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Biphasic effect of gonadotropin releasing hormone on progestin secretion by rat granulosa cells

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Biphasic effect of gonadotropin releasing hormone on progestin secretion by rat granulosa cells
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  BIOLOGYOFREPRODUCTION28,591-597(1983) 591 Biphasic Effect of GonadotropinReleasingHormoneon Progestin Secretion by Rat Granulosa Cells C.S.SHEELARANt,’C.EKHOLM,H.BILLIG,C.MAGNUSSONandT.HILLENSJO2 DepartmentofPhysiologyUniversityofGOteborgS-40033Goteborg,Sweden ABSTRACTTheeffectofanagonisticgonadotropinreleasinghormone(GnRH)-analog(D-Ala’,des-Gly’ {176}NH3-GnRH-ethylamide,GnRHa)ongranulosacellsteroidogenesisinthepresenceorabsenceoffollicle-stimulatinghormone(FSH)orluteinizinghormone(LH)wasstudied.Granulosacells,isolatedfrompreovulatoryfolliclesofpregnantmare’s serum gonadotropin (PMSG) -treatedimmatureratsorfromthelessmaturefolliclesofuntreatedimmaturerats,wereculturedforaperiodof72hwithdailychangesofmedium,andprogesteroneanditsmetabolite,20a-dihydro - progesterone(20o-OHP),wereassayedin themedium.In granulosacellsfrompreovulatoryfollicles,LHandFSHcausedamuchgreaterstimulationofsteroidogenesisthandidGnRHa.ThereappearedtobenointeractionbetweenGnRHaandFSHduringthefirst10h,butat24handlaterthepresenceofGnRHaclearlyinhibitedthesteroidogenicresponsetoLHandFSH.SteroidogenesisingranulosacellsfromimmatureratswasconsiderablylowerandtheeffectsofGnRHaandFSHalonelesspronounced.Inthesecells,FSH {149}stimulatedprogesteronesecretionwasinhibitedbyGnRHaonlyat72h.Incontrast,20o-OHPsecretioninthesamecultureswaspotentiatedbythecombinedpresenceofFSHandGnRHa.Inconclusion,itseemsasthoughtheeffectsofGnRHaongranulosacellsteroidogenesisvaries with exposuretime,theinitialresponsebeingstimulatoryandthelaterinhibitory.Further - more,the response isalsotosomeextentdeterminedbythematurationalstageofthegranulosacells. INTRODUCTION Inadditiontotheireffectsonthepituitary, GnRH anditspotentsyntheticanalogs(GnRHa)areknowntohavedirectactions on thegonads(reviewedbyHsuehandJones,1981;Sharpe,1982).Therehavebeenreports of both stimulatory(Clarketal.,1980;Ekholm et al.,1981,1982;Clark,1982)andinhibitory (Hsueh andErickson,1979,Hsueheta ., 1980;Knechtetal.,1981)effectsofGnRHon granulosa cellsteroidogenesis.However, differentmodelsystemshavebeenusedinthesestudies.Thus,wheninhibitoryeffectswereobserved,granulosacellsfromimmaturehypophysectomized,diethylstilbestrol(DES)   treatedrats had been usedand exposedtoAcceptedNovember 4,1982. ReceivedAugust 9,1982. ‘ address: Dept. of Biochemistry,IndianInstituteofScience,Bangalore 560012,India.Sup - portedby a ResearchTraining Grantfrom theWorld Health Organization.2Reprint requests: Dr. T.Hillensj {246},Dept.of Physiology, University ofGoteborg,Box33031, S-400 33 Goteborg,Sweden. GnRHforaperiodofnotlessthan2days,usuallyinthepresenceofFSH.Ontheother hand,stimulatoryeffectswereobservedwhengranulosacellsfrompreovulatoryfolliclesofintact,PMSG - treatedratswereincubatedforshortperiods(upto 5h)withGnRH alone, andundertheseconditionsGnRHdidnotmodifytheresponsetoFSHorLH.ItseemedlikelythattheresponseofgranulosacellstoGnRH(stimulationorin-hibition)varieswithtypeofgranulosacell usedand withtime.Inthepresentstudy,carriedouttotest thesepossibilities, granu- losacellsofdifferent stageswerecultured forupto 3dayswithGnRHa,bothinthe presence andabsenceofFSH orLH. Theculturemediumwaschangeddailyandpro-gesteroneand 20a - dihydroprogesterone(20 z   OHP)wereassayedinthemedium. MATERIALSANDMETHODS AnimalsandCultureProcedure Twenty-eight-day-old,femaleSprague-Dawleyrats(AnticimexLtd.,Stockholm)wereusedeither  592 SHEELARANIETAL. InteractionsbetweenGnRHaand FSH 20 w x  GnRHo z llllhlkflt   10  {149} nRHa.Anf Mi 20 15 II FIG.1.EffectofGnRHa (100ng/mI)and GnRH antagonist (Ant,10Mg/mI)onprogestinaccumulationduringa24-hcultureofgranulosacells obtained fromPMSG - treatedrats.Mean ± SEMof 5 observa - tionsareshown.xx=P<0.01vs.control  C .  1i NH3)GnRH-ethylamide,SigmaChemicalCo.,St.Louis,MO)weredilutedwithculturemediumtothedesiredconcentrationbeforeuse.GnRHantagonist((D-pGLU’, D-Phe2,D-Trp3”J LRF,Boehringer-MannheimBiochemicals,Indianapolis,IN)wasdissolvedinphosphate-bufferedsalinepriortouseanddilutedwithculturemedium.3H-labeledprogesteroneand20a-OHPusedforradioimmuno-assay (RIA) werefromNewEnglandNuclear,Boston, MA. OtherchemicalswerepurchasedfromSigmaChemical Co. SteroidAssay Radioimmunoassayforprogesteroneand20o- OHPemployedspecificantisera (LindnerandBauminger,1974)kindlydonatedbyDrs.LindnerandKohen,TheWeizmannInstituteofScience,Rehovot,Israel. RIAwasperformedwithoutprior extractionafterdilution of theculturemediumwithdistilledwaterasdescribedpreviously(Hillensj {246}etal.,1981).  tatistical nalyses The datapresentedaremeans ± SEMfromat least three differentexperiments,thenumberofreplicatesin each experimentbeing 4-6.Analysis ofvariancewasfollowedbyStudent-Newman- Keulstestanda P value lessthan0.05was considered significant. RESULTS GranulosaCellsfromPMSG - TreatedRats withoutany pretreatmentorafteras.c.injectionof 10IUPMSG2 daysearlier.Theratswerekilled by cervicaldislocation,theovariesremoved,cleanedofadherenttissue and placedinsterileculturemedium(seebelow).Thefollicles wereincisedwitha micro-knifeunderastereomicroscopeandthegranulosacellsexpressed into themedium.Cellswerecollected andwashedoncewithfreshmediumafter centrifuga-tion for 5mmat50-100Xg.Afterdetermining the numberofcellsand checkingtheir viability withthecrypanbluedyeexclusiontest(50-60%viablecellsineachexperiment),thecellsuspensionwas pipettedinto multi -wellcultureplates(Falcon). Approximately 70-100X io granulosa cellswerecultured in 500 slmedium.Oocyte-cumuluscom-plexes werecollectedseparatelyand distributed10perwellinMicrotestplates(Falcon),200Mlfinalvolume.Thecultureswerekeptat37 {176}Cn humidified airfor10or72hwithchangesofmediaat24 and48 h. CultureMedium Eagle’sminimalessentialmediumwithEarle’ssalts,10mMHepes,10%fetalbovine serum andgentamicin(50 g/ml)wasused. HormonesandCbemicals Stock solutions(0.1Mg/mi)ofLH(NIH-S19), FSH(NII-I-S13) andGnRHa((D-Ala’,des-Gly10- Ingranulosacellsculturedfor24h,GnRHa(100ng/ml)causeda2.5-foldincreasein progesteroneanda 4-foldincreasein20a- OHPsecretioncomparedtocontrolcultures(Fig.1).OtherexperimentsshowedthatGnRHa concentrations between1 and 100 ng/mlproducedsimilarlevelsofstimulationofprogesterone,while0.1ng/mlhadnosignifi-canteffect(Table1).TheantagonisticanalogofGnRH(10i.eg/ml)hadnointrinsicactivity, butwhenpresenttogetherwithGnRHait completelyabolishedthestimulationbyGnRHa(Fig. 1).GnRHastimulatedprogesteronesecretion witha maximalresponseat24h,butastimula- tion couldalreadybeseenat10h(Fig.2).FSH and LH(100ng/ml)bothcausedamorepronouncedstimulationofprogesteronethan did GnRHaatall times ofculture.Therate of progesteronesecretiondeclinedwithtime and theamountofprogesteronesecretedinresponsetoFSHandLH was loweronDays2 and 3ofculturethanduringthefirst 24-hperiod.  50 a a DC  {149}  LHlal z 0I’-,c/) o5 * GnRHO -+-+   -+-+-+-+-+-+-+-+ I: EFFECTOFGnRHONGRANULOSACELLPROGESTINSECRETION 593 cp<OOS O-IOhO-24h 24-48h 48-72h FIG.2.ProgesteroneaccumulationduringcultureofgranulosacellsobtainedfromPMSG - treatedrats. Thecells wereculturedincontrolmedium  C , withFSH(100ng/ml)or LH (100ng/ml),without (-) orwith(+)GnRHa (100 ng/ml).The culturesdesignated 0-10b wereterminatedat10h,whereastheothercultures werecontinuedfor72hwithchangesofmediaat24and48h.Mean ± SEM of 6-15 observations. x P<0.05 vs.correspondingcontrol(withoutGnRHaandgonadotropin).xx P<0.01,*p< )5vs. corresponding group withoutGnRHa,and =P<0.01. orLHwerestudiedbycombiningGnRHa and eithergonadotropinfromthestartof culture. Theprogesterone accumulationduring 10hofculturewithFSH was notdifferentwhenGnRHaalsowaspresent. However, at24 h GnRHasignificantlyreducedthe gonadotropin   stimulatedprogesteroneproduc - tion(Fig.2).Maximal inhibitionofFSH-stimulatedprogesteronesecretion(60%)had developedafter48hand wassustainedat 72h.SignificantinhibitionoftheFSH re-sponseat24hwasobtainedwithGnRHaat10ng/mlwhilelowerconcentrationswereineffective(Table1),butonDay2significantinhibitionwasobtainedwith1ng/ml(40%inhibition).Thepatternof20a-OHPsecretioninre-sponsetohormonestimulationresembled that ofprogesterone,althoughthelevelswereseveralfoldhigher(Fig.3).GnRHacausedamaximalstimulationat24h,butafterthestimulationdeclined and ceased at72h.FSHcausedamoderatestimulationat10h, and at24hapronouncedstimulation wasseenwithbothgonadotropins,sustainedatlater times.Nointeractionwasfound between GnRHaandFSHduringthefirst48hof TABLE1.Effect ofdifferentconcentrationsof GnRHaonprogesteroneaccumulationduringa 24-hcultureofgranulosacellsobtainedfromPMSG - treatedrats. GnRHa(ng/ml) - FSH + FSH (100 ng/mI)05.6 ± o.loa49.0± 3.33 0.11.0 10.0 100.06.4 ± 0.38 11.8 ± 1 21b11.5 ± 090b14.2 ± 1 {149}08b42.1 ± 3.6239.6 ± 2.95 28.8 ± 329baValuesshownarethemean ± SEMof4-5ob-servations. bp<ooi versus appropriatecontrol(GnRHa, 0ng/ml)withanalysisof variance afterlogtrans - formation.  DC  {149}  LH a a a a -+-+-+-+-+-+-+-+-+-+-4. 594 SHEELARANIETAL. 0- 0h 0-24h24-48h48-72h FIG.3. 20a -OHPaccumulationduringcultureofgranulosacellsobtainedfromPMSG-treatedrats.CultureconditionswerethesameasgiveninFig.2.xx=P<0.01vs.correspondingcontrolgroup(withoutGnRHaandgonadotropin). =p<0.05vs.correspondinggroupwithoutGnRHa,and =P<0.01. culturebut a slight inhibitionoccurredat 72h.TheLH responsewasalreadyinhibitedbyGnRHaat48hand thisinhibitionwas morepronouncedat72h(Fig.3). CumulusCells CumuluscellmucificationandprogesteronesecretionwereinitiatedbyFSH(10-100ng/ml)asreportedearlier(Hillensj#{246}etal.,1981).NoeffectofGnRHa(10-100ng/ml)wasfoundonanyoftheseparametersunderbasalorFSH-stimulatedconditionsatculturetimesvaryingbetween10and48h(datanotshown). GranulosaCellsfromImmatureRats Granulosa cellsfromuntreatedimmature rats had alowrateofsteroidogenesisand theeffectsofthehormoneswerelesspro- nounced. GnRHacausedatransientstimulationofprogesteronesecretion(Fig.4),whileFSHstimulatedprogesteronesecretionduringtheentire3-daycultureperiod,thisresponse beingincreasedbetween48and72h.Presenceof GnRI-ladecreasedthegonadotropin-stimu- latedprogesteronesecretionat48horlater.20a-OHPwasstimulatedbyGnRHafor48h.However, thecombinationofGnRHaand FSHcausedasynergisticstimulationafter24hofculture(Fig.4). DISCUSSION Inthepresentstudy,GnRHastimulated bothprogesteroneand 20a-OHPproduction bygranulosacellsfrombothpreovulatoryandimmatureratovariesduringthefirst24-48hofculturebutnotlater.Within-creasingculturetime,at24h and beyond,GnRHa partlyinhibitedtheprogesterone secretionstimulatedbyFSHorLH.Thus, GnRHaappears tohaveabiphasicortime-dependenteffecton granulosacellprogestin production.ThefindingthatGnRHaby itselfcan cause stimulation ofprogesteroneproduction inrat granulosa cells invitroisinaccordance withearlierreportsonpreovulatoryrat granu - losacellsorwholefollicles.Theseearlierreportsincludedonlyshort-term(upto 5 h)exposures(Clarketal.,1980;Ekholmetal.,  GnRHa Uffihl SH GnRHa FSH 2000-150010005000 r*I r 0-24h 24-48h 48-72hU I- cF 1W o c’J FIG.4.Accumulationofprogesterone  top and20o -OHP  bottom duringcultureofgranulosacellsob- tainedfromuntreatedimmaturerats.Thecellswereculturedwithouthormone  C , orwithGnRHa(100ng/ml)and/orFSH(100ng/ml).Themediawerechangedafter24and48hofculture.Mean ± SEMof10-12observa - tions.x=P<0.05 vs.correspondingcontrolgroup,andxx=P<.01, =P<0.01vs.FSHalone. O-24h24-48h48-72h EFFECTOFGnRHONGRANULOSACELLPROGESTINSECRETION 595 Lai z 0Co..,.00 1981;Clark,1982;Hillensjo et al.,1982).Recently,an intrinsic stimulationofbasalprogestinsecretionbyGnRHwasalsore-portedforimmaturegranulosacellsculturedfor2days(Jones and Hsueh,1982a,b).Inoneofthesestudies(JonesandHsueh,1982b),GnRH was foundtohaveastimulatoryeffectonbasalpregnenolonesynthesiswhileitin- hibitedtheFSHstimulationonthisparameter. Furthermore,theseauthorssuggestedthatthestimulatoryeffectrequiredhighercon-centrationsofGnRHathandidtheinhibitoryeffect.Weobservedthatduringthefirst24hofculturetheconcentrationofGnRHare-quiredtoelicititsstimulatoryeffectonbasalprogesteronewaslowerthanthatrequiredtoelicititsinhibitionoftheFSHresponse.However,lowerconcentrationswereinhibi-toryafterprolongedculture.TheinhibitoryeffectsofGnRHaongranulosacellsteroido-genesishavepreviouslybeenreportedmainlyfortheFSH-responsivesystem,consistingofcellsfromimmature,hypophysectomized,DES-treatedratovariesculturedfor48hormore(HsuehandErickson,1979;Hsuehetal.,1980;Jones and Hsueh,1981a;Knecht
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