disparate embryonic/fetaltissues;and(4)anendocrine/paracrine a roleinpreventing immunologicalrejection ofgenetically cell invasion; (2)anutritive roleforthedeveloping embryo;(3) Gellersen, 2006):(1)aprotective rolein controlling trophoblast been attributed todecidua(Bell,1983;Aplin,2000; Brosens and maternal environment. Anumberofimportant functionshave the interface separatinginvading trophoblastcellsfromthe Gellersen, 2006).Duringgestation,decidualcellsarelocated at 1995; Braretal.,1997;Dimitriadis2005;Brosens and of cyclic AMP/proteinkinaseA(Tang etal.,1994;Lydon etal., regulatory actionsofprogesterone,interleukin-11,and activators Decidual celldifferentiation isexquisitely sensitive tothe pregnancy (DeFeo,1967;Parr andParr, 1989;Aplin,2000). of decidualcellsisonetheearliestuterineadaptationsto modified uterineendometrialstromalcells.Thedifferentiation activities ofthematernalcompartment.Decidualcellsare are likely toprovide thesignalingsystemthatcoordinates exchange ofnutrientsandwastes. Decidualandtrophoblastcells Carson etal.,2000).Thiscloseconnectionfacilitates the between maternalandfetaltissues(EndersWelsh, 1993; rodents, resultsintheestablishmentofacloseconnection Hemochorial placentation,whichoccursinbothprimatesand The establishmentofpregnancy requiresmaternaladjustments. INTRODUCTION Accepted 14November 2006 Departments of KEY WORDS: stressor. placenta morphogenesis.TheobservationssuggestthatDPRPparticipatesinpregnancy-dependentadaptationstoaphysiological was associatedwithaberrationsinmesometrialdecidualcells,vascularintegrity, anddisruptionsinchorioallanto deficiency interferedwithpregnancy-dependentadaptationstohypoxiaresultinginpregnancyfailure.Termination ofpregnancy to 6ofthe the mouse (DPRP). DPRPisaheparin-bindingcytokine,whichabundantlyexpressedinuterinedecidua.Inthisinvestigation,wehave Among theirmanyfunctionsistheproductionofcytokinesrelatedtoprolactin(PRL),includingdecidualprolactin-relatedprot embryo. Decidualcellsarecruciallyinvolvedincreatingtheintrauterineenvironmentconducivetoembryonicdevelopment. In themouse,decidualcellsdifferentiate fromuterinestromalcellsinresponsetosteroidhormonesandsignalsarising KhorshedAlam S. M. dependent adaptationstoaphysiologicalstressor A uterinedecidualcellcytokineensurespregnancy- (2007)doi:10.1242/dev.02743 Development 134,407-415 *Author forcorrespondence (e-mail:[email protected]) USA. Developmental Biology, UniversityofKansasMedicalCenter, KansasCity, KS66160, Gynecology, Biology, InstituteofMaternal-Fetal DivisionofCancerand and Therapeutics, lnR Godwin R. Alan sizes. Aprominentphenotypewasobservedwhenpregnant null mutantmaleandfemalemicewereviable,exhibitednormalpostnatalgrowthrates,fertileproducedlitter expressed EGFPwithindecidualtissueinlocationsidenticaltoendogenous Heterozygous intercrossbreedingofthemutantmiceyieldedexpectedmendelianratio.Pregnantheterozygotefemales Dprp Dprp 1 Dprp Pathology andLaboratoryMedicine, 3 Molecular andIntegrativePhysiology, and gene withanin-frameenhancedgreenfluorescentprotein( gene, characterizeditsstructureandevaluatedbiologicalrole. ( 3 Dtprp and MichaelJ.Soares ), Decidua,Pregnancy, Uterus,Nullmutation,Adaptationstohypoxia,Mouse 1 , Toshihiro Konno 2 Pharmacology, Toxicology, 1,3,4, 4 1 Obstetrics and GouliDai , * 2 Dprp , Lu -null micewereexposedtoaphysiologicalstressor. DPRP 1990; Cohicketal.,1997;Müller1998),PLP-J Mouse GenomeInformatics)(Duckworth etal.,1988;Croze etal., Orwig etal.,1997b),prolactin-like proteinB(PLP-B;PRLPB – Mouse GenomeInformatics)(Robyetal.,1993;Lin1997; decidua: decidualprolactin-relatedprotein(DPRP;DTPRP – four membersofthePRLsuperfamily areexpressed inuterine et al.,2003;Soares,2004;Alam2006).Intheratandmouse, with anarrayofdifferent biologicaltargets andactions(Wiemers structurally-related hormones/cytokines (thePRLsuperfamily) and Critchley, 2001).PRLisamemberoflarger collectionof 1994; Orwigetal.,1997c;Telgmann andGellersen,1998;Jabbour production ofcytokines relatedtoprolactin(PRL)(Tang etal., limited. understanding specializeddecidualcellfunctionshasbeen et al.,1998;RobbMantena2006).Progressin are notcompatiblewithpregnancy (Lydon etal.,1995;Bilinski specialized functions.Disruptionsindecidualcelldevelopment dependent upondecidualcellacquisitionofeachthese establishment andmaintenanceofpregnancy. Pregnancy is role incontrollingmaternaladaptationsrequiredforthe through investigation ofthe In thisreport,weexplore thebiologyofuterinedecidualcells 2000). Littleisknown aboutthephysiologicalactionsofDPRP. 1996; Rasmussenetal.,1997; Orwigetal.,1997b;Wang etal., high affinity toheparin-containingmolecules(Rasmussenetal., and residesinthedecidualextracellular matrixwhereitbindswith downstream mediatorofintrauterineprogesteroneaction. these decidualPRLfamily cytokines canbeviewed asa itself (Prigent-Tessier etal., 1999;Kimuraetal.,2001).Eachof Imai, 1999;Toft andLinzer, 1999;Daietal.,2000)andprolactin Mouse GenomeInformatics 1 , DanhuaWang Decidual cellsignalingismediated,atleastinpart,throughthe DPRP issecretedasaglycoprotein byuterinedecidualcells Dprp EGFP mRNA andproteinexpression.Homozygous ) geneandaneomycin( Dprp -null miceweremadebyreplacingexons2 1 , JudyH.Dunmore ) Dprp (Hiraoka etal.,1999;Ishibashiand -null mouse. RESEARCH ARTICLE neo ) resistancecassette. 3 , ( PRLPJ – the Dprp olated ic ein 407 -

DEVELOPMENT Plp-b Plp-j was routinelyusedtoidentifyoffspring withwild-typeand and transferredintopseudopregnant (C57BL/6 signal. ChimerasweregeneratedbyinjectionintoC57BL/6blastocysts homozygous mutantalleleswerecharacterizedbya5.5kbhybridization type alleleswerecharacterizedbya21kbhybridizationsignal; Southern blotswereperformedwithaprobederived fromintronA.Wild- isolated, digestedwith homologous recombinationwiththetargeting vector. GenomicDNA was analysis was usedtoidentifyclonesthatappropriatelyunderwent were selectedbyexposure toG418andgangcyclovir. Southernblot Lunenfeld ResearchInstitute,Toronto, Canada)byelectroporation.Cells cells (Nagyetal.,1993)(agenerousgiftfromDrJanetRossant,Samuel in Fig.1.Thetargeting vector was introducedintoR1embryonicstem representation ofthemouse restriction enzymeandDNA sequenceanalyses.Aschematic kinase gene.Theaccuracy ofvector constructionwas verified by the located immediatelydownstream ofexon 6was subcloneddownstream of fragment ofthe genomic construct,was subclonedupstream of 5 sites (Godwinetal.,1998).A6.6kbDNA fragment,containing3.8kbof fluorescent protein( replacing exons 2-6ofthemouse Kits (Foster City, CA).The sequencer andAppliedBiosystemsDyeTerminator CycleSequencing sequencing was performedwithan AppliedBiosystemsModel310 designed andusedtosequenceexons andexon-intron boundaries.DNA reverse oligonucleotide primersetsbasedonthemouse and usedtoinoculateLE392 mouse eeFradpie Reverseprimer Forwardprimer Gapdh Dprp Gene Table 1.Primersetsusedforanalysis ofdecidualPRLfamilytranscripts Genetics (Houston,TX).Approximately1 packaged intheLambdaFIXIIvector was agenerousgiftofLexicon A genomicDNA librarygeneratedfroma129/SvEvstrainmouseliver and Gene targeting MATERIALS ANDMETHODS and of wild-type(+/+),heterozygous (+/–)andnull(–/–)alleles.( expression. Fig. 1.Mouse 408 Ј flanking DNA and2.8kbofexon 1andintronAofthemouse MC1neo Dprp Dprp RESEARCH ARTICLE -null (–/–)mice.( cDNA (Orwigetal.,1997b).Positive plaqueswereamplified cassette andupstreamofaherpessimplex virusthymidine ( A Dprp ) Exons2-6ofthemouse Dprp EGFP gene, constructionofa genomic constructcontaining3 Sal D ) geneand Dprp ) WesternblotanalysisofDPRPprotein ingestationday7.5deciduafrom wild-type(+/+)and I, andfractionatedin0.8%agarosegels. Dprp Escherichia coli. 5 5 5 5 Ј Ј Ј Ј -GCACTTCAAAAGCCTGATACT-3 -TATGACCGGAAATCCAATGAA-3 -ACCACACTCCATGCCATCAC-3 -TGAATGTCAAACAGGAGAGAA-3 gene andthetargeting vector areshown Dprp targeting vector was constructedby MC1neo Dprp gene withtheenhancedgreen ϫ 10 6 ϫ gene were replaced withanin-frame cassette flanked by Dprp pfu werescreenedwitha A seriesofforward and CBA) F1females.PCR EGFP -null mutanttargetingvector, genotypeanalysis,and Dprp . A6.0kbDNA Ј flanking DNA Dprp C cDNA were ) RT-PCR analysisof Ј mutant Ј Ј Dprp loxP Ј the with reverse primers correspondingtonucleotidesequencesinintronBof EGFP A ofthe alleles. Aforward primercorresponding toanucleotidesequenceinintron 2006). Undertheseconditions,airiscirculatedatabarometricpressure of beginning onday5.5ofgestation,aspreviously described(Ho-Chenetal., Female C57BL/6pregnant micewereplacedinhypobaric chambers Hypobaric hypoxia experimentation withrodents. Care andUseCommitteeapproved allprocedures forhandlingand Arroyo etal.,2005).TheUniversity ofKansasMedicalCenterAnimal deciduomal tissueweremeasuredaspreviously described(Soares,1987; et al.,2006;Ain2006).Alkalinephosphataseactivities in until used.Protocolsfortheabove procedures have beendescribed(Deb frozen indryice-cooledheptane.Alltissuesampleswerestoredat–80°C For insituhybridizationandimmunohistochemicalanalyses,tissueswere tissues weresnap-frozeninliquidnitrogenforRNA andproteinanalyses. pseudopregnancy byinjecting25 males. Deciduomalreactionswereinducedonday3.5of animals. Pseudopregnancy was inducedbymatingwithvasectomized uterus, decidual,andplacentaltissues,weredissectedfrompregnant mice was designatedasday0.5ofpregnancy. Placentationsites, including males. Thedaywhenaseminalplugwas foundinthevagina offemale Timed matingsofanimalswereconductedbyplacingfemaleswithfertile lights onfrom0600-2000h,andallowed freeaccesstofoodandwater. ME). Micewerehousedinanenvironmentally controlledfacility, with C57BL/6 micewereobtainedfromJacksonLaboratories(BarHarbor, Animals andtissuepreparation with EthidiumBromide.Micethe mutant allele,1148bp)wereseparatedon1%agarosegelsandstained extension, 72°Cfor1.5minutes.PCRproducts(wild-typeallele,676bp; 4 minutes;denature,94°Cfor1minute;anneal,60°Cand conducted for30cycles underthefollowing conditions:preheat,94°Cfor for sixgenerationstoC57BL/6or129SvJgeneticbackgrounds. Dprp Dprp EGFP Dprp gene (5 gene (5 transcripts ingestationday7.5deciduafrom wild-type(+/+) gene followedbyan gene (5 5 5 5 5 Ј Ј Ј Ј -GTGCCCATGTATGCCAGTTTG-3 -GGTTTTGATTTTGCCATGCTT-3 -CAATCTTGCCCAGTTATGCGG-3 -TCCACCACCCTGTTGCTGTA-3 Ј Ј -GTATGGCTGATTATGATCTAGA-3 -GTGTGCTAAATGAACGTAGT-3 Ј -GAGCTTAAACTTCAATGTAAGT-3 ␮ l ofsesameoil/uterinehorn.Harvested MC1neo Dprp Dprp mRNA andprotein cassette. ( mutation werebackcrossed Dprp Ј Development 134(2) Ј Ј Ј -null (–/–)mice. B Ј ) andwithinthe ) PCRanalysis Ј ). PCRwas Ј ) was used

DEVELOPMENT 2/v ae25.26±2.21( Male 129/SvJ 5B/ ae24.67±1.74 ( C57BL/6 Male Microsystems GmbH,Welzlar, Germany). Leica MZFLIIIstereomicroscopeequippedwithaCCDcamera(Leica All processedtissuesectionswereexamined andimagesrecordedwitha Science, Penzberg, Germany) accordingtothemanufacturer’s instructions. performed withtheInSituCellDeathDetectionKit(RocheApplied CD31 antibody(BDPharmingen,FranklinLakes, NJ).TUNELassayswere Hybridoma Repository, Iowa City, IA)andaratmonoclonalanti-mouse monoclonal anti-mouseendoglinantibody(Developmental Studies Repository, Iowa City, IA).Endothelialcellswerelocalizedusingarat cytokeratin antibody(TROMA-1; Developmental Studies Hybridoma Trophoblast cellsweremonitoredwitharatmonoclonalanti-mouse polyclonal anti-perforin1antibody(Torrey PinesBiolabs,Houston,TX). International, Temecula, CA).NKcellsweredetectedwitharabbit immunoreactivity withrabbit anti-GFPpolyclonalantibodies(Chemicon M.J.S.,unpublished).GFPwas monitoredbyfluorescence and and and endothelialcells(Ainetal.,2003)(T.K., L.A.Rempel,J.Arroyo determine thedistribution ofGFP, naturalkiller(NK)cells,trophoblastcells immunocytochemistry. Immunocytochemical analyseswereusedto Laboratories, Peterborough,UK)histochemistry, orusedfor (Invitrogen, Carlsbad,CA).Total RNA (15 1990). Total RNA was extracted fromtissuesusing TRIzolreagent Northern blotanalysiswas performedasdescribedpreviously (Faria etal., Northern blotanalysis array assaywas performedaspreviously described(Daietal.,2002). superfamily mini- monitor thephenotypesofdeciduaandplacenta.ThePRL superfamily (Daietal.,2002).Theassayhasbeeneffectively usedto simultaneously monitoringexpression ofeachmemberthePRL superfamily mini-array assayisahybridization-basedtoolfor The PRL PRL superfamilymini-arrayassay to biotinylated subjected a cryostat.SectionswerestainedwithHematoxylinandEosin,or Analyses wereperformedon10 Histological analyses protein assay(Bio-Rad,Hercules,CA). concentrations weredeterminedforeachsampleusingtheBio-RadDC previously described(Rasmussenetal.,1996;Orwig1997b).Protein DPRP proteinwas detectedintissueextracts byimmunoblottingas Western blotanalysis Phenotypic analysesoftheuteroplacental compartment ~420 Torr, whichresultsinaninspiredPO Pregnancy-dependent adaptationstohypoxia tan / / –/– +/– +/+ 29.02±2.67( the mean±s.d. There are no significant differences inbodyweight(g)amongthe maleorfemalewild-type(+/+),heterozygous (+/–)or Male Mixed Strains Table 3.Bodyweightat55daysofage 107:116 119:110 106:100 Male:Female 57 56 47 –/– 106 109 105 +/– 60 also comparabletoeachother. 64 54 ratio ofwild-type(+/+),heterozygous (+/–)and Heterozygous breedings yieldedtheexpectedmendelian ratio(1:2:1).Male:female +/+ 129/SvJ C57BL/6 Mixed Strains heterozygous matings Table 2.Genotypicdistributionofoffspring from breathing 11%O cages andreplenishfoodwater (15-20minutes). Griffonia simplicifolia 2 at sealevel. Thechamberswereopeneddailytoclean eae20.65±1.82 ( Female eae18.18±1.56 ( Female eae22.17±2.90 ( Female ␮ m tissuesectionspreparedwiththeaidof Dprp ␮ lectin IisolectinB g perlane)was resolved in1% 2 -null mutant(–/–)micewere of ~78Torr, equivalent to n n n n n n 2)19.76±2.21 ( =24) 2)24.67±2.63 ( 18.63±1.11 ( =20) =22) 2)24.83±2.09 ( 22.17±3.04( =27) =48) 3)28.54±3.80( =36) 4 (Vector staining. resolved byelectrophoresisin1%agarosegelsandEthidiumBromide seconds; extension, 72°Cfor1minute).Theamplified productswere performed for30cycles (denature,95°Cfor45seconds;anneal,55°C and conducted usingPlatiniumTaq DNA HighFidelitypolymerase(Invitrogen) reactions withSuperScriptIIreverse transcriptase(Invitrogen). PCRwas 2/v 6.6±1.69( 129/SvJ RNA (2 was isolatedfromuterinetissuesdays5.5to7.5ofgestation.Total different animalswereanalyzedwitheachprobefortimepoint. loading ofRNA samples.Atleastthreedifferent tissuesamplesfromthree dehydrogenase ( metallothionein-I ( 5B/ 8.71±1.58( C57BL/6 Dprp RT-PCR analysis et al.,1997b), crosslinked. Blotswereprobedwith[ formaldehyde-agarose gels,transferredtonylon membranesand targeting strategies culminatinginthereplacementofaregion of et al.,2006). (Dai etal.,1996;Orwig1997a;Wiemers etal.,2003;Alam the PLP-C(PRLPC–MouseGenomeInformatics)subfamily a 6-exon organization, similartorat entire codingsequenceformouse cDNA resultedintheisolationofaphageclonecontaining Screening ofamousegenomiclibrarywiththe Generation ofa RESULTS determined bytheNewman-Keuls Test. The datawereanalyzedbyanalysisofvariance andposthoccomparisons Statistical analysis Molecular Biochemicals,Indianapolis,IN). labeled riboprobesaccordingtothemanufacturer’s instructions(Roche were usedastemplatestosynthesizesenseandantisensedigoxigenin- cDNAs formouse tissues werepreparedandstoredat–80°Cuntilused.Plasmidscontaining previously (Ainetal.,2003; Weimers etal.,2003).Cryosections (10 The localizationofmRNAs withintissueswas performedasdescribed In situhybridization Values are expressed asthemean±s.d. +/+ 7.92±2.35( Mixed Strains Table 4.Reproductive performanceof Dprp- , Plp-j ␮ , g) and0.5 and Plp-j- n n n n n n Plp-j 5)18.98±1.44 ( =53) 4)23.65±1.61 ( 18.63±1.74 ( =43) =42) 4)23.57±1.75 ( 21.41±3.32( =47) =82) 10 28.49±4.06 ( =100) Plp-b Dprp Gapdh , Mt1 Dprp Plp-b- ϫ (Dai etal.,2000), mRNA levels wereestimatedbyRT-PCR. Total RNA ␮ / +/– +/+ -null mutantmiceweregeneratedbygene- ) (Liangetal.,1996).Glyceraldehyde-3-phosphate ) cDNA was usedtoevaluate theintegrity andequal g ofoligodTwereusedforreverse transcription n n n Dprp 1)6.41±1.47( =16) 2)8.24±2.53( =21) 1)9.08±2.19( =12) and Dprp or Gapdh Plp-j -null mutant(–/–)mice.Values are expressed as -null mouse Breeding combinations (Orwig etal.,1997b;Dai2000) -specific primers(Table 1).PCRwas ␣ 32 Dprp P]-labeled cDNAs for RESEARCH ARTICLE Plp-b ϫ Dprp / –/– +/– . The Dprp n n n 2)7.27±1.18( =22) 2)8.66±1.8( =21) 1)7.83±1.59( =12) (Müller etal.,1998)and n n n n n n and othermembersof =28) =21) =27) =26) =54) =61) Dprp mutant mice gene possesses Dprp ϫ –/– n (Orwig ␮ n n =16) Dprp m) of =18) =12) 409

DEVELOPMENT determined bySouthernblottingandPCRanalyses.Two mutant Correcthomologousrecombinationwas are shown inFig.1. representation ofthemouse 10 aminoacidsoftheDPRPsignalpeptide.Aschematic sequence remaininginthemutatedgene(exon 1)encodes thefirst and an homozygous mutant(C,F)mice.( implantation sitesofwild-type(A,D),heterozygous (B,E)and ( and compartment inimplantationsitesofwild-type,heterozygous the Fig. 2. 410 is locatedatthetopofeachimage.Scalebars:1mm. (counterstain, Propidium Iodide).Themesometrialregion oftheuterus heterozygous mutant(H)andhomozygous(I)mice sections from gestationday7.5implantationsitesofwild-type(G), D-F Dprp ) wasperformedonfrozen sectionsfrom gestationday7.5 Dprp Dprp RESEARCH ARTICLE MC1neo -null mutantmice. gene (exons 2through6)withanin-frame and Dprp cassette. Theportionofthe GFP allele expression intheuteroplacental Immunostaining forDPRP( Dprp G-I ) EGFPfluorescence isshownin gene andthetargeting vector Dprp A-C EGFP ) andGFP coding gene The 1996; Rasmussenetal.,1997;LinOrwig1997b). from bothpregnant andpseudopregnant animals(Rasmussenetal., DPRP isknown tobeexpressed indecidualanddeciduomaltissues Dprp Characterization oftheuterinecompartmentin successfully disrupted analyses areshown inFig.1.Thegenetargeting strategy with the background didnotsignificantly affect thephenotypeofmice postnatal growth ratesandwerefertile(Tables 3and4).Genetic 129SvJ geneticbackgrounds.Theoffspring exhibited normal genetic backgroundandfollowing transferto C57BL/6and and femalemicewereviableonamixed 129SvJandC57BL/6 generations ofbackcrosses.Homozygous moved totwo inbredstrains(C57BL/6and129SvJ)following six from theexpected mendelianratio(Table 2).Themutationwas resulted inoffspring genotypesthatdidnotsignificantly deviate 44. Breedingofmiceheterozygousforthe offspring. Subsequentanalyseswerederived frommouselineNo. placenta ondays12.5or17.5ofgestation(Fig.4B). expression ofothermembersthePRLsuperfamily withinthe products, not significantly affect theexpression oftwo otherdecidual were decreasedin the decidualPRLsuperfamily, DPRP deficiency influencedtheexpression ofanothermember on day11.5ofgestation(Fig.3). of wild-typemice(Fig.2).Thisdifference continuedtobeevident tissue distribution ofDPRPproteininthemesometrialcompartment in themesometrialcompartmentof type (+/+)females(Fig.2).However, thetissuedistribution ofEGFP locations similartoendogenousDPRPexpression inpregnant wild- females faithfully expressed EGFPwithindecidualtissuein locus. Pregnant heterozygous(+/–)andhomozygous null(–/–) and successfullytransmittedthe both theNo.44and96lineswerebredtoC57BL/6females chimerism, andafemaleof60%chimerism.Malechimerasfrom gave risetothreechimeras,includingtwo malesof40%and75% 44 celllinegave risetoa>95%malechimera.TheNo.96cellline injected intoblastocysts inordertogeneratechimeras.TheNo. ES celllines(No.44andNo.96)withanormalkaryotypewere Pregnancy proceededintheabsenceofdetectable Dprp mutant mice Dprp -null allelecontainsanEGFPgeneinsertedintothe Plp-b -null mutation.Genotypingand bars: 250 comparable inwild-typeand contrast, theanti-mesometrialdecidual regions appear expression inthemesometrialdeciduaofBandC.By mesometrial decidua.NotetheminimalGFP arrowheads inA-Cindicatethelocationof using anti-GFP(B,E)andfluorescence (C,F).The tissues (A,D),andforGFPinDPRP-null(–/–) monitored forDPRPexpression inwild-type(+/+) ( 11.5 wild-typeand and anti-mesometrialdeciduaofgestationday Fig. 3.Histologicalexaminationofmesometrial and A-C Dprp ) andanti-mesometrial( Mt1 Dprp -null mutantdecidua.DPRPdeficiency did ␮ (Fig. 4A),anddidnotsignificantly affect m. mRNA andproteinexpression. Plp-j Dprp Dprp Dprp (Fig. 4A). -null micewas lessthanthe -null mice. D-F Dprp Dprp mutant alleletotheir Development 134(2) ) deciduawere Dprp Plp-j -null tissues.Scale -null mutantmale Dprp -null mutation Mesometrial Dprp mRNA levels expression mRNA. Dprp

DEVELOPMENT Dprp Pregnancy-dependent adaptationstohypoxia GFP in expressed perbodyweight(Fig.5D).DPRPproteinin wild-type and modest but significant decreaseindeciduomalweight,when mice ofbothgenotypeswithonlysubtledifferences, includinga pregnancy, mesometrial deciduomalcompartment(Fig.5F-H).Similarto and We next examined decidualizationinpseudopregnant wild-type Dprp -null deciduoma(Fig.5I). Dprp -null mice(Fig.5).Deciduomaformationwas similarin Plp-j -null micelocalizedpredominantlytotheanti- mRNA expression was alsodown-regulated in husbandry conditions. and theorganization ofthematernal-fetalinterface understandard consequences fortheestablishmentandmaintenanceofpregnancy days 7.5and9.5(datanotshown). wild-type and (perforin 1)markers andTUNELactivity did notdiffer between Distributions ofendothelial(endoglinandCD31)NKcell Overall, theDPRPdeficiency appearedtohave onlymodest The organization ofthematernal-fetalinterface was examined. located atthetopofeachimage.Scalebars:1mm. (data notshown).Themesometrialregion oftheuterusis The senseprobes didnotdemonstratespecificstaining digoxigenin-labeled senseandanti-senseRNAprobes. plasmids were usedastemplatesforthesynthesisof (–/–; D,F)miceonday7.5ofgestation. implantation sitesofwild-type(+/+;C,E)and ( salmon spermDNAwere usedascontrols. used tomakeprobes byreverse-transcription. RNA from day12.5or17.5placentaltissueswere superfamily were spottedontonylonmembranes.Total assay. cDNAsforallmembersofthemousePRL mouse placentasusingthePRLsuperfamilyminiarray mice onday7.5ofgestation. decidual tissuesofwild-type(+/+)and Mt1 ( demonstrate integrityoftheRNAandloadingaccuracy. null mice. Fig. 4.Expression analysisofwild-typeand C-F B Dprp ) PRLsuperfamilyexpressionwere patterns examinedin Localizationof ) top ofeachimage.( mesometrial region ofthe uterus islocatedatthe decidualized uterusfrom fluorescence intheday7.5 pseudopregnant- uterus from of GFPintheday7.5pseudopregnant-decidualized type (+/+)mice.( 7.5 pseudopregnant-decidualized uterusfrom wild- Immunocytochemical localizationofDPRPintheday Plp-j Dprp from day7.5pseudopregnant wild-type(+/+)and n pseudopregnant deciduomafrom wild-type(+/+; Alkaline phosphatase(AP)activitiesofday7.5 expressed byratiotobodyweight.*, wild-type (+/+; pseudopregnant deciduomaweightresponses from Dprp weight responses from wild-type(+/+; mice. ( pseudopregnant wild-type(+/+)and artificially decidualizeduterifrom day7.5 and Fig. 5.Decidualizationresponses inwild-type in decidualtissues.Total RNAwasisolatedfrom =7) and -null uteroplacentalcompartmentsongestation , Dprp -null (–/–)mice.Scale bars:1mm. -null (–/–; Plp- C ( A ) Day7.5pseudopregnant deciduoma ) Northern analysisfor ) Northern b Dprp -null mice. , Mt1 Dprp -null (–/–; n n Dprp =9) mice.( and =7) and G -null (–/–)mice.( ) Immunocytochemicallocalization RESEARCH ARTICLE I ) Northern blotanalysisof ) Northern Gapdh (C,D) and ( A , n Dprp B Dprp Gapdh =7) mice.( ) Gross appearanceof D expression indeciduoma ) Day7.5 -null (–/–; -null (–/–)mice.The Plp-j Dprp was usedto H Dprp Dprp ) GFP (E,F) mRNAsin F , Dprp P ) Plp-j n <0.01. ( -null (–/–) =7) and n and Dprp =9) mice Gapdh -null (–/–) , Dprp Plp-b Plp-j -null E Dprp ) 411 - and and ,

DEVELOPMENT type pregnant femalemice, type and dying/resorbed conceptusesare significantlydifferent betweenwild- (–/–; pregnancy outcomesinwild-type(+/+; dp effectively tohypoxia. adapt and appearance ofarepresentative uterusfrom pregnant wild-type(+/+) stressors. Hypoxia was selectedasaphysiological stressorbecause regulation ofpregnancy-dependent adaptationstophysiological et al.,2006).Theseinsightsledus toexamine aroleforDPRPinthe stressors (DorshkindandHorseman, 2001;Ainetal.,2004;Soares to participateintheregulation ofadaptationstophysiological superfamily hasbeenpostulated challenging conditions.ThePRL genes thatspecifically permitreproductioninphysiologically performance. Thisoptimizationislikely toincludetheevolution of Successful speciesdevelop strategies tooptimizetheirreproductive phenotype hypoxiaonthe Impact ofmaternal conditions andexaminedonday17.5ofgestation.( gestation. Afterday11.5,theanimalswere returnedtoambient hypobaric hypoxia(equivalentof11%oxygen)from days5.5to11.5of expression byRT-PCR analysis.( hypoxia. Fig. 6.Pregnancies in 412 Dprp n =10) miceexposedtohypoxia.Numbersofhealthyand RESEARCH ARTICLE Dprp -null (–/–)miceexposedtohypoxia.( ( A ) DeterminationoftheontogenydecidualPRLfamily -null mutantpregnancies; Dprp Dprp -null miceare vulnerabletomaternal B ) Exposure ofpregnant femalesto -null pregnant femalemicedonot n =19) and P <0.01. Notethatunlikewild- E ) Quantificationof Dprp Dprp C , D -null mutant ) Gross -null prominent hemorrhagicareas inthe dissected uteroplacental compartments.Notethepresence of hemorrhagic regions are encircled (yellowbroken line)withinthe uteroplacental compartmentswere dissected.Thelocationsof gestation. Micewere sacrificedonday11.5ofgestationand exposed totheequivalentof11%oxygenfrom days5.5to11.5of hypoxia. wild-type and Fig. 7.Gross inspectionofdecidua-placentalcompartmentsfrom ( challenge, wefirst examined theontogeny ofdecidualPRLfamily Fryer andSimon,2006;Myatt,2006). tissue remodelingatthematernal-fetalinterface (Zamudio,2003; it iswellestablishedthatlow oxygentensionpromotes extensive mice fromdays5.5to11.5(durationofdecidual Consequently, wechallengedpregnant wild-typeand initiated betweendays5.5and6.5ofgestation(Fig.6A). Analysis of the maternal-fetal interfacein Analysis ofthematernal-fetal adaptations tohypoxia. these observations thatDPRPparticipatesinpregnancy-dependent were dyingorresorbinginthe gestation inwild-typeanimals;whereasmostfetal-placentalunits (Fig. 6C-E).Mostfetal-placentalunitswerehealthyonday17.5of Dprp gestation (Fig.6B).Pregnant femalemicepossessing themutant were returnedtoambientconditionsandexamined on day 17.5of oxygen isambientatsealevel). Afterhypoxiaexposure, animals during normalpregnancy) withtheequivalent of11%oxygen(21% evident byday9.5ofgestationin histological examination indicatedthatonlymodesteffects were exposed tohypoxiawereunique.Initialgrossinspection and The defectsresponsibleforpregnancy terminationin null miceexposedtohypoxia or decidualEGFPexpression in significantly affect decidual uteroplacental compartments(Fig.7).Maternalhypoxiadid not uteroplacental compartments,but werenotevident inwild-type the mesometrial-anti-mesometrialjunctionof macroscopic lesionswerediscernibleinthemesometrialregion orin However, notablepathologieswereidentified byday11.5of gestation; potentially maladaptive responsesinthe increased depthofendovascular trophoblastcellinvasion. The compartment includedcompression ofthemesometrialdeciduaand maternal hypoxiaobserved inthewild-typeuterinemesometrial type and a rangeofpotentiallymaladaptive responses tohypoxiainthewild- uteroplacental compartmentsrevealed prominent adaptive aswell exaggerated compressionofthemesometrial decidua(Fig.8C,F); including trophoblastgiantcell overgrowth (Fig.8B,E);(3) 8A,D); (2)distortedchorioallantoic placentalorganization, compartment included:(1)enlarged mesometrialbloodspaces (Fig. Dprp In ordertodeterminethetimecourseforphysiological Histological examination oftissuesectionsthroughthe gene didnotadapttohypoxiaaswellwild-typemice , Plp-j Wild-type (+/+)and Wild-type Dprp and Dprp -null mice(Figs8,9).Theadaptive responses to Plp-b -null pregnant miceexposedtohypobaric ) geneexpression. Dprp Dprp Dprp Dprp Dprp -null (–/–)pregnant micewere gene expression inwild-typemice Dprp -null mice.We concludefrom -null mice(datanotshown). -null miceexposed tohypoxia. -null (–/–)tissues. Dprp Dprp Development 134(2) -null mesometrial Dprp expression was Dprp expression Dprp Dprp Dprp -null mice -null -null -

DEVELOPMENT and have utilizedastandardsinglegenemutationapproach.Based the biologyofmembersexpanded mousePRLsuperfamily gained insightsintothePRLsuperfamily throughanexamination of respect tothisclassichormone/cytokine isunknown. We have et al.,2006).Whymammaliangenomesevolved differently with superfamily hasbut asingleconstituent(Wiemers etal.,2003;Alam two dozengenes,whereasinotherspecies(e.g.humananddog)the rat thePRLsuperfamily hasexpanded, consistingofapproximately specific (Forsyth andWallis, 2002;Soares,2004).Inthemouseand hormones/cytokines. secretory productisamemberofthePRLsuperfamily of pregnancy-dependent adaptationstohypoxia.Thedecidualcell product oftheuterinedeciduaisfundamentaltoregulation of in itsabsence.Inthisreport,weprovide evidence thatasecretory development anditisestablishedthatpregnancy doesnotproceed a supportive structurethatfacilitates placentationandembryonic species withhemochorialplacentation(Aplin,2000).Itfunctionsas Decidua isaspecializeduterinestromalcellmodification foundin DISCUSSION maintenance ofpregnancy. the placentation-specific adaptationstohypoxiarequiredensure Dprp aberrations mayberelatedtothealteredmesometrialdeciduain and (4)decreasedendovascular trophoblastinvasion (Fig.9).These Pregnancy-dependent adaptationstohypoxia The compositionofthePRLsuperfamily isdiverse andspecies- -null mousenotedabove (Fig.3).Thenetresultisafailure in couple ofdayslater, whichisassociatedwith aseriesofanomalies early interactionwiththematernalenvironment but collapsesa Dprp leading togrowth restriction(Ainetal.,2004).Thehypoxia-exposed trophoblast-vascular interactions,disruptingnutrientdelivery and conditions, theabsenceofPLP-Aobstructsearlystages adaptive responsestohypoxiaandresultinfetalloss.Underhypoxic mutations ineitherthe vasculature, includingitsinteractionswithtrophoblastcells.Null mesometrial deciduaandalterationsintheuterine maternal-fetal interface. Mostnotableareacompressionofthe the pregnancy-dependent adaptationsareevents occurringatthe the timing,durationandmagnitudeofhypoxicexposure. Among fetal loss(Ho-Chenetal.,2006).Adaptationsaredependentupon to hypoxia. both PLP-AandDPRPmodulatepregnancy-dependent adaptations subtle influencesunderordinaryhusbandryconditions.However, superfamily producedbyuterinedecidualcells,DPRP, alsohas In thecurrentstudy, wehave shown thatanothermemberofthePRL laboratory housingconditions(Mülleretal.,1999;Ain2004). only modesteffects onthebiologyofpregnancy understandard Mouse GenomeInformatics),targets uterineNKcellsandimposes trophoblast cell-derived PRLfamily member, PLP-A(PRLPA – pregnancy (Soares,2004).Previously, wedemonstratedthata on geneexpression patterns,thePRLsuperfamily islinked to Wild-type pregnant micecaneffectively adapttohypoxiawithout -null placentaisabletosatisfactorily progressthroughthis (+/+) and( normoxia orhypobarichypoxia. trophoblast invasion(arrowheads inC,F)the E, respectively. Notethedecreased endovascular images oftheareas delineatedbytheboxesinBand immunostaining. CandFare highmagnification Trophoblast cellswere identifiedbycytokeratin normoxia (A,D)orhypobarichypoxia(B,C,E,F). wild-type and within day11.5uteroplacental compartmentsof Fig. 9.Invasivetrophoblast celldistribution bars: 1mmforA,B,D,E of theuterusislocatedattop ofeachimage.Scale null miceexposedtohypoxia.The mesometrialregion decidual layer)inthe lines demarcate thethicknessofmesometrial chorioallantoic placenta(CversusF, dashedblack compressed mesometrialdeciduaandenlarged giant cells(BversusE,arrowheads), andthe arrowheads), theovergrowth oftrophoblast mesometrial bloodspaces(AversusD, hypoxia. and uteroplacental compartmentsofwild-type Fig. 8.Histologicalexaminationofday11.5 B Hematoxylin andEosin(A,B,D,E)orbyisolectin sectionswere stainedwith hypoxia. Tissue null (–/–)micewere exposedtohypobaric the topofeachimage.Scalebars:500 mesometrial region oftheuterusislocatedat 4 Plp-a itceity(,) Notetheenlarged histochemistry (C,F). Dprp D-F ) gene orthe Dprp ( -null miceexposedtohypobaric Dprp A-C ) Wild-type (+/+)and( ) Wild-type -null (–/–)micewere exposedto RESEARCH ARTICLE -null miceexposedto Dprp Dprp -null tissues.The gene interferewith ( A-C ) Wild-type D-F ␮ ) m. Dprp Dprp 413 - -

DEVELOPMENT Ain, R.,Dai,G.,Dunmore, J.H.,Godwin,A.R.andSoares, M.J. Ain, R.,Canham,L.N.andSoares, M.J. References HD39878, HD48861,HD49503)andtheHallFamilyFoundation. supported bygrantsfrom the NationalInstitutesofHealth(HD20676, with DrNorbertoC.Gonzalezregarding thebiologyofhypoxia.Thisworkwas and mouseMt1cDNA,respectively. We alsoacknowledgehelpfuldiscussions We thankDrsJanetRossant andGlenK.Andrews forthemouseR1EScellline trophoblast invasion characterize the distorted chorioallantoicplacentas,anddecreasedendovascular appearance ofvascular lesions,enlarged mesometrialbloodspaces, in theuterinemesometrialcompartmentandplacenta.The 414 Ain, R.,Konno,T., Canham,L.N. and Soares, M.J. unknown. Someinsightsintothe hypoxia. Thespecific aberrationthatleadstopregnancy failure is a key tounderstandingthephenotypeof coordinating uteroplacentaladaptationstohypoxiaandmayprovide DPRP. Thismesometrialdecidualstructuremaybecrucialin place moresignificance onthemesometrialdecidualcellsourceof compartments ofwild-typeand By contrast,prominentdifferences werenotedinthemesometrial tissues from organization ofanti-mesometrialdeciduaorinitsneighboring mouse. Inthepresentstudy, abnormalities werenotobserved inthe expression isinitiatedbetweendays5.5and6.5ofgestationinthe 1997b; Rasmussenetal.,1997)(Figs2,3).Decidualcell proximal tothedeveloping chorioallantoicplacenta(Orwigetal., in asmallerpopulationofmesometrialdecidualcellssituated Gibori, 1995).DPRPisexpressed inanti-mesometrialdeciduaand structurally andfunctionally(Krehbiel,1937;Bell,1983;Gu compartment. Mesometrialandanti-mesometrialdeciduadiffer opposite sideoftheuterusisreferredtoasanti-mesometrial entry isreferredtoasthemesometrialcompartment,and the entrysiteofvasculature. Theregion associatedwithvascular chorioallantoic placenta. deduced frominspectionofdecidualtissueadjoiningthedeveloping placentation. as atoolforelucidatingintrinsicregulatory processescontrolling stressors, andhasdemonstratedtheeffectiveness ofinvivo hypoxia of mechanismscontrollingdecidualcelladaptationstophysiological physiological stressorsremainstobedetermined. functionally overlaps withDPRPandfacilitates adaptationsto Critchley, 2001).WhetherhumanPRLproducedbydecidualcells al., 1999),anditstargets arelikely tobeintrauterine(Jabbourand human decidualcells,possessesanaffinity forheparin(Khuranaet vascular-trophoblast interactions.Interestingly, PRLisproducedby Alternatively, DPRPmayindependentlymodulatemesometrial mesometrial decidua(assuggestedbythepresentstudy). activity requiredforthedifferentiation and/orsurvival ofthe action ofDPRPisunknown but mayincludeanautocrine/paracrine signaling pathway (Rasmussenetal.,1996).Themechanismof structurally relatedtoPRL,DPRPdoesnotutilizethePRL-receptor the decidualextracellular matrix.AlthoughtheDPRP proteinis suggests thatDPRPdoesnotcirculatebut insteadisdepositedwithin structures (Rasmussenetal.,1996;Wang etal.,2000).Evidence exposed tohypoxia. phenotype andregulation. intrauterine trophoblast cellinvasionintheratandmouse:novelendocrine stressor. prolactin familyparalogregulates reproductive adaptationstoaphysiological The orientationofthepost-implantationuterusisdeterminedby Investigation ofthe DPRP isacytokine possessinganaffinity forheparin-containing RESEARCH ARTICLE Proc. Natl.Acad.Sci. USA Dprp -null miceundernormoxicorhypoxicconditions. Dprp Dev. Biol. -null mousehaspermittedadissection 101 Dprp 260 , 16543-16548. (2003). Gestationstage-dependent , 176-190. Dprp -null mice.Suchobservations Dprp -null phenotypemaybe -null mutantresponseto (2006). Phenotypic analysis Dprp -null mouse (2004). A Dprp Dorshkind, K.andHorseman,N.D. Alam, S.M.K.,Ain,R.,Konno,T., Ho-Chen,J.K.andSoares, M.J. Godwin, A.R.,Stadler, H.S.,Nakamura,K.andCapecchi,M. R. Faria, T. N.,Deb,S.,Kwok,S.C.M.,Talamantes, F. andSoares, M.J. Enders, A.C.andWelsh, A.O. Duckworth, M.L.,Peden,L.andFriesen,H.G. Aplin, J. Hiraoka, Y., Ogawa,M.,Sakai,Y., Takeuchi, Y., Komatsu,N.,Shiozawa,M., Gu, Y. andGibori,G. Fryer, B.H.andSimon,M.C. Forsyth, I.A.andWallis, M. Ishibashi, K.andImai,M. Ho-Chen, J.K.,Ain,R.,Alt,A.,Wood, J.G.,Gonzalez,N.C.and Soares, M.J. Arroyo, J.A.,Konno,T., Khalili,D.andSoares, M.J. Bilinski, P., Roopenian,D.andGossler, A. Bell, S.C. Deb, K.,Reese,J.andParia,B.C. Brosens, J.andGellersen,B. Brar, A.K.,Frank,G.R.,Kessler, C.A.,Cedars,M.I.andHandwerger, S. Dimitriadis, E.,White,C.A.,Jones,R.L.andSalamonsen,A. DeFeo, V. J. Dai, G.,Lu,L.,Tang, S.,Peal,M.J.andSoares, M.J. Dai, G.,Wang, D., Liu,B.,Kasik,J.W., Müller, H.,White,R.A.,Hummel,G. Croze, F., Kennedy, T. G.,Schroedter, I.C.andFriesen,H.G. Dai, G.,Liu,B.,Levan,Szpirer, C.,Kwok,S.C.M.andSoares, M.J. Cohick, C.B.,Xu,L.andSoares, M.J. Carson, D.D.,Bagchi,I.,Dey, S.K.,Enders,A.C.,Fazleabas,T., Lessey, B. of theratplacenta.In uterus duringhemochorialplacentaformation. and immunesystemhomeostasis. Mamm. Genome The ratprolactin genefamilylocus:species-specificexpansion. (ed. M.J.Soares andJ.S.Hunt),pp.295-313.Totowa, NJ:HumanaPress. Natl. Acad.Sci.USA Detection oftargetedGFP-Hoxgenefusionsduringmouseembryogenesis. 495-498. developing ratplacenta. 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DEVELOPMENT Müller, H.,Ishimura,R.,Orwig,K.E.,Liu,B.andSoares, M.J. Jabbour, H.N.andCritchley, H.O.D. Pregnancy-dependent adaptationstohypoxia Müller, H.,Liu,B.,Croy, B.A.,Head,J.R.,Hunt,S.,Dai,G.andSoares, M. Myatt, L. Mantena, S.R.,Kannan,A.,Cheo,Y.-P., Li,Q.,Johnson,P. F., Bagchi,I.C.and Lydon, J.P., DeMayo,F. J.,Funk,C.R.,Mani,S.K.,Hughes,A. Krehbiel, R.H. Kimura, F., Takakura, K.,Takebayashi, K.,Ishikawa,H.,Goto,S.andNoda,Y. Khurana, S.,Kuns,R.andBen-Jonathan,N. Orwig, K.E.,Ishimura,R.,Müller, H.,Liu,B.andSoares, M.J. Nagy, A.,Rossant,J.,Nay, R.,Abramow-Newerly, W. andRoder, J.C. Lin, J.,Poole,J.andLinzer, D.I.H. Liang, L.,Fu,K.,Lee,D.Sobieski,R.J.,Dalton,T. P. andAndrews, G.K. Orwig, K.E.,Rasmussen,C.A.andSoares, M.J. Orwig, K.E.,Dai,G.,Rasmussen,C.A.andSoares, M.J. Reprod. Homologues forprolactin-like protein-A andBare present inthemouse. 572 prolactin inearlypregnancy. cytokine, prolactin-like protein A. J. Acad. 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