The skeletal muscle fusogen myomerger as a contributor to sperm:egg fusion

Abstract

Disclosed are methods for selecting or identifying fusion-competent gametes. The methods can include providing a sample including gametes obtained from a subject; and contacting the sample with a reagent capable of binding Myomerger expressed on the surface of the gametes. The methods can also include isolating gametes bound by the reagent capable of binding Myomerger from gametes not bound by the reagent capable of binding Myomerger. Also disclosed are methods for contraception that include administering a reagent capable of binding Myomerger expressed on the surface of gametes to a subject. Also disclosed are compositions that include a reagent capable of binding Myomerger and a reagent capable of binding phosphatidylserine, as are kits that include the disclosed compositions.

Description

[0001] Attorney Docket No. 3062 / 202 PCT

[0002] DESCRIPTION

[0003] THE SKELETAL MUSCLE FUSOGEN MYOMERGER AS A CONTRIBUTOR TO SPERM:EGG FUSION

[0004] CROSS-REFERENCE TO RELATED APPLICATION

[0005] This application claims the benefit of and priority to U.S. Provisional Patent Application Serial No. 63 / 679,446, filed August 5, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0006] REFERENCE TO SEQUENCE LISTING XML

[0007] The Sequence Listing XML associated with the instant disclosure has been electronically submitted to the United States Patent and Trademark Office via the Patent Center as a 17,693 byte UTF-8-encoded XML file created on June 5, 2025 and entitled “3062_202_PCT_ST26.xml”. The Sequence Listing submitted via Patent Center is hereby incorporated by reference in its entirety.

[0008] GRANT STATEMENT

[0009] This invention was made with government support under GM064709 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0010] TECHNICAL FIELD

[0011] The presently disclosed subject matter relates to compositions and methods useful for assessing fusion competency in male and female gametes. In some embodiments, methods, kits, and compositions for identifying, selecting, and functionally evaluating fusion competent gametes are provided.

[0012] BACKGROUND

[0013] Besides the binding between sperm and oocytes, an important step in successful fertilization is the fusion between the cellular membranes of the two gametes. In the last two decades, major progress has been made in identifying several molecules expressed on the sperm and oocytes that contribute to mammalian fertilization (including Izumol, Juno, CD9, SPACA6, TMEM95, SOF1, FIMP, MAIA / FcRL5, and DCST1 and 2). While some of these molecules are linked to recognition, the fusogen(s) involved in sperm: egg fusion remain(s) incompletely understood.

[0014] SUMMARY

[0015] This summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment Attorney Docket No. 3062 / 202 PCT can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this summary does not list or suggest all possible combinations of such features.

[0016] In some embodiments, a method of selecting fusion-competent gametes is provided. In some embodiments, the method comprises: (a) providing a sample comprising gametes obtained from a subject; (b) contacting the sample with a reagent capable of binding Myomerger expressed on the surface of the gametes; and (c) isolating gametes bound by the reagent capable of binding Myomerger from gametes not bound by the reagent capable of binding Myomerger. In some embodiments, the gametes are sperm cells or oocytes. In some embodiments, the reagent capable of binding Myomerger comprises an anti-Myomerger antibody or a fragment thereof, or a peptide comprising a Myomerger ectodomain. In some embodiments, the contacting comprising mixing.

[0017] In some embodiments, the gametes comprise sperm cells, and wherein the method further comprises contacting the sample with a reagent capable of binding phosphatidylserine on the surface of the sperm cells and isolating sperm cells bound by the reagent capable of binding phosphatidylserine. In some embodiments, the reagent capable of binding phosphatidylserine comprises Annexin V or GST-BAI1-TSR. In some embodiments, the contacting comprising mixing.

[0018] In some embodiments, the subject is a human subject or a non-human animal subject. In some embodiments, the non-human animal subject is selected from the group consisting of bovine, equine, porcine, ovine, canine, feline, and avian.

[0019] In some embodiments, a method of identifying fusion-competent gametes is provided. In some embodiments, the method comprises: (a) providing a sample comprising gametes obtained from a subject; and (b) contacting the sample with a reagent capable of binding Myomerger expressed on the surface of the gametes; wherein gametes that express Myomerger on their surface are fusion-competent. In some embodiments, the contacting comprising mixing.

[0020] In some embodiments, the subject is a human subject or a non-human animal subject. In some embodiments, the non-human animal subject is selected from the group consisting of bovine, equine, porcine, ovine, canine, feline, and avian.

[0021] In some embodiments, a method of contraception is provided. In some embodiments, the method comprises administering a reagent capable of binding Myomerger expressed on the surface of gametes to a subject. In some embodiments, the subject is a human subject or a non- Attorney Docket No. 3062 / 202 PCT human animal subject. In some embodiments, the non-human animal subject is selected from the group consisting of bovine, equine, porcine, ovine, canine, feline, and avian.

[0022] In some embodiments, provided is a composition comprising a reagent capable of binding Myomerger and a reagent capable of binding phosphatidylserine. In some embodiments, the reagent capable of binding Myomerger comprises an anti-Myomerger antibody or a fragment thereof, or a peptide comprising a Myomerger ectodomain; and / or wherein the reagent capable of binding phosphatidylserine comprises Annexin V or GST- BAI1-TSR. In some embodiments, a kit comprising a composition of the presently disclosed subject matter and / or for carrying out a method of the presently disclosed subject matter is provided.

[0023] Accordingly, it is an object of the presently disclosed subject matter to provide methods, compositions, and kits for identifying, selecting, and functionally evaluating fusion competent gametes. This and other objects are achieved in whole or in part by the presently disclosed subject matter. Further, an object of the presently disclosed subject matter having been stated above, other objects and advantages of the presently disclosed subject matter will become apparent to those skilled in the art after a study of the following description, Figures, and Examples.

[0024] BRIEF DESCRIPTION OF THE FIGURES

[0025] FIGURES 1A - IK. Myomerger is expressed in male gametes. FIG. 1A: Relative expression of Myomerger (Mymx I beta-actin) evaluated by RT-PCR on C2C12 Myoblasts differentiated for 0, 1 or 3 days (d) in differentiated medium (DM), testes and dissociated germ cells (dGC). Each dot is one mouse (testis and dGC) or one experiment (myoblasts). FIG. IB: Immunoblot showing the detection of Myomerger (~15 KDa) and beta actin (42 KDa) on C2C12 myoblasts differentiated for 1 day (DMdl) or dissociated germ cells (dGC). Each line is an individual mouse (n=6). Quantification was performed as the ratio of Myomerger to Actin using ImageJ and values are depicted below the immunoblots. FIG. 1C- FIG. ID: Immunolocalization of Myomerger on testicular sections of WT adult mice. In C, low magnification, it is possible to appreciate that not all the seminiferous tubules show Myomerger staining. Arrows point the seminiferous tubules expressing Myomerger. (When shown in color: Myomerger: green, PNA: red, Hoechst: blue). Scale bar: FIG. 1C: 100 pm; FIG. ID: 50 pm. FIG. IE: Immunolocalization of Myomerger on dissociated germ cells smears. Arrows show the staining of Myomerger in the neck of elongated spermatids. (When shown in color: Myomerger: green, PNA: red, Hoechst: blue). Scale bar: 10 pm. FIG. 1F- FIG. 1H: Immunolocalization of Myomerger on the acrosome of fixed epididymal (caudal) Attorney Docket No. 3062 / 202 PCT sperm. Scale bar: 10 pm. FIG. 1G-FIG. 1H photos are zoom in from FIG. IF. (When shown in color: Myomerger: green, PNA: red, Hoechst: blue). FIG. 1I-FIG. 1J: Immunolocalization of Myomerger on the head of sperm isolated from the caudal epididymis. Live sperm was incubated with the Myomerger antibody (When shown in color: green), washed and then fixed and stained with PNA (rhodamine, when shown in color: red) and Hoechst (DNA, when shown in color: blue). Note that Myomerger+ sperm has undergone acrosome reaction (PNA negative). Scale bar: 10 pm. FIG. IK: Quantification of Myomerger+ sperm stained as described in FIG. 1I-FIG. 1J after incubation in PBS (NC, not capacitated), TYH+BSA medium (CAP, capacitated) or A23187+TYH+BSA (AR, acrosome reaction).

[0026] FilGURES 2A - 21. Myomerger expression in female gametes. FIG. 2A: Relative expression of Myomerger (Mymx / beta-actin) evaluated by RT-PCR on C2C12 myoblasts differentiated for 1 or 2 days (d) in differentiated medium (DM) and ovaries from WT females. Each dot is one mouse (ovaries) or one experiment (myoblasts). FIG. 2B: Immunoblot showing the detection of Myomerger (~15 KDa) and beta actin (42 KDa) on C2C12 myoblasts differentiated for 1 day (DMdl) or ovaries from WT females. Each line is an individual mouse (n=6). Quantification was performed as the ratio of Myomerger to Actin using ImageJ and values are depicted below the immunoblots. FIG. 2C-FIG. 2F: Immunolocalization of Myomerger on WT adult ovarian sections. FIG. 2C: Schematic representation of an ovarian follicle. FIG. 2D-FIG. 2E: Myomerger localization on the oocyte (when shown in color, red), surrounded by the zona pellucida (when shown in color, green, ZP3). FIG. 2F: Preabsorption of the Myomerger antibody (Ab) with the specific peptide removes the oocyte staining. Scale bars: 50 pm. FIG. 2G-FIG. 21: Immunolocalization of Myomerger on Metaphase II zona pellucida-free oocytes isolated from WT females. Live oocytes were incubated with a Myomerger antibody (FIG. 2G, FIG. 2H) or Myomerger and Juno antibodies (FIG. 21) for 1 hour in TYH+BSA medium at 37C, 5%CO2, washed and fixed. FIG. 2G and FIG. 2H are different z-stack of the same oocytes showing the surface staining or the Hoechst+ spindle with or without the DIC, respectively. Scale bars: 20 pm.

[0027] FIGURES 3A - 31. Role of Myomerger in sperm-egg and sperm-myoblast fusion. FIG. 3 A-FIG. 3B : Schematic representation and results of the in vitro fertilization assays using ZP-intact oocytes and sperm pre-incubated with 10 ug / ml Sheep IgG, Myomerger antibody (Ab) or left untreated. FIG. 3B: Results are expressed as percentage of oocytes with DAPI+ decondensed sperm DNA. FIG. 3C-FIG. 3D. Schematic representation and results of the in vitro fertilization using ZP-free oocytes and sperm co-incubated with Myomerger26’84peptide. FIG. 3D: Results are expressed as percentage of oocytes with DAPI+ decondensed sperm Attorney Docket No. 3062 / 202 PCT

[0028] DNA. FIG. 3E-FIG. 3F: Schematic representation and fusion index of undifferentiated WT, Myomerger KO or Myomaker KO myoblasts co-incubated with Calcein-AM labeled sperm for 4 hours. n=3 independent experiments. FIG. 3G: Relative expression of Myomerger (Mymx / beta-actin) during myoblast differentiation for 0-3 days (d) by RT-PCR. n=2-4 independent experiments. FIG. 3H-FIG. 31: Schematic representation and fusion index of undifferentiated and differentiated WT and Myomerger KO myoblasts co-incubated with Calcein-AM labeled sperm for 4 hours. The percentage of Calcein+ myoblasts was normalized to WT myoblasts (fusion index). n=4-5 independent experiments.

[0029] FIGURES 4A - 4B. Expression of Myomerger on C2C12 myoblasts. FIG. 4A: Immunoblot of Myomerger (~15 KDa) and beta actin on myoblasts incubated with differentiation media (DM) for 1, 2 or 3 days (d). n=3 independent experiments per time point. Quantification was performed as the ratio of Myomerger to Actin using ImageJ and values are depicted below the immunoblots. FIG. 4B: Immunolocalization of Myomerger on differentiated WT or Myomerger KO myoblasts. (When shown in color: Myomerger: red; Hoechst: blue; no red is seen on the right panel of FIG. 4B). Scale bars: 50 pm.

[0030] FIGURES 5A-5B. Co-localization of Myomerger and alpha-tubulin on testicular sections and dissociated germ cells smears. FIG. 5A: Immunolocalization of alpha-tubulin (green when shown in color, left panel), Myomerger (red when shown in color, middle panel) and merged image (right panel) on testicular sections of WT adult mice. Scale bars: 20 pm. FIG. 5B: Immunolocalization of alpha-tubulin (green when shown in color, left panel), Myomerger (red when shown in color, middle panel) and merged image (right panel) on dissociated germ cells from WT adult testis. Scale bars: 20 pm. Hoechst staining in blue when shown in color.

[0031] FIGURES 6A - 6C. Pre-absorption of Myomerger antibody with Myomerger peptide shows specificity of staining. Immunolocalization of Myomerger (green when shown in color) and PNA (red when shown in color) after preabsorption of the Myomerger antibody with a Myomerger peptide shows that the Myomerger antibody staining is specific on testicular sections (FIG. 6A, scale bar: 50 pm), dissociated germ cells smears (FIG. 6B, scale bar: 10 pm) or epididymal sperm isolated from the cauda (FIG. 6C, scale bar: 10 pm). Negative control was performed omitting the Myomerger antibody (right panel in FIG. 6C). Hoechst staining in blue when shown in color.

[0032] FIGURES 7A - 7D. Co-localization of Myomerger and Annexin V on sperm (live staining). FIGS. 7A - 7D. Co-localization of Myomerger (red when shown in color) and Annexin V-FITC (green when shown in color) on sperm isolated from cauda epididymis Attorney Docket No. 3062 / 202 PCT incubated in TYH+BSA medium. Both stainings were performed on live sperm. Different patterns were observed (arrows). Scale bar: 10 pm. Hoechst staining in blue when shown in color.

[0033] FIGURES 8A - 8E. Myoblast-sperm fusion. FIG. 8A: Dot plots showing the gating strategy for the analysis of the myoblast-sperm fusion by flow cytometry. After the coincubation of Calcein-AM labeled sperm with myoblasts, cells were washed with PBS, trypsinized and analyzed by flow cytometry. Myoblasts were gated according to their forward and size scatter (FSC and SSC) and dead cells were excluded using Sytox live / dead dye. The percentage of Calcein+ live myoblasts is indicated in each dot plot. In some experiments, WT myoblasts were pre-incubated with Cytochalasin D (CytoD). Myomerger KO showed a reduction of Calcein-AM acquisition indicating less fusion with sperm. FIG. 8B: Myoblastsperm fusion is partially reduced by the pre-incubation of myoblasts with Cytochalasin D (CytoD). Each dot is one experiment, n=2 experiments. FIG. 8C: Myoblast-sperm fusion using different ratios of sperm and undifferentiated WT, Myomerger KO or Myomaker KO myoblasts (i.e. IxlO4myoblasts and 2.5xl05sperm, 10K:250K ratio). The percentage of Calcein+ myoblasts was normalized to WT myoblasts (fusion index). FIG. 8D: Relative expression of MyoG, Myomaker (Mymk) and Myosin heavy chain (Myh4) relative to betaactin on C2C12 myoblasts differentiated for 0, 1, 2 and 3 days (d) was determined by RT- PCR. FIG. 8E: Myomerger26’84ectodomain peptide has no detrimental effects on sperm motility, p=0.07.

[0034] DETAILED DESCRIPTION

[0035] While several molecules linked to fertilization have now been described on sperm and oocytes, successful fertilization depends on the fusion between the gametes and the fusogen(s) responsible for the fusion of the sperrmegg cell membranes remain to be fully defined. Disclosed herein is the identification that Myomerger, a fusogen involved in myoblast cell fusion, is expressed on both male sperm cells and female oocytes, and that Myomerger has a role in sperrmegg fusion.

[0036] In some embodiments, the presently disclosed subject matter demonstrates that Myomerger, an 84-amino acid protein that plays a role in myoblast fusion, is also expressed on the surface of both murine sperm and oocytes. Interestingly, Myomerger is displayed on the head region of acrosome-reacted sperm, and on the microvillar region of oocytes, both of which are areas previously known to be involved in sperm-egg fusion. Functionally, blocking with Myomerger antibody or a Myomerger blocking peptide during in vitro fertilization assays reduced fertilization. Using a synthetic sperm fusion assay, the presently disclosed subject matter also demonstrates that Myomerger is important for ce 11 :ce 11 fusion. Collectively, these Attorney Docket No. 3062 / 202 PCT data indicate that Myomerger is expressed on female and male gametes, and contributes to sperrmegg fusion.

[0037] Aspects of the presently disclosed subject matter thus include that Myomerger but not Myomaker is expressed in sperm; oocytes also express Myomerger; and in functional assays, the blockade of Myomerger reduces cellular fusion.

[0038] The presently disclosed subject matter now will be described more fully hereinafter, in which some, but not all embodiments of the presently disclosed subject matter are described. Indeed, the presently disclosed subject matter can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.

[0039] I. GENERAL CONSIDERATIONS

[0040] Juno, and its partner Izumol, as well as CD9 are essential for fertilization [1-4]. More recently, based on an unbiased approach, the sperm molecules SOF1, TMEM95, SPACA6, FIMP and DCST1 and 2 have also been reported as key players in fertilization [5-10]. However, the molecular mechanism for sperm-egg fusion is still incompletely understood; most of the molecules linked to fertilization are either primarily involved in sperrmegg binding or have no known fusogenic roles in cellular ‘fusion’. It has been postulated that a fusogen is recruited after Izumol multimerization to promote pore formation necessary for the fusion of the plasma membranes of sperm and the egg [11-13]. Very recently, a member of the immunoglobulin superfamily, MAIA / Fc receptor-like 3, has been reported to bind to Izumol, and promote fusion of human sperm with transfected Chinese ovarian cells (CHO) or human embryonic kidney 293T cells (HEK293T) cells, suggesting a role in fusion during fertilization

[0014] . Using a baby hamster kidney fibroblast (BHK) cell line, Brukman et al

[0015] recently suggested that Izumol itself may induce fusion, likely by interacting with other proteins.

[0041] Another context where fusion of membranes and the mixing of the nuclear materials occur is during skeletal myogenesis to form multi-nucleated muscle cells. Extensive efforts have gone into defining the molecular identify of fusogens involved in myoblast fusion [16, 17]. Two molecules key to myoblast fusion and mammalian myogenesis are Myomaker and Myomerger (also called Myomixer / Minion) [18-21]. These two fusogens work independently. Myomaker is critical for hemifusion between the membranes of two fusing cells, while Myomerger is involved in the formation of the ‘fusion pore’ that then leads to the mixing of contents between the fusing cells

[0022] . Interestingly, myoblasts undergoing differentiation also express phosphatidylserine (PtdSer), and this PtdSer externalization is functionally linked to myoblast fusion [23-25]. Recently, it was shown that Myomerger functionality in ‘pore formation’ is dependent on lipid composition of the membranes and was linked to the Attorney Docket No. 3062 / 202 PCT phospholipid PtdSer

[0026] . We have previously demonstrated that live and motile sperm express PtdSer on their surface, and that sperrmegg fusion depends on sperm-expressed PtdSer binding to certain receptor molecules on oocytes

[0027] . The PtdSer-BAIl-Elmo signaling pathway is involved in both sperrmegg fusion and formation of myotubes. As proof-of- principle in that work, we also found that sperm cells can fuse with myoblasts in a PtdSer- dependent manner.

[0042] II. DEFINITIONS

[0043] While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0044] All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. Mention of techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art. Thus, unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the presently disclosed subject matter. Although any compositions, methods, kits, and means for communicating information similar or equivalent to those described herein can be used to practice the presently disclosed subject matter, particular compositions, methods, kits, and means for communicating information are described herein. It is understood that the particular compositions, methods, kits, and means for communicating information described herein are exemplary only and the presently disclosed subject matter is not intended to be limited to just those embodiments.

[0045] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, in some embodiments the phrase “a peptide” refers to one or more peptides.

[0046] The term “about”, as used herein to refer to a measurable value such as an amount of weight, time, dose (e.g., therapeutic dose), etc., is meant to encompass in some embodiments variations of ± 20%, in some embodiments ± 10%, in some embodiments ± 5%, in some embodiments ± 1%, in some embodiments ± 0.5%, in some embodiments ± 0.1%, and in some embodiments ± 0.01% from the specified amount, as such variations are appropriate to perform the disclosed methods.

[0047] As used herein, the term “and / or” when used in the context of a list of entities, refers to the entities being present singly or in any and every possible combination and Attorney Docket No. 3062 / 202 PCT subcombination. Thus, for example, the phrase “A, B, C, and / or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D. It is further understood that for each instance wherein multiple possible options are listed for a given element (i.e., for all “Markush Groups” and similar listings of optional components for any element), in some embodiments the optional components can be present singly or in any combination or subcombination of the optional components. It is implicit in these forms of lists that each and every combination and subcombination is envisioned and that each such combination or subcombination has not been listed simply merely for convenience. Additionally, it is further understood that all recitations of “or” are to be interpreted as “and / or” unless the context clearly requires that listed components be considered only in the alternative (e.g., if the components would be mutually exclusive in a given context and / or could not be employed in combination with each other).

[0048] As used herein, the term “subject” refers to an individual (e.g., human, animal, or other organism) to be assessed, evaluated, and / or treated by the methods or compositions of the presently disclosed subject matter. Subjects include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and includes humans. As used herein, the terms “subject” and “patient” are used interchangeably, unless otherwise noted.

[0049] As used herein, the terms “effective amount” and “therapeutically effective amount” are used interchangeably and refer to the amount that provides a therapeutic effect, e.g., an amount of a composition that is effective to treat or prevent one or more conditions in a subject.

[0050] As used herein, the term “adjuvant” as used herein refers to an agent which enhances the pharmaceutical effect of another agent.

[0051] As used herein, amino acids are represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated in the following table: Attorney Docket No. 3062 / 202 PCT

[0052] The expression “amino acid” as used herein is meant to include both natural and synthetic amino acids, and both D- and L- amino acids. “Standard amino acid” means any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid residue” means any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or derived from a natural source. As used herein, “synthetic amino acid” also encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and substitutions. Amino acids contained within the peptides of the presently disclosed subject matter, and particularly at the carboxy- or amino- terminus, can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change the peptide's circulating half-life without adversely affecting their activity. Additionally, a disulfide linkage can be present or absent in the peptides of the presently disclosed subject matter.

[0053] The term “amino acid” is used interchangeably with “amino acid residue”, and can refer to a free amino acid and to an amino acid residue of a peptide. It will be apparent from Attorney Docket No. 3062 / 202 PCT the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.

[0054] The term “antibody”, as used herein, refers to an immunoglobulin molecule which is able to specifically or selectively bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the presently disclosed subject matter may exist in a variety of forms. The term “antibody” refers to polyclonal and monoclonal antibodies and derivatives thereof (including chimeric, synthesized, humanized and human antibodies), including an entire immunoglobulin or antibody or any functional fragment of an immunoglobulin molecule which binds to the target antigen and or combinations thereof. Examples of such functional entities include complete antibody molecules, antibody fragments, such as Fv, single chain Fv, complementarity determining regions (CDRs), VL (light chain variable region), VH (heavy chain variable region), Fab, F(ab’)2 and any combination of those or any other functional portion of an immunoglobulin peptide capable of binding to target antigen.

[0055] Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab’)2 a dimer of Fab which itself is a light chain joined to VH -CHI by a disulfide bond. The F(ab’)2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab’)2 dimer into an Fabi monomer. The Fabi monomer is essentially an Fab with part of the hinge region (see Paul, 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies.

[0056] An “antibody heavy chain”, as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules.

[0057] An “antibody light chain”, as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules.

[0058] The term “single chain antibody” refers to an antibody wherein the genetic information encoding the functional fragments of the antibody are located in a single Attorney Docket No. 3062 / 202 PCT contiguous length of DNA. For a thorough description of single chain antibodies, see Bird et al., 1988; Huston et al., 1988).

[0059] The term “humanized” refers to an antibody wherein the constant regions have at least about 80% or greater homology to human immunoglobulin. Additionally, some of the nonhuman, such as murine, variable region amino acid residues can be modified to contain amino acid residues of human origin. Humanized antibodies have been referred to as “reshaped” antibodies. Manipulation of the complementarity-determining regions (CDR) is a way of achieving humanized antibodies. See for example, Jones et al., 1986; Riechmann et al., 1988, both of which are incorporated by reference herein. For a review article concerning humanized antibodies, see Winter & Milstein, 1991, incorporated by reference herein. See also U.S. Patent Nos. 4,816,567; 5,482,856; 6,479,284; 6,677,436; 7,060,808; 7,906,625; 8,398,980; 8,436,150; 8,796,439; and 10,253,111; and U.S. Patent Application Publication Nos. 2003 / 0017534, 2018 / 0298087, 2018 / 0312588, 2018 / 0346564, and 2019 / 0151448, each of which is incorporated by reference in its entirety.

[0060] By the term “synthetic antibody” as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

[0061] The term “antigen” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. An antigen can be derived from organisms, subunits of proteins, whole cells, cellular components or cellular lysates.

[0062] As used herein, the term “antisense oligonucleotide” means a nucleic acid polymer, at least a portion of which is complementary to a nucleic acid which is present in a normal cell or in an affected cell. The antisense oligonucleotides of the presently disclosed subject matterinclude, but are not limited to, phosphorothioate oligonucleotides and other modifications of oligonucleotides. Methods for synthesizing oligonucleotides, phosphorothioate oligonucleotides, and otherwise modified oligonucleotides are well known in the art (see e.g., U.S. Patent No. 5,034,506 to Summerton and Weller; Nielsen et al. (1991) Science 254:1497-1500). The term “antisense” refers particularly to the nucleic acid sequence of the non-coding strand of a double stranded DNA molecule encoding a protein, or to a Attorney Docket No. 3062 / 202 PCT sequence which is substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule. The antisense sequence can be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences.

[0063] As used herein, the term “biologically active fragments” or “bioactive fragment” of a polypeptide encompasses natural or synthetic portions of the full-length protein that are capable of specific binding to their natural ligand or of performing the function of the protein.

[0064] “Complementary” refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. As used herein, the terms “complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, for the sequence “A- G-T”, is complementary to the sequence “T-C-A.”

[0065] The term “complex”, as used herein in reference to proteins, refers to binding or interaction of two or more proteins. Complex formation or interaction can include such things as binding, changes in tertiary structure, and modification of one protein by another, such as phosphorylation.

[0066] A “compound”, as used herein, refers to any type of substance or agent that is commonly considered a chemical, drug, or a candidate for use as a drug, as well as combinations and mixtures of the above. The term compound further encompasses molecules such as peptides and nucleic acids.

[0067] As used herein, a “derivative” of a compound refers to a chemical compound that can be produced from another compound of similar structure in one or more steps, such as in replacement of H by an alkyl, acyl, or amino group. Similarly, a “derivative” of a peptide (or of a polypeptide) is a compound that can be produced from or has a biological activity similar to a peptide (or a polypeptide) but that differs in the primary amino acid sequence of the peptide (or the polypeptide) to some degree. By way of example and not limitation, a derivative of a subject peptide of the presently disclosed subject matter is a peptide that has a similar although not identical primary amino acid sequence as the subject peptide (for example, has one or more amino acid substitutions) and / or that has one or more other modifications (e.g., N-terminal, C-terminal, and / or internal modifications) as compared to the Attorney Docket No. 3062 / 202 PCT subject peptide. Thus, the term “derivative” compasses the term “modified peptide” and vice versa, in the context of peptides. In some embodiments, a derivative of a peptide is a C- terminal amidated peptide. Derivatives of antibodies can include but are not limited to antibody fragments, and chimeric, synthesized, humanized and human antibodies.

[0068] As used herein, a “detectable marker” or a “reporter molecule” is an atom or a molecule that permits the specific detection of a compound comprising the marker in the presence of similar compounds without a marker. Detectable markers or reporter molecules include, e.g., radioactive isotopes, antigenic determinants, enzymes, nucleic acids available for hybridization, chromophores, fluorophores, chemiluminescent molecules, electrochemically detectable molecules, and molecules that provide for altered fluorescence-polarization or altered light-scattering.

[0069] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

[0070] In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

[0071] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0072] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA can include introns.

[0073] As used herein, an “essentially pure” preparation of a particular protein or peptide is a preparation wherein at least about 95%, and preferably at least about 99%, by weight, of the protein or peptide in the preparation is the particular protein or peptide. Attorney Docket No. 3062 / 202 PCT

[0074] A “fragment” or “segment” is a portion of an amino acid sequence, comprising at least one amino acid of the amino acid sequence, or a portion of a nucleic acid sequence comprising at least one nucleotide. The terms “fragment” and “segment” are used interchangeably herein. In some embodiments, a “fragment” is a “biologically active fragment” or “bioactive fragment”.

[0075] As used herein, a “functional” biological molecule is a biological molecule in a form in which it exhibits a property or activity by which it is characterized. A functional enzyme, for example, is one which exhibits the characteristic catalytic activity by which the enzyme is characterized.

[0076] The terms “formula” and “structure” are used interchangeably herein.

[0077] The term “identity” as used herein relates to the similarity between two or more sequences. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100 to achieve a percentage. Thus, two copies of exactly the same sequence have 100% identity, whereas two sequences that have amino acid deletions, additions, or substitutions relative to one another have a lower degree of identity. Those skilled in the art will recognize that several computer programs, such as those that employ algorithms such as BLAST (Basic Local Alignment Search Tool, Altschul et al. (1993) J Mol Biol 215:403-410) are available for determining sequence identity.

[0078] In some embodiments, “identity” can be expressed as a “percent identity”. As used herein, the phrase “percent identity” in the context of two nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have in some embodiments 60%, in some embodiments 70%, in some embodiments 75%, in some embodiments 80%, in some embodiments 85%, in some embodiments 90%, in some embodiments 92%, in some embodiments 94%, in some embodiments 95%, in some embodiments 96%, in some embodiments 97%, in some embodiments 98%, in some embodiments 99%, and in some embodiments 100% nucleotide or amino acid residue identity, respectively, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. The percent identity exists in some embodiments over a region of the sequences that is at least about 50 residues in length, in some embodiments over a region of at least about 100 residues, and in some embodiments, the percent identity exists over at least about 150 residues. In some embodiments, the percent identity exists over the entire length of the sequences.

[0079] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if Attorney Docket No. 3062 / 202 PCT necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0080] Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm disclosed in Smith & Waterman (1981) 2 Adv Appl Math 482-489; by the homology alignment algorithm disclosed in Needleman & Wunsch (1970) 48 J Mol Biol 443-453; by the search for similarity method disclosed in Pearson Pearson & Lipman (1988) Proc Natl Acad Sci U S A 85:2444-2448; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG® WISCONSIN PACKAGE®, available from Accelrys, Inc., San Diego, California, United States of America), or by visual inspection. See generally, Altschul et al. (1990) 215 J Mol Biol 403- 410; Ausubel et al. (2002) Short Protocols in Molecular Biology, Fifth ed. Wiley, New York, New York, United States of America; and Ausubel et al. (2003) Current Protocols in Molecular Biology, John Wylie & Sons, Inc, New York, New York, United States of America.

[0081] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. (1990) 215 J Mol Biol 403-410. Software for performing BLAST analysis is publicly available through the website of the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. See generally, Altschul et al. (1990) 215 J Mol Biol 403-410. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M = 5, N = 4, and a comparison of both strands. For amino acid sequences, the BLASTP Attorney Docket No. 3062 / 202 PCT program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. See Henikoff & Henikoff (1992) 89 Proc Natl Acad Sci U S A 10915-10919.

[0082] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see e.g., Karlin & Altschul (1993) 90 Proc Natl Acad Sci U S A 5873-5877). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. Lor example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is in some embodiments less than about 0.1, in some embodiments less than about 0.01, and in some embodiments less than about 0.001.

[0083] The term “inhibit”, as used herein, refers to the ability of a compound or any agent to reduce or impede a described function or pathway. Lor example, inhibition can be by at least 10%, by at least 25%, by at least 50%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 97%, by at least 99%, or more.

[0084] As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the presently disclosed subject matter in the kit for effecting alleviation of the various diseases or disorders recited herein. Optionally, or alternately, the instructional material can describe one or more methods of assessing fertilization competency of a gamete from a subject. The instructional material of the kit of the presently disclosed subject matter can, for example, be affixed to a container which contains a reagent of the presently disclosed subject matter or be shipped together with a container which contains the reagent. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the reagent be used cooperatively by the recipient.

[0085] An “isolated” compound / moiety is a compound / moiety that has been removed from components naturally associated with the compound / moiety. Lor example, an “isolated nucleic acid” refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, Attorney Docket No. 3062 / 202 PCT into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

[0086] The term “modulate”, as used herein, refers to changing the level of an activity, function, or process. The term “modulate” encompasses both inhibiting and stimulating an activity, function, or process.

[0087] The term “oligonucleotide” typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T.”

[0088] As used herein, the term “purified” and like terms relate to an enrichment of a molecule or compound relative to other components normally associated with the molecule or compound in a native environment. The term “purified” does not necessarily indicate that complete purity of the particular molecule has been achieved during the process. A “highly purified” compound as used herein refers to a compound that is greater than 90% pure.

[0089] As used herein, the term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions such as an oil / water or water / oil emulsion, and various types of wetting agents. The term also encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in an animal. In some embodiments, a pharmaceutically acceptable carrier is pharmaceutically acceptable for use in a human.

[0090] The term “polypeptide” refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non- naturally occurring analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.

[0091] The term “protein” typically refers to large polypeptides (e.g., a polypeptide of in some embodiments at least 50 amino acids, in some embodiments at least 75 amino acids, in some embodiments at least 100 amino acids, in some embodiments at least 200 amino acids, in some embodiments at least 300 amino acids, in some embodiments at least 500 amino acids, and in some embodiments more than 500 amino acids).

[0092] A peptide encompasses a sequence of 2 or more amino acids wherein the amino acids are naturally occurring or synthetic (non-naturally occurring) amino acids. Attorney Docket No. 3062 / 202 PCT

[0093] The term “linked” or like terms refers to a connection between two entities. The linkage can comprise a covalent, ionic, or hydrogen bond or other interaction that binds two compounds or substances to one another.

[0094] As used herein the term “peptidomimetic” refers to a chemical compound having a structure that is different from the general structure of an existing peptide, but that functions in a manner similar to the existing peptide, e.g., by mimicking the biological activity of that peptide. The term “modified peptide” encompasses a peptidomimetic. Peptidomimetic s typically comprise naturally-occurring amino acids and / or unnatural amino acids, but can also comprise modifications to the peptide backbone. For example, a peptidomimetic can include one or more of the following modifications:

[0095] 1. Peptides wherein one or more of the peptidyl -C(O)NR- linkages (bonds) have been replaced by a non-peptidyl linkage such as a -CFF-carbamate linkage (- CH2OC(O)NR-), a phosphonate linkage, a -CFF-sulfonamide (-CH2-S(O)2NR-) linkage, a urea (-NHC(O)NH-) linkage, a -CPF-secondary amine linkage, an azapeptide bond (CO substituted by NH), or an ester bond (e.g., depsipeptides, wherein one or more of the amide (- CONHR-) bonds are replaced by ester (COOR) bonds) or with an alkylated peptidyl linkage (-C(O)NR-) wherein R is Ci-Ce alkyl;

[0096] 2. Peptides wherein the N-terminus is derivatized to a -NRR1 group, to a - NRC(O)R group, to a -NRC(O)OR group, to a -NRS(O)2R group, to a -NHC(O)NHR group where R and R1 are hydrogen or Ci-Ce alkyl with the proviso that R and R1 are not both hydrogen;

[0097] 3. Peptides wherein the C terminus is derivatized to -C(O)R2 where R2 is selected from the group consisting of Ci-Ce alkoxy, and -NR3R4 where R3 and R4 are independently selected from the group consisting of hydrogen and C1-C4 alkyl;

[0098] 4. Modification of a sequence of naturally-occurring amino acids with the insertion or substitution of a non-peptide moiety, e.g., a retroinverso fragment.

[0099] The term “permeability”, as used herein, refers to transit of fluid, cell, or debris between or through cells and tissues.

[0100] A “sample”, as used herein, refers preferably to a biological sample from a subject, including, but not limited to, normal tissue samples, diseased tissue samples, biopsies, blood, saliva, feces, semen, tears, and urine. A sample can also be any other source of material obtained from a subject which contains cells, tissues, gamete cells, or fluid of interest. A sample can also be obtained from cell or tissue culture.

[0101] By the terms “specifically binds” or “selectively binds”, as used herein, is meant a compound which recognizes and binds a specific target molecule, but does not substantially Attorney Docket No. 3062 / 202 PCT recognize or bind other molecules in a sample, or it means binding between two or more molecules as in part of a cellular regulatory process, where said molecules do not substantially recognize or bind other proteins in a sample.

[0102] The term “standard”, as used herein, refers to something used for comparison. For example, it can be a known standard agent or compound which is administered or added to a control sample and used for comparing results when measuring said compound in a test sample. Standard can also refer to an “internal standard”, such as an agent or compound which is added at known amounts to a sample and is useful in determining such things as purification or recovery rates when a sample is processed or subjected to purification or extraction procedures before a marker of interest is measured.

[0103] The term “symptom”, as used herein, refers to any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by the patient and indicative of disease. In contrast, a sign is objective evidence of disease. For example, a bloody nose is a sign. It is evident to the patient, doctor, nurse and other observers.

[0104] As used herein, the term “treating” includes prophylaxis of the specific disorder or condition, or alleviation of the symptoms associated with a specific disorder or condition and / or preventing or eliminating said symptoms. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

[0105] A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs.

[0106] As used herein an “amino acid modification” refers in some embodiments to a substitution, addition, or deletion of an amino acid, and includes substitution with, or addition of, any of the 20 amino acids commonly found in human proteins, as well as unusual or non- naturally occurring amino acids such as but not limited to D-amino acids. Commercial sources of unusual amino acids include Sigma-Aldrich (Milwaukee, Wisconsin, United States of America), ChemPep Inc. (Miami, Florida, United States of America), and Genzyme Pharmaceuticals (Cambridge, Massachusetts, United States of America). Unusual amino acids can be purchased from commercial suppliers, synthesized de novo, or chemically modified or derivatized from naturally occurring amino acids. Amino acid modifications include linkage of an amino acid to a conjugate moiety, such as a hydrophilic polymer, acylation, alkylation, and / or other chemical derivatization of an amino acid. The term “modified peptide” encompasses any amino acid modification as described herein. Attorney Docket No. 3062 / 202 PCT

[0107] Modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine .

[0108] Also included are polypeptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids. The peptides of the presently disclosed subject matter are not limited to products of any of the specific exemplary processes listed herein.

[0109] Substitutions can be designed based on, for example, the model of Dayhoff et al. (in Atlas of Protein Sequence and Structure 1978, National Biomedical Research Foundation, Washington D.C., United States of America).

[0110] In some embodiments, an amino acid substitution is a conservative amino acid substitution. As used herein, the term “conservative amino acid substitution” is defined in some embodiments as exchanges within one of the following five groups:

[0111] I. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, Gly;

[0112] II. Polar, charged residues and their amides: Asp, Asn, Glu, Gin, His, Arg, Lys;

[0113] III. Large, aliphatic, nonpolar residues: Met Leu, He, Vai, Cys

[0114] IV. Large, aromatic residues: Phe, Tyr, Trp

[0115] Conservative substitutions are likely to be phenotypically silent. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Vai, Leu, and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al. (1990) Science 247:1306-1310.

[0116] For example, the hydropathic index of amino acids may be considered (Kyte & Doolittle (1982) J Mol Biol 157:105-132). The relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the Attorney Docket No. 3062 / 202 PCT interaction of the protein with other molecules. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doolittle (1982) J Mol Biol 157:105-132), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (- 3.9); and arginine (-4.5). In making conservative substitutions, the use of amino acids whose hydropathic indices are within + / -2 is preferred, within + / -1 are more preferred, and within + / - 0.5 are even more preferred.

[0117] Amino acid substitution may also take into account the hydrophilicity of the amino acid residue (e.g., U.S. Patent No. 4,554,101). Hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-0.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). Replacement of amino acids with others of similar hydrophilicity is preferred.

[0118] Other considerations include the size of the amino acid side chain. For example, in some embodiments an amino acid with a compact side chain, such as glycine or serine, would not be replaced with an amino acid with a bulky side chain, e.g., tryptophan or tyrosine. The effect of various amino acid residues on protein secondary structure is also a consideration. Through empirical study, the effect of different amino acid residues on the tendency of protein domains to adopt an alpha-helical, beta-sheet, or reverse turn secondary structure has been determined and is known in the art (see e.g., Chou & Fasman (1974) Biochemistry 13:222- 245; Chou & Fasman (1978) Ann Rev Biochem 47: 251-276; Chou & Fasman (1979) Biophys J 26:367-384).

[0119] Based on such considerations and extensive empirical study, tables of conservative amino acid substitutions have been constructed and are known in the art. By way of example and not limitation, the following substitutions can be made: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Alternatively, Table 1 lists exemplary conservative amino acid substitutions.

[0120] Table 1

[0121] Exemplary Conservative Amino Acid Substitutions Attorney Docket No. 3062 / 202 PCT

[0122] In some embodiments, another consideration for amino acid substitutions include whether or not the residue is located in the interior of a protein or is solvent exposed. For interior residues, conservative substitutions can include in some embodiments: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala and Gly; He and Vai; Vai and Leu; Leu and He; Leu and Met; Phe and Tyr; Tyr and Trp. For solvent exposed residues, conservative substitutions can include in some embodiments: Asp and Asn; Asp and Glu; Glu and Gin; Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Vai and Leu; Leu and He; He and Vai; Phe and Tyr. Various matrices have been constructed to assist in selection of amino acid substitutions, such as the PAM250 scoring matrix, the Dayhoff matrix, the Grantham matrix, the McLachlan matrix, the Doolittle matrix, the Henikoff matrix, the Miyata matrix, the Fitch matrix, the Jones matrix, the Rao matrix, the Levin matrix, and the Risler matrix (summarized in, for example, Johnson & Overington (1993) J Mol Biol 233:716-738; see also the PROWL resource available at the website of The Rockefeller University, New York, New York, United States of America).

[0123] In determining amino acid substitutions, one may also consider the existence of intermolecular or intramolecular bonds, such as formation of ionic bonds (salt bridges) between positively charged residues (e.g., His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) or disulfide bonds between nearby cysteine residues.

[0124] Methods of substituting any amino acid for any other amino acid in an encoded peptide sequence are well known and a matter of routine experimentation for the skilled artisan, for example by the technique of site-directed mutagenesis or by synthesis and assembly of oligonucleotides encoding an amino acid substitution and splicing into an expression vector construct. Attorney Docket No. 3062 / 202 PCT

[0125] III. METHODS, COMPOSITIONS, DEVICES, AND KITS

[0126] Fertilization has been studied for decades, yet the understanding of the molecular events responsible for the incorporation of the sperm into the oocyte is incomplete. While several molecules linked to fertilization have now been described on sperm and oocytes, successful fertilization depends on the fusion between the gametes and the fusogen(s) responsible for the fusion of the sperm: egg cell membranes remain to be fully defined. The presently disclosed subject matter now identifies that Myomerger, a fusogen involved in myoblast cell fusion, is expressed on both male sperm cells and female oocytes, and that there is a role for Myomerger in sperrmegg fusion.

[0127] The term “Myomerger” is used herein to refer in some embodiments to a polypeptide having the following amino sequence: mptpllplll rlllsclllp aarlarqyll pllrrlarrl gsqdmreall gcllfilsqr hspdageasr vdrlerrerl gpqk (SEQ ID NO: 1). Also, provided and falling within the term “Myomerger” is a sequence having at least about 60% percent identity, in some embodiments at least about 70% percent identity, in some embodiments at least about 75% percent identity, in some embodiments at least about 80% percent identity, in some embodiments at least about 85% percent identity, in some embodiments at least about 90% percent identity, in some embodiments at least about 92% percent identity, in some embodiments at least about 94% percent identity, in some embodiments at least about 95% percent identity, in some embodiments at least about 96% percent identity, in some embodiments at least about 97% percent identity, in some embodiments at least about 98% percent identity, in some embodiments at least about 99% percent identity, or in some embodiments at least about 100% percent identity thereto, or fragment or derivative thereof, for any peptide sequence set forth herein, as are substantially homologous amino acid sequences of any of the foregoing sequences. In some embodiments, the amino acid sequence comprises at least one modification selected from the group consisting of an amino acid deletion, an amino acid addition, an amino acid substitution, and combinations thereof.. A Myomerger ectodomain (e.g., soluble Myomerger; amino acids 22-84 of SEQ ID NO: 1; ((26) rqyll pllrrlarrl gsqdmreall gcllfilsqr hspdageasr vdrlerrerl gpqk (84); SEQ ID NO: 2), [22, 43]) is an exemplary fragment. Sequences having the sequence of a Myomerger ectodomain, or a sequence having at least about 60% percent identity, in some embodiments at least about 70% percent identity, in some embodiments at least about 75% percent identity, in some embodiments at least about 80% percent identity, in some embodiments at least about 85% percent identity, in some embodiments at least about 90% percent identity, in some embodiments at least about 92% percent identity, in some embodiments at least about 94% Attorney Docket No. 3062 / 202 PCT percent identity, in some embodiments at least about 95% percent identity, in some embodiments at least about 96% percent identity, in some embodiments at least about 97% percent identity, in some embodiments at least about 98% percent identity, in some embodiments at least about 99% percent identity, or in some embodiments at least about 100% percent identity thereto, as defined herein above and derivatives of this fragment are also encompassed by the term “Myomerger”, as are substantially homologous amino acid sequences of any of the foregoing sequences. In some embodiments, the amino acid sequence comprises at least one modification selected from the group consisting of an amino acid deletion, an amino acid addition, an amino acid substitution, and combinations thereof.

[0128] The term “Myomerger” also includes the following: also known as Myomixer and Minion. MYMX myomixer, myoblast fusion factor [Homo sapiens (human)] Gene ID: 101929726 and orthologs and isoforms thereof, including orthologs or isoforms thereof corresponding to the animal subjects described elsewhere herein.

[0129] The term “Myomerger” also includes the following: Protein myomixer [Homo sapiens] NCBI Reference Sequence: NP_001302423.1: mptpllplll rlllsclllp aarlarqyll pllrrlarrl gsqdmreall gcllfilsqrhspdageasr vdrlerrerl gpqk (SEQ ID NO: 1); protein myomixer isoform 1 [Mus musculus] NCBI Reference Sequence: NP_001170939.1: mpvpllpmvl rsllsrlllp varlarqhll pllrrlarrl ssqdmreall scllfvlsqq qppdsgeasr vdhsqrkerl gpqk (SEQ ID NO: 3).

[0130] A nucleic acid sequence encoding Myomerger is also provided in accordance with the presently disclosed subject matter. A representative sequence is: ATGCCCACGCCACTGCTCCCGCTGCTGCTTCGATTGCTGCTGTCCTGCCTGCTGCT GCCTGCTGCCCGCCTGGCCCGCCAATACCTCCTGCCCCTGCTGCGCCGATTGGCCC GCCGCCTGGGCTCCCAGGACATGCGAGAGGCTTTGCTGGGCTGTCTGCTGTTCATT CTCAGCCAGCGACACTCGCCAGACGCTGGGGAGGCCTCAAGAGTGGACCGCCTG GAGAGGAGGGAGAGGTTAGGCCCCCAAAAGTGA (SEQ ID NO:4). Also, provided is a sequence having at least about 60% percent identity, in some embodiments at least about 70% percent identity, in some embodiments at least about 75% percent identity, in some embodiments at least about 80% percent identity, in some embodiments at least about 85% percent identity, in some embodiments at least about 90% percent identity, in some embodiments at least about 92% percent identity, in some embodiments at least about 94% percent identity, in some embodiments at least about 94% percent identity, in some embodiments at least about 96% percent identity, in some embodiments at least about 97% Attorney Docket No. 3062 / 202 PCT percent identity, in some embodiments at least about 98% percent identity, in some embodiments at least about 99% percent identity, or in some embodiments at least about 100% percent identity thereto, for any nucleotide sequence set forth herein.

[0131] Representative sequences include:

[0132] NCBI Ref. Sequence: CCDS83093.1 [Homo sapiens] Nucleotide Sequence (255 nt)(SEQ ID NO: 4): ATGCCCACGCCACTGCTCCCGCTGCTGCTTCGATTGCTGCTGTCCTGCCTGCTGCT GCCTGCTGCCCGCCTGGCCCGCCAATACCTCCTGCCCCTGCTGCGCCGATTGGCCC GCCGCCTGGGCTCCCAGGACATGCGAGAGGCTTTGCTGGGCTGTCTGCTGTTCATT CTCAGCCAGCGACACTCGCCAGACGCTGGGGAGGCCTCAAGAGTGGACCGCCTG GAGAGGAGGGAGAGGTTAGGCCCCCAAAAGTGA

[0133] NCBI Ref. Sequence: CCDS50119.1 [Mas musculus] (255 nt) (SEQ ID NO:5): ATGCCCGTTCCATTGCTCCCGATGGTGCTTCGATCGCTGCTGTCCCGCCTGCTGCT GCCTGTTGCCCGCCTGGCCCGGCAGCACCTCCTGCCCTTGCTGCGCCGGCTGGCCC GCCGACTGAGCTCCCAAGACATGAGAGAGGCTCTGCTGAGCTGTCTGCTCTTTGT CCTCAGCCAGCAACAGCCACCGGATTCTGGAGAGGCCTCCAGA GTGGACCACTCCCAGAGGAAGGAGAGATTGGGCCCCCAGAAGTGA

[0134] The term “Myomerger” also includes the proteins, polypeptides, peptides and nucleic acids disclosed in the following journal article, herein incorporated by reference in its entirety: Chen B, You W, Wang Y, Shan T. The regulatory role of Myomaker and Myomixer- Myomerger-Minion in muscle development and regeneration. Cell Mol Life Sci. 2020 Apr;77(8):1551-1569. doi: 10.1007 / s00018-019-03341-9. Epub 2019 Oct 23. PMID: 31642939; PMCID: PMC11105057.

[0135] The term “Myomerger” also includes the following, and orthologs and isoforms thereof, including orthologs or isoforms thereof corresponding to the animal subjects described elsewhere herein, and derivatives and fragments thereof as defined herein:

[0136] Homo sapiens myomixer, myoblast fusion factor (MYMX), transcript variant 1, mRNA: Accession No. NM_001315494.2 of the GENBANK® biosequence database

[0137] (SEQ ID NO:6)

[0138] 1 gcaaatccag ccagagactg attctgagca gcagttctgc ccggcactga ctcactggcc

[0139] 61 ctgccatgcc cacgccactg ctcccgctgc tgcttcgatt gctgctgtcc tgcctgctgc

[0140] 121 tgcctgctgc ccgcctggcc cgccaatacc tcctgcccct gctgcgccga ttggcccgcc

[0141] 181 gcctgggctc ccaggacatg cgagaggctt tgctgggctg tctgctgttc attctcagcc

[0142] 241 agcgacactc gccagacgct ggggaggcct caagagtgga ccgcctggag aggagggaga

[0143] 301 ggttaggccc ccaaaagtga ggccacaagt cctggcagca gctgtatcca caaaatgctt

[0144] 361 tcttttggag taggataatc ctggcaccag cactgaccga agcctgccca gtggacagaa Attorney Docket No. 3062 / 202 PCT

[0145] 421 gatatagtga gggttgtgca tgagagggat ctgccacaga catgcctctc cactcccaac

[0146] 481 agaaatgtct ttctggaaga atgccttgca tctagcacaa aactgattat tgcccctctg

[0147] 541 tcctccagca gttcctccca aagaccactc ctaatcacct ctggcctcag gcgggagggg

[0148] 601 aactaacacc cacccacccc tgccctccct gcaaatggga acatcaaggt tcccagtgct

[0149] 661 taactgaggg acaagtgaca atttagcaga gaggcaagat ttgaatccag actgtcttcc

[0150] 721 agactcagga cctaccttaa aataatatct gagttgctta tggaggcaga cctgcctgca

[0151] 781 aagcccagca ctcagcaagt gctcaataaa tatttgattt gaattctt

[0152] Which encodes:

[0153] 1 mptpllplll rlllsclllp aarlarqyll pllrrlarrl gsqdmreall gcllfilsqr 61 hspdageasr vdrlerrerl gpqk ( SEQ ID NO : 1 )

[0154] Homo sapiens myomixer, myoblast fusion factor (MYMX), transcript variant 2, mRNA: Accession No. NM_001347931.2 of the GENBANK® biosequence database (SEQ ID NO: 7)

[0155] 1 gcaaatccag ccagagactg attctgagca gcagttctgc ccggtgagag ctgccgtgga

[0156] 61 ttggtggggg cactgactca ctggccctgc catgcccacg ccactgctcc cgctgctgct

[0157] 121 tcgattgctg ctgtcctgcc tgctgctgcc tgctgcccgc ctggcccgcc aatacctcct

[0158] 181 gcccctgctg cgccgattgg cccgccgcct gggctcccag gacatgcgag aggctttgct

[0159] 241 gggctgtctg ctgttcattc tcagccagcg acactcgcca gacgctgggg aggcctcaag

[0160] 301 agtggaccgc ctggagagga gggagaggtt aggcccccaa aagtgaggcc acaagtcctg

[0161] 361 gcagcagctg tatccacaaa atgctttctt ttggagtagg ataatcctgg caccagcact

[0162] 421 gaccgaagcc tgcccagtgg acagaagata tagtgagggt tgtgcatgag agggatctgc

[0163] 481 cacagacatg cctctccact cccaacagaa atgtctttct ggaagaatgc cttgcatcta

[0164] 541 gcacaaaact gattattgcc cctctgtcct ccagcagttc ctcccaaaga ccactcctaa

[0165] 601 tcacctctgg cctcaggcgg gaggggaact aacacccacc cacccctgcc ctccctgcaa

[0166] 661 atgggaacat caaggttccc agtgcttaac tgagggacaa gtgacaattt agcagagagg

[0167] 721 caagatttga atccagactg tcttccagac tcaggaccta ccttaaaata atatctgagt

[0168] 781 tgcttatgga ggcagacctg cctgcaaagc ccagcactca gcaagtgctc aataaatatt

[0169] 841 tgatttgaat tctt

[0170] Which encodes:

[0171] 1 mptpllplll rlllsclllp aarlarqyll pllrrlarrl gsqdmreall gcllfilsqr

[0172] 61 hspdageasr vdrlerrerl gpqk ( SEQ ID NO : 1 )

[0173] PREDICTED: Homo sapiens myomixer, myoblast fusion factor (MYMX), transcript variant XI, mRNA: Accession No. XM_024446300.2 of the GENBANK® biosequence database

[0174] (SEQ ID NO: 8):

[0175] 1 aaagctccac cctccaggaa gccagctctc acttttcaca cattggccaa gtgtcctgga 61 ctcctaaggt gcaggccatt tctttttctg tcttgtaatt tcctgtttcc tcctgcctct Attorney Docket No. 3062 / 202 PCT

[0176] 121 cctactgacc atatcttgag agcaggactg tctttttctc tgatgtactt cgagagcctg

[0177] 181 cacaagagtg tgttgactaa agcaaccaat ggcggtccca gctaacgtga ccgcttccta

[0178] 241 ttttgaactt caaagctctc catccaggat gcctcttaag ggccaaagtg ccccagctgg

[0179] 301 aattggaacc ctcccaggca tatccagtgt gtgtcccact gcccggtcgc ctgtgcatgg 361 gtctatcatt gcctggaatt ggtgggcaga ggaatgatgg tcatggataa gggaccatcc

[0180] 421 ctccaaagcg catcacctct tctttcagtg gtcacattca ggccctagct cctcactcag

[0181] 481 gtaggtggtt tctggcctgt tgtcccaggc agaaaggcac tgtgattatg acagggctga

[0182] 541 gctcccagtt cctgggctag aactccgccc attccccacc cacactgcct atacctggct

[0183] 601 ggctctggtt cctggcccca aggtctcccc acctgctcct tggtaacatc accccttgac 661 acaccctgcc cttgatgagg cctccctggc tccttgtcag aactccttgt ttaggggtca

[0184] 721 gagctgtcct caacctccca caaacaggaa gggccccaca ccaatgctga tttcccagag

[0185] 781 gacaacaggc cagtgaaaca tgacagagcg aggggccaca gcctccgcag gcctcccttg

[0186] 841 attcttcaag cattaacaga gccccagcct ctgatctaag tgctgcccat gagcagcagg

[0187] 901 gtgccttgca tcaggcctcc aaggagcagc aagttctctg ctggcctctg acataagggt 961 ggaaggaagg aaactaatat ttatgagaca cctacctggt agccaaactc tgagcctcac

[0188] 1021 attctcctca tctctttatc tggtcagctg taggtattat tataattccc attttaggga

[0189] 1081 gggggaccac cccatagcac agctccggaa ggtgccactc tcaactcccg ctgtgcaata

[0190] 1141 tacagcctgc tctggcatcc agaggggccc tgagagaggg tcccagagcc agcatgtggt

[0191] 1201 tccagagcga ccaactcaga agcacctcca gccaacttca cgagctggga cccagacaca 1261 tgtttttaat gcaagtcatg cggaggcagt ggcctgtcgc agcatgctct tgagcgcctg

[0192] 1321 ggtggagaca tgcagaaggg gtgggaagag caggactctc ggtgcccgtg ggtctggcaa

[0193] 1381 gaacccatta gctctagtgc aactcttgct atcattcctg ctttccccat ctgctgtctg

[0194] 1441 tcactctggt ggctcttggt gtgtctgtcc tcttcaccat tgacccagag ccttgcacgg

[0195] 1501 gctttcacac agttggcaca caatgtatgt tgtcatgtct ggcccgatta actgatgcct 1561 gtgtatgggc atcatccaga atgtttctgg atggccctgg gcatctgggg agtggaccac

[0196] 1621 aaaggcagat cccaggggcc acttagtgcc agcccacagt cctcaccttc acaggttggt

[0197] 1681 atccagatac tatttttttt ttttttcaat tgaccttaag gaaacaggag atttggtcag

[0198] 1741 tttcctcaaa gttgcctgca gagcatggga ggcagaggtc agggaagtgc aggaaggatc

[0199] 1801 catgacacaa ggctaaactg ggaactggaa gaaactgctc cctggcggct ttgttcctga 1861 cgaagatggt tatttttcca ggaatgctga ggaaatccac agcagcttta gtgttcccgc

[0200] 1921 cttggagaaa atgtctgggg caggcaaggg aggtggggtg gagctaatgg aagtgaggat

[0201] 1981 gggaaggtgg tggagagggc agttaatccc aggaagagtc aggggttaga gagtaaacta

[0202] 2041 agactagatc cattccttgg agccagccac tgcccctccc ccacctccct gcccaaaccc

[0203] 2101 ctctggagtt ccccattgtt cccagagaca atagctagtt tcctttacct gcctcatcat 2161 cccaggccca ggaatctctc actctgttta caagaggcag cggtggggta ggagtatctg

[0204] 2221 ggccttaccc cagggtccaa ggatctcacc gctggaagat cccacagcta tggaaactga

[0205] 2281 ggctgggagg agtgggatgt gacttagcca aggtcacagc aaccacagat gtgggccagt

[0206] 2341 gcttggtggg gcctccacgg ttccccgtgg agtacgcact ttggtggttc tcagagggag

[0207] 2401 ccatcagggc ctgccgttcc tcccctcgag actctctcca gctgtctgtg cagcccaggg 2461 gttctcacac acatcctggg agtgtgttta gatcagtgga gccctggaac ccagctgcac Attorney Docket No. 3062 / 202 PCT

[0208] 2521 agactctggg gcaagcaaac ctgggtttgg atcctgccac agaaccttca tttatttgtt

[0209] 2581 ccgtaaacca aaaaaaaagt gtgtgagaca gatctccgtc aacttagcgg tttattttgc

[0210] 2641 caaggttgac gataagcctg ggaaaaagag acaaaagtta cagaaggctc tgtggcctgt

[0211] 2701 cctttttcca aagacagttt caggatttcc attcttaaag gggaaagagc agcaggaggg

[0212] 2761 aaaagaggaa cagtccatta ctggtgagat tcaacagaac ttggccttag ggtgaggact

[0213] 2821 ttggagccca caaggaattc ctcctgagca atttgtgaga aaggtcacct ggggaaatat

[0214] 2881 gcggccttcc attgctgcag ctgtctgttt aggaaccaaa ataaggcagt ttttgtgtga

[0215] 2941 ctcagttccc aagcttaact tttccctttg tgagtttggg gtctcttttc ctttcacatt

[0216] 3001 tcttacaaca atttttacta gtatttattg ccaagcactg ggccacaaca aaattgttcc

[0217] 3061 tactctcaag tcccttctct caaggaactt atgttttatt tctttatttt tatttattta

[0218] 3121 tttatttttg agacagagtc tcgctctgtc acccaggctg gagtgcagtg gcatgatctt

[0219] 3181 ggcacactgc aacctctgcc tcccgggttc aagagattct tctgcctcag cctcctgagt

[0220] 3241 agctgggatt acaggcacgc atcgccatgc ccggctaatt ttttgtattt ttaatagaga

[0221] 3301 ctgggtttcc atgttggccc agctggtgtc tcaaactcct gacctcaggt gatccacccg

[0222] 3361 ccttggtctc ccaaagtgct gggtttacag gagtgagtca ctgcgcctgg tggaacttac

[0223] 3421 gttttagtaa gggaggcaga taaccaacaa gataaggaag taaaatatat agcatgtctg

[0224] 3481 acaatgattt agcagagagg taaagaaagt atggcagggc agctggaggt

[0225] 3541 ggggtggaag gactgtacat ttaaatagga tggttgggta atatttgagc aaagatttga

[0226] 3601 aggaggtcac agatgaagcc acgtagattt ctggataaag acccttttgg taggtgcttg

[0227] 3661 agcctgagta ggggctcagg acataccact ccaaaacaga actgccagtg actagactgt

[0228] 3721 gccaccccaa aatatacctc tttgggatat tgattatttc aagctggtta ttttaagaaa

[0229] 3781 ctgtagacat gggagttgct ctgaaaagct gttcttttat aaaggaaatc tacatttaac

[0230] 3841 aaggaaattt tcacgggaga gggtatctat atcaggaaga gatttactcc agacaatttt

[0231] 3901 tatcacctga gagacttgta tcttacatag caaggcacac tttattcacc acccatttcc

[0232] 3961 tcccctcacc ctcttatgtc tccatcaccc ctagaggccc caaaccccta ttgtgtagct

[0233] 4021 caggatagta cataagcctt aatttttggg ttgtttcttg gggtcttata tttttgtggg

[0234] 4081 actcctgtgc atacatatga aattaaaaat gtttttctcc tgtaaatctg tctatgtcaa

[0235] 4141 tttaattcat agacctgcca aagaacttag aagggtacca agaagcaata tttttttctc

[0236] 4201 ttctatagtt gtagaacccc aaaactcagt ttgagtacct gatgtgcagg aagccaaaca

[0237] 4261 gtgacacatc agtgcttcag aagagagaat ggtttatttg atttggccaa cggtaatata

[0238] 4321 ggctctgtta caaagggacc ttactgtctg ggacctgcta gctccagtac tgccacaatg

[0239] 4381 caggattcca ggcactgact cactggccct gccatgccca cgccactgct cccgctgctg

[0240] 4441 cttcgattgc tgctgtcctg cctgctgctg cctgctgccc gcctggcccg ccaatacctc

[0241] 4501 ctgcccctgc tgcgccgatt ggcccgccgc ctgggctccc aggacatgcg agaggctttg

[0242] 4561 ctgggctgtc tgctgttcat tctcagccag cgacactcgc cagacgctgg ggaggcctca

[0243] 4621 agagtggacc gcctggagag gagggagagg ttaggccccc aaaagtgagg ccacaagtcc

[0244] 4681 tggcagcagc tgtatccaca aaatgctttc ttttggagta ggataatcct ggcaccagca

[0245] 4741 ctgaccgaag cctgcccagt ggacagaaga tatagtgagg gttgtgcatg agagggatct

[0246] 4801 gccacagaca tgcctctcca ctcccaacag aaatgtcttt ctggaagaat gccttgcatc

[0247] 4861 tagcacaaaa ctgattattg cccctctgtc ctccagcagt tcctcccaaa gaccactcct Attorney Docket No. 3062 / 202 PCT

[0248] 4921 aatcacctct ggcctcaggc gggaggggaa ctaacaccca cccacccctg ccctccctgc

[0249] 4981 aaatgggaac atcaaggttc ccagtgctta actgagggac aagtgacaat ttagcagaga

[0250] 5041 ggcaagattt gaatccagac tgtcttccag actcaggacc taccttaaaa taatatctga

[0251] 5101 gttgcttatg gaggcagacc tgcctgcaaa gcccagcact cagcaagtgc tcaataaata

[0252] 5161 tttgatttga attctt

[0253] Which encodes:

[0254] 1 mptpllplll rlllsclllp aarlarqyll pllrrlarrl gsqdmreall gcllfilsqr 61 hspdageasr vdrlerrerl gpqk ( SEQ ID NO : 1 )

[0255] MOUSE SEQUENCES protein myomixer isoform 1 [Mus musculus]: Accession No. NP_001170939.1 of the GENBANK® biosequence database, encoded by Accession No. CCDS50119.1 of the GENBANK® biosequence database (identical to SEQ ID NO: 3). protein myomixer isoform 2 [Mus musculus]: Accession No. NP_001170941.1 of the GENBANK® biosequence database, encoded by Accession No. CCDS50120.1 of the GENBANK® biosequence database (identical to SEQ ID NO: 3).

[0256] All references listed above to database entries (including but not limited to UniProt, EMBL, and GENBANK® biosequence database entries and including all annotations available therein) are incorporated herein by reference in their entireties.

[0257] By “fusion-competent” in some embodiments, it is meant a sperm capable of fusion with an egg and / or an egg capable of fusion with a sperm. In some embodiments, “fusion- competent” further includes the subsequent fertilization of the egg by the sperm.

[0258] Representative sequences for GST-BAI1-TSR (Park et al. 2007. Nature 450:430- 434):

[0259] GST (SEQ ID NO:9):

[0260] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGL EFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGV SRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALD VVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGD HPPK

[0261] BAI1-TSR (residues 202-585 of Accession No. XP_006520332 of the GENBANK® biosequence database) (SEQ ID NO: 10)

[0262] SSHPCGIMQTPCACLGGDVGDPASSPLVPRGDVCLRDGVAGGPENCLTSLT QDRGGHGSAGGWKLWSLWGECTRDCGGGLQTRTRTCLPTLGVEGGGCEGVLEE Attorney Docket No. 3062 / 202 PCT

[0263] GRLCNRKACGPTGRSSSRSQSLRSTDARRREEFGDELQQFGFPSPQTGDPAAEEWS PWSVCSSTCGEGWQTRTRFCVSSSYSTQCSGPLREQRLCNNSAVCPVHGAWDEW SPWSLCSSTCGRGFRDRTRTCRPPQFGGNPCEGPEKQTKFCNIALCPGRAVDGNW NEWSSWSTCSASCSQGRQQRTRECNGPSYGGAECQGHWVETRDCFLQQCPVDG KWQAWASWGSCSVTCGGGSQRRERVCSGPFFGGAACQGPQDEYRQCGAQRCPE PHEICDEDN

[0264] In some embodiments, the presently disclosed subject matter provides derivatives of GST-BAI1-TSR that comprise isolated and purified peptides that comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 and 10, a fragment thereof, a peptide having an amino acid sequence that is at least about 60% percent identity, in some embodiments at least about 70% percent identity, in some embodiments at least about 75% percent identity, in some embodiments at least about 80% percent identity, in some embodiments at least about 85% percent identity, in some embodiments at least about 90% percent identity, in some embodiments at least about 92% percent identity, in some embodiments at least about 94% percent identity, in some embodiments at least about 95% percent identity, in some embodiments at least about 96% percent identity, in some embodiments at least about 97% percent identity, in some embodiments at least about 98% percent identity, in some embodiments at least about 99% percent identity, or in some embodiments at least about 100% percent identity thereto, as defined herein above and fragments and / or derivatives thereof are also encompassed by the term “GST-BAI1-TSR”, as are substantially homologous amino acid sequences of any of the foregoing sequences and / or a derivative of any of the foregoing sequences. In some embodiments, the amino acid sequence comprises at least one modification selected from the group consisting of an amino acid deletion, an amino acid addition, an amino acid substitution, and combinations thereof. See also, PCT International Publication No. WO 2020 / 167789, published on August 20, 2020, incorporated herein by reference in its entirety.

[0265] III.A. METHODS OF AND KITS FOR SELECTING FUSION-COMPETENT GAMETES

[0266] In some embodiments, a method of selecting fusion-competent gametes is provided. In some embodiments, the method comprises: (a) providing a sample comprising gametes obtained from a subject; (b) contacting the sample with a reagent capable of binding Myomerger expressed on the surface of the gametes; and (c) isolating gametes bound by the reagent capable of binding Myomerger from gametes not bound by the reagent capable of Attorney Docket No. 3062 / 202 PCT binding Myomerger. In some embodiments, the gametes are sperm cells or oocytes. In some embodiments, the reagent capable of binding Myomerger comprises an anti-Myomerger antibody or a fragment thereof and / or a derivative thereof, or a peptide comprising a Myomerger ectodomain or a fragment thereof and / or a derivative thereof. In some embodiments, the contacting comprising mixing.In some embodiments, the reagent that is capable of binding Myomerger is labeled for detection (such as fluorescence, biotinylation, etc.).

[0267] In some embodiments, the presently disclosed subject matter provides a kit for selecting fusion-competent gametes in a sample. In some embodiments, the kit comprises a reagent capable of binding Myomerger expressed on the surface of the gametes; and instructional material for selecting fusion-competent gametes in a sample. In some embodiments, the gametes are sperm cells or oocytes. In some embodiments, the reagent capable of binding Myomerger comprises an anti-Myomerger antibody or a fragment thereof and / or a derivative thereof, or a peptide comprising a Myomerger ectodomain or a fragment thereof and / or a derivative thereof. In some embodiments, the kit includes a detectable label. In some embodiments, the reagent that is capable of binding Myomerger is labeled for detection (such as fluorescence, biotinylation, etc.).

[0268] Representative antibodies against Myomerger are disclosed in the Examples presented herein below. Additional representative antibodies are commercially available and can be identified and obtaining using a reference website such as biocompare.com. Particular nonlimiting examples of antibodies including the following: Product No. E3508-35 (USBiological Life Sciences; sheep antibody); NBP3-17169-100ul (Novus Biologicals; human antibody); AF4580 (R&D Systems; human, mouse, transgenic mouse) ab214934 (Abeam; mouse); and PA5-140314 (biocompare; human). Additional antibodies and fragments thereof and / or derivatives thereof can be prepared based on the Myomerger sequence data disclosed herein, using techniques disclosed herein. The Myomerger ectodomain, and representative fragments and / or derivatives thereof are disclosed herein, and techniques for modifying polypeptides and peptides are also disclosed herein.

[0269] In some embodiments, the gametes comprise sperm cells. In this case, the method can further comprise contacting the sample with a reagent capable of binding phosphatidylserine on the surface of the sperm cells and isolating sperm cells bound by the reagent capable of binding phosphatidylserine. The kit can further comprise such a reagent. In some embodiments, the contacting comprising mixing. In some embodiments, the reagent capable of binding phosphatidylserine comprises Annexin V or GST-BAI1-TSR. In some embodiments, the reagent that is capable of binding phosphatidylserine is chosen from the Attorney Docket No. 3062 / 202 PCT group consisting of Annexin V, GST-BAI1-TSR, an anti-phosphatidylserine antibody, and fragments and / or derivatives thereof. In some embodiments, the reagent that is capable of binding phosphatidylserine is labeled for detection (such as fluorescence, biotinylation, etc.). In some embodiments, a kit of the presently disclosed subject matter comprises a reagent capable of binding phosphatidylserine. In some embodiments, the reagent that is capable of binding phosphatidylserine is labeled for detection (such as fluorescence, biotinylation, etc.). In some embodiments, the reagents capable of binding Myomerger and capable of binding phosphatidylserine are provided in a single composition.

[0270] In some embodiments, the sample is formed by concentrating a semen sample from the subject. Approaches, reagents, and / or devices for concentrating a semen sample are disclosed elsewhere herein and include, but are not limited to, enriching sperm in the sample using an isolation gradient. Indeed, any suitable approach, reagent, and / or devices for concentrating sperm in a sample as would be apparent to one of ordinary skill in the art upon a review of the instant disclosure is provided in accordance with the presently disclosed subject matter. The sperm swim-up technique is another method for enriching sperm that is recognized in the art. Briefly, the sperm swim-up technique involves liquefaction of a semen sample followed by centrifugation of the semen sample and discarding of the supernatant. The pellet is suspended in a pre-warmed medium, such as Ham's F-10 culture medium, and then centrifuged again. Medium is gently over-layered on the resulting pellet in the tube, which is sealed. The tube is inclined at 45 degrees and kept at 37°C for 60-90 minutes under carbon dioxide, such as 5% CO2. A supernatant containing actively motile sperms is removed by sterile techniques, such as via a sterile Pasteur pipette. See Jameel, T., J Pak Med Assoc. 2008 Feb;58(2):71-4; Volpes et al., J Assist Reprod Genet. 2016 Jun; 33(6): 765-770. In some embodiments, the kit comprises a reagent and / or device for concentrating sperm cells in a sample.

[0271] In some embodiments, isolating gametes bound by the reagent capable of binding Myomerger from gametes not bound by the reagent capable of binding Myomerger and / or isolating sperm cells bound by the reagent capable of binding phosphatidylserine comprises employing flow cytometry (FCM) or fluorescence-activated cell sorting (FACS). Any suitable FCM or FACS technique as would be apparent to one of ordinary skill in the art upon a review of the instant disclosure is provided in accordance with the presently disclosed subject matter. By way of example and not limitation, sperm cells can be labeled with Annexin V as described herein and FACS performed.

[0272] In some embodiments, the reagent capable of binding Myomerger expressed on the surface of the gametes and / or the reagent capable of binding phosphatidylserine further Attorney Docket No. 3062 / 202 PCT comprises a capture moiety and isolating the gametes comprises isolating the capture moiety. In some embodiments, the capture moiety comprises a substrate. The reagent binding can be reversible. Thus, in some embodiments, the reagent capable of binding Myomerger expressed on the surface of the gametes and / or the reagent capable of binding phosphatidylserine is / are bound to the substrate. In some embodiments, the substrate comprises a microbead or a nanobead. In some embodiments, the method further comprises eluting the gametes from the reagent capable of binding Myomerger expressed on the surface of the gametes and / or the reagent capable of binding phosphatidylserine. By way of example and not limitation, a gamete -microbeads suspension is loaded on a column containing a coated cell-friendly matrix containing iron balls, which was fitted in a magnet (MiniMACS; Miltenyi Biotec). The power of magnetic field is measured as about 0.5 Tesla between the poles and up to about 1.5 Tesla within the iron globes of the column. In embodiments where the gametes are sperm cells, the fraction containing the phosphatidylserine + sperm (due to binding to Annexin V beads) is retained in the column, and the then sperm will be eluted with a Ca++free buffer. As the binding of Annexin V to phosphatidylserine is Ca++dependent, the Ca++free buffer releases the phosphatidylserine+ sperm from the Annexin microbeads.

[0273] In some embodiments, the kit comprises one or more reagents for carrying out flow cytometry (FCM) or fluorescence-activated cell sorting (FACS). In some embodiments, the reagent capable of binding Myomerger expressed on the surface of the gametes and / or the reagent capable of binding phosphatidylserine further comprises a capture moiety and isolating the gametes comprises isolating the capture moiety. In some embodiments, the capture moiety comprises a substrate. The reagent binding can be reversible. Thus, in some embodiments, the reagent capable of binding Myomerger expressed on the surface of the gametes and / or the reagent capable of binding phosphatidylserine is / are bound to the substrate. In some embodiments, the substrate comprises a microbead or a nanobead. In some embodiments, the kit further comprises a reagent and an apparatus (e.g., buffer and column) for eluting gametes from the reagent capable of binding Myomerger expressed on the surface of the gametes and / or the reagent capable of binding phosphatidylserine.

[0274] In some embodiments, the subject is a human subject or a non-human animal subject. In some embodiments, the non-human animal subject is selected from the group consisting of bovine, equine, porcine, ovine, canine, feline, and avian.

[0275] In some embodiments, the method further comprises using the results of the gamete analysis to determine an assisted reproductive technology (ART) treatment pathway (e.g., intra-uterine insemination (IUI), in vitro fertilization (IVF), or intra-cytoplasmic sperm injection (ICSI)) for a subject. In some embodiments, the instruction material includes Attorney Docket No. 3062 / 202 PCT instructions for determining an assisted reproductive technology (ART) treatment pathway (e.g., intra-uterine insemination (IUI), in vitro fertilization (IVF), or intra-cytoplasmic sperm injection (ICSI)) for a subject. Thus, the presently disclosed subject matter provides tools to assess fusion competency to improve the quality of information that millions of couples currently lack when determining their ART treatments. In some embodiments, the method further comprises discussing and / or suggesting additional health care interventions to a subject.

[0276] III.B. METHODS OF AND KITS FOR IDENTIFYING FUSION-COMPETENT GAMETES

[0277] In some embodiments, a method of identifying fusion-competent gametes is provided. In some embodiments, the method comprises: (a) providing a sample comprising gametes obtained from a subject; and (b) contacting the sample with a reagent capable of binding Myomerger expressed on the surface of the gametes; wherein gametes that express Myomerger on their surface are fusion-competent. In some embodiments, the gametes are sperm cells or oocytes. In some embodiments, the reagent capable of binding Myomerger comprises an anti-Myomerger antibody or a fragment thereof and / or a derivative thereof, or a peptide comprising a Myomerger ectodomain or a fragment thereof and / or a derivative thereof. In some embodiments, the contacting comprising mixing. In some embodiments, the reagent that is capable of binding Myomerger is labeled for detection (such as fluorescence, biotinylation, etc.).

[0278] In some embodiments, the presently disclosed subject matter provides a kit for identifying fusion-competent gametes in a sample. In some embodiments, the kit comprises a reagent capable of binding Myomerger expressed on the surface of the gametes; and instructional material for selecting fusion-competent gametes in a sample. In some embodiments, the gametes are sperm cells or oocytes. In some embodiments, the reagent capable of binding Myomerger comprises an anti-Myomerger antibody or a fragment thereof and / or a derivative thereof, or a peptide comprising a Myomerger ectodomain or a fragment thereof and / or a derivative thereof. In some embodiments, the kit includes a detectable label. In some embodiments, the reagent that is capable of binding Myomerger is labeled for detection (such as fluorescence, biotinylation, etc.).

[0279] Representative antibodies against Myomerger are disclosed in the Examples presented herein below. Additional representative antibodies are commercially available and can be identified and obtaining using a reference website such as biocompare.com. Particular nonlimiting examples of antibodies including the following: Product No. E3508-35 (USBiological Life Sciences; sheep antibody); NBP3-17169-100ul (Novus Biologicals; Attorney Docket No. 3062 / 202 PCT human antibody); AF4580 (R&D Systems; human, mouse, transgenic mouse) ab214934 (Abeam; mouse); and PA5-140314 (biocompare; human). Additional antibodies and fragments thereof can be prepared based on the Myomerger sequence data disclosed herein, using techniques disclosed herein. The Myomerger ectodomain, and representative fragments thereof and / or derivatives thereof are disclosed herein above, and techniques for modifying polypeptides and peptides are also disclosed herein.

[0280] In some embodiments, the gametes comprise sperm cells. In this case, the method can further comprise contacting the sample with a reagent capable of binding phosphatidylserine on the surface of the sperm cells and isolating sperm cells bound by the reagent capable of binding phosphatidylserine. The kit can further comprise such a reagent. In some embodiments, the contacting comprising mixing. In some embodiments, the reagent capable of binding phosphatidylserine comprises Annexin V or GST-BAI1-TSR. In some embodiments, the reagent that is capable of binding phosphatidylserine is chosen from the group consisting of Annexin V, GST-BAI1-TSR, an anti-phosphatidylserine antibody, and fragments and / or derivatives thereof. In some embodiments, the reagent that is capable of binding phosphatidylserine is labeled for detection (such as fluorescence, biotinylation, etc.). In some embodiments, a kit of the presently disclosed subject matter comprises a reagent capable of binding phosphatidylserine. In some embodiments, the reagent that is capable of binding phosphatidylserine is labeled for detection (such as fluorescence, biotinylation, etc.). In some embodiments, the reagents capable of binding Myomerger and capable of binding phosphatidylserine are provided in a single composition.

[0281] In some embodiments, the sample is formed by concentrating a semen sample from the subject. Approaches, reagents, and / or devices for concentrating a semen sample are disclosed elsewhere herein and include, but are not limited to, enriching sperm in the sample using an isolation gradient. Indeed, any suitable approach, reagent, and / or devices for concentrating sperm in a sample as would be apparent to one of ordinary skill in the art upon a review of the instant disclosure is provided in accordance with the presently disclosed subject matter. The sperm swim-up technique is another method for enriching sperm that is recognized in the art. Briefly, the sperm swim-up technique involves liquefaction of a semen sample followed by centrifugation of the semen sample and discarding of the supernatant. The pellet is suspended in a pre-warmed medium, such as Ham's F-10 culture medium, and then centrifuged again. Medium is gently over-layered on the resulting pellet in the tube, which is sealed. The tube is inclined at 45 degrees and kept at 37°C for 60-90 minutes under carbon dioxide, such as 5% CO2. A supernatant containing actively motile sperms is removed by sterile techniques, such as via a sterile Pasteur pipette. See Jameel, T., J Pak Med Assoc. Attorney Docket No. 3062 / 202 PCT

[0282] 2008 Feb;58(2):71-4; Volpes et al., J Assist Reprod Genet. 2016 Jun; 33(6): 765-770. In some embodiments, the kit comprises a reagent and / or device for concentrating sperm cells in a sample.

[0283] In some embodiments, isolating gametes bound by the reagent capable of binding Myomerger from gametes not bound by the reagent capable of binding Myomerger and / or isolating sperm cells bound by the reagent capable of binding phosphatidylserine comprises employing flow cytometry (FCM) or fluorescence-activated cell sorting (FACS). Any suitable FCM or FACS technique as would be apparent to one of ordinary skill in the art upon a review of the instant disclosure is provided in accordance with the presently disclosed subject matter. By way of example and not limitation, sperm cells can be labeled with Annexin V as described herein and FACS performed.

[0284] In some embodiments, the reagent capable of binding Myomerger expressed on the surface of the gametes and / or the reagent capable of binding phosphatidylserine further comprises a capture moiety and isolating the gametes comprises isolating the capture moiety. In some embodiments, the capture moiety comprises a substrate. The reagent binding can be reversible. Thus, in some embodiments, the reagent capable of binding Myomerger expressed on the surface of the gametes and / or the reagent capable of binding phosphatidylserine is / are bound to the substrate. In some embodiments, the substrate comprises a microbead or a nanobead. In some embodiments, the method further comprises eluting the gametes from the reagent capable of binding Myomerger expressed on the surface of the gametes and / or the reagent capable of binding phosphatidylserine. By way of example and not limitation, a gamete -microbeads suspension is loaded on a column containing a coated cell-friendly matrix containing iron balls, which was fitted in a magnet (MiniMACS; Miltenyi Biotec). The power of magnetic field is measured as about 0.5 Tesla between the poles and up to about 1.5 Tesla within the iron globes of the column. In embodiments where the gametes are sperm cells, the fraction containing the phosphatidylserine + sperm (due to binding to Annexin V beads) is retained in the column, and the then sperm will be eluted with a Ca++free buffer. As the binding of Annexin V to phosphatidylserine is Ca++dependent, the Ca++free buffer releases the phosphatidylserine+ sperm from the Annexin microbeads.

[0285] In some embodiments, the kit comprises one or more reagents for carrying out flow cytometry (FCM) or fluorescence-activated cell sorting (FACS). In some embodiments, the reagent capable of binding Myomerger expressed on the surface of the gametes and / or the reagent capable of binding phosphatidylserine further comprises a capture moiety and isolating the gametes comprises isolating the capture moiety. In some embodiments, the capture moiety comprises a substrate. The reagent binding can be reversible. Thus, in some Attorney Docket No. 3062 / 202 PCT embodiments, the reagent capable of binding Myomerger expressed on the surface of the gametes and / or the reagent capable of binding phosphatidylserine is / are bound to the substrate. In some embodiments, the substrate comprises a microbead or a nanobead. In some embodiments, the kit further comprises a reagent and an apparatus (e.g., buffer and column) for eluting gametes from the reagent capable of binding Myomerger expressed on the surface of the gametes and / or the reagent capable of binding phosphatidylserine.

[0286] In some embodiments, the subject is a human subject or a non-human animal subject. In some embodiments, the non-human animal subject is selected from the group consisting of bovine, equine, porcine, ovine, canine, feline, and avian.

[0287] In some embodiments, the method further comprises using the results of the gamete analysis to determine an assisted reproductive technology (ART) treatment pathway (e.g., intra-uterine insemination (IUI), in vitro fertilization (IVF), or intra-cytoplasmic sperm injection (ICSI)) for a subject. In some embodiments, the instruction material includes instructions for determining an assisted reproductive technology (ART) treatment pathway (e.g., intra-uterine insemination (IUI), in vitro fertilization (IVF), or intra-cytoplasmic sperm injection (ICSI)) for a subject. Thus, the presently disclosed subject matter provides tools to assess fusion competency to improve the quality of information that millions of couples currently lack when determining their ART treatments. In some embodiments, the method further comprises discussing and / or suggesting additional health care interventions to a subject.

[0288] III.C. CONTRACEPTIVE METHODS, COMPOSITION, AND DEVICES

[0289] In some embodiments, a method of contraception and / or a composition for contraception and / or a device for contraception is / are provided. In some embodiments, the method comprises administering a reagent capable of binding Myomerger expressed on the surface of gametes to a subject. In some embodiments, the reagent capable of binding Myomerger expressed on the surface of gametes is administered in an effective amount. In some embodiments, the subject is a human subject or a non-human animal subject. In some embodiments, the non-human animal subject is selected from the group consisting of bovine, equine, porcine, ovine, canine, feline, and avian. In some embodiments, the method further comprises administering a reagent capable of binding phosphatidylserine on the surface of the sperm cells in an effective amount. Thus, one or more compositions comprising the reagent or reagents for use in contraception are provided. In some embodiments, the composition or compositions and / or kit comprising the composition can further comprise a pharmaceutically acceptable carrier so that the composition or compositions can be for use in a medical or Attorney Docket No. 3062 / 202 PCT veterinary application. In some embodiments, the administering is topically to a subject at a time of sexual intercourse.

[0290] In some embodiments, the reagent capable of binding Myomerger comprises an antiMyomerger antibody or a fragment thereof and / or a derivative thereof, or a peptide comprising a Myomerger ectodomain or a fragment thereof and / or a derivative thereof. Representative antibodies against Myomerger are disclosed in the Examples presented herein below. Additional representative antibodies are commercially available and can be identified and obtaining using a reference website such as biocompare.com. Particular non-limiting examples of antibodies including the following: Product No. E3508-35 (USBiological Life Sciences; sheep antibody); NBP3-17169-100ul (Novus Biologicals; human antibody); AF4580 (R&D Systems; human, mouse, transgenic mouse) ab214934 (Abeam; mouse); and PAS- 140314 (biocompare; human). Additional antibodies and fragments thereof and / or derivatives thereof can be prepared based on the Myomerger sequence data disclosed herein, using techniques disclosed herein. The Myomerger ectodomain, and representative fragments thereof and / or derivatives thereof are disclosed herein, and techniques for modifying polypeptides and peptides are also disclosed herein. In some embodiments, the reagent that is capable of binding phosphatidylserine is chosen from the group consisting of Annexin V, GST-BAI1-TSR, an anti-phosphatidylserine antibody, and fragments and derivatives thereof.

[0291] In some embodiments, the method comprises administering a composition comprising the reagent or reagents topically to a subject at a time of sexual intercourse. Thus, one or more compositions comprising the reagent or reagents are provided. In some embodiments, the reagents are provided in a single composition. In some embodiments, the administering comprising applying the composition or compositions comprising the reagent or reagents to a contraceptive device. In some embodiments, either or both reagent(s) comprise(s) a modified bead, such as a carboxylate modified bead.

[0292] In some embodiments, the presently disclosed subject matter provides a contraceptive device comprising a coating or lubricant comprising a reagent capable of binding Myomerger expressed on the surface of gametes. In some embodiments, the contraceptive device comprises a coating or lubricant comprising a reagent capable of binding phosphatidylserine on the surface of the sperm cells in an effective amount. In some embodiments, the coating or lubricant comprises both reagents. Representative contraceptive devices include but are not limited to a diaphragm, male condom, female condom, or other device as would be apparent to one of ordinary skill in the art upon a review of the instant disclosure. In some embodiments, either or both reagent(s) comprise(s) a modified bead, such as a carboxylate modified bead. Attorney Docket No. 3062 / 202 PCT

[0293] Thus, the disclosed reagents can be employed by administration to a subject in need thereof. In some embodiments, the disclosed reagents can be prepared as pharmaceutical compositions can be administered in vivo in a pharmaceutically acceptable carrier. By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject, along with a reagent of the presently disclosed subject matter, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. The materials can be in solution and / or in suspension. Suitable carriers and their formulations are described in Remington et al. (1975) Remington's Pharmaceutical Sciences, 15th ed., Mack Pub. Co., Easton, Pennsylvania, United States of America. It will be apparent to those persons skilled in the art that certain carriers can be selected depending upon, for instance, the route of administration and / or concentration of composition being administered.

[0294] Pharmaceutical compositions can include carriers, thickeners, diluents, buffers, preservatives, surface active agents, and the like, in addition to the molecule of choice. Pharmaceutical compositions can also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, and the like.

[0295] III.D. COMPOSITIONS COMPRISING REAGENT CAPABLE OF BINDING MYOMERGER AND A REAGENT CAPABLE OF BINDING PHOSPHATIDYLSERINE

[0296] In some embodiments, provided is a composition comprising a reagent capable of binding Myomerger and a reagent capable of binding phosphatidylserine. Such a composition can be used in any of the methods disclosed herein. In some embodiments, the reagent capable of binding Myomerger comprises an anti-Myomerger antibody or a fragment thereof and / or a derivative thereof, or a peptide comprising a Myomerger ectodomain or a fragment thereof and / or a derivative thereof; and / or wherein the reagent capable of binding phosphatidylserine comprises Annexin V or GST-BAH-TSR. In some embodiments, the reagent that selectively binds phosphatidylserine is chosen from the group consisting of Annexin V, GST-BAH-TSR, an anti-phosphatidylserine antibody, and fragments and / or derivatives thereof. In some embodiments, the reagent capable of binding Myomerger and / or the reagent that selectively binds phosphatidylserine is labeled for detection (such as fluorescence, biotinylation, etc.). Attorney Docket No. 3062 / 202 PCT

[0297] In some embodiments, a kit comprising a composition comprising a reagent capable of binding Myomerger and a reagent capable of binding phosphatidylserine; and instructions for using the composition, is provided. In some embodiments, a kit comprising a reagent capable of binding Myomerger and a reagent capable of binding phosphatidylserine is provided. In some embodiments, the kit comprises additional reagents and instructions for making and using a composition comprising a reagent capable of binding Myomerger and a reagent capable of binding phosphatidylserine. In some embodiments, the composition and / or kit can further comprise a pharmaceutically acceptable carrier so that the composition can be for use in a medical or veterinary application, including those disclosed elsewhere herein. In some embodiments, the kit comprises a detectable label, and additional reagents and / or instructions for modifying or detecting the composition comprising a reagent capable of binding Myomerger and a reagent capable of binding phosphatidylserine.

[0298] Representative antibodies against Myomerger are disclosed in the Examples presented herein below. Additional representative antibodies are commercially available and can be identified and obtaining using a reference website such as biocompare.com. Particular nonlimiting examples of antibodies including the following: Product No. E3508-35 (USBiological Life Sciences; sheep antibody); NBP3-17169-100ul (Novus Biologicals; human antibody); AF4580 (R&D Systems; human, mouse, transgenic mouse) ab214934 (Abeam; mouse); and PA5-140314 (biocompare; human). Additional antibodies and fragments thereof and / or derivatives thereof can be prepared based on the Myomerger sequence data disclosed herein, using techniques disclosed herein. The Myomerger ectodomain, and representative fragments thereof and / or derivatives thereof are disclosed herein, and techniques for modifying polypeptides and peptides are also disclosed herein.

[0299] III.E. ANTIBODY FORMATS AND PREPARATION THEREOF

[0300] Antibodies directed against proteins, polypeptides, or peptide fragments thereof of the presently disclosed subject matter may be generated using methods that are well known in the art. For instance, U.S. Patent No. 5,436,157, which is incorporated by reference herein in its entirety, discloses methods of raising antibodies to peptides. For the production of antibodies, various host animals, including but not limited to rabbits, mice, and rats, can be immunized by injection with a polypeptide or peptide fragment thereof. To increase the immunological response, various adjuvants may be used depending on the host species, including but not limited to Freund’s (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Attorney Docket No. 3062 / 202 PCT

[0301] In some embodiments, one or more antibodies or fragments thereof are used. In some embodiments, one or more antibodies are single chain, monoclonal, bi-specific, synthetic, polyclonal, chimeric, human, or humanized, or active fragments or homologs thereof. In some embodiments, the antibody binding fragment is scFV, F(ab’)2, F(ab)2, Fab’, or Fab.

[0302] For the preparation of monoclonal antibodies, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be utilized. For example, the hybridoma technique originally developed by Kohler & Milstein, the trioma technique, the human B-cell hybridoma technique (Kozbor & Roder, 1983), and the EBV- hybridoma technique (Cole et al., 1985) may be employed to produce human monoclonal antibodies. In some embodiments, monoclonal antibodies are produced in germ-free animals.

[0303] In accordance with the presently disclosed subject matter, human antibodies may be used and obtained by utilizing human hybridomas (Cote et al., 1983) or by transforming human B cells with EBV virus in vitro (Cole et al., 1985). Furthermore, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984; Neuberger et al., 1984; Takeda et al., 1985) by splicing the genes from a mouse antibody molecule specific for epitopes of SLLP polypeptides together with genes from a human antibody molecule of appropriate biological activity can be employed; such antibodies are within the scope of the presently disclosed subject matter. Once specific monoclonal antibodies have been developed, the preparation of mutants and variants thereof by conventional techniques is also available.

[0304] Various techniques have been developed for the production of antibody fragments of humanized antibodies. Traditionally, these fragments were derived via proteolytic digestion of full-length antibodies (see e.g., Morimoto & Inouye, 1992; Brennan et al., 1985). However, these fragments can now be produced directly by recombinant host cells. Alternatively, Fab’- SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab’)2 fragments (Carter et al., 1992a). According to another approach, F(ab’)2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv). See PCT International Patent Application Publication No. WO 1993 / 16185; U.S. Patent Nos. 5,571,894; 5,587,458. The antibody fragment may also be a “linear antibody”, e.g., as described in U.S. Patent No. 5,641,870, for example. Such linear antibody fragments may be monospecific or bispecific.

[0305] Humanized (chimeric) antibodies are immunoglobulin molecules comprising a human and non-human portion. More specifically, the antigen combining region (or variable region) of a humanized chimeric antibody is derived from a non-human source (e.g., murine) and the constant region of the chimeric antibody (which confers biological effector function to the Attorney Docket No. 3062 / 202 PCT immunoglobulin) is derived from a human source. The humanized chimeric antibody should have the antigen binding specificity of the non-human antibody molecule and the effector function conferred by the human antibody molecule. A large number of methods of generating chimeric antibodies are well known to those of skill in the art (see e.g., U.S. Patent Nos. 4,975,369; 5,075,431; 5,081,235; 5,169,939; 5,202,238; 5,204,244; 5,231,026; 5,292,867; 5,354,847; 5,472,693; 5,482,856; 5,491,088; 5,500,362; and 5,502,167). Detailed methods for preparation of chimeric (humanized) antibodies can be found in U.S. Patent No. 5,482,856. A “humanized” antibody is a human / non-human chimeric antibody that contains a minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR residues are those of a human immunoglobulin sequence. The humanized antibody can optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see e.g., Jones et al., 1986; Riechmann et al., 1988; Presta, 1992, PCT International Patent Application Publication No. WO 92 / 02190, U.S. Patent Application Publication No. 2006 / 0073137, and U.S. Patent Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,761; 5,693,762; 5,714,350; 5,766,886; 5,770,196; 5,777,085; 5,821,123; 5,821,337; 5,869,619; 5,877,293; 5,886,152; 5,895,205; 5,929,212; 6,054,297; 6,180,370; 6,407,213; 6,548,640; 6,632,927; 6,639,055; and 6,750,325.

[0306] In some embodiments, this presently disclosed subject matter provides for fully human antibodies. Human antibodies consist entirely of characteristically human polypeptide sequences. The human antibodies of this presently disclosed subject matter can be produced in using a wide variety of methods (see e.g., U.S. Patent No. 5,001,065, for review).

[0307] Typically, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers Attorney Docket No. 3062 / 202 PCT

[0308] (Jones et al, 1986; Riechmann et al., 1988); Verhoeyen et al., 1988), by substituting hypervariable region sequences for the corresponding sequences of a human “acceptor” antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (see e.g., U.S. Patent Nos. 4,816,567 and 5,482,856) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0309] Another method for making humanized antibodies is described in U.S. Patent Application Publication No. 2003 / 0017534, wherein humanized antibodies and antibody preparations are produced from transgenic non-human animals. The non-human animals are genetically engineered to contain one or more humanized immunoglobulin loci that are capable of undergoing gene rearrangement and gene conversion in the transgenic non-human animals to produce diversified humanized immunoglobulins.

[0310] In some embodiments, the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against a library of known human variable-domain sequences or a library of human germline sequences. The human sequence that is closest to that of the rodent can then be accepted as the human framework region for the humanized antibody (Sims et al., 1993; Chothia & Lesk, 1987). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., 1992b; Presta et al., 1993). Other methods designed to reduce the immunogenicity of the antibody molecule in a human patient include veneered antibodies (see e.g., U.S. Patent No. 6,797,492 and U.S. Patent Application Publication Nos. 2002 / 0034765 and 2004 / 0253645) and antibodies that have been modified by T-cell epitope analysis and removal (see e.g., U.S. Patent Application Publication No. 2003 / 0153043 and U.S. Patent No. 5,712,120).

[0311] It is important that when antibodies are humanized they retain high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that Attorney Docket No. 3062 / 202 PCT illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0312] The hybrid antibodies and hybrid antibody fragments include complete antibody molecules having full length heavy and light chains, or any fragment thereof, such as Fab, Fab’, F(ab’)2, Fd, scFv, antibody light chains and antibody heavy chains. Chimeric antibodies which have variable regions as described herein and constant regions from various species are also suitable. See for example, U.S. Patent Application No. 2003 / 0022244.

[0313] Fragments within the scope of the term “antibody” include those produced by digestion with various proteases, those produced by chemical cleavage and / or chemical dissociation and those produced recombinantly, so long as the fragment remains capable of specific binding to a target molecule. Among such fragments are Fab, Fab’, Fv, F(ab’)2, and single chain Fv (scFv) fragments.

[0314] In some embodiments, the specific binding molecule is a single-chain variable analogue (scFv). The specific binding molecule or scFv may be linked to other specific binding molecules (for example other scFvs, Fab antibody fragments, chimeric IgG antibodies (e.g., with human frameworks)) or linked to other scFvs of the presently disclosed subject matter so as to form a multimer which is a multi-specific binding protein, for example a dimer, a trimer, or a tetramer. Bi-specific scFvs are sometimes referred to as diabodies, tri-specific such as triabodies and tetra-specific such as tetrabodies when each scFv in the dimer, trimer, or tetramer has a different specificity. Diabodies, triabodies and tetrabodies can also be monospecific, when each scFv in the dimer, trimer, or tetramer has the same specificity.

[0315] In some embodiments, techniques described for the production of single-chain antibodies (U.S. Patent No. 4,946,778, incorporated by reference herein in its entirety) are adapted to produce protein-specific single-chain antibodies. In some embodiments, the techniques described for the construction of Fab expression libraries (Huse et al., 1989) are utilized to allow rapid and easy identification of monoclonal Fab fragments possessing the desired specificity for specific antigens, proteins, derivatives, or analogs of the presently disclosed subject matter. Attorney Docket No. 3062 / 202 PCT

[0316] Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab’)2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab’ fragments which can be generated by reducing the disulfide bridges of the F(ab’)2 fragment; the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent; and Fv fragments.

[0317] The generation of polyclonal antibodies is accomplished by inoculating the desired animal with the antigen and isolating antibodies which bind the antigen therefrom at any epitopes present therein.

[0318] Monoclonal antibodies directed against full length or peptide fragments of a protein or peptide may be prepared using any well known monoclonal antibody preparation procedures, such as those described, for example, in Harlow & Lane, 1988; Tuszynski et al., 1988). Quantities of the desired peptide may also be synthesized using chemical synthesis technology. Alternatively, DNA encoding the desired peptide may be cloned and expressed from an appropriate promoter sequence in cells suitable for the generation of large quantities of peptide. Monoclonal antibodies directed against the peptide are generated from mice immunized with the peptide using standard procedures as referenced herein.

[0319] Exemplary complementarity-determining region (CDR) residues or sequences and / or sites for amino acid substitutions in framework region (FR) of such humanized antibodies having improved properties such as, e.g., lower immunogenicity, improved antigen-binding or other functional properties, and / or improved physicochemical properties such as, e.g., better stability, are provided.

[0320] The presently disclosed subject matter encompasses more than the specific fragments and humanized fragments disclosed herein. In some embodiments, the antibody is selected from the group consisting of a single chain antibody, a monoclonal antibody, a bi-specific antibody, a chimeric antibody, a synthetic antibody, a polyclonal antibody, or a humanized antibody, or active fragments or homologs thereof.

[0321] A nucleic acid encoding the monoclonal antibody obtained using the procedures described herein may be cloned and sequenced using technology which is available in the art, and is described, for example, in Wright et al., 1992) and the references cited therein. Further, the antibody of the presently disclosed subject matter may be “humanized” using the technology described in Wright et al., 1992 and in the references cited therein, and in Gu et al., 1997.

[0322] To generate a phage antibody library, a cDNA library is first obtained from mRNA which is isolated from cells, e.g., the hybridoma, which express the desired protein to be Attorney Docket No. 3062 / 202 PCT expressed on the phage surface, e.g., the desired antibody. cDNA copies of the mRNA are produced using reverse transcriptase. cDNA which specifies immunoglobulin fragments are obtained by PCR and the resulting DNA is cloned into a suitable bacteriophage vector to generate a bacteriophage DNA library comprising DNA specifying immunoglobulin genes. The procedures for making a bacteriophage library comprising heterologous DNA are well known in the art and are described, for example, in Green & Sambrook, 2012.

[0323] Bacteriophage which encode the desired antibody, may be engineered such that the protein is displayed on the surface thereof in such a manner that it is available for binding to its corresponding binding protein, e.g., the antigen against which the antibody is directed. Thus, when bacteriophage which express a specific antibody are incubated in the presence of a cell which expresses the corresponding antigen, the bacteriophage will bind to the cell. Bacteriophage which do not express the antibody will not bind to the cell. Such panning techniques are well known in the art.

[0324] Processes such as those described above, have been developed for the production of human antibodies using M13 bacteriophage display (Burton & Barbas, 1994). Essentially, a cDNA library is generated from mRNA obtained from a population of antibody-producing cells. The mRNA encodes rearranged immunoglobulin genes and thus, the cDNA encodes the same. Amplified cDNA is cloned into M13 expression vectors creating a library of phage which express human Fab fragments on their surface. Phage which display the antibody of interest are selected by antigen binding and are propagated in bacteria to produce soluble human Fab immunoglobulin. Thus, in contrast to conventional monoclonal antibody synthesis, this procedure immortalizes DNA encoding human immunoglobulin rather than cells which express human immunoglobulin.

[0325] In accordance with the presently disclosed subject matter, human antibodies may be used and obtained by utilizing human hybridomas (Cote et al., 1983) or by transforming human B cells with EBV virus in vitro (Cole et al., 1985). Furthermore, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984; Neuberger et al., 1984; Takeda et al., 1985).

[0326] The procedures just presented describe the generation of phage which encode the Fab portion of an antibody molecule. However, the presently disclosed subject matter should not be construed to be limited solely to the generation of phage encoding Fab antibodies. Rather, phage which encode single chain antibodies (scFv / phage antibody libraries) are also included in the presently disclosed subject matter. Fab molecules comprise the entire Ig light chain, that is, they comprise both the variable and constant region of the light chain, but include only the variable region and first constant region domain (CHI) of the heavy chain. Single chain Attorney Docket No. 3062 / 202 PCT antibody molecules comprise a single chain of protein comprising the Ig Fv fragment. An Ig Fv fragment includes only the variable regions of the heavy and light chains of the antibody, having no constant region contained therein. Phage libraries comprising scFv DNA may be generated following the procedures described in Marks et al., 1991. Panning of phage so generated for the isolation of a desired antibody is conducted in a manner similar to that described for phage libraries comprising Fab DNA.

[0327] The presently disclosed subject matter should also be construed to include synthetic phage display libraries in which the heavy and light chain variable regions may be synthesized such that they include nearly all possible specificities (Barbas, 1995, ; de Kruif et al., 1995).

[0328] In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g., ELISA (enzyme-linked immunosorbent assay). Antibodies generated in accordance with the presently disclosed subject matter may include, but are not limited to, polyclonal, monoclonal, chimeric (i.e., “humanized”), and single chain (recombinant) antibodies, Fab fragments, and fragments produced by a Fab expression library.

[0329] III.F. PEPTIDE MODIFICATION AND PREPARATION

[0330] Peptide preparation is described in the Examples. It will be appreciated, of course, that the proteins or peptides of the presently disclosed subject matter may incorporate amino acid residues which are modified without affecting activity. For example, the termini may be derivatized to include blocking groups, i.e. chemical substituents suitable to protect and / or stabilize the N- and C-termini from “undesirable degradation”, a term meant to encompass any type of enzymatic, chemical or biochemical breakdown of the compound at its termini which is likely to affect the function of the compound, i.e. sequential degradation of the compound at a terminal end thereof.

[0331] Blocking groups include protecting groups conventionally used in the art of peptide chemistry which will not adversely affect the in vivo activities of the peptide. For example, suitable N-terminal blocking groups can be introduced by alkylation or acylation of the N- terminus. Examples of suitable N-terminal blocking groups include C1-C5 branched or unbranched alkyl groups, acyl groups such as formyl and acetyl groups, as well as substituted forms thereof, such as the acetamidomethyl (Acm) group. Desamino analogs of amino acids are also useful N-terminal blocking groups, and can either be coupled to the N-terminus of the peptide or used in place of the N-terminal reside. Suitable C-terminal blocking groups, in which the carboxyl group of the C-terminus is either incorporated or not, include esters, ketones or amides. Ester or ketone-forming alkyl groups, particularly lower alkyl groups such as methyl, ethyl and propyl, and amide-forming amino groups such as primary amines (-NH2), Attorney Docket No. 3062 / 202 PCT and mono- and di-alkylamino groups such as methylamino, ethylamino, dimethylamino, diethylamino, methylethylamino and the like are examples of C-terminal blocking groups. Descarboxylated amino acid analogues such as agmatine are also useful C-terminal blocking groups and can be either coupled to the peptide’s C-terminal residue or used in place of it. Further, it will be appreciated that the free amino and carboxyl groups at the termini can be removed altogether from the peptide to yield desamino and descarboxylated forms thereof without affect on peptide activity.

[0332] Acid addition salts of the presently disclosed subject matter are also contemplated as functional equivalents. Thus, a peptide in accordance with the presently disclosed subject matter treated with an inorganic acid such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like, or an organic acid such as an acetic, propionic, glycolic, pyruvic, oxalic, malic, malonic, succinic, maleic, fumaric, tataric, citric, benzoic, cinnamie, mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salic yc lie and the like, to provide a water soluble salt of the peptide is suitable for use in the presently disclosed subject matter.

[0333] The presently disclosed subject matter also provides for analogs of proteins. Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. For example, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function. To that end, 10 or more conservative amino acid changes typically have no effect on peptide function.

[0334] Modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine .

[0335] Also included are polypeptides which have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or non-standard synthetic amino acids. The peptides of the presently disclosed subject matter are not limited to products of any of the specific exemplary processes listed herein. Attorney Docket No. 3062 / 202 PCT

[0336] The presently disclosed subject matter includes the use of beta-alanine (also referred to as ^-alanine, (3- A I a, bA, and (3 A , having the structure: beta alanine

[0337] Sequences are provided herein which use the symbol “|3A”, but in the Sequence Listing submitted herewith “|3A” is provided as “Xaa” and reference in the text of the Sequence Listing indicates that Xaa is beta alanine.

[0338] It will be appreciated, of course, that the peptides or antibodies, derivatives, or fragments thereof may incorporate amino acid residues which are modified without affecting activity. For example, the termini may be derivatized to include blocking groups, i.e. chemical substituents suitable to protect and / or stabilize the N- and C-termini from “undesirable degradation”, a term meant to encompass any type of enzymatic, chemical or biochemical breakdown of the compound at its termini which is likely to affect the function of the compound, i.e. sequential degradation of the compound at a terminal end thereof.

[0339] Blocking groups include protecting groups conventionally used in the art of peptide chemistry which will not adversely affect the in vivo activities of the peptide. For example, suitable N-terminal blocking groups can be introduced by alkylation or acylation of the N- terminus. Examples of suitable N-terminal blocking groups include C1-C5 branched or unbranched alkyl groups, acyl groups such as formyl and acetyl groups, as well as substituted forms thereof, such as the acetamidomethyl (Acm) group. Desamino analogs of amino acids are also useful N-terminal blocking groups, and can either be coupled to the N-terminus of the peptide or used in place of the N-terminal reside. Suitable C-terminal blocking groups, in which the carboxyl group of the C-terminus is either incorporated or not, include esters, ketones or amides. Ester or ketone-forming alkyl groups, particularly lower alkyl groups such as methyl, ethyl and propyl, and amide-forming amino groups such as primary amines (-NH2), and mono- and di-alkylamino groups such as methylamino, ethylamino, dimethylamino, diethylamino, methylethylamino and the like are examples of C-terminal blocking groups. Descarboxylated amino acid analogues such as agmatine are also useful C-terminal blocking groups and can be either coupled to the peptide’s C-terminal residue or used in place of it. Further, it will be appreciated that the free amino and carboxyl groups at the termini can be removed altogether from the peptide to yield desamino and descarboxylated forms thereof without affect on peptide activity. Attorney Docket No. 3062 / 202 PCT

[0340] Other modifications can also be incorporated without adversely affecting the activity and these include, but are not limited to, substitution of one or more of the amino acids in the natural L-isomeric form with amino acids in the D-isomeric form. Thus, the peptide may include one or more D-amino acid resides, or may comprise amino acids which are all in the D-form. Retro-inverso forms of peptides in accordance with the presently disclosed subject matter are also contemplated, for example, inverted peptides in which all amino acids are substituted with D-amino acid forms.

[0341] Substantially pure protein obtained as described herein may be purified by following known procedures for protein purification, wherein an immunological, enzymatic or other assay is used to monitor purification at each stage in the procedure. Protein purification methods are well known in the art, and are described, for example in Deutscher et al., 1990.

[0342] As discussed, modifications or optimizations of peptides of the presently disclosed subject matter are within the scope of the application. Modified or optimized peptides are included within the definition of peptide binding ligand. Specifically, a peptide sequence identified can be modified to optimize its potency, pharmacokinetic behavior, stability and / or other biological, physical and chemical properties.

[0343] In certain embodiments, the disclosed methods and compositions may involve preparing peptides with one or more substituted amino acid residues.

[0344] In various embodiments, the structural, physical and / or therapeutic characteristics of peptide sequences may be optimized by replacing one or more amino acid residues.

[0345] Other modifications can also be incorporated without adversely affecting the activity and these include, but are not limited to, substitution of one or more of the amino acids in the natural L-isomeric form with amino acids in the D-isomeric form. Thus, the peptide may include one or more D-amino acid resides, or may comprise amino acids which are all in the D-form. Retro-inverso forms of peptides in accordance with the presently disclosed subject matter are also contemplated, for example, inverted peptides in which all amino acids are substituted with D-amino acid forms.

[0346] The skilled artisan will be aware that, in general, amino acid substitutions in a peptide typically involve the replacement of an amino acid with another amino acid of relatively similar properties (i.e., conservative amino acid substitutions). The properties of the various amino acids and effect of amino acid substitution on protein structure and function have been the subject of extensive study and knowledge in the art.

[0347] For example, one can make the following isosteric and / or conservative amino acid changes in the parent polypeptide sequence with the expectation that the resulting polypeptides would have a similar or improved profile of the properties described above: Attorney Docket No. 3062 / 202 PCT

[0348] Substitution of alkyl-substituted hydrophobic amino acids: including alanine, leucine, isoleucine, valine, norleucine, S-2-aminobutyric acid, S -cyclohexylalanine or other simple alpha-amino acids substituted by an aliphatic side chain from Cl-10 carbons including branched, cyclic and straight chain alkyl, alkenyl or alkynyl substitutions.

[0349] Substitution of aromatic-substituted hydrophobic amino acids: including phenylalanine, tryptophan, tyrosine, biphenylalanine, 1 -naphthylalanine, 2-naphthylalanine, 2- benzothienylalanine, 3 -benzothienylalanine, histidine, amino, alkylamino, dialkylamino, aza, halogenated (fluoro, chloro, bromo, or iodo) or alkoxy-substituted forms of the previous listed aromatic amino acids, illustrative examples of which are: 2-, 3- or 4-aminophenylalanine, 2-,3- or 4-chlorophenylalanine, 2-, 3- or 4-methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-, 5-chloro-, 5-methyl- or 5-methoxytryptophan, 2’-, 3’-, or 4’-amino-, 2’-, 3’-, or 4’- chloro-, 2,3, or 4-biphenylalanine, 2’, -3’,- or 4’-methyl-2, 3 or 4-biphenylalanine, and 2- or 3- pyridylalanine.

[0350] Substitution of amino acids containing basic functions: including arginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid, homoarginine, alkyl, alkenyl, or arylsubstituted (from Cl -CIO branched, linear, or cyclic) derivatives of the previous amino acids, whether the substituent is on the heteroatoms (such as the alpha nitrogen, or the distal nitrogen or nitrogens, or on the alpha carbon, in the pro-R position for example. Compounds that serve as illustrative examples include: N-epsilon-isopropyl-lysine, 3-(4-tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)-alanine, N,N-gamma, gamma’ -diethyl -homoarginine. Included also are compounds such as alpha methyl arginine, alpha methyl 2,3-diaminopropionic acid, alpha methyl histidine, alpha methyl ornithine where alkyl group occupies the pro-R position of the alpha carbon. Also included are the amides formed from alkyl, aromatic, heteroaromatic (where the heteroaromatic group has one or more nitrogens, oxygens, or sulfur atoms singly or in combination) carboxylic acids or any of the many well-known activated derivatives such as acid chlorides, active esters, active azolides and related derivatives) and lysine, ornithine, or 2,3-diaminopropionic acid.

[0351] Substitution of acidic amino acids: including aspartic acid, glutamic acid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, and heteroaryl sulfonamides of 2,4- diaminopriopionic acid, ornithine or lysine and tetrazole-substituted alkyl amino acids.

[0352] Substitution of side chain amide residues: including asparagine, glutamine, and alkyl or aromatic substituted derivatives of asparagine or glutamine.

[0353] Substitution of hydroxyl containing amino acids: including serine, threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromatic substituted derivatives of serine Attorney Docket No. 3062 / 202 PCT or threonine. It is also understood that the amino acids within each of the categories listed above can be substituted for another of the same group.

[0354] For example, the hydropathic index of amino acids may be considered (Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (- 0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). In making conservative substitutions, the use of amino acids whose hydropathic indices are within + / -2 is preferred, within + / -1 are more preferred, and within + / - 0.5 are even more preferred.

[0355] Amino acid substitution may also take into account the hydrophilicity of the amino acid residue (e.g., U.S. Patent No. 4,554,101). Hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-0.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). Replacement of amino acids with others of similar hydrophilicity is preferred.

[0356] Other considerations include the size of the amino acid side chain. For example, it would generally not be preferred to replace an amino acid with a compact side chain, such as glycine or serine, with an amino acid with a bulky side chain, e.g., tryptophan or tyrosine. The effect of various amino acid residues on protein secondary structure is also a consideration. Through empirical study, the effect of different amino acid residues on the tendency of protein domains to adopt an alpha-helical, beta-sheet or reverse turn secondary structure has been determined and is known in the art (see e.g., Chou & Fasman, 1974, Biochemistry, 13:222- 245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979, Biophys. J., 26:367-384).

[0357] Based on such considerations and extensive empirical study, tables of conservative amino acid substitutions have been constructed and are known in the art. For example: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R) gin, asn, lys; Asn (N) his, asp, lys, arg, gin; Asp (D) asn, glu; Cys (C) ala, ser; Gin (Q) glu, asn; Glu (E) gin, asp; Gly (G) ala; His (H) asn, gin, lys, arg; Ile (I) val, met, ala, phe, leu; Feu (E) val, met, ala, Attorney Docket No. 3062 / 202 PCT phe, ile; Lys (K) gin, asn, arg; Met (M) phe, ile, leu; Phe (F) leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W) phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

[0358] Other considerations for amino acid substitutions include whether or not the residue is located in the interior of a protein or is solvent exposed. For interior residues, conservative substitutions would include: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala and Gly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr; Tyr and Trp. (See e.g., PROWL Rockefeller University website). For solvent exposed residues, conservative substitutions would include: Asp and Asn; Asp and Glu; Glu and Gin; Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and Ile; Ile and Val; Phe and Tyr. Various matrices have been constructed to assist in selection of amino acid substitutions, such as the PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlan matrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix, Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

[0359] In determining amino acid substitutions, one may also consider the existence of intermolecular or intramolecular bonds, such as formation of ionic bonds (salt bridges) between positively charged residues (e.g., His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) or disulfide bonds between nearby cysteine residues.

[0360] Methods of substituting any amino acid for any other amino acid in an encoded peptide sequence are well known and a matter of routine experimentation for the skilled artisan, for example by the technique of site-directed mutagenesis or by synthesis and assembly of oligonucleotides encoding an amino acid substitution and splicing into an expression vector construct.

[0361] EXAMPLES

[0362] The following Examples provide illustrative embodiments. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

[0363] EXAMPLE 1

[0364] Myomerger is expressed in male germ cells

[0365] When we assessed the expression of Myomaker (Mymk) and Myomerger (Mymx) via RT-PCR, we detected Myomerger expression in the whole testis and also in dissociated germ cells (FIG. 1A). In contrast, Myomaker was undetectable in these same samples. Interestingly, the level of mRNA for Myomerger in the testis and day 1 differentiated C2C12 myoblasts was Attorney Docket No. 3062 / 202 PCT nearly comparable, despite the myoblasts used being a pure population versus the whole testes (FIG. 1A). Of interest, Myogenin, important in the induction of Myomerger [19, 21], was also detected in the testis (via RT-PCR). Immunoblots for Myomerger protein also confirmed the presence of Myomerger in dissociated germ cells, and the signal intensity was similar to day 1 differentiating C2C12 myoblasts (FIG. IB, FIG. 4A).

[0366] We next addressed the expression of Myomerger in testicular sections by immunofluorescence. At any given time, several cycles of spermatogenesis coexist within the germinal epithelium of the mammalian testis

[0028] . Thus, a unique combination of germ cell subtypes (spermatogonia, spermatocytes and spermatids) can be observed on a cross section of the seminiferous tubules. This repetitive appearance of specific cell associations is referred to as stages of the spermatogenic cycle. To help identify the different germ cells, we used peanut agglutinin (PNA) and alpha-tubulin. PNA is a plant lectin that binds glycoproteins of the acrosome in early ‘round spermatids’ through late ‘elongated spermatids’, as well as in mature spermatozoa

[0029] . The acrosome is an endoplasmic reticulum / Golgi derived organelle, containing multiple enzymes essential for sperm penetration of oocyte layers [30, 31]. alphatubulin is a component of the ‘manchette,’ present in the spermatids

[0032] . The manchette is a transitory tubular structure involved in protein trafficking and nuclear remodeling during sperm elongation [33-36]. Testicular immunolocalization of Myomerger showed expression in some but not all seminiferous tubules (arrows, FIG. 1C, FIG. ID), suggesting a stage-specific staining. In fact, Myomerger colocalized with alpha-tubulin but not PNA (FIG. 1C, FIG. ID, and FIG. 5A), indicating that Myomerger was associated with the manchette of spermatids. To gain a closer visualization, we dissociated germ cells from adult WT testis and placed them on slides (smears). After fixation and permeabilization, we could observe that Myomerger did not colocalize with PNA but was found on the necks of spermatids-like cells in association with alpha-tubulin (FIG. IE, FIG. 5B). We confirmed the specificity of this staining by preabsorbing the Myomerger antibody with Myomerger peptide in testicular sections and in dissociated germ cells smears (FIGs. 6A-6B). In addition, immunostaining of Myomerger KO myoblasts with this Myomerger antibody did not result in any significant staining (FIG. 4B). Because Myomerger was present in the manchette, a transient structure involved in protein / vesicle transport towards the nucleus and the developing tail [34, 36, 37], we investigated whether Myomerger was relocalized to another cellular compartment during sperm differentiation. For this, we isolated sperm from the cauda epididymis of WT mice. After fixation and permeabilization, Myomerger colocalized with PNA (FIGs. 1F-1H), suggesting that Myomerger was likely transported from the manchette to the acrosome. Again, Attorney Docket No. 3062 / 202 PCT pre-absorption of the Myomerger antibody with Myomerger peptide or the omission of primary antibody showed that this staining was Myomerger-specific (FIG. 6C).

[0367] Izumol, the binding partner of Juno, and an essential player in fertilization, is initially present in the acrosome, and then translocates to the plasma membrane during acrosome reaction [4, 38]. Thus, we asked whether Myomerger could follow the same pattern. Caudal sperm were incubated under non-capacitating (PBS) and capacitating (TYH+BSA) conditions in the presence of Myomerger antibody or isotype control (Sheep IgG). After capacitation, we also induced acrosome reaction with A23187 (which we confirmed in parallel by Coomassie blue staining, data not shown). Sperm was then washed, fixed, and stained with the secondary antibody and streptavidin-Texas Red / Hoechst. PNA was added in the last step to identify sperm with an intact acrosome (after releasing acrosome contents, sperm becomes PNA negative). Of note, Myomerger was detected on the surface of some sperm, but it did not colocalize with PNA since the staining was performed with live sperm and the antibody cannot penetrate cellular membranes (FIGs. 1I-1J). Interestingly, surface Myomerger was only observed in PNA-negative sperm, suggesting that it translocated to the plasma membrane after acrosome reaction (similar to Izumol). Further, we detected a greater percentage of Myomerger+ cells in acrosome -reacted sperm compared to capacitated-only sperm, again suggesting that Myomerger was mobilized to the plasma membrane during acrosome reaction (FIG. IK). Although we see some Myomerger+ sperm in capacitating vs non-capacitating conditions, this is likely due to the spontaneous acrosome reaction that occurs under capacitating conditions (NC vs CAP, FIG. IK).

[0368] We have previously shown that PtdSer (normally thought as an apoptotic cell marker) is also expressed on live sperm and contributes to fertilization

[0027] . Therefore, we performed live staining of sperm with Myomerger antibody and Annexin V (FIGs. 7A-7D). Consistent with our previous observations, PtdSer exposure was seen on the head and midpiece of caudal sperm. Myomerger was also localized in punctate fashion on the sperm head FIGs. 7A-7D). Collectively, these data suggest that Myomerger is first localized to the manchette on the testicular spermatids, and likely mobilized to the acrosome in epididymal sperm; once acrosome reaction is triggered, Myomerger is relocalized to the surface of the sperm head, where it may play role in fusion with the egg.

[0369] EXAMPLE 2

[0370] Myomerger is also expressed in female germ cells

[0371] In the context of myoblast fusion, Myomerger can be expressed on both fusing entities, such as the founder and the fusion competent cells [19-21, 39]. Thus, we asked whether Myomerger was also expressed on mouse oocytes. When we analyzed ovarian tissues Attorney Docket No. 3062 / 202 PCT from WT mice, mRNA for Myomerger (Mymx) was detected at low levels (FIG. 2A). However, immunoblots also indicated the presence of Myomerger in ovaries (FIG. 2B). Further, in frozen sections of murine ovaries, Myomerger immunolocalization was surrounded by ZP3 staining (FIGs. 2C-2E), indicating that Myomerger is localized in oocytes in preMetaphase II stages. This staining was not observed when the Myomerger antibody was preabsorbed with the immunizing peptide (FIG. 2F). Immunostaining of Metaphase II oocytes (live, and not fixed), with a Myomerger antibody showed expression of Myomerger in a microvillar pattern (FIGs. 2G, 2H), a region that is well documented to both bind and fuse with sperm. Moreover, when we tested staining for Juno on oocytes, we noticed colocalization of Juno and Myomerger (FIG. 21). These data suggested that Myomerger is expressed on the surface of both male and female gametes.

[0372] EXAMPLE 3

[0373] Role of Myomerger in fertilization

[0374] To investigate the possible role of Myomerger in fertilization, we performed in vitro fertilization assays in the presence of Myomerger antibody; specifically, we used the decondensation of sperm DNA after entry into the oocytes as a readout [27, 40-42]. Sperm were preincubated with Myomerger antibody or isotype control (Sheep IgG), and then mixed with oocytes that were pre-loaded with the DNA staining dye DAPI and preincubated with Myomerger antibody; after 3.5h of coincubation, we evaluated the percentage of oocytes with decondensed sperm (FIG. 3A). A significant reduction in fertilized oocytes was noted after Myomerger Ab treatment compared to the isotype control (FIG. 3B). It has been previously reported that the Myomerger peptide (ectodomain) can block myoblast fusion when used at high concentrations [22, 43]. The incubation of sperm and oocytes with this Myomerger ectodomain peptide also resulted in reduced fertilization (FIGs. 3C, 3D). This effect cannot be explained by a detrimental effect in sperm motility (FIG. 8E).

[0375] Myoblasts share certain features of the fusion machinery with oocytes such that sperm can fuse with myoblasts in vitro, and we found that this can be used as a surrogate assay for sperrmegg fusion

[0027] . Specifically, when sperm labeled with the cytoplasmic dye Calcein- AM is added to myoblasts, sperrmmyoblast fusion occurs and the myoblasts turn ‘red’, which we could visualized via microscopy. We used this sperrmmyoblast fusion assay to evaluate whether the genetic loss of Myomerger had an impact on sperm fusion. To analyze a larger number of events (compared to microscopy), we designed a flow cytometry-based spermmyoblast fusion assay, where we evaluated the percentage of Calcein-AM+ myoblasts among the live myoblasts (FIG. 3E, FIG. 8A). Consistent with our previous work, sperm did transfer Calcein-AM to myoblasts and the fusion was blocked when myoblasts were treated with Attorney Docket No. 3062 / 202 PCT

[0376] Cytochalasin D (preventing cytoskeletal reorganization, and in turn, fusion) (FIGs. 8A, 8B). Next, we tested the fusion of Calcein-AM+ sperm with WT, Myomerger KO and Myomaker KO undifferentiated C2C12 myoblasts (FIGs. 3E, 3F, FIGs. 8A, 8C). A small but significant reduction in fusion was detected in Myomerger KO C2C12 cells compared to WT (FIG. 3F). In contrast, this was not observed in Myomaker KO myoblasts (FIG. 3F).

[0377] As Myomerger, as well other markers, increase during myoblast differentiation [19-21, 39], we next reasoned that sperm-myoblast fusion might increase in differentiating myoblasts compared to undifferentiated C2C12 cells. Using standard protocols [18, 24, 44], we differentiated WT and Myomerger KO myoblasts for 1-2 days in fusion media containing 2% horse serum (FIG. 3H). First, we confirmed that Myomerger expression, as well as Myogenin (MyoG), Myomaker (Mymk) and Myosin heavy chain 4 (Myh4), were indeed increased in WT C2C12 myoblasts (FIG. 3G, FIG. 8D). After co-culture of Calcein-AM labeled sperm with differentiating myoblasts, we made two key observations. First, we noted a significantly increased percentage of fusion in differentiating WT myoblasts compared to undifferentiated WT C2C12 myoblasts (“-” and “+”columns on the left side, FIG. 31). Second, when we tested Myomerger KO myoblasts under the same differentiation conditions as WT myoblasts, the Myomerger KO myoblasts did not have the same increase in fusion, i.e. showed similar fusion indexes before and after differentiation (“-” and “+”columns on the right side, FIG. 31). Collectively, these data suggest that Myomerger contributes to sperm-myoblast fusion.

[0378] DISCUSSION OF THE EXAMPLES

[0379] While a number of elegant pieces of work have identified molecules on the surfaces of sperm and egg that mediate their binding and specific recognition [1-10], the players involved in fusion of the membranes of the two gametes are less defined. Aspects of the current study include the identification of the expression of Myomerger, a muscle fusogen, on both female and male gametes, and the specific localization of Myomerger to areas dedicated to sperm-egg fusion, suggesting Myomerger as a putative fusogen in fertilization. Interestingly, Myomerger localization changes during sperm differentiation: from the manchette in the testicular elongated spermatid to the acrosome in epididymal acrosome reacted sperm. Although several molecules have been identified in the intra-manchette transport [33-35], it is still not clear how the trafficking of proteins is coordinated to deliver proteins to specific locations

[0036] . Thus, the mechanisms of Myomerger relocalization from the manchette to the acrosome remains to be clarified in the future. More importantly, our functional studies suggest that Myomerger contributes to the fusion between the plasma membrane of the oocyte and sperm. Interestingly, the functionality of Myomerger in latter stages of membrane fusion was also linked to the lipid PtdSer as a positive regulator

[0026] . Given our previous work demonstrating a role for PtdSer Attorney Docket No. 3062 / 202 PCT in sperm:egg fusion

[0027] , the identification of Myomerger on both sperm and eggs, the colocalization of Myomerger and PtdSer on the sperm head further support their role in sperrmegg fusion. An important step in sperm structural change prior to sperrmegg fusion is the acrosome reaction

[0011] . We find that while Myomerger is clearly present on the sperm head, the number of Myomerger+ cells substantially increase after acrosome reaction. Moreover, Myomerger co-localized with PtdSer, a phospholipid involved in fertilization

[0027] .

[0380] Whereas Myomerger might function as a common fusogen in the muscle and in fertilization, some key differences can be pinpointed between these two systems. First, we have not detected Myomaker, another essential fusogen for differentiating muscle cells

[0018] , on sperm and oocytes. However, it has been reported that Myomerger and Myomaker function independently: while Myomaker is responsible for the initiation of fusion and formation of hemifusion intermediates, Myomerger is involved in the formation and expansion of the membrane pore

[0022] . In addition, in the fibroblast-erythrocyte fusion mediated by hemagglutinin A, Myomerger promoted this fusion in the complete absence of Myomaker

[0022] . Second, several studies have revealed that Myomaker is required in both fusing cells, while Myomerger only needed on one cell side to complete fusion [20, 39]. In the case of the gametes, both sperm and eggs express Myomerger on their membranes, opening up the possibility for a role of Myomerger on both cells. We surmise that Myomerger may be expressed on both gametes perhaps to ensure a successful fertilization, which is vital for species survival.

[0381] Myomerger was also detected on oocytes pre-ovulation (oocytes within the ovary) and post-ovulation oocytes (Metaphase II oocytes). This suggests, that Myomerger, as other important molecules for fertilization, is readily on the egg surface before ovulation. On Metaphase II oocytes, Myomerger was detected specifically on the microvilli region, area where fusion with sperm occurs, colocalizing with Juno. We envision a model where Juno- Izumol, as well as other molecules, lock the interaction between the egg and spermatozoa, maintaining the immediate contact of the plasma membranes for enough time to allow fusogens, such as Myomerger, to form membrane pores promoting gamete fusion. As Myomerger global knockout mice are embryonic lethal, future studies via tissue specific deletion in gametes would be useful.

[0382] MATERIALS AND METHODS EMPLOYED IN THE EXAMPLES

[0383] Mice. C57BL / 6 mice (stock 000664) were purchased from the Jackson Laboratory and bred in our facilities. All animal procedures were approved by and performed according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) at the University of Virginia. Attorney Docket No. 3062 / 202 PCT

[0384] Myoblast differentiation. C2C12 murine skeletal muscle myoblasts (ATCC) were maintained at sub-confluent densities in Dulbecco's modified Eagle's medium (DMEM) medium supplemented with 20% heat inactivated fetal bovine serum (FBS) and antibiotics at 37 °C, 8.5% CO2 atmosphere. Myomerger- / - and Myomaker- / - C2C12 cells were generated as previously described [18, 20] and kindly provided by Dr. Millay (Cincinnati Children’s Hospital Medical Center, Ohio). For myoblast differentiation, C2C12 cells were grown to confluence and switched to DMEM supplemented with 2% heat inactivated horse serum and antibiotics for 1-3 days [18, 24, 44]. For immunostaining, WT and Myomerger- / - C2C12 cells were grown and differentiated in four-well EabTek II Permanox chamber slides.

[0385] Collection of gonadal tissues and gametes. Testes from >10 week old male mice were dissected and snap frozen in liquid nitrogen for immunostaining, lysed for western blot or dissociated for the enrichment of germ cells as previously described

[0045] . Briefly, testes were decapsulated and seminiferous tubules were mechanically dispersed and allowed to settle in HBSS. The supernatant containing mainly interstitial cells was discarded and the seminiferous tubules were incubated with type II collagenase (100 U / ml, Worthington Biochemical Corp) and DNAse (10 ug / ml, Sigma) for 15 min at 34 °C on shaker. The enzymatic digestion was stopped by dilution and cells and remaining tubules were passed thru a cell strainer and centrifugated for 10 minutes at 500 g. The pellet containing mainly dissociated germ cells was resuspended in HBSS and cells were placed on slides for smears or lysed for western blot. Smears were then fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature and used for immunostaining (see below).

[0386] Cauda epididymes were also dissected and nicked to allow sperm to swim in capacitating media [TYH media (119 mm NaCl, 4.7 mm KC1, 1.71 mm CaCh, 1.2 mm KH2PO4, 25.1 mm NaHCCF, 5.56 mm glucose, 0.51 mm Na pyruvate, 1% phenol red) supplemented with 4 mg / ml bovine serum albumin (BSA), penicillin, and streptomycin

[0046] ] for 20 min at 37 °C, 5% CO2 and placed on slides for smears (immunostaining). Slides were dried and fixed in 4% PFA for 15 minutes at room temperature (“Fixed sperm smears”).

[0387] For live staining, sperm was allowed to swim in PBS or HBSS (non capacitating media; NC), counted, and 5xl05sperm cells were transferred to tubes with NC or capacitating (Cap) media. Myomerger Ab (10 ug / ml, Invitrogen PA5-47639) or Sheep IgG (10 ug / ml, Jackson Immunoresearch, 013-000-003) were added and incubated for Ih at 37 °C, 5% CO2. To induce acrosome reaction

[0047] , 20 uM A23187 (Sigma, C7522) was added to some tubes with sperm previously incubated in Cap media and all the tubes were incubated for an additional hour at 37 °C, 5% CO2. After washing with PBS and centrifugation, sperm were fixed with 4% PFA (15 minutes, room temperature) and placed on slides (“Five staining Attorney Docket No. 3062 / 202 PCT sperm”). In some cases, Annexin V (conjugated with FITC, BD 556416) was added during the incubation with the antibodies.

[0388] Ovaries were dissected from 4-6 week old female mice and snap frozen in liquid nitrogen for immunostaining or lysed for western blot.

[0389] Quantitative RT-PCR (qPCR). Total RNA was extracted from WT C2C12 cells at different stages of differentiation, testis, dissociated germ cells, and ovaries using NucleoSpin RNA columns (Machery-Nagel) and the cDNA was synthesized using QuantiTect Reverse Transcription Kit (Qiagen) according to manufacturer’s instructions. qPCR for mouse Mymx, Mymk, MyoG, Myh4, or housekeeping gene beta actin was performed using Taqman probes (Applied Biosystems) using StepOnePlus Real Time PCR System (ABI).

[0390] Western Blot. Testis, ovaries, dissociated germ cells and cultured C2C12 cells were washed, trypsinized, pelleted and resuspended in RIPA lysis buffer (Pierce) with proteases inhibitors (Sigma) and sonicated (5 second pulses, three times). Lysates were centrifuged and protein concentration was determined in the supernatants by the BCA protein assay (Pierce). Samples were boiled for 5 min in the presence of loading buffer and b-mercaptoethanol and loaded into Miniprotean TGX Precast gels (10-100 kD, BioRad 4569033). Gels were subsequently transferred to a PDVF membrane, blocked in 5% milk in Tris-buffered saline / 0.1% Tween-20 (TBS-T) and incubated with Myomerger Ab (1:200; Invitrogen) for 18- 20 h at 4°C. Membranes were then washed with TBS-T and incubated with a secondary antibody conjugated with HRP for Ih at room temperature. After washing again, membranes were developed using ECL (Perkin Elmer). Bands were visualized using the Biorad Chemidoc Touch system. Beta-actin directly conjugated to HRP (Sigma, S3854) was used as a loading control.

[0391] Immunostaining. All the steps were performed at room temperature except specified. Testicular sections, and sperm or TC smears were fixed in 4% PFA for 15 min. Permeabilization was performed with 0.1% triton for 10 min. Non specific binding was blocked by incubation with 5% BSA in PBS followed by the Avidin / Biotin blocking kit (Vector Labs). Slides were then incubated with anti Myomerger antibody (0.5 ug / ml, Invitrogen, PA5-47639). For fixed sperm we used lOug / ml) for 20 h at 4°C. After washing with PBS, a biotinylated antibody against sheep IgG (Vector BA-6000) was added and incubated for Ih. Slides were washed again and incubated with Neutralite avidin (NA)-Texas Red (2.5 ug / ml, SouthernB iotech 7200-07)) for Ih. DNA was stained with Hoechst. For testicular sections, sperm and dissociated germ cell smears, PNA- Rhodamine (Vector, RL- 1072), was added during the staining with Hoechst. Testicular sections and dissociated germ cell smears were also incubated with Myomerger and alpha-tubulin (1.5 ug / ml, ProteinTEch, Attorney Docket No. 3062 / 202 PCT

[0392] 11224-1-AP) antibodies (20h, 4°C) followed by a biotinylated anti sheep IgG and anti rabbit IgG (Invitrogen, A21206) conjugated with AlexaFluor 488, NA-Texas Red and Hoechst.

[0393] For live staining, sperm (0.5xl06) were incubated with Myomerger antibody or Sheep IgG (10 ug / ml) for Ih at 37°C, 5% CO2 as described above. After washing and fixing, sperm smears were incubated with a biotinylated anti sheep IgG, followed up by washes, Streptoavidin (SA)-FITC (BD 554060) and Hoechst and PNA-Rhodamine. Four different fields were photographed and the percentage of Myomerger+ sperm was quantified in each frame. The average of 4 frames is presented.

[0394] WT and Myomerger- / - C2C12 cells were differentiated for 3 days, washed, fixed with 4% PFA and permeabilized with 0.1% triton for 10 min. After blocking with 5% BSA and avidin / biotin kit, cells were incubated with 1 ug / ml Myomerger antibody, followed by a biotinylated anti sheep antibody and Streptavidin-Texas Red. Hoechst was used to stain the nucleus.

[0395] Ovarian frozen sections were fixed in 3.7% formaldehyde for 10 min and immunostaining was performed as described above with a few modifications. Briefly, a mouse antibody against murine ZP3 (1G2 clone; kindly provided by Dr. Tung,

[0048] ) was added during the incubation with the biotinylated anti sheep IgG and an antibody against mouse IgG conjugated with FITC was included during the staining with NA-Texas Red. Hoechst was also used for DNA staining.

[0396] Metaphase II zona pellucida free oocytes were obtained from superovulated WT females (see below) and stained with 10 ug / ml Myomerger and / or 1 ug / ml Juno antibodies (Biolegend, 125102) for 30 min in TYH+BSA medium at 37°C, 5% CO2. Oocytes were then washed and fixed in 4% PFA for 30 min. A biotinylated anti sheep IgG and anti rat IgG conjugated with Alexa Fluor 488 (Invitrogen, A21470) were used as secondary antibodies followed by Streptavidin-Texas Red and Hoechst.

[0397] The specificity of the Myomerger antibody was tested by preabsorption with the Myomerger ectodomain (soluble Myomerger26’84, [22, 43]). Briefly, Myomerger antibody was incubated with 10X peptide for 20 h at 4°C with rotation. Another tube of Myomerger antibody alone served as positive control. The Myomerger antibody + peptide mix was used to stain testicular, ovarian sections or sperm or dissociated germ cell smears as indicated above. Tissues and cells were visualized using the Axio Imager 2 with Apotome (Zeiss) and LSM 700 and 880 confocal microscopes (Zeiss).

[0398] In vitro fertilization (IVF) assays. IVF was performed as previously described [27, 49]. In brief, 4-5 weeks old female mice were super-ovulated by intraperitoneal injections of 5 IU of pregnant mare serum gonadotropin (Prospec) and 5 IU of human chorionic Attorney Docket No. 3062 / 202 PCT gonadotropin (hCG, Sigma) 48 h later. Zona Pellucida (ZP) intact Metaphase II oocytes were obtained 13 h after hCG injection and treated with 3 mg / ml hyaluronidase (Sigma) in 0.01% poly vinyl alcohol / FHM medium (EMD Millipore) to remove the cumulus. ZP-intact oocytes were incubated with 10 ug / ml Myomerger antibody (Invitrogen, PA5-47639), Sheep IgG (isotype control, Jackson Immunoresearch) or left untreated for 30 min in 5% FBS+TYH+BSA at 37 °C, 5% CO2. During the last 20 min, oocytes were loaded with lO pg / ml DAPI (BioRad), and washed several times in TYH+BSA [1, 27, 40-42] before transferring them to the fertilization drop. Caudal sperm were allowed to swim in TYH+BSA for 20 minutes at 37 °C, 5% CO2 and counted. Sperm was then incubated with 10 ug / ml Myomerger Ab, Sheep IgG or left untreated. Oocytes were inseminated for 3 h at 37 °C, 5% CO2, washed and fixed with 0.25% glutaraldehyde and 0.1% paraformaldehyde for 20 min at 4 °C. The percentage of oocytes with DAPI+decondensed sperm nuclei (typically 1-2 / oocyte) was evaluated by microscopy.

[0399] ZP-free oocytes were obtained from superovulated female mice after removal of cumulus cells and the ZP via treatment with hyaluronidase and Tyrode’s solution (Sigma, T1788), respectively. The Myomerger ectodomain 26-84 (5 uM, LifeTein) was added during oocyte-sperm co-incubation (Ih) under paraffin oil at 37°C, 5% CO2. Fusion was evaluated as described above.

[0400] Sperm: myoblast fusion experiments. Sperm was stained with the cytoplasmic dye Calcein red orange AM (Invitrogen, C34851) as described before [27, 50]. Briefly, caudal sperm were allowed to swim in TYH + BSA for 20 min at 37°C, 5% CO2and incubated with 8 pM Calcein red orange -AM (Invitrogen) for 30 min in PBS at room temperature. Cells were washed with PBS, counted and resuspended in DMEM medium supplemented with 20% heat inactivated FBS.

[0401] Undifferentiated vs differentiated C2C12 cells were trypsinized, counted and plated in 24 well plates (2xl05cells / well). After 18-20h, Calcein- AM+ sperm (0.25xl06cells / well) were added and incubated for 4 h at 37 °C, 8.5% CO2. Myoblasts were then washed with PBS and harvested by trypsinization for flow cytometry. A live / dead dye (Sytox green, Invitrogen S7029) was added. Cells were run on an Attune Nxt (Fife Technologies) and data was analyzed using Flowjo. The percentage of Calcein-AM+ cells within the live gate is depicted. The fusion index was calculated as a percentage of Calcein-AM+ cells in Myomerger- / - or Myomaker- / - myoblasts normalized to WT myoblasts.

[0402] Statistical analysis. Statistical significance was determined using GraphPad Prism 5 or 6 using unpaired Student’ s two-tailed t test, one-sample t test, Mann-Whitney test, or oneway analysis of variance as according to test requirements. No inclusion / exclusion criteria Attorney Docket No. 3062 / 202 PCT were pre-established. A p value of < 0.05 (*), < 0.01 (**), or < 0.001 (***) were considered significant.

[0403] REFERENCES

[0404] All references listed in the instant disclosure, including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and database entries (including but not limited to UniProt, EMBL, and GENBANK® biosequence database entries and including all annotations available therein) are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, and / or teach methodology, techniques, and / or compositions employed herein. The discussion of the references is intended merely to summarize the assertions made by their authors. No admission is made that any reference (or a portion of any reference) is relevant prior art. Applicants reserve the right to challenge the accuracy and pertinence of any cited reference.

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[0413] 9. Inoue et al. (2021). Evolutionarily conserved sperm factors, DCST1 and DCST2, are required for gamete fusion. Elife 10.

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[0454] It will be understood that various details of the presently disclosed subject matter can be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.

Claims

1. Attorney Docket No. 3062 / 202 PCTCLAIMSWhat is claimed is:

1. A method of selecting fusion-competent gametes, the method comprising:(a) providing a sample comprising gametes obtained from a subject;(b) contacting the sample with a reagent capable of binding Myomerger expressed on the surface of the gametes; and(c) isolating gametes bound by the reagent capable of binding Myomerger from gametes not bound by the reagent capable of binding Myomerger.

2. The method of claim 1, wherein the gametes are sperm cells or oocytes.

3. The method of claim 1 or claim 2, wherein the reagent capable of binding Myomerger comprises an anti-Myomerger antibody or a fragment thereof, or a peptide comprising a Myomerger ectodomain.

4. The method of any one of claims 1-3, wherein the gametes comprise sperm cells, and wherein the method further comprises contacting the sample with a reagent capable of binding phosphatidylserine on the surface of the sperm cells and isolating sperm cells bound by the reagent capable of binding phosphatidylserine.

5. The method of claim 4, wherein the reagent capable of binding phosphatidylserine comprises Annexin V or GST-BAI1-TSR.

6. The method of any one of claims 1-5, wherein the subject is a human subject or a nonhuman animal subject.

7. The method of claim 6, wherein the non-human animal subject is selected from the group consisting of bovine, equine, porcine, ovine, canine, feline, and avian.

8. A method of identifying fusion-competent gametes, the method comprising:(a) providing a sample comprising gametes obtained from a subject; and(b) contacting the sample with a reagent capable of binding Myomerger expressed on the surface of the gametes; wherein gametes that express Myomerger on their surface are fusion-competent.

9. The method of claim 8, wherein the gametes are sperm cells or oocytes.

10. The method of claim 8 or claim 9, wherein the reagent capable of binding Myomerger comprises an anti-Myomerger antibody or a fragment thereof, or a peptide comprising a Myomerger ectodomain.

11. The method of any one of claims 8-10, wherein the gametes comprise sperm cells, and wherein the method further comprises contacting the sample with a reagent capable of binding phosphatidylserine on the surface of the sperm cells and isolating sperm cells bound by the reagent capable of binding phosphatidylserine.Attorney Docket No. 3062 / 202 PCT12. The method of claim 11, wherein the reagent capable of binding phosphatidylserine comprises Annexin V or GST-BAI1-TSR.

13. The method of any one of claims 8-12, wherein the subject is a human subject or a non-human animal subject.

14. The method of claim 13, wherein the non-human animal subject is selected from the group consisting of bovine, equine, porcine, ovine, canine, feline, and avian.

15. A method of contraception comprising administering a reagent capable of binding Myomerger expressed on the surface of gametes to a subject.

16. The method of claim 15, wherein the subject is a human subject or a non-human animal subject.

17. The method of claim 16, wherein the non-human animal subject is selected from the group consisting of bovine, equine, porcine, ovine, canine, feline, and avian.

18. A composition comprising a reagent capable of binding Myomerger and a reagent capable of binding phosphatidylserine.

19. The composition of claim 18, wherein the reagent capable of binding Myomerger comprises an anti-Myomerger antibody or a fragment thereof, or a peptide comprising a Myomerger ectodomain; and / or wherein the reagent capable of binding phosphatidylserine comprises Annexin V or GST-BAI1-TSR.

20. A kit comprising the composition of claim 18 or claim 19.

21. A kit comprising:(a) a reagent capable of binding Myomerger;(b) a reagent capable of binding phosphatidylserine;(c) instructions for preparing the reagents of (a) and (b) into a single composition; and(d) optionally, one or more additional reagents for use in preparing and / or modifying the composition.

22. A kit suitable for use in carrying out a method of any one of claims 1-17.

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