Patent Application
20140315185
This application relates to methods for enhancing the functionality of sperm. More particularly, this application relates to methods for reducing the number of sperm required in livestock artificial insemination (AI) in particular for application with flow cytometry semen sexing. The methods may also be employed to increase the fertility of sperm in some human male individuals with sub-optimal fertility.
The ability to identify and select male and female sperm has great value in the livestock industries, where there is an established market in AI of over US$2 billion per annum in the Organization for Economic Cooperation and Development (OECD) countries. This is particularly true in the dairy industry where over 80% of dairy farmers in key OECD markets impregnate their cows through AI. Sexed semen provides the opportunity to increase farmer productivity and income. For example, the availability of sexed semen has significant impact in reducing and/or eliminating the minimal returns of male dairy calves as compared to female calves.
Currently, the only commercial technique for semen sexing uses flow cytometry to sort sperm on the basis of DNA content. Bovine sperm bearing the Y chromosome have approximately 4% less DNA than sperm bearing the X chromosome. This difference, in combination with a fluorescent DNA binding dye (for example Hoechst 33342) and flow cytometry, permits purification of X chromosome bearing sperm to greater than 90% (Johnson et al., 1989). However, the ability to sort bovine sperm is currently limited to a rate of 8000 s−1 which, when each straw, or dose, contains 2×106 sperm, translates to 14 straws/hour (Sharpe and Evans, 2009). As a result, sexed semen straws generally incorporate ten-fold less sperm than unsexed straws. In addition, the sorting process itself has a negative effect on the fertility of the sperm. The reduction in the number of sperm per straw, together with reduction in sperm fertility due to the sorting process, causes a significant reduction of 14% in the conception rate for sexed sperm compared to unsexed sperm (Frijters et al., 2009). The sexed semen also has a significant price premium over unsexed sperm due to the high cost of sorting even the sub-optimal number of sperm used in the sexed semen straws. A valuable addition to the semen sexing technology would be a method to enhance the fertility of sperm so that a dose of considerably less than the approximately 2×106 sperm per straw currently used for sexed semen would achieve the same conception rate as the normal, unsexed, straws.
Such methods would also have application in swine AI where much higher doses of sperm are used in the standard AI methodology than with bovine, namely approximately 2500×106 sperm per straw. Recent work suggests that more sophisticated techniques involving deep intrauterine insemination can lower this requirement to 50-70 million (Vazquez et al., 2005; Vazquez et al., 2008). However, this reduced dose is still beyond the present commercial capability of flow cytometry sorted sperm.
Sperm are highly specialized cells that deliver the haploid male genome to the haploid female genome contained in the oocyte. Despite this seemingly simple mission, the path to achieving this goal is highly complex. Extraordinarily large numbers of sperm are inseminated in a natural mating, for example approximately four billion sperm/oocyte in the cow. The inseminated sperm spend a variable period of time, ranging from hours to days in the different regions of the female reproductive tract (FRT). The environments that sperm encounter from ejaculation to fertilization of the oocyte also vary considerably. These environments range from the complex molecular mix added to sperm at ejaculation by the male to the various female secretions and different cell surfaces of the female epithelia (Drobnis and Overstreet, 1992).
Once sperm are deposited in the FRT, a combination of active sperm migration and female uterine muscle contraction propels the sperm to the oocyte. During the journey through the FRT, sperm can be retained in specialized regions, most notably the cervix and oviduct (Drobnis and Overstreet, 1992). This retention may increase the probability that at least some sperm will be present in the oviduct at the same time as ovulation occurs. However, for the cervix it is more likely that the restriction of entry and trapping acts as a negative selection imposed against sperm by the female. In fact, one of the major innovations that launched the modern bovine AI industry was the finding that considerably less sperm were required when sperm were passed through the cervix and deposited into the body of the uterus (Foote, 2002). The final phase of the sperm journey in the oviduct involves release of sperm from the isthmus region (controlled by the female) and travel to the ampulla for fertilization of the oocyte. At this time near unitary numbers of sperm are present (Drobnis and Overstreet, 1992). Fertilization itself is again a complex phenomena involving penetration of the cumulus oophorus and subsequently the zona pellucida (Katz et al., 1989). Although the sperm journey through the FRT is broadly similar between mammalian species, various aspects do differ.
Sperm also undergo a maturational change while resident in the FRT, known as capacitation. When sperm are ejaculated, they are not capable of fertilizing the oocyte. However, during passage through the FRT sperm gain the capacity to fertilize. Changes to sperm during passage through the FRT include alterations in membrane properties, enzyme activities and motility (Salicioni et al., 2007). One such important change is the loss of cholesterol from the outer sperm surface membrane (Flesch et al., 2001; Osheroff et al., 1999; Visconti et al., 1999). Ultimately these changes enable sperm to respond to stimuli that induce the acrosome reaction and penetration of the egg. One of the important changes that occur during capacitation is alterations in the surface properties of sperm. A specialized protein-carbohydrate coating stabilizes the surface membrane (Schroter et al., 1999), regulates capacitation (Topfer-Petersen et al., 1998), facilitates transport through the FRT (Toliner et al., 2008b), and enables attachment at the oviduct (Tollner et al., 2008a). In different species, essentially the same functions are carried out by the surface coatings, however the specific molecular components vary (Calvete and Sanz, 2007; Tollner et al., 2008a; Topfer-Petersen et al., 1998).
In a natural bovine mating, approximately 4 billion sperm are inseminated yet less than 10,000 get to the oviduct and less than 10 get through to the oocyte (Mitchell et al., 1985). Why there are such large losses is poorly understood. Following coitis, greater than 80% of sperm are lost through vaginal discharge (Mitchell et al., 1985). The remaining sperm form a gradient in concentration from the cervix to the oviduct (Hawk, 1983; Hunter, 2003; Mitchell et al., 1985). In bovine, approximately 10,000 sperm arrive at the oviduct 6-8 hours after insemination (Mitchell et al., 1985). By 12 to 24 h after insemination, sperm have either been lost through back flushing, eliminated by phagocytosis or reached the oviduct (Hawk, 1983). In pigs, there is strong evidence for phagocytosis of sperm by polymorphonuclear neutrophils, with a massive infiltration of neutrophils occurring in the uterine lumen shortly after insemination (Matthijs et al., 2003). Recently, similar evidence that neutrophils infiltrate the uterine lumen after insemination in cows has been published (Alghamdi et al., 2009).
Experimental evidence suggests that both motile and damaged (immotile and/or dead) sperm are lost by discharge (Lightfoot and Restall, 1971; Oren-Benaroya et al., 2007). In contrast, in vitro evidence indicates that live sperm are preferentially phagocytosed by neutrophils (Woelders and Matthijs, 2001). Several phenomena contribute to sperm damage from the FRT, although the mechanism and significance are poorly understood. Such phenomena include:
Sperm also cause damage to themselves through generation of reactive oxygen species (ROS) mainly as a by-product of mitochondrial function (de Lamirande and Gagnon, 1995; Koppers et al., 2008; Vernet et al., 2001). ROS cause loss of sperm motility and lipid peroxidation. The latter damage leads to alteration of the membrane properties such as flexibility and fluidity, and can also lead to lack of membrane integrity and/or decreased chromatin quality (Storey, 1997). Sperm are particularly sensitive to ROS-induced damage because of their membrane composition and their limited antioxidant defenses. In particular, the high proportion of polyunsaturated fatty acids (PUFA) in the surface membrane makes this membrane highly susceptible to oxidation (Jones et al., 1979). The nature of the sperm cell, with limited cytoplasmic fluid, also constrains the availability of intracellular antioxidants (Koppers et al., 2008, & ref within). In human sperm at least, there exists a strong relationship between ROS production and antioxidant protection for determining the lifespan of sperm in the absence of external damaging agents (Alvarez and Storey, 1985; Storey, 1997, 2008).
Sperm from human (Bell et al., 1992), bovine (Bilodeau et al., 2002), equine (Baumber et al., 2000) and porcine (Awda et al., 2009) all show loss of motility when exposed to H2O2 concentrations in the mico molar range, however where examined this loss of motility has not been associated with loss of viability (Awda et al., 2009; Bell et al., 1992). In addition this ROS-induced loss of motility is specific to H2O2 and not other ROS like .O2− (Awda et al., 2009; Baumber et al., 2000; Bilodeau et al., 2002). The mechanism for H2O2-induced loss of motility is currently unknown, however it may be related to the observation that, unlike other ROS, H2O2 is able to pass through the cell membrane (Bienert et al., 2006). This membrane passage by H2O2 has so far been shown to be facilitated by aquaporin membrane proteins. Sperm express surface membrane aquaporin pumps, which are thought to be associated with sperm volume regulation by being able to pump H2O through the cell membrane (Chen and Duan, 2011; Chen et al., 2011; Yeung, 2010).
Sensitivity of sperm motility to H2O2 may also occur in the FRT. It has been shown that dead sperm in combination with aromatic amino acids produce H2O2 (Shannon and Curson, 1972; Tosic and Walton, 1950) and dead sperm are abundant in the FRT. H2O2 may also result from bacterial organisms present in the FRT. In particular, Lactobacillus acidophilus is known to produce H2O2 and is frequently present in both the human (Klebanoff and Smith, 1970) and the cattle vagina (Otero and Nader-Macias, 2006).
In all species where sperm suffer H2O2-induced loss of motility, antioxidants such as catalase and glutathione are able to rescue the loss of motility if added in sufficient concentration and simultaneously with the H2O2 (Baumber et al., 2000; Bilodeau et al., 2002). In contrast, antioxidants reactive against .O2− such as superoxide dismutase (SOD) could not rescue motility (Baumber et al., 2000; Bilodeau et al., 2002; Lapointe and Sirard, 1998).
Surface Properties of Sperm May have a Significant Influence on Fertility
The specialized protein-carbohydrate coating that facilitates transport through the FRT (Tollner et al., 2008b), may influence fertility by protecting sperm from mucus capture in the FRT or assisting the motion of sperm through mucus. An example of a protein that coats the surface of sperm and facilitates travel through the FRT is β-defensin 126 in macaque monkeys. This highly sialylated glycoprotein coats macaque sperm and is a major component of the sperm glycocalyx (Yudin et al., 2003; Yudin et al., 2005). Importantly, this glycoprotein facilitates movement of sperm through cervical mucus (Tollner et al., 2008b) as does the human β-defensin 126 on human sperm through the cervical mucus surrogate, hyaluronic acid (Tollner et al., 2011). Men that are homozygous for a deletion mutation of β-defensin 126 exhibit impaired sperm function and subfertility (Tollner et al., 2011). Macaque β-defensin 126 has extensive O-linked-glycosylation in the carboxy-terminal portion of the protein and a significant amount of sialic acid on the carbohydrate-terminal residues (Yudin et al., 2005). The negatively charged sialic acid residues from β-defensin 126 contribute the majority of the charge on the macaque sperm surface (Yudin et al., 2005) and presumably also on human sperm (Tollner et al., 2011). These sperm surface charges may well be responsible for penetration through the negatively charged cervical mucus or substitutes with similar properties of charge and viscosity like hyaluronic acid (Aitken et al., 1992; Tang et al., 1999). If surface charge is important for sperm movement through mucus, changing either the actual surface charge or the distribution of charge may affect sperm motion in uterine mucus and fertility.
In summary, the FRT is hostile to sperm, in particular selecting for motile non-damaged sperm but also removing the vast majority of sperm. While in the FRT, sperm have to deal with a wide variety of physiological environments, mature particularly at the cell surface and respond appropriately to signals at the right time and place. Thus despite the sperm's simple mission and relatively simple construction, successful sperm have the characteristics of at least reaching the upper uterine horn, remaining undamaged (mainly a surface phenomena), not being phagocytosed, remaining motile (a function of mitochondria, glycolytic enzymes and flagella components), avoiding capture by mucus and being able to respond to signals appropriately (a surface phenomena but also involving signal transduction and effector pathways). Treatments to sperm that enhance the ability of sperm to remain undamaged, motile, not phagocytosed and functionally competent could therefore reduce the number of sperm required for insemination.
Polyethylene glycol (PEG) has the general formula: H(OCH2CH2)nOH with typical molecular weights of 500-20,000 daltons. It is non-immunogenic and soluble in aqueous solutions. The polymer is nontoxic and generally does not harm active proteins or cells.
Pegylation of proteins has been shown to improve solubility and vascular longevity, and decrease the immunogenicity of xenogeneic proteins while retaining normal protein function (Abuchowski et al., 1977a; Abuchowski et al., 1977b; Jackson et al., 1987; Senior et al., 1991; Zalipsky et al., 1994). Pegylation has also been used directly on cells to provide immune camouflage, initially for transfusion of red blood cells (Chen and Scott, 2001; Scott et al., 1997) and subsequently for other tissues such as pancreatic beta islet cells (Chen and Scott, 2001; Teramura and Iwata, 2009). For both red blood cells and pancreatic beta islet cells, the respective cell functions were preserved despite the pegylation.
The present disclosure provides methods and conjugates for improving the functionality of cells, such as sperm. More specifically, the present disclosure provides conjugates that can be employed to attach functional molecules of interest, such as proteins or carbohydrates, to cells. The disclosed methods and compositions are effective in improving the functionality and/or fertility of sperm in the FRT, for example by preventing loss of motility, protecting against phagocytosis by neutrophils or other immune attack, facilitating sperm movement through the FRT by aiding movement or avoiding capture by mucus and thus in general extending the lifespan of sperm in the FRT and/or improving functionality. The disclosed methods and compositions can be employed in AI, for example, to reduce the number of sperm needed for insemination and to improve conception rates. Addition of proteins to cells other than sperm using the disclosed conjugates can also be used in other applications, such as transplantation protection.
In one aspect, the present disclosure provides conjugates that can be employed to improve the functionality of cells, such as sperm, by attaching a functional molecule of interest, such as a protein, to the surface of the cells. The disclosed conjugates comprise, or consist of, four components: a membrane anchoring component, such as a lipid; a spacer and/or solubilizing component, such as PEG; an attachment group or linker; and a functional molecule of interest that is attached to the spacer and/or solubilizing component via the attachment group.
Lipids that can be effectively employed in the disclosed conjugates include cholesterol, diacylglycerolipids, dialkylglycerolipids, glycerophospholipids, sphingosine derived diacyl- and dialkyl-lipids, ceramide, phosphatidate, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol and phosphatidyl glycerol. Examples of lipid-PEG-attachment group structures employed in the disclosed conjugates include those provided in
In certain embodiments, functional molecules of interest employed in the disclosed conjugates are able to increase the lifespan of sperm in the FRT by at least 20%, 30%, 40% or 50% compared to untreated sperm. Examples of such molecules include, but are not limited to, amino acids and their derivatives, glutathione, CD55, CD59, CD73, SPAM1, DNaseI, catalase, and variants thereof. The amino acid sequences of CD55, CD59, CD73, SPAM1, DNase1L3 and catalase from bovine are provided in SEQ ID NO: 1-6, respectively. Seminal plasma proteins that bind to the surface of sperm or other sperm surface proteins can also be used as the functional molecules of interest employed in the disclosed conjugates. In certain embodiments, the functional molecules of interest are polypeptides selected from the group consisting of SEQ ID NO: 7-163 and variants thereof.
In another aspect, compositions comprising one or more of the conjugates disclosed herein and a physiologically acceptable carrier are also provided, together with preparations comprising at least one such composition and live sperm. In certain embodiments, the live sperm bear the X chromosome. Such preparations can be effectively employed in AI or in vitro fertilization.
In a further aspect, methods for improving the functionality and/or fertility of sperm are provided, such methods comprising contacting the sperm with an effective amount of a conjugate or composition disclosed herein. Such methods can be effectively employed with sperm from cows, pigs, sheep, goats, humans, camels, horses, deer, alpaca, dogs, cats, rabbits and rodents. In certain embodiments, the sperm are sorted into X and Y chromosome-bearing sperm either prior to or after contact with the conjugate or composition.
In yet another aspect, the present disclosure provides methods for making a preparation for use in AI or in vitro fertilization, such methods comprising obtaining sperm from a mammal, optionally separating the sperm into X chromosome-bearing and Y chromosome-bearing sperm, and contacting the sperm with an effective amount of a composition and/or conjugate disclosed herein. Methods for separating X chromosome-bearing sperm from Y chromosome-bearing sperm are known to those of skill in the art, and include, for example, flow cytometry. Such methods include, but are not limited to, those described in U.S. Pat. Nos. 5,135,759, 5,985,216, 6,149,867 and 6,263,745.
Methods for the cryopreservation of sperm are also provided by the present disclosure. Such methods comprise: (a) contacting the sperm with a cryoprotectant and an effective amount of a composition and/or conjugate disclosed herein, and (b) storing the sperm and the composition/construct at a temperature of about 4° C. to about −196° C., wherein the effective amount of the composition/conjugate is sufficient to increase the functionality and/or fertility of the sperm relative to sperm stored without the composition/conjugate. Examples of cryoprotectants that can be effectively employed in such methods include, but are not limited to, PEG, dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, polyvinyl pyrrolidone (PVP), polyethylene oxide, raffinose, lactose, trehalose, melibiose, melezitose, mannotriose, stachyose, dextran, hydroxy-ethyl starch, sucrose, maltitol, lactitol and glycerol. In related aspects, preparations comprising cryogenically preserved sperm and a composition and/or conjugate disclosed herein are provided. Methods for cryopreserving sperm are well known in the art and include those disclosed, for example, in U.S. Pat. No. 7,208,265 and US Patent Application Publication no. US 2007/0092860.
The methods, compositions and constructs disclosed herein are particularly advantageous in the preparation of semen for use in AI of mammals including, but not limited to, cows, pigs, sheep, goats, humans, camels, horses, deer, alpaca, dogs, cats, rabbits and rodents. Semen used in such methods may be either fresh ejaculate or may have been previously frozen and subsequently thawed.
These and additional features of the present invention and the manner of obtaining them will become apparent, and the invention will be best understood, by reference to the following more detailed description.
As outlined above, the present disclosure provides methods for improving the functionality and/or fertility of sperm, together with compositions and conjugates for use in such methods. In certain embodiments, the methods and compositions disclosed herein enhance sperm motility, protect sperm from phagocytosis, aid sperm in avoiding capture by mucus, extend the lifespan of sperm in the FRT, and/or enhance the function of a necessary sperm characteristic. The ability of a composition or conjugate to increase the functionality and/or fertility of sperm may be determined by contacting sperm with the composition or conjugate; measuring parameters such as the motility, resistance to neutrophil attack, membrane integrity and/or presence of sperm surface markers indicative of capacitation and acrosome status on the treated sperm and ability to recover from cryopreservation; and comparing the results with those obtained for untreated sperm. The functionality of sperm can also be determined by investigating their interaction with cervical mucus/explants or synthetic analogues, and/or their ability to capacitate, acrosome react and fertilize in vitro. Techniques for measuring the above parameters are well known in the art and include those described below. In certain embodiments, the disclosed methods comprise contacting the compositions and/or conjugates disclosed herein with either sorted or unsorted sperm.
The present disclosure provides methods for adding a functional molecule of interest to the surface of cells, such as sperm, using a conjugate including a membrane anchoring agent, PEG and the molecule of interest. Such conjugates provide protection or enhancement of sperm functionality while at the same time allowing sperm to maintain the array of molecular and cellular interactions that occur in ascent through the FRT. In certain embodiments, the disclosed conjugates have the following general structure: membrane anchoring agent-PEG-X-functional molecule, where X is any reactive group (referred to herein as an attachment group) that allows conjugation of at least one functional molecule of interest.
As used herein, the term “membrane anchoring agent” or “membrane anchoring component” refers to a molecule that is known to spontaneously and stably incorporate into lipid bilayers, including cell membranes. Examples of such molecules include, but are not limited to, the synthetic molecules described in US Patent Publication no. US 2007/0197466, the disclosure of which is hereby incorporated by reference. In certain embodiments, the membrane anchoring agent is a lipid. Lipids that may be effectively employed in the disclosed methods include, but are not limited to, diacyl- and dialkylglycerolipids, including glycerophospholipids and sphingosine derived diacyl- and dialkyl lipids, including ceramide. In certain embodiments, the lipid is selected from the group consisting of: cholesterol, diacylglycerolipids, phosphatidate, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol and phosphatidyl glycerol. The lipid may be derived from one or more cis-desaturated fatty acids.
Cholesterol is considered to be a desirable lipid membrane anchoring agent as this lipid is the most abundant molecule in sperm surface membranes (Flesch et al., 2001) and is lost upon capacitation in the oviduct. Thus the same mechanism that removes endogenous cholesterol (cholesterol chelating agents such as bovine serum albumin; BSA) may also remove some of the added conjugate before fertilization.
PEGs having a wide range of lengths, or molecular weights, and a varying number of branches can be effectively employed in the disclosed conjugates. For example, in certain embodiments the PEG has a molecular weight in the range of about 200 to about 40,000 daltons. PEGs contemplated for use in the conjugates disclosed herein include, but are not limited to, PEGs having one or more amine reactive groups that allow conjugation to a protein, and include linear and branched chain PEGs. As will be appreciated by those of skill in the art, when a PEG having an amine reactive group is employed in the conjugate, the attachment group (X) is the amine reactive group.
In certain embodiments, the attachment group (X) is an amine reactive group, however the attachment group can be any group that reacts with —COOH, —OH and/or —SH groups as well as disulfide (—S—S—) bonds and oxidized carbohydrates, on proteins or small molecules (Greenwald et al., 2003; Roberts et al., 2002). Examples of reactive groups that have previously been used to attach PEG to proteins or peptides are shown in Table 1 below. Alternatively, the membrane anchoring agent-PEG backbone can be linked to the functional molecule using a biotin-streptavidin linkage or click chemistry (Lutz and Zarafshani, 2008).
An example of the general structure of a cholesterol-PEG-attachment group starting tripartite molecule is shown in
Functional molecules of interest that can be added to sperm using the disclosed conjugates include amino acids and their derivatives, polyamino acids, peptides, enzymes, adhesion molecules, immune proteins and antigens. Specific examples of functional molecules of interest include antioxidants such as catalase and glutathione. Catalase and glutathione both protect sperm from H2O2, and if sperm are exposed to H2O2 in the FRT, membrane attached oxidation protection would aid sperm motility.
Other examples of functional molecules that can be effectively employed in the disclosed methods and conjugates include molecules that potentially protect sperm from immune attack, including CD55 (decay factor), CD59 and CD73 (Kirchhoff and Hale, 1996); or entrapment by neutrophils, such as DNase1 (Alghamdi and Foster, 2005). Membrane bound CD55 and CD59 inhibit the formation of complement induced membrane attack complex and could protect cells or sperm from complement attack (Fraser et al., 2003). CD55 and CD59 are glycosylphosphatidylinositol (GPI) linked proteins present on the surface of sperm and have specific roles in sperm function (Donev et al., 2008). Addition of proteins such as CD55 and CD59 to the cell surface also has application in other uses, such as transplantation protection (Hill et al., 2006). DNase1 present in bovine seminal fluid is known to be associated with sperm fertility (Bellin et al., 1998; McCauley et al., 1999).
Other molecules that are important for fertility and that can also be employed in the disclosed methods include SPAM1, which is also GPI linked and present in the epididymis (Kirchhoff et al., 1997). SPAM1 is a potential sperm adhesion molecule and hyaluronidase that enables sperm to penetrate through the hyaluronic acid-rich cumulus cell layer surrounding the oocyte (Lathrop et al., 1990).
If sperm surface charge is important for movement of sperm through the FRT then altering surface charge or charge distribution could improve fertility. Using the conjugate described here and a functional group composed of amino acids, amino acid derivatives, polymeric amino acids or peptides enables sperm surface charge to be manipulated. For example, reacting an amine reactive cholesterol-PEG with glycine would allow addition of one negative charge per conjugate, reaction with glutamic acid adds two negative charges per conjugate, γ-carboxy-glutamic acid adds three negative charges per conjugate, and poly(L-glutamic acid) in defined numbers of residues, such as 20 or 50 (available from Almanda Polymers), allows addition of 21 or 51 negative charges per conjugate, respectively.
In general, apart from sperm charge modification, the seminal fluid (Novak et al., 2010a; Novak et al., 2010b; Rodriguez-Martinez et al., 2011) or epididymal proteins (Belleannee et al., 2011) that bind to sperm, or proteins present on sperm, and are correlated with high fertility (D'Amours et al., 2010; Novak et al., 2010b) represent functional molecules that can be employed in the disclosed methods and conjugates for addition to sperm.
The addition of GPI lipid anchored proteins to sperm during sperm maturation occurs at least partly through a mechanism where epididymosomes transfer such proteins to the sperm surface (Frenette et al., 2006; Kirchhoff and Hale, 1996). Epididymosomes are small membranous vesicles secreted by epithelial cells within the luminal compartment of the epididymis (Girouard et al., 2009). The inventors believe that the addition of a cholesterol-PEG-functional group construct to sperm is analogous to the transfer of such GPI-linked proteins that occurs in the epididymis.
Proteins and/or polypeptides employed in the disclosed methods, compositions and conjugates can be isolated from seminal fluid (Kelly et al., 2006; Novak et al., 2010a; Novak et al., 2010b), epididymis or accessory sex glands (Moura et al., 2006a, 2007; Moura et al., 2006b) or other sources (e.g. catalase from bovine liver (Summer and Dounce, 1937)), or are commercially available. Alternatively, such proteins and/or polypeptides can be prepared recombinantly by inserting a polynucleotide that encodes the protein into an expression vector and expressing the antigen in an appropriate host. Any of a variety of expression vectors known to those of ordinary skill in the art may be employed. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. Preferably, the host cells employed are E. coli, mycobacteria, insect, yeast or a mammalian cell line such as COS or CHO.
The proteins and/or polypeptides employed in the methods, compositions and conjugates disclosed herein are isolated and purified, as those terms are commonly used in the art. Preferably, the proteins and/or polypeptides are isolated to a purity of at least 80% by weight, more preferably to a purity of at least 95% by weight, and most preferably to a purity of at least 99% by weight. In general, such purification may be achieved using, for example, the standard techniques of ammonium sulfate fractionation, SDS-PAGE electrophoresis, and affinity chromatography.
The conjugates and compositions disclosed herein encompass variant polypeptide sequences that have been modified by one or more amino acid deletions, additions and/or substitutions. Variant sequences preferably exhibit at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably yet at least 95%, and most preferably at least 98% identity to a specific polypeptide sequence disclosed herein. The percentage identity is determined by aligning the two sequences to be compared as described below, determining the number of identical residues in the aligned portion, dividing that number by the total number of residues in the inventive (queried) sequence, and multiplying the result by 100. In addition to exhibiting the recited level of sequence identity, variant sequences preferably exhibit a functionality that is substantially similar to the functionality of the specific sequences disclosed herein. Preferably a variant polypeptide sequence will have at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably yet at least 95%, and most preferably 100% of the sperm fertility enhancing activity possessed by the specifically identified polypeptide sequence in one or more sperm fertility assays, such those described below. Such variants may generally be identified by modifying one of the polypeptide sequences disclosed herein, and evaluating the properties of the modified polypeptide using, for example, the representative procedures described herein.
In certain embodiments, variant sequences differ from the specifically identified sequence only by conservative substitutions, deletions or modifications. As used herein, a “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. In general, the following groups of amino acids represent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Variants may also, or alternatively, contain other modifications, including the deletion or addition of amino acids that have minimal influence on the antigenic properties, secondary structure and hydropathic nature of the polypeptide. For example, a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide in the conjugate.
Polypeptide sequences may be aligned, and percentages of identical amino acids in a specified region may be determined against another polypeptide, using computer algorithms that are publicly available, such as the BLASTP algorithm. BLASTX and FASTX algorithms compare nucleotide query sequences translated in all reading frames against polypeptide sequences. The use of the BLAST family of algorithms is described at NCBI's website and in the publications of Altschul et al. (Altschul et al., 1990; Altschul et al., 1997). The “hits” to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, FASTA, or a similar algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.
In certain embodiments of the disclosed methods, sperm are purified by a single density layer (Percoll™ PLUS, GE Healthcare, see protocol below). Sperm are then incubated in a suitable media with an effective amount of one or more of the compositions and/or conjugates disclosed herein for a short period of time, followed optionally by the addition of a suitable extender to enable immediate use or freezing. Alternatively, the compositions and/or conjugates are added directly to the ejaculate and, after slight dilution, a short incubation (15-30 minutes) and the addition of extender, the resulting mixture is either cooled or frozen for storage. In another method, the compositions and/or conjugates are added to extended semen. In other embodiments, sperm are sexed by flow cytometry and are collected in media containing an effective amount of one or more of the disclosed compositions and/or conjugates. Alternatively, once sufficient sorted sperm are collected, the composition and/or conjugate is added and the resulting mixture is incubated in a suitable media for a short period of time, followed by the addition of extender and then either immediate use or freezing.
As used herein, the term “effective amount” of a composition and/or conjugates disclosed herein refers to that amount sufficient to enhance sperm motility, protect sperm from phagocytosis, allow sperm to avoid capture by mucus, extend the lifespan of sperm in the FRT, and/or increase sperm functionality by at least 5-50% compared to untreated sperm.
Those of skill in the art will appreciate that for use in the disclosed methods, the compositions and conjugates disclosed herein may be present in compositions including one or more physiologically acceptable carriers or diluents, such as water or saline. Such compositions may additionally contain other components, such as preservatives, stabilizers, buffers and the like. Carriers, diluents and other components suitable for use in the present compositions are well known to those of skill in the art and include those currently used in preparations for AI.
All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, non-patent publications, tables, sequences, web pages, or the like referred to in this specification, are incorporated herein by reference, in their entirety. The following examples are intended to illustrate, but not limit, this disclosure.
Equal volumes of 2 mM cholesterol-PEG5000-NHS-FITC (Nanocs) and 20 μM bovine catalase (Sigma; 100:1 ratio of cholesterol-PEG5000-NHS-FITC to bovine catalase) in phosphate buffered saline (PBS) were mixed by rotation for 3 hr at room temperature. The mixed solution was then dialysed into PBS using a 50 kDa molecular weight cut off (MWCO) membrane at 4° C. overnight. The free cholesterol-PEG5000-NHS-FITC that had not reacted with the catalase was removed using an ammonium sulphate precipitation where 200 μl of 4.1 M saturated ammonium sulphate solution was added slowly into 500 μl of the cholesterol-PEG5000-NHS-FITC/catalase mixture. Centrifugation of the sample mixture for 20 min at 20,000×g separated the free cholesterol-PEG5000-NHS-FITC as a pellet, and the reacted cholesterol-PEG5000-FITC-catalase as supernatant. Both supernatant and pellet were then dialysed into PBS using a 10 kDa MWCO membrane at 4° C. overnight. Dialysed samples were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The resulting gel was subjected to fluorescence imaging using an ImageQuant LAS-4000 (GE Healthcare), and stained with the Coomassie Brilliant Blue R-250 to visualize the protein bands. This combined gel analysis indicated that the catalase had been labeled with cholesterol-PEG and that the cholesterol-PEG that had not reacted with catalase was removed by precipitation (cholesterol-PEG-catalase>95% purity). The average number of cholesterol-PEG molecules added per catalase monomer was determined by measuring the fluorescence of known protein amount of cholesterol-PEG5000-NHS-FITC and employing a standard curve of free cholesterol-PEG5000-NHS-FITC concentration versus fluorescence. Depending upon the preparation of cholesterol-PEG-catalase, the range varied from 4.0 to 5.4 molecules of cholesterol-PEG per catalase monomer or 4× this number for the intact tetramer.
Recovery of the cholesterol-PEG5000-FITC-catalase was determined by Coomassie staining and catalase activity assay. For the catalase activity assay, 0.5 μl of test samples and serially diluted bovine catalase (Sigma) were placed in a microtitre plate, and incubated with 50 μl of 10 ng/ml of streptavidin-HRP (Biosource) and 50 μl of TMB substrate (Invitrogen) for 5 min. The plate was read at 450 nm on an EnVision plate reader (PerkinElmer) after stopping the reaction with 50 μl of 2 M sulfuric acid. Catalase activity of the test sample was calculated from the standard curve generated with known concentrations of bovine catalase. The catalase activity assay showed 85% recovery (equivalent to 1.11 mg; 44,406 units) of catalase in the form of cholesterol-PEG5000-FITC-catalase.
1.5 ml of bovine sperm in liquid extender was carefully placed on top of 4 ml of 60%_Percoll™ PLUS (GE Healthcare) column, and centrifugated for 20 min at 700×g at 20° C. The resulting purified sperm pellet was resuspended in non-capacitating media (NCM; see Table 2) containing 0.1 mg/ml of BSA to a cell concentration of 5×107 ml. 200 μl of the purified sperm was mixed with an equal volume of 3 mg/ml cholesterol-PEG5000-FITC-catalase, and incubated light protected at 37° C. for 30 min. The unbound cholesterol-PEG5000-FITC-catalase was removed by layering 400 μl of sperm/cholesterol-PEG5000-FITC-catalase mixture on the top of 500 μl of 60% Percoll™ PLUS column, followed by centrifugation for 20 min at 700×g at 20° C. The sperm pellet was resuspended in NCM containing 0.1 mg/ml of BSA to a total volume of 400 μl. The binding of cholesterol-PEG5000-FITC-catalase to sperm was analysed by flow cytometry and catalase activity assay. For flow cytometry analysis, the cholesterol-PEG5000-FITC-catalase bound sperm were diluted to 5×106 cells/ml in NCM containing 0.1 mg/ml of BSA, and stained with 0.2 μg/ml of a viability dye (Hoechst 33258). Significant binding (approx. 10 fold over background) of cholesterol-PEG5000-FITC-catalase to live sperm (Hoechst 33258 negative) was observed (see
Jurkat cells (ATCC, TIB-152; human T lymphocyte cell line) were cultured in RPMI-1640 media with 10% fetal calf serum. For analysis, cells were removed from culture, centrifuged at 700×g for 5 min, the culture media was removed and cells were resuspended at a concentration of 5×107 cells/ml in PBS. 100 μl of the Jurkat cell suspension was mixed with 100 μl of 2 mg/ml cholesterol-PEG5000-FITC-catalase and incubated light protected at 37° C. for 30 min. Cells were centrifuged for 5 min at 700×g, supernatant removed, and resuspended in 400 μl of fresh PBS. The Jurkat cells were analyzed by both flow cytometry and catalase activity assay.
For flow cytometry analysis, the Jurkat cells were diluted in 200 μl of PBS to a concentration of 5×106 cells/ml. 0.2 μg/ml of Hoechst 33258 (Invitrogen, H21491) was then added to each sample (see
The catalase activity of the treated Jurkat cells was measured as described in Example 1. 50 μl of 5×107 cells/ml of each test sample was analysed in the assay and the catalase activity contained in each of the Jurkat samples was calculated from the standard curve. The catalase activity assay indicated: 0 U of catalase in freshly washed Jurkat cells, 44 U in Jurkat cells with catalase alone without linker, 66 U in Jurkat cells with cholesterol-PEG5000-FITC-catalase (units per 2.5×106 cells).
In this experimental configuration, a concentration of H2O2 is chosen that causes sperm to rapidly lose motility (30-60 minutes) unless oxidation protection like catalase is present. Sperm that have had cholesterol-PEG-catalase added and been washed so that no remaining free cholesterol-PEG-catalase remains are compared with sperm that have been exposed to a similar molar amount of catalase as contained in the cholesterol-PEG-catalase and then washed, and also with sperm exposed to no catalase, for their ability to resist H2O2 induced motility loss. Sperm motility is determined by a quantification system such as QualiSperm™ (Biophos).
Bovine semen samples were collected from three bulls using an artificial vagina and pooled. The pooled semen was centrifugated for 15 min at 1500×g to remove sperm cells and the resulting supernatant was further centrifugated for 15 min at 15000×g to remove any particulates. Complete mini protease inhibitor cocktail was added to the cleared seminal plasma before storage at −20° C. Protein concentration was measured by bicinchoninic acid (BCA) protein assay kit (Pierce).
Two methods were used to prepare peptides from bovine seminal plasma for proteomic analysis. The first method used standard in-solution digestion of proteins. Briefly, the seminal plasma was diluted in lysis buffer consisting of 7 M urea, 2 M thiourea, 4% CHAPS and 2 mM DTT, and incubated for 1 hr at 4° C. with constant rotation. Following centrifugation at 14000×g for 5 min at 4° C., an aliquot was removed for protein estimation by EZQ™ protein quantitation kit (Molecular Probes). 30 μl of cleared seminal plasma containing approximately 100 μg of proteins was alkylated for 30 min with 2-fold molar excess of iodoacetamide relative to the DTT. Proteins in the seminal plasma were then precipitated with methanol/chloroform. The resulting protein pellet was reconstituted in 0.5 M TEAB and 1 M urea containing 0.1 mg/ml trypsin, and incubated overnight at 37° C. Filter-aided sample preparation method (FASP II) (Wisniewski et al., 2009) was the second method used for peptide preparation from seminal plasma.
Acetonitrile was added to 5% to all peptide samples, before acidifying the peptides to pH 2 to 3 with formic acid. Peptides were then desalted on Sep-Pak Vac tC18 solid phase extraction cartridge (Waters), and completely dried in vacuum concentrator.
Dried peptide samples were sent to the Australian proteome analysis facility (APAF), provided by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS). At APAF, high sensitivity amino acid analysis was carried out to accurately measure the amount of peptides in each sample. Peptide samples were loaded onto a Capillary LC system coupled to an MS/MS instrument. The peptides were separated using a reverse phase C18 column and directly eluted into a Q-STAR mass spectrometer. 1D-LC-ESI-MS data acquired was analysed by ProteinPilot software 3.0 (ABI) to identify the proteins. A thorough identification search was conducted in ProteinPilot. The International Protein Index (IPI) Bos taurus database (v3.49) was used for all searches. Proteins identified from two samples prepared by two different peptide preparation methods were compared to each other. Ensembl genome browser (www.ensembl.org) was used to check the presence of transmembrane domains and signal peptide sequence in each identified protein.
A list of 73 seminal plasma proteins that may enhance functionality and/or fertility of sperm is provided in Table 3. The sequences for these proteins are provided in SEQ ID NO: 7-79, respectively. These 73 proteins were detected in two independently prepared seminal plasma samples, and also only those predicted to have signal peptide sequences were selected. Three seminal plasma proteins that were detected and that had multiple transmembrane domains were excluded from the list.
200 μl of extended bovine semen was loaded onto 2 ml 50% Percoll™ PLUS column, and centrifugated at 1200×g for 20 min at room temperature. 5×106 purified sperm cells resuspended in 1 ml of NCM (Table 2) were incubated with 10 μg/ml of biotinylated WGA (Vector Laboratories) for an hour at 28° C. on a rotating platform. 25 μl of washed streptavidin Dynabeads™ (Invitrogen) were then incubated with the sperm for an hour at 28° C. Dynabeads™/sperm complex was placed on magnet and washed three times with NCM. A minimal number of sperm was found in the supernatant, indicating that most sperm (>95%) formed a complex with the Dynabeads.
b) Peptide Preparation for iTRAQ Labeling
500 μl of lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS and 13 mM DTT was added to the Dynabeads™/5×106 sperm complex, vortexed, and incubated for 1 hr at 4° C. on a rotating platform. Dynabeads™/sperm complex was then removed by magnet, and the supernatant was centrifugated at 14000×g for 5 min to remove any remaining insoluble material. Protein estimation was performed using an EZQ™ protein quantitation kit (Molecular Probes). Filter-aided sample preparation method (FASP II) (Wisniewski et al., 2009) was then used to prepare peptides from the sperm lysate. Peptide samples were added to 5% acetonitrile, and acidified with formic acid to pH 2 to 3. Samples were then desalted on Sep-Pak Vac tC18 solid phase extraction cartridge (Waters), and completely dried in a vacuum concentrator.
c) iTRAQ Proteome Analysis
Dried peptide samples were sent to the APAF in Sydney for high sensitivity amino acid analysis and mass spectrometry analysis. Isobaric tags for relative and absolute quantitation (iTRAQ) was used for simultaneous identification and quantification of multiple peptide samples. 4-plex or 8-plex iTRAQ reagents (SCIEX) were used to analyse 4 or 8 different biological samples in a single experiment, respectively. In each iTRAQ experiment, an equal amount of each peptide sample was labeled with a different iTRAQ reagent. Mixed iTRAQ-labeled peptide sample was then loaded onto a strong cation ion exchange (SCX) column and fractionated into 20 fractions. Each fraction was separated by reverse-phase gradient and injected into a Q-STAR Elite mass spectrometer. 2D-nanoLC-ESI-MS/MS data acquired was then analysed by ProteinPilot software 3.0 (ABI), and relative quantitation and protein identification were obtained. Paragon algorithm was used to perform database matching for protein identification, protein grouping to remove abundant hits, and comparative quantitation. A thorough identification search was conducted in ProteinPilot. The IPI Bos taurus database (v3.49) was used for all searches. The data were normalized for loading error by bias correction using ProteinPilot. Proteins identified in multiple iTRAQ experiments were compared to each other, and also to the proteins identified in seminal plasma samples. Ensembl genome browser (www.ensembl.org) was used to check the presence of transmembrane domains and signal peptide sequence in each identified protein.
A list of 84 proteins in bovine sperm that may enhance sperm functionality and are likely to be on the sperm surface is provided in the Table 4. The sequences for these 84 proteins are provided in SEQ ID NO: 80-163, respectively. These proteins were selected from a total of 2206 proteins identified across 19 different sperm lysates by the following criteria: unique proteins with a signal sequence that occurred in at least two experiments and that were not listed in Table 3, and additionally, proteins with known mitochondrial subcellular location or more than one transmembrane domain were omitted.
A series of experiments are performed in vitro to determine the ability of a cholesterol-PEG-functional group conjugate to improve various measures of sperm functionality. Treated and untreated sperm are compared for changes in the following characteristics: motility; membrane integrity; mitochondrial membrane potential; membrane fluidity; chromatin integrity; lipid peroxidation; capacitation; acrosome reaction; binding of antibodies, heparin and lectins to the sperm surface (or modified sperm surface proteins); ability of sperm to migrate in the FRT; the resistance of sperm to phagocytosis; and the ability of sperm to fertilize in vitro (see Table 5 for details).
In this model, as detailed below, bovine sperm are incubated overnight in NCM under non-capacitating conditions (simulating the conditions sperm experience for the majority of the journey in the FRT, starting cell viability approximately 90%). Following overnight incubation, sperm are diluted in capacitating media (CM; Table 6), triggering capacitation with high efficiency and minor loss of viability (cell viability in the 75-85% range). In typical experiments, when bovine sperm are capacitated with caffeine, db-cAMP and IBMX (3-isobutyl-1-methylxanthine), greater than 95% of viable cells capacitate as assessed by WGA-fluorescein/Annexin V or merocyanine 540 binding (see Table 5; WGA staining is the most sensitive, with approximately 10-fold shift in the staining upon capacitation). When cells are capacitated in vitro they also gain the capacity to acrosome react (Table 5). Although the combination of caffeine, db-cAMP and IBMX is an efficient inducer of capacitation, when more in vivo like capacitation induction is required, heparin is used. Sperm treated with cholesterol-PEG-functional molecule are compared with untreated sperm for their ability to capacitate, in particular using heparin induction method.
A 90% Percoll™ PLUS solution is made by adding 10×NCM to Percoll™ PLUS. A 60% single layer gradient is then made by dilution with 1×NCM. In the standard method, 4 ml of 60% Percoll™ PLUS/NCM is added in a 15 ml tube, 1.5 ml of ejaculate in liquid extender (standard tris-egg yolk, extension 1:4 egg yolk-citrate-glycerol) is then gently loaded on top, and centrifuged at 700×g for 20 min at room temperature. The pellet is removed and washed once in 8 ml of NCM by centrifugation for 5 min at 700×g. The supernatant is then removed and the pellet resuspended in 1 ml of NCM. Capacitation treatment tubes are set up at a sperm concentration of 5×107 cells/ml.
Samples are prepared for flow cytometry analysis as follows. The components shown in Table 7 below are incubated with 5×105 Percoll™ PLUS-purified bovine sperm in a final volume of 200 μl at room temperature for 10 min, while propidium iodide (PI) is added just before analyzing by flow cytometry.
Percoll™ PLUS-purified bovine sperm at 5×107 cells/ml concentration are incubated in NCM overnight in a 28° C. water bath. The sperm are then visually assessed under inverted bright field microscope and/or using QualiSperm prior to inducing capacitation.
d) Day 2—Transition of Cells from Non-Capacitating Media to Capacitating Media
After overnight incubation, the cells are diluted in to CM (Table 2). Specifically, 1 ml of overnight incubated sperm is diluted 1:1 with 1 ml of CM media. Activators for capacitation, specifically caffeine and db-cAMP are added at a final concentration of 1 mM (˜16 hours after incubation started), and IBMX is added at a final concentration of 100 mM. Alternatively, bovine sperm capacitation is induced using heparin or methylbeta cyclodextrin (cholesterol acceptor). Samples are then incubated for an hour at 37° C.
Similar to day 1, bovine sperm samples are then incubated with fluorescently labeled SBTI, PNA and WGA for 10 min and PI added just prior to flow analysis.
Achieving pregnancy is dependent upon both the male and female fertility, and also upon other factors (such as management of animals, parity, age, environment, insemination procedure etc.) and thus analysis of male fertility usually requires large numbers of animals in trials (Amann and Hammerstedt, 2002). At least for the bovine, the large number of sperm/ejaculate and also careful study design mean that many sources of variation can be controlled. In cattle, AI trials have been conducted to look at number of sperm required for insemination either alone (Den Daas et al., 1998) or in conjunction with other variables such as flow cytometry sorting (Bodmer et al., 2005), extender composition or other modification (Amann et al., 1999). The basic design is a sperm dose response using several bulls and a large number of cows (Den Daas et al., 1998).
In alternative studies, heterospermic inseminations with mixtures of treated and non-treated (control) sperm are employed to quickly determine functionality and/or fertility of the treated sperm. In this experimental design, two distinguishable types of sperm are inseminated simultaneously, with the aim being to compare the different types of sperm and thus remove female fertility as an experimental variable. Previous reports have described heterospermic insemination using sperm from multiple bulls (Dziuk, 1996; Flint et al., 2003), and a few methods have been developed together with various techniques to assess the success of the sperm (Flint et al., 2003; Parrish and Foote, 1985).
In specific studies, semen is collected from a single bull and sperm are either treated with a cholesterol-PEG-functional molecule or left untreated. Treated and untreated (control) sperm are labelled with two different fluorescent dyes (such as Hoechst 33342 and Vybrant DyeCycle stains) to enable the control and treated sperm to be distinguished. Equal amounts of the treated and control sperm are then simultaneously inseminated into the same cow, and reciprocal studies are also carried out to ensure effects on sperm transport are not due to the marker fluorescent dye. Twelve to sixteen hours after heterospermic insemination the cow is slaughtered, the uterus and oviduct removed, and the ratio of treated and control sperm in the upper uterine horn and oviduct is determined. Significantly increased number of treated sperm are present in the upper uterine horn and oviduct compared to untreated (control) sperm when the treatment successfully improves sperm functionality.
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, method, method step or steps, for use in practicing the present invention. All such modifications are intended to be within the scope of the claims appended hereto.
SEQ ID NO: 1-163 are set out in the attached Sequence Listing. The codes for nucleotide sequences used in the attached Sequence Listing, including the symbol “n,” conform to WIPO Standard ST.25 (1998), Appendix 2, Table 1.
Aitken, R. J., Bowie, H., Buckingham, D., Harkiss, D., Richardson, D. W., and West, K. M. (1992). Sperm penetration into a hyaluronic acid polymer as a means of monitoring functional competence. J Androl 13, 44-54.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/NZ2012/000140 | 8/8/2012 | WO | 00 | 5/1/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61522609 | Aug 2011 | US |
