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Reproduction, Fertility and Development http://dx.doi.org/10.1071/RD15231

Effect of seminal plasma proteins on the motile sperm subpopulations in ram ejaculates Carolina Luna A,*, Marc Yeste B,C,*, Marı´a M. Rivera del Alamo B, Juan Domingo A, Adriana Casao A, Joan E. Rodriguez-Gil B, Rosaura Pe´rez-Pe´ A, Jose´ A. Cebria´n-Pe´rez A and Teresa Muin˜o-Blanco A,D A

University of Zaragoza, C/Miguel Servet, 177, 50013-Zaragoza, Spain. Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, Travessera dels Turons, s/n. 08193 Cerdanyola, Barcelona, Spain. C Present address: Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Level 3, Women’s Centre, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, United Kingdom. D Corresponding author. Email: [email protected] *These authors contributed equally to this work. B

Abstract. It has been proposed that seminal plasma proteins (SPP) support survival of ram spermatozoa, exerting a dual effect, both capacitating and decapacitating. In this study, changes in motility patterns of ram spermatozoa capacitated in the presence of epidermal growth factor (EGF) were evaluated. Clustering procedures were used to determine the presence of sperm subpopulations with specific motion characteristics. Four sperm subpopulations (SP) were defined after the application of a principal component analysis procedure. Progressive spermatozoa with high straightness (STR) were found in SP1, reflected in the high linearity (LIN) and STR values and low amplitude of lateral head movement (ALH; rapid, non-hyperactivated spermatozoa). SP2 spermatozoa seemed to be starting to acquire hyperactivated motility, while the SP3 group consisted of rapid, hyperactivated spermatozoa. SP4 showed less-vigorous spermatozoa, with non-linear motility. The addition of SPP before in vitro capacitation with EGF induced a decrease in SP1 and an increase in SP3. However, a reduction in the chlortetracycline-capacitated sperm rate and protein tyrosine phosphorylation was found, which corroborates with the hypothesis that the SPP protective effect on spermatozoa is related to their decapacitating role. These findings allow us to deduce that ram spermatozoa are able to undergo capacitation with no hyperactivation and that SPP are able to induce hyperactivation in spermatozoa but maintain them in a decapacitated state. Additional keywords: capacitation, motility pattern, tyrosine phosphorylation.

Received 10 April 2015, accepted 21 July 2015, published online 24 August 2015

Introduction Seminal plasma is an essential key modulator of mammalian sperm motility (Baas et al. 1983; Graham 1994; Bernardini et al. 2011), viability (Ashworth et al. 1994; Maxwell et al. 1998, 1999, 2007) and function (reviewed by Muin˜o-Blanco et al. 2008; Caballero et al. 2012). Seminal plasma proteins (SPP) are able to increase sperm resistance against cold shock (Garcı´aLo´pez et al. 1996a; Barrios et al. 2000; Pe´rez-Pe´ et al. 2001; Cola´s et al. 2009) or detergent treatment (Ollero et al. 1997) and stimulate sperm function and fertilising ability (Maxwell et al. 2007). It has already been shown that SPP support survival of ram spermatozoa acting not only at the plasma membrane but also by inhibition of capacitation (Desnoyers and Manjunath 1992; Barrios et al. 2005) and apoptosis-like changes (Mendoza et al. 2013). In a very recent study, we have proposed that SPP Journal compilation Ó CSIRO 2015

could exert a dual effect, capacitating and decapacitating. Thus, SPP would contribute to maintaining the structure of the sperm membrane until it receives the necessary stimulus that triggers the physiological changes leading the spermatozoon to bind the oocyte (Luna et al. 2015). While the effects of SPP upon ram sperm capacitation have been widely studied (Maxwell et al. 1998, 1999, 2007; Barrios et al. 2005; Muin˜o-Blanco et al. 2008; Leahy and Gadella 2011; Rodrı´guez-Martı´nez et al. 2011; Caballero et al. 2012; Mendoza et al. 2013; Luna et al. 2015) their influence on ram sperm motility is yet to be determined. The characteristic sperm motility pattern defined as hyperactivation (Yanagimachi 1970) displays a high amplitude of flagellar beating (reviewed in Kay and Robertson 1998). This movement has been characterised after incubating spermatozoa of different species www.publish.csiro.au/journals/rfd

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under capacitating conditions (Mortimer and Maxwell 1999; Marquez and Suarez 2007; McPartlin et al. 2008, 2009; Suarez 2008; Goodson et al. 2011). It is worth noting that although capacitation and hyperactivation can be concomitant processes (Fraser 1977; Suarez et al. 1987; Ramio´ et al. 2008; Goodson ´ lvarez et al. 2014), they may not neceset al. 2011; Garcı´a-A sarily be interdependent (de Lamirande et al. 1997; Mortimer 1997; Mortimer et al. 1998; Cola´s et al. 2010). Therefore, assessing the effects of SPP on sperm motility patterns during in vitro capacitation of ram spermatozoa could contribute to further the knowledge about the sperm-protecting mechanism mediated by SPP and the relationship between hyperactivation and capacitation. Mammalian ejaculates are very heterogeneous and contain sperm subpopulations with important differences in their kinematic characteristics (Holt 1995; Abaigar et al. 1999; Mortimer 2000; Abaigar et al. 2001; Quintero-Moreno et al. 2003, 2004, 2007; Cremades et al. 2005; Nu´n˜ez-Martı´nez et al. 2006; Flores et al. 2008). The presence of these sperm subpopulations in ejaculates might be related to their functionality (Flores et al. 2008) or fertilising ability (Quintero-Moreno et al. 2003) and appears to involve genetic factors (Abaigar et al. 2001). In addition, semen handling has been reported to affect the motility patterns defining each subpopulation and their relative proportions within a given ejaculate (Cremades et al. 2005; Nu´n˜ez-Martı´nez et al. 2006; Flores et al. 2008). The computer-assisted sperm analysis (CASA) system allows for differentiation of individual spermatozoa according to their motility characteristics and is one of the most reliable methods for studying sperm subpopulations (Abaigar et al. 1999, 2001; Thurston et al. 2001). Given that data are not ‘normally’ distributed, mean values represent inappropriate estimations, so that a great deal of information is ignored and the evaluation of sperm quality can be inaccurate (Abaigar et al. 1999; Verstegen et al. 2002; Holt and Van Look 2004; Holt et al. 2007). Conversely, multiparametric kinematic definitions precisely define the exact movement of each individual spermatozoon and allow their assignment into a specific subpopulation according to the different trajectories obtained from the analysis of a sample (ESHRE Andrology Special Interest Group 1998). Therefore, studying sperm subpopulations may contribute to better understanding the motility changes that take place during in vitro capacitation of spermatozoa. Several studies have provided direct evidence that specific components of seminal plasma, particularly proteins, are adsorbed onto the sperm surface of several species (Metz et al. 1990; Watson 1995; Garcı´a-Lo´pez et al. 1996a; Ollero et al. 1997; Barrios et al. 2000; Thomas et al. 2003; Cola´s et al. 2009; Mendoza et al. 2013) and a specific sperm surface receptor for a seminal plasma protein complex has been identified in rabbit spermatozoa (Minelli et al. 2001a, 2001b). Thus, one could suggest that SPP may have a different effect upon a particular sperm subpopulation (Ollero et al. 1994, 1996a, 1997, 1998; Garcı´a-Lo´pez et al. 1996a; Mendoza et al. 2013). In this context, clustering procedures may shed light on the effects of SPP upon sperm motility because they may reveal not only differences in the overall sperm motility but also in the characteristics and proportions of each sperm subpopulation.

C. Luna et al.

Therefore, the main aim of this study was to evaluate the effects of SPP upon motility of ram spermatozoa freed from seminal plasma by swim-up and in vitro capacitated in the presence of epidermal growth factor (EGF; Luna et al. 2015). With this purpose, we first determined the sperm subpopulation structure following the swim-up and we then assessed the changes that this structure underwent in response to in vitro capacitation and addition of SPP. Materials and methods Sperm preparation All experiments were carried out with fresh semen obtained from eight mature Rasa Aragonesa rams (2–4 years old), using an artificial vagina. All the rams belonged to the National Association of Rasa Aragonesa Sheep Breeders and were housed under uniform nutritional conditions at the Experimental Farm of the University of Zaragoza (Spain) in compliance with the requirements of the European Union Directive for Scientific Procedures. This breed corresponds to a local Spanish genotype with a short seasonal anoestrus between May and July. The sires were divided into two groups and, based on the positive results from a previous study, two successive ejaculates were collected every third day to avoid deterioration of spermatozoa (Ollero et al. 1996b). The experiments were performed between October and May 2014 at the Veterinary School (University of Zaragoza, Spain). For every experiment, the first and second ejaculates from each group (four rams) were pooled separately and used for each assay in order to eliminate individual differences. A seminal plasma-free sperm population was obtained by a dextran swim-up procedure (Garcı´a-Lo´pez et al. 1996b) performed by using a swim-up medium consisting of 200 mM sucrose, 50 mM NaCl, 18.6 mM sodium lactate, 21 mM hydroxyethyl piperazineethanesulfonic acid (HEPES), 10 mM KCl, 2.8 mM glucose, 0.4 mM MgSO4, 0.3 mM sodium pyruvate, 0.3 mM K2HPO4, 5 mg mL1 bovine serum albumin (BSA), 1.5 IU mL1 penicillin and 1.5 mg mL1 streptomycin, pH 7.2 (adjusted by NaOH addition) and devoid of CaCl2 and NaHCO3 (Grasa et al. 2006). Sperm motility evaluation Motility kinematics parameters were evaluated using a CASA system (ISAS 1.0.4; Proiser SL, Valencia, Spain) that consisted of a video camera (Basler A312f; Basler Vision Components, Exton, PA, USA) mounted on a microscope (Nikon eclipse 50i; Nikon, Tokyo, Japan). The microscope was equipped with a 10 negative phase-contrast lens and a 10 projection ocular. Samples (6 mL) were placed onto pre-warmed slides then covered with standard coverslips (20  20 mm) and maintained at 378C during the analysis by a heated slide holder. Recording was performed at 25 frames s1 and 25 consecutive digitalised images were taken from a single field. The kinematic parameters recorded for each spermatozoon [19,20] were curvilinear velocity (VCL, mm s1: the average path velocity of the sperm head along its actual trajectory), straightline velocity (VSL, mm s1: the average path velocity of the sperm head along a straight line from its first to its last position), average path velocity (VAP, mm s1: the average velocity of the

SPP and sperm subpopulations

sperm head along its average trajectory), percentage linearity (LIN, %: the ratio between VSL and VCL), percentage straightness (STR, %: the ratio between VSL and VAP), wobble coefficient (WOB, %: the ratio between VAP and VCL), mean amplitude of lateral head displacement (ALH, mm: the average value of the extreme side-to-side movement of the sperm head in each beat cycle), beat-cross frequency (BCF, Hz: the frequency with which the actual sperm trajectory crosses the average path trajectory), combination of the lateral and forward movement of the head (DANCE, mm2 s1: product of VCL and ALH) and mean angular displacement (MAD, degrees: time average of absolute values of the instantaneous turning angle of the sperm head along its curvilinear trajectory; Verstegen et al. 2002). In vitro capacitation For the induction of in vitro capacitation, aliquots of 1.6  108 cells mL1 were incubated for 3 h at 398C in a humidified incubator with 5% CO2 in air. Incubations were performed in complete Tyrode’s albumin lactate pyruvate medium (TALP; Parrish et al. 1988) containing 100 mM NaCl, 3.1 mM KCl, 25 mM NaHCO3, 0.3 mM NaH2PO4, 21.6 mM Na lactate, 3 mM CaCl2, 0.4 mM MgCl2, 10 mM HEPES, 1 mM Na pyruvate, 5 mM glucose and 5 mg mL1 BSA, pH 7.2 (adjusted using NaOH) for the control sample, or with added 100 nM EGF (Abcam, Cambridge, UK; Luna et al. 2012), for the capacitated sample. To analyse the involvement of ram SPP on in vitro sperm capacitation, 1.7 mg SPP obtained as described by Barrios et al. (2000) were added to 500 mL of swim-up-obtained samples containing 8  107 spermatozoa diluted in TALP with 100 nM EGF. The proteins were added immediately after preparing the aliquots (0 h) and the samples were incubated for 3 h simultaneously with the controls. Analyses were performed at 30 min, 1 h 30 min and 3 h of incubation in capacitating conditions. The capacitation status was determined through chlortetracycline (CTC) fluorescence assay, previously validated for the evaluation of capacitation and acrosome reaction-like changes in ram semen (Grasa et al. 2006). Each spermatozoon was classified into one of the following three CTC-patterns (Gillan et al. 1997): non-capacitated (even distribution of fluorescence over the head, with or without a bright equatorial band), capacitated (with fluorescence in the anterior portion of the head) and acrosome-reacted cells (showing no fluorescence on the head). The samples were examined within 12 h using a Nikon Eclipse E-400 microscope (Nikon Corporation, Tokyo, Japan) under epifluorescence illumination with a V-2A filter (Nikon Corporation). All samples were processed in duplicate and at least 150 spermatozoa were scored per slide. No fluorescence was observed when CTC was omitted from the preparation. Extraction of ram sperm proteins Aliquots containing 3  107 sperm cells from control or EGFcapacitated samples were resuspended in 100 mL of an extraction medium, made up of 2% sodium dodecyl sulfate (SDS) (w/v), 0.0626 mM TRIS HCl (pH 6.8), 0.002% bromophenol blue in 10% glycerol (final glycerol concentration 1%) and protease and phosphatases inhibitors (Sigma Chemical, Madrid,

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Spain), as described in Colas et al. (2008). Samples were immediately incubated for 5 min at 1008C. After centrifugation at 7500g and room temperature (RT) for 5 min, the supernatant was recovered and 2-mercaptoethanol and glycerol were added to a final concentration of 5% and 1%, respectively. The protein concentration was determined using the Bradford assay (Bradford 1976) and lysates were stored at 208C. SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting For detection of tyrosine-phosphorylated proteins, spermextracted proteins (20 mL) were separated through onedimensional SDS-PAGE (separating gel: 10%) following the Laemmli method (Laemmli 1970) and using a Mini-PROTEAN II vertical slab gel electrophoresis apparatus (Bio-Rad, Hercules, CA, USA). Electrophoresis was performed at 130 V and 48C for 90 min. A mixture of pre-stained molecular weight markers ranging from 10 to 250 kDa (Bio-Rad) was used as a standard. Gels were stained with 0.1% Coomassie R (Serva, Heidelberg, Germany). For western blot analysis, separated proteins were blotted onto 0.2-mm polyvinylidene fluoride membranes (Bio-Rad) at 2.5 A constant up to 25 V for 10 min, using the Trans-Blot Turbo unit (Trans-Blot Turbo Transfer System; Bio-Rad). For the detection of phosphorylated proteins, the blots were incubated as described in Luna et al. (2012). Briefly, nonspecific sites on the membranes were blocked with 5% BSA (w/v) in phosphate-buffered saline (PBS: 136 mM NaCl, 0.2 g L1 KCl, 1.44 g L1 Na2HPO4 and 0.24 g L1 KH2PO4, pH 7.4) at RT for 1 h. The blots were then incubated with a mouse monoclonal anti-phosphotyrosine antibody (Monoclonal Antibody, clone 4G10, 1 : 1000; Millipore, Temecula, CA, USA) at 48C overnight. Next, the blots were incubated with a secondary antimouse horseradish peroxidase-conjugated immunoglobulin G antibody (1 : 40000; GE Healthcare – Amersham, Little Chalfont, UK) at RT for 1 h. After extensive washing, the proteins that bound the antibody were visualised by chemiluminescence procedures (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific, Waltham, MA, USA). Western-blot images were quantified using Quantity One software (Bio-Rad) to determine the peak intensity of the tyrosine-phosphorylated protein bands. The total intensity signal of each lane was evaluated as the summation of the peak intensity of all bands detected in the lane. The experiment was performed three times and a representative membrane is shown. To prove that the signal was specific, western blotting omitting either primary or secondary antibodies was performed (data not shown). Statistical analysis On the one hand, statistical analyses were performed to determine whether CTC-capacitation status and levels of protein tyrosine phosphorylation differed between control and treated samples. This was performed using GraphPad InStat software (3.01; GraphPad Software, Inc., San Diego, CA, USA). Briefly, data distribution was first analysed through the Shapiro–Wilks test. After that, differences between experimental groups were

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analysed by means of the Kruskal–Wallis test, followed by Mann–Whitney’s test. A P value of 0.05 or lower was considered to be statistically significant. Results are shown as mean  standard error of the mean (s.e.m.) of three assays. On the other hand, the present study also determined the effects of EGF and EGF plus SPP upon in vitro capacitation of ram spermatozoa. This was performed using IBM SPSS statistical software (V 21.0; IBM Corp., Chicago, IL, USA). Briefly, all sperm motility and kinematic parameters obtained from CASA assessments (i.e. VSL, VCL, VAP, LIN, STR, WOB, ALH, BCF, DANCE and MAD) were used to run a principal component analysis (PCA). Data were first checked through Shapiro–Wilks and Levene tests and percentages were arcsine transformed. All sperm parameters were sorted into two PCA components and the data matrix obtained was rotated using the Varimax procedure with the Kaiser normalisation. Following this analysis, only those variables with a positive loading factor (aij) higher than 0.6 with its respective component and lower than 0.3 with respect to the others in the rotated matrix were selected from the linear combination of j variables (x) in each component yi (yi ¼ ai1x1 þ ai2x2 þ y þ aijxj). For each spermatozoon, which was considered as an independent statistical case, a regression score was calculated per component. Following PCA, each spermatozoon in each treatment and time point was classified using a cluster analysis. This classification was made upon the regression scores per PCA component obtained in the previous step, using the between-groups linkage method based on the squared Euclidean distance. A total of four subpopulations were obtained. After this, the effects of in vitro capacitation treatment (control, EGF and EGFþSPP) upon the ram sperm subpopulation distribution were determined through a linear mixed model followed by a Bonferroni’s post hoc test. In this model, the incubation time point was the intra-subject factor and the treatment (control, EGF and EGFþSPP) was the fixedeffects factor. All sperm motility parameters were considered as dependent variables. Furthermore, correlations between ALH and the other sperm motility parameters were calculated within each subpopulation using Pearson’s coefficient. In all cases, the level of significance was set at P # 0.05. Results Sperm subpopulation analysis in the samples obtained by swim-up The kinematic characteristics of the spermatozoa selected by dextran–swim-up procedure are summarised in Table 1. The principal component analysis from all kinematic parameters showed that two extracted components accounted for 78.31% of the total variance (Table 2). The first principal component was mainly related to straight-line velocity, straightness and beatcross frequency (VSL, VAP, LIN, STR, WOB, BCF, MAD), whereas the second one was highly related to curvilinear velocity and amplitude of head displacement (VCL, ALH, DANCE). Fig. 1 shows the relationship between the two principal components (first component in x-axis, second component in y-axis), the original kinematic sperm parameters and the four sperm subpopulations. The original kinematic parameters are

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depicted as vectors and the vector components represent the loading factors against each principal component (as aforementioned, the predominant component is shown in Table 2). Longer vectors indicate higher loading factors for the x (first component) or y (second component) axis. Fig. 1 also shows the four sperm subpopulations as ellipses. In this case, the regression factors for each individual spermatozoon after the PCA were used to represent each ellipse (size according to the percentage of spermatozoa within the subpopulation and range of regression factors as limits of the ellipse). Although this is only a schematic representation, it seeks to show the predominant factors and vectors defining each sperm subpopulation in a bidimensional plot.

Table 1. Characteristics of the sample obtained by swim-up based on motility descriptors obtained from CASA analysis VCL, curvilinear velocity; VSL, straight-line velocity; VAP, average path velocity; LIN, linearity; STR, straightness; WOB, wobble (VAP/VCL); ALH, amplitude of lateral head displacement; BCF, beat-cross frequency; DANCE (VCL  ALH); MAD (angular degrees). Percentages were arcsine transformed. Values are means  s.e.m. (n ¼ 6) Kinematic parameter

Swim-up sample

1

131.91  0.40 52.45  0.62 88.01  0.52 44.18  0.51 65.25  0.63 76.94  0.56 5.09  0.02 6.61  0.05 732.03  2.04 87.02  0.7

VCL (mm s ) VSL (mm s1) VAP (mm s1) LIN (%) STR (%) WOB (%) ALH (mm) BCF (Hz) DANCE (mm2 s1) MAD

Table 2. Detail of PCA based on motility descriptors obtained from CASA analysis The loading factor (a2ij) represents the highest association between a given sperm motility parameter and the corresponding principal component. VSL, straight-line velocity; VAP, average path velocity; LIN, linearity; STR, straightness; WOB, wobble (VAP/VCL); BCF, beat-cross frequency; MAD (angular degrees); VCL, curvilinear velocity; ALH, amplitude of lateral head displacement; DANCE (VCL  ALH) Principal component

Variance

Parameter

Component 1

39.88%

Component 2

38.43%

VSL VAP LIN STR WOB BCF MAD VCL ALH DANCE

Total

78.31%

a2ij 0.81 0.48 0.89 0.69 0.78 0.26 0.32 0.89 0.85 0.96

SPP and sperm subpopulations

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Second principal component

Four sperm subpopulations were defined after running principal component and cluster analyses from the aforementioned kinematic parameters, using a total of 182 362 ram spermatozoa previously selected by swim-up (Table 3). Subpopulations 1, 2 and 3 (SP1, SP2 and SP3) represented fast spermatozoa with very high values of VCL (97.78  0.29, 137.30  0.33 and 134.68  0.30 mm s1, respectively), VSL (64.25  0.32, 83.99  0.44 and 37.12  0.26 mm s1, respectively) and VAP (81.51  0.32, 109.21  0.41 and 83.16  0.25 mm s1, respectively), while subpopulation 4 (SP4) presented lower values of these kinematic parameters. Percentages of linearity (LIN) and straightness (STR) were much lower in SP3 and SP4 than in SP1 and SP2. The ALH value was higher in SP2 and SP3 (4.40  0.02 and 5.59%  0.01, respectively) and lower in

ALH DANCE

E

SP1 and SP4. The highest DANCE (VCL  ALH) value was found in SP3. Correlation coefficients between ALH with all other kinematic parameters are shown in Table 4. Besides the negative, significant correlation between ALH and LIN in SP1, SP2 and SP3, a positive, significant correlation between ALH and VCL was also found, with SP3 presenting the highest value. Influence of in vitro capacitation with EGF on sperm motility characteristics and evolution of sperm subpopulations In vitro capacitation with EGF induced a decrease in VCL, VSL, VAP and ALH and increased LIN, BCF and WOB in SP1, mainly after 3 h of incubation. Smaller changes were also identified in SP3 (Table 5). However, the linear mixed model revealed that this effect was not reflected in any significant change in the subpopulation distribution at any time of incubation in capacitating conditions (Fig. 2).

VCL 3

MDA

2 VAP

BCF

VSL

4 1 First principal component

%STR %WOB

Table 4. Correlation coefficients between ALH and other kinematic parameters of the four sperm subpopulations identified in ram sperm samples selected by swim-up VCL, curvilinear velocity; VSL, straight-line velocity; VAP, average path velocity; LIN, linearity; STR, straightness; WOB, wobble (VAP/VCL); ALH, amplitude of lateral head displacement; BCF, beat-cross frequency; DANCE (VCL  ALH). Statistical significances are denoted with asterisks as follows: * P , 0.05, ** P , 0.01, *** P , 0.005 Parameter

%LIN

Fig. 1. Diagram showing the two principal components (first in x-axis, second in y-axis) and loading factors of the original sperm kinematic parameters (VSL, VCL, VAP, LIN, STR, WOB, ALH, BCF, DANCE, MAD), which are represented as vectors. Subpopulations are represented as ellipses, according to their respective regression factors against the two extracted components.

VCL VSL VAP LIN STR WOB BCF DANCE

SP1

SP2

SP3

SP4

0.44* 0.16 0.04 0.64** 0.38* 0.71** 0.01 0.91***

0.23* 0.32* 0.26* 0.55** 0.19 0.70** 0.12 0.86***

0.70** 0.11 0.09 0.39* 0.04 0.51** 0.21* 0.92***

0.91*** 0.45* 0.71** 0.09 0.18 0.36* 0.41* 0.93***

Table 3. Kinematic characteristics (mean values ± s.e.m.) of the four sperm subpopulations identified in ram sperm samples selected by swim-up Subpopulations include the whole population of motile spermatozoa. SP1, rapid, non-capacitated spermatozoa; SP2, rapid, spermatozoa starting capacitation; SP3, rapid, non-linear spermatozoa with high ALH and low LIN (capacitated spermatozoa); SP4, less-rapid, non-linear spermatozoa with medium ALH. VCL, curvilinear velocity; VSL, straight-line velocity; VAP, average path velocity; LIN, linearity; STR, straightness; WOB, wobble (VAP/VCL); ALH, amplitude of lateral head displacement; BCF, beat-cross frequency; DANCE (VCL  ALH) Parameter No. cells Percentage VCL (mm s1) VSL (mm s1) VAP (mm s1) LIN (%) STR (%) WOB (%) ALH (mm) BCF (Hz) DANCE (mm2 s1)

SP1

SP2

SP3

SP4

47 192 25.87 97.78  0.29 64.25  0.32 81.51  0.32 66.02  0.27 79.06  0.24 82.91  0.16 3.03  0.01 8.05  0.04 300.68  1.64

28 613 15.69 137.30  0.33 83.99  0.44 109.21  0.41 60.99  0.26 76.75  0.24 79.22  0.18 4.40  0.02 7.81  0.05 607.32  3.06

52 259 28.65 134.68  0.30 37.12  0.26 83.16  0.25 27.48  0.18 44.78  0.28 61.83  0.14 5.59  0.01 6.27  0.03 765.17  3.59

54 298 29.77 76.60  0.51 21.47  0.22 48.70  0.39 27.49  0.27 44.12  0.42 60.61  0.32 3.35  0.02 6.34  0.05 279.50  2.61

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C. Luna et al.

Table 5. Kinematic characteristics (mean values ± s.e.m.) of the four sperm subpopulations identified in ram sperm samples selected by swim-up after incubation in capacitating conditions with either EGF or EGF1SPP VCL, curvilinear velocity; VSL, straight-line velocity; VAP, average path velocity; LIN, linearity; STR, straightness; WOB, wobble (VAP/VCL); ALH, amplitude of lateral head displacement; BCF, beat-cross frequency. Significant differences between columns a,b: P , 0.05; b,c: P , 0.01; a,c: P , 0.005. Significant differences between lines 1,2: P , 0.05; 2,3: P , 0.01; 1,3: P , 0.005 Parameter

Treatment

SP1 0.50 h

VCL

VSL

VAP

LIN

STR

WOB

ALH

BCF

Control EGF EGFþSPP Control EGF EGFþSPP Control EGF EGFþSPP Control EGF EGFþSPP Control EGF EGFþSPP Control EGF EGFþSPP Control EGF EGFþSPP Control EGF EGFþSPP

SP3

1.50 h

95.73  0.31 93.79  0.34a,b,1 92.76  0.26b,1 66.37  0.35a,1 65.17  0.37b,1 63.96  0.28a,b,1 81.75  0.33a,1 79.90  0.35a,b,1 78.81  0.27b,1 69.28  0.26a,1 69.55  0.27a,1 69.30  0.23a,1 80.81  0.21a,1 81.26  0.22a,1 81.19  0.20a,1 84.90  0.16a,1 84.78  0.17a,1 84.63  0.14a,1 2.87  0.01a,1 2.84  0.01a,1 2.81  0.01a,1 7.63  0.04a,1 7.59  0.04a,1 7.91  0.03b,1 a,1

3.00 h

91.81  0.57 95.95  0.47b,1 90.07  0.25a,1 71.65  0.61a,2 72.44  0.51a,2 61.42  0.25b,1 82.62  0.60a,1 85.05  0.49b,2 75.70  0.25c,2 77.64  0.39a,2 75.39  0.34a,2 68.65  0.21b,1 86.22  0.29a,2 84.82  0.28a,2 81.13  0.17b,1 89.21  0.25a,2 88.11  0.20a,2 83.96  0.13b,1 2.40  0.02a,2 2.58  0.02b,2 2.78  0.01c,1 7.94  0.07a,2 8.22  0.06b,2 7.93  0.03a,1

a, 2

0.50 h

87.63  1.30 74.65  1.71b,2 79.79  0.37c,2 65.89  1.22a,1 59.40  1.51b,3 54.67  0.30c,2 74.91  1.28a,2 66.90  1.64b,3 66.80  0.33b,3 75.03  0.67a,2 79.52  0.69b,3 69.70  0.27c,1 87.50  0.44a,2 88.65  0.46a,3 82.25  0.22b,1 85.33  0.54a,1 89.32  0.49b,2 84.15  0.18a,1 2.61  0.04a,3 2.15  0.05b,3 2.60  0.02a,2 8.60  0.14a,3 8.04  0.16b,3 7.46  0.04c,2

To verify that in vitro capacitation was achieved, we determined the CTC-capacitation patterns and tyrosine phosphorylation after incubation of samples obtained by swim-up with 100 nM EGF in capacitating conditions. As expected, a significant increase in both the proportion of capacitated sperm patterns (Fig. 3a) and total signal intensity of phosphotyrosine (Fig. 3b, 3c) was found. Influence of seminal plasma proteins (SPP) on sperm motility characteristics and subpopulation evolution during in vitro capacitation The addition of SPP before capacitation accounted for significant changes in SP1 and SP3 (Table 5). A significant decrease in motility descriptors related to velocity, straightness and beatcross frequency (VSL, VAP, LIN, STR, WOB and BCF) was found in SP1 when capacitation was carried out in the presence of SPP, while VCL, ALH, DANCE and MAD, indicative of curvilinear velocity and sinuous trajectory, increased after 3 h of incubation in EGF-containing samples. In addition, higher values of VAP and WOB and lower scores for ALH, DANCE and MAD were found in SP3 in these conditions. The percentage of spermatozoa in SP1 was significantly reduced after 1.5 h incubation with SPP, when compared with control and capacitated samples (Fig. 2a). Likewise, there was a

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136.53  0.31 136.98  0.37a,1 137.97  0.27a,1 37.21  0.24a,1 36.89  0.28a,1 36.06  0.21a,1 81.33  0.26a,1 80.84  0.29a,b,1 78.84  0.22b,1 27.22  0.16a,1 26.96  0.19a,1 26.12  0.14a,1 45.73  0.26a,1 45.67  0.30a,1 45.54  0.23a,1 59.65  0.14a,1 59.20  0.16a,1 57.30  0.12a,1 5.84  0.01a,1 5.89  0.02a,1 6.01  0.01a,1 6.02  0.03a,1 6.04  0.04a,1 5.92  0.03a,1 a,1,2

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135.10  0.78 135.87  0.91a,1 135.28  0.28a,1,2 37.23  0.62a,1 37.90  0.73a,1 35.57  0.22a,1 79.42  0.64a,1,2 84.80  0.82b,2 78.19  0.23a,1 27.60  0.43a,1 27.92  0.50a,1 26.24  0.15a,1 46.91  0.68a,1 45.28  0.80a,1 45.33  0.25a,1 58.88  0.35a,1 62.38  0.45b,1 57.87  0.13a,1 5.91  0.04a,1 5.60  0.04b,1 5.87  0.01a,b,2 6.48  0.08a,2 6.29  0.10a,b,2 6.08  0.03b,2 a,1

139.58  1.81a,2 137.15  2.88a,b,1 133.41  0.41b,2 41.17  1.53a,2 33.09  1.81b,2 34.98  0.32b,1 76.22  1.39a,2 68.39  2.24b,3 75.44  0.33a,2 29.98  1.11a,1 24.24  1.18b,2 26.20  0.22a,b,1 53.93  1.74a,2 48.08  2.05b,1 46.11  0.36b,1 54.74  0.81a,2 49.90  1.18b,2 56.60  0.19a,1 6.17  0.09a,2 6.28  0.11a,2 5.93  0.02b,2 6.47  0.24a,2 6.13  0.24b,1,2 6.02  0.05b,1,2

concomitant increase in the percentage of cells in SP3 after 1.5 h of incubation (Fig. 2c). In contrast, no significant differences in the percentages of SP2 and SP4 spermatozoa were found throughout the incubation time (Fig. 2b, 2d). Since results from chlortetracycline analysis revealed that data were non-normally distributed, they are presented as boxwhisker plots (Fig. 3). As shown in Fig. 3a, SPP were able to maintain a higher proportion of non-capacitated sperm patterns in both control and EGF-containing samples. Furthermore, the addition of SPP resulted in a significant decrease in protein tyrosine phosphorylation (Fig. 3b, c). Discussion Sperm subpopulations with specific motility characteristics have been identified within ejaculates from bull (Muin˜o et al. 2008), boar (Abaigar et al. 1999; Cremades et al. 2005), goat (Abdelwahab et al. 2006; Dorado et al. 2010), stallion (Quintero-Moreno et al. 2003), donkey (Miro´ et al. 2005), dog (Martı´nez et al. 2006), gazelle (Abaigar et al. 2001), deer (Martinez-Pastor et al. 2005; Esteso et al. 2009), mouse (Goodson et al. 2011) and human (Buffone et al. 2004). In ram semen, the presence of subpopulations based on differences in ´ lvarez et al. 2014) and motility (Bravo et al. 2011; Garcı´a-A morphometric characteristics (Maroto-Morales et al. 2012;

SPP and sperm subpopulations

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Incubation time (hh:mm) Fig. 2. Effect of incubation in capacitating conditions with either EGF or EGFþSPP on the evolution of sperm subpopulations identified in ram sperm samples selected by swim-up.

Ramo´n et al. 2013) has also been reported. In this study, a principal component analysis followed by a multivariate clustering procedure separated four subpopulations of motile spermatozoa from a sample obtained by swim-up. While previous studies have found up to five motile subpopulations in fresh ram ejaculates (Quintero-Moreno et al. 2003; Bravo et al. 2011), the present work revealed only four. A possible explanation for this difference could be that sperm subpopulations in our study were defined from a swim-up-selected sample rather than from fresh ejaculates. As expected for a sample selected by a technique based on sperm motility, the four subpopulations of the swim-up sample could be classified as rapid, since they presented values of VCL and VAP higher than 75 and 45 mm s1, respectively, which have been previously set as minimum thresholds for rapid spermatozoa (Marco-Jime´nez et al. 2005; Cola´s et al. 2010; Bravo et al. 2011). The fact that velocity values are lower than those reported in other studies could be due to the use of slides instead of specific chambers (Mortimer and Maxwell 1999, 2004). However, PCA and cluster analyses revealed important differences between these subpopulations. On the one hand, SP1 was constituted by rapid, progressive spermatozoa with high linearity and straightness (LIN and STR) and low ALH.

Considering the threshold values for hyperactivation previously established for ram spermatozoa, LIN # 45% and ALH $ 3.5 mm (Cola´s et al. 2010), it can be inferred that SP1 represents non-hyperactivated spermatozoa. In this context, it is worth remembering that the term ‘hyperactivation’ refers to a characteristic motility pattern that spermatozoa acquire during the process of capacitation (Topper et al. 1999; Ho and Suarez 2001). These threshold values are less strict than those defined for Merino rams using a 30-mm-deep chamber instead of a slide. This, together with the use of different capacitation conditions (Mortimer and Maxwell 1999, 2004), could explain the differences obtained in kinematic parameters. Therefore, SP1 in our study included rapid, non-capacitated spermatozoa. In contrast, SP3 contained spermatozoa with the lowest LIN and STR percentages together with the highest ALH and DANCE values. Furthermore, SP3 presented the highest correlation between ALH and VCL, which indicates a correlation between the head movement of these spermatozoa and the flagellar movement. These results led us to implement the criteria for hyperactivated ram spermatozoa including STR # 45% and DANCE $ 760 mm2 s1. Thus, SP3 included rapid, hyperactivated spermatozoa, potentially capacitated, which would match with the hyperactivated sperm subpopulation described by other authors

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C. Luna et al.

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Fig. 3. Whisker-box plots showing the effect of SP proteins on the capacitation status, evaluated by chlortetracycline staining. Boxes enclose the 25th and 75th percentiles, the dot is the median and whiskers represent the minimum and maximum value in each case. Controls (0 h and 3 h) and EGF-containing samples (500 mL containing 8  107 spermatozoa) without and with 1.7 mg SP proteins were incubated in capacitating conditions for 3 h (n ¼ 3). (a) Non-capacitated, (b) capacitated and (c) acrosome-reacted spermatozoa. (d) Protein tyrosine phosphorylation (e) quantified by densitometry; average values of the total peak intensity signal of each lane (n ¼ 3). Different letters indicate significant differences between treatments and times.

(Mortimer and Mortimer 1990). Our data also showed important differences between SP2 and SP3. Not only were velocity parameters higher, but also LIN and STR were significantly higher in SP2 than in SP3, whereas ALH was significantly lower. These differences could indicate different degrees of hyperactivation between the spermatozoa of these two subpopulations. In addition, one could suggest that spermatozoa of SP2 would be starting to acquire their hyperactivated motility, thereby indicating that they would be on-going capacitated. It is also worth pointing out that although SP1 and SP4 showed very low values of ALH and DANCE, LIN was the lowest in SP4 and the highest in SP1. Furthermore, whereas SP1 presented the maximum value of STR, SP4 showed a very low STR and the lowest values of velocity parameters. These data indicate that SP4 contains the less vigorous spermatozoa, with nonlinear motility. The cluster procedure performed in the present study revealed that the sample obtained by swim-up was constituted not only by rapid but also functionally heterogeneous spermatozoa, with cells of each subpopulation showing differences in motility. We suggest that these differences could correspond to different degrees of capacitation. Given that capacitation is a progressive, time-developing process, all the associated sperm

changes, including the acquisition of the hyperactivated motility pattern, are also gradual in the overall sample. Therefore, our results match with previous reports indicating through techniques independent from sperm motility that samples obtained by swim-up are heterogeneous (Garcı´a-Lo´pez et al. 1996a; Ollero et al. 1997; Grasa et al. 2005). Next, we investigated whether the induction of capacitation with EGF affected the subpopulation structure. Surprisingly, we found no change in the subpopulation distribution, despite sperm capacitation being effectively induced as the significant increases in the CTC-capacitated pattern and protein tyrosine phosphorylation proved. In contrast to our data, previous results showed that in vitro capacitation of ram spermatozoa led to an increase in the sperm subpopulation characterised by a ´ lvarez et al. hyperactivated-like motility pattern (Garcı´a-A 2014). These differences could be due to the different procedure used to induce sperm capacitation in vitro. Indeed, while Garcia-Alvarez et al. (2014) used synthetic oviductal fluid supplemented with 2% oestrous sheep serum, the present study used EGF. This suggests differences in the molecular mechanisms of both protocols. One of the most interesting findings of the present study was that supplementing the capacitating medium with SPP resulted

SPP and sperm subpopulations

in a significant reduction in both the CTC-capacitated sperm rate and protein tyrosine phosphorylation, with regard to the control. These findings corroborate previous reports showing that the protective effect exerted by SPP upon spermatozoa is related to their ability to prevent capacitation (decapacitating role; Desnoyers and Manjunath 1992; The´rien et al. 2001; Manjunath et al. 2002; Barrios et al. 2005). In spite of this, SPP-treated samples showed a decrease in the proportion of non-hyperactivated spermatozoa (SP1) and a concomitant increase in those that were hyperactivated (SP3). These results suggest that SP1 is a heterogeneous subpopulation, with some spermatozoa being stimulated by SPP and acquiring hyperactivated movement. This hypothesis could explain why such spermatozoa change from SP1 to SP3 and is in agreement with previous studies showing that spermatozoa may pass from one subpopulation to another in response to some treatments (Ramio´ ´ lvarez et al. 2014). et al. 2008; Garcı´a-A The overall results indicate that ram spermatozoa are able to undergo capacitation with no hyperactivation. These findings are consistent with previous results showing that ram spermatozoa can be capacitated in a medium with cAMP-elevating agents without displaying hyperactivated movement (Ishijima and Witman 1987; Cola´s et al. 2010), similar to bull (Marquez and Suarez 2004) and stallion (McPartlin et al. 2009) spermatozoa. Furthermore, we have also reported that the inhibition of protein kinase A reduced both capacitation and protein tyrosine phosphorylation although hyperactivation increased (Cola´s et al. 2010). These results indicate an essential difference between hamster (White and Aitken 1989), mouse (Marquez et al. 2007) and rabbit (de Lamirande et al. 1983) spermatozoa, which become hyperactivated in capacitating conditions, and other species including human (Bajpai and Doncel 2003), bull (Marquez and Suarez 2004) and ram, which can be capacitated in vitro without displaying hyperactivated movement. In conclusion, the present results confirm that dextran– swim-up samples obtained from fresh ram ejaculates contain four sperm subpopulations with specific movement patterns. Incubation of these samples with EGF in capacitating conditions did not produce any change in the subpopulation distribution. The addition of SPP before the treatment induced changes consistent with a decrease in the sperm subpopulation characterised by a non-hyperactivated motility pattern, as well as an increase in the proportion of hyperactivated spermatozoa. However, a reduction in both the CTC-capacitated sperm rate and protein tyrosine phosphorylation was found, which corroborates the hypothesis that the SPP protective effect on spermatozoa is related to their decapacitating role. Therefore, we can conclude that ram spermatozoa are able to undergo capacitation with no hyperactivation and that SPP are able to induce hyperactivation of spermatozoa but maintain them in a decapacitated state, thereby ensuring that functionally competent spermatozoa meet the ovulated egg at the site of fertilisation (Kumar et al. 2009). It is worth pointing out that seminal plasma not only contributes to sperm transport but also to overall sperm function, particularly their interactions with the tubular genital tract and the oocyte (see Rodrı´guez-Martı´nez et al. 2011 for review). Since the excess of seminal plasma is removed from the uterus (Troedsson et al. 2005), only specific seminal plasma components, mainly

Reproduction, Fertility and Development

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proteins adhered to spermatozoa, remain on the sperm surface. These sperm-coating components are involved in sperm capacitation, interaction with the zona pellucida, the acrosomal reaction and oocyte penetration (Kumar et al. 2009). The removal of these components before spermatozoa undergo the acrosome reaction is a prerequisite for fertilisation (Yanagimachi 1994; Manjunath et al. 2002). Therefore, most of these SPP are removed from the sperm membrane during capacitation (Desnoyers and Manjunath 1992; Cross 1996; Cross and Mahasreshti 1997; Mortimer et al. 1998; Bedu-Addo et al. 2005), which must take place inside the oviduct. Thus, we have identified two specific proteins in ram seminal plasma that are partially removed from the sperm membrane during in vitro capacitation and the acrosome reaction and redistributed towards the equatorial segment, which suggests their role in capacitation and sperm–egg interactions (Barrios et al. 2005). Further studies about the significance of this hyperactivated movement in response to SPP are warranted, since unveiling whether SPP are essential for spermatozoa releasing from the oviductal reservoir at the time of ovulation could help to better understand ram sperm physiology. Acknowledgements Supported by grants CICYT-FEDER AGL 2011–25850 and DGA A- 26FSE. C. L. was financed by FPU AP2009–1298. The authors thank ANGRA for supplying the sires and S. Morales for the collection of semen samples.

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Effect of seminal plasma proteins on the motile sperm subpopulations in ram ejaculates.

It has been proposed that seminal plasma proteins (SPP) support survival of ram spermatozoa, exerting a dual effect, both capacitating and decapacitat...
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