Reproduction in Domestic Animals

180 R Fayrer-Hoskenet al. 2000a,b, 2002; Barber et al. 2001). The use ofheterologous ZP antigens allows for standardization ofa vaccine’s dose and adjuvant as well as its deliverymethod. In addition, the use of heterologous ZPobviates the necessity for a homologous source ofantigens, which for most exotic species would beimpossible. The successful use of heterologous ZPglycoproteins suggests that the mechanism of sperm-ZP interaction might have some commonality betweenspecies. For the same reason, it works as an immunocontraceptivethat affects multiple species, it also questionsthe precise mechanism of the species-specificity ofsperm-ZP interaction. For much of the publishedliterature, the pZP has been the heterologous antigenof choice. To harvest enough native ZP for laboratoryor field studies has been labour-intensive and investigatorshave considered alternative sources of ZP. Thehorse is one of the best-studied mammals as a model forimmunocontraception. The prototypical studies in thehorse were completed by Dr Irwin Liu (Liu and Shivers1982; Liu et al. 1989), who demonstrated the efficacyand reversibility of the pZP vaccine. The horse has beenused as both a laboratory model (Liu et al. 1989; Williset al. 1994) and an ecological model (Kirkpatrick andTurner 2003). These immunocontraceptive studies in thehorse have shown that the pZP vaccine is both safe andeffective. Long-term vaccinations appear to have effectson the functionality of the ovary (Turner and Kirkpatrick2002), but these effects appear to be reversible withtime.Structure and origin of the ZPThe ZP matrix consists of 3–4 glycoproteins (ZP1, ZP2,ZP3 and ZP4) depending on the species. At a ratio of1 : 1 (Green 1997), ZP2 and ZP3 are inter-twined toform glycoprotein filaments that are cross-linked by ZP1(Wassarman and Mortillo 1991; Jovine et al. 2002). TheZP glycoproteins are combined into a microfibrillarmatrix. The ZP matrix is secreted around the oocyte andhas significant elastic properties. In some species, theindividual glycoproteins are produced in the ooplasmwithin the Golgi apparatus (Wassarman et al. 2005) andsubsequently moved through the cytoplasm and releasedoutside the oolemma into the perivitelline space.For the mare, the target antigens (eqZP) andvaccinational antigens (pZP) have been well characterized.The equine research shows that there are threeequine ZP (eqZP) glycoproteins using silver-stained2D-PAGE. The glycoprotein families have molecularweights of 93–120 K, 73–90 K and 45–80 K (Milleret al. 1992). Common epitopes on the eqZP (Milleret al. 1992) were detected by rabbit antiserum againstheat-solubilized pZP (RaHSPZ) (Dunbar and Raynor1980), and guinea pig antiserum against rabbit ZP(R55K) (Lee and Dunbar 1993; Lee et al. 1993).RaHSPZ recognized all three eqZP glycoprotein familiesand R55K only recognized the lowest molecularweight eqZP glycoprotein family eqZP3 (Miller et al.1992). This molecular data is complimented by thesuccess of mare immunocontraception in the field (Liuet al. 1989; Kirkpatrick et al. 1992). In addition, asanti-total-pZP antibodies and anti-R55K recognizeeqZP3, they could serve as a specific immunocontraceptiveantigen. This is important in targeting the ZP3molecule or portion of the molecule and would be thebest immunological approach for immunocontraceptionusing a synthetic ZP.The formation of the eqZP is a collaborative processbetween the oocyte and cumulus cells (Kolle et al. 2007),and the final external layer of eqZP is a function of thecumulus cells. The collaborative secretion of ZP glycoproteinsis also seen in pig, cow, dog, rabbit, marmosetand rhesus monkey (Kolle et al. 2007). In other species,e.g. the cat and mouse, only the oocyte secretes ZP. Thetemporal secretion of ZP glycoproteins is variablebetween species relative to oogenesis. In mouse, ZP2 isdeposited in the primordial follicle’s perivitelline space,but ZP3 and ZP4 only appear with the formation of theprimary oocyte and the initiation of its growth (Millaret al. 1993; Epifano et al. 1995). Therefore, there aredifferences in the cellular origin of the secretion of ZPglycoproteins, the temporal secretion of the ZP glycoproteinsand the manifestation of the antigenic determinantson the ZP glycoproteins.Although the precise molecular mechanism for theimmunocontraceptive effect of pZP remains to bedetermined, the carbohydrate portion of the ZP glycoproteinhas been implicated as an important aspect(Paterson et al. 1992). If immunocontraception is in partmediated by role of terminal carbohydrates, theirdistribution and configuration might hold the answer.Possible molecular explanations for the biochemicalmechanism of immunocontraception could includeblocking sperm binding sites (Barber and Fayrer-Hosken2000b) or structural changes in the ZP. When amare is successfully immunocontracepted, there are highcirculating levels of both IgG and IgM. The follicularIgG levels are dependent on the peripheral circulatinglevels (Hussein and Bourne 1984; Widders et al. 1984).IgG is probably the primary antibody to mediateimmunocontraception (Barber and Fayrer-Hosken2000b) as declining serum IgG levels are associated withreturn to fertility of the mare (Liu et al. 1989; Barberand Fayrer-Hosken 2000b). Another possible explanationfor the immunocontraceptive effects of pZP immunizationin the mare is that during the deposition offibrils of eqZP, anti-pZP IgG molecules might beintercalated into the matrix. The combination of theIgG and eqZP matrix could cause the anti-pZP antibodiesto act like cross-linkers (e.g. autoimmune reactions)and this could mimic the zona block. Theseimmunocontracepted zona-blocked oocytes would preventfertilization and this effect would remain untilsystemic levels of IgG fall low enough to allow conception.This mechanism would be most relevant inanimals, where cumulus cells participate in secretingZP. This would provide a reasonable explanation as towhy animals, e.g. cats whose ZP is derived completelyfrom the oocyte, are unaffected by heterologous pZPvaccination (Jewgenow et al. 2000).Another possible mechanism for the immunocontraceptiveeffect of pZP (Barber and Fayrer-Hosken 2000b)is an affect on the sperm-zona binding through therecognition of carbohydrate epitopes on the glycoproteinsof the ZP. It is clear that the ZP proteins areÓ 2008 The Author. Journal compilation Ó 2008 Blackwell Verlag