【word】 发情期蓝狐卵泡发育的显微结构观察

【word】 发情期蓝狐卵泡发育的显微结构观察 发情期蓝狐卵泡发育的显微结构观察 Agricultural, Science&Technology,2012,13(3):644—648,685 Copyright2012,InformationInstituteofHAAS.Allrightsreserved MiCroStrUCtUreObServatiOnontheFollicular DevelopmentofBlueFoxduringtheEstrous Period KaiCUl’,MingyuTIAN,ZefangMA1,YuyingLIANG’,YanpengLI’,ShansongGAO’,Shutian NIU.,HaitaoWANG.,JunwenLIANG.,ShuhuiYANG 1.CollegeofAnimalScienceandVeterinaryMedicine,QingdaoAgriculturalUniversity,Qingdao266109,China; 2.CollegeofWildlifeResources,No~heastForestryUniversity,Harbin150040,China; 3.AnimalHealthSupervisionInstituteofShandongProvince,Jinan250022,China Abstractf0b1microslructure.iectiveTheovaryandthechangesofthefollicleduring thedevelopmentperiodofbluefoxduringtheestrousperiodwerestudied.fMethod1 TenIeftovarieswerecollectedfrom10femalebluefoxesduringtheestrousperiod. ThehistologicaIstructureoftheovaryandfolliculardevelopmentprocesswereob— servedbyusingopticaImicroscope.And30primordialfollicles.30primaryfollicles, 15growingfollicles.15vesicularfollicle.15maturefolliclesandtheoocytesinthe folliclesofdifferentdevelopingstagesweremeasuredandphotographedbyeyepiece micrometerandphotomicrographictechniquerespectively.fResult]Theovaryofblue foxconsistedofgerminaIepithelium,tunicaalbuginea,codexandmedulla.Thecor- tex.whichwasJocatedattheperipheryoftheovary.containedmanyinterstitiaIcells andfolliclesatdifferentdevelopingstages.MedullawasIocatedbelowthecodex, withaIotofbloodvessels.Thediametersoffolliclesatdifferentdevelopmental stagesandOOCyteshadgreaterdifferencesduringthedevelopmentalperiodoffolli— cle.Thediameteroffollicleswas45.45—974.55umandthediameterofOOC yteswas 30.23-147-27Hm.ThezonapellucidaappearedattheprimaryfoIliclestage.Thea— tresiaoffolliclesoccurredatalIthedevelopmentalstagesoffoIlicleandthemain manifestatiOnsweretheshrunkOOCyteandkaryon.andlooseanddegradedgranular cells.fConclusion1Theresearchprovidedhistologicalbasisforrevealingtherepro- ductivemechanismoffemalebluefox. KeywordsOestrusperiod;Bluefox;Ovary;Follicle;Microstructure Inrecentyears,thepropagation Itechniquesofbluefoxiscontinu一 0ouslyupdatedandadvancedwith therapidextensionofthefoxfarmina industry.TheresearchonanimaI’s ovarytissuecanfurtherpromotethe developmentofitspropagationtech- niques.Takingthehistologicalstudies onthereproductivesystemofbluefox asthetheoreticaIbasis.tomasterthe statusofovarianfolliclesateachgrade canbettergetknowofthereproduc- tivemechanismoffemalebluefox. ThehistologicaIobservationstudieson thereproductiveorgansoffemaleblue foxathomeandabroadwasfew.es- peciallythestudiesonovarianfollicles. InChina.LIUYutangeta1.【1】reported thehistologicalanatomyontherepro- ductiveorgansoffemalebluefoxand LIUBingxuetaLreportedthehisto- fogicaIobservationontheovaryinthe anestrousphaseofbluefoxafterSU- perovulation.Atabroad,TedY. MashimaetaL嘲reportedtheovary studiesoffoxintheresearchofsteroid celItumorintheovumofOtocyon megaIotis.Westudiedtheovarianhis- tologicalstructureandthechangesof follicleduringthedevelopmentaIpro- cessofbluefoxinoestrusperiod,so astoprovidehistologicaIbasisforre- vealingthereproductivesystemoffe— malebluefox. MaterialsandMethods Maininstruments Microtome(ZhejiangJinhuaRadio Factory):0pticalmicroscopeOlympus CKX41:eyepiecemicrometer(Wu- han);microscopecamera(Olympus); temperature electrothermaIconstant— waterbathSY21一NifBeijingChang- fengInstrument&MeterCompany); electrothermaIconstant—temperature blastdHingovenDGG-9140AfShang— haiSumsungExperimentaIlnstrument Co.,Ltd). Maindrugsandreagentpreparation MaindrugsAnhydrousalcoho1.fOrm— aldehyde,hematoxylin,mercuricoxide, qlaciaIaceticacid.eosinandxylene werealIdomesticanalyticalreagents. Mainreagentspreparation(10% neutraIformaldehydesolutionwas preparedwith1O0mIof40% formaldehydeaqueoussolutionand 900mlofdistilledwater;hema- toxylinsolutionwaspreparedasfOI- IOWS:200mIof10%potassiumalum aqueoussolutionwasheatedanddis- solvedinerlenmeyerflask.then1O% hematoxylinalcohoIsolutionthatwas dissolvedwaspouredandboiledfor oneminute.Afterceasefire.0.5qmer- curicoxidewasslowlyadded.Then thesolutionwasboiledforanother minute.theerlenmeyerflaskwas cooledincoldwater.Finally.1Om10f glaciaIaceticacidwasadded;(3)1% eosinaqueoussolutionwasprepared with1geosinand90mlofdistilled water. Thecollectionandprocessingof ovary TenIeftovarieswerecollected 2O12 fr0mhe. alt c h 0 y n f v e e m n a ti l 0 e n b a l I uefoxthatw fr e O r m e fedunderconditions DayuFoxFarminWeifangCityof ShandongProvinceonApril4.2011. Afterthefoxeswerekilledbyusing electricimpactmethod,thefurwas shuckedoffimmediately.TheIeft ovariesweretakenoutandputintothe culturedisheswithdouble-resistance normaIsaline.Aftertheovarieswere bisectedintotwohalves,theovaries werefixedwith10%neutraIformalin solution.Theovarieswerebrought andsavedinthelaboratoryatnormaI temperatureforthepreparationoftis- suesections. Thefolliclemicrostructureobser- vationofovaries Aftertheovarieswerefixedinfixa- tionliquidfor48hours,theovaries werewashed,driedandembedded withparaffin.Throughcontinuoussec- tion,thethicknessofsectionswas5 um.Continuous5-10sectionswere selectedfrOm10sections.Afterthe sectionswerestainedbvHEdyeing andsealed.thehistologicaIstructure ofovariesandfollicles,ateachgrade wereobservedbyusingopticaImicro- scope.1—3completefolliclesateach gradewereselectedfrOmeachovary. And30primordiaIfoIlicles.30primary follicles,15growingfollicles,15vesic— ularfolliclesand15maturefollicles weretotallyobserved.Thediameterof folliclesandooc~esweremeasured. Referringtotheclassificationmethod 0ffoIliclesbyLIYunlong~.thefoIlicles wereclassifiedintoprimordiaIfollicles. primaryfollicles,growingfollicles, vesicularfoIliclesandmaturefoIlicles accordingtothesizeoffoIlicles,the fOrmationofzonapellucida.theap- pearingtimeoffollicularcavityandthe developmentaldegreeofooc~es. ResultsandAnalysis ThegeneralmorphoIogicaIatruc- tureofovary Theovarywascomposedofger- minaIepithelium.tunicaalbuginea, codexandmedulla(Fig.1).Thegermi- nalepithelium.whichwascomposed ofmonolavercubicepitheliaIcells,was Iocatedonthesuffaqeofovary.The nextwastunicaalbuginea,whichwas composedofspindle--shapedconnec?- tivetissuecellsinthecloseanctparal— IeIarrangement.Thecodexwasinthe peripheryofovary.Andtherewerefol- Iiclesandatresicfolliclesatdifferent developmentaIstagesintheinterstitiaI cells.TheinterstitiaIcellsweremostly spindle-shapedorelliptica1.Themed- ullaofovarywascomposedofloose connectivetissues.includingsmooth muscles,nerves,bloodvesselsand Iymphaticvessels.Theprimordialfolli- clesandprimaryfolliclesweremainly distributedintheshallowlayerofcor- tex.Thegrowingfolliclesandvesicular folliclesweremostlyIocatedinthe deeplayerofthecodexandthecon- nectivetissuethatsurroundedfollicles obviouslydifferentiatedandformedto thecafolliculi.Thematurefollicles movedtowardstheshallowlayerof thecodexofovary. Themicrostructureofthefollicle development PrimordiaIfolliclesMostprimordial foIlicleswereIocatedintheshallow Iayeroftheovariancodex.Theprima— ryooc~eswereIocatedInthecentraI part,monolayercompleteorincom- pleteflatgranulosacellswereinthe s~roundingpartandafewooc~es weresurroundedbymonolayergranu- losacellsandcubicgranulosacells (Fig.2).Therewerealargeamountof primordialfoIliclesintheintheshallow layeroftheovariancodexandthe distributionwasuneven.Theprimor- diaIfoIliclesshowedsingleorclustered distribution.Thegranulosacellshad deepdyeingandtheywerecloselyIo- catedtoooc~es.Thecellnuclearwas Iargeandcircular,therewasfewhete- rochromatininthecelInuclearandthe nucleoluswaslargeandclear.The fOrmationofzonapellucidasub- stanceswerenotseeninthisstage. PrimaryfolliclesPrjmaryfollicles weremainlyintheshallowIayerofthe ovariancodexandweresurrounded bymonolayercolumnarormonolayer cubicgranulosacells.Thevolumeof ooc~esincreased.buttheIncreasing speedoffollicles’volumewasslow. ThebasaImembraneappearedinthis stage.whichseparatedfolliclesfrOm theovary’sbase.Thezonapellucida formed.butitwasthinandincomplete. Thezonapellucidawasacidophiland showedincarnadineafterdyeing(Fig.3). GrowingfolliclesTheooc~esof growingfolliclesincreasedquicklyand thediameteroffolliclesalsoincreased. Thelayernumberofgranulosacells obviouslyincreasedandwasabout morethan3.Thefollicularismem- braneobviouslythickened,complete zonapellucidawasseenandgranu- IosacellsIooselyarranged(Fig.4). Thereweresomelacunaamonggran- ulosacells,butobviousfollicularcavity wasnotseen.Thefolliclesmovedto— wardsthedeepIayeroftheovarian codex. VesicularfolliclesAsthegranulosa cellsappearedobviousIacuna.the Iayernumberofgranulosacellswas morethan4andthediameteroffolli- clesobviouslyincreased.Theooc~es hadthetrendofmovingtowardsone side.thecellnuclearthatwasIocated inthemarginwasIargeandcircular andthenucleoluswasclearlyseen (Fig.5a).Inthisstage,thefolliclewall wasthicker.thegranulosacellsjnin- nerIayerhaddeepercolorafterdye- ing.Thefolliclemembranelayerwas outsideofthefolliclewalIanditwas differentiatedfrOmconnectivetissue. Thezonapellucidawasthickenedand itshowedgarnetafterdyeing.With fudherdevelopmentoffoIlicles.smalI IacunaofgranulosacellsformedIarger follicularcavity.Andfollicularfluidfilled thefollicularcavityfFig.5b).Inthis stage,thecircumstancethatthere weretwoooc~esinonefolliclewas seldomseen(Fig.5c). MaturefolliclesMaturefoIlicles wereIocatedinshallowIayeroftheo- vary’scodexregion.ThefcIIlicular cavityenlargedandtherewasfullOf follicularfluidinthecavity.Thefollicu- Iarcavityshowedcircularorelliptica1. Theooc~eswerelocatedononeside Off0?icIes,andtheVandtheirsur- roundinggranuJ0saceIJsfom1edcu— muIus.Therewere3—4layersOfgran- u10SaCe?SOnthet0D0fCUmUlUS.The f0lIicIewal1wasc0mpOsed0fmultiIay- ergranu10saCelIsandhighIy—vasCu- IarisedfollicuIarismembrane(Fig.6). Thecircumstance廿1atthereweret,『lf0 OOc,,tesinOnef0I?cIewasalsOseen. TheIOcatjOn0fmaturefOllicIeSwaSdif- ferentfrOmthal0f0theranImaJsand fOllicIesthatwereexlrudedfrOmtl1e surface0f0Vary. ThediameterOffO?jcIesand OOcytes Table1shOwedII;atthedIameter OffOIIIcIesandOOcvtesincreasedbe- V01.13,N0.3,2012AgricuIturaIScience&TechnOlOgy 2O12 foretheoccurrenceofgrowingfolli— cles.AsthesmalIIacunasappeared amonggranulosacells,thestageof growingfolliclecame.Theincreasing speedofthediameteroffolliclesquick- enedinthisstage.buttheIncreasin# speedofoocytes’diameterwasstilI slow.Especiallyinthematurefollicle stage.thediameteroffollicleswas ?,tunica;?,thepartofcortex;?,thepart ofmedulla. Fig.1Thetunicaandcortexofovary(10x20 times) ?,granularcell;?,cellnucleolusandcel caryon. Fig.2Theprimordialfollicles(10x40times) ,monolayergranularcell;?,occurrence ofzonapellucida;?,basementmembrane. Fig.3Theprimaryfollicle(10x20times) , incrassatethecaoffollicle;?,three— layergranularcell;?,unbrokenzonapel- Iucida. Fig.4Thegrowingfollicles(10x20times) evenabout7timesasthatofOOCytes. Themicrostructureofatreticfol?- cles TheatreticprimordiaIfolliclehad itsgranulosacellsanditsarrangement wasirregular.TheOOCytesshrunkand deformedandthecelInuclearwasPY- knotic(Fig_7).Whenprimaryfollicles wereatretic,theoocytesshrunkand deformed.thecelInuclearwasPY— knotic.zonapellucidadeformed,gran- ularcelllayerwasIooseandthebasal membranedisappeared(Fig.8).When growingfollicleswereatretic,the ooc~esdeformedseriously.thezona pellucidabasicallydisappeared,the cellularIayeroffolliclescollapsed,the granulosacellsintheInnerlayerof follicularwallloosenedandshedinto follicularcavity.butthebasaImem— branewasstilIcomplete(Fig.9).The atresiaofsaccularfolliclewascomplex anditwasacontinuousatresiapro- cess.Andtheprocesscouldbedivid- edintothreestages:earlystage.mid— diestageandanaphase. Theatreticfolliclesatearlystage showedthebasaImembraneoffolli— clesbegantoruptureandthepartnear tobasaImembraneportionvascular- abC a,?standsforthegranularcellwithmorethan4layersand?standsforTheoccurrence ofsmalllacuna(10x40times);b,?standsforfulloffollicularfluidand? standsforthe incrassateandcrimsonzonapellucida(10x20times);c,twoooc~esinafollicle (10x20 times). Fig.5Thevesicularfollicles ?,thefollicularfluid;?,thecumulus oophorus;?,thethecaoffollicle. Fig.6Thematurefollicle(10xl0times) ?,theloosegranularcell;?,thetransm— utativezonapellucida;?,thebasement membranedisappeared. Fig.8Theatresicprimaryfolliclef10x20 times) ?,theincompletegranularcell’slayer;?, theshrunkOOCytecaryon. Fig.7Theatresicprimordialfollicle(10x20 times) ?,thegranularcell’slayercollapsed;?, thezonapellucidawasnotclear;?,the karyondisappeared. Fig.9Theatresicgrowingfollicles(10x40 times) Table1Thediameteroffollicleandooc~eofBlueFoxpm AgriculturalScience&TechnologyVo1.13,No.3,2012 2012647 ized.ThegranulosacelIIayerinside thefollicularwalfbecameuneven.The #ranulosacellsneartofollicularcavity werelooseandsomegranulosacells begantodegenerateandwither.The zonapellucidafoldedanddeformed, buttheoocyteswerestilIparceledby 1—2Iayerofgranulosacells(Fig.10). Withthefurtheratresiaoffollicles,the wholefollicularcavityofatreticfollicles atmiddlestagedeformedandthebro— kenbasaImembranedisappeared,the unevengranulosacelllayerbecame thinnerandthinnerandshedintothe follicularcavity.Theepitheliajnside 1,.thebasementmembranewasfrac— tured;),theloosegranularcell’slayer; . thezonapellucidawasseriouslyshrunk anddeformed. Fig.10Theprophaseofatresicvesicular folliclef10x20times) l1).1—2layersgranularcells;(,thegran— ularcel1wasshed;(,theepithelialcellwas multiplicated;.thebasementmembrane disappeared. Fig.11Themetaphaseofatresicvesicular follicle(10x40times) . theintimaincrassatedandepithelial cellsinsteadofgranulecells;),thefoIlicu— Iarantrumwasshrunkandlheooc~edis— appeared Fig.12Thetelophaseofatresicvesicular folliclef10x40times1 thefollicularwalIbegantoproliferate, buttheoocytesstillmaintainedthe stateofatreticfolliclesatearlystage (Fig.11).1ntheanaphaseoffolliculara— tresiajtcouldbeclearlyobservedthat thefollicularintimathickened.the granulosacellsinthefollicularwall completelydisappearedandthegran— ulosacellswerecompletelyreplaced byepithelioidcells.TheoocytesalI disappearedandthewholefollicular cavityobviouslyshrunk(Fig.121. Discussion ThehistologicaIstructureofovary inbluefox Theovaryofbluefoxwascom— posedofgerminaIepithelium,tunica albuginea,cortexandmedulla.The substrateofovarywascomposedof connectivetissueandtheinterstitiaI cellsexisted.Theinterstitialcellsjnthe corticalzoneofthebluefox’sovary weredevelopedandsegmentedinto cord.-likesubstancebyconnectivetis?- sueandbloodvesse1.Buttheconnec— tivetissuewasIess.Theseresults weresimilartotheresearchresultsof Mustelavison[5】andobviouslydifferent frOmtheresultsofthestudiesonHo— mosapiens[0】andCervuselaphusI. GAOMao-ying[5~reportedthattherole ofinterstitialcellswastoexcretethe estrogen.Inthisexperiment.1twasob— servedthattheinterstitialglandwas developedintheovaryofbluefox. whichindicatedthattheestrogenin bluefoxinoestrumstagewasmainly frOminterstitiaIcells.Andagreat amountofestrogenthatwasexcreted inthisstagewasmainlytopromotethe folliculardevelopmentan