WO2010025548A1

WO2010025548A1 - Sperm fertility biomarkers and uses thereof

Info

Publication number: WO2010025548A1
Application number: PCT/CA2009/001213
Authority: WO
Inventors: Olivier D'amours; Robert Sullivan
Original assignee: UNIVERSITé LAVAL; L'alliance Boviteq Inc.
Priority date: 2008-09-03
Filing date: 2009-09-03
Publication date: 2010-03-11

Abstract

The invention relates to biomarkers associated with fertility, and methods of uses thereof. The invention describes sperm proteins associated with fertility and methods for evaluating a mammalian sperm sample. Preferred aspects relates to evaluating the fertility of bulls by measuring in spermatozoa the amount of one or more proteins selected from AK1, PEBP1, CCT5, CCT8, ELSPBP1, PSMA6 and BSP1. Also described are methods for assigning a fertility index to a mammalian semen sample, methods for enhancing the fertility of a mammalian semen sample and semen extenders for increasing fertility of mammalian spermatozoa.

Description

SPERM FERTILITY BIOMARKERS AND USES THEREOF

RELATED APPLICATION

[0001] The present application claims priority of U.S. application serial No. 61/093,847 filed on September 03, 2008, the content of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates to the field of biology. More particularly, it concerns sperm proteins associated with fertility, and methods of uses thereof.

BACKGROUND OF THE INVENTION [0003] Traditionally, assessment of male fertility relies on a descriptive analysis of the semen, where the principal parameters are the numbers of spermatozoa present in the ejaculate and the percentage that are motile and morphologically normal (WHO 2000, WHO manual for the standardized investigation, diagnosis and management of the infertile male. Cambridge: Cambridge University Press). However, the predictive or diagnosis value of these parameters in animal productions or in men is questioned due to extensive overlaps between fertile and infertile male in all three semen parameters. Based on those parameters, 15% cases of male subfertility/infertility remain unexplained (WHO 1987, lnt J Androl 7(suppl.):1-53). The limitation of the conventional semen parameters to diagnose idiopathic infertility is due to the lack of information regarding functional deficiencies of sperm cells.

[0004] Sperm should be able to reach, recognize, bind to, and penetrate the oocyte in order to deliver its genome. Each of the single steps leading to fertilization represents critical points where defect could alter semen fertility. Over the years, a range of in vitro tests have been developed to determine sperm functions, including cervical mucus penetration, capacitation, sperm-zona pellucida recognition and interaction, acrosome reaction, and sperm-oocyte fusion. As such functional tests can be time consuming or quite expensive for routine procedures into an industrial context, alternatives to monitor sperm functional deficiencies have to be developed. The fertilization process is a net result of a series of molecular events. Based on their implication in particular steps of the fertilization process, many sperm compounds of lipidic, proteomic, ionic, and molecular nature have been shown to vary in quantity or in quality with the fertility status in many mammalian species. [0005] Bovine sperm proteome is composed of more than 3500 different proteins (Peddinti et al., 2008, BMC Syst Biol 2:19). Although lots of them remain unknown, many are implied in particular steps of the fertilization process. In this point of view, absence, presence, low, or high abundance of particular proteins could alter sperm functionalities, endangering his fertilizing abilities, thus lowering the semen fertility. In bovines, amount and/or presence of such proteins have been associated with bull fertility indexes (Bellin et al., 1998, J Anim Sci 76(8):2032-2039; Parent et al., 1999, MoI Reprod Dev 52:57-65; Peddinti et al., 2008, supra). Bovine sperm fertility proteins have also been disclosed by the inventors in a poster after the development of the invention (D'Amours et al., Sperm bull proteins associated with fertility indexes. 9^th International Congress of Andrology, Barcelona (Spain), 7-10 March 2009). Fluids from cauda epididymidis and accessory sex glands instead of sperm have also yield to the identification of proteins that correlate with fertility data in dairy bulls (Moura et al. 2006, J Androl 27 534-541 ; Moura et al. 2006, J Androl 27 201-211).

[0006] Today, in the western world, about one couple out of 12 is sub-fertile and in about half of all sub-fertile couples, male sub-fertility is observed (WHO 1993, WHO manual for the standardized investigation and diagnosis of the infertile couple. Cambridge University Press, Cambridge). Therefore, diagnosis and management of infertility is a growing prevalent health concern and there is a strong need for markers that can help to assess the quality of the semen. It would also be desirable to be provided with methods for assessing male fertility.

[0007] Similarly, the artificial insemination industry is a growing global and fiercely competitive industry. It encompasses a host of mammals, including humans, bovines, equines, ovines, swine, and endangered mammals such as non-human primates, elephants, rhinos, and the like. Thus, any method or composition that improves the fertility of the semen collected is highly coveted and manifestly useful.

[0008] The present invention addresses these needs as it will be apparent from review of the disclosure, drawings and description of the features of the invention below.

BRIEF SUMMARY OF THE INVENTION [0009] The invention relates to biomarkers and methods for evaluating a mammalian sperm sample, e.g. human sperm, bovine sperm, porcine sperm, equine sperm, dog sperm, cat sperm, rat sperm, mouse sperm, etc. In preferred embodiments, the mammalian sperm sample comprises bovine spermatozoa. [00010] The invention also relates to methods for evaluating at least one fertility- related parameter such as semen quality, sperm survival, semen fertility, semen resistance to cryopreservation, sensibility to cryo-capacitation, etc.

[00011] The invention also encompasses the use of the sperm fertility proteins described herein wherein semen is preserved in a liquid state (i.e. liquid phase semen).

[00012] Preferred aspects relates to evaluating the fertility of bulls (e.g. Non-Return Rate (NRR), fertility solution (SOL)) after processing, diluting, freezing, and thawing their semen.

[00013] In one embodiment, the methods of the invention comprises measuring in spermatozoa the amount and/or activity of one or more selected proteins and/or of a fragment, isoform or homolog thereof. Preferably, the protein is a sperm fertility protein selected from the group consisting of AK1 , PEBP1 , CCT5, CCT8, ELSPBP1 , PSMA6 and BSPl

[00014] The invention further relates to a method of in vitro fertilization and/or artificial insemination comprising: a) screening a sample of mammalian spermatozoa for assessing in sperm cells the amount and/or activity of a sperm fertility protein (and/or of a fragment, isoform or homolog thereof), the sperm fertility protein being selected from the group consisting of AK1 , PEBP1 , CCT5, CCT8, ELSPBP1 , PSMA6 and BSP1 ; and b) selecting for in vitro fertilization and/or artificial insemination a sub- population of spermatozoa of higher fertility potential based upon the results of the screen.

[00015] The invention further relates to a method for assigning a fertility index to a mammalian semen sample comprising: a) measuring in the semen sample the amount and/or activity of a sperm fertility protein (and/or of a fragment, isoform or homolog thereof), the sperm fertility protein being selected from the group consisting of AK1 , PEBP1 , CCT5, CCT8, ELSPBP1 , PSMA6 and BSP1 ; and b) assigning a fertility index to said semen sample based upon the results of the measurement. [00016] The invention also relates to a method for enhancing the fertility of a mammalian semen sample comprising: a) removing from the semen sample sperm cells for which amount and/or activity of a first sperm fertility protein, fragment, isoform or homolog thereof is higher than a predetermined acceptable threshold value, wherein the first protein is selected among CCT5, CCT8, ELSPBP1 , PSMA6 and BSP1 ; and/or b) enriching from the semen sample sperm cells for which amount and/or activity of a second protein sperm fertility, fragment, isoform or homolog thereof is lower than a predetermined acceptable threshold value, wherein said second sperm fertility protein is AK1 or PEBP1.

[00017] According to a further aspect, the invention relates to a semen extender for increasing fertility of mammalian spermatozoa. In one embodiment, the extender comprises an acceptable sperm diluent and a compound inhibiting activity of a sperm fertility protein selected from the group consisting of CCT5, CCT8, ELSPBP1 , PSMA6 or BSP1 ; and/or a compound enhancing activity of a sperm fertility protein consisting of AK1 or PEBP1. In another embodiment, the semen extender comprises an acceptable sperm diluent and a sperm fertility protein consisting of AK1 or PEBP1 , and/or a fragment, isoform or homolog thereof. Such extenders can be contacted with a semen sample for the purpose of improving or treating male sub-fertility.

[00018] According to a further aspect, the invention relates to a commercial package or kit for evaluating in a mammalian sperm sample at least one fertility-related parameter. In some embodiments the kit is for evaluating semen quality, sperm survival, semen fertility, semen resistance to cryopreservation a sperm sample. In preferred embodiments, the kit is particularly useful for evaluating the Non-Return Rate (NRR) or fertility solution (SOL)) after in vitro processing, diluting, freezing, and thawing of semen. The kit may comprise elements for components for measuring in the mammalian sperm sample the amount and/or activity of a sperm fertility protein (and/or of a fragment, isoform or homolog thereof). Advantageously, the protein is selected among AK1 , PEBP1 , CCT5, CCT8, ELSPBP1 , PSMA6 and BSP1. For instance, the kit may comprise on or more specific antibodies directed against the sperm fertility sperm fertility protein.

[00019] An advantage of the present invention is that it provides effective means for promptly evaluating and/or predicting sperm quality, integrity and/or fertility. The invention also provides means for improving the fertility index of a mammalian sperm sample by selecting spermatozoa having a preferred fertility profile.

[00020] Additional aspects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments which are exemplary and should not be interpreted as limiting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[00021] Figure 1 is a picture of a scanned gel of 1 % Triton X-100™ extracted bovine sperm proteins labeled with Cy2 and submitted to 2-D SDS-PAGE electrophoresis. DIGE analysis by Image Master™ (GE Healthcare) revealed significant differences between high fertility and low fertility bull groups for nine of these spots (identified as Numbeπname). Eight of these spots were analyzed and identified by LC-MS/MS.

[00022] Figure 2 are bar graphs showing volume ratio (±SEM) of protein spots identified as CCT5, CCT8, ELSPBP1 isoforms, PSMA6, BSP1 , AK1 , and PEBP1 on two- dimensional maps of 1 % Triton X-100™ sperm extracts from Holstein sires and quantified by DIGE. Bulls were grouped based on their fertility solution (SOL): High fertility (HF; SOL>3.0; n=6), Medium-high fertility (MHF; 2.9>SOL>2.0; n=5), Medium-low fertility (MLF; -2.8>SOL>-4.9; n=8), and Low fertility (LF; SOL<-5.0; n=4). Values followed by different letters are significantly different (p<0.05).

[00023] Figure 3 are bar graphs and pictures of ECL detected bands showing Western blot confirmation of BSP1 (spot 836), ELSPBP1 (spot 576), AK1 (spot 686), and PEBP1 (spot 740) for three bulls from the high fertility (HF) and low fertility (LF) groups. Equal amounts of protein for each sample were loaded on gel. Histograms show the corresponding spot quantification by DIGE for each of those bulls.

[00024] Figure 4 are pictures of ECL detected bands showing Western blot detection of α-Tubuline, ELSPBP1 , PSMA6, AK1 , PEBP1 , and BSP1 in the immotile (I) and motile (M) sperm subpopulations of frozen-thawed semen from three bulls. Immotile and motile sperm subpopulations were separated following centrifugation of frozen-thawed semen on discontinuous Percoll gradient. Qualitative sperm motility was assessed visually. Each line contains protein extracts from 3x10⁶ cells. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[00025] The present invention is based on the identification of biomarkers in the field of medicine and biology of the reproduction. Surprisingly, the inventors have found that the amount of selected proteins in spermatozoa (referred to hereinafter as "sperm fertility proteins") can be correlated with fertility indexes of sperm samples

[00026] As used herein, the term "sperm" or "spermatozoa" generally refers to mature ejaculated male gametes which are able to fertilize the counterpart female gamete. Nevertheless, the invention is not limited to mature ejaculated sperm and, in some embodiments, it may encompass the use of non-ejaculated sperm and sperm at different stages of their maturation, including but not limited to spermatogoniums (types A and B), primary and secondary spermatocytes and spermatids. The term "sperm" or "spermatozoa" also encompasses subpopulations of sperm (e.g. sorted sperm, marked sperm, purified sperm, motile sperm subpopulation, etc.).

[00027] In general, the principles of the present invention are applicable to mammalian sperm. The term "mammal" or "mammalian" includes cats, dogs, horses, pigs, cows, goats, sheep, rodents (e.g., mice or rats), rabbits, primates (e.g., chimpanzees, monkeys, gorillas, and humans)) and endangered mammals such as non- human primates, elephants, rhinos, bears). Preferably, the mammalian sperm are human spermatozoa or bovine spermatozoa, most sperm from bulls which semen is use in the artificial insemination industry.

[00028] In some embodiments, the sperm fertility protein is selected amongst the proteins known as AK1 , PEBP1 , CCT5, CCT8, ELSPBP1 , PSMA6 and BSPl The GeneBank™ (NCBI) accession number of the protein sequence in various species, including bovine, and human is provided hereinafter in Table 4. Also provided is a sequence listing comprising the complete amino acid sequence of those seven (7) proteins in the bovine species (SEQ ID NOs 1 to 7) and humans (SEQ ID NOs 8 to 14). The sperm fertility protein may also be another protein part of a larger protein complex for which the seven above-mentioned proteins are a member (e.g. "CCT Complex" for CCT5 and CCT8 and "proteasome" for PSMA6).

[00029] In some embodiments the spermatozoa are bovine spermatozoa and the sperm fertility protein comprises an amino acid sequence selected from SEQ ID NO: 1 (CCT5), SEQ ID NO: 2 (CCT8), SEQ ID NO: 3 (ELSPBP1), SEQ ID NO: 4 (PSMA6), SEQ ID NO: 5 (AK1), SEQ ID NO: 6 (PEBP1) and SEQ ID NO: 7 (BSP1). [00030] In other embodiments, the spermatozoa are human spermatozoa and wherein the sperm fertility protein comprises an amino acid sequence selected from SEQ ID NO: 8 (CCT5), SEQ ID NO: 8 (CCT8), SEQ ID NO: 10 (ELSPBP1), SEQ ID NO: 11 (PSMA6), SEQ ID NO: 12 (AK1), SEQ ID NO: 13 (PEBP1) and SEQ ID NO: 14 (BSP1).

[00031] According to some embodiments, it is the amount and/or activity of fragment(s), isoform(s) or homolog(s) of the sperm fertility proteins described herein which is measured. Examples of protein fragments can be found in the tables of Figures 3 to 10 of U.S. application serial No. 61/093,847 which is incorporated herein by reference, those figures listing for each of the sperm fertility proteins many different peptide sequences. Additional suitable fragments can easily be identified by skilled artisans. Those skilled in the art can also readily identify isoforms or homologs of the sperm fertility proteins identified herein, if any, by searching for sequence similarities and/or by using sequence databases such as GenBank™. Furthermore, a function and/or biological activity in spermatozoa of most, if not all, of the sperm fertility proteins is known and has already been described in the scientific literature.

[00032] Accordingly, one aspect of the present invention relates to a method of evaluating the fertility of mammalian spermatozoa by measuring in a sample of mammalian spermatozoa the amount and/or activity of a sperm fertility protein (and/or of a fragment, isoform or homolog thereof).

[00033] In one embodiment, the method comprises comparing the measured amount and/or activity of the sperm fertility protein (and/or a fragment, isoform or homolog thereof) to one or more threshold values; and evaluating fertility based upon such a comparison. The threshold value may be a predetermined value that has been calculated (e.g. using a population of spermatozoa of a given known or predicted fertility) or it can be a value that is measured every time (e.g. control measurement of the amount of a protein in a given sample of spermatozoa having a known or predicted fertility).

[00034] In one preferred embodiment, the protein which is examined is AK1 and/or PEBP1 (and/or a fragment, isoform or homolog thereof), and a higher amount and/or activity in the sample, when compared to the threshold value or control sample, is indicative of a higher or increased fertility potential. On the opposite and a lower amount and/or activity in the sample, when compared to the threshold value or control sample, may be indicative of a lower or decreased fertility potential. In various embodiments, the measured level of AK1 and/or PEBP1 (and/or a fragment, isoform or homolog thereof) in the sample, when compared to the threshold value or measured level in control(s), is higher by 10%, 20%, 30%, 40%, 50%, 75%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, 500% or more, such a higher level being predicative of an increased or higher fertility potential.

[00035] In another preferred embodiment, the examined protein is CCT5, CCT8, ELSPBP1 , PSMA6 and/or BSP1 (and/or a fragment, isoform or homolog thereof), and a higher amount and/or activity in the sample, when compared to the threshold value or control sample, is indicative of a decreased or lower fertility potential. On the opposite and a lower amount and/or lower activity in the sample, when compared to the threshold value or control sample, may be indicative of a higher or increased fertility potential. In one embodiment, the measured level of CCT5, CCT8, ELSPBP1 , PSMA6 and/or BSP1) (and/or a fragment, isoform or homolog thereof) in the sample, when compared to the threshold value or measured level in control(s), is higher by 10%, 20%, 30%, 40%, 50%, 75%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, 500% or more, such a lower level being predicative of a lower or decreased fertility potential.

[00036] Given the correlation between the amount of AK1 , PEBP1 , CCT5, CCT8, ELSPBP1 , PSMA6 and BSP1 and fertility potential, and it is also possible to assign a fertility index to a semen sample. This can be achieved by measuring in a mammalian semen sample the amount and/or activity of a sperm fertility protein (and/or of a fragment, isoform or homolog thereof); and assigning a fertility index to the semen sample based upon the measurement.

[00037] As described hereinafter in the Examplification, the inventors have also found that ELSPBP1 , PSMA6, PEBP1 , and BSP1 are associated either with the motile or immotile subpopulations following cryopreservation, when considering abundance of these biomarkers. Therefore, the invention encompasses the use of the sperm fertility proteins described herein for assessing viability of spermatozoa and/or for assessing motility of spermatozoa, including but not limited to assigning a motility index of a semen sample.

[00038] The reliability of the evaluation methods of the invention may be improved by combining the results of measurements of two or more of the sperm fertility proteins described herein. In one embodiment, the measured level of AK1 is combined with the measured level of at least one other fertility protein. In another embodiment, the measured level of CCT5 is combined with the measured level of at least one other fertility protein. In a preferred embodiment, the invention comprises assessing the amount and/or activity of both, CCT5 and AK1. [00039] The sperm fertility proteins described herein may also be useful to complement various assisted reproduction techniques, including but not limited to in vitro fertilization (IFV), artificial insemination (Al), gamete intrafallopian transfer (GIFT), embryo transfer, intracytoplasmic sperm injection (ICSI), etc. One particular aspect of the present invention concerns a method of in vitro fertilization and/or artificial insemination. The method comprises screening a sample of mammalian spermatozoa for assessing in sperm cells the amount and/or activity of a sperm fertility protein as defined herein (and/or of a fragment, isoform or homolog thereof); and selecting for in vitro fertilization and/or artificial insemination a sub-population of spermatozoa of increased fertility potential based upon the screening. In one embodiment, the method further comprises removing from the sample the sperm cells for which a measured amount and/or activity of CCT5, CCT8, ELSPBP1 , PSMA6, BSP1 (or of fragment, isoform or homolog thereof) is higher than one or more predetermined threshold values. Similarly, it is also conceivable to enrich the sample with sperm cells for which a measured amount of for which a measured amount and/or activity of CCT5, CCT8, ELSPBP1 , PSMA6, BSP1 (or of fragment, isoform or homolog thereof) is lower than one or more predetermined threshold values.

[00040] In another embodiment, the method of in vitro fertilization and/or artificial insemination comprises enriching the sample with sperm cells for which a measured amount and/or activity of AK1 or PEBP1 (or of fragment, isoform or homolog thereof) is higher than one or more predetermined threshold values. Similarly, it is also conceivable to deplete the sample from sperm cells for which a measured amount and/or activity of

AK1 or PEBP1 (or of fragment, isoform or homolog thereof) is lower than one or more predetermined threshold values.

[00041] Different methods and tools can be used for measuring the content (i.e. level or amount) and/or the activity of the sperm fertility proteins of the invention (and/or fragments, isoforms or homologs thereof). For instance, specific antibodies directed against the bovine and human (and other species) AK1 , PEBP1 , CCT5, CCT8, ELSPBP1 , PSMA6, and BSP1 are available. Examples of available antibodies against the bovine proteins are provided hereinafter in Table 2. Alternatively, other monoclonal or polyclonal antibodies may be raised or developed against any of the sperm fertility proteins described herein. Therefore, immunodetection and/or quantification can be done using different methods, including but not limited to flow cytometry and densitometric analysis of western blot. Those skilled in the art will know how to use such specific antibodies to make different tolls useful in the immunodetection and quantification, including but not limited to antibody coated colored beads, antibody coated magnetic beads, a dip stick coated with antibodies, etc

[00042] It is also conceivable according to the invention, that the biological activity of the sperm fertility proteins described herein be correlated with fertility indexes of sperm samples. Therefore, in some embodiments, it is activity of one or more of the sperm fertility proteins which is assessed using methods and techniques known to those skilled in the art. For instance, the catalytic activity of AK1 could be assayed by monitoring ATP production using a luciferase assay from added ADP in the reaction medium. The catalytic activity of the sperm proteasome (or of any of its subunit, including PSMA6) could be monitored, for instance, by using proteasome activity assay kits (e.g. Chemicon™'s kit based on detection of the fluorophore 7-Amino-4-methylcoumarin (AMC) after cleavage from the labeled substrate LLVY-AMC). It is also conceivable to measure the content and/or the activity of the sperm fertility proteins of the invention by measuring a parameter of sperm function likely affected by such treatment (e.g. motility, survival, capacitation state, fertility, etc.).

[00043] Another aspect of the present invention concerns a composition (e.g. a semen extender) for preserving or for increasing fertility of a sperm sample, the composition comprising an effective amount of a compound modulating the biological activity of a sperm fertility protein. In one embodiment, the compound inhibits the activity of CCT5, CCT8, ELSPBP1 , PSMA6 and/or BSP1 (and/or a fragment, isoform or homolog thereof). In another embodiment, the compound enhances the activity of AK1 or PEBP1 (and/or a fragment, isoform or homolog thereof). For instance, PSMA6 is a structural component of a larger functional complex called proteasome, which is involved in protein degradation. Although there are no known specific inhibitors of PSMA6, the whole proteasome activity can be inhibited by, but is not limited to, Lactacystin (NPI- 0052), MG132, Bortezomib, and Epoxomicin (PR-171). It has been reported that Epoxomicin can be used in human spermatozoa at a dose of 10 μM (see Morales et al., 2004, MoI Reprod Dev. 68: 1 15-124).

[00044] It is also conceivable to use inhibitors of CCT5 and/or CCT8. For instance, it has been shown that the activity of the CCT complex (which comprises the CCT5 and

CCT8 sub-units) can be inhibited using CCT5 specific antibodies (see Grantham et al.,

2006, Experimental Cell Research 312: 2309 - 2324). Therefore, the present invention encompasses the use of currently known and future inhibitors of CCT5, CCT8,

ELSPBP1 , PSMA6 and/or BSP1 and/or enhancers of AK1 or PEBP1. Such compounds may be useful for increasing fertility of mammalian spermatozoa. Given their correlation with increased fertility, it is also conceivable to add the AK1 and/or PEBP1 proteins (or a biologically functional fragment, isoform or homolog thereof) into a semen extender for increasing fertility of mammalian sperm. It is within the knowledge of those skilled in the art to identify and use suitable modulators (e.g. enhancers, inhibitors, competitors) of the sperm fertility proteins described herein, according to desired particular purposes.

[00045] Alternatively, the activity of any of the sperm fertility protein of the invention could be modulated in methods of contraception, for instance by inhibiting activity of AK 1 or PEBP1 and/or by enhancing activity of CCT5, CCT8, ELSPBP1 , PSMA6 and/or or BSP1. Therefore, the present invention encompasses the use of currently known and future enhancers of CCT5, CCT8, ELSPBP1 , PSMA6 and/or BSP1 and/or inhibitors of AK1 or PEBP1 , for reducing the fertility of mammalian spermatozoa.

[00046] A related aspect concerns the use of modulating compounds having beneficial effects on sperm fertility for the manufacture of a semen extender. In one embodiment, the semen extender is for semen preserved in a liquid state (i.e. liquid phase semen). In another embodiment, the semen extender is for use during any of the steps of in vitro fertilization in which the sperm are manipulated. In another embodiment, the semen extender is for use during the cryopreservation of the sperm. The amount of the modulating compound(s) in the composition of the present invention is an effective amount. An effective amount of modulating compound(s) is that amount necessary so that modulation of the activity of the targeted protein(s) can be assessed in a sperm sample. The exact amount of compound(s) to be used will vary according to factors such as the number of spermatozoa in the sample, the final sperm concentration, sperm species, the type of manipulation, as well as the other ingredients in the composition.

[00047] In addition to the active agents, the compositions of the invention may also contain metal chelators (proteinic or not), metal scavengers (proteinic or not), coating agents, preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts, buffers, coating agents and/or antioxidants. For preparing the composition of the invention, methods well known in the art may be used. For instance, semen extenders typically comprise egg yolk, glycerol, tris, acid citric monohydrate, glucose and antibiotics, and they have an osmotic pressure varying from 280 to 300 mOsmol, and a pH varying from 6.7 to 7.4.

[00048] Accordingly, a related aspect of the invention concerns a method of treating male sub-fertility, comprising contacting a semen sample from a male in need thereof with a compound modulating the biological activity of a sperm fertility protein and/or with a semen extender comprising such a compound as defined herein.

[00049] It is understood that suitable modulators (e.g. enhancers, inhibitors, competitors) of the sperm fertility proteins described herein may be used as various stages of the semen processing and/or fertilization processes. For instance, it is not only conceivable according to the invention to contact spermatozoa with desired modulator(s) in the semen extender, but also at different moment when processing, diluting, freezing, and/or thawing the semen. The spermatozoa could also be contacted with the modulator(s) at the fertilization or insemination stage. It is within the knowledge of those skilled in the art to identify the proper moment according to a particular purpose.

[00050] Given the correlation which exists between the presence of the sperm fertility proteins and semen quality, an additional aspect of the invention relates to kits for evaluating at least one fertility-related parameter such as semen quality, sperm survival, semen fertility, semen resistance to cryopreservation, semen freezability, sperm sensibility to cryo-capacitation, etc. Kits according to the invention may also be useful for assessing the impact of manipulation, preparation, dilution, freezing, thawing, cell- sorting, and/or sex-sorting etc., on at least one fertility-related parameter such as semen quality, sperm survival, semen fertility, semen resistance to cryopreservation, fertilization rates, etc.

[00051] Therefore, a further aspect of the invention relates to a commercial package or kit. A kit of the invention may comprise one or more of the following elements: a buffer for the homogenization of the sperm sample(s), purified sperm fertility proteins (and/or a fragment, isoform or homolog thereof) to be used as controls, incubation buffer(s), substrate and assay buffer(s), modulator buffer(s) and modulators (e.g. enhancers, inhibitors), standards, detection materials (e.g. antibodies, fluorescein-labelled derivatives, luminogenic substrates, detection solutions, scintillation counting fluid, antibody coated beads, etc.), laboratory supplies (e.g. desalting column, reaction tubes or microplates (e.g. 96- or 384-well plates)), a user manual or instructions, etc. Preferably, the kit and methods of the invention are configured such as to permit a quantitative detection or measurement of the protein(s) of interest (e.g. cytofluorimetry, Luminex™, ECL Plex™ Western Blotting Detection System, etc.). In a particular embodiment, the kit comprises a dip stick coated with one or more antibodies specific for the sperm fertility proteins. [00052] The kits of the invention may be particularly useful for applications in animals and humans according to the evaluation methods described hereinbefore. More particularly, the kits disclosed may be helpful for laboratory and diagnostic purposes in humans during artificial insemination procedures. The kits and methods of the invention may be used in combination with previously described assay kits.

[00053] Although the disclosure of the present invention focuses primarily on improving sperm fertility, those skilled in the art will also understand that the principles of the present invention can be readily applied for opposite purposes. For instance, it is conceivable according to the invention to use compounds capable of (1) enhancing in mammalian sperm activity of CCT5, CCT8, ELSPBP1 , PSMA6 or BSP1 ; and/or (2) inhibiting in mammalian sperm activity of AK1 or PEBP1 as spermicidal agents. Such spermicidal agents could be incorporated in gels, creams or in condoms. Therefore, the invention also encompasses those concepts (i.e. contraceptive methods, spermicidal compositions and the like).

[00054] Although the Examplification section of the present invention focuses primarily on bovine spermatozoa, those skilled in the art will recognize that the invention is not so limited and that it may be adapted to others species, and more particularly humans. Therefore, the invention encompasses diagnosis and management of human male fertility (e.g. fertility clinics) using any of the methods, techniques and compositions described herein.

[00055] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of this invention and covered by the claims appended hereto. The invention is further illustrated by the following examples, which should not be construed as further limiting.

EXAMPLE

[00056] EXAMPLE 1 : Identification of proteins from bull spermatozoa associated with fertility indexes

[00057] Spermatozoa intrinsic factors such as proteins modulate the fertilizing ability of the male gamete. The present study was carried out to compare protein composition of sperm from bull with different fertility indexes in order to identify protein markers of male fertility. [00058] Materials and Methods

[00059] Animals and fertility data: Frozen semen from 23 Holstein bulls with proven fertility was generously provided by I'Alliance Boviteq inc. (Saint-Hyacinthe, Qc). Bulls were housed at the Centre d'insemination artificielle du Quebec (CIAQ; Saint-Hyacinthe, Qc) and at Gencor (Guelph, Ont.). Fertility indexes for each bull were evaluated by the Canadian dairy network (CDN) as the non-return rate (NRR). NRRs are based on the number of cows that does not return to service 56 days after the first insemination. Ninety-five to 31 905 first inseminations were used to calculate NRRs, giving an accuracy of the fertility indexes ranging from 29% to 99%. Twenty of these bulls had a REL above 60%. NRRs are adjusted by a linear statistical model to include effects of age of the inseminated cows, month of insemination, technician, herd, and price of the bull semen. NRRs were converted to another index called the «fertility solution» (SOL), where zero is the average of the population at a given location for a specific period of time. According to their SOL, bulls were divided into four groups: high fertility (HF; SOL>3.0; n=6), average fertility (AF; 2.9>SOL>2.0; n=5), medium-low fertility (MLF; - 2.8>SOL>-4.9; n=8), and low fertility (LF; SOL<-5.0; n=4). Nineteen bulls were from the local progeny testing program and four were commercially used bulls. There was a commercially used bull in each of the four fertility groups. Average age at collection was 965 days ± 759 for the HF group, 1279 days ± 1180 for the MHF group, 771 days ± 962 for the MLF, and 841 days ± 788 for the LF group. Such heterogeneity in age at collection for each fertility group should avoid any bias regarding effect of age with fertility data.

[00060] Sperm sample preparation: Semen straws stored in liquid nitrogen were thawed in a water bath at 37°C for 1 min. Frozen-thawed spermatozoa were first washed by centrifugation at 85Og for 20 min. on isotonic 45% Percoll in phosphate buffered saline (PBS; 137mM NaCI, 3mM KCI, 8mM Na₂PO₄ ^«7H₂O, 1 ,5mM KH₂PO₄, pH 7.3). Sperm pellets were washed twice by centrifugation at 85Og for 10 min. with 10 ml of PBS. Proteins were extracted by ressuspending sperm pellets in 450 μl of 1% (w/v) Triton X-100™ in water (containing protease inhibitors: 5 μg/ml pepstatine A, 10 μg/ml leupeptine and PMSF 1 mM) for 15 min. at room temperature. Extracted spermatozoa were pelleted by centrifugation at 4000g for 5 min. and supernatant was assayed for protein content according to Lowry et al. (Lowry et a/., 1951 , J Biol Chem 193(1):265- 275). Proteins were precipitated for 2 hours at -2O⁰C with 9 volumes of ice cold acetone and pelleted by centrifugation for 15 min. at 800Og, air dried, and resuspended in lysis buffer (7M Urea, 2M Thiourea, 30 mM Tris, 4% CHAPS (w/v), pH 8.5) to a final concentration of 2 μg/μl for two-dimensional electrophoresis and in Laemmli sample buffer (2% SDS, 2% β-mercaptoethanol, 50 mM Tris, 20% glycerol) to a final concentration of 1 μg/μl for one-dimensional electrophoresis. Samples were kept frozen at -80⁰C until used.

Two-dimensional difference gel electrophoresis (2D DIGE) and gel analysis

[00061] Experimental design: 2D DIGE development has made bidimensionnal electrophoresis more accurate and reliable to monitor differences in protein abundance between two distinct biological samples. 2D DIGE allows multiplexing, i.e. co-migration of up to three different samples on the same 2D gel. This is made possible by prelabeling each extract with one of the three spectrally distinct, charge and mass- matched cyanine fluorescent dyes known as Cy2, Cy3, and Cy5. The sample labelled with Cy2 serves as an internal standard and is made from an equal protein amount of all biological samples evaluated in an experiment. Thus, each spot from individual biological samples can be measured as a ratio to its corresponding spot in the internal standard, significantly reducing effects of gel-to-gel variations on protein spots quantification (Alban et al. 2003, Proteomics 3 36-44; Van den Bergh et al. 2003, J Neurochem 85 193-205). Moreover, CyDye DIGE fluors offer sensitivity as low as 0.025 ng and a dynamic range above 3.6 orders of magnitude (Marouga et al. 2005, Anal Bioanal Chem 382 669-678), giving great accuracy to protein quantification. In the present study, samples from bulls with SOL>0 and SOL<0 were alternatively labelled with Cy3 and Cy5 to prevent dye- specific protein labelling.

[00062] Protein Labeling with CyDye DIGE fluors: Prior to labelling, equal amount of proteins from two to three ejaculates were pooled together for a total of 50 μg protein per bull. Each sample was labelled with the CyDye DIGE fluors developed for fluorescence 2D DIGE technology (GE Healthcare), according to the manufacturer's instructions. Briefly, 50 μg of each sample was minimally labelled by incubation with 400 pmol of amine-reactive cyanine dyes, Cy3 or Cy5, on ice for 30 min. in the dark. At the same time, the internal standard sample was generated by combining equal amounts of proteins from each bull and labelled with Cy2 for a ratio of 50 μg protein / 400 pmol Cy2. Reactions were quenched by incubation with 1 μl of 10 mM lysine/50 mg protein (Sigma- Aldrich). Samples labelled with Cy3 were mixed with samples labelled with Cy5 and 50 μg of Cy2 labelled internal standard, and rehydratation solution was added (7M Urea, 2M Thiourea, 2% CHAPS (w/v), 2% Pharmalyte 3-10™ (Pharmacia biotech, Uppsala, Sweden), 0,28% DTT (w/v)) to a total volume of 250 μl. [00063] Two-dimensional SDS-PAGE: Precast immobilized pH gradient strips (pH 3-10 L, 13 cm) were used for isoelectric focusing and carried out on an IPGphor Il IEF™ system (GE Healthcare). Gels were subjected to 30 V for 12 hours, 250 V for 30 minutes, 500V for 1 hour, 1000 V for 1 hour, 3000 V for 1 hour, and 8000 V until they reached 16 000 Vh. Strips were equilibrated in SDS equilibration buffer (6 M urea 57 mM Tris-HCI, pH 8.8, 29.3% glycerol, 2% SDS (w/v), and trace of bromophenol blue) containing 10 mg/mL DTT for 15 min. and then in SDS equilibration buffer containing 25 mg/ml iodoacetamide for 15 min., and sealed to 4% acrylamide stacking gels using 0.7% agarose in standard Tris-glycine electrophoresis buffer. Second dimension 12.5% SDS- PAGE were run at 35 mA/gel until the tracking dye had run off the gel.

[00064] Ge/ imaging and data analysis: After SDS-PAGE, CyDye labelled proteins were visualised by scanning with specified excitation and emission filter wavelengths using an Ettan DIGE Imager™ (GE Healthcare). All gels were scanned at a resolution of 100 μm. Gel images were processed using ImageMaster 2D Platinum 6.0™ (GE Healthcare), which allowed gel matching and spot quantification. A master gel was generated by the software, which is one of the internal standard images with the most and best resolved spots. Gel images were automatically matched to the master gel by ImageMaster™ and manually verified. Only spots whose match was beyond any doubt were conserved for DIGE analysis. Spots were quantified as their volume ratio, i.e. volume of the spot divided by its corresponding volume spot on the internal standard co- migrated on the same gel. After DIGE analysis, gels were stained with Coomassie brilliant blue R-250™ (Bio-Rad). Spots presenting significant differences between fertile and subfertile bull groups were manually excised, trypsin digested, and identified by liquid chromatography tandem mass spectrometry (LC-MS/MS).

[00065] Statistical analysis: Differences in protein abundance among the four groups (HF, MHF, MLF, LF) were evaluated by a one-way ANOVA followed by the Duncan statistical test. Differences were considered to be significant at P<0.05. Spots presenting significant differences between HF and LF groups were identified by LC- MS/MS and used as independent variables in regression models (Statistical Analysis Systems™ (SAS), version 9.1.3).

[00066] Protein identification by mass spectrometry: The in-gel digest and mass spectrometry experiments were performed by the Proteomics platform of the Eastern Quebec Genomics Center, Quebec, Canada. [00067] In-gel protein digestion: Tryptic digestion of protein excised spot was performed on a MassPrep™ liquid handling robot (Waters, Milford, USA) using Shevchenko's protocol (Shevchenko et al., 1996, Anal Chem 68(5):850-858) modified by Havlis et al. (Havlis et al., 2003, Anal Chem 75(6): 1300-1306). Briefly, proteins were reduced with 10 mM DTT and alkylated with 55mM iodoacetamide. Trypsin digestion was performed using 105 mM of modified porcine trypsin (Sequencing grade, Promega, Madison, Wl) at 58°C for 1 h. Digestion products were extracted using 1% formic acid, 2% acetonitrile followed by 1 % formic acid, 50% acetonitrile. The recovered extracts were pooled, vacuum centrifuge dried and then resuspended into 8 μl of 0.1% formic acid and 4 μl were analyzed by mass spectrometry.

[00068] Mass spectrometry: Peptide samples were separated by online reversed- phase (RP) nanoscale capillary liquid chromatography (nanoLC) and analyzed by electrospray mass spectrometry (ES MS/MS). The experiments were performed with a Thermo Surveyor™ MS pump connected to a LTQ™ linear ion trap mass spectrometer (Thermo Electron, San Jose, Ca USA) equipped with a nanoelectrospray ion source (Thermo Electron, San Jose, Ca USA). Peptide separation took place on a PicoFrit™ column BioBasic C18™, 10 cm x 0.075 mm internal diameter, (New Objective, Woburn, MA) with a linear gradient from 2-50% solvent B (acetonitrile, 0.1% formic acid) in 30 minutes, at 200 nL/min (obtained by flow-splitting). Mass spectra were acquired using a data dependent acquisition mode using Xcalibur™ software version 2.0. Each full scan mass spectrum (400 to 2000 m/z) was followed by collision-induced dissociation of the seven most intense ions. The dynamic exclusion (30 sec exclusion duration) function was enabled, and the relative collisional fragmentation energy was set to 35%.

[00069] Database searching: All MS/MS samples were analyzed using Mascot™ (Matrix Science, London, UK; version 2.2.0). Mascot™ was set up to search the bovine

UnireflOO™ database (release 13.2) assuming the digestion enzyme trypsin. Mascot was searched with a fragment ion mass tolerance of 0.50 Da and a parent ion tolerance of 2.0 Da. Iodoacetamide derivative of cysteine was specified as a fixed modification and oxidation of methionine was specified as a variable modification. Two missed cleavage were allowed.

[00070] Criteria for protein identification: Scaffold™ (version Scaffold-2_00_06, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet™ algorithm (Keller et al., 2002, Anal Chem 74(20):5383-5392). Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003, Anal Chem 75(17):4646-4658). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.

[00071] Motile and immotile sperm separation: lmmotile and motile sperm subpopulations were separated on Percoll discontinuous gradient according to Parrish et al. (1995, Theriogenology 44 859-869). Briefly, 0.5 ml aliquots of thawed semen were layered on a discontinuous gradient of 45% and 90% (v/v) isotonic Percoll. The gradient consisted of 2 ml of 45% Percoll and 2 ml of 90% Percoll in a 15 ml conical plastic tube. Sperm suspensions were layered on the top of the gradient and centrifuged at 700 g for 20 min. Immotile spermatozoa were recovered at 45%-90% Percoll interface, while the motile spermatozoa were recovered in the pellet at the bottom of the gradient. Qualitative sperm motility was assessed visually. Separated subpopulations were washed by centrifugation in PBS and then ressuspended in Laemmli sample buffer, heated for five minutes, centrifuged, and supernatant kept frozen until used on 12.5% SDS-PAGE, and immonublots were probed for the detection of α-Tubuline, ELSPBP1 , PSMA6, AK1 , PEBP1 , and BSP1. Laemmli sample buffer was used in order to extract α-Tubuline, used as a housekeeping protein. ELSPBP1 , AK1 , PEBP1 , and BSP1 are fully extracted by the Triton X-100™ treatment while PSMA6 is partly extracted by Triton X-100™ (data not shown). Thus, PSMA6 was extracted with Triton X-100™ as described above.

[00072] One-dimensional gel electrophoresis and western blot analysis: As neither β-Actin nor α-Tubulin, which are usually used as ubiquitous proteins, seem not to be extracted uniformly by Triton X-100™, protein concentrations in sample buffer were verified by amido black staining of dot blots. For Triton X-100™ protein extracts, fifteen μg of protein/sample were loaded on gel and protein from 3x10⁶ cells were loaded when extracted directly with Laemmli sample buffer. Proteins were separated by 12.5% SDS- PAGE and transferred onto nitrocellulose membranes. Nitrocellulose membranes were stained with Ponceau Red to verify uniformity of transfer. Membranes were blocked for 1 h in PBS 0.1 % Tween-20™ (PBS-T) containing 5% (w/v) defatted milk. Western blots were performed to detect BSP1 , ELSPBP1 , AK1 , PEBP1 and PSMA6. Membranes were incubated with 1/500 rabbit anti-CE12 (ELSPBP1 ; generously provided by C. Kirchhoff from Institute for Hormone and Fertility Research, Hamburg, Germany; Saalmann et al., 2001 , MoI Reprod Dev 58(1):88-100), 1/5000 anti-BSP-A1/A2 (BSP1 ; generously provided by P. Manjunath from University of Montreal, Montreal, Canada; Manjunath and Sairam, 1987, Biochem J 241(3):685-692), 1/200 goat anti-AK1 (S16) (Santa Cruz Biotechnology Inc., Santa Cruz, CA), 1/200 rabbit anti-RKIP (PEBP1 ; Santa Cruz Biotechnology Inc., Santa Cruz, CA) and 1/200 rabbit anti-PSMA6 (H-135) (PSMA6; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). When proteins were directly extracted in Laemmli sample buffer, α-Tubulin monoclonal antibody (Sigma, ON, Canada) was used as housekeeping protein. Membranes were then incubated with HRP-conjugated goat anti-rabbit or rabbit anti-goat IgG (dilution 1 :5000) in 5% defatted milk in PBS-T for 1 h at room temperature. Immunological complexes were visualised using the ECL detection kit (Amersham Biosciences, Baie d'Urfe, Qc).

[00073] RESULTS

[00074] An average of 861 ± 45 spots were detected on internal standard gel images (Figure 1). Of these, 567 ± 143 spots were matched to the master gel. Only matched spots present on all internal standard images were considered for DIGE analysis. None of these spots were unique to any of the four fertility bull groups, but DIGE analysis by ImageMaster™ revealed significant differences for nine of these spots between the high fertility (HF) and low fertility (LF) bull groups (Figure 1).

[00075] Two spots (686 and 740 on Figure 1) were predominantly abundant in the higher fertility groups. Mass spectrometry analysis identified them as Adenylate kinase isoenzyme 1 (AK1) and Phosphatidylethanolamine-binding protein 1 (PEBP1), which were 1.27 (PO.01) and 1.34 (PO.01) fold greater in high fertility bulls than in high subfertility bulls, respectively. AK1 and PEBP1 were both identified with 15 unique peptides with more than 95% confidence for a sequence coverage of 68% and 84% respectively. AK1 and PEBP1 were both identified with 15 unique peptides with more than 95% confidence for a sequence coverage of 68% and 84% respectively (Table 1).

[00076] The seven other spots (214, 522, 531 , 576, 582, 640, and 836 on Figure 1) were predominantly abundant in the LF groups. Spots number 214, 522, 576, 640, and 836 were identified as T-complex protein 1 subunit epsilon (CCT5), T-complex protein 1 subunit theta (CCT8), epididymal sperm binding protein E12 (ELSPBP1), Proteasome subunit alpha type-6 (PSMA6), and Binder of sperm 1 (BSP1 ; also known as seminal vesicle secretory protein 109 (PDC-109)), respectively. Spot 582 has also been identified as ELSPBP1. Only spot 531 was not identified, as it could not be visualized on Coomassie blue stained gel. All these spots were identified with at least nine unique peptides with more than 95% confidence, covering a minimum of 35% of amino acid sequences. Table 1 shows LC-MS/MS identifications and DIGE results for those nine spots. Figure 2 shows graphically DIGE results and western blot examples for BSP1 , ELSPBP1 , AK1 , and PEBP1.

[00077] As shown in Table 1 , CCT5 (214), CCT8 (522), ELSPBP1 isoforms (576, and 582), PSMA6 (640), and BSP1 (836) were about 1.65 (PO.01), 1.79 (PO.01), 2.62 (PO.01), 2.58 (PO.01), 2.45 (PO.01), 1.62 (PO.01), and 2.01 (PO.01) fold greater in low fertility (LF) than in high fertility (HF) bull groups, respectively.

[00078] Antibodies against BSP1 , ELSPBP1 , AK1 , and PEBP1 were available and used to confirm the LC-MS/MS identification. Details on the antibodies used for that confirmation are given hereinafter in Table 2. Western blot analysis showed visually the same tendencies as DIGE results.

Table 1 : Sperm proteins associated with high and low fertility in Holstein bulls as determined by 2D-DIGE analysis of sperm Triton-X-100™ 1% extract and identified b LC-MS/MS.

Spot numbers refer to those shown in bull sperm protein 2D map in Figure 1 Boldface vol. ratios represent the fertility group were spot vol. ratios were the highest.

Table 2: Antibodies used for mass spectrometry confirmation

Adenylate kinase isoenzyme 1 (AK1)

Supplier: Santa Cruz Biotechnology Inc., Santa Cruz, CA, U.S.A.

Product name: AK1 (S-16) (http://datasheets.scbt.com/sc-22567.pdf)

Product number: sc-22567

Clonality: Polyclonal

Host: Goat

Phosphatidylethanolamine-binding protein 1 (PEBP1)

Supplier: Santa Cruz Biotechnology Inc., Santa Cruz, CA, U.S.A.

Product name: RKIP (FL-187) (http://datasheets.scbt.com/sc-28837.pdf)

Product number: sc-28837

Clonality: Polyclonal

Host: Rabbit

Epididymal sperm binding protein E12 (ELSPBPI)

Supplier: C. Kirchhoff, Institute for Hormone and Fertility Research,

Hamburg, Germany.

Product name: anti-CE12-3 (Saalmann et al. (2001) MoI. Reprod. Dev. 58:

88-100)

Clonality: Polyclonal

Host: Rabbit

Seminal plasma protein BSP1 (BSP1)

Supplier: P. Manjunath, University of Montreal, Montreal, QC, Canada.

Product name: anti-BSP-A1/A2 (Manjunath et Sairam (1987) Biochem. J.

241 : 685-692)

Clonality: Polyclonal

Host: Rabbit

Proteasome subunit alpha type-6 (PSMA6)

Supplier: Santa Cruz Biotechnology Inc., Santa Cruz, CA, U.S.A.

Product name: 2OS Proteasome α6 (H-135) (http://datasheets.scbt.com/sc-

67343.pdf)

Product number: sc-67343

Clonality: Polyclonal

Host: Rabbit

[00079] Western blot analysis showed visually the same tendencies as DIGE results for the 23 bulls, which confirm LC-MS/MS identification. Figure 3 shows an example of western blot detection of BSP1 , ELSPBP1 , AK1 , and PEBP1 with corresponding quantification by DIGE for three bulls from the HF group and three bulls from the LF group. [00080] In order to assess relationship between bull fertility and abundance of the seven identified proteins, a regression model using SOL as a dependent variable and the seven proteins as independent variables has been established using SAS™ software The STEPWISE approach was used to select independent variables that explain the most the variation of fertility The regression model established that CCT5 and AK1 explained a significant proportion of the variation in fertility scores (R²=0 6394, P<0 0001) Using the RSQUARE approach to select independent variables, addition of a third protein to the model increased R² of about 2%, which is negligible Also, the use of the seven proteins for the model increased R² of only 10% Table 3 shows regression models established with both CCT5 and AK1 and with the seven proteins Finally, since bulls used in this study had not the same reliability for their SOL, a balanced regression model with the REL variable was established in order to give more weight to bulls with a high REL Addition of the individual REL variable gave a similar model to the one established with AK1 and CCT5

Table 3: Regression models of bull fertility based on volume ratio Ot spots in 2D

DIGE protein maps of sperm Triton X-100™ 1% extracts.

Regression equations R 2 P value

SOL = -9 23346 - 4 95663^*(CCT5) + 10 67058^*(AK1 ) 0 6394 P<0 0001

SOL = -11 26895 + 11 16030^*(AK1) - 4 02233^*(CCT5) -

0 21990^*(ELSPBP1) + 4 13624^*(BSP1) + _{p=n n9£-y}

2 54656*(PEBP1 ) + 0 03277*(CCT8) - ^{U / ύZ0} r u uzo^/

4 82768^*(PSMA6)

SOL indicates the fertility solution, a normalized non return rate, (CCT5) volume ratio of spot 214, (CCT8) volume ratio of spot 522, (ELSPBP1) volume ratio of spot 576 (PSMA6) volume ratio of spot 640, (AK1) volume ratio of spot 686 (PEBP1) volume ratio of spot 740 (BSP1) volume ratio of spot 836 as shown in Figure 1

[00081] Following sperm separation on Percoll discontinuous gradient, both immotile and motile subpopulations were assayed for the presence of α-Tubulιne, ELSPBP1 , PSMA6, AK1 , PEBP1 , and BSP1 by western blot (Figure 4) Abundance of AK1 did not change between the two subpopulations However, ELSPBP1 and BSP1 were in higher abundance in the immotile subpopulation and PEBP1 was in higher abundance in the motile subpopulation PSMA6 was found in higher abundance in the immotile subpopulation for two bulls but was not detectable for the third one ELSPBP1 , PSMA6, PEBP1 , and BSP1 are associated with either motile or immotile sperm subpopulations Therefore, abundance of these proteins in the whole sperm extract indicates the importance of either the motile or immotile sperm subpopulations Therefore, abundance of these proteins constitute a biomarker for assessing motility of spermatozoa, including but not limited to assigning a motility index of a semen sample and/or assessing viability of spermatozoa. These may particularly be helpful for assessing subpopulations of sperm following cryopreservation.

[00082] Altogether, the results presented herein show clearly that fertility correlates with the amount of selected sperm proteins. Thus, it opens gates to many practical applications such as in the areas of diagnostics, therapeutics, and contraception by modulating the amount and/or activity of those proteins, not only in bovine but in other species, including humans. In some embodiments, the present invention encompasses the use of proteins from mammalian species other than bovine and more particularly human proteins, as suitable fertility markers according to the invention. Table 4 hereinafter provides GeneBank™ (NCBI) accession numbers of sperm fertility protein amino acid sequence in various species. The SEQ ID NO corresponding to the enclosed sequence listing is also provided for the bovine and human sperm fertility proteins. Identification of additional accession numbers and/or the sequence information of the sperm fertility proteins encompass by the present the invention is within the knowledge of those skilled in the art.

[00083] Headings are included herein for reference and to aid in locating certain sections These headings are not intended to limit the scope of the concepts described therein under, and these concepts may have applicability in other sections throughout the entire specification Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

[00084] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a sperm fertility protein" includes one or more of such proteins, and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[00085] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the present invention and scope of the appended claims. Table 4: GeneBank™ (NCBI) accession number of sperm fertility protein sequences in various species

Claims

CLAIMS:

1. A method for evaluating the fertility of mammalian spermatozoa, comprising: assessing in a sample of mammalian spermatozoa the amount and/or activity of a sperm fertility protein and/or of a fragment, isoform or homolog thereof; wherein said sperm fertility protein is selected from the group consisting of AK1 , PEBP1 , CCT5, CCT8, ELSPBP1 , PSMA6, BSP1 and combinations thereof; and wherein said amount and/or activity is predicative of said fertility.

2. The method of claim 1 , wherein said spermatozoa are bovine spermatozoa and wherein the sperm fertility protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (CCT5), SEQ ID NO: 2 (CCT8), SEQ ID NO: 3 (ELSPBP1), SEQ ID NO: 4 (PSMA6), SEQ ID NO: 5 (AK1), SEQ ID NO: 6 (PEBP1) and SEQ ID NO: 7 (BSP1 ).

3. The method of claim 1 , wherein said spermatozoa are human spermatozoa and wherein the sperm fertility protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8 (CCT5), SEQ ID NO: 8 (CCT8), SEQ ID NO: 10 (ELSPBP1), SEQ ID NO: 11 (PSMA6), SEQ ID NO: 12 (AK1 ), SEQ ID NO: 13 (PEBP1 ) and SEQ ID NO: 14 (BSP1).

4. The method of any one of claims 1 to 3, further comprising the steps of: comparing said amount and/or activity of said sperm fertility protein, fragment, isoform or homolog thereof to one or more predetermined threshold values; and evaluating fertility of the mammalian spermatozoa based upon said comparison.

5. The method of claim 4, wherein the protein is AK1 or PEBP1 and wherein a higher amount and/or activity in the sample compared to a predetermined threshold value is indicative of a higher fertility potential.

6. The method of claim 4, wherein the protein is CCT5, CCT8, ELSPBP1 , PSMA6 or BSP1 and wherein a higher amount and/or activity in the sample compared to a predetermined threshold value is indicative of a lower fertility potential.

7. The method of claim 1 , comprising assessing the amount and/or activity of at least two of said sperm fertility proteins, fragment, isoform or homolog thereof.

8. The method of claim 7, wherein said at least two sperm fertility proteins consist of CCT5 and AK1.

9. A method for evaluating the fertility of ejaculated bovine spermatozoa, comprising assessing in a sample of bovine spermatozoa the amount of one or more sperm fertility protein, said sperm fertility protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 (CCT5), SEQ ID NO: 2 (CCT8), SEQ ID NO: 3 (ELSPBP1), SEQ ID NO: 4 (PSMA6), SEQ ID NO: 5 (AK1), SEQ ID NO: 6 (PEBP1) and SEQ ID NO: 7 (BSP1).

10. The method of any one of claims 1 to 9, wherein said sample of spermatozoa comprises spermatozoa which have been submitted to in vitro processing, diluting, freezing, and thawing.

1 1. A method of in vitro fertilization and/or artificial insemination comprising:

(a) screening a sample of mammalian spermatozoa for assessing in sperm cells the amount and/or activity of a sperm fertility protein and/or of a fragment, isoform or homolog thereof, said sperm fertility protein being selected from the group consisting of AK1 , PEBP1 , CCT5, CCT8, ELSPBP1 , PSMA6 and BSP1 ; and

(b) selecting for in vitro fertilization and/or artificial insemination a sub- population of spermatozoa of higher fertility potential based upon said screening.

12. The method of claim 11 , comprising removing from the sample of mammalian spermatozoa sperm cells for which a measured amount and/or activity of the sperm fertility protein, or of fragment, isoform or homolog thereof, is higher or lower than a predetermined acceptable threshold value.

13. The method of claim 11 , comprising removing from the sample of mammalian spermatozoa sperm cells for which a measured amount and/or activity of CCT5, CCT8, ELSPBP1 , PSMA6, BSP1 , or of a fragment, isoform or homolog thereof, is higher than one or more predetermined acceptable threshold values.

14. The method of claim 11 , comprising enriching the sample of mammalian spermatozoa with sperm cells for which a measured amount and/or activity of AK1 or PEBP1 , or of fragment, isoform or homolog thereof, is higher than one or more predetermined acceptable threshold values.

15. A method for assigning a fertility index to a mammalian semen sample comprising:

(a) measuring in the semen sample the amount and/or activity of a sperm fertility protein, and/or of a fragment, isoform or homolog thereof, said sperm fertility protein being selected from the group consisting of AK1 ,

PEBP1 , CCT5, CCT8, ELSPBP1 , PSMA6 and BSP1 ; and

(b) assigning a fertility index to said semen sample based upon said measurement.

16. A method for enhancing the fertility of a mammalian semen sample comprising at least one of the following steps:

(a) removing from the semen sample sperm cells for which amount and/or activity of a first sperm fertility protein, fragment, isoform or homolog thereof is higher than a predetermined acceptable threshold value, wherein said first sperm fertility protein is selected from the group consisting of CCT5, CCT8, ELSPBP1 , PSMA6 and BSP1 ;

(b) enriching the semen sample with sperm cells for which amount and/or activity of a second sperm fertility protein, fragment, isoform or homolog thereof is lower than a predetermined acceptable threshold value, wherein said second sperm fertility protein is AK1 or PEBP1.

17. The method according to any one of claims 11 to 16, wherein said mammalian spermatozoa are bovine spermatozoa.

18. A semen extender for increasing fertility of mammalian spermatozoa, the extender comprising an acceptable sperm diluent and at least one of:

(a) a compound inhibiting activity of a sperm fertility protein selected from the group consisting of CCT5, CCT8, ELSPBP1 , PSMA6 and BSP1 ;

(b) a compound enhancing activity of a sperm fertility protein consisting of AK1 or PEBPl

19. A semen extender for increasing fertility of mammalian spermatozoa, the extender comprising: (i) an acceptable sperm diluent; and (ii) at least one sperm fertility protein consisting of AK1 ,PEBP1 , and/or a fragment, isoform or homolog thereof.

20. A method of treating male sub-fertility in a mammal, comprising contacting a semen sample from a mammalian male in need thereof with a semen extender according to claim 18 or 19.

21. A method of contraception in mammal, the method comprising at least one of the following steps:

(a) inhibiting in mammalian sperm activity of a sperm fertility protein consisting of AK1 or PEBP1 ; (b) enhancing in mammalian sperm activity of a sperm fertility protein selected from the group consisting of CCT5, CCT8, ELSPBP1 , PSMA6 or BSP1.

22. A kit for evaluating in a mammalian sperm sample a fertility-related parameter, the kit comprising a user manual or instructions and kit components for measuring in the mammalian sperm sample the amount and/or activity of a sperm fertility protein and/or of a fragment, isoform or homolog thereof, said sperm fertility protein being selected from the group consisting of AK1 , PEBP1 , CCT5, CCT8, ELSPBP1 , PSMA6 and BSP1.

23. The kit of claim 22, wherein said kit components comprise specific antibodies directed against said sperm fertility protein.

24. The kit of claim 22 or 23, wherein the fertility-related parameter is Non-Return Rate (NRR) or fertility solution (SOL)) after in vitro processing, diluting, freezing, and thawing of semen.