The membranes of mammalian sperm are usually rich in polyunsaturated fatty acids, a property which predisposes them to damage by oxygen-free radicals which causes increased membrane permeability, loss of sperm motility and morphological abnormalities, all leading to a low level of fertilising capacity.

When sperm are first produced in the testis they are effectively incapable of fertilisation, and are said to be immature. Sperm then leave the testis and pass down a ductoral system called the epididymis, where they undergo a number of changes, collectively known as sperm maturation, during which they acquire the ability to fertilise an egg.

Apart from its role in sperm maturation, the epididymis is also a site of sperm storage prior to ejaculation and it is during this period of storage in the cauda region of the epididymis that sperm are particularly susceptible to damage by free radicals. Several years ago. using the rat and macaque monkey (Macaca fascicularis) models, the inventors demonstrated that the epididymis is the site of expression of two enzymes which are important in controlling free radicals and that these two enzymes are secreted by the epididymis into the epididymal fluid which contains the stored sperm. One of these enzymes is a superoxide dismutase which is also secreted by other tissues, for example the placenta, whereas the second enzyme is a novel isoform of glutathione peroxidase which does not appear to be produced by any other tissues and is specific to the epididymis. The genes encoding both enzymes have been cloned from both the rat and macaque monkey, and their sequences are published (A C F Perry, R Jones, L S P Niang, R M Jackson and L Hall (1992) Biochemical Journal, 285,863-870). The abundant expression of these two enzymes strongly suggests that they are the major enzymes responsible for controlling free radical levels in the epididymis.

Recently, the inventors have used the Polymerase Chain Reaction (PCR) to identify and specifically amplify human eGPX cDNA which has subsequently been completely sequenced. Unexpectedly, it was found that the majority of human eGPX transcripts were abnormally processed and would not produce a functional enzyme. This is not observed in the rat or monkey model systems where only normal transcripts have been observed.

However, despite these major processing problems in humans, a small amount of correctly processed transcript (almost certainly less than 5%) is produced, so some functional enzyme should be produced.

Accordingly, a first aspect of the invention provides an isolated polypeptide or functional equivalent wherein the function is preventing lipid peroxidation of sperm membranes, or hydrogen peroxide scavenging, the polypeptide or functional equivalent comprising the amino acid sequence of Fig. 1. Prevention of lipid peroxidation, of sperm membranes may be measured for example as described in Aitken, R. J., Harkiss D., Buckingham D. (1993) J. Reprod Fertil. 98,257-265.

According to a second aspect of the invention there is provided a DNA molecule comprising a DNA sequence selected from: (a) the DNA sequence of Fig. 1, (b) DNA sequences which hybridise under stringent conditions i. e. specific to hybridise to eGPX but not other GPX isoforms to the DNA sequences of Fig. 1 and which code for a polypeptide of the eGPX-type.

(c) DNA sequences which are degenerate as a result of the genetic code to the DNA sequence defined in (a) and (b) and which code for a polypeptide of the eGPX-type.

Polypeptides of the eGPX type may be considered to be those having a function of eGPX such as prevention of lipid peroxidation of sperm membranes or hydrogen peroxide scavenging.

According to a third aspect of the invention, there is provided a plasmid or other vector including a DNA sequence according to the second aspect of the invention.

According to a fourth aspect of the invention there is provided a polypeptide complex comprising a polypeptide according to the first aspect of the invention or a polypeptide complex according the fourth aspect of the invention.

According to a fifth aspect of the invention there is provided an antibody raised against a polypeptide according to the first aspect of the invention.

According to a sixth aspect of the invention there is provided a host cell containing a DNA sequence according to the second aspect of the invention or a plasmid or vector according to the third aspect of the invention. The host cell may be mammalian such as human, or the host cell may be bacterial.

According to a seventh aspect of the invention there is provided a method of producing a polypeptide comprising inserting a DNA sequence according to the second aspect of the invention or a plasmid or other vector according to the third aspect of the invention into a host cell. The host cell may be a mammalian cell such as a human cell, or the host cell may be bacterial. Alternatively, a polypeptide according to the first aspect of the invention may be produced bv chemical means.

According to an eighth aspect of the invention there is provided an organism engineered to contain, express or overexpress a polypeptide according to the first aspect of the invention.

The organism may be a mammal, or may be bacteria.

According to a ninth aspect of the invention there is provided an enzyme inhibitor specific to a polypeptide according to the first aspect of the invention.

According to a tenth aspect of the invention there is provided a composition comprising the whole or part of a polypeptide capable of eliciting an anti-eGPX response in a subject.

According to an eleventh aspect of the invention there is provided a method of contraception comprising supplying the whole or part of a polypeptide according to the first aspect of the invention to a subject in order to elicit an anti-eGPX response. According to a twelfth aspect of the invention there is provided a method of contraception comprising supplying an inhibitor of eGPX activity to a subject.

According to a thirteenth aspect of the invention there is provided a diagnostic probe comprising the whole or part of a polypeptide according to the first aspect of the invention or a DNA molecule according to the second aspect of the invention. This probe may be used in diagnostic tests, assays or monitoring methods.

According to a fourteenth aspect of the invention there is provided a method of supplying wild-type eGPX function to a subject lacking such wild-type function comprising supplying to that subject a DNA sequence according to the second aspect of the invention or a polypeptide according to the first aspect of the invention The preparation of heGPX will now be described, by way of example only, with reference to the accompanying Figures 1 to 3 in which:- Figure 1 shows the DNA sequence and deduced amino acid sequence of heGPX; Figure 2 shows the results of Western blot analysis; and Figure 3 shows agarose gel electrophoresis of PCR products.

Cloning of heGPX.

In order to study the expression of heGPX, establish its presence in human seminal plasma, and look for a possible correlation with elevated levels of the active oxygen species in the mammalian male reproductive tract, it was first necessary to identify and clone heGPX as a prelude to raising specific antisera. A reverse transcriptase-PCR-based approach (RT-PCR) was used to clone heGPX cDNA using all the nucleotides based on the known macaque monkey cDNA sequence.

A commercial preparation (Clontech Labs., Palo Alto, CA, USA) of human testis total RNA (a pooled sample from 29 sudden-death victims, ages 23 to 65) was used as a template for oligo-dT-directed cDNA synthesis. The inventors had previously established that this source of RNA also contained epididymis-specific sequences, suggesting that the epididymides had not been removed from the testes prior to RNA isolation.

Initial attempts to clone the entire coding region of heGPX cDNA utilised a PCR-based approach with specific primers (5'-CAG ACT AGC ATC TAC AAA CAC TAG-3'and 5'CCT GCC CTT AAC TCC ACC TT-3') based on the cloned macaque (Macaca fascicularis) eGPX sequence (Perry, A. C. F., Jones, R., Niang, L. S. P., Jackson, R. M. and Hall, L. (1992) Biochem. J. 285). Whilst PCR products were readily obtained, they were consistently about 120 bp shorter than that predicted from the known macaque sequence (Fig. 3 in Perry, A. C. F. et al supra). Similar results were obtained using a variety of primer pairs. To determine the identity of the apparently truncated PCR product, it was gel-purified, cloned, and several of the resulting clones sequenced using an ABI377 automated DNA sequencer. Alignment of the sequences of these putative heGPX clones with that of macaque eGPX cDNA confirmed that they were eGPX orthologues. but that the cloned human PCR products all possessed a deletion of 118bp within the coding region when compared to the macaque sequence.

In order to establish whether all heGPX transcripts possessed a deletion, attempts were made to"force"the PCR amplification of a full-length heGPX transcript using a primer (5'GCG CAA TAT CCT GAA CTA AAT GCA-3') which straddled the site of deletion and contained, at its 3'base end, 11 bases derived from the deleted region (based on the macaque sequence). Hence this primer could only bind to, and produce an amplified product from, a full-length cDNA template. When this primer was used in conjunction with a suitable downstream primer designed against the heGPX cDNA or host vector sequence, a very faint PCR product of the expanded (macaque) size was obtained with human cDNA, indicating the presence of some correctly-sized transcripts. This PCR product was subsequently cloned and sequenced using an ABI377 automated DNA sequencer.

By combining overlapping sequence data from the deleted PCR products and the"forced" PCR product, a full-length sequence could be derived, but this did not provide formal proof that a full-length transcript exists. It was therefore important to establish that a complete, full-length heGPX transcript could be amplified as a single product by PCR. This was achieved using a more concentrated, better quality cDNA template, using the macaque-based primers which flank the entire eGPX coding region, and fractionating the resulting PCR products on a low melting temperature agarose gel. This time two bands were observed (Fig. 3) ; a major band representing the deleted product and a very minor band, consistent with the expected size of the full-length heGPX coding sequence. This larger product from the minor band was excised, cloned and sequenced using an ABI377 automated DNA sequencer, and found to contain the expected full-length heGPX cDNA coding sequence (Fig. 1).

Sequence of deleted and full-length heGPX cDNA clones Amplification of heGPX transcripts from a commercial testis RNA preparation by RT-PCR has indicated that the majority of transcripts contain a 118bp deletion within the coding region which would render these transcripts non-functional. Nevertheless, some full-length, functional transcripts do appear to be produced, albeit at a very much reduced level.

Since the above results were obtained with"testicular"RNA pooled from several individuals, it was important to establish whether the full-length and deleted heGPX transcripts were both present within a single individual. With this in mind, RT-PCR experiments were carried out on an epididymal RNA sample prepared from a single patient who had undergone surgery. As with the pooled RNA sample, the majority of heGPX PCR products possessed the 118bp deletion but a small amount of full-length heGPX PCR product could be"forced"using the deletion straddling primer (see above), indicating that both deleted and full-length transcripts were present in epididymal RNA from one individual. In contrast, comparable RT-PCR analyses of macaque and rat testis RNA, as well as Northern blot analyses (Perry, A. C. F., Jones, R., Niang, L. S. P., Jackson, R. M. and Hall, L. (1992) Biochem. J. 285), indicated the absence of deleted forms of eGPX in these species, with all the eGPX transcripts being of the expected full-length size. In the mouse, the eGPX gene is split between five exons (Ghyselinck, N. B., Dufaure, I., Rigaudiere, N., Mattei, M., Dufaure, J. P. (1993) Mol. Endocrinol. 7,258-272), the equivalent positions of the introns in the human sequence being indicated in Fig. 2 of that paper. In addition, the inventors have isolated the heGPX gene from a genomic library and the macaque eGPX introns have been amplified and cloned using a PCR-based approach.

DNA sequence analyses have confirmed an identical exon/intron organisation in the mouse, macaque and human eGPX genes. From this organisation it can be seen that the region of eGPX cDNA deleted from the majority of transcripts corresponds precisely to exon 3. strongly suggesting that the heGPX deleted transcripts arise by incorrect splicing of the corresponding human mRNA, with exon 2 being spliced onto exon 4 and a concomitant elimination of exon 3.

Detection of eGPX by Western blot analysis These findings would imply that the level of functional heGPX protein in the human epididymis must be extremely low in comparison to that in other species such as rat, mouse and macaque.

In order to confirm that functional heGPX protein levels are indeed substantially decreased in the human, polyclonal antibodies were raised against a maltose binding protein (MBP) heGPX recombinant fusion protein containing the first 70 amino acid residues of the mature heGPX protein deduced from its nucleotide sequence. By limiting the immunogen to the first 70 amino acid residues, cross-reactivity with other eGPX isoforms could be minimised.

The DNA construct for expression of recombinant MBP-heGPX was prepared by PCR amplification, using the full-length heGPX cDNA transcript as the template. The resulting fusion protein was used to immunise two rabbits for the production of polyclonal antisera.

Antisera from both rabbits (L16 and L17) recognised the MBP-heGPX fusion protein on Western blots, whilst the pre-immune sera gave no reaction.

Because of the scarcity of normal human epididymal tissue, macaque and rat epididymal homogenates and sperm membrane extracts were Western-blotted alongside human seminal plasma and human sperm membrane extracts, in order to investigate the presence of eGPX within these species. As Fig. 2 shows, it was found that the antiserum raised against the heGPX fusion protein cross-reacted with rat eGPX and that eGPX is abundant in rat epididymis (both caput and cauda), epididymal fluid and cauda sperm membrane extracts as previously observed (Williams, Frayne and Hall, unpublished data). eGPX is also abundant in macaque epididymis (caput, corpus and cauda) as well as in macaque cauda sperm membrane extracts. In contrast, heGPX was undetectable in human ejaculated sperm membrane extracts, or in human seminal plasma from five fertile semen donors or one vasectomized donor, indicating a very low abundance in these samples and consistent with the observed low level of correctly-processed heGPX transcripts. However, the unavailability of normal, fresh, human epididymal tissue precluded comparative analyses of epididymal homogenates or cauda epididymal sperm.

The heGPX polypeptide or nucleic acid sequences may be used as probes or diagnostic tools to detect the presence of interacting polypeptides or homologous nucleic acid sequences respectively. Further, antibodies raised against heGPX can be used as probes to identify the presence of heGPX in a sample.

Expression or overexpression of heGPX in a suitable host organism can provide a convenient source of heGPX polypeptide for pharmaceutical use or research use, and the heGPX gene sequence itself could be used in gene therapy techniques.

It is reasonably well established that in many men suffering from reduced fertility, there is an elevated level of free radicals in the male reproductive tract and this may well be the cause, or a contributing factor, to the reduced level of fertility. In view of the results regarding heGPX, it seems reasonable to suppose that the reduced level of functional glutathione peroxidase in the epididymis may well explain the elevated free radical levels in some men, if the level of this enzyme has reached a critically low concentration and is unable to adequately contain free radical levels. This in turn would imply the heGPX may be an excellent potential contraceptive target if the low enzyme levels (due to aberrant processing) are already reducing the effectiveness of this protective enzyme system in maintaining sperm viability during epididymal storage. At least two contraceptive strategies could be envisaged. Firstly, immunocontraception in which a synthetic peptide, or part of the heGPX protein, would be used to elicit an immune response in the reproductive tract. thereby reducing heGPX enzyme activity substantially further. Whilst most immunocontraceptive strategies of this type to date appear to be based on testicular antigens, many feel that epididymal antigens (like heGPX) are less likely to produce unacceptable"side effects"such as local inflammation, making them better potential candidates. The alternative strategy would be to identify or produce an enzyme inhibitor, specific to the heGPX isoform. This would inhibit the heGPX polypeptide from scavenging free radicals, thereby raising the levels of free radicals present in epithelial tissues and subsequently reducing fertility.