N OCIETY' - the Society for Reproductive Biology

. INTERSPECIFIC VARIATION IN GLYCOCONJUGATES OF THE ZONAE PELLUCIDAE OF SEVERALMARSUPIAL SPECIESLA. Chapman, O.W. Wiebkin l and.W.G. BreedDepartment of Anatomical Sciences, University of Adelaide, SA 5005 and IDepartment ofMedicine, University ofAdelaide,.SA 5005'I "-" :Jl' ,"'; C' 1.-...-"!\v J ,.:,> .:..y_.J\Introduction .jThe eggs of all vertebrates are surrounded by a glycoprotein coat known as the zona pellucida (ZP). It is to thisstructure that sperm must fIrst bind, and then penetrate, before fertilisation can occur. Sperm-ZP binding is facilitated bythe glycoconjugates of the ZPs oligosaccharides (1). While the protein components of the mammalian ZP glycoproteinshave been shown to be highly homologous, there appear to be interspecifIc differences in glycoconjugate content (2).While we have previously shown interspecific variation in thicknesses of marsupial ZPs, ranging from IJllD in theopossum to 8-10JllD in the koala (3), almost no work has been performed on characterising the glycoconjugatecomposition of marsupial ZPs. The aim of this study, therefore, was to determine and characterise, by the use ofdifferential lectin binding together with enzyme elimination and saponification, the saccharide components of the ZPsof follicular oocytes of several marsupial species each representing a different family within the marsupial infrac1ass.Materials and MethodsOvaries of brushtail possums (Trichosurus vuIpecuIa), fat-tailed dunnarts (Sminthopsis crassicaudata), koalas(Phascolarctos cinereus), eastern grey kangaroos (Macropus giganteus) and grey short-tailed opossums (Monodelphisdomestica) were fixed in Rossman's fluid (90% ethanol saturated with picric acid and 10% formalin). They were thenroutinely processed for light microscopy, embedded in paraffIn wax and sectioned. Sections were then deparaffinised,rehydrated, blocked with 1% BSA in Tris-buffered saline and incubated with one of 7 FITC-conjugated lectins andviewed under a fluorescent microscope. To detect sugar residues masked by sialic acid or O-acetyl groups, sectionswere first either desialylated (4) or saponified (5) respectively, prior to incubation with the lectins, as previouslydescribed. Specific controls involved incubating the lectins with 0.2-0AM of their inhibitory sugar prior to applicationto the ovary sections. Results were recorded as positive (+) or negative (-) binding, with positive binding beingsubjectively categorised into mild (+), strong (++) and intense (+++).Results and DiscussionThere is considerable variation in glycoconjugate content of marsupial ZPs. The ZPs of the fat-tailed dunnartsdisplayed the most deviation in their saccharides from those of other marsupial ZPs, with positive reactivity to alliectinsoccurring after desialyation (Table 1) and saponification (Table 2). Most signifIcantly, a-D-mannose, as demonstratedby Con A, was found only in the saponified ZPs of the fat-tailed dunnarts and no other species. The presence ofmannose may indicate, for the fIrst time, the presence ofN-linked oligosaccharides in the ZPs of dunnarts.ZPs of all species, except those of the opossum, appeared sialylated with increases in binding for f3-Gal(I-3)GaINAc(PNA) and a-D-Gal(NAc) (SBA) after removal of sialic acid with neuraminidase. Other glycoconjugates like thedisaccharides f3-Gal(1-4)GlcNAc and f3(1-4)-D-GlcNAc, as demonstrated by the lectins ECA and WGA respectively,appear to be common components within the ZPs of marsupials. The former, however, also appears to be masked tosome extent by O-acetyl groups, with increases in binding to ECA after saponification.Table 1. Lectin binding to ZPs of 5 marsupial speciesbefore and, in brackets, after desialyationBrushtall Fat-tailedPossum Dunnart Koala Kane:aroo OoossumPNA ++(+++) - (++) ++(+++) ++(+++) +(+)RCA·I1 -(-) +(++) +(+) - (+) -(-)WGA +++(+++) ++(+++) +++(+++) +++(+++) ++(++)SNA 0(+) +(++) ++(++) 0(+) -(-)SBA - (+) 0(+) - (++) -C++) -(-)Table 2. Lectin binding to ZPs of 5 marsupial speciesbefore and, in brackets, after saponificationBrushtail Fat-tailedPossum Dunnart Koala Kanl!aroo OoossumPNA ++(+++) - (+) ++(++) ++(+++) +(++)ECA - (+++) - (+) +(++) +(++) +(++)Con A 0(_) - (+++) -(-) - (0) -(-)RCA-II 0(_) +(+) +(+) - (+) -(-). WGA +++(+++) ++(++) +++(+++) +++(+++) ++(+++)SNA - (0) +(+) ++(++) - (++) - (++)SBA -(-) - (++) -(-) 0(0) -(-)ConclusionWhile marsupial ZPs contain common saccharide components such as GlcNAc and GalNAc, which are also commonin eutherian mammalian ZPs (2), they also display considerable interspecific variation in their glycoconjugate content.Whether this variation relates to the species-specificity of sperm-ZP binding is unknown. Further work is required toelucidate this.References1. Florman, H.M., and Wassarman, P.M. (1985) Ce1141:313-324.2. Skutelsky, E., et al., (1994) J Reprod Fertill00:35-41.3. Chapman, lA., and Breed, W.G. (1998) Aust Mamm Soc conference proceedings, Perth Australia:23.4. Riley, C.B., and Elhay, MJ. (1994) Ch. 3. In I Laboratory Histopathology: A Complete Reference'68IMMUNOCONTRACEPTION FOR WILDLIFE CONTROL:THE ZONA PELLUCIDA AS A TARGET IN MACROPODS.A.L. Kitchener, DJ. Kay and L.M. EddsMarsupial CRC, Unive:sity of Newcastle, Callaghan NSW 2308, Australia.IntroductionBy altering the landscape for agriculture andgrazing we have allowed .the numbers of. somespecies of macropods to Increase dramatIcally.This has lead to environmental degradation andcompetition for resources. Contraceptivevaccines are being investigated as a means ofcontrolling problem population of macropods(1). The zona pellucida (ZP) is beingconsidered as a target as it performs essentialfunctions in fertilisation' (2). This study hasbeen conducted to provide proof of concept forZP molecules as potential target antigens in acontraceptive vaccine for macropods.Materials & Methods16 female Tammar wallabies (Macropuseugenii) were divided evenly into two treatmentgroups. The first group was immunised on 5occasions, at fortnightly intervals, with l00J.lgof whole porcine zona pellucida (PZP)emulsified in Freunds complete adjuvant for theinitial injection and incomplete Freunds for thesecond and subsequent injections. Thevaccinations were distributed over 4 sites, twosubcutaneous and two intramuscular. Thecontrol group received saline and Freundsadjuvant in the same manner. Blood sampleswere collected each time the animals wereimmunised and thereafter at monthly intervals.After the fifth injection the animals were placedin a breeding trial with two PZP and two controlanimals placed with two stud males in eachbreeding group. Success of natural mating andartificial insemination after super-ovulation (3)were used to assess the fertility of animals ineach group. Animals were sacrificed at the endof the breeding trial and the reproductive tractscollected for immunohistology. Immuneresponses were evaluated using ELISAs andwestern blots (4).ResultsImmunised wallabies produced strong andsustained immune responses to PZP (Figure 1).67% of control animals produced offspring bynormal mating. PZP immunised animals failedto produce any offspring. Fertilised ova wererecovered from all control animals that had beenartificially inseminated, showing that 100% ofcontrol animals were fertile. Artificial ~.nsemination demonstrated that PZP immunised\~allabies failed to ovulate. The ovaries of PZP .. )_immunised super-ovulated wallabies were...'1\smaller, 0.239g +/- 0.15,3g co~pared to 0.781g.! +/_ 0.275g, and had fewer folhcles