Immunological studies of canine ovarian antigens

THERIOGENOLOGY

~LOGICAL

STUDIESOF CANINE OVARIANANTIGENS"'

StevenY. W. Chan3, D. E. Wildt, and P. K. Chakraborty Departmentof Physiology,Baylor Collegeof Medicine,Houston,Texas 77030 and the VeterinaryResourcesBranch,Divisionof ResearchServices, NationalInstitutesof Health,Bethesda,Maryland 20205 Receivedfor publication:l.l8/8l, accepted: 4/24/81 ABSTRACT Studieswere conductedto determinethe feasibilityof producing canine ovary antiserumin the rabbit and to characterizethe antirrenic composition.of the canine ovary. Ovariesfrom dogs were obtained-and corporalutea (CL)macroscopically removed. Followinghomogenization of -ovaries, adult male rabbitswere innmmizedby injectingthe ovarian preparation. Unabsorbedantiserumcross-reacted with canine ovarian extractand with other reproductiveand non-reproductive organ extracts to form precipitinbands. Speciescross-reactivity was also observed by testingthe unabsorbedantiserumwith extractsfrom organs of the cat and rat. Absorptionof antiserumwith canine liver and lung extracts removed antibodiesnot specificto the canine ovary. One band was observedwhen such absorbedantiserumwas allowedto react with the canineovarian extract. The absorbedantiserumproducedno bands with extractsfrom other canine reproductiveand non-reproductive organs tested. These experimentssuggestedthat the canine ovary containedat least 1 organ-specific antigen. This organ-specific antigenwas locatedin the isolatedcanine ovariancells by the inmnmofluorescence technique.

1 The authorsare indebtedto Andrew Stewartand BarbaraAlford for their technicalassistance,Dr. D. V. Ablashi of the NationalCancer Institute for the immunofluorescence test, and the Animal Birth ControlClinic of Houston for their generousdonationof canine ovarianmaterial. Portionsof this study were conductedat the Instituteof ComparativeMedicine,Baylor College of Medicine,Houston,Texas, with the partial financialsupportof the Ralston Purina Company,St. Louis,MO and Ohio Animal Health Foundation. 2 Names of commercialmanufacturersand trade names are providedfor identification only and inclusiondoes not imply endorsementby the NationalInstitutesof Health,US Public Health Service,or US Department of Health and Human Services. 3 Presentaddress: VeterinaryResourcesBranch,Divisionof Research Services,National Institutesof Health, Bethesda,Maryland 20205.

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INIR0DUCTION Recentinterestin an innnmologic approachfor fertilitycontrol has stimulatedresearchfor the detectionof antigensspecificto the femalereproductive system. Studieson the inmnmogenicity of mammalian ovarieshave shown that heteroinnmmization with ovariantissueyields antibodies which can be employedto detectovarianantigensimmunechemically.In severalspeciesincludingthe hamster,mouse,guineapig and rabbit,ovarianantigenshave been demonstrated in the atreticfollicle as well as the zona pellucidaand theta internacomponentsof the ovum (l-6). Very littleinformation is availableconcerningthe mogenicity of the canineovary and other than surgicalsterilization, therehave been few successful methodsfor controlling reproduction in the dog, a species currentlyproducinga sociological problemdue to overpopulation (7). This laboratory has recentlyinitiateda comprehensive investigation into inmnmolorric annroachesfor the controlof fertilitvin this snecies. The purposeoz the-present investigation was to studythe feasibility of the oroductionof canineovary antiserumin the rabbitand to characterize the antigeniccomposition of the'canineovary,using agar-geldiffusion and innnunofluorescence techniques. MATERIALSANDMETHODS Preparation of ovarianextract(antigens):Ovarieswere removed from adult femaledogs by routinelaparotomy proceduresand immediately kept frozenat -2OW until used. Warian materialwas laterthawedand grosslyvisiblelutealtissueremoved. The remainingovariantissue was washed 5 times in cold phosphate-buffered saline (PBS;0.14MNaCl, 0.Ol.M NaP04,PI-E 7.0). Threehundredgm of wet tissuewere homogenized in cold PBS in an ultra-Turrax Tissumizerand then centrifuged at 2100 x g for 30 min. The supernatant was collected,snap frozenin a dry ice-acetone bath and then lyophilized. a A totalof 24.94gm of lyophilized materialwas obtained. Proteincontentof this materialwas measuredby the Bio-Radproteinassaymethod.b Innnediately beforeinmnmization of rabbitsfor antibodyproduction,the lyophilized materialwas suspended in PBS. Inmnmization procedureand serumcollection:Antiserumwas prepared by innmmizing2 male rabbitswith the lyophilized ovarianextract. Rabbitswere bled by marginalear vein punctureto obtainpre-immune normalserum. For the first immunization, 1 ml of canineovarianextract (13mg protein)was emulsified with an equalvolumeof Freund'scomplete adjuvant,and 2 ml of this emulsionwere injectedboth subcutaneously and intradermally at S-10 sites in the abdominalwall. The secondinuaunization was givensubcutaneouslv and intradennallv in the abdominalwall 21 days lgterand consistedof 2 ml of equalvolumeof ovarianextract (6.5mg protein/ml) and Freund'sincompleteadjuvant. The third inmnmization consistedof 2 ml of equalvolumeof ovarianextract a VirtisLyophilizer Model lo-146MR-BA,NY b Bio-RadProteinAssay TechnicalBulletin,1051,Bio-RadLab; Chem.Div.

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(3.25mg protein/ml)and Freund'sincompleteadjuvant. It was given subcutaneously and intrademnallyin the abdominalwall 14 days after the second GmnuniZation.Booster injections,consistingof 1 m.l.ofovarian extract (3.25mg protein/nil) and no adjuvant,were given once every 2 months after the third immunization.Each rabbit receiveda total of 5 immunizations. Rabbitswere bled weekly (10 ml) followingthe second immmization and the bleedingscontinueduntil 4 weeks after the final iamnmization. Detectionof the antigen-antibody complex: The inmunological responses of innnrnized rabbitsand the organ- and species-specificity of the canine ovary antiserumwhich was producedwere evaluatedby Ouchterlony'sdouble diffusionmethod (8). Diffusionplates were preparedby pouring 6 ml of 1% solution (weight/volume) of agaroseCin PBS onto 75x50nunglass plates. The gel was cut with a templateto create a centralwell and 6 peripheral wells. After the additionof the antiserum(volume0.05 ml) into the peripheral wells, diffusionwas allowedto progressat 4OC in a humiditychamberfor 5 days. Only a single applicationof the antiserumand the antigenicpreparation was used in the proc$dure. Resultingprecipitinpatternswere stainedwith Amid0 Black solution (1 5 of Amid0 Black 1OB in 900 ml of acetatebuffer) for examination. The titres of ovary antiseracollectedduring successive bleedingswere monitoredqualitativelyby reactingthe undilutedantiserum (0.05ml) with differentconcentrations of the canine ovarianextract (0.02ml, protein content:13, 6.5, 3.3, 1.6, 0.8 and 0.4 mg/ml, respectively)and observingthe maximum n~ber of precipitinbands developedin agar-gelplates. Distinctprecipitinbands were first observedin the diffusiontest using sera that were collectedfrom both innmmizedrabbits 2 weeks after the second immunization.Furthermore,successivebleedingsthroughoutthe experimental neriod uroducedno additiondlorecioitinbands. For these two reasons it was rabbits decide&to pool all the sera that were collectedfrom both innnunized after the second imnunizationon an equal quantitybasis (2 ml antisenan/weekly of the pooled antiserum bleeding/rabbit).The organ- and species-specificity were evaluatedby reactingthe undilutedpooled antiserum(0.05ml) with differentorgan crude extracts (0.02ml, protein content13 mg/ml) which were preparedby homogenization of the tissuesin cold PBS, centrifugation (ZlOOxg,30 min) and finallyadjustmentof the protein contentof the supernatantscollectedto 13 mg/ml. Antiserumabsorptionwith non-reproductive organ extracts: Absorption of the antiserumby non-reproductive organ extractwas necessaryto obtain antiserumspecificto the reproductiveorgans. Lyophilizedcanine liver and lung extractswere preparedas followsand used to remove the non-specific antibodiesin the Dooled antiserum. Canine liver (45.6am wet weight) and lung (31.3gm wet weight) tissueswere homogenized-in 105 ml cold FBS; respectivelyand then centrifugedat ZlOOxg for 30 min. Ihe supernatants were collectedand lyophilized; Absorptionof the pooled antiserumwas performedaccordingto Sacco and Shivers (4). Liver-absorbed antiserumwas

cdAgarose Type II, Sigma ChemicalCo., St. Louis,W. Sigma ChemicalCo., St. Louis,MO.

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preparedby adding10 mg of lyophilized canineliverextract(totalprotein content5.25mg) to 3 ml of the pooledcanineovaryantiserum.The mixture was incubatedat 37'C for 1 hr, then at 4'C overnightand was then centrifuged at 21000x g for 30 min. The resultingprecipitate was discardedand the super-r&ant was used as liver-absorbed antiserum. Liver-lungabsorbedantiserumwas preparedby the additionof 4 mg of lyophilized caninelung extract(totalproteincontent9.2 mg) to 3 ml of the liver-absorbed antiserumand the mixturewas incubatedat 37'C for 1 hr and then overnightat 4OC. Remainingmethodswere identicalto thosewhich were describedfor the liver-absorbed antiserum. Agar-geldiffusiontechniquewas used to confirmthe effectiveness of each absorption.Antiserumwas consideredcompletelyabsorbedif no precipitinbandswere formedwhen allowedto reactwith absorbingorgancrude extracts. Immunofluorescence test - Demonstration of the bindingof antibodiesto isolatedovariancells: The indirectinmnmofluorescence t&t describedby Johnsonet al. (9)was used to demonstrate the bindingof the develoned antibodiesin the-pooledantiserumto isolatedcanine-ovarian cells.-Isolated ovariancellswere collectedby trypsinization of the ovarianmaterials. Two freshcanineovarieswere obtainedfrom a matureanestrousbitch by laparotomy, washed 5 times in cold PBS, and mincedinto smallpieces.-Trypsin solution(enzymatic activity10~)~was added to the ovariantissuein a ratio of 20 to 1 (volume/volume) and incubatedfor 15 min at room temperature. The trypsinization processwas terminatedat the end of the incubation period by transferring the solutionto an ice-waterbath. Isolatedovariancells were collectedby centrifugation at 4OC (15OOxg,15 min). The isolated-cell preparation was washed3 times in cold PBS to removethe tryps$ examined with an opticalmicroscope,and transferred to Multispotslides for indirect inmunofluorescence test. Isolatedovariancellswere air-driedand fixed in a mixtureof acetone-methanol (1:lvolume/volume; 4OC) for 10 min. Dnabsorbedand liver-lung-absorbed canineovary antisera(1:lOdilutionin PBS) were added to the fixed-cell preparations and incubatedin a c02-02-humidity incubator the slideswere washed (37OC,98% Mdity) for 45 min. After incubation? in PBS at 37'C for 30 min, with 3 changesof solution,to removeunreacted IgG fraction antibodies, and then air-dried.A fluorescein-conjugated of goat anti-rabbit IgG solutiongwas added to the slides,and the reaction was allowedto progressin a C02-02-htiditychamberfor 30 min. Dnreacted fluorescein-conjugated antibodies were removedby washingin PBS (37OC),with mountedin a occasionalshaking,for 1 hr. The slideswere air-dried, elvcerol-PBS mixture (1:lvolume/volume). and examinedwith a Leitz-Weltzer &lux microscopewith an ultraviolet light source. Preinmnmenormalrabbit serumratherthan canineovary antiserumwas used as the control. e

-GibcoLaboratory, Grand IslandBiologicalCo., Grand Island,NY. f Roboz SurgicalInstrument Co., Inc.,Washington, D.C. gCappelLaboratories, Cochranville, PA.

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RESULTS The unabsorbed, pooledantiserumreactedwith the canineovarianextract to form 4 precipitinbands on the agar-gelplates (Fig.la). The minimum proteinconcentration of the ovarianextractfor reactionwith the unabsorbed and undilutedantiserumwas 1.6 mg/ml. The precipitinlinesdecreasedin numberand becamefainterwhen higherdilutionsof antiserum(1:2,1:4) were pooledantiserumalso cross-reacted with the extracts used. This unabsorbed, of caninereproductive and non-reproductive organs. The numberof precipitin bandswhich were formedbetweenthe unabsorbedantiserumand variousorgan extractsare presentedin Table 1. All organsproducedprecipitinbands, rangingfrom 1 distinctcomplex(thyroid,stomachand testis)to 5 (senrm, with extracts plasmaand liver). The unabsorbedantiserumalso cross-reacted of severalfelineorganstestedincluding'ovary, CL, heart,skeletalmuscle, uterus,plasmaand serumto form 2 precipitinbands. No reactions,however, were observedwith unabsorbedantiserumand the extractsof any rat organs testedincludingserum,plasma,ovary,liver,kidney,lung,stomach,heart and spleen. The liver-absorbed antiserumreactedwith serum,plasma,and extractsof canineovary,testis,kidney,lung,small intestine,stomach,skeletalmuscle, bladderand uterusto form l-2 precipitinbands (Tablel), Additionof the lyophilized caninelung extractto the liver-absorbed antiserumremovedthe remainingantTbodiesexceptthose reactingwith the ovary,giving1 precipitin band (Fig.lb). The liver-lungabsorbedantiserumformedno precipitinband with serum,plasmaor extractsof 13 other tissues(Table1). The liver-and liver-lunn-absorbed antiserafailedto reactwith extractsof any of the cat and rat o,ganstestedto form precipitinbands. Resultsof fluorescent antibodylocalization of antigensin isolated canineand felineovariancell preparations are shown in Fig. 2. Isolated canineovariancells treatedwith unabsorbedand liver-lungabsorbedantisera treatedwith the normalrabbit fluoresced(Fig.2 a 6 b). Cell preparations serumwere negative,givinga faintbackgroundof fluorescence (Fig.2~). Fluorescence stainingof the isolatedcell preparations was confinedto the cell surface. DISCUSSION The resultsindicatedthat immunization of rabbitswith an extract of canineovarianhomogenate,exclusiveof macroscopically-removed CL, resultedin the productionof antibodiesdirectedagainstthe innmmizing agent. At least4 ovarianantigenswere detected.-Absorption studiesalso revealedthat the canineovary sharesconnnon antigenswith othercanineand felineorganstested. These commonantigensstimulatedthe inmnmizedrabbits to producethe antibodiesthat cross-reacted with extractsfrom otherorgans. Within the sensitivity limitof agar-geldiffusiontechnique,there was at least1 antigenspecificto the canineovary,which was not detected in other reproductive tissuesincludingthe CL and uterus.

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Figurela.

Figurelb. Figure1

Precipitin bands formedby canineovarianextractstested againstunabsorbedand liver-lung-absorbed canineovary antiserain agar-geldoublediffusiontest.

la.

Centralwell - unabsorbedcanineovaryantiserum; Wells 1 to 6 canineovaryextractwith proteinconcentration 13 mg/ml, 6.5 mg/ml 3.3 mg/ml,1.6 mg/ml,0.8 mg/ml and 0.4 mg/ml .respectively.

lb.

Centralwell - liver-lung-absorbed canineovaryantiserum; Well 1 -canineliver;Well 2 - caninelung;Well 3 - canine ovary;Well 4 - canineCL; Well 5 - canineserum;Well 6 caninetestis. Proteinconcentration of all extracts= 13 ng/ml.

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rable 1. Number of precipitinbands formed by various canine organ extractstested againstcanine ovary antiserm in agar-gel diffusiontest.

btigen bg

Unabsorbed antiserum

Liver-absorbed antiserum

4

1

ovary (withCL macroscopically removed) corpus lutem (CL) serum plasma testis liver kidney lmg small intestine heart stcmach spleen skeletalmuscle thyroid bladder uterus

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Liver-Lung absorbed antiserum 1

2

8 0 0 0 0 0

0” 8 0 4

0 0 0

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Figure 2a.

Figure 2b. Figure 2a. Isolatedcanineovariancells treatedwith unabsorbed canine ovary antiserum(1:lOdilution) Figure 2b. Isolatedcanine ovariancells treatedwith liver-lung absorbedcanine ovary antiserum(1:lOdilution)

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Figure 2c.

Figure 2c. Isolatedcanine ovariancells treatedwith normal rabbit serum (1:lOdilution)

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Similarfindingsof antigensspecificto manmmlianovarieshave been reported. Porter et al. (2,3)reported1 or more common gonadalspecific antigen(s)in the guineapig ovary and testis. Sacco g Shivers (4;5)reported at least 2 specificantigensin the rabbit ovary,which were not present in oviductand uterus. Hamster and mouse ovarieswere reportedto contain3 and 2 organ-specific antigens,respectively(10,ll). Some workershave been unable to detect ovary-specific antigens. Isojima& Stepus (12),using both rabbitsand guineapigs as antibodyproducers,found no organ-specific antigensin the mouse ovary. Porter et al. (2,3)suggestedthat the failureof earlier workers to demonstrateovary-specific antigenswas likely relatedto a low titre of ovarianantibodiesin the antiserumprepared. The presenceof few canine ovarianantigensdetectedby the unabsorbed antiserumin this study may in part be attributedto the exclusionof most of the luteal tissue in the preparationof the ovarianextract. Fifteen and 8-9 antigenswere detectedin rabbit and hamster ovaries.resoectivelv. with unabsorbedantiserumagainstwhole ovarianextracts (4?6). ?here have been no reportsconcerningthe localizationof ovarian-specific antigensin the CL. Since the latter is an inroortant endocrineorgan instrumentalin maintainingoreanancv.it would be-of interestto stud; its antipenicitvand potential;Is‘e in fertilityregulation. The present results indicatethat the CL reactedwith the canine ovary antiserumto form 2 precipitinbands. Preliminarvstudiesin this laboratbrvhave demonstratedthat the crude canine lutkal extractcan stimulateantibodyformationin the rabbit and that CL antiserumreactswith luteal extractto form at least 2 bands in agar-geldiffusionplates. Since absorbedcanine ovary antiserumdid not react with the feline and rat ovariesthe resultssuggestthat the 1 canine ovarianantigen detectedmay be species-specific.Sacco E Shivers (13) reportedthat absorbedantiserumto rabbit ovary did not cross-reactwith mouse, rat, guineapig or human ovary but did cross-reactwith the ovary of 2 other breeds of rabbits. Garavagnoet al. (14) reportedthat anti-hamsterovary antiserumdid not cross-reactwith zona antigenof mouse and rat eggs. Shivers (15) impliedthat the zona antigenof the ovary from severalspecies includingthe hamster,mouse and rat was species-specific. test in the present The resultsof the indirectimmunoflucrescence studiesindicatethat the isolatedcanine ovariancells containantigens capableof bindingwith antibodiesin the preparedantisera. This confirms the resultsof agar-geldiffusiontest, in which solubleovarianantigens combinedwith ovary antibodiesto form precipitinbands. The use of liver-lungabsorbedcanine ovary antiserumin this procedurealso demonstratesthat the 1 specificantigendetectedby the agar-geldouble diffusiontest can be located in the isolatedcanine ovariancells. Appropriatecontrol (normalrabbit serum) indicatestit the fluorescenceobservedwas due to antigen-antibody interactions Since isolatedovariancells were used in this study, the exact location of the ovarianantigensin the whole ovary is presentlyunknown. Localization of the ovarianantigensat the tissue levelwill reauirefuture investieations involvingpreparati& of histologicalsectionsof the ovary or separatiznof differentovariancomponentsto determinethe applicabiltiyof such antigen(s) in the immunologiccontrolof the unwantedcaninepopulation.

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1. PorterCW. Ovarianantibodiesin femaleguineapigs. Int. J. Fert. 10:257-260(1965). 2.

PorterCW, HighfillD, WinovichR. Guineapig ovary and testis: demonstration of commongonadalspecificantigensin the ovary and testis.rnt. J. Fert. 15:171-176(1970).

3. PorterCW, HighfillD, WinovichR. Guineapig ovary and testis: localization of connnon gonadalspecificantigens. Int.J. Fert. 15:177-181(1970). 4. SaccoAG, ShiversCA. Antigensof the rabbitovary,oviductand uterus.J. Reprod.Fert. 32:403-417(1973). of tissue-specific antigensin 5. SaccoAG, ShiversCA. Localization the rabbitovary,oviductand uterusby the fluorescent antibody technique. J. Reprod.Fert. 32:415-420(1973). of hamster, 6. TsunodaY, ChangMC. In vivo and in vitro fertilization rat and mouse eggs a?Xer treatmezsanti-hamster ovaryantiserum.J. Exp. Zool.195:409-416(1976). controlin the dog 7. Wildt DE, Kinneyc51,SeagerSWJ. Reproduction and cat: an examination and evaluationof currentand proposed methods. J. Am. Anim. Hosp.Assoc. 13(2):223-231 (1977). 8.

Ouchterlony 0. Antigen-antibody reactionsin gels. Acta. Pathol. &and. 26:505-513(1949).

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JohnsonGD, HolborowET, DarlingJ. Inmunofluorescence and immunoenzyme techniques, in Handbookof Experimental Immunology U.K., 1978. 7.1 - 7.11,Weir F%l,Ed, BlackwellScientific,

10. Ownby CL, ShiversCA. Antigensof the hamsterovary and effectsof anti-ovaryserum on eggs. Biol.Reprod.6:310-318(1972). AB, FranklinLE, et al.. Inhibition of sperm11. ShiversCA, Dudkiewicz egg interaction by specificantibody. Science178:1211-1213 (1972). of guineapig testisand ovary. 12. IsojimaS, StepusS. Antigenicity Int.Arch.AllergyApl. hmmmology 15:350-359(1975). 13. Sacco AG, Shivers CA. Comparisonof antigensin the ov;ryie;viriuct and uterusof the rabbitand othermammalianspecies. . . Fert. 32:421-427(1973). of the 14. GaravagnoA, PosadaJ, BarrosC, et al.. Some characteristics zonapellucidaantigenin the hamster. J. Exp. Biol. 189:37-50(1974). interference with fertilization, in 15. ShiversCA. Bmnunological Immunological Approachesto FertilityControl,223-244, DiczfalusyE, Ed. KarolinskaInstitute,Stockholm(1974).

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