WO1999064457A1

WO1999064457A1 - Mammalian sperm protein pkdrej with homology to polycystin-1

Info

Publication number: WO1999064457A1
Application number: PCT/GB1999/001839
Authority: WO
Inventors: Peter Charles Harris; James Raymond Hugues; Christopher James Ward
Original assignee: Medical Research Council
Priority date: 1998-06-10
Filing date: 1999-06-10
Publication date: 1999-12-16
Also published as: GB9812534D0; AU4281099A

Abstract

The present invention relates to a functional mammalian acrosome reaction protein or functional fragment thereof having the sequence given in figure 1. The present invention also relates to nucleic acid sequences encoding the protein and to assays using the protein or nucleic acid sequences as well as therapeutic uses of the protein or nucleic acid sequences.

Description

MAMMALIAN SPERM PROTEIN PKDREJ WITH HOMOLOGY TO POLYCYSTTN-1

The present invention relates to a sperm protein, which is associated with recognising the egg and in triggering the acrosome reaction. The present invention also relates to nucleic acid sequences encoding the protein and to assays using the protein or nucleic acid sequences as well as therapeutic uses of the protein or nucleic acid sequences.

When a sperm contacts an egg, the acrosome reaction takes place. The acrosome reaction is an exocytic remodelling of the sperm outer membrane necessary for fertilisation and is associated with an influx of Ca²⁺ triggered by interaction with the egg glycoprotein coat (1,2).

The initial interactions at the surface of the egg essentially go unnoticed by the egg; however, the acrosome reaction that they initiate is critical for transforming the sperm into a fusogenic cell. The acrosome reaction brings about remodelling of the sperm surface, with a portion of the original plasma membrane being replaced by newly exposed inner acrosomal membrane and releasing hydrolytic enzymes, presumed to be required for the sperm to penetrate the zona pellucida (ZP). The exact mechanism by which the acrosome reaction occurs is not known. Several sperm proteins have been implicated in the initial recognition event (4-7) but the evidence for each remains unproven and controversial (2, 8-10 and 12).

In sea urchin, fertilisation is better characterised. A 210 kDa sperm integral membrane protein has been found to be the receptor for egg jelly (suREJ) and triggers the acrosome reaction on contact with egg glycoprotein (13, 14). There is no suggestion in the prior art that a corresponding protein exists in mammals.

The suREJ protein does not have significant homology with the protein of the present invention. In fact the most similar region of the suREJ protein to the protein of the present invention has less than 30% homology. Furthermore, one skilled in the art trying to identify proteins having significant homology with the suREJ would have identified numerous other proteins not involved in sperm/egg interactions having greater homology than the protein of the present invention. The present invention was arrived at in the course of work relating to identifying polycystin-like molecules based on homology of voltage-gated channel-like subunits of polycystin-1 and -2. Polycystin -1 and -2 are autosomal dominant polycystic kidney disease (ADPKD) proteins and are functionally totally unrelated to proteins involved in sperm/egg interactions.

As indicated herein, a protein was identified having a homologous integral membrane section comprising what appear to be voltage gated channel subunits; however, the protein was not expressed ubiquitously in human organs like the polycystin molecules. It was surprisingly found that the protein was expressed in the testis. At this point the function of the protein was further investigated and the protein was found to be a mammalian acrosome reaction protein.

The present invention provides a functional mammalian acrosome reaction protein or functional fragment thereof having an amino acid sequence at least 30% homologous to the amino acid sequence of figure 1.

The term "a functional mammalian acrosome reaction protein " means a protein associated with the mammalian acrosome reaction and preferably means a protein sufficient and/or necessary for the mammalian acrosome reaction to occur. The acrosome reaction is the remodelling of the acrosome layer of the sperm head that contains acid hydrolases and leads to the breakdown of the outer membrane of the egg. In a preferred embodiment of the present invention the functional mammalian acrosome reaction protein is involved in binding the sperm to the egg and/or triggering the acrosome reaction. In a further preferred embodiment of the present invention, the functional mammalian acrosome reaction protein is necessary for the binding of the sperm to the egg and/or for the remodelling of the acrosome layer.

The term "mammalian" as used herein encompasses all mammals including humans, rodents such as mice and rats, cats, dogs and cattle. Preferably, the term means humans and mice. The term "a functional fragment" as used herein means a part of the acrosome reaction protein that still retains at least part of the function of the complete acrosome reaction protein. Preferably the functional fragment is at least 20%, more preferably, at least 40% and most preferably at least 50% of the size of the complete acrosome reaction protein.

The term "30% homologous" as used herein means the acrosome reaction protein of the present invention has at least 30% homology at the amino acid level with the protein defined by the amino acid sequence of figure 1. Sequence homologies can be determined using commercially available software packages such as BLAST, FASTA and BESTFIT. Preferably the acrosome reaction protein of the present invention has at least 50%, more preferably at least 60%, still more preferably at least 80%, and most preferably, at least 90% homology with the protein defined in figure 1. A preferred acrosome reaction protein having at least 30% homology to the protein defined in figure 1 is the protein defined in figure 2, which has 64% homology.

Preferably the acrosome reaction protein having at least 30% homology to the protein defined in figure 1 is a species, allelic or splice variant of the protein defined in figure 1. The acrosome reaction protein having at least 30% homology to the protein defined in figure 1 may also be a protein wherein conservative amino acid changes have been made to the protein defined in figure 1. Conservative changes are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided in four families: (1) acidic - aspartate, glutamate; (2) basic - lysine, arginine, histidine; (3) non-polar - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar - glycine, asparagine, glutamine, cystine, serine, threonine and tyrosine. Phenylalanine, tryptophan and tyrosine are sometimes classified jointly as aromatic amino acids. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an asprartate with a glutamate, a threonine with a serine, or similar conservative replacements of an amino acid with a structurally related amino acid will not have a major effect on the biological activity of the protein. The functional acrosome reaction protein of the present invention may be obtained by the method described herein or may be obtained by standard recombinant DNA technology using standard procedures known to those skilled in the art. The amino acid sequence of two functional acrosome reaction proteins are given in figures 1 and 2, and the corresponding nucleotide sequences encoding the proteins are given in figures 3 and 4 respectively.

Functional fragments of the acrosome reaction protein can be obtained by standard recombinant DNA technology techniques known to those skilled in the art. The functional fragments can also be generated by enzymatic digestion or chemical cleavage of the complete acrosome reaction protein. The functional fragments can also be generated by chemical synthesis using commercially available synthesisers.

In vivo the acrosome reaction protein, or functional fragments thereof, of the present invention is likely to form part of a protein complex which is capable of binding the sperm to the egg and/or triggering the acrosome reaction. Accordingly, the present invention provides a protein complex comprising the acrosome reaction protein or a functional fragment thereof, of the present invention, which is capable of binding the sperm to the egg and/or remodelling of the acrosomal layer.

The present invention also provides an antibody having affinity for the acrosome reaction protein of the present invention or a functional fragment thereof. Preferably, the antibody is a monoclonal antibody, advantageously of IgG class, or an antigen binding fragment thereof, such as a Fab, F(ab')₂, Fv, single chain Fv or multivalent Fv. Methods for the preparation of antibodies and antibody fragments are well known in the art, for example, see Antibodies: A Laboratory Manual, 1988, eds. Harlow and Lane, Cold Spring Harbor Laboratories Press.

The antibodies of the present invention can be used in an assay for a functional acrosome reaction protein or a functional fragment thereof. A variety of assay techniques can be used and are well known to those of skill in the art. A number of suitable assays are described in US patent No. 3817837, 4006360 and 3996345. _ . The present invention also provides a method for identifying proteins, antibodies, peptides and other molecules which block the acrosome reaction by, for example, binding to the acrosome reaction protein and/or by inhibiting the ability of sperm to undergo the acrosome reaction on contact with ZP. The acrosome reaction can be measured in accordance with the procedures outlined by Harmann (3) and Vacquier (11).

In a preferred embodiment, the present invention provides a method for identifying proteins, antibodies, peptides and other molecules which block the acrosome reaction comprising: contacting either

(a) sperm containing the fimctional acrosome reaction protein; or

(b) mammalian ZP, with a candidate molecule under conditions so that the candidate molecule can bind to the sperm or the mammalian ZP; adding mammalian ZP to (a) or adding sperm containing functional mammalian acrosome reaction protein, to (b); and determining if there is a reduction in the acrosome reaction of the sperm.

If there is a reduction in the acrosome reaction of the sperm, then the candidate molecule blocks the acrosome reaction.

Proteins or molecules that block the acrosome reaction may be used as contraceptives.

The present invention also provides a method for identifying a molecule that triggers the acrosome reaction in the absence of ZP. Preferably, the method comprises adding a candidate molecule to sperm comprising the functional acrosome reaction protein of the present invention, and measuring any acrosome reaction in the absence of ZP. Preferably, the molecule also binds the acrosome reaction protein of the present invention.

The present invention also provides a method for identifying a molecule that enhances or improves the efficacy of the acrosome reaction protein of the present invention in initiating the acrosome reaction. Preferably, the method comprises adding a candidate molecule to sperm comprising the acrosome reaction protein of the present invention, wherein mammalian ZP may be present with the sperm prior to addition of the candidate molecule or may be added after the addition of the candidate molecule, and measuring for any improvement or enhancement of the acrosome reaction.

The molecule that enhances or improves efficacy of the acrosome reaction protein can be used to increase fertility.

The present invention also provides a nucleic acid sequence encoding the acrosome reaction protein of the present invention or a fragment thereof. The nucleic acid sequence may be isolated by screening a testis cDNA library with nucleic acid probes prepared by obtaining a peptide sequence from the acrosome reaction protein of the present invention. Expression of the nucleic acid sequence in an expression system selected from those available in the art results in the production of the acrosome reaction protein in pure form.

Preferably, the nucleic acid sequence is a mammalian sequence, more preferably a human or murine sequence.

Preferably, the nucleic acid sequence has the nucleotide sequence of figure 3 or figure 4, or a fragment thereof which encodes a functional fragment of the acrosome reaction protein of the present invention.

Preferably, the nucleic acid sequence of the present invention is DNA.

The present invention also provides a method for identifying nucleic acid sequences encoding mutated forms of the acrosome reaction protein of the present invention using fragments of the nucleic acid sequence of the present invention as probes or primers. Mutated forms of the acrosome reaction protein of the present invention can be defined as forms which have no function or have a reduced function leading to infertility, or an enhanced or altered function. The present invention further provides a nucleic acid vector for the expression of the acrosome reaction protein of the present invention, or a fragment thereof, comprising a promoter and a nucleic acid sequence encoding the acrosome reaction protein, or a fragment thereof.

The vector may additionally comprise other control elements such as enhancers, termination sequences, etc.

The present invention also provides a process for the production of the acrosome reaction protein of the present invention, or a fragment thereof, by recombinant DNA technology comprising: transforming a host cell with a nucleic acid vector of the present invention; expressing the nucleic acid sequence encoding the acrosome reaction protein of the present invention, or a fragment thereof, in the host cell; and recovering the acrosome reaction protein, or a fragment thereof, from the host cell or the host cell's medium.

The acrosome reaction protein of the present invention, or a fragment thereof, and/or mRNA encoding the protein, or a fragment thereof, is useful in in vitro fertilisation techniques performed in humans and other animals.

The present invention further provides the acrosome reaction protein of the present invention, or a fragment thereof, and/or the nucleic acid sequence of the present invention, for use in therapy.

The present invention further provides the use of the acrosome reaction protein of the present invention, or a fragment thereof, and/or the nucleic acid sequence of the present invention, in the manufacture of a composition for use in a fertility treatment. Preferably, the fertility treatment is IVF.

The present invention further provides a method for treating a mammal in need of treatment by administration of an effective amount of the acrosome reaction protein of the present invention, or a fragment thereof, and/or the nucleic acid sequence of the present invention. o

In a further aspect of the present invention a composition is provided comprising the acrosome reaction protein of the present invention, or a fragment thereof, and/or the nucleic acid sequence of the present invention, and a pharmaceutically acceptable diluent, carrier and/or excipient.

The present invention further provides the antibody of the present invention or a protein, peptide or other molecule capable of blocking the acrosome reaction for use in therapy.

The present invention further provides the use of the antibody of the present invention or a protein, peptide or other molecule capable of blocking the acrosome reaction in the manufacture of a composition for use as a contraceptive.

The present invention further provides a method for treating a mammal in need of treatment by administration of an effective amount of the antibody of the present invention or a protein, peptide or other molecule capable of blocking the acrosome reaction.

The present invention further provides a composition comprising the antibody of the present invention or a protein, peptide or other molecule capable of blocking the acrosome reaction, and a pharmaceutically acceptable diluent, carrier and/or excipient.

The present invention further provides a molecule that triggers the acrosome reaction in the absence of ZP for use in therapy.

The present invention further provides the use of a molecule that triggers the acrosome reaction in the absence of ZP in the manufacture of a composition for use in a fertility treatment.

The present invention further provides a method for treating a mammal in need of treatment by administration of an effective amount of a molecule that triggers the acrosome reaction in the absence of ZP. The present invention further provides a composition comprising a molecule that triggers the acrosome reaction in the absence of ZP and a pharmaceutically acceptable diluent, carrier and/or excipient.

The present invention further provides a molecule that enhances or improves the efficacy of the acrosome reaction protein for use in therapy.

The present invention further provides the use of a molecule that enhances or improves the efficacy of the acrosome reaction protein in the manufacture of a composition for use in a fertility treatment.

The present invention further provides a method for treating a mammal in need of treatment by administration of an effective amount of a molecule that enhances or improves the efficacy of the acrosome reaction protein.

The present invention further provides a composition comprising a molecule that enhances or improves the efficacy of the acrosome reaction protein and a pharmaceutically acceptable diluent, carrier and/or excipient.

The present invention also provides the use of the acrosome reaction protein of the present invention as an immunogen to raise an antibody response.

The present invention also provides a transgenic animal expressing the human acrosome reaction protein or a functional fragment thereof. Preferably the transgenic animal is a rat or a mouse. The transgenic animal can be used in in vivo screening procedures for molecules that block the acrosome reaction. Preferably, the transgenic animal is a "knock out" for the endogenous acrosome reaction protein, i.e. does not produce the endogenous acrosome reaction protein.

A further object of the present invention is the use of the nucleic acid sequence of the present invention, or a fragment thereof, in gene therapy. Preferably, the nucleic acid sequence encoding the acrosome reaction protein of the present invention, or a fragment thereof, is comprised within an expression vector which comprises a promoter and allows the expression of the protein, or a fragment thereof, in mammalian cells.

The expression vector is preferably a nucleic acid vector comprising DNA. The vector may be of linear or circular configuration and can be adapted for episomal or integrated existence in the host cell, as set out on the extensive body of literature known to those skilled in the art. Vectors can be delivered to cells using viral or non-viral delivery systems. The choice of delivery vehicle determines whether the nucleic acid sequence to be delivered is to be incorporated into the cell genome or remain episomal.

Preferably, the vector is to be delivered to cells involved in sperm cell production such as spermatogonia, spermatocytes, spermatids and spermatozoa. It is further preferred that the vector additionally comprises a locus control region (LCR), thereby ensuring that if the DNA is inserted in to the cell's genome it is inserted in a open state, allowing expression of the DNA sequence contained in the vector.

The present invention is now described below, for the purposes of exemplification only, with reference to the figures.

FIGURES:

Figure 1 shows the amino acid sequence of the human acrosome reaction protein.

Figure 2 shows the amino acid sequence of the murine acrosome reaction protein.

Figure 3 shows the nucleotide sequence encoding the human acrosome reaction protein.

Figure 4 shows the nucleotide sequence encoding the murine acrosome reaction protein.

Figure 5 shows the structure and expression of the acrosome reaction protein (also referred to as P3 or the Polycystic Kidney Disease and Receptor for Egg Jelly protein (PKDREJ)). (a) Map of the genomic P3 locus in humans (top) and mouse (bottom) showing restriction sites for BarnH I and Sac II; cosmid (shaded) and plasmid (open boxes) genomic probes including the 'intronic' probes (Int) to test alternative splicing; GC content illustrating CpG islands at the 5' ends of the genes; the open reading frames (ORFs; solid boxes) in genomic DNA and 3'UTR (open boxes); cDNA clones (open boxes) including alternatively spliced clones (hatched).

(b) Northern blot (2 μg poly A⁺ RNA per lane) hybridised with a P3 probe (REJ) and control PKD1 probe (3 A3) against adult human tissues: peripheral blood leukocytes (L); colon (C); small intestine (I); ovary (O); testis (T); prostate (P); thymus (Th) and spleen (S). P3 was found to be expressed as a ~8 kb mRNA only in testis. Negative results were also obtained for P3 in adult heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, and fetal brain, lung, liver and kidney (data not shown).

(c) 20 μg total mouse testis RNA from newborn (NB), 5 (5D),7 (7D), 10 (10D), 14 (14D) and 21 day (2 ID), 6 weeks (6W) and adult hybridised with an adult mouse P3 probe, 2.1 (left) and rehybridised with β actin control (right).

Figure 6 shows the alignment of the REJ modules of the human P3, suREJ (Q26627) and human polycystin- 1 (Q15140) showing identity (solid box) and similarity to identity (shaded boxes). BEST FIT analysis of this area showed identity between P3 and suREJ of 22.7% and similarity of 46.2%, corresponding figures for P3 and polycystin- 1 were 21 % and 43%.

Figure 7 shows hydropathy plots of the homologous areas of suREJ, P3 (human and mouse) and polycystin- 1 and -2 (human) using the Kyte-Doolittle method with a 20 nt window. Predicted transmembrane domains (TM) are numbered and aligned. Sequence alignment with P3 across the transmembrane area showed identity of 23% and similarity of 46.2% with polycystin- 1 over 1045 amino acids; corresponding figures with polycystin-2 were 24.2% and 52.7% over 539 amino acids.

Figure 8 shows (A) light microscope (B) dark field image of antisense PKDREJ mRNA probe hybridised to adult mouse testis. Signal is seen in the developing spermatocytes in the seminiferous tubes.

METHODS RNA was isolated from snap frozen human or mouse testis tissue using the SV Total RNA Isolation System (Promega). RT-PCR was performed as previously described (19), or modified in GC rich areas by using the RT enzyme Superscript II (Gibco) at 50°C in the presence of 10% DMSO with gene specific primers. To avoid confusion from genomic contamination of RNA all samples were DNased prior to reverse transcription and a RT negative control was included during each amplification. Mouse Northern blots were prepared as previously described (19) and Clontech human filters were employed.

Genomic and cDNA clones

The cosmids cN5H6 (Z93024), cB5E3 (Z79998) and cN98Gl (a bridging clone) were identified, characterised and sequenced as part of the chromosome 22 genome project. The human genomic clones REJ, 16, Intl and Int2 were amplified from cosmid DNA and contain the following areas of the P3 transcript: REJ (1992 bp) 2772-4743 nt; 16 (1012 bp) 5332-6343 nt; Intl (707 bp) 2963-3669 nt and Int2 (216 bp) 4106-4321 nt. 5SE is a Sac I - Eco RI fragment (1965 bp) 338-2302 nt. The cDNAs plO (2469 bp: 4711-7179 nt), p7 (1041 bp: 1837-2877 nt) and pD (1172 bp: 410-1581 nt) were obtained by screening 1.5 x 10⁶ pfu of a human adult testis cDNA library (5 ' STRETCH PLUS cDNA library in gtlO; Clontech) by standard hybridisation procedures with the genomic probes 16, REJ and 5SE, respectively. The clones gl.5 (1184 bp: 1496-2679 nt), g2 (1052 bp: 2786-3837 nt), g3 (1052 bp: 3768-4819 nt), 5p3 (636 bp: -27-611 nt) alt g2 (285 bp: 2786-2947 and 3716-3877 nt) and alt g3 (779 bp: 3768-4083 and 4357- 4819 nt) were generated by RT-PCR . The 3' cDNA rl (937 bp: 6723 nt - 7659 nt) was isolated by 3'RACE using the primer 5'-CAGAGAAGAACAGCCACCGTTATC-3' (6683-6706 nt) and testis cDNA (Marathon-Ready; Clontech) with the Expand Long Template PCR System (Boehringer Mannheim). The 3 A3 PKD1 probe has been described previously (20). The mouse PI (ICRFP70302313Q5) was isolated by hybridising the mouse PI library #703 (21) with the human clone 16 at reduced stringency. An 11 kb Not I/Eco RI (11NE) fragment containing the P3 was subcloned and Bam HI fragments of ~2. L kb and ~1.8 kb were subcloned as hybridisation probes. The end of the PI interrupts the 3'UTR of 3.

Sequencing

Human cDNAs and where necessary genomic clones were sequenced from plasmids using DyeDeoxy Terminator Cycle Sequencing (TaqFS or BigDye; ABI) and ABI 373A or 377 Sequencers. The mouse genomic clone 11NE was sonicated and subcloned as ~2 kb fragments into Ml 3 and sequenced as above. Contigs were assembled with the programme Sequencher. Sequence was analysed with MacVector and homologies sought in the GenEMBL and Tremble databases using BLAST, FASTA and BESTFIT. Alignment of REJ modules was prepared with MegAlign using the Clustal Method. SIGCLEAVE was used to predict signal peptides and cleavage sites. Potential transmembrane regions and protein topology were predicted with alignment with existing proteins (16, 17) and using the PredictProtein programme.

mRNA In Situ Hybridisation

The method of mRNA in situ hybridisation has been used to localise the mouse orthologue ofPKDREJ mRNA in mouse testis tissue. Sense and antisense [³⁵S] UTP labelled RNA probes of 200bp were generated using SP6 polymerase, to two areas of the mouse PKDREJ transcript. One probe contains a region of the 5' part of the gene (nucleotides 1118-1317) and the other matched part of the 3' UTR (nucleotides 6573- 6772). These RNA probes were hybridised to lOμm cyro-sections of adult and 10 day old mouse testis. Sections were fixed in 4% paraformaldehyde treated with proteinase K and further prepared for hybridisation as previously described in Maxwell et al. (1997). The sections were hybridised overnight at 60°C with antisense and sense control probes, washed and exposed to emulsion for 2 weeks as set out in Maxwell et al.

Generation of Monoclonal Antibodies

A number of regions of the human PKDREJ gene have been cloned into expression vectors to generate protein to raise antibodies. A 566bp fragment that encodes the first intracellular loop ofPKDREJ (amino acids 1203-1370) has been cloned into commercially available vectors pET32A+ and pMALc2 (obtainable from Novagen, Madison, WI, USA and New England Biolabs, Beverley, MA, USA, respectively) to generate thiorodoxin and maltose binding fusion proteins, respectively. In addition the region encoding the REJ module ofPKDREJ has been cloned into the pEAK 10 vector (Edge BioSystems) and expressed in the eukaryotic PEAK cell system to generate appropriately modified protein. These proteins have been used to immunise mice to raise monoclonal antibodies.

EXAMPLE

The present invention was arrived at by looking for polycystin-like proteins having similar integral membrane sections defining voltage-gated channel subunits to those of polycystin-1 and -2. Surprisingly, a protein having acrosome reaction functions was identified.

The similarity of integral membrane sections of the autosomal dominant polycystic kidney disease (ADPKD) proteins, polycystin-1 and -2 and their homology with voltage-gated channel subunits (16, 17) suggested the possibility of other polycystin- like molecules. BLAST and FASTA analysis of the EMBL database with polycystin-1 and -2 sequence revealed significant hits in two cosmids located in chromosome region 22ql3. Further mapping information and analysis by PCR determined that these cosmids lay immediately end to end and contained a single region of homology with polycystin-1 extending over 6 kb (Fig. 5a). The contiguous nature of the homology between genomic DNA and PKDl protein sequence initially suggested a pseudo-gene, however, further analysis indicated a functional locus. An open reading frame (ORF) of 6,759 bp was maintained in genomic DNA and the 5' end of the ORF lay at a GC rich, CpG island (Fig. 5a). Southern analysis showed that the sequence was single copy and Northern blotting revealed expression, however, unlike the ADPKD genes that are ubiquitously expressed in human organs, the P3 mRNA was restricted to testis (Fig. 5b). The observed size of the transcript, ~8 kb was consistent with the detected ORF plus 5' and 3' UTRs. To better understand the significance of this unusual locus a PI clone containing the mouse orthologue was identified, subcloned and the gene-containing region sequenced. This revealed a similar structure to that found in man with a CpG island and ORF in genomic DNA of 6,378 bp (Fig. 5a), transcribing a testis expressed mRNA. To confirm the apparent intronless nature of .P3 a cDNA contig encompassing most of the human ORF and 3 ' UTR was constructed by screening a testis cDNA library and RT-PCR from testis RNA. A cDNA contig of 7,250 bp was assembled including a 3' UTR of 900 bp terminating with the atypical polyadenylation signal ATTAAA and a poly A tail. The first in- frame Met codon in the ORF is proposed as the start codon because of the Kozak consensus and conservation between man and mouse. The majority of the cDNAs confirmed the intronless nature of the gene although two possible alternatively spliced products were characterised (Fig. 5 a). The clones alt g2 and alt g3 contain in- frame deletions of 768 nt and 273 nt, respectively, and in each case the deleted areas are flanked by consensus splice sites. To determine if these clones represented the normal major product of this gene 'intronic' probes were designed and hybridised to a Northern blot. In both cases these probes detected the ~8 kb transcript (data not shown) confirming that these regions are usually present in the P3 mRNA, although they may generate a minor alternatively spliced product (see Fig. 5b). Analysis of P3 during mouse maturation showed expression in testis starting at ~2 weeks and continuing into adult life, mirroring the production of mature spermatozoa (18) (Fig. 5c).

The predicted human P3 protein has 2253 amino acids and a calculated unglycosylated molecular mass of 255 kDa; the corresponding mouse protein is 2126 amino acids and 241 kDa. The human and murine proteins show 64 % identity and 78% similarity. The P3 proteins have predicted signal peptides and sequence similarity of -1000 amino acids with polycystin-1 and the suREJ protein, corresponding to the previously defined REJ module (Fig. 6) (15, 16). The C terminal half of the protein is similar to polycystin-1 and the C terminal quarter to polycystin-2 (see Fig. 7 legend). Analysis of hydrophobicity through this region indicated a similar integral membrane structure to polycystin-1 with eleven transmembrane regions proposed; six corresponding to those found in polycystin-2 (Fig. 7). The suREJ has just a single transmembrane domain, corresponding to the first found in the P3 and polycystin-1 proteins. No clear homology has been found with the N-terminal 170 amino acids of human P3 between the signal peptide and REJ module, and this region differs between humans and mouse with the murine protein 113 amino acids shorter.

The results of the in situ hybridisation studies are shown in Figure 8. Identical signal was seen with only the antisense probes and only on adult tissue. Figure 8 shows that the hybridisation pattern typically observed indicating that the expression is in the developing spermatocytes in the seminiferous tubules and not in the supporting Sertoli cells or the surrounding interstituium. These results further indicate that PKDREJ is a sperm protein.

The role of the suREJ protein in fertilisation has been shown both by evidence of binding to egg jelly and that a mAb to suREJ induces the acrosome reaction. Sequence similarity and expression temporally and spatially coincident with sperm production indicate that P3 is a mammalian sperm factor having a role in triggering the acrosome reaction. However, the overall structure of P3 is closer to polycystin-1 than the suREJ protein. P3 does not contain the lectins found in the suREJ, which interact with carbohydrates in the egg glycoprotein coat, so it is not clear whether P3 interacts directly with mammalian glycoprotein coat, the zona pellucida (ZP), or whether it may act via another protein or have a protein ligand.

References

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20. European Chromosome 16 Tuberous Sclerosis Consortium. Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell 75, 1305-1315 (1993). 21. Lehrach, H. et al. Hybridization fingerprinting in genome mapping and sequencing, in Genome Analysis : Genetic and Physical Mapping (eds. Davies, K.E. &

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22. Maxwell et al., Hypoxia-inducible factor- 1 modulates gene expression in solid tumours and influences both angiogenesis and tumour growth. PNAS USA 94, 8104- 8109 (1997).

Claims

1. A functional mammalian acrosome reaction protein or functional fragment thereof having an amino acid sequence at least 30% homologous to the amino acid sequence of figure 1.

2. The functional acrosome reaction protein or functional fragment thereof of claim 1 which has an amino acid sequence at least 60% homologous to the amino acid sequence of figure 1.

3. An antibody having affinity for the acrosome reaction protein of claim 1 or claim 2 or a functional fragment thereof.

4. A method for identifying proteins, antibodies, peptides and other molecules which block the acrosome reaction comprising: contacting either

(a) sperm containing the functional acrosome reaction protein; or

5. A method for identifying a molecule that triggers the acrosome reaction in the absence of ZP comprising adding a candidate molecule to sperm comprising the functional acrosome reaction protein of the present invention and measuring any acrosome reaction in the absence of ZP.

6. A method for identifying a molecule that enhances or improves the efficacy of the acrosome reaction protein of the present invention in initiating the acrosome reaction comprising adding a candidate molecule to sperm comprising the acrosome . reaction protein of the present invention, wherein mammalian ZP may be present with the sperm prior to addition of the candidate molecule or may be added after the addition of the candidate molecule, and measuring for any improvement or enhancement of the acrosome reaction.

7. A nucleic acid sequence encoding the acrosome reaction protein of claim 1 or claim 2.

8. The nucleic acid sequence of claim 7 wherein the nucleic acid sequence is that shown in figure 3 or figure 4.

9. The acrosome reaction protein of claim 1 or claim 2, or a fragment thereof, and/or the nucleic acid sequence of claim 7 or 8, for use in therapy.

10. The use of the acrosome reaction protein of claim 1 or claim 2, or a fragment thereof, and/or the nucleic acid sequence of claim 7 or claim 8, in the manufacture of a composition for fertility treatment.

11. A composition comprising the acrosome reaction protein of claim 1 or claim 2, or a fragment thereof, and/or the nucleic acid sequence of claim 7 or claim 8, and a pharmaceutically acceptable diluent, carrier and/or excipient.

12. The antibody of claim 3 or a protein or molecule capable of blocking the acrosome reaction for use in therapy.

13. The use of the antibody of claim 3 or a protein or molecule capable of blocking the acrosome reaction in the manufacture of a composition for use as a contraceptive.

14. A composition comprising the antibody of claim 3 or a protein or molecule capable of blocking the acrosome reaction, and a pharmaceutically acceptable diluent, carrier and/or excipient.

15. A molecule that triggers the acrosome reaction in the absence of ZP for use in . therapy.

16. The use of a molecule that triggers the acrosome reaction in the absence of ZP in the manufacture of a composition for use in a fertility treatment.

17. A method for treating a mammal in need of treatment by administration of an effective amount of a molecule that triggers the acrosome reaction in the absence of ZP.

18. A composition comprising a molecule that triggers the acrosome reaction in the absence of ZP and a pharmaceutically acceptable diluent, carrier and/or excipient.

19. A molecule that enhances or improves the efficacy of the acrosome reaction protein for use in therapy.

20. The use of a molecule that enhances or improves the efficacy of the acrosome reaction protein in the manufacture of a composition for use in a fertility treatment.

21. A method for treating a mammal in need of treatment by administration of an effective amount of a molecule that enhances or improves the efficacy of the acrosome reaction protein.

22. A composition comprising a molecule that enhances or improves the efficacy of the acrosome reaction protein and a pharmaceutically acceptable diluent, carrier and/or excipient.

23. The use of the nucleic acid sequence of claim 7 or claim 8, in gene therapy.