Cryopreservation of ram sperm alters the dynamic changes associated with in vitro capacitation

Journal Pre-proof Cryopreservation of ram sperm alters the dynamic changes associated with in vitro capacitation Patricia Peris-Frau, Alicia Martín-Maestro, María Iniesta-Cuerda, Irene SánchezAjofrín, Andreina Cesari, J. Julián Garde, Margarita Villar, Ana J. Soler PII:

S0093-691X(20)30051-0

DOI:

https://doi.org/10.1016/j.theriogenology.2020.01.046

Reference:

THE 15343

To appear in:

Theriogenology

Received Date: 6 August 2019 Revised Date:

8 December 2019

Accepted Date: 21 January 2020

Please cite this article as: Peris-Frau P, Martín-Maestro A, Iniesta-Cuerda Marí, Sánchez-Ajofrín I, Cesari A, Garde JJuliá, Villar M, Soler AJ, Cryopreservation of ram sperm alters the dynamic changes associated with in vitro capacitation, Theriogenology (2020), doi: https://doi.org/10.1016/ j.theriogenology.2020.01.046. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.

Author contribution statement PP-F, AM-M, MI-C, IS-A and AC collected samples and run the experiment. PP-F drafted the manuscript and performing the statistical analysis being supervised by MV, JJG and AJS. AJS designed and coordinated the study. All authors read and approved the final manuscript.

1

Cryopreservation of ram sperm alters the dynamic changes associated

2

with in vitro capacitation

3

Patricia Peris-Frau1, Alicia Martín-Maestro1, María Iniesta-Cuerda1, Irene Sánchez-

4

Ajofrín1, Andreina Cesari2, J Julián Garde1, Margarita Villar1, Ana J Soler1*.

5

1

SaBio IREC (CSIC-UCLM-JCCM), Albacete, Spain.

6

2

Biología de Microorganismos y Gametas, Instituto de Investigaciones Biológicas

7

CONICET, Universidad Nacional de Mar del Plata, Funes 3250, 7600, Mar del Plata,

8

Argentina.

9

Running title: Sperm cryopreservation and capacitation

10

*Correspondence: Dr AJS ([email protected])

11

ETSIAM.UCLM, Campus Universitario s/n. 020071. Albacete

12

13

14

15

16

17

18

19

20

21 1

22

Abstract

23

The aim of this study was to investigate the dynamic changes that ram sperm experience

24

during in vitro capacitation before and after cryopreservation. Using flow cytometry and

25

computer assisted sperm analysis system (CASA), protein tyrosine phosphorylation and

26

several functional parameters were evaluated in fresh and cryopreserved ram sperm

27

incubated under capacitating and non-capacitating conditions at 0, 1, 5, 15, 30, 60, 120,

28

180 and 240 min. A short incubation period (5-30 min) under capacitating conditions

29

was enough to increase mitochondrial activity and tyrosine phosphorylation in

30

cryopreserved sperm, inducing also changes in the motility pattern, which could be

31

related to hyperactivation. However, fresh sperm required a longer incubation (180-240

32

min) under capacitating conditions to undergo similar modifications. In both types of

33

samples, tyrosine phosphorylation increased in a sequential manner in the midpiece,

34

principal piece and tail at specific time points during in vitro capacitation. Moreover,

35

the proportion of viable sperm with intact acrosome begun to decrease during

36

capacitation, occurring before in cryopreserved sperm. Our findings suggest that

37

cryopreserved ram sperm become competent for fertilization after a short exposure to

38

capacitating conditions as a result of drastic changes inflicted by the freezing-thawing

39

procedure, while prolonged incubations after cryopreservation severely impair sperm

40

quality.

41

Keywords: cryopreservation, capacitation, tyrosine phosphorylation, ram sperm.

42

43

1. Introduction

44

Mammalian sperm acquire their fertilizing ability after a set of physiological and

45

biochemical changes collectively termed sperm capacitation [1]. In vivo, capacitation 2

46

occurs in the oviduct [2], but it can be achieved in vitro after incubating sperm in a

47

medium that mimic the composition of the oviductal fluid. In addition, each species has

48

its specific requirements, being oestrous sheep serum a key-effector for in vitro

49

capacitation in small ruminants [3,4]. During this time-dependent event, sperm undergo

50

a plasma membrane rearrangement, hyperpolarization, actin polymerization, alterations

51

of intracellular ion concentrations, energy metabolism increment and activation of

52

cAMP-dependent protein phosphorylation [5–9]. Besides these variations, sperm

53

motility begins to change during capacitation leading to sperm hyperactivation [10–12],

54

a concomitant event necessary for oocyte penetration together with the acrosome

55

exocytosis.

56

The response to capacitation not only differs between males [13] or within the same

57

ejaculate [14], but also after cryopreservation [15]. Freezing-thawing procedures induce

58

several injuries in a considerable proportion of sperm, reducing their lifespan. Among

59

the sublethal damages produced during cryopreservation, modifications in membrane

60

composition and permeability, excessive production of reactive oxygen species (ROS),

61

changes in protein content, lipid peroxidation, impairment of mitochondrial activity that

62

drives to ATP depletion and sperm motility reduction have been reported [16–19].

63

Moreover, as a result of membrane fluidity changes and calcium influx increment, the

64

surviving sperm population experience capacitation-like changes (cryo-capacitation)

65

immediately after thawing [20,21] or become capacitated after a shorter exposure to

66

capacitating conditions in comparison with fresh sperm [21,22].

67

Some sperm modifications previously documented during capacitation differ

68

between species and after cryopreservation. In addition, these changes are also time-

69

dependent, highlighting the importance of chronological studies in different species to

70

improve our actual knowledge about capacitation. However, few works have

3

71

investigated these capacitation-associated changes at different time points, evaluating

72

some of them only cryopreserved sperm while others compared fresh and cryopreserved

73

sperm [23–28].

74

To provide new insights into how cryopreservation could alter sperm capacitation

75

in small ruminants, a deeper dynamic study was carried out in fresh and cryopreserved

76

ram sperm incubated under capacitating and non-capacitating conditions. We

77

hypothesize that cryo-damage have a severe impact on ram sperm, changing their

78

capacitation behaviour in relation to fresh sperm. Therefore, the aim of the present study

79

was to better characterise the temporal changes that several functional sperm parameters

80

undergo during in vitro capacitation before and after cryopreservation. Moreover, a

81

novel technique, image-based flow cytometry, was employed to measure the principal

82

signalling pathway involved in sperm capacitation (protein tyrosine phosphorylation)

83

and some sperm parameters related to this event, such as mitochondrial activity and

84

acrosome integrity.

85

2. Material and methods

86

2.1 Chemicals and reagents

87

Fluorescent probes were acquired from Invitrogen (Barcelona, Spain); Biladyl®

88

was obtained from Minitube (Tiefenbach, Germany); anti-phosphotyrosine monoclonal

89

antibody Clone 4G10 was purchased from Merck-Millipore (Madrid, Spain) and all

90

other chemicals were acquired from Sigma-Aldrich (Madrid, Spain).

91 92 93

2.2 Sperm collection and cryopreservation Animal procedures were carried out by the Reproduction Biology Group of

94

University of Castilla-La Mancha (UCLM, Spain), which is officially authorized for

95

collecting and storing semen from sheep (ES07RS02OC) following the RD 841/2011. 4

96

Semen was collected from four Manchega rams (> 3 years of age) housed at the

97

experimental farm of UCLM via artificial vagina. Immediately after collection,

98

individual sperm motility and wave motion were assessed subjectively using bright field

99

and phase contrast microscopy, respectively (Eclipse 50i Nikon; Tokyo, Japan). Only

100

ejaculates with sperm motility higher than 65% and wave motion values of 3.5 were

101

chosen and pooled to avoid individual variability. Sperm concentration (mean value of

102

the pool: 3,810 x 106 sperm/ml) was calculated using a Makler counting chamber and

103

the Sperm Class Analyzer software (SCA®) (Microptic, Barcelona, Spain). The

104

experiment was repeated four times using one pool of four ejaculates every time. Each

105

pool was divided into two fractions, one fraction was handled as fresh sample, while the

106

other fraction was cryopreserved.

107

Semen diluted to 200 x 106 sperm/ml in Biladyl® (with 20% egg yolk and 7%

108

glycerol) was slowly cooled during 2 h from 30 °C to 5 °C and allowed to equilibrate

109

for 2 h at 5 °C. At the end of equilibration period, samples were automatically packed

110

into 0.25 ml straws, frozen in a programmable biofreezer (Planer Kyro 10 Series III;

111

Planer PLC, London, United Kingdom) following a freezing curve ( -20 ºC/min from 5

112

ºC to -100 ºC and -10 ºC/min from -100 ºC to -140 ºC) and stored in a liquid nitrogen

113

container. Straws were thawed in a water bath at 37 ºC for 30 s.

114 115

2.3 Experimental design

116

Fresh and cryopreserved sperm were centrifuged through single columns of

117

Percoll® 45% (700 x g; 10 min) to eliminate extenders, seminal plasma and debris.

118

Sperm pellets were diluted to 10 x 106 sperm/ml and incubated for 240 min under

119

capacitating or non-capacitating conditions at 38.5 ºC under 5% CO2. To achieve sperm

120

capacitation, sperm were incubated in synthetic oviductal fluid (SOF) enriched with

5

121

oestrous sheep serum (2% of ESS; CAP conditions), an indispensable substance that

122

promote acrosome reaction and allow optimal IVF rates in small ruminants [3,4].The

123

SOF was formulated as previously described by Takahashi and First [29] but without

124

adding BSA (107.70 mM NaCl, 7.16 mM KCl, 1.19 mM KH2PO4, 1.71 mM CaCl2,

125

0.49 mM MgCl2, 25.07 mM NaHCO3, 3.30 mM Na Lactate, 0.30 mM Na pyruvate, 200

126

mM glutamine, phenol red 1.30 µg/ml and 100 U/ml penicillin).

127

Additionally, as a negative control, sperm were incubated in a serum-free SOF

128

(SOF supplemented with 0.1% polyvinyl alcohol; NC conditions). Different sperm

129

parameters were analysed in fresh and cryopreserved sperm after 1, 5, 15, 30, 60, 120,

130

180 and 240 min of incubation in CAP and after 0, 15 and 240 min of incubation in NC

131

conditions. At each time point, sperm viability, motility, mitochondrial activity, intact

132

acrosomes, ROS production and protein tyrosine phosphorylation were evaluated.

133 134 135

2.4 Sperm kinematics Sperm motility parameters were estimated using SCA® as reported by García-

136

Álvarez et al. [4]. Settings were adjusted to ram spermatozoa and at least ten fields for

137

each sample were analysed. The following kinematic parameters were statistically

138

evaluated in the present study for each sample: percentage of motile sperm (total and

139

progressive), curvilinear velocity (VCL; µm s-1), linearity (LIN; %) and amplitude of

140

lateral head displacement (ALH; µm). We selected VCL, LIN, and ALH among other

141

kinematic parameters evaluated (VSL (µm/s), VAP (µm/s), STR (%),WOB (%) and

142

BCF (Hz)) because previous studies reported that these parameters can be a valuable

143

tool to detect hyperactivated motility [14,30,31].

144 145

6

146 147

2.5 Flow cytometry analyses In order to avoid overlapping measures between incubation times and obtain an

148

accurate assessment of those capacitation-associated changes over time, two flow

149

cytometers were simultaneously employed in our study. Sperm viability and ROS

150

production were evaluated using a Cytomics FC500 flow cytometer (Beckman Coulter,

151

Brea, CA, USA) controlled with the MXP software (v.3). While acrosome integrity,

152

mitochondrial activity and protein tyrosine phosphorylation were measured using a

153

FlowSight® imaging flow cytometer (Amnis, Merck-Millipore, Germany) controlled

154

with the INSPIRE® software (v.3). Raw data were analysed with the WEASEL software

155

(WEHI, Melbourne, Australia) in the first cytometer and with the IDEAS® software in

156

the second cytometer. In all cases, 10,000 events were acquired per sample. Mitotracker

157

Deep Red was excited with a 633 nm helium-neon laser and all other fluorochromes

158

were excited with a 488 nm laser. Dot plots with forward-scatter light and side-scatter

159

light or aspect ratio and area were employed in the respective cytometers to exclude

160

debris from sperm population.

161 162

2.5.1 Assessment of sperm viability, acrosome integrity, mitochondrial activity and ROS

163

production

164

Samples were diluted to 1 x 106 sperm/ml in different staining solutions

165

prepared with SOF-PVA-HEPES and processed immediately, except those samples

166

used for evaluating mitochondrial activity, which were incubated for 15 min in the dark

167

at 37 ºC. The staining solution for sperm viability was 12 µM of PI and 50 nM of YO-

168

PRO-1; for acrosome integrity 3 µM of PI and 0.5µg/ml of PNA-FITC; for

169

mitochondrial activity 25 nM of YO-PRO-1 and 100 nM of Mitotracker Deep Red and

170

for ROS production 12 µM of PI and 5 µM of CM-H2DCFDA. The percentage of sperm

7

171

YO-PRO-1−/PI− represented the proportion of viable sperm, the percentage of sperm

172

Mitotracker+/ YO-PRO-1− represented the proportion of viable sperm with active

173

mitochondria and the percentage of sperm PNA-FITC−/PI− represented the proportion

174

of viable sperm with intact acrosome. Finally, ROS production was measured only in

175

viable sperm (PI−) using CM-H2DCFDA, an indicator that is oxidised in the presence of

176

intracellular ROS.

177

2.5.2 Assessment of protein tyrosine phosphorylation

178

The proportion of fresh and cryopreserved sperm with tyrosine phosphorylated

179

proteins and the phosphorylation of different sperm regions were studied during the

180

incubation period with an imaging flow cytometer. After the addition of 3 µM of PI to

181

the samples collected at different time points of incubation, to discriminate between live

182

and dead sperm, samples were centrifuged and fixed with 2% (v/v) paraformaldehyde in

183

PBS at room temperature (RT). 10 min later, samples were washed in PBS (5,000 x g; 5

184

min) and resuspended in 0.1% (v/v) BD FACS™ Permeabilizing Solution for 10 min at

185

RT. Then, sperm were washed in PBS and non-specific binding sites were blocked with

186

10 % (w/v) BSA in PBS (blocking buffer) for 30 min at 38.5 °C. After washing again

187

with PBS, sperm were incubated with the first antibody (anti-phosphotyrosine

188

monoclonal antibody Clone 4G10; 1:300 dilution in blocking buffer) for 1 h at 38 °C.

189

Subsequently, samples washed in PBS were incubated with FITC-conjugated anti-

190

mouse IgG antibody (1:300 dilution in blocking buffer) for 1 h in the dark at 38 °C and

191

run through the imaging flow cytometer. A negative control was obtained by incubation

192

with IgG1-FITC (isotype from murine myeloma, clone MOPC 21; 1:300 dilution)

193

instead of the primary antibody.

194 195

The proportion of live sperm showing positive fluorescence (PI−, phosphorylated sperm) was recorded for each sample. Once phosphorylated sperm were

8

196

detected in every sample, the localization of tyrosine-phosphorylated proteins was

197

investigated in the different subcellular sperm regions following the segmentation

198

method reported by Matamoros-Volante et al. [32] with the IDEAS® software in all

199

samples. To detect how tyrosine phosphorylation changes in different sperm regions

200

over the incubation period under CAP and NC conditions, samples collected at different

201

time intervals were all normalized to the NC conditions of 0 min. For this purpose, in

202

each sample, the mean fluorescence of each sperm region was divided by the mean

203

fluorescence of the same region at 0 min in NC conditions.

204 205

2.6 Statistical analyses

206

Statistical analyses were performed using SPSS v. 23.0 (IBM corp., Chicago, USA).

207

A general lineal model (GLM) was employed to evaluate the effect of incubation media

208

(CAP and NC) and treatment (cryopreserved and fresh sperm) on diverse sperm

209

parameters. Four biological replicates were included in the analysis and significance

210

was set at P < 0.05. Moreover, the effect of incubation time on sperm physiology was

211

studied separately in fresh and cryopreserved samples by another GLM to investigate

212

how fresh and cryopreserved sperm respond to in vitro capacitation after short and long

213

incubation periods. When a significant effect was observed, post hoc comparisons with

214

Bonferroni correction were carried out. Results were expressed as least square means ±

215

SEM.

216

3. Results

217

3.1 Effect of cryopreservation and capacitation on sperm quality

218

When a global comparison between fresh and cryopreserved samples was

219

performed, several sperm parameters were found to be adversely affected by

220

cryopreservation (Table 1 and table 2). Among them, total motility (TM), progressive 9

221

motility (PM), sperm viability, acrosome integrity and mitochondrial activity were

222

significantly reduced (P < 0.05) in cryopreserved sperm compared to fresh sperm.

223

However, cryopreservation significantly increased (P < 0.05) ROS production in

224

comparison with fresh samples (Table 2).

225

Substantial differences were also found in some sperm parameters depending on

226

the incubation media (Table 1 and table 2). Diverse motility parameters (TM, PM, VCL

227

and ALH) were significantly enhanced (P < 0.05) when sperm were incubated under

228

CAP conditions as well as sperm viability, mitochondrial activity and protein tyrosine

229

phosphorylation. Contrarily, LIN and ROS levels significantly increased (P < 0.05)

230

when sperm were incubated under NC conditions.

231 232

3.2 Evolution of sperm kinematic parameters during incubation of fresh and

233

cryopreserved samples under capacitating and non-capacitating conditions

234

Incubation of fresh and cryopreserved sperm for 240 min under NC conditions

235

declined the proportion of motile sperm in a time-dependent manner (Fig. 1A). In

236

contrast, when both types of samples were incubated under CAP conditions for 240

237

min, sperm motility was barely affected in fresh samples throughout incubation time,

238

while the percentage of motile sperm in cryopreserved samples decreased drastically (P

239

< 0.05) after 60 min.

240

Regarding the other motility parameters (Fig. 1B-D) measured in the motile sperm

241

subpopulation, LIN was significantly higher (P < 0.05) at 0 min, either in fresh or

242

cryopreserved sperm but then decreased, regardless of incubation media. Although the

243

lowest values of LIN were obtained at specific time points during in vitro capacitation.

244

VCL and ALH were low and remained unchanged through the entire incubation under

245

NC conditions in both types of samples. However, incubation of fresh and 10

246

cryopreserved sperm under CAP conditions significantly increased (P < 0.05) VCL and

247

ALH at different time intervals, occurring at 180-240 min in the former and at 5-30 min

248

in the latter.

249 250

3.3 Evolution of several functional sperm parameters during incubation of fresh and

251

cryopreserved samples under capacitating and non-capacitating conditions

252

Incubation under NC conditions induced a rapid reduction of sperm quality over

253

time in fresh and cryopreserved samples, manifested by a significant decrease (P < 0.05)

254

in sperm viability, mitochondrial activity and acrosome integrity (Fig. 2). In contrast,

255

incubation under CAP conditions barely changed the percentage of viable sperm over

256

time in fresh samples, preserving sperm viability of cryopreserved samples for a longer

257

incubation time (120 min) than NC conditions (15 min) (Fig. 2A).

258

The proportion of live sperm with active mitochondria in fresh samples (Fig. 2B)

259

showed fluctuations during the entire incubation under CAP conditions, decreasing

260

from 30 min to 120 min and increasing again at 180-240 min. However, in

261

cryopreserved samples, mitochondrial activity was higher after 15-30 min under CAP

262

conditions and then went down.

263

The percentage of viable sperm with intact acrosome (Fig. 2C) in fresh samples

264

significantly decreased (P < 0.05) at 60, 120 and 240 min of incubation under CAP

265

conditions compared to 0 min. Whereas in cryopreserved samples, acrosome integrity

266

began to decline after 30 min of incubation under CAP conditions compared to 0 min.

267

ROS levels were lower when fresh and cryopreserved sperm (Fig. 2D) were

268

incubated under CAP conditions in comparison with those sperm incubated under NC

269

conditions. Despite this, prolonged incubations under CAP conditions raised ROS

270

production in cryopreserved samples. 11

271

3.4 Dynamics of protein tyrosine phosphorylation during incubation of fresh and

272

cryopreserved samples under capacitating and non-capacitating conditions

273

Figure 3 shows that the freezing-thawing procedure induced a premature

274

phosphorylation in cryopreserved sperm at 0 min. However, incubation under CAP

275

conditions significantly increased (P < 0.05) the proportion of live tyrosine

276

phosphorylated sperm over time in relation to sperm incubated under NC conditions in

277

both type of samples, reaching the highest values at 240 min in fresh sperm and at 15

278

min in cryopreserved sperm with regard to 0 min (Fig. 3).

279

Tyrosine phosphorylation was also further studied in different subcellular sperm

280

regions in those phosphorylated sperm: a) head (includes sperm with fluorescence in the

281

entire head, acrosome or equatorial segment), b) midpiece, c) principal piece and d) tail

282

(includes sperm with fluorescence in the midpiece and principal piece) (Fig. 4). Head

283

phosphorylation appeared in fresh and cryopreserved sperm incubated in both media

284

and was practically constant during the incubation period, decreasing after 240 min

285

under NC conditions only in cryopreserved sperm (Fig. 5A).

286

However, tail phosphorylation increased exclusively at specific incubation times

287

under CAP conditions, occurring in fresh sperm later than cryopreserved sperm (at 120-

288

240 min and at 15-60 min, respectively) (Fig. 5B). In a high percentage of sperm, tail

289

phosphorylation was combined with head phosphorylation. Besides, phosphorylation

290

appeared first in the midpiece and then progressed to the principal piece (Fig. 5C-D). In

291

fresh sperm, the fluorescence intensity in the midpiece was significantly superior (P <

292

0.05) at 180-240 min of incubation under CAP conditions and at 240 min in the

293

principal piece, whereas in cryopreserved sperm, the fluorescence intensity in the

294

midpiece was significantly higher (P < 0.05) at 15-30 min and at 30 min in the principal

295

piece.

12

296

4. Discussion

297

Although the main goal of cryopreservation is to preserve sperm quality and

298

fertility at similar levels of those found in fresh sperm for long-term, several factors

299

during freezing-thawing procedures induce a severe cellular stress, altering diverse

300

sperm parameters [16,17,20]. Therefore, it was not unexpected that in our study,

301

cryopreservation of ram sperm increased ROS production and simultaneously declined

302

viability, motility, mitochondrial function and acrosome integrity in comparison with

303

fresh sperm. Similar results were found in other reports [18,33–36] and led us to

304

investigate whether cryopreservation could alter or not sperm functionality during

305

capacitation. To our knowledge, this is the first time that several dynamic changes

306

associated with sperm capacitation have been characterised throughout incubation time

307

in fresh and cryopreserved ram sperm.

308

A different behaviour between both types of samples was observed during the

309

incubation period. In fresh sperm, motility and viability barely changed over the entire

310

incubation under CAP conditions, while in cryopreserved sperm, both parameters

311

decreased after 60 and 120 min, respectively. Comparisons with other studies are

312

difficult since methodologies, media and species greatly differ, however, some authors

313

demonstrated that cryopreservation declines the proportion of viable and motile sperm

314

during in vitro capacitation in a time-dependent manner [23,24,26]. In addition,

315

incubation under NC conditions diminished these two parameters at a faster rate than

316

CAP conditions.

317

We hypothesized that the presence of ESS in the capacitating medium could

318

play a crucial role in these differences, since it has been documented that this fluid

319

contains energy metabolites (pyruvate and lactate), several antioxidants and a specific

320

glycoprotein that stimulates and sustains sperm motility [37–39]. Moreover, Del Olmo 13

321

et al. [40] proposed that the antioxidant capacity of ESS might control ROS production,

322

keeping their levels low during in vitro capacitation, which is indispensable to drive the

323

signalling pathways involved in sperm capacitation [41,42]. This was in line with our

324

findings and it would explain that the absence of excessive ROS levels preserves better

325

sperm viability over time when fresh and cryopreserved samples were incubated under

326

CAP conditions compared to NC conditions. However, the proportion of viable and

327

motile sperm in cryopreserved samples still decreased after a period of time under CAP

328

conditions, suggesting that, apart from oxidative stress, modifications in the membrane

329

structure and mitochondrial function during cryopreservation severely impair sperm

330

viability and motility [19].

331

To successfully reach the oocyte and penetrate zona pellucida, sperm develop a

332

high-amplitude and asymmetric flagellar movement at some point during capacitation

333

[43], known as hyperactivation [11,12]. In our study, incubation under CAP conditions

334

induced changes in the flagellar movement, characterised by a low LIN and a high ALH

335

and VCL, which could be related with the acquisition of hyperactivated motility as

336

previous works reported [14,31]. Such variations appeared before in cryopreserved

337

sperm, whereas in fresh sperm occurred after a longer incubation. Moreover, the lack of

338

this specific motility pattern when both types of samples were incubated under NC

339

conditions suggests that ESS could be responsible for those changes.

340

During sperm capacitation a high supply of energy is required [44,45], being

341

mitochondrial oxidative phosphorylation and glycolysis the main metabolic pathways

342

involved in energy production in ram sperm [46]. Therefore, it was not surprising that in

343

the present study, sperm mitochondrial function increased at certain incubation times

344

during in vitro capacitation but not in NC conditions. Besides, a short exposure to CAP

345

conditions was enough to raise the mitochondrial activity of cryopreserved sperm, while 14

346

fresh sperm showed oscillations in their mitochondrial activity during the entire

347

incubation, increasing again at 180-240 min. This latter increment might be associated

348

with the acquirement of capacitated state, obtaining Betarelli et al. [47] similar data

349

during in vitro capacitation of fresh boar sperm. In addition, the presence of energy

350

substrates and antioxidants in ESS could have helped to delay the damaging effects of

351

cryopreservation on these organelles up to 60 min, obtaining the opposite result under

352

NC conditions.

353

At the same time interval that mitochondrial function increased, sperm

354

underwent changes in their flagellar movement and tyrosine phosphorylation was more

355

intense. All these variations could point out the optimal incubation time in which ram

356

sperm acquire their fertilizing ability under CAP conditions. This phenomenon seems to

357

happen at 15-30 min in cryopreserved sperm and at 180-240 min in fresh sperm. In

358

addition, it is known that capacitation prepares sperm for the subsequent acrosome

359

reaction [48]. However, our findings revealed that the acrosome integrity of

360

cryopreserved sperm begins to decay at 30 min of in vitro capacitation and in fresh

361

sperm after 60 min. A possible explanation could be that the removal of sterol during

362

capacitation promotes in parallel the initiation of fusion events between the outer

363

acrosome and plasma membrane, as have been proposed in mice [49], which in turn,

364

decreases acrosome stability. In contrast, incubation under NC conditions accelerated

365

the loss of acrosome integrity over time in both types of samples, which is supported by

366

an earlier study in cryopreserved ram sperm [14] and might indicate that incubation in a

367

serum-free medium drastically reduces sperm quality through the time.

368

The increase in protein tyrosine phosphorylation detected in our study during in

369

vitro capacitation has been observed earlier in ram and other species [8,26,27,47,50].

370

Chatterjee et al. [51] showed a gradual increment of tyrosine phosphorylation over the 15

371

course of capacitation in fresh goat sperm, reaching the highest values at 240 min,

372

which supports our results in fresh ram sperm. Similar data were previously found in

373

fresh ram sperm by Pérez-Pé et al. [52], although the highest values were reached at 360

374

min. Differences in methodology and samples used probably altered the time point in

375

which the maximum levels of tyrosine phosphorylation were obtained.

376

On the other hand, changes in membrane permeability during cryopreservation

377

lead to increase calcium influx, which in turn activates adenylyl cyclase, initiating a

378

premature cAMP/PKA/tyrosine phosphorylation cascade [53]. These capacitation-like

379

changes known as cryo-capacitation were immediately observed at 0 min in our study,

380

when a higher percentage of cryopreserved sperm was phosphorylated in comparison

381

with fresh sperm, which is in line with another report [54]. Notwithstanding, incubation

382

of cryopreserved sperm under CAP conditions rapidly increased the proportion of live

383

tyrosine phosphorylated sperm, as has been described in cryopreserved boar sperm [23],

384

obtaining the highest values at 15 min.

385

Additionally, tyrosine phosphorylated proteins showed a different distribution

386

during the time course of capacitation. While phosphorylation of head proteins seems

387

necessary for sperm-oocyte interaction and fusion events, phosphorylation of flagellum

388

proteins has been related to hyperactivation [53]. In agreement with Matamoros-Volante

389

et al. [32], sperm incubated under NC conditions only showed fluorescence in the head.

390

But incubation under CAP conditions induced, besides head labelling, a gradual

391

fluorescence in the tail over time, which is in line with previous studies [27,55]. Our

392

results also revealed that tail phosphorylation increased in cryopreserved sperm earlier

393

than fresh sperm whereas head phosphorylation was constant in both samples.

394

Moreover, phosphorylation augmented first in the midpiece and then in the principal

395

piece during capacitation. However, in mouse and boar sperm, the principal piece was

16

396

phosphorylated before the midpiece [13,56]. Although the underlying reason for these

397

dissimilarities must be further investigated, apparent discrepancies could be due to

398

differences among species.

399

In summary, our results demonstrated that cryopreserved ram sperm require a

400

shorter exposure to capacitating conditions than fresh ram sperm to achieve their

401

fertilizing potential because of cryodamage. This study provides evidences that ram

402

sperm undergo changes in their motility pattern at the same time that mitochondrial

403

activity and tyrosine phosphorylation increase during in vitro capacitation. Moreover,

404

the presence of ESS in the capacitating medium seems to reduce ROS production to

405

optimal levels. A further understanding of the molecular mechanisms of cryodamage as

406

well as those substances from ESS that benefit sperm would help to prolong the fertility

407

of cryopreserved sperm.

408

5. Conflict of interest

409

The authors declare that there is no conflict of interest that could be perceived as

410

prejudicing the impartiality of the research reported.

411

Acknowledgments

412

PP-F was supported by UCLM fellowships. MV was supported by the Research Plan of

413

University of Castilla- La Mancha (UCLM, Spain). MI-C was supported by a Ministry

414

of Economy and Competitiveness fellowship.

415

Author contribution statement

416

PP-F, AM-M, MI-C, IS-A and AC collected samples and run the experiment. PP-F

417

drafted the manuscript and performing the statistical analysis being supervised by MV,

418

JJG and AJS. AJS designed and coordinated the study. All authors read and approved

419

the final manuscript. 17

420

References

421

[1]

Austin CR. The capacitation of the mammalian sperm. Nature 1952;170:326.

422

[2]

Rodriguez-Martinez H. Role of the oviduct in sperm capacitation. Theriogenology 2007;68:138–46. doi:10.1016/j.theriogenology.2007.03.018.

423 424

[3]

Huneau D, Crozet N, Ahmed-Ali M. Estrous sheep serum as a potent agen for

425

ovine IVF: effect on cholesterol efflux from spermatozoa and the acrosome

426

reaction. Theriogenology 1994;42:1017–28.

427

[4]

García-Álvarez O, Maroto-Morales A, Jiménez-Rabadán P, Ramón M, Del Olmo

428

E, Iniesta-Cuerda M, et al. Effect of different media additives on capacitation of

429

frozen-thawed ram spermatozoa as a potential replacement for estrous sheep

430

serum. Theriogenology 2015;84:948–55.

431

doi:10.1016/j.theriogenology.2015.05.032.

432

[5]

Brener E, Rubinstein S, Cohen G, Shternall K, Rivlin J, Breitbart H. Remodeling

433

of the actin cytoskeleton during mammalian sperm capacitation and acrosome

434

reaction. Biol Reprod 2003;68:837–45. doi:10.1095/biolreprod.102.009233.

435

[6]

during capacitation. Int J Dev Biol 2008;52:473–80. doi:10.1387/ijdb.082583bg.

436 437

Gadella BM, Tsai P, Boerke A, Brewis IA. Sperm head membrane reorganisation

[7]

Visconti PE, Krapf D, De La Vega-Beltrán JL, Acevedo JJ, Darszon A. Ion

438

channels, phosphorylation and mammalian sperm capacitation. Asian J Androl

439

2011;13:395–405. doi:10.1038/aja.2010.69.

440

[8]

Grasa P, Cebrián-Pérez JA, Muiño-Blanco T. Signal transduction mechanisms

441

involved in in vitro ram sperm capacitation. Reproduction 2006;132:721–32.

442

doi:10.1530/rep.1.00770.

443 444

[9]

Leemans B, Stout TAE, Schauwer C De, Heras S, Nelis H, Hoogewijs M, et al. Update on mammalian sperm capacitation: how much does the horse differ from

18

445

other species? Reproduction 2019;157:181–97. doi:10.1530/REP-18-0541.

446

[10] Freitas MJ, Vijayaraghavan S, Fardilha M. Signaling mechanisms in mammalian

447 448

sperm motility. Biol Reprod 2017;96:2–12. doi:10.1095/biolreprod.116.144337. [11] Cummins JM. Hyperactivated motility patterns of ram spermatozoa recovered

449

from the oviducts of mated ewes. Gamete Res 1982;6:53–63.

450

doi:10.1002/mrd.1120060107.

451 452 453

[12] Yanagimachi R. The Movement of Golden Hamster before anf after capacitation. J Reprod Fertil 1970;23:193–6. [13] Kumaresan A, Johannisson A, Humblot P, Bergqvist AS. Oviductal fluid

454

modulates the dynamics of tyrosine phosphorylation in cryopreserved boar

455

spermatozoa during capacitation. Mol Reprod Dev 2012;79:525–40.

456

doi:10.1002/mrd.22058.

457

[14] García-Álvarez O, Maroto-Morales A, Ramón M, Del Olmo E, Jiménez-Rabadán

458

P, Fernández-Santos MR, et al. Dynamics of sperm subpopulations based on

459

motility and plasma membrane status in thawed ram spermatozoa incubated

460

under conditions that support in vitro capacitation and fertilisation. Reprod Fertil

461

Dev 2014;26:725–32. doi:10.1071/RD13034.

462

[15] Pérez LJ, Valcárcel A, De Las Heras MA, Moses D, Baldassarre H. Evidence that

463

frozen/thawed ram spermatozoa show accelerated capacitation in vitro as

464

assessed by chlortetracycline assay. Theriogenology 1996;46:131–40.

465

doi:10.1016/0093-691X(96)00148-3.

466

[16] Bailey JL, Bilodeau JF, Cormier N. Semen cryopreservation in domestic animals:

467

a damaging and capacitating phenomenon. J Androl 2000;21:1–7.

468

doi:10.1002/j.1939-4640.2000.tb03268.x.

469

[17] Pini T, Leahy T, de Graaf SP. Sublethal sperm freezing damage: Manifestations

19

470

and solutions. Theriogenology 2018;118:172–81.

471

doi:10.1016/j.theriogenology.2018.06.006.

472

[18] Gürler H, Malama E, Heppelmann M, Calisici O, Leiding C, Kastelic JP, et al.

473

Effects of cryopreservation on sperm viability, synthesis of reactive oxygen

474

species, and DNA damage of bovine sperm. Theriogenology 2016;86:562–71.

475

doi:10.1016/j.theriogenology.2016.02.007.

476

[19] Grötter LG, Cattaneo L, Marini PE, Kjelland ME, Ferré LB. Recent advances in

477

bovine sperm cryopreservation techniques with a focus on sperm post-thaw

478

quality optimization. Reprod Domest Anim 2019;54:655–65.

479

doi:10.1111/rda.13409.

480

[20] Watson PF. Recent developments and concepts in the cryopreservation of

481

spermatozoa and the assessment of their post-thawing function. Reprod Fertil

482

Dev 1995;7:871–91.

483 484 485 486 487

[21] Gillan A, Evans G, Maxwell WMC. Capacitation status and fertility of fresh and frozen–thawed ram spermatozoa. Reprod Fertil Dev 1997;9:481–7. [22] Garde JJ, Gutiérrez-Adán A, Artiga CG, Vázquez I. Influence of freezing process on “in vitro” capacitation of ram semen. Theriogenology 1993;39:225. [23] Kumaresan A, González R, Johannisson A, Berqvist A. Dynamic quantification

488

of intracellular calcium and protein tyrosine phosphorylation in cryopreserved

489

boar spermatozoa during short-time incubation with oviductal fluid.

490

Theriogenology 2014;82:1145–53. doi:10.1016/j.theriogenology.2014.07.029.

491

[24] Kumaresan A, Johannisson A, Humblot P, Bergqvist A. Effect of bovine

492

oviductal fluid on motility, tyrosine phosphorylation, and acrosome reaction in

493

cryopreserved bull spermatozo. Theriogenology 2019;124:48–56.

494

doi:10.1016/j.theriogenology.2018.09.028.

20

495

[25] Pons-Rejraji H, Bailey JL, Leclerc P. Cryopreservation affects bovine sperm

496

intracellular parameters associated with capacitation and acrosome exocytosis.

497

Reprod Fertil Dev 2009;21:525–37. doi:10.1071/rd07170.

498

[26] Satorre MM, Breininger E, Beconi MT, Beorlegui NB. Protein tyrosine

499

phosphorylation under capacitating conditions in porcine fresh spermatozoa and

500

sperm cryopreserved with and without alpha tocopherol. Andrologia

501

2009;41:184–92. doi:10.1111/j.1439-0272.2009.00915.x.

502

[27] Pommer AC, Rutllant J, Meyers SA. Phosphorylation of Protein Tyrosine

503

Residues in Fresh and Cryopreserved Stallion Spermatozoa under Capacitating

504

Conditions. Biol Reprod 2003;68:1208–14. doi:10.1095/biolreprod.102.011106.

505

[28] Peris SI, Morrier A, Dufour M, Bailey JL. Cryopreservation of Ram Semen

506

Facilitates Sperm DNA Damage: Relationship between Sperm Andrological

507

Parameters and the Sperm Chromatin Structure Assay. J Androl 2004;25:224–33.

508

doi:10.1002/j.1939-4640.2004.tb02782.x.

509

[29] Takahashi Y, First NL. In vitro development of bovine one-cell embryos:

510

Influence of glucose, lactate, pyruvate, amino acids and vitamins.

511

Theriogenology 1992;37:963–78.

512

[30] Mortimer ST, Maxwell WMC. Kinematic definition of ram sperm

513

hyperactivation. Reprod Fertil Dev 1999;11:25–30. doi:10.1071/rd99019.

514

[31] Mortimer ST, Mortimer D. Kinematics of human spermatozoa incubated under

515

capacitating conditions. J Androl 1990;11:195–203. doi:10.1002/j.1939-

516

4640.1990.tb03228.x.

517

[32] Matamoros-Volante A, Moreno-Irusta A, Torres-Rodriguez P, Giojalas L,

518

Gervasi MG, Visconti PE, et al. Semi-automatized segmentation method using

519

image-based fl ow cytometry to study sperm physiology : the case of

21

520

capacitation-induced tyrosine phosphorylation. Mol Hum Reprod 2018;24:64–73.

521

doi:10.1093/molehr/gax062.

522

[33] Connell MO, Mcclure N, Lewis SEM. The effects of cryopreservation on sperm

523

morphology , motility and mitochondrial function. Hum Reprod 2002;17:704–9.

524

[34] He Y, Wang K, Zhao X, Zhang Y, Ma Y, Hu J. Differential proteome association

525

study of freeze-thaw damage in ram sperm. Cryobiology 2016;72:60–8.

526

doi:10.1016/j.cryobiol.2015.11.003.

527

[35] Partyka A, Nizanski W, Łukaszewicz E. Evaluation of fresh and frozen-thawed

528

fowl semen by flow cytometry. Theriogenology 2010;74:1019–27.

529

doi:10.1016/j.theriogenology.2010.04.032.

530

[36] Mostek A, Dietrich MA, Słowińska M, Ciereszko A. Cryopreservation of bull

531

semen is associated with carbonylation of sperm proteins. Theriogenology

532

2017;92:95–102. doi:10.1016/j.theriogenology.2017.01.011.

533

[37] Brown D V, Senger PL. Influence of homologous blood serum on motility and

534

head-to-head agglutination in nonmotile ejaculated bovine spermatozoa. Biol

535

Reprod 1980;23:271–5.

536

[38] Mandal M, Banerjee S, Majumder GC. Stimulation of forward motility of goat

537

cauda epididymal spermatozoa by a serum glycoprotein factor. Biol Reprod

538

1989;41:983–9. doi:10.1095/biolreprod41.6.983.

539

[39] Hussein HA, Ellah MRA, Derar DRI. Antioxidant concentrations in serum ,

540

follicular fluid , and corpus luteum of cyclic buffalo cows. Comp Clin Path

541

2013;22:829–33. doi:10.1007/s00580-012-1485-7.

542

[40] Del Olmo E, García-Álvarez O, Maroto-Morales A, Ramón M, Iniesta-Cuerda

543

M, Martinez-Pastor F, et al. Oestrous sheep serum balances ROS levels to supply

544

in vitro capacitation of ram spermatozoa. Reprod Domest Anim 2016;51:743–50.

22

545 546

doi:10.1111/rda.12741. [41] Aitken RJ. Reactive oxygen species as mediators of sperm capacitation and

547

pathological damage. Mol Reprod Dev 2017;84:1039–52.

548

doi:10.1002/mrd.22871.

549

[42] Rivlin J, Mendel J, Rubinstein S, Etkovitz N, Breitbart H. Role of hydrogen

550

peroxide in sperm capacitation and acrosome reaction. Biol Reprod

551

2004;70:518–22. doi:10.1095/biolreprod.103.020487.

552

[43] Suarez SS. Control of hyperactivation in sperm. Hum Reprod 2008;14:647–57.

553

[44] Ferramosca A, Zara V. Bioenergetics of mammalian sperm capacitation. Biomed

554 555

Res Int 2014;2014:8. doi:10.1155/2014/902953. [45] Luna C, Serrano E, Domingo J, Casao A, Pérez-Pé R, Cebrián-Pérez JA, et al.

556

Expression, cellular localization, and involvement of the pentose phosphate

557

pathway enzymes in the regulation of ram sperm capacitation. Theriogenology

558

2016;86:704–14. doi:10.1016/j.theriogenology.2016.02.024.

559

[46] Losano J, Angrimani D, Dalmazzo A, Rui B, Brito M, Mendes C, et al. Effect of

560

mitochondrial uncoupling and glycolysis inhibition on ram sperm functionality.

561

Reprod Domest Anim 2017;52:289–97. doi:10.1111/rda.12901.

562

[47] Betarelli RP, Rocco M, Yeste M, Fernández-Novell JM, Placci A, Azevedo

563

Pereira B, et al. The achievement of boar sperm in vitro capacitation is related to

564

an increase of disrupted disulphide bonds and intracellular reactive oxygen

565

species levels. Andrology 2018;6:781–97. doi:10.1111/andr.12514.

566

[48] Stival C, Molina CP, Paudel B, Buffone MG, Visconti PE, Krapf D. Sperm

567

capacitation and acrosome reaction in mammalian sperm. Sperm Acrosome Biog.

568

Funct. Dur. Fertil., vol. 220, 2016, p. 93–106. doi:10.1007/978-3-319-30567-7.

569

[49] Asano A, Nelson-harrington JL, Travis AJ. Phospholipase B is activated in

23

570

response to sterol removal and stimulates acrosome exocytosis in murine sperm.

571

J Biol Chem 2013;288:28104–15. doi:10.1074/jbc.M113.450981.

572

[50] Visconti PE, Bailey JL, Moore GD, Pan D, Olds-clarke P, Kopf GS. Capacitation

573

of mouse spermatozoa I . Correlation between the capacitation state and protein

574

tyrosine phosphorylation. Development 1995;121:1129–37.

575

[51] Chatterjee M, Nandi P, Ghosh S, Sen PC. Regulation of tyrosine kinase activity

576

during capacitation in goat sperm. Mol Cell Biochem 2010;336:39–48.

577

doi:10.1007/s11010-009-0261-8.

578

[52] Pérez-Pé R, Grasa P, Fernández-Juan M, Peleato ML, Cebrián-Pérez JÁ, Muiño-

579

Blanco T. Seminal plasma proteins reduce protein tyrosine phosphorylation in the

580

plasma membrane of cold-shocked ram spermatozoa. Mol Reprod Dev

581

2002;61:226–33. doi:10.1002/mrd.1152.

582

[53] Naresh S, Atreja SK. The protein tyrosine phosphorylation during in vitro

583

capacitation and cryopreservation of mammalian spermatozoa. Cryobiology

584

2015;70:211–6. doi:10.1016/j.cryobiol.2015.03.008.

585

[54] Kadirvel G, Kathiravan P, Kumar S. Protein tyrosine phosphorylation and zona

586

binding ability of in vitro capacitated and cryopreserved buffalo spermatozoa.

587

Theriogenology 2011;75:1630–9. doi:10.1016/j.theriogenology.2011.01.003.

588

[55] Grasa P, Colas C, Gallego M, Monteagudo L, Muiño-Blanco T, Cebrián-Pérez

589

JÁ. Changes in content and localization of proteins phosphorylated at tyrosine,

590

serine and threonine residues during ram sperm capacitation and acrosome

591

reaction. Reproduction 2009;137:655–67. doi:10.1530/REP-08-0280.

592 593

[56] Urner F, Sakkas D. Protein phosphorylation in mammalian spermatozoa. Reproduction 2003;125:17–26.

594

24

595

25

596

Table 1. Influence of cryopreservation and capacitation on several motility parameters in ram sperm.

597

Data represent the least square means ± SEM for fresh or cryopreserved sperm and for sperm incubated in

598

CAP or NC conditions, considering all the incubation times.

Motility parameters

Treatments

TM (%)

VCL (µm s-1)

LIN (%)

ALH (µm)

48.08 ± 3.52 40.66 ± 2.48 Fresh Cryopreservation Cryopreserved 22.36 ± 2.66 15.01 ± 2.48 < 0.001 < 0.001 P values

60.72 ± 8.49 55.42 ± 7.14 0.137

63.88 ± 2.3 1.70 ± 0.18 60.25 ± 2.05 1.81 ± 0.23 0.240 0.662

48.30 ± 2.06 30.47 ± 1.90 25.14 ± 3.37 17.18 ± 3.11 < 0.001 < 0.001

92.48 ± 5.56 32.66 ± 5.56 < 0.001

54.46 ± 1.79 2.48 ± 0.14 71.67 ± 2.92 1.12 ± 0.22 < 0.001 < 0.001

Incubation media

CAP NC P values

599 600 601 602 603 604

PM (%)

P < 0.05 indicates significant differences among treatments. Data are expressed as least square means ± SEM. Total motility (TM; %) and progressive motility (PM; %) were evaluated in the whole sperm population. Curvilinear velocity (VCL; µm s-1), linearity (LIN; %) and amplitude of lateral head displacement (ALH; µm) were estimated only in motile sperm.

605 606 607 608 609 610 611 612 613 614 615 616 617 618

26

619 Table 2. Influence of cryopreservation and capacitation on several flow cytometry parameters in ram sperm. 620 Data represent the least square means ± SEM for fresh or cryopreserved sperm and for sperm incubated in 621 CAP or NC conditions, considering all the incubation times. Treatments Fresh Cryopreservation Cryopreserved P values

Sperm viability (%) 49.1 ± 2.85 20.05 ± 2.12 < 0.001

Sperm parameters Acrosome Mitochondrial integrity (%) activity (%) 35.42 ± 1.95 31.25 ± 1.68 18.97 ± 1.89 18.70 ± 1.68 < 0.001 < 0.001

ROS levels pY (%) (MFI) 82.48 ± 1.72 49.01 ± 4.01 103.80 ± 2.29 53.10 ± 4.01 0.143 < 0.001

CAP 43.01 ± 2.70 38.78 ± 1.49 38.62 ± 2.31 51.08 ± 1.34 63.01 ± 2.43 Incubation media NC 35.82 ± 2.70 35.61 ± 1.46 19.40 ± 2.13 115.21 ± 2.19 39.05 ± 3.97 P values 0.275 0.026 < 0.001 < 0.001 < 0.001 622 623 P < 0.05 indicates significant differences among treatments. Data are expressed as least square means ± 624 SEM. Sperm viability (%) represents the proportion of live sperm from the whole population, acrosome 625 integrity (%) illustrates the percentage of viable sperm with intact acrosome, mitochondrial activity (%) 626 indicates the proportion of viable sperm with active mitochondria. ROS levels (Mean Fluorescence 627 Intensity (MFI)) and the percentage of sperm showing tyrosine phosphorylation (pY) were estimated only in viable cells. 628 629

27

630

Figure legends

631

Figure 1. Kinematic changes in fresh and cryopreserved ram sperm during the

632

incubation period under CAP and NC conditions. (A) Sperm motility (%) illustrates the

633

proportion of motile sperm from the whole sperm population. The kinematic parameters

634

of (B) linearity (LIN; %), (C) curvilinear velocity (VCL; µm s-1) and (D) amplitude of

635

lateral head displacement (ALH; µm) were evaluated in motile sperm. Values are

636

expressed as least square means ± SEM. Different letters denote significant differences

637

(P < 0.05) among incubation times. Uppercase letters were used for cryopreserved

638

sperm and lowercase letters for fresh sperm.

639 640

Figure 2. Functional changes in fresh and cryopreserved ram sperm during the

641

incubation period under CAP and NC conditions. (A) Sperm viability (%) shows the

642

percentage of live cells from the whole sperm population, (B) mitochondrial activity

643

(%) illustrates the proportion of viable sperm with active mitochondria, (C) acrosome

644

integrity (%) indicates the percentage of viable sperm with intact acrosome and (D)

645

ROS levels (Mean Fluorescence Intensity (MFI)) were measured only in viable sperm.

646

Values are expressed as least square means ± SEM. Different letters indicate significant

647

differences (P < 0.05) among incubation times. Uppercase letters were used for

648

cryopreserved sperm and lowercase letters for fresh sperm.

649 650

Figure 3. Dynamics of global tyrosine phosphorylation (pY) during incubation of fresh

651

and cryopreserved ram sperm under CAP and NC conditions. The pY was evaluated in

652

live sperm. Values are expressed as least square means ± SEM. Different letters denote

653

significant differences (P < 0.05) among incubation times. Uppercase letters were used

654

for cryopreserved sperm and lowercase letters for fresh sperm.

28

655

Figure 4. Brightfield and fluorescence images of ram sperm presenting protein tyrosine

656

phosphorylation (pY) in different subcellular regions. The brightfield images (top

657

panels) highlight the masks created with the IDEAS® software to detect the different

658

phosphorylated sperm regions ((A) Head, (B) midpiece, (C) principal piece and (D) tail)

659

while the fluorescent images (down panels) illustrate the sperm region of the mask

660

where pY staining is observed (note that pY staining could appeared in other regions

661

outside the mask, but the fluorescence intensity was only measured in the region of

662

interest selected with the mask). The tail mask (D) analysed the whole tail region,

663

including pY staining of the midpiece and principal piece.

664 665

Figure 5. Dynamics of tyrosine phosphorylation (pY) in different subcellular sperm

666

regions during incubation of fresh and cryopreserved ram sperm under CAP and NC

667

conditions. (A) Head, (B) tail, (C) midpiece and (D) principal piece. For each time, the

668

mean fluorescence of each sperm region was normalized to the mean fluorescence of

669

the same region at 0 min in NC conditions. The pY of different sperm regions was only

670

measured in phosphorylated sperm. Data with different letters indicate significant

671

differences (P < 0.05) among incubation times. Uppercase letters were used for

672

cryopreserved sperm and lowercase letters for fresh sperm.

673

29

Figure 1. A Fresh sperm

Sperm motility (%)

70 60

ab

ab

ab

a

a b

50

b

Cryopreserved sperm a

ab

b c

40 30

A AB

20

AB

AB

AB

d

BD CD

10

E

E CE

CE

E

0 0

1

5

15

NC

30

60

120

180

240

0

CAP

15

240

NC

Incubation time (min)

B

LIN (%)

Fresh sperm 100 90 80 70 60 50 40 30 20 10 0

Cryopreserved sperm

a

a b c

A

c

bc

A d

B

BC

bc

c

BC E

BC

0 NC

1

B D

D

5

15

d

DC

30

60

120

CAP

Incubation time (min)

B e

e

180

240

0

15 NC

240

C

VCL (µm s-1)

Fresh sperm 180 160 140 120 100 80 60 40 20 0

D C

Cryopreserved sperm e

e

A

A

d

C

cd bc

b

B a

A

A

B

a

a

a a 0

1

5

15

30

NC

60

120

180

240

a

A

A

A

0

15

240

CAP

NC

Incubation time (min)

D

ALH (µm)

Fresh sperm 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

c

C

c

BC B

A a 0

NC

A

b a

a

A

a

A

a

1

Cryopreserved sperm

A

A

a

5

15

30

60

a

a A

120 180 240

CAP

Incubation time (min)

a

0

A

A 15

NC

240

Figure 2. A Fresh sperm

Cryopreserved sperm

Sperm viability (%)

80 70 60

a

ab

ab

a ac

50

ac

bc

c

ac

c

c

40 30

A

A

20

A

A

A A

d

AB

10

B

C

B

C

C

0 0

1

5

15

30

NC

60

120 180 240

0

CAP

15

240

NC

Incubation time (min)

B

Mitochondrial activity (%)

Fresh sperm

Cryopreserved sperm

60 b

50 40

a

ab

ab

ab

b a

C c

C

30 20

AB

AB

c

c

AC c

B

10

DE D

0 0 NC

1

5

15

30

60

D

D

120 180 240

CAP

Incubation time (min)

d

AB

0

15 NC

E 240

C

Acrosome integrity (%)

Fresh sperm

Cryopreserved sperm

60 50

a

a

a

ab

ab

ac

bc

40 30

ab

bc

cd A AB

20

d

A AB

e

AB BC

10

E

E

CD

CD

E

DE

0 0

1

5

15

NC

30

60

120 180 240

0

15

CAP

240

NC

Incubation time (min)

D Fresh sperm

ROS levels (MFI)

120

Cryopreserved sperm E

A

A

100 C 80 60

B

B

B

B

a

40 20

b

b

b

1

5

15

b

AD

CD

B

c

B b

a b

b

ac

b

0 0 NC

30

60

120 180 240

CAP

Incubation time (min)

0

15 NC

240

Sperm with tyrosine phosphorylation (%)

Figure 3.

Fresh sperm 100 90 80 70 60 50 40 30 20 10 0

B A

A

b

AB

f AB

e A

b

Cryopreserved sperm

C

bc b

A

d

c

a

C D

D

a

a

a E

0 NC

1

5

15

30

60

120 180 240

CAP

Incubation time (min)

0

15 NC

240

D) Tail

C) Principal piece B) Midpiece A) Head Figure 4.

Figure 5. A Fresh sperm

Normalized pY fluorescence

Head

Cryopreserved sperm

2.5 2 1.5

A

A

A

ab

A

ab

b

b

B

B

AB ab

1

ab

A

a

a

a

AB

0.5

AB

AB

a

a

0

15

a

D

0 0

1

5

15

30

NC

60

120 180 240

CAP

240

NC

Incubation time (min)

B Fresh sperm

Normalized pY fluorescence

Tail

Cryopreserved sperm

6 5

C

C C

4

c B

3 2 1

d d

A

A

a

a

0

1

ab

a

5

15

ab

bc

A

A A

A

a A

a

a A

0 NC

30

60

120 180 240

CAP

Incubation time (min)

0

15 NC

240

C Fresh sperm

Normalized pY fluorescence

Midpiece

Cryopreserved sperm

7 6 C

5

C d

4 B

3 2 1 0

B

d

c A

A

b

a

a

a

ab

0

1

5

15

NC

30

b 60

A

A

A

120 180 240

a

A

a

aA

A 0

CAP

15

240

NC

Incubation time (min)

D Fresh sperm

Normalized pY fluorescence

Principal Piece

Cryopreserved sperm

7 6

D d

5 3 2 1 0

C

C

4

b

c

A

A

B AB

AB

a

a

ab

ab

ab

0

1

5

15

30

NC

b

a 60

a

AB A

120 180 240

CAP

Incubation time (min)

0

A 15 NC

a A 240

Highlights: Sperm cryopreservation changes the dynamic events occur during in vitro capacitation Cryopreserved sperm is capacitated before than the fresh sperm Tyrosine phosphorylation increased in midpiece followed by piece principal and tail in both samples