separate336.com

BioMedCentralPage1of10(pagenumbernotforcitationpurposes)BMCDevelopmentalBiologyOpenAccessResearcharticleAroleforsperminregulationofegg-layingintheNematodeC.
elegansMarieMcGovern1,3,LingYu1,MaryKosinski2,DavidGreenstein2andCathySavage-Dunn*1Address:1DepartmentofBiology,QueensCollege,andTheGraduateSchoolandUniversityCenter,CUNY,Flushing,NY11367,USA,2DepartmentofGenetics,CellBiology,andDevelopment,UniversityofMinnesota,Minneapolis,MN55455,USAand3DepartmentofBiology,NewYorkUniversity,NewYork,NY10003,USAEmail:MarieMcGovern-mariemcgov@yahoo.
com;LingYu-vortexyin@yahoo.
com.
cn;MaryKosinski-marykosinski@mcmail.
vanderbilt.
edu;DavidGreenstein-green959@umn.
edu;CathySavage-Dunn*-cathy.
savagedunn@qc.
cuny.
edu*CorrespondingauthorAbstractBackground:Ininsectsandinmammals,malespermandseminalfluidprovidesignalingfactorsthatinfluencevariousaspectsoffemalephysiologyandbehaviortopromotereproductivesuccessandtocompetewithothermales.
Itislessapparenthowimportantsuchsignalingisinthecontextofaself-fertilehermaphroditespecies.
WehaveaddressedthisquestioninthenematodeCaenorhabditiselegans,whichcanreproduceeitherbyhermaphroditeself-fertilizationorbymale-hermaphroditemating.
Results:Wehavestudiedtheegg-layingdefectivemutant,egl-32,andfoundthatthecellularbasisoftheegl-32egg-layingphenotypeislikelyadefectinsperm.
First,thetimeofegl-32actioncoincideswiththetimingofspermatogenesisinthehermaphrodite.
Second,egl-32interactswithgenesexpressedinsperm.
Third,matingexperimentshaverevealedthatwild-typespermcanrescuetheegg-layingdefectofegl-32mutantanimals.
Mostimportantly,introductionofmutantegl-32spermintowild-typehermaphroditesorfemalesissufficienttoinduceanegg-layingdefectivephenotype.
Conclusion:PreviousworkhasrevealedthatC.
elegansspermreleasefactorsthatstimulateoocytematurationandovulation.
Herewedescribeevidencethatspermalsopromoteegglaying,thereleaseofembryosfromtheuterus.
BackgroundEgglayinginC.
elegansisacarefullymonitoredprocess.
Geneticstudiesofegg-layingdefective(Egl)mutantshaverevealedsignificantinsightintotheneuronal,develop-mental,andenvironmentalcontrolofegglayingbehavior[1].
Egglayingisbiphasic,alternatingbetweenanactivephasewheneggsarelaid,andaninactivephase[2].
Thehormoneserotoninisnecessarytoinitiatetheactivephaseofegglaying.
Serotoninisproducedbythehermaphro-ditespecificneurons(HSNs)[3].
Hermaphroditesinte-gratemanyinternalandexternalcuesindecidingwhetherornottoentertheactivephase.
Wild-typehermaphro-ditesunderlaboratoryconditionsreleaseembryosfromtheuterusatasimilarstageintheirdevelopment.
Retain-ingfertilizedeggsfortoolongendangersthemother'slife,aslarvaethathatchinternallywillconsumeanddestroyPublished:1May2007BMCDevelopmentalBiology2007,7:41doi:10.
1186/1471-213X-7-41Received:10November2006Accepted:1May2007Thisarticleisavailablefrom:http://www.
biomedcentral.
com/1471-213X/7/412007McGovernetal;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
BMCDevelopmentalBiology2007,7:41http://www.
biomedcentral.
com/1471-213X/7/41Page2of10(pagenumbernotforcitationpurposes)themother.
Therearecircumstances,however,suchasintheabsenceoffood,whereeggretentionmaybedesirable,asthisincreasestheoddsthattheprogenywillbelaidinafavorableenvironment.
Inaddition,wheneggsareretained,theyeventuallyhatchinternallyanddevourthehermaphrodite'sbodybeforeescapingthecuticle,allow-ingmaternalresourcestobesacrificedtotheprogeny.
Accordingly,intheabsenceoffood,wormsentertheactivephaselessfrequently,andanimalsbegintobloatwitheggs[4].
Neurotransmittersthatinhibitegglayingarealsoknown.
Forexample,theendocrineuv1cellsproducetyraminewhichactstoinhibitegglaying[5].
Thisinhibi-torypathwaymayallowegg-layingbehaviortorespondtotheinternalcueofthepresenceoffertilizedeggsintheuterus[6].
Instudiesoftheegg-layingdefectivemutantegl-32,wehavenowuncoveredanotherinternalcue,thepresenceofsperm,whichstimulatesegglaying.
C.
eleganshermaphroditesproducespermduringthefourthlarval(L4)stage[7].
Thespermareamoeboidnotflagellated,andtheyarestoredinthespermathecae.
Whenthewormsundergotheirfinalmolttobecomeadultstheyswitchtoproducingexclusivelyoocytesandwilldosofortheremainderoftheirlives.
Astheoocytesmaturetheyareovulatedandpassthoughthespermathecaewheretheycomeincontactwithsperm.
Theoocytesarefertilized,passintotheuterus,andareeventuallylaid.
Unlikeinhumans,almostallofthesperminthehermaphroditeareusedtofertilizeoocytes[8].
Thenumberofspermlimitsthenumberofpotentialoffspringaself-fertilizingher-maphroditeiscapableofproducing,whilematingswithmaleswillincreasethenumberofprogeny[9].
Malespro-duceexclusivelyspermbeginningintheL4stageandthroughouttheiradultlives.
Ifamaleisallowedtomatewithahermaphroditehisspermwillcrawlintothesper-mathecawheretheywillbepreferentiallyusedtofertilizetheoocytes[8].
PreviousworkhasrevealedthatC.
elegansspermreleasefactorsthatstimulateoocytematurationandovulation[10,11].
Herewedescribeevidencethatspermalsopromoteegglaying,thereleaseofembryosfromtheuterus.
ResultsCharacterizationoftheegg-layingdefectsofegl-32mutantsInlarge-scalegeneticscreensforegg-layingdefective(Egl)mutants,asingletemperature-sensitivealleleofthegeneegl-32,n155,wasisolated[12].
PharmacologicaltestsdesignedtocharacterizegeneactivityrelativetotheHSNsdidnotidentifythecellularoranatomicalbasisfortheegl-32egg-layingdefect.
Herewepresentevidencethatthecellularbasisoftheegl-32egg-layingphenotypeisadefectinsperm.
Weusedtwoassaystoquantifyegglaying,aneggretentionandanegglayingassay(Methods).
Intheeggretentionassay,mutantegl-32animalsretainabouttwiceasmanyeggsaswild-typeanimals(Figure1a).
Intheegg-layingassay,mutantegl-32animalslayabouthalfasmanyeggspertimeperiodaswild-typeanimals(Figure1b).
Becausefoodcuesplayanimportantroleinegglaying,wewereinterestedindeterminingwhetheregl-32mutantani-malshaveanormalresponsetofood.
Intheabsenceoffoodwild-typeanimalslayfewereggs.
Thisisalsotrueofegl-32animals(Figure1b)suggestingthatthedefectinegl-32isnotatthelevelofchemosensationorsensoryprocessingoffoodcues.
Theoneexistingalleleofegl-32,n155,displaysatemperature-sensitiveegg-layingdefectatthenonpermissivetemperatureof25°C.
Weperformedatemperatureshiftassaytodeterminethecriticalperiodforegl-32activity.
Thetemperaturesensitiveperiodisbetween24and48hoursaftereggcollectionat25°C,whichcorrespondstotheL4stageofdevelopment(Figure2).
Thisresultwasinterestingbecauseegglayingdoesnotoccuratthisstage.
However,thisisthetimeduringwhichvulvalmorphogenesisoccursandtheHSNsinnervatetheirtargets.
Moreover,itistheoneandonlytimeduringwhichhermaphroditesproducesperm[7].
egl-32interactswithT08G11.
2Intransformationexperiments,weidentifiedacosmid,C26G6,whichcouldrescuetheegl-32phenotype.
Sub-clonesofthiscosmidcontainingthepredictedopenread-ingframe(ORF)T08G11.
2canalsorescueegl-32(Figures3a&3b).
Severallinesofevidence,however,suggestthategl-32doesnotencodeT08G11.
2,butratherthattheyareinteractingloci.
First,whenwesequencedT08G11.
2fromegl-32(n155)mutantswecouldfindnochangetothegenome.
WeextendedoursequencingofthisregionfromthepreviousORFtothenextORFandstillcouldnotfindachangetothegenome.
Second,SNPmappingwasper-formed(datanotshown),thatconfirmedthategl-32isseparablefromT08G11.
2.
Third,weperformedacomple-mentationtestbetweenegl-32andaT08G11.
2deletionallele,tm336.
Thetrans-heterozygotestm336/n155retainedanaverageof13eggs,significantlyfewerthann155homozygotes(p=1.
74*10-14).
Thus,tm336com-plementsegl-32,supportingtheconclusionthattheyareseparatebutgeneticallyinteractinggenes.
ABLASTsearchrevealedthatT08G11.
2hasthreehomo-logues,B0207.
11,F44F4.
10andF42G4.
6inC.
elegans(Figure3c).
T08G11.
2anditsthreehomologueshaveallbeenidentifiedasspermenrichedtranscripts[13].
T08G11.
2anditsclosesthomologue,B0207.
11,haveabouta10,000-foldenrichmentinsperm.
Asimilarfoldenrichmentisfoundinthespermspecificproteins(ssp-11,ssp-16,ssp-19,ssq-1,ssq-2,sss-1andsss-2).
T08G11.
2encodesasmallnovelproteinof282aminoacids.
Itcon-tainsanSH2-likedomain.
However,itlacksthecriticalBMCDevelopmentalBiology2007,7:41http://www.
biomedcentral.
com/1471-213X/7/41Page3of10(pagenumbernotforcitationpurposes)argininepresentinfunctionalSH2domains.
Tocharacter-izefurtherthebiologicalrolesofthesespermenrichedproteins,weobtaineddeletionallelesfromtheC.
elegansknockoutconsortium.
tm336containsadeletioninT08G11.
2,tm322intheORFB0207.
11,andtm332intheORFF44F4.
10.
Theseanimalsallretainedfarfewereggsthanwild-typeanimals(Figure1a),suggestingtheabsenceofanegg-layingdefect.
However,olderembryoswereoftenseeninmutanthermaphrodites,whichwouldbeconsistentwithanegg-layingdefect.
Wethereforedeterminedtheegg-layingratefortm336,theT08G11.
2mutant,toresolvethisissue(Figure1c).
Thedatademon-stratethattm336is,infact,egg-layingdefectivebasedonratesofegglaying.
Thedecreasedeggretentionisthereforenotduetoincreasedegglaying,butmayreflectareducedovulationrateinstead(seebelow).
MatingexperimentsWebecameinterestedinthepossibilitythatspermplayanactiveroleinegglayingwhenwediscoveredthattheegl-32interactinggeneT08G11.
2anditsthreehomologuesareallhighlyexpressedinsperm.
Thishypothesisissup-portedbythetemperatureshiftassay,whichrevealedthattheL4stageofdevelopment,thestageatwhichhermaph-roditesproducesperm,isthecriticalperiodforegl-32activity(Figure2).
Weinvestigatedthepossibilitythatspermareplayinganactiveroleinegglayingbyperform-ingmatingexperiments.
Inalloftheseexperiments,themutationhim-5wasincludedinthegeneticbackgroundtofacilitaterecoveryofmales.
Maleswerestainedwithavitaldyeforaminimumof2hoursandallowedtomatewithstage-matchedyoungadulthermaphrodites.
Her-maphroditeswerethenexaminedforthepresenceorEggRetentionFigure1EggRetention–(a)egl-32(n155)animalsretainabouttwiceasmanyeggsaswildtypeanimals.
AnimalscontainingadeletioninT08G11.
2(tm336)andanimalscontainingdeletionsintwoofT08G11.
2'shomologues(tm332andtm322)retainfarfewereggsthanwild-typeanimals.
36wild-typeN2animals,36egl-32(n155)animals,35tm336animals,67tm332animalsand144tm322animalswereusedinthisassay.
*Statisticallysignificantdifference.
Studentt-testvalue<0.
05.
Errorbarsinthisandallfiguresshowstandarddeviation.
(b)–Mutantegl-32animalslayabouthalfasmanyeggsaswild-typeanimals.
However,mutantegl-32animalsdorespondnormallytofoodcues.
Theybegintolayevenfewereggsintheabsenceoffood.
26N2animalsinthepres-enceoffood,39N2animalsintheabsenceoffood,26egl-32animalsinthepresenceoffood,and38egl-32animalsintheabsenceoffoodwereusedinthisassay.
*Statisticallysignificantdifference.
Studentt-testvalue<0.
05.
(c)–T08G11.
2deletionmutants(tm336)laysignificantlyfewereggsthanwild-typeanimals.
However,theyretainlatestageembryos.
*Statisticallysig-nificantdifference.
Studentt-testvalue<0.
05.
BMCDevelopmentalBiology2007,7:41http://www.
biomedcentral.
com/1471-213X/7/41Page4of10(pagenumbernotforcitationpurposes)absenceoffluorescentspermasanindicatorofthesuccessorfailureofmating.
Iftheegl-32egg-layingdefectisduetoadefectinthesperm,thenwemightexpectthattheintroductionofwild-typespermcouldcorrectthisdefect.
Consistentwiththishypothesis,theexperimentsrevealedthattheintroductionofwild-typesperm,viamating,intoegl-32mutantanimalsrescuedtheegg-layingdefect(Table1).
Morestrikingly,theintroductionofmutantegl-32spermintowild-typeanimalswassufficienttoinduceanegg-layingdefectivephenotype(Table1).
Thepresenceofmalesinitselfdidnotleadtothischangeinegg-layingbehavior,sincecontrolanimalsonthesameplateswith-outdetectablestainedspermwerenotaffectedsignifi-cantly.
Inaddition,incontrolexperimentswithegl-32;him-5hermaphroditesmatedwithegl-32;him-5males,thehermaphroditesretainedanaverageof31eggs.
Thisincreaseineggretentionwasnotstatisticallysignificant,anddemonstratesthatmatingpersedoesnotrescuetheegg-layingdefect.
Todeterminetheeffectofegl-32mutantspermintheabsenceofendogenoushermaphroditicspermweper-formedmatingexperimentsusingfemalefog-2animals.
Thesemutantsmakeoocytesbutnotsperm,andsoarenotself-fertile[14].
Consistentwithourpreviousmatingexperiments,fog-2animalsmatedwithegl-32malesretainedsignificantlymoreeggsthanfog-2femalesmatedwithwild-typemales(Table1).
Toconfirmthattheegg-layingdefectisduetotheinactivationofegl-32,werepeatedtheseexperimentsat20°C,apermissivetemper-ature.
Atthistemperature,thereisnodifferencebetweenfemalesmatedwithegl-32andthosematedwithwild-typemales,consistentwiththeconclusionthategl-32providestherelevantactivity.
Finally,wedeterminedthattheintro-ductionofspermcannotrescueeveryegg-layingdefect.
Wetestedegl-33mutantanimalsbyintroducingwild-typespermintoegl-33hermaphrodites(Table1).
Theanimalscontainingstainedspermdidnotretainfewereggsthanthosenotmatedwithwild-typemales.
TemperatureShiftAssayFigure2TemperatureShiftAssay–Temperatureshiftassayrevealsthat24to48hoursisthecriticalperiodforegl-32activity.
Thiscor-respondstotheL4stageofdevelopment.
Time0isthetimeatwhichparentaladulthermaphroditeswerepicked.
Progenywerescoredafter72hoursat25°Corequivalentdevelopmentaltime.
10–30animalswereusedateachtimepoint.
BMCDevelopmentalBiology2007,7:41http://www.
biomedcentral.
com/1471-213X/7/41Page5of10(pagenumbernotforcitationpurposes)RescueExperimentsFigure3RescueExperiments–(a)Cosmidsfromtheregionthoughttobeassociatedwithegl-32wereinjectedintomutantegl-32(n155)animals.
Onecosmidfromthisinterval,C26G6,whichoverlapsthesequencedcosmidT08G11,rescuedtheegl-32phenotype.
Amongseveralsubclonesofthiscosmid,onecontainingtwopredictedORFsT08G11.
2andT08G11.
3,butnotonecontainingonlyT08G11.
3,alsorescuedegl-32.
(b)–Injectionofthesubclonealongwithrol-6rescuestheegg-layingdefectofegl-32.
*Statisticallysignificantdifference.
Studentt-testvalue<0.
05.
(c)–AlignmentofT08G11.
2withB0207.
11andF44F4.
10.
Identicalaminoacidresiduesarehighlightedinblack,similaraminoacidsingray.
TheSH2-likedomainishighlightedinyellow.
Sequencesdeletedindeletionallelesareoutlinedinred.
FortheinternaldeletionsinB0207.
11andF44F4.
10wehavenotdeterminedwhetheranin-frameproductiscreated.
BMCDevelopmentalBiology2007,7:41http://www.
biomedcentral.
com/1471-213X/7/41Page6of10(pagenumbernotforcitationpurposes)Thesematingexperimentsestablishedtheexistenceofasperm-derivedsignal,dependentonegl-32activity,whichpromotesegglaying.
Thebestcharacterizedspermsignal-ingmoleculesaretheMajorSpermProteins(MSPs),whicharenecessaryforoocytematurationandovulation[11].
Inmutantfemaleanimalsproducingonlyoocytes,theoocyteswillnotmatureandovulationoccursataverylowrate.
But,whenMSPsareintroduced,theoocytesareinducedtomatureandthesheathcellssurroundingtheovaryarestimulatedtocontractresultinginmorefre-quentovulations.
Sinceasperm-derivedsignalregulatesovulationrates,wetestedwhetherourmutantshavealteredovulationrates.
Wehavefoundthattheovulationrateinegl-32(n155)mutantanimalsisslightly,butsignif-icantlyreduced(Figure4).
Theovulationrateintm336mutants,inwhichtheegl-32interactinggene,T08G11.
2isdeleted,isevenmoresignificantlyreduced(Figure4).
Fertilizationisrequiredforegl-32activityInourmatingexperiments,wefoundthategl-32mutantmalespermcansuppressegglayingeveninthepresenceofwild-typehermaphroditesperm.
Thequestionarisesastowhythehermaphroditespermarenotsufficienttopro-motenormalegg-layingrates.
Onepossibleexplanationisthategglayingiscoupledtofertilization.
Sincemalespermout-competehermaphroditespermforfertilizationofoocytes,inmatingswithegl-32malesthedefectivemalespermwillbepreferentiallyusedforfertilizationeveninthepresenceofwild-typehermaphroditesperm.
Wewerethereforeinterestedindeterminingiffertilizationisrequiredforthesperm-derivedsignalregulatingegglay-ing.
Todetermineiffertilizationisrequiredwematedegl-32hermaphroditeswithfertilization-defectivemales.
Spermfromspe-9andspe-38mutantsarestructurallynor-mal,cancrawlintothespermatheca,butareincapableofbindingtoandfertilizinganoocyte[15,16].
Inthesemat-ingexperiments,wefoundthatneitherspe-9norspe-38mutantspermcouldrescuetheegl-32defect,unlikewild-typesperm(Table1).
Thisresulteliminatesthepossibilitythattherescuingactivityisprovidedeitherbyseminalfluidorbythephysicalactofmatingratherthanbysomefactorinthesperm.
Weproposeinsteadthatfertilizationiscoupledtotheregulationofegg-layingrates.
MSPlocalizationinegg-layingdefectivemutantsInterestingly,spe-9spermarecapableofsignalingtopro-motemeioticmaturation[15-17],butwefindtheminsuf-ficienttopromoteegglaying.
Thus,thesetwospermfunctionsareseparablegenetically.
SinceMSPsaretheactivefactorinpromotingmeioticmaturation,weaskedwhetheradefectinMSPproductionorlocalizationcouldalsounderlietheegg-layingdefectsofegl-32(n155)andtm336mutants.
MSPhaspreviouslybeenfoundtobepresentintheuterus,whereitcouldtheoreticallybeinvolvedinregulatingegglaying.
MSPlocalizationwasvisualizedbyimmunohistochemistry(Figure5).
Wefoundthatinbothmutantbackgrounds,detectablelevelsofMSParereleasedandarepresentintheuterus,arguingagainstaroleforMSPinregulatingegglayingdown-streamofegl-32andT08G11.
2.
AlthoughwecannotexcludetheexistenceofsubtledifferencesinMSPlocaliza-tionoraccessibility,ortheexistenceofisoform-specificdifferencesinthesemutants,ourresultssuggestthattheegg-layingdefectsarenotduetoalossofMSPsignaling.
Table1:MatingExperimentsHermaphroditeMaleEggsretainednegl-32;him-5notmated22±689egl-32;him-5him-5(matingnotdetected)19±560egl-32;him-5him-513*±377egl-32;him-5egl-32;him-531±839egl-32;him-5spe-38;him-524±310egl-32spe-9(matingnotdetected)24±420egl-32spe-9;him-524±320him-5notmated11±371him-5egl-32;him-5(matingnotdetected)13±480him-5egl-32;him-520*±838fog-2him-513±429fog-2egl-32;him-518*±538fog-2him-5(20°C)7±3104fog-2egl-32;him-5(20°C)7±384egl-33him-5(matingnotdetected)27±1059egl-33him-528±1029*Statisticallysignificantdifferencebetweenmatedandcontrolanimals,p<0.
05.
Allexperimentsareperformedat25°C,unlessotherwiseindicated.
Datarangesgivestandarddeviation.
BMCDevelopmentalBiology2007,7:41http://www.
biomedcentral.
com/1471-213X/7/41Page7of10(pagenumbernotforcitationpurposes)Thisconclusionisconsistentwiththeobservationthatregulationofmeioticmaturationandofegglayingaregeneticallyseparable.
DiscussionMutantegl-32hermaphroditesretainabouttwiceasmanyeggs,layabouthalfasmanyeggsandovulateataslightly,butsignificantlyslowerratethanwild-typeanimals.
Thereductioninegglayingrateisnotduetoadefectinchem-osensationorsensoryprocessingoffoodcuesasegl-32animalsrespondnormallytofoodcues.
Severallinesofevidencesuggestthatthedefectinegl-32isduetoanabnormalityofthesperm.
First,theegl-32geneinteractswithT08G11.
2,agenethatishighlyexpressedinsperm,topromoteproperegglayinginC.
elegans.
InjectionofT08G11.
2intoegl-32(n155)mutantanimalsrescuestheiregg-layingdefectivephenotypes.
Second,egl-32(n155)isatemperature-sensitivemutation.
TemperatureshiftassaysrevealthattheL4stageofdevelopmentisthecriticalperiodforEGL-32activity.
Thiswasaninterestingfindingbecausethisisnotwheneggsarebeinglaid.
However,itistheoneandonlytimethatahermaphroditeproducessperm.
Third,matingexperimentsrevealthatwild-typespermintroduced,viamating,intoegl-32animalsrescuestheiregg-layingdefectivephenotype.
Thisisnottrueofallegg-layingdefectivemutants,astheegg-layingdefectofegl-33couldnotberescuedviamating.
Fourth,egl-32mutantspermaresufficienttoconfertheeggretentionphenotypeinbothwild-typehermaphroditesandfemales.
Wehavealsodeterminedthatfertilizationofoocytesisrequiredtomediateegl-32activity.
Mutantspe-9andspe-38sperm,whichareincapableoffertilizingoocytes,wereunabletoreducethenumberofeggsretainedbymutantegl-32hermaphroditeswhenintro-ducedviamating.
Thus,weconcludethategl-32activityisrequiredforasperm-derivedsignalthatpromotesegglay-ingandactsatorafterfertilization.
ThissignalisunlikelytobeMSP,sinceMSPlocalizationisnotdetectablyalteredinegl-32.
Furthermore,theregulationofmeioticmatura-tion/ovulationandegglayingareatleastpartiallysepara-blegeneticallyasseeninegl-32(n155)mutants,whichhavesignificantdefectsinegglayingbutonlyaslightlyreducedovulationrate(thisstudy),andinspe-9mutants,whicharedefectiveinpromotingegglayingbutnotinpromotingmeioticmaturation[17].
Aroleforseminalfluidinregulatingfemaleoocytematu-rationandovulation,amongotherbehaviors,hasbeenwellestablishedinDrosophila[18].
Inparticular,thesem-inalfluidhormoneAcp70A(sexpeptide)stimulatesoog-enesisandAcp26Aa(ovulin)stimulatesovulation.
Similarly,factorsinmammalianseminalfluid,suchasTGFβandprostaglandin,promotechangesinthefemaletofacilitateembryosurvivalandimplantation[19].
Therapidevolutionofmaleseminalfluidproteinssuggestsaroleinmale-femalesexualconflict,inwhichthemalebenefitsmostbyincreasingthequantityofoffspring,whilethefemalebenefitsmorebyselectinghighqualitymates[20,21].
InC.
elegans,althoughcompetitionbetweenhermaphroditeandmalespermoccurs[8]itmightbeexpectedthatthehermaphrodite'sspermwouldplayalimitedroleinregulatingitsownreproductivebehaviorsduetothelackofsexualconflict.
This,appar-ently,isnotthecase.
Instead,hermaphroditespermactivelysignaltopromoteoocytematurationandovula-tionviatheMSPs[11]and,asshownhere,topromoteegglayingviaanegl-32-dependentactivity.
Spermsignalinginthehermaphroditemayexisttoensureprudentinvest-mentofresources.
Intheabsenceofsperm,ovulationwouldbeanenergeticallywastefulbehavior.
Similarly,asperm-derivedsignalmayexisttocoordinatetherateoffertilizationtotherateofegglaying.
Aslongasspermarepresenttherateofegglayingisclosetotherateofovula-tionresultinginafairlyconstantnumberofeggspresentintheuterus.
Older,wild-typewormsnormallybloatwitheggs.
Theseolderhermaphroditesmaybecomedefectiveinegglayingbecausetheynolongercontaintheinternalcuepresentinspermtopromoteegglaying.
Conversely,egglayinginyoungadultsisinhibiteduntiltheuterusbecomessufficientlyfilledwitheggs,possiblybyaninhib-itoryeffectoftheuv1cellsthatisblockedwhenmechan-icaldeformationbyeggsoccurs[6].
Itispossiblethatthespermsignalmodifiesorcoordinateswiththeuterine-OvulationRateAssayFigure4OvulationRateAssay–Mutantegl-32;him-5animalsovulateataslightly,butsignificantly,slowerratethanhim-5animals.
AnimalscontainingadeletioninT08G11.
2(tm336)ovulateatamuchslowerratethanwild-typeanimals.
68wild-typehim-5animals,100egl-32;him-5animalsand53tm336animalswereusedinthisassay.
*Statisticallysignificantdifference.
Studentt-testvalue<0.
05.
BMCDevelopmentalBiology2007,7:41http://www.
biomedcentral.
com/1471-213X/7/41Page8of10(pagenumbernotforcitationpurposes)occupancysignaltoeffecttheappropriaterateofegglay-ingthroughoutthenematode'slifehistory.
ConclusionOurdatademonstratetheexistenceofanovelsperm-derivedsignalthatregulatesratesofegglayinginthenem-atodeC.
elegans.
ThissignalisunlikelytobedependentontheknownspermsignalingmoleculeMSP.
Thenovelsig-nalrequiresspermfertilizationofoocytestofunction.
MethodsC.
elegansstocksandgeneticsThefollowingwormstocksusedinthisstudywereobtainedfromtheCaenorhabditisGeneticsCenter(CGC):egl-32(n155)I,spe-9(hc88)I,him-5(e1490)V,fog-2(q71)V.
ThefollowingdeletionalleleswereobtainedfromDr.
ShoheiMitani,NationalBioresourceProjectforthenematode,TokyoWomen'sMedicalUniversitySchoolofMedicine,Japan:tm336I,tm322I,tm332II.
Inadditiontophenotypesreportedhere,tm322,thedeletionalleleofB0207.
11,hasanUncphenotypethatcosegregateswiththedeletion.
Thespe-38I;him-5VanimalswereobtainedfromAndrewSingson.
Wegeneratedegl-32;him-5andspe-9;him-5bystandardtechniques.
Wormswereraisedat25°C,unlessotherwisenoted,andgrownonEZwormplates(E.
Lambie,personalcommunication;Perliter:550mgTris-Cl,240mgTris-OH,3.
1gBactoPeptone,8mgcholesterol,2.
0gNaCl,200mgstreptomycinsulfate,20gagar).
Egg-layingassaysL4larvaewereplacedonplateswithfoodandallowedtomatureat25°Covernighttobecomeyoungadults.
Theyoungadultsweremovetoindividualplatesthateithercontainedorlackedfood.
Thewormswereallowedtolayeggsat25°Cfor2hours.
Thenumberofeggs(orprogeny,assometimeswasthecasewiththeegg-layingdefectiveanimals)presentontheplateafter2hourswasthencounted.
EggretentionassayL4larvaewereplacedonplateswithfoodandallowedtomatureat25°Covernightintoyoungadults.
Theyoungadultwormswerethenplacedonanagarpadonaglassslideinadropof10mMsodiumazide.
Thewormswerethenviewedundera40*objectivelens(ZeissAxioskop).
Thenumberofeggsretainedintheuteruswascounted.
OvulationrateassayL4larvaewereplacedonplateswithfoodandallowedtomatureat25°Covernightintoyoungadults.
WormswerethenplacedinasmalldropofOP50onanagarpad.
Thenumberofeggs/oocytescontainedintheuterusandsper-mathecaewerecounted.
Thewormswerethentransferredtoindividualplatescontainingfoodandallowedtolayeggsat25°Cfor5hours.
ThewormswerethenagainplacedonaslidewithanagarpadinasmalldropofOP50andtheeggsretainedwereagaincounted.
Thenumberofeggspresentontheplatewasalsocounted.
Theovulationratewasdeterminedbytakingthenumberofeggsretainedoriginallyandsubtractingitfromthenumberofeggsretainedafter5hoursplusthenumberofeggslaidduringthe5hours.
Theaveragenumberofeggs/oocytesovulatedin1hourandpergonadarmwasthencalculated.
DetectionofExtracellularMSPintheUterusFigure5DetectionofExtracellularMSPintheUterus–Adulthermaphroditeswerestainedinwhole-mountwithmonoclonalanti-MSP(red).
DNAwasdetectedwithDAPI(blue).
Brightredstainingspermatozoa(arrows)arelocatednearsmallredpunctaanddiffusepatchesofstaining,whichlikelycorrespondtoextracellularMSPvesiclesandfreeMSP,respectively[17].
Whilethepat-ternsofspermlocalizationandMSPstainingareslightlyvariableintheuterus,thestainingobservedintheegl-32(n155)andtm336mutantswasqualitativelywithinthenormalrangeseeninthewildtype.
Spermareapproximately4μminsize.
BMCDevelopmentalBiology2007,7:41http://www.
biomedcentral.
com/1471-213X/7/41Page9of10(pagenumbernotforcitationpurposes)TemperatureshiftassayEggswereplacedonaplateandallowedtohatchateither15°Cor25°C.
Atvarioustimesafterhatchingthewormswhereshiftedtoahigherorlowertemperature[22];thewormsthathatchedat25°Cwereshifteddownto15°C;thewormshatchedat15°Cwereshiftedupto25°C.
Thenumberofeggsretainedintheyoungadultswasthencountedasabove.
10–30animalswerescoredateachtimepoint.
Twohoursofdevelopmentat15°Cwascon-sideredequivalenttoonehourofdevelopmentat25°C.
RescuebyinjectionThefollowingclonesandsubcloneswereinjected,alongwithrol-6asamarkergene,intothegonadofegl-32(n155)animals:C45D10,C26G6,F53B6,H14D01,F32H2,W06D4,F36F2,W10D5,ZK858,H07C08,H32K16,F25H5,C45G3,H07C08andsubclonesofC26G6.
Wescreenedforrollinganimalsthatwerenolongeregg-layingdefective.
MatingexperimentsAllanimalsusedinmatingexperimentsincludedthehim-5mutationinthegeneticbackground.
Thehermaphro-dites,orfemalefog-2animals,usedinthematingexperi-mentswereselectedasL4larvaewereallowedtomatureovernightat25°C.
Themalesusedinthematingexperi-mentswerestainedwithSYTO-17[23]asfollows:WormswerewashedoffplateswithM9solutionandtransferredtoa1.
5mlmicrocentrifugetube.
Thewormswerepelletedbymicrocentrifugation.
Thesupernatantwasremoved.
Thedyewaspreparedbytaking3μlof5mMstocksolutioninto1497μlM9tomakea10μMsolu-tion.
(occasionallyasolutionasconcentratedas100μMwasused.
Thiswasdoneonlywhenthedyewasoldandappearedtobelosingitsabilitytofluorescestrongly).
500μlofdyewasaddedtothewormpellet.
250μlofdyedwormsweretransferredtotwocleandryplates.
Theplateswerewrappedinaluminumfoilandplacesat25°Cforaminimumof2hours.
Toremovethedyesolutionthewormswerepelletedinafresh1.
5mlmicrocentrifugetube.
Thesupernatantwasremoved.
ThewormswereresuspendedinafewdropsofM9andtransferredtoafreshplateforrecovery.
Oncetheplatesweredrystainedmaleswerepickedtoplatescontainingtheyoungadulthermaphroditesandallowedtomateforaminimumof2hours.
Thehermaphroditeswerethentransferredtoanagarpadonamicroscopeslideandplacedinadropof10mMsodiumazide.
Thenumberofeggsretainintheuteruswasthencountedundera40*objectivelens.
MSPlocalizationImmunohistochemistryusinganti-MSPmonoclonalanti-bodieswasperformedasdescribedpreviously[17].
Authors'contributionsMMcarriedoutthephenotypicstudies,participatedintheanalysisofthestudy,anddraftedthemanuscript.
LYcar-riedoutthetemperatureshiftassaysandthemicroinjec-tionexperiments.
MKcarriedouttheimmunohistochemistry.
DGparticipatedinthedesignandanalysisofthestudy.
CSDconceivedofthestudy,andparticipatedinitsdesignandcoordinationandhelpedtodraftthemanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsWethankDr.
ShoheiMitani,NationalBioresourceProjectforthenema-tode,TokyoWomen'sMedicalUniversitySchoolofMedicine,Japan,fortm322,tm336,andtm336deletionmutants.
SomeC.
elegansmutantstrainswereobtainedfromtheCaenorhabditisGeneticsCenter,whichissup-portedbytheNIHNationalCenterforResearchResources(NCRR).
ThisworkwassupportedinpartbyresearchgrantsPOWRE:9805868fromtheNSF,1-FY00-798fromtheMarchofDimesBirthDefectsFoundationandagrantfromPSC-CUNY.
ThisworkwascarriedoutinpartialfulfillmentoftherequirementsforthePh.
D.
degreefromtheGraduateCenterofCityUniversityofNewYork(M.
McGovern).
References1.
SchaferWF:Geneticsofegg-layinginworms.
AnnuRevGenet2006,40:487-509.
2.
WaggonerLE,ZhouGT,SchaferRW,SchaferWR:ControlofalternativebehavioralstatesbyserotonininCaenorhabditiselegans.
Neuron1998,21(1):203-214.
3.
HorvitzHR,ChalfieM,TrentC,SulstonJE,EvansPD:SerotoninandoctopamineinthenematodeCaenorhabditiselegans.
Science1982,216(4549):1012-1014.
4.
TrentC:Geneticandbehaviorstudiesoftheegg-layingsys-temofCaenorhabditiselegans.
Cambridge,MassachusettsInsti-tuteofTechnology;1982.
5.
AlkemaMJ,Hunter-EnsorM,RingstadN,HorvitzHR:TyramineFunctionsindependentlyofoctopamineintheCaenorhabdi-tiselegansnervoussystem.
Neuron2005,46(2):247-260.
6.
JoseAM,BanyIA,ChaseDL,KoelleMR:ASpecificSubsetofTransientReceptorPotentialVanilloid-TypeChannelSubu-nitsinCaenorhabditiselegansEndocrineCellsFunctionasMixedHeteromerstoPromoteNeurotransmitterRelease.
Genetics2007,175(1):93-105.
7.
HirshD,OppenheimD,KlassM:Developmentofthereproduc-tivesystemofCaenorhabditiselegans.
DevBiol1976,49(1):200-219.
8.
WardS,CarrelJS:FertilizationandspermcompetitioninthenematodeCaenorhabditiselegans.
DevBiol1979,73(2):304-321.
9.
HodgkinJ:SexdeterminationinthenematodeC.
elegans:analysisoftra-3suppressorsandcharacterizationoffemgenes.
Genetics1986,114(1):15-52.
10.
McCarterJ,BartlettB,DangT,SchedlT:OnthecontrolofoocytemeioticmaturationandovulationinCaenorhabditiselegans.
DevBiol1999,205(1):111-128.
11.
MillerMA,NguyenVQ,LeeMH,KosinskiM,SchedlT,CaprioliRM,GreensteinD:Aspermcytoskeletalproteinthatsignalsoocytemeioticmaturationandovulation.
Science2001,291(5511):2144-2147.
12.
TrentC,TsuingN,HorvitzHR:Egg-layingdefectivemutantsofthenematodeCaenorhabditiselegans.
Genetics1983,104(4):619-647.
13.
ReinkeV,SmithHE,NanceJ,WangJ,VanDorenC,BegleyR,JonesSJ,DavisEB,SchererS,WardS,KimSK:Aglobalprofileofgerm-linegeneexpressioninC.
elegans.
MolCell2000,6(3):605-616.
14.
SchedlT,KimbleJ:fog-2,agerm-line-specificsexdetermina-tiongenerequiredforhermaphroditespermatogenesisinCaenorhabditiselegans.
Genetics1988,119(1):43-61.
PublishwithBioMedCentralandeveryscientistcanreadyourworkfreeofcharge"BioMedCentralwillbethemostsignificantdevelopmentfordisseminatingtheresultsofbiomedicalresearchinourlifetime.
"SirPaulNurse,CancerResearchUKYourresearchpaperswillbe:availablefreeofchargetotheentirebiomedicalcommunitypeerreviewedandpublishedimmediatelyuponacceptancecitedinPubMedandarchivedonPubMedCentralyours—youkeepthecopyrightSubmityourmanuscripthere:http://www.
biomedcentral.
com/info/publishing_adv.
aspBioMedcentralBMCDevelopmentalBiology2007,7:41http://www.
biomedcentral.
com/1471-213X/7/41Page10of10(pagenumbernotforcitationpurposes)15.
SingsonA,MercerKB,L'HernaultSW:TheC.
elegansspe-9geneencodesaspermtransmembraneproteinthatcontainsEGF-likerepeatsandisrequiredforfertilization.
Cell1998,93(1):71-79.
16.
ChatterjeeI,RichmondA,PutiriE,ShakesDC,SingsonA:TheCaenorhabditiselegansspe-38geneencodesanovelfour-passintegralmembraneproteinrequiredforspermfunctionatfertilization.
Development2005,132(12):2795-2808.
17.
KosinskiM,McDonaldK,SchwartzJ,YamamotoI,GreensteinD:C.
elegansspermbudvesiclestodeliverameioticmaturationsignaltodistantoocytes.
Development2005,132(15):3357-3369.
18.
WolfnerMF:Thegiftsthatkeepongiving:physiologicalfunc-tionsandevolutionarydynamicsofmaleseminalproteinsinDrosophila.
Heredity2002,88(2):85-93.
19.
RobertsonSA:Seminalplasmaandmalefactorsignallinginthefemalereproductivetract.
CellTissueRes2005,322(1):43-52.
20.
ChapmanT:Seminalfluid-mediatedfitnesstraitsinDro-sophila.
Heredity2001,87(Pt5):511-521.
21.
ClarkNL,SwansonWJ:Pervasiveadaptiveevolutioninprimateseminalproteins.
PLoSGenet2005,1(3):e35.
22.
HirshD,VandersliceR:Temperature-sensitivedevelopmentalmutantsofCaenorhabditiselegans.
DevBiol1976,49(1):220-235.
23.
HillKL,L'HernaultSW:AnalysesofreproductiveinteractionsthatoccurafterheterospecificmatingswithinthegenusCaenorhabditis.
DevBiol2001,232(1):105-114.