WO2011108946A1 - Methods for improving sperm functionality - Google Patents

Abstract

Methods for improving the functionality and/or fertility of sperm, for example, by enhancing motility and extending the lifespan of sperm in the female reproductive tract, are provided, such methods comprising contacting semen and/or sperm with an effective amount of a composition comprising a mitochondria targeted antioxidant.

Description

METHODS FOR IMPROVING SPERM FUNCTIONALITY

FIELD OF THE INVENTION

This application relates to methods for enhancing the functionality of sperm. More particularly, this application relates to methods for reducing the number of sperm used in livestock artificial insemination (AI), especially 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.

BACKGROUND

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). 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 (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 limited to a rate of 8000 s^"1, and when each straw contains 2 x 10⁶ sperm, this rate 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 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 x 10⁶ sperm 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, namely approximately 2500 x 10⁶ sperm. 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 commercial capability of flow cytometry sorted sperm.

The sperm journey in the female reproductive tract

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 one billion sperm per 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 muscle contractions 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 presumably increases the probability that at least some sperm will be present in the oviduct at the same time as ovulation occurs. However, such retention may also act as a negative selection imposed against sperm by the female. 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 this complex journey 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 cannot fertilize 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). 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 (Schroter et al., 1999) stabilizes the surface membrane, regulates capacitation (Topfer-Petersen et al., 1998), facilitates transport through the FRT (Tollner 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 molecular components do vary (Calvete and Sanz, 2007; Tollner et al., 2008a; Topfer-Petersen et al., 1998).

The attrition of sperm in the female reproductive tract

In a natural bovine mating, approximately one 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 only 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 bovines, only 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 (PMNs), with a massive infiltration of PMNs occurring in the uterine lumen shortly after insemination (Matthijs et al., 2003). Recently, similar evidence that PMNs also infiltrate the uterine lumen after insemination in cows has been published (Alghamdi et al., 2009).

How sperm are damaged during passage through the female reproductive tract

Experimental evidence suggests that both damaged/immotile/dead and intact sperm are lost by discharge and phagocytosis (Lightfoot and Restall, 1971; Oren-Benaroya et al., 2007) (Oren-Benaroya et al., 2007). Several phenomena contribute to sperm damage from the FRT, although the mechanism and significance are poorly understood. Such phenomena include:

• Adhesive properties of female epithelia capturing sperm and/or damaging the sperm surface, particularly mucus laden surfaces such as the cervix. This occurs by both biochemical and physical shearing (Katz et al., 1989; Mullins and Saacke, 1989).

• Female secretions modulating/damaging the sperm surface or functionality such as flagella activity, capacitation and acrosome status. Such secretions include antibodies, molecular species affecting energy and osmotic homeostasis, signaling molecules particularly for capacitation, and also catabolic entities.

• 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 later damage leads to alteration of 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). The use of fluorescent probes specific for mitochondria membrane potential, such as rhodamine 123, JC-1 and Mitotracker™ deep red 633, has demonstrated that in bull sperm there is a strong relationship between mitochondrial membrane potential and motility (Garner et al., 1997; Hallap et al., 2005). However, in boar sperm the inhibitory effects of ROS on motility indicate a mitochondria independent mechanism (Guthrie et al., 2008). Thus, the damaging effects of ROS on sperm motility may not directly reduce mitochondria membrane potential and hence oxidative phosphorylation and ATP production. Instead the mechanism of action may operate through impairment of ATP utilization or the contractile apparatus of the flagellum (Guthrie et al., 2008).

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 remaining undamaged (mainly a surface phenomenon), not being phagocytosed, remaining motile (a function of mitochondria, glycolytic enzymes and flagella components), and being able to respond to signals appropriately (a surface phenomenon but also involving signal transduction and effector pathways). Thus treatments to sperm that enhance the ability of sperm to remain undamaged, motile, not phagocytosed and functionally competent could reduce the number of sperm required for insemination.

SUMMARY

In one aspect, methods for improving the functionality and/or fertility of sperm, for example, by enhancing motility and extending the lifespan of sperm in the FRT, are provided, such methods comprising contacting semen and/or sperm with an effective amount of a composition comprising a mitochondria targeted antioxidant. The disclosed methods may be used in AI, for example, to reduce the number of sperm needed for insemination and to improve conception rates.

In one embodiment, the mitochondria targeted antioxidant comprises an antioxidant conjugated to a carrier that specifically targets mitochondria. In certain embodiments, the carrier is a lipophilic cation such as, but not limited to, a triphenylphosphonium cation, an alkyl-triphenylphosphonium cation, a tribenzyl ammonium cation or a phosphonium cation. Other lipophilic cationic molecules that can act as carriers include fluorescent dyes that are known to accumulate in the mitochondria, such as rhodamine 123 (Antonenko et al., 2008; Chen, 1988), and carbocyanine based dyes such as JC-1 (Rottenberg and Wu, 1998). For a review see (Plasek and Sigler, 1996). The antioxidant may be, for example, ubiquinone, tocopherol, nitroxide, a superoxide dismutase mimetic (such as M40403), a glutathione peroxidase mimetic, ebselen, plastoquinone, lipoic acid (Antonenko et al., 2008; Smith et al., 2008), or a derivative thereof. In specific embodiments, the mitochondria targeted antioxidant is mitoquinone, 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SKQ1), 10-(6'-plastoquinonyl) decylrhodamine 19 (SKQR1), 10-(6'-plastoquinonyl) decylcarnitine (SKQ2), 10-(6'-plastoquinonyl) decylmethylcarnitine (SKQ2M), 10-(6'-methyl-plastoquinonyl) decyltriphenylphosphonium (SKQ3), 10-(6'-plastoquinonyl) decyl-tributylammonium (SKQ4), or 5-(6'-plastoquinonyl) amyltriphenylphosphonium (SKQ5). Those of skill in the art will appreciate that other carriers and antioxidants known in the art may be effectively employed in the disclosed methods.

In another embodiment, the mitochondria targeted antioxidant comprises an aromatic-cationic peptide such as, but not limited to, H-Tyr-D-Arg-Phe-Lys-NH₂, H-Dmt- D-Arg-Phe-Lys-NH₂, H-D-Arg-Dmt-Lys-Phe-NH₂ or H-Phe-D-Arg-Phe-Lys-NH₂.

In another aspect, methods for preparing a composition for use in AI or in vitro fertilization are provided. Such methods comprise obtaining a semen sample from a mammal, and contacting the semen with a mitochondria targeted antioxidant for an amount of time sufficient to increase the functionality and/or fertility of sperm. In one embodiment, the mitochondria targeted antioxidant is added to seminal fluid and extender, and incubated. The resulting solution is then aliquoted and frozen until use. In certain embodiments, the semen is sorted to separate X chromosome bearing sperm from Y chromosome bearing sperm, using methods known to those of skill in the art, such as flow cytometry. Methods to sort semen for sperm bearing the X or Y chromosome using a flow cytometer include those described, for example, in US Patents No. 5,135,759, 5,985,216, 6,149,867 and 6,263,745. The mitochondria targeted antioxidant can be added to the solution that the sorted sperm are collected in and incubated, prior to being aliquoted and frozen.

In a related aspect, preparations for use in AI or in vitro fertilization are provided. Such preparations comprise live sperm and a mitochondria targeted antioxidant. The mitochondria targeted antioxidant is generally present in an amount sufficient to increase the functionality and/or fertility of the sperm. In certain embodiments, preparations for use in AI contain significantly more X chromosome bearing sperm than Y chromosome bearing sperm.

In yet a further aspect, methods for the cryopreservation of sperm are provided. Such methods comprise (a) contacting the sperm with a cryoprotectant and a composition comprising an effective amount of a mitochondria targeted antioxidant, and (b) storing the sperm and the composition at a temperature of about 4°C to about -196°C, wherein the effective amount of the mitochondria targeted antioxidant is sufficient to increase the functionality, fertility and/or motility, and/or to extend the lifespan of the sperm relative to sperm stored without mitochondria targeted antioxidant. Examples of cryoprotectants that can be effectively employed in such methods include, but are not limited to, polyethylene glycol, DMSO, ethylene glycol, propylene glycol, polyvinyl pyrrolidone, polyethylene oxide, rafiinose, lactose, trehalose, melibiose, melezitose, mannotriose, stachyose, dextran, hydroxy-ethyl starch, sucrose, maltitol, lactitol and glycerol. In related aspects, compositions comprising cryogenically preserved sperm and a mitochondria targeted antioxidant are provided. Methods for cryopreserving sperm are well known in the art and include those disclosed, for example, in US Patent 7,208,265 and US Patent Application Publication no. US 2007/0092860.

The methods 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.

In a further aspect, methods are provided for determining the ability of a composition to modify the functionality of sperm, the methods comprising: (a) contacting sperm obtained from a male with the composition to provide treated sperm; (b) labeling the treated sperm with a first detection agent, such as a fluorescent dye, to provide labeled treated sperm; (c) labeling non-treated sperm obtained from the male with a second, different, detection agent to provide labeled non-treated sperm; (d) simultaneously inseminating a female with the labeled treated sperm and the labeled non-treated sperm; and (e) determining a ratio of labeled treated sperm to labeled non-treated sperm present in the oviduct of the female.

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.

DETAILED DESCRIPTION

As outlined above, the present disclosure provides methods for improving the functionality and/or fertility of sperm, together with compositions for use in such methods.

In bovine sperm, a high level of lipid peroxidation for each ejaculate is negatively correlated with competitive indices for siring calves (Kasimanickam et al., 2007). While sperm and seminal fluid contain antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase and catalase (Alvarez et al., 1987; Drevet, 2006; Mennella and

Jones, 1980; Ursini et al., 1999), and small molecule antioxidants such as glutathione (de

Lamirande and Gagnon, 1995), the action of such antioxidant compounds appears to be limited for most sperm (Alvarez et al., 1987). However, the importance of such protection in the development of sperm has recently been demonstrated. For example mice deficient for the glutathione peroxidase 4 gene are completely infertile (Imai et al., 2009; Schneider et al., 2009). It has been shown that a very strong relationship exists between the amount of human sperm SOD activity and time to total loss of sperm motility (Alvarez et al., 1987). Further, a portion of some human sperm ejaculates have been shown to have tenfold higher SOD activity and correspondingly longer periods of time before total loss of motility (Storey, 1997). The contrary also appears to be true, namely defects in sperm antioxidant defenses appear to result in significant loss of sperm fertility (Chabory et al., 2009; Storey, 1997). . Thus it appears that fertility of sperm would be enhanced if additional antioxidants were delivered and carried with sperm, particularly if those antioxidants are targeted to the location of the majority of ROS production in sperm.

Studies have shown that mitochondria are the site for the majority of ROS production in sperm (Boveris and Chance, 1973; Koppers et al., 2008; Vernet et al., 2001). These studies are supported by the fact that mitochondria are strictly segregated to the mid- piece of sperm, and that lipid peroxidation is most prominent at the mid-piece and virtually absent from the head in bovine sperm (Brouwers and Gadella, 2003). ROS cause loss of mitochondrial membrane potential which is known to be vital to the maintenance of mitochondrial function and integrity (Koopman et al., 2010).

Although ROS are responsible for damage in sperm, low levels are believed to specifically mediate signaling in the process of capacitation, particularly triggering and modulating protein phosphorylation (O'Flaherty et al., 2006). The location of these events appears to be restricted to the surface membrane and fibrous sheath (O'Flaherty et al., 2006). Thus the inventors believe that, due to the unusual compartmentalization of the sperm cell, in particular segregation of the mitochondria to the sperm mid-piece, provision of an appropriate concentration of mitochondria targeted antioxidant will result in lowered mitochondrial ROS while still mamtaining the ability of the sperm to capacitate in response to the appropriate signals.

^' The inventors believe that mitochondria targeted antioxidants will minimize mitochondrial oxidative stress, enhance mitochondrial energy production and prevent cell death (Suleiman et al., 1996; Szeto, 2008). Such improved sperm function will result in both enhanced motility and extended lifespan of sperm in the FRT, and therefore result in less sperm being required for Al. This reduction in required sperm numbers will have two components: first that less sperm will lose motility and be phagocytosed, and second that if sperm have an extended lifespan in the FRT there is more chance that sperm will be present at ovulation.

DNA damage in the male germ line is a major contributor to infertility, miscarriage and birth defects in offspring (Aitken et al., 2009). In humans, it is speculated that a large percentage of such DNA damage results from oxidative damage, particularly in sperm with poorly protaminated chromatin generated as a result of defective spermiogenesis (Aitken and De Iuliis, 2009). Mitochondria targeted antioxidant compounds may therefore also be employed to help human males that have infertility problems due to low antioxidant levels, excessive production of oxidants or poor packing of their sperm chromatin (Aitken and De Iuliis, 2009). In one embodiment disclosed herein, sperm from a human male are incubated with a mitochondria targeted antioxidant compound and then administered by AL

Cryopreservation is a common storage method employed in the preservation of sperm. However considerable loss of fertility results from the procedure (Meseguer et al., 2004). At least part of the damage appears to result from ROS production (Brouwers and Gadella, 2003; Park et al., 2003; Sariozkan et al., 2009). Although data obtained to date has shown improved in vitro sperm parameters upon addition of antioxidants such as cysteine, this has not translated into improved conception rates (Sariozkan et al., 2009). The inventors believe that mitochondria targeted antioxidants, by nature of the specific targeting, have advantages that other antioxidants do not and thus may be employed to reduce the loss of fertility currently associated with sperm cryopreservation.

The methods disclosed herein employ compositions including mitochondria targeted antioxidants to increase the functionality and/or fertility of sperm. In certain embodiments, such compositions enhance motility and extend the lifespan of sperm in the FRT. As used herein, the term "effective amount" of a mitochondria targeted antioxidant refers to that amount sufficient to enhance sperm motility and/or extend the lifespan of sperm in the FRT, by at least 5-50% compared to untreated sperm.

The ability of a composition to increase the functionality and/or fertility of sperm may be determined by contacting sperm with the composition; measuring parameters such as the motility, membrane integrity, mitochondrial membrane potential, ROS generation, lipid peroxidation, and/or presence of sperm surface markers indicative of capacitation and acrosome status on the treated sperm, and comparing the results with those obtained for untreated sperm. Techniques for measuring such parameters are well known in the art and include those described below in Example 2. As used herein, the term "mitochondria targeted antioxidant" refers to a compound that has antioxidant activity and is capable of passing through the cell membrane and specifically targeting the mitochondria. Using such compounds, an antioxidant moiety can be transported through the mitochondrial membrane and accumulate within the mitochondria of intact cells.

A first class of compounds that can be effectively employed as mitochondria targeted antioxidants comprise a lipophilic cation that targets the mitochondria, conjugated, or covalently coupled, to an antioxidant moiety. Examples of antioxidants that can be effectively employed in such compounds include, but are not limited to, ubiquinone, tocopherol, nitroxide, superoxide dismutase mimetics (such as M40403), glutathione peroxidase mimetics (see, for example, US Patent no. 7,109,189), ebselen, plastoquinone, lipoic acid and derivatives thereof. Examples of lipophilic cations that can be effectively employed in such compounds include triphenylphosphonium, alkyl-triphenylphosphonium, tribenzyl ammonium and phosphonium cations, fluorescent dyes that are known to accumulate in the mitochondria (such as rhodamine 123) (Antonenko et al., 2008; Chen, 1988), and carbocyanine based dyes, such as JC-1. In certain embodiments, the antioxidant moiety is linked to the lipophilic cation by a carbon chain having 1 to 30 carbon atoms. The most studied of these compounds is the ubiquinone-containing mitoquinone or MitoQ™ (Smith et al., 2008). Methods for the preparation of such compounds are known to those of skill in the art and include those described in US Patents 6,331,532 and 7,232,809, and US patent application publication no. US 2008/0275005, the disclosures of which are hereby incorporated by reference. In certain embodiments, the mitochondria targeted antioxidant comprises plastoquinone linked to a phosphonium or rhodamine moiety, for example by a decane or pentane linker. Examples of such compounds include 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SKQ1), 10-(6'- plastoquinonyl) decylrhodamine 19 (SKQR1), 10-(6'-plastoquinonyl) decyl-carnitine (SKQ2), 10-(6'-plastoquinonyl) decylmethylcarnitine (SKQ2M), 10-(6'-methyl- plastoquinonyl) decyltriphenylphosphonium (SKQ3), 10-(6'-plastoquinonyl) decyl- tributylammonium (SKQ4), and 5-(6'-plastoquinonyl) amyltriphenylphosphonium (SKQ5). Methods for the preparation of such compounds are well known to those of skill in the art and include, for example, those described in US Published Patent Application no. US 2008/0176929, the disclosure of which is hereby incorporated by reference.

A second class of compounds that can be effectively employed as mitochondria targeted antioxidants are peptide-based compounds known as SS (Szeto-Schiller) peptides (Szeto, 2008), or aromatic-cationic peptides. These compounds have been shown to target the mitochondrial inner membrane, accumulating at levels up to 5000 times the concentration found in the surrounding media (Zhao et al., 2004). The structural motif of the SS peptides centers on alternating aromatic residues and basic amino acids. Their antioxidant activity is believed to be due to the tyrosine or dimethyltyrosine (Dmt) residue, with the specific location of the tyrosine or Dmt residue not being important. Examples of such peptides that can be effectively employed as mitochondria targeted antioxidants include those described in US Patent Publication no. US 2009/0221514, the disclosure of which is hereby incorporated by reference. In specific embodiments, the mitochondria targeted antioxidants disclosed herein comprise, or consist of, an aromatic-cationic peptide selected from the group consisting of: H-Tyr-D-Arg-Phe-Lys-NH₂; H-Dmt-D-Arg-Phe- Lys-NH₂; H-D-Arg-Dmt-Lys-Phe-NH₂; and H-Phe-D-Arg-Phe-Lys-NH₂. The SS peptides have been shown to protect cells from induced apoptosis and external added oxidation agents (Szeto, 2008; Whiteman et al., 2008; Zhao et al., 2004). The first class of compounds is dependent upon membrane potential for uptake to the mitochondria, and also accumulates in the cell cytoplasm because of the cell membrane potential. In contrast, the SS peptides do not require mitochondrial membrane potential for accumulation in the mitochondria (Zhao et al., 2004).

In one embodiment, sperm are collected by artificial vagina and are immediately purified by a single density layer (Percoll™ PLUS, GE Healthcare, see protocol below). Sperm are then incubated in a suitable media with mitochondria targeted antioxidant reagent(s) for a short period of time, followed by the addition of a suitable extender to enable either immediate use or freezing. In another embodiment, sperm are sexed by flow cytometry and are collected in a media containing mitochondria targeted antioxidant reagent(s). Alternatively, once sufficient sorted sperm are collected, the mitochondria targeted antioxidant reagents are added and incubated for a short period of time, followed by the addition of extender and then either immediate use or freezing.

Example 1 - Sperm Maturation Model

In this model, as detailed below, bovine sperm are incubated overnight in non- capacitating media (NCM; Table 1; 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 2), 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 db-cAMP, IBMX (3-isobutyl-l-methylxanthine) and caffeine, greater than 95% of viable cells capacitate as assessed by WGA-fluorescein, Annexin V or merocyanine 540 binding (see Table 4 below; 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 4). Although the combination of db-cAMP, IBMX and caffeine is an efficient inducer of capacitation, when more in vivo like capacitation induction is required, heparin is used. Sperm treated with mitochondria specific antioxidants are compared with untreated sperm for their ability to mature, in particular using the heparin induction method. In addition, the effects of the antioxidants are determined when sperm are stressed by temperature, oxygen levels, media components affecting osmolarity and/or oxidant promoting agents.

Table 1: lx NCM (non-capacitating media. pH 7.4)

Table 2: lx CM (capacitating media. pH 7.4)

Component Concentration

NaH₂P0₄ 0.3 mM

KC1 3.1 mM

MgCl₂ 0.4 mM

Sodium pyruvate 1 mM

HEPES 20 mM

NaCl 100 mM

Lactate (85%) 21.7 mM

NaHCOs 60 mM

CaCl₂ 3.9 mM

Gentamicin 50 μ^πύ Component Concentration

Fatty acid-poor BSA 2 mg/ml

a Day 1 - Percoll PLUS purification of bovine sperm cells

A 90% Percoll™ PLUS solution is made by adding 10x NCM to Percoll™ PLUS (GE Healthcare). A 60% single layer gradient is then made by dilution with 1 ^χ NCM. 1.5 ml of bovine sperm in liquid extender (standard tris-egg yolk-citrate-glycerol extender, extension ~ 1 :3) is carefully placed on top of 4 ml of 60% Percoll™ PLUS column. Samples are then centrifuged for 20 min at 700xg at 20°C. The pellet is carefully removed and washed once in 8 ml of l x NCM by centrifugation for 5 min at 700xg at 20°C. The supernatant is discarded and the pellet resuspended in 1 ml of NCM. Capacitation treatment tubes are set up at a sperm concentration of 5 ^χ 10⁷ cells/ml. b Day 1 - Flow cytometry analysis of non-capacitated sperm

Samples are then prepared for flow cytometry analysis as follows. The components shown in Table 3 below are incubated with 5 10⁵ Percoll™ PLUS-purified bovine sperm in a final volume of 200 μΐ. Fluorescently labeled SBTI, WGA and PNA are incubated with the Percoll™ PLUS-purified sperm at room temperature for 10 min, while propidium iodide (PI) is added just before analyzing by flow cytometry.

Table 3

c Day 1 to 2 - Incubation of bovine sperm sample overnight

Percoll™ PLUS-purified bovine sperm at 5 x 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. After addition of 1 : 1 ratio of CM to NCM, CaCl₂ will be at a concentration of 1.95 mM. 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 methyl-beta- cyclodextrin (cholesterol acceptor). Samples are then incubated for an hour at 37°C. e) Day 2 - Flow cytometry analysis of capacitated sperm

Similar to day 1, bovine sperm samples are then incubated with fluorescently labeled SBTI, PNA and WGA for 10 min and PI is added just prior to flow analysis.

Example 2 - In vitro Sperm Testing

A series of experiments are performed in vitro to determine the characteristics of sperm treated with mitochondria targeted antioxidants versus untreated sperm in various measures of sperm functionality in the sperm maturation model of Example 1. Sperm are compared with and without antioxidants 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/or the ability of sperm to fertilize in vitro (see Table 4 below for details).

Table 4

CHARACTERISTIC ASSAY NOTES REFERENCES

Motility & QualiSperm^1M and Enables quick quantitative (Tejerina et al., morphology Bright field microscopy motility analysis for 1000s of 2008)

cells. Can also indicate

capacitation (hypermotility)

Viability/ ^' Flow cytometry (FC)/ Depending upon the experiment, See (Gillan et al., Membrane integrity Fluorescent microscopy different vital dyes are used 2005) for a

(FM) using a range of depending upon their properties review and dyes including (all available from Invitrogen). references within Propidium iodide, Yo These dyes are used alone but pro-l, Hoechst 33258 also in combination with other

(H33258), FC/FM assays described below.

LIVE/DEAD fixable far Overall allows quantification of red and SYBR 14 cells with permeant membranes

Shape and FC/FM Enables quantification of size (Gillan et al., granularity and cellular aggregation changes 2005)

Example 3 - Effects of the Mitochondria Targeted Antioxidants SKOl and SKORl on in vitro Motility of Bull Sperm

The effects of the mitochondrial targeted antioxidants SKQl (10-(6'-plasto- quinonyl) decyltriphenylphosphonium) and SKQRl (10-(6'-plastoquinonyl) decyl- rhodamine 19) on in vitro motility of sperm was examined as follows.

SKQl and SKQRl were prepared as described by Antonenko et al. (2008). The chemical structures for these two compounds are as shown below:

In a first experiment, bovine sperm cells from four bulls (Bandana, Zealot, Newman and Strike) were purified through a 60% single gradient Percoll™ PLUS column and washed once in NCM. Washed sperm cells were diluted to 5 x 10⁶/ml cell concentration in NCM containing either no SKQRl (control), 4 nM, 20 nM or 100 nM SKQRl, in the presence or absence of 5 mM glucose. The assay was set up in a 96 well flat bottom plate at room temperature in a total volume of 200 μΐ, and the sperm motility was assessed. After the initial motility assessment, the assay plates were incubated at either 28°C or 37°C, arid the sperm motility was assessed again. All motility assessment was performed by a single operator. The data is provided in Table 5 below, wherein 0 indicates no movement; 1 indicates slow movement; 2 indicates medium movement; 3 indicates fast movement; and 4 indicates very fast movement. The data demonstrates that the antioxidant SKQRl enhanced the motile life of sperm in vitro for sperm from most bulls. Table 5

Day 1

2 PM Concentration of SKQRl

Room

temperature O nM 4 nM 20 nM 100 nM

(Control)

Bandana no glucose 4 4 4 4

5 mM glucose 4 4 4 4

Zealot no glucose 4 4 4 4

5 mM glucose 4 4 4 4

Newman no glucose 4 4 4 4

5 mM glucose 4 4 4 4

Strike no glucose 4 4 4 4

5 mM glucose 4 4 4 4

Day 2

10 AM Concentration of SKQRl

28°C

O nM 4 nM 20 nM 100 nM (Control)

Bandana no glucose 2 3 3 3

5 mM glucose 1 2 2 2

Zealot no glucose 2 2 2 1

5 mM glucose 2 2 2 2

Newman no glucose 2 3 3 3

5 mM glucose 2 3 3 3

Strike no glucose 1 2 2 2

5 mM glucose 1 1 1 2

Day 2

10 AM Concentration of SKQRl

37°C

O nM 4 nM 20 nM 100 nM (Control)

Bandana no glucose 1 1 1 1

5 mM glucose 2 3 3 3

Zealot no glucose 1 1 1 1

5 mM glucose 1 2 2 2

Newman no glucose 1 1 1 1

5 mM glucose 1 2 2 2

Strike no glucose 1 1 1 1 5 mM glucose 1 1 1 2

Day 3

10 AM Concentration of SKQR1

28°C

O nM 4 nM 20 nM 100 nM (Control)

Bandana no glucose 0 0 0 0

5 mM glucose 0 1 1 1

Zealot no glucose 0 0 0 0

5 mM glucose 1 1 1 1

Newman no glucose 1 2 2 2

5 mM glucose i 2 2 2

Strike no glucose 1 1 1 1

5 mM glucose 1 1 1 1

Day 3

10 AM Concentration of SKQR1

37°C

O nM 4 nM 20 nM 100 nM (Control)

Bandana no glucose 0 0 0 0

5 mM glucose 0 1 1 1

Zealot no glucose 0 0 0 0

5 mM glucose 0 1 1 1

Newman no glucose 0 0 0 0

5 mM glucose 0 1 1 1

Strike no glucose 0 0 0 0

5 mM glucose 0 1 1 1

In a second experiment, bovine sperm cells from two bulls (Ez4me and Pamment) were purified through a 60% single gradient Percoll™ PLUS column and washed once in NCM. Washed sperm cells were diluted to 25 ^χ 10⁶/ml cell concentration in NCM containing 5 mM glucose and either no antioxidant (control) or various concentrations of the antioxidants SKQ1 or SKQR1. The assay was set up in a 96 well flat bottom plate at room temperature in a total volume of 200 μΐ. The assay plates were incubated at 37°C and assessed for sperm motility. The data (provided below in Table 6) demonstrates that both SKQ1 and SKQR1 extended motile life span in vitro for sperm from most bulls. Table 6

Example 4 - Binding of the Mitochondria Targeted Antioxidant SKORl

to Live Sperm

The ability of the mitochondria targeted antioxidant SKQRl to bind to live bovine sperm was examined as described below. a) Examination of live bovine sperm stained with SKQRl and Hoechst 33258 by fluorescent microscopy Percoll™-purified sperm from two bulls (Zealot and Bandana) were diluted to 1 ^χ 10 cells/ml in NCM and stained with 1 μί*/πι1 of the viability stain Hoechst 33258 and 1.3 μΜ mitochondrial antioxidant SKQR1. A parallel set of samples were incubated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) at 100 μΜ for 10 min prior to the addition of SKQR1 and Hoechst 33258. Five minutes after addition of SKQR1 and Hoechst 33258, the sperm were examined by fluorescent microscopy.

A highly specific SKQR1 signal was detected at the mid-piece of Zealot sperm at a minimal exposure time. The pattern of staining in Bandana sperm was similar to that of Zealot although some sperm heads were also stained. Hoechst 33258 specifically stained the heads of dead sperm, while dead sperm showed no SKQR1 staining. Pre-incubation with CCCP (100 μΜ) also abolished the SKQR1 signal. The signal intensity of Zealot sperm stained with SKQR1 was more intense than that of Bandana.

(b) Examination of live bovine sperm stained with SKQR1 by flow cytometry

(i) Staining of Percoll™-purified sperm with SKOR1

Bovine sperm cells were purified through a 60% single gradient Percoll™ PLUS column and washed once in NCM. Washed sperm cells were then diluted to 5 ^χ 10⁶/ml or 400 x 10⁶/ml cell concentration in NCM and incubated with the specified concentration of SKQR1 for 30 min at 37°C in a 96 well V-bottom plate, and with Hoechst 33258 (20 ng/ml) just prior to flow cytometry analysis . Following incubation, live sperm (Hoechst negative) were assessed for SKQR1 fluorescence on a FACSAria™ flow cytometer (BD Sciences). The results are shown below in Table 7.

Table 7

(ii) Staining of non-purified sperm with SKQRl

Fresh non-extended bovine ejaculate was diluted to 400 ^χ 10⁶/ml sperm cell concentration with NCM. 0.5 mg/ml of BSA, 5 mM glucose and 500 nM SKQRl were added to 1 ml of the diluted sperm and incubated for 15 min at 38°C. After incubation, diluted sperm was extended 1:1 with egg yolk extender, and SKQRl -treated or control (non-treated) sperm were Percoll™ PLUS-purified through a 60% single gradient, washed once in NCM, and stained with Hoechst 33258 (20 ng/ml) prior to analyzing live sperm on the FACSAria™ flow cytometer for SKQRl fluorescence. The results are shown in Table 8 below.

Table 8

Example 5 - In vivo Field Artificial Insemination Trials

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 mitochondria targeted antioxidant (such as SKQ1 or SKQR1) 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, reciprocal studies are also carried out to ensure effects on sperm transport are not due to the marker fluorescent dye. Twelve to fourteen 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. Treated sperm having increased functionality compared to untreated sperm will be present in the upper uterine horn and oviduct in larger amounts compared to untreated (control) sperm.

Example 6 - Crvopreservation of sperm

Mitochondria targeted antioxidants are added to compositions including bovine sperm prior to crvopreservation using standard techniques (Watson, 1990). Subsequent to freezing, treated and untreated sperm are compared for the following characteristics: motility, viability (membrane integrity), mitochondrial membrane potential, ROS generation, lipid peroxidation, and the presence of sperm surface markers indicative of capacitation and acrosome status, as described above. For a similar examination of cryopreserved sperm, see (Setyawan et al., 2009).

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Claims

We claim:

1. A method for improving functionality and/or fertility of sperm, comprising contacting the sperm with an effective amount of a composition comprising a mitochondria targeted antioxidant, wherein the effective amount of the composition is sufficient to increase the functionality and/or fertility of the sperm.

2. The method of claim 1, wherein the composition comprises an antioxidant conjugated to a carrier that specifically targets mitochondria.

3. The method of claim 2, wherein the carrier is a lipophilic cation.

4. The method of claim 3, wherein the lipophilic cation is selected from the group consisting of: triphenylphosphonium cations; alkyl-triphenylphosphonium cations; tribenzyl ammonium cations; phosphonium cations; carbocyanine based dyes; and rhodamines.

5. The method of claim 2, wherein the antioxidant is selected from the group consisting of: ubiquinone; tocopherol; nitroxide; superoxide dismutase mimetics; glutathione peroxidase mimetics; ebselen; plastoquinone; lipoic acid; and derivatives thereof.

6. The method of claim 2, wherein the composition comprises mitoquinone.

7. The method of claim 1, wherein the mitochondria targeted antioxidant is selected from the group consisting of: 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SKQ1); 10-(6'-plastoquinonyl) decylrhodamine 19 (SKQR1); 10-(6'-plastoquinonyl) decylcarnitine (SKQ2); 10-(6'-plastoquinonyl) decylmethylcarnitine (SKQ2M); 10-(6'- methylplastoquinonyl) decyltriphenylphosphonium (SKQ3); 10-(6'-plastoquinonyl) decyltributylammonium (SKQ4); and 5-(6'-plastoquinonyl) amyltriphenylphosphonium (SKQ5).

8. The method of claim 1, wherein the composition comprises an aromatic-cationic peptide.

9. The method of claim 8, wherein the aromatic-cationic peptide is selected from the group consisting of: H-Tyr-D-Arg-Phe-Lys-NH₂; H-Dmt-D-Arg-Phe-Lys-NH₂; H-D- Arg-Dmt-Lys-Phe-NH₂; and H-Phe-D-Arg-Phe-Lys-NH₂.

10. A method for preparing a composition for use in artificial insemination or in vitro fertilization, comprising:

(a) obtaining sperm from a mammal; and

(b) contacting the sperm with an effective amount of a mitochondria targeted antioxidant,

wherein the effective amount of the mitochondria targeted antioxidant is sufficient to increase the functionality and/or fertility of the sperm.

.

11. The method of claim 10, further comprising sorting the sperm to separate X- chromosome bearing sperm from Y-chromosome bearing sperm.

12. The method of claim 10, wherein the mitochondria targeted antioxidant comprises an antioxidant conjugated to a carrier that specifically targets mitochondria.

13. The method of claim 12, wherein the carrier is a lipophilic cation.

14. The method of claim 13, wherein the lipophilic cation is selected from the group consisting of: triphenylphosphonium cations; alkyl-triphenylphosphonium cations; tribenzyl ammonium cations; phosphonium cations; carbocyanine based dyes and rhodamines.

15. The method of claim 12, wherein the antioxidant is selected from the group consisting of: ubiquinone; tocopherol; nitroxide; superoxide dismutase mimetics; glutathione peroxidase mimetics; ebselen; plastoquinone; lipoic acid; and derivatives thereof.

16. The method of claim 10, wherein the mitochondria targeted antioxidant is mitoquinone.

17. The method of claim 10, wherein the mitochondria targeted antioxidant is selected from the group consisting of: 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SKQl); 10-(6'-plastoquinonyl) decylrhodamine 19 (SKQRl); 10-(6'-plastoquinonyl) decylcarnitine (SKQ2); 10-(6'-plastoquinonyl) decylmethylcarnitine (SKQ2M); 10-(6'- methylplastoquinonyl) decyltriphenylphosphonium (SKQ3); 10-(6'-plastoquinonyl) decyltributylammoniuni (SKQ4); and 5-(6'-plastoquinonyl) amyltriphenylphosphonium (SKQ5).

18. The method of claim 10, wherein the mitochondria targeted antioxidant comprises an aromatic-cationic peptide.

19. The method of claim 18, wherein the aromatic-cationic peptide is selected from the group consisting of: H-Tyr-D-Arg-Phe-Lys-NH2; H-Dmt-D-Arg-Phe-Lys-NH₂; H-D- Arg-Dmt-Lys-Phe-NH₂; and H-Phe-D-Arg-Phe-Lys-NH₂.

20. The method of claim 10, wherein the mammal is human, bovine, porcine or equine.

21. A preparation for use in artificial insemination or in vitro fertilization, comprising:

(a) live sperm; and

(b) an effective amount of a mitochondria targeted antioxidant,

wherein the effective amount of the mitochondria targeted antioxidant is sufficient to increase the functionality and/or fertility of the sperm.

22. The preparation of claim 21, wherein the sperm are X-chromosome bearing sperm.

23. The preparation of claim 21, wherein the mitochondria targeted antioxidant comprises an antioxidant conjugated to a carrier that specifically targets mitochondria.

24. The preparation of claim 23, wherein the carrier is a lipophilic cation.

25. The preparation of claim 24, wherein the lipophilic cation is selected from the group consisting of: triphenylphosphonium cations; alkyl-triphenylphosphonium cations; tribenzyl ammonium cations; phosphonium cations; carbocyanine based dyes; and rhodamines.

26. The preparation of claim 23, wherein the antioxidant is selected from the group consisting of: ubiquinone; tocopherol; nitroxide; superoxide dismutase mimetics; glutathione peroxidase mimetics; ebselen; plastoquinone; lipoic acid; and derivatives thereof.

27. The preparation of claim 21, wherein the mitochondria targeted antioxidant is mitoquinone.

28. The preparation of claim 21, wherein the mitochondria targeted antioxidant is selected from the group consisting of: 10-(6'-plastoquinonyl) decyltriphenyl- phosphonium (SKQ1); 10-(6'-plastoquinonyl) decylrhodamine 19 (SKQR1); 10-(6'- plastoquinonyl) decylcarnitine (SKQ2); 10-(6'-plastoquinonyl) decylmethylcarnitine (SKQ2M); 10-(6'-methyl-plastoquinonyl) decyltriphenylphosphonium (SKQ3); 10-(6'- plastoquinonyl) decyltributylammonium (SKQ4); and 5-(6'-plastoquinonyl) amyl- triphenylphosphonium (SKQ5).

29. The preparation of claim 21, wherein the mitochondria targeted antioxidant comprises an aromatic-cationic peptide.

30. The preparation of claim 29, wherein the aromatic-cationic peptide is selected from the group consisting of: H-Tyr-D-Arg-Phe-Lys-NH₂; H-Dmt-D-Arg-Phe-Lys-NH₂; H-D-Arg-Dmt-Lys-Phe-NH₂; and H-Phe-D-Arg-Phe-Lys-NH₂.

31. The method of claim 21 , wherein the sperm is human, bovine, porcine or equine.

32. A method for cryopreserving sperm comprising:

(a) contacting the sperm with a cryoprotectant and a composition comprising an effective amount of a mitochondria targeted antioxidant; and

(b) storing the sperm at a temperature of about 4°C to about -196 °C,

wherein the effective amount of the mitochondria targeted antioxidant is sufficient to increase the functionality and/or fertility of the sperm relative to sperm stored without mitochondria targeted antioxidant.

33. The method of claim 32, wherein the cryoprotectant is selected from the group consisting of: polyethyleneglycol; DMSO; ethylene glycol; propylene glycol; polyvinvyl pyrrolidone; polyethylene oxide; raffmose; lactose; trehalose; melibiose; melezitose; mannotriose; stachyose; dextran; hydroxy-ethyl starch; sucrose; maltitol; lactitol; and glycerol.

34. A composition comprising cryogenically preserved sperm and a mitochondria targeted antioxidant.

35. A method for determining the ability of a composition to modify the functionality of sperm, comprising:

(a) contacting sperm obtained from a male with the composition to provide treated sperm;

(b) labeling the treated sperm with a first detection agent to provide labeled treated sperm;

(c) labeling non-treated sperm obtained from the male with a second, different, detection agent to provide labeled non-treated sperm;

(d) simultaneously inseminating a female with the labeled treated sperm and the labeled non-treated sperm; and

(e) determining a ratio of labeled treated sperm to labeled non-treated sperm present in the oviduct of the female.

36. The method of claim 35, wherein at least one of the first and second detection agents is a fluorescent dye.