WO2003087151A1

WO2003087151A1 - A sperm polypeptide

Info

Publication number: WO2003087151A1
Application number: PCT/AU2003/000422
Authority: WO
Inventors: Deborah Maree Hickox; David Morritz De Kretser; Moira Kathleen O'bryan
Original assignee: Monash University
Priority date: 2002-04-08
Filing date: 2003-04-08
Publication date: 2003-10-23
Also published as: AUPS160402A0

Abstract

The present invention relates to a sperm polypeptide in particular a sperm polypeptide that binds to the tpx-1 gene, nucleic acids encoding the polypeptide, processes for production of recombinant forms of the polypeptide, antibodies generated against the polypeptide and uses of the polypeptides, particularly in regulating sperm function. The invention also includes compositions comprising the polypeptide, in particular compositions which can affect functions of the tpx-1 gene.

Description

A SPERM POLYPEPTIDE TECHNICAL FIELD

BACKGROUND ART

Many fertility-related problems in men arise as a result of abnormal sperm development and/or function. Unfortunately, scientific research is yet to determine the underlying cause of many such defects, primarily because sperm, although structurally well-characterised, are yet to be understood at a molecular level, especially with respect to the sperm tail.

The CRISP protein family (Cysteine Rich Secreted Protein) have been found to be involved in sperm development. These proteins have the presence of 16 conserved cysteinyl residues, 14 of which are in the carbonyl half of the proteins. These proteins have been identified in higher eukaryotes as well as in lower eukaryotes and plants, implicating a diverse involvement of multiple functions. Already, there have been CRISP genes identified in sperm development including the CRISP-1 gene in the epididymis of the mouse and rat and in the sub-mandibular gland in the mouse. The CRISP-2 gene is found in the testis of the human, guinea pig and mouse and the CRISP-3, gene is expressed in the sub-mandibular gland of the mouse and human.

Other CRISP proteins have been identified and implicated in sperm development. Tpx-1 protein is localized to the acrosome and the outer dense fibres (ODF) of the sperm tail and has been implicated in cell adhesion between spermatids and Sertoli cells. Tpx-1 is a cysteine-rich secretory protein (CRISP), however the functional significance of this family of proteins remains to be determined. The tpx-1 gene is a 1.6kb transcript and encodes a protein of 25kDa and 27kDa. Characterising the tpx-1 gene and identifying tpx-1 interacting proteins can provide insight into the processes underlying fertility-related problems.

Accordingly, it is an object of the present invention to provide a protein which is implicated in the fertilisation process and which can provide insight into the fertilisation process particularly sperm function.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

DESCRIPTION OF THE INVENTION Throughout the description and claims of this specification, the word

"comprise" and variations of that word, such as "comprising" and "comprises" are not intended to exclude other additives, steps or integers.

In a first aspect of the present invention there is provided an isolated nucleic acid molecule which encodes a polypeptide that binds to a tpx-1 gene, said molecule comprising a nucleic acid sequence selected from the group including:

(a) SEQ ID NO:1 , SEQ ID NO: 4, or fragments thereof;

(b) nucleic acid sequence which encodes SEQ ID NO:2, SEQ ID NO:5 or fragments thereof; (c) mutants and variants of (a) or (b); and

(d) nucleic acid sequences capable of hybridising to the nucleic acid sequences of (a), (b) or (c).

Data obtained so far suggests that tpx-1 plays a role in a complex process, or perhaps a number of processes, given the identification of several types of interacting proteins. This is consistent with the localization of tpx-1 protein in diverse sites within spermatids and spermatozoa.

The nucleic acid sequences of the present invention have been identified as encoding proteins which bind to the tpx-1 gene. The sequences were identified using a yeast two-hybrid approach. The region coding for the mature tpx-1 protein was used as bait to screen a testis expression library.

This was achieved by co-transformation of a fpx-7-containing plasmid and a mouse testis cDNA library into yeast, followed by plating on nutrient deficient media. Interactions were identified by growth of yeast colonies on histidine and adenine deficient media, which indicated expression of the reporter genes HIS and ADE respectively. Positive yeast clones were amplified, cloned, sequenced and then analysed using BLAST and various other sequence analysis tools on the web. To determine the temporal expression of putative tpx-1 -interacting proteins, developmental Northern blots were performed. Additionally, where antibodies were available, immunohistochemistry and co-immunoprecipitation studies were performed to further validate the binding of the candidate proteins with tpx-1. Deletion constructs were designed to determine regions of the proteins that are necessary for protein-binding.

Several positive clones were identified by this technique, however, the nucleic acid sequence which encodes the polypeptide that binds tpx-1 has been identified as being different to other tpx-1 binding proteins selected from the group including ranbp-9, Basigin (a protein shown to be involved in spermiogenesis) and msj-1 (a testis-specific DNA J class heat shock protein).

Data obtained so far suggests that tpx-1 plays a role in a complex process, or perhaps a number of processes, given the identification of several types of interacting proteins. This is consistent with the localization of tpx-1 protein in diverse sites within spermatids and spermatozoa. The classes of the tpx-1 -interacting proteins identified include: a novel protocadherin, which is in accordance with the hypothesized role of tpx-1 in spermatid-SertoIi cell adhesion; a CRISP protein referred to herein as CRISPY, thus representing the first example of CRISP proteins interacting with each other and the protein of the present invention; a Ran binding protein indicating a potential role in microtubule assembly; and msj-1 which is interesting as heat shock protein which has been implicated within non- mammalian species in intraflagellar protein transport. The mechanisms for this in mammalian flagellae are largely unknown. Further, the heat shock proteins hsp90 and hsp70-2 are found within sperm tails and around the acrosome respectively, consistent with the localization of tpx-1.

Hence the nucleic acid and polypeptides encoded by the nucleic acids may be strongly implicated in sperm function due to their interactions with tpx-1. However, to further show this interaction, the polypeptide encoded by SEQ ID NO:1 or SEQ ID NO:4 shows expression patterns consistent with that of the tpx-1 gene in the testis (ie post-meiotic) as shown in Northern blot analysis.

Preferably, the nucleic acid sequence is 893bp with an open reading frame of 236 amino acids. More preferably the nucleic acid sequence is a cDNA sequence which encodes a protein of 27.1 K with a pi of 8.92. The protein may be cleaved to provide a 24.3K protein with a pi of 8.4. The protein also preferably contains a consensus site for N-linked glycosylation (amino acids NESK), a protein kinase C phosphorylation site (amino acids 153-155), casein kinase II phosphorylation sites including amino acids 35-38, 115-118, 137-140, 191-194, potential N-myristoylation sites including amino acids 157-162, 172-177, 182-187, 220-225, RGD cell attachment sequences including amino acids 192-194 and like all members of the CRISP family SCP/tpx-1/Ag5/PR-1/Sc7 signature sequences preferably between amino acids 143-153 and 173-184.

The "isolated nucleic acid molecule" of the present invention is isolated and free of contaminating endogenous material.

A "nucleotide sequence" refers to a polynucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. The nucleic acid molecule may be derived from DNA or RNA isolated at least once in substantially pure form and in a quantity or concentration enabling identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (such as those outlined in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd sed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). Such sequences are preferably provided and/or constructed in the form of an open reading frame uninterrupted by internal nontranslated sequences, or introns, that are typically present in eukaryotic genes. Sequences of non- translated DNA can be present 5¹ or 3' from an open reading frame, where the same do not interfere with manipulation or expression of the coding region.

Nucleic acid molecules of the invention include DNA in both single- stranded and double-stranded form, as well as the RNA complement thereof. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. Genomic DNA may be isolated by conventional techniques such as using the cDNA of SEQ ID NO: 1 , SEQ ID NO: 4, or a suitable fragment thereof, as a probe.

The DNA molecules of the invention include full length genes as well as polynucleotides and fragments thereof. The full length gene may include the N-terminal signal peptide.

The nucleic acids of the invention are preferentially derived from human sources, but the invention includes those derived from non-human species, as well, such as mice and rats.

Due to the known degeneracy of the genetic code, wherein more than one codon can encode the same amino acid, a DNA sequence can vary from that shown in SEQ ID NO: 1 , or SEQ ID NO: 4, and still encode a polypeptide having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5. Such mutants and variant DNA sequences can result from silent mutations (e.g., occurring during PCR amplification), or can be the product of deliberate mutagenesis of a sequence. Such changes in the nucleic acid sequence may include substitutions of different nucleotides resulting in the same or a functionality equivalent gene product. The term "functional equivalent" as used herein means a sequence which functions in a similar way but may have deletions, additions or substitutions that do not substantially change the activity or function of the sequence.

The nucleic acid sequences of the present invention include those sequences in (d) that are capable of hybridising to the sequences of (a), (b) or (c) above. Preferably moderate to high stringency conditions are employed. These conditions may be readily determined by those of ordinary skill in the art based on, for example the length of DNA. The basic conditions are set forth by Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989), and may include use of a prewashing solution for the nitrocellulose filters 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridisation conditions of about 50% formamide, 6X SSC at about 42°C (or other similar hybridisation solution, such as Stark's solution, in about 50% formamide at about 42°C), and washing conditions of about 60°C, 0.5X SSC, 0.1% SDS. However, the stringency conditions are not limited to these conditions. Conditions of high stringency can also be readily determined by the skilled artisan based on, for example, the length of the DNA. Generally, such conditions are defined as hybridisation conditions as above, and with washing at approximately 68° C, 0.2X SSC, 0.1% SDS. The skilled artisan will recognize that the temperature and wash solution salt concentration can be adjusted as necessary according to factors such as the length of the probe.

In another preferred aspect the nucleic acid molecules of the invention also comprise nucleotide sequences that are at least 80% identical to SEQ ID NO: 1 or SEQ ID NO: 4. Also contemplated are embodiments in which a nucleic acid molecule comprises a sequence that is at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to SEQ ID NO: 1 or SEQ ID NO: 4.

The percent identity may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two nucleic acid sequences can be determined by comparing sequence information using computer programs available to the skilled person.

The nucleic acid sequences of the present invention may be obtained from any source. Preferably they are obtained from sources in which the tpx- 1 gene is expressed. Most preferably, they are obtained from the testis, and sperm.

The invention also provides isolated nucleic acids useful in the production of polypeptides. Such polypeptides may be prepared by any of a number of conventional techniques. A DNA sequence encoding a tpx-1 binding polypeptide, or desired fragment thereof may be subcloned into an expression vector for production of the polypeptide or fragment. The DNA sequence advantageously is fused to a sequence encoding a suitable leader or signal peptide. Alternatively, the desired fragment may be chemically synthesized using known techniques. DNA fragments also may be produced by restriction endonuclease digestion of a full length cloned DNA sequence, and isolated by electrophoresis on agarose gels. If necessary, oligonucleotides that reconstruct the 5' or 3' terminus to a desired point may be ligated to a DNA fragment generated by restriction enzyme digestion. Such oligonucleotides may additionally contain a restriction endonuclease cleavage site upstream of the desired coding sequence, and position an initiation codon (ATG) at the N-terminus of the coding sequence.

The polymerase chain reaction (PCR) procedure also may be employed to isolate and amplify a DNA sequence encoding a desired protein fragment.

Also provided are expression vectors and cloning vectors as well as host cells which can harbour the expression vectors for expressing a protein encoded by the nucleic acid.

The present invention provides an expression vector comprising a nucleic acid molecule which encodes a polypeptide that binds to a tpx-1 gene, said molecule comprising a nucleic acid sequence selected from the group including:

(a) SEQ ID NO: 1 , SEQ ID NO: 4, or fragments thereof;

(b) nucleic acid sequence which encodes SEQ ID NO:2, SEQ ID NO:5 or fragments thereof;

(c) mutants and variants of (a) or (b); and

(d) nucleic acid sequences capable of hybridising to the nucleic acid - sequences of (a), (b) or (c).

The nucleic acid sequence may be inserted into an expression vector or cloning vector. A large number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pBR322 or pUC plasmid derivatives. The insertion into a cloning vector can, for example be accomplished by ligating the DNA fragment into the cloning vector or the expression vector which has complimentary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) into the DNA termini; these ligated linkers may comprise specific chemically synthesised oligonucleotides encoding restriction endonuclease restriction sequences. In an alternative method, the cleaved DNA may be modified by homopolymeric tailing.

The present invention also provides a host cell transformed or transfected with an expression vector or cloning vector described above.

Recombinant molecules such as expression vectors, cloning vectors or plasmids can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.

Any suitable expression system may be employed. The vectors include a DNA encoding a polypeptide or fragment of the invention, operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences which control transcription and translation initiation and termination. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA sequence. Thus, a promoter nucleotide sequence is operably linked to a DNA sequence if the promoter nucleotide sequence controls the transcription of the DNA sequence. An origin of replication that confers the ability to replicate in the desired host cells, and a selection gene by which transformants are identified, are generally incorporated into the expression vector.

In addition, a sequence encoding an appropriate signal peptide (native or heterologous) can be incorporated into expression vectors. A DNA

Substitute Sheet sequence for a signal peptide (secretory leader) may be fused in frame to the nucleic acid sequence of the invention so that the DNA is initially transcribed, and the mRNA translated, into a fusion protein comprising the signal peptide. A signal peptide that is functional in the intended host cells promotes extracellular secretion of the polypeptide. The signal peptide is cleaved from the polypeptide upon secretion of polypeptide from the cell.

Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors or cloning vectors can be propagated and prepared in quantity.

Suitable host systems to express the nucleotide sequence may include, but are not limited to, mammalian cell systems and microorganisms such as yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA.

A method for producing polypeptides comprises culturing host cells transformed with a recombinant expression vector encoding the polypeptide, under conditions that promote expression of the polypeptide, then recovering the expressed polypeptides from the culture. The skilled artisan will recognize that the procedure for purifying the expressed polypeptides will vary according to such factors as the type of host cells employed, and whether the polypeptide is membrane-bound or a soluble form that is secreted from the host cell.

In another aspect of the present invention there is provided a polypeptide comprising an amino acid sequence selected from the group including:

(a) SEQ ID NO: 2, SEQ ID NO:5 or variants thereof;

(b) fragments of SEQ ID NO:2, or SEQ ID NO:5 wherein the fragment binds to the tpx-1 gene. In yet another aspect there is provided a polypeptide encoded by a nucleic acid molecule which encodes a polypeptide that binds to a tpx-1 gene, said molecule comprising a nucleic acid sequence selected from the group including: (a) SEQ ID NO:1 , SEQ ID NO:4 or fragments thereof;

(b) nucleic acid sequence which encodes SEQ ID NO:2 or SEQ ID NO:5 or fragments thereof;

(c) mutants and variants of (a) or (b); and (d) nucleic acid sequences capable of hybridising to the nucleic acid sequences of (a), (b) or (c).

The invention encompasses polypeptides and fragments thereof in various forms, including those that are naturally occurring or produced through various techniques such as procedures involving recombinant DNA technology. Such forms include, but are not limited to, derivatives, variants, and oligomers, as well as fusion proteins or fragments thereof.

Preferably the polypeptide co-expresses with tpx-1 both temporally and/or locally. This may be confirmed by methods available to the skilled addressee including immunoprecipitation studies, pull down assays and/or Far Westerns, Northern and Western blots and in situ hybridisation studies.

Preferably the polypeptide is homologous to the CRISP family of proteins. More preferably the polypeptide is 27.1 K and has a pi of 8.92. If cleaved, the polypeptide is 24.3K with a pl of 8.4. The polypeptide may also contain a consensus site for N-linked glycosylation (amino acids NESK), a protein kinase C phosphorylation site including amino acids 153-155, casein kinase II phosphorylation sites including amino acids 35-38, 115-118, 137-

140, 191-194, potential N-myristoylation sites including amino acids 157-162,

172-177, 182-187, 220-225, a RGD cell attachment sequences including amino acids 192-194 and like all members of the CRISP family SCP/tpx- 1/Ag5/PR-1/Sc7 signature sequences preferably between amino acids 143-

153 and 173-184.

Naturally occurring variants as well as derived variants of the polypeptides and fragments are provided herein.

Variants may exhibit amino acid sequences that are at least 80% identical. Also contemplated are embodiments in which a polypeptide or fragment comprises an amino acid sequence that is at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least

99.9% identical to the preferred polypeptide or fragment thereof. Percent identity may be determined by visual inspection and mathematical calculation.

Among the variant polypeptides provided herein are variants of native polypeptides that retain the native biological activity or the substantial equivalent thereof.

Variants include polypeptides that are substantially homologous to the native form, but which have an amino acid sequence different from that of the native form because of one or more deletions, insertions or substitutions. Particular embodiments include, but are not limited to, polypeptides that comprise from one to ten deletions, insertions or substitutions of amino acid residues, when compared to a native sequence.

A given amino acid may be replaced, for example, by a residue having similar physiochemical characteristics. Examples of such conservative substitutions include substitution of one aliphatic residue for another, such as lie, Val, Leu, or Ala for one another; substitutions of one polar residue for another, such as between Lys and Arg, Glu and Asp, or Gin and Asn; or substitutions of one aromatic residue for another, such as Phe, Trp, or Tyr for one another. Other conservative substitutions, e.g., involving substitutions of entire regions having similar hydrophobicity characteristics, are well known. Similarly, the DNAs of the invention include variants that differ from a native DNA sequence because of one or more deletions, insertions or substitutions, but that encode a biologically active polypeptide.

The polypeptide of the present invention may be purified using standard protein or peptide purification techniques including but not limited to electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography, affinity chromatography, immunoadsorbent affinity chromatography, and gel permeation high performance liquid chromatography, isoelectric focussing, and variations and combinations thereof. The polypeptide may be isolated from recombinant sources and may be generated by gene expression systems and host systems as described above. The present invention also provides fusion proteins comprising the tpx-1 binding protein or variant thereof and another protein. Preferably the fusion protein is used to immunise animals such as rabbits and mice to produce antibodies specific to the tpx-1 binding protein. In another aspect of the present invention, there is provided an antibody that specifically binds to the tpx-1 binding protein or polypeptide or variant of the present invention.

For the production of antibodies, isolated or preferably purified preparations of the polypeptide or protein may be used as immunogens. The immunogens may be conjugated to a carrier molecule, preferably a carrier protein. Carrier proteins may be any commonly used in immunology including, but not limited to, bovine serum albumin (BSA), chicken albumin, keyhole limpet hemocyanin (KLH) and the like.

Polyclonal and monoclonal antibodies are included in the scope of the present invention. Immunization regimes for production of both polyclonal and monoclonal antibodies are well known in the art. The immunogen may be injected by any number of routes including subcutaneous, intravenous, intraperitoneal, intradermal, intramuscular, mucosal, or a combination of these. The immunogen may be injected in soluble form, aggregate form, attached to a physical carrier, or mixed with an adjuvant, using methods and materials known in the art. The antibodies may be purified by methods known in the art.

The antibodies of the invention may be used to facilitate isolation and purification of the polypeptide or protein. They may also be used as probes for identifying clones in expression libraries that have inserts encoding the polypeptide or variants thereof. Such antibodies would have use in the purification of molecules during the production of recombinant polypeptides for example.

In a preferred embodiment antibodies are bound to chromatography resins. Passing a protein mixture through a column of the resin allows the cognate antigen to adhere. Non-target molecules may be flushed through the column to leave purified polypeptide which is then eluted. In another embodiment antibodies can be used to purify protein by the process of immunoprecipitation. Antibodies can also be used to probe for polypeptide and closely related proteins in cDNA expression libraries and cell extracts. The skilled addressee will understand that antibody fragments (such as Fab fragments) will also be useful in the context of the present invention. In another aspect of the present invention there is provided a therapeutic or prophylactic composition including a pharmaceutical composition comprising any of the nucleic acids, polypeptides or antibodies described above along with a carrier.

Polynucleotides and polypeptides of the invention may be used in developing treatments for any disorder mediated (directly or indirectly) by defective, excessive or insufficient amounts of the tpx-1 binding protein or polypeptide.

Isolated and purified polypeptides or a fragment thereof can also be useful as a therapeutic agent in treating sperm functions. Such therapeutic uses can involve their administration by the introduction of the tpx-1 binding polypeptide or fragment into the intracellular environment by well-known means. One such means is by encasing the protein in liposomes or coupling it to a monoclonal antibody targeted to a specific cell type.

The polypeptides may also be employed in inhibiting a biological activity of sperm functions, in in vitro or in vivo procedures. For example, a purified polypeptide or soluble fragment thereof may be used to inhibit binding of tpx-1 protein to the gene. Biological effects that result from the protein binding to the gene thus are inhibited.

In addition, tpx-1 binding polypeptides may be administered to a mammal to treat a disorder mediated by the tpx-binding protein. Such disorders include conditions caused (directly or indirectly) or exacerbated by the tpx-1 binding protein. For instance, modified tpx-1 binding polypeptides which compete with tpx-1 binding proteins may be used.

Compositions of the present invention may contain a polypeptide in any form described herein, such as native proteins, variants, derivatives, oligomers, and biologically active fragments.

Compositions comprising an effective amount of the polypeptide or fragment thereof of the present invention, in combination with other components such as a physiologically acceptable diluent, carrier, or excipient, are provided herein. The polypeptides can be formulated according to known methods used to prepare pharmaceutically useful compositions. They can be combined in admixture, either as the sole active material or with other known active materials suitable for a given indication, with pharmaceutically acceptable diluents (e.g., saline, Tris-HCl, acetate, and phosphate buffered solutions), preservatives (e.g., thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/or carriers. Suitable formulations for pharmaceutical compositions include those described in Remington 's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Company, Easton, PA.

In addition, such compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application. The compositions of the invention can be administered in any suitable manner, e.g., topically, parenterally, or by inhalation. The term "parenteral" includes injection, e.g., by subcutaneous, intravenous, intracellular or intramuscular routes, also including localized administration, e.g., at a site of disease or injury. Sustained release from implants is also contemplated. One skilled in the pertinent art will recognize that suitable dosages will vary, depending upon such factors as the nature of the disorder to be treated, the patient's body weight, age, and general condition, and the route of administration. Preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration is performed according to art-accepted practices.

Compositions comprising nucleic acids in physiologically acceptable formulations are also contemplated. DNA may be formulated for injection, for example. Other uses of the polynucleotides or polypeptide or antibodies of the present invention include use as a research tool for studying the biological effects that result from the tpx-1 binding protein and tpx-1 gene interactions on different cell types. Polypeptides also may be employed in in vitro assays for detecting tpx-1 binding protein or the interactions thereof.

Polypeptides, and antibodies against the tpx-1 binding protein can be used as reagents in a variety of research protocols.

The present invention provides for an insight into sperm function which will aid in both infertility and contraception development. By determining and understanding the significance of the tpx-1 gene and the protein which binds to it, further uses of the nucleic acid sequence, polypeptides and antibodies of this invention may help in understanding any one or all of the following:

- the development and regulation of sperm motility and as such male fertility - mutations may be causal in human male fertility

- the regulation of flagellar function in other tissues eg. the respiratory tract, the nervous system, the eye, ovarian function, the ear.

The invention also includes antagonists to the tpx-1 binding protein. These antagonists may act to inhibit activity of the tpx-1 gene or the binding protein generally by blocking its action.

Antagonists may include antibodies or small molecules directed to the tpx-1 binding protein. These may be used to understand the function of tpx-1 in sperm function or they may be used to treat sperm conditions or functions.

Alternatively, agonists which can trigger sperm function and which can be targeted to enhance the interactions between tpx-1 gene and tpx-1 binding proteins are also included within the scope of this invention.

In yet another aspect of the present invention there is provided a method of monitoring sperm function and development, said method comprising monitoring expression of a nucleic acid molecule which encodes a polypeptide that binds to a tpx-1 gene, said molecule comprising a nucleic acid sequence selected from the group including:

(a) SEQ ID NO: 1 , SEQ ID NO: 4, or fragments thereof; (b) nucleic acid sequence which encodes SEQ JD NO:2, SEQ ID NO:5 or fragments thereof;

(c) mutants and variants of (a) or (b); and

Expression of the molecule is preferably monitored in a sample that contains sperm including spermatids, spermatozoa, and testicular samples. By monitoring the levels of this molecule an indication of the degree to which this molecule can contribute to sperm development can be made. The levels may be compared to normal levels in normal sperm samples, or they may be compared against each other over a time period to monitor progress of sperm development and function.

In a preferred aspect of the present invention there is provided a method of monitoring sperm function and development, said method comprising monitoring expression of a polypeptide encoded by a nucleic acid molecule which encodes a polypeptide that binds to a tpx-1 gene, said molecule comprising a nucleic acid sequence selected from the group including: (a) SEQ ID NO: 1 , SEQ ID NO: 4, or fragments thereof;

(c) mutants and variants of (a) or (b); and

The polypeptide may be monitored using any method available to the skilled addressee. Preferably, the polypeptide is monitored using antibodies to the tpx-1 binding protein.

These methods are preferably used to monitor for defective sperm that may be defective in their development of sperm tails. Accordingly, in a further preferred embodiment, the invention provides a method of determining male fertility, said method comprising monitoring sperm function and development as described above. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1 shows the cDNA and protein sequence of mouse CRISPY.

Figure 2 shows the cDNA and protein sequence of human CRISPY. Figure 3 shows a phylogenetic tree identifying CRISPY as a distinct subset of the CRISP family.

Figure 4 shows the expression of CRISPY within the developing testis and adult mouse tissues.

BEST METHOD OF CARRYING OUT THE INVENTION

The present invention will now be more fully described with reference to the accompanying examples and figures. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction of the generality of the invention described.

EXAMPLES

Example 1 : Identification of a tpx-1 binding protein

(a) Animals Adult male Balb/c mice (36-60 days old) were obtained from the

Monash University Central Animal House, and were maintained under standardized conditions of lighting (12 h light: 12 h dark) and nutrition (food and water ad libitum) throughout the experimental period. Studies were performed in accordance with the NH&MRC Guidelines on Ethics in Animal Experimentation and were approved by the Monash Medical Centre Animal Experimentation Ethics Committee. Mice were killed by cervical dislocation and tissues were either snap frozen for mRNA collection or immersion fixed in Bouin's fixative and processed into paraffin blocks as described previously [O'Bryan et al, 2001]. For immunofluorescence studies, sperm were collected from the caput epididymis and prepared as described previously [O'Bryan et al, 2001]. (b) Yeast two hybrid library screening

A mouse testis expression library cloned into the pACT plasmid (Clontech, CA, U.S.A.) was screened for tpx-1 interacting proteins using the Matchmaker GAL4 Two-hybrid System 3 as recommended by the manufacturer (Clontech). Briefly the coding region for mature mouse tpx-1 protein ie. amino acids 22-243, was cloned into pAS2-1 and co-transfected with the testis expression library into the yeast strain PJ694A, followed by sequential plating onto histidine then adenine deficient media. Positive interactions were identified by growth of yeast colonies on histidine and adenine deficient media. Positive clones were picked and plasmids were amplified, cloned and sequenced. Following the isolation and sequencing of potentially positive clones, insert sequences were analysed using the BLAST algorithm to search for protein homology and expression data.

Screening of a mouse testis expression two hybrid library with the mature tpx-1 sequence identified a potential tpx-1 interacting protein which was found to contain a sequence with homology to the CRISP family of proteins. As such, this sequence which was originally identified as Y2H3 was renamed CRISPY. Cloning of the entire CRISPY cDNA sequence using a combination of EST database searching and RT-PCR revealed a sequence of 893bp with an open reading frame of 236 amino acids (Figure. 1). Blast analysis revealed that CRISPY is most closely related to the human proteins GLIPR (accession number JC4131) and RTVP-1 (accession number JC5309) and the mouse protein cripi (accession number BAB03398) for which it showed 31.7%, 34.3% and 33.5% identity respectively. Mouse CRISPY cDNA is predicted to encode a protein of 27.1 k with a pi of 8.92 with a hydrophobic leader sequence like region at the NH₂ terminus (amino acids 1-24). If cleaved, 'mature' CRISPY is predicted to be a protein of 24.3k with a pi of 8.4. CRISPY also contains a consensus site for N-linked glycosylation (amino acids NESK), a protein kinase C phosphorylation site (amino acids 153-155), 4 casein kinase II phosphorylation sites (amino acids 35-38, 115- 118, 137-140, 191-194), 4 potential N-myristoylation sites (amino acids 157- 162, 172-177, 182-187, 220-225), a RGD cell attachment sequence (amino acids 192-194) and like all members of the CRISP family SCP/tpx-1/Ag5/PR- 1/Sc7 signature sequences between amino acids 143-153 and 173-184.

(c) Cloning of human CRISPY The human orthologue was virtually cloned using a combination of search data derived from the public and Celera databases.

Through analysis of publicly available sequence databases it was possible to identify the orthologous human CRISPY sequence (Figure 2) ie.

ESTs with accession numbers BC14603 and AAH14603. Human CRISPY was identified as a cDNA of 980bp with an open reading frame of 233 amino acids.

Sequence alignments and construction of a phylogenetic tree, revealed that CRISPY forms a distinct sub-family of the CRISP family (Figure

3).

(d) Northern blotting

In order to assess the possibility of tpx-1 and putative interacting proteins, Y2H3, interacting in vivo, the expression was determined by Northern blotting. Interactions were only deemed biologically significant if the partner mRNA occurred in the same or interacting cell type at the same time as tpx-1 , that is, spermatids or Sertoli cells respectively. A mouse CRISPY specific cDNA was obtained by PCR from the isolated pAS2-1 clone using the forward primer 5'-CTGGCAAAACTAGCGAAAGC-3' (SEQ ID NO 6) and the reverse primer 5'-CCTGCTGGCGAATAATTACT-3' (SEQ ID NO 7). The resultant cDNA, corresponded to nucleotides 336-668 (Figure 1) and was designated clone probe 3. The developmental expression of CRISPY within mouse testis and adult mouse tissues was determined by Northern blotting as described previously [O'Bryan et al, 2001]. In addition the expression of CRISPY mRNA was assessed using a BLAST search of the EST databases. Northern blot analysis detected a single CRISPY transcript of ~1 b solely within the testis (Figure 4). No CRISPY mRNA was detected in any of epididymis, ovary, lung, liver, kidney, spleen, stomach, large intestine, small intestine, brain, heart, adipose tissue or skeletal muscle. A search of the EST database confirmed that CRISPY is a testis specific transcript.

Example 2: Synthesis of the tpx-1 binding protein and antibody production thereto

(a) CRISPY peptide synthesis and antibody production

A synthetic peptide was prepared based on differences between the predicted mouse CRISPY protein and other CRISP sequences. The mouse peptide (CRPMNRKTPHHK) (SEQ ID NO:3) designated DH5 had 46.7% sequence identity with the most closely related mouse CRISP. The peptide was prepared using solid-phase Fmoc-based solid phase peptide synthesis protocols and was purified and characterized based on procedures described elsewhere [O'Bryan et al, 2001]. The DH5 peptide was conjugated to keyhole limpet hemocyanin and used to immunize New Zealand White female rabbits to generate a high titer anti-DH5 sera [O'Bryan et al, 2001]. Specific anti-CRISPY immunoglobulins were purified over a Sepharose 4B- DH4 peptide column as described previously [Harlow and Lane, 1999].

(b) Sequence homology Analysis of the primary CRISPY protein sequence was carried out using the suit of tools available through the All-in-one-sequence-analyzer suit of programs (http://www-personal.umich.edu/~ino/blast.html). Sequence comparisons and a phylogenetic tree for of the entire vertebrate CRISP family was achieved using the Multiple program which is based on the sequence alignment with hierarchical clustering methods

(http://prodes.toulouse.inra.fr/multalin/multalin.html) (Corbet, 1988). Additional non-published CRISP sequences were identified by searching the Ensembl Genome Server (http://www.ensembl.org) database using the BLAST algorithm. Extracted cDNA sequences were translated into putative proteins using the Translate program (http://au.expasy.org/tools/dna.html) and the longest open reading frame taken as the most likely protein sequence. Finally it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.

REFERENCES

O'Bryan MK, Sebire K, Meinhardt A, Edgar K, Keah HH, Hearn MT, De Kretser DM. Tpx-1 is a component of the outer dense fibers and acrosome of rat spermatozoa. Mol Reprod Dev. 2001 Jan;58(1):116-25

Harlow E, Lane, D. (1999) Using antibodies, a laboratory manual. Cold Spring Harbor Press, New York, pp340-341.

Corpet F. Multiple sequence alignment with hierarchical clustering Nucleic Acids Res. 1988 Nov 25;16(22):10881-90.

Claims

CLAIMS:

1. An isolated nucleic acid molecule which encodes a polypeptide that binds to a tpx-1 gene, said molecule comprising a nucleic acid sequence selected from the group including: (a) SEQ ID NO:1 , SEQ ID NO: 4, or fragments thereof;

(c) mutants and variants of (a) or (b); and

2. An isolated nucleic acid molecule according to claim 1 wherein the nucleic acid sequence is 893bp with an open reading frame of 236 amino acids.

3. An isolated nucleic acid molecule according to claim 1 or claim 2 wherein the nucleic acid sequence is a cDNA sequence which encodes a protein of 27.1 K with a pi of 8.92.

4. An isolated nucleic acid molecule according to claim 3 wherein the protein may be cleaved to provide a 24.3K protein with a pi of 8.4.

5. An isolated nucleic acid molecule according to claim 3 wherein the protein contains a consensus site for N-linked glycosylation (amino acids

NESK), a protein kinase C phosphorylation site (amino acids 153-155), casein kinase II phosphorylation sites including amino acids 35-38, 115-118, 137-140, 191-194, potential N-myristoylation sites including amino acids 157-162, 172-177, 182-187, 220-225, RGD cell attachment sequences including amino acids 192-194 and SCP/tpx-1/Ag5/PR-1/Sc7 signature sequences preferably between amino acids 143-153 and 173-184.

6. An isolated nucleic acid molecule according to claim 1 derived from human sources.

7. An isolated nucleic acid molecule according to claim 1 derived from non-human species such as mice and rats.

8. A nucleic acid molecule according to claim 1 wherein the sequence is capable of hybridising to a sequence of (a), (b) or (c) under moderate to high stringency conditions.

9. A nucleic acid molecule according to claim 1 wherein the nucleic acid molecule comprises nucleotide sequences that are at least 80% identical to SEQ ID NO: 1 or SEQ ID NO: 4.

10. A nucleic acid molecule according to claim 9 wherein the nucleic acid molecule comprises nucleotide sequences that are at least 90% identical to

SEQ ID NO: 1 or SEQ ID NO: 4.

11. A nucleic acid molecule according to claim 10 wherein the nucleic acid molecule comprises nucleotide sequences that are at least 95% identical to SEQ ID NO: 1 or SEQ ID NO: 4.

12. A nucleic acid molecule according to claim 11 wherein the nucleic acid molecule comprises nucleotide sequences that are at least 98% identical to SEQ ID NO: 1 or SEQ ID NO: 4.

13. A nucleic acid molecule according to claim 12 wherein the nucleic acid molecule comprises nucleotide sequences that are at least 99% identical to SEQ ID NO: 1 or SEQ ID NO: 4.

14. A nucleic acid molecule according to claim 13 wherein the nucleic acid molecule comprises nucleotide sequences that are at least 99.5% identical to SEQ ID NO: 1 or SEQ ID NO: 4.

15. An expression or cloning vector comprising a nucleic acid molecule which encodes a polypeptide that binds to a tpx-1 gene, said molecule comprising a nucleic acid sequence selected from the group including:

(a) SEQ ID NO: 1 , SEQ ID NO: 4, or fragments thereof;

(d) nucleic acid sequences capable of hybridising to the nucleic acid sequences of (a), (b) or (c), together with vector nucleic acid sequence.

16. A host cell transformed or transfected with an expression or cloning vector according to claim 15.

17. A method for producing a polypeptide comprising culturing a host cell according to claim 16 transformed with an expression vector according to claim 15 encoding the polypeptide, under conditions that promote expression of the polypeptide, then recovering the expressed polypeptides from the culture.

18. A polypeptide comprising an amino acid sequence selected from the group including: (a) SEQ ID NO: 2, SEQ ID NO:5 or variants thereof;

(b) fragments of SEQ ID NO:2, or SEQ ID NO:5 wherein the fragment binds to the tpx-1 gene.

19. A polypeptide encoded by a nucleic acid molecule which encodes a polypeptide that binds to a tpx-1 gene, said molecule comprising a nucleic acid sequence selected from the group including:

(a) SEQ ID NO:1 , SEQ ID NO:4 or fragments thereof;

20. A polypeptide according to claim 18 or claim 19 which co-expresses with tpx-1 temporally and/or locally.

21. A polypeptide according to claim 18 or claim 19 which is homologous to the CRISP family of proteins.

22. A polypeptide according to claim 21 which is 27.1 K and has a pi of 8.92.

23. A polypeptide according to claim 22 which when cleaved is 24.3K with a pi of 8.4.

24. A polypeptide according to claim 18 or claim 19 which contains a consensus site for N-linked glycosylation (amino acids NESK), a protein kinase C phosphorylation site including amino acids 153-155, casein kinase II phosphorylation sites including amino acids 35-38, 115-118, 137-140, 191- 194, potential N-myristoylation sites including amino acids 157-162, 172-177, 182-187, 220-225, a RGD cell attachment sequences including amino acids 192-194 and SCP/tρx-1/Ag5/PR-1/Sc7 signature sequences preferably between amino acids 143-153 and 173-184.

25. A polypeptide according to claim 18 or claim 19 wherein the polypeptide molecule comprises polypeptide sequences that are at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 5.

26. A polypeptide molecule according to claim 25 wherein the polypeptide molecule comprises polypeptide sequences that are at least 95% identical to

SEQ ID NO: 2 or SEQ ID NO: 5.

27. A polypeptide molecule according to claim 26 wherein the polypeptide molecule comprises polypeptide sequences that are at least 98% identical to SEQ ID NO: 2 or SEQ ID NO: 5.

28. A polypeptide molecule according to claim 27 wherein the polypeptide molecule comprises polypeptide sequences that are at least 99% identical to SEQ ID NO: 2 or SEQ ID NO: 5.

29. A polypeptide molecule according to claim 28 wherein the polypeptide molecule comprises polypeptide sequences that are at least 99.5% identical to SEQ ID NO: 2 or SEQ ID NO: 5.

30. A fusion protein comprising a polypeptide or variant thereof according to claim 18 or claim 19 and another protein.

31. Use of a fusion protein according to claim 30 to immunise an animal such as a rabbit or mouse to produce an antibody specific to a polypeptide according to claim 18 or claim 19.

32. An antibody or fragment thereof that specifically binds to a polypeptide or variant according to claim 18 or claim 19.

33. Use of an antibody or fragment thereof according to claim 32 to facilitate isolation and purification of a polypeptide or protein according to claim 18 or 19 or as a probe for identifying clones in expression libraries that have inserts encoding the polypeptide or variants thereof.

34. An antibody or a fragment thereof according to claim 32 bound to a chromatography resin.

35. A therapeutic or prophylactic composition comprising a nucleic acid according to claim 1 , a polypeptide according to claim 18 or claim 19 or an antibody according to claim 32 together with a carrier.

36. A composition according to claim 35 which composition is a pharmaceutical composition.

37. Use of a composition according to claim 36 in treatment for a disorder mediated (directly or indirectly) by defective, excessive or insufficient amounts of the tpx-1 binding protein or polypeptide.

38. Use of an isolated and purified polypeptide according to claim 18 or claim 19 or a fragment thereof as a therapeutic agent in treating sperm function.

39. Use of an isolated and purified polypeptide according to claim 18 or claim 19 or a fragment thereof in inhibiting a biological activity of sperm function, in an in vitro or in vivo procedure.

40. Use according to claim 39 wherein a purified polypeptide or soluble fragment thereof is used to inhibit binding of tpx-1 protein to the gene.

41. Administration of polypeptide according to claim 18 or claim 19 to a mammal to treat a disorder mediated by the tpx-binding protein.

42. Use of a polynucleotide according to claim 1 or a polypeptide according to claim 18 or 19 or an antibody according to claim 32 as a research tool for studying biological effects that result from tpx-1 binding protein and tpx-1 gene interactions on different cell types.

43. Use of a polypeptide according to claim 18 or 19 in an in vitro assay for detecting tpx-1 binding protein or the interactions thereof.

44. An antagonist to a polypeptide according to claim 18 or 19.

45. An antagonist according to claim 44 wherein the antagonist is an antibody or a small molecule directed to the tpx-1 binding protein.

46. An agonist which can trigger sperm function and which can be targeted to enhance interactions between the tpx-1 gene and tpx-1 binding proteins.

47. A method of monitoring sperm function and development, said method comprising monitoring expression of a nucleic acid molecule which encodes a polypeptide that binds to a tpx-1 gene, said molecule comprising a nucleic acid sequence selected from the group including:

(a) SEQ ID NO: 1 , SEQ ID NO: 4, or fragments thereof;

48. A method according to claim 47 wherein expression of the molecule is monitored in a sample that contains sperm.

49. A method according to claim 48 wherein the sample contains spermatids, spermatozoa, or is a testicular sample.

50. A method according to claim 47 which provides an indication of the degree to which this molecule can contribute to sperm development.

51. A method of monitoring sperm function and development, said method comprising monitoring expression of a polypeptide encoded by a nucleic acid molecule which encodes a polypeptide that binds to a tpx-1 gene, said molecule comprising a nucleic acid sequence selected from the group including:

(a) SEQ ID NO: 1 , SEQ ID NO: 4, or fragments thereof;

(c) mutants and variants of (a) or (b); and (e) nucleic acid sequences capable of hybridising to the nucleic acid sequences of (a), (b) or (c).

52. A method according to claim 51 wherein the polypeptide is monitored using antibodies to the tpx-1 binding protein.

53. Use of a method according to claim 50 to monitor for defective sperm that may be defective in their development of sperm tails.

54. A method of determining male fertility, said method comprising monitoring sperm function and development by a method according to claim 51.