Egg serpins: The chicken and/or the egg dilemma

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Egg serpins: The chicken and/or the egg dilemma

Clara Dombre, Nicolas Guyot, Thierry Moreau, Philippe Monget, Mylène da

Silva, Joël Gautron, Sophie Réhault-Godbert

To cite this version:

Clara Dombre, Nicolas Guyot, Thierry Moreau, Philippe Monget, Mylène da Silva, et al.. Egg serpins:

The chicken and/or the egg dilemma. Seminars in Cell and Developmental Biology, Elsevier, 2017,

62, pp.120-132. �10.1016/j.semcdb.2016.08.019�. �hal-01595204�

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SeminarsinCell&DevelopmentalBiology62(2017)120–132

ContentslistsavailableatScienceDirect

Seminars

in

Cell

&

Developmental

Biology

jo u r n al h om ep age : w w w . e l s e v i e r . c o m / l o c a t e / s e m c d b

Review

Egg

serpins:

The

chicken

and/or

the

egg

dilemma

Clara

Dombre

a,b,c,d

_,

_Nicolas

_Guyot

e

_,

_Thierry

_Moreau

f

_,

_Philippe

_Monget

a,b,c,d

_,

Mylène

Da

Silva

e

_,

_Joël

_Gautron

e

_,

_Sophie

_{Réhault-Godbert}

e,∗

a_INRA,_UMR85_Physiologie_de_la_Reproduction_et_des_{Comportements,}_F-37380_Nouzilly,_France b_CNRS,_UMR6175_Physiologie_de_la_Reproduction_et_des_{Comportements,}_F-37380_Nouzilly,_France c_Université_Franc¸_ois_Rabelais_de_Tours,_F-37041_Tours,_France

d_IFCE,_F-37380_Nouzilly,_France

e_INRA,_UR83_Recherches_Avicoles,_F-37380_Nouzilly,_France

f_CEPR,_UMR_INSERM_U1100,_Faculté_de_Médecine,_Université_de_Tours,₁₀_Bd._Tonnellé,_F-37032_Tours_Cedex,_France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received15March2016

Receivedinrevisedform22June2016 Accepted22August2016

Availableonline24August2016 Keywords: Serpins Birds Eggformation Reproduction Development

a

b

s

t

r

a

c

t

Twenty-sevenserpinsbelongingtocladeA,B,C,D,E,F,G,HandIserpinsarecurrentlyreferencedin chickengenomedatabases.Phylogeneticanalysisofchickenserpinsrevealedthatovalbumin(Serpinb14) anditsparalogsovalbumin-relatedproteinY(Serpinb14b)andovalbumin-relatedproteinX(Serpinb14c) arefoundinbirdspecies.ThesecladeBserpinsarespecificallyexpressedinreproductivetissuesand exportedintheeggwheretheyconstitutemajorproteincomponents.Thesedatasuggestthatthese threeparalogshaveprobablyappearedinbirdstofacenewenvironmentsandensuretheextra-uterine developmentofanembryoinashellegg.Twelveotherserpinshavebeenidentifiedinthenewlyproduced egg,someofthemhavingaspecificdistributionintherespectiveeggstructures(eggshell,eggwhite, vitellinemembraneandeggyolk).Thephysiologicalroleoftheseeggserpinsremainlargelyunexplored, butthereisincreasingevidenceinliteratureorbyhomologieswiththeirmammaliancounterparts,that someofthemparticipateincellproliferation,tissueremodelingand/orangiogenesisassociatedwith folliculogenesisanddevelopmentofextraembryonicstructures,eggshellbiomineralization,eggdefense andnutritionoftheembryo.Abetterknowledgeofthephylogeneticevolutionofthese15serpinsin otheroviparousspecies,ontheireggdistribution,ontheirregulationduringembryonicdevelopment (activation/degradation/transfer)andontheirfunctionalspecificity,isneededtobetterappreciatetheir roleandtheirbird-specificity.Thesereviewshedlightonthemultiplepossibilitiesthatoffertheavian eggmodeltostudytheroleofserpinsinreproductionanddevelopmentalbiology.

Contents

1. Evolutionanddistributionofeggserpins...121

1.1. Phylogeneticanalysisofchickenserpins...121

1.2. Tissuedistributioninchicken(thechickenand/ortheeggquestion)...123

2. Eggserpins:anupdate...123

2.1. Biologicalsigniﬁcanceofeggyolkserpins ... 124

2.2. Vitellinemembraneserpins:apredictedroleinfolliculogenesis,defenseandangiogenesis...126

2.3. Eggwhiteserpins:aroleinnutritionanddefense.Whatelse?...127

2.4. Eggshellserpinsasregulatorsofbiomineralizationprocess...127

2.5. Serpinsinextraembryonictissues...130

2.5.1. Amnioticﬂuid...130

Abbreviations:ACC,amorphouscalciumcarbonate;CAM,chorioallantoicmembrane;ESM,eggshellmembrane;OVAX,ovalbumin-relatedproteinX;OVAY, ovalbumin-relatedproteinY.

∗ Correspondingauthorat:INRACentreValdeLoire,UR83RecherchesAvicoles-FunctionandRegulationofEggProteins,37380Nouzilly,France. E-mailaddress:srehault@tours.inra.fr(S.Réhault-Godbert).

http://dx.doi.org/10.1016/j.semcdb.2016.08.019

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2.5.2. Chorioallantoicmembraneandallantoicﬂuid...130

2.5.3. Eggshellmembranes...130

2.5.4. Yolkandyolksac...131

3. Conclusions...131

Conﬂictofinterest ... 131

Acknowledgements...131

References...131

1. Evolutionanddistributionofeggserpins

SystematicanalysisofNationalCenterforBiotechnology Infor-mationandchickenEnsembldatabasesforidentifyingserpinsin chickenspeciesrevealedthepresenceof27membersofthis fam-ily(Table1, Fig.1).Amongthese,16 serpinsare stillpredicted andhave beenreferenced indatabasesbyautomated computa-tionalanalysisofgenomicchickensequencesannotatedusinggene predictionmethods,whichsometimescanleadtodiscrepancies betweengeneandproteinnames(asanexample,Serpinb2gene correspondstoapredictedSerpinb10protein,Table1).However, therelevanceof5ofthemhavebeenrecentlyvalidated,as ser-pinsa8,c1,d1andf1wereunambiguouslyidentifiedintheegg (see§1.2).Thus,11oftheserpinsidentifiedtodatefromgenome analysis, need further validation and the information available abouttheirphysiologicalfunctionsisthereforeverypartial,even inexistent. Surprisingly enough, Serpine1was not identified in this analysis.To investigatewhether Serpine1 existsas a pseu-dogeneinchickenorwhetheritwasnotannotatedcorrectlyin databases,Serpine1wassearched inthechicken genomeusing reciprocaltBLASTn.Theserpinwasnotfoundandtheclosestgene identifiedbyreciprocaltBLASTnagainsthumangenomereferred toSERPINE2,which wasactually identifiedinboth Pubmedand Ensembldatabasesandreferenced inTable1.Thus,todate,we cannotascertainthatthisgeneisabsentduetoerrorsingenome annotationorwhetheritactuallydisappearedinchickensduring evolution.Chickenov-serpinsarelargelyrepresentedas10clade Bserpinscouldbeidentified[1,2].Theseov-serpinsareclustered ona150kblocuschromosome2q(Fig.1B)andcompriseSerpinb1, Serpinb2,Serpinb5,Serpinb6,twoSerpinb10homologs(Serpinb10, Serpinb10b/MENT),Serpinb12 andSerpinb14,Serpinb14b and Ser-pinb14cnamelyovalbuminanditsrelatedgenesY(OVAY)andX (OVAX).Anotherclusteronchromosome5wasidentified contain-ing7membersoftheSerpinafamily(Fig.1E).Thisclusterincludes5 homologsofalpha1-antitrypsin/alpha1-proteinaseinhibitors, Ser-pina1,Serpina3,Serpina4,Serpina5,Serpina9,whichcorrespondto humanantitrypsin, alpha1-antichymotrypsin,kallistatin, Protein Cinhibitor,andSERPINA9,respectively.Outofthese27serpins, only15areactuallyrecoveredinthechickenegginwhichthe bio-logicalsignificanceandbiologicalactivityintimatelydependson theprocessofeggformationandontheirsubsequentlocalization (eggshell/eggwhite/vitellinemembrane/yolk).Thisfirstpartwill reviewtheevolutionofthesechickenserpinsinvertebratespecies andtheirdistributioninthevariouseggcompartments.

1.1. Phylogeneticanalysisofchickenserpins

Inserpingenes,somehaveshownastrongcorrelationbetween genomicorganization,patternsofaminoacidsatspeciﬁcsites,and insertion/deletionpatterns,whichcontributedtoidentifyserpin groupsandtodeciphervertebrateserpinevolution[3,4].Serpin geneshaverapidlyevolved;ahighsequencedivergenceisfound betweenallserpinclades,thesequenceidentityvaryingfrom22% to29%.

Phylogeneticanalysisofthe27serpins foundinthechicken genomeshowsthatserpingeneshavebeenoriginatedand

dupli-catedbeforethedivergence ofteleosteans.Anotherduplication eventoccurredafterdivergencebetweenspecies,forexample,the cladeAofGallusgallusencompassessevenSerpinageneswitha sequenceidentityaround47%.Thesamephenomenonisobserved formammalandﬁsh.

UsingthephylogenetictreesavailableinEnsembl(http://www. ensembl.org),andbecauseofthestringencyofthemethodused, threeserpinsgroupsaredistinguished.Theﬁrstphylogenetictree containsserpinsfromcladeB,C,EandI(Fig.2),thesecondrefersto serpinsfromcladeA,D,F,GandH(Fig.3),andthelasttree,contains onlyonecladeAserpin,Serpina8.

ThecladeBserpin,present inthefirsttree,contains ovalbu-mingene(Serpinb14)anditsrecentlyduplicatedOVAY(Serpinb14b) andOVAX(Serpinb14c)[5–7].OVAX,isnotannotatedinEnsembl butbyusingreciprocaltBLASTnalignment method(http://blast. ncbi.nlm.nih.gov/Blast.cgi),thisgenecouldbeeasilyidentifiedas the closestneighbor of OVAY in chromosome2 witha 73% of proteinsequenceidentityaspreviouslyshown [1,2].The evolu-tionofcladeBserpinsstartsbeforethesplitofbonyfishesand tetrapods,450millionsyearsago,leadingtoatleastsixcladeB serpingenesfoundinmammaland birdgenomes.TheG.gallus genomecontainstencladeBserpingenesonchromosome2in thesamesynteniclocus(Figs.1and2).Sixofthemcouldalsobe foundinHomosapiensgenome(SERPINB1,B2,B5,B6,B10,B12). ChickencladeBserpins(Figs.1and2)aresyntenicwithtwolocion chromosome6and18inhumanandothermammaliangenomes, whichsuggestsasplitresultingtoabreakofsyntenyinmammals

[2].Recentduplicationseventsoccurredindependentlyinfishes, mammalsandbirds,afterdivergencebetweenthesespecies.Due torecent duplication,avian Serpinb14 (ovalbumin),Serpinb14b (OVAY)andSerpinb14c (OVAX),havenohumanorother mam-malianspeciesorthologuesandseemtobespecifictooviparous species[1,8,9].Threeorthologuestoovalbuminarereferencedin duck,flycatcherandturkeyinEnsembldatabasebutinformation islackingforotheroviparousspecies.However,itisnoteworthy thatwefoundapotentialorthologousofSerpinb14c inAlligator mississippiensis(A0A0Q3ZV65),sharing56%proteinsequence iden-titywiththechickenhomolog.Theseovalbumingenesadaptedto oviparousspecies,aresupposedtohavelosttheirproteaseinhibitor activity[1],and potentiallyacquiredspecificpropertiesadapted tothedevelopmentofanembryoinaneggexposedtoterrestrial environments[8,9].

CladeA,D,F,GandHserpinsarefoundinthesecond phyloge-netictree.AsshowninFig.3,exceptcladeAserpins,eachofthese serpinshasanorthologueinbothG.gallusandBostaurus.

SimilarlytocladeBserpins,andforbothspecies(G.gallus;B. taurus), duplicationevents ledtothepresence of severalclade A serpinsafterspecies divergence(Fig.4).ConcerningG. Gallus genome,thisduplicationgaverisetoSerpina1,a3,a4,a5,a9,a10 anda12.Theseserpinshavesimilarpeptidaseinhibitorfunction and areessentiallyexpressedbytheliver.Thesynteny analysis (http://www.genomicus.biologie.ens.fr/)revealsthatcladeAgenes arelocalizedinageneclusterinchickenchromosome5(Fig.3). ThesamephenomenonisobservedinB.Taurus,whereallcladeA serpinsarelocalizedinauniquegeneclusteronchromosome21. CladeAserpinsshowmultiplerecentduplicationsleadingtoten

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122 C.Dombreetal./SeminarsinCell&DevelopmentalBiology62(2017)120–132

Fig.1.Chromosomelocalizationofchickenserpins.Serpinsandﬂankinggeneswiththeirrespectiveorientation(backward/forward)aredrawntoscale.Assignmentofgene nameswasestablishedbasedonEnsembldatabaseinformationorbycomparisonwithhumanorthologs.ThenameMENT(GeneID:1017749622)hasbeenreplacedby Serpinb10inaccordancewith[1],basedonthehighsequenceidentityofSerpinb10andSerpinb10b,suggestingthattheyareparalogs.Accessionnumbersareindicatedin Table1.WiththeexceptionofSerpinb14c,allgeneswerefoundinEnsembldatabase.

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Table1

Chickenserpins.AnalysisofPubmeddatabase(http://www.ncbi.nlm.nih.gov/pubmed/)using“serpin”and“gallus”askeywordsin“Protein”databaseresultedin93proteins hits;whereasusing“serpin”tosearchrelatedproteinsinChickenEnsembldatabase(http://www.ensembl.org/Gallusgallus/)gave83hits(01-18-2016).Manualcompilation andintegrationofresultsbyremovingredundancytokeeponlygeneIDsresultedinatotalof27distinctserpingenes.

ProteinName[Gallusgallus] Proteinaccessionnumber Genesymbol GeneID Geneaccessionnumber serpinpeptidaseinhibitor,cladeA(alpha-1antiproteinase,

antitrypsin),member1precursor

NP001264422.1 Serpina1 423434 ENSGALG00000023070 PREDICTED:proteinZ-dependentproteaseinhibitor XP015143253.1 Serpina10 423432 ENSGALG00000020391 PREDICTED:serpinA3-4-likeisoformX1 XP015143255.1 Serpina12 107049127 ENSGALG00000028598 PREDICTED:alpha-1-antiproteinase XP015143260.1 Serpina3 772339 ENSGALG00000020388 serpinpeptidaseinhibitor,cladeA(alpha-1antiproteinase,

antitrypsin),member4precursor

NP 001264421.1 Serpina4 423433 ENSGALG00000010969 PREDICTED:alpha-1-antitrypsinisoformX1 XP421344.1 Serpina5 423435 ENSGALG00000020390 PREDICTED:angiotensinogen XP004935550.1 Serpina8 421543 ENSGALG00000011117 PREDICTED:alpha-1-antitrypsin XP004941955.1 Serpina9 423436 ENSGALG00000020389 PREDICTED:leukocyteelastaseinhibitorisoformX1 XP015137679.1 Serpinb1 420894 ENSGALG00000019555 PREDICTED:heterochromatin-associatedproteinMENT XP 004939740.1 Serpinb10 101749622 ENSGALG00000019554 heterochromatin-associatedproteinMENT O73790.1 Serpinb10b 395715 ENSGALG00000019553 PREDICTED:serpinB12 XP418985.2 Serpinb12 420899 ENSGALG00000012872

ovalbumin AAB59956.1 Serpinb14 396058 ENSGALG00000012869

ovalbumin-relatedY NP 001026172 Serpinb14b 420897 ENSGALG00000019551 ovalbumin-relatedproteinX AGN32861.1 Serpinb14c 420898 NM001276386.1 PREDICTED:serpinB10 XP418982.1 Serpinb2 420896 ENSGALG00000019552 PREDICTED:serpinB5 XP418986.3 Serpinb5 420900 ENSGALG00000012873

serpinB6 NP001006377.1 Serpinb6 420895 ENSGALG00000012866

PREDICTED:antithrombin-III XP422282.3 Serpinc1 424440 ENSGALG00000004591 PREDICTED:heparincofactor2 XP001232767.1 Serpind1 395877 ENSGALG00000001396 SERPINE2serpinpeptidaseinhibitor,cladeE(nexin,plasminogen

activatorinhibitortype1),member2

E1BWU2 Serpine2 424805 ENSGALG00000005135 PREDICTED:serpinE3 XP015131346.1 Serpine3 418875 ENSGALG00000017017 PREDICTED:pigmentepithelium-derivedfactorisoformX1 NP001244218.1 Serpinf1 417561 ENSGALG00000003015 serpinpeptidaseinhibitor,cladeF(alpha-2antiplasmin,pigment

epitheliumderivedfactor),member2

XP015151529.1 Serpinf2 100857105 ENSGALG00000002987 plasmaproteaseC1InhibitorPrecursor XP003641424.1 Serping1 423132 ENSGALG00000007381 PREDICTED:serpinH1isoformX1 XP015136453.1 Serpinh1 396228 ENSGALG00000011214 neuroserpinprecursor NP001004411.1 Serpinh1 425002 ENSGALG00000009470

Serpina(Serpina1,a3,a4,a5,a6,a7,a10,a11,a12,a14).Sixofthem belongtothesubgroupofSerpina3(Serpina3-1,a3-5,a3-6,a3-7, a3-7-like,a3-8),andsharealmost70%ofidentitywithhuman SER-PINA3members[10].ThesyntenyanalysisofcladeAserpinsfor B.taurusshowsthepresenceofageneclusterinchromosome21, similartotheclusteronchromosome5foundinG.gallus.Inboth G.gallusandB.taurusloci,serpinsfromcladeAarelocalizedinthe samesynteniclocus.

Inthethird phylogenetictree,onlySSerpina8ispresentand couldbefoundinallspecies.Incontrasttotherecentpublication whereauthorsusedsyntenyandsignaturesequencestoanalyse Serpina8gene inotherspecies[11],Ensemblphylogenetictools revealsthattheduplicationofSerpina8occurredbeforedivergence ofteleosteans.Thisserpinhasalsodivergedrapidlyasopposedto theotherserpins.ComparedwithothercladeAinhibitoryserpins, thisSERPINA8alsonamedangiotensinogenhasaveryspeciﬁcrole invertebrates,inthatit isproteolyticallyprocessedbyreninto generateangiotensinI,whichisfurthertrimmedintovasoactive angiotensinIIthatregulatesbloodpressure.

Toconclude,serpinshavebeenduplicatedbeforethedivergence ofteleosteangivingriseto16clades(fromAtoP)[12,13].Nine cladesarepresentin theaviangenomeandtwoofthem (clade AandB)havebeenrecentlyduplicatedleading,amongothers,to ovalbumin,OVAXandOVAY,whicharespeciﬁctobirdspecies.A betterunderstandingofthefunctionoftheseproteinsisnecessary tohighlightthereasonoftheirduplicationandspeciﬁcity. 1.2. Tissuedistributioninchicken(thechickenand/ortheegg question)

Thereisveryfewinformationontheexpressionandbiological activitiesof chickenserpins.Chickens,byhomologywith

mam-mals areexpectedtoexpressserpins ata basal statetoensure basicbiological processes suchascoagulation/hemostasis (PRE-DICTED:angiotensinogen,PlasmaProteaseC1InhibitorPrecursor, PREDICTED:antithrombin-III,PREDICTED:heparincofactor2),cell proliferation(SERPINB5)althoughcontroversial[14],inflammation (PREDICTED:leukocyteelastaseinhibitor),etc.Atsexualmaturityof thepullets,thesecretionofovariansteroidhormonesbythetheca ofthegrowingfollicletriggersthedevelopmentand differentia-tionofthehenoviduct.Thisdevelopmentisconcomitantwiththe formationofthefirsteggthatwillcontainnutrientsandbioactive proteinstosupportembryonicdevelopment[15].Theformation ofchickeneggisaspatialandtemporalprocessthatreliesonthe ovary(siteofsexsteroidsynthesis,gametogenesisandyolk for-mation)andtheoviduct,whichreceiptstheovulatedmatureyolk andwherethewhite,theshellmembranesandtheshellare suc-cessivelydepositedinveryspecializedregions,themagnum,the isthmusandtheuterus/vagina,respectively(Fig.5)[15].Analyses andintegrationofthevariousproteomicdatapublishedonthe cuti-cle/eggshell,eggwhite,vitellinemembraneandeggyolkrevealed thepresenceof15serpinsinthefreshlylaidegg[16–28](Fig.5).The eggshellcontains14differentserpins,withSerpinb6andSerpini1 beingspecificallyfoundinthiscompartment.Theeggwhite recov-ers6serpinsincludingonewhichhasbeenidentifiedonlyinthis compartment(Serpinb5).Thevitellinemembranepossesses5 ser-pinswhicharealsocomponentsoftheeggwhiteandtheeggshell, andtheeggyolkcontains10serpinsthatarealsolistedintheother eggcompartments(Fig.5).Aspreviouslydiscussed,3oftheseegg serpins(Serpinb14,Serpinb14b,Serpinb14c)mightbespecifically associatedwithbirdspeciesandtheextra-uterinedevelopmentof theembryowithinanegg.

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Fig.2. PhylogenetictreeofSERPINB,C,EandIandlocirepresentation.Phylogenetictree,generatedbyEnsembl,mergingmaximumlikehoodandneighborjoiningtrees, showsserpinsorthologsfoundforchickenandcow.AllG.galluscladeBserpinsarelocalizedinthesamegeneclusteronchromosome2.

*TheSerpinb14c,notannotatedinEnsemblwasmanualyaddedtothisﬁgure.Theproteinsequencesofthe27serpinsgenesfoundinthechickengenegenomewerealigned andaserpinphylogenetictreewasgenerated(usingthewebsitehttp://www.phylogeny.fr),inordertocomparevalidatethepositionofSerpinb14cinthephylogenetictree.

2. Eggserpins:anupdate

Withtheexceptionofovalbumin (Serpinb14)and itsrelated proteinX(Serpinb14c)andY(Serpinb14b),mosteggserpins recov-ered in the egg are not speciﬁcally expressed to support egg formation.Therefore,toappreciatetheirrespectivephysiological activityinegg,itisimportanttohaveagoodrepresentationofthe processofeggformationinmind,astheirfunctionisintimately linkedtotheirlocalizationwithintheegg.

2.1. Biologicalsigniﬁcanceofeggyolkserpins

Major proteins of the egg yolk, with the exception of immunoglobulins,aresynthesizedbytheliveroflayinghensin whichproteinsynthesisandlipogenesisarestimulated15–20fold atsexualmaturity.Eggyolkproteinsresultfromthestimulation ofhepaticexpressionofpreexistingproteinsandneosynthesisof speciﬁceggcomponents.Oncesecretedintotheblood,eggyolk

precursorssuchasvery-lowdensitylipoproteinsaretransported totheovarianfollicleandincorporatedinthegrowingyolky folli-cles,viareceptor-mediatedendocytosis[29].Meanwhile,theliver continuestoexpressmanyproteinswhicharenotrelatedto vitel-logenesisand which canbeunselectively incorporatedintothe eggyolkbypassivebindingtoeggyolk-specificproteins.Eleven serpins havebeenidentified intheegg yolk:Serpina1 (alpha1-antitrypsin), Serpina4 (kallistatin), Serpina8 (angiotensinogen), Serpinb14(ovalbumin),Serpinb14b(OVAY),Serpinb14c(OVAX), Serpinc1(antithrombinIII),Serpind1(heparincofactorII),Serping1 (plasma Protease C1 Inhibitor), Serpinf1 (pigment epithelium-derivedfactor)andSerpinf2(alpha2-antiplasmin)[25,27].Except Serpina1,Serpinb14,Serpinb14b,Serpinb14c,serpinsidentifiedin eggyolkareexpressedbythechickenliver,regardlessofthe sex-ualmaturityofthehens[30],corroboratingthattheseserpinsare notspecificallyexpressedtosupportvitellogenesis.Serpina1, Ser-pinb14, Serpinb14b,Serpinb14c might beexpressedwithinthe ovarybysurroundingcellsincludinggranulosacellsandthecato

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Fig.3.ComparativeanalysesofGallusgallusandBosTauruslociincludingcladeA,D,F,G,andHserpins.PhylogenetictreesaregeneratedbyEnsembl.CladeAserpinsare foundinthesamesyntenicclusterinbothG.gallusandB.taurus.TheB.taurusgenomewaspreferredforthisstudyasitiswellreferencedforthislocus.Moreover,serpinsfrom cladeBfoundinB.taurusgenomepossessmoreduplications(aschickenSerpinb14forexample)ascomparedwithhumangenome.Thephylogenetictreeswereprocessed byENSEMBL,withthecompletesequencesincludingamino-andcarboxy-terminalextensions.

Fig.4.EvolutionaryscenarioofSERPINfromcladeA,D,F,GandHformammal,birdsandﬁshes.LastSERPINAduplicationappears,formammals,birds,andﬁshes,after divergencebetweenspecies.Speciationbranchesarerepresented:forexample,forSERPING1andSERPINF2thenoderepresentsthelastcommonancestor,andthedivergence beforespeciationbetweendifferentspecies.Thereddotrepresentsparalogduplicationafterdivergencebetweenspecies.ThisphylogenetictreewasmadeusingPRANK (http://www.ebi.ac.uk/goldman-srv/prank/)toalignsequencesandRAxML(http://sco.h-its.org/exelixis/software.html)tobuildthetree.

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Fig.5.Schematicrepresentationofeggformationandserpinsidentiﬁedineacheggcompartments.Foreachcompartment,thesizeofthefontroughlyindicatestherelative quantityofoneserpintoanother.Biggerlettercorrespondstoabundanttoveryabundantproteins,smallerletterscorrespondtolowabundantproteinsandnormalletters toproteinswithintermediateabundance.Notethatcompartmentsarenotcomparable,astherelativequantityintimatelydependsontheintrinsiccompositionofeach compartment.Datawereextractedfrom[25,27](eggyolk),[26](vitellinemembrane),[22,28](eggwhite),and[23,80](eggshell).

beincorporatedin theyolk,orberecoveredintheeggyolk by passivediffusionfromtheeggwhitewheretheyaremajor com-ponents.Actually,theexhaustiveanalysisofserpinsexpressionin chickenmaleorfemaletissueshasneverbeeninvestigatedandin females,exceptforSerpinb14,Serpinb14bandSerpinb14c,their expressiondoesnotseemtobehormone-regulated[30].The func-tionalannotationof eggyolkproteinshasrevealedthat serpins identiﬁedintheeggyolkareessentiallyknownactorsof coagu-lation/ﬁbrinolysiscascades[24].Thisconsideration,togetherwith theirverylowabundanceineggyolk,questionsthebiological rel-evanceoftheseelevenserpinsintheeggyolk.

2.2. Vitellinemembraneserpins:apredictedrolein folliculogenesis,defenseandangiogenesis

Thevitellinemembraneis theacellularproteinaceous mem-braneat theinterfacebetweentheeggwhiteand theeggyolk. Thismembraneiscomposedoftwodistinctlayers(outerandinner layers)separatedbyathincontinuousmembrane[31].Theinner layer,incontactwiththeyolkandtheoocyteandcorresponding tothezonapellucidainmammals,isconstitutedofinterlaced pro-teinaceousfibersmostlikelyproducedbygranulosacellsand/or livercells,duringvitellogenesis/folliculogenesis.Theouterlayer containsproteinsformingalatticenetworkoffinefibrils,secreted bytheinfundibulum(Fig.5).Fertilizationoftheoocytebyasperm celloccursintheinfundibulum,presumablypriortothesecretion ofthevitellinemembraneouterlayer.Thevitellinemembranehas differentrolesinavianreproduction.Theinnerlayerencloseszona

pellucidaproteinsknownfortheirroleinsperm-egginteraction duringfertilization[32].Theinnersurfaceofvitellinemembrane promotescellgrowth[33]followingeggfertilization,whichinturn progressivelydegradesthevitellinemembranetoforma vascular-izedtissuearoundtheyolk,namelytheyolksac[34].Thevitelline membraneactsasa natural ﬁlterbarriertoseparateeggwhite fromyolkcomponents,andtopreventmicrobialcontaminationof yolkpotentiallycomingfromthealbumen.Theouterlayerofthe vitellinemembraneisalsorichinantimicrobialproteins(lysozyme, ovotransferrin,avianbetadefensin11).Proteomicanalysisofhen eggvitellinemembranerevealedthepresenceof137proteins[26]

including5serpins:Serpinb14(ovalbumin),Serpinb14b(OVAY), Serpinb14c(OVAX),Serpine2(glia-derivednexin/protease nexin-1) and Serpinf2 (alpha2-antiplasmin). The distributionof these avian serpins within the various layers of the vitelline mem-braneremainsunknowntodateandtheirbiologicalrolesarestill unclear.Aspreviouslymentioned,Serpinb14,Serpinb14band Ser-pinb14careegg-speciﬁcproteins.Theirrolewithintheeggandthe vitellinemembranestillremainselusivealthoughtheyconstitute majorcomponentsofthiscompartment[26](Fig.5).No protease-inhibitingactivityhasbeenfoundfortheseserpinstodate.Arecent study demonstrated that Serpinb14c possesses heparin-binding properties (Fig.6)and antibacterialactivities, in contrasttoits homologSerpinb14[35].Serpinb14cmightthereforecontributeto eggdefensetogetherwithotheractiveantimicrobialspresentin thevitellinemembrane.

By comparison with theirmammalian homologs, avian Ser-pine2 and Serpinf2 are presumed to have important functions

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ineggformation,especiallyduringfolliculardevelopmentand/or ovulation phases. Both serpins are expressed in bovine gran-ulosa cells [36,37] and temporal expression of Serpine2 gene canbeobservedduringfollicular growth:highexpressionlevel isindeed associatedwithlargegrowingfollicles whereaslower expression is rather observed in small growing follicles or in pre-ovulatoryfollicles[36].Plasmin,thecognateserineprotease of Serpine2 and Serpinf2 [38,39], is involved in the extensive remodeling of the follicular connective tissue and degradation of the basal lamina during follicular expansion. Ovulation also involvesproteolyticeventsatthefollicularapexresponsiblefor theformationofthestigmaandthereleaseofthematureoocyte. Plasminogen activators/plasmin system is believed to partici-pateinfollicularmaturationinchickens[40].Serpine2possesses glycosaminoglycan-bindingproperties[38]andknowingthatthere aresome pre-ovulatorychanges of glycosaminoglycanscontent (i.e. degradation) in the stigma, the area of the ovarian sur-facewherethematurechickenfolliclewillburstthroughduring ovulation [41],glycosaminoglycansproduced bygranulosa cells and their degradation might regulate theproteolytic processes duringthefolliculargrowthand/ortheovulation,viathe inter-actionwithfollicularserpinsandthemodulationoftheiractivity

[42].

Besides follicular growth, the vitelline membrane also sup-portscellproliferationandangiogenesisassociatedwithembryonic development. In chicken, Haas and Spratt have reported that componentsof theinner membrane promotethe outgrowthof extraembryonic tissues onto the vitelline membrane [33]. Ser-pinsfromvitellinemembranemightparticipateintheseprocesses viaboth antiprotease-dependentand independentmechanisms. SERPINE2 is known to interact with several modulators of angiogenesis,suchasproteases(thrombin,plasmin,plasminogen activators),extracellularmatrixproteinsandglycosaminoglycans

[43,44]. Antiangiogenic properties have been reported in vitro and in vivo for SERPINE2, which has been demonstrated to inhibit vascular epithelial growthfactor activity onendothelial cells(includingproliferationandmigration)andtodecreasecell spreading onvitronectin [45]. Interestingly, its anti-angiogenic effects do not involve its inhibitory site but rather rely onits glycosaminoglycan-bindingproperties[45].Serpine2might there-foreregulateangiogenesiswhichguidesthedevelopmentofyolk sacbytargetingpivotalcellsignalingpathwayssuchasthe Hedge-hogpathway[46].

2.3. Eggwhiteserpins:aroleinnutritionanddefense.Whatelse? Eggwhiteis anaqueoussolutionmainlycomposedofwater (88%), proteins(90%dry matter),minerals (6%dry matter) and freeglucose (3.5%drymatter). Thedominantphysiological role of egg white is assumed to provide nutrients for the embryo and protection against microbial contamination. Clade B ser-pins including ovalbumin (Serpinb14), OVAY (Serpinb14b) and OVAX(Serpinb14c)aremajorcomponentsoftheeggwhite[28], withovalbuminaccountingfor54%ofeggwhiteproteins(about 50mg/mL).Allthreeproteinsaremainlyproducedbytheoviduct and more speciﬁcally by tubular gland cells of the chicken’s magnum,responsibleforeggwhiteformation[8](Fig.5).The addi-tionalmajor proteinsfoundin this compartment arelysozyme, ovotransferrin,whichbothpreventbacterialproliferationand dis-semination. Eggwhite is alsocharacterized by thepresence of numerous active protease inhibitors including ovomucoid and ovoinhibitor (Kazal-like proteins), cystatin and ovostatin [47], which are assumed to protect egg white proteins from inap-propriate/early proteolytic events. Non-inhibitory properties of certainserpinsincludingovalbumin,ovalbumin-relatedproteinX ormaspin(Serpinb5)canbepossiblyexplainedbymultiple

devi-ationsinthehingeregionofthereactivecenterloop,compared withtheconsensus sequence for inhibitoryserpins [1] (Fig. 6). Thephysiologicalfunctionofallthreeparalogsisstillunclear.The on-goinghypothesisisthatitwouldserveasasourceof amino-acidsforthedevelopingchickenembryo fromtheeleventhday ofincubationwhiletheeggwhitemigratestotheamnioticfluid tobeorallyabsorbedbytheembryo.Uptothatstage,eggwhite proteinsareprobablyprotectedfromproteolysisthankstomajor eggwhiteactiveantiproteases.Indeed,eggwhiteproteinsremain essentiallyuncleavedduringthefirsthalfincubation,asrevealed by proteomicapproaches [48]. It is noteworthythat Serpinb14 naturallyundergoessomeconformationalchangesduringegg incu-bation,toconverttoaheat-stableformnamedS-ovalbumin[49]. ThisS-ovalbuminischaracterizedbychemicalinversionsof ser-ineresiduesintotheDconfiguration,andothersubtlechanges, that are supposed to give a thermodynamic advantage to the structuralstabilityofS-ovalbumin[50].Interestingly,once swal-lowed fromtheamnioticfluid,ovalbumin doesnotseem tobe fullyalteredinthegastrointestinaltractoftheembryo[49]. Oval-bumin is recovered in the extracts of many embryonic organs includingthehead,eye,heart,liver,intestine,spinal cord, mus-cle,dermis,andbone[49].Surprisinglyenough,thepresenceof uncleavedovalbuminpersistsinembryonicorganssuggestingthat atleastafractionofovalbumin moleculescouldbetransported intacttoembryonicorgans [49].Thisobservationtogetherwith theabsenceofovalbuminmRNAexpressionintheseorgansand withthefactthattheneonateorgansarenolongerpositivefor ovalbumin shortly afterhatching [49], suggeststhat egg white ovalbuminmaynotmerely serveasa sourceofamino acidbut mayalso havea more active/directfunctionon developing tis-sues.

WithregardtoOVAYandOVAX,thereisnoinformation avail-ableabouttheirsusceptibilitytoconverttoaS-form,similarlyto ovalbumin,norabouttheirpresenceinembryonictissuesduring incubation.But,theabundanceofOVAYineggwhitewasshown tobesigniﬁcantlyaffectedduringincubation [48].Its predicted Lys-His reactive site suggeststhat OVAY couldinhibit trypsin-like proteases [1] and thus possibly gastrointestinal proteases of theembryo and/oryolk proteases.ConsideringOVAX,it was shown tolack inhibitory activityagainst trypsin/chymotrypsin-likeproteases [35].However, itexhibits antimicrobialactivities against two pathogens, Listeria monocytogenes and Salmonella enterica Enteritidis via its heparin-binding domain [35]. These activities suggest a role for OVAX at least in innate defense. Similar function has been proposed for mammalian heparin-binding serpins including Heparin cofactor II/SERPIND1 [51], which is also present in theegg white. The expressionof Ser-pind1 and Serping1 in the oviduct and more particularly in the magnum has not been investigated yet. Their presence in the egg white could be the consequence of oviducal expres-sionbut couldalsoresultfrompassivediffusionfromtheyolk. Concerning Serpinb5, itsrole in the eggwhite willnot be fur-therdiscussedsinceit wasidentiﬁedasveryminorcomponent

[52].

2.4. Eggshellserpinsasregulatorsofbiomineralizationprocess Thecalcifiedchickeneggshellisanaturalenvelopewhich pro-tectsthedevelopingembryofromphysicalandmicrobialassaults. Itiscomposedof95%calciumcarbonate(calcitepolymorph),1.5% waterand3.5%proteins,polysaccharidesandproteoglycans[53]. Avianeggshellisaporousminerallayerwithawell-defined struc-turalpolycrystallineorganization(Fig.7A).Biomineralizationmay bedefinedastheproductionofthehardtissuecharacterizedbya specificminerals/organicmatrixframework,byalivingorganism. Eggshellproteinsandproteoglycansplayakeyroleinshell

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forma-128 C.Dombreetal./SeminarsinCell&DevelopmentalBiology62(2017)120–132

Fig.6.Three-dimensionalstructuresofcladeAandcladeBserpins.

(A)Cartoonrepresentationofhumanalpha-1antitrypsin/SerpinaA1(1QLP)showingtheexposedreactivesiteloopthatinteractswiththeproteaseactivesiteviathe P1-P1residues(Met358-Ser359)toformaMichaeliscomplex.Thisloopisfurthercleavedbytheproteaseandleadstotheformationofanirreversible,covalentcomplex betweentheserpinanditscognateprotease.(BandC)3Dstructuresofovalbumin/Serpinb14andOVAX/Serpinb14c,respectively.Althoughthestructuresofovalbumin andovalbumin-relatedproteinXarehighlysimilartothatofknowninhibitoryserpins,theirreactivesiteloopareunlikelytointeractwithproteases[35,81],which canbeexplainedbymultipledeviationsinthehingeregionofthereactivecenterloop,comparedwiththeconsensussequenceforinhibitoryserpins[1].Thesolvent-accessible

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Fig.7. Structureoftheeggshellandeffectofovalbuminoncalciumcarbonatecrystals.(A)Ultrastructuralstructureofchickeneggshell(scanningelectronmicroscopy).(B) Controlcrystal.(C)Crystalobtainedinthepresenceofovalbumin(133␮g/mL).Themorphologyandthesizeofcrystals(halfreduction)aresigniﬁcantlymodiﬁedinthe presenceofovalbumin.Unpublisheddata(J.Gautron,S.Solomon,M.Bain,Y.Nys).1bar=100␮m.

tion[54].Thiscontrolledprocessoccursinaconfinedspace(lumen oftheuterus)whereionicconcentrations(calciumand bicarbon-ates)arehighlysupersaturated[15].Recently,theinvestigationof earlyshellmineralizationmechanismshighlightedtheimportance oftheformationofatransientamorphouscalciumcarbonate min-eral(ACC)attheinitialstageofeggshellmineralization[55].The ACCmineralfirstaccumulatesoneggshellmembranesandon spe-cificnucleationsites(mammillaryknobs)(Fig.7A).ACCdeposited aroundthesesitesdissolvesrapidly,providingacontinuoussupply ofionstoformcalcitecrystalsonspecificnucleationsites.These unitscoalescetoformlargercrystalsinthemammillarylayer,and thenduringthenextrapidgrowthphase,theyformthecompact shellpalisadelayercharacterizedbycolumnarcrystalswitha pre-ferredorientation(Fig.7A).Calcitecrystalsresultfromaggregation ofACCparticlesthatsupportrapidmineralizationoftheeggshell andthereisevidencethatthisnon-crystallineformofcalcium car-bonateispresentthroughoutthevariousphasesofshellformation

[55].Duringthesedistinctphases,matrixproteinsplayakeyrole tostabilizethistransientformofcalciumcarbonate[55]butalso influencetheselectionofthecalcitepolymorphintowhichitis ulti-matelyconvertedandthepreferentialorientationofcalcitecrystals intheeggshell[53,56].Thisinteractionleadstotheeggshell ultra-structureanditsassociatedmechanicalproperties[53,54].Many effortsaredriventoidentifyandcharacterizetheroleofeggshell matrixproteinsintheeggshellbiomineralizationprocess. Ovalbu-min/Serpinb14wasthesecondproteinandthefirsteggshellserpin identifiedintheshellmatrix[57].Itspresenceinthemammillary bodiesofdecalcifiedshellwasconfirmedby immunohistochem-istry,indicatingthatovalbuminispresentduringtheinitialphaseof shellformationandbecomesincorporatedintotheproteinmatrix ofthemammillarybodies[57].Thenumerouseggshellproteomics studiesperformedinthelastdecade,widelyconfirmedthe pres-enceofovalbuminineggshellasanabundantproteinandidentified 13additionalserpinsinthisbiomineral[17–21,23,58].

Ovalbumin/Serpinb14isbelievedtoplayacrucialrolein cal-ciumcarbonateformationandACCstabilization[59,60].Calcium bindstoovalbuminandthisaccumulationcreatesanucleation cen-terfortheminerals[60].Calciumionsareboundtotheproteinby complexationviaacidicgroupsleadingtoproteinstructural rear-rangements[59].Thecalciumcationsarethestartingpointsforthe subsequentformationofACCnuclei,whichthenundergoaseries oftransitionphasestothestablecrystallinepolymorphs[59].In arecentstudy,ovalbuminwasreportedasamajorproteinatall keytimeeventsofshellmineralization.Furthermore,ovalbuminis overabundantwhenlargercalcitecrystalunitsaregrowingonthe seedingsitesofshell(mammillaryknobs)[18]anditcontrolsboth calcitecrystalmorphologyandsize(Fig.7BandC).

Serpinf2, wasidentiﬁedas a proteinof intermediate[23] or major abundance in the shell [18]. Serpinb14b, Serpine2, Ser-pinf1andSerpini1areeggshellmatrixproteinwithintermediate abundance.Serpinb14c,Serpina8,Serping1,Serpinc1,Serpind1are presentintheshellatlowabundanceorintermediateabundance

[18,23].Theremaining4eggshellserpins(Serpinb6,Serpina1, Ser-pina4andSerpinb14c)wereidentiﬁedinverylowconcentrationin theshell.Thefunctionofalltheseserpinshasnotbeenexploredyet. Aspotentialantiproteases,theycouldparticipateincontrollingthe calciﬁcationprocessbylimiting proteolyticdegradation[61,62]. OthereggshellserpinssuchastheantibacterialSerpinb14ccould alsoparticipateineggdefense[35]withintheeggshelland/or dur-ingtheprocessofitsformation.Itisalsonoticeablethatseveralof theseeggshellserpinsareactuallyglycosaminoglycanbinding pro-teins(Serpinb14c,Serpind1,Serpinc1,Serpine2).Consideringthat eggshell matrix contains numerous glycosaminoglycans includ-ingkeratansulfate,chondroitinsulfate,hyaluronanandheparan sulfate[63],theseheparin-bindingproteinsmayalsotriggerthe interactionwiththemineralphaseand/ortheothereggshell pro-teins.

surfaceofbothserpinsisshownandcoloredaccordingtovaluesofelectrostaticpotentials(blue:positivecharges;red:negativecharges).Theclusterofpositivechargesin OVAXcorrespondingtotheputativeheparin-bindingsiteissurroundedbyablackcircle.ThisclusterisnotpresentinovalbuminnorinOVAYdespitetheirhighsequence identitywithOVAX.Atomiccoordinatesofovalbumin(1OVA)andthoseofOVAXmodelwereobtainedbycomparativemodelingbasedonovalbuminstructureusing Swiss-Modelserver(swissmodel.expasy.org).TheﬁgurewaspreparedwithPYMOLsoftware.

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Fig.8. Schematicrepresentationofafertilizedeggatday16ofincubationandserpinsidentifiedineacheggstructureduringchickendevelopment.Foreachcompartment,the sizeofthefontroughlyindicatestherelativequantityofoneserpintoanother.Biggerlettercorrespondstoabundanttoveryabundantproteins,smallerletterscorrespondto lowabundantproteinsandnormalletterstoproteinswithintermediateabundance.Notethatcompartmentsarenotcomparable,astherelativequantityintimatelydepends ontheintrinsiccompositionofeachcompartment.*Datarelatedtotherelativeabundanceofserpinsinthechorioallantoicmembranewerenotavailable[71].Serpinsfound amnioticfluidcorrespondtoeggwhiteproteinsthatpresumablytransfertoamnioticfluidfromday11onwards(thecompositionofeggwhiteremainsgloballyunchanged duringthefirsthalfofincubation[48]).Datawereextractedfrom[71](shellmembraneandchorioallantoicmembrane),[76](yolk),and[22,28,52](eggwhite).

2.5. Serpinsinextraembryonictissues

Duringthe21-dayincubation, theextraembryonicstructures thatincludetheamniotic,chorioallantoicandyolksacs,are essen-tialduringembryonicdevelopment(Fig.8).Amnioticsacappears earlyinthedevelopmentandembracestheembryotoprotectit frommechanicalshocks,dehydrationandadhesion[64].In paral-lel,theallantoisexpandsfromthehindgutoftheembryoatday3 ofthedevelopment(E3)andfuseswiththechorion,an extraem-bryonicmembranelyingundertheeggshellmembranes(Fig.8). Theclosecontactbetweenthechorioallantoicmembrane(CAM) andtheeggshellallowsoxygenationoftheembryo,aswellas cal-ciumintakeforitsskeletaldevelopment[64].Thisstructurealso providesareservoirfordisposalwastesproducedbythe embry-onicmetabolism,someofitscomponentsbeingreabsorbedbythe CAMandusedbytheembryoforitsgrowth.Theyolksacbeginsto formfromtheembryo’sgutandenclosestheyolkduringincubation whilethevitellinemembraneisdisrupted(Fig.8).Thisresulting membranesupportsyolk nutrientsdigestionandtheirtransport throughthebloodsystemtotheembryo[65,66].Bothyolksacand CAMsupportangiogenesis.Meanwhile,extraembryonicﬂuidsare transferredfromonecompartmenttoanotherduringincubation. Thus,eggwhiteanditscomponentsaremovingtotheamniotic sacfromE11onward,andareorallyabsorbedbytheembryo[64]

beforereachingtheyolksac[67].

Exceptovalbuminwhichhasbeendetectedinmany extraem-bryonicandembryonictissues,thereislittleinformationrelated totheanalysisofserpinsinthevariousstructuresandﬂuidsof incubatedeggs.

2.5.1. Amnioticﬂuid

Giventhateggwhitetransfersintotheamnioticsac,ovalbumin (Serpinb14)areothereggwhiteserpinsareassumedtobefound intheamnioticﬂuidfromday11onward[68],intheembryonic serumandorgans[49].TheantimicrobialpropertiesofOVAX (Ser-pinb14c)couldprotecttheembryoduringitsdevelopment[35],but theimpactofthechangingenvironment(i.e.transfertothe amni-oticﬂuid)onitspropertieshasnotbeenexploredyet.Asregards

totheSerpinb14b,itsroleinembryonicdevelopmentisstillnot known.Theplasma proteaseC1inhibitor/Serping1 and heparin cofactorII/Serpind1aseggwhiteproteinsaresupposedtobe sim-ilarlytransferredintotheamnioticﬂuid(Fig.8).Theformerhas beendetectedinthewomanamnioticﬂuid[69]andhasevenbeen describedassynthesizedbytheamnion[70].

2.5.2. Chorioallantoicmembraneandallantoicﬂuid

Exhaustive analysis of allantoic ﬂuid using proteomic tools hasnotbeeninvestigatedyetwhereastheproteomeoftheCAM allowedtheidentiﬁcationofthreeserpins:ovalbumin(Serpinb14) anditsrelatedproteinSerpinb14b(OVAY),andSerpinb14c(OVAX)

[71].Serpinb14hasbeenfoundinthechorioallantoicfluidfromE6 toE12[68]andintheCAMandthebloodatE19[71].Serpinb14c andSerpinb14bhavealsobeendetectedintheCAMatE19[71], butfurtherstudiesareneededtoidentifytheirorigins,evenifthe albumen–amnioticfluid–gastrointestinaltract–chorioallantoic fluid–CAMisassumedtobethemainroute.

2.5.3. Eggshellmembranes

Duringtheembryonicdevelopment,there isaglobal enrich-ment in serpins’ amount in the eggshell membranes (ESM). Serpinb14, Serpinb14b, and Serpinb14c has been described as higherinabundanceintheESMoffertilizedeggscomparedtothe ESMofunfertilizedsampleallalongtheincubation.However, Ser-pinb14candSerpinb14bhavenotbeendetectedintheembryonic bloodatE19,whichsuggestsalocalexpressionbythe chrorioallan-toicmembraneoragradualsolubilizationfromtheupperpartofthe eggshell.Serpinb10wasfoundtobeenrichedintheESMfrom fer-tilizedeggsatE3.Thisserpinismainlyknownasanuclearprotein convertingDNAintoacompacttranscriptionallyinert heterochro-matin[72].Ithasbeenshowntoberegulatedduringdevelopment toaccumulateinadultchickenerythrocytenuclei[73]andcould also be involved in the chicken host defense [74]. In contrast, neuroserpin/Serpini1,plasmaproteaseC1inhibitor/Serping1and alpha2-antiplasmin/Serpinf2wereonlyincreasedinthethirdpart of thedevelopmentjust beforepipping (E19).Thisdistribution impliesthateachserpinislikelytohaveaspeciﬁcroleintheESM

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duringincubation.Serpini1isinvolvedinneuronaldevelopment andsynapticplasticity,andisrestrictedtothenervoussystemof thechicken[75].Thebiologicalsigniﬁcanceofitspresenceinthe ESMatE15remainsunclear.Oftheeightserpinsidentiﬁedinthe ESMduringembryonicdevelopment,Serpinb6istheonlyoneto decreaseinabundance.Itsroleinthiscompartmentisnotknown todate.

2.5.4. Yolkandyolksac

Ovalbumin/Serpinb14hasbeenfoundintheyolkatE18[49].It hasbeenproposedthatthisproteincouldbedigestedbyenzymes oftheeggyolktoreleaseamino-acidsandsomespeciﬁcpeptides withadditionalbiologicalactivities(antioxidant,antihypertensive etc.)[8].

Asmallamountofangiotensinogen/Serpina8hasbeenfound in theyolk sacatE0 and decreasesuntilE12 [76]. Theprotein isexpressedbythechickenyolksacmembranefromE2onward

[77],indicatingapotentialfunctionoftheproteininthe regula-tionofhomeostasisandprimitiveerythropoiesisintheyolksac. Moreover,somehaveshownthattargeteddeletionofthegenes encodingSERPINA8producesspeciﬁcrenalabnormalitiesin mam-malianembryossuggestingacrucialroleofSERPINA8inkidney development[78]. Plasmaprotease C1inhibitor/Serping1, hep-arincofactor/Serpind1,andalpha-1antitrypsin/Serpina1havebeen detectedin very small amountin the yolk of unfertilized eggs

[25,27],which question theirbiological signiﬁcance (§2.1). Ser-pind1andSerpina4havebeendescribed atlowerabundancein theeggyolkoffertilizedeggsafterE12[76],andthus,mightbe usedbytheembryoortheextraembryonicstructuresduringthis period.

3. Conclusions

Outofthe27serpinsidentifiedinthechickengenome,only15 havebeendetectedintheegg.Theirlocalizationinthedifferentegg compartmentssuggestsaspecificroleeitherduringeggformation orduringembryonicdevelopment.Thebiologicalfunctionofmost eggserpinsisstillunknownbuttheavailabledatasuggeststhat theyparticipateintissueremodelingandangiogenesis (folliculo-genesis,developmentofyolk,amnioticandallantoicmembranes), eggdefenses (antibacterialserpins, eggshellserpins) and nutri-tion(asasourceofamino-acids).Amongstthe15serpinspresent inthe chickenegg,chicken cladeBovalbumin (Serpinb14)and itsrelatedproteins(Serpinb14b,Serpinb14c)retainmuch inter-estastheseserpinsarefoundexclusivelyintheeggandastheir functionsremainslargelyunknown.Thisobservationsuggeststhat Serpinb14membersmighthavecontributedtoevolutionofegg lay-ingspecies,similarlytoeggyolkvitellogenins,whichhavebeenlost inmammals,concomitantlywiththedevelopmentofplacentation ineutherianandmarsupialanimals[79].Additionalevidencefrom otherspecieslayingeggswouldbeveryinformativetostrengthen this hypothesis/conjecture.Allthree Serpinb14paralogs display subtleconformationalandphysicochemicaldifferences,whichmay affecttheirrespectiveactivity.Uniquelyintheserpinsworld, oval-buminisconvertedtoaheat-stableformwhilemigratingtothe extraembryonic fluidsand embryonic organs where it recovers asanuncleavedprotein.Thisobservationdoesnotactually sup-portitsuniqueroleasasourceofamino-acidsandsuggestssome moredirectfunctionondevelopingorgans.Ovalbuminalso con-tributestoshapeeggshellultrastructurebyinteractingwiththe mineralphase.Comprehensiveandfunctionalanalysesofegg ser-pinsinembryonicandextraembryonictissuesislackingtodate. Thisreviewhighlightsthatchickeneggserpinsoffermultiplesaxes ofresearchinthefieldofdevelopmentalbiology.

Conﬂictofinterest

Allauthorsdeclarenoconﬂictofinterest.

Acknowledgements

WethankAurélienBrionne(UR83RecherchesAvicoles)forhis remarkableworktointegrateallpublishedproteomicdataonthe chickenegg.WethankRegionCentre–ValdeLoire,FranceforM. DaSilvaPhDfellowship.

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