WO1999034825A1 - Vaccin anti-fecondite et mode d'utilisation - Google Patents

Vaccin anti-fecondite et mode d'utilisation Download PDF

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Publication number
WO1999034825A1
WO1999034825A1 PCT/US1998/027658 US9827658W WO9934825A1 WO 1999034825 A1 WO1999034825 A1 WO 1999034825A1 US 9827658 W US9827658 W US 9827658W WO 9934825 A1 WO9934825 A1 WO 9934825A1
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WO
WIPO (PCT)
Prior art keywords
vaccine
fertility
impairing
carnivore
zona pellucida
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PCT/US1998/027658
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English (en)
Inventor
Richard A. Fayrer-Hosken
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The University Of Georgia Research Foundation, Inc.
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Publication date
Application filed by The University Of Georgia Research Foundation, Inc. filed Critical The University Of Georgia Research Foundation, Inc.
Priority to AU20961/99A priority Critical patent/AU2096199A/en
Publication of WO1999034825A1 publication Critical patent/WO1999034825A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0006—Contraceptive vaccins; Vaccines against sex hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/18—Feminine contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • Surgical spaying is a laborious procedure, requiring the initial induction of the animal, gas anesthesia during surgery, a surgical pack with suture materials and post-operative medications.
  • Common surgical complications include problems associated with the procedure itself, allergic reactions to anesthetics or post-operative medications, and adverse local or systemic effects during the recovery period. Examples include ovarian remnant syndrome, where dogs continue to cycle despite being spayed, uterine infections, abdominal hemorrhage, and premature opening of the suture line. A substantial recovery period is typically needed even after an uncomplicated procedure. Surgical spaying is also expensive, and pet owners are often unwilling to assume the costs.
  • Hormonal therapies have also been used to curb pet overpopulation. However these methods usually require daily administration of the drug, and they only result in temporary infertility. Furthermore, most protracted hormonal therapies have undesirable side effects such as uterine infections, mammary cancer, and diabetes.
  • a vaccine comprising porcine zona pellucida and an adjuvant comprising synthetic trehalose dicorynomycolate has been successfully used to cause immunocontraception in horses (P. Willis et al., J. Equine Vet. Sci., 364- 370 (1994)) and elephants (R. F-H., Wildlife Soc. Bull., 25(1):18-21 (1997)).
  • Dunbar et al. e.g., EP 599822, U.S. Pat. No. 5,637,300 have experimented with reproductive control in non-rodent mammals using a recombinant zona pellucida protein. Due to limitations imposed by recombinant DNA technology and available expression systems, however, the recombinant protein lacks the glycosylation pattern of the native glycoprotein.
  • the present invention provides a vaccine and a method for impairing fertility in an animal.
  • the method for impairing fertility in the animal comprises administering to the animal a vaccine comprising substantially pure, nonrecombinant zona pellucida glycoprotein, or an antigenic fragment thereof.
  • the vaccine is administered in a manner and an amount effective to cause fertility impairment in the animal.
  • the fertility impairment vaccine causes temporary, reversible infertility in the animal.
  • the fertility impairing vaccine causes permanent, irreversible infertility in the animal.
  • the animal to which the vaccine is administered is a carnivore.
  • the carnivore is a dog or a cat; more preferably, the carnivore is a dog.
  • the vaccine preferably does not cause abnormal estrus cycles in a vaccinated dog.
  • the fertility impairing vaccine of the invention preferably comprises porcine zona pellucida glycoprotein, and optionally includes an immunological adjuvant comprising an immunostimulant, preferably synthetic trehalose dicorynomycolate (STDCM). Also optionally, the vaccine contains an oil, preferably squalene oil. In a preferred embodiment, the fertility impairing vaccine is an immunosterilant vaccine.
  • the immunosterilization method of the invention is far preferable to surgical sterilization and hormone regimens as a population control tool for domestic dogs and cats, and can further be used to control ferrel dog and cat populations, for example by development of a species-specific oral delivery vehicle.
  • Figure 1 shows one version of the oocyte purification apparatus of the invention.
  • Figure 2 is a graph depicting serum anti-porcine zona pellucida antibody titers in experimental dogs (subjects 9727, 9728, 9729 and 9731) and clinical dogs (subjects 2-5) during the course of vaccination with a porcine zona pellucida (pZP) vaccine.
  • pZP porcine zona pellucida
  • the fertility impairing vaccine of the invention comprises an antigen comprising zona pellucida glycoprotein, preferably substantially pure zona pellucida glycoprotein, or an antigenic fragment thereof.
  • zona pellucida glycoprotein is a total porcine zona pellucida glycoprotein.
  • a total zona pellucida glycoprotein preparation obtained from pig ovaries includes all three major heavily glycosylated porcine zona pellucida glycoproteins: pZPl, pZP3 ⁇ and pZP3 ⁇ .
  • pZP3 ⁇ and pZP3 ⁇ each have reported molecular weights of about 55 kD
  • pZPl has a reported molecular weight of about 82 kD.
  • the amino acid sequences of these three glycoproteins are known (J.D. Harris et al.,
  • Purity of the zona pellucida glycoprotein can be evaluated analytically using a combination or series of two-dimensional sodium dodecyl sulfate polyacrylamide gels (SDS-polyacrylamide gel electrophoresis, or SDS-
  • Glycoproteins typically migrate electrophoretically in gels as broad smears rather than narrow bands, as a result of the variable levels of negative charge present in the constituent oligosaccharide chains.
  • a "substantially pure" total zona pellucida glycoprotein preparation isolated from pig ovaries migrates as two distinct smears in the gel electrophoretic experiments (one smaller smear representing pZP 1 , and one larger smear representing pZP3 ⁇ and pZP3 ⁇ ), and shows immunological reactivity in Western blot analysis using a polyclonal antibody raised in rabbits to highly purified total porcine zona pellucida glycoprotein.
  • a substantially pure zona pellucida glycoprotein preparation used for fertility impairment there are no detectable contaminating proteins.
  • An antigenic fragment of a zona pellucida glycoprotein is a peptide fragment, preferably a glycosylated peptide fragment, that elicits an immune response characterized by detectable anti-pZP antibody levels in the subject using ELISA as described in Example II.
  • the peptide fragment preferably contains more than seven amino acids, more preferably at least about 10 amino acids, most preferably at least about 20 amino acids.
  • the zona pellucida glycoprotein used in the present vaccine is preferably a naturally occurring glycoprotein or a chemically or enzymatically synthesized glycoprotein.
  • the glycoprotein is preferably not a recombinant glycoprotein, but use of a recombinant glycoprotein in the present vaccine is not necessarily excluded in alternative embodiments of the invention.
  • the vaccine of the invention preferably additionally includes an immunological adjuvant to enhance the immunological response of the subject to the glycoprotein antigen.
  • adjuvants include Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, and an adjuvant comprising an immunostimulant such as synthetic trehalose dicorynomycolate (STDCM) and an oil such as squalene oil (see P. Willis et al., J. Equine Vet. Sci., 14, 364-370 (1994)).
  • STDCM synthetic trehalose dicorynomycolate
  • An adjuvant comprising synthetic trehalose dicorynemycolate, squalene oil, and a surfactant such as lecithin is preferred.
  • Lecithin typically includes phosphatidyl choline.
  • the vaccine comprises oil, preferably a biodegradable oil such as squalene oil, in an amount of about 2.5% to about 15%, preferably about 8% to about 12%.
  • oil preferably a biodegradable oil such as squalene oil
  • the vaccine is prepared using an adjuvant concentrate which contains lecithin (about 5% to about 15 % wt/vol, preferably about 12% wt /vol) and STDCM (preferably about 25 mg/mL to about 50 mg mL) in squalene oil.
  • lecithin about 5% to about 15 % wt/vol, preferably about 12% wt /vol
  • STDCM preferably about 25 mg/mL to about 50 mg mL
  • % wt/vol means grams per 100 mL of liquid.
  • the aqueous solution containing the isolated pZP glycoprotein is typically a phosphate-buffered saline (PBS) solution, and additionally preferably contains Tween 80 (about 0.2% vol/vol to about 0.8% vol/vol, preferably about 0.4% vol/vol).
  • PBS phosphate-buffered saline
  • Tween 80 about 0.2% vol/vol to about 0.8% vol/vol, preferably about 0.4% vol/vol.
  • Homogenization of the oily adjuvant concentrate with the aqueous pZP solution can be accomplished using any convenient means known in the art, such that the oil disperses within the aqueous solution to form an oil in water emulsion. Oil droplet sizes of about 200 nm or less are particularly preferred as they produce a more uniform and stable suspension.
  • a particularly preferred vaccine comprises predetermined amounts of pZP and STDCM in an emulsion containing about 10% squalene oil and about 90% aqueous phase.
  • the invention further includes a method for administering a fertility impairing vaccine as described herein in a manner effective to cause impaired fertility in an animal, preferably a carnivore (i.e., a member of the order
  • Carnivora Preferably the carnivore is not a primate, and is a dog or a cat, more preferably a dog. Impairment of fertility in an animal in accordance with the invention can take the form of either immunocontraception and immunosteriliztion. Immunosterilization means permanent, irreversible infertility, in contrast to immunocontraception wherein infertility is temporary or transient, and reversible.
  • Immunocontraception and immunosterilization are both dependent on the antibody titer level in the serum of the subject, but immunosterilization is typically the result of ovarian pathology caused by vaccine administration and high titers of anti-pZP antibodies, as evidenced by, for example, total destruction of the zona pellucida glycoproteins and/or influx of leukocytes into the follicles. Reducing the number of boosters leads to lower antibody titers which results in immunocontraception (i.e., infertility that is temporary and reversible) instead of immunosterilization.
  • the vaccine is administered in a manner and an amount effective to cause the desired infertility in the mammalian subject.
  • the vaccine is preferably administered in the form of a plurality of doses (typically about 1.0 mL for a dog, 0.5 mL for a cat), each dose containing zona pellucida glycoprotein, or an antigenic fragment thereof, in an amount of about 100 ⁇ g to about 2 mg, more preferably about 200 ⁇ g to about 400 ⁇ g.
  • An immunostimulant such as STDCM is typically present in a per dose amount of about 50 ⁇ g to about 5 mg, preferably in an amount of about 1 mg to about 3.5 mg, more preferably in an amount of about 2 mg to about 3 mg.
  • the animal is given an initial dose, usually via intramuscular injection although subcutaneous injection can also be used.
  • the initial injection is followed by two or more booster injections at two to four week intervals, although the boosters can be administered from about 9 days to about twelve months following the previous vaccination.
  • the body's immunological response to the vaccine at this dosing regimen appears to render the ovaries permanently inactive as a result of, for example, follicle disruption or destruction, as evidenced by immunocytochemical analysis and histological evaluation of the ovarian tissue of vaccinated subjects.
  • Sterility is permanent and irreversible. Immunosterilization of carnivores in accordance with the present method typically does not cause abnormal estrus cycles or other significant undesirable side effects in the vaccinated subjects.
  • the vaccine When the vaccine is administered to a dog or a cat as described above, but with only one booster instead of two or more boosters, the vaccine typically results in immunocontraception (i.e., temporary or transient, reversible infertility) rather than immunosterilization.
  • Buffers Saline buffer (40 L) was made by addition 4 L of the following solution: 0.9% NaCl, 0.01 M dibasic sodium phosphate, 0.01 M monobasic sodium phosphate, and 0.002 M sodium citrate dihydrate, pH 7.2, in triple distilled water, to 36 L of triple distilled water.
  • Tris detergent buffer (1L) was made by combining 2 mL of NP-40 (Cat. No. N-6507, Sigma Chemical Co., St. Louis, MO) with 998 mL Tris buffer.
  • the oocyte purification apparatus (Fig. 1) consisted of three chambers. Each chamber consisted of a stainless steel wire mesh container (Home Depot) suspended inside a buffer container set on an orbital shaker (shown in Fig. 1 ) or a rotary washing system with an internal agitator.
  • the pore size of the wire mesh used to form the wire mesh containers in the first, second, and third chambers was 1000 ⁇ m, 500 ⁇ m, and 150 ⁇ m, respectively.
  • Tubing connecting the chambers allowed fluid transfer from the buffer space external to the wire mesh of one chamber to a collection or holding carboy, or, alternatively, to the inside of the next succeeding downstream wire mesh container in a continuous flow process, as shown in Fig. 1.
  • Peristaltic pumps are used to effect fluid movement within the tubing between chambers
  • Pig ovaries were obtained from pig slaughterhouses.
  • Porcine ovaries (5-6 lbs.) were twice ground through a commercial meat grinder (Hobart), and the homogenate was collected.
  • the homogenate and grinder were rinsed with 4L of saline buffer, and the homogenate solution was placed in the wire mesh container of the first chamber of the purification apparatus.
  • the three buffer containers of the purification apparatus were filled with saline buffer.
  • the shakers were operated at an orbital shaker rotation speed of about 20 revolutions per minute during the oocyte purification process. Periods of rotary agitation were alternated with periods of fluid removal from the region surrounding the mesh container.
  • Filtered oocytes together with a small amount of tissue, passed through the 1000 ⁇ m mesh and were thus pumped from the buffer space of the first chamber into a collection carboy or into the wire mesh container in the second chamber.
  • the filtered oocytes are subsequently pumped into the wire mesh container in the second chamber.
  • the oocytes were then passed through the 500 ⁇ m mesh of the wire mesh container of the second chamber while the fibrous tissue remained in the mesh container.
  • the oocytes and saline buffer were then pumped from the buffer space of the second chamber into a collection carboy or directly into the 150 ⁇ m wire mesh container in the third chamber.
  • Rotary agitation was continued in the third chamber and the solution surrounding the wire mesh (containing the oocytes) was removed.
  • the solution containing the oocytes was then passed over a 75 ⁇ m screen (l / 4 inches or 2 y inches in diameter).
  • the oocytes were collected on the 75 ⁇ m screen and were then backwashed into a 100 mL beaker using Tris buffer.
  • the 100 mL solution was divided into 2 x 50 mL vials and homogenized at 15,000 rpm for 3 to 5 minutes in a Powergen 700D (Fisher) homogenizer.
  • Powergen 700D Powergen 700D
  • the zona fragments were then poured onto a 1 3 / 4 inches or 2 y inches diameter, 0.040mm (40 ⁇ m) filter screen and washed with Tris detergent buffer.
  • the zona fragments were removed from the screen by backwashing with Tris detergent buffer into a small polypropylene beaker, then incubated at 4°C with constant mechanical stirring to dissociate any undesired proteins, such as albumin.
  • the zona material is preferably handled in polypropylene or siliconized glass beakers to prevent adherence to surfaces which results in loss of the material.
  • the zona fragments were again poured a 1 3 / 4 inch diameter, 0.040mm (40 ⁇ m) filter screen and washed with Tris buffer to remove any protein contaminants.
  • the zona fragments retained on the screen were collected by spooning or backwashing (using Tris buffer) into a small polypropylene beaker to a maximal volume of 25 mL.
  • the beaker was covered and placed in a 75-76°C water bath and incubated for 20 minutes to solubilize the zona protein such that the temperature of the zona protein- containing solution was 73 ⁇ 1°C.
  • the mixture was centrifuged at 21,000 rpm for 25 minutes or until a pellet was observed at the base of the tube. The supernatant was collected, and protein concentration was estimated. The supernatant was aliquoted (3mg/vial), lyophilized, and stored under N 2 gas in a desiccator at 4°C.
  • the vaccine was prepared by homogenizing a concentrated oily adjuvant concentrate with an aqueous antigen solution containing isolated pZP glycoprotein.
  • the oily adjuvant concentrate contained a surfactant, lecithin, and an immunostimulant, synthetic trehalose dicorynomycolate (SDTCM), in squalene oil.
  • SDTCM synthetic trehalose dicorynomycolate
  • a typical adjuvant concentrate contained about 12% wt/vol (grams/100 mL) lecithin and about 25-50 mg/mL STDCM in squalene oil.
  • the aqueous antigen solution contained the pZP glycoprotein preparation in saline or phosphate buffered saline (PBS) and Tween 80.
  • the aqueous composition When prepared for use in combination with an adjuvant concentrate to yield the vaccine composition, the aqueous composition typically contained 0.4% (vol/vol) Tween 80 and an amount of pZP calculated to yield a dose of about 200 ⁇ g to about 400 ⁇ g per vaccination. Vaccine doses for dogs were about 1 mL in volume.
  • Homogenizing was accomplished by combining adjuvant concentrate (to a final concentration of no greater than 10% vol/vol) with aqueous pZP solution and emulsifying using a Powerjam 700D homogenizer at 15,000 rpm for 6 minutes.
  • the resulting emulsion is then homogenized with phosphate buffered saline (PBS) (containing 0.4% vol/vol Tween 80) at 20,000 for 8-12 minutes.
  • PBS phosphate buffered saline
  • the homogenization process resulted in a vaccine composition that is an oil-in-water emulsion or possibly a water-in-oil-in-water emulsion.
  • the inventors do not intend that the invention be bound by any particular scientific theory, it is believed that the STDCM, an amphiphilic glycolipid, partitions to the oil/water interfaces in the emulsion, and that the antigen is attracted to and associates with the STDCM at these interfaces.
  • Vaccinations Four experimental dogs were vaccinated, and an FDA approved clinical trial has begun in which privately owned dogs in Clark and Walton Counties, Georgia, have also been vaccinated. To date, 43 dogs (four experimental dogs and 39 privately owned dogs) have been through the series of injections and have had serum antibody levels determined.
  • STDCM synthetic trehalose dicorynomycolate
  • Example II Hamilton, MT, and the vaccines were prepared as described in Example II.
  • the dogs were vaccinated consecutive boosters (containing the same amount of pZP, 200 ⁇ g) administered at 30-day intervals.
  • boosters containing the same amount of pZP, 200 ⁇ g
  • vaccinations were delivered to dogs intra-muscularly in the longissimus muscle (loin area), although subcutaneous vaccination is also acceptable.
  • follow up booster injections were administered on the contra-lateral side. No pain or adverse reactions were observed at the injection sites. In some cases boosters were administered subcutaneously with equivalent results.
  • Antibody titers Blood was drawn from each dog weekly, and serum antibody titers were determined using an enzyme linked immunosorbant assay (ELISA). Adjacent wells of a microwell plate were coated with 2 ⁇ g pZP, and incubated for 6 hours. The wells were then blocked with 5% bovine serum albumin (Sigma Chemical Co., St. Louis, MO) in TBST (Tris-buffered saline + 5% Tween-20) and incubated overnight. Wells were then loaded with the primary antibody (canine serum) in TBST at a 1 :500 and 1 : 1 ,000 dilution and incubated for 4 hours.
  • ELISA enzyme linked immunosorbant assay
  • the wells were then washed and loaded with 50 ⁇ l of the secondary antibody (rabbit anti-dog IgG) and incubated for 2 hours. Color change was observed after the addition of /?-nitrophenyl phosphate for 30 minutes and the reaction terminated by the addition of 3 M NaOH. The optical density was read at a 405-492 nm range on a Spectramax spectrophotometer. The dogs pre-immune serum served as the negative controls.
  • the secondary antibody rabbit anti-dog IgG
  • ELISA trials revealed that there was a similar antibody profile in all eight dogs (experimental and clinical) characterized by a significant rise in antibody titers between the first and second booster. Antibody levels rose slightly after the initial vaccination and then significantly (p ⁇ 0.05) after the first and second boosters. The rise in titer was the greatest in the clinical trial dogs (trials 2-5). These data clearly show that the there is a significant immune response to the pZP vaccine and synthetic adjuvant.
  • the immunological response was further investigated by treating formalin fixed, paraffin embedded ovarian sections with anti-pZP antibodies raised in rabbits against highly purified pZP, and incubating for 1 hour.
  • the sections were then treated with biotin-conjugated anti-rabbit IgG (Sigma Chemical Co., St. Louis, MO), followed by avidin-conjugated horseradish peroxidase (Sigma Chemical Co., St. Louis, MO). Finally, the sections were stained with diaminobenzidine and counterstained with Mayer's hematoxylin.
  • the integrity of all ovarian follicles was found to have been breached, and no immunodetectable zona material was present on the ovarian sections.
  • normal dog ovaries have distinct oocytes with a zona pellucida.

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Abstract

L'invention concerne un vaccin comprenant un antigène dérivé d'une glycoprotéine de l'aire pellucide, ce vaccin permettant d'inhiber la fécondité chez les animaux, de préférence carnivores. Ce vaccin peut être utilisé comme immunostérilisant et comme immunocontraceptif.
PCT/US1998/027658 1998-01-02 1998-12-30 Vaccin anti-fecondite et mode d'utilisation WO1999034825A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU20961/99A AU2096199A (en) 1998-01-02 1998-12-30 Fertility impairing vaccine and method of use

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US7037598P 1998-01-02 1998-01-02
US60/070,375 1998-01-02
US7140698P 1998-01-15 1998-01-15
US60/071,406 1998-01-15
US7636898P 1998-02-27 1998-02-27
US60/076,368 1998-02-27

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WO1999034825A1 true WO1999034825A1 (fr) 1999-07-15

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7019114B2 (en) 1998-02-19 2006-03-28 Eastern Virginia Medical School Recombinant, biologically active human zona pellucida protein 3 (HZP3) to test male fertility
US7037663B2 (en) 1998-02-19 2006-05-02 Eastern Virginia Medical School Human zona pellucida protein 3 and uses thereof
US7148021B2 (en) 2001-08-02 2006-12-12 Eastern Virginia Meical School Human zona pellucida proteins and methods of their use in diagnosing male infertility

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989003399A1 (fr) * 1987-10-07 1989-04-20 Zonagen, Inc. Procede de preparation et utilisation d'antigenes de zona pellucida et anticorps pour la sterilisation et la contraception
WO1993014786A1 (fr) * 1992-02-04 1993-08-05 Colorado State University Research Foundation Composition et procede anticonceptionnels ou de sterilisation pour animaux
WO1994011019A1 (fr) * 1992-11-09 1994-05-26 Zonagen, Inc. Materiaux et procedes utiles pour l'immunocontraception

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989003399A1 (fr) * 1987-10-07 1989-04-20 Zonagen, Inc. Procede de preparation et utilisation d'antigenes de zona pellucida et anticorps pour la sterilisation et la contraception
WO1993014786A1 (fr) * 1992-02-04 1993-08-05 Colorado State University Research Foundation Composition et procede anticonceptionnels ou de sterilisation pour animaux
WO1994011019A1 (fr) * 1992-11-09 1994-05-26 Zonagen, Inc. Materiaux et procedes utiles pour l'immunocontraception

Non-Patent Citations (4)

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Title
DIETL J. ET AL: "Immunogenic potency of the zona pellucia", EXPERIENTIA, vol. 38, 1982, pages 502 - 503, XP002100172 *
EAST I J ET AL: "MONOCLONAL ANTIBODIES TO THE MAJOR PROTEIN OF THE MURINE ZONA PELLUCIDA: EFFECTS ON FERTILIZATION AND EARLY DEVELOPMENT", DEVELOPMENTAL BIOLOGY, vol. 104, no. 1, July 1984 (1984-07-01), pages 49 - 56, XP002052324 *
PATERSON M ET AL: "Analysis of the contraceptive potential of antibodies against native and deglycosylated porcine ZP3 in vivo and in vitro", BIOLOGY OF REPRODUCTION, vol. 46, 1992, pages 523 - 534, XP002100174 *
SACCO A G ET AL.: "Heteroimmunization of squirrel monkeys (Saimiri sciureus) with a purified porcine zona antigen (PPZA): immune response and biologic activity of antiserum", FERTILITY AND STERILITY, vol. 39, no. 3, March 1983 (1983-03-01), pages 350 - 358, XP002100173 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7019114B2 (en) 1998-02-19 2006-03-28 Eastern Virginia Medical School Recombinant, biologically active human zona pellucida protein 3 (HZP3) to test male fertility
US7037663B2 (en) 1998-02-19 2006-05-02 Eastern Virginia Medical School Human zona pellucida protein 3 and uses thereof
US7148021B2 (en) 2001-08-02 2006-12-12 Eastern Virginia Meical School Human zona pellucida proteins and methods of their use in diagnosing male infertility

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