ZP Proteins Research Articles

The immune response and fertility of koalas (Phascolarctos cinereus) immunised with porcine zonae pellucidae or recombinant brushtail possum ZP3 protein

To evaluate the potential contraceptive effect of immunisation with zona pellucida antigens, 50 free-ranging koalas were immunised with either porcine zonae pellucidae (PZP), recombinant brushtail possum ZP3 (recBP-ZP3) or buffer, in complete Freund's adjuvant. A single booster immunisation in incomplete Freund's adjuvant was administered 3-5 months later. Where possible animals were recaptured, reproductive status was assessed and blood was collected at 1-3-month intervals for the next 33 months. Forty-three koalas were recaptured at least three times allowing reliable assessments of their fertility. Fourteen animals were observed never to have a pouch young. Of the remaining 29 animals the reproductive productivity of PZP treated females was reduced compared with control and recBP-ZP3 treated females, in terms of both total number of young produced, and failure to produce further young in females of proven fertility. One month after the initial immunisation, serum antigen-specific antibody titres were higher in animals immunised with PZP or recBP-ZP3 compared to controls, and reached a plateau by 4 months. Antibody against the relevant immunising antigen was also detected in ovarian follicular fluid, uterine fluid and vaginal secretions. Epitope analysis suggested that immune responses other than antibodies directed against the ZP3 amino acid sequence were responsible for mediating infertility. The results demonstrate that the fertility of female koalas can be compromised by immunisation against zona pellucida antigens. However, unlike in the eastern grey kangaroo and the brushtail possum, immunisation with bacterial recombinant brushtail possum ZP3 did not compromise fertility in the koala.

Evidence for the Participation of Sperm Proteasomes in Zona Pellucida Degradation and Zona-Induced Acrosomal Exocytosis During Fertilization in the Domestic Pig.

Realization of importance of the ubiquitin-proteasome pathway (UPP) in mammalian fertilization is continuing to grow with every new publication. The UPP is known to participate in multiple steps of mammalian fertilization, including sperm capacitation, acrosomal exocytosis (AE), and sperm-zona pellucida (ZP) penetration. In the UPP, a chaperone protein ubiquitin binds in tandem fashion to proteins that need to be recycled, and targets them for proteolysis by a multi-subunit protease, the 26S proteasome. We have been studying the participation of the 26S proteasome in the ZP-induced AE and sperm-ZP penetration during porcine fertilization. The 26S proteasome is a ~2 MDa, complex consisting of a 19S regulatory complex (role: ubiquitinated substrate recognition, deubiquitination and unfolding) and the 20S core (role: substrate degradation). Three distinct proteolytic activities reside in the 20S core: trypsin-like, chymotrypsin-like and caspase-like. In the presence of proteasomal inhibitors, porcine sperm are unable to fertilize oocytes, which may be due either to inhibition of acrosomal exocytosis and/or degradation of the ZP matrix. To understand this, fresh, capacitated boar spermatozoa were incubated with solubilized porcine ZP proteins with or without proteasomal inhibitors MG 132 (inhibits chymotrypsin-like core activity), clasto-lactacystin β-lactone (targets trypsin-like activity) and epoxomicin (targets chymotrypsin-like activity). After two-hour incubation, soluble ZP proteins and exocytosed acrosomal ghosts were separated from sperm pellets by centrifugation. They were analyzed for evidence of ZP protein degradation by the sperm proteasomes and for the accumulation of undegraded sperm acrosomal proteins. Results show that solubilized ZP proteins readily bind to acrosomal caps of the capacitated boar spermatozoa and induce AE. Presence of proteasomal inhibitors during sperm-ZP coincubation protected the zona glycoprotein ZPB from being degraded, without affecting its deglycosylation. Additionally, release of the 26S proteasomes with the acrosomal ghosts was reduced in the presence of proteasomal inhibitors. Acrosomal ghost-associated proteins such as Sperm Adhesion Molecule 1 (SPAM1), Lactadherin/SP47, Beta-Acrosin, Zona Pellucida-Binding Protein 2 (ZPBP2/IAM38) and Proacrosin-binding Protein (SP32), were protected from proteasomal degradation and accumulated in the ghost fraction. Thus, it appears that proteasomes are released from the sperm acrosome during ZP-induced acrosomal exocytosis. However, since acrosomal ghost remains zona bound and surrounds the sperm head during early stages of sperm-ZP penetration, the resident acrosomal proteasomes could assist the spermatozoon during the formation of the fertilization slit. A second pool of proteasomes remains associated with the inner acrosomal membrane (IAM) of the exocytosed spermatozoa; it may provide the fertilizing spermatozoon with a mean to digest the zona, as it advances through the fertilization slit. Collectively these results explain why the inhibition of sperm proteasomal activity prevents porcine fertilization. This model provides tools to modulate fertilization in vitro and points to a new direction in which to contemplate non-hormonal contraceptive design. Supported by USDA-NRI grant #2007-01319 and by seed funding from the F21C Program of UM (PS). (platform)

Immunocontraception of Eastern Grey kangaroos ( Macropus giganteus) with recombinant brushtail possum ( Trichosurus vulpecula) ZP3 protein

This study examined the potential of a recombinant marsupial zona pellucida 3 protein as a contraceptive vaccine for the Eastern Grey kangaroo, a marsupial that is locally overabundant in several regions of eastern Australia. First, a pilot study using porcine zona pellucidae (PZP) demonstrated that ZP proteins, primarily the ZP3 component of PZP, are highly immunogenic in the grey kangaroo and produce a long-lasting humoral response to a single immunisation, as found in other marsupials. Immunisation with 300 μg of a non-glycosylated recombinant brushtail possum ZP3 (recBP-ZP3) protein in complete Freund's adjuvant produced a similar, significant and sustained antibody response, and none of the immunised kangaroos ( n = 7) produced offspring during the following breeding season compared with four out of the six control animals. An epitope analysis of the B-cell response to recBP-ZP3 using a brushtail possum ZP3 identified numerous B-cell epitope regions clustered around the N- and C-terminal regions of the protein. Two regions of interest for further fertility vaccine development based on their immunogenicity and fertility trials and functional studies in other species were found to be immunogenic. These results suggest that immunocontraception based on targeting the ZP3 protein within the zona pellucida may be an effective strategy for fertility reduction in Eastern Grey kangaroos.

Prior infection with murine cytomegalovirus (MCMV) limits the immunocontraceptive effects of an MCMV vector expressing the mouse zona-pellucida-3 protein

We have developed a murine cytomegalovirus (MCMV)-vectored vaccine expressing the mouse zona-pellucida-3 gene (rMCMV-ZP3), which successfully induces infertility in experimentally inoculated laboratory or wild-derived mice. However, the future success of this vector as a fully disseminating vaccine in free-living mice may be compromised by pre-existing immunity since there is a high prevalence of naturally acquired MCMV infection in these mice. To evaluate the effect of prior immunity to MCMV on vaccine efficacy, we constructed two new biologically effective recombinant MCMV vectors expressing the mouse ZP3 protein from two MCMV strains (N1 and G4) derived from free-living mice. In wild mice, mixed MCMV infection is common and could be acquired either by simultaneous coinfection or sequential infection with different MCMV strains. Interestingly, while coinfection with both wild-type and rMCMV-ZP3 via the intraperitoneal route reduced the impact of the rMCMV-ZP3, prior infection with the same wild-type strain as that used to construct the rMCMV-ZP3 abrogated the immunocontraceptive effects of either N1-ZP3 or G4-ZP3. However, prior infection with G4 28 days before the introduction of N1-ZP3 had a reduced influence on the efficacy of the rMCMV-ZP3. Thus, the strain of virus and the timing of prior infection are factors that may influence the efficacy of the rMCMV-ZP3. Given that mixed infection of mice with MCMV is common, it is possible that prior immunity acquired by natural mucosal infection may have less a less inhibitory effect on the immunocontraceptive outcome.

Reproductive protein evolution within and between species: maintenance of divergent ZP3 alleles in Peromyscus

In a variety of animal taxa, proteins involved in reproduction evolve more rapidly than nonreproductive proteins. Most studies of reproductive protein evolution, however, focus on divergence between species, and little is known about differentiation among populations within a species. Here we investigate the molecular population genetics of the protein ZP3 within two Peromyscus species. ZP3 is an egg coat protein involved in primary binding of egg and sperm and is essential for fertilization. We find that amino acid polymorphism in the sperm-combining region of ZP3 is high relative to silent polymorphism in both species of Peromyscus. In addition, while there is geographical structure at a mitochondrial gene (Cytb), a nuclear gene (Lcat) and eight microsatellite loci, we find no evidence for geographical structure at Zp3 in Peromyscus truei. These patterns are consistent with the maintenance of ZP3 alleles by balancing selection, possibly due to sexual conflict or pathogen resistance. However, we do not find evidence that reinforcement promotes ZP3 diversification; allelic variation in P. truei is similar among populations, including populations allopatric and sympatric with sibling species. In fact, most alleles are present in all populations sampled across P. truei's range. While additional data are needed to identify the precise evolutionary forces responsible for sequence variation in ZP3, our results suggest that in Peromyscus, selection to maintain divergent alleles within species contributes to the pattern of rapid amino acid substitution observed among species.

Zona pellucida components are present in human fetal ovary before follicle formation

The zona pellucida is a glycoprotein matrix surrounding oocytes and early-stage embryos in mammals. To elucidate the roles of the zona pellucida glycoproteins ZP1 and ZP3 and their key regulatory factor FIGLA in human ovarian development and folliculogenesis, their expression and localization was studied in human fetal and adult ovaries. FIGLA mRNA and ZP3 mRNA/protein were localized mainly in the oocytes, and during fetal development their maximal expression was observed around the 20th week, the time of follicle formation. The expression of ZP1 mRNA was low both in fetal and adult ovaries. Present findings demonstrate that ZP3 and FIGLA transcripts are expressed in the oocytes from early ovarian development. The function of ZP proteins during early fetal life is not clear, but the simultaneous expression of FIGLA and ZP3 suggests, that they may have a role in the development of primordial follicle before zona pellucida formation.

The mRNA for zona pellucida proteins B1, C and D in two genetic lines of turkey hens that differ in fertility

The avian inner perivitelline layer (IPVL) contains zona pellucida protein-B1 (ZPB1), zona pellucida protein-C (ZPC) and zona pellucida protein-D (ZPD). These three proteins may be involved in sperm binding to the IPVL. ZPB1 is produced by the liver and transported to the developing preovulatory follicle, while ZPC and ZPD are synthesized and secreted by the granulosa cells of the preovulatory follicle. The mRNA of ZPB1, ZPC, and ZPD was investigated in two lines of turkey hens selected for over 40 generations for either increased egg production (E line) or increased body weight (F line). Total RNA was extracted from the liver and from 1 cm 2 sections of the granulosa layer around the germinal disc and a nongerminal disc area of the F 1 and F 2 follicles of hens from each genetic line. Northern analysis was performed using chicken cDNA probes for all three ZP proteins. Hepatic mRNA for ZPB1 was greater ( P < 0.05) in turkey hens from the E line than the F line. Although, there was no difference in ZPC mRNA between the germinal disc and nongerminal disc region of the two largest follicles in E line hens, ZPC mRNA was greater in the nongerminal disc region compared to the germinal disc region in the two largest follicles obtained from the F line hens. There were no differences in ZPD mRNA between the germinal disc and nongerminal disc regions of the F 1 and F 2 follicles for either genetic line. The results suggest that the greater rates of fertility previously observed in eggs from the E line hens compared with the F line of hens may be related to differential amounts of the potential sperm binding proteins ZPB1 and ZPC.

Identification of differentially expressed ovarian genes during primary and early secondary oocyte growth in coho salmon, Oncorhynchus kisutch

BackgroundThe aim of this study was to identify differentially expressed ovarian genes during primary and early secondary oocyte growth in coho salmon, a semelparous teleost that exhibits synchronous follicle development.MethodsReciprocal suppression subtractive hybridization (SSH) libraries were generated from ovaries with perinucleolus (P) or cortical alveolus (CA) stage follicles and selected genes were assessed with quantitative PCR (qPCR). An assessment of changes in RNA composition during oocyte growth and its relationship to transcript levels was also conducted.ResultsSSH revealed several differentially expressed genes during early oogenesis, some which will not likely be utilized until 1–3 years later in salmon. Zona pellucida glycoprotein (zp) genes, vitellogenin receptor (vldlr) isoforms, cathepsin B (ctsba), cyclin E (ccne), a DnaJ transcript (dnaja2), and a ferritin subunit (fth3) were significantly elevated at the P stage, while a C-type lectin, retinol dehydrogenase (rdh1), and a coatomer protein subunit (cope) were upregulated at the CA stage. Putative follicle cell transcripts such as anti-Müllerian hormone (amh), lipoprotein lipase (lpl), apolipoprotein E (apoe), gonadal soma-derived growth factor (gsdf) and follicle-stimulating hormone receptor (fshr) also increased significantly at the CA stage. The analysis of RNA composition during oocyte growth showed that the total RNA yield and proportion of messenger RNA relative to non-polyadenylated RNAs declined as oogenesis progressed. This influenced apparent transcript levels depending on the type of RNA template used and normalization method.ConclusionIn coho salmon, which exhibit a dramatic change in oocyte size and RNA composition during oogenesis, use of messenger RNA as template and normalization of qPCR data to a housekeeping gene, ef1a, yielded results that best reflected transcript abundance within the ovarian follicle. Synthesis of zp transcripts and proteins involved in yolk incorporation and processing occurred during primary growth, while increased expression of a CA component and genes related to lipid incorporation occurred concomitant with the appearance of CA, but prior to lipid accumulation. Significant increases in transcripts for fshr, gsdf, and amh at the CA stage suggest a role of FSH and TGFβ peptides in previtellogenic oocyte growth and puberty onset in female salmon.

Development of a contraceptive vaccine for the marsupial brushtail possum (Trichosurus vulpecula): lack of effects in mice and chickens immunised with recombinant possum ZP3 protein and a possum ZP3 antifertility epitope

Zona pellucida fertility-control vaccines are being developed in New Zealand to control an introduced marsupial pest, the brushtail possum (Trichosurus vulpecula). In this study recombinant possum ZP3 protein (rZP3) and a possum ZP3 peptide (amino acids 334–361) (both known to block fertility in possums) were examined for their potential to induce species-specific, or at least marsupial-specific, infertility. Laboratory mice (a ‘model’ eutherian mammal species) and domestic chickens (a ‘model’ bird species) immunised with possum rZP3 or possum-infertility ZP3 peptide in Freund’s adjuvants showed no reduction in a range of parameters indicative of reproductive performance. The lack of contraceptive effects on mouse and chicken fertility is an encouraging result in terms of rZP3 and ZP3 peptide specificity, and these promising antigens are to be expressed in a bacterial ghost vaccine system for mucosal delivery to possums and the effects on possum fertility evaluated. Ultimately, a much wider range of non-target species will need to be screened and tested once the antigens have been successfully formulated in their final delivery vehicle.

Spermatozoa from a marsupial, the brushtail possum, contain β1,4-galactosyltransferase

Beta1,4-galactosyltransferase-I (GalTase-I) is one of the key molecules on the sperm surface of eutherian mammals that is likely to be involved in binding to the egg coat, the zona pellucida, to mediate sperm-egg interaction. In laboratory mice, the species for which most data are available, this protein functions as a receptor for the zona pellucida protein ZP3 of the oocyte and, upon binding, triggers the sperm acrosome reaction. In the present study, we investigated the presence and abundance of GalTase-I in epididymal sperm extracts of a marsupial, the brushtail possum, Trichosurus vulpecula. For this, spermatozoa were collected from cauda epididymides and the amount of beta1,4-galactosyltransferase activity in washed sperm extracts was compared with that of porcine spermatozoa. Overall beta1,4-galactosyltransferase enzyme activity was found to be more abundant in possum sperm extracts than those from porcine spermatozoa (P<0.05). Immunoblots with an antibody to mouse GalTase-I revealed that the molecular weight of possum spermatozoa GalTase-I was 66 kDa, which is similar to the molecular weight of GalTase-I in spermatozoa from eutherian mammals. The molecular weight of GalTase-I was the same in sperm extracts collected from the caput and cauda epididymides. These results demonstrate that GalTase-I is indeed present in possum spermatozoa and thus it may be a gamete receptor molecule on the sperm surface of marsupials as well as those of eutherian mammals.

Towards a ZP‐based contraceptive for marsupials: Comparative analysis and developmental expression of marsupial ZP genes

Fertility control in the form of a zona pellucida (ZP)-based immunocontraceptive has shown potential as a humane form of control for overabundant marsupials including the brushtail possum and macropods. Further refinement and development of a ZP-based vaccine requires detailed knowledge of the protein structure and expression in order to ensure maximum efficacy and specificity. Sequencing and comparative analysis of the ZP3 protein from three marsupial orders in this study found a high overall level of conservation; within order Diprotodontia, the ZP3 protein is 86.9-98.9% identical. ZP3 identity falls to 56.6-57.2%, when the grey, short-tailed opossum (a Didelphimorphian) is compared to dasyurid and diprotodontan marsupials. This is similar to its amino acid identity with ZP3 from eutherian species (50.7-52.8%). Comparison of a 21 amino acid epitope in marsupial ZP3 that has shown contraceptive effects, reveals 95-100% identity between the four macropodid species, 81-86% amino acid identity between brushtail possum and the macropods and 67-71% identity between the diprotodontans and the fat-tailed dunnart (a dasyurid). This is comparable to the level of identity between related eutherian mammals. The expression pattern of three ZP genes during brushtail possum and tammar wallaby pouch young development was examined by RT-PCR. This analysis of ZP gene expression has confirmed that ZP mRNA transcription begins in the ovary during pouch young development by about 51 days of age. The presence of ZP transcripts at this stage in pouch young development suggests that marsupial ZP gene transcription begins before the onset of follicular development.

Isolated ZP-N domains constitute the N-terminal extensions of Zona Pellucida proteins

Zona Pellucida (ZP) domains have been found in a wide variety of extracellular proteins, in which they play essential role for polymerization. They are shared by the ZP proteins, which constitute the extracellular coat of animal eggs. Except from ZP3, constituting the primary sperm receptor, the ZP proteins possess, in addition to their C-terminal ZP domains, N-terminal extensions, which are thought to play an important role in the species-specific gamete recognition. Here, we show that these extensions are made of single or multiple copies of a small globular domain, which can be significantly related to the N-terminal region of ZP domains (ZP-N domains). This finding brings new insights into the molecular evolution of ZP proteins, which may have evolved around a common ZP-N architecture, and more generally into the noticeable sequence diversity of ZP-N domains, which can be found as isolated subunits or tightly associated with ZP-C domains to form complete, canonical ZP domains.

Egg extracellular coat proteins: from fish to mammals.

The extracellular coat surrounding fish (vitelline envelope; VE) and mammalian (zona pellucida; ZP) eggs is composed of long, interconnected filaments. Fish VE and mammalian ZP proteins that make up the filaments are highly conserved groups of proteins that are related to each other, as well as to their amphibian and avian egg counterparts. The rainbow trout (O. mykiss) egg VE is composed of 3 proteins, called VEalpha (approximately 58 kDa), VEbeta (approximately 54 kDa), and VEgamma (approximately 47 kDa). The mouse (M. musculus) egg ZP also is composed of 3 proteins, called ZP1 (approximately 200 kDa), ZP2 (approximately 120 kDa), and ZP3 (approximately 83 kDa). Overall, trout VE and mouse ZP proteins share approximately 25% sequence identity and have features in common; these include an N-terminal signal sequence, a ZP domain, a consensus furin cleavage-site, and a C-terminal tail. VEalpha, VEbeta, and ZP1 also have a trefoil or P-type domain upstream of the ZP domain. VEalpha and VEbeta are very similar in sequence (approximately 65% sequence identity) and are related to ZP1 and ZP2, whereas VEgamma is related to ZP3 (approximately 25% sequence identity). Mouse ZP proteins are synthesized and secreted exclusively by growing oocytes in the ovary. Trout VE proteins are synthesized by the liver under hormonal control and transported in the bloodstream to growing oocytes in the ovary. The trout VE is assembled from VEalpha/gamma and VEbeta/gamma heterodimers. The mouse ZP is assembled from ZP2/3 heterodimers and crosslinked by ZP1. Despite approximately 400 million years separating the appearance of trout and mice, and the change from external to internal fertilization and development, trout VE and mouse ZP proteins have many common structural features; as do avian and amphibian egg VE proteins. However, the site of synthesis of trout and mouse egg extracellular coat proteins has changed over time from the liver to the ovary, necessitating some changes in the C-terminal region of the polypeptides that regulates processing, secretion, and assembly of the proteins.

P-838: Induction of apoptosis and functional deterioration of human sperm by lipid aldehydes

Low concentrations (5-10μM) of reactive oxygen species (ROS) are required for regulatory mechanisms that control sperm primary functions such as capacitation, hyperactivation, the acrosome reaction, and sperm-oocyte interactions via the oocyte protein ZP3. Such processes must occur for the sperm to acquire the capacity to fertilize an egg. Although low levels of ROS are necessary for proper sperm function, higher levels (50μM and greater) are believed to have detrimental effects on sperm. Oxidative stress is widely accepted as a primary cause of infertility; however the related mechanism(s) are poorly understood.

Mechanical Analysis of Chorion Softening in Prehatching Stages of Zebrafish Embryos

During early development, the chorion envelope of the zebrafish embryo undergoes a thinning process called "chorion softening," which has so far only been characterized chemically. In this study, a micromechanical force sensing system was used to characterize and quantitate mechanical modifications of the zebrafish embryo chorion during early development. Quantitative relationships between applied forces and chorion structural deformations were established at various embryonic stages. The measured penetration force into the chorion at the blastula stage was 1.3-fold greater than those at the prehatching stage. Furthermore, chorion elastic modulus values were determined by using a biomembrane elastic model. The elastic modulus of the chorion at the blastula stage was 1.66-fold greater than that at the prehatching stage, thus indicating that the chorion envelope become mechanically "softened" at the prehatching stage. The experimental results quantitatively describe "chorion softening," which is most likely due to proteolytic activities at the prehatching stage. Gradual chorion softening during embryonic development was also artificially achieved by treating blastula chorion with pronase, a proteolytic enzyme. The forces required to penetrate the pronase-treated chorion were similar to those at the prehatching stage. This similarity suggests that "chorion softening" may be induced by the release of protease from the embryos, and the chemical nature of the process involves proteolytic fragmentation of the ZP2 protein.

Tandem-Repeated Zebrafish zp3 Genes Possess Oocyte-Specific Promoters and Are Insensitive to Estrogen Induction1

Zona pellucida (Zp) proteins are glycoproteins in fish chorion and are encoded by multiple gene families, including zp1, zp2, zp3, and potentially other zp genes. Expression of zp genes in teleosts is either in the liver under the induction of estrogen or in the ovaries. In the present study, we isolated and characterized a zebrafish zp3 genomic clone and found three tandem-repeated zp3 genes with high sequence identities. We estimated that there were 10-15 zp2 and 17-21 zp3 genes in a haploid genome. We also found some variant zp2 and zp3 subfamilies, and each subfamily may have multiple members. These zp2 and zp3 genes are distributed in several different chromosomes. Ontogenetic expression of zp2 and zp3 mRNAs was first detected at 3 wk postfertilization, which was about 5 wk earlier than initial vtg1 expression, indicating that ovary development was earlier than vitellogenesis. Both zp2 and zp3 mRNAs were expressed specifically in early-growing oocytes and are insensitive to estrogen induction. Because zp3 genes are organized in tandem repeats, to investigate whether an individual zp3 promoter is capable of driving oocyte-specific expression, green fluorescence protein (gfp)-transgenic zebrafish were developed by using a 3.8-kb zp3 5' upstream region, and we found that the gfp reporter gene was faithfully expressed in developing oocytes in zp3:gfp transgenic females. Thus, the new transgenic line not only provided a convenient living marker for monitoring female gonad development, but also demonstrated that a single zp3 gene promoter is sufficient for oocyte-specific transcription.

Fertility Control for Wildlife Management The Brushtail Possum in New Zealand

Author(s): Cowan, Phil; Duckworth, Janine; Cui, Xianlan; Molinia, Frank; Lubitz, Werner; Walcher, Petra | Abstract: Fertility control vaccines are under development for a number of pest wildlife species, mostly based on whole zona pellucida (ZP) or individual ZP proteins. Such vaccines must be effective, long lasting, cheap and readily deployed. One approach to deployment is oral delivery in baits. This is one strategy being taken in New Zealand for control of brushtail possums, 2- to 4-kg marsupials introduced from Australia in the 1850s, and now major pests of both conservation and agricultural production. New Zealand has highly effective aerial and ground systems for delivery of toxic baits to possums that could be adapted readily to deliver vaccine baits. Recent trials in captivity where female possums were immunised with recombinant possum ZP3 and ZP2 proteins demonstrated 70-75% reductions in fertility in natural and assisted breeding trials. Immunisation with possum-specific epitopes of the ZP2 and ZP3 proteins has also proved effective at reducing the numbers of fertilised eggs recovered from immunised females. For field delivery of an oral vaccine, we are investigating the use of bacterial “ghosts”. These are the empty cell walls of bacteria that have been modified to express possum ZP proteins in their cell walls. The possum’s immune system recognises the bacterial ghost as foreign, and produces antibodies against them. At the same time, it is tricked into developing antibodies against the possum egg proteins, causing a contraceptive effect. In a recent proof of concept trial, female possums immunised with a possum ZP2-bacterial ghost vaccine by nasal spray showed a significant reduction in fertility. Oral delivery may require protection of vaccine ghosts from degradation. We have developed a protective system and are currently repeating this trial using oral delivery of the ZP2 ghost vaccine. Our future priority is increasing the vaccine efficacy and longevity ahead of limited field trials in 2009.

Mapping of B cell epitopes on the zona pellucida 2 protein of a marsupial, the brushtail possum (Trichosurus vulpecula)

Immunocontraceptive vaccines against zona pellucida (ZP) proteins are being developed for brushtail possum (Trichosurus vulpecula) management in New Zealand. Mapping of B cell epitopes on the ZP2 protein of possums was undertaken in this study to define the antigenic regions that may be crucial to sperm-egg binding. The amino acid sequence of the full-length possum ZP2 protein (712 amino acids) was used to synthesize a complete set of 71 (15-mer) biotinylated peptides with an offset of five amino acids. The peptides were used in a modified enzyme-linked immunosorbent assay (ELISA) to identify continuous epitopes recognized by antibodies in the sera of possums immunized with recombinant possum ZP2 (rZP2) constructs. Seventeen continuous epitopes were located on possum ZP2 protein. Comparisons of the peptide binding pattern of antibodies in individual sera with the fertility status of the same immunized possums revealed three significant infertility-relevant peptide epitopes (amino acids 111-125, 301-315, and 431-445). One of these (amino acids 431-445) bound to possum spermatozoa from the caudal epididymis. The implications of these findings for developing immunocontraceptive vaccines for possum control are discussed.

Contraceptive Vaccines

The world's population is growing at a tremendous rate, affecting growth and development. Apart from this population growth, unintended pregnancies resulting in elective abortions continue to be a major public health issue. In over half of these unintended pregnancies, the women have used some type of contraception. Thus, there is an urgent need for a better method of contraception that is acceptable, effective and available. The contraceptive choices available to women at this time include steroid contraceptives, intrauterine devices, barrier methods, spermicides, natural family planning, male and female sterilisation, and recently available emergency contraceptives. Contraceptive vaccines (CVs) may provide viable and valuable alternatives that can fulfill most, if not all, properties of an ideal contraceptive. Since both the developed and most of the developing nations have an infrastructure for mass immunisation, the development of vaccines for contraception is an exciting proposition. The molecules that are being explored for CV development either target gamete production (gonadotropin releasing hormone, follicle-stimulating hormone and luteinising hormone), gamete function (zona pellucida [ZP] proteins and sperm antigens) or gamete outcome (human chorionic gonadotropin [hCG]). Disadvantages of CVs targeting gamete production are that they affect sex steroids and/or show only a partial effect in reducing fertility. CVs targeting gamete function are better choices. Vaccines based on ZP proteins are quite efficacious in producing contraceptive effects. However, they invariably induce oophoritis affecting sex steroids. Sperm antigens constitute the most promising and exciting targets for CVs. Several sperm-specific antigens have been delineated in several laboratories and are being actively explored for CV development. Antisperm antibody-mediated immunoinfertility provides a naturally occurring model to indicate how an antisperm vaccine will work in humans. Vaccines targeting gamete outcome primarily focus on the hCG molecule. The hCG vaccine is the first vaccine to undergo phase I and II clinical trials in humans. Both the efficacy and the lack of immunotoxicity have been reasonably well demonstrated for this vaccine. The present studies focus on increasing the immunogenicity and efficacy of this birth control vaccine.

A synthetic decapeptide from a conserved ZP3 protein domain induces the G protein-regulated acrosome reaction in bovine spermatozoa

In some animal species, the zona pellucida protein 3 (ZP3) plays a central role during fertilization, functioning as a specific receptor for sperm and as an inducer of the acrosome reaction. On the other hand, the zona pellucida protein 2 (ZP2) acts as a secondary receptor, binding to acrosome-reacted sperm. The objective of these studies was to identify ZP2 and ZP3 domains that may be of importance for the induction of the acrosome reaction. For this purpose, we synthesized a number of ZP2 and ZP3 peptides that were either conserved among species or that were species-specific according to their respective primary structures. We identified a defined, conserved ZP3 decapeptide (ZP3-6 peptide) that bound to the surface of the acrosomal region and induced the acrosome reaction in a concentration-dependent manner in capacitated bovine sperm; this effect was significant in the nanomolar range. Pertussis toxin inhibited the ZP3-6 peptide-induced acrosome reaction but had no effect on the progesterone-induced exocytotic event. Our data are in accordance with previous studies showing that progesterone induces acrosomal exocytosis via a different pathway than ZP3 and strengthen the hypothesis that the effect of ZP3-6 peptide upon acrosomal exocytosis is G protein regulated. Despite the commonly accepted idea that glycosylation of ZP proteins is required for successful sperm–oocyte interaction, we found that acrosomal exocytosis can be induced by a synthetic ZP3 peptide that is not glycosylated. The results presented in this study may be useful for the investigation of the molecular mechanisms of sperm–egg interaction in bovine and other species.