Constitutive Notch Signaling Causes Abnormal Development of the Oviducts, Abnormal

12 Pages·2016·2.92 MB·English

BIOLOGY OF REPRODUCTION (2016)94(3):67,1–12
Published online beforeprint 3February 2016.
DOI 10.1095/biolreprod.115.134569
Constitutive Notch Signaling Causes Abnormal Development of the Oviducts,
Abnormal Angiogenesis, and Cyst Formation in Mouse Female Reproductive Tract1
Lydia Ferguson,3 Elena M. Kaftanovskaya,3 Carmen Manresa,3 Agustin M. Barbara,3 Robert J. Poppiti,4,5
Yingchun Tan,3,6 and Alexander I. Agoulnik2,3,7
3Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International
University, Miami, Florida
4Department of Pathology, Mount Sinai Medical Center, Miami Beach, Florida Do
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5Department of Pathology, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida n
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6Department of Gynecology, Shandong Qianfoshan Hospital, Shandong University, Jinan, China ad
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7Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas d
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ABSTRACT transmembrane receptor essential for local juxtacrine cell-to- h
cellsignaling,andisfoundinamultitudeofcelllineages.The ttp
ofTmheanNyottcihssusiegsnaalinndgpoartghawnasyiinsctrhiteicaelmfobrrytoh.edTioffesrteundtyiattiohne NceOlluTlCarHdifsfiegrneanltiinagtiopnaathnwdapyrolhifaesratbioeenn,apshoopwtonsist,oanddecteerllmfiantee s://ac
consequences of Notch1 gain-of-function signaling on female in both adult and embryonic life. In particular, the heart, ad
e
reproductivetractdevelopment,weusedacre-loxPstrategyand prostate, kidney, mammary gland, and stem cell systems mD
Amhr2-cre transgene to generate mice with conditionally ico
activated Notch1 (RosaNotch1). The Amhr2-cre transgene is depend on NOTCH signaling, and defects in this pathway can .ouw
leadtotumorgrowth[1].ThecrucialroleofNOTCHsignaling pn
expressedinthemesenchymeofdevelopingfemalereproductive in the regulation of angiogenesis and vascularization was .colo
tract and in granulosa cells in the ovary. Double transgenic ma
Amhr2-cre, RosaNotch1 females were infertile, whereas control revealedinmousemodelsoftargeteddisruptionorconstitutive /bd
opRrroogsaganrNesostsciihnn1gmmwiucitetahnhtaasgdes.nhooTrwhmeeadlmfhueetramtinloittryro.hvaAidglluincfgtesmodafildeblrnoeooptdroddveuevcsetslieovlpes sRibgpnTjahlgeineNgneOosTf[C2N–Ho7t]cr.ehc1e,pNtoortcbhin1d/Nsoatcmhe4m, Jbaragn1e,-Dbollu4n,dHliegya1n/2d,oannda iolreproded fro
cmouillitnipgl,eanbdlowckeargeeisnsitneadthleoolpuemdeanroaulnodngthteheovaorvyi.duTchterelenwgetrhe, nuenidgehrbgooreisngtwoceslel.quAenftteiarltchleisavcaognetsabcty,AthDeANMOpTroCtHeasereacnepdtocr- /articlem w
creating a barrier for sperm or oocyte passage. Mutant females secretase, thus releasing the active intracellular domain -aw
bw
demonstratedinflameduteriwithincreasedvascularizationand (NICD). The NICD then travels to the nucleus, where it s
adneveinloflpuexdoofvairniaflna,momviadtuocrytalc,ealnlsd. uAtderdiintieoncyaslltys,. Tohldeesrignfeimficaalnest MintAerMacAtsLw1it(hMtahsetetrrmaninscdr-ilpiktieon1f)a,ctroerspRoBnsPiJbjleanfodrcoacatcitvivataitnogr tract/94.biolr
change in gene expression was detected in the mutant oviduct downstreamtargets[8].Inmammals,thetwomaintargetgenes /3e
expression of Wnt4, essential for female reproductive tract most commonly activated are Hes and Hey, which are key /67pr
dperevveilooupsmlyenitn. SmimicielarwoitvhiduaccttaivlaptehdenoSmtyopesanhdavienbbeeenta-dceatteecntiend, tranInscaridpdtiiotinonfatoctoitrss.ownregulatorynetwork,NOTCHinteracts , 1-12od.o
Wnt4, Wnt7a, and Dicer conditional knockouts, indicating a withthetransforminggrowthfactorbeta(TGFb)andtheWnt/ /24rg
common regulatory pathway disrupted by these genetic abnor- b-catenin pathways [9]. The intersection of NOTCH and b- 34.
malities. 4
catenin pathways has been well documented, particularly in 5
8
angiogenesis, female reproductive tract, infertility, Notch1, stemcelldifferentiation[10],embryonicvasculardevelopment b
y
oviduct [11], and cardiac progenitor cell differentiation [12]. The two g
u
signaling pathways undergo crosstalk at many different stages es
INTRODUCTION of signaling, and direct interaction has been demonstrated by t o
n
the binding of unphosphorylated b-catenin directly to the 0
TheNOTCHsignalingpathwayishighlyconservedinmost 1
NICD [13]. Collision of the NOTCH signaling pathway and A
multicellularorganismsand,inmammals,fourNOTCHgenes, p
NOTCH1–4, have been identified. NOTCH is a type-1 tphaattteronfsiTnGaFnbumisbeerxopfedcteevde,logpimveenntathlepiarthmwiarryosreadndecxepllretysspieosn. ril 2
0
Coregulation of NOTCH and TGFb target genes has been 19
1Supported by the Herbert Wertheim College of Medicine, Florida frequently described: the NOTCH target gene Hes1 was
InternationalUniversity.
2Correspondence: Alexander I. Agoulnik, Department of Human and upregulated by TGFb in keratinocytes [14]; such interactions
MolecularGenetics,HerbertWertheimCollegeofMedicine,Miami,FL havealsobeendescribedinsmoothmusclecells[15,16]andT
33199.E-mail:[email protected] cells [17, 18].
ThedifferentiationoftheMu¨llerianductisinitiatedinmice
atEmbryonicDay(E)11.5byinvaginationoftheepitheliumof
Received:14August2015.
Firstdecision:31August2015. the anterior mesonephros [19]. In females, an absence of
Accepted:25January2016. testosterone leads to the regression and disintegration of the
(cid:2)2016bytheSocietyfortheStudyofReproduction,Inc.Thisarticleis Wolffian duct beginning at E13.5 [20]. In the absence of
available under a Creative Commons License 4.0 (Attribution-Non- Mu¨llerianinhibitingsubstance,theMu¨llerianductcontinuesto
Commercial),asdescribedat http://creativecommons.org/licenses/by-nc/
differentiate, eventually forming the oviducts, uterus, cervix,
4.0
and upper region of the vagina by birth; however, further
eISSN:1529-7268http://www.biolreprod.org
ISSN:0006-3363 maturation of these tissues continues for several weeks. The
1 Article 67
FERGUSON ETAL.
TABLE1. PCRprimersusedforgenotypingandqRT-PCR.
Gene Forward Reverse
Ccnd2 CTGTGCATTTACACCGACAAC CACTACCAGTTCCCACTCCAG
Cdkn1a(P21) CAGATCCACAGCGATATCCA GGCACACTTTGCTCCTGTG
Cdk6 GCCCTTACCTCGGTGGTC ACAGGGGTGGCATAGCTG
Fn1 CGGAGAGAGTGCCCCTACTA CGATATTGGTGAATCGCAGA
Gli1 CTGACTGTGCCCGAGAGTG CGCTGCTGCAAGAGGACT
Hes1 ACACCGGACAAACCAAAGAC CGCCTCTTCTCCATGATAGG
Myc CCTAGTGCTGCATGAGGAGA TCTTCCTCATCTTCTTGCTCTTC
Ptch1 GGAAGGGGCAAAGCTACAGT TCCACCGTAAAGGAGGCTTA
Smo GCAAGCTCGTGCTCTGGT GGGCATGTAGACAGCACACA
Wnt4 CTGGACTCCCTCCCTGTCTT ATGCCCTTGTCACTGCAAA
Wnt5a ACGCTTCGCTTGAATTCCT CCCGGGCTTAATATTCCAA
Wnt7a CGCTGGGAGAGCGTACTG CGATAATCGCATAGGTGAAGG D
o
b-Actin CCCAACTTGATGTATGAAGG TTGTGTAAGGTAAGGTGTGC w
n
cre ATCAACGTTTTCTTTTCGG ATTTGCCTGCATTACCGGTC lo
DNotch1 AAAGTCGCTCTGAGTTGTTAT CGGAACTTCTTGGTCTCCAG ad
Notch1(NICD) GGACATGCAGAACAACAAGG CAGTCTCATAGCTGCCCTCA ed
Gapdh AACGACCCCTTCATTGAC TCCACGACATACTCAGCAC fro
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ttp
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oviductsdevelopfromthecranialregionoftheMu¨llerianduct, Carolina,ChapelHill,supportedbyNIH.ThemicehaveIRES-Cre-pAFRT- ://a
with coiling completed by E15 [21]; however, at birth, no flankedPgk-neo-bpAcassetteintroducedintoexon5oftheAmhr2locus,and c
a
morphological differences in the oviductal epithelial cells are t[h3u2s].trAamnshgre2n-iccreCrmeaelexpmreiscseionwefraeithcfruolslysedrepwroitdhucReossAaNmohtcrh21 gfeemnealeexpmreicsesioton demD
apparent. The first ciliary cells are visible from 5 days after generate Amhr2-cre, RosaNotch1 females and RosaNotch1 female littermates, ico
birth, and secretory cells are mature by Day 23 [22]. The whichwereusedascontrols. .ouw
doiffftehreenteixatpiroenssoifontheoMf u¨mllaenryiangdeuncets.reTquhieresWanptrefacmiseilyintgerepnleasy, PCR Genotyping p.comnloa
specifically, Wnt4 and Wnt7a, expressed in the mesenchyme /bd
asenxd-seppeitchifeilciudmev[e2l3o]pmareenitmopfotrhtaenMt fuo¨lrletrhieanindituiaclt.foLrimm1atieoxnpraensd- bplroootGodceoanlno.dmMitciiscDseuNewAkeriwtea(gQseienaxogtteryanpc,eteVddauflersoninmcgiam,porCiumAsee)rtsaicssscpuoeercdsiiufnisgcintfgoortthhceerQemiaaangndeunfaaDccttNiuvreaeatres’yds iolreproed fr
sioninitiatesintheMu¨llerian ductepithelium,butlaterrefines do
Notch1transgene(DNotch1)(Table1). /am
dtcooefnittnrhiinbegutpitorhneecsuarnfsrtooemrrioart-nhpdeostdHeirofiofmerreeoanxbtioisaxtAoinfgt(hHeoovxdiaed)vuecfltaomp[iin2lyg4],rgeepanrneods- Histology and Immunohistochemistry rticle-a ww
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dDcreruiicccteietcpirvatole[r2[-1t8dr9,ar]ic2v.t9e,Cn]oownrCdirrtieehtitoirAnneomacilochmdr2eablc-eicintdriaeos,sneisagn[no2daf5lWic]n,ongnbtd7-aciantaidowteninEathilmnpaxc[r22ot,i6gv,eaasrt2eitoe7rn]aolnsoooefr tPainsBsdSuFeeoasmnrwdbeeaesdcrtehdoerfaedixgdeeiindngirp7noa0urB%apof,fuieanitnhmaasinnonodliumltsaiueotmcn4ti8oooCnvfeetodrhvnreieagretnh6iatg,nhtoihmtr.eanT7lshwlewamtsaih.ssseSaudlneitadshleyrwezseeeerdceti.tmipEorenxosstcrieanwscste1eer3dde stract/94/3.biolre
deparaffinized,rehydratedingradedethanol,andstainedwithhematoxylinand /6p
t[h3e0]siaglnlalledtratonsadudceevreslompomoethnetnoefdugnecnoei,leSdmoov,iwduitchtsAimnhmr2u-tcarnet eDoisaignno(Hst&icsE,)A. Stleacnttiao,nGsAw)ebreefdoerehymdorautnetdinignweitthhanaocloavnedrslHipi.stoclear (National 7, 1-1rod.
females. Immunohistochemistry(IHC)wasperformedonmouseorgansfixedin4% 2o
In this study, we generated a mouse model with condition- paraformaldehyde,washedwithPBS,treatedwithserialdilutionsofethanol, /24rg
ally activated NOTCH signaling in the Mu¨llerian duct to epmerbfoerdmdeedd iunsinpgaratfhfien,aanntid-NsOecTtCioHne1daunstiinbgodystan(1d:a4r0d0,praobto2c7o5l2s.6;IHACbcwamas, 3445.
determine its impact on the development of the female Cambridge, MA) recognizing the C-terminal part of the protein. Protein 8 b
reproductive tract and fertility. Analysis of mutant females detection was performed using a Vectastain ABC kit (Vector Laboratories, y
showedthattheactivationoftheNOTCH1pathwayresultedin Burlingame, CA) as recommended. The color was developed with diamino- gu
e
their infertility associated with the abnormal development of benzidine as chromogen. Samples were counterstained with Harris hematox- s
the oviducts, inflamed uterus, and increased vascularization ylin.StainedslideswereexaminedwithaCarlZeissAxioA1Microscopeand t on
aOnlddehremfeomrrahlaegsingdeovfelbolpoeodd vuetsesreilnseinvethine rtehprroomdubcotisvise,traanctd. iMmiacgroesscowpeyr,eLLcaCp,tuTrhedornbwyooadn,NAYxi)o.Cam MRc5 CCD camera (Carl Zeiss 01 A
Theproliferationofthesomaticcellsinmutantandwild-typePostnatalDay p
oviductal, ovarian and uterine cysts. (P) 100 oviducts was analyzed using rabbit polyclonal to Ki67 (1:1000; ril 2
ThermoScientific,FairLawn,NJ).ThepercentageofKi67-positivecellswas 0
1
MATERIALS AND METHODS evaluatedinatleast500cellsindifferentpartsoftheoviductsin3femalesper 9
group,andtheaveragenumberswerecomparedusingStudentt-test.
Mouse Strains and Breeding Thenumberofendometrialglandswasanalyzedinuterinesagittalsections
isolated from four P100 Amhr2-cre, RosaNotch1 females and four RosaNotch1
Thisstudywascarriedoutinstrictaccordancewiththerecommendationsin femalelittermates.Atotalof20randomviewsat320magnificationwasused
the Guide for the Care and Use of Laboratory Animals of the National forcountingineachsample,andtheaveragenumberofglandsperviewwas
Institutes of Health (NIH). The protocol was approved by the Institutional usedfortheStudentt-testanalysis.
Animal Care and Use Committee of Florida International University. Gt
(ROSA)26Sortm1(Notch1)Dam/J(RosaNotch1)micewereobtainedfromtheJackson RNA Isolation and Real-Time Quantitative RT-PCR
Laboratory(BarHarbor,ME).RosaNotch1micecontainasequenceencodingan
intracellular portion of the mouse Notch1 gene, but lacking the C-terminal Total RNA was extracted from tissue samples with a Trizol reagent
PEST domain, inserted into the GT (ROSA)26Sor locus. Expression of the (Ambion,Austin,TX).Afteradditionofchloroformandcentrifugation,thetop
Notch1fragmentisblockedbyaloxP-flankedSTOPfragment.Thetruncated aqueouslayercontainingtheRNAwasremovedandaddedtoanequalvolume
cytoplasmic fragment encoded by the Notch1 sequence causes constitutive ofisopropanol,mixedbyinversion,andincubatedatroomtemperaturefor10
signalingactivity[31].Amhr2tm3(cre)Bhr(Amhr2-cre)micewereobtainedfrom min.SampleswerethentransferredtoanRNeasyspincolumn(QiagenRNeasy
the Mutant Mouse Regional Resource Center at the University of North Kit) and centrifuged for 15 sec at 10000 rpm before resuming the Qiagen
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FIG.1. Activation of Notch transgene inAmhr2-cre,RosaNotch1 femalescauses abnormalreproductive tractvascularization and absenceofoviduct /am
ccferomeilaitnrlaegns..sAgNe)onAercetacivnoadmteGbdianapaldlteiholenpDrwimNasoedrtscehtde1ecrwtievadesdinpfrraeonsmeynoatfistnihnetghlteeissoeuxveoisdnufrcaotsm(aORcdoo)s,nautNrtooetlrcuhfo1srf(eUDmtN)a,Alaensq.duCaorlevit-aysr.pyBe(c)OiIfn)i,ccbrpeuraitmsneeodrtsviawnsecthrueelauhrsieezadarttitoo(Hnc)oongffefienrmmomaplirecesrDeepnNrcoAedoouffcttthihveeeAmomrughtaarn2ns-t rticle-ab www
(arrowheads)andinflameduteriinP100virginmutants.Uncoiledoviduct(Od;dashedlinearea)circlesaroundtheovary(Ov)inmutant. stra.b
RNeasykitprotocolfromthestepatwhichbufferRW1isadded.Qualityand females,whereasnosuchbandwasdetectedinmutantheartor ct/94/3iolre
concentration of RNA was analyzed with a Nanovue (GE Healthcare inanyofthesiblingRosaNotch1controlmousetissues(Fig.1A). /6p
7r
BwPMCiaaosRdscissiy(eoqnnnPtc,hCeesRWs,i)zPIe)iwdttaasubsnsudiprngegghrfe,aonrPeVmA-see)prd,seocturiefscaiiDcntegNdpAGrfiomokrTietDarsq(NTaihqnsePerCmacRooRnSMteacamailesPintneltearifxtii2cMo)n.iM,xQaau(nsPadtenrortccimtDyaectNiglveAaer, TRlehoaesdarienNfgootcrtheo1,aainnlletahlceetiwvparaetsisosenunccocefeosNsffouAtlcmhinhrtfr2ea-mncsragele,enrreeec.pormodbuinctaitvioenorogfatnhse, , 1-12/24od.org
(Eppendorf, Westbury, NY) according to the manufacturers’ instructions. Amhr2-cre, RosaNotch1 female mice (n ¼ 12) were mated 34.
4
ThreedilutionsofRNAwereusedasstandards,andvalueswerenormalizedto with wild-type males of proven fertility for 3 mo, but failed to 58
bcsoo-afmtcwptiaanrr.eatTi(vGheerCaprteh(l2Pa–atiDdvDeCSto)fofmtlwdeathrceohdaI.nngRce.e,sLiunlatsmJwoRlelNrae,AaCnAlael)vyezfleodrwusatssainticgsatiGlccaruallpashtiegPdnaidfbiPycarintshcmee fpermodaulecseshanoyweodffnsoprrminagl.feTrthieliitry,laitstedrmesactreibecdonptrreovlioRuosslyaN[o3tc1h]1. by gue
usingStudentt-test.ThesequencesoftheprimersareshowninTable1. Docicssuercretinocne ooffvfaivgeinaPl1p0l0ugmsuretavnetalefedmthalaetsno1n0edoafytsheaffteemratlhees st on
RESULTS waspregnant.Therewerenoimplantationsites,andtheiruteri 01
appeared to be enlarged, highly vascularized, and dark purple A
p
Amhr2-cre, RosaNotch1 Females Are Infertile in color. In five control female mice, multiple implantation ril 2
sites were identified 10 days after vaginal plug detection. 0
Transgenic mice with NOTCH1 gain of function in the 1
9
stromalandmusclecellsofthefemalereproductivetractandin Analysisoffivevirginmutantfemalesofthesameagerevealed
the gonads were generated by using a cre-loxP strategy. that the uterus enlargement and excessive vascularization was
Transgenic Amhr2-cre mice express Cre recombinase in alsopresent,andthuswasindependentofcopulation(Fig.1B).
granulosa cells in ovary and in the developing (from E12.5) Abnormal vascularization in reproductive organs was detected
and adult female reproductive tract in endometrial stromal even in newborn females (Fig. 2B). The most remarkable
cells,aswellasinmyometrium,butnotinluminalorglandular featureinthefemalereproductivetractwastheanatomyofthe
epithelium [26, 30, 32–34]. In diheterozygous Amhr2-cre, mutantoviducts.Normalmouseoviductwaspresentincontrol
RosaNotch1 transgenic females, the Cre-driven recombination RosaNotch1 females as a complex, coiled, tubal structure with
betweentwoloxPlociledtotheexcisionoftheSTOPcassette several longitudinal folds (Figs. 1B and 2A). The mutant
andthusactivationoftheNotch1transgene.ThePCRanalysis oviducts did not develop coils, and had instead encircled the
confirmed the presence of a 650 bp PCR amplicon derived ovary; they were shorter and thicker than normal. As in the
fromtheactivatedRosaNotch1transgeneinDNAextractedfrom uterus, hemorrhaging blood vessels were clearly visible in
uterus,ovary,andoviductof27-day-oldAmhr2-cre,RosaNotch1 mutant females (Figs. 1B and 2A).
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FIG.2. UncoiledoviductsinAmhr2-cre,RosaNotch1mutantfemales.A)Controlandmutantoviducts(Od;dashedlinedarea)atP2,P27,andP100.In , 1-12od.o
controlRosaNotch1females,theoviductformscoilsandislocatedbetweentheovary(Ov)anduterus(Ut).Mutantoviductcirclesaroundtheovary.Notean /24rg
excessivevascularizationwithhemorrhagingbloodvessels(arrowheads).B)AbnormaldevelopmentoftheisthmusanduterotubaljunctioninAmhr2-cre, 34.
RosaNotch1mutantoviducts.Histologicalsectionsofwild-typeandmutantuterotubaljunctionsatdifferentages.Inthetoppanel,theP27oviductsare 45
8
markedwithadashedline.Mutantisthmusopensdirectlytotheuterinelumen(areamarkedwitharectangle).Thereisaprogressiveoutgrowthofthe b
mutant longitudinal folds (dashed line in second to fourth rows) leading to constrictions (Co) of the lumen and the formation of cysts (C). Inf, y g
infundibulum;Od,oviduct;Ov,ovary;Uo,uterotubaljunction.Noteanexcessivevascularizationwithhemorrhagingbloodvessels(arrowheads).Bars¼1 u
e
mm(A),500lm(first,third,andfourthrowsinB),and200lm(secondrowinB). s
t o
n
0
In order to determine the cause of the observed female Abnormal Oviduct Development in Female Mice with 1 A
infertility, four mutant females were separated from the male p
following identification of a plug, and then dissected after 4 Constitutive Activation of NOTCH1 Signaling ril 2
days. In all four females, the reproductive tract appeared Groups of three Amhr2-cre, RosaNotch1 and control 01
9
enlarged, highly vascularized, but anatomically well devel- RosaNotch1 female littermates were dissected at different time
oped, except for the uncoiled oviduct. The oviducts from the points after birth to determine the impact of Notch1 activation
females were dissected and then attempted to be flushed to on the reproductive tract condition (Fig. 2). At all ages in the
release any trapped blastocysts. However, these efforts failed mutants, prominent hemorrhage of the blood vessels was
due to apparentblockage of theoviduct lumen preventing free detectedalongthereproductivetract.Eveninneonatalfemales,
flowoftheinjectedmedia.Thus,theobservedinfertilitymight leaking blood vesselswereclearlyvisible. Most strikingly,the
be, at least in part, the result of either the blockage of sperm mutant oviducts did not develop normal coils as seen in the
transportintotheoviductfromtheuterusorofoocytesfromthe wildtype,andwereinsteadloopedaroundtheovary,encircling
ovary. it(Fig.2A).Thehistologicalanalysisoftheoviductsincontrol
and mutant animals revealed further abnormalities (Fig. 2B).
Despite the aberrant formation of the oviduct, the three major
structuresoftheoviduct(infundibulum,ampulla, andisthmus)
could be identified in the mutants (Fig. 2B). The uterotubal
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FIG.3. HistologicalanalysisofmutantoviductsatDays100and200.A–C)ControlandmutantoviductsatP100andP200.Theinfundibulum(Inf)and
theampullaarepresentinmutantoviducts.B)Bendingofmutantoviductformsconstrictions(Co;arrows)intheoviductlumen.Hemorrhageisshown
withthearrowheads(BandC).MutantP200ovaryhasalargeovariancyst(OvC)(C).DandE)Progressivedisorganizationofciliatedandsecretory
epithelialcells(arrowheads)inthemutantampulla.Alargenumberofcellularvacuoles(arrows)andthelossofciliaonepithelialcellscanbeseenin
P200 mutants (F). NOTCH1 immunostaining in control (G and J) and mutant P100 (H and K) and P200 (I and L) oviducts. Arrows show nuclear
localizationofNOTCH1instromalcellsofthemutantoviducts(H,J,andL).ArrowheadsinG–LshowoviductalepitheliumandendothelialNOTCH1
staining.Bars¼500lm(A–C),25lm(D–F),50lm(G–L).Ut,uterus.
5 Article 67
FERGUSON ETAL.
oviducts in mesenchymal cells, and nuclear staining was
present in both stromal and epithelial cells (Fig. 3, G–L), as
well as in the endothelial cells of blood vessels of normal
oviductsandinhemorrhagingbloodvesselsinmutants(Fig.3,
G–L).
The histological appearance suggested an overgrowth of
cells in mutant oviduct (Fig. 4). The Ki67 staining in mutant
oviducts was mainly present in dense cell areas within the
stroma. Comparisons of cell proliferation using Ki67 antibody
revealed a significant increase (P¼0.0015) of Ki67-positive
stromal cells in P100 mutant oviducts (Fig. 4).
Mutant Females Develop Uterine and Ovarian Cysts and D
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In younger animals, the mutant uteri, ovaries, and vagina a
d
were of normal size; however, even newborn mutants ed
displayed disorganized vascularization of all reproductive fro
organs, with extensive collapsed and leaky blood vessels m
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causing pronounced hemorrhage. In younger mutant females, ttp
tthheatoovfecraolnltraonlatfoemmiaclaels.uTtehreinneusmtrbuecrtuoref ewnadsomcoemtripaalragblalendwsitihn s://ac
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theuteriwascomparablebetweenthetwogroupsatP100(48.3 d
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63.6endometrialglandsperviewinwildtypeand39.567.2 mD
inmutants;P¼0.32).Inolderfemales(P200),normalstructure ic.oow
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of the myometrium and endometrium was progressively pn
affected by an increased number of vascular abnormalities .colo
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(Fig. 5). There was no NOTCH1 expression detected in /bd
endometrial glandular epithelium, but strong staining was ioe
present in the endothelial cells of blood vessels in normal lrepd
females and in dilated blood vessels of mutants (Fig. 5, C–F). rodfro
In mutant females, the ovarian histology revealed normal /am
developmentinP2,P27,andP100animals,withfolliclesatall rticle w
developmental stages and corpora lutea (Fig. 6). Increased -aw
vascularization and dilated blood vessels were detected in all bsw
mutant ovaries. In 8-mo-old mutant females the ovary often tra.b
had cysts. ct/94iolr
Analysisofthereproductivetractof27-,100-,and200-day- /3e
old females revealed the presence of uterine and ovarian cysts /67pr
FfeIGm.al4e.s.CInocnrteraosle(dAsatnrodmCa)lacnedllApmrohlirf2e-rcarteio,nRoinsamNoutctah1ntmouvtiadnutc(tBinanPd10D0) iufnetmerooalldteuebrmaalicnjeiumnaacnltdsio.nfTivhaereewamiloodfs-tttyhfpreeequcuteoernnuttsrlyo(lFsaifwgfe.ecr2teeBda).gaeFrdeivateowm1as-uytatehnaert , 1-12/24od.org
oviducts(Od)stainedwithKi67.Arrowheadsshowstromalareasusedto and killed. All mutant females exhibited abnormal uterine 34.
4
count the positive cells. C) The difference between control (n¼3) and morphology; three females also had ovarian cysts (Fig. 7A). 58
mutant (n¼3) in Ki67-positive stromal cells is significant (P , 0.01). Gross anatomy of the reproductive tract appeared abnormal, b
Columnsrepresentthemean6SEM(n¼3foreachgenotype).Ov,ovary. with large uterine growths, uncoiled oviducts, and ovaries that y g
Bars¼500lm(AandB)and20lm(CandD). were dark purple in color. ues
H&E staining of a uterine swelling revealed banded t o
n
junction in wild-type mice is a small opening of the oviduct staining, characteristic of an acute thrombus (Fig. 7B), but no 0
1
into the uterine horn; in mutants, this simple connection was evidence of a hemangioma. Other cysts were filled with cells. A
p
suetevreursely(Fdigis.o2rgBa)n.iSzeedctiaonndsthoef itshtehmovuisdoupctesnefrdomdirePc1t0ly0inmtoutathnet S1tianindiincagtewditthhaatntthibeoudtieersinfeorgrsomwotohthwmasunscoltedaecrtiivnedanfdrocmaveeiothlienr ril 20
1
females showed that the oviduct was bent in several places smoothmuscleorendothelialcells(datanotshown);however, 9
alongitslength(Fig.3).Attheseparts,theoviductlumenwas these cells showed a strong staining for NOTCH1 (Fig. 7Bd).
often blocked due to the overgrowth of oviduct longitudinal
folds(Fig.3B).Theregularmuscularislayerthatwasvisiblein Activation of Notch1 Causes Up-Regulation of Wnt4
normaloviductwasalsogrosslyrearrangedinmutantfemales.
The WNT family ligands, WNT4, WNT5A, and WNT7A,
With age, fragmentation of the oviduct tubule progressed, and
are expressed in the female reproductive organs and play a
in P200 females we observed oviducts consisting entirely of prominent role in their differentiation. The expression of these
small cysts (Fig. 3C). While both ciliated and secretory genes was measured by quantitative RT-PCR (qRT-PCR) in
epithelial cells were present in mutant oviducts, their normal theuteri,oviducts(Fig.8),andovaries(datanotshown)in27-
structurewasprogressivelydisorganizedinolderfemales,with day-old Amhr2-cre, RosaNotch1 mice, and compared to the
a large number of cellular vacuoles in secretory cells and a littermate controls. The most dramatic change was detected in
decrease in the number of cilia on the ciliary cells (Fig. 3F). the expression of Wnt4, which was significantly higher (P ,
NOTCH1expressiondetectedbyIHCwasincreasedinmutant 0.05) in the mutant oviducts (Fig. 8). There was about a 50%
6 Article 67
NOTCH OVERACTIVATION INFEMALE REPRODUCTIVE TRACT
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FIG.5. Increasedvascularizationandhemorrhagingbloodvessels(arrowheads)intheP100Amhr2-cre,RosaNotch1mutantuterus.Controluterusshows gu
e
normalstructureoftheuterus(A,C,andE).Themutantmyometriumisseverelydisorganized,buttheendometrialglands(Eg)arenormallydevelopedin s
mutants(B,D,andF).TheinsetsinCandDshowtheabsenceofNOTCH1expressioninglandularepithelium.NOTCH1expressioninendothelialcells t o
n
ofnormalbloodvesselsincontrol(E)andinhemorrhagingbloodvesselsinmutant(F)isindicatedbyarrowheads.Bars¼are200lm(AandB)and50lm 0
(C–F). 1 A
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increase in Notch1 expression in mutant oviduct; however, it split-1)weresignificantlyalteredinthemutants,althoughboth 0
1
was not statistically significant. No difference was detected in showed a trend of higher expression levels in the mutant 9
the expression of Notch1 in mutant uteri. oviduct. No change was detected in Myc, Gli1, and Ptch1
The expression of Wnt5a and Wnt7a was unchanged in between the mutant and control oviducts. P21 (Cdkn1a) is
mutant mice compared to sibling controls. The expression of another downstream target of Notch1, and its expression was
cyclinD2,(Ccnd2),apositiveregulatorofG1progression,was unaltered in the mutant oviducts, as was also the case with
cyclin-dependent kinase 6 (Cdk6) (Fig. 8).
slightly lower (P , 0.05) in the mutant oviducts compared to
the littermate controls (Fig. 8). Transcriptional targets in the
hedgehog signaling pathway were also measured, as crosstalk DISCUSSION
betweenpathwayshasbeenpreviouslynotedandtheactivation We investigated the effect of conditional activation of
of Smo (Smoothened) in female reproductive tract caused Notch1 signaling on the development of the female
uncoiled oviducts [30]. Smo was unchanged between mutant reproductive tract by generating Amhr2-cre, RosaNotch1
and control in oviduct and uterus. Neither fibronectin nor the females. These mice express the NOTCH1 intracellular
downstream target of NOTCH1, Hes1 (hairy and enhancer of domain in ovarian granulosa cells, in the mesenchyme of
7 Article 67
FERGUSON ETAL.
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9
FIG.6. DevelopmentofovariancystsinAmhr2-cre,RosaNotch1mutantfemales.Shownarecontrolandmutantovariesofdifferentages.NotethatP3and
P27mutantovariescontainanormalnumberofantralfollicles(F)atdifferentstagesofdifferentiation.Corporalutea(Cl)arealsopresentinmutants.Large
ovariancyst(markedwith*)ispresentin8-mo-old(8M)mutantovary.Bars¼100lmforP3and500lmforP27,P100,and8M.
the developing Mu¨llerian duct, and then in the oviduct and and uncoiled. Our results revealed that the induction of
uterine stromal cells. Analysis of females revealed a number Notch1 signaling results in the upregulation of Wnt4 in the
of anatomical and histological abnormalities in the reproduc- oviducts of the mutant females, indicating crosstalk between
tive tracts. The most noticeable change was in the formation the NOTCH and WNT pathways. We also observed a
of the oviducts, which lacked the coiling normally seen in decrease in the cell cycle checkpoint regulator Ccnd2 in the
wild-type females, and were instead looped around the ovary oviducts of young mutant females, but an increase of Ki67-
8 Article 67
NOTCH OVERACTIVATION INFEMALE REPRODUCTIVE TRACT
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FcyIGst.i7n.b.UBt)erVinareiocuysstustienriAnmehcyr2st-scr(em,aRrokseadNwotcihth1m*)uwtainthtfceemlladleesb.rAis)(Ua)t,ereionsein(UoptChiilnicas)eacnredtioovnar(iba)n,o(OrdvCenisnebc)ecllysgtrsoiwntmhu(cta)nwteferemiadleens.tiHfieCd,.h(edm)NorOrhTaCgHin1g 7, 1-1rod.
expressionwasdetectedinthecellswithinthecystshowninc.Bars¼200lm(a–c),20lm(insetinb),and50lm(d). 2/2or
4g
34.
4
positive stromal cells in older mutant oviducts, suggesting development of the embryos. The latter could be further 5
8
higher cell proliferation rates. analyzed by embryo transfer accompanied by the analysis of b
y
Conditional activationof theNotch1pathwayin thefemale steroid production in mutant females. g
u
reproductivetractresultedininfertility,andexaminationofthe The role of Notch1 expression in the uterus has been found e
s
uterus revealed an acute inflammatory response in the uterine to be critical for completion of decidualization. Reduction in t o
n
horns of both virgin females and females mated with males, Notch1expressioninfemaleswithafloxedNotch1alleleanda 0
1
with no evidence of embryos or implantation sites. In aged cre transgene driven by a progesterone receptor (Pgr-cre) A
mlarugteantutfeerminaelesth,rwoemobbosseersv,ehdeemnolarrrgheadgirnegprobdlouocdtivveetsrsaecltss,waintdh wcahuiscehd aareredkuncotiwonnintoexrpergeuslsaitoen sotfroWmnatl4daencdidBumalpiz2a,tiboont.h Ionf pril 2
0
ovarian cysts. The infertile phenotype seen in the Amhr2-cre, addition, many G1/S cell cycle checkpoint genes were 19
RosaNotch1 females appear to be primarily a result of the deregulated, and a number of proapoptotic factors increased
abnormal oviduct development, although abnormal ovulation [35]. Pgr-cre is expressed in all postnatal uterine cells in
and uterine decidualization might also contribute to infertility. contrast to Amhr2-cre, which is absent from uterine epithelial
The malformed structure of the oviduct appears to restrict the cells and active only in uterine stroma cells [26, 36]. Notably,
sperm or oocyte movement, or, if fertilization does occur, the no implantation sites were found in the uterus of the mutant
migration of embryos to the uterus for implantation. The females in our experiments. Moreover, we observed uterine
presenceofnormalfolliclesandcorporalutea,atleastinyoung enlargement, characteristic of inflammation even in virgin
females,indicatesthattheovaryisfunctional,althoughamore females. This leads us to suggest that the decidualization
detailed analysis of the functions of granulosa cells where process is sensitive to expression levels of Notch1.
Amhr2-creisexpressed[33]willbeneeded.Thissuggeststhat The extensive disorganized vasculature and hemorrhage
the primary cause of infertility appears to be mechanical and leading to thrombosis in mutant females indicates that the
priortotheimplantationstage.Itisnotclear,however,whether activation of NOTCH1 in stromal endometrial cells or in
the mutant uterus is able to support implantation and myometriumcellswhereAmhr2-creisexpressedmaysignalto
9 Article 67
FERGUSON ETAL.
presented data do not pinpoint the exact target cells or tissues
responsible for oviductal and uterine abnormalities, as Amhr2-
cre is expressed in various sites of the reproductive tract. It is
not clear either at what level NOTCH1 needs to be
overexpressed to cause the mutant phenotype observed here.
A more detailed analysis of NOTCH1 expression at earlier
stages of development might answer these questions.
The oviductal phenotype seen in the reproductive tract of
Amhr2-cre, RosaNotch1 females is similar to that seen in mice
with a conditional b-catenin knockout, driven by Amhr2-cre.
Ablation of b-catenin resulted in uncoiled oviducts and a
reductioninthesizeofuterinehorns[27].Inthosemutants,no
differences in the Wnt5a and Wnt7a expression were noted by D
o
whole-mount in situ hybridization on E15.5 and E19.5 w
n
mesonephroi, although no data on Wnt4 were supplied [27]. lo
a
Interestingly, unlike the other models, the ovary of the d
e
conditional b-catenin knockout mutant was encased in a d
fluid-filled (believed to be follicular fluid) cyst [27]. Uterine fro
m
glandswereunaffectedincontrasttoWnt7aknockoutandmice h
withconstitutive activation ofSmogene[25,38–40].Analysis ttp
s
of the Dicer knockout females also revealed abnormal oviduct ://a
coiling, as seen in our mutant females. Many of these mutants c
a
had fluid-filled cysts forming from P28 in the isthmus region de
mD
[28,29].InsituhybridizationofWntgenesidentifiedabnormal
ico
expression of Wnt4 and Wnt5a in the luminal epithelium and .ow
u
Wnt4, Wnt5a, and Wnt11 in the glandular epithelium of the pn
uterus [28]. Another study identified significantly elevated .colo
ma
expression of Wnt4, Wnt5a, and Wnt7a in the mutant oviducts /bd
[29]. iolreed
Abnormalities of Mu¨llerian-derived structures were also p
seen in females exposed in utero to diethylstilbestrol (DES), a rodfro
FIG. 8. Gene expression analysis in Amhr2-cre, RosaNotch1 mutant sdyenscthriebtiecdehsterroeg,enDE[2S1-,tre4a1t]e.dSfimemilaalrestohtahde umnuctoainletdf,emshaolerst /articlem w
females. A) Real-time qRT-PCR analysis of Wnt ligands, cyclin D2, oviducts wrapped around the ovary, no clear demarcation -aw
smoothened,andNotch1inthereproductivetractof27-day-oldAmhr2- bw
betweentheoviductanduterus,andaproliferationofcolumnar s
Hvcareelud,egRsehoaosraegNnsooitcgrhnm1aalfilenizmgeadplaettoshwt(hnaey¼eixnp7rt)eh.sesBioo)nvAidonufacblty-asoicfsti2no7fg-degnaeyen-oeanlddtafrpegrmeetsaselenoste.fdTthhaees ecpelilthinelfiiultmratliionnin.gGethneeleuxmperens,scioynstafnoarlmysaitsioonf,uatnedrininefRlaNmAmaftroormy tract/94.biolr
thepercentageofthegeneexpressionin controltissues(100%)(n¼7). DES-treatedfemalesshowedadecreaseinexpressionofWnt7a /3e
Columnsrepresentthemean6SEM.*P,0.05. and Wnt4, but an increase in Wnt5a expression [42] mediated /67pr
tthhraotuNgOhTeCstHro1gerengruelcaetepstoarssuEbSseRt1ofoErSERS1R-2ta.rIgtehtagsenbeeseninsbhroewasnt , 1-12od.o
theendothelialcellsofbloodvessels,resultingintheobserved cancer cells [43]. Further analysis of ESR/NOTCH crosstalk, /24rg
inflammatory phenotype and dilated blood vessels. Interest- as well as the detailed analysis of steroid production and 34.
ingly a somewhat similar phenotype was recently described in 4
mice with constitutive NOTCH signaling in endothelial cells sreigqnuairleidn.g in females with NOTCH1 overexpression, is 58 b
[7].However,itisalsopossiblethattheappearanceofthecysts In contrast to Amhr2-cre, RosaNotch1 females, no defects in y g
andotherabnormalitiesinoldermutantfemalesissecondaryto the formation of the oviducts in Wnt4 conditional knockout ues
the infertile phenotype and possible hormonal changes. females using Amhr2-cre were noted [44]. The latter mutants t o
We have detected an increasein NOTCH1 immunostaining had smaller ovaries than their sibling controls and were 50% n 0
in stromal cells in mutant uterus and in oviducts. While no less fertile. Histologically, the ovaries had fewer follicles and 1 A
increased staining was seen in uterine glandular or luminal p
epithelium, there were variations of NOTCH1 expression in wanedrelamrgoereanattrreatlicf,olwliictlhesre[d4u4c]e.dInnucmonbdeirtsioonfalbomthutcaonrtspoorfaWluntet4a ril 20
oviductal epithelium. As Amhr2-cre was not reported to be 1
generated with progesterone receptor-driven Cre, there was a 9
expressed in epithelial cells, it is possible that detected significant reduction of uterine glands [45]. The differences in
variations reflect endogenous NOTCH1 expression changes uterine expression of Pgr-cre and Amhr2-cre may account for
throughout the estrous cycle, as recently described [37]. the unaffected uterine glands in the Amhr2-cre, RosaNotch1
Surprisinglywe didnotdetect asignificant increaseofNotch1 females described here. The Pgr-cre transgene is expressed in
expression or its target genes in the P27 female reproductive epithelial cells, whereas Amhr2-cre is not. It appears that, in
tractbyqRT-PCR.TheexpressionofendogenousNotch1gene our mice, aberrant activation of NOTCH1 in the mesenchyme
in RNA isolated from total organs might mask the differences alters the expression of Wnt4 to which the Mu¨llerian duct is
betweenmutantandwild-typeanimals.Itcanbealsosuggested highly sensitive.
thatthepreviouslyreported[26,27]lowefficiencyorchimeric It was shown that Wnt4 plays a crucial role in the
expression of Amhr2-cre might be responsible. The more development of the Mu¨llerian duct, as Wnt4 knockout females
severephenotypeseeninolderanimalscanbe,infact,aresult werecompletelymasculinizedanddonothaveMu¨llerianducts
of a gradual accumulation of cells with the recombinant [23]. Given the misexpression of Wnt4 in both Dicer and Smo
transgene and hence increased NOTCH1 expression. The conditionalmousemodels[28,30],itisthereforepossiblethat
10 Article 67