EP1085903A1 - Oberflächenproteine von eiern und methoden zur modulation der fruchtbarkeit - Google Patents

Oberflächenproteine von eiern und methoden zur modulation der fruchtbarkeit

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Publication number
EP1085903A1
EP1085903A1 EP99930430A EP99930430A EP1085903A1 EP 1085903 A1 EP1085903 A1 EP 1085903A1 EP 99930430 A EP99930430 A EP 99930430A EP 99930430 A EP99930430 A EP 99930430A EP 1085903 A1 EP1085903 A1 EP 1085903A1
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EP
European Patent Office
Prior art keywords
egg
plc
sperm
egg surface
protein
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EP99930430A
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English (en)
French (fr)
Inventor
John C. Herr
Scott A. Coonrod
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UVA Licensing and Ventures Group
University of Virginia UVA
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University of Virginia UVA
University of Virginia Patent Foundation
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Publication of EP1085903A1 publication Critical patent/EP1085903A1/de
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/367—Infertility, e.g. sperm disorder, ovulatory dysfunction

Definitions

  • the present invention is directed to egg surface antigens useful for producing 15 antibodies which bind epitopes on the egg surface and modulate fertility.
  • the invention encompasses compositions and methods for immunizing an individual for production of antibodies against egg surface antigens.
  • the invention is based on the discovery of egg surface antigens involved in egg-sperm binding and fusion. Methods are provided for the use of such antigens in methods for sterilization of female animals. Methods are further 20 provided for the use of egg surface antigens to generate antibodies useful for temporary, reversible contraception methods. Methods are further provided for the use of anti-idiotypic monoclonal antibodies which mimic egg surface epitopes to actively immunize a mammal against pregnancy.
  • contraceptive vaccines would be useful for permanent sterilization regimes useful for pest control, such as for sterilization of rodents or other unwanted populations. While there has been much interest in the development of immunocontraceptives, the focus has been, until recently, on the development of immunocontraceptives directed against sperm surface antigens, or on already known peptide hormones such as human chorionic gonadotropin and follicle stimulating hormone. One obstacle to the development of an effective egg surface antigen based immunocontraceptive vaccine has been the lack of knowledge regarding the molecular identities of egg surface proteins known to be directly involved in the fertilization process.
  • Mammalian fertilization may be defined as a series of gametic interactions in which capacitated sperm must first penetrate the cumulus cells and zona pellucida (the egg vestments), then bind to and fuse with the egg plasma membrane (oolemma).
  • the initial binding event between gametes is known as primary binding and occurs, in the mouse model, when the zona pellucida protein, ZP3, binds to a receptor(s) on the sperm (reviewed in (McLeskey et al., 1998, Int. Rev. of Cytol. 177: 57-113).
  • This binding event also initiates the acrosome reaction in which hydrolytic enzymes are released from the acrosomal compartment and act on the zona pellucida to facilitate penetration ofthe zona pellucida by sperm.
  • Zona penetration is known as secondary binding and is mediated by the zona protein, ZP2, and one or more molecules on the inner acrosomal membrane (reviewed in Snell and White, 1996, Cell 85: 629-637).
  • sperm Upon emergence from the zona pellucida, sperm then cross the perivitelline space and bind to and fuse with the oolemma.
  • the molecular basis of sperm-oolemma binding and fusion has yet to be fully elucidated; however, recent evidence has demonstrated that integrins are involved in the interaction. Almeida et al. (1995, Cell 81 : 1095-1104) found that when oocytes were treated with monoclonal antibodies against the egg surface integrin ⁇ 6 ⁇ l, mouse sperm-oolemma binding was reduced. Further, these investigators reported that somatic cells which express c ⁇ l bind mouse sperm avidly while somatic cells that lack ⁇ 6 or ⁇ l do not.
  • a proposed sperm surface ligand for ⁇ l is fertilin.
  • Fertilin contains a domain homologous to a family of integrin ligands known as disintegrins (Blobel et al., 1992, Nature 356: 248-252), which suggest a cell adhesion function for the molecule.
  • recombinant fertilin is known to bind to the oolemma (Evans et al., 1997, Dev. Biol. 187:79-93), with both monoclonal antibodies to fertilin (Primakoff et al., 1987, J. Cell. Biol.
  • protein DE protein DE
  • Glycosyl-phosphatidylinositol (GPI)-anchored proteins may play a key role in gamete interaction.
  • GPI-anchored proteins possess a covalently linked glycosylated phosphatidylinositol moiety which serves to attach the protein portion ofthe molecule to the cell surface lipid bilayer (Low and Saltiel, 1988, Science, 239: 268-275).
  • Proteins linked to the cell surface via a phosphatidylinositol anchor are known to be involved in a wide variety of cellular functions including T cell activation, hydrolysis of extracellular matrix proteins, transduction of extracellular stimuli, and cell-cell adhesion (reviewed in Low and Saltiel, 1988, Science, 239: 268-275).
  • GPI-anchored proteins can be released from the cell surface by treatment of cells with the highly specific enzyme phosphatidylinositol- specific phospholipase C (PI-PLC) (Low and Finean, 1978, Bioch. Biophys. Acta. 508: 565-570). Therefore, treatment of intact cells with PI-PLC has become a useful tool to characterize the released proteins and to investigate the role of GPI-anchored proteins in cell function.
  • PI-PLC highly specific enzyme phosphatidylinositol- specific phospholipase C
  • Mouse sperm surface hyaluronidase (also known as PH-20) is a GPI- anchored protein and is thought to aid sperm in passage through the cumulus oopherous and possibly the zona pellucida by hydrolyzing the extracellular matrix protein, hyaluronic acid (Gmachl and Kreil, 1993, Proc. Nat. Acad. Sci. USA 90: 3569-3573); (Myles and Primakoff, 1997, Biol. Reprod., 56: 320-327).
  • SAGA-1 is another sperm surface protein which has been shown to be a GPI-linked.
  • GPI-anchored proteins on oocytes.
  • the hamster oocyte is unique in that zona-free eggs from other species such as the mouse, rat, and guinea pig do not incorporate heterologous sperm as readily
  • the invention further encompasses compositions and methods for ⁇ immunizing an individual for production of antibodies against egg surface antigen(s).
  • the invention is based on the discovery of a conserved set of egg surface antigens, a subset of which are released from the follicular membrane upon treatment ofthe membrane with PI- PLC.
  • Methods are also provided for the use of antibodies against such antigens for active immunization, or sterilization, of female animals, by induction of a T-cell attack on the egg. 15 These methods are particularly useful in cases where permanent sterilization is desired, for example, for sterilization of animal populations.
  • Methods are further provided for the use of egg surface antigens to generate antibodies useful for passive immunization that can be used for reversible contraception methods.
  • Methods are further provided for the use of anti- idiotypic monoclonal antibodies which mimic egg surface epitopes to actively immunize a
  • the invention comprises a monoclonal antibody against an egg surface protein, novel hybridoma cells which express such antibodies; and methods for immunocontraception utilizing such monoclonal antibodies.
  • compositions comprising
  • Figure 1 PI-PLC treatment of gametes has no effect on sperm-zona pellucida binding, however, fertilization of zona-intact oocytes is blocked. Both sperm and eggs were separately treated with 1 U per ml PI-PLC for 30 min followed by gamete co-incubation in the presence of PI-PLC. To evaluate the effects of PI-PLC on sperm-zona binding, the ⁇ ⁇ oocytes were washed following 1 h gamete co-incubation and prepared for observation (see methods in Example in Section 6).
  • a single focal plane for each oocyte was selected in which the widest diameter ofthe zona pellucida could be visualized and the number of sperm in that focal plane was determined. No difference in sperm-zona pellucida binding was noted between the control group treated with inactivated PI-PLC (A) and the PI-PLC treated group (B). To evaluate the effects of PI-PLC on fertilization, the oocytes were washed following 2 h gamete co-incubation and incubated overnight. Following 24 h incubation, a significant decrease in the number of fertilized oocytes (as determined by cleavage) was noted in the treatment group (D) compared to the control group (C). In Figs.
  • Figure 2 Treatment of sperm with PI-PLC prior to incubation with zona-free oocytes has no effect on sperm-egg binding and fusion whereas treatment of eggs with PI-PLC inhibits sperm-egg binding and fusion.
  • Zona-free oocytes were pre-loaded with 1 ⁇ m Hoechst #33342 for 10 min and washed.
  • either sperm (A,B) or zona-free eggs (C,D) were treated with 1 U per ml PI-PLC for 30 min, washed, and the gametes were co- incubated for 40 min. The oocytes were then gently washed and prepared for observation (see methods in the Example given in Section 6).
  • Zona-free eggs were treated with either 5U per ml of heat inactivated PI-PLC, no PI-PLC, or increasing concentrations of PI-PLC for 30 min, washed and co-incubated with untreated sperm for 40 min. The oocytes were gently washed and prepared for observation (see methods in the Example in Section 6).
  • FIG. 5 Mouse eggs treated with PI-PLC can be artificially activated with calcium ionophore A23187.
  • Zona-free eggs were pre-loaded with Hoechst #33342, washed, and treated with PI-PLC for 30 min.
  • a small number of oocytes from the control group (A, inset) and treatment group (B, inset) were observed to ensure that oocytes had remained in metaphase II arrest following treatment.
  • Oocytes were then activated with 0.5 ⁇ m calcium ionophore A23187 for 5 min, washed and cultured for 40 min.
  • Figure 6 Treatment of zona-free oocytes with PI-PLC does not affect recognition of the ⁇ 6 ⁇ l integrin by its cognate antibody. Fluorescent beads (l ⁇ M) were coated with 6 ⁇ l antibodies then incubated with zona-free eggs that were either untreated or treated with 1 U er ml PI-PLC. No difference was observed in the number of anti- ⁇ l coated beads bound per egg when comparing the control group (A) with the PI-PLC treated group (B). Few beads bound when beads were coated with an equivalent concentration of control antibody and incubated with untreated eggs (C).
  • Figure 10 (A-B) Zona-free hamster oocytes can be artificially activated following PI-PLC treatment.
  • zona- free eggs were preloaded with Hoechst #33342, washed, and treated with either 1 U/ml PI- PLC or 1 U/ml heat inactivated PI-PLC (95 °C, 5 min) for 30 min.
  • Gocytes were then activated with 0.5 ⁇ m calcium ionophore A23 187 for 5 min, washed incubated at 37 °C and 5% CO 2 for 3h.
  • Fi ure 11 Treatment ofzona-free hamster oocytes with PI-PLC releases a 25-40 kDa (pi 5-6) protein cluster from the oolemma.
  • Zona-free hamster oocytes were biotinylated, separated into two groups (130 oocytes per group), and either mock treated or treated with 1 U/ml PI-PLC.
  • the supernatants were collected, the eggs were washed, and the egg proteins were extracted in Celis buffer.
  • the egg protein extracts and the proteins from the supernatants were separated by 2-D electrophoresis and electroblotted to nitrocellulose membranes. The membranes were stained with Protogold to visualize the egg proteins (red staining).
  • C The repertoire of surface labeled oolemmal proteins following PI-PLC treatment. Arrows labeled c2 represent the location ofthe 25-40 kDa (pi 5-6) protein cluster which was prominent in extracts of mock treated oocytes.
  • D Supernatant from PI-PLC treated oocytes. Arrows indicate a 25-40 kDa (pi 5-6) protein cluster which has a mass and isoelectric point similar to that which is released from the oocyte following PI-PLC treatment. Asterisk indicates PI-PLC isoforms.
  • the present invention relates to identification of egg surface proteins, that can be used as immunogens in a vaccine preparation to aid in the modulation of fertility.
  • the invention is directed to mouse egg plasma membrane polypeptides, herein called M70 polypeptides, that are involved in sperm-egg fusion.
  • M70 polypeptides ofthe invention encompass polypeptides which can be isolated from mouse egg which: 1) are located on the egg plasma membrane; 2) have a molecular weight of 70kDa; 3) have a pi of 5; 4) possess a covalently linked glycosylated phosphatidylinositol moiety; and 5) are specifically released from the egg plasma membrane upon treatment with phosphatidylinositol-specific phospholipase C (PI-PLC).
  • Other activities associated with such M70 polypeptides are antigenicity (ability to bind to an anti-M70 antibody or compete with M70 for binding), immunogenicity (ability to generate antibody which binds to M70).
  • H25/40 polypeptides migrate on polyacrylamide gels as a protein with a molecular weight of between 25 and 30 kDa, or between 30 and 35kDa, or between 36 and 40 kDa.
  • the H25/40 polypeptides migrate on polyacrylamide gels as a protein with a molecular weight of 25 kDa, 26kDa, 27 kDa, 28 kDa, 29 kDa, 30 kDa, 31 kDa, 32kDa, 33 kDa, 34 kDa, 35 kDa, 36kDa, 37 kDa, 38 kDa, 39 kDa, and 40 kDa.
  • the invention relates to GPI-linked ZP3 polypeptide, or fragment or analog thereof.
  • ZP3 is a well known, highly conserved, zona pellucida polypeptide.
  • GPI-linked ZP3 is useful as a vaccine for sterilization or contraception of an animal or human subject.
  • An antigenic fragment of GPI-linked ZP3 can comprise a GPI moiety linked to 1-10 amino acids, 10-30 amino acids, 30- 80 amino acids, 50-100 amino acids, 100-200 amino acids, or 200-450 amino acids of ZP3.
  • the invention provides isolated M70, M35/40, and H25/40 polypeptide antigens.
  • the invention provides purified M70, M35/40, and H25/40 polypeptide antigens.
  • the GPI-linked ZP3 ofthe invention encompasses a GPI-linked ZP3 homolog or ortholog from any species.
  • a GPI-linked egg surface protein can comprise (A) the amino acid sequence shown in Fig. 12B (SEQ ID NO:2), or, (B) an amino acid sequence encoded by a DNA sequence that hybridizes to the complement ofthe DNA sequence of Fig.
  • egg plasma membrane polypeptides such as M70, M35/45, H25/40, and GPI-linked ZP3, bind to a sperm protein to facilitate sperm-egg fusion.
  • Egg plasma membrane polypeptides involved in sperm-egg fusion can be prepared for a variety of uses.
  • such polypeptides, or peptide fragments thereof can be used for the generation of antibodies, for use in diagnostic and therapeutic assays, or for the identification of agents or small molecules that modulate sperm-egg fusion process required for fertilization.
  • M70, M35/45, H25/40, and GPI-linked ZP3 can be purified from any cell type that expresses such polypeptides, such as mouse or hamster egg cells.
  • the M70, M35/40, and H25/40 polypeptides are purified from a fraction of an extract of such cells enriched for cell membrane components.
  • egg plasma membrane polypeptides, such as M70, M35/40, and H25/40 proteins can be solubilized from cells, or cell extracts, such as a cell membrane fraction.
  • protein "spots" that appear in samples from normal eggs, but are absent in PI-PLC treated eggs can be analyzed further. Differences can be detected by visual inspection of gels, or by using densitometry and computerized image analysis thereby facilitating spot detection, background subtraction and spot matching (see Pennigton et al., 1997, Trends Cell Biol. 7: 168-73). Further, egg plasma membrane polypeptides, such as M70, M35/40, or H25/40 protein can be detected by Western Blot analysis of 2D gels, if antibody is available (Harlow and Lane, 1988, supra).
  • the molecular weight (MW) and the isoelectric point (pi) of an egg surface protein can be determined by calibrating its position relative to known standards run in parallel on 2D gels. Specific proteins can then be purified, and their sequence determined, or a portion of their sequence determined, by techniques well known in the art, such as Edman degradation sequencing (Edman and Begg, 1967, Eur. J. Biochem. 1 :80-91; see, e.g., Creighton, 1983, "Proteins: Structures and Molecular Principles," W.H. Freeman & Co., N.Y., pp.34-49), automated by electroblotting onto polyvinylidene difluoride (PVDF) membranes using Edman degradation chemistry
  • proteins and peptides can be characterized by mass spectrometry, using peptide-mass fingerprinting or protein sequencing methodologies to identify sequence information and post-translational modifications (Dainese et al., 1997, Electrophoresis,
  • the amino acid sequence obtained may be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for gene sequences encoding such proteins.
  • sequences of egg plasma membrane polypeptides involved in sperm-egg fusion can be derived by deduction from the DNA sequence if such is available, or alternatively, by direct sequencing ofthe protein, e.g., with an automated amino acid sequencer.
  • the protein sequences can be further characterized by a hydrophilicity analysis (Hopp and Woods, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-
  • a method ofthe invention for isolating M70, M35/40, or H25/40 protein comprises the steps of: a) preparing an extract of egg cells; b) contacting a M70, M35/40, or H25/40 specific antibody with the extract for a time period sufficient for the M70, M35/40, or H25/40 in the extract to bind the antibody; and c) recovering the bound antibody.
  • the method can also be used with an antibody that comprises an affinity tag. Accordingly, the method further comprises incubating the extract and the tagged antibody to the M70, M35/40, or H25/40 protein with a solid phase surface containing a binding partner ofthe affinity tag for a time period sufficient to allow binding ofthe egg plasma membrane polypeptides, such as M70, M35/40, or H25/40 to the solid phase surface prior to the recovery step. The isolated protein can then be eluted from the antibody.
  • Egg plasma membrane polypeptides such as M70, M35/40, and H25/40 can be purified by isolating cell membranes and purifying the egg plasma membrane polypeptides from other membrane components.
  • Membranes can be isolated from the egg cells ofthe invention, prepared according to the methods described in Section 6, below. Cells can be lysed and the plasma membrane fraction can be isolated from cells using procedures known in the art, such as dextran/polyethylene glycol biphase separation.
  • Plasma membranes can be treated with a buffer which dissociates membrane-associated proteins from the lipid bilayer (e.g., a buffer containing a non-ionic detergent such as Nonidet P-40TM, Triton X-100TM, or sodium deoxycholate). Proteins can be purified away from membrane lipids using conventional dialysis procedures.
  • the crude dialysed protein preparation can be applied to an affinity column composed of antibody or antiserum stabilized on an appropriate matrix.
  • Membrane-associated proteins other than M70, M35/40, or H25/40 will pass through the column, while M70, M35/40, or H25/40 will remain bound.
  • Non-specific binding of other membrane components can be reduced by increasing the salt concentration and varying the pH ofthe buffer in which the crude protein preparation is dissolved. Thorough washing of the column after application ofthe crude protein preparation can further reduce binding of non-specific proteins.
  • egg plasma membrane polypeptides such as M70, M35/40, or H25/40 protein may be purified using specific antibodies, previously generated against M70, M35/40, or H25/40 protein (see Section 5.7, infra), or cell membranes.
  • the crude protein preparation from membrane dialysis is applied to an antibody affinity column which is composed of M70, M35/40, or H25/40-specific antibody, stabilized on an appropriate matrix.
  • Antibody-coupled resin, or filter methods can also be used, or other antibody affinity techniques known in the art (see, for e.g., Harlow and Lane, 1988, supra).
  • the column or resin can be washed with buffer to remove proteins which bind non- specifically.
  • the protein which remains bound to the column is eluted by conventional procedure such as washing with a buffer containing high salt or low pH.
  • egg plasma membrane polypeptides such as
  • egg plasma membrane polypeptide nucleotide sequences ofthe invention can be isolated directly from mRNA, cDNA or from a cDNA or genomic library.
  • egg plasma membrane polypeptide cDNA can be isolated indirectly by first isolating and characterizing the egg plasma membrane polypeptide protein, and subsequently using the egg plasma membrane polypeptide sequence to identify gene sequences in a cDNA or genomic library. Details of such methods are fully described herein.
  • Nucleic acids encoding egg plasma membrane polypeptides involved in sperm-egg fusion can be isolated by a variety of methods well known to those of skill in the art, including, but not limited to: screening a cDNA expression library in mammalian cells using egg plasma membrane polypeptide specific antibodies, or "panning"; screening a cDNA expression library in bacterial cells; or differential expression methods such as screening a subtracted cDNA library with an egg plasma membrane polypeptide nucleic acid probe or a specific antibody.
  • oligo-dT matrices in different configurations may also be used, including but not limited to, simple gravity columns, para-magnetic particles, and spin columns.
  • Substituted oligo-dT such as biotinylated oligo-dT, may also be used.
  • the quantity and quality of RNA thus obtained may be determined by methods such as formaldehyde agarose gel electrophoresis. The use of RNA enriched for poly-A+ RNA is most preferred.
  • RNA into double-stranded cDNA can be accomplished by a number of different procedures well known in the art. See for example, Okayama and Berg, 1982, Mol. Cell Biol. 2:161-170; Gubler and Hoffman, 1983, Gene 25:263-269; and Huse and Hansen, 1988, Strategies (Stratagene) 1:1-3.
  • the first step in the making of cDNA involves the oligonucleotide-primed synthesis of a first strand cDNA by reverse transcriptase.
  • mRNA hybridized to an oligo-dT primer can be copied into DNA by a reverse transcriptase, such as AMV reverse transcriptase, MMLV reverse transcriptase, or Superscript (Kotewicz et al, 1988, Nucleic Acid Res. 16:265-277). Random hexamers may be used to prime first-strand synthesis from internal sites within the mRNA instead of oligo-dT primers resulting in shorter cDNAs which are enriched for the 5' ends of long messenger RNAs.
  • a reverse transcriptase such as AMV reverse transcriptase, MMLV reverse transcriptase, or Superscript (Kotewicz et al, 1988, Nucleic Acid Res. 16:265-277). Random hexamers may be used to prime first-strand synthesis from internal sites within the mRNA instead of oligo-dT primers resulting in shorter cDNAs which are enriched for the 5' ends of long messenger RNAs.
  • the next step in the process involves synthesizing the second strand cDNA
  • the second strand cDNA may be synthesized using E. coli DNA polymerase I, Klenow fragment using the RNA-DNA as a template.
  • the RNA in the RNA-DNA hybrid can be removed with RNase H, and gaps in the newly synthesized second strand cDNA can be filled in by E. coli DNA polymerase I.
  • the fragments of second strand cDNAs thus
  • E. coli DNA ligase are ligated with E. coli DNA ligase to form a contiguous second strand cDNA.
  • the double stranded cDNA requires further repair with enzymes, such as RNase H, RNase A, T4 DNA polymerase and E. coli DNA ligase, to form perfectly matched strands (i.e., having "flush” or "blunt” ends).
  • the cDNA can be amplified in vitro, by nucleic acid amplification methods known in the art, such as polymerase chain reaction (PCR) and ligation-mediated chain reaction (LCR).
  • PCR polymerase chain reaction
  • LCR ligation-mediated chain reaction
  • first strand oligo-dT primed cDNA obtained by a standard method is extended with a oligo-dG tail by terminal transferase, and a second primer containing a oligo-dC segment is used to prime second strand synthesis with a thermostable DNA
  • RT-PCR can be used to generate amplified cDNAs from the RNAs (see, e.g., Domec et al, 1990, Anal Biochem, 188:422-426; Van Gelder et al, 1990, Proc. Natl. Acad. Sci., 87:1663-1667).
  • oligonucleotides are end-labeled and used as primers in reverse-transcriptase polymerase chain reactions (RT-PCR) to generate a population of specific cDNAs. Products of such RT-PCR reactions are displayed on a sequencing gel. Using mRNAs derived from different populations of cells, the pattern of displayed products can be compared to identify bands that are unique to different cell types (Liang and Pardee, 1995, Curr. Opin. Immunol, 7:274-280; McClelland, M. et al., 1995, Trends Genet., 11, 242-246). mRNA from eggs is compared to mRNA from control cells by differential display.
  • Novel sequences can be chosen as potential egg plasma membrane polypeptide candidates. Such gene products can then be isolated from the cDNA population using standard cloning techniques (Ausubel et al, eds., 1992, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, New York), and can be tested for their ability to bind egg plasma membrane polypeptide antibodies.
  • nucleic acid array technology can be used to identify egg plasma membrane protein-specific sequences. Such micro-arrays of cDNA probes have been successfully used to compare the expression patterns of different cell types (DeRisi, et al, 1996, Nat. Genet.14:457-460).
  • mRNA derived from egg plasma membrane polypeptide positive hybrid cells and control cells is
  • cDNAs can be inserted into an appropriate cloning vector, and introduced into an appropriate host organism for propagation. Such cDNA libraries may then be used for preparation of "subtracted" cDNA libraries, or for direct and expression screening for egg plasma membrane polypeptide gene sequences. Methods for such procedures are well known in the art, and are described in standard treatises, e.g. , Methods in Enzymology,
  • a labeled degenerate oligonucleotide can be designed. Protein sequence information is used to design degenerate oligonucleotides containing all possible codons for egg plasma membrane polypeptide amino acids. Sequence information from various regions ofthe protein can be used to generate a series of such degenerate pools of oligonucleotides, where each oligonucleotide pool contains some sequences that are complementary to egg plasma membrane polypeptide gene sequences.
  • each oligonucleotide pool contains some sequences that are complementary in its entirety to egg plasma membrane polypeptide gene sequences.
  • degenerate oligonucleotide pools can be used to screen a gene library, prepared as described herein, supra.
  • the method comprises (a) incubating a labeled nucleic acid probe with DNA molecules derived from recombinant cells containing a plurality of DNA molecules from egg plasma membrane polypeptide positive hybrid cells, for a time period sufficient to allow hybridization ofthe labeled probe to the DNA molecules, wherein the labeled probe having a nucleic acid sequence that comprises a sequence that encodes egg plasma membrane polypeptide or a fragment thereof; (b) identifying the recombinant cell containing the DNA molecule to which the labeled probe bound; (c) recovering the DNA molecule present in the recombinant cell.
  • An expression construct refers to a polynucleotide comprising egg plasma membrane polypeptide positive hybrid cell derived cDNA sequences operably associated with one or more regulatory regions which enables expression ofthe library of cDNAs in an appropriate host cell. "Operably-associated” refers to an association in which the regulatory regions and the cDNA sequence to be expressed are joined and positioned in such a way as to permit transcription, and ultimately, translation.
  • the regulatory regions necessary for transcription of the cDNA library can be provided by an expression construct.
  • a translation initiation codon (ATG) may also be provided if the cDNA fragments without their cognate initiation codon are to be expressed.
  • Such a library of cDNA expression constructs can be amplified and maintained in vitro, without the use of DNA sequences that propagate the polynucleotide within living cells. Depending on needs, an aliquot ofthe cDNA expression library can be thawed and introduced directly into host cells. Such expression constructs can be used for expression of cDNAs transiently in recombinant host cells.
  • An expression vector is a cloning vector that can be used for maintenance and expression of cDNA library in an appropriate host cell. Any cloning vector known in the art can be used to propagate the cDNA library.
  • a variety of cloning vectors may be used in the present invention which include, but are not limited to, plasmids, cosmids, phage, phagemids, or modified viruses.
  • such cloning vectors comprise a functional origin of replication for propagation ofthe vector in an appropriate host cell, one or more restriction endonuclease sites for insertion ofthe cDNA library, and one or more selection markers.
  • the cloning vector must be used with a compatible host cell which may be derived from a prokaryotic or an eukaryotic organism including but not limited to bacteria, yeasts, insects, mammals, and humans.
  • Host cells broadly encompass cells of unicellular organisms, such as bacteria, fungi, and yeast, and of multicellular organisms, such as insects and animals including but not limited to birds, mammals and humans. Host cells may be obtained from private laboratory deposits, public culture collections such as the American Type Culture Collection, or from commercial suppliers.
  • cDNA expression cloning in a eukaryotic host is advantageous because egg plasma membrane polypeptide(s) can be post-translationally modified and correctly inserted into the plasma membrane. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of cDNA-encoded proteins may enhance egg plasma membrane polypeptide activity.
  • an exemplary expression host-vector system is ⁇ DR2 which is a lambda bacteriophage-based cloning vector coupled with a mammalian expression plasmid.
  • Advantages of this system include the utilization of highly efficient lambda in vitro packaging systems for initially generating a library in E. coli hosts. Size selection may not be required since the packaging system only accepts inserts in a certain size range. Lambda vectors generally provide greater ease in amplification and storage.
  • the initial library in E. coli may be amplified to produce supercoiled plasmid DNA which may be used in high efficiency transformation methods for introduction into other expression host organisms.
  • cDNA expression in a host cell may be enhanced by the inclusion of appropriate transcription enhancer elements in the expression vector, such as those found in SV40 virus, Hepatitis B virus, cytomegalovirus, immunoglobulin genes, metallothionein, ⁇ -actin (see Bittner et al, 1987, Methods in Enzymol. 153:516-544; Gorman, 1990, Curr. Op. in Biotechnol. 1:36-47).
  • appropriate transcription enhancer elements such as those found in SV40 virus, Hepatitis B virus, cytomegalovirus, immunoglobulin genes, metallothionein, ⁇ -actin (see Bittner et al, 1987, Methods in Enzymol. 153:516-544; Gorman, 1990, Curr. Op. in Biotechnol. 1:36-47).
  • adenine phosphoribosyltransferase (Lowy et al, 1980, Cell 22:817) genes can be employed in tk “ , hgprt " or aprt " cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for dihydrofolate reductase (dhfr), which confers resistance to methotrexate (Wigler et al, 1980, Natl. Acad. Sci. USA 77:3567; O'Hare et al, 1981, Proc. Natl. Acad. Sci.
  • vectors based on the Epstein-Barr virus (EBV) origin (OriP) and EBV nuclear antigen 1 (EBNA-1; a trans-acting replication factor) can be used.
  • EBV Epstein-Barr virus
  • EBNA-1 EBV nuclear antigen 1
  • Such vectors can be used with a broad range of human host cells, e.g., EBO-pCD (Spickofsky et al, 1990, DNA Prot Eng Tech 2:14-18); pDR2 and ⁇ DR2 (available from Clontech Laboratories).
  • Autographa californica nuclear polyhydrosis virus (AcNPV) a baculovirus
  • AcNPV Autographa californica nuclear polyhydrosis virus
  • the cDNA sequences may be cloned into non-essential regions (for example the polyhedrin gene) ofthe virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • AcNPV promoter for example the polyhedrin promoter
  • the recombinant host cells may be cultured under standard conditions of temperature, incubation time, optical density, and media composition.
  • Expression constructs containing cloned cDNA can be introduced into the host cell by a variety of techniques known in the art, including but not limited to, for prokaryotic cells, ⁇ -phage packaging and infection, bacterial transformation (Hanahan, 1985, in DNA Cloning, A Practical Approach, 1:109-136), and for eukaryotic cells, calcium phosphate mediated transfection (Wigler et al, 1977, Cell 11:223-232), liposome-mediated transfection (Schaefer-Ridder et al, 1982, Science 215:166-168), electroporation (Wolff et al, 1987, Proc. Natl. Acad. Sci. 84:3344), and microinjection (Cappechi, 1980, Cell 22:479-488).
  • ovarian-derived cell line may be used. It is preferable that the type of host cell is non-adherent to surfaces of cell culture containers, such as plastic, so as to facilitate screening methods and harvesting ofthe cells.
  • an SV40 vector and control sequences are utilized, and the resulting cDNA library is introduced into African green monkey cells (COS cells).
  • the cDNA library can be constructed in a vector containing viral control regions, and introduced in mammalian cells by transfection or infection with viral vectors. Cells are distributed on microtiter dishes for screening.
  • the cDNA library can be transiently expressed in mammalian cells.
  • the cDNA used in constructing the library is prepared from mRNA isolated from the egg plasma membrane polypeptide positive hybrid cells ofthe invention.
  • each oligonucleotide pool contains some sequences that are
  • Non-limiting examples of regulatory regions that can be used for expression in E. coli may include but not limited to lac, trp, lpp, phoA, recA, tac, T3, T7 and ⁇ P L (Makrides, 1996, Microbiol Rev, 60:512-538).
  • Non-limiting examples of prokaryotic expression vectors may include the ⁇ gt vector series such as ⁇ gtl 1 (Huynh et al, 1984 in "DNA Cloning Techniques", Vol. I: A Practical Approach (D. Glover, ed.), pp. 49-78, IRL
  • Expression constructs containing cloned cDNA can be introduced into the prokaryotic host cell by a variety of techniques known in the art, including but not limited to, ⁇ -phage packaging and infection, transduction and transformation (Hanahan, 1985, in DNA Cloning, A Practical Approach, 1 : 109- 136). Bacteria is infected with phage or transformed with plasmid carrying the cDNA library, plated on LB agar plates, and induced to express cDNA inserts.
  • a potential drawback of a prokaryotic host-vector system is the inability to perform many ofthe post-translational processing, folding and insertion into membranes normally required of cell surface receptors.
  • a specific cDNA insert can be detected and isolated by inducing expression ofthe cDNA inserts and utilizing screening methods that rely on detection of protein activity. Such methods include filter binding to a labeled ligand or immunological methods to detect antibody binding.
  • nucleotide sequence coding for an egg surface polypeptide protein or a functionally active analog or fragment or other derivative thereof can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation ofthe inserted protein-coding sequence.
  • the necessary transcriptional and translational signals can also be supplied by the native egg surface polypeptide gene and/or its flanking regions.
  • host- vector systems can be utilized to express the protein-coding sequence.
  • any ofthe methods previously described for the insertion of DNA fragments into a vector can be used to construct expression vectors containing a chimeric gene comprising of appropriate transcriptional/translational control signals and the protein coding sequences. These methods can include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of a nucleic acid sequence encoding an egg surface polypeptide protein or peptide fragment can be regulated by a second nucleic acid sequence so that the egg surface polypeptide protein or peptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of an egg surface polypeptide protein can be controlled by any promoter/enhancer element known in the art.
  • a host cell strain can be chosen which modulates the expression ofthe inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression ofthe genetically engineered egg surface polypeptide protein can be controlled.
  • different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g. , glycosylation, phosphorylation of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing ofthe foreign protein expressed. For example, expression in a bacterial system can be used to produce a non-glycosylated core protein product. Expression in yeast will produce a glycosylated product.
  • polyclonal antibodies to an egg surface protein or derivative or analog thereof may be obtained.
  • various host animals including but not limited to rabbits, mice, rats, etc can be immunized by injection with the native egg surface proteins, or a synthetic version, or derivative (e.g., fragment) thereof.
  • Antibody fragments which contain the idiotype ofthe molecule can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion ofthe antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges ofthe F(ab') 2 fragment, the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent, and Fv fragments.
  • Antibodies specific to a domain of an egg surface proteins are also provided.
  • the foregoing antibodies can be used in methods known in the art relating to the localization and activity ofthe egg surface proteins ofthe invention, e.g., for imaging these proteins, measuring levels thereof in appropriate physiological samples, in diagnostic methods, etc.
  • the egg surface proteins ofthe invention are involved in mediating sperm- egg fusion via a direct interaction between egg surface proteins and a sperm ligand.
  • the present invention relates to in vitro and in vivo assay systems, described in the subsections below, which can be used to identify compounds or compositions that modulate the activity of egg surface polypeptide and its interaction with a sperm ligand.
  • Such molecules such as peptides or non-protein molecules, including organic or inorganic small molecules, large molecules, antibodies, and nucleotide sequences may bind egg surface polypeptide with differing affinities.
  • Such molecules can serve as powerful modulators of fertilization in vivo, and can be used therapeutically to modulate the fertility.
  • the screening assays ofthe present invention may also be used to identify compounds or compositions that modulate the interaction of egg surface polypeptides with its binding partners, as identified herein.
  • recombinant cells expressing egg surface polypeptide nucleic acids can be used to recombinantly produce an egg surface polypeptide in these assays, to screen for molecules that bind to the egg surface polypeptide. Similar methods can be used to screen for molecules that bind to egg surface polypeptide derivatives or nucleic acids. Methods that can be used to carry out the foregoing are commonly known in the art.
  • Sequences for more than one oligonucleotide can be combined to test for even higher affinity binding to egg surface polypeptide. Knowing which amino acid sequences confer the strongest binding to egg surface polypeptide, computer models can be used to identify the molecular contacts between egg surface polypeptide and ligand. This will allow the design of non- protein compounds which mimic those contacts. Such a compound may have the same activity ofthe peptide and can be used therapeutically, having the advantage of being efficient and less costly to produce.
  • host cells to which the test compound is added may be genetically engineered to express egg surface polypeptide and its target interactor (such as an egg surface polypeptide antibody or an egg surface polypeptide ligand) which may be transient, induced or constitutive, or stable.
  • target interactor such as an egg surface polypeptide antibody or an egg surface polypeptide ligand
  • a wide variety of host cells may be used including, but not limited to, tissue culture cells, mammalian cells, yeast cells, and bacteria. Each cell type has its own set of advantages and drawbacks.
  • Mammalian cells, such as egg cells which express the egg surface polypeptides ofthe invention may be a preferred cell type in which to carry out the assays ofthe present invention. Bacteria and yeast are relatively easy to cultivate but process proteins differently from mammalian cells.
  • the invention provides for a method for detecting the interaction between egg surface polypeptide and a known potential ligand, such as a sperm integrin.
  • a known potential ligand such as a sperm integrin.
  • Insect cells can be infected with baculoviruses co-expressing egg surface polypeptide and the known sperm integrin, and cell extracts can be prepared and analyzed for protein-protein interactions. Protein-protein interactions can be analyzed by methods known in the art, such as Western blotting or immune precipitation using egg surface polypeptide specific antibodies together with an anti-integrin antibody, and analyzing complexes by polyacrylamide gel electrophoresis.
  • Purified or partially purified components which have been determined to interact with one another by the methods described above can be placed under conditions in which the interaction between them would normally occur, with and without the addition of the test agent, and the procedures previously established to analyze the interaction can be used to assess the impact ofthe test agent.
  • the purified or partially purified components may be prepared by fractionation of extracts of eggs, or they may be obtained by expression of cloned genes or cDNAs or fragments thereof, optionally followed by purification ofthe expressed material.
  • Mouse or hamster egg surface proteins can be used in any of a variety of vaccines in human and non-human animals.
  • the present invention encompasses vaccines useful for contraception.
  • egg surface polypeptides involved in sperm-egg fusion such as M70, M35/45, H25/40, and GPI-linked ZP3, are delivered to a subject to elicit an active immune response.
  • the vaccine acts as a temporary and reversible antagonist ofthe function ofthe egg surface proteins ofthe invention.
  • such vaccines could be used for active immunization of a subject, to raise an antibody response to temporarily block the sperm's access to the egg plasma antigen.
  • an antigen could be administered at a certain period ofthe month, for example during ovulation of a female subject to block fertilization.
  • Pharmaceutically acceptable carriers include but are not limited to saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof.
  • a physiologically balanced culture medium containing one or more stabilizing agents such as stabilized, hydrolyzed proteins, lactose, etc.
  • the carrier is preferably sterile. The formulation should suit the mode of administration.
  • composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • an ampoule of sterile diluent can be provided so that the ingredients may be mixed prior to administration.
  • the vaccine formulations ofthe present invention can also be used to produce antibodies for use in passive immunotherapy, in which short-term protection of a host is achieved by the administration of pre-formed antibody directed against a heterologous organism.
  • the antibodies generated by the vaccine formulations ofthe present invention can also be used in the production of antiidiotypic antibody.
  • the antiidiotypic antibody can then in turn be used for immunization, in order to produce a subpopulation of antibodies that bind the initial antigen ofthe pathogenic microorganism (Jerae, 1974, Ann. Immunol. (Paris) 125c:373; Jerne, et al, 1982, EMBO J. 1:234).
  • the amount of immunogen to be used and the immunization schedule will be determined by a physician skilled in the art and will be administered by reference to the immune response and antibody titers ofthe subject.
  • the gametes were co-incubated for 1 h, washed gently 3 times in M199, and fixed in PBS containing 4% paraformaldehyde for 1 h.
  • the oocytes were placed in a phosphate buffered saline (PBS)/4% paraformaldehyde solution between a microscope slide and an elevated cover slip.
  • PBS phosphate buffered saline
  • the oocytes were visualized at 200X using a light microscope (Zeiss Axioplan) and a single focal plane for each oocyte was selected in which the widest diameter ofthe zona pellucida could be visualized. The number of bound sperm in that focal plane was then determined.
  • the eggs were then split into two groups of 100 and either mock treated or treated with lU/ml PI-PLC in 20 ⁇ l drops for 30 min.
  • the supernatants were removed, the eggs were washed six times, and the oocytes and the oocyte supernatants were then frozen at -70 °C in BWW/PVA containing protease inhibitors (CompleteTM, Boehringer Mannheim, Mannheim, Germany).
  • the maximal inhibitory effect on sperm-egg binding was reached at 5U/ml while the maximal inhibitory effect on fusion occurred at lU/ml.
  • the recommended dose for releasing most GPI-anchored proteins from intact cells using B. thuringiensis-de ⁇ ved PI-PLC is lU/ml.
  • the dose-dependent inhibition of both sperm-egg binding and fusion by PI-PLC supports the hypothesis that treating oocytes with PI-PLC releases GPI-anchored proteins from the oolemma which are required for fertilization.
  • a bead binding experiment was performed to determine if PI-PLC treatment of zona-free mouse oocytes affected the antibody recognition of a well characterized egg surface integrin. Fluorescent beads were coated with ⁇ 6 ⁇ l antibodies and incubated with either untreated or PI-PLC treated oocytes. No difference was observed in the number of ⁇ l antibody-coated beads bound per oocyte between the control (Fig. 6A) and treatment group (Fig. 6B). Minimal bead binding was observed when beads were coated an with equivalent concentration of an irrelevant antibody and incubated with untreated eggs (Fig. 6C).
  • PI-PLC sensitive proteins The precise function of these PI-PLC sensitive proteins in the fertilization process is unknown at this time.
  • One possibility is that the molecules are involved in the block to polyspermy as is the Ascidian GPI-anchored molecule, N-acetylglucosaminidase (Lambert, 1989, Development 105: 415-420).
  • Ascidians sperm first bind to the ligand, N-acetylglucosamine, on the vitelline coat (VC), then penetrate through the VC and the perivitelline space to reach the egg surface, where sperm-egg fusion then occurs (Rosati and De Santis, 1980, Nature 283: 762-764).
  • sperm-egg binding and fusion - Following gamete co- incubation loosely bound sperm were removed from the oocytes by gentle pipetting. The eggs were then treated with ImM acridine orange-3% in DMSO (Sigma) for 15 sec to stain the chromatin and washed through three 20 ⁇ l microdrops. To quantitate binding, the oocytes were placed between a microscope slide and an elevated cover slip, the oocytes were visualized at 200X using a light microscope (Zeiss Axioplan) and the number of sperm bound per oocyte was recorded. The number of sperm fused per egg was scored by counting the number of acridine orange-stained decondensed sperm heads within each oocyte using fluorescent microscopy. Artificial activation of oocytes
  • the supernatants were collected from the two groups, the eggs were washed, and the egg proteins were extracted.
  • the egg protein extracts and the proteins from the supernatants were separated by 2-D electrophoresis and electroblotted to nitrocellulose membranes.
  • the membranes were then stained with Protogold to visualize the egg proteins.
  • the membranes were probed with strepavidin-HRP and the biotinylated egg surface proteins were visualized using TMB membrane peroxidase substrate.
  • the repertoire of zona-free hamster egg proteins is shown in Fig. 11 A with over one hundred egg proteins being resolved following Protogold staining (red staining).
  • the enzyme appears to be specifically affecting the 25-40 kDa protein cluster (c2) because the staining intensity ofthe remaining surface labeled proteins in Fig. 11C is similar to that which is seen in the untreated eggs in Fig. 11A.
  • the predominant protein cluster (c2) is PI-PLC sensitive while the less prominent protein cluster (cl) is not affected by PI-PLC treatment (see Fig. 1 IB).
  • both the 70 kDa and 35-40 kDa protein clusters are PI-PLC sensitive (see Example in Section 6).
  • ZP3 is responsible for primary binding ofthe egg to a receptor(s) on the sperm (reviewed in McLeskey et al., 1998, Int. Rev. of Cytol. 177: 57-113). This binding event also initiates the acrosome reaction in which hydrolytic enzymes are released from the acrosomal compartment and act on the zona pellucida to facilitate penetration ofthe zona pellucida by sperm.
  • M70 is a membrane-associated GPI-linked form of ZP3.
  • Either zona-free mouse oocytes or mouse zona pellucidae were extracted in Triton-Xl 14 and the detergent and aqueous phases were isolated. The samples were subjected to SDS-PAGE and Western blot analysis using the monoclonal antibody (mAb) (IE- 10).
  • mAb monoclonal antibody
  • Figure 13 A the oolemmal form of ZP3 partitions in the detergent phase. This indicates that oolemmal ZP3 is associated with the membrane and contains a membrane-associated domain.
  • a novel sequence of a novel egg surface protein has been identified. These sequences can be used as antigenic molecules for the compositions and methods ofthe present invention.
  • Microsequence data was obtained using LC-MS tandem mass spectrometry (Wilm et al . 1996). To ensure that sufficient protein concentrations would be available for this sequencing experiment, proteins from approximately 1300 zona-free oocytes were separated on a 2-D minigel and the gel was Coomassie-stained. The selected surface- labeled proteins spots were then cored from the minigel and submitted to the Mass Spectrometry Laboratory for microsequence analysis.
  • the specificity ofthe rabbit polyclonal antibody to full length recombinant calreticulin used for the subsequent assays was confirmed by probing a 2-D blot of zona- free mouse oocytes. The blot was first stained with Protogold to visualize oocyte proteins (Fig. 3A). Immunoblotting revealed that the calreticulin antibodies are highly specific and recognize only two oocyte proteins, calreticulin and calnexin (Fig. 3B, arrows). It is not surprising that the calreticulin polyclonal antibody also recognized calnexin because calnexin is the membrane bound homologue of calreticulin.
  • calreticulin antibodies were then investigated in an in vitro fertilization assay.
  • Cumulus-free zona-intact mouse oocytes were either not treated with antibodies or treated with a 1 :50 dilution of normal rabbit sera or anti-calreticulin antibodies.
  • the eggs were then washed and inseminated with untreated mouse epididymal sperm. Following overnight incubation, oocytes which had not undergone cleavage were scored as unfertilized and oocytes which bad cleaved were scored as fertilized.
  • Significantly fewer (p ⁇ 0.05) oocytes underwent cleavage following incubation with calreticulin antibodies (1.3%) when compared to oocytes which were either not treated (68%) or treated with normal rabbit sera (75%).

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