Method for enhancing sperm survival

This invention relates to maintenance of sperm viability to increase the success rate of artificial insemination (AI). AI is when spermatozoa are introduced into a female's reproductive tract using artificial means rather than by natural copulation.

AI is used in animals to propagate desirable characteristics of one male to one or more females or to overcome breeding problems. AI is now a fundamental technology for the intensive breeding of domestic animals, in human infertility treatments and in wildlife conservation programmes for the breeding of threatened species. Nevertheless, it has become clear that current semen preservation techniques severely compromise the spermatozoa's survival in the female reproductive tract and hence limit the successful application of the technique.

Sperm survival is particularly compromised when spermatozoa cannot be delivered directly into the uterus because the cervical anatomy is too complex, for example in sheep. This significantly reduces the efficiency of AI. Large numbers of viable spermatozoa must be used to maximize the chance of fertilization, therefore making this technique uneconomical. Surgical intrauterine insemination by laparoscopy is an efficient way of solving this problem and through use of this method conception rates of better than 60% are now common in sheep and other species. However, this method is increasingly regarded as unacceptable for routine agricultural use on grounds of welfare; in some countries routine use of this surgical approach is expected to be curtailed within a relatively short period.

Means to improve the success rate of non-surgical methods is therefore urgently required. One means of achieving this is by extending the lifespan of spermatozoa in the female reproductive tract.

Following mating (natural insemination), inseminated mammalian spermatozoa are transported to the oviduct where a reservoir of spermatozoa is formed. Studies in several species have shown that the reservoir is limited to the caudal isthmus. The spermatozoa are held in the isthmus until ovulation, when a small number are released to meet the

egg(s). During storage in the isthmus, many spermatozoa attach to the oviductal epithelial cells. Attachment to oviductal epithelial cells is important in maintaining sperm viability both in vivo and in vitro. Sperm attachment to oviductal epithelial cells is initiated by uncapacitated spermatozoa. The process of capacitation, along with the switch to the hyperactivated flagellar beating pattern, appears to coincide with the ability of spermatozoa to be released from the oviductal reservoir.

Co-culture of spermatozoa with whole oviductal epithelial cells in vitro improves the viability of spermatozoa from a number of species including rabbit, ox, sheep, horse, pig and human. It seems this is a widespread characteristic of oviductal cells however the mechanism by which oviductal cells maintain sperm viability is unknown. Both oviductal secretory products and direct membrane contact between spermatozoa and oviductal epithelial cell membranes have been reported to bestow this beneficial effect.

Despite the importance of direct membrane contact between oviduct epithelia and spermatozoa, and the possible involvement of oviductal membrane proteins in mis interaction, many studies in the past have only investigated the role of oviductal secretory products (proteins) on spermatozoa. This was related to the ease of obtaining oviduct secretory products from in vivo, and the fact that exposure to an epithelial membrane fraction invariably exposed spermatozoa to secretory proteins at the same time. The development of a membrane fractionation protocol (Smith and Nothnick, 1997) facilitated the investigation of the role of apical plasma membranes (APM) in improving sperm viability by providing the first in vitro model for studying interactions between spermatozoa and oviductal APM without the interference of oviduct secretions.

US 7,070,918, (herein incorporated by reference in its entirety) discloses that the soluble apical plasma membrane (sAPM) fraction of oviductal epithelial cells is associated with the maintaining/impiOving/prolonging activity of sperm viability.

In a study carried out to investigate normal and polyspermic fertilization rates of spermatozoa exposed to oviductal epithelial APM proteins (Satake et ah), proteins were extracted and solubilised from the APM of the oviduct, prior to co-culture with oocytes in in vitro fertilisation (FVF). The overall fertilization rate was significantly higher (78%

vs. 86%) when spermatozoa were incubated in the presence of APM proteins (P <0.05). Moreover, polyspermia fertilisation was significantly reduced (47% vs. 21%) when spermatozoa were exposed to APM proteins (P <0.01). The results of this study suggest that exposure of spermatozoa to APM proteins prior to IVF increases fertilization rate and decreases the incidence of polyspermic penetration.

Thus, the APM of oviductal epithelial cells appears to contain one or more factors which can not only prolong/improve sperm viability, but also increase rates of fertilisation in IVF (and reduce polyspermic fertilisation).

Further experimental methodology has been developed and utilised by the present inventors to identify one or more candidate proteins within sAPM which are responsible for the maintaining/improving/prolonging activity of sperm viability. The inventors have now identified a molecular chaperone, Hsc70, which is present within the sAPM of oviductal epithelial cells, and which possesses the ability to maintain and/or improve and/or prolong the viability of spermatozoa.

According to a first aspect of the present invention, there is provided a spermatozoa diluent comprising isolated Hsc70 protein, in which the Hsc70 has sperm viability improving and/or prolonging activity.

"Diluent" in this context includes any liquid or solid material used to dilute and/or carry the spermatozoa.

"Isolated" means a protein, which is substantially free of other cellular proteins found with that protein in nature.

"Hsc70" includes whole Hsc70 protein, or one or more fragments, domains, or peptides derived from Hsc70 which retain the ability to improve and/or prolong sperm viability.

By "viability improving activity" is meant an ability to increase the proportion of spermatozoa which are viable in comparison with control spermatozoa.

By "viability prolonging activity" is meant an ability to maintain the viability of spermatozoa for a longer time period than the normal lifespan of control spermatozoa which are not contacted with the Hsc70 protein. This longer time period preferably extends for from one day to three days, or greater than three days.

Molecular chaperones are ubiquitous proteins, the major classes of which include the six families of heat-shock proteins (Hsps). These proteins, so named because temperature elevation (heat shock) was the first described inducer of the response, have been found in virtually all organisms and are highly conserved phylogenetically. The Hsps most involved in protein folding are members of the Hsp40, HspόO and Hsp70 families. Each Hsp gene family comprises different forms of the protein; for example, most organisms have around 12 iso forms of the 7OkDa Hsp family, with members found in various cellular compartments, and having different expression patterns. Some isoforms are constitutively expressed (e.g. Hsc70) and others are stress-induced (e.g. Hsp70).

There is provided a spermatozoa diluent comprising Hsc70 protein, in which the Hsc70 has sperm viability improving and/or prolonging activity.

The concentration of the Hsc70 protein may be between approximately 0.1 μg/L and 10 g/L.

Preferably, the spermatozoa diluent or additive is synthetic. By synthetic we mean the diluent or additive is synthesised de novo. The advantage of synthetic diluents or additives is that these substantially eliminate the risk of transmitting viruses or other contaminants which might be associated with products obtained directly from mammalian tissue.

There is provided a spermatozoa diluent comprising Hsc70 protein, in which the Hsc70 has sperm viability improving and/or prolonging activity, wherein the concentration of the Hsc70 protein is between approximately 0.1 μg/L and 10 g/L.

The concentration of the Hsc70 protein in the diluent may be between approximately 0.1 μg/L to 10 g/L, or between 1 μg/L to 1 g/L, or between 10 μg/L to 500 mg/L, or between

10 μg/L to 200 mg/L, or between 100 μg/L to 200 mg/L, or between 200 μg/L to 200 mg/L, or between 500 μg/L to 200 mg/L, or between 500 μg/L to 100 mg/L.

Preferably a concentration of between approximately 0.05 mg/L and approximately 10 mg/L is used. More preferably a concentration of between approximately 0.1 mg/L and approximately 5 mg/L is used. More preferably still, the concentration used is between approximately 1 mg/L and approximately 4 mg/L. Optimally, the concentration of the Hsc70 protein in the diluent is about 1 mg/L (or 1 μg/ml), or about 2 mg/L (or 2 μg/ml), or about 3 mg/L (or 3 μg/ml), or about 4 mg/L (or 4 μg/ml).

The Hsc70 protein may be contained within a membrane fraction or membranes, which may be from oviductal epithelial cells. The Hsc70 may be contained within an apical membrane fraction. The membrane fraction may be soluble or insoluble. The membrane fraction may be enriched for Hsc70, i.e. contain a greater concentration of Hsc70 than is present endogenously in apical membranes of oviductal epithelial cells. The concentration of the Hsc70 protein in the membrane fraction or membranes, or the diluent may be between approximately 0.1 μg/L and 10 g/L. Preferably a concentration of between approximately 5 μg/L and approximately 400 μg/L is used. More preferably the concentration used is between approximately 25 μg/L and approximately 200 μg/L.

The diluent may comprise further additives. The diluent may comprise any mixed salt and/or sugar solution with an isosmolarity similar to or greater than the osmolality of seminal fluid which is supportive to sperm viability. The diluent may also comprise additional proteins, peptides or organic molecules, and/or water-soluble polymers and/or cryoprotective agents. The diluent may comprise one or more antibiotics.

The diluent may comprise a semen extender. The diluent may be a semen extender. Semen extenders are variable in actual ingredients, but all contain ingredients that serve a common purpose, i.e. nourish the spermatozoa, buffer the pH, protect the spermatozoa during temperature change, and kill microorganisms (e.g. bacteria) within the semen. The semen extender may comprise one or more nutrients, buffers, cryoprotective agents, and/or antibiotics.

The nutrient may be a sugar, such as glucose or sucrose, which serves to provide energy source for the sperm. Buffers are added to balance pH and osmolality of the solution, and the sugars may also serve this purpose. The cryoprotective agent may be glycerol or DMSO. The extender may comprise egg yolk, which is a common ingredient in a wide variety of frozen semen extenders.

The diluent or extender may be Beltsville Thawing Solution. The diluent may comprise one or more of glucose, sodium citrate dihydrate, EDTA, sodium bicarbonate, potassium chloride, and water.

Other diluents and extenders are commercially available and will be well known to a person skilled in the art of AI.

In another aspect the Hsc70 protein may comprise the sequence of SEQ ID NO: 1.

The Hsc70 protein may have at least 80% identity with the protein listed in SEQ ID NO.

1, or at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% identity with the protein listed in SEQ ID NO.

1.

The Hsc70 protein maybe from any animal which is propagated by AI.

The Hsc70 protein may be selected from the group consisting of: human, pig, sheep, ox, camel, horse, dog, cat, poultry, rabbit, buffalo, elephant, llama, guanaco, alpaca, and vicuna.

The cat may be any cat contained within the family Feliάae, and may therefore include members of the Panthera genus such as lion, tiger, leopard, and jaguar. The cat maybe a domestic cat.

The camel may be a Bactrian camel, a Dromedary camel, or a hybrid camel.

The buffalo may be a bison, a water buffalo (also known as Swamp Buffalo and Indian Buffalo), or a Cape buffalo.

The Hsc70 protein may be bovine Hsc70.

The Hsc70 protein may be isolated and/or purified from an animal or human source, or it may be produced using recombinant DNA technology, or synthesised chemically. Such techniques are well established and will be known to a person skilled in the art.

The Hsc70 protein may be linked to an inert polymer. By "linked" it is meant that the polymers are joined to the protein; the join may be through one or more ionic or covalent bonds. Such linking is well known in the art and can result in the advantages of increased efficiency and reduced toxicity.

The polymer may be hydrophilic. Hydrophilic polymers are defined as polymers having a solubility of greater than 10g/L in an aqueous solution, at a temperature between 0 to 50 deg C. The aqueous solution may include small amounts of water-soluble organic solvents, such as dimethylsulfoxide, dimethylformamide, alcohols or acetone. The polymer may be polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, hydroxylated celluloses, polypeptides, polysaccharides such as polysucrose or dextran and alginate.

The polymer may be amine and carbonyl-reactive dextran.

The concentration of Hsc70 protein in the diluent may be equal to or greater than the concentration of membrane bound Hsc70 in oviductal epithelial cells. The concentration of Hsc70 protein in the diluent may be equal to or greater than the total concentration of Hsc70 contained within oviductal epithelial cells.

The diluent may further comprise spermatozoa.

The spermatozoa may be selected from the group consisting of: human, pig, sheep, ox, camel, horse, dog, cat, poultry, rabbit, buffalo, elephant, llama, guanaco, alpaca, and vicuna.

The camel may be a Bactrian camel, a Dromedary camel, or a hybrid camel.

The buffalo may be a bison, a water buffalo, or a Cape buffalo.

The cat may be any cat contained within the family Felidae. The cat may be a domestic cat.

The conservation within Hsc70 proteins across different species means that Hsc70 protein from one species may be used to improve/prolong sperm viability for another species, whether closely related, or distantly related. The Hsc70 protein may be from one species, and the spermatozoa may be from a different species. The Hsc70 protein and the spermatozoa may be from the same species, or from species within the same genus, or sub-family, or family, or order, or class, or phylum.

The Hsc70 protein may be bovine Hsc70 and the spermatozoa may be porcine, ovine or bovine spermatozoa.

The spermatozoa may be microencapsulated. By "microencapsulated", is meant that the spermatozoa are enclosed within a semi-permeable membrane. Examples of membranes which can be used include beeswax, starch, gelatine, and polyacrylic acid and polylysine.

The spermatozoa may be microencapsulated in a semi-permeable membrane. The semipermeable membrane may comprise poly-lysine.

The spermatozoa may be microencapsulated in a gel, which may be a sodium alginate gel.

According to a further aspect of the present invention, there is provided a kit of parts comprising the diluent of the first aspect of the invention, and spermatozoa.

According to a further aspect of the present invention, there is provided a composition comprising spermatozoa and Hsc70 protein.

According to a further aspect of the present invention, there is provided a use of the diluent of the first aspect of the invention to improve and/or prolong sperm viability.

According to a further aspect of the present invention, there is provided a use of Hsc70 protein to improve and/or prolong spermatozoa viability.

The use may be carried out in vivo or in vitro.

According to a further aspect of the present invention, there is provided a use of Hsc70 protein in the manufacture of a medicament to improve and/or prolong sperm viability.

According to a further aspect of the present invention, there is provided a use of Hsc70 in the artificial insemination of a human or animal.

According to a further aspect of the present invention, there is provided a method of improving and/or prolonging sperm viability which comprises contacting spermatozoa with Hsc70 in vitro.

The method may be performed during in vitro fertilisation.

There is provided a method of in vitro fertilisation, comprising contacting spermatozoa with Hsc70 and contacting the spermatozoa with one or more oocytes or IVF droplets comprising one or more oocytes.

According to a further aspect of the present invention, there is provided a method of artificially inseminating an animal, the method comprising contacting spermatozoa with Hsc70 and inseminating the animal.

Numerous IVF and artificial insemination methodologies exist, and these are considered to be well known to a person skilled in the art.

The spermatozoa may be chilled, frozen or at ambient temperature or body temperature. The spermatozoa may be stored frozen or at any temperature in the range -9 deg C to 45 deg C. The spermatozoa may be in the vitrified state.

The Hsc70 may be provided in a diluent according to the first aspect of the present invention.

The spermatozoa may be cryopreserved prior to contact with Hsc70.

The spermatozoa may be contacted with Hsc70 during cryopreservation.

The method may be carried out before or after sex-sorting of the spermatozoa for X- (female) and Y-bearing (male) spermatozoa.

A method as claimed in any one of claims 27-30, the Hsc70 and spermatozoa being contacted for between about 30 seconds and 14 days.

The spermatozoa and Hsc70 may be contacted for between about 30 seconds and 14 days, or between about 30 seconds and 12 days, or between about 30 seconds and 10 days, or between about 30 seconds and 8 days, or between about 1 minute and 6 days, or between about 1 minute and 3 days, or between about 1 minute and 2 days, or between about 1 minute and 24h, or between about 1 minute and 12h, or between 1 minute and 6h, or between about 5 minutes and 3h, or between about 10 minutes and Ih.

The present invention will be further apparent from the following description, which shows, by way of example only, specific embodiments of the use of Hsc70 and experimentation therewith.

Brief description of the figures

Figure 1 shows the viability index (mean ± SEM) of boar spermatozoa incubated with different concentrations of antibodies;

Figure 2 shows the viability index (mean ± SEM) of boar spermatozoa incubated with different concentrations of bovine Hsc70;

Figure 3 shows the viability index (mean ± SEM) of boar spermatozoa incubated with bovine Hsc70, oviductal APM proteins, and TALP medium;

Figure 4A shows the viability index (mean ± SEM) of boar spermatozoa incubated for 24 h at 39 ⁰ C in the presence of soluble apical plasma membrane protein fraction and either anti-hsp70 or anti-hsc70 antibody,

Figures 4B and 4C show the viability index (mean ± SEM) of boar spermatozoa incubated for 24h at 39°C in the presence of TLP, sAPM (200μg/mL) or two concentration ranges of recombinant Hsc70;

Figure 5 shows the viability index (mean ± SEM) of bull spermatozoa incubated for 48h with various concentrations of recombinant bovine Hsc70;

Figure 6 shows the viability index (mean ± SEM) of ram spermatozoa incubated for 48h with various concentrations of recombinant bovine Hsc70; and

Figure 7 shows the scores table from a ClustalW alignment of 33 Hsc70 protein sequences.

Experimental

Methods and Materials

Oviduct tissue preparation

Porcine oviduct tissues were obtained and oviducts (attached to ovaries) were cleaned and washed with cold PBS. The oviducts were divided into two groups designated: FOL

(follicular) and LUT (luteal), based on the appearance of the associated ovaries. Those oviducts attached to ovaries containing large follicles (8-12 mm in diameter) with signs of recent ovulation and no corpora lutea were assigned to the FOL group, those with ovaries containing several corpora lutea, without large follicles were assigned to the LUT group. Oviducts in both groups were trimmed from the ovaries and washed by passing four times through PBS. Each oviduct was divided into three sections; the first, designated as ampulla, was a section between the fimbria and the middle (thicker part) of the oviductal tube. The second section designated as isthmus, consisted of 1-2 cm of the

caudal part of the uterine horn, the uterotubal junction, and up to nearly the middle (thinner part) of the oviductal tube. Finally, a section around the junction of the thin and thick part of the oviductal tube, approximately 2-3 cm long, was excised and discarded to assure differentiation of isthmic and ampullar parts of the oviduct. Each oviduct section (isthmic or ampullar) was processed separately. They were opened longitudinally and epithelia were scraped into a Petri dish using a clean glass microscope slide. Scraped tissues collected from approximately 8-12 oviduct sections were collected separately (FOL isthmus, FOL ampulla, LUT isthmus and LUT ampulla) into 20 ml of cold PBS and kept on ice. These suspensions were centrifuged for five minutes at 200 g. The supernatants were discarded and pellets were resuspended in 20 ml of buffer 1 containing 60 niM mannitol, 5 mM EGTA, 1 μM phenylmethylsulfonylfluoride (PMSF), Tris base (pH 7.4). Suspensions (5 ml) were snap frozen in liquid nitrogen and stored at -80 deg C. until subsequent use for APM preparation.

sAPM Preparation

Tissue homogenates were thawed and homogenized on ice for one minute using a small homogeniser (Silverson, Waterside, UK). Two hundred microlitre aliquots of this initial homogenate were snap-frozen in liquid nitrogen and stored at -80 deg. C. for subsequent analyses. The homogenate was supplemented with 200μl of IM MgCl ₂ followed by 30 minutes incubation on ice. Thereafter the homogenate was centrifuged for 15 minutes at 3000 g. The pellet was discarded and the supernatant was centrifuged for 30 minutes at 90,000 g. After centrifugation, the pellet was resuspended in 20 ml of buffer 2 containing 60 mM mannitol, 7 mM EGTA, Tris base (pH 7.4) with ten strokes of a Potter S homogenizer. The homogenate was supplemented with 200 μl IM MgCl ₂ and incubated on ice for 30 minutes. Afterwards, the mixture was centrifuged at 3000 g for 15 minutes. The pellet was discarded and the supernatant was centrifuged at 90,000 g for 30 minutes. The pellet, following ultracentrifugation, was resuspended in 20 ml of a modified Tyrode's medium containing 2 mM CaCl ₂ , 3.1 mM KCl, 0.4 mM MgCl ₂ .6H ₂ O, 100 mM NaCl, 25 mM NaHCO ₃ , 0.3 μM NaH ₂ PO ₄ 2H ₂ O, 10 mM HEPES, 21.6 mM Sodium lactate and 1 mM sodium pyruvate with ten strokes of a Potter S homogenizer. The suspension was centrifuged for 30 minutes at 90,000 g. The supernatant was discarded and the pellet was resuspended in 900 μl of the modified Tyrode's medium by aspiration

through a 0.9x90 mm Yale spinal needle (Becton Dickinson, Oxford, UK). This fraction was portioned, snap-frozen in liquid nitrogen and stored at -80 deg. C.

Protein and [gamma] -glutamyl Transpeptidase Activity Analysis Protein concentrations of initial homogenates, final APM preparations from different tissues, and peripheral membrane protein fractions obtained from oviductal APM, were quantified using a Bio-Rad Protein Assay kit (Bio-Rad, Hemel Hempstead, UK). The kit is based on a dye-binding assay, in which the colour of the dye changes differentially, in response to change in protein concentration. [Gamma]-glutamyl transpeptidase has previously been shown to reside mainly in the APM of polarized epithelial cells. The activity of [gamma] -glutamyl transpeptidase in the initial homogenate and in the APM preparations was measured calorimetrically, using the Sigma diagnostic kit 545 (Sigma, Poole, Dorset, UK). The assay is based on the transfer of the glutamyl group from L- glutamyl-p-nitroanilide to glycylglycine catalyzed by [gamma] -glutamyl transpeptidase. The liberated p-nitroaniline is diazotized by the addition of Sodium Nitrite and Ammonium Sulfamate. The absorbance of the pink azo-dye resulting from the addition of N-(l-napthyl)-ethylenediamine, measured at 530-550 nm, is proportional to [gamma]- glutamyl transpeptidase activity. The degree of enzyme enrichment was expressed as fold increase in [gamma] -glutamyl transpeptidase activity in the final APM preparations compared to the initial homogenate. This demonstrated the success of the method employed to isolate APM preparations from the initial homogenates. In addition, distinct differences in the protein profile of APM preparations were observed compared to that of original homogenates. Three proteins diminished and three were enriched in APM preparations compared to that of the initial oviductal homogenates.

Semen preparation

Boar semen, diluted and stored for 24 hrs in Beltsville thawing solution was obtained, and the semen (45 ml) washed three times with PBS by centrifugation and resuspension (600 g for 10 min). After the last centrifugation the supernatant was discarded, and the pellet was resuspended in modified Tyrode's medium supplemented with 12 mg/ml BSA (TALP), 200 U/ml penicillin, 200 μg/ml streptomycin and 0.5 μg/ml amphotericin B (Life Technologies, Paisley, UK) (supplemented Tyrode's medium). One ml of washed semen sample was overlaid with 500 μl of supplemented Tyrode's medium in a test tube.

The tube was placed at a 45 degree angle in an incubator held at 39 deg. C. in a humidified atmosphere saturated with 5% CO ₂ . After one hour the top 0.5 ml of medium containing the swim-up spermatozoa fraction was collected. Spermatozoa concentration was measured using a counting chamber.

Sperm viability was assessed using a combination of Ethidium homodimer-1 (ETHD-I; Molecular Probes, Leiden, The Netherlands) and SYBR- 14 (Molecular Probes, Leiden, The Netherlands). One μl of 2 mM ETHD-I and 2.5 μl of 20 pM SYBR-14 were diluted in 1 ml of PBS. An equal volume of the dye mixture was added to the semen sample and incubated for 20 minutes at 39 deg. C. An aliquot of this preparation was placed on a slide and evaluated by epifluorescence microscopy (x40 objective). Viable spermatozoa with intact membranes excluding ETHD-I demonstrated green fluorescence over the nucleus due to SYBR-14 staining. Spermatozoa with disrupted membranes showed red nuclear fluorescence due to ETHD-I staining. Two hundred spermatozoa were evaluated by fluorescence microscopy and classified as membrane intact (green) or membrane damaged (red). The same methodology was used for the preparation of ram and bull semen.

Gel electrophoresis Gel electrophoresis procedures were carried out using a Bio-Rad Modular Mini Electrophoresis System (Bio-Rad Labs, Hemel Hempstead, Herts, UK) using the discontinuous buffer system (Laemmli, 1970). 5μg protein of original homogenate and purified APM preparations obtained from FOL isthmic, FOL ampullar, LUT isthmic, LUT ampullar and lung tissues were loaded on SDS-polyacrylamide gels (12% separation, 5% stacking). Proteins were fractionated by electrophoresis for between approximately 45 mins to 1 hr at between approximately 180 to 200 volts. Following electrophoresis the gels were fixed and then stained with Brilliant Blue G-Colloidal concentrate (Sigma, Poole, Dorset, UK). A digital image was produced from stained gels using a Hewlett Packard ScanJet 6200c scanner (CA, USA). The image was further analyzed using Scion image Beta 4.0.2 software program (Scion Corporation, MD, USA). Protein profiles of oviduct peripheral membrane proteins, pellet left after the recovery of peripheral membrane proteins, and oviductal APM were produced and analyzed using the methodology described above.

Labelling and detection of sAPM proteins

The lumena of sow oviducts were treated with the biotinylation reagent Sulfo-NHS-LC- biotin so that proteins lining the oviduct that would normally come into contact with spermatozoa were labelled. The method for purification and solubilisation of the sAPM was carried out as described above. Semen was washed through a Percoll density gradient and the resulting spermatozoa were incubated with the labelled sAPM. Unbound sAPM was removed and the membranes of spermatozoa were solubilised in SDS. The mixture was centrifuged to isolate the surface proteins from the spermatozoa and any sAPM proteins that may have bound to them. The proteins were then fractionated by SDS-PAGE, transferred to a nylon membrane by electroblotting, and the biotinylated proteins detected using an Avidin horse-radish peroxidise conjugate, NeutrAvidin/HRP.

Identification of candidate sAPM proteins using proteomic analysis Samples of speraiatozoa/sAPM were separated by SDS-PAGE and the proteins stained with Coomassie blue. Using a western blot for comparison, stained protein bands of interest were cut from the stained gel. Each gel band was diced into approximately lmm ³ pieces and then destained with 20OmM ammonium bicarbonate with 40% (v/v) acetonitrile. The gel pieces were incubated overnight in a trypsin solution of 0.4 μg trypsin (Promega, Southampton, UK) and 50 μl of 5OmM ammonium bicarbonate. The next day, peptides were extracted in four sequential extraction steps using 30 μl of 25mM NH ₄ CO ₃ (lOmin, room temperature), 30 μl acetonitrile (15min, 37°C), 50 μl of 5% (v/v) formic acid (15 min, 37 ⁰ C), and finally, with 30 μl acetonitrile (15 min, 37 ⁰ C). All extracts were pooled and dried in a vacuum centrifuge.

Dried peptides were resuspended in 0.1% (v/v) formic acid in 3% acetonitrile. The mixture was separated on a PepMap Cl 8 RP capillary column (LC-Dionex, Leeds, UK), and eluted in a 60-minute gradient via a LC packings Ultimate nanoLC directly into the mass spectrometer. The compositions of the hydrophilic and hydrophobic solvents were 5% (v/v) acetonitrile, 0.1% (v/v) formic acid and 95% (v/v) acetonitrile, 0.1% (v/v) formic acid. An Applied Biosystems/MDS-Sciex QStarXL electrospray ionisation

quadrapole time-of-fiight tandem mass spectrometer (ESI-qQ-TOF) was used for mass spectrometric analysis. Analyst Qs software (Applied Biosystems) was used for data acquisition and data analysis. The data acquisition was performed in the positive ion mode using Information dependent Acquisition (IDA). After each TOF-MS scan, three peaks with charge states two or three were selected for tandem mass spectrometry.

The peptide-based protein matches were obtained using MASCOT 2.0 software (www.matrixscience.com). A sequence query search was performed using the Mass Spectrometry protein sequence DataBase (MSDB, Nov 2004). The taxonomy was limited to filter for only mammalian matches, and trypsin was used as enzyme, with one missed cleavage site allowed. The peptide tolerance was set to 1.0Da and the MS/MS tolerance was set to 0.25Da.

Hsc70 protein Recombinant bovine Hsc70 protein (Product number SPP-751) was purchased from Assay Designs, 5777 Hines Drive, Ann Arbor, MI 48108, USA.

Antibodies

Antibodies having specificity for each of the proteins Hsp90, Hsp70, Hsc70 and Clusterin were utilised in the experimental procedures.

Spermatozoa-APM and spermatozoa-Hsc70 co-incubation

Swim-up spermatozoa fractions (50 x 10 ⁶ spermatozoa/ml) in 25 μl aliquots were added to 25 μl of APM or Hsc70 protein (variable concentrations depending on experimental design). Spermatozoa-APM and spermatozoa-Hsc70 co-incubation droplets were covered with mineral oil, incubated at 39 deg. C, in an atmosphere containing 5% CO ₂ for 24 hrs. After co-incubation 50 μl of PBS containing 20 μM SYBR-14 and 2 μM ETHD-I was added to each droplet and further incubated for 15 min. Thereafter the sperm viability was assessed as described above.

Sperm viability assays

Boar sperm viability in the presence of antibody-treated sAPM

Antibody-treated sAPM (200 μg/mL) was prepared by incubating sAPM with antibodies for 2 hours on a rotary shaker at room temperature. A series of preliminary experiments was undertaken to determine approximately how much antibody was required to prevent binding of FITC-5 labelled sAPM to washed boar spermatozoa. For this purpose boar spermatozoa were incubated with antibody-treated sAPM. Antibodies to Hsc70, Hsp70 and Hsp90 (Abeam PIc, Cambridge, UK) were supplied at 0.2 mg protem/mL and diluted in TLP (1 :200 v/v). Semen from 8 boars was individually washed with Percoll- saline as previously described and each sample (in TALP) was adjusted to give a final concentration of 25 x 10 ⁶ 10 spermatozoa /mL. 15 μL droplets of sperm suspension and antibody-treated sAPM (with untreated sAPM and TLP as control treatments) were mixed 1:1 in 20 mm diameter wells in 4 well Multi-dish plates (Sigma Aldrich, Poole, Dorset, UK) and then covered with mineral oil. They were incubated at 39 ⁰ C in 5% CO2 for 24 hours. The viability of spermatozoa was assessed at zero hours and after 24 hours.

Dose response effect of Hs c70 protein on sperm viability

Boar spermatozoa were prepared as previously described. Recombinant bovine Hsc70

(Stressgen, Ann Arbor, Mi, USA; SPP-751) was diluted in TLP to give final concentrations of 8, 2, 0.5 and 0.25 μg/mL (series 1, Figure 4B) and 4, 0.5 and 0.1 μg/mL (series 2, Figure 4C) and when incubated with equal volumes of spermatozoa.

Control samples were incubated in TLP only or TLP + sAPM (200 μg/mL). The samples were incubated at 39°C in 5% CO ₂ for 24 hours. Sperm viability was examined as described previously.

Effect of Hsc70 protein on bull sperm viability

Serial dilutions of bovine recombinant Hsc70 and a control protein alpha tubulin were prepared in TALP to give final concentrations of 4, 1, 0.5 and 0.1 μg/mL (Figure 5).

Spermatozoa were added to droplets of TALP, TALP + alpha tubulin (Abeam pic, Cambridge, UK) or TALP + recombinant Hsc70 in equal volumes of 25 μL each. The 50 μL droplets were covered with mineral oil and incubated at 37°C in a 5% CO2 incubator for 48 hours. The viability of samples was assessed at zero hours and then at 48 hours following the same procedure as that for boar spermatozoa.

Effect of Hsc70 protein on ram sperm viability

Freshly collected and washed ram spermatozoa were co-incubated for up to 48 h with seven different concentrations of bovine recombinant Hsc70 and without protein (Figures 6A-6C). The final concentrations of Hsc70 were: 16, 8, 4, 2, 1 ₅ 0.5, and 0.25 μg of protein/ml. The viability of the spermatozoa co-incubated with or without Hsc70 was determined at 24 h and 48 h. This experiment was replicated using spermatozoa from eight rams.

Microencapsulation of spermatozoa

Suspensions of spermatozoa in physiological saline containing 1% sodium alginate (w/v), pH 6.8, were passed through a syringe pump to form droplets having a mean diameter of between 0.75 and 1.5 mm. Briefly, the spermatozoa suspension within a 10 ml syringe was forced through a 19 gauge hypodermic needle contained within an encapsulating jet at a rate of approximately 1.5 ml/min to form droplets which were collected in a beaker containing aqueous solution (80 ml) of 1.5% CaCb-Hepes buffer (50 mM) pH 6.8. Immediately on contact with the CaCl ₂ -Hepes buffer solution, the droplets absorb calcium ions, which cause solidification of the entire-cell suspension resulting in a shape-retaining, high viscosity microcapsule. To form a semi-permeable membrane on the surface of the microcapsules, the microcapsules were rinsed three times with physiological saline and suspended in physiological saline containing 0.4% polylysine having a molecular weight range of 25 to 50 kDa. The excess polylysine was aspirated and the microcapsules rinsed with 0.1% CHES buffer, pH 8.2. After three rinses with physiological saline, the alginate gel inside the microcapsules was liquefied by suspending the capsules in isotonic 3% sodium-citrate saline solution, pH 7.4 for approximately 5 minutes.

Cryopreservation of spermatozoa

Collected semen was allowed to cool slowly to room temperature over a period of around 2 hours. Semen was aliquoted into tubes containing approximately 6 x 10 ⁹ spermatozoa and centrifuged at room temperature for 10 minutes at 300 g. The supernatant was removed by aspiration and the spermatozoa resuspended into Beltsville F5 extender (5 ml).

The tubes containing the extended spermatozoa were then placed in a beaker containing water (50 ml) at room temperature, which was then placed into a refrigerator and cooled to 5 deg. C. over a two hour period. After the spermatozoa were cooled, 5 ml of Beltsville F5 extender containing 2% glycerol was added to each tube. The contents of the tubes were mixed by immersion and frozen immediately into pellets of 0.15 ml to 0.2 ml on dry ice. The pellets were then transferred to liquid nitrogen for storage.

SILAC and parallel purification and analysis of individual cellular compartments Stable isotope Labelling with Amino acids in Cell culture (SILAC) combined with the parallel purification and analysis of individual cellular compartments (cell-surface and intracellular compartments) was employed to investigate protein trafficking in response to gamete presence in the oviduct. Oviductal epithelial cells (OEC) were cultured in a medium containing stable isotope labelled arginine to create an isotope labelled OEC population otherwise identical to an unlabelled OEC population. Both populations were co-incubated with spermatozoa for 6 hours. However spermatozoa were in direct physical contact with the labelled OEC, and separated from direct physical contact with unlabelled OEC by a diffusible membrane. Cell-surface proteins were then biotinylated, and affinity-purified using immobilised avidin, to create a 'cell-surface' protein fraction. Non-biotinylated proteins were collected to create an 'intracellular' protein fraction.

Proteins were separated by ID gel electrophoresis, and subjected to Liquid

Chromatography/Mass Spectrometry/Mass Spectrometry (LC-MS/MS).

In vitro fertilization technique Sow ovaries were obtained from a local abattoir (A & G Barber, Purleigh, Essex, UK); these were placed in 0.9% saline solution supplemented with 100 IU/mL penicillin-G and 100 μg/mL streptomycin sulphate solution (pen/strep; Invitrogen Life Technologies, Paisley, UK) and maintained in a thermos flask at 25-3O ⁰ C for transportation to the laboratory. On arrival the ovaries were washed in lukewarm water then rinsed in warm saline solution with pen/strep supplements.

Follicular fluid from 3-6 mm diameter ovarian follicles were aspirated using an 18.5 gauge needle attached to a 1OmL disposable syringe containing 2mL of collection

medium 1 (TCM 199 supplemented with 5mM NaHCO ₃ , 15mM Hepes (Na ⁺ salt or free acid), 0.05g/L kanamycin sulphate, 0.4% (w/v) BSA fraction V and 0.04g/L heparin). The aspirated fluid was then filtered through a 70 μm cell strainer to harvest the oocytes. The oocytes were washed and collected into pre- warmed collection medium 2 (TCM 199 (with Hank's salts and sodium bicarbonate) supplemented with 0.1% (w/v) polyvinyl alcohol, 3.05mM DlO glucose, 0.9ImM sodium pyruvate and pen-strep solution).

Cumulus-oocyte complexes (COCs) were collected under a stereomicroscope; oocytes had at least two complete layers of cumulus cells and evenly granulated ooplasm. The selected COCs were washed in collection medium 2 and transferred in groups of 50 COCs into pre-equilibrated 50OmL wells of IVM medium (TCM 199 (with Earle's salts, L-glutamine and sodium bicarbonate) and supplemented with 0.1 % polyvinyl 5 alcohol, 3.05mM D-glucose, 0.9ImM sodium pyruvate, 0.57μM cysteine and IOng/mL epidermal growth factor and pen-strep solution and pre-equilibrated). Before oocyte transfer 0.5μg/mL LH and 0.5μg/mL FSH were added to the 500 μL maturation droplets. The preparations were incubated for 48h. After IVM incubation 0.1% (w/v) hyaluronidase was added for 30 min to the maturation droplets to loosen the cumulus cells. COCs were then washed in pre-equilibrated mTBM (modified Tris-buffered medium (mTBM: 113.ImM NaCl ₅ 3mM KCl, 2OmM Tris-base, HmM D-glucose and 5mM sodium pyruvate supplemented with 5mM NaHCO3 and 0.4% (w/v) BSA). After equilibration of the medium, 1OmM CaC12 was added to avoid precipitation). Groups of 30-40 COCs were placed in pre-equilibrated 98μL droplets of mTBM covered in mineral oil. Approximately 3 x 10 ⁶ mL of Percoll-washed spermatozoa were incubated for 10 min in ImL of pre-equilibrated mTBM containing 1 μg/ mL recombinant Hsc70 or 50 μg/mL sAPM as appropriate. 2 μL sperm suspension were added to the IVF droplets, to give a final concentration of 6 x 104 spermatozoa /mL. The oocytes and spermatozoa were co- incubated for 6 h.

Following sperm-oocyte co-incubation, the presumptive zygotes were collected into ImL of preequilibrated NCSU23 medium (North Carolina State University-23 medium: 108.73mM NaCl, 4.78mM 20 KCl, 1.19mM K2PO4, 1.19mM MgSO4.7H2O, ImM L- glutamine, 5.55mM D-glucose, 7mM taurine, 5mM hypotaurine, 25.07mM NaHCO3 and 1.7ImM CaC12 supplemented with 0.4% (w/v) BSA and pen-strep solution) in a 5 mL

glass tube and vortexed for 2 min to remove loosely bound spermatozoa and cumulus cells. The presumptive zygotes were then washed twice in fresh pre-equilibrated 40 μL droplets of NCSU23 and transferred to 20 μL of the same medium in groups of 20-25. They were cultured for 48h and then fixed in 2.5% paraformaldehyde fixative with 0.5% triton X-100 for 30 min at 39 ⁰ C. The embryos were washed (x 3) in warm PBS and stained with 10 μg/mL PI for 10 min before being mounted in mounting medium (Vectashield, Vector Laboratories). They were examined by confocal microscopy (Zeiss LSM 510, Germany) to identify fertilisation status. The oocytes/embryos 5 were classified as (1) unfertilised oocytes (the presence of metaphase plates or female pronuclei), (2) fertilised, with male and female pronuclei, (3) one cell embryos, (4) cleaved embryos with two or more cells (5) embryos with more than 4 cells.

Statistical Analysis

The data were expressed as mean viability index ± SEM. Viability index was defined as percentage of viable spermatozoa after 24 hours incubation in comparison to that of the initial viability of the same semen sample at the beginning of incubation period. Sperm viability data were tested for normal distribution. Analysis of variance was used for the statistical analysis of the data. The level of significance was considered P = 0.05.

Results

Labelling and detection of sAPM proteins

The inventors have previously shown that incubating boar spermatozoa with sAPM proteins from sow oviducts enhances the viability of the spermatozoa. In US 2004/0086842, the inventors have also previously demonstrated the transfer of several labelled sAPM proteins to spermatozoa protein profiles. Since the labelling of the sAPM proteins was performed after purification and solubilisation, it was conceivable that the labelled protein fraction may not be exclusively surface proteins, meaning that their biological relevance was questionable.

The inventors therefore devised a method to distinguish surface-located proteins of oviductal origin based on the biotin/avidin interaction (Holt et al., 2005), as described

previously. Labelled protein bands on the blots were seen in the spermatozoa protein profiles. These were most likely to be oviductal proteins.

Identification of candidate sAPM proteins using proteomic analysis Further samples of spermatozoa/sAPM were separated by SDS-PAGE and the proteins stained within the gels. Proteins in four of these excised bands were subjected to proteomic analysis as described previously. Approximately 30 proteins were identified by the use of bioinformatic analysis, and of these, 19 were selected for further investigation based on their species specificity, tissue specificity, and putative or known activity. This selection of 19 was logically reduced by further bioinformatic analysis to 4 proteins (Hsp90, Hsp70, Hsc70 and Clusterin), all of which belong to the heat shock protein family.

Identification ofHsc70 as a sAPM protein having sperm viability improving properties Having narrowed the activity of the sAPM fraction to four proteins, further investigation was then undertaken to determine which protein was responsible for the improved viability of spermatozoa. Commercial antibodies having specificity for each of the proteins Hsp90, Hsp70, Hsc70 and Clusterin were purchased. Bioassays of boar sperm survival were performed as detailed above, whereby the respective antibodies were included within the media in order to neutralise specific protein functions of the sAPM fraction.

Figure 1 shows the viability index (Mean ± SEM) of boar spermatozoa incubated with different concentrations of antibodies. Column 1 : No antibody, no treatment with sAPM fraction

Column 2: sAPM fraction, showing increase in sperm viability

Column 3: sAPM fraction and anti-Hsp70 antibody

Column 4: sAPM fraction and anti-Hsc70 antibody

Column 5: anti-Hsp70 antibody in Tyrode's TALP medium Column 6: anti~Hsp70 antibody in Tyrode's TALP medium.

Viability index is the percentage of viable spermatozoa after 24 hours incubation in comparison to that of the initial viability of the same semen sample at the beginning of incubation period. The greatest effect was obtained with the anti-Hsc70 antibody

(column 4), where the viability enhancement effect of sAPM was significantly reduced (P=0.00057).

When the 24 h bioassays were repeated using sAPM and antibody-treated sAPM, it was found that the beneficial effect of sAPM on sperm survival was negated only by the action of anti-Hsc70 (P = 0.013; Fig. 4A) . Antibodies against Hsp70 and Hsρ90 did not reduce the sAPM effect and there was no deleterious effect on spermatozoa of anti- Hsc70 antibody treatment alone (P>0.05). This outcome indicated that Hsc70 was a particularly interesting protein worthy of further investigation.

Characterization ofHsc70 and sperm viability

Co-incubation studies were undertaken to investigate the effect of Hsc70 on sperm viability, by utilising various concentrations of the recombinant Hsc70 protein. These data are shown in Figures 2, 4B, 4C and 5. As shown in Figure 2, bovine Hsc70 protein was effective with boar spermatozoa at concentrations between 2- 16μg/ml.

The other three proteins investigated in parallel with Hsc70 did not confer any protection to boar spermatozoa (see Figure 1). Combinations of Hsc70 and the other proteins also failed to provide enhanced sperm survival and even caused deleterious changes.

Incubation of boar spermatozoa with the Hsc70 protein instead of sAPM showed improved viability over the control (TALP medium only) after 24h incubation (as shown in Figure 3).

To test the effects of Hsc70 itself on boar sperm survival at 39°C under capacitating conditions, washed spermatozoa were incubated in TLP containing 0, 2, 4, 8 and 16 μg/mL Hsc70 for 24h and then evaluated for plasma membrane integrity (data not shown). There was a significant overall enhancement of sperm survival, represented by a 15% increase in viability index, due to the presence of Hsc70 (P=O.007). However, this beneficial effect' appeared to be optimal at 2 μg/mL (P=0.0007; Fl/24 = 14.9) and there was no further benefit to be gained by further increasing the concentration of protein.

This outcome was pursued further by repeating the bioassays, but using lower concentrations of Hsc70 (0, 0.25, 0.5, 2 and 8 μg/mL - see Figure 4B). As an additional comparison, the spermatozoa were also incubated in the presence of 200 μg/mL sAPM to evaluate the effectiveness of Hsc70 against the original oviductal preparation. The beneficial effects of Hsc70 were evident at both 0.25 and 0.5 μg/mL (P = 0.034 for the comparison between TLP and 0.25 μg/mL and P<0.001 for the specific comparison between TLP and both concentrations together). Both of the higher concentrations (2 and 8 μg/mL) were less effective at maintaining sperm survival and it was concluded that the optimal concentration must be lower than 2 μg/mL.

A further series of bioassays was therefore undertaken using an even lower range of Hsc70 concentrations (0.1, 0.5, 1 and 4 μg/mL - see Figure 4C). As before, sAPM (200 μg/mL) treatment was also included. Beneficial effects on sperm survival over a 24 h period were evident with Hsc70 at 0.1 μg/mL (P = 0.005), 0.5 μg/mL (P = 0.0001) and 1 μg/mL (P = 0.0001).

Effects ofHsclO on spermatozoa from other species

As Hsc70 is so highly conserved (see below, and alignment in Figure 7) the inventors hypothesized that its role in sperm viability maintenance should apply to mammals other than the pig. To test this hypothesis sperm viability experiments were set up using bull or ram spermatozoa, instead of boar spermatozoa.

When bull spermatozoa were incubated in the presence of Hsc70 for 48h at 37°C under capacitation conditions, approximately 50% remained viable. However, the inclusion of Hsc70 in the TLP medium increased the rate of sperm survival in a dose dependent manner up to an optimal value at 1 μg/mL (TLP control v 1 μg/mL Hsc70; P=0.0013). Although increasing the dose of Hsc70 further (to 4μg/mL) _, still resulted in a beneficial effect on sperm survival (P=O.006) compared to TLP alone, the higher dose was less effective at maintaining sperm survival. When spermatozoa were incubated in media containing tubulin as a control protein treatment, there was no significant difference in outcome with respect to the TLP control. These results are summarised in Figure 5.

Ram spermatozoa co-incubated with Hsc70 for 48h were more viable than those in medium without protein (Fmn ^~ 3.92; P = 0.05; Figure 6A). Although intermediate (1-2 μg of protein/ml) concentrations of Hsc70 maintained ram sperm viability significantly (Fi _/64 - 4.74; P < 0.04) better than medium without protein, the optimal concentration of Hsc70 for maintaining sperm viability was 4 μg of protein/ml (Fy ₆₄ = 5.67; P < 0.03) (Figure 6B). Lower (0.25-0.5 μg of protein/ml) and higher (8-16 μg of protein/ml) concentrations of Hsc70 did not maintain sperm viability significantly better than medium without protein. A significant log-linear relationship (Fy ₄₈ = 10.61; P < 0.003) was observed between sperm viability and HSc70 dose over the low to intermediate protein concentrations (0.25-4 μg of protein/ml; Figure 6C).

In summary Hsc70 prolongs ram sperm survival significantly better than the control (TLP+BSA medium without Hsc70) over a 48 h in vitro co-incubation period. These results are consistent with those found with boar and bull spermatozoa.

In vitro fertilization assays

Spermatozoa were briefly (10 min) exposed to 1 μg/mL of recombinant bovine Hsc70 protein before being diluted (1: 200) into the final fertilisation droplet. A control treatment with no protein exposure was also set up. The summarised IVF results are shown in Table 1.

Table 1: Effect of exposing spermatozoa to Hsc70 protein for 10 minutes prior to transfer into the IVF droplet.

Treatment No. Degenerate Failed Fertilised Monospermic 2PN stage 3 or 4 cell 7/8 cell oocytes oocytes to (%) (%) or later (% embryos or embryos fertilize of later (% of or later fertilized) fertilized) (% of fertilized)

Control 72 26 17 29 (40.3) 17 (58.6) 16 (55.1) 11 (37.9) 1 (3.5) Hsc70 72 35 10 27 (37.5) 24 (88.9)" 27 (100) 20 (74.1)* 6 (22.2)*

Total 144 61 27 56 41 43 31 7

^a Significantly higher than the corresponding control value (P < 0.05). * Significantly higher than the corresponding control value (P < 0.05).

Exposure to Hsc70 did not enhance the fertilization rate compared to the control, but it significantly reduced the extent of polyspermy (control v Hsc70; 41.4% v 11.1%; P <

0.05). Exposing spermatozoa to Hsc70 also appeared to increase the rate of embryonic development so that by 60 hours of incubation a higher proportion of embryos had reached or passed the 3 or 4 cell stage (control v Hsc70; 41.4% v 96.3%: P< 0.05).

Hsc70 prolongs and maintains the viability of spermatozoa from other species

Further experiments (described above) using ram and bull spermatozoa and bovine Hsc70 have confirmed that the protein is also effective at prolonging the viability of spermatozoa within sheep and ox (cattle). The protein enhanced survival at 48h, and proved effective and dose-dependent at lower doses than in the pig (in the range 0.5 - 4 μg/ml). In addition to the effect with boar, bull and ram spermatozoa, a porcine APM fraction has been shown to improve and/or prolong the viability of elephant spermatozoa (data not shown). There is therefore good evidence to suggest that the effect of the protein is exerted across a wide range of species, most likely due to the very high degree of conservation exhibited between members of the Hsc70 family (see below, and Figure 7).

Hsc70 is a major protein constituent ofsAPM

Western blots of sAPM from both pig and sheep have confirmed that Hsc70 is a major protein constituent of s APM (data not shown).

Conservation ofHsc70 across species

The ClustalW algorithm (Higgins et al.) (http://www.ebi.ac.uk/clustalw/) was utilised to align 33 Hsc70 protein sequences to determine their protein identity. ClustalW is a general purpose multiple sequence alignment program which produces biologically meaningful multiple sequence alignments of divergent sequences. Pairwise scores are calculated as the number of identities in the best alignment divided by the number of residues compared (gap positions are excluded).

The scores table from the ClustalW alignment is shown in Figure 7. The table compares a first named sequence (SeqA) against a second named sequence (SeqB). The length (in amino acid residues) is listed for each of SeqA and SeqB. As can be seen from the table, Hsc70 is very highly conserved between species. Taking the bovine protein as an example (seqA 15), this protein shares 99% identity with human, orang-utan, Chinese hamster, and rat Hsc70, 97% identity with chicken Hsc70, and 94-95% identity with

zebrafish Hsc70, and 92% identity with Xenopus Hsc70. The very high degree of identity between species is further evidence that Hsc70 protein from one species could be utilised to prolong/improve/maintain sperm viability from another species.

Hsc70 expression in the sAPMis increased in response to the presence of spermatozoa in the oviduct

The interaction between Hsc70 and spermatozoa has been validated through further experimentation to investigate gene expression and protein secretion in response to gamete presence in the oviduct.

In nearly all biological systems, eukaryotic cells regulate the abundance and distribution of cell-surface proteins in response to contact with an adjacent cell. The export and internalisation of whole receptor molecules after ligand binding is a well characterised phenomenon. Surface protein trafficking from and towards internal cellular compartments can not be identified using gene expression technologies. These changes are not necessarily the result of changes in gene expression and protein production, but rather the result of protein dynamics and interactions. The present inventors developed a technique for investigating global cell-surface protein trafficking (rather than just investigating specific protein trafficking). The methodology utilised SILAC combined with the parallel purification and analysis of individual cellular compartments (cell- surface and intracellular compartments). Approximately 30% of identified proteins were quantified according to the relative abundance of labelled compared to unlabelled MS spectra. Direct contact of spermatozoa with oviductal epithelial cells (OEC) altered the abundance of Hsc70 on the OEC cell surface, while the presence in the intracellular compartment did not show any change. This suggests that the alteration of the protein was not due to a change in protein synthesis, but rather, the result of its transport to the cell surface.

Conclusions From these data the present inventors conclude that the principal protein responsible for the protective action of sAPM with spermatozoa is Hsc70, and that Hsc70 expression on the surface of oviductal epithelial cells is modulated through contact of the cell with spermatozoa.

Summary

The inventors have identified Hsc70 as a protein present with the sAPM of oviductal epithelial cells which can maintain and prolong the viability of spermatozoa. These data show an important role for Hsc70 in the maintenance of sperm survival in vitro and imply that the mechanism of action should be highly conserved across species. The experiments demonstrating that recombinant bovine Hsc70 was beneficial to bull and ram, as well as boar spermatozoa in vitro indicates that this highly conserved protein could be a useful additive to sperm extenders used for artificial insemination. Interspecies variability in effectiveness is a major difficulty for the development of sperm extenders in mammals, but the present inventors believe that such a highly conserved protein as Hsc70 may be useful in overcoming this problem.

The present invention provides an effective diluent additive which enables AI centre operators to extend the shelf-life of diluted semen beyond the timeframe currently guaranteed, which is for example 3-5 days for boar semen. Further, the present invention enables cryopreservation of semen without loss of fertility. In addition, the present invention enables increased dilution of semen without loss of fertility and further the present invention provides a means of increasing fertility. The present invention also provides means for sperm viability to be higher following cryopreservation, thus enabling efficient freezing and subsequent provision of high numbers of viable spermatozoa following freezing.

References

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