Evidence of Altered Brain Sexual Differentiation in Mice Exposed Perinatally to Low, Environmentally Relevant Levels of Bisphenol A

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Evidence of Altered Brain Sexual Differentiation in Mice Exposed Perinatally to Low, Environmentally Relevant Levels of Bisphenol A
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  Evidence of Altered Brain Sexual Differentiation in MiceExposed Perinatally to Low, Environmentally RelevantLevels of Bisphenol A  Beverly S. Rubin, Jenny R. Lenkowski, Cheryl M. Schaeberle, Laura N. Vandenberg, Paul M. Ronsheim,and Ana M. Soto Tufts University, School of Medicine, Boston, Massachusetts 02111 Humans are routinely exposed to bisphenol A (BPA), an es-trogenic chemical present in food and beverage containers,dental composites, and many products in the home and work-place. BPA binds both classical nuclear estrogen receptorsand facilitates membrane-initiated estrogenic effects. Herewe explore the ability of environmentally relevant exposureto BPA to affect anatomical and functional measures of braindevelopmentandsexualdifferentiation.Anatomicalevidenceof alterations in brain sexual differentiation were examinedin male and female offspring born to mouse dams exposed to0, 25, or 250 ng BPA/kg body weight per day from the evening of d 8 of gestation through d 16 of lactation. These studiesexamined the sexually dimorphic population of tyrosine hy-droxylase (TH) neurons in the rostral periventricular preop-tic area, an important brain region for estrous cyclicity andestrogen-positive feedback. The significant sex differences inTH neuron number observed in control offspring were dimin-ished or obliterated in offspring exposed to BPA primarilybecause of a decline in TH neuron number in BPA-exposedfemales. As a functional endpoint of BPA action on brain sex-ual differentiation, we examined the effects of perinatal BPA exposure on sexually dimorphic behaviors in the open field.Datafromthesestudiesrevealedsignificantsexdifferencesinthe vehicle-exposed offspring that were not observed in theBPA-exposed offspring. These data indicate that BPA may becapableofalteringimportanteventsduringcriticalperiodsof brain development. (  Endocrinology  147: 3681–3691, 2006) B ISPHENOL A (BPA) is an environmental estrogenicchemical that has been attracting increased attention because of its high potential for human exposure. BPA isusedinthemanufactureofpolycarbonateplasticsandepoxyresins, and it has been shown to leach from food and bev-eragecontainers(1,2)anddentalsealantsandcomposites(3)under normal conditions of use (4). BPA is also used in themanufacture of many other products present in the homeandworkplace(5)thatwouldbeexpectedtofurtherincreaseroutineexposuretothiscompound.Althoughassessmentsof currentexposurelevelsarelimited,severalstudieshavenowconfirmeddetectablelevelsofBPAinhumanpopulations.Ina recent study of a reference human population (394 sub- jects), BPA was detected in 95% of the urine samples ana-lyzed(6).Smallerstudieshavealsoreporteddetectablelevelsof BPA in all or most individuals examined (7–9). Perhapsmost relevant to the studies to follow, BPA has been mea-sured in amniotic fluid, maternal and fetal plasma, and pla-cental tissue at birth (10, 11) and in breast milk of lactatingmothers(12).Thesedataindicatetheveryrealpossibilitythatthe developing human fetus and neonate may be exposed toBPA.Results of animal studies in our laboratory and othersindicate that the developing fetus is more sensitive to BPArelative to the adult (13, 14), a finding that may reflect thelimitedcapacityofthefetustometabolizeBPArelativetotheadult (10, 15–17). The exposure of pregnant female rodentsto low doses of BPA results in pleiotropic effects in theoffspring.Manyofthedefectsthathavebeendescribedoccurlong after BPA exposure has ended. We have reported al-terations in body weight, mammary gland development,reproductive tract tissue, gonadotropin levels, and estrouscyclicity in offspring exposed perinatally to BPA (13, 14,18–20). Other laboratories have reported effects of prenatalexposure to low doses of BPA on the prostate, preputialglands, and epididymis in males (21, 22) and on the time of vaginal opening and the time from vaginal opening to firstestrus in females (23). In addition, behavioral alterationshave been reported in offspring born to rodents treated withBPAduringgestationand/orlactation(24–32).Inthestudiesto follow, we have examined the offspring of pregnant miceexposed from the evening of d 8 of pregnancy through d 16of lactation to vehicle or to one of two doses of BPA thatshould fall within the range of potential human environ-mental exposure (7).BPA is an estrogen agonist that can bind to both classicalnuclear estrogen receptors (ERs), ER   and ER   (33, 34). Inaddition, data from recent studies have revealed that lowlevels of BPA can induce rapid, membrane-initiated estro-genic effects (35, 36), suggesting that low levels of BPA ex-posuremightinterferewithnormalestrogenicsignaling.Thedeveloping brain is exquisitely sensitive to estrogen andtherefore might be particularly vulnerable to xenoestrogenexposure. Various mechanisms facilitate precise temporal First Published Online May 4, 2006 Abbreviations: Afp,   -Fetoprotein; AVPV, anteroventral periven-tricular preoptic area; BPA, bisphenol A; BST, bed nucleus of the striaterminalis; BW, body weight; ER, estrogen receptor; ERKO, estrogenreceptor knockout; TH, tyrosine hydroxylase. Endocrinology ispublishedmonthlybyTheEndocrineSociety(http:// www.endo-society.org), the foremost professional society serving theendocrine community. 0013-7227/06/$15.00/0 Endocrinology 147(8):3681–3691  Printed in U.S.A.  Copyright © 2006 by The Endocrine Societydoi: 10.1210/en.2006-0189 3681  and spatial control of estrogen action during critical periodsof brain development (37). These include the transient ex-pression of both estrogen receptors and aromatizing en-zymes that correspond to transient increases in testosteroneproduction in the developing male. In rodents and otheranimal models, numerous studies have documented thattestosterone secreted by the testes in the developing male isconverted to estradiol  in situ  by aromatase, which is presentin specific brain regions during critical periods of perinataldevelopment. This conversion of testosterone to estradiolplays an important role in the sexual differentiation of the brain (for review see Refs. 37 and 38). Unlike the testes, theovaries of the developing female rodent are quiescent peri-natally (39). Recent data have verified the importance of   -fetoprotein(Afp)inprotectingthebrainofthefemalefetusfrom defeminization or masculinization by binding circulat-ing estradiol from the mother or neighboring male litter-mates (40). Afp, which is abundant during fetal and earlyneonatal life (41, 42), binds estradiol in rodents with highaffinity. Nonsteroidal estrogens, such as BPA, that exhibit aloweraffinityforplasmaestrogen-bindingproteins(43)may be able to evade this protective mechanism.In the present studies, we explore the ability of chroniclow-levelperinatalexposuretoBPAtoaffectanatomicalandfunctional measures of brain development and sexual dif-ferentiation. First, we searched for anatomical evidence of alterations in brain sexual differentiation. For these studies,we concentrated on a prominent sexually dimorphic regionof the brain in the rostral periventricular preoptic area. Theanteroventral periventricular preoptic area (AVPV) is im-portant for cyclic gonadotropin release and for the LH surgerequiredforovulation(44,45).Neuronsinthisregioncontainsexually dimorphic patterns of steroid receptor distribution(46) and peptide expression in rats (47–49). One robust sexdifference that has been observed in the AVPV of both ratsand mice is the sexually dimorphic population of tyrosinehydroxylase (TH) neurons (50–52). TH is the rate-limitingenzyme for dopamine synthesis. The number of TH-positiveneurons in the AVPV is significantly higher in female ratsandmicerelativetomales,andthesexualdimorphismofthispopulation of neurons appears to be dependent on perinatallevels of gonadal steroids (50, 51). Studies of estrogen re-ceptor knockout (ERKO) mice indicate that the significantdecline in TH neuron number in the male AVPV is depen-dent upon the presence of ER   (52). To determine whetherperinatalBPAexposurecanalterarobustanatomicalmarkerof brain sexual differentiation in mice, we compared THneuronnumberintissuesectionsthroughtherostralperiven-tricular preoptic area in male and female littermates born tomothersexposedtoBPAortovehicleonly.Hereinwereporteffects of perinatal BPA exposure on the expected sex dif-ferences in TH neuron number.As a second and functional endpoint of BPA action on brainsexualdifferentiation,weexaminedpotentialeffectsof developmental exposure to BPA on sexually dimorphic be-haviors in the open field. Strain-dependent sex differenceshave been reported in open-field behaviors in rats and mice(53, 54), and data from the majority of studies reveal higherlevels of activity in females relative to males (54–56). Al-though circulating hormones in adulthood can affect these behaviors, differential exposure to gonadal steroids duringthe perinatal period plays an important role in the develop-ment of sexually dimorphic behaviors in the open field (55).More specifically, data from studies of male ERKO micesuggest that masculinization of open-field behavior requiresestrogenactionduringdevelopment(57).Theresultsofthese behavioralstudiesfurthersuggestthatearlyexposuretoBPAmay alter important events during critical periods of braindevelopment in mice and indicate the need for careful studyof the potential effects of developmental exposure to thiscompound in humans. Materials and Methods  Animals Animalsforthesestudiesweremaintainedintemperature-andlight-controlled (14 h light, 10 h dark, lights on at 0400 h) conditions at theTufts–New England Medical Center Animal Facility. The cages and bedding used for housing tested negligible for estrogenicity using theE-SCREEN assay (58). The food (rodent diet 2018; Harlan Teklad, St.Louis,MO)wasextractedandassayedaspreviouslydescribed(58),andit also tested negligible for estrogenicity (20 fmol estradiol equivalents/g). Water was supplied by glass bottles only. All experimental proce-dures were approved by the Tufts University-New England MedicalCenter Animal Research Committee in accordance with the Guide forCare and Use of Laboratory Animals.To generate offspring for these studies, female and male CD-1 micewere purchased (Charles River Laboratories, Wilmington, MA) at 8–10wkofage.Femalebreederswerehousedbetweencagesofbreedermalesfor a minimum of 1 wk after arrival. Two days before introduction of amale into the cage, females were exposed to male bedding to stimulateestrous cyclicity and ovulation. Males were placed in the female cages,and the morning on which a vaginal plug was observed was designatedd 1 of pregnancy. On the evening of d 8 of pregnancy, dams wereweighed and implanted sc with Alzet osmotic pumps (Alza Corp., PaloAlto, CA) that were designed to deliver either 50% dimethylsulfoxide(vehicle control) or BPA dissolved in 50% dimethylsulfoxide at the rateof 25 ng or 250 ng BPA/kg body weight (BW) per day (Sigma ChemicalCo., St. Louis, MO) throughout the remainder of the pregnancy andthrough d 16 of lactation. Dams were allowed to deliver naturally, andthe litters were culled to eight pups per mother (four males and fourfemales) 1 d after birth. Litters were weaned on postnatal d 22–24. Onlylitters with normal distributions of males and females were included inthese studies. The average number of pups and mean percentageof female pups in each litter examined in these studies are shown inTable 1.Vehicle-exposed and BPA-exposed offspring for anatomical studieswere examined before puberty (22–24 d). For behavioral studies, off-spring were initially examined at 6–9 wk of age. However, because of concerns regarding potential effects of differences in circulating hor-mone levels in postpubertal animals, additional offspring were exam-ined before puberty (27–29 d of age). It should be noted that vaginalsmear records from our colony indicate that 6- to 9-wk-old group-housed females do not exhibit regular 4- to 5-d estrous cycles (Rubin,B. S., and A. M. Soto, unpublished data). This observation probablyreflects the importance of pheromones to the maintenance of regular TABLE 1.  Composition of the litters used for the studiesdescribed BPA treatmentMean no. of pupsper litterProportion of femalepups per litter Control 12.17  0.58 0.591  0.02625 ng 11.30  0.423 0.524  0.052250 ng 11.27  0.506 0.524  0.034No significant differences were noted between any experimentalgrouprelativetothecontrols.Alllitterswereculledtoeightpups(fourmales and four females) 1 d after birth. 3682  Endocrinology, August 2006, 147(8):3681–3691 Rubin  et al.  • BPA Alters Brain Sexual Differentiation  estrouscyclicityinmice,whichisenhancedbyexposuretomalesormaleurine and can be suppressed by exposure to other females (59–61). TH neuron number Tissue preparation.  Two male and two female littermates from each litterwere killed at 22–24 d of age. Prepubertal animals were chosen forexamination because of evidence of a potential influence of circulatingsteroid levels on TH expression in adult animals (50). Animals wereinjected with an overdose of pentobarbital and perfused intraventricu-larly with heparinized saline followed by a solution containing 4%paraformaldehyde and 3.0% acrolein. Brains were removed from thecalvarium, placed into phosphate buffer, and stored at 4 C. Brains froma matched set of male and female littermates from seven to eight dif-ferentlitterswereanalyzedforeachofthethreeexposurelevels(rangingfrom 0–250 ng BPA/kg BW/d) as detailed above.  Immunocytochemistry protocols Brainswereblocked,and40-  msectionswerecutinthecoronalplaneon a Vibratome (Technical Products International, St. Louis, MO). Sec-tions were collected beginning rostrally at the diagonal band of Brocaandcontinuingcaudallythroughthearcuatenucleus.Consecutivebrainsections were collected into two numbered tissue boats with each boatreceivingeveryothersectionthroughtheregionofinterest,thuslimitingthe number of tissue sections in each boat to facilitate antibody incu- bations.Bothtissueboatsforeachanimalweretreatedidenticallysothatevery section through the areas of interest was available for analysis.After pretreatment to remove residual aldehydes, tissues were rinsed,and free-floating sections were incubated for 48 h at 4 C in anti-THmonoclonal antibody (MAB318; Chemicon, Temecula, CA) diluted at1:5000. Secondary antibody was biotin-SP-conjugated donkey anti-mouse (no. 715-066-151; Jackson ImmunoResearch, West Grove, PA).DetectionwascompletedwithVectastainABCreagent(PK-4000;VectorLaboratories, Burlingame, CA) and diaminobenzidine. A total of sevenchemistries were performed, and each immunocytochemistry run in-cluded tissues from matched male and female littermates from thedifferent treatment groups.  Data analysis Tissue sections through the AVPV were identified using the MouseBrain Atlas of Paxinos and Franklin (62) as a guide to define the pa-rameters of the region of interest. The region analyzed corresponds tothatidentifiedastheAVPVinFigures26–30intheatlasandisindicated by the  shaded areas  in Fig. 1. As depicted, this region extends frominteraural 4.42 mm to interaural 3.94 mm, with the consecutive sectionsexaminedbeginningrostrallyattheleveloftheorganumvasculosumof the lamina terminalis and extending caudally to the level just before thecrossing of the anterior commissure. TH neurons were identified usinga Zeiss Axioscope (  10 and   40 objectives), and counted by threedifferentobserversblindtothesexandtreatmentgroupsoftheanimals. F IG . 1. Sections through the rostral preoptic area. The drawings depicted were adapted from the Mouse Brain Atlas by Paxinos and Franklin(62), and the AVPV, as defined by the atlas, appears  shaded . As shown, these drawings span the region extending from interaural 4.42 mmrostrally through interaural 3.94 mm caudally. Actual tissue sections through most (nine of 11) of the rostral-caudal extent of the AVPV fromone representative female in the study are also shown below the adapted atlas diagrams. The tissue sections depicted here span an areabeginningintherostralAVPVthroughtheregionbeforethecrossingoftheanteriorcommissure.3V,Thirdventricle;aca,anteriorcommissure; VOLT, organum vasculosum of the lamina terminalis; MPA, medial preoptic area; MnPO, medial preoptic nucleus. Rubin  et al.  • BPA Alters Brain Sexual Differentiation Endocrinology, August 2006, 147(8):3681–3691  3683  BecausethetotalnumberofsectionsthroughtheAVPVwasnotidenticalfor all animals in the study, TH neuron number was also assessed inseven consecutive sections extending from the caudal AVPV (rostral tothe level of the crossing of the anterior commissure) through the mid-regionoftheAVPVrostrallyineachanimal.THneuronnumberwasalsoassessed in two matched sections through the arcuate nucleus of thehypothalamus.  Behavioral studies Open-field test at 6–9 wk of age.  At 6–9 wk of age, male and femaleoffspring of pregnant dams treated with 0, 25, or 250 ng BPA/kg BW/dwere observed in a novel open field. The open-field apparatus was alargeplastictubmeasuring16  24incheswithawallheightof11inches.The floor of the tub was divided into squares to facilitate behavioralmeasurements. The apparatus was carefully cleaned with ethanol,rinsed with water, and then dried after each behavioral test. Two iden-tical open fields were available for testing and were rotated during thetesting period. All behavioral tests were conducted in the same room atthe same time of day (1200–1400 h) and were scored by three observers.Beforethestartofdatacollection,3dofbehavioraltestswereconductedwithanadditionalcohortofanimalstofamiliarizetheobserverswiththe behaviors to be recorded and to standardize the scoring of behaviors. Atotal of 94 animals were tested in the open field, including 14–17 malesandfemalesfromeachofthethreetreatmentgroups.Onlyasinglemaleand female from each litter were examined in these studies to eliminatepotential litter effects. At the start of each test, the mouse was carefullyplaced into the center of the open field and his/her movements wererecorded over the next 5 min. Measurements included number of rears,time spent in center, time stopped, time grooming, and number of fecalpellets. Initial attempts to score the number of squares entered to assessdistance traveled in a given testing period were abandoned because theanimals moved too quickly to obtain accurate numbers without digitalrecordings. Open-field tests in prepubertal animals.  Additional open-field tests wereperformed in younger prepubertal animals at 27–29 d of age. Theseanimals were examined before vaginal opening and before the estab-lishment of adult gonadal hormone levels. For these studies, controloffspring (n  10 animals per sex) and offspring born to females treatedwith 250 ng BPA/kg BW/d (n    12 animals per sex) were examined.Animals were tested in the open field as described above with onechange to the protocol. Because the prepubertal animals were veryactive, we chose to detain each subject in the center of the open fieldcovered by the transfer beaker for a period of 5 sec before the start of the test to facilitate accurate scoring of behaviors. This method has beenused by other laboratories. Unfortunately, this procedural change mayhave influenced the time-in-center measurement such that this param-eter was not comparable in the two age groups examined.  Statistics The data were analyzed using both parametric and nonparametricstatistics. Both showed similar significant differences. The parametricanalyses are presented, and the data are graphed as mean  sem . Theanatomical data and the behavioral data from the 6- to 9-wk animalswere analyzed by two-way ANOVA with sex and BPA as the twoparameters. Planned comparisons of male and female offspring fromeach treatment group were evaluated by  t  tests, and planned same-sexcomparisons across treatment groups were assessed by ANOVA fol-lowed by Bonferroni  post hoc  tests. Because the behavioral data for theprepubertal animals contained only a single BPA dose, planned com-parisons of male and female offspring in the control and BPA-exposedgroup and same-sex comparisons across the two treatment groups wereanalyzed by  t  tests. Results TH neuron number in control and BPA-exposed offspring  MeannumberoftissuesectionsthroughtheAVPV. Comparisonsof the total number of tissue sections through the AVPV(based on the delineation of this region in the Paxinos andFranklin atlas and independent of the presence or absence of TH neurons) by two-way ANOVA revealed overall signifi-cance ( P  0.039) and a significant interaction between vari-ables (sex  BPA,  P  0.014). The number of tissue sectionsdiffered significantly in male and female offspring born tovehicle-treated mothers ( P  0.003,  t  test; see Fig. 2 ) . Tissuesection number did not differ significantly in male and fe-maleoffspringfromeitheroftheBPA-exposedgroups.Thesedata are consistent with an increase in the rostral-caudalextent of the AVPV in control females relative to controlmales and a loss of this sex difference in offspring of BPA-treated dams. Comparison of tissue section number by sexacrosstreatmentgroupsrevealedadifferenceinfemales( P  0.020, ANOVA), and  post hoc  analysis revealed significantdifferencesbetweencontrolfemalesandthoseexposedto250ng BPA ( P  0.023, Bonferroni). TH-positive cell number.  Analysis (two-way ANOVA) of totalTH cell number revealed an overall significance ( P  0.001)and differences by sex ( P    0.001), BPA ( P    0.020), andBPA  sex ( P  0.014). Subsequent analyses ( t  test) revealedthat the total number of TH-positive neurons counted insections through the AVPV differ in male and female off-spring born to vehicle-treated mothers ( P    0.001; controlmale TH neuron number  46% of control females; see Figs.3 and 4) and to mothers exposed to the lowest dose of BPA,25 ng/kg BW/d ( P  0.024; male TH neuron number  65%of females). No significant sex differences were observed inoffspringexposedto250ngBPA/kgBW/d(maleTHneuronnumber  80%offemales).Moreover,comparisonoffemalesacross treatment groups was significant ( P    0.005,ANOVA), and  post hoc  analysis revealed that females ex-posed to the higher dose of BPA exhibited a significant de-crease in the total number of TH-positive cells relative tocontrol females ( P  0.004, Bonferroni). Females exposed tothe low dose of BPA revealed a 24% decrease in TH neuronnumberandthoseexposedtothehigherdoserevealeda41% F IG . 2. Number of sections through the AVPV. The average numberof 40-  m tissue sections through the AVPV are shown here for eachtreatment group.  Solid bars  represent mean (  SEM ) female values(n  7–8 per treatment), and  gray bars  represent mean (  SEM ) male values(n  7–8pertreatment).Asignificantsexdifferenceinsectionnumberisobservedonlyincontroloffspring(**,  P  0.003,female vs. male), and the number of sections were decreased in female offspring exposed to the higher dose of BPA relative to controls (*,  P  0.020).Thex-axislabelreferstothelevelofBPAexposureofthemothers(perkg BW per day). 3684  Endocrinology, August 2006, 147(8):3681–3691 Rubin  et al.  • BPA Alters Brain Sexual Differentiation  decline in TH neuron number relative to controls. No sig-nificant differences in TH cell number were noted acrosstreatment groups in the males.Measurements of total cell counts included assessment of TH neurons in every section through the AVPV in eachanimal and therefore did not include equal numbers of sec-tions from every brain. When TH-positive cell counts wererestrictedtosevenconsecutivesectionsthroughtheAVPVof eachbrain(beginningcaudallyjustbeforethecrossingoftheanterior commissure and extending rostrally through themidregion of the AVPV), analysis (two-way ANOVA) re-vealed overall significance ( P    0.001) and significant dif-ferences by sex ( P  0.001) and by BPA ( P  0.032). Addi-tional analyses revealed significant sex differences in controloffspring ( P  0.002,  t  test), but not in the offspring born tomotherstreatedwitheitherdoseofBPA(Fig.5A).THneuronnumber differed significantly across treatment groups in fe-males ( P    0.011, ANOVA) and more specifically betweenfemales in the control and 250-ng groups ( P    0.009,Bonferroni).When the data from these seven sections was further di-videdtoassesscellcountsinfoursectionsthroughthemedialaspect of the AVPV (Fig. 5B) and three sections through themore caudal aspect of the AVPV (Fig. 5C), it appeared thatBPAexertedamoreprofoundinfluenceonTH-positiveneu-ron number in the medial part of the nucleus (overall  P   0.004;sex P  0.026;BPA P  0.033;andsex  BPA P  0.040,two-way ANOVA). A robust sex difference in TH neuronnumber was noted in the mid-AVPV in the control group( P  0.001, t test)butnotineitheroftheBPA-exposedgroups.Same-sex comparisons across treatment groups revealed asignificant difference in the females ( P    0.012, ANOVA),and  post hoc  tests revealed that TH neuron number wassignificantly decreased in females born to mothers treatedwith the 250-ng dose of BPA relative to controls ( P  0.010,Bonferroni). Comparisons of TH-positive neuron number inthe more caudal aspect of the AVPV revealed overall sig-nificance and differences by sex ( P    0.001, two-wayANOVA). Significant sex differences were present in controloffspring ( P    0.001,  t  test) and offspring exposed to thelower dose of BPA ( P  0.005). The sex difference in animalsexposed to 250 ng BPA/kg BW/d approached significance( P  0.059). TH-positive neurons in the arcuate nucleus.  No significant sexdifferences in the number of TH-positive cells per sectionwere observed in the arcuate nucleus of control or BPA-exposedoffspring.Therewerealsonosignificantdifferencesin cell number in BPA-exposed relative to control offspring(see Fig. 6).  Behavioral tests Open-field tests in 6- to 9-wk-old animals.  Male and femaleoffspringborntomothersexposedto0,25,or250ngBPA/kg F IG . 3. Photomicrographs of sections through the rostral periven-tricular preoptic area of control and BPA-exposed mice. TH-positiveneurons are shown in representative sections through the mid AVPV (A) and through the caudal AVPV (B) of female and male offspring born to control dams and born to dams treated with 250 ng BPA/kg BW/d. III V, Third ventricle; OC, optic chiasm.  Bar , 100   m.F IG . 4. Total number of TH-positive neurons in sections though the AVPV. A, Data are shown for offspring born to control dams and offspring born to dams treated with two doses of BPA (mean  SEM ). Significant sex differences are noted in TH neuron number in control offspring (***,  P  0.001) as well as offspring born to dams treated with the lowest dose of BPA (*,  P  0.024). A significant decline in TH neuron numberis noted in 250-ng females relative to controls (**,  P    0.004).  Black bars , females (n    7–8 per treatment);  gray bars , males (n    7–8 pertreatment). B, The mean female to male ratio of the total TH-positive neuron number was calculated for each pair of littermates examined. As shown, control females have approximately twice the number of TH-positive neurons in the AVPV relative to control males, and the femaleto male ratio is markedly reduced with exposure to the higher dose of BPA. The x-axis label refers to the level of BPA exposure of the mothers(per kg BW per day). Rubin  et al.  • BPA Alters Brain Sexual Differentiation Endocrinology, August 2006, 147(8):3681–3691  3685
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