WO2016135284A1

WO2016135284A1 - Sperm extender comprising a rac inhibitor

Info

Publication number: WO2016135284A1
Application number: PCT/EP2016/054074
Authority: WO
Inventors: Vincent Sauzeau; Marie LIABEUF
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Université de Nantes; Centre National De La Recherche Scientifique (Cnrs)
Priority date: 2015-02-27
Filing date: 2016-02-26
Publication date: 2016-09-01

Abstract

The present invention relates to a sperm extender composed of a base medium comprising a Rac inhibitor. The invention also relates to the use of a Rac inhibitor for preserving sperm and/or for maintaining fertility potential for artificial insemination.

Description

SPERM EXTENDER COMPRISING A RAC INHIBITOR

FIELD OF THE INVENTION:

The invention belongs to the field of artificial insemination. The invention more specifically consists of a sperm extender comprising a Rac inhibitor and the use thereof for sperm preservation and maintaining its fertility potential.

BACKGROUND OF THE INVENTION:

With the development of reproduction biotechnologies, artificial insemination techniques are commonly used in numerous animal species, including the caprine, ovine, porcine, bovine and equine species. However, spermatozoa are complex and highly specialized cells that can quickly lose their fertilizing power. And even under these optimized conditions, the fertilizing power of spermatozoa after thawing remains relatively weak.

Thus, the long-term preservation of cooled (positive temperatures) or frozen sperm while maintaining its fertilizing power presents a problem. Indeed, one of the biggest problems today in artificial insemination techniques is the preservation of sperm (or semen) and its fertilizing power until the moment of insemination arrives. To solve this problem a medium referred to as "extender" in which the sperm is diluted immediately after being collected, is commonly used. It is therefore preserved until the moment of insemination. Semen for artificial insemination is often preserved by freezing. Freezing semen is an effective preservation method, but there is a problem with maintaining potency after thawing. Indeed, the extreme stress of freezing and thawing treatments negatively affect the original functions of the cell by increasing production of ROS which, in turn, can affect original cell functions^1"3. As observed in human semen, stallion semen docs not survive the cryoprcservation process as well as other species (most notably the bull) leading to significantly reduced conception rates following insemination with cooled or frozen-thawed semen. Sperm extenders are frequently used to preserve the viability of the sperm after thaw. High e tender/semen dilution ratios are known to reduce the deleterious effects of seminal plasma on cooled, stored spermatozoa.

However, the standard protocols used to process semen often fail to provide sufficient number of progressively motile sperm for insemination and even under these optimized conditions, the fertilizing power of spermatozoa after thawing remains relatively weak. It is therefore highly desirable to increase the sperm motility and/or sperm viability by improving compositions of sperm extender. SUMMARY OF THE INVENTION:

In a first aspect, the invention relates to a sperm extender composed of a base medium comprising a Rac inhibitor.

In a second aspect, the invention relates to a sperm preparation intended for artificial insemination, characterized in that said sperm is diluted in a sperm extender of the invention.

In a third aspect, the invention relates to a method for increasing sperm motility and/or sperm viability comprising a step of diluting sperm in a sperm extender of the invention.

In a fourth aspect, the invention relates to a method for preserving sperm and/or for maintaining fertility potential for artificial insemination comprising a step of diluting sperm in a sperm extender of the invention.

DETAILED DESCRIPTION OF THE INVENTION:

The inventors have now surprisingly established that a Rac inhibitor added to a sperm extender such as the INRA96® sperm extender useful for preserving stallion sperm by considerably improving the sperm motility and/or sperm viability.

In a first aspect, the invention thus relates to a sperm extender composed of a base medium comprising a Rac inhibitor.

As used herein, the terms "sperm extender" or "semen extender" are interchangeably and refer to a sperm dilution medium used for the long-term preservation of fresh sperm notably by preserving good fertilizing capability of the spermatozoa during freezing. Such an sperm extender is for instance sperm extender commercially available for bull sperm (Bioxcell®) or for stallion sperm (INRA96®) purchased by IMV, L'Aigle, France. The sperm extender is indeed suitable for preserving sperm of mammals, such as for example the caprine, ovine, bovine and equine species.

In one embodiment of the invention, the sperm is mammalian sperm. In a particular embodiment of the invention, the sperm is a stallion sperm or a bull sperm. In one embodiment of the invention, the ba.se medium comprises yolk or yolk plasma. Commercial semen extender utilizing raw egg yolk is commercially available (Biladyl® from Minitube GmbH). In general, the egg yolk is added to the base medium just prior to the addition of the semen. See, U.S. Pat. No. 6,130,034 to Aitken; U.S. Pat. No. 3,444,039 to Rajamannan; U.S. Pat. No. 3,718,740 to Hafs et al.; and U.S. Pat. No. 3,973,003 to Kolas.

In one embodiment of the invention, the base medium is a milk-based diluent.

Milk-based semen diluents are known to be practical and effective in protecting spermatozoa such as equine spermatozoa during storage before artificial insemination.

The base medium thus comprises a milk product such as buttermilk, whole or skim milk, or whey. The milk product may be in either a dried or liquid form but, if dried, must be fluid in the sense that it is pourable and easily dissolvable in an aqueous solution.

Additionally to the milk product, the base medium may also comprise a sugar nutrient which has been found to be particularly desirable in achieving the extension of the life of the sperm cells. The sugar nutrient may be in either a dried or liquid form but, if dried, must be fluid in the sense that it is pourable and easily dissolvable in an aqueous solution. Such sugar nutrients include, but are not limited to, mono- and disaccharides, among which may be included dextrose, fructose, glucose, lactose and sucrose.

Such milk-based diluents include, but are not limited to, the skim milk diluents INRA82® or Kenney's diluents. For instance, the diluent INRA82® has been described by Palmer, E. (Proc. 10th International Congress on Animal Reproduction and Artificial Insemination, Urbana-Champaign, 111. USA, 3:377, 1984) and amended by Magistrini et al. (Acta Veterinaria Scandinavica, 1st European Symposium on Production, Evaluation and Preservation of Stallion Semen, Oct. 1-2, 1992, Uppsala, Sweden, Suppl. 88, 97-110). The INRA82® sperm extender is a mixture of 0.5 liter of a base medium (saline-glucose solution: glucose 25 g-L^-1, lactose 1.5 g-L^-1, raffmose 1.5 g-L^-1, dehydrated sodium citrate 0.25 g-L^-1, potassium citrate 0.41 g-L^-1, hepes buffer 4.76 g-L¹) with 0.5 liter of milk at pH 6.8.

Alternatively, the base medium is chemically defined which is composed of efficient milk components (milk micellar proteins) and optimized for sperm survival and storage temperature. Accordingly, in one embodiment of the invention, the base medium comprises a purified fraction of native phosphocaseinate and/or β-lactoglobulin.

In one embodiment of the invention, the base medium comprises between 1 and 100 g/L native phosphocaseinate, preferably between 10 and 50 g/L native phosphocaseinate.

Such base media and sperm extenders comprising thereof (including the INRA96® sperm extender) are extensively described in the international patent application WO98/37904 as well as in the publication by Batellier et al, 1997 Theriogenology, 48-3, 391-410).

The INRA96® sperm extender is composed of a base medium (HGGL medium, composed of Hank's salts supplemented with hepes buffer, lactose and glucose) and 27 g/L of native phosphocaseinate. The INRA96® sperm extender is purchased by IMV Technologies. The base medium may also include additives such as antibiotics or antifungal agents

(e.g. the base medium contains 50,000 IU L ¹ of penicillin and 50 mg L^-1 of gentamicin).

The INRA82® and INRA96® media may be also supplemented with egg yolk dry matter and glycerol. For instance said media comprise 2% centrifiiged egg yolk (at 600 ^N g for 10 minutes to eliminate possible contamination by egg white, the chalaza or shell debris) and 2.5% glycerol as described in the international patent application WO2009/103908.

As used herein, the term "Rac" refers to Rho-related Rac GTPases, which are molecular switches that control signaling pathways regulating cytoskeleton reorganization, gene expression, cell cycle progression, and other cellular processes. In an active state, Rho- related Rac GTPases bind to GTP and transduce signals of other proteins in signal transduction pathways. In their inactive state, they are bound to GDP. The Rho-related Rac GTPases family comprises 3 isoforms (Racl, Rac2 and Rac3). As used herein, the term "Rac inhibitor" refers to an inhibitor or antagonist of Rac

GTPase (Racl, Rac2 and/or Rac3) activity. A Rac inhibitor or antagonist is a natural or synthetic compound that reduces or blocks the activity of Rac. More preferred Rac inhibitors are compounds that inhibit Rac activation by its GEFs (guanine nucleotide exchange factor) in an exchange assay and/or that inhibit the activity of Rac (e.g. activation of Pakl by its phosphorylation). A compound also able to reduces or abolish Rac gene expression is also considered as Rac inhibitor. As used herein, the term "inhibitor of gene expression" refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene including for instance siRNA, a shRNA, an antisense oligonucleotide and a ribozyme.

A person skilled in the art can easily determine whether a compound is capable of inhibiting Rac activity. Assays for evaluating Rac activity are for example, described in Pellegrin & Harry (2008) Curr. Protoc. Cell Biol. 38:14.8.1-14.8.19 and in international patent application WO2007/016539. Rac Activation Assays are also commercially available.

Rac inhibitors are well known in the art. For instance, a typical example of Rac inhibitors includes N6-(2-((4-(diethylamino)-l-methylbutyl)amino)-6-methyl-4-py-rimidinyl)- 2-methyl-4,6-quinolinediamine (also known as NSC 23766) described in international patent application WO2007/016539. Said compound specifically and reversibly inhibits Rac GDP/GTP exchange activity by interfering Rac interaction with Rac-specific GEFs.

Another typical example of Rac inhibitors includes EHT 1864 described in international patent application WO2004/076445. EHT 1864 is a small molecule that blocks the Racl signaling pathways. Other examples of Rac inhibitors include W56, sold by Tocris Biosciences (Ellisville, Mo.) or the inhibitors described in Yuan Gao, et al. PNAS, May 18, 2004, vol. 101, 7618-7623, N⁴-(9-Ethyl-9H-carbazol-3-yl)-N2-(3-morpholin-4-yl-propyl)- pyrimidine-2,4-diamine (also known as EHop-016) disclosed in Montalvo-Ortiz et al., Characterization of EHop-016 J Biol Chem. 2012 April 13;287(16):13228-13238 and in the US patent application N° US2013/172552, as well as those described in EP2433636, WO2005/051392, WO2007/031878, WO2007/016539 and WO2009/007457. embodiment of the invention, the Rac inhibitor is a compound having the formula (I):

(I) wherein: Ri to R2 are independently: H, halo, (G-C4) alkyl, branched (C3-C4) alkyl, halo (Ci- C₄) alkyl, (C1-C4) alkoxy, NO2, NH₂, -X-Alk, -X-Alk-X, -X-Y-X, -NRe or 0-R₆> wherein X is O, NR6, or CR7R8; Alk is a C2-C18 saturated or unsaturated hydrocarbon chain, straight chain or branched, optionally substituted with halo, halo (C1-C4) alkoxy, (C3-C8) cycloalkyl, hydroxy, or acetyl; Y is an alkylene chain 2 to 8 carbon atoms long, that optionally includes an O, S, SO, SO2, or NR6 group, and optionally includes a saturated or unsaturated carbocyclic ring comprising three to seven carbon atoms, and optionally is substituted with (C1-C3) alkyl, (C2-C4) phenyl, (C3-C8) cycloalkyl, hydroxy, halo, or (C1-C4) acyl; and Ar is 1,3-benzodioxolyl fluorenyl, pyridyl substituted pyridyl, indolyl, furanyl, substituted furanyl, thienyl, optionally substituted with CH2 or CI, thiazolyl, cyclopentyl, 1-methylcyclopentyl, cyclohexenyl (tetrahydrophenyl), cyclohexyl (hexahydrophenyl), naphthyl, substituted naphthyl, dihydronaphthyl, tetrahydronaphthyl, or decahydronaphthyl; R6 is H, (C1-C4) alkyl, or acetyl; R7 and Rs are independently H, (C1-C4) alkyl, (C1-C4) acyl, halo, -OH, O-Y-Ar, or - NR9 -Y-Ar; and R9 is H, (C1-C4) alkyl, or acetyl; or a salt of a compound of formula (I).

In one embodiment of the invention, the Rac inhibitor is a compound having the f rmula (II):

wherein: Rio to R12 are independently selected from the group consisting of H, halo, (C1-C4) alkyl, branched (C3-C4) alkyl, halo (C1-C4) alkyl, (C1-C4) alkoxy, NO2, and NH2; or a salt of a compound of formula (II).

In one embodiment of the invention, the Rac inhibitor is NSC 23766.

According to some embodiments, the Rac inhibitor is provided at a concentration range of about 10^~7 M (0,1 μΜ), preferably about 10^"6 M (1 μΜ), more preferably about 10^"5 M (10 μΜ), and still more preferably 10^~4 M ( 100 μΜ). In a second aspect, the invention relates to a sperm preparation intended for artificial insemination, characterized in that said sperm is diluted in a sperm extender composed of a base medium comprising a Rac inhibitor. This invention may be used in connection with artificial insemination in different species of mammals, particularly in the caprine, ovine, porcine, bovine and equine species and in a particularly advantageous manner in the bovine and equine species.

In a third aspect, the invention relates to a method for increasing sperm motility and/or sperm viability comprising a step of diluting sperm in a sperm extender composed of a base medium comprising a Rac inhibitor.

In a fourth aspect, the invention relates to a method for preserving sperm and/or for maintaining fertility potential for artificial insemination comprising a step of diluting sperm in a sperm extender composed of a base medium comprising a Rac inhibitor.

In one embodiment of the invention, the Rac inhibitor improves preservation and maintains the fertility potential up to 48 hours, preferably up to 72 hours. In a fifth aspect, the invention relates to a method for preserving sperm by freezing comprising a step of diluting sperm in in a sperm extender composed of a base medium comprising a Rac inhibitor.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES: Figure 1: Representative Z-projection images of stallion semen.

Figure 2: Survival and sperm motility in fresh stallion semen. A- Representative Z-projection images at indicated time in control condition or treated with a Rac inhibitor (10^" ⁵M). B- Quantification of viability and sperm mobility at indicated time and different Rac inhibitor concentrations. Results are expressed in %±SEM. *, p<0.05. n=3-4.

Figure 3: Survival of frozen/thawed stallion semen. A- Representative Z-projection images at indicated Rac inhibitor concentrations in fresh or frozen/thawed stallion semen. B- Quantification of sperm viability at indicated Rac inhibitor concentrations in fresh or frozen/thawed stallion semen. Results are expressed in %±SEM. *, p<0.05. n=3.

EXAMPLE: Material & Methods

Semen collection: Three fertile stallions were used in this study. All stallions were allowed daily paddock exercise and were fed sufficient coastal bermudagrass hay and grain mix to maintain good body condition.

Three ejaculates from each of the stallions were used to investigate the effects of Rac inhibitor. Ejaculates were collected using an artificial vagina. Following collection, the gel fraction was removed and spermatozoa! concentration in gel-free semen was determined photometrically. Then, semens were diluted with the sperm extender INRA96 (1MV Technologies, France) to obtain a final concentration of 20x 10⁶ sperm/ml. The Rac inhibitor (NSC23766, Tocris) was added to obtain a final concentration of 10 ⁷, 10^"6, 10^~5 and 10^~4 M.

Conservation, freezing and thawing process: All semen samples were diluted with warmed diluents (30°C) in a water bath. The diluted semen were gradually cooled to 4°C. For fresh condition, samples were stored at 4°C during 0, 24, 48 and 72h). For freezed condition, samples were diluted at a final concentration of 100^χ 10⁶ sperm- ml and were packed in 0.25 ml straw. The straws were kept at 4°C until freezing, which was carried out by placing the samples in liquid nitrogen (-125°C) until examination. Frozen spermatozoa were thawed in a water bath at 37°C during 1 min. Viability and motility analyses: Progressive motility and viability were assessed using a microscope (Leica Z16 Plan APO 5.0x/0.50 LWD), with a warm stage maintained at 37 °C. A wet mount was made using a 20 μί drop of semen placed directly on a microscope slide and covered with a coverslip. A high-speed video camera (ORCAD2, HAMAMATSU) mounted on the microscope projected the image onto a color monitor and the images (n=500) were recorded for offline analysis. Sperm motility and viability estimations were performed in five microscopic fields (~50 spermatozoa/field) for each semen sample. The video images were processing and analyzed with the software ImageJ/Fiji, an open source image processing software. A Z-projection was applied to each video to evaluate semen parameters. In Z- projections images, white dots correspond to dead sperm and straight/curve lines to motile spermatozoa. Motility is evaluated by the ratio (number of spermatozoa with a rapid and progressive direction) / (number of motile spermatozoa).

Statistic: Two-way ANOVA statistical analysis method was used to assess differences between each experimental conditions. Values are therefore expressed as medium±SEM range. Statistical differences were considered to be significant if p<0.05. All analyses were performed using the Prism/GraphPad statistical software (version 5.0).

Results

Rac inhibition improves survival and sperm motility in fresh stallion semen: To evaluate the impact of Rac inhibition on survival and sperm motility, fresh stallion semens were diluted with a sperm extender containing the Rac inhibitor NSC23766 (10^~4 M to i0^~7 M). As expected in control condition, sperm motility and viability decrease dramatically after ejaculation (Figure 1). 48-72h after ejaculation sperm viability is approximatively -35% and only 40% of spermatozoa have a normal, motility suggesting a weak probability of conception rates following insemination. However, pretreatment of samples with Rac inhibitor (10 and 100 μΜ) provides protection of sperm quality. Indeed, pretreated spermatozoa show a significant increase in viability and motility (over 50%). This protection activated by the Rac inhibitor appears 24h after ejaculation and is maintained over time. These results suggest that addition of Rac inhibitor in fresh semen preserves sperm quality and will improve the probabi lity of conception rates following insemination.

Rac inhibition improves survival of frozen/thawed stallion semen: Freezing/thawing of sperm sample is routinely performed in cattle breeding industries in order to perform artificial insemination. These procedures are known to produce ROS in sperm samples. During cryopreservation, semen is exposed to cold shock and atmospheric oxygen, which in turn increases the susceptibility to lipid peroxidation due to higher production of ROS leading to a significant decrease in sperm viability and a highly reduced fertilizing capacity. Indeed, frozen/thawed stallion semen induced a decrease of =50% of sperm viability (Figure 2). Semens preatreated with a Rac inhibitor (>10^"6M) are protected of post-thaw damages (Figure 3). REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

1. Gil L, Olaciregui M, Luno V, Malo C, Gonzalez N, Martinez F. Current status of freeze-drying technology to preserve domestic animals sperm. Reproduction in domestic animals = Zuchthygiene. 2014;49 Suppl 4:72-81

2. Santiani A, Evangelista S, Sepulveda N, Risopatron J, Villegas J, Sanchez R. Addition of superoxide dismutase mimics during cooling process prevents oxidative stress and improves semen quality parameters in frozen/thawed ram spermatozoa. Theriogenology. 2014;82:884-889

3. Zribi N, Chakroun NF, Ben Abdallah F, Elleuch H, Sellami A, Gargouri J, Rebai T, Fakhfakh F, Keskes LA. Effect of freezing-thawing process and quercetin on human sperm survival and DNA integrity. Cryobiology. 2012;65:326-331

Claims

CLAIMS:

1. A sperm extender composed of a base medium comprising a Rho-related Rac GTPases (Rac) inhibitor.

2. The sperm extender according to claim 1, wherein the Rac inhibitor is NSC 23766.

3. The sperm extender according to claim 1 or 2, wherein the base medium is a milk- based diluent.

4. The sperm extender according to any one claims 1 to 4, wherein the base medium comprises a purified fraction of native phosphocaseinate and/or β-lactoglobulin.

5. A sperm preparation intended for artificial insemination, characterized in that said sperm is diluted in a sperm extender according to any one claims 1 to 4.

6. A method for increasing sperm motility and/or sperm viability comprising a step of diluting sperm in a sperm extender according to any one claims 1 to 4.

7. A method for preserving sperm and/or for maintaining fertility potential for artificial insemination comprising a step of diluting sperm in a sperm extender according to any one claims 1 to 4.