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Journal of Reproduction and Fertility (2000) 120, 19–32

Effectiveness of zona pellucida protein ZPB as an immunocontraceptive antigen

M. L. Martinez and J. D. Harris*

Zonagen, Inc., 2408 Timberloch Place, B4, The Woodlands, TX 77380, USA

Immunization of female mammals with native zona pellucida (ZP) proteins is known to cause infertility. Since each human ZP protein is now available as a puriﬁed recombinant protein, is it possible to compare the immunocontraceptive potential of each ZP protein. A breeding study was conducted in cynomolgus monkeys (Macaca fasicularis) after immunization with recombinant human ZP (rhZP) proteins (ZPA, ZPB, ZPC) separately and in combinations. This study demonstrated that immunization with recombinant human ZPB (rhZPB) protein caused cynomolgus monkeys to become infertile for 9–35 months. A second study was conducted in baboons (Papio cynocephalus), which yielded a similar result. The baboons immunized with rhZPB became infertile for 9 to > 20 months. During the time of maximum antibody titre, some animals experienced disruption of the menstrual cycle, but eventually all of the animals resumed normal menstrual cycles. Control animals and animals immunized with other rhZP proteins all became pregnant before any of the rhZPB-treated animals. This is the ﬁrst study in which a recombinant ZP protein has consistently induced infertility in a primate without permanent disruption of the normal menstrual cycle.

Introduction Table 1. Examples of zona pellucida nomenclature

The use of the zona pellucida (ZP) proteins as immuno- Other mammalsa Mice Rabbits Pigsb Humans contraceptive antigens has a long and diverse history. The use of crude extracts of oocytes from pig ovaries has been ZPA ZP2 rec75 ZP1, ZP2, ZP4 ZP2 ongoing for the past two decades (Mahi-Brown et al., 1982; ZPB ZP1 rec55 ZP3α ZPB Kirkpatrick et al., 1996). ZPC ZP3 ZPC ZP3β ZP3 Purified recombinant proteins are available for use as vaccine antigens (Harris et al., 1999). There are three classes aIncludes clones of the following species: cattle, cat, dog, cynomolgus monkey (Harris et al., 1994). of ZP proteins (Harris et al., 1994): ZPA (also called ZP1, ZP2 b α These refer to proteins separated by gel electrophoresis (Sacco, et al. 1989 and or ZP4), ZPB (also called ZP1 or ZP3 ) and ZPC (also called Hedrick and Wardrip, 1987); the other columns refer to clones. The individual ZP3 or ZP3β). The variety of nomenclature is presented clones were assigned ZPA, ZPB and ZPC (Harris et al., 1994). (Table 1). This multiplicity exists because the first Additional references: mice: ZP2, Bleil et al., 1988; ZP1, Epifano et al., 1995a; identification of zona pellucida proteins was made using ZP3, Ringuette et al., 1988; rabbits: rec75, Lee et al., 1993; rec55, Schwoebel et al., 1991; ZPC, Harris et al., 1994; humans: ZP2, Liang and Dean, 1993; ZPB, Harris PAGE: the largest proteins were assigned ZP1, next largest, et al., 1994; ZP3, Chamberlin and Dean, 1990. ZP2, and so on. However, due to post-translational modification, the species to species correlation is not exact. Thus, pig ZP1 and ZP2 are derived from the same gene and are related to mouse ZP2, but not to mouse ZP1. been discouraging, the zona pellucida offers a variety of Furthermore, a human homologue to mouse ZP1 has been antigens that could prove to be more effective targets. identified (Hughes and Barratt, 1999), which implies that the VandeVoort et al. (1995) vaccinated cynomolgus monkeys with mouse ZP1 is not homologous to the other mammalian ZPB bacterially expressed proteins derived from two partial genes. In this paper the ZPA, ZPB and ZPC nomenclature complementary DNA rabbit ZPA clones or a full-length rabbit will be used. ZPB clone. Since no breeding data were published with this Paterson et al. (1996) used recombinant human ZPC (rhZPC) study, it was not possible to predict whether ZPAor ZPB would protein to immunize marmoset monkeys, but infertility was be effective immunocontraceptive agents. only achieved in animals in which the ovarian cycle was Kerr, et al. (1999) reported that alloimmunization of rabbits completely disrupted. Although the results using rhZPC have with recombinant rabbit ZPB induced infertility in most of the treated animals. However, the rabbit ZPA and ZPC *Correspondence. proteins were not tested and thus could not be compared Received 9 November 1999. with ZPB.

Because a breeding study compares fertility versus study, but as she became pregnant, the illness did not appear infertility directly, the best way to measure the effect of an to impair her reproductive capabilities. immunocontraceptive vaccine on fertility is to conduct breeding trials in a well-controlled environment. No breeding study has ever compared the immunocontraceptive Vaccine preparation potential of all three proteins. A breeding study was performed of cynomolgus monkeys The recombinant glycoproteins, which were used as vaccinated with the three rhZP proteins, alone and in various antigens, were expressed in Chinese hamster ovary (CHO) combinations. These results showed that rhZPB can be used cells. These glycoproteins were prepared and characterized alone as an immunocontraceptive antigen in cynomolgus as described by Harris et al. (1999), and PAGE analysis of the monkeys. A breeding study was also conducted using purified proteins is shown (Fig. 1). Vaccine consisted of baboons that had been vaccinated with individual rhZP 500 µg heat solubilized pig zona pellucida (HSPZ) or 250 µg proteins and did not include any antigen combinations to purified protein (250 µg of each protein was used when a test whether the result could be repeated in a second primate combination was given) formulated to a maximum volume species. The vaccination regimen was reduced since it had of 1 ml with adjuvant. A variety of adjuvants was used in the become obvious that extremely high antibody titres against cynomolgus monkey study because the aim was to compare the ZPB were not necessary to achieve infertility. complete Freund’s adjuvant with two adjuvants developed at Zonagen, ImmuMax and ImmuMaxSR. The CFA was modified as described by Paterson et al. (1992) and was used as the adjuvant for 16 cynomolgus monkeys: the four Materials and Methods controls, the four monkeys immunized with HSPZ, four of the monkeys that received rhZPB and four of the monkeys Animals that received rhZPC. For the modified complete Freund’s All animals were maintained in accordance with the adjuvant vaccine, each 1 ml final vaccine contained 80 µl National Institutes of Health guidelines for the care and use Bacillus Calmette Guerin vaccine, 125 µl aluminium of laboratory animals. All the cynomolgus monkey (Macaca hydroxide gel, 500 µl non-ulcerative Freund’s adjuvant and a fasicularis) females were nulliparous and 3–4 years of age total of 355 µl of antigen plus PBS. Boosters for these animals when the study was commenced. All the females were were formulated in incomplete Freund’s adjuvant. domestic, born in the Philippines and were routinely tested ImmuMax SR adjuvant (Zonagen, The Woodlands, TX), a negative for tuberculosis, filovirus, herpes B virus, simian chitosan-based oil emulsion formulation, was used with two retrovirus, simian immunodeficiency virus and herpes of the cynomolgus monkeys that were immunized with simplex I virus. Each animal received a complete physical rhZPB vaccine and with two of the monkeys that were examination by a veterinarian and was determined to be in immunized with rhZPC vaccine. ImmuMax adjuvant good health before she was accepted for study. Monkeys (Zonagen, The Woodlands, TX), a chitosan–zinc chelating were housed in outdoor cribs in south Texas. Males were introduced into stable female groups, consisting of four to seven females each belonging to a different vaccination ZPA ZPB ZPC ZPA ZPB ZPC group, to normalize any possible male effect on fertility. They kDa remained in continuous co-habitation throughout the study. 250 The numbers of pregnancies were comparable between cribs, indicating that there was no significant male effect on fertility rates. Males cannot be rotated between cribs, as this would disrupt the necessarily stable social groups that are 98 established under these breeding conditions. 64 For the baboons (Papio cynocephalus), the four vaccination groups of females were assigned so that the groups were 50 comparable for weight and age. Baboons were housed in groups at a facility in San Antonio, Texas. When assigned to 36 the two different breeding groups, the baboons ranged in age 30 from 7.7 to 15.3 years. The 20 animals in the study were placed 16 into two breeding groups with males with good breeding 6 records so that the four different vaccination groups were 4 represented comparably in both groups. All of the females had Coomassie stain Silver stain previously given birth and the number of offspring ranged Fig. 1. An SDS-PAGE gel of recombinant human zona pellucida from one to nine (correlated with the age of the animal). All (ZP) proteins rhZPA, rhZPB and rhZPC stained with either animals received a complete physical examination by a Coomassie G-250 (BioRad, Hercules, CA) or silver stain (Novex, San veterinarian and were determined to be in good health before Diego, CA). In the Coomassie stained gel each lane has 2 µg, whereas they were accepted for study. One of the ZPB-vaccinated in the silver stained gel each lane has 0.5 µg. The markers are animals, number 4587, contracted histoplasmosis and Seablue molecular mass markers (Novex, San Diego, CA). required hospitalization, treatment and surgery during the Reproduced from Harris et al., 1999.

Downloaded from Bioscientifica.com at 09/26/2021 02:22:48PM via free access Effectiveness of ZPB as an immunocontraceptive antigen 21 formulation designed for use with histidine-6 tagged females was observed each day to monitor for sexual recombinant proteins, was used with the remainder of the receptivity and pregnancy. Animals were examined by vaccines for the cynomolgus monkeys and all of the baboons. ultrasonography to conﬁrm pregnancy approximately 1 month after their sex skin colour indicated pregnancy.

Immunization and sampling protocols for the cynomolgus monkeys ELISA for zona pellucida antibodies Thirty-six young adult female cynomolgus monkeys were Serum titres of antibodies against the immunizing antigen immunized with either complete or incomplete Freund’s were measured using an ELISA. Flat-bottom polyvinyl adjuvant alone (negative controls) or with HSPZ, rhZPA, chloride 96-well assay plates (Falcon No. 3912) were coated rhZPA + rhZPB, rhZPB, rhZPB + rhZPC, or rhZPC. The (50 µl per well) with an optimal concentration of antigen immunization protocol is summarized (Table 2). The (usually 1 µgml–1) in 0.2 mol carbonate–bicarbonate l–1 schedule of immunizations for the cynomolgus monkeys is buffer, pH 9.6. After overnight incubation at 4ЊC, the plates shown (Fig. 2). The rhZPA immunizations (Fig. 2a) were were blocked with 2% (w/v) non-fat dry milk (NFDM) in started 6 months after the other immunizations (Fig. 2b) carbonate–bicarbonate buffer for 2 h at 37ЊC to inhibit non- were started. This procedure was necessary because of an specific binding. Blocked plates were emptied and washed unanticipated delay in producing sufficient rhZPA antigen to four times with 0.15 mol PBS l–1 containing 0.05% (v/v) begin all immunizations simultaneously. All animals Tween 20 (PBS-T). Plates were stored with 100 µl PBS-T received three immunizations before males were introduced. in each well at –20ЊC, thawed and emptied completely Subsequent vaccinations were administered to the females before use. Fifty microlitres of sample or control sera diluted while they were co-habitating with the males. Blood samples optimally (based on preliminary titration) in PBS-T were taken twice a week from females from the femoral vein containing 2% (w/v) NFDM was added to each well and to obtain serum. After introduction of the males, females diluted serially across the plate. The plates were covered and were manually palpated for pregnancy once a week. incubated for 2 h at 37ЊC, emptied and washed four times with PBS-T. Biotin-labelled goat anti-human IgG (H + L) antibody (Zymed, S. San Francisco, CA), 50 µl of a 1:1000 dilution in PBS-T, was added to each well. Plates were Immunization and sampling protocols for the baboons covered and incubated for 1 h at 37ЊC. The plates were Four groups each of five baboons were immunized either emptied, washed four times with PBS-T, and 50 µl of a with ImmuMax adjuvant only (control) or with rhZPA, 1:1000 dilution of horseradish peroxidase conjugated to rhZPB or rhZPC. Each immunization consisted of 250 µg streptavidin (Zymed, S. San Francisco, CA) in PBS-T was antigen combined with ImmuMax adjuvant to a maximum added to each well. After incubation at 37oC for 1 h, the volume of 1 ml. Three immunizations were spaced 3–4 plates were emptied and washed four times with PBS-T, and weeks apart. Males were introduced to the groups of female 50 µl of a 400 µg O-phenylenediamine ml–1 substrate solution baboons 7 weeks after the third immunization. Blood was added. Plates were developed in the dark at room samples were taken every 2 weeks. The sex skin of the temperature for 10 min and the absorbance at 450 nm was read on an Emax reader. End-point titres were determined as the reciprocal dilution the absorbance of which exceeded (a) twice the background (that is the absorbance obtained using 12 3 4 sera from adjuvant control monkeys).

(b) 1 2 34 56 7 Progesterone assay

Selected Selected Serum progesterone concentrations were determined animals animals for every serum sample with the use of the Coat-a-Count® non-extraction solid-phase radioimmunoassay (DPC, Los 0 4 8 12 16 20 24 28 32 36 40 44 48 Angeles, CA). Time after primary immunization (weeks) Fig. 2. Schematic diagram of immunization regimens for the cynomolgus monkeys immunized with recombinant human zona pellucida (rhZP) proteins A, B and C. Numbers above the lines are Results vaccination numbers; 1 indicates the primary vaccination and the other numbers indicate boosters. Arrowheads indicate introduction Cynomolgus monkey studies of males; arrows indicate continued vaccinations of selected animals (see text). (a) Timeline for immunization of monkeys vaccinated with This study was designed to compare different antigens rhZPA or with rhZPA + rhZPB (started February 1997). (b) Timeline combined with different adjuvants. During the ﬁrst 6 months for immunization of control monkeys and monkeys vaccinated with of the study before the rhZPA was ready for use, it became heat solubilized pig zona pellucida (HSPZ), rhZPB, rhZPC or rhZPB apparent that the different adjuvants induced similar + rhZPC (started August 1996). antibody titres (see below). Thus, when the rhZPAwas ready,

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Table 2. Immunization of cynomolgus monkeys with recombinant human zona pellucida proteins

Cycle in which Number of Months with animal became Total number of Vaccine antigen injections Animal number Adjuvant male pregnant infertile cyclesa

None 5 CQ8118 CFA/IFA 1 1 – 5 CQ8120 CFA/IFA 9 3 – 5 CQ8121 CFA/IFA 1 1 – 5 CQ8151 CFA/IFA 17 12 –

HSPZ 5 CQ8116 CFA/IFA 4 4 – 7 CQ8117 CFA/IFA 10 6 – 7 CQ8119 CFA/IFA 13 8 – 5 CQ8140 CFA/IFA 6 6 – rhZPA 4 CQ8125 ImmuMax < 1 Died before 1b – 4 CQ8133 ImmuMax 1 1 – 4 CQ8137 ImmuMax 5 3 – 4 CQ8143 ImmuMax < 1 Died before 1b – rhZPA+ rhZPB 4 CQ8128 ImmuMax 12 Died after 1c – 4 CQ8146 ImmuMax 17 8 – 4 CQ8156 ImmuMax 10 4 – 4 5027 ImmuMax 9 5 – rhZPB 5 CQ8122 CFA/IFA 19 Died after 5d – 5 CQ8126 CFA/IFA 35 – 18 6 CQ8131 CFA/IFA 35 – 19 5 CQ8152 CFA/IFA 35 – 14 6 CQ8130 ImmuMax SR 7 Died after 4d – 5 CQ8155 ImmuMax SR 35 – 15 5 CQ8135 ImmuMax 35 – 27 6 CQ8163 ImmuMax 18 Died after 2e – rhZPB + rhZPC 7 CQ8129 ImmuMax 14 8 – 7 CQ8132 ImmuMax 17 Died after 4f – 6 CQ8139 ImmuMax 14 7 – 6 CQ8161 ImmuMax 24 10 – rhZPC 5 CQ8124 CFA/IFA 3 3 – 6 CQ8127 CFA/IFA 13 8 – 6 CQ8138 CFA/IFA 7 6 – 5 CQ8153 CFA/IFA 4 4 – 7 CQ8134g ImmuMax SR 19 7 – 5 CQ8164 ImmuMax SR 2 2 – 5 CQ8136 ImmuMax 4 4 – 5 CQ8165 ImmuMax 4 4 – aAn infertile cycle is defined as a cycle during which the animal did not get pregnant, and the infertile cycles given for non-pregnant animals are not necessarily consecutive. bDied of enteritis before 3 months. cDied of retroperitoneal fibromatosis in month 12. dDied of trauma: one in month 7 and one in month 20. eDied of peritonitis in month 19. fBecame pregnant, aborted after 3 months, and died of enteritis 1 week later. gReceived a single rhZPB/ImmuMaxSR immunization for first booster. The mortality rate is within the normal limits for a population of this size maintained under these conditions for over 2 years. CFA: complete Freund’s adjuvant; HSPZ: heat-solubilized pig zona pellucida; IFA: incomplete Freund’s adjuvant; rhZP: recombinant human zona pellucida (A, B and C indicate individual proteins). only ImmuMax was used with this antigen since it is the of the animals died of causes unrelated to the experimental easiest to prepare and administer, and it produces virtually procedure. According to the Director of the animal facility, no site reaction. this mortality rate was within the normal limits for a A summary of the results from the cynomolgus monkeys population of this size maintained under these conditions for is shown (Table 2). During the first 2 years of the study, seven over 2 years. The monkeys receiving rhZPA were each

Downloaded from Bioscientifica.com at 09/26/2021 02:22:48PM via free access Effectiveness of ZPB as an immunocontraceptive antigen 23 injected four times. All of the other monkeys were each months are months 1–4 (and months 13–16). For all of the injected at least ﬁve times. Some animals were injected six or other monkeys the summer months are months 6–9 (and seven times in an effort to keep serum antibody titres high. months 18–21). In the control animals and the animals By the time the rhZPA monkeys were added to the study, the receiving HSPZ, rhZPA or rhZPC alone, some menstrual extra injections were deemed unnecessary. cycles were missed, but these corresponded to the summer The cyclic patterns of the surviving cynomolgus monkeys months in every case. However, all of the monkeys that during 17 months of co-habitation are summarized (Table 3). received rhZPB as the vaccine antigen, either alone or in Monkeys that became pregnant were removed from the combination with rhZPA or rhZPC, experienced temporary study. Two of the control monkeys, two monkeys immunized disruptions of their menstrual cycles, as determined by the with HSPZ and one of the monkeys that received rhZPA pattern of serum progesterone. The disruption of the experienced some disruption of the menstrual cycle, which menstrual cycle varied from 2 to 16 months (average 8 may be due to heat stress that occurs during summer in months), and every animal eventually resumed a normal southern Texas. For the rhZPA-treated monkeys the summer menstrual cycle.

Table 3. Pattern of menstrual cycles of cynomolgus monkeys immunized with recombinant human zona pellucida proteins

Months with male Vaccine Animal antigen number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Adjuvant control CQ8118 Preg CQ8120 + – + – – – – – Preg CQ8121 Preg CQ8151 – + + + + + – – + + + – + – + + Preg

HSPZ CQ8116 + + + Preg CQ8117 + + + + + – – – – Preg CQ8119 + + + + – – – – – + + + Preg C18140 + + + + + Preg rhZPA CQ8133 Preg CQ8137 – + – + Preg rhZPA+ rhZPB CQ8146 – + – – – – + + + – + + – – + – Preg CQ8156 – – – – – + + + – Preg 5027 – – – – + + + + Preg rhZPB CQ8126 + + + – – + –+–++–– ++–– CQ8131 + + + – – – ––––––– ––– – CQ8152 + – + – – – ––––––– ––– – CQ8155 + – + – – – ––––––– ––– – CQ8135 – + + – – + – – + + + – + + + – – rhZPB + rhZPC CQ8129 + + + – – + – + – – – + + Preg CQ8139 + + + – + – ––––+–+Preg CQ8161a + + + – –+ – – – –+–+ ++– – CQ8134b + + + – +–––––––– –+–– rhZPC CQ8124 + + Preg CQ8127 + + + – + + – – – + – + Preg CQ8138 + – + + + + Preg CQ8153 + + + Preg CQ8164 + Preg CQ8136 + + + Preg CQ8165 + + + Preg aAnimal CQ8161 experienced a menstrual cycle in months 21 and 24 and became pregnant in month 24. bAnimal CQ8134 received only one injection of rhZPB. This animal experienced a menstural cycle in months 18 and 19 and became pregnant in month 19. +: indicates the animal experienced a menstrual cycle sometime during the month; –: indicates there was no menstrual cycle during that month; Preg: indicates the animal became pregnant during that month. Since individual monkeys can vary from 4–6 weeks in the duration of a normal menstrual cycle, a normal menstrual cycle is not necessarily one cycle per month, but could be nine or ten cycles per year (12 months). It is not uncommon for normal untreated animals to experience periods of 1–3 months without a menstrual cycle. HSPZ: heat-solubilized pig zona pellucida; IFA: incomplete Freund’s adjuvant; rhZP: recombinant human zona pellucida (A, B and C indicate individual proteins).

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Table 4. Antibody titres in cynomolgus monkeys immunized with recombinant human zona pellucida proteins

Anti-HSPZ Anti-rhZPA Anti-rhZPB Anti-rhZPC

Vaccine Animal At At At At antigen number Maximum pregnancy Maximum pregnancy Maximum pregnancy Maximum pregnancy

HSPZ CQ8116 1:256 1:16 CQ8117 1:512 1:6.4 CQ8119 1:512 1:0.2 C18140 1:512 1:8 rhZPA CQ8133 1:512 1:4 CQ8137 1:512 1:1.6 rhZPA+ rhZPB CQ8146 1:1000 1:0.2 1:1000 1:0.2 CQ8156 1:64 1:0.2 1:256 1:0.2 5027 1:256 1:3.2 1:512 1:0.8 rhZPB + rhZPC CQ8129 1:64 1:0.2 1:64 1:0.2 CQ8139 1:1000 1:1.6 1:256 1:0.2 CQ8161 1:64 1:0.2 1:32 1:0.2 CQ8134 1:32 1:0.2 1:128 1:0.2 rhZPC CQ8124 1:256 1:2 CQ8127 1:256 1:0.4 CQ8138 1:256 1:128 CQ8153 1:128 1:16 CQ8164 1:256 1:256 CQ8136 1:128 1:32 CQ8165 1:32 1:8

Titres are ϫ 1000. HSPZ: heat-solubilized pig zona pellucida; rhZP: recombinant human zona pellucida (A, B and C indicate individual proteins).

signiﬁcant antibody titres to the antigen (Fig. 4), but some 600 animals did not respond to every boost. This is particularly true of the fourth injection with rhZPA (Fig. 4a). The level of 500 CQ8116 the antibody titre in the case of rhZPC seems to have no effect CQ8117 400 on fertility (Fig. 4b and Table 4). 1000) CQ8119

× Combining rhZPB with rhZPA (Fig. 5) or rhZPB with CQ8140 300 rhZPC (Fig. 6) produced a marked effect. Pregnancy in these two groups was delayed relative to rhZPA or rhZPC alone by 200 at least 4–5 months, but every animal in the combination groups eventually became pregnant once the antibody titres ELISA titres ( 100 had dropped to very low levels (Table 4). Although animal CQ8134 was initially assigned to the group scheduled to 0 receive rhZPC alone, she erroneously received a single injection of rhZPB + ImmuMax SR® as the second immunization in September 1996. She mounted a strong 02/09/9602/10/9602/11/9602/12/9602/01/9702/02/9702/03/9702/04/9702/05/9702/06/9702/07/9702/08/9702/09/9702/10/9702/11/9702/12/9702/01/98 immune response to rhZPB (Fig. 4d) and became acyclic for 8 Fig. 3. Antibody titres for heat solubilized pig zona pellucida months (Table 3). This animal resumed a normal menstrual (HSPZ), which were measured once a week. Asterisks indicate cycle in February 1998 and became pregnant in her seventh immunizations. The males were introduced in November 1996 cycle, 19 months after the introduction of the male. She (indicated by arrow). remained infertile 6 months longer than any other rhZPC- vaccinated animal, which was 12 months longer than the average for this group. The pattern of infertility observed in The ranges of the antibody responses to the antigens were CQ8134 was similar to that seen in animals that received similar (Table 4; Figs 3–6). The titres to HSPZ tended to rise rhZPB + rhZPC, and for the purpose of analysis, she has been and fall more quickly than the titres to other antigens (Fig. 3). added to this group. Three of these animals became pregnant even when the After almost 3 years of continuous co-habitation with antibody titres to the antigen were high (Table 4). males, none of the animals immunized with rhZPB alone All of the animals receiving rhZPA or rhZPC generated (Fig. 7) ever became pregnant despite their lack of signiﬁcant

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(a) 600

500 CQ8133

1000) 400

× CQ8137

300

200

ELISA titres ( 100

17/03/9731/03/9714/04/9728/04/9712/05/9726/05/9709/06/9723/06/9707/07/9721/07/9704/08/9718/08/9701/09/9715/09/9729/09/9713/10/9717/11/9715/12/97

(b) 300

250 CQ8124 CQ8127 CQ8138 200 CQ8153 1000)

× CQ8164 150 CQ8136 CQ8165

100 ELISA titres (

02/09/9616/09/9630/09/9614/10/9628/10/9611/11/9622/11/9629/11/9606/12/9613/12/9630/12/9613/01/9727/01/9710/02/9724/02/9710/03/9724/03/9707/04/9721/04/9705/05/9719/05/9702/06/9716/06/9730/06/9714/07/97

Fig. 4. Antibody titres for (a) recombinant human zona pellucida protein A (rhZPA) and (b) rhZPC. The antibody titres were measured once a week. Immunizations are indicated by ᭹. The males were introduced (indicated by arrow) in June 1997 (a) and in November 1996 (b).

antibody titres for 12–15 months. This finding indicates that sustained the highest antibody titre to rhZPB, had high serum antibody titres do not correlate with infertility measurable titres for the greatest length of time, and and that hyperimmunizing to sustain high antibody titres is experienced the longest disruption to the menstrual cycle. not necessary to maintain infertility. However, the duration Monkeys CQ8126 and CQ8131 have profiles similar to that of of high anti-ZPB antibody titres does correlate generally with CQ8155 (Fig. 7d and 7e). Although all rhZPB-immunized the duration of disruption of the menstrual cycle. In all animals have returned to a normal menstrual cycle, none monkeys receiving rhZPB, peak titres у 1:500 000 were became pregnant. achieved, but the duration of the high antibody titre varied At the end of the first year of the study it was obvious that among the animals in this group (Fig. 7). Monkey CQ8135 immunization with rhZPB induced infertility whereas (Fig. 7a) had high antibody titres for the shortest period, immunization with rhZPA or rhZPC did not. Most of the experienced the shortest interruption of the menstrual cycle, control animals, the HSPZ animals and the animals treated resumed a normal menstrual cycle in the presence of the with rhZPA or rhZPC alone animals were already pregnant antibody and remained infertile throughout the study. (Table 3). Only two of the animals (CQ8156 and 5027) that Monkey CQ8155 (Fig. 7b) sustained measurable antibody had received rhZPB, alone or in any combination had titres for a longer period and took longer to resume a normal become pregnant. rhZPB appeared to be the best antigen menstrual cycle than CQ8135. Monkey CQ8152 (Fig. 7c) candidate.

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1200 (a)

1000

800 1000) ×

600

400 ELISA titres (

200

17/03/9724/03/9731/03/9707/04/9714/04/9721/04/9728/04/9705/05/9712/05/9719/05/9726/05/9702/06/9709/06/9716/06/9723/06/9730/06/9707/07/9714/07/9721/07/9728/07/9704/08/9711/08/9718/08/9725/08/9701/09/9708/09/9715/09/9722/09/9729/09/9706/10/9713/10/9720/10/9727/10/9703/11/9710/11/9717/11/97

300 (b)

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200 1000)

× 150

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ELISA titres ( 50

17/03/9731/03/9714/04/9728/04/9712/05/9726/05/9709/06/9723/06/9707/07/9721/07/9704/08/9718/08/97 01/09/9715/09/97 29/09/9713/10/9727/10/9710/11/97

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Fig. 5. Antibody titres for recombinant human zona pellucida proteins rhZPA ( ) and rhZPB (- - - -). The antibody titres were measured once a week for animals (a) CQ8146, (b) CQ8156 and (c) 5027. Immunizations are indicated by ᭹. The males were introduced (indicated by arrow) in June 1997.

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(b) (a) 1200 70

60 1000 50 1000) 800 1000) × × 40 600 30 400 20 ELISA titres ( ELISA titres ( 10 200

0 0

02/09/9623/09/9614/10/9604/11/9625/11/9613/12/9606/01/9727/01/9717/02/9710/03/9731/03/9721/04/9712/05/9702/06/9723/06/9714/07/9704/08/9725/08/9715/09/9706/10/9717/11/9712/01/98 02/09/9630/09/9628/10/9622/11/9606/12/9630/12/9627/01/9724/02/9724/03/9721/04/9719/05/9716/06/9714/07/9711/08/9708/09/9706/10/9708/12/97

02/09/9616/09/9630/09/9614/10/9628/10/9611/11/9625/11/9609/12/9623/12/9606/01/9720/01/9703/02/9717/02/9703/03/9717/03/9731/03/9714/04/9728/04/9712/05/9726/05/9709/06/9723/06/9707/07/9721/07/9704/08/9718/08/9701/09/9715/09/9729/09/9713/10/9727/10/9710/11/97 02/09/9630/09/9628/10/9622/11/9606/12/9630/12/9627/01/9724/02/9724/03/9721/04/9719/05/9716/06/9714/07/9711/08/9708/09/9706/10/9708/12/97

Fig. 6. Antibody titres for recombinant human zona pellucida proteins rhZPB (——) and rhZPC (-----). The antibody titres were measured once a week for animals (a) CQ8129, (b) CQ8139, (c) CQ8161 and (d) CQ8134. Immunizations are indicated by ᭹. The males were introduced (indicated by arrow) in November 1996. In (d), ᭺ indicates that only the rhZPB immunization was given.

cynomolgus monkeys (Table 6). As before, the antibody titres Baboon study against rhZPA and rhZPB tended to be higher than the Another breeding study was commenced using baboons antibody titres against rhZPC. The baboons that were to test the hypothesis that rhZPB was the best antigen immunized with rhZPA became pregnant when the titres fell candidate. For the baboon study, a single adjuvant was below 1:8000, and the baboons that were immunized with chosen since there appeared to be no difference in antibody rhZPC became pregnant when the titres fell below 1:1000. titres induced in the cynomolgus monkeys by the different The antibody titres in the rhZPB-immunized baboons adjuvants. Complete Freund’s adjuvant, ImmuMaxSR or returned to baseline levels at the same rate as the antibody ImmuMax produced similar titres, and the ImmuMax titres in the rhZPA- and rhZPC-immunized baboons, but produced the smallest injection site reaction. No antigen there was a delay of at least 4 months before the first of the combinations were used since rhZPB alone worked well in five baboons immunized with rhZPB became pregnant. the cynomolgus monkeys. In both studies, despite some disruption of the menstrual The results of the baboon study correlate well with the cycle of some of the animals vaccinated with the rhZPB results obtained with cynomolgus monkeys (Table 5). antigen, all animals returned to a normal menstrual cycle. Immunization with rhZPB alone resulted in prolonged The duration of disruption of the menstrual cycles in infertility, whereas immunization with rhZPA or rhZPC had the cynomolgus monkeys and the baboons was correlated no effect on fertility. All of the control animals and all of the with the maximal antibody titre achieved (Table 7) and animals immunized with rhZPA or rhZPC were pregnant persistence of measurable antibody titre: the higher and within 5 months after the males were introduced. more sustained the titre, the greater the disruption. For both The controls and the rhZPA- and rhZPC-immunized species, the antibody titres eventually decreased to < 1:2000. baboons experienced no significant disruption of their The titres dropped to this level within 7 months after the normal monthly menstrual cycles throughout this seventh booster in the cynomolgus monkeys and within 5 experiment. The antibody titres induced by all three antigens months after the third booster in the baboons. in the baboons were similar to those produced in the In the baboon study, the animals with the highest titres did

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Table 5. Pattern of menstrual cycles of baboons immunized with recombinant human zona pellucida proteins

Months with male Vaccine Animal antigen number 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Adjuvant control 4598 + Preg 6684 + Preg 6758 + + + Preg 8412 + Preg 8785 – + + + Preg rhZPA 4586 + + Preg 6740 + Preg 8487 + + Preg 8865 + – + – Preg 9714 + Preg rhZPB 4587 – – + + + – + + Preg 4603 – – – – – – + + + Preg 6729 – + + + + + + + – + + – + + 8816 – – – – + – – – + + + + + Preg 8911––– – +––– – – – –++ rhZPC 4324 + + Preg 6838 + – – + Preg 8114 + + + Preg 8244 + Preg 8569 – + + + Preg

+: indicates the animal experienced a menstrual cycle sometime during the month; –: indicates there was no menstrual cycle during that month; Preg: indicates the animal became pregnant during that month. rhZP: recombinant human zona pellucida (A, B and C indicate individual proteins).

Table 6. Antibody titres in baboons immunized with recombinant human zona pellucida proteins

Anti-rhZPA Anti-rhZPB Anti-rhZPC

Datea 4586 6740 8487 8865 9714 4587 4603 6729 8816 8911 4324 6838 8114 8244 8569

03/11/97 0.8 0.8 0.8 0.2 0.2 1.6 3.2 6.4 6.4 3.2 0.1 0.1 0.1 0.1 0.1 17/11/97 0.8 0.4 0.4 0.4 1.6 0.4 3.2 3.2 3.2 3.2 0.1 0.1 0.1 0.1 0.8 01/12/97 128 128 64 32 128 64 256 64 256 512 8 256 128 128 64 15/12/97 256 128 256 32 256 32 128 64 128 128 16 128 16 64 64 29/12/97 1024 512 1024 256 512 128 256 64 256 1024 32 64 512 256 256 12/01/98 256 32 512 32 128 16 64 16 32 256 8 16 16 8 16 09/02/98b 16 4 16 4 8 2 16 4 8 16 2 2 2 2 2 09/03/98 16 8 8 4 8 0.5 8 0.5 2 8 0.5 0.5 0.5 0.5 0.5 08/04/98 8 Preg 4 2 Preg 0.5 2 0.5 1 8 0.5 0.5 0.5 Preg 0.5 04/05/98 Preg Preg 2 1 4 2 2 8 Preg 2 0.5 2 29/06/98 1 0.25 2 0.5 0.5 4 1 Preg 0.5 27/07/98 Preg 0.25 1 0.25 1 2 Preg Preg 11/98 Preg 12/98 Preg 04/99 Preg aInjections administered 20/10/97, 17/11/97, 15/12/97. bMales introduced 16/2/98. Titres are ϫ 1000. rhZP: recombinant human zona pellucida (A, B and C indicate individual proteins). not resume a normal menstrual cycle immediately (Fig. 8). 8816, which achieved a higher titre, missed three or four Cessation of the menstrual cycle was not observed in baboon menstrual cycles (Fig. 8b); and baboon 8911, which achieved 6729, which had the lowest antibody titre (Fig. 8a); baboon the highest titre among the baboons, missed seven or eight

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(a) (b) ) )

600 60 –1 600 90 –1 80 500 50 500 70 1000) 400 40 1000) × 400 60 × 50 300 30 300 40 200 20 200 30 20

ELISA titres ( 100 10 100 ELISA titres ( 10 0 0 0 0 Serum progesterone (ng ml Serum progesterone (ng ml

30/08/9604/11/9616/12/9620/01/9724/02/9731/03/9705/05/9713/06/9718/07/9722/08/9726/09/9731/10/9705/12/9709/01/9813/02/9820/03/9824/04/9829/05/9803/07/9807/08/9811/09/9816/10/9820/11/9825/12/9829/01/99 30/08/9604/11/9616/12/9620/01/9724/02/9731/03/9705/05/9709/06/9714/07/9718/08/9722/09/9727/10/9701/12/9705/01/9809/02/9816/03/9820/04/9825/05/9829/06/9803/08/9807/09/9812/10/9816/11/9821/12/9825/01/9901/03/99

600 100 ) 600 100 –1 90 –1 90 500 500 80 80 70 70 1000) 1000) 400 400 × × 60 60 300 50 300 50 40 40 200 200 30 30 ELISA titres ( ELISA titres ( 100 20 100 20 10 10 Serum progesterone (ng ml 0 0 Serum progesterone (ng ml 0 0

30/08/9604/11/9616/12/9620/01/9724/02/9731/03/9705/05/9709/06/9714/07/9718/08/9722/09/9727/10/9701/12/9705/01/9809/02/9816/03/9820/04/9825/05/9829/06/9803/08/9807/09/9812/10/9816/11/9821/12/9825/01/9901/03/99 30/08/9604/11/9616/12/9620/01/9724/02/9731/03/9705/05/9709/06/9718/07/9722/08/9726/09/9731/10/9705/12/9709/01/9813/02/9820/03/9824/04/9829/05/9803/07/9807/08/9811/09/9816/10/9820/11/9825/12/9829/01/9905/03/9909/04/99

(e) )

600 70 –1 500 60

1000) 400 50 × 40 300 30 200 20 100 ELISA titres ( 10 0 0 Serum progesterone (ng ml

Fig. 7. Antibody titres ( ) and progesterone concentrations (᭜) in sera of cynomolgus monkeys vaccinated with recombinant human zona pellucida protein rhZPB: (a) CQ8135, (b) CQ8155, (c) CQ8152, (d) CQ8126 and (e) CQ8131. The antibody titres for anti-rhZPB were measured once a week by ELISA and serum progesterone concentrations were measured twice a week. An animal was considered to have ovulated if the serum progesterone was > 5 ng ml–1. Each of these animals received inoculations as indicated by ᭹. The males were introduced in November 1996 (indicated by arrow). menstrual cycles (Fig. 8c). Three of the five baboons Immunization of cynomolgus monkeys with rhZP proteins immunized with rhZPB became pregnant (baboon 4587, expressed in bacteria, yeast or insect cells induced antibody November 1998; baboon 4603, December 1998; and baboon titres that were low and of short duration. The fertility status 8816, April 1999) after 9, 10 and 14 months with the males, of the immunized animals did not differ compared with the respectively. The antibody titre profiles and the menstrual control animals. In a study using marmoset monkeys cycle patterns for baboon 4587 and baboon 4603 were similar immunized with CHO-expressed rhZPC, Paterson et al. to those for baboon 8816 (Fig. 8d,e). (1996) achieved high antibody titres with two inoculations. Therefore, in the present study in cynomolgus monkeys CHO-expressed proteins were used. The CHO-expressed proteins induced higher antibody responses of a greater Discussion duration in cynomolgus monkeys than had been observed In a study using bacterially expressed recombinant rabbit ZP previously. protein, monthly immunizations were necessary to achieve CHO-expressed rhZPC induces the acrosome reaction high antibody titres (VandeVoort et al., 1995). Mixed results (Van Duin et al., 1994), and the rhZPC produced in this were obtained with the partial ZPA proteins: one disrupted laboratory also induces the acrosome reaction (Harris et al., the menstrual cycle and caused ovarian pathology in most of 1999), indicating that these proteins may be biologically the animals, whereas the other fragment had neither of these active. Proteins expressed in bacteria, yeast or insect cells effects. Immunization with ZPB did not disrupt the were not soluble without 6 mol urea l–1 (Harris et al., 1999), so menstrual cycle nor did it cause ovarian pathology. The it was impossible to test for biological activity. This finding antibodies from these animals were found to inhibit may indicate that native conformation is an important sperm–egg fusion, but no breeding data were presented. element for a ZP antigen, either because conformational

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Table 7. Comparison of animals receiving recombinant Therefore, the immunization schedule for the baboon human zona pellucida rhZPB (no combination) study was significantly reduced. The results from the baboons are in agreement with those from the cynomolgus Maximum monkeys for the administration of rhZPA and rhZPC. The Animal antibody titre Months with Number of result using rhZPB is the major difference when the two number (ϫ 1000) males menstrual cycles studies are compared. None of the cynomolgus monkeys Cynomolgus that received rhZPB alone became pregnant, but three of five monkeys baboons that received rhZPB did become pregnant. The large CQ8135 512 35 27 number of missed menstrual cycles in the rhZPB groups CQ8126 512 35 18 probably indicates significant ovarian pathology. The CQ8131 512 35 19 monkeys missed many more menstrual cycles than the CQ8155 1000 35 15 baboons and probably had more damage leading to apparent CQ8152 1000 35 14 permanent infertility. This issue requires more extensive investigation. Baboons The finding of a shorter duration of infertility with a 6729 64 19 14 combination of antigens was unexpected, and further 4587a 128 9 6 8816b 256 14 6 investigation is needed to confirm and explain this effect. 4603c 256 10 3 The profile of monkey CQ8134 is similar to those of animals 8911 1000 19 5 that consistently received the combination of rhZPB and rhZPC. This animal was supposed to receive only rhZPC but aThis baboon became pregnant in November 1998. received rhZPB for the second booster (of seven given). She bThis baboon became pregnant in December 1998. remained infertile 6–17 months longer than the monkeys that c This baboon became pregnant in April 1999. received rhZPC alone. This observation indicates that a single injection of rhZPB may be sufficient to induce infertility, a possibility that needs to be tested. More studies will be necessary to determine whether certain effects are epitopes are important or because a folded protein is associated with specific combinations of antigens. A study of generally more stable than a denatured protein. antigen combinations could also examine the effect of The design of the cynomolgus monkey study was based administration of different antigens simultaneously or in on Paterson et al. (1996). These authors used marmoset various sequences. monkeys and rhZP3 (rhZPC) in a modified complete The HSPZ-treated group was included as a positive Freund’s adjuvant. They began using three monkeys that control, but sustaining the titres required constant booster received only two injections each of 25 µg. The monkeys immunizations. The animals regained fertility very quickly produced antibody titres to the antigen of > 1:20 000. When when the titres decreased to background levels. Since only the mating study was commenced, two of the three animals antibodies to ZPB are effective, this finding indicates that the quickly became pregnant. The results of the present study antibodies induced by the native pig ZP do not crossreact as using rhZPC administered to cynomolgus monkeys and effectively with the primate ZPB as the antibodies induced baboons are very similar to those of the earlier study. by rhZPB. Paterson et al. (1996) concluded that the titres were too low in The aim of the present study was to identify a zona pellucida the first group of marmoset monkeys. Therefore the second protein that could serve as a target for immunocontraception. group of three marmosets was immunized using a similar This aim was achieved. Every animal in two different groups of regimen except that they continued to receive 25 µg booster primates that was inoculated with rhZPB experienced long injections of rhZP3 at intervals of 2 months. This procedure periods of infertility. Furthermore, every animal that was not ensured that antibody titres were maintained before mating inoculated with rhZPB became pregnant before any animal studies commenced at about 9 months after the primary that was inoculated with rhZPB. immunization. When the mating study commenced, the What is not clear is why this effect is sustained after the animals were no longer showing normal menstrual cycles, antibody titres have dropped to levels that could be yet one of the three animals became pregnant. Again, the considered insignificant. This finding infers that even though results of the present study using rhZPC are consistent with the serum antibody titres are very low, there may be a higher those of Paterson et al. (1996). relative amount of antibody sequestered within the ovary. Paterson et al. (1992 and 1996) demonstrated that the Alternatively, it is possible that even a very small number of disruption of the normal progesterone cycle could be antibodies to ZPB may be enough to interfere with the correlated with ovarian pathology. Therefore, in designing function of ZPB. the immunization protocol for the cynomolgus monkeys, the Analysis of the structure of the mouse zona pellucida has aim was to use enough antigen and immunizations to determined that ZP2 (ZPA) and ZP3 (ZPC) are present in produce an effective titre without causing cessation of the approximately equal amounts and make up most of the menstrual cycle. Except for many of the rhZPB-treated structure (Wassarman et al., 1989; Epifano et al., 1995b). animals, the results were generally successful, but it was ZP1 (considered to be the mouse equivalent of ZPB) concluded that the immunization schedule was unnecessarily comprises only about 9% of the total structure of the zona aggressive. pellucida (Wassarman et al., 1989; Green, 1997). Other studies

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(a) (b) 300 35 25 )

70 ) –1 –1 60 250 30 20 25 50 200 1000) 1000) × × 40 15 20 150 15 30 10 100 20 10 ELISA titres ( ELISA titres ( 5 10 50 5 Serum progesterone (ng ml 0 0 Serum progesterone (ng ml 0 0

20/10/9717/11/9715/12/9712/01/9809/02/9809/03/9806/04/9804/05/9801/06/9829/06/9827/07/9824/08/9821/09/9819/10/9816/11/9814/12/9811/01/9908/02/9908/03/9905/04/9903/05/9901/06/99 20/10/9717/11/9715/12/9712/01/9809/02/9809/03/9806/04/9804/05/9801/06/9829/06/9827/07/9824/08/9821/09/9819/10/9816/11/9814/12/9811/01/9908/02/9908/03/9905/04/9903/05/99 (c) 1200 25 (d) )

140 40 ) –1 –1 35 1000 20 120 30 800 100 1000) 1000)

× 15 25 × 80 600 20 10 60 400 15 40 10 ELISA titres (

5 ELISA titres ( 200 20 5 Serum progesterone (ng ml 0 0 0 0 Serum progesterone (ng ml

20/10/9703/11/9717/11/9701/12/9715/12/9729/12/9712/01/9826/01/9809/02/9823/02/9809/03/9823/03/9806/04/9820/04/9804/05/9818/05/9801/06/9815/06/9829/06/9813/07/9827/07/9810/08/9824/08/9808/09/9821/09/9805/10/9819/10/9802/11/9816/11/9830/11/9814/12/98 20/10/9717/11/9715/12/9712/01/9809/02/9809/03/9806/04/9804/05/9801/06/9829/06/9827/07/9824/08/9821/09/9819/10/9816/11/9814/12/9811/01/9908/02/9908/03/9905/04/9903/05/9901/06/99 )

300 (e) 35 –1

250 30 25 200 1000)

× 20 150 15 100 10 50

ELISA titres ( 5

0 0 Serum progesterone (ng ml

20/10/9717/11/9715/12/9712/01/9809/02/9809/03/9806/04/9804/05/9801/06/9829/06/9827/07/9824/08/9817/09/9824/09/9801/10/9808/10/9815/10/9822/10/9829/10/9816/11/9814/12/9811/01/99

Fig. 8. Serum antibody titres (vertical bars) and serum progesterone concentrations (᭜) of individual baboons (a) 6729, (b) 8816, (c) 8911, (d) 4587 and (e) 4603 vaccinated with recombinant human zona pellucida protein rhZPB. The antibody titre scales are different in each panel. An animal was considered to have ovulated if the serum progesterone was > 5 ng ml–1 (horizontal line). Each animal received three inoculations (indicated by ᭹) before males were introduced in February 1998 (indicated by arrow).

demonstrate that if either the ZPA protein or the ZPC protein important factor. The ZPB molecules might occupy some is missing, no zona pellucida layer is produced (Tong et al., junctional position that is critical to the maintenance of the 1995; Liu et al., 1996; Rankin et al., 1996). Absence of ZP1 in structure or to fertilization. The results of the present study mice weakens but does not eliminate the zona pellucida imply that the ZPB molecule may be more critical to the layer (Rankin et al., 1999). Since ZP1 makes up only a small structure of the zona pellucida structure than has been part of the zona pellucida layer, its absence does not have the assumed. It is possible that some cases of unexplained same marked effect as the absence of either of the other two infertility in women are due to the presence of what would structural proteins. otherwise be considered insigniﬁcant concentrations of ZPB The ﬁnding by Hughes and Barratt (1999) that mouse ZP1 antibodies. A better understanding of the function of ZPB is and human ZPB are not homologous casts doubt on using a needed as it relates to oocyte structure and maturation and to mouse model for the structure of the primate zona pellucida. fertilization. However, if ZPB has a corresponding role in the structure of the primate zona pellucida as does ZP1 in mice, it is The authors thank Joe Podolski for advice and support and possible that production of antibodies to ZPB has the more Lizette Bravo for technical assistance. pronounced effect because ZPB represents a smaller more Note added in press: since this manuscript went to press the following focused target. That is, the greater relative abundance of ZPA paper was published Govind CK and Gupta SK (2000) Failure of and ZPC requires a much greater abundance of antibody female baboons (Papio anubis) to conceive following immunization molecules. Fewer antibodies to ZPB would be necessary per with recombinant non-human primate zona pellucida glycoprotein B oocyte. It is also possible that the relative position within the expressed in Esherichia coli. Vaccine 18 2970–2978. The study described zona pellucida layer of ZPA and ZPC versus ZPB is an in this paper is similar to one of the studies in the present manuscript.

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