METABOLIC ACTIVATORS FOR ENHANCING SPERM CAPACITATION IN MAMMALS

Abstract

The present invention relates to a SIRT1 activator for use in promoting and/or enhancing sperm capacitation process in mammals, a specific process for these cells. The disclosure also relates to a compound and a kit comprising the SIRT1 activator. The present invention further discloses the use of a commercially available SIRT1 activator, YK-3-237, as an additive or as a new ingredient to any sperm media already existing in the market to promote the sperm capacitation process, promoting fertilization in mammals.

Claims

1. SIRT1 activator for use in promoting and/or enhancing sperm capacitation process in mammals.

2. SIRT1 activator according to claim 1, wherein the SIRT1 is at a concentration from 5-30 μM.

3. SIRT1 activator according to claim 1, wherein the SIRT1 activator is at a concentration of 10 μM.

4. SIRT1 activator according to claim 1, wherein the SIRT1 activator is B-[2-Methoxy-5-[(1E)-3-oxo-3-(3,4,5-trimethoxyphenyl)-1-propen-1-yl]phenyl]boronic acid or YK-3-237.

5. SIRT1 activator according to claim 1 for use in assisted reproductive technology.

6. SIRT1 activator according to claim 5, wherein the assisted reproductive technology is carried out with low quality oocytes.

7. SIRT1 activator according to claim 5, wherein assisted reproductive technology is intra-uterine insemination, in vitro fertilization or intracytoplasmic sperm injection.

8. SIRT1 activator according to claim 1 for use in gradient sperm selection.

9. SIRT1 activator according to claim 1 for use in idiopathic infertility or in male infertility associated to sperm capacitation disorders.

10. SIRT1 activator according to claim 1, wherein it is combined with a further sperm promoter and/or enhancer.

11. SIRT1 activator according to claim 1, wherein it is combined with a commercially available sperm media, preferably sperm washing media, cryopreservation media, thawing media or combinations thereof.

12. SIRT1 activator according to claim 11, wherein the media comprises spermatozoa previously washed and diluted.

13. SIRT1 activator according to claim 1, wherein it induces maximum tyrosine phosphorylation levels on human spermatozoa after incubation in a capacitating media containing HCO.sub.3 and a sterol removal component, in particular Bovine Serum Albumin.

14. SIRT1 activator according to claim 1 for use in humans.

15. SIRT1 activator according to claim 1 for use in non-human mammals, particularly equines, more particularly in horses.

16. Pharmaceutical compound comprising the SIRT1 activator according to claim 1.

17. Kit comprising the SIRT1 activator according to claim 1.

Description

DETAILED DESCRIPTION OF FIGURES

[0064] FIG. 1. Effect of YK-3-237 on protein phospho-tyrosine level in human sperm. Human spermatozoa were incubated for 6 hours at 37° C. in capacitating conditions (BWW-modified media supplemented with HCO.sub.3 25 mM and BSA 26 mg/mL) in presence or absence of different concentrations of YK-3-237. A) Left panel: a representative western blot using anti-phospho-tyrosine substrate of human sperm incubated at different concentrations of YK-3-237 in capacitating conditions. Right panel: western blots were analyzed using ImageJ (n=7). For comparison between blots, pixels for each lane were quantified and normalized using the CAP (0 μM) lane as reference (100%). B) Sperm viability of human spermatozoa incubated at different concentrations of YK-3-237 determined by eosine-nigrosine stain (n=4). Dotted line shows initial sperm viability before to initiate sperm incubation. C) Example of human spermatozoa stained with eosine-nigrosine stain. Sperm with white background it is an example of alive spermatozoa where spermatozoa with pink background shows a dead spermatozoa. Bars represent the average±SEM. Data were analyzed statistically by one-way analysis of variance (ANOVA). Differences between concentrations were analyzed by Tukey post-hoc test, *p<0.05 and ***p<0.0005 indicate differences versus capacitating (0 μM) conditions.

[0065] FIG. 2. Role of HCO.sub.3 and BSA on the induction of tyrosine phosphorylation level by YK-3-237 (10 μM) in human sperm. Human spermatozoa were incubated for 6 hours at 37° C. in different conditions that support or not human sperm capacitation. A) A representative western blot using anti-phospho-tyrosine substrate of human sperm incubated in different conditions (presence or absence of HCO.sub.3.sup.− 25 mM and BSA 26 mg/mL) with or without YK-3-237 for 6 hours at 37° C. (n=3). B) Western blots were analyzed using ImageJ (n=3). For comparison between blots, pixels for each lane were quantified and normalized using the CAP (YK-3-237 0 μM) lane as reference (100%). Bars represent the average±SEM. Data were analyzed statistically by one-way analysis of variance (ANOVA). Tukey post-hoc test was used to analyzed differences between the presence of absence of YK-3-237 (10 μM), **p<0.005 and ****p<0.0001 indicate differences versus YK-3-237 (10 μM) conditions. Different superscript a,b show differences between treatments p<0.05.

[0066] FIG. 3. Time curve of tyrosine phosphorylation levels of human spermatozoa incubated in presence or absence of YK-3-237 (10 μM). A) A representative western blot using anti-phospho-tyrosine substrate of human sperm incubated at different time points in capacitating conditions (HCO.sub.3−25 mM and BSA 26 mg/mL) in presence or absence of YK-3-237 (10 μM) (n=3). For comparison between blots, pixels for each lane were quantified and normalized using the CAP (YK-3-237 0 μM) lane as reference (100%). Bars represent the average±SEM. Data were analyzed statistically by one-way analysis of variance (ANOVA). Tukey post-hoc test was used to analyzed differences between the presence of absence of YK-3-237 (10 μM), **p<0.005 and ***p<0.0005 indicate differences versus YK-3-237 (10 μM) conditions at different time points. Different superscript a,b,c show differences between treatments YK-3-237 (10 μM) along the time. Different superscript z,y show differences between control conditions (0 μM) along the time.

[0067] FIG. 4. Effect of YK-3-237 (10 μM) on human sperm viability and lipid peroxidation levels (4-HNE). Human spermatozoa were incubated for 6 hours at 37° C. in conditions that support sperm capacitation (HCO.sub.325 mM and BSA 26 mg/mL) in presence or absence of YK-3-237 (10 μM). A) Sperm viability was determined by eosine-nigrosine of spermatozoa incubated in presence or absence of YK-3-237 (10 μM) for 6 hours at 37° C. (n=11). B) Left panel: a representative western blot using anti-4 Hydroxynonenal (4-HNE) antibody used to measure lipid peroxidation levels (n=4). B) Western blots were analyzed using ImageJ (n=4). Right panel: for comparison between blots, pixels for each lane were quantified and normalized using the CAP (YK-3-237 0 μM) lane as reference. Bars represent the average±SEM. Differences between conditions were analyzed using two-tailed, paired t-test *p<0.05 shows differences versus YK-3-237 (10 μM).

DETAILED DESCRIPTION OF THE INVENTION

[0068] The present invention enhances the sperm capacitation process through the use of a metabolic activator. The SIRT1 activator has the ability to synchronize sperm capacitation process independently of the initial ejaculate status.

[0069] Moreover, the SIRT1 activator brings forward sperm capacitation events (tyrosine phosphorylation). After 6 hours of capacitation in combination with the metabolic activator, tyrosine phosphorylation levels triplicate in comparison with control, showing that more spermatozoa might be ready to fertilize the oocyte.

[0070] In an embodiment, the present disclosure uses a SIRT1 activator to enhance sperm capacitation for further use in reproductive technologies (ART). ART consists in collecting and handling oocytes, sperm and embryos in in vitro conditions designed with the aim of increasing the reproductive outcome.

[0071] In another embodiment, the activator is used in combination with standardized IUI protocol to increase the number of IUI performed as expenses of IVF/ICSI. These last 2 procedures are more expensive, complex and less friendly to women.

[0072] In a particular embodiment, a specific SIRT1 activator, YK-3-237 is used in others ART as IVF with the aim to maximize the number of spermatozoa capacitated increasing thus the chances that fertilization occurs.

[0073] The present invention is also for veterinary application. The present results show that it is to be applied to a wide spectrum of species with fertility problems associated to inability to display sperm capacitation status, with special interest on those species with economic interest and endangered/exotic animals. For instance, animal farm industry is suitable to exploit the beneficial effects of enhance sperm capacitation process; for example, equine in vitro fertilization fails mainly due to incomplete activation of spermatozoa because of inadequate capacitating media (Leemans et al., 2016).

[0074] In an embodiment, the activator is used in any ART which implies obtention and processing sperm in in vitro conditions.

[0075] The present invention provides the use of an activator to enhance the sperm capacitation process. The activator synchronizes sperm capacitation process independently of the initial ejaculate status. Thus, with the present activator, which is also a metabolic enhancer, it is possible to promote capacitation events and increase the number of spermatozoa ready to fertilize an oocyte. This invention is useful to be applied in spermatozoa from men unable to conceive a child due to problems associated to sperm capacitation events.

[0076] In an embodiment, the activator is used in combination with standardized intrauterine insemination (IUI) protocol to increase the number of IUI performed as expenses of IVF/ICSI. These last 2 procedures are more expensive, complex protocol and less friendly to women.

[0077] The present invention is also for veterinary use. For instance, animal farm industry is suitable to exploit the beneficial effects of enhancing sperm capacitation process. The equine sector does not have available a successful in vitro fertilization protocol due to an incomplete activation of spermatozoa associated to an inadequate capacitating media. Therefore, the use of the activator disclosed in the present disclosure provides beneficial effects for this specific industry.

[0078] Here, we provide an invention based on the supplementation of a metabolic enhancer to induce maximum tyrosine phosphorylation levels on human spermatozoa after 6 hours of co-incubation in a defined capacitating media containing HCO.sub.3 and a sterol removal. Both components are universal between all the commercial in vitro fertilization media available nowadays. Thus, after 6 hours of incubation in capacitating conditions (bicarbonate and BSA), the best concentration of YK-3-237 which enhanced tyrosine phosphorylation levels was of 10 μM (Figure-la). Moreover, none of the concentration tested had detrimental effect of sperm viability (Figure-1B). After that, the inventors decided to test with one of the components of capacitating media (bicarbonate or BSA) to determine which one is responsible for the increase of tyrosine phosphorylation in presence of YK-3-237 (FIG. 2). Although co-incubation of YK-3-237 in presence of bicarbonate increases the tyrosine phosphorylation levels, this increase was further improved when there was a co-incubation of YK-3-237 in a complete capacitating media containing bicarbonate and BSA (Figure-2). Interestingly, after just 1 hour of co-incubation of spermatozoa with YK-3-237 in capacitating conditions, we achieved the same levels of tyrosine phosphorylation as control samples after 6 hours (Figure-3) illustrating that we found a faster way to induce and achieve capacitation status. Moreover, after 6 hours of incubation, YK-3-237 tripled the levels of tyrosine phosphorylation in comparison with control 6 hours (Figure-3) which shows that more spermatozoa might be ready for fertilization. This means that the present invention identified a way to have more spermatozoa ready for fertilization in a faster way. The inventors checked the spermatozoa viability for the best/selected concentration of YK-3-237, 10 μM, and found that it did not affect sperm viability nor induced any change on markers associated to oxidative stress (lipid peroxidation (4-HNE)) even after 6 hours (Figure-4).

[0079] YK-3-237 allows to hasten the capacitation signaling pathway understood as increased levels of protein tyrosine phosphorylation. Currently, the gold standard technique to measure sperm capacitation status or ability to undergo capacitation process is determining the protein tyrosine phosphorylation levels by western blotting. Although western blotting is a standardized procedure used in molecular biology there is not a routine probe performed in fertility clinics. Moreover, it is not characterized by obtaining results very fast, an average of 3 days entailing: incubation in capacitation conditions by at least 6 hours, protein extraction and concentration determination, plus the timing involved in the western blotting procedure, are needed. Using the activator YK-3-237, there is no need to evaluate the ability of the ejaculate to capacitate.

[0080] Hence, the present invention brings forward this waiting time and the present findings show it is possible to avoid the uncomfortable proceeding of a second semen donation and the annoying displacement to the fertility clinics.

[0081] In a particular embodiment, the present invention discloses not only an increase of the protein tyrosine phosphorylation levels but so bring forward this event. Within one hour of co-incubation we achieved the same levels that are only reached after 6 hours of incubation in control capacitating conditions. Moreover, after 6 hours of sperm co-incubation with YK-3-237, the tyrosine levels are tripled in comparison with control. Consequently, the present disclosure increased the number of spermatozoa in an optimal status to accomplish fertilization increasing their chances to fertilize and oocyte. One cannot neglect the use of YK-3-237 (which enhances capacitation events associated) in combination with other procedures that improve sperm motility as for example with a method using mTOR activators (MHY-1485).

[0082] In one embodiment YK-3-237 is added to the media where spermatozoa are washed and diluted before to perform an intrauterine insemination.

[0083] In a further embodiment, YK-3-237 is added to the sperm capacitation media previously to the coincubation with the oocyte. Although there is an established system to provide spermatozoa from healthy donors, mostly from cryobanks, it is well known that couples under ART treatment only use it when there are no other possible chances to use their own genetic material. Hence, any chance to increase the opportunity to achieve pregnancy using their own genetic material, as the inventors disclose, will be very welcome.

[0084] In another embodiment, YK-3-237 is used alone or in combination with other sperm enhancers. For instance, YK-3-237 is used in combination with other methods which improve sperm motility that would further increase the sperm ability to fertilize and oocyte.

[0085] For sperm incubations Biggers-Whitten-Whittingham (BWW) media described by Biggers J D et al. (1971) with slightly modifications was used. BBW-washed (BWW-W): 94.5 mM NaCl, 4.8 mM KCl, 1.7 mM CaCl.sub.2×2H2O, 1.17 mM KH.sub.2PO.sub.4, 1.22 mM MgSO.sub.4×7H.sub.2O, 20 mM HEPES. Non capacitation media (NC) contains all the component of BWW-washed plus 5.56 mM glucose and gentamycin 10 mg/mL. Capacitating media (CAP): contains all the component of NC plus 25 mM of HCO.sub.3 and 26 mg of Bovine Serum Albumin (BSA). All the media were balanced to a final pH 7.2-7.4

[0086] For sperm preparation, semen samples were collected by masturbation into sterile cups after 2-4 days of sexual abstinence. Samples were left to liquefy for up 2 hours at 37° C. to complete liquefaction prior to processing as described below. Only ejaculates whose semen parameters (total fluid volume, sperm concentration, motility and morphology) meet the (2010) normality criteria established by the WHO (2010) were processed. Ejaculates were submitted to discontinuous gradient centrifugation (Sperm Wash Gradient Set (45% and 90%)) for 20 min at 300 g at room temperature. The bottom gradient layer (purified populations of highly motile sperm) was recovered and washed (500 g for 5 min at room temperature) with modified BWW-W media that does not contain any energy substrate. The pellet was then resuspended in 1 mL of non-capacitating (NC) media and sperm concentration was determined using a Neubauer counting chamber under an optical microscope (×100 magnification). Due to the low number of spermatozoa obtained in each ejaculate, different ejaculates were pooled for different assessments. Finally, depending on experimental design spermatozoa were diluted in 1 ml of capacitating (CAP media containing 26 mg/mL of BSA and 25 mM of bicarbonate) or NC at a final concentration of 20×106/ml for 6 hours at 37° C. in presence or absence of the activator. YK-3-237 was added at the referred concentration at time 0 of incubation and keep along the time of sperm incubation.

[0087] For determination of sperm viability, the instructions by the WHO (2010) were followed, sperm viability was assessed by eosin-nigrosin staining technique. Briefly, an aliquot of semen was mixed with an equal volume of eosin-nigrosin suspension. This suspension was used to make a smear on a glass slide. A total of 200 spermatozoa were counted in random fields under a bright-field microscope. Dead spermatozoa stained pink, as the loss of membrane integrity allows the cells to take up eosin, whereas live cells appear white. Nigrosin stains the background in a dark violet colour for a better visualization of the cells.

[0088] For the Western blot analysis, spermatozoa were washed twice in phosphate-buffered saline (PBS) by centrifugation at 5000 g for 3 min at room temperature. The pellet was resuspended in 2×Laemmli sample buffer and incubated over night at 4° C. The samples were then centrifuged again (10000 g, 15 min, 4° C.) and the protein concentration was measured using a Bio-Rad DC Protein Assay. Samples were boiled for 5 min at 95° C. in presence of 2-mercaptoethanol (2.5%), and 25 μg of each sample was subjected to 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis at constant voltage (100 V) before being transferred to PVDF membranes (Biorad semi-dry system, protocol mix for 8 min). The phosphorylation state of the sperm proteins was analysed by overnight incubation at 4° C. with an anti-phosphotyrosine monoclonal antibody (mAb; Clone 4G10, Millipore; diluted 1:3000, v/v, in Tris-buffered saline-Tween 20 solution (TBST) containing 3% milk). The levels of sperm lipid peroxidation was evaluated by overnight incubation at 4° C. with an anti-4-hydroxynonenal (4HNE) antibody (pAb; Millipore; diluted 1:3000, v/v, in Tris-buffered saline-Tween 20 solution (TBST) containing 3% BSA. Membranes incubated with anti-phosphotyrosine were incubated for 60 min at room temperature with a horseradish peroxidase (HRP)-conjugated secondary anti-mouse antibody (diluted 1:5000, v/v, in TBST containing or 3% milk). Membranes incubated with anti-4HNE were incubated for 60 min at room temperature with a horseradish peroxidase (HRP)-conjugated secondary anti-goat antibody (diluted 1:5000, v/v, in TBST containing or 3% BSA). Finally, membranes were washed 3 times for 5 min in TBST and incubated for 5 min with the Clarity™ Western ECL Substrate.

[0089] Membranes' fluorescence was read with the BioRad FX-Pro-plus (Bio-Rad, Hemel Hempstead, UK). Image analysis was conducted using the Image Lab vs 5.2 (Bio-Rad). Western blotting regions of interest (ROIs) used for quantification are indicated by a vertical bar on the right of the respective western blot. Images shown are representative of experiments repeated three times (n=3) using a pool of three different donors.

[0090] As will be clear to one skilled in the art, the present invention should not be limited to the embodiments described herein, and a number of changes are possible which remain within the scope of the present invention.

[0091] Of course, the preferred embodiments shown above are combinable, in the different possible forms, being herein avoided the repetition all such combinations.

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