1 Laboratory of Cellular and Developmental Biology, NIDDK National Institutes of Health, Bethesda, Maryland 20892 and 2 Department of Obstetrics and Gynecology, George Washington University, Washington, DC 20037, USA
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Abstract |
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Key words: contraception/human ZP3//knockout mice/monoclonal antibodies/spermatozoonovum interactions
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Introduction |
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Fertilization results from the fusion of a spermatozoon and egg in the ampulla of the oviduct. In mammals, this interaction is initiated by the binding of capacitated, acrosome-intact spermatozoa to the zona pellucida, an extracellular matrix surrounding the egg (Yanagimachi, 1994). Although considerable progress has been made in understanding the molecular basis of spermatozoonegg interactions, controversy remains over the role of candidate molecules (Snell and White, 1996
). However, despite these uncertainties, immunological reagents targeting antigens on the surface of either male and female gametes have been successful in preventing fertilization (Primakoff et al., 1988
; Millar et al., 1989
). The zona pellucida is highly immunogenic, and the relatively small number of eggs makes it a particularly attractive target for immunocontraception. Antibodies directed against the zona pellucida were first shown to inhibit fertilization in the 1970s (Shivers et al., 1972
) and, as understanding of the molecular biology of the zona pellucida has progressed, immunological targeting of the zona pellucida has become increasingly sophisticated.
Candidate protein epitopes in the mouse zona pellucida have been identified and the administration of monoclonal antibodies specific to them results in long-term, reversible contraception (East et al., 1984, 1985
). Although vaccination with the same epitope can inhibit fertility, the efficacy is not 100%, and can result in autoimmune oophoritis in inbred mouse lines, a problem that may be avoided by insightful molecular design (Millar et al., 1989
; Rhim et al., 1992
; Lou et al., 1995
). The long-term goal of these and other studies in identifying zona epitopes and determining their biological efficacy has been to provide a rational basis for the development of immunocontraception in humans. However, the translation of the accumulated knowledge has been hampered by using laboratory animals such as mice that have a zona that is only distantly related to that of humans (Chamberlin and Dean, 1990
; Liang and Dean, 1993
; Harris et al., 1994
) or using primates (VandeVoort et al., 1995
; Mahi-Brown, 1996
; Paterson et al., 1996
; Afzalpurkar et al., 1997
; Bagavant et al., 1997
) which is limited because of prohibitive costs and availability. Transgenesis in mice could circumvent these issues by creating inexpensive laboratory animals that express individual human zona proteins and provide model systems for identifying and testing potential epitopes for immunocontraceptive efficacy.
The human and mouse zonae pellucidae are each composed of three glycoproteins (ZP1, ZP2, ZP3), the primary structures of which have been deduced from full-length cDNA (Ringuette et al., 1988; Chamberlin and Dean, 1990
; Liang et al., 1990
; Liang and Dean, 1993
; Harris et al., 1994
; Epifano et al., 1995
). Of the three zona proteins, ZP1 is the least conserved; human ZP1 (540 amino acids) contains 83 fewer amino acids compared with the mouse homologue (623 amino acids) and, when aligned, only 43% of the amino acids are identical. Human and mouse ZP2 contain 745 and 713 amino acids, respectively, which are 61% identical, whereas human and mouse ZP3 both have polypeptide chains of 424 amino acids that are 67% identical (77% similar). Each zona protein is post-translationally modified. The apparent molecular mass of the three proteins in the mouse and human zona matrices are quite different (Bleil and Wassarman, 1980b
; Sacco et al., 1981
; Shimizu et al., 1983
; Shabanowitz and O'Rand, 1988
), which has led to controversy over the nomenclature of human ZP1 and ZP2, but not ZP3 (Barriere et al., 1998
). To date, relatively little is known about the three-dimensional structure of individual zona proteins, their formation into an extracellular zona matrix, and the potential effect of the supramolecular structure of the zona pellucida on sperm binding (Green, 1997
).
Genetically altered female mice that carry an insertional mutation in the Zp3 locus (Zp3tm/tm) have been developed and shown to express ZP1 and ZP2, but not ZP3 (Liu et al., 1996; Rankin et al., 1996
). These animals lack a zona matrix and are infertile. We have reversed this phenotype by crossing transgenic mice expressing human ZP3 into mouse Zp3 null mice (Rankin et al., 1998
). HuZP3 rescue females reconstitute a chimeric zona pellucida composed of mouse ZP1, mouse ZP2 and human ZP3. Human ZP3 expressed in mouse oocytes has an apparent mass (64 kDa) that is indistinguishable from that of native human ZP3 and distinct from mouse ZP3 (83 kDa). Nevertheless, despite the replacement of mouse ZP3 with human ZP3, these females are as fertile as their normal litter mates. To determine if huZP3 rescue mice provide a useful experimental model to investigate zona epitopes for efficacy in immunocontraception, we have assayed mutant and normal mice for sperm binding and fertility after administration of monoclonal antibodies specific to either mouse or human ZP3.
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Materials and methods |
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To determine the affect of mAb on fertilization, additional mAb was administered to create the same four groups described above. In these experiments, each animal also received 5 IU of pregnant mare serum gonadotrophin (PMSG) coincident with antibody injection and, 48 h later, 5 IU of human chorionic gonadotrophin (HCG) to induce ovulation. At the time of HCG administration, females were placed with fertile males (two females, one male) and five females from each group with vaginal plugs (indicative of coitus) were selected. At 20 h after administration of HCG, eggs/1-cell zygotes were recovered from their oviducts and fertilization was determined by the presence of two pronuclei. All experiments were conducted in compliance with the guidelines of the Animal Care and Use Committee of the National Institutes of Health under a Division of Intramural Research, NIDDK approved animal study protocol.
Immunohistochemistry
Isolated ovaries were fixed (3 h) in 3% glutaraldehyde, 0.1 M sodium cacodylate, pH 7.2 and transferred to 70% ethanol. After embedding in DB4-equivalent plastic (American Histolabs, Gaithersburg, MD, USA), 4 µm longitudinal sections were cut, preincubated (30 min) with 3% H2O2 and blocked (1 h) with 10% goat serum in phosphate-buffered saline (PBS). Sections from mice immunized with mAb H3.1 (specific to human ZP3) or mAb IE-10 (specific to mouse ZP3) were incubated (1:30, 1 h, 20°C) with a biotinylated anti-mouse or anti-rat IgG antibody (Vector Laboratories, Burlingame, CA, USA), respectively and visualized according to the manufacturer's instructions (Zymed Laboratories, San Francisco, CA, USA). Sections were counterstained (15 min) with Harris Modified Hematoxylin (Fisher Scientific, Fair Lawn, NJ, USA). In positive controls, sections from huZP3 rescue (injected with mAb specific to mouse ZP3) and normal females (injected with mAb to human ZP3) were blocked with goat 10% serum (1 h) and incubated (1:100, 1 h, 20°C) with mAb H3.1 and IE-10, respectively. In negative controls, the biotinylated antibody was omitted. Photographs were obtained with a Leitz Diaplan microscope (Bunton Instrument Company, Rockville, MD, USA) equipped with a didymium filter.
Detection of post-fertilization zona modification by Western blot
Mouse spermatozoa bind to eggs but not to 2-cell embryos, a physiological change that is correlative with proteolytic cleavage of ZP2. The molecular mass of mouse ZP2 from ovulated eggs is 120 kDa on SDSPAGE in the presence or absence of 50 mM ß-mercaptoethanol. Mouse ZP2 from 2-cell embryos is also 120 kDa in the absence of ß-mercaptoethanol, but in the presence of ß-mercaptoethanol it migrates as a 90 kDa band, plus a set of smaller fragments (2030 kDa). This suggests that the post-fertilization block to polyspermy results in cleavage of ZP2 into a 90 kDa subunit and additional smaller subunits linked by disulphide bond(s) (Moller and Wassarman, 1989). Western blot analysis was used to assay this post-fertilization modification of ZP2 using a monoclonal antibody (East and Dean, 1984
) to mouse ZP2 (713 amino acids) that recognizes a linear peptide epitope (amino acids 114129; J.Dean, unpublished observations) near the N-terminus.
Ovulated eggs were harvested from cumulus masses and 2-cell embryos were flushed from mouse oviducts using Whitten's medium with bovine serum albumin (BSA; 3 mg/ml) and 4-(2-aminoethyl)-benzenesulphonyl fluoride (AEBSF; 0.4 mM). Eggs or embryos were washed briefly with Whitten's medium lacking BSA/AEBSF and prepared with and without 50 mM ß-mercaptoethanol for 420% SDSPAGE (Laemmli, 1970), after which proteins were transferred to nitrocellulose membranes (Burnette, 1981
). Blots were incubated (1:1000, 1.5 h, 20°C) with ascites containing the primary antibody and binding was detected with horseradish peroxidase-conjugated goat anti-rat IgG (1:1000, 1.5 h, 20°C) and chemiluminescence (Amersham, Arlington Heights, IL, USA) using X-Omat AR film (Kodak, Rochester, NY, USA). Unlabelled proteins (BioRad, Hercules, CA, USA) were used as standards for molecular mass.
Sperm binding
Ovulated eggs from huZP3 rescue and normal virgin mice were obtained as described above. 2-cell embryos were recovered (40 h after HCG treatment) from the oviduct of superovulated normal mice mated with fertile males. Epididymal mouse spermatozoa were isolated from retired male breeders and capacitated by incubation (30 min, 37°C) with Eagle's minimum essential media (MEM) supplemented with 3% BSA. To assay sperm binding, unfertilized eggs (from huZP3 rescue and normal females) and normal 2-cell embryos were incubated with 1.5x106/ml of motile mouse spermatozoa in 30 µl of modified MEM under mineral oil in 5% CO2 for 30 min. The eggs were washed to remove non-adherent spermatozoa with a 0.009 inch (230 µm) pipette until the 2-cell, control embryos possessed 26 spermatozoa/embryo, then fixed for 2 h in 1% formaldehyde/2% polyvinylpyrrolidone (Sigma, St Louis, MO, USA) in PBS and mounted to quantify adherent spermatozoa by difference interference contrast microscopy (Bleil and Wassarman, 1980a). The average number of spermatozoa (range 26) per 2-cell embryo was subtracted from the average number of attached spermatozoa per egg. Each experiment was performed twice.
Early development
To determine the effect of mAb on early embryonic development, huZP3 rescue mice and normal (6-week-old) female mice were superovulated as described and caged with males proven to be fertile. After overnight mating, females were separated from the males and mAb to human ZP3 (H3.1) or to mouse ZP3 (IE-10) were administered at the 2-cell embryo stage (40 h after HCG). At 17 days after HCG, females were killed. The pregnancy rate and the number/weight of fetuses in utero was determined.
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Results |
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Antibody localization within the ovary
To confirm intra-ovarian localization of administered antibodies, ovaries were isolated from two huZP3 rescue females after treatment with mAb to human or mouse ZP3. Plastic-embedded sections were stained with a peroxidase-labelled secondary antibody to identify mAb within the ovary. In huZP3 rescue females treated with mAb to human ZP3, the antibody was detected throughout the zonae pellucidae surrounding growing oocytes and the remaining follicular architecture appeared normal (Figure 2A). No antibody was detected in the ovaries isolated from females treated with mAb to mouse ZP3 (Figure 2B
), unless the sections were first incubated with the mAb to human ZP3 as a control (Figure 2C
). Thus, the presence of the mAb to human ZP3 corresponded to the observed infertility in the huZP3 rescue females (Figure 1
). Furthermore, after giving birth to their first litter, huZP3 rescue females no longer had detectable antibodies in their zonae pellucidae (Figure 3A
) unless first incubated with the mAb to human ZP3 as controls (Figure 3B
). Therefore, the loss of the mAb from the ovary was correlative with the reversibility of the contraceptive effect.
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Effect of antibodies on sperm binding
To determine if the absence of live births was associated with inhibition of sperm binding, ovulated eggs were isolated from six huZP3 rescue females that had been injected with mAb either to human or mouse ZP3, but not mated. Capacitated, epididymal spermatozoa were incubated with eggs (4448) and control embryos in vitro for 30 min and washed until no more than 26 spermatozoa were bound to 2-cell embryos (Figure 4, insets). Although there was considerable variation in the number of bound spermatozoa (321/egg), on average fewer (Student's t-test, P <0.001) spermatozoa bound to the huZP3 rescue eggs obtained from females treated with mAb to human ZP3 (11.5 ± 0.7) than from females treated with mAb to mouse ZP3 (20.8 ± 1.6) (Figure 4A and B
). A corresponding effect was observed in normal female mice. There were fewer spermatozoa (7.5 ± 0.7 versus 20.1 ± 1.4, P <0.001) binding to eggs obtained from females treated with mAb to mouse ZP3 (Figure 4C
) than from those treated with mAb to human ZP3 (Figure 4D
).
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Discussion |
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After administration, the monoclonal antibodies to human ZP3 localize in the zonae of growing oocytes of huZP3 rescue mice. The contraceptive effect lasts for eight mouse oestrus cycles (on average), but whether the duration can be modulated by adjustment of the dose of antibody remains to be determined. All of the animals eventually regain fertility and, after the first delivery, have litter sizes that are indistinguishable from those of controls. This resumption of fertility is associated with the absence of antibody coating the zona matrix and presumably reflects the normal physiological turnover of germ cells during oogenesis. To wit, the smallest (primordial) oocytes within the ovary do not have a zona pellucida, an extracellular matrix synthesized only when oocytes enter a 2-week growth phase prior to meiotic maturation and ovulation. Thus, as the antibody is cleared from the circulation and new waves of oocytes begin to form zonae pellucidae, their zonae are not coated with antibody, and ovulated eggs are fertilized normally.
The duration of infertility varies (3588 days) among the huZP3 rescue females injected with the mAb to human ZP3. The kinetics of oogenesis and the half-life of the antibody most likely affect the period of infertility, but whether the observed variation is reproducible within a particular animal is not yet known. Additionally, the contraceptive effect is shorter in huZP3 rescue females treated with antibody to human ZP3 (32 days) than in normal mice treated with antibody to mouse ZP3 (60 days), even though the mAb are to homologous ZP3 epitopes. It seems unlikely that this reflects a reduced circulating half-life of the mouse mAb to human ZP3 compared with the rat mAb to mouse ZP3. More likely, the mAb to human ZP3 has a lower affinity for its target protein than the mAb to mouse ZP3, allowing more rapid clearance from the ovary. Alternatively, if the zona pellucida acts as a reservoir and antibody that dissociates from the matrix of one oocyte can bind to neighbouring zonae, then the thinner zona pellucida in huZP3 rescue females (Rankin et al., 1998) would accumulate a smaller reserve of antibodies with which to coat oocytes with newly synthesized zonae pellucidae. The more rapid depletion of the smaller reserve could account for shorter duration of the contraceptive effect.
Prior to antibody administration, genetically engineered huZP3 rescue female mice are as fertile as normal litter mates and, despite the presence of human ZP3, human spermatozoa do not bind to the `humanized' zona pellucida (Rankin et al., 1998). The binding of the human ZP3 antibody to huZP3 rescue eggs results in a decrease in the number of mouse spermatozoa which bind in vitro, but does not induce the biochemical changes of zona proteins known to prevent sperm binding after fertilization. This decrease in sperm binding suggests that at least some binding sites are unavailable. However, because only one spermatozoon is required for fertilization, it seems likely that steric hindrance is the major mechanism by which fertilization is prevented in vitro, although prevention of the acrosome reaction by the antibody could play a role. Normally, spermatozoonegg binding takes place in the ampulla of the oviduct where relatively few spermatozoa (<100) are available for interactions with ovulated eggs. Thus, it may be that in vivo, steric hindrance and inhibition of sperm binding act in concert to prevent fertilization.
Folliculogenesis and ovulation are not disrupted by the binding of antibody to the zona matrix. Follicles of all stages are present within the huZP3 rescue ovaries including corpora lutea (indicative of past ovulations) and the number of eggs obtained after hormonal stimulation is comparable with that in controls. It is unlikely that the initial small litter size is due to impaired early development or implantation, because neither fertility nor litter size is affected when antibody is administered at the 2-cell embryo stage. Rather, the small first litter size may reflect ovulation of a mixed pool of eggs (some coated with more antibodies than others) that is not present in subsequent matings and which results in litters of normal size. Normal and huZP3 rescue females treated with either antibody mate normally, and pups born after the contraceptive effect has dissipated are indistinguishable from controls.
These data extend earlier studies which demonstrated that monoclonal antibodies to five different epitopes on mouse ZP2 and ZP3 can inhibit fertilization (East et al., 1984, 1985
). As in these previous studies, no ovarian histopathology is noted after administration of monoclonal antibodies to the zona pellucida in the huZP3 rescue mice. The current results suggest that a systematic analysis for contraceptive efficacy of overlapping epitopes on the human zona proteins could be undertaken in a rodent model. However, the `humanized' zonae in mice may differ structurally from native human zonae, and immunological responses to human zona epitopes may vary in the two species. Thus, while the rodent model may be useful for screening epitopes, successful candidates will need considerably more evaluation including testing in primatesan experimental system that more closely approximates human biology. It is important to emphasize that vaccination with zona pellucida-derived peptides can lead to significant ovarian histopathology (Tung et al., 1997
), and can cause a decrease in the pool of primordial oocytes (Paterson et al., 1996
). Thus, the identification of candidate human zona pellucida peptides must be considered a first step in the development of a contraceptive vaccine. Considerable research will be required to ensure that candidate peptides do not cause adverse immune responses within the ovary or elsewhere in vaccinated animals. Whether such safeguards can be assured remains an open question.
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Acknowledgments |
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Notes |
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References |
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Submitted on September 25, 1998; accepted on December 1, 1998.