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Small
Ruminant
Research
j o ur n a l ho m e p age : w w w . e l s e v i e r . c o m / l o c a t e / s m a l l r u m r e s
Effect
of
roscovitine
and
cycloheximide
on
ultrastructure
of
sheep
oocytes
Letícia
Ferrari
Crocomo
∗,
Wolff
Camargo
Marques
Filho,
Mateus
José
Sudano,
Daniela
Martins
Paschoal,
Fernanda
da
Cruz
Landim
Alvarenga,
Sony
Dimas
Bicudo
DepartmentofAnimalReproductionandVeterinaryRadiology,SchoolofVeterinaryMedicineandAnimalScience,SãoPauloStateUniversity,UNESP,Distrito deRubiãoJuniors/no,18603-970,Botucatu,SP,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received31January2012
Receivedinrevisedform1May2012 Accepted9July2012
Available online 4 August 2012
Keywords: Ovine Oocyte
Meiosisinhibitors Cytoplasmicmaturation
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Itisbelievedthetemporarymeiosisarrestwithroscovitineorcycloheximidemayimprove theinvitrodevelopmentalcompetenceofoocytesindifferentanimalspecies.However, lit-tleisknownabouttheeffectsoftheseinhibitorsonultrastructureofovinescumulus–oocyte complexes(COCs).Theaimofthisstudywastoevaluatetheprogressionof cytoplas-micmaturationandtheultrastructuralchangesinsheepCOCsexposedtoroscovitine orcycloheximide,atacceptableconcentrations.COCswereinvitroculturedfor24hin maturationmedium(controlgroup)containing100Mroscovitineor1g/mL cyclohex-imide(treatmentgroups).Afterthistime,someCOCswereculturedforfurther22hin inhibitor-freemedium.TheultrastructureorganizationofCOCswasevaluatedby trans-missionelectronmicroscopybefore(immaturegroup)andafterinvitroculturefor24and 46h.Asexpected,signsofimmaturityandmaturitywereobservedinimmatureandcontrol groups,respectively.Intreatmentwithroscovitine,therewerecumuluscellsdegeneration, swellingofmitochondrias,fewcorticalgranulesandmanyvesicleswithelectron-dense material.However,incycloheximidetreatmenttherewerenotsignsofdegenerationor cellularsenescence.Metabolicunitsandmitochondrialpleomorphismwerefoundinall experimentalgroups.Theseevidencesdemonstratethatroscovitinepromotedirreversible ultrastructuralchangeswhilecycloheximidedidnotaffectthecytoplasmicmaturation. However,theimplicationsonembryodevelopmentarestillunclear.
1. Introduction
Theefficiencyofinvitroembryoproduction(IVP),the developmentand survivalof embryo,theestablishment andmaintenanceofpregnancyandthefetaldevelopment dependontheoocytequality. Thisoocyte developmen-talcompetenceisacquiredduringfolliculogenesisandis directly related to nuclear and cytoplasmic maturation (Krisher,2004).
∗ Correspondingauthor.Tel.:+551438824743;fax:+551438824743. E-mailaddress:[email protected](L.F.Crocomo).
Theprocess bywhich theoocytecompletesthe first meioticdivisionandprogressestometaphaseIIisknown asnuclearmaturation(Mehlmann, 2005).However,the correct dynamic of chromosome segregationin itself is notenoughtoensuresubsequentembryodevelopment. Theoocytealsoneedstoundergoultrastructural modifica-tionstoattainthefulldevelopmentalcompetence.These cytoplasmicchangesincludemRNAtranscription,protein translationandorganellesredistribution(Meirellesetal., 2004).Inthis processofoocytematuration,the bidirec-tional communication betweenthe oocyte and its sur-roundingcumuluscellsisofvitalimportance.Throughthe gapjunctions,cumuluscellskeeptheoocyteundermeiotic arrest,inducing themeiosisresumptionand supporting
0921-4488
http://dx.doi.org/10.1016/j.smallrumres.2012.07.006
© 2012 Elsevier B.V. Open access under the Elsevier OA license.
oocytes (Le Beux et al., 2003). Roscovitine, a specific inhibitorofcyclin-dependentkinasesandcycloheximide, aproteinsynthesisinhibitorhavebeenextensively stud-iedindifferentanimalspecies(bovine:Albarracinetal., 2005; Takayama et al., 2006; pig: Coy et al., 2005; Ye etal.,2005;goat:Hanetal.,2006;cat:Sananmuangetal., 2010).However,thereisnoinformationabouttheeffectsof theseinhibitorsonnuclearmaturationandultrastructure ofovinesCOCs.
In addition, despite of the reports of efficiency and reversibilityofmeiosisarrest,thereisdiscrepancybetween authorsregardingthesubsequentembryonic developmen-talpotentialandlittleisknownaboutthemorphological changesinCOCsexposedtothesemeiosisinhibitors.Thus, in the present study, we evaluated the characteristics, distribution and ultrastructure changes of cytoplasmic organelles and cumulus cells in ovines COCs exposed, for24h,toeitherroscovitineorcycloheximide,followed by in vitro culture for additional 22h in inhibitor free medium.
2. Materialsandmethods
Unlessotherwiseindicated,allchemicalswerepurchasedfromSigma (Sigma–AldrichCorp.,St.Louis,MO,USA).
2.1. COCscollectionandmeioticinhibition
Sheepovarieswerecollectedatslaughterandtransportedtothe labo-ratoryinsterilesalinesolution(0.9%NaCl)at35◦C.Allvisiblefollicleswere
aspiratedwitha20-gaugeneedleattachedtoa10mLsyringe contain-ing0.5mLpre-incubatedTCM199HEPESsupplementedwith50IU/mL heparin(ShiraziandSadeghi,2007).Groupsof20COCsgradesIandII, withmorethantwolayersofcompactcumuluscellsandhomogeneous ooplasm(Mortonetal.,2005),wereculturedin90Ldropletsof
matura-tionmedium(controlgroup)containing100Mroscovitineor1g/mL
cycloheximide(treatmentgroups),undermineraloil,for24hat38.5◦C
ina5%CO2saturatedhumidityairatmosphere.Thematurationmedium wascomprisedofTCM199withEarle’ssaltssupplementedwith0.1IU/mL FSH(Folltropin®,BionicheCo.);0.1IU/mLLH(Lutropin-V®,BionicheCo.,
Belleville,ON,Canada),0.3mMsodiumpyruvate,75g/mLpenicillin,10%
fetalcalfserumand100Mcysteamine.Stocksolutionsof1g/L
roscov-itineand20mg/Lcyclohexemidewerepreparedindimethylsulphoxide andinTCM199,respectively,aliquotedandstoredat−20◦Cuntiluse.
2.2. Reversionofmeioticinhibition
Aftermeioticinhibitionfor24h,COCswerewashedseveraltimesin TCM199HEPESforremovalofinhibitorsandcultured,forafurther22h,
(1)Immature:immediatelyaftertheharvest,COCswerefixedforTEM. (2)Control24h:COCsfixedafter24hofinvitrocultureininhibitor-free
medium.
(3)Roscovitine:COCsfixedafter24hofinvitroculturewith100M
roscovitine.
(4)Cycloheximide:COCsfixedafter24hofinvitroculturewith1g/mL
cycloheximide.
(5)Control(46h):COCsfixedafter46hofinvitrocultureininhibitor-free medium.
(6)Roscovitinereversibility:COCstreatedwithroscovitinefor24hwere fixedafterinvitrocultureforadditional22hininhibitor-freemedium. (7)Cycloheximidereversibility:COCstreatedwithcycloheximidefor24h werefixedafterinvitrocultureforadditional22hininhibitor-free medium.
Eachexperimental group consisted of100 COCs, approximately, whichwereobtainedin5replicatesforeachgroupperformedondifferent days.AllCOCswerepreparedasdescribedinSection2.3.Theultra-thin sectionswererandomlyselectedandabout10COCspergroupwere ana-lyzedintransmissionelectronmicroscope.
3. Results
3.1. Group(1):immatureCOCs
Fig.1. ImmaturesheepCOCs(group1).(A)Notethecompactcumuluscells(cc),narrowperivitellinespace,thelargeamountoflipiddropletsandvesicles spreadthroughouttheooplasm;(B)detailofthecumuluscellsprojections(pc)throughthezonapellucid(zp)andgapjunctions(jg);(C)clustersof mitochondria(m)rounded,elongatedorwithanarc-likeprojectionlocatedmainlyintheooplasmperiphery.Someoftheminassociationwithlipid droplets(L),formingmetabolicunits.WelldevelopedGolgicomplex(G)andfewcorticalgranules(cg)heterogeneousinsizeandcolorationcanalsobe observed.
sizesandelectrodensityandsomemyelinfigureswerealso found.
3.2. Group(2):control24h
COCsfixedafter24hofIVMininhibitor-freemedium showedmaturitysignscharacterizedbyfullexpansionof thecumuluscells(Fig.2A);retractionofcytoplasmic projec-tionsemittedbycumuluscells(Fig.2A),withsomeofthem remainedintheperivitellinespace,anddisjunctionof junc-tional complexes(Fig.2B).Perivitelline spacewasmore developedandtherewereseveralmicrovillionthesurface ofthecytoplasmicmembrane(Fig.2B).Manypleomorphic mitochondria(Fig.2C),withthesameaspectfromthose ofimmatureCOCs,werespreadthroughouttheooplasm. Therewerefewlipiddroplets(Fig.2C)butmanyvesicles andmyelinfigures(Fig.2C).AsinimmatureCOCs,SERwas eitherasisolatedaggregationsorascomplexassociation
withmitochondriaandlipiddroplets.Therewas substan-tialreductionofquantityandsizeofGolgicomplexes.Large amountof homogeneouscorticalgranuleswereisolated andalignedwiththecytoplasmicmembrane(Fig.2B).
3.3. Group(3):roscovitine
COCs treated with roscovitineshowed some charac-teristics ofmaturity and immaturityas:partialcumulus
Fig.2.SheepCOCsinvitroculturedfor24hwithoutinhibitors(group2). (A)Notethefullexpansionofthecumuluscells(cc)andtheabsenceof projectionsthroughthezonapellucid(zp);(B)observethelargeamount ofcorticalgranules(cg)inclosecontactwiththecytoplasmicmembrane, theabsenceofjunctionalcomplexes,theperivitellinespacewell devel-opedandfilledbymicrovilli(mv);(C)detailoftheooplasm:pleomorphic mitochondria(m),fewlipiddroplets,manyvesiclesandmyelinicfigures (arrows)canbeobservedspreadthroughouttheooplasm.COCstreated for24hwithcycloheximide(group4)showedthesesamecharacteristics.
Fig.4. SheepCOCsinvitroculturedfor46hwithoutinhibitors(group5).(A)Observethefullexpansion,dispersionandlossofcumuluscells(cc);(B)detail oftheareasofcytoplasmicmatrixabsence(arrows)thatrepresentdegenerationsignsintheooplasm.COCstreatedwithroscovitineandtheninvitro cultureforadditional22hininhibitor-freemedium(group6)showedthesesamecharacteristics.
(Fig.3C).Thereweremanylipiddropletsandvesicleswith different size and electrodensity, as in immature COCs (Fig.3C).SERwaseitherassociatedwithmitochondriasor dispersedintheooplasm.Thereweremoremyelinfigures thaningroup2.Golgicomplexeswerenotfoundandthere werefewcorticalgranulesinclosecontactwiththe cyto-plasmicmembrane(Fig.3B).Signsofdegeneration,suchas pyknoticnucleuswereidentifiedincumuluscells(Fig.3A), butnotintheooplasmandorganelles.
3.4. Group(4):cycloheximide
COCstreatedwithcycloheximidehadthesame charac-teristicsofmaturityobservedinthegroup(2)(Fig.2A–C).
3.5. Group(5):control46h
COCs fixedafter 46hof invitroculture in inhibitor-free mediumrevealedmaturitysigns similartoCOCs of
Fig.5. SheepCOCstreatedwithcycloheximideandtheninvitrocultureforadditional22hininhibitor-freemedium(group7).(A)Detailofthefullcumulus (cc)expansionwithoutsubstantiallossofcells;(B)notethelargeamountoflipidgranules(L)andvesiclesspreadthroughouttheooplasm.
group(2).However,besidesthefullcumulus expansion, thereweredispersionandsubstantiallossofcells(Fig.4A). Moreover, there were few myelin figures and dilated SERinassociationwithmitochondriaandlipiddroplets. Evidencesofcellularsenescenceand degenerationwere observed in theooplasm, such as areas of cytoplasmic matrixabsence(Fig.4B),andincumuluscells,characterized bypyknoticnucleus.
3.6. Group(6):roscovitinereversibility
cat:Sananmuangetal.,2010).However,thereisnomuch informationabouttheeffectoftheseinhibitorsontheCOCs ultrastructure.Here,wereportthemorphologicalchanges insheepCOCsinvitroculturedwithroscovitineand cyclo-heximide,atacceptableconcentrations.
ThesignsofimmaturityandmaturityobservedinCOCs analyzedbefore(group1)andafter(group2)theinvitro
culture for 24h in inhibitor-free medium, respectively, areconsistentwiththeexpectedand withthe observa-tionsof earlier workers. It is assumed that the invitro
cultureconditionswereadequateandallowedthe progres-sionofcytoplasmicmaturation.Thesefindingsaresimilar withtheothersmammalianspecies,likebovine(Nagano etal.,2006);ovine(O’Brienetal.,2005);equine(Alvarenga, 2006)andpig(Cran,1985).
Thestudieswithmeiosisinhibitorsincattleproposea periodof24hformeioticinhibitionfollowedby18–24h forresumptionandprogressionofmeiosis(AdonaandLeal, 2004;Albarracinetal.,2005).Thisexperimentalmodelwas carriedoutbecausetherewerenostudiesconductedwith meioticinhibitorsinsheep.But,theevidencesofcellular senescence,suchasdegenerationsignsintheooplasmand
cumuluscells,afterinvitroculturefor46hininhibitor-free medium,indicatethatthisperiodwasexcessive.According tosomeresearchers,theadequatetimeforIVMofsheep COCsvariesfrom24h(Shietal.,2009)to26–27h(Shirazi andSadeghi,2007).Máximoetal.(2011)alsoreportedthat 18hwasenoughfortheoocytetocompletematuration.
AccordingtoIsobeetal.(1998),themeiotic resump-tionisdirectlyrelatedtothedisruptionofgapjunctions between the cumulus cells and the oocyte. Thus, it is believed that the partial cumulus expansion and pres-enceofsomejunctional complexesin theCOCs treated for24hwithroscovitine(group3)aredirectlyrelatedto theefficiencyofmeioticarrestpromotedbythisinhibitor, sincethecumulus cellsareinvolved intheregulationof oocytematuration. However,roscovitine treatmentalso resultedincumuluscellsdegenerationandsubstantialloss ofcellsafterinvitrocultureforadditional22hin inhibitor-freemedium.InbovineCOCs,Lonerganetal.(2003)also reportedabsenceofcumulusexpansion,prematurelossand degenerationofcumuluscellsevenafterinvitroculturein absenceofroscovitine.
Incontrast,treatmentwith1g/mLcycloheximidedid
notinterfereincumuluscells,whichwerefullyexpanded
So,thenumberandthemovementoftheseorganellesto theareas withhighenergy consumptionare crucial for oocyteandembryoduringthecriticalperiodsof develop-ment.Inthisstudy,mitochondriamigratedfromperipheral positioninimmatureCOCs(group1)toamoredisperse dis-tributionthroughouttheooplasminmatureCOCs(group 2),asdescribedbyHytteletal.(1986).
Themitochondrialmigrationwasnotimpairedbyboth meiosisinhibitorsstudied,buttheroscovitinetreatment for24haffectedtheelectrondensityofmitochondriaand caused the swelling of them as reported by Lonergan et al. (2003). These changes were not reversible even afterinvitrocultureforadditional22hininhibitor-free medium.
In all experimental groups there was mitochondrial pleomorphism whichwasalsodescribed bySenger and Saacke(1970),inbovine,Luccietal.(2001),ingoat,and
Máximoetal.(2011)inovineoocytes.AccordingtoFair etal.(1995),theroundshape ofmitochondriaindicates immaturity.Withtheprogression ofoocyte maturation, theybecomeelongated.Moreover,thepresenceofarc-like projectionincreasesthemitochondrialsurfaceandmaybe relatedwithhighoocytemetabolismduringthe matura-tion(Máximoetal.,2011).
Functionalormetabolicunitswerealsoobservedinthis study.Thesestructuresareinvolvedwiththemetabolism oflipidandsynthesisoflipoproteins,nutrientsandgrowth factorsduringthecriticalperiodsofoocytedevelopment, fertilization and neogenesis of membranes in the first embryoniccleavages(Mottaetal.,2000).
Asexpected,duringtheoocytematuration,therewas reduction in the number of lipid droplets and increase of themyelin figures,which represent digestivevesicle responsibleformetabolismoflipidanddegradationofaged and non-functional cellular structures (Kacinskis et al., 2005).ThelargeamountoflipiddropletsobservedinCOCs treatedwithcycloheximideattheendofinvitroculturefor 46hprobablycorrespondstoanindividualcharacteristic ofexaminedoocytes.Itisnoteworthythat,inthisstudy, ovaries were randomlycollectedat theslaughterhouse, regardlessofphysiologicalfemaleconditions.
Theyappeartobeinvolvedwithstorageandtransportation ofmetabolites(Mottaetal.,2000).
AsdescribebyHytteletal.(1989),welldevelopedGolgi complexeswerefoundonlyinimmatureCOCs(group1) and,afterinvitromaturationfor24h(group2),the cor-ticalgranuleswereinclosecontactwiththecytoplasmic membraneformingamonolayerstrategicallytoprevent polyspermy. In comparison, COCs treated with roscovi-tinehadreductioninthenumberofcorticalgranulesand increaseofelectron-densematerialintheirvesicles,after
invitroculturefor24and46h.
5. Conclusion
Thesefindingsdemonstratethetreatmentwith100M
roscovitineresultedinharmfuland irreversiblechanges in oocytesand cumuluscells. While thetreatmentwith 1g/mLcycloheximidedidnotimpaircytoplasmic
matu-rationofsheepCOCswhichshowedmaturitysignswithout ultrastructurechangesanddegenerationsigns.However theimplicationsonembryodevelopmentremaintobe elu-cidated.
Acknowledgement
ThisworkwassupportedbytheFundac¸ãodeAmparoa PesquisadoEstadodeSãoPaulo(FAPESP),Brazil.
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