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RESEARCH |
Department of Zoology, The University of Melbourne, Parkville, 3010 Victoria, Australia
Correspondence should be addressed to A Nation; Email: a.nation{at}pgrad.unimelb.edu.au
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Abstract |
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Introduction |
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The zona pellucida (ZP) plays a critical role in mammalian fertilization, hence ZP glycoproteins are the most frequently proposed candidates for ovarian-based immunocontraception (Barber & Fayrer-Hosken 2000). Numerous trials have validated the contraceptive potential of various antigens (Frank et al. 2005); however, fertility reduction is frequently accompanied by ovarian pathology (Mahi-Brown et al. 1988, Curtis et al. 2007) and hormonal disturbance (Skinner et al. 1984, Stoops et al. 2005). An ideal ovarian immunological agent would inhibit fertility without causing ovarian dysfunction.
A major obstacle in developing contraceptive vaccines for the regulation of wildlife populations is the risk of inadvertently affecting non-target species. The situation in New Zealand is unusual in that the country's major mammalian pest is an introduced marsupial, the common brushtail possum (Trichosurus vulpecula). As New Zealand has no native marsupials, the development of a marsupial-specific contraceptive vaccine, derived from a unique feature of marsupial development, such as oocyte–conceptus polarity, would eliminate the threat to endemic species.
In marsupials, polarity is initially established in primary oocytes by the eccentric, peripheral location of the nucleus (Selwood 1992). Polarity is further enhanced in many species by the accumulation of conspicuous membrane-bound electron-lucent vesicles opposite the nucleus, either during oogenesis or following fertilization (Selwood 1994). Oocyte vesicles originate via the fusion of multivesicular bodies, derived from endoplasmic, endocytotic, and Golgi vesicles in Monodelphis domestica (Falconnier & Kress 1992) as well as coated and heterogeneous vesicles in Isoodon macrourus (Ullmann & Butcher 1996). Although the chemical composition of these vesicles remains largely unknown, there is some evidence they contain glycoproteins (Kress 1996) and saccharide residues (Breed 1996). Oocyte and zygote polarity is maintained by species-specific cytoskeletal architecture (Breed 1994, Merry et al. 1995, Frankenberg & Selwood 1998).
During early cleavage, vesicular contents are generally shed in a polarized fashion into the perivitelline space (Breed & Leigh 1990, Taggart et al. 1993, Sathananthan et al. 1997), contributing to blastocoel formation (Frankenberg & Selwood 2001). Cell-zona attachment first occurs in the opposite hemisphere to vesicle discharge (Selwood 1992, 2001, Merry et al. 1995), where the pluriblast (future embryonic) cells develop. Trophoblast (future placental) cells line the opposite hemisphere and continue to secrete vesicular matter into the cleavage cavity. In a number of marsupials, T. vulpecula (Frankenberg & Selwood 1998), Sminthopsis macroura, and Antechinus stuartii (Selwood & Smith 1990), cytoplasmic vesicles are unevenly distributed in the first two cell lineages of the embryo, and the polarized discharge of extracellular material appears essential for normal development (Frankenberg & Selwood 1998, Kress & Selwood 2003, 2004).
Recently, a number of vesicle-associated molecules (VAMs) have been identified in the vesicle-rich hemisphere of the T. vulpecula oocyte (Selwood et al. 1999). VAMs are associated with oocyte polarity, cell lineage allocation, and are believed to be essential for normal oogenesis, cleavage, and blastocyst formation. Vesicle-associated protein 1 (VAP1), a previously characterized ovarian-specific protein, has been proposed as a potential immunocontraceptive target for possum control in New Zealand (Cui et al. 2005). This study was conducted to assess the ability of VAP1 immunization to elicit an immune response in T. vulpecula and to examine the effect of vaccination on female fertility.
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Results |
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Estrous cycling activity and fertility
To determine whether female fertility was affected by VAP1 immunization, estrous cycling activity, mating, and birth data were collected from the same cohort of animals (n=7), pre- and post-VAP1 immunization. In general, most females cycled continuously between March and September. Following immunization, however, animals were more likely to continue cycling during the months of October and November, indicating that VAP1 immunization may prolong the breeding season in some females. Female fertility (the percentage of estrous cycles resulting in birth) showed a monthly decline following immunization, with later months of the breeding season (May to October–November) most affected (Fig. 1). Despite continuing to cycle during October–November, no immunized females produced young during this period. Following immunization, individual fertility levels dropped in six of the seven treated animals (Fig. 2), and mean female fertility (±S.E.M.) declined significantly (t=10.0, df=6, P
0.01). Changes in fertility were not significantly correlated with animal age or estrous cycle stage at immunization (P>0.05).
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Degradation bodies first became apparent in primary oocytes after the transition of primordial to the primary follicle stage (Fig. 6A and B). As follicles developed, the inclusions increased in size and accumulated to various extents within the ooplasm (Fig. 6C–F). Analysis of primary oocyte cross-sections (from VAP1-immunized and non-immunized females) revealed that the number of lipid inclusions (degradation bodies and normal lipid droplets) per µm2 oocyte cytoplasm was significantly greater in primary (t=3.37, df=44, P=0.02) and antral (t=4.28, df=15, P=0.02) follicle oocytes of VAP1-immunized animals. The number of vesicles (greater or equal in size to the largest observed lipid inclusion) per µm2 oocyte cytoplasm was significantly reduced in primary follicle oocytes (t=4.45, df=44, P=0.12) of VAP1-immunized females but not in antral follicle oocytes (P<0.05). Degradation bodies remained prominent in secondary oocytes and appeared largest in the most mature oocytes.
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Discussion |
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VAP1 is encoded by a unique gene with no significant homologies (Cui et al. 2005). Some regions of the protein, however, show structural similarity to the active regions of cystatin C, an endogenous cysteine proteinase inhibitor (Barrett et al. 1984, Turk & Bode 1991, Dieckmann et al. 1993). Cystatin C, a member of the type 2 cystatin superfamily (Abrahamson et al. 2003), regulates cysteine proteinase activity in intra- and extracellular biological reactions (Barrett 1981). In addition to similar spatial form, the conserved presence of all five cystatin C functional motifs (disulfide-paired cysteine residues) within the VAP1 sequence implies a homologous role. If so, VAP1 may contribute to extracellular matrix (ECM) stability during oogenesis and early development by allowing the accumulation and stable storage of proteins within the oocyte cytoplasm, and/or stabilizing proteins as they are secreted into the blastocoel during cleavage (Cui et al. 2005).
Sparsely distributed lipid droplets have been described in the ooplasm of T. vulpecula (Frankenberg & Selwood 2001), I. macrourus, I. obesulus, and Perameles nasuta (Ullmann 1981, Lyne & Hollis 1983) and various eutherian species (Fleming & Saacke 1972, Tesoriero 1981, Homa et al. 1986, Kikuchi et al. 2002). Although rare, degradation bodies have been observed in T. vulpecula zygote cytoplasm (Frankenberg & Selwood 1998) but not during normal oogenesis (Frankenberg & Selwood 2001). Degradation bodies of the size and magnitude observed here are previously unreported.
Despite the unusual oocyte changes induced by VAP1 immunization, local inflammatory cell levels were not elevated, therefore the ovarian immune response appears systemic. In contrast to ZP-based immunocontraceptives (Kitchener et al. 2002), VAP1 immunization affected only the germ cells of the ovary. The preservation of surrounding somatic tissue and normal ovarian morphology strengthens previous speculation that Vap1 expression may be oocyte specific (Cui et al. 2005). Following immunization, degradation bodies first became apparent in primary oocytes after the transition of primordial follicles to the primary follicle stage, correlating with a period of high Vap1 expression (Cui et al. 2005), and the initiation of oocyte vesicle formation (Frankenberg et al. 1996, Frankenberg & Selwood 2001). The accumulation of degradation bodies in affected oocytes was associated with decreased vesicle number, suggesting that immunization compromises normal vesicular accumulation during oogenesis.
If VAP1 plays a role akin to cystatin C, then the transfer of VAP1 antibodies into the developing oocyte via serum would disrupt the normal cysteine proteinase-inhibitor balance, effectively allowing uncontrolled proteolytic activity of this type within the oocyte. In humans, imbalances between cathepsins of the cysteine proteinase family and their endogenous inhibitors are related to a variety of pathological conditions (Lenarcic et al. 1988, Henskens et al. 1996, Cimerman et al. 2001, Kos et al. 2001). We propose that the degradation bodies observed in this study are a direct result of increased proteolysis of oocyte storage proteins and/or ECM molecules due to VAP1 inhibition. During early development in T. vulpecula, the polarized discharge of ECM material appears important for embryonic–abembryonic patterning (Frankenberg & Selwood 1998, Kress & Selwood 2003, 2004) and vesicles are preferentially distributed in the first two cell lineages (Frankenberg & Selwood 1998). Decreased ECM stability, change in vesicular secretion and disruption of cell lineage allocation may all contribute to reducing embryonic success following immunization.
In addition to the antibody-mediated ovarian response, a small number of VAP1-immunized females demonstrated uterine changes consistent with endometrial cystic hyperplasia (De Bosschere et al. 2001), raising the possibility that a uterine immune response may contribute to this condition. The presence of macrophage clusters within enlarged endometrial glands suggests a cell-mediated reaction, possibly in response to the presence of a failed conceptus. All conceptuses obtained from the uteri of VAP1-immunized females showed cytoplasmic fragmentation; therefore, it appears probable that the macrophages observed were attacking the remains of a disintegrated oocyte or conceptus. There was no evidence to suggest that macrophages attack the intact conceptus, implying that the shell coat may have immunoprotectant properties. The absence of T lymphocytes within VAP1-immunized uteri is not unusual as experiments were generally scheduled during early pregnancy (prior to shell coat loss) in the hope of obtaining a conceptus. Due to the limited number of animals available, we were unable to examine all stages of pregnancy.
In conclusion, VAP1 immunization induces a novel oocyte-specific systemic immune response, which decreases female fertility without detrimental ovarian pathology. A secondary cell-mediated uterine response may also contribute to embryonic failure. Passive immune transfer (Adamski & Demmer 2000), from immunized mothers to female young during lactation, may impart a generational effect. The ovary has a finite reserve of oocytes. As VAP1 targets all stages of oogenesis, immunization has the potential to impair fertility throughout an animal's lifespan. With the marsupial-specific VAP1 protein identified and shown to have an antibody mediated response to oocytes in vivo, VAP1 antibodies can now be used to carry fertility control agents into developing oocytes of the marsupial ovary.
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Materials and Methods |
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2:1) was preserved throughout the experimental period, and the colony contained an average of 18 individuals at any given time.
Reproductive monitoring
Adult females were monitored from March to November to establish the reproductive status. Estrous cycle stage was determined by cytology and mating success by the presence of spermatozoa in daily urine samples (Duckworth et al. 1999). T. vulpecula are monovular, polyoestrous seasonal breeders (Pilton & Sharman 1962). Following RPY females return to estrus within 7–15 days (Duckworth et al. 1998). Estrus occurs 1–3 days prior to ovulation (Shorey & Hughes 1973), and young are born 17.3 (±0.2) days post-coitus (Duckworth et al. 1998). Experimental females were monitored for 6–18 months before VAP1 immunization. Data obtained during this period served as respective (pre-immunized) controls for each individual, enabling the comparison of pre- and post-immunization activity. Immunizations were undertaken during the breeding season and post-immunization data were collected during the remainder of that season and the subsequent breeding season. The length of time animals were monitored post-immunization varied between individuals.
Immunization
Animals were randomly assigned to VAP1-immunized (n=7) or control-immunized (n=3) groups. The immunization antigen, recombinant GST-VAP1 fusion protein, was prepared according to a previous paper (Cui et al. 2005). The immunization protocol comprised an initial dose of 300 µg GST-VAP1 in 500 µl magnesium- and calcium-free PBS– emulsified with 500 µl Freund's Complete Adjuvant (Sigma–Aldrich). Two booster vaccines followed at 28-day intervals, each containing 300 µg GST-VAP1 in 500 µl PBS– in the presence of 500 µl Freund's Incomplete Adjuvant (Sigma–Aldrich). Four animals received a further booster between 6 and 18 months from the initial immunization. Control-immunized animals were treated over the same period with equivalent volumes of antigen-free PBS– (n=2) or GST in PBS– (n=1) using the same adjuvants. Immunizations were delivered to multiple sites by s.c. injection. Prior to immunization and/or blood collection animals were sedated via i.m. injection with 10 mg/kg Zoletil (Virbac Animal Health, Peakhurst, NSW, Australia). Blood samples were collected from the lateral tail vein or the basilic forearm vein, prior to the initial injection (pre-immune control), 7–10 days after booster injections (Cooper & Paterson 2008), and at 3 monthly intervals throughout the remaining experimental period. Prior to autopsy, animals were anesthetized with 15 mg/kg Zoletil delivered intramuscularly. The blood was collected via heart puncture and animals euthanized by intracardial injection of 150 mg/kg sodium pentobarbitone (Lethabarb; Virbac Animal Health). The blood samples were centrifuged at 2130 g for 10 min. Sera were removed and stored at –20 °C for analysis.
ELISA
The titer of polyclonal antibodies (against GST-VAP1) in the sera of VAP1-immunized (n=7) and control-immunized (n=3) animals was determined by ELISA, with pre-immune samples serving as negative controls for each individual. Assays were performed in 96-well ELISA plates (Immunlon 4 HBX; Thermo Scientific, Noble Park, VIC, Australia) using recombinant GST-VAP1 as the antigen. Non-specific binding was blocked with 3% albumin bovine serum (Sigma–Aldrich) and plates were washed between incubations with a buffering solution of 0.05% Tween-20 (Selby Scientific, Notting Hill, VIC, Australia) in PBS–. Serial dilutions of the primary antibody, T. vulpecula-raised polyclonal antisera, were detected by application of the secondary antibody, horseradish peroxidase (HRP)-conjugated protein A (GE Health Care, Castle Hill, NSW, Australia), diluted 1:5000 in PBS–. Incubation with tetramethyl benzidine substrate (Sigma–Aldrich), which reacts with HRP, produced an insoluble compound. Reactions were terminated after 30 min by the addition of 0.5 M sulfuric acid. Color absorbance was measured at 450 nm using an ELISA reader (Multiscan EX; Thermo Electron Corporation, Vantaa, Finland). The three negative controls included on all plates comprised no primary antibody incubation, no secondary antibody incubation, and no primary or secondary antibody incubation.
Endpoint antibody titers were obtained from the lowest dilution factor at which the absorbance of post-immunization sera remained greater than the absorbance of pre-immunization sera (1:200 dilution). For analysis, endpoint dilution factors were expressed as log2 values (Doolin et al. 2002).
Histology
Tissue segments (heart, kidney, liver, lung, spleen, lymph node, ovary, oviduct, and uterus) were collected from all immunized animals and fixed by immersion in 4% paraformaldehyde (PFA; Merck) in PBS– for 24 h, dehydrated in ethanol, embedded in paraffin, serially sectioned at 6–8 µm, mounted, and stained with Harris’ Hematoxylin and Putt's Eosin. Tissue morphology was compared between VAP1-immunized (n=7) and control-immunized (n=3) animals. The tissue from healthy, non-immunized colony animals (n=5) was also collected, processed in the same manner, and used as a reference for normal morphology.
Transmission electron microscopy
Ovarian follicles, oocytes, and conceptuses were immersed in Superfix (2.5% glutaraldehyde (ProSciTech, Thuringowa, QLD, Australia), 3% PFA, 0.2 M sodium cacodylate buffer) at RT for 2 h (pH 7.4). After three rinses in 0.1 M sodium cacodylate buffer (pH 7.4), post-fixation took place in 1% osmium tetroxide (ProSciTech) diluted with 0.2 M sodium cacodylate buffer for 2 h at RT. Following rinsing in 0.1 M sodium cacodylate buffer, the tissue was dehydrated in an ethanol series, infiltrated with a propylene oxide–Epon araldite mixture, embedded in pure Epon araldite, and polymerized at 60 °C for 48 h. The resin consisted of a mixture of 25 ml Procure 812, 15 ml Araldite 502, 55 ml dodecenyl succinic anhydride, and 1.25 ml benzyldimethylamine (ProSciTech).
Semi-thin sections (1 µm) were cut with glass knives on an ultramicrotome (Ultracut E; Reichert-Jung, Heidelberg, Germany) and stained with 1% toluidine blue and 1% sodium borate in distilled water at 90 °C for 1–2 min. At least three resin-embedded ovarian segments were serially sectioned, for each VAP1-immunized (n=7) and non-immunized (n=5) animal. Primary oocyte diameters, in primary and antral follicles, were measured at their largest cross-section, and counts made of the number of lipid inclusions (degradation bodies and normal lipid droplets) and the number of vesicles (greater or equal in size to the largest lipid inclusion) within the oocyte cytoplasm.
Thin sections (70 nm), cut with a diamond knife, were contrasted with 3% uranyl acetate and 0.6% lead citrate, mounted on coated grids and examined with a transmission electron microscope (CM 10; Philips, Eindhoven, The Netherlands).
Statistical analysis
Paired-samples t-tests were used to compare female fertility (i.e., the percentage of estrous cycles resulting in birth), pre- and post-VAP1 immunization. The correlation between individual fertility reduction and maximum immune titer reached was determined by linear regression. Independent-samples t-tests compared the number of lipid inclusions and the number of vesicles per µm2 oocyte cytoplasm between VAP1-immunized and non-immunized animals.
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Declaration of interest |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestFundingAcknowledgementsReferences |
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Funding |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestFundingAcknowledgementsReferences |
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Acknowledgements |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestFundingAcknowledgementsReferences |
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Received 9 April 2008
First decision 14 May 2008
Accepted 18 August 2008
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References |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsDeclaration of interestFundingAcknowledgementsReferences |
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