-Kiss mrna at proestrus was higher than those at estrus (P<0.0; Figure F) and diestrus (P<0.0), which was further elevated by the BPA-application (P<0.0). The level of ARC-Kiss mrna at proestrus was less than those at estrus (P<0.0; Figure G) and diestrus (P<0.0), which were hardly affected by the BPA-application (P>0.0). Similarly, the level of AVPV-kisspeptin protein at proestrus was increased approximately 0% by the BPA-application (P<0.0; Figure H), while the BPA-application did not alter the level of ARC-kisspeptin protein at diestrus (P>0.0). In addition, at h after the BPA-application the number of AVPV-kisspeptin positive (kisspeptin + ) cells at proestrus was not altered compared to controls (P>0.0, n=; Figure I), whereas the AVPV-kisspeptin + cells showed darker staining than that in control mice. The

14 Page of results indicate that the exposure to the low-dose BPA at proestrus is able to enhance the activation of HPG axis and AVPV-kisspeptin neurons. Influence of BPA-application (i.c.v.) on kisspeptin expression and E-involvement To determine whether BPA acts directly on AVPV-kisspeptin neurons, a single intracerebroventricular (i.c.v.) injection of BPA at various doses of 0.0, 0.,, 0 and 00 nm/mouse was given at proestrus and diestrus, respectively (see the time chart of experimental procedure; Figure A). At h after the BPA-injection (i.c.v.), the AVPV and ARC regions were harvested to measure the level of Kiss mrna. The BPA-injection at proestrus could dose-dependently increase the AVPV-Kiss mrna with EC0. nm (F (, 0)=.0, P<0.0; Figure B), whereas it did not affect the level of ARC-Kiss mrna (F (, 0)=0., P>0.0; Figure C). By contrast, the BPA-injection at diestrus failed to affect the levels of AVPV-Kiss mrna (F (, 0) =0., P>0.0; Figure D). The ARC-kisspeptin expression at diestrus was slightly inhibited only at higher doses of BPA than 0 nm (P<0.0, n=; Figure E). In addition, the application of E was able to increase the AVPV-Kiss mrna in OVX-mice (F (, ) =., P<0.0; Figure F). However, the BPA-injection (i.c.v.) could only enhance the AVPV-kisspeptin expression in OVX-mice treated with E (F (, ) =., P<0.0), but not in OVX-mice (P>0.0). In contrast, the BPA-injection (i.c.v.) had no effects on the ARC-kisspeptin expression in OVX-mice or OVX-mice treated with E (F (, ) =0.0, P>0.0; Figure G), although alone treatment with E in OVX-mice could reduce the level of ARC-Kiss mrna (F (, ) =.0, P<0.0). The results indicate that, in the presence of high-concentration E, BPA can enhance the AVPV-kisspeptin expression. Influence of BPA-injection (i.c.v.) on activation of HPG axis and GPR-dependent

15 Page of In accordance with the BPA-enhanced AVPV-kisspeptin expression at proestrus, at h after BPA-injection (i.c.v.), the levels of GnRH mrna (F (, 0) =0.0, P<0.0; Figure A), plasma LH (F (, 0) =.0, P<0.0; Figure B) and E (F (, 0) =., P<0.0; Figure C) showed a dose-dependent increase. More importantly, the treatment (i.c.v.) with the GPR blocker p ( nmol/mouse) at 0 min prior to the BPA-injection could perfectly abolish the BPA-induced increase in the GnRH expression (P<0.0, n=; Figure D) and the release of LH and E (P<0.0, n=; Figure E&F), even though the increased AVPV-Kiss mrna was not affected (P>0.0, n=; Figure G). The results indicate that BPA enhances the release of hypothalamic-pituitary-gonadal hormones through stimulating AVPV-kisspeptin neurons. Influence of ER antagonists on BPA-stimulated AVPV-kisspeptin neurons E-driven AVPV-kisspeptin biosynthesis depends on the ERα-activation (Smith, et al. 00). To confirm targets of the BPA-stimulated AVPV-kisspeptin neurons, the ERα antagonist MPP or the ERβ antagonist PHTPP was used (i.c.v.) at 0 min prior to BPA-injection (i.c.v.). At proestrus, the administration of MPP alone, but not PHTPP, could attenuate the level of AVPV-Kiss mrna (F (, ) =0., P<0.0; Figure A). By contrast, the enhancing effects of BPA (i.c.v.) on AVPV-kisspeptin expression was abolished by the treatment with MPP (F (, ) =., P<0.0). As expected, the treatment with MPP could perfectly block the BPA-induced increase in GnRH mrna and plasma LH and E (P<0.0, n=; Figure B-D). At h after the BPA-injection (i.c.v.), the levels of ERα and ERβ mrna in AVPV and ARC had no significant difference from controls (P>0.0, n=; Figure E&F). The results suggest that BPA-stimulated AVPV-kisspeptin neurons depend on the

16 Page of ERα-activation. Influence of BPA-injection (i.c.v.) on E-enhanced AVPV-kisspeptin expression The application (s.c.) of E (0, 0.,, 0, 00, 00 and 00 µg/kg) in OVX-mice induced a dose-dependent increase on the levels of AVPV-Kiss mrna within h with a sigmoidal-shaped curve with EC0.0 µg/kg (F (, ) =., P<0.0; Figure ), indicative of E-induced positive feedback. Interestingly, the BPA-injection (i.c.v.) could leftward shift the curve of E-increased AVPV-Kiss mrna (EC0. µg/kg, F (, ) =., P<0.0). The results further demonstrate that BPA can enhance E-induced positive feedback in AVPV-kisspeptin neurons. Influence of BPA-enhanced AVPV-kisspeptin on LH-surge The AVPV-kisspeptin neurons, as a target of E positive feedback, are involved in the generation of GnRH surge. Final experiment was designed to examine whether the BPA-injection (i.c.v.) at proestrus affects the production of LH-surge (see the time chart of experimental procedure; Figure A). Repeated-measures ANOVA revealed that the basal levels of plasma LH were increased by BPA-injection (i.c.v.) (F (, ) =., P<0.0). As shown in Figure B, in mice of proestrus, a distinct peak of plasma LH concentration was observed during h (n=0), showing an apparent LH-surge. By contrast, the basal level of plasma LH during h was elevated by the BPA-application (P<0.0, n=0), which led to a decrease in the amplitude of LH-surge, a difference between the baseline of LH in 00 h and the peak concentration of LH. However, the BPA-application failed to alter the peak timing and the peak concentration of LH-surge relative to controls (P>0.0, n=0). The results indicate that the acute application of BPA does not affect the generation of LH-surge.

17 Page of Discussion The present study provides, for the first time, in vivo evidence that exposure of adult female mice to a low dose of BPA enhances ERα-mediated positive regulation in AVPV-kisspeptin expression and release. BPA stimulates AVPV-kisspeptin neurons leading to potentiation of HPG axis One of the principal observations in this study is that the low-dose BPA can selectively stimulate AVPV-kisspeptin neurons in adult female mice. This conclusion is deduced mainly from the following observations. () The oral administration of BPA (0 µg/kg) could increase the level of AVPV-Kiss mrna and kisspeptin protein, whereas it hardly affected the ARC-kisspeptin expression. () A single injection (i.c.v.) of BPA could dose-dependently increase the levels of AVPV-Kiss mrna within h. () Either the BPA-administration (p.o.) or the BPA injection (i.c.v.) could increase the plasma LH, FSH and E levels, and the level of GnRH mrna within h, which was sensitive to GPR antagonist. A recent study (Lee, et al. 0) has reported that in adult female rats the BPA exposure at doses of and 00 µg/kg increases serum LH levels and LH protein content in pituitary gland. Thus, the results indicate that the BPA-application increases the activation of HPG axis through enhancing AVPV-kisspeptin expression and release. () Consistent with the effects of BPA on ERα-mediated gene expression (Matthews, et al. 00), the enhancing effects of BPA on the AVPV-kisspeptin neurons and the activation of HPG axis were blocked by the ERα antagonist. () The BPA injection (i.c.v.) potentiated the E-induced AVPV-kisspeptin expression, indicating that BPA can enhance the E-induced positive feedback in

18 Page of AVPV-kisspeptin neurons. The results from a wide variety of studies support the indication that kisspeptin stimulates gonadotrophin secretion via the activation of GnRH neurons expressing GPR (Kinoshita et al. 00). Irwig et al. (Irwig, et al. 00) have demonstrated that the central administration of kisspeptin induces Fos expression of GnRH neurons in rats. The level of ARC-Kiss mrna at proestrus was lower than that at diestrus and was reduced by the treatment with E in OVX-mice, indicating that the ARC-kisspeptin neurons in adult female mice receive the E-induced negative feedback regulation. The molecular mechanisms by which E produces reverse effects on Kiss mrna expression in AVPV and ARC are unknown. Whereas E apparently acts through the same ERα to produce opposite results in AVPV and ARC, it seems likely that something intrinsic to the two cell types causes the opposing regulatory effects of E. For example, E acting through ERα recruits co-activators of transcription in AVPV-kisspeptin neurons and co-repressors of transcription in ARC (Gottsch, et al. 00; Klinge, et al. 00). Interestingly, our results show that the ARC-kisspeptin expression was hardly affected by the BPA-application, although the high-dose BPA caused a slight decline in the ARC-Kiss mrna. The ratio of ERα to ERβ in ARC-kisspeptin neurons is less than that in AVPV, because the E-induced decline in ARC-Kiss mrna appears greater in ERβ knockout mice than in control mice (Smith et al. 00). The BPA-stimulated AVPV-kisspeptin neuron depends on the activated ERα by high-concentration of E. ERβ can oppose ERα-mediated gene transcription (Matthews and Gustafsson 00). Taking into account of our data, as well as other studies, we propose that the different expression of ERα and ERβ is a possible molecular basis for the selective action of BPA on AVPV-kisspeptin neurons.

19 Page of Possible molecular mechanisms of BPA-enhanced activity of AVPV-kisspeptin neurons The next question we should address may be the molecular mechanism(s) of the BPA-effects on E-induced positive feedback in AVPV-kisspeptin neurons. Generally, environmental and exogenous E enhances the functioning of E by at least four possible ways: by binding to ER to mimic the action of endogenous E; by modulating ER to enhance the E-induced responses; by increasing the expression of ER; or by modifying the synthesis, transport, metabolism and excretion of E. First, the BPA-enhanced AVPV-kisspeptin expression and release only occurred at proestrus, but not at diestrus or in OVX-mice. BPA s binding affinity for ERα has been demonstrated to be over 000-fold lower than that of E (Gould, et al. ). Therefore, it is conceivable that the BPA itself has no E-like effects or a very weak estrogenic activity, which is insufficient to trigger the activation of ERα. Interestingly, in the presence of E, high concentration BPA is able to exert the enhancing effect to further increase the E-induced AVPV-kisspeptin expression. However, the BPA-application does not alter the ERα expression in AVPV and ARC. Thus, it is highly likely that BPA, as a positive modulator of ERα activated, augments the E-induced response. In fact, BPA has been reported to enhance the ERα-mediated response through rapid activation of ERK/ signaling pathway (Gertz, et al. 0; Li, et al. 0), as well as the generation of second messengers such as camp, cgmp and intracellular Ca + (Alonso-Magdalena, et al. 0; Belcher, et al. 0), or the activation of transcription factor camp response element-binding protein (CREB) (Quesada et al. 00). On the other hand, BPA has been reported to have a directly adverse effect on the regulation of E production. We observed that at estrus, the acute application of BPA could increase E levels, which was

20 Page 0 of not associated with an increase in LH level and AVPV-kisspeptin neurons. An earlier study reported that the treatment of cultured theca-interstitial cells with BPA increased steroidogenic acute regulatory protein (StAR) and P0 side-chain cleavage (P0scc) expression (Zhou, et al. 00). However, there is an apparently conflicting report describing that exposure to BPA for 0 days in adult female rats significantly decreases E serum concentration through reducing P0arom and StAR protein levels (Lee et al. 0). This discord may arise from the difference in treatment duration and doses of BPA. In addition, a BPA metabolite, -methyl-, -bis (-hydroxyphenyl) pent--ene (MBP), has been demonstrated to have transcriptional activity at nm concentrations, which is 000-fold higher than the estrogenic activity of BPA (Baker and Chandsawangbhuwana 0). Although our results do not support the metabolism of BPA by injection (i.c.v.), further studies are needed to clarify the effects of MBP on AVPV-kisspeptin neurons. Acute application of BPA does not affect LH-surge generation The expression of Kiss mrna in the AVPV tends to be the highest at :00, around the time of the LH-surge peak (Adachi et al. 00). At the time of preovulatory LH-surge, the levels of Fos protein in AVPV-kisspeptin neurons are increased in an E-dependent manner (Smith, et al. 00). The responsiveness of the AVPV kisspeptin neurons to E has been demonstrated to be critical for the generation of LH-surges (Ishii, et al. 0), although AVPV-kisspeptin neurons can well maintain their circadian activation when lacking E does not display LH-surges. It is proposed that preovulatory LH-surge generates only when E-induced kisspeptin production was sufficiently high enough to drive GnRH neurons (e.g. on proestrus). Local injection of a kisspeptin-specific monoclonal antibody to the preoptic 0

21 Page of 0 0 area (POA) of female rats during :00-:00 h on the day of proestrus completely blocks the preovulatory LH-surge (Kinoshita et al. 00) or E-induced LH-surges (Gottsch et al. 00). A single injection of kisspeptin can induce a large and sustained LH release (Matsui, et al. 00; Shahab, et al. 00). Interestingly, we observed that the BPA injection (i.c.v.) could elevate the basal level of plasma LH to reduce the amplitude of LH-surge through stimulating the AVPV-kisspeptin neurons, however it did not alter the timing and peak concentration of LH-surge generation. Thus, we consider that at h after the BPA-application the generation of LH-surge is not affected. Chronic high level of LH in female ERα knockout mice is reported to reduce the size of LH-surge, which was accompanied by the ovarian dysfunction and subsequent infertility (Couse and Korach ). Therefore, further studies are needed to investigate the chronic effects of BPA-administration (p.o.) on the AVPV-kisspeptin expression and the LH-surge generation. Conclusion The present study provides in vivo evidence that the acute exposure of adult female mice to low-dose of BPA can stimulate AVPV-kisspeptin neurons to induce the hyper-activation of HPG axis. Although much is to be done, our results suggest that the exposure to low-dose of BPA during adulthood through enhancing ERα-mediated positive regulation in AVPV-kisspeptin neurons can disrupt hypothalamic-pituitary-gonadal reproductive endocrine, which might subsequently affect the estrous cyclicity and ovarian function. 0 Declaration of interest The authors declare that there is no conflict of interest that could be perceived as

22 Page of 0 prejudicing the impartiality of the research reported. Funding This work was supported by grants for Programme for Major State Basic Research Development Programme of China (0CB0); National Natural Science Foundation of China (0, 0) to Chen L. 0 0 References Adachi S, Yamada S, Takatsu Y, Matsui H, Kinoshita M, Takase K, Sugiura H, Ohtaki T, Matsumoto H, Uenoyama Y, et al. 00 Involvement of anteroventral periventricular metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats. J Reprod Dev -. Alonso-Magdalena P, Ropero AB, Soriano S, Garcia-Arevalo M, Ripoll C, Fuentes E, Quesada I & Nadal A 0 Bisphenol-A acts as a potent estrogen via non-classical estrogen triggered pathways. Mol Cell Endocrinol 0-0. Baker ME & Chandsawangbhuwana C 0 D models of MBP, a biologically active metabolite of bisphenol A, in human estrogen receptor alpha and estrogen receptor beta. PLoS One e0. Belcher SM, Chen Y, Yan S & Wang HS 0 Rapid estrogen receptor-mediated mechanisms determine the sexually dimorphic sensitivity of ventricular myocytes to beta-estradiol and the environmental endocrine disruptor bisphenol A. Endocrinology -0.

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25 Page of Harrington WR, Sheng S, Barnett DH, Petz LN, Katzenellenbogen JA & Katzenellenbogen BS 00 Activities of estrogen receptor alpha- and beta-selective ligands at diverse estrogen responsive gene sites mediating transactivation or transrepression. Mol Cell Endocrinol 0 -. Ikezuki Y, Tsutsumi O, Takai Y, Kamei Y & Taketani Y 00 Determination of bisphenol A concentrations in human biological fluids reveals significant early prenatal exposure. Hum Reprod -. Irwig MS, Fraley GS, Smith JT, Acohido BV, Popa SM, Cunningham MJ, Gottsch ML, Clifton DK & Steiner RA 00 Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS- mrna in the male rat. Neuroendocrinology 0 -. Ishii MN, Matsumoto K, Matsui H, Seki N, Matsumoto H, Ishikawa K, Chatani F, Watanabe G & Taya K 0 Reduced responsiveness of kisspeptin neurons to estrogenic positive feedback associated with age-related disappearance of LH surge in middle-age female rats. Gen Comp Endocrinol C -. Kandaraki E, Chatzigeorgiou A, Livadas S, Palioura E, Economou F, Koutsilieris M, Palimeri S, Panidis D & Diamanti-Kandarakis E 0 Endocrine disruptors and polycystic ovary syndrome (PCOS): elevated serum levels of bisphenol A in women with PCOS. J Clin Endocrinol Metab E0-. Kinoshita M, Tsukamura H, Adachi S, Matsui H, Uenoyama Y, Iwata K, Yamada S, Inoue K, Ohtaki T, Matsumoto H, et al. 00 Involvement of central metastin in the regulation of preovulatory luteinizing hormone surge and estrous cyclicity in female rats. Endocrinology -.

26 Page of Klinge CM, Jernigan SC, Smith SL, Tyulmenkov VV & Kulakosky PC 00 Estrogen response element sequence impacts the conformation and transcriptional activity of estrogen receptor alpha. Mol Cell Endocrinol -. Lee SG, Kim JY, Chung JY, Kim YJ, Park JE, Oh S, Yoon YD, Yoo KS, Yoo YH & Kim JM 0 Bisphenol A exposure during adulthood causes augmentation of follicular atresia and luteal regression by decreasing beta-estradiol synthesis via downregulation of aromatase in rat ovary. Environ Health Perspect -. Li Y, Burns KA, Arao Y, Luh CJ & Korach KS 0 Differential estrogenic actions of endocrine-disrupting chemicals bisphenol A, bisphenol AF, and zearalenone through estrogen receptor alpha and beta in vitro. Environ Health Perspect Markey CM, Rubin BS, Soto AM & Sonnenschein C 00 Endocrine disruptors: from Wingspread to environmental developmental biology. J Steroid Biochem Mol Biol -. Matsui H, Takatsu Y, Kumano S, Matsumoto H & Ohtaki T 00 Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat. Biochem Biophys Res Commun 0 -. Matthews J & Gustafsson JA 00 Estrogen signaling: a subtle balance between ER alpha and ER beta. Mol Interv -. Matthews JB, Twomey K & Zacharewski TR 00 In vitro and in vivo interactions of bisphenol A and its metabolite, bisphenol A glucuronide, with estrogen receptors alpha and beta. Chem Res Toxicol -. Mayer C & Boehm U 0 Female reproductive maturation in the absence of

27 Page of kisspeptin/gpr signaling. Nat Neurosci 0-0. Ohkura S, Uenoyama Y, Yamada S, Homma T, Takase K, Inoue N, Maeda K & Tsukamura H 00 Physiological role of metastin/kisspeptin in regulating gonadotropin-releasing hormone (GnRH) secretion in female rats. Peptides 0 -. Okada H, Tokunaga T, Liu X, Takayanagi S, Matsushima A & Shimohigashi Y 00 Direct evidence revealing structural elements essential for the high binding ability of bisphenol A to human estrogen-related receptor-gamma. Environ Health Perspect -. Penatti CA, Oberlander JG, Davis MC, Porter DM & Henderson LP 0 Chronic exposure to anabolic androgenic steroids alters activity and synaptic function in neuroendocrine control regions of the female mouse. Neuropharmacology -. Pinilla L, Aguilar E, Dieguez C, Millar RP & Tena-Sempere M 0 Kisspeptins and reproduction: physiological roles and regulatory mechanisms. Physiol Rev -. Quennell JH, Howell CS, Roa J, Augustine RA, Grattan DR & Anderson GM 0 Leptin deficiency and diet-induced obesity reduce hypothalamic kisspeptin expression in mice. Endocrinology -0. Quesada I, Fuentes E, Viso-Leon MC, Soria B, Ripoll C & Nadal A 00 Low doses of the endocrine disruptor bisphenol-a and the native hormone beta-estradiol rapidly activate transcription factor CREB. FASEB J -. Rubin BS, Murray MK, Damassa DA, King JC & Soto AM 00 Perinatal exposure to low doses of bisphenol A affects body weight, patterns of estrous cyclicity, and plasma LH levels. Environ Health Perspect 0-0. Shahab M, Mastronardi C, Seminara SB, Crowley WF, Ojeda SR & Plant TM 00 Increased

28 Page of hypothalamic GPR signaling: a potential mechanism for initiation of puberty in primates. Proc Natl Acad Sci U S A 0 -. Smith JT, Cunningham MJ, Rissman EF, Clifton DK & Steiner RA 00 Regulation of Kiss gene expression in the brain of the female mouse. Endocrinology -. Smith JT, Popa SM, Clifton DK, Hoffman GE & Steiner RA 00 Kiss neurons in the forebrain as central processors for generating the preovulatory luteinizing hormone surge. J Neurosci -. Stathatos N, Bourdeau I, Espinosa AV, Saji M, Vasko VV, Burman KD, Stratakis CA & Ringel MD 00 KiSS-/G protein-coupled receptor metastasis suppressor pathway increases myocyte-enriched calcineurin interacting protein expression and chronically inhibits calcineurin activity. J Clin Endocrinol Metab 0-0. Strock J & Diverse-Pierluissi MA 00 Ca+ channels as integrators of G protein-mediated signaling in neurons. Mol Pharmacol 0-0. Sugiura-Ogasawara M, Ozaki Y, Sonta S, Makino T & Suzumori K 00 Exposure to bisphenol A is associated with recurrent miscarriage. Hum Reprod 0 -. Washington W, Hubert L, Jones D & Gray WG 00 Bisphenol a binds to the low-affinity estrogen binding site. In Vitr Mol Toxicol -. Welshons WV, Nagel SC & vom Saal FS 00 Large effects from small exposures. III. Endocrine mechanisms mediating effects of bisphenol A at levels of human exposure. Endocrinology S-. Wetherill YB, Akingbemi BT, Kanno J, McLachlan JA, Nadal A, Sonnenschein C, Watson CS, Zoeller RT & Belcher SM 00 In vitro molecular mechanisms of bisphenol A action.

29 Page of 0 0 Reprod Toxicol -. Wozniak AL, Bulayeva NN & Watson CS 00 Xenoestrogens at picomolar to nanomolar concentrations trigger membrane estrogen receptor-alpha-mediated Ca+ fluxes and prolactin release in GH/B pituitary tumor cells. Environ Health Perspect -. Xi W, Lee CK, Yeung WS, Giesy JP, Wong MH, Zhang X, Hecker M & Wong CK 0 Effect of perinatal and postnatal bisphenol A exposure to the regulatory circuits at the hypothalamus-pituitary-gonadal axis of CD- mice. Reprod Toxicol 0-. Xu J, Osuga Y, Yano T, Morita Y, Tang X, Fujiwara T, Takai Y, Matsumi H, Koga K, Taketani Y, et al. 00 Bisphenol A induces apoptosis and G-to-M arrest of ovarian granulosa cells. Biochem Biophys Res Commun -. Zhou W, Liu J, Liao L & Han S 00 Effect of bisphenol A on steroid hormone production in rat ovarian theca-interstitial and granulosa cells. Mol Cell Endocrinol -. 0 Figure legends Figure Influence of BPA-application (p.o.) on activation of HPG axis and kisspeptin neurons. (A) Time chart of experimental procedure. The numbers in hollow arrow indicate the experimental time. BPA or vehicle (control) was administered at diestrus (D), proestrus (P) or estrus (E), respectively. Blood and brain samples were harvested at h after oral administration of BPA (B-E) Each point represents mean value of E, LH and FSH levels, and GnRH mrna in POA. *P<0.0 and **P<0.0 vs. controls. ## P<0.0 vs. diestrus mice; + P<0.0 and ++ P<0.0 vs. proestrus mice (two-way ANOVA). (F) Levels of Kiss mrna in AVPV are expressed as percentage of control values. **P<0.0 vs. controls. ## P<0.0 vs. diestrus mice; ++ P<0.0 vs. proestrus mice (two-way ANOVA). (G) Levels of Kiss mrna in

30 Page 0 of 0 ARC are expressed as percentage of controls. ## P<0.0 vs. diestrus mice; ++ P<0.0 vs. proestrus mice (two-way ANOVA). (H) Western blot analysis of kisspeptin in AVPV and ARC. *P<0.0 vs. controls (t test). (I) Representative picture of kisspeptin neurons (black arrows) in AVPV of control and BPA-mice (left panels). Scale bars = 00 µm. Bars indicate the number of kisspeptin positive cells in AVPV (t test). 0 Figure Influence of BPA-injection (i.c.v.) on kisspeptin expression and E-involvement. (A) Time chart of experimental procedure. The numbers in hollow arrow indicate the time of various experiments. (B&C) At proestrus BPA-injection (0.0~00 nm// µl/mouse)-induced changes in AVPV- and ARC-Kiss mrna. Each point represents mean value of AVPV- and ARC-Kiss mrna. **P<0.0 vs. vehicle-treated group (one-way ANOVA). (D&E) At diestrus BPA-injection (0.0~00 nm// µl/mouse)-induced changes in AVPV- and ARC-Kiss mrna. *P<0.0 vs. vehicle-treated group (one-way ANOVA). (F&G) E-dependency of BPA-regulated AVPV- and ARC-kisspeptin expression. Bars represent mean levels of AVPV- or ARC-Kiss mrna after h of BPA-injection (i.c.v.) in OVX-mice treated with vehicle or E. **P<0.0 vs. OVX-mice; ## P<0.0 vs. E-treated OVX-mice (two-way ANOVA). 0 Figure Influence of BPA-injection (i.c.v.) at proestrus on activation of HPG axis and GPR-dependent. (A-C) Each point represents the mean level of GnRH mrna, and plasma mean levels of LH and E after h of BPA-injection (0.0~00 nm/ µl/mouse). **P<0.0 vs. vehicle-treated group (one-way ANOVA). (D-G) Effects of the GPR antagonist on 0

31 Page of 0 BPA-injection (i.c.v.)-enhanced HPG axis. The GPR antagonist p was injected (i.c.v.) at 0 min prior to BPA-injection (i.c.v.). Bars show plasma mean levels of LH and E, and the levels of GnRH mrna and AVPV-Kiss mrna. **P<0.0 vs. BPA-treated group (t test). 0 Figure Influence of ER antagonists on BPA-enhanced AVPV-kisspeptin expression and activity of HPG axis. The ERα antagonist MPP or the ERβ antagonist PHTPP was injected (i.c.v.) at 0 min prior to BPA-injection (i.c.v.). The blood and brain samples were harvested at h after BPA injection (i.c.v.). (A) Bars show the mean levels of AVPV-Kiss mrna. **P<0.0 vs. vehicle-treated group; ## P<0.0 vs. BPA-treated group (two-way ANOVA). (B-D) Bars show the level of GnRH mrna and plasma mean levels of LH and E. **P<0.0 vs. BPA-treated group (t test). (E&F) Influence of BPA on expression of ERα and ERβ in AVPV and ARC. Bars show the mean levels of ERα and ERβ mrna at proestrus in vehicle and BPA-treated group. Figure Influence of BPA-injection (i.c.v.) on E-enhanced AVPV-kisspeptin expression. Open points and black points represent the mean level of AVPV-Kiss mrna in OVX-mice treated with alone E (0~00 µg/kg) or E/+BPA ( nm// µl/mouse). * P<0.0 and **P<0.0 vs. E-treated group; # P<0.0 and ## P<0.0 vs. vehicle-treated group (two-way ANOVA). 0 Figure Influence of BPA-injection (i.c.v.) on LH-surge. (A) Time chart of experimental procedure. The numbers in hollow arrow indicate the experimental time. (B) Hourly changes