Stallion spermatozoa surviving freezing and
thawing experience membrane depolarization
and increased intracellular Na+
C. Ortega Ferrusola, L. Anel-Lopez, J. M. Ortiz-Rodriguez, P. Martin Munoz, M. Alvarez, P. de Paz, J. Masot, E. Redondo, C. Balao da Silva, J. M. Morrell, Heriberto Rodriguez-Martinez, J. A. Tapia, M. C. Gil, L. Anel and F. J. Pena
The self-archived postprint version of this journal article is available at Linköping University Institutional Repository (DiVA):
http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-143075
N.B.: When citing this work, cite the original publication.
Ortega Ferrusola, C., Anel-Lopez, L., Ortiz-Rodriguez, J. M., Martin Munoz, P., Alvarez, M., de Paz, P., Masot, J., Redondo, E., Balao da Silva, C., Morrell, J. M., Rodriguez-Martinez, H., Tapia, J. A., Gil, M. C., Anel, L., Pena, F. J., (2017), Stallion spermatozoa surviving freezing and thawing experience membrane depolarization and increased intracellular Na+, Andrology, 5(6), 1174-1182.
https://doi.org/10.1111/andr.12419 Original publication available at: https://doi.org/10.1111/andr.12419 Copyright: Wiley (12 months) http://eu.wiley.com/WileyCDA/
Stallion spermatozoa surviving freezing and thawing
experience membrane depolarization and increased
intracellular Na
+4Ortega Ferrusola C, 4Anel-López L, 1Ortiz- Rodriguez JM, 1Martin Muñoz P, 4Alvarez M, 5de Paz P, 1Masot J, 1Redondo E, 3Morrell JM, 2 Rodriguez Martinez H, 1Gil MC, 4Anel L, 1Peña FJ*
1Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching
Hospital, University of Extremadura, Cáceres, Spain. 2 Department of Clinical and
Experimental Medicine, Faculty of Medicine & Health Sciences, Linköping University, Linköping, Sweden. 3Division of Reproduction, Faculty of Veterinary Medicine and Animal Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden; 4
Reproduction and Obstetrics Department of Animal Medicine and Surgery, University of León, Spain,. 5Department of Molecular Biology, University of León, Spain
*Correspondence to Dr. FJ Peña, Veterinary Teaching Hospital, Laboratory of Equine Spermatology and Reproduction, Faculty of Veterinary Medicine University of Extremadura Avd de la Universidad s/n 10003 Cáceres Spain. E-mail
fjuanpvega@unex.es phone + 34 927-257167 fax +34 927257102
Acknowledgements
The authors received financial support for this study from the Ministerio de Economía y Competitividad-FEDER, Madrid, Spain, grant AGL2013-43211-R, Junta de Extremadura-FEDER (GR 15029) and the Swedish Research Councils VR (521-2011-6353) and Formas (221-2011-512). PMM is supported by a pre-doctoral grant from the Ministerio de Educación, Cultura y Deporte, Madrid Spain FPU13/03991. COF is supported by a post-doctoral grant from the Ministerio de Economía y Competitividad “Juan de la Cierva” IJCI-2014-21671.
ABSTRACT
In order to gain insight of the modifications that freezing and thawing cause to the surviving population of spermatozoa, changes in the potential of the plasma membrane (Em) and intracellular Na+ content of stallion spermatozoa were investigated using flow cytometry. Moreover, changes in tyrosine phosphorylation and caspase 3 activity were also investigated. Cryopreservation caused a significant (p<0.001) increase in the subpopulation of spermatozoa with depolarized sperm membranes, concomitantly with an increase (p<0.05) of intracellular Na+. These changes occurred in relation to
activation of caspase 3 (p<0.001) without changes in tyrosine phosphorylation. When thawed spermatozoa were supplemented with ATP, intracellular Na+ was reduced (p<0.05) in an sperm subpopulation .Inhibition of the Na+-K+ ATPase pump with ouabain induced caspase 3 activation. It is concluded that inactivation of Na+-K+ ATPase occurs during cryopreservation, an inhibition that could explain the accelerated senescence of the surviving population of spermatozoa, caused by activation of caspase 3.
Key words: sperm, cryopreservation, flow cytometry, ATP, Na+-K+ ATPase, horse.
INTRODUCTION
Cryopreservation of stallion semen is the ultimate goal in the equine breeding industry [1]. However, this technology is still flawed by a number of drawbacks, the major ones probably being the lack of standardization, and stallion-to-stallion variability.
Moreover, the surviving population in the mare´s reproductive tract has a reduced lifespan, and a reduced capability to replenish the sperm reservoir in the oviduct [2], compared with raw or fresh semen. This latter fact increases the costs related to artificial insemination (AI) due to the more intense mare management necessary to perform AI close enough to ovulation to compensate this reduced lifespan. In many instances, deep horn intrauterine insemination or even video-endoscopic assisted AI are required. Accelerated senescence of cryopreserved spermatozoa has been attributed to capacitation-like changes [3, 4], or, more recently, apoptotic changes or “spermptosis” as it has recently been termed [5, 6]. Capacitation is a redox activated process, with increased production of reactive oxygen species (ROS) activating a soluble adenylyl cyclase (sAC) that stimulates cAMP and protein kinase A (PKA), that in turn activates SRC kinases; leading to a dramatic up-regulation of tyrosine phosphorylation,
characteristic of capacitation. Recently, plasma membrane potential (Em)
hyperpolarization [7, 8] and decreased intracellular sodium [9] have been described as hallmarks of capacitation. Initiation of capacitation involves ion fluxes, with influx of HCO3- activatingthe soluble adenylyl cyclase and subsequently protein kinase A
(PKA). Permeability to Na+ is also reduced during sperm capacitation leading to Em hyperpolarization [9]. We hypothesized that if changes in the cryo-surviving sperm population are capacitating, increased tyrosine phosphorylation, Em hyperpolarization and reduced intracellular sodium should occur in thawed spermatozoa. On the other hand, apoptosis is characterized by cell shrinkage caused by disruption in maintenance of the normal physiological concentrations of K(+) and Na(+) and intracellular ion
homeostasis. The disrupted ion homeostasis leads to depolarization and apoptosis [10, 11]. Thus, following the spermptosis theory, similar changes may occur in thawed spermatozoa. In this study, we used flow cytometry to qualitatively analyze changes in intracellular Na+ concentration in fresh stallion spermatozoa and in the same ejaculates after thawing. Moreover, we also used the anionic fluorescent voltage-sensitive probe DiSBAC2 (3) [7] to investigate changes in sperm Em as consequence of
cryopreservation. Changes in tyrosine phosphorylation and activation of caspase 3 were also determined. Overall, our observations provide new evidence linking
cryopreservation and apoptotic changes.
MATERIAL AND METHODS Reagents and media
CellRox Deep Red Reagent (Excitation: 644 nm; Emission: 655nm) (Ref: C10422); Cell Event Caspase-3/7 Green Detection Reagent (Excitation, 502 nm; Emission: 530 nm) (Ref: C10423); ethidium homodimer (Excitation, 528 nm; Emission, 617 nm) (Ref: E1169); LIVE/DEAD® Fixable Violet Dead Cell Stain Kit (Excitation: 405 nm, Emission: 451 nm) (Ref: L34955), Sodium Green cell permeant indicator Ref S-6901 (Excitation 488nm Emission 532 nm), DisBAC2(3) (Excitation 488 Emission 525 nm)
(Ref B413) Mitotracker deep Red (Excitacion 644 Emission 655) (Ref M22426) were purchased from ThermoFisher Scientific (Molecular Probes) (Waltham, Massachusetts, USA). Phospho-Tyrosine Mouse mAb (P-Tyr-100) (PE conjugate) (Ref: 14967) was acquired from Cell Signaling technology (Danvers, Massachusetts, USA) Anti CD 44 APC/Fire antibody was purchased from Biolegend (Excitation 650, Emission 787) Ref 103061. ATP, Ouabain and all other chemicals were purchased from Sigma-Aldrich (Madrid, Spain).
Semen collection and processing
Samples from 6 fertile stallions were obtained on a regular basis (three collections /week) during the 2016 breeding season. Stallions were maintained according to institutional and European regulations (Spanish Law 6/2913 June 11th and European Directive 2010/63/EU). Ejaculates were obtained using a pre-warmed (45ºC), lubricated Missouri model artificial vagina with an inline filter to eliminate the gel fraction. The semen was immediately transported to the laboratory for evaluation and processing. The ejaculates were separated into two aliquots, extended 1:2 in INRA96 (IMV, L’Aigle, France), and centrifuged at 600g for 10 min at room temperature. One of the aliquots was further extended in INRA 96 after centrifugation to obtain a final concentration of 100 x 106 spermatozoa/ml and was kept at room temperature (22ºC) for 1 hour for analysis as controls. The other aliquot was extended in the freezing medium Cáceres (University of Extremadura Cáceres, Spain) to 100x106 spermatozoa/ml. After loading the extended
semen into 0.5-mL straws (IMV, L’Aigle, France), the straws were ultrasonically sealed with UltraSeal 21® (Minitube of America MOFA, Verona, Wisconsin, USA) and immediately placed in an IceCube 14S (SY-LAB Neupurkersdorf, Austria) programmable freezer. The following freezing curve was used: straws were kept for 15 min at 20ºC, and they were then slowly cooled from 20ºC to 5ºC at a cooling rate of 0.1 ºC/min. Thereafter the freezing rate was increased to -40ºC/min from 5ºC to -140ºC. The straws were then plunged into liquid nitrogen and stored until analysis. For the analysis, two straws per stallion and freezing operation were thawed in a water bath at 37ºC for at
least 30 sec and extendedin pre-warmed INRA-96 extender to a final concentration of 50 x 106 spermatozoa/ml. All analyses were conducted immediately post-thaw.
Flow cytometry
Flow cytometry analyses were conducted using a MACSQuant Analyzer 10 (Miltenyi Biotech), flow cytometer equipped with three lasers emitting at 405, 488, and 635 nm and 10 photomultiplier tubes (PMTs): V1 (excitation 405 nm, emission 450/50 nm), V2 (excitation 405nm, emission 525/50 nm), B1 (excitation 488 nm, emission 525/50nm), B2 (excitation 488 nm, emission 585/40 nm), B3 (excitation 488 nm, emission 655-730 nm (655LP + split 730), B4 (excitation 499 nm, emission 750 LP), R1 (excitation 635 nm, emission 655-730 nm (655LP+split 730), and R2 (excitation 635 nm, emission filter 750 LP. The system was controlled using MACS Quantify software. The quadrants or regions used to quantify the frequency of each sperm subpopulation depended on the particular assay. Forward and sideways light scatter were recorded for a total of 50,000 events per sample. Gating the sperm population after Hoechst 33342 staining eliminated non-sperm events. The instrument was calibrated daily using specific calibration beads provided by the manufacturer. A compensation overlap was performed before each particular experiment. The data were analyzed using FlowjoV 10.2 Software (Ashland, OR, USA). Unstained, single-stained and Fluorescence Minus One (FMO) controls were used when appropriate to determine compensations and positive and negative events, as well as to set regions of interest as described in previous publications from our laboratory [6, 12, 13].
Em assessment in fresh and frozen thawed stallion spermatozoa
The potential of the sperm membrane (Em) of stallion spermatozoa was measured using flow cytometry, following published protocols [7] adapted to equine species in our laboratory. DiSBAC2 (3) belong to the family of Voltage Sensor Probes (VSPs), awe used a Fluorescence Resonance Energy Transfer (FRET)-based voltage-sensing assay technology to measure changes in cellular membrane electrical potential. Voltage Sensor Probes (VSPs) represent a Fluorescence Resonance Energy Transfer (FRET)-based voltage-sensing assay technology used to measure changes in cellular membrane electrical potential. The VSP-FRET pair consists of a mobile, voltage sensitive acceptor and an outer membrane bound donor in resting cells (with a relative negative internal potential); both members of the FRET pair bind to the outer surface resulting in efficient FRET. When the cells are depolarized, the donor remains in the outer surface but the mobile acceptor rapidly translocates to the inner surface of the membrane resulting in diminished FRET. Stallion
spermatozoa were loaded with DiSBAC2 (3) 15 µM for 30 min at 37ºC; dead
spermatozoa were gated out after staining with LIVE/DEAD® Fixable Violet Dead
Cell Stain Kit (1 µL/ 1mL of sperm suspension)
Determination of intracellular Na+
The amount of intracellular Na+ in stallion spermatozoa was determined flow cytometrically [14] after loading the cells with Sodium Green Permeant (5 µM), and incubation at r.t. for 30 min. Finally, samples were washed in PBS and dead spermatozoa excluded from the analysis after ethidium homodimer staining (0.35µM at r.t. for 5min).
Simultaneous flow cytometric assessment of caspases 3 and 7 activity, viability and production of superoxide anion (O2-)
CellEvent Caspase-3/7 Green Detection Reagent consists of a four-amino-acid peptide (DEVD) conjugated to a nucleic acid-binding dye. This cell-permeant substrate is intrinsically non-fluorescent because the DEVD peptide inhibits the ability of the dye to bind to DNA. After activation of caspase 3 and caspase 7 in apoptotic cells, the DEVD peptide is cleaved, enabling the dye to bind to DNA and produce a bright, fluorogenic response with absorption/emission maxima of 502/530 nm. Spermatozoa exhibiting oxidative stress emit fluorescence in the far red spectrum after staining with CellRox deep red Reagent, whereas Hoechst 33342-positive sperm emit blue fluorescence. The semen samples were diluted in PBS to a final concentration 5 x 106 spermatozoa/ml. Thecells were stained with 1µl of CellRox (5μM), 1µl CellEvent Caspase-3/7 Green Detection Reagent (2mM) and 0.3 µl of Hoechst 33342 (0.5μM). After thorough mixing, the sperm suspension was incubated at r.t. in the dark for 25 min, washed in PBS, and loaded with 0.3µl ethidium homodimer (1.167 mM in DMSO). After incubation for a additional 5 min, the samples were immediately run on the flow cytometer. The controls consisted of unstained and single stained controls to properly set gates and compensations. The positive controls for oxidation and caspase activation were samples supplemented with 800 µM SO4Fe2 and 1.7 M H2O2 (Sigma) to induce the Fenton reaction [6]. The debris
was gated out based on the DNA content of the events after Hoechst 33342 staining. Representative cytograms of the assay and gating strategies performed are given in fig 1.
Simultaneous detection of Em, Caspase 3 activity, live and dead spermatozoa, mitochondrial activity and CD 44 expression
For staining, stallion spermatozoa were washed in PBS and resuspended in PBS to a final concentration of 4 x106/ml. Stallion spermatozoa were loaded with DiSBAC2 (3) 15 µM for 30 in at 37ºC, dead spermatozoa were gated out after staining with LIVE/DEAD® Fixable Violet Dead Cell Stain Kit (1 µL/ 1mL of sperm suspension), 1µl CellEvent Caspase-3/7 Green Detection Reagent (2mM), and 1µl of Mitotracker deep red (stock solution 10 µM) and incubated at rt for 20 min. Then samples were washed in PBS and
resuspended in 100 µl of PBS-1%BSA , the samples were incubated with anti-CD44
APC/Fire antibody 1:100 for 20 minutes at r.t, washed in PBS-1%BSA and resuspended in 1 ml of PBS for flow cytometry analysis
Assessment of tyrosine phosphorylation.
The anti phospho-Tyrosine Mouse mAb (P-Tyr-100) (PE conjugate) was used to detect the phosphorylation of tyrosine. The samples (1 ml) containing 10 x 106 spermatozoa/mL in PBS were stained with 1 μL of LIVE/DEAD violet Fixable Dead Cell stain kit Solution. After thorough mixing, the samples were incubated at room temperature (22ºC) for 30 min in the dark. Then, the spermatozoa were washed with PBS (1x) and fixed with 900 μL of 4% paraformaldehyde and incubated for 10 minutes at 37ºC. Afterwards, samples were incubated 1 minute on ice. The aliquots were washed by centrifugation twice more. For the permeabilization, pre-chilled cells were slowly resuspended in ice-cold 100% methanol, to a final concentration of 90% methanol. The samples were incubated 30 minute on ice. Then, the spermatozoa were washed in incubation buffer (0.5g Bovine Serum Albumin (BSA) in 100 ml 1x PBS) twice. The cells were loaded with 2 µL/ml Phosphor-Tyrosine Mouse mAb (P-Tyr-100) (PE conjugate) (50µg/ml # 14967) at room
temperature (22ºC) in the dark for 30 min. Then, the spermatozoa were washed in 500 µL PBS. Finally, the samples were washed again in PBS before reading in the flow cytometer
Automatic classification of cellular expression by nonlinear stochastic embedding (ACCENSE)
Flow cytometry data are usually analyzed using a series of 2D plots and manual gating. However, the augmentation in the number of parameters measured increase the number of 2D plots to display for every marker combination; for example a combination of four colors will require 30 2D plots. To overcome these problems, computational methods to automatically identify populations in multidimensional flow cytometry data have now been developed [15]. We used the ACCENSE software available at http://www.cellaccense.com. Compensated data of the multi-parametric assay described in material and methods were exported as FCS files from the flow cytometer, and loaded in ACCENSE for computational analysis, data were concatenated and single cell events analyzed. Flow cytometry data were analyzed using automatic classification of cellular expression by non-linear stochastic embedding (ACCENSE). The Barnes –Hut implementation of t-SNE was used for low dimensional embedding to perform dimensional reduction of cytometry data; classification of cells was based in K means techniques with the significance level set at p=0.0001. Data of all subpopulations generated were exported as FCS files. ACCENSE identifies clusters within multidimensional data without losing single cell resolution [16, 17], allowing automatic gating of cells.
Statistical analysis
Three ejaculates were collected from each of 7 individual stallions. All experiments were repeated at least three times with independent samples (three separate ejaculates from each of the seven stallions). The normality of the data was assessed using the Kolmogorov-Smirnoff test. Since the data showed equivalence of variance, the results were analyzed using a paired t test (fresh versus FT spermatozoa) (SPSS 19.0 for Mac). Differences were considered significant when P < 0.05, and are indicated as; * p<0.05 and ** p<0.01. Results are displayed as means ± SEM. Correlations between assays were investigated using the Pearson correlation test.
RESULTS
Cryopreservation and thawing cause Em depolarization in a subpopulation of surviving spermatozoa
Changes in Em in individual spermatozoa were investigated using the anionic bis oxonol dye DiSBAC2 (3). In order to restrict the assay to live spermatozoa the Live Dead Violet
fixable dye was used. This combination of probes has minimal spectral overlay. DiSBAC2 (3) enters depolarized cells where they bind to the intracellular proteins of
membranes and exhibit enhanced fluorescence and red-orange spectral shifts. Increased depolarization results in more influx of the anionic dye and thus an increase in fluorescence. Conversely, hyperpolarization is indicated by a decrease in fluorescence. In contrast to cationic carbocyanines, anionic bis-oxonols are largely excluded from mitochondria and are primarily sensitive to plasma membrane potential [18]. Initially a gate was applied to the live subpopulation of spermatozoa to restrict the analysis to live
sperm (Fig 2 A). In fresh semen, two sperm subpopulations were easily identified, with a population showing increased DiSBAC2 (3) fluorescence (Fig 2B) representing
depolarized spermatozoa with respect to the general population of spermatozoa. When stallion semen was frozen and thawed, significant changes occurred in the distribution of sperm subpopulations with respect to their Em. In thawed semen there was a marked change in the distribution of Em of the surviving spermatozoa (Fig 2 C) with a significant increase in the number of spermatozoa showing depolarized membranes (p<0.001) and a significant decrease in the percentage of hyperpolarized membranes (p<0.001) compared to fresh sperm (fig 2D).
Cryopreservation and thawing causes a increase in [Na+] in a subpopulation of the surviving spermatozoa
Since the depolarization of a subpopulation of spermatozoa can be attributed to intra-cellular increase in Na+, changes in sodium content in the population of spermatozoa surviving freezing and thawing were monitored using flow cytometry. The analysis was restricted to live spermatozoa gating out dead sperm after ethidium homodimer staining (fig 3 A). In fresh sperm, a single sperm subpopulation in terms of Na+ content was identified (fig 4 A-C). In contrast, thawed spermatozoa showed two subpopulations showing high (H) and low (L) Na+ content, respectively (fig 3F). Overall, cryopreserved thawed spermatozoa showed an increase in intracellular sodium (fig 3 g).
Cryopreservation modifies the sperm subpopulation structure, activates caspase 3 and reduces ROS
ACCENSE identified sperm subpopulations in the stallion ejaculate based on caspase expression, live and dead spermatozoa and production of reactive oxygen species (ROS). In the 2D t-SNE maps obtained, each point represent a spermatozoon in the ejaculate (n= 10,000) after down sampling of the original data set in fresh (fig 4 A) and frozen-thawed spermatozoa (fig 5 D). As shown in B and E, the structure of the sperm subpopulations changed after cryopreservation, the structure of thawed samples being more complex than fresh semen. In fig 4 B (fresh) and E (frozen thawed) subpopulation of cells are presented. Effects of cryopreservation were studied in relation to spermptosis and production of reactive oxygen species. For this purpose a multi-parameter panel was designed and stallion spermatozoa were analyzed to determine live and dead spermatozoa, caspase 3 activity and production of ROS (fig 1). Also the different expression of caspase 3 (fig 4 C and F) and production of ROS (fig 5 C and F) were investigated at a single cell level using ACCENSE. Particular attention was paid to the expression of caspase 3 in the subpopulation of spermatozoa surviving cryopreservation. Traditional flow cytometry also revealed that the population of spermatozoa that survives cryopreservation showed increased caspase 3 activity (p<0.001) (fig 4 G) and reduced production of ROS (fig 7 B). Moreover activation of caspase 3 was studied using computational flow cytometry. The 2D t –SNE map of caspase 3 activity showed an increase in caspase 3 expression in thawed samples as indicated in the heat maps (fig 4 C-F). The ACCENSE analysis also revealed a reduced production of O2•- after cryopreservation (fig 5 C and F).
Cryopreservation does not modify tyrosine phosphorylation of sperm proteins
Since Tyrosine phosphorylation of sperm proteins is a hallmark of capacitation of mammalian spermatozoa, changes after cryopreservation were investigated using flow
cytometry and anti phosphotyrosine monoclonal antibodies. Cryopreservation did not modify tyrosine phosphorylation of sperm proteins (fig 7 A).
Relationships among DiSBAC2 (3) fluorescence, caspase 3 activity and tyrosine phosphorylation in thawed stallion spermatozoa
Significant correlations were found among depolarized sperm membranes and cells producing ROS (p<0.01) in Caspase 3 positive spermatozoa (P<0.01) (Table 1). A There was also a significant correlation between depolarized cells and tyrosine phosphorylation (p<0.05). The opposite situation was observed among hyperpolarized spermatozoa where negative correlations with ROS positive, Caspase 3 + and ROS positive and tyrosine phosphorylation were observed (Table 1).
Relations among caspase 3 and Na+ in raw semen and depolarized and caspase 3, and depolarized membranes in thawed sperm
Intracellular Na+ correlated with caspase 3 activity in fresh sperm (R= 0.662, p<0.05), and caspase 3 activity also correlated with an increase in the number of spermatozoa with depolarized membranes in thawed spermatozoa (R= 0.770, P<0.01) (table 2)
The subpopulation of depolarized spermatozoa is also caspase 3 positive
To further investigate the relationship between sperm membrane depolarization and caspase 3, 2D plots were constructed to simultaneously evaluate DiSBAC 2 (3) and
caspase 3 fluorescence. Caspase 3 fluorescence co-localized with the subpopulation of depolarized spermatozoa (fig 7 A). In addition, most spermatozoa with high mitochondrial activity corresponded to spermatozoa with hyperpolarized membranes (fig 7 B). Also most CD 44 positive spermatozoa corresponded to hyperpolarized and low caspase 3 spermatozoa (fig 7 C and D). When the same relation was investigated at the single cell level using ACCENSE, the t-SNE maps for each of the channels similar findings were evident, with more intense DiSBAC 2 (3) fluorescence (depolarized)
corresponding also with more intense Caspase 3 florescence (fig 7 E and F).
Supplementation of thawed spermatozoa with ATP reduces intracellular Na+ in a subpopulation of spermatozoa
If increments in intracellular Na+ causing membrane depolarization in thawed stallion spermatozoa are due to inhibition of the Na+-K+ pump due to low ATP levels, a restoration of ATP should decrease Na+. To test this hypothesis, thawed spermatozoa were incubated in BWW medium supplemented with ATP (0, 0.5, 2.5, 5 and 10 mM) at 37ºC. At the beginning of the incubation period and after 30 min, intracellular Na+ was measured in the surviving sperm subpopulation. Two subpopulations were identified (fig 8 C-F), corresponding to spermatozoa with high (H) and low (L) Na+. When both subpopulations were considered, at the beginning of the incubation period, supplementation with ATP reduced intracellular Na at concentrations of 0.5mM, but increased Na+ at concentrations of 10mM (fig 8 A). After 30 min of incubation, ATP at 5 and 10 mM increased Na+. However, when the analysis was performed independently in the subpopulations of high and low Na+, interesting results were observed. Concentrations of ATP higher than 2.5 mM reduced Na+ in the L-Na+ subpopulation at the beginning of the incubation period (p<0.05) (fig 8 B) and all concentrations except
ATP 5mM, reduced Na+ after 30 minutes of incubation (p<0.05) (fig 8 E). In the subpopulation of H-Na+ ATP at 0.5 and 1mM reduced Na + at the beginning of the incubation period (p<0.05) (fig 4 B).
Inhibition of the Na+-K+ ATPasa activates Caspase 3 in fresh and frozen-thawed spermatozoa
To provide further evidence linking dysfunction of the Na+-K+ ATPase pump and apoptotic changes, stallion spermatozoa were incubated in presence of 0, 10, 100, 1000 or 2000 μM ouabain for 5 h. At 0, 2, and 5 h of incubation, 10 μl of semen were drawn from each treatment group and caspase 3 activation was measured using flow cytometry. At the beginning of the incubation period, and after 3 hours of incubation, there were no effects of ouabain on the percentage of live sperm or in caspase activation (fig 9 A and B). However, after 5 hours of incubation, ouabain reduced the percentage of live spermatozoa and increased caspase 3 activation (fig 9 C).
DISCUSSION
In the present study, we evaluated changes induced by cryopreservation in the surviving population of spermatozoa. Recent research indicates that capacitation is accompanied by the hyperpolarization of the sperm plasma membrane in a sperm subpopulation [7]. This hyperpolarization is linked to decreased intracellular Na+ [9].
In an attempt to disclose if spermatozoa surviving cryopreservation experience accelerated capacitation-like changes, tyrosine phosphorylation, changes in intracellular Na+ and the polarization of the plasma membrane were monitored before and after cryopreservation. We describe, for the first time, membrane depolarization and increased intracellular Na+ in relation to cryopreservation. This novel finding provides new evidence indicating that molecular changes induced by cryopreservation do not mimic capacitation, and support the theory of spermoptosis [14]. Caspase 3 was activated after thawing, confirming previous reports [5, 19], and, more interestingly, co-localized in the population showing membrane depolarization. It was determined previously that improvement of current cryopreservation protocols depends on proper knowledge of the molecular injuries occurring during this process [1, 20]. Identification of molecular targets may pave the way for new strategies of sperm conservation, as has been recently reported for stallion conservation at ambient temperature [21, 22].
Changes in intracellular Na+ have been described in relation to apoptosis in numerous cellular models [23, 24], linked to dysfunction of the Na+, K+ ATPase pump. The Na +-K+ pump is an essential heterodimeric membrane protein, which maintains electrochemical gradients for Na+ and K+ across cell membranes in all tissues [25]. Active transport mediated by this pump maintains the electrochemical gradients for Na+ and K+ across cell membranes and is estimated to consume up to 20-30% of cell ATP [26]. In relation to this, stallion spermatozoa depends primarily on OXPHOS in the mitochondria to obtain energy for motility [27-30], and cryopreservation has a major impact on mitochondria, leading to mitochondrial dysfunction [31-33]. Both factors might explain the increased Na+ observed in thawed samples, linking inhibition of the Na+-K+ ATPase and sperm dysfunction post-haw. To test this hypothesis, we supplemented thawed samples with ATP and Na+ was measured again; a population of spermatozoa responded to ATP supplementation with reduced Na+ suggesting that
reduced ATP may cause increased Na+ in a subpopulation of cryo-surviving spermatozoa. However, not all subpopulations responded to exogenous ATP; this can be explained by cryopreservation-induced damage of the protein itself. Cryopreservation increases the formation of 4-hydroxynonenal (4-HNE) [5, 19], that can lead to degradation of proteins through post-translational modifications. Interestingly, it has been demonstrated that 4-HNE is able to alter the function of diverse trans-membrane transporters [34], providing a plausible explanation for our findings. On the other hand, the Na+ K+ ATPase pump is a redox regulated protein [25], and thawed stallion spermatozoa experiences a massive redox deregulation [1, 33, 35].
Increased intracellular Na+ relate to apoptosis in somatic cells [36-39], and in our study cryopreservation caused membrane depolarization, increased Na+ and activation of caspase 3. These findings support the hypothesis linking increased Na+ and apoptotic changes also in spermatozoa. In this regard, significant correlations were observed between caspase 3 activity and Na+ content. Further evidence linking malfunction of the Na+-K+ ATPase increased sodium and apoptosis was the ability of ouabain to activate caspase 3 in spermatozoa. Similar findings have been recently reported in other mammalian spermatozoa linking Na+-K+ dysfunction increased Na+ and membrane depolarization [40-42].
Interesting additional findings relate to differences in the phenotype of cryo-surviving spermatozoa, stressing the already documented heterogeneous nature of the ejaculate [43-45]. Flow cytometry has been extensively used for the assessment of sperm functionality and to determine the quality of a semen sample [46, 47]. In addition, flow cytometry has been shown to be a powerful tool for the study of sperm biology [6, 7, 9, 14]. In our study, its ability to provide information at the single cell level was especially relevant. Changes in the polarization of the membrane appeared only in a subpopulation of spermatozoa, depolarization occurred in a subpopulation of spermatozoa after thawing. The bimodal response of spermatozoa has been previously reported [7] and capacitation in vivo occurs only in a specific sperm subpopulation [48, 49], representing a small percentage of all the spermatozoa in the ejaculate. Also, the increase in intracellular Na+ was more evident in a subpopulation of spermatozoa, as was the response to exogenous ATP. In our study, we also employed computational flow cytometry to obtain information at the single cell level. This approach provided interesting information, disclosing the complex structure of the ejaculate and showing that the cryopreservation process even increases this complexity. This technique also allows for a clear visualization of changes of the sperm phenotype. In our experiment, the analysis of the sperm phenotype allowed changes associated with cryopreservation to be identified more accurately at the single cell level, Interestingly, an increase in the expression of caspase 3 was more evident than ethidium uptake. Another interesting finding was the novel application of a 5 colors panel in flow cytometry applied to spermatozoa, demonstrating, for example, the relation between caspase 3 and membrane depolarization, or the utility of the CD44 marker as a possible tool for the identification of fertilizing spermatozoa [50-55]; the ability to bind hyaluronic acid is considered to be a hallmark of spermatozoa that have completed the spermiogenic process of sperm plasma remodeling, cytoplasmatic extrusion and nuclear histone – protamine replacement and have unreacted acrosomes [55]. Our findings support this hypothesis since CD44 was present in caspase 3 negative, hyperpolarized spermatozoa.
In conclusion, this study provided new evidence linking apoptotic changes and cryopreservation as opposed to capacitation-like changes. Moreover, we provide for the
first time evidence linking malfunction of Na+-K+ ATPase and the apparition of apoptotic changes in cryopreserved stallion spermatozoa, suggesting a new target for the development of strategies to improve current cryopreservation protocols.
REFERENCES
1. Pena FJ, Garcia BM, Samper JC, Aparicio IM, Tapia JA, Ferrusola CO. Dissecting the molecular damage to stallion spermatozoa: the way to improve current cryopreservation protocols? Theriogenology 2011; 76:1177-1186. 2. Abad M, Sprecher DJ, Ross P, Friendship RM, Kirkwood RN. Effect of sperm
cryopreservation and supplementing semen doses with seminal plasma on the establishment of a sperm reservoir in gilts. Reprod Domest Anim 2007; 42:149-152.
3. Sieme H, Harrison RA, Petrunkina AM. Cryobiological determinants of frozen semen quality, with special reference to stallion. Anim Reprod Sci 2008; 107:276-292.
4. Schembri MA, Major DA, Suttie JJ, Maxwell WM, Evans G. Capacitation-like changes in equine spermatozoa throughout the cryopreservation process. Reprod Fertil Dev 2002; 14:225-233.
5. Munoz PM, Ferrusola CO, Lopez LA, Del Petre C, Garcia MA, de Paz Cabello P, Anel L, Pena FJ. Caspase 3 Activity and Lipoperoxidative Status in Raw Semen Predict the Outcome of Cryopreservation of Stallion Spermatozoa. Biol Reprod 2016; 95:53.
6. Gallardo Bolanos JM, Balao da Silva CM, Martin Munoz P, Morillo Rodriguez A, Plaza Davila M, Rodriguez-Martinez H, Aparicio IM, Tapia JA, Ortega Ferrusola C, Pena FJ. Phosphorylated AKT preserves stallion sperm viability and motility by inhibiting caspases 3 and 7. Reproduction 2014; 148:221-235. 7. Escoffier J, Navarrete F, Haddad D, Santi CM, Darszon A, Visconti PE. Flow
cytometry analysis reveals that only a subpopulation of mouse sperm undergoes hyperpolarization during capacitation. Biol Reprod 2015; 92:121.
8. McPartlin LA, Visconti PE, Bedford-Guaus SJ. Guanine-nucleotide exchange factors (RAPGEF3/RAPGEF4) induce sperm membrane depolarization and acrosomal exocytosis in capacitated stallion sperm. Biol Reprod 2011; 85:179-188.
9. Escoffier J, Krapf D, Navarrete F, Darszon A, Visconti PE. Flow cytometry analysis reveals a decrease in intracellular sodium during sperm capacitation. J Cell Sci 2012; 125:473-485.
10. Suzuki-Karasaki Y, Suzuki-Karasaki M, Uchida M, Ochiai T. Depolarization Controls TRAIL-Sensitization and Tumor-Selective Killing of Cancer Cells: Crosstalk with ROS. Front Oncol 2014; 4:128.
11. Suzuki-Karasaki M, Ochiai T, Suzuki-Karasaki Y. Crosstalk between
mitochondrial ROS and depolarization in the potentiation of TRAIL-induced apoptosis in human tumor cells. Int J Oncol 2014; 44:616-628.
12. Pena FJ, Johannisson A, Wallgren M, Rodriguez-Martinez H. Assessment of fresh and frozen-thawed boar semen using an Annexin-V assay: a new method of evaluating sperm membrane integrity. Theriogenology 2003; 60:677-689. 13. Martin Munoz P, Ortega Ferrusola C, Vizuete G, Plaz Davila M, Rodriguez
Martinez H, Pena Vega FJ. Depletion of Intracellular Thiols and Increased Production of 4-Hydroxynonenal That Occur During Cryopreservation of
Stallion Spermatozoa Leads to Caspase Activation, Loss of Motility, and Cell Death. Biol Reprod 2015.
14. Ortega Ferrusola C, Anel-Lopez L, Martin-muNoz P, Ortiz Rodriguez JM, Gil MC, Alvarez M, De Paz P, Ezquerra J, Masot J, Redondo E, Anel L, Pena FJ. Computational flow cytometry reveals that cryopreservation induces
spermptosis. Reproduction 2016.
15. Mair F, Hartmann FJ, Mrdjen D, Tosevski V, Krieg C, Becher B. The end of gating? An introduction to automated analysis of high dimensional cytometry data. Eur J Immunol 2016; 46:34-43.
16. Shekhar K, Brodin P, Davis MM, Chakraborty AK. Automatic Classification of Cellular Expression by Nonlinear Stochastic Embedding (ACCENSE). Proc Natl Acad Sci U S A 2014; 111:202-207.
17. Chester C, Maecker HT. Algorithmic Tools for Mining High-Dimensional Cytometry Data. J Immunol 2015; 195:773-779.
18. Dasheiff RM. A new method of monitoring membrane potential in rat
hippocampal slices using cyanine voltage-sensitive dyes. J Neurosci Methods 1985; 13:199-212.
19. Martin Munoz P, Ortega Ferrusola C, Vizuete G, Plaza Davila M, Rodriguez Martinez H, Pena FJ. Depletion of Intracellular Thiols and Increased Production of 4-Hydroxynonenal that Occur During Cryopreservation of Stallion
Spermatozoa Lead to Caspase Activation, Loss of Motility, and Cell Death. Biol Reprod 2015; 93:143.
20. Kopeika J, Thornhill A, Khalaf Y. The effect of cryopreservation on the genome of gametes and embryos: principles of cryobiology and critical appraisal of the evidence. Hum Reprod Update 2015; 21:209-227.
21. Swegen A, Lambourne SR, Aitken RJ, Gibb Z. Rosiglitazone Improves Stallion Sperm Motility, ATP Content, and Mitochondrial Function. Biol Reprod 2016; 95:107.
22. Gibb Z, Lambourne SR, Quadrelli J, Smith ND, Aitken RJ. L-carnitine and pyruvate are prosurvival factors during the storage of stallion spermatozoa at room temperature. Biol Reprod 2015; 93:104.
23. Bortner CD, Cidlowski JA. Uncoupling cell shrinkage from apoptosis reveals that Na+ influx is required for volume loss during programmed cell death. J Biol Chem 2003; 278:39176-39184.
24. Kawazoe N, Aiuchi T, Masuda Y, Nakajo S, Nakaya K. Induction of apoptosis by bufalin in human tumor cells is associated with a change of intracellular concentration of Na+ ions. J Biochem 1999; 126:278-286.
25. Figtree GA, Liu CC, Bibert S, Hamilton EJ, Garcia A, White CN, Chia KK, Cornelius F, Geering K, Rasmussen HH. Reversible oxidative modification: a key mechanism of Na+-K+ pump regulation. Circ Res 2009; 105:185-193. 26. Jorgensen PL, Hakansson KO, Karlish SJ. Structure and mechanism of
Na,K-ATPase: functional sites and their interactions. Annu Rev Physiol 2003; 65:817-849.
27. Davila MP, Munoz PM, Bolanos JM, Stout TA, Gadella BM, Tapia JA, da Silva CB, Ferrusola CO, Pena FJ. Mitochondrial ATP is required for the maintenance of membrane integrity in stallion spermatozoa, whereas motility requires both glycolysis and oxidative phosphorylation. Reproduction 2016; 152:683-694. 28. Plaza Davila M, Martin Munoz P, Tapia JA, Ortega Ferrusola C, Balao da Silva
CC, Pena FJ. Inhibition of Mitochondrial Complex I Leads to Decreased Motility and Membrane Integrity Related to Increased Hydrogen Peroxide and
Reduced ATP Production, while the Inhibition of Glycolysis Has Less Impact on Sperm Motility. PLoS One 2015; 10:e0138777.
29. Ortega Ferrusola C, Gonzalez Fernandez L, Salazar Sandoval C, Macias Garcia B, Rodriguez Martinez H, Tapia JA, Pena FJ. Inhibition of the mitochondrial permeability transition pore reduces "apoptosis like" changes during
cryopreservation of stallion spermatozoa. Theriogenology 2010; 74:458-465. 30. Gibb Z, Lambourne SR, Aitken RJ. The paradoxical relationship between
stallion fertility and oxidative stress. Biol Reprod 2014; 91:77.
31. Gonzalez-Fernandez L, Morrell JM, Pena FJ, Macias-Garcia B. Osmotic shock induces structural damage on equine spermatozoa plasmalemma and
mitochondria. Theriogenology 2012; 78:415-422.
32. Garcia BM, Moran AM, Fernandez LG, Ferrusola CO, Rodriguez AM, Bolanos JM, da Silva CM, Martinez HR, Tapia JA, Pena FJ. The mitochondria of stallion spermatozoa are more sensitive than the plasmalemma to osmotic-induced stress: role of c-Jun N-terminal kinase (JNK) pathway. J Androl 2012; 33:105-113.
33. Pena FJ, Plaza Davila M, Ball BA, Squires EL, Martin Munoz P, Ortega Ferrusola C, Balao da Silva C. The Impact of Reproductive Technologies on Stallion Mitochondrial Function. Reprod Domest Anim 2015; 50:529-537. 34. Jovanovic O, Pashkovskaya AA, Annibal A, Vazdar M, Burchardt N, Sansone
A, Gille L, Fedorova M, Ferreri C, Pohl EE. The molecular mechanism behind reactive aldehyde action on transmembrane translocations of proton and potassium ions. Free Radic Biol Med 2015; 89:1067-1076.
35. Neagu VR, Garcia BM, Sandoval CS, Rodriguez AM, Ferrusola CO, Fernandez LG, Tapia JA, Pena FJ. Freezing dog semen in presence of the antioxidant butylated hydroxytoluene improves postthaw sperm membrane integrity. Theriogenology 2010; 73:645-650.
36. Honisch S, Alkahtani S, Kounenidakis M, Liu G, Alarifi S, Al-Yahya H, Dimas K, AlKahtane AA, Prousis KC, Al-Dahmash B, Calogeropoulou T,
Alevizopoulos K, et al. A steroidal Na+/K+ ATPase inhibitor triggers pro-apoptotic signaling and induces apoptosis in prostate and lung tumor cells. Anticancer Agents Med Chem 2014; 14:1161-1168.
37. Englund UH, Gertow J, Kagedal K, Elinder F. A voltage dependent non-inactivating Na+ channel activated during apoptosis in Xenopus oocytes. PLoS One 2014; 9:e88381.
38. Sapia L, Palomeque J, Mattiazzi A, Petroff MV. Na+/K+-ATPase inhibition by ouabain induces CaMKII-dependent apoptosis in adult rat cardiac myocytes. J Mol Cell Cardiol 2010; 49:459-468.
39. Fang KM, Lee AS, Su MJ, Lin CL, Chien CL, Wu ML. Free fatty acids act as endogenous ionophores, resulting in Na+ and Ca2+ influx and myocyte apoptosis. Cardiovasc Res 2008; 78:533-545.
40. Jimenez T, Sanchez G, Wertheimer E, Blanco G. Activity of the Na,K-ATPase alpha4 isoform is important for membrane potential, intracellular Ca2+, and pH to maintain motility in rat spermatozoa. Reproduction 2010; 139:835-845. 41. Anzar M, Buhr MM. Spontaneous uptake of exogenous DNA by bull
spermatozoa. Theriogenology 2006; 65:683-690.
42. Thundathil JC, Anzar M, Buhr MM. Na+/K+ATPase as a signaling molecule during bovine sperm capacitation. Biol Reprod 2006; 75:308-317.
43. Pena FJ, Saravia F, Nunez-Martinez I, Johannisson A, Wallgren M, Rodriguez Martinez H. Do different portions of the boar ejaculate vary in their ability to sustain cryopreservation? Anim Reprod Sci 2006; 93:101-113.
44. Nunez-Martinez I, Moran JM, Pena FJ. Identification of sperm morphometric subpopulations in the canine ejaculate: do they reflect different subpopulations in sperm chromatin integrity? Zygote 2007; 15:257-266.
45. Ortega-Ferrusola C, Macias Garcia B, Suarez Rama V, Gallardo-Bolanos JM, Gonzalez-Fernandez L, Tapia JA, Rodriguez-Martinez H, Pena FJ. Identification of sperm subpopulations in stallion ejaculates: changes after cryopreservation and comparison with traditional statistics. Reprod Domest Anim 2009; 44:419-423.
46. Pena FJ, Ortega Ferrusola C, Martin Munoz P. New flow cytometry approaches in equine andrology. Theriogenology 2016; 86:366-372.
47. Martinez-Pastor F, Mata-Campuzano M, Alvarez-Rodriguez M, Alvarez M, Anel L, de Paz P. Probes and techniques for sperm evaluation by flow cytometry. Reprod Domest Anim 2010; 45 Suppl 2:67-78.
48. Eisenbach M, Giojalas LC. Sperm guidance in mammals - an unpaved road to the egg. Nat Rev Mol Cell Biol 2006; 7:276-285.
49. Eisenbach M. Why are sperm cells phagocytosed by leukocytes in the female genital tract? Med Hypotheses 2003; 60:590-592.
50. Ranganathan S, Bharadwaj A, Datta K. Hyaluronan mediates sperm motility by enhancing phosphorylation of proteins including hyaluronan binding protein. Cell Mol Biol Res 1995; 41:467-476.
51. Meyers SA. Equine sperm-oocyte interaction: the role of sperm surface hyaluronidase. Anim Reprod Sci 2001; 68:291-303.
52. Pena FJ, Johannisson A, Wallgren M, Rodriguez-Martinez H. Effect of hyaluronan supplementation on boar sperm motility and membrane lipid architecture status after cryopreservation. Theriogenology 2004; 61:63-70. 53. Szucs M, Osvath P, Laczko I, Jakab A. Adequacy of hyaluronan binding assay
and a new fertility index derived from it for measuring of male fertility potential and the efficacy of supplement therapy. Andrologia 2015; 47:519-524.
54. Myles DG, Primakoff P. Why did the sperm cross the cumulus? To get to the oocyte. Functions of the sperm surface proteins PH-20 and fertilin in arriving at, and fusing with, the egg. Biol Reprod 1997; 56:320-327.
55. Huszar G, Ozenci CC, Cayli S, Zavaczki Z, Hansch E, Vigue L. Hyaluronic acid binding by human sperm indicates cellular maturity, viability, and unreacted acrosomal status. Fertil Steril 2003; 79 Suppl 3:1616-1624.
Figure Legends
Figure 1.- Simultaneous determination of live and dead spermatozoa, caspase 3 expression and reactive oxygen species. Stallion spermatozoa were stained with H33342, CellEvent Caspase-3/7 Green Detection Reagent, CellRox deep red and ethidium homodimer as described and material and methods. Gating strategy applied, A) control of the quality of flow using time as a parameter. B) Elimination of debris and cell clumps from the analysis after H33342 analysis and FSC-A C) Identification of live
and dead sperm after H33342/Eth-1 staining, three distinct populations are identified, live sperm, dead sperm and an intermediate population named “?” D) The population “?” is further investigated for the presence of caspase 3, showing intense signal for this activated caspase E) Identification of active caspase 3, three subpopulations are
identified “live sperm” “caspase 3” positive sperm and dead spermatozoa. F) The population of live sperm in E is investigated for the production of reactive oxygen species showing high production G) Investigation of ROS production in Caspase 3 positive spermatozoa H) investigation of ROS production in dead spermatozoa. I) 2D plot showing active caspase 3 versus reactive oxygen species production J)
Investigation of dead cells in the subpopulation of CellRox+ events, most of the population are live (ethidium negative) spermatozoa K) Investigation of live an dead spermatozoa in the population of low ROS production L) Investigation of live an dead spermatozoa in the population of Caspase 3 positive spermatozoa.
Figure 2.- Effect of cryopreservation in the potential of the membrane (Em) of stallion spermatozoa. Aliquots of Stallion spermatozoa were loaded with the anionic bis oxonol dye DiSBAC2 (3). In order to restrict the assay to live spermatozoa the Live Dead
Violet fixable dye was used. A) Orange fluorescence of DiSBAC2 (3) is plotted against
Live Dead Violet fixable dye blue fluorescence; a gate is applied to live spermatozoa (low blue fluorescence) B) The population of live spermatozoa gated in A is further studied in B (raw sperm) and C ( frozen thawed sperm); two subpopulations are identified representing depolarized sperm (high DiSBAC2 (3) fluorescence) and
hyperpolarized sperm (low DiSBAC2 (3) fluorescence) D) Modification in the Em of
the stallion spermatozoa during the process of cryopreservation, results are expressed as means ± SD, *** p<0.01
Figure 3.- Effect of cryopreservation in the intracellular concentration of Na+ in stallion spermatozoa. A) Stallion spermatozoa were loaded with Sodium green as described in material and methods; dead spermatozoa were excluded from the assay after ethidium homodimer staining. B) Representative cytogram showing live spermatozoa loaded with Sodium green C) Histogram overlays of fresh and frozen thawed sperm. D)
Representative cytogram of raw sperm, only one subpopulation in terms of Na+ concentration is evident F) Evaluation of intracellular Na+ content in frozen thawed sperm, two subpopulations are evident, L low Na+ content and H, high Na+ content G) Changes in intracellular Na+ induced by cryopreservation * p< 0.05
Figure 4.- ACCENSE applied to high dimensional multicolor cytometry data. A and D. The 2D tSNE maps of stallion spermatozoa after multicolor staining (H 33342, caspase 3 eth and cell rox) in fresh (A) and frozen thawed spermatozoa (D). Each point
represent a cell derived by downsampling from the original dataset. B-E composite map depicting the complex structure of the stallion ejaculate; circles represent centers of phenotypically distinct subpopulations (p=0.001) C and F Density map representing the intensity of caspase 3 activity in raw (C) and thawed (F) stallion spermatozoa, caspase activity increased after cryopreservation (see heat maps on the right). G) Changes in caspase 3 expresion after cryopreservation after conventional flow cytometry analysis ** p<0.01
Figure 5.- ACCENSE derived t-SNE maps depicting channels for each phenotype marker in stallion spermatozoa in raw and thawed stallion spermatozoa. A and D H33342. B-E ethidium homodimer C-F CellRox.
Figure 6.- Effect of cryopreservation on tyrosine phosphorylation and production of reactive oxygen species by stallion spermatozoa. Stallion ejaculates were obtained and processed as indicated in material and methods. A) Changes in tyrosine phosphorylation B) Changes in the percentage of spermatozoa producing ROS *** p<0.01
Figure 7.- Relation between the potential of the sperm membrane, caspase 3 mitochondrial membrane potential and CD44 positive spermatozoa. A) Dot plot showing caspase 3 versus DiSBAC 2 (3) caspase 3 positive sperm was in the region of high
DiSBAC 2 (3)fluorescence (depolarized membranes)Q2. B) Dot plot showing DiSBAC 2 (3) vs. Mitotracker deep Red fluorescence, the population of high mitochondrial
potential correspond to hyperpolarized membranes (low DiSBAC 2 (3) fluorescence). C
and D, CD44 positive spermatozoa appears in the regions of low caspase 3 activity C, and hyperpolarized membranes D. ACCENSE derived t-SNE maps depicting the intensity of fluorescence of caspase 3 ( E), DiSBAC 2 (3) fluorescence (F), CD44 (G) and
Mitotracker deep red (H).
Figure 8. Effect of ATP supplementation on the Na+ intracellular content of thawed stallion spermatozoa. Stallion semen doses were thawed and processed as described in material and methods A and D) Effect of ATP supplementation on Na+ content in the whole population of thawed spermatozoa at the beginning of the incubation period (A) and after 30 minutes of incubation (D). B and E effect of ATP supplementation considering the subpopulation of high and low Na+ at the beginning of the incubation period (B) and after 30 minutes of incubation (E). C and F, representative cytograms showing the changes induced by ATP supplementation C) Controls F) ATP 10mM L, low Na+ H high Na+ * p<0.05
Figure 9. Effect of uoabain on the percentage of live spermatozoa and caspase 3 activation. Stallion semen was processed as described in material and methods incubated at 37ºC up to 5 hours, split samples were supplemented with ouabain ( 1, 10, 100, 1000 and 2000µM) at 0, 2 and 5 hours of incubation samples were taken for evaluation of caspase 3 activation A) At the beginning of the incubation period B) After 2 hours of incubation C) After 5 hours of incubation * p<0.05.
Table 1.- Significant correlations between DiSBAC2 (3) fluorescence (DiSBAC2 (3)
-H, depolarized sperm; DiSBAC2 (3) -L hyperpolarized sperm), caspase 3 activity and
tyrosine phosphorylation in frozen thawed stallion spermatozoa
Casp3+ CellRox+ CellRox+ PhosphoTyr DiSBAC2 (3) -H (Dep) R= 0.681* R= 0.936** R=0.778* DiSBAC2 (3)- L Hyper R= -0.685* R=-0.891** R=-0.783* *P<0.05, **P<0.01
Table 2.- Significant correlations among caspase 3 positive sperm and Na+ in raw spermatozoa, and caspase 3 positive and DiSBAC2 (3) fluorescence (DiSBAC2 (3) -H,
depolarized sperm; DiSBAC2 (3) -L hyperpolarized sperm) in thawed stallion
spermatozoa Caspase 3 (raw sperm) DiSBAC2 (3) -H (Dep) (Thawed) R= 0.770** DiSBAC2 (3)- L Hyper (Thawed) R =-0.794** Na+ (Raw sperm) R= 0,662* *P<0.05, **P<0.01
