Sexually Dimorphic Effects of Ancestral Exposure to Vinclozolin on Stress Reactivity in Rats

14 Pages·2014·0.78 MB·English

ENDOCRINE-DISRUPTING CHEMICALS
Sexually Dimorphic Effects of Ancestral Exposure to
Vinclozolin on Stress Reactivity in Rats
Ross Gillette, Isaac Miller-Crews, Eric E. Nilsson, Michael K. Skinner,
Andrea C. Gore, and David Crews
InstituteforCellularandMolecularBiology(R.G.,I.M.-C.,A.C.G.,D.C.),DivisionofPharmacologyand
D
Toxicology(A.C.G.,D.C.),andDepartmentofIntegrativeBiology(D.C.),TheUniversityofTexasat o
w
Austin,Austin,Texas78712;andCenterforReproductiveBiology(E.E.N.,M.K.S.),SchoolofBiological n
lo
Sciences,WashingtonStateUniversity,Pullman,Washington99164 a
d
e
d
fro
m
Howanindividualrespondstotheenvironmentdependsuponbothpersonallifehistoryaswell
h
as inherited genetic and epigenetic factors from ancestors. Using a 2-hit, 3 generations apart ttp
s
model,wetestedhowF3descendantsofratsgiveninuteroexposuretotheenvironmentalen- ://a
docrine-disruptingchemical(EDC)vinclozolinreactedtostressduringadolescenceintheirown ca
d
lives, focusing on sexually dimorphic phenotypic outcomes. In adulthood, male and female F3 e
m
vinclozolin-orvehicle-lineagerats,stressedornonstressed,werebehaviorallycharacterizedona ic
.o
batteryoftestsandtheneuthanized.Serumwasusedforhormoneassays,andbrainswereused up
for quantitative PCR and transcriptome analyses. Results showed that the effects of ancestral .co
m
exposuretovinclozolinconvergedwithstressexperiencedduringadolescenceinasexuallydi- /e
n
morphicmanner.Debilitatingeffectswereseenatalllevelsofthephenotype,includingphysiol- d
o
ogy,behavior,brainmetabolism,geneexpression,andgenome-widetranscriptomemodifications /a
inspecificbrainnuclei.Additionally,femalesweresignificantlymorevulnerablethanmalesto rticle
transgenerationaleffectsofvinclozolinonanxietybutnotsocialitytests.Thisfundamentaltrans- -a
b
s
formationoccursinamannernotpredictedbytheancestralexposureortheproximateeffectsof tra
c
stress during adolescence, an interaction we refer to as synchronicity. (Endocrinology 155: t/1
3853–3866,2014) 55
/1
0
/3
8
5
Thelegacyofexposuretoenvironmentalchemicals,in- zation,fromgeneregulationtobehavioralinteractionsof 3/2
4
cluding endocrine-disrupting chemicals (EDCs), has conspecifics(13,16–18). 2
3
1
permanentlyalteredthepresentandfuturehealthofhu- Environmental and social stressors are an immediate 7
2
mansandwildlife(1–4).DirectexposuretoEDCsaffects andprimarysourceofcontext-dependentepigeneticmod- b
y
g
thelifehistoryofindividualsandtheirdescendants.Such ifications(19–22).Chronicorexcessivestressduringsen- u
e
s
context-dependentepigeneticmodificationsarenotheri- sitive periods such as prenatal development or the early t o
n
tablepersebecausethegermcellsarenotaffected.Other postnatalperiodcanpredisposefordiseaseanddisorder 0
4
epigenetic modifications can affect the germline (germ- later in life, a phenomenon known as the fetal basis of A
p
line-dependent)andthusmanifestineachgenerationeven adult disease (23, 24). Later developmental periods, in- ril 2
intheabsenceofthecausativeagent(1–9).Thisisthecase cluding adolescence, also have enduring effects that in- 0
1
9
forseveralEDCs,notably,vinclozolin(10–12),bisphenol cludeneuralremodeling,sensitivitytodrugsofabuse,im-
A (BPA; 13, 14), and tributyltin (15). Such transgenera- pairedlearningandmemory,andemotionaldisordersin
tionalmodificationsaffectalllevelsofbiologicalorgani- adulthood(24–31).
ISSNPrint0013-7227 ISSNOnline1945-7170 Abbreviations:BLA,basolateralamygdaloidnucleus;BnST,bednucleusofthestriater-
PrintedinU.S.A. minalis;BPA,bisphenolA;CeAmy,centralamygdaloidnucleus;C-NS,controlnonstress;
Copyright©2014bytheEndocrineSociety CO,cytochromeoxidase;CoAmy,anteriorcorticalamygdaloidnucleus;CRS,chronicre-
ReceivedMarch24,2014.AcceptedJuly10,2014. straintstress;C-S,controlstress;CV,coefficientofvariation;DAB,3,3(cid:2)-diaminobenzidine;
FirstPublishedOnlineJuly22,2014 EDC,endocrine-disruptingchemical;MeAmy,medialamygdaloidnucleus;mPOA,medial
preopticarea;PCB,polychlorinatedbiphenyl;PND,postnatalday;qPCR,quantitativePCR;
TLDA,TaqManlow-densityPCRarray;VMN,ventromedialhypothalamicnucleus;V-NS,
vinclozolinnonstress;V-S,vinclozolinstress.
doi:10.1210/en.2014-1253 Endocrinology,October2014,155(10):3853–3866 endo.endojournals.org 3853
3854 Gilletteetal SexDifferences,AncestralEDC,andStress Endocrinology,October2014,155(10):3853–3866
Most neurobehavioral, neurological, and addiction lifetimeresultsinprofoundsexdifferencesinthescopeand
disordersexhibitsignificantsexdifferencesinrelativerisk natureofreactivity.
level and severity. Women have higher levels of diseases
anddisorderssuchasAlzheimer’sdisease,dementia,ma-
jor depressive disorder, posttraumatic stress disorders, Materials and Methods
and anxiety and panic disorders (32); whereas autism-
Animals
spectrumdisorderandattentiondeficithyperactivitydis-
MaleandfemaleSprague-Dawleyrats,vinclozolin-andve-
order predominate in men (33). Developmental sex dif-
hicle(dimethylsulfoxide)-lineage,werebredatWashingtonState
ferencesinadrenalandreproductivehormonesmustplay
University(10).AnF0generationofpregnantmothers(vinclo-
aroleintheiretiologybecause,inmanyinstances,disor- zolin,n(cid:3)3;control,n(cid:3)2)wasinjectedipwithvinclozolin(100
D
ders manifest after adrenarche/puberty, fluctuate during mg/kg/d)ordimethylsulfoxide(vehicle)onembryonicdays8to o
w
menstrualcycles,andchangeprevalence/severityatmeno- 14(Figure1A).Subsequentgenerationswerebredsuchthatthere nlo
pauseorduringtimesofstress,suggestinginvolvementof wasnosiblingorcousininbreeding.F3-generationpupswithno ad
e
chemicalbodyburdenwereweanedatpostnatalday(PND)21, d
adrenalandgonadalsteroidhormones(34–38).Environ- implanted sc with an identification microchip (AVID), and fro
mental exposures to compounds that disrupt these hor- m
shippedtotheUniversityofTexasatAustinthefollowingday. h
monalsystemshavehadamajorimpactonhealthandare AllanimalprotocolswereapprovedatbothWashingtonState ttp
s
believedtobeacontributingfactortotheincreasedinci- UniversityandtheUniversityofTexasatAustin.Uponarrival, ://a
denceofobesity,illness,andaffectivedisordersinhumans animalswerepair-housedwithasame-sexindividualoftheop- ca
d
posing lineage (eg, a control-lineage male with a vinclozolin- e
(1,39,40). m
lineage male). Cage-mates were all within 1 day separation in ic
Vinclozolin is a commonly used fungicide with dem- .o
age.Dyadswerehousedinstandardtranslucentpolycarbonate u
p
onstrated antiandrogenic properties (41, 42). When ad- ratcages(46(cid:4)24(cid:4)20.5cm)withadlibitumaccesstotapwater .c
o
ministeredtohumancancercelllinesinvitro,itprevents andstandardratchow(RMH1800fromPurina)andenviron- m
/e
the androgen receptor from binding androgen response mentalenrichment(7-cmdiameterPVCpipe).Thecolonyroom nd
elements,therebyinhibitingtheandrogenreceptor’sabil- wasona14-hourlight,10-hourdarkcyclewithlightsoffat o/a
ity to act as a transcription factor (43). Vinclozolin ad- (8S:u3p0pAleMm.eAntnailmFaiglsurwee1r)e. handled and weighed twice a week rticle
ministeredbyeitherintraperitonealinjectionororallyis -ab
s
metabolizedintotwoprimarymetabolites(M1andM2), Chronic restraint stress in adolescence and tra
c
bothofwhichareeffectiveagainstpreventingtheandro- behavioral testing in adulthood t/1
5
genreceptorfrombindingitstargetedandrogenresponse Chronicrestraintstress(CRS)duringadolescenceandbehav- 5/1
0
elements (42). Exposure to vinclozolin during organiza- ioral testing of adults were conducted in a separate room as /3
8
tionalperiodsofembryonicdevelopmentresultsinfemi- describedindetail(45),transportedincoveredcages,andtested 53
underdimredlightorwhitelightdependingonthetest.CRSwas /2
nizedmaleswithreducedanogenitaldistanceandhypos- 4
conducted for 6 hours daily for 21 days from PND 23 to 44 23
padias (41); as adults, these males exhibit a reduced 1
(Figure1A). 7
2
number of testis cords, increased apoptosis of immature Behavioraltestswereconductedinacounterbalancedorder b
y
sperm,anddecreasingspermmotility,allofwhichresult andwereadministeredtoeachindividualbeginningonPND90. g
u
in decreased fertility (44). Some aspects of this disease Behavioral tests consisted of open-field, sociability, and social es
noveltyasdescribedpreviouslyinearliervinclozolinwork(45) t o
phenotypepersistforupto4generationsintheabsenceof n
(Supplemental Methods). In addition, the light-dark box and 0
furtherexposure(10). elevated plus maze were added to provide more thorough be- 4 A
p
Previousworkfromourlaboratoriesrevealsignificant havioralcharacterization.Thetestforsociabilitywasperformed ril 2
interactionsoftransgenerationalvinclozolineffectsinher- tomeasuresocialapproach,anxiety,andexploration.Thesocial 0
1
9
itedfromthegreat-grandparentswithstressexperienced novelty test immediately followed, enabling animals to differ-
inadolescence(45).Thatworkwaslimitedtomales,over- entiatebetweenthenowfamiliarconspecificandanovel,unfa-
miliarconspecificasatestforarat’spreferenceforsocialnovelty
looking fundamental sex differences in reproductive in-
(46,47).Theopen-fieldtestisastandardmeasureofanxiety,
vestment,behavior,andsurvival.Inadditiontotheindi-
takingadvantageofarat’snaturalaversiontoexplorationofa
vidual effects of ancestral exposure to vinclozolin and centrallitareaandthepreferencetospendtimearoundtheedges
stressduringadolescence,wewereparticularlyinterested (48,49).Similarly,alight-darkboxtestmeasuresanxiety-like
in the combination of these 2 events, a phenomenon we behaviorsandaversiontoexplorationinabrightlylitcompared
withadarkchamber(50).Theelevatedplusmazeallowsaratto
refer to as synchronicity. We demonstrate here that the
spendtimeinanenclosedrunwaycomparedwithanopenrun-
sexesdiffermarkedlyintheirresponsestothese2typesof
waythatisraisedofftheground,againprovidinginformation
epigeneticmodificationsandthatthesynchronicityofan- aboutanxiety-likebehaviors(51).Detailsforhowthesestandard
cestral exposure and proximate stress during one’s own behaviors were performed are provided in Supplemental
doi:10.1210/en.2014-1253 endo.endojournals.org 3855
A
Birth/
Treatment Breeding Weaning Adolescence Early Maturity Adulthood
E 8-14 3 Generations PND 0-22 PND 23-43 PND 44-111 PND 112-120
DMSO One Animal of each
Injection lineage pair housed
~~ ~~
21 Days, 6 hrs/day CRS
~~ ~~
F0~~ F1~~ F2~~ F3 Euthanasia and
Bi-Weekly Handling & Weighing Behavioral Battery
F0~~ F1~~ F2~~ F3 Tissue Collection
~~ ~~
Daily Handling
~~ ~~
Vinclozolin Injection Animals Shipped from
100 mg/kg/day WSU to UT at Weaning D
o
w
n
lo
a
d
B Stress during e
d
adolescence of F3 fro
m
Non- Effect of h
Stress Stress Stress ttp
s
exposurens earlier Control C-NS C-S 1 ://academ
ne atio Vinclozolin V-NS V-S 2 ic.o
Germli 3 gener ELifnfeecatg oef 5 SAynnccehsrotrnailc aityn do f up.com/e
Proximate Experience nd
3 4 o
/a
rtic
Figure1. ExperimentalmodelandcategorizationofeffectsofancestralvinclozolinvscontrollineageandCRSinrats.A,Experimentalflowchart. le
Ratswereexposedtovinclozolinorvehicle(dimethylsulfoxide)control3generationspreviously,andtheF3generationusedforCRSduring -a
b
ashdoowlensc,ewnictehocromnopastrriseossn.sA1l,la3naimndal5swtheerefotchaelngbroeuhpavoiofrtahlelystteusdteyd. asadults.B,Experimentalgroupsandcomparisons.Plannedcomparisonsare strac
t/1
5
Methods.Stimulusratsusedforsocialtestswerenaive,intact, Table 1). CO histochemistry is well-established as relating to 5/1
same-sex, and age-matched Sprague-Dawley rats (Harlan). overall metabolic history within a cell, as has been shown for 0/3
All behaviors were quantified using the video tracking system learningandmemory,sexualactivity,anddevelopmentalexpe- 85
3
(ANY-maze)andapparatusfromStoelting.Afterallbehavioral rience(53).Aspublishedpreviously(45),inbrief,fresh-frozen /2
4
testingwascompleted,betweenPND118and124,animalswere braintissuewascryosectionedandmounted.Slicescontaining 2
3
euthanizedbydecapitationasperapprovedprotocols. nuclei of interest were incubated in a heated bath containing 17
3,3(cid:2)-diaminobenzidine (DAB) and saturated with oxygen. Cy- 2 b
y
Brain collection and selection of brain regions tochromeCwithinthetissueoxidizesDAB,whichturnsfrom g
u
Thebrainwasquicklyremovedandprocessedaspreviously clear to light brown. The slices were imaged (Javelin) using a es
described(45).Briefly,eachbrainwassplitinthesagittalplane, constant-intensitylightbox(NorthernLightR95)andtheODof t o
n
withonesagittalhalffrozenforsectioningandcytochromeox- definednucleiwasmeasured.TheabundanceofcytochromeCin 0
4
idase(CO)histochemistryandtheothercut(2mm)onachilled brain tissue has been tightly linked to metabolic activity and A
p
rat brain matrix for later micropunching of specific nuclei for therefore neuronal activity. Thus, the amount of DAB that is ril 2
mRNAandtranscriptomeanalyses.Thefollowingregionsofthe oxidizedanditsresultingODisusedasanindexofmetabolic 0
1
amygdala,hippocampus,andhypothalamuswerethefocusof history. 9
thisstudy,selectedfortheirinterconnectivityandfunctionalre-
lationshipsinthecontrolsofthebehaviorsstudiedherein:ba- Gene expression analysis
solateralamygdaloidnucleus(BLA),centralamygdaloidnucleus Brainnucleiwerepunchedfromfrozencoronalhemisections
(CeAmy),medialamygdaloidnucleus(MeAmy),anteriorcor- usinga1-mmdiameterPalkovitspunch.Tenregionswereused
ticalamygdaloidnucleus(CoAmy),CA1andtheCA3ofthe forquantitativePCR(qPCR),andofthese,4wereusedforfur-
hippocampus, medial preoptic area (mPOA), ventromedial ther transcriptome analysis (Supplemental Table 1). RNA ex-
hypothalamic nucleus (VMN), and bed nucleus of the stria traction, purification, and preparation for the cDNA reaction
terminalis(BnST). were performed based on standard procedures, as detailed in
SupplementalMethodsandaspublishedpreviously(54).Gene
Brain metabolic activity expressionanalysiswasperformedusinga48-geneOD(qPCR)
CO histochemistry, a measure of brain metabolic activity platform, the TaqMan low-density PCR array (TLDA) (Life
(52),wasmeasuredin13discreteneuralnuclei(Supplemental Technologies)thatenablesdetectionofall48genesinmicrodis-
3856 Gilletteetal SexDifferences,AncestralEDC,andStress Endocrinology,October2014,155(10):3853–3866
1(cid:2)Lserumforfemalesand25(cid:2)Lformales(CaymanChemical).
Table 1. GenesontheTLDA
The intra-assay CV was 4.9%, and assay sensitivity was 7.8
FunctionalGroup Genes pg/mL.Serumtestosteronewasmeasuredbyenzymeimmuno-
assay(CaymanChemical)using200(cid:2)Lserumforfemalesand
Housekeepinggenes 18S,Gapdh
6(cid:2)Lformales.Theintra-assayCVwas7.6%,andassaysensi-
Epigeneticmodifiers Dnmt1,Dnmt3a,Dnmt3b,Dnmt3l,
tivitywas3.9pg/mL.
Hdac1,Mbd2
Stresssignaling Crh,Crh1,Gmeb2,Mc3r,Mcr4r,
Mc5r,Nr3c1,Pomc Statistics
Steroidogenicenzymes Cyp19a1,Hsd11b2,Srd5a1 Initial analyses of individual datasets were performed first,
Sexsteroidhormone Ar,Esr1,Esr2,Gnrhr,Gper,Pgr andthisrevealedthatdatasetshadnon-normaldistributionsand
receptors heterogeneous variance, determined by Shapiro-Wilk test for
Dopaminergic Comt,Drd2,Drd4,Th
normalityandLevene’stestfortheequalityofvariance,respec- D
Serotonergic Slc6a4 o
tively. Therefore, Kruskal-Wallis nonparametric analysis was w
Glutamatergic Gria1,Gria2,Grik2,Grin1,Grin2a, n
performedtodetermineamaineffectofEDClineage(vinclozolin lo
Grin2b,Grin2c,Grin2d a
vscontrol)andofstress(vsnonstress)foreachendpoint.Sub- d
GABAergic Gad1,Gad2 e
Neruercoeppetoprtsidesand AvOpx,tAr,vTpa1ca2,Kiss1,Kiss1r,Lepr,Oxt, speeqrufoernmtecdovmiapaarpisaoirn-swiwseitMhinannse-xWhaintdneybeUtwteesetnsbgertowuepesniwnderee- d from
Growthfactors BdNnef,gCr1tg,rP,tgIgdfs1,,SIg1f010ra,4Ig,fTbgpf2a,,ITggfbfbp15, pmeinndi-eHnotcghrobuerpgscaonrdreacptpiornopfroiratmeluyltciopnletrcoollmedpaforirsounsisnwghaeBreenajpa-- https
Transcriptionfactors Nfkb1,Nrf1,Per2 propriate.Functionallandscapeanalysisofcorrelatedtraitswas ://a
c
ThegenesmeasuredbyqPCRonacustom-designedTLDAarelisted performedasperRef.62toprovideavisualrepresentationofthe ade
byfunctionalgroup.FullgenenamescanbefoundinSupplemental relationshipsamongmultiplevariablesbetweengroupsofindi- m
Table2. viduals, allowing for a more concise presentation of data. As ic.o
such,thiscapturesthenatureofrelationshipspictorially.How- up
ever, this analysis is in no way intended to replace individual .co
sectedbrainregionsofindividualrats(54).Thechoiceofgenes m
fortheqPCRpanelwasbasedonpreviousworkfromthislab- statisticalanalyses.Itisimportanttonotethatinthebehavioral /e
n
tests,eachnodewithinthelandscaperepresentsacompositeof d
oratoryandothersandarelistedinTable1,withfullgenenames o
severalmeasures,whereasthebargraphsrepresentanindividual /a
in Supplemental Table 2 (55, 56). Specific gene selection was measure within a task. This is not the case for the functional rtic
based on a priori hypotheses about molecules involved in the le
neurobiological responses to EDCs, predicted sex differences, landscapesofbrainmetabolism. -a
b
s
and relationships between selected genes and behaviors mea- tra
suredinthesamerats.Selectedgenesfellinseveralfunctional c
t/1
groups including epigenetic modification, stress signaling, ste- Results 5
5
roidhormones,andgrowthfactors.TLDAqPCRresultswere /1
0
quantified using a ViiA7 PCR machine (Life Technologies) as FivesetsofcomparisonswereperformedasshowninFig- /3
8
published(54).Asasecondarylevelofanalysis,transcriptome 5
ure 1B: control nonstress (C-NS) vs control stress (C-S; 3
analysiswasconductedonpooledsamplesin4ofthebrainre- /2
gions(BLA,CeAmy,BnST,andCA3)usingtheAffymetricRat comparison 1); C-NS vs vinclozolin nonstress (V-NS; 423
Geneversion1.0STarrays,withallmethodsandanalysesiden- comparison3);andC-NSvsvinclozolinstress(V-S;com- 17
2
ticaltothosepublishedbyourlaboratories(45)(Supplemental parison5).Comparisons1,3,and5areconsideredfirst- b
y
Methods). order effects. Comparison 1 gives an index of stress vs gu
e
Hormone assays ncoonntsrtorelsvssinvcinocnltorzoollrinatsl;inceoamgepairnisonnon3stgrievsessedanraintsd;exanodf st on 0
A terminal trunk blood sample was collected, centrifuged, 4
comparison 5 represents the synchronicity of stress and A
and frozen for hormone analyses, each performed following p
manufacturers’protocols.The3sexsteroidhormones(estradiol, vinclozolincomparedwithC-NSrats.Forgrapheddata, ril 2
progesterone,andtestosterone)wereselectedbasedonprevious statisticaldetailsarepresentedinthefiguresandtables;for 01
9
workfortheirdisruptionbyvinclozolin(45,57,58).Cortico- datanotshown,Pvaluesareindicatedinthetext.
sterone was also measured because a primary dependent end-
pointofthisstudywasCRS,whichaffectsadrenalglucocorticoid
Hormones and adrenal weight
levelsandissexuallydimorphic(59–61).Foreachassay,stan-
Stress and ancestral vinclozolin had effects on serum
dardswereprocessedinduplicateandsamplesintriplicate.Ina
few cases, outliers within a triplicate were removed using testosteroneandprogesteroneinasex-dependentmanner
Grubb’stest.Serumcorticosteronewasmeasuredin10(cid:2)Lserum (Figure2).Estradiolwasunaffected(Figure2B).Fortes-
fromindividualratsinasingleRIA(MPBiomedicals).Theintra- tosterone,inthenonstressedfemales,vinclozolin-lineage
assaycoefficientofvariation(CV)was3%,andassaysensitivity
rats had lower testosterone than control-lineage females
was25ng/mL.Serumestradiolwasmeasuredin200(cid:2)Lserum
(comparison3,Figure2A).Serumprogesteroneshoweda
in a single RIA (Beckman-Coulter). The intra-assay CV was
6.7%, and assay sensitivity was 5 pg/mL. Progesterone serum maineffectofstressinfemalesonly,withhigherproges-
concentrationsweredeterminedviaanenzymeimmunoassayin terone in stressed than nonstressed females of both lin-
doi:10.1210/en.2014-1253 endo.endojournals.org 3857
Females Males Females Males
A B
Testosterone Estradiol
500 40
Non-Stressed
3000 Stressed
mL)400 mL) 30
g/ g/
n (p300 2000 n (p
ntratio200 pC =o m0.p0 437 ntratio 20
ce 1000 ce
n n 10
Co100 Co
0 0 0 D
Control Vinclozolin Control Vinclozolin Control Vinclozolin Control Vinclozolin o
w
C D n
Progesterone Corticosterone lo
a
100k 1000 d
e
d
mL)75k 2k mL)750 from
g/ g/ h
ntration (p50k 1k ntration (n500 ttps://ac
e e a
Conc25k Conc250 dem
ic
.o
0 0 0 u
Control Vinclozolin Control Vinclozolin Control Vinclozolin Control Vinclozolin p
.c
E Adrenal Weight om
/e
.08 n
Comp 5 d
p = 0.045 o
Comp 5 /a
.06 p = 0.038 rtic
le
g) -a
ht ( .04 bs
eig tra
W c
t/1
.02 5
5
/1
0
0 Control Vinclozolin Control Vinclozolin /385
3
Figure2. Serumtestosterone(A),estradiol(B),progesterone(C),corticosterone(D),andadrenalweights(E)areshown.Significantpair-wise /2
comparisonsareshownwithinthefigure.Asmaineffects,progesterone(C)wasincreasedinfemalesduetoCRS(P(cid:3).048),andadrenalweight 42
3
wasdecreasedinbothfemalesandmalesduetovinclozolinlineage(P(cid:3).008andP(cid:3).025respectively). 1
7
2
b
y
g
eages(Figure2C).Finally,adrenalweightwasmeasured aspect. Therefore, the measure that best discriminated the u
e
s
becauseitissexuallydimorphicandhasbeenshowntobe experimentalgroupswaschosenforpurposesofgraphicpre- t o
n
affected by vinclozolin treatment (57, 63) and because sentationandusedinthefunctionallandscapes. 0
4
CRSiswell-establishedasaffectingadrenalweightaswe Open-field, light-dark transition, and elevated plus Ap
previously reported in an earlier study on males (45). In mazebehaviorsweredifferentiallyaffectedinmalesand ril 2
0
bothsexes,thereweremainlineageeffects,withadrenal 1
femalesbystressandlineage.Representativedataofone 9
weights lower in vinclozolin- than control-lineage rats
individualcomponentforeachbehaviorareshowninFig-
(females P (cid:3) .008 and males P (cid:3) .025, Figure 2E).
ure3.Forcornertimeintheopen-fieldtest,thesynchron-
Furthermore, in both sexes, V-S rats had significantly
icityofstressandlineage(comparison5)wasaffectedin
loweradrenalweightscomparedwithC-NS(compari-
females, with V-S spending more time in corners com-
son 5) counterparts.
paredwithC-NS(Figure3A).Cornertimeinmaleswas
Behaviors affectedonlyinthecontrollineage(comparison1),with
TheStoeltingAny-Mazeapparatusesallowfortheobser- C-SshowinglowercornertimethanC-NS.
vationandquantificationofmultiplemeasureswithineach Forthelight-darkbox,thenumberofentriesintothe
specificbehavioraltest.Althoughnumerousbehaviorswere lightchamberwasgreaterinmaleC-SthanC-NS(com-
quantified,thelargenumberprecludespresentationofevery parison 1, Figure 3B). Vinclozolin-lineage males and fe-
3858 Gilletteetal SexDifferences,AncestralEDC,andStress Endocrinology,October2014,155(10):3853–3866
Females Males sociability,bothmalesandfemalespreferredtoassociate
A Open Field with an animal as opposed to an empty cage (P (cid:5) .001,
Non-Stressed SupplementalFigure2A).Inatestforsocialnovelty,fe-
300 Stressed
Comp 5, p = 0.04 malespreferredtospendmoretimewithanovelanimalas
s) Comp 1 opposed to a familiar one (P (cid:3) .005). Males showed no
e ( p = 0.02
Tim 200 preferenceforsocialnovelty(SupplementalFigure2B).
er Functional landscape analysis was performed to pro-
n
Cor videanintegrativeperspectiveofbehaviorsrelatedtoanx-
n 100
a iety, activity, and sociability for the first-order compari-
e
M
sons1,3,and5(Figures4,A,C,andE,respectively).For
D
0 comparison1,therewerefewdifferencesbetweenC-Sand o
Control Vinclozolin Control Vinclozolin w
C-NS females, whereas males differed for composite be- n
B Light-Dark haviorsforlight-darkandopen-fieldtests.Moreeffectsof load
e
ox Entries 11802 Comp 1, p < 0.001 spatfarfreeiscsstoewnde3irn.eFtsheineeanslyliynn,cffehemrmoanaleliecssitcyaonmmdpomadreaelldeoswfwcitoehmrmepdaalirefifsseofronern5tc.ioamlly- d from https
ean Light-B 46 BraCinOmhiesttoacbhoelmismistrywasusedasanindexofbrainme- ://academ
M 2 tabolism(45,52),anddataaregraphedasfunctionalland- ic.o
0 Control Vinclozolin Control Vinclozolin scapesinFigure4,B,D,andF(comparisons1,3,and5, up.c
respectively).Forcomparison1,whichfocusesoneffects o
m
C Elevated Plus Maze ofstressincontrolrats(C-SvsC-NS;Figure4B),C-Smales /e
n
d
Time (s) 300 Comp 5, p = 0.02 smshhaoolwweseeddiniinnctchrreeeaasBseenddSmTaect(taPivbio(cid:3)tylicc.a0oc2mt)i,vpiawtryehcdeormweapistahrfeeCdm-wNalieStshiCn(C-Nt-hSSe) o/article-a
m 200 mPOA(P(cid:3).03)andBnST(P(cid:3).04).Forcomparison3, bs
ean Closed Ar 100 eFpfaifgreuecdrteswo4ifDthv)i,VnCc-Nl-oNSzSomlmianlaeilnsesinnsohtnhosewtCreeAdss1dedeacnrrdaetaCsseA(Cd3-aN(cPtSi(cid:3)vvist.y0V4c-oaNmnSd-; tract/155/10
M P(cid:3).02),whereasV-NSfemalesshowedincreasedactivity /38
0 Control Vinclozolin Control Vinclozolin compared with C-NS females in the MeAmy (P (cid:3) .04), 53/2
Figure3. Behavioralmeasuresofinterest.A,Open-fieldtest,with CoAmy(P(cid:3).02),andVMN(P(cid:3).04). 42
3
timespentinthecornerofthearenaindicatingdecreasedexploration 1
7
andincreasedanxiety.B,Light-darkbox,withnumberofentriesinto Gene expression and transcriptome 2 b
thelightboxindicatingincreasedexplorationanddecreasedanxiety. y g
C,Elevatedplusmaze,withtimespentintheclosedarmindicating BrainmicropuncheswereanalyzedbyTLDA,andthe u
e
decreasedexplorationandincreasedanxiety. relative expression of 48 selected genes was analyzed st o
n
(listed in Supplemental Table 2) in each of 10 selected 0
4
malesshowedadelayedinitialentryintothedarkcham- brainregions(SupplementalTable1).Resultsofaffected A
p
ber,comparedwithcontrolcounterparts(datanotshown; genesareshownforthefemaleCA3,CA1,andBnST(Fig- ril 2
P (cid:5) .01). Also, V-S males exhibited a longer latency to ure5),chosenbecausetheseregionshadthemostrobust 01
9
enterthedarkchambercomparedwithC-NSmales(com- effectsforcomparisons1,3,and5.Asummaryofiden-
parison5;datanotshown,P(cid:3).03). tified genes and directionality of effect for these 3 com-
Elevatedplusmazebehaviorwasaffectedonlyinmales parisonsispresentedinFigure6,showingsex-andregion-
(Figure 3C), with V-S spending more time in the closed specific differences in affected genes. Additional gene
armthanC-NS(comparison5).Furthermore,V-Smales expression data are shown in Supplemental Figure 3 for
spentmoretimeintheclosedarmoftheelevatedplusmaze the male BLA and for the male and female lateral
(P(cid:3).02),displayedaslowermeanspeed(P(cid:3).02),and hypothalamus.
coveredashorterdistance(P(cid:3).02)thantheirC-NScoun- In the female CA3 (Figure 5A), expression of a large
terparts(comparison5,datanotshown). numberofgeneswassignificantlydifferentbetweenV-S
Behaviorsinthesociabilityandsocialnoveltytestwere andC-NS(comparison5):Crhr1,Drd2,Tgfa,Esr1,and
also quantified (Supplemental Figure 2). In the test for Esr2(geneabbreviationsarelistedinSupplementalTable
doi:10.1210/en.2014-1253 endo.endojournals.org 3859
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
c
a
d
e
m
ic
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u
p
.c
o
m
/e
n
d
o
/a
rtic
le
-a
b
s
tra
c
t/1
5
5
/1
0
/3
8
5
3
/2
4
2
3
1
7
2
Figure4. Functionallandscapesofbehaviorsandbrainmetabolism(COactivity)areshownforcomparison1(AandB),comparison3(CandD), b
andcomparison5(EandF),respectively,inmaleandfemalerats.Arelativeincreaseforonegroupovertheotherisshownbythedirectionality y g
andheightofapeakorvalley.Forbehaviors(A,C,andE),theclockwisenodesare:light-darkbox(LD),elevatedplusmaze(EP),open-field(OF), u
e
sociability(SOC),andsocialnovelty(SOCNOV).Forbrainmetabolism(B,D,andF),theclockwisenodesshownareBLA,CeAmy,MeAmy,CoAmy, st o
CA1andCA3ofthehippocampus,mPOA,VMN,andBnST. n
0
4
A
p
2).Inallcases,expressionwashigherinV-SthanC-NS.In response to ancestral vinclozolin (64) and CRS in males ril 2
addition, there was significantly higher expression of 3 (45). In the current study, we performed transcriptome 01
9
genes in V-NS than C-NS (comparison 3): Esr1, Gnrhr, analysisinaliquotsofRNAfromthesamesamplesused
andPtgds. forTLDAin4selectedbrainnuclei:BLA,BnST,CeAmy,
In the female CA1 (Figure 5B), expression of Ar was andCA3(SupplementalTable1)tocomparewithTLDA
lower in C-S than C-NS (comparison 1), and for Esr1, results(Figure7).Acutoffofa1.2-foldchange(transcrip-
expressionwashigherinV-NSthanC-NS(comparison3). tome)anda1.5-foldchange(TLDA)wasappliedforiden-
Finally,3geneswereaffectedinthefemaleBnST(Figure tified genes with the goal of supporting the results ob-
5C).Mc4randPomcwerelowerinV-SthanC-NSfemales tained from the more targeted TLDA analysis and to
(comparison 5) and Th was higher in V-NS than C-NS identify candidate networks for future analysis. Because
females(comparison3). sampleswerepooledfortranscriptomeanalysis,butwere
Previouslypublishedresearchonasimilarexperimen- individuallyassayedbyTLDA,onlygeneralinformation
talmodelhasreportedin-depthtranscriptomeanalysisin aboutdirectionalityofchangewithreferencetoC-NScan
3860 Gilletteetal SexDifferences,AncestralEDC,andStress Endocrinology,October2014,155(10):3853–3866
A Female CA3
Crhr1 Drd2 Tgfa Esr1
n 5 Comp 5, p = 0.01 5 Comp 5, p = 0.01 4 Comp 5, p = 0.03 5 Comp 5, p = 0.02
o
pressi 4 4 3 4 Cp o=m 0p.0 32
Ex 3 3 3
e
ang 2 2 2 2
h
C
old 1 1 1 1
F
0 0 0 0
Control Vinclozolin Control Vinclozolin Control Vinclozolin Control Vinclozolin
D
o
Esr2 Gnrhr Ptgds w
n
4 3 10.0 lo
Fold Change Expression 123 Comp 5, p = 0.02 12 Cp o=m 0p.0 33 257...505 Cp o=m 0p.0 32 aded from https://a
c
0 Control Vinclozolin 0 Control Vinclozolin 0 Control Vinclozolin ade
m
ic
.o
u
p
B Female CA1Ar Esr1 Non−Stress .com
2.0 Comp 3 Stress /en
Expression1.5 Comp 1, p = 0.02 23 p = 0.03 do/article
nge 1.0 -ab
a s
Fold Ch0.5 1 tract/1
0 Control Vinclozolin 0 Control Vinclozolin 55/1
0
/3
8
5
3
C Female BnST /24
Mc4r Pomc Th 2
3
1
n1.25 Comp 5, p = 0.01 2.0 Comp 5, p = 0.01 3 Cp o=m 0p.0 34 72 b
nge Expressio001...570050 11..05 2 y guest on
ha 1 0
C 4
Fold 0.25 0.5 Ap
0 Control Vinclozolin 0 Control Vinclozolin 0 Control Vinclozolin ril 20
1
Figure5. GeneexpressionresultsofaffectedgenesidentifiedbyTLDAqPCRassaysareshownforthefemaleCA3(A),CA1(B),andBnST(C).A, 9
IntheCA3,5geneshadhigherexpressioninV-SthanC-NSfemales(comparison5)and3geneshadhigherexpressioninV-NSthanC-NSfemales
(comparison3).B,IntheCA1,ArmRNAlevelswerelowerinC-SthanC-NSfemales(comparison1)andEsr1mRNAwashigherinV-NSthanC-S
females(comparison3).IntheBnST,Mc4randPomcmRNAlevelswerelowerinV-SthanC-NSfemales(comparison5)andforTh,mRNAlevels
werehigherinV-NSthanC-NSfemales(comparison3).
be drawn. Patterns of expression were very different be- Discussion
tweenthesexes(Figure7C).TheTLDAanalysisidentified
Vinclozolin is a well-established endocrine disruptor
thefemaleCA3ashavinganoverrepresentednumberof
affectedgenes,especiallyforcomparisons3and5,anef- when administered during critical developmental life
fectthatwaslargelyconfirmedbytranscriptomeanalysis, stages. Due to its antiandrogenic properties, male off-
albeitwiththecaveatthatsampleswerepooledfortran- springofdamstreatedwithvinclozolinduringpregnancy
scriptomeanalysis. showademasculinizedmorphological(reducedanogeni-
doi:10.1210/en.2014-1253 endo.endojournals.org 3861
Males
CA3 CA1 CeA BLA BnST LH
Stress Vincl V&S Stress Vincl V&S Stress Vincl V&S Stress Vincl V&S Stress Vincl V&S Stress Vincl V&S
Comparison 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5
Gene
Avp 0.03 0.03
Bdnf 0.02
Oxt 0.02
Negr1 0.02
Pgr 0.02
Females
CA3 CA1 CeA BLA BnST LH
Stress Vincl V&S Stress Vincl V&S Stress Vincl V&S Stress Vincl V&S Stress Vincl V&S Stress Vincl V&S
Comparison 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5 1 3 5
Gene
D
Ar 0.02 o
Crhr1 0.01 w
n
Drd2 0.01 lo
Esr1 0.02 0.02 0.03 a
d
Esr2 0.02 e
d
GLnerhprr 0.03 0.02 fro
m
PPMotgcmd4csr 0.02 00..0011 0.03 0.03 https
TgTfha 0.03 0.04 ://ac
a
Figure6. GeneExpression.SummaryofmRNAexpressionoftargetedgenes,selectedfortheirrolesinsocial,affiliative,andanxiety-related de
m
behavioraltestsandpredictedtoberegulatedbyendocrinedisruptorsand/orstress,forcomparisons1,3,and5.Sixhypothalamicand ic
hippocampalnucleiwithsignificantgeneexpressioneffectsareshown.DifferencesareshownrelativetotheC-NSgroupandareatleast2-foldin .o
u
magnitude.Notethateachregionhasuniquegeneexpressionchangesbysex,treatment,andstress.Uparrowsindicateasignificantincreaseand p
.c
downarrowsrepresentasignificantdecreaserelativetotheC-NSgroup.LH,lateralhypothalamicnuclei;Vincl,vinclozolin. o
m
/e
n
d
tal distance, nipple retention, ectopic testis, vaginal littleisknownaboutsex-specifictransgenerationalEDC o
/a
pouches,andagenesisoftheprostate)andbehavioral(fail- effects.This,togetherwithobservationsthatCRSelicits rtic
ure to attain intromission or ejaculate with a receptive sex-specificresponsesinphysiologyandbehavior,andex- le-a
b
female)phenotype(42).Femaleratsdisplayreducedano- pression of genes and proteins in related brain regions s
tra
genitaldistance(41);laterinlife,theyexhibituterinehem- (78–80),ledtoourcurrentworkcombiningtransgenera- c
t/1
orrhaging and anemia during pregnancy and increased tional effects of vinclozolin with CRS in males and 5
5
tumorformation(65). females. /10
/3
Inadditiontoitsdirecteffects,vinclozolinwasthefirst 8
5
environmentalEDCshowntohavetransgenerationalef- Behavioral effects of proximate adolescent stress 3/2
4
fects (10, 16, 64, 66). Although another laboratory re- and ancestral exposure to vinclozolin 2
3
1
portedtransgenerationalactionsofvinclozolinonexpres- In our study, males exposed to CRS in adolescence 7
2
sionofimprintedgenes(12),otherlaboratorieshavenot showeddecreasedanxietybehaviorsinadulthood.Results by
g
replicatedthesefindings(67,68),underscoringtheneed of the open-field and light-dark box tests demonstrated u
e
s
for more research and careful attention to experimental greaterexplorationanddecreasedaversiontopotentially t o
n
differences.However,inter-andtransgenerationaleffects dangerous and stressful situations. No such differences 0
4
of other endocrine disruptors, such as BPA (13, 69), di- causedbyCRSwerefoundinfemales.Theseresultssug- A
p
ethylstilbestrol(inmouse[70]andhuman[71]),andpoly- gest that males and females differ in the compensatory ril 2
chlorinated biphenyls (PCBs) (72) have been reported, strategiesacquiredwithearlylifestress. 01
9
addingfurtherevidencethatEDCsaffectmultiplegener- Bycontrast,ancestralexposuretovinclozolinwasas-
ations,uptoandbeyondtheF3generation. sociated with few behavioral effects in control (non-
Previously,weshowedthatancestralexposuretovin- stressed) males or females, a finding that was surprising
clozolincausedbehavioraldifferencesintheF3malede- becausepreviousworkshowedthatancestralvinclozolin
scendants when challenged with CRS (45). The present increased anxiety behaviors in young females in an ele-
studywaspredicatedonourunderstandingoffundamen- vatedplusmazeanddecreasedanxietybehaviorsofyoung
talsexdifferencesinthedevelopingnervoussystemgov- males in a light-dark box (64). Differences between the
erned by steroid exposure and functional outcomes on laboratoryenvironments(currentworkconductedatUni-
brain structure, neurochemistry, and numerous behav- versity of Texas Austin, past work at Washington State
iors, both reproductive and otherwise (73). Although University)andageattestingmaycontributetodivergent
many effects of prenatal EDCs are sex-specific (74–77), experimentaloutcomes.Additionally,thelimitednumber
3862 Gilletteetal SexDifferences,AncestralEDC,andStress Endocrinology,October2014,155(10):3853–3866
A Females B Males
Females BLA BnST CA3 CeA Males BLA BnST CA3 CeA
StressVincl V&SStressVincl V&SStressVincl V&SStressVincl V&S StressVincl V&SStressVincl V&SStressVincl V&SStressVincl V&S
Comparison 1 3 5 1 3 5 1 3 5 1 3 5 Comparison 1 3 5 1 3 5 1 3 5 1 3 5
Gene Gene
18s 18s
Ar Ar
Avp Avp
Avpr1a Avpr1a
Bdnf Bdnf
Comt Comt
Crh Crh
Crhr1 Crhr1
Ctgf Ctgf
Dnmt1 Dnmt1
Dnmt3a Dnmt3a
Dnmt3b Dnmt3b
Drd2 Drd2
Esr1 Esr1
Esr2 Esr2 D
GGmaepbd2h GGmaepbd2h own
Gria1 Gria1 lo
Gria2 Gria2 a
HGdarikc21 HGdraikc21 ded
Hsd1I1gbf12 Hsd1I1gbf12 fro
Igf1r Igf1r m
Igfbp2 Igfbp2 h
IgMMLfbbecpd3p52rr IgMMLfbbecpd3p52rr ttps://a
NMegc4r1r NMegc4r1r cad
Nfkb1 Nfkb1 e
Nr3c1 Nr3c1 m
Nrf1 Nrf1 ic
Oxt Oxt .o
PPOoPemxrgt2crr PPOoPemxrgt2crr up.com
S1P0t0gad4s S1P0t0gad4s /en
Tgfa Tgfa d
TgfTbh1 TgfTbh1 o/artic
C Concordance Summary le-a
b
s
BLA BnST CA3 CeA tra
COMPARISON 1 - CRS3 - Vincl 5 - V&S 1 - CRS3 - Vincl 5 - V&S 1 - CRS3 - Vincl 5 - V&S 1 - CRS3 - Vincl 5 - V&S SUM c
t/1
Females 5
Concordant - UP 0 2 2 0 1 0 3 6 13 0 0 0 27 5
Concordant - DOWN 1 0 0 0 0 1 2 0 0 0 1 2 7 /1
Discordant 0 0 2 0 0 0 1 0 1 1 0 0 5 0/3
Males 8
5
Concordant - UP 2 3 3 3 2 0 0 0 1 0 1 2 17 3
Concordant - DOWN 0 2 1 0 1 0 3 9 2 3 2 0 23 /2
Discordant 0 0 1 0 1 1 1 1 4 4 2 2 17 4
2
Figure7. TLDAandtranscriptomecomparisons.CorrespondencebetweenexpressionpatternsingenesidentifiedbyPCR-basedTLDA(black)and 31
genome-widetranscriptome(gray)analysisisshownforaffectedgenesinthe4brainregionsusedforbothTLDAandtranscriptome(BLA,BnST, 72
CeAmy,andCA3ofthehippocampus).Dataareshownforfemales(A)andmales(B).ColumnsshowdifferenceswithC-NSindividualsasthe by
referencegroup.Uparrowsindicateupregulation,whereasdownarrowsindicatedownregulation.Cutoffcriteriawere(cid:6)1.5-foldchange(TLDA) g
u
and(cid:6)1.2-foldchange(transcriptome).C,Summaryofconcordance,definedasmeasuresthatwereinthesamedirection(upregulatedor es
downregulated).Discordantchangeswerethoseforwhichthe2measuresdidnotagree. t o
n
0
4
of animals used in the current study, derived from rela- ioraloutcomes:first,measuresthatwereinfluencedbyCRS A
p
tivelyfewlitters,likelyplaysaroleindifferences. thatwereabolishedbyvinclozolin,andsecond,thosemea- ril 2
Other endocrine disruptors have been implicated in suresthatneitherCRSnorancestralexposuretovinclozolin 01
9
modifying anxiety responses. BPA consistently increases alonepredicted.Inthecaseoftheopen-fieldtest,synchron-
anxietybehaviorinratsasmeasuredbytheelevatedplus icitywaslimitedtofemales,withincreasedanxietybehavior
andopen-field,regardlessofsex,inF1animals(81,82). intheV-SgroupcomparedwithC-NSgroup.Synchronicity
Theseresultshavebeenreplicatedinmiceandextendedto inmaleswasseenintheelevatedplusmaze,againwithin-
social behaviors and the light-dark box (83). Although creasedlevelsintheV-Sgroup.Neitheroftheseoutcomes
BPAhasadifferentmechanismofaction,targetingdiffer- couldbepredictedbyeitherancestralEDCexposureorCRS.
entsteroidhormonereceptors,theseresultsprovidefur- Socialbehaviorwasnotstronglyaffectedbyvinclozolin
ther support for transgenerational effects of EDCs on a or CRS, nor was there evidence of synchronicity. In the
suiteofbehaviors. sociabilitytestinwhichratsdistinguishedbetweenacon-
Thecombinedeffectofancestralvinclozolinandproxi- specific and an empty cage, both males and females
mateCRS,ortheirsynchronicity,fellinto2generalbehav- showedincreasedtimeinthesocialchambercompared