WO1996025495A1 - Sperm factor - Google Patents

Abstract

A cytosolic sperm factor is provided which is capable of inducing Ca2+ oscillations in mammalian oocytes. Antibodies having affinity for the cytosolic sperm factor, nucleic acid sequences encoding the cytosolic sperm factor, processes for producing the cytosolic sperm factor and uses of the cytosolic sperm factor are also provided.

Description

SPERM FACTOR

The present invention relates to a cytosolic sperm factor capable of causing Ca²* oscillations in mature mammalian oocytes (referred to herein as eggs) . The factor is responsible for activation of eggs at fertilisation.

At fertilization in mammals, sperm-egg interaction gives rise to a prolonged series of Ca²⁺ spikes, or transient oscillations, which is an essential prerequisite for egg activation and the further development of the egg (Kline and Kline, 1992, Dev. Biol., 149. 80-89; Miyazaki et al . , 1993, Science, 257. 62-78; Swann and Ozil, 1994, Int. Rev. Cytol., 152- 283-222) . There are two main hypothesis to explain how sperm-egg interaction causes the release of Ca²⁺ from intracellular stores in eggs. The first and most preferred hypothesis is the receptor hypothesis, wherein the sperm binds to a ligand receptor on the egg plasma membrane and via a series of messengers leads to the release of Ca from intracellular stores. The second hypothesis, which has received little acceptance amongst the scientific community, is the sperm factor hypothesis. Basically, a sperm factor is considered to diffuse from the sperm into the egg after gamete membrane fusion, and the factor then triggers Ca²⁺ release from intracellular stores.

A paper by Dale et al . (1985, Experientia, 4-1, 1068-1070) presents evidence that following the injection of a sperm extract into sea urchin eggs the cortical reaction is triggered. However, there have been no reports confirming this data. Dale's collaborators subsequently presented evidence and argued that the factor responsible is inositol 1,4,5-triphosphate (InsP₃) (Iwasa et al . , 1990, Biochem. Biophys. Res. Comm. , 172. 932-938).

Evidence that a factor obtained from rabbit sperm activated mammalian eggs was presented by Stice & Robl (1990, Mol. Repro. and Dev. , J2j5, 272-280) . The factor was not characterised or isolated and it was not shown that the factor caused Ca²⁺ oscillations. The experiments have never been repeated or extended by that group.

The majority of text books and reviews on the subject of fertilization only refer to the receptor hypothesis. The main hypothesis is that the sperm mediates its effects via a receptor which leads to the generation of InsP-. (Berridge, 1993, Nature, 361. 315-325; Berridge, 1995, Bioessays, 17. 491-500) . The hypothesis that the sperm binds to a ligand receptor on the egg plasma membrane which stimulates a phospholipase C, resulting in the production a Ca ⁺ mobilising messenger, namely InsP₃, has also been put forward (Jaffe, 1990, J. Reproduct. Fert., 4-2. (Suppl.), 107- 116; Foltz and Shilling, 1993, Zygote, 1, 276-279). Evidence for this hypothesis is based upon the finding that injection of G-protein activators, or sustained injection of InsP₃, can trigger Ca²⁺ oscillations in both hamster and mouse eggs. Stimulating endogenous or exogenous plasma membrane receptors can also cause Ca²⁺ increases and, in some circumstances, egg activation, and a monoclonal antibody to the InsP₃ receptor has been found to block Ca²⁺ release in mouse and hamster eggs. In addition, an egg- activating protein present on the surface of the sperm has been described for the marine worm Urechis, (Gould et al . , 1986, Devi. Biol. 117, 306-318).

In a review by Fewtrell (1993, Ann. Rev. Physiol., J55, 427- 454) it is assumed that fertilization uses the same signalling pathway as acetylcholine.

The sperm factor idea is briefly mentioned by Miyazaki et al. (1993, Devi. Biol., 158. 62-78) and it is speculated that the factor is a kinase. It is also stated that the sperm factor mechanism would be a secondary mechanism for maintaining Ca²⁺ oscillations. The possibility of a sperm factor is also mentioned in papers by Ohlendieck & Lennarz (1995, TIBS, ____., 29-32) and Shen (1992, Current Opinions in Genetics and Development, 2, 642-646) . No information is given concerning the possible identity of the factor.

It has been found that the Ca ⁺ oscillations induced by stimulating InsP₃ production are noticeably distinct from those seen at fertilization (Swann & Ozil, 1994). Furthermore, there is no direct evidence for a sperm-bound agonist that can stimulate phosphoinositide turnover via a plasma membrane interaction in sea urchin, frog fish or mammalian eggs.

Consistent with the sperm factor hypothesis is the observation that sperm-egg membrane fusion appears to occur before initiation of Ca²⁺ release in all species examined thus far. Direct evidence supporting this hypothesis in mammals was demonstrated by injection of cytosolic sperm extracts into hamster, mouse and human eggs causing activation and producing Ca²⁺ oscillations remarkably similar to those seen after fertilization in these species (Swann, 1990, Development 110. 1295-1302; Swann, 1994, Cell Calcium ___, 331-339; Homa and Swann, 1994, Human Reproduction (in press)). In addition, microinjection of intact human sperm directly into human oocytes has been shown to cause activation and Ca ⁺ oscillations after a period of delay (Tesarik et al . , 1994, Human Reproduction _ , 511-518)

The putative sperm factor responsible for producing Ca²⁺ oscillations in eggs is ineffective when applied outside the egg, and Ca²⁺ releasing activity is not associated with cytosolic extracts made from other tissues such as brain and liver (Swann, 1990) . Early observations had suggested that the egg-activating sperm factor is protein based (Swann, 1990) .

Despite the lack of credence given to the sperm factor theory it has now been determined that a protein factor, which appears as a low molecular weight polypeptide (35KD) on SDS-PAGE, is responsible for inducing Ca²⁺ oscillations in eggs. This 35KD protein therefore constitutes, or it is the principal component of, the factor responsible for egg activation after gamete fusion.

According to the first aspect of the invention there is provided a cytosolic sperm protein capable of inducing Ca²⁺ oscillation in eggs upon intracellular microinjection, having an amino acid sequence that is at least 55% homologous to the amino acid sequence of SEQ ID NO. 1, or a functionally equivalent fragment thereof.

The cytosolic protein capable of inducing Ca ⁺ oscillations, or a fragment thereof, may be obtained by recombinant DNA technology using standard procedures known to those skilled in the art and described in numerous text books such as Maniatis et al., (1982), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, or may be isolated by the purification procedure as described herein, following fractionation of cytosolic sperm extracts on adsorption columns.

Preferably, the columns used in the preparation of the cytosolic protein capable of inducing Ca oscillations, or a fragment thereof, are a concentrating membrane, such as Centricon C-30 (Amicon) , a blue dye affinity column such as a Cibacron blue F3GA column (BioRad) , an anion exchange column such as a MonoQ (Pharmacia) column which is subjected to FPLC, for example using a Pharmacia LCC-500 FPLC system, and a protein chromatography column such as a hydroxyapatite column, for example Econo-HTP (BioRad) . Fractions are selected for application to successive columns by their ability to cause Ca²⁺ oscillation in eggs.

If the eluate from the columns is separated by high resolution SDS-PAGE a 35KD moiety is isolated. The 35KD moiety has been shown to be the cytosolic protein capable of inducing Ca²⁺ oscillations. A fragment of the cytosolic protein capable of inducing Ca²⁺ oscillations can be formed from the cytosolic protein by using standard techniques such as enzyme digestion.

The cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, may be prepared by other means, for example by chemical synthesis. The invention comprises the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, prepared by any and all means, including chemical synthesis.

Preferably, the cytosolic protein capable of inducing Ca²⁺ oscillations has an amino acid sequence which is at least 55%, preferably 60%, more preferably 70%, still more preferably 85% and most preferably 90% homologous to the amino acid sequence shown in SEQ ID NO. 1.

The cytosolic protein capable of inducing Ca²⁺ oscillations having at least 55% homology to the amino acid sequence shown in SEQ ID NO. 1, is preferably a species or allelic variant of the protein having the amino acid sequence shown in SEQ ID NO. 1

Preferably, the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, is of mammalian origin. It is also preferred that the cytosolic protein capable of inducing Ca²⁺ oscillations appears as a 35KD band on SDS- PAGE.

In vivo , however, it is believed to oligomerise and behaves as a polypeptide of high molecular weight. It is believed that the cytosolic protein capable of inducing Ca²⁺ oscillations forms a ultimeric protein having a molecular weight of greater than 100KD.

According to a second aspect of the invention, there is provided a multimeric protein comprising the cytosolic protei .n capable of i .nduci .ng Ca 2+ osci .llations, or a fragment thereof. Preferably, the multimeric protein has a molecular weight greater than 100KD.

According to a third aspect of the present invention, there is provided a fraction of a cytosolic extract of sperm which is enriched for the cytosolic protein capable of inducing Ca²⁺ oscillations.

It has been observed that the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, is active in causing calcium release in tissues other than eggs (for example, liver and neuronal tissues) and it is postulated that the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, may be a member of a family of calcium-releasing proteins. It has further been found that the cytosolic protein capable of inducing Ca²⁺ oscillations has 53.6% similarity with a glucosamine-6- phosphate isomerase found in prokaryotes (EMBL Accession No. M19284) , suggesting that other similar calcium-releasing proteins exist.

The Ca²⁺ oscillations induced by the cytosolic protein capable of inducing Ca oscillations, or a fragment thereof, are typical of sperm-egg interaction in mammals and appear as a prolonged series of spikes in Ca²⁺ concentration. The spikes are similar in amplitude and terminate by a sudden decline in frequency. As with naturally-induced Ca 2+ oscillations, the first spike is usually larger and longer lasting than subsequent spikes (see Swann, 1994) .

The cytosolic protein capable of inducing Ca oscillations, or a fragment thereof, gives rise to the Ca²⁺ oscillations observed only when microinjected into the cytoplasm of mature mammalian eggs, to mimic the natural delivery of the protein to eggs by sperm. Suitable microinjection techniques are known in the art and preferred techniques are described herein. Details of the preferred techniques have appeared in the literature (Swann, 1994).

The Ca²⁺ oscillations are important for egg activation and the number of Ca²⁺ spikes effects the rate of pronuclear development. The formation of pronuclei is the criterion for successful fertilization. It has been found that by using a heavy metal chelator, for example N,N,N',N'- tetrakis(2-pyridyl-methyl)ethylenediamine (TPEN) , it is possible to inhibit Ca²⁺ oscillations. By inhibiting one or more of the Ca²⁺ spikes it has been possible to determine the optimal number of Ca spikes required for efficient fertilization. The optimal number of Ca²⁺ spikes is about 8 or 9 but can be as many as 20 without any deleterious effects occurring. It is preferred that the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, gives rise to the optimal number of Ca spikes for efficient fertilization. See also Ozil and Swann, 1995, J. Physiol., 483. 331-346.

The cytosolic protei .n capable of i.nduci.ng Ca2+ oscillations, or a fragment thereof, is preferably provided in substantially pure form, and can be isolated from a cytosolic extract of sperm as described above. Alternative methods of purification from sperm cytosol, such as immunopurification, are contemplated.

In a fourth aspect of the present invention there is provided an antibody having affinity for the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof. Preferably, the antibody is a monoclonal antibody, advantageously of IgG class, or a 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.

Antibodies according to the invention localise the cytosolic protein capable of inducing Ca oscillations, or a fragment thereof, to the equatorial segment inside mammalian sperm. Accordingly, the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, is preferably derived from the equatorial segment of mammalian sperm.

The antibodies of the present invention can be used in an assay for the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof. A variety of assay techniques can be used and are well known to those skilled in the art. A number of suitable assays are described in US patent No. 3817837, 4006360 and 3996345.

The cytosolic protein capable of inducing Ca oscillations, or a fragment thereof, activates eggs. However, it is possible that additional factors are required to constitute the fully active sperm factor. Accordingly, in a fifth aspect of the present invention, there is provided a cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, in association with additional factors as required to constitute the fully active sperm factor.

The cytosolic protein capable of inducing Ca oscillations. or a fragment thereof, may be derived from any source, including non-mammalian sources such as frog and sea urchin. However, mammalian sources are preferred. It has been demonstrated that the egg activating sperm factor is not species-specific. Sperm extract from hamster, mouse, human and rat are generally able to cross-activate . eggs. Moreover, factors have been identified in sperm from human, hamster and boar. Preferably, however, the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, is for medical or veterinary use and accordingly is preferably derived from the species it is required to treat. For example, the protein may be derived from domestic mammals such as bulls, rams or boars. Advantageously, the protein is a human protein.

In a sixth aspect of the invention, there is provided a nucleic acid sequence encoding the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof. The nucleic acid sequence may be isolated by screening a sperm cDNA library with nucleic acid probes prepared by obtaining peptide sequence from the cytosolic protein capable of inducing Ca²⁺ oscillations. Expression of the nucleic acid sequence in an expression system selected from those available in the art results in the production of the cytosolic protein capable of inducing Ca oscillations, or in pure form.

Preferably, the nucleic acid sequence is a human nucleic acid sequence. Sequences derived from other sources, including non-mammalian sources, are however envisaged.

Preferably, the nucleic acid sequence of the present invention has the nucleotide sequence of SEQ ID NO. 1, or a fragment thereof which encodes a functionally equivalent fragment of the natural protein.

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

Preferably, the nucleic acid sequence of the present invention is mRNA. When the nucleic acid sequence of the present invention is a mRNA sequence encoding the cytosolic protein capable of inducing Ca oscillations, or a fragment thereof, it can be injected into mammalian eggs where it is translated in to the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, and leads to Ca ⁺ oscillations. When the nucleic acid sequence is mRNA encoding the cytosolic protein capable of inducing Ca ⁺ oscillations, or a fragment thereof, it may be used in a fertility treatment either with or in place of the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof.

In a seventh aspect of the present invention, a fragment of the nucleic acid sequence of the present invention is provided which can be used as a probe for a nucleic acid species which encodes the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof. The present invention also provides a fragment of the nucleic acid sequence of the present invention which can be used as a primer for amplifying a nucleic acid species which encodes the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof. Preferably, these nucleic acid fragments are at least 5, more preferably at least 10 and most preferably at least 20 nucleotides in length.

In an eighth aspect of the present invention there is provided a method for identifying nucleic acid species encoding the cytosolic protein capable of inducing Ca²⁺ oscillations, or similar proteins in a body fluid or tissue sample using the nucleic acid fragments of the present invention as probes or primers.

In a ninth aspect of the present invention there is provided a nucleic acid vector for the expression of the cytosolic protein capable of inducing Ca oscillations, or a fragment thereof, comprising a promoter and a nucleic acid sequence encoding the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof.

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

In a tenth aspect of the present invention there is provided a process for the production of the cytosolic protein capable of inducing Ca²⁺ oscillations, 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 cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, in the host cell; and recovering the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, from the host cell or the host cell's medium.

The cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, and/or mRNA encoding the protein, or a fragment thereof, is useful for increasing the efficiency of in vitro fertilisation techniques performed in humans and other animals.

Preferably, the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, and/or mRNA encoding the protein is delivered to eggs together with genetic material from sperm. For example, eggs may be microinjected with the cytosolic protein capable of inducing Ca ⁺ oscillations simultaneously with or after gamete fusion has taken place. Alternatively, the protein and/or mRNA encoding the protein may be injected into eggs to generate gynogenetic embryos.

Alternatively, where the sperm is itself microinjected into the cytoplasm of the egg, for example, using the intracytoplasmic sperm injection (ICSI) technique (Tesarik et al . , 1994; Tesarik et al . , 1994, Human Reproduction 9__/

977-978; Swann et al , 1994, Human Reproduction , 978-980;

Van Steirtegham, 1995, Human Reproduction, 2__, 2527-2528), the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, may be injected with the sperm in order to increase egg activation efficiency and embryo development. Furthermore, mRNA encoding the cytosolic protein capable of inducing Ca ⁺ oscillations, or a fragment thereof, may be additionally or alternatively injected into the egg in order to increase egg activation efficiency.

The effective amount of the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, and/or mRNA encoding the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, which is injected into an egg in order to maximise fertilisation efficiency, will be determined empirically.

The desired amount of the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, and/or mRNA encoding the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, will preferably give rise to about 8 or 9 Ca spikes and may give rise up to about 20 spikes.

The cytosolic protein capable of inducing Ca oscillations, or a fragment thereof, and/or mRNA encoding the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, is also useful for increasing the efficiency of development of non-human embryos generated by nuclear transfer protocols designed to clone embryos.

Accordingly, in an eleventh aspect of the present invention there is provided the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, and/or mRNA encoding the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, for use in medicine or veterinary science.

In a twelfth aspect of the present invention there is provided the use of the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, and/or mRNA encoding the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, in the manufacture of a composition for fertility treatment. Preferably, the fertility treatment is ICSI.

In a still further aspect of the invention, there is provided a method for treating a mammal in need of treatment by administration of an effective amount of the cytosolic protein capable of inducing Ca ⁺ oscillations, or a fragment thereof, and/or mRNA encoding the cytosolic protein capable of inducing Ca ⁺ oscillations, or a fragment thereof, or an antibody having affinity for the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof. Preferably, the method comprises the administration to a mammalian egg in vivo or in vitro .

In a further aspect of the present invention there is provided a method for treating a mammalian egg in vitro comprising administering to the egg an effective amount of the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, and/or mRNA encoding the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, or an antibody having affinity for the cytosolic protein capable of inducing Ca oscillations, or a fragment thereof.

In a further aspect of the present invention a composition is provided comprising the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof; and/or mRNA encoding the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof; and a pharmaceutically acceptable diluent, carrier and/or excipient. Preferably, the diluent, carrier and/or excipient are especially suitable for microinjection into egg cells.

A further aspect of the present invention is a kit comprising the cytosolic protein capable of inducing Ca ⁺ oscillations, or a fragment thereof, and/or mRNA encoding the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, or the composition of the present invention; and reagents and equipment for facilitating egg or embryo handling and microinjection. The reagents and equipment may comprise injection pipettes, holding pipettes, buffers, reagents and other equipment intended to facilitate egg or embryo handling and microinjection.

In a further aspect of the present invention there is provided a kit according to the invention which comprises additional pharmaceutically active substances which may be administered before, in conjunction with or after the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, and/or mRNA encoding the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof. The various pharmaceutically active substances may have additive or synergistic effects.

The invention therefore provides the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, and/or mRNA encoding the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, and one or more further agents for separate, simultaneous or sequential administration.

Kits may be provided comprising an antibody according to the invention, for example for use as an assay for the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, in association with further reagents and components as desired, for example secondary antibodies, histochemical stains, viability stains and the like.

Kits may also be provided comprising a fragment of the nucleic acid sequence of the present invention useful as a probe or as a primer, for example for use as a diagnostic, in association with further reagents as necessary, for example polymerase chain reaction reagents.

A further object of the present invention is the use of the nucleic acid sequence encoding the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, in gene therapy.

A further object of the present invention is the use of the nucleic acid sequence encoding the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, in the manufacture of a composition for a fertility treatment.

Preferably, the nucleic acid sequence encoding the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, is comprised within an expression vector which comprises a promoter and allows the expression of the cytosolic protein capable of inducing Ca oscillations, 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.

In a final aspect of the present invention there is provided an agent capable of decreasing the activity of the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof. The agent can be used to decease the activity of the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, in vivo or in vitro . Preferably, the agent is delivered to the egg using standard techniques known to those skilled in the art. Preferably, the agent is the antibody or antibody fragment of the present invention or any other molecule which can bind to the cytosolic protein capable of inducing Ca²⁺ oscillations, or a fragment thereof, and decrease its activity.

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

FIGURES: Fig.l

Purification of the sperm factor on a blue dye affinity column, (a) UV absorption trace - vertical axis: OD260, Cone. KC1 (M) (stepped trace) ; Horizontal axis: Concentrates FT, 120 mM wash, 1M eluate, final wash with 2 M guanidine (b) Coomassie SDS-PAGE gel: Horizontal axis - Lane 1 - molecular weight markers; lane 2 - crude sperm extract; lane 3 - crude sperm extract after concentration on Centricon C- 30 ultrafiltration membranes; lane 4 - flow through from blue dye affinity column; lane 5 - 120 mM wash; lane 6 - 1 M eluate; lane 7 - molecular weight markers (lanes 2 to 6 all 25 μg per lane) . Vertical axis shows position of molecular weight markers.

Fig. 2 Injection of sperm extracts into unfertilized mouse eggs. Ca²⁺ is measured with intracellular fluo3 and increases in cytoplasmic Ca²⁺ are indicated by increases in fluorescence. Injections are made into individual mouse eggs at the time indicated by the arrows. The fluorescence trace is briefly interrupted during the injection. Each trace has a time bar that indicates 5 minutes. A: crude >30KD fraction; B: flow- through from blue dye affinity column; C: 1M eluate from blue dye affinity column.

Fig. 3

Purification of the sperm factor on a MonoQ ion exchange column. A: UV absorption trace: Vertical axis: OD260, Cone. KC1 (M) (gradient trace) ; Horizontal axis: fractions FT, A, B, C, D, E, F, G, H. B: SDS-PAGE as in Fig. 1 of corresponding fractions from MonoQ column.

Fig. 4 Injection of sperm extracts and measurement of intracellular calcium as in Fig. 2. Fractions A - G of the MonoQ column eluate are shown. Fig. 5 Purification of the sperm factor on a hydroxyapatite column. A: UV absorption trace - Vertical axis: OD260, Cone. KH₂P0₄ (mM) (gradient trace); horizontal axis: concentrates FT, A, B, C, D, E, F, G. B: Coomassie SDS-PAGE gel: Horizontal axis - Lane 1 - molecular weight markers; lanes 2 to 9 - fractions in order listed above (lanes 2 to 9 all 5 μl/50 μl of concentrate per lane) ; lane 10 - molecular weight markers. Asterisks at either side of lanes 5 and 6 indicate position of 35 KDDa protein. Vertical axis show position of molecular weight markers.

Fig. 6

Injection of fractions eluted from the hydroxyapatite column into unfertilized mouse eggs. The conditions are otherwise the same as in Fig. 2. The time bar represents 5 minutes, shown are typical examples of the effects of injecting fractions A to E that are eluted from the hydroxyapatite column as indicated in Fig. 5a.

Fig.7

Immunohistochemical staining using an (FITC conjugated secondary antibody) of sperm with antibody to cyytosolic protein capable of inducing Ca oscillations, or, localising the protein to the equatorial band. A: Hamster sperm; B: Human sperm.

Fig.8

Isoelectric focusing profile of soluble sperm extracts on a pH 3-10 gradient and immunoblot analysis of fractions with monoclonal antibody to the 35kD protein. Fig. 9

Primary structure of the hamster sperm 35kD protein. A: Nucleotide sequence of 1,191-base-pair (bp) hamster testis cDNA clone with deduced amino acid sequence. The N-terminal sequence obtained by automated amino acid microsequence analysis of sperm 35kD protein is underlined. B: Sequence alignment of hamster sperm 35kD protein with E.coli glucosamine-6-phosphate isomerase.

Fig.10

The relationship between the rate of pronuclear formation (= % activation) of mouse eggs at fertilization and the numbers of Ca²⁺ spikes before adding TPEN. Female pronuclei (closed squares) and male pronuclei (open squares) .

Fig 11.

Ca²⁺ spikes produced in mouse eggs injected with spermatogenic cell mRNA.

EXPERIMENTAL PROCEDURES Protein Purification

Motile sperm from male Syrian golden hamsters were prepared by incubation of dissected epididymis in M2 medium (see Swann, 1990; 1992) for 20 min at 37°C. The intact sperm were then washed twice with extraction buffer (120mM KC1, 20 mM HEPES, lmM EDTA, 200μM PMSF, 2μg/ml leupeptin, 2μg/ml pepstatin, pH7.5, all from Sigma) at 23^βC by centrifugation for 10 min, 800g and resuspension. All subsequent steps were carried out at +4°C. Cytosolic sperm extracts were prepared by homogenisation of the resuspended sperm with an Ultra-turrax (IKA Labortechnik, Staufen, Germany) for 2 x 600 sec, followed by centrifugation for 60 min at 100,000g to remove insoluble cell debris. The supernatant fraction was concentrated on Centricon C-30 membranes (A icon) for 60 min at 4000g, diluted ten-fold in extraction buffer containing lOmM KC1, then applied to a 5ml Cibacron blue F3GA column (Econo-Blue dye affinity column, BioRad) using a BioRad Econo-System.

Proteins passing through the column, and those eluting with 120 mM KC1 and 1M KC1 in the extraction buffer, were individually collected and concentrated on C-30 membranes as described above.

The concentrated 1M eluate from the Cibacron blue column was diluted twenty-fold into a buffer comprising 20 mM HEPES, lmM EDTA, 200μM PMSF, pH '7.5, and then loaded onto a 1ml Mono Q anion exchange column using a Pharmacia LCC-500 FPLC system. The Mono Q column was washed with 20ml of the same buffer, followed by 10ml of the buffer containing 150mM KC1. Proteins remaining on the column were then eluted with a 30ml linear gradient from 150 mM to 450 mM KC1, and finally with 10ml of buffer containing 1M KC1. The fractions observed to possess oscillogen activity (see egg microinjection assay details below) from the Mono Q column, were concentrated on C-30 membranes and diluted twenty-fold into a buffer containing 100 mM KC1, 20 mM HEPES, 200 μM PMSF, pH 7.5, and loaded onto a 1 ml hydroxyapatite column (Econo-HTP, BioRad) using a BioRad Econo-System.

The HTP column was washed with 10 ml of the same buffer, then eluted with a 20 ml linear gradient from 0 mM to 50 mM phosphate, and a final 200 mM phosphate step. fractions were collected and individually concentrated on C-30 membranes. The entire separation procedure, from the initial homogenisation of sperm to concentration of HTP fractions was completed in less than 20 hours. Microinjection assays for oscillogen activity were performed, as described below, within 28 hours of sperm homogenisation.

Isoelectric focusing of sperm extracts was performed for 5 hours at 4°C with 2% a pholytes 3/10 (Biorad) using a Rotofor Preparative IEF Cell (Biorad) in a pH3 to 10 gradient.

PROTEIN ANALYSIS

Protein concentrations were determined using the bicinchoninic acid protein assay (Pierce) with bovine serum albumin as standard. Samples for SDS-polyacrylamide gel electrophoresis were denatured prior to electrophoresis in 60 mM Tris-HCl pH 6.8, 5% ,9-mercaptoethanol, 2% SDS, 10% glycerol, 0.001% bromophenol blue for 5 min at 95^βC. Electrophoresis was performed (Laemmli, 1970, Nature 227. 680-685) using 1 mm thick polyacrylamide inigels, with a 5% stacking and 17.5% separating gel, for 2-3 hours at 20 mA constant current. Gels were stained overnight in 0.25% Coomassie Brilliant Blue R250, 15% acetic acid, 10% methanol and were destained in the same solution without Coomassie R250.

PREPARATION OF EGGS

Mature metaphase II mouse oocytes were used for all injection experiments and are referred to herein as eggs. Female mice (21-24 day old FI hybrid crosses of C57Bl/6JLac and CBA/CaLac) were superovulated by serial injections of 7 IU of PMSG and HCG, and eggs were collected 12-15 hours after HCG injection. Cumulus masses were dispersed by treatment with hyaluronidase and eggs were maintained in M2 medium containing 4 mg/ml BSA, at 37°C. Eggs were always microinjected within 4 hours of collection.

CALCIUM MEASUREMENTS AND MICROINJECTION ASSAYS

Intracellular Ca²⁺ was monitored with the dye fluo-3 (Sigma) , which shows an increase in fluorescence upon binding Ca ⁺. Eggs were incubated in 50 μM fluo-3 AM for 10 min, the zona pellucida was removed by brief treatment with acid Tyrode's solution followed by washes in M2, then placed in 400 μl drops of M2 medium in a chamber maintained at 30- 33°C on a microscope stage. The M2 recording medium contained 200 nM fluo-3 AM to help maintain a constant level of fluorescence in eggs (Carroll and Swann, 1992, J. Biol. Chem. 267. 11196-11201) . The base of the chamber consisted of a polylysine-coated coverslip to which eggs spontaneously adhered, eggs were microinjected with broken-tipped glass micropipettes filled with sperm extracts. Pipettes were inserted into eggs by overcompensation of the negative capacitance on an electrical amplifier in circuit with the back of the pipette (Swann, 1990) .

Solutions were pressure-injected into the eggs and the volume of the injection (approximately 1% of egg volume) estimated by the size of a balloon of solution observed with an eyepiece graticule. Pipettes were withdrawn from the eggs immediately after injection.

SEQUENCE AND STRUCTURE DETERMINATION

Two different preparations of hamster sperm 35kD protein were isolated as described previously, electrophoretically transferred onto Problott membrane (Applied Biosystems) , and subjected to microsequence analysis by automated Ed an degradation chemistry and conversion to phenylthiohydantoin derivatives (Applied Biosystems 470A) to yield the N terminal 27 amino acid residues. Oligonucleotide primers for polymerase chain reaction (PCR, 5'-CTCATCATCC TGGAGCAC-3¹ and 5'-ATCCTCGTCACACACAAA-3•) were synthesized (Applied Biosystems 377) based on the analysed peptide sequence (EMBL D31776) , to amplify a 719-bp fragment (30 cycles, with one cycle consisting of 1 min at 94 C, 1 min at 65 C and 2 min at 72 C) from isolated hamster spermatid messenger RNA by reverse transcriptase-PCR (Microfast Track & cDNA Cycle Kit, Invitrogen) . The digoxigenin-labelled 719-bp fragment (DIG High Prime, Boehringer Mannheim) was used to screen by phage plaque hybridisation approximately 1 x 10⁶ clones of a Uni-ZAP XR cDNA library derived from hamster testis mRNA (Stratagene no. 937915) at high stringency according to manufacturers instructions, yielding 28 independent clones. After in vivo excision to form the pBluescript phagemid, one of these clones, containing a 5'- end Kozak initiation sequence and a 3*-end poly(A) tail, was sequenced on both strands with internal oligonucleotide primers (Applied Biosytems 373A) . An open reading frame of 867 bp encodes a 289 amino acid protein, with a calculated Mr of 32, 610.

IMMUNOHISTOCHEMICAL STAINING

Epididymal hamster sperm, ejaculated boar sperm (JSR HealthBred Ltd. Wiltshire) and ejaculated human sperm were washed in phosphate buffered saline (PBS) . Sperm were fixed in 3% paraformaldehyde in PBS for 1 hour at room temperature, washed again in PBS and smeared onto polylysine coated coverslips. Sperm were permeabilized with 0.5% Triton X100 in PBS for 10 mins and sequentially treated with (i) mouse monoclonal antibodies to the 27kD, 35kD and 40kD proteins (0.2% ascites fluid in PBS) and then (ii) Texas red (1:20 dilution; Calbiochem) or FITC labeled (1:50 dilution; Sigma) anti-mouse immunoglobulin antibody with 1% BSA, 5% milk protein in PBS for 1 hour each. Finally sperm were stained with Hoechst 33342 at 10 ug/ml in PBS for 10 mins. Slides were mounted in 50% glycerol and examined under epifluorescence.

EXAMPLE l: SEPARATION OF SPERM EXTRACTS

The sperm factor has been shown to be of high molecular weight (Swann, 1990) and although the extracts taken straight from the initial 100,000 g centrifugation caused Ca²⁺ oscillation in eggs, extracts were routinely concentrated on Centricon C-30 membranes (with a 30KD molecular weight cutoff) . The first chromatographic separation was performed on a blue dye affinity column. The protein which bound to the column was eluted with a series of increasing salt steps and protein elution was monitored by measuring absorbency. Fractions corresponding to the different regions of the purification profile were pooled and concentrated, then assayed for Ca 2+ releasing activity and for protein content, both quantitatively using the bicinchoninic acid protein assay and qualitatively on coomassie-stained SDS-PAGE gels (Fig. lb) . Ca²⁺ releasing activity was consistently found in IM KCl (Fig. 2) and not in the material that flowed through the column. The degree of purification achieved between the >30 KD cytosolic sperm extract and the IM eluate was found to be 4.8 fold. At this stage of purification, as monitored on coomassie-stained SDS-PAGE gels, active IM eluate still contains a large number of different protein moieties (Fig. lb) .

The next chromatographic separation was based on ionic charge. Concentration samples eluted from the Blue-dye affinity column were diluted into a buffer containing 10 mM KCl, 20 mM HEPES and applied to a 1 ml MonoQ column. The protein which bound to the column was eluted with a 150 mM KCl salt step and then with a salt gradient of from 150 mM KCl, followed by a final IM KCl salt step. The best type of gradient for optimal protein separation had been previously worked out using a series of different purification profiles (data not shown) . As before, protein elution was monitored by measuring UV absorbency (Fig.3a). Measurement of Ca²⁺ releasing activity and analysis of protein content was performed as in the previous step. No activity was detected in the flow-through material. In contrast, moderate Ca²⁺ releasing activity was consistently found in fractions C to E, which correspond to a salt concentration range of from 230 mM to 310 mM KCl (Figs 3a and 4). Injection of this material into mouse eggs typically induce multiple Ca²⁺ spikes. Occasionally what may have represented weak activity was indicated on either side of this elution region by the ability of fractions above and below this elution region to cause a single Ca²⁺ spike. However, only fractions that could cause Ca²⁺ oscillations were used for subsequent steps. The degree of purification achieved between the dye-affinity IM eluate load and the active MonoQ fractions was 3.9 fold. On coomassie-stained SDS-PAGE gels the active MonoQ fractions clearly contained fewer protein moieties than the load material.

A greater separation was achieved with the next chromatographic step which was performed on an hydroxyapatite column. The protein which bound to the column was eluted with a phosphate gradient of from OmM to 50 mM KH₂P0₄, followed by a final 200 MM KH₂P0₄ salt step. As in the previous step, the best gradient for optimal separation had been previously worked out in a series of experiments (data not shown) . As before, protein elution was monitored by measuring UV absorbency (Fig. 5a). Measurement of Ca²⁺ releasing activity and analysis of protein content was performed as in the previous step. Ca²⁺ releasing activity was not found in the flow through material but activity was detected in fractions 15 to 18 (Figs. 5a and 6) , which correspond to a phosphate concentration range of from 16 mM to 26 mM KH₂P0₄. The degree of purification achieved between the MonoQ active fractions load and the active hydroxyapatite fractions was 44.5 fold. On coomassie-stained SDS-PAGE gels of fractions from the hydroxyapatite column only very few protein moieties are associated with activity. One of these, a 35KD protein (indicated by an asterisk in Fig. 5b) , consistently correlated with Ca²⁺ releasing activity. The final protein concentration of these extracts that caused Ca²⁺ oscillations was lmg/ml. These data suggest that the 35KD protein is the sole or principal component of the cytosolic sperm factor.

Isoelectric focusing in a pH 3-10 gradient revealed the 35KD protein to migrate with a pi of 6 following immunoblot analysis with a monoclonal antibody to the 35KD protein which correlated precisely with fractions that displayed oscillogen activity.

EXAMPLE 2: THE 35KD MOIETY INDUCES Ca²⁺ OSCILLATION SDS-PAGE of fractions C and D of the HTP column (Fig. 5b) consistently produces three bands at 40, 35 and 27KD. In order to determine definitively whether one or all of the protein species represented by these bands is responsible for the Ca²⁺ oscillation observed in the oscillation assay, each band was isolated from the gel and used to prepare a monoclonal antibody specific thereto. The monoclonal antibodies were then used to remove the appropriate band from semi-purified cytosolic sperm extract.

Semi-purified cytosolic sperm extract was depleted using monoclonal antibodies reactive with the 40, 35 and 27KD bands in conjunction with secondary antibodies bound to a separable label. Removal of the 35KD species by immunoseparation resulted in loss of ability to induce a Ca oscillation response.

By immunohistochemical staining using the anti-35KD antibody, the 35KD protein was localised to the intracellular region of the equatorial segment of mammalian sperm (Fig. 7) . In contrast, antibodies to the 40KD and 27KD proteins localise to completely different regions. In mammals, the equatorial segment of the sperm is the first region to fuse with the egg and is the expected localisation for a sperm factor involved in fertilisation.

EXAMPLE 3 : THE NUCLEOTIDE AND AMINO ACID SEQUENCE OF THE 35KD PROTEIN

N-terminal amino acid sequence analysis of the 35KD hamster sperm protein enabled the elucidation of the primary structure by cDNA cloning and sequencing. A 1.2kb clone with a 3' poly(A) tail, isolated from a hamster testis cDNA library, contained an open reading frame that encoded a 289 amino acid polypeptide with a calculated Mr of 32,610, and predicted pi of 6.4 (Fig. 9A) , consistent with our observations of the sperm-derived protein. Sequence identity of 53.6% was observed with a glucosamine-6- phosphate isomerase isolated from E.coli (Fig. 9B) .

EXAMPLE 4 : INHIBITING Ca²⁺ OSCILLATION AND DETERMINING OPTIMAL NUMBER OF Ca²⁺ SPIKES

Mouse eggs were collected from superovulated MF1 female mice and oocytes separated from cummulus masses by incubation in hyaluronidase in M2 media (Fulton and Whittingham, 1978, Nature, 273. 149-150) . The zona pellucidas were removed by treatment with acid Tyrode's solution. Eggs were loaded with fura red by incubation for 10 mins in the presence of 2μm fura red-AM (Molecular Probes, Eugene OR, USA) and then they were washed in M2 and attached to a polylysine coated coverslip that formed the base of heated chamber mounted on a microscope stage (Swann, 1994, Cell Calcium, 15., 331-339). Fluorescence measurements used an epifluorescence microscope and the Newcastle Multipoint System (Newcastle Photometries, Newcastle upon Tyne, UK) . Fluorescence ratio measurements of fura red were used to indicate intracellular Ca2+ concentrations (Kurebayashi, et al . , 1993, Biophys. Journal, 64. 1934-1960) . Sperm were capacitated before fertilization (Quinn et al . , 1982, J. Reprod. and Fert., __& , 161-168) and added to the bath containing the eggs. Ca2+ changes were monitored continuously and at various times after the start of Ca2+ oscillations a solution of M2 containing 40 μM TPEN plus 1 mM dithiothreitol (DTT) (both from Sigma) was added to the eggs. This solution blocked Ca2+ oscillations. The eggs were monitored for up to 10 hours after adding TPEN plus DTT to assess the rates of pronuclear formation.

EXAMPLE 5 : INJECTING SPERMATOGENIC CELL mRNA INTO MAMMALIAN EGGS

Immature mouse oocytes were collected from PMSG primed female MF1 mice and processed as in Example 4 except they were incubated for 7 hours in Ml6 (Fulton and Whittingham, Nature, 273. 149-150) in a 5% C0₂ incubator to allow them to undergo spontaneous maturation (Edwards, Nature, 1965, 208. 349-351) . Oocytes were then transferred to drops of M2 media, loaded with fura red and then infected with -lOpl of a solution containing the mRNA in a KCl buffer (as in Swann, 1990, Development, 110. 1295-1302). Intracellular Ca2+ was monitored by fura red fluorescence as in Example 4. Spermatogenic cells were collected from freshly dissected hamster testis (Belive. A.R. , 1993, Methods in Enzymology, 225. 161-113) . Spermatogenic cell suspensions were passed through -50uM nitex mesh and cells collected log centrifugation and washed into PBS plus BSA before being frozen by immersion in liquid nitrogen. Polyadenylated messenger RNA was prepared from 200mg of hamster testis- derived spermatogenic cells and other hamster tissues (brain, liver etc.) by selection on oligo(dT)-cellulose using a commercially available kit (Invitrogen Microfast Track) . The mRNA yield was determined by OD260 absorbance and diluted to -lug/uL for microinjections.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: MEDICAL RESEARCH COUNCIL

(B) STREET: 20 PARK CRESCENT

(C) CITY: LONDON (E) COUNTRY: UNITED KINGDOM

(F) POSTAL CODE (ZIP): WIN 4AL

(ii) TITLE OF INVENTION: SPERM FACTOR

(iϋ) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: li

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1191 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATIO :21..887 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CACGAGGCCT CCTGGAGGAG ATG AAG CTG ATT ATC CTG GAA CAC TAC TCT 50

Met Lys Leu lie lie Leu Glu His Tyr Ser 1 5 10

CAG GCC AGT GAG TGG GCG GCC AAG TAC ATT AGG AAC CGC ATC ATC CAG 98

Gin Ala Ser Glu Trp Ala Ala Lys Tyr lie Arg Asn Arg lie lie Gin 15 20 25

TTT AAC CCA GGG CCG GAC AAA TAC TTC ACC ATG GGG CTC CCC ACT GGG 146

Phe Asn Pro Gly Pro Asp Lys Tyr Phe Thr Met Gly Leu Pro Thr Gly 30 35 40

AGC ACC CCA CTT GGC TGC TAC CAG AAG CTG ATT GAG TAC TAT AAG AAT 194

Ser Thr Pro Leu Gly Cys Tyr Gin Lys Leu lie Glu Tyr Tyr Lys Asn 45 50 55

GGG GAC CTG TCC TTT AAA TAT GTG AAA ACC TTC AAC ATG GAC GAG TAT 242

Gly Asp Leu Ser Phe Lys Tyr Val Lys Thr Phe Asn Met Asp Glu Tyr 60 65 70

GTG GGT CTT CCT CGA GAG CAC CCA GAG AGC TAC CAC TCC TTC ATG TGG 290

Val Gly Leu Pro Arg Glu His Pro Glu Ser Tyr His Ser Phe Met Trp 75 80 85 90

AAT AAT TTC TTC AAG CAC ATT GAC ATC CAT CCT GAA AAC ACC CAC ATT 338

Asn Asn Phe Phe Lys His lie Asp lie His Pro Glu Asn Thr His lie 95 100 105 CTG GAT GGG AAT GCG GCT GAC CTG CAG GCT GAG TGT GAT GCC TTT GAG

386

Leu Asp Gly Asn Ala Ala Asp Leu Gin Ala Glu Cys Asp Ala Phe Glu 110 115 120

GAG AAG ATC CGG GCT GCA GGC GGG ATC GAA CTC TTT GTT GGA GGC ATC

434

Glu Lys lie Arg Ala Ala Gly Gly lie Glu Leu Phe Val Gly Gly He 125 130 135

GGC CCC GAT GGA CAC GTT GCT TTC AAT GAG CCG GGT TCC AGT CTG GTG

482

Gly Pro Asp Gly His Val Ala Phe Asn Glu Pro Gly Ser Ser Leu Val 140 145 150

TCC AGG ACC CGT GTG AAG ACT CTG GCC ATG GAC ACC ATC CTG GCC AAT

530

Ser Arg Thr Arg Val Lys Thr Leu Ala Met Asp Thr He Leu Ala Asn

155 160 165 170

GCT AGA TTC TTT GAT GGC GAT CTT GCC AAG GTG CCC ACC ATG GCC CTG

578

Ala Arg Phe Phe Asp Gly Asp Leu Ala Lys Val Pro Thr Met Ala Leu 175 180 185

ACG GTG GGC GTA GGC ACT GTG ATG GAT GCT AGA GAG GTG ATG ATT CTC

626

Thr Val Gly Val Gly Thr Val Met Asp Ala Arg Glu Val Met He Leu 190 195 200

ATC ACA GGC GCT CAC AAG GCT TTT GCT CTG TAC AAG GCC ATT GAG GAA

674

He Thr Gly Ala His Lys Ala Phe Ala Leu Tyr Lys Ala He Glu Glu 205 210 215

GGC GTG AAC CAT ATG TGG ACT GTG TCC GCC TTC CAG CAG CAT CCC CGT

722

Gly Val Asn His Met Trp Thr Val Ser Ala Phe Gin Gin His Pro Arg 220 225 230

ACC GTG TTT GTG TGT GAC GAG GAT GCC ACC TTG GAG CTG AAA GTG AAG 770 Thr Val Phe Val Cys Asp Glu Asp Ala Thr Leu Glu Leu Lys Val Lys 235 240 245 250

ACG GTC AAG TAT TTC AAA GGT TTA ATG CTT GTT CAT AAC AAG TTG GTG 818 Thr Val Lys Tyr Phe Lys Gly Leu Met Leu Val His Asn Lys Leu Val

255 260 265

GAC CCC CTG TAT AGT ATC AAG GAG AAG GAA ATT CAG AAA AGC CAG GCT 866 Asp Pro Leu Tyr Ser He Lys Glu Lys Glu He Gin Lys Ser Gin Ala 270 275 280

GCT AAG AAG CCA TAC AGC GAC TAGCCTGTGC CAAGTCACGA GTACCTCCGA 917 Ala Lys Lys Pro Tyr Ser Asp 285

GCGAGACAGG CAGGTCTTTC TGGAAACTGT AAGAGAGTAG GATTACTTCG CTTCAGTTCA 977

CTGTGGTTGC TGCAGACCCT TTTGGCTAGG AACATACTGG CTGTGGGGAA CATTGAGTTT 1037

AGCTATGGAG AACAGTGTTT ATAACTTTTA CCTTTTTCAG TCCTGGGGTT TGAACCTAGG 1097

GCCTTGCACA TGCCAGGCAA GTGCTCTACT GAGCTATGTC TCCAACTTGT TTACAATGTG 1157

ATATTTTCTA TTAAATCTAA TATTTTCAAA AAAA 1191 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 289 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Lys Leu He He Leu Glu His Tyr Ser Gin Ala Ser Glu Trp Ala 1 5 10 15

Ala Lys Tyr He Arg Asn Arg He He Gin Phe Asn Pro Gly Pro Asp 20 25 30

Lys Tyr Phe Thr Met Gly Leu Pro Thr Gly Ser Thr Pro Leu Gly Cys 35 40 45

Tyr Gin Lys Leu He Glu Tyr Tyr Lys Asn Gly Asp Leu Ser Phe Lys 50 55 60

Tyr Val Lys Thr Phe Asn Met Asp Glu Tyr Val Gly Leu Pro Arg Glu 65 70 75 80

His Pro Glu Ser Tyr His Ser Phe Met Trp Asn Asn Phe Phe Lys Hie 85 90 95

He Asp He His Pro Glu Asn Thr His He Leu Asp Gly Asn Ala Ala 100 105 110

Asp Leu Gin Ala Glu Cys Asp Ala Phe Glu Glu Lys He Arg Ala Ala 115 120 125

Gly Gly He Glu Leu Phe Val Gly Gly He Gly Pro Asp Gly His Val 130 135 140

Ala Phe Asn Glu Pro Gly Ser Ser Leu Val Ser Arg Thr Arg Val Lys 145 150 155 160

Thr Leu Ala Met Asp Thr He Leu Ala Asn Ala Arg Phe Phe Asp Gly 165 170 175

Asp Leu Ala Lye Val Pro Thr Met Ala Leu Thr Val Gly Val Gly Thr 180 185 190

Val Met Asp Ala Arg Glu Val Met He Leu He Thr Gly Ala His Lys 195 200 205

Ala Phe Ala Leu Tyr Lys Ala He Glu Glu Gly Val Asn His Met Trp 210 215 220

Thr Val Ser Ala Phe Gin Gin His Pro Arg Thr Val Phe Val Cys Asp 225 230 235 240

Glu Asp Ala Thr Leu Glu Leu Lys Val Lys Thr Val Lys Tyr Phe Lys 245 250 255

Gly Leu Met Leu Val His Asn Lys Leu Val Asp Pro Leu Tyr Ser He 260 265 270

Lys Glu Lys Glu He Gin Lys Ser Gin Ala Ala Lys Lys Pro Tyr Ser 275 280 285

Asp

Claims

_________

1. A cytosolic protein capable of inducing Ca²⁺ oscillations in eggs upon intracellular microinjection, having an amino acid sequence that is at least 55% homologous to the amino acid sequence of SEQ ID NO. l, or a functionally equivalent fragment thereof.

2. The protein of claim 1 which is of mammalian origin.

3. The protein of claim 1 or claim 2 which has a molecular weight on an SDS-PAGE gel of 35KD.

4. A multimeric protein comprising the protein of any one of the previous claims.

5. A cytosolic sperm extract enriched for the protein of any one of claims 1 to 4.

6. The protein of any one of claims 1 to 4 in association with additional factors as required to constitute the fully active sperm factor.

7. An antibody or an antibody fragment having affinity for the protein of any one of claims 1 to 4.

8. The use of the antibody of claim 7 in an assay for the protein of any one of claims 1 to 4.

9. A nucleic acid sequence encoding the protein of any one of claims 1 to 3.

10.The nucleic acid sequence of claim 9 which has the nucleotide sequence of SEQ ID NO. l.

11. The nucleic acid sequence of claim 9 which is DNA.

12.The nucleic acid sequence of claim 9 which is mRNA.

13. The use of a fragment of the nucleic acid sequence of any one of claims 9 to 12 as a probe or a primer for identifying nucleic acid species encoding the protein of any one of claims 1 to 3.

14.A nucleic acid vector comprising a promoter and the nucleic acid sequence of any one of claims 9 to 12.

15.A process for the production of the protein of any one of claims 1 to 3 comprising transfecting a host cell with the vector of claim 14, expressing the nucleic acid sequence and harvesting the protein from the host cell or the host cell's medium.

16.The protein of any one of claims l to 3 and/or the mRNA of claim 11 for use in medicine and veterinary science.

17. The use of the protein of any one of claims l to 3 and/or the mRNA of claim 11 in the manufacture of a composition for use in a fertility treatment.

18.The use of claim 17 wherein the fertility treatment is intracytoplasmic sperm injection (ICSI) .

19.A composition comprising the protein of any one of claims 1 to 3 and/or the mRNA of claim 11, and a pharmaceutically acceptable diluent, carrier and/or excipient.

20.A method for treating a mammalian egg in vitro comprising administering to the egg the protein of any one of claims 1 to 3 and/or the mRNA of claim 11, or the antibody or antibody fragment of claim 7.

21.A kit comprising the protein of any one of claims 1 to 3 and/or the mRNA of claim 11, or the composition of claim 19, and reagents and equipment for facilitating egg or embryo handling and microinjection.

22. The kit of claim 21 which additionally comprises pharmaceutically active substances which may be administered before, in conjunction with, or after the protein of any one of claims 1 to 4.

23. The nucleic acid sequence of claim 9 for use in gene therapy.

24. The use of the nucleic acid sequence of claim 9 in the manufacture of a composition for use in a fertility treatment.

25.An agent capable of decreasing the activity of the protein of any one of claims 1 to 4.