Inhibitors of serine proteases decrease sperm penetration during porcine fertilization in vitro by inhibiting sperm binding to the zona pellucida and acrosome reaction

Accepted Manuscript Inhibitors of serine proteases decrease sperm penetration during porcine fertilization in vitro by inhibiting sperm binding to the zona pellucida and acrosome reaction J. Beek, H. Nauwynck, R. Appeltant, D. Maes, A. Van Soom PII:

S0093-691X(15)00380-5

DOI:

10.1016/j.theriogenology.2015.07.022

Reference:

THE 13269

To appear in:

Theriogenology

Received Date: 28 December 2014 Revised Date:

11 May 2015

Accepted Date: 17 July 2015

Please cite this article as: Beek J, Nauwynck H, Appeltant R, Maes D, Van Soom A, Inhibitors of serine proteases decrease sperm penetration during porcine fertilization in vitro by inhibiting sperm binding to the zona pellucida and acrosome reaction, Theriogenology (2015), doi: 10.1016/ j.theriogenology.2015.07.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

Revised non-highlighted

ACCEPTED MANUSCRIPT 1

Inhibitors of serine proteases decrease sperm penetration during porcine fertilization in

2

vitro by inhibiting sperm binding to the zona pellucida and acrosome reaction

3

5 6

a

7

Ghent University, B-9820 Merelbeke, Belgium.

8

b

9

Ghent University, B-9820 Merelbeke, Belgium.

RI PT

J. Beeka, H. Nauwynckb, R. Appeltanta, D. Maesa, A. Van Sooma

Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine,

Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine,

M AN U

10

SC

4

11

Corresponding author: Josine Beek, Department of Reproduction, Obstetrics, and Herd

12

Health, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium. Fax:

13

0032 9264 7779; Tel: 0032 92647550; e-mail: [email protected]

EP

16

Involvement of serine proteases in porcine IVF

AC C

15

TE D

14

[Type here]

ACCEPTED MANUSCRIPT

Abstract

18

Serine proteases are involved in mammalian fertilization. Inhibitors of serine proteases can be

19

applied to investigate at which point these enzymes exert their action. We selected two serine

20

protease inhibitors, 4-(2-Aminoethyl)benzene sulfonyl fluoride hydrochloride (AEBSF, 100

21

µM) and Soybean Trypsin Inhibitor from Glycine Max (STI, 5 µM) via previous dose-

22

response IVF experiments and sperm toxicity tests. In the present study, we evaluated how

23

these inhibitors affect porcine fertilization in vitro as calculated on total fertilization rate,

24

polyspermy rate and the sperm number per fertilized oocyte of cumulus-intact, cumulus-free

25

and zona-free oocytes. In the control group (no inhibitor), these parameters were 86%, 49%

26

and 2.2 for cumulus-intact oocytes and 77%, 43% and 2.2 for cumulus-free oocytes (6 h

27

gamete incubation period, 1.25x105 spermatozoa/mL). AEBSF and STI significantly reduced

28

total fertilization and polyspermy rate in cumulus-intact and cumulus-free oocytes (P<0.05).

29

Total fertilization rates were respectively 65% and 53% (AEBSF) and 36% and 17% (STI).

30

Inhibition rates were higher in cumulus-free than cumulus-intact oocytes, indicating that

31

inhibitors exerted their action after sperm passage through the cumulus. AEBSF but not STI

32

reduced sperm binding to the zona pellucida. The acrosome reaction was significantly

33

inhibited by both inhibitors. Only 40.4% (AEBSF) and 11.4% (STI) of spermatozoa

34

completed a calcium-induced acrosome reaction compared to 86.7% of spermatozoa in the

35

control group. There was no effect on sperm binding or fertilization parameters in zona-free

36

oocytes. In conclusion, sperm-zona binding and acrosome reaction were inhibited by serine

37

protease inhibitors during porcine IVF.

AC C

EP

TE D

M AN U

SC

RI PT

17

38 39

Key words: serine protease; inhibitor; porcine; IVF.

40

2

[Type here] 41

ACCEPTED MANUSCRIPT

1. Introduction Mammalian fertilization requires the successful completion of a number of steps in a

43

compulsory order [1, 2]. Previous studies have demonstrated that the important players

44

involved in fertilization, i.e. the sperm cell, the oocyte and its surrounding cumulus cells, all

45

contain and/or secrete several proteases [3-9]. The family of serine proteases, named after the

46

serine residue in the active site of the enzyme, is the largest family of proteases and based on

47

the current knowledge also the most represented on gametes [5].

48

The serine protease acrosin and its precursor proacrosin are found specifically within the

49

acrosome of mammalian spermatozoa [10]. Basic residues on the surface of (pro)acrosin can

50

interact with polysulphate groups on glycoproteins of the zona pellucida (ZP) [11-13]. The

51

binding of proacrosin to the ZP potentiates the conversion of proacrosin to acrosin [14, 15].

52

Acrosin is associated with several steps of fertilization, including sperm binding to the ZP,

53

dispersal of acrosomal content, subsequent zona lysis and activation of oocyte transmembrane

54

receptors [10, 16, 17]. Next to acrosin, numerous other sperm serine proteases have been

55

described, not only in the acrosomal content but also on acrosomal and sperm membranes [5].

56

Serine protease activity derived from the cortical granules has been shown to contribute to the

57

oocyte’s defense mechanism against polyspermy in the hamster and in the mouse [18, 19].

58

Similarly, a role has been proposed for serine protease activity in the regulation of sperm

59

penetration in bovine oocytes, mediated by the release of tissue-type plasminogen activator

60

(tPA) from the oocyte and activation of plasminogen into the serine protease plasmin [20].

61

Recent studies have described how the plasminogen-plasmin system may contribute to the

62

regulation of sperm penetration during porcine fertilization [9, 21]. As proposed by Coy et

63

al.[9], the porcine oocyte releases tPA and urokinase-type plasminogen activator (uPA) upon

64

contact with spermatozoa. They also suggested that these plasminogen activators convert

65

plasminogen into plasmin, which protease activity will lead to detachment of spermatozoa

AC C

EP

TE D

M AN U

SC

RI PT

42

3

[Type here]

ACCEPTED MANUSCRIPT

previously attached to the ZP. This model is supported by the detection of plasminogen in

67

porcine oviductal fluid [21]. The precursor of plasmin is thus present at the site of gamete

68

interaction in vivo. In addition, a decrease in sperm-ZP binding and sperm penetration was

69

observed when plasminogen was supplemented to the in vitro fertilization (IVF) medium in

70

concentrations similar to those detected in oviductal fluid [21].

71

Next to that, in several mammalian species including the pig, oocytes are still surrounded by

72

the cumulus oophorus at the time of fertilization in vivo [22, 23]. The presence of cumulus

73

cells during porcine IVF has beneficial effects on fertilization outcome [23-25]. In the mouse,

74

cumulus cells have been shown to synthesize and secrete several proteases, including tPA and

75

uPA [26]. The secretion of plasminogen activators by cumulus cells rapidly increases when

76

cumulus expansion is completed and matrix disassembly begins. Activation of the

77

plasminogen-plasmin system is thus proposed to play a regulatory role in cumulus matrix

78

disaggregation.

79

Serine proteases might be thus involved in several aspects of gamete interaction and in

80

mechanisms that work either conceptive or contraceptive. These features make them

81

interesting candidates for further research on how serine protease inhibition may affect

82

porcine fertilization in vitro. Up to now, the high degree of polyspermic fertilization during

83

porcine IVF hampers large scale in vitro production of porcine embryos. Specific protease

84

inhibitors could be useful as regulators of sperm penetration during porcine IVF, lessening the

85

problem of polyspermy. The present study investigated which fertilization step(s) during

86

porcine IVF are affected by serine protease inhibitors 4-(2-Aminoethyl) benzene sulfonyl

87

fluoride hydrochloride (AEBSF) and Soybean Trypsin Inhibitor from Glycine Max (STI).

88

These two inhibitors were previously tested in our laboratory and were shown not to

89

compromise membrane integrity and motility of porcine spermatozoa [27].

AC C

EP

TE D

M AN U

SC

RI PT

66

90 4

[Type here]

91

ACCEPTED MANUSCRIPT

2. Materials and methods Five experiments were performed to study the effects of serine protease inhibitors during the

93

sequential steps of porcine in vitro fertilization.

94

2.1 Media

95

All chemicals used in this study were purchased from Sigma-Aldrich (Bornem, Belgium),

96

unless otherwise stated.

RI PT

92

SC

97

2.2 Protease inhibitors

99

Protease inhibitors were purchased from Sigma-Aldrich (Bornem, Belgium). Inhibitor STI

100

(T6522) was stored desiccated and dissolved in fertilization medium prior to use. Inhibitor

101

AEBSF (A8456) was dissolved in deionized water to a stock solution of 10 mM. The

102

supplementation of AEBSF stock solution with deionized water to the fertilization medium

103

was corrected by the addition of equal volume of medium with twofold concentration of

104

medium components. Inhibitor AEBSF was added to the fertilization medium resulting in a

105

final concentration of 100 µM. The final concentration of STI in the fertilization medium was

106

5 µM. These concentrations were chosen based on previous dose-response IVF experiments

107

and were shown to have no adverse effects on sperm membrane integrity and sperm motility

108

[27].

TE D

EP

AC C

109

M AN U

98

110

2.3 Oocyte collection

111

Ovaries of prepubertal gilts (Piétrain [boar line] × commercial hybrid [sow line]) were

112

collected at a local slaughterhouse. Immature cumulus-oocyte-complexes (COCs) were

113

aspirated from follicles with a diameter ranging from 3 to 6 mm. Then COCs were selected

114

based on a homogeneous ooplasm and a multilayered cumulus. The basic medium used for

5

[Type here]

ACCEPTED MANUSCRIPT

115

the collection and washing of cumulus-oocyte- complexes (COCs) was a modified HEPES-

116

buffered Tyrode balanced salt solution (HEPES-TM) with 10 µg/mL gentamycin sulfate, 10

117

mM HEPES and 3 mg/mL BSA.

118

2.4 In vitro maturation and fertilization of porcine oocytes

120

Immature oocytes were matured in BSA-free ‘North Carolina State University’ 23 (NCSU23)

121

[28] supplemented with 0.57 mM cysteine, 10 ng/mL EGF (E9644), 10 IU/mL eCG

122

(Folligon®, Intervet, The Netherlands), 10 IU/mL hCG (Chorulon®, Intervet, The

123

Netherlands) and 10% (v/v) porcine follicular fluid. Follicular fluid was collected from 5-6

124

mm follicles of prepubertal gilts. After centrifugation of follicular fluid (100 X g, 10 min), the

125

supernatant was filtered (0.22 µm) and stored at -80ºC until use. Groups of 100 immature

126

COCs were placed into 500 µl maturation medium and cultured for the first 22 h (39°C, 5%

127

CO2). Subsequently, COCs were cultured in hormone-free maturation medium for 22 h.

128

The basic medium for IVF was Tyrode’s albumin lactate pyruvate medium (TALP medium)

129

[29] supplemented with 0.3% (w/v) BSA (FERT-TALP). Protease inhibitor dilutions were

130

prepared in concentrated stocks and diluted with FERT-TALP to obtain the final

131

concentration required in fertilization droplets of 100 µL covered with mineral oil.

132

Approximately 10 min after the addition of protease inhibitors, the matured COCs were

133

randomly assigned to fertilization droplets of 90 µL FERT-TALP (45-50 COCs/droplet unless

134

stated otherwise). Cryopreserved epididymal spermatozoa were used from one Landrace boar

135

with known fertility in IVF and cryopreserved according to the protocol of Rath and Niemann

136

[30]. Frozen epididymal semen was thawed for 60 sec in a water bath at 38°C and

137

spermatozoa were washed by centrifugation (3 min at 390 X g) in 9.5 mL of Androhep

138

extender consisting of 27 mM Tri-natriumcitrate, 2.7 mM Titriplex III EDTA, 2.5 mg/mL

139

BSA (fraction V), 14 mM NaHCO3, 38 mM Hepes, 144 mM D(+)-glucose.H2O and 50

AC C

EP

TE D

M AN U

SC

RI PT

119

6

[Type here]

ACCEPTED MANUSCRIPT

µg/mL gentamycine sulfate. The sperm pellet was resuspended in 1 mL FERT-TALP and

141

sperm concentration was assessed using a Bürker counting chamber. Spermatozoa (10 µL)

142

were added to the 90 µL droplets containing the COCs resulting in a final concentration of

143

0.25x105 (zona-free oocytes) or 1.25x105 spermatozoa/mL (cumulus-intact and cumulus-free

144

oocytes), as indicated in the experimental design. The control COCs were fertilized in

145

fertilization medium without addition of a protease inhibitor. After a co-incubation period of 6

146

h (39°C, 5% CO2), the presumed zygotes were vortexed during 3 min in 2.5 mL HEPES-

147

buffered TALP medium (HEPES-TALP), i.e. TALP medium with 25 mM HEPES, to remove

148

loosely bound spermatozoa. Subsequently, presumed zygotes were washed three times in

149

embryo culture medium, NCSU23 with 0.4% (w/v) BSA, and cultured for 18 h in droplets of

150

50 µL embryo culture medium covered by mineral oil (modular incubator chamber, 39°C, 5%

151

CO2).

M AN U

SC

RI PT

140

152

2.5 Assessment of fertilization parameters

154

Approximately 24 h after insemination, presumed zygotes were washed in 0.1% (w/v) poly-

155

vinyl pyrrolidone (PVP) in phosphate buffered saline (PBS). Subsequently, the zygotes were

156

fixed overnight with 4% paraformaldehyde in PBS and stained with 10 µg/mL bis-benzimide

157

(Hoechst 33342; Molecular Probes, Leiden, The Netherlands) for 10 min. Zygotes were

158

mounted in a droplet of glycerol with (25 mg/mL) 1,4-diazabicyclo (2.2.2) octane (DABCO;

159

Acros, Ghent, Belgium) and nuclear DNA was visualized using a Leica DMR fluorescence

160

microscope (Leica Microsystems, Belgium). Oocytes with a metaphase plate and a polar body

161

were classified as being in the MII stage. The presence of two pronuclei or cleaved embryos

162

with two equally sized blastomeres was indicative for normal fertilization, whereas zygotes

163

with more than two pronuclei or more than one decondensed sperm head were classified as

164

polyspermic. In case of polyspermic fertilization, the number of penetrated spermatozoa was

AC C

EP

TE D

153

7

[Type here]

ACCEPTED MANUSCRIPT

counted. The following fertilization parameters were assessed: total fertilization (%),

166

polyspermy (%) and the sperm number per fertilized oocyte.

167

2.6 Assessment of sperm acrosome integrity

168

Acrosome integrity was evaluated by fluorescence microscopy using fluorescein

169

isothyiocyanate-conjugated Pisum sativum agglutinin (PSA-FITC; L0770, Sigma-Aldrich,

170

Bornem, Belgium) in combination with Hoechst 33342 (Molecular Probes, Leiden, The

171

Netherlands). First, 1 µL of Hoechst was added to the sperm aliquot and incubated for 3 min

172

(37°C, incubator IN, Memmert GmbH + Co.KG, Germany). After centrifugation at 720 X g

173

for 10 min, the supernatant was removed and the sperm pellet was resuspended in 50 µL of

174

96% ethyl alcohol and incubated for 30 min at 4°C. Then, 15 µL of each sperm aliquot was

175

smeared on a glass slide and air-dried. Afterwards, 15 µL of PSA-FITC (2 mg PSA-FITC

176

diluted in 2 mL PBS) was added. The glass slides were kept for 15 min at 4°C, washed 5

177

times with deionized water and air-dried. Then, each sperm suspension was evaluated using a

178

Leica DMR fluorescence microscope. All spermatozoa were stained with Hoechst 33342. The

179

acrosomal region of acrosome-intact spermatozoa was PSA-FITC positive and labeled green,

180

while acrosome-reacted spermatozoa retained only an equatorial labeled band with little or no

181

labeling of the anterior head region (supplemental figure).

EP

TE D

M AN U

SC

RI PT

165

AC C

182 183

2.7 Experimental design

184

2.7.1 Experiment 1: effect of serine protease inhibitors on sperm penetration through the

185

cumulus oophorus

186

In vitro matured COCs were randomly assigned to 6 groups of approximately 50 COCs (3

187

replicates). Three groups of COCs were denuded by vortexing with 0.1% (w/v) hyaluronidase

188

in HEPES-TM (cumulus-free or CF), the other groups were kept cumulus-intact (CI). Both

189

CF and CI oocytes were fertilized in standard fertilization medium and in fertilization medium 8

[Type here]

ACCEPTED MANUSCRIPT

supplemented with either AEBSF or STI. The CF and CI oocytes were co-incubated with

191

1.25x105 spermatozoa/mL for 6 h. Total fertilization, polyspermy rate and the sperm number

192

per fertilized oocyte were compared between CI and CF oocytes to evaluate the effect of

193

inhibitors on sperm penetration through the cumulus oophorus.

194

2.7.2 Experiment 2: effect of serine protease inhibitors on sperm-zona binding

195

In vitro matured COCs were denuded by vortexing with 0.1% (w/v) hyaluronidase in HEPES-

196

TM and randomly assigned to 2 groups of 20 oocytes (3 replicates for each inhibitor). The

197

first group was co-incubated with spermatozoa in standard fertilization medium (control), the

198

second group in fertilization medium supplemented with protease inhibitor, either AEBSF or

199

STI. For unambiguous counting of the number of spermatozoa bound to the ZP, oocytes were

200

co-incubated with a lower sperm concentration than in the fertilization experiments, more

201

specifically 0.25x105 spermatozoa/mL (39°C, 5% CO2). After 4 h of co-incubation, oocytes

202

were washed five times to remove loosely bound spermatozoa, and subsequently fixed and

203

stained with Hoechst 33342. Of each oocyte, the average of two counts was taken into

204

account as the number of sperm bound to the ZP.

205

2.7.3 Experiment 3: effect of serine protease inhibitors on sperm-oolemma binding and

206

fertilization of zona-free oocytes

207

In vitro matured COCs were denuded by vortexing with 0.1% (w/v) hyaluronidase in HEPES-

208

TM during 3 min. Subsequently, the CF oocytes were washed in PBS and shortly incubated in

209

a 0.1% (w/v) pronase solution in PBS [31] to dissolve their ZP. Oocytes were continuously

210

observed under a stereomicroscope for dissolution of ZP. After a recovery period of 30 min in

211

FERT-TALP, the ZP-free oocytes (45 to 50 oocytes per group) were randomly assigned to 3

212

different media: standard fertilization medium and fertilization medium supplemented with

213

either AEBSF or STI. The ZP-free oocytes were co-incubated with spermatozoa at a final

214

concentration of 0.25x105 spermatozoa/mL for unambiguous counting of the number of

AC C

EP

TE D

M AN U

SC

RI PT

190

9

[Type here]

ACCEPTED MANUSCRIPT

spermatozoa bound to the oolemma. In fertilization experiments with ZP-free oocytes,

216

spermatozoa are not stimulated to undergo capacitation and the acrosome reaction by contact

217

with the ZP. Boar spermatozoa are relatively sensitive to other stimuli, including medium

218

components such as calcium, bicarbonate, caffeine and BSA [32]. In order to allow

219

capacitation, the sperm dilution was incubated in FERT-TALP (39°C, 5% CO2) for 30 min

220

prior to insemination. One hour after insemination, 10 oocytes from each group were washed

221

five times to remove loosely bound spermatozoa, fixed and stained with Hoechst 33342. Per

222

presumed zygote, the number of spermatozoa bound to the oolemma was counted. All other

223

oocytes were co-incubated with spermatozoa for 6 h and fertilization parameters were

224

assessed.

225

2.7.4 Experiment 4: effect of pre-incubation of gametes with serine protease inhibitors

226

For the pre-incubation experiment, in vitro matured COCs were divided into four groups of

227

approximately 50 COCs. For pre-incubation of female gametes, COCs of group 1 and group 2

228

were incubated in standard fertilization medium and in fertilization medium with protease

229

inhibitor (either AEBSF or STI), respectively, for 30 min prior to fertilization. Subsequently,

230

COCs were washed, transferred to standard fertilization medium and co-incubated with

231

1.25x105 spermatozoa/mL (39°C, 5% CO2) during 6 h. The two other groups of COCs, group

232

3 and group 4, were used to evaluate pre-incubation of male gametes. Both groups were co-

233

incubated with 1.25x105 spermatozoa/mL in standard fertilization medium, but with

234

differently prepared spermatozoa. Group 3 was inseminated with spermatozoa previously

235

incubated in standard fertilization medium for 30 min, whereas group 4 was inseminated with

236

spermatozoa previously incubated for 30 min in fertilization medium supplemented with

237

protease inhibitor.

238

2.7.5 Experiment 5: effect of serine protease inhibitors on the acrosome reaction

AC C

EP

TE D

M AN U

SC

RI PT

215

10

[Type here]

ACCEPTED MANUSCRIPT

Spermatozoa were washed by centrifugation (3 min at 390 X g) and the sperm pellet was

240

resuspended in HEPES-TM to obtain a sperm concentration of 2x106 sp/mL and divided into

241

aliquots of 1 mL. Aliquot 1 without inhibitor was used as negative control. A second aliquot

242

was supplemented with 1 µM A23187 calcium ionophore as full response control. Sperm

243

aliquots 3 and 4 were treated with 100 µM AEBSF, aliquots 5 and 6 were treated with 5 µM

244

STI. Sperm aliquots 4 and 6 were then supplemented with 1 µM A23187 calcium ionophore

245

to evaluate the response in the presence of a protease inhibitor. All incubations were carried

246

out for 1 h at 37°C (incubator IN, Memmert GmbH + Co.KG, Germany). Subsequently,

247

acrosome integrity was evaluated using PSA-FITC staining as described in 2.6. For each

248

replicate, the average of two counts of 100 spermatozoa was calculated per sperm aliquot.

249

Three replicates were performed.

250

2.8 Statistical analysis

251

Differences in total fertilization and polyspermy rate between control and treatment group or

252

between cumulus-intact and cumulus-free oocytes within a group were analyzed by means of

253

binary logistic regression. ANOVA was used to evaluate the sperm number per fertilized

254

oocyte, the differences in mean number of spermatozoa bound to the ZP and the oolemma,

255

and the percentage of acrosome intact spermatozoa. In all experiments, the control group was

256

compared with each treatment group, no direct comparisons between treatment groups were

257

analyzed. Hypothesis testing was performed using a significance level of 5% (SPSS 21.0).

259

SC

M AN U

TE D

EP

AC C

258

RI PT

239

3. Results

260

3.1 Effect of serine protease inhibitors AEBSF and STI on sperm penetration through the

261

cumulus oophorus during porcine IVF

11

[Type here]

ACCEPTED MANUSCRIPT

In comparison with the control group, total fertilization and polyspermy rate decreased

263

significantly in both CI and CF oocytes when either inhibitor AEBSF or STI was added to the

264

fertilization medium (Fig. 1). Total fertilization rate was reduced more in CF than in CI

265

oocytes by both inhibitors. Polyspermy rate decreased in medium with AEBSF independent of

266

the presence of the cumulus cells, inhibition rates compared to the control group were 67%

267

and 70% for CI and CF oocytes, respectively. The presence of STI in the fertilization medium

268

resulted in a significant reduction of polyspermic fertilization in CI oocytes compared to the

269

control group (-93%). Its inhibitory effect on polyspermy was higher in CF oocytes (-99%

270

compared to the polyspermy rate in the control group) (P<0.05). The sperm number per

271

fertilized oocyte was reduced by AEBSF as well as STI in both CI and CF oocytes, with a

272

similar degree of reduction independent of the presence of cumulus cells (Fig. 2).

273

3.2 Effect of serine protease inhibitors AEBSF and STI on sperm binding to the ZP in vitro

274

The number of spermatozoa bound to the ZP decreased significantly when AEBSF was added

275

to the fertilization medium. After 4 h of gamete co-incubation, the average number of

276

spermatozoa bound to the ZP was 92 ± 8.7 (Mean ± SD) in the control group and 75 ± 6.5 in

277

the presence of AEBSF. The supplementation of STI to the fertilization medium did not

278

decrease the number of sperm bound to the ZP when compared to the respective control group

279

(Fig. 3).

280

3.3 Effect of serine protease inhibitors AEBSF and STI on sperm-oolemma binding and

281

fertilization of zona-free oocytes

282

Supplementation of either AEBSF or STI did not significantly influence sperm-oolemma

283

binding. However, small numerical differences were observed between groups. Compared to

284

the control group (8.6 ± 1.0; mean ± SD), the number of sperm bound per oocyte slightly

285

increased to 8.9 ± 1.0 (STI) and 11 ± 1.2 (AEBSF). Serine protease inhibitors AEBSF and

AC C

EP

TE D

M AN U

SC

RI PT

262

12

[Type here]

ACCEPTED MANUSCRIPT

STI did not inhibit total fertilization, polyspermy rate nor the sperm number per fertilized

287

oocyte in zona-free oocytes.

288

3.4 Effect of pre-incubation of gametes with serine protease inhibitors AEBSF and STI on

289

fertilization parameters after porcine IVF

290

The results of the pre-incubation experiments are presented in Fig. 4. Pre-incubation of COCs

291

with AEBSF for 30 min prior to fertilization significantly decreased total fertilization rate

292

compared to the control group: 83.0% (AEBSF) versus 93.5% (control). Polyspermy rate was

293

also significantly lower when COCs were pre-incubated in fertilization medium with AEBSF.

294

Pre-incubation of spermatozoa with AEBSF did not affect fertilization parameters. When

295

COCs were pre-incubated with STI, only 46% of the COCs became fertilized during

296

subsequent IVF, compared to 75% of the COCs in the respective control group. Similarly,

297

polyspermy rate and the number of penetrated sperm per fertilized oocyte were lower in

298

COCs pre-incubated in STI compared to COCs pre-incubated in medium without protease

299

inhibitor (P<0.05). Pre-incubation of spermatozoa with STI resulted in a significant reduction

300

of total fertilization rate and polyspermy rate (Fig. 4) as well as sperm number per fertilized

301

oocyte (Fig. 2).

302

3.5 Effect of serine protease inhibitors on the calcium-induced acrosome reaction

303

The acrosomal status of spermatozoa in medium without calcium-ionophore was not

304

influenced by incubation with AEBSF or STI when compared to the control group (Fig. 5).

305

Treatment with calcium ionophore induced the acrosome reaction in the majority of

306

spermatozoa in the full response control, with only 13.3 ± 5.7% (mean ± SD) acrosome intact

307

spermatozoa after 1 h of incubation. Inhibitors AEBSF and STI significantly inhibited the

308

calcium-induced acrosome reaction (P<0.05). In the presence of AEBSF, 59.6 ± 8.5% of the

309

spermatozoa did not complete the acrosome reaction in response to the inducing agent. In

310

STI, 88.6 ± 5.6% of the spermatozoa remained acrosome intact.

AC C

EP

TE D

M AN U

SC

RI PT

286

13

[Type here]

ACCEPTED MANUSCRIPT

311 312

4. Discussion Serine proteases are generally classified according to their proteolytic activity. Three main

314

families can be distinguished: trypsin-like, chymotrypsin-like and elastase-like serine

315

proteases [33]. The present study investigated the effect of two serine protease inhibitors,

316

AEBSF and STI, during the sequential steps of porcine IVF. Inhibitor AEBSF is a broad

317

spectrum inhibitor and inhibits serine proteases of all three families irreversibly. The trypsin

318

inhibitor from Glycine max (Soybean) Type I-S (STI) inhibits proteases with mechanisms

319

similar to trypsin, so-called trypsin-like serine proteases in a reversible manner [34].

320

The functional significance of proteases in sperm passage through the cumulus matrix is

321

largely unknown, whereas sperm hyaluronidase and sperm motility are generally accepted to

322

be imperative [1, 2]. Metalloprotease activity was shown to be involved in sperm passage

323

through the cumulus matrix during porcine IVF [35]. In the present study, sperm passage

324

through the cumulus was not hindered by serine protease inhibitors AEBSF and STI as the

325

inhibitory effect was less pronounced in CI oocytes than in CF oocytes. Nevertheless,

326

fertilization rate was decreased in both CI and CF oocytes. Sperm-oolemma binding and IVF

327

of zona-free oocytes were unaltered in the presence of AEBSF or STI, which indicates that

328

serine protease inhibitors have their effect prior to sperm-oolemma interaction. As sperm

329

passage through the cumulus was not hindered by AEBSF or STI, our results strongly indicate

330

that inhibitors influenced fertilization parameters via an effect on sperm-ZP binding and/or

331

zona penetration. Previous studies have described similar effects of trypsin-like serine

332

protease inhibitors during fertilization in rabbit [36], hamster [37] and mouse [38]. Moreover,

333

these early reports of decreased zona penetration in the presence of serine protease inhibitors,

334

the lytic effect of acrosomal extracts on the ZP and the necessity of the acrosome reaction for

AC C

EP

TE D

M AN U

SC

RI PT

313

14

[Type here]

ACCEPTED MANUSCRIPT

successful fertilization, are the foundation of the paradigm in which zona lysis by acrosomal

336

proteases enables sperm to penetrate the ZP [2].

337

The present study demonstrated an inhibitory effect on sperm-ZP binding by AEBSF. Based

338

on previous research, hampered sperm motility can be excluded with a high degree of

339

certainty as reason for the decrease in sperm-ZP binding [27]. More specifically, total and

340

progressive motility were shown not to be different when spermatozoa were incubated in

341

fertilization medium supplemented with 100 µM AEBSF or standard fertilization medium.

342

The effect of AEBSF in the sperm-ZP binding experiment is in line with previous studies

343

reporting interference of sperm-ZP binding by other serine protease inhibitors in the pig [11,

344

12, 39]. Analysis of the crystal structure of porcine acrosin showed the presence of an effector

345

side that could act as receptor for ZP glycoproteins in secondary sperm binding to the ZP [10].

346

Surprisingly, inhibitor STI showed no significant effect on sperm-ZP binding, whereas

347

inhibitors AEBSF and STI are both able to inhibit members of the family of trypsin-like

348

serine proteases [33]. Different characteristics of AEBSF and STI may have caused the

349

discrepancy. Similar to our findings, Müller-Esterl et al. (1983)[40] described that low

350

molecular weight inhibitors inhibited porcine acrosin more effectively than inhibitors with a

351

high molecular weight and furthermore, that irreversible inhibitors appeared to perform better

352

than reversible inhibitors.

353

Sperm penetration of the ZP can be divided in 1) binding of the sperm cell to the ZP (starting

354

with a loose attachment followed by species-specific tight binding), 2) the acrosome reaction

355

with release of acrosomal enzymes at the surface of the zona pellucida and subsequently 3)

356

zona lysis and sperm penetration of the zona pellucida. The observed inhibition of zona

357

penetration in the presence of serine protease inhibitors can be due to inhibition of one or

358

more of these steps. As in mouse [41], human [42] and bovine [43], serine protease inhibitors

359

may interfere with the acrosome reaction and delay zona penetration during porcine IVF. The

AC C

EP

TE D

M AN U

SC

RI PT

335

15

[Type here]

ACCEPTED MANUSCRIPT

effect of AEBSF and STI on the acrosome reaction of porcine spermatozoa was therefore

361

investigated. In the presence of AEBSF or STI in the medium, less than 50% of spermatozoa

362

completed the acrosome reaction in response to treatment with calcium ionophore.

363

Furthermore, the inhibitory effect of STI on the acrosome reaction induced by calcium

364

ionophore was about 1.5 times stronger than the inhibitory effect of AEBSF. Exocytosis of

365

the acrosomal content is a prerequisite for successful fertilization as only acrosome-reacted

366

spermatozoa are able to penetrate the ZP [2]. Therefore, we consider inhibition of the

367

acrosome reaction by serine protease inhibitors a major reason for the decrease in sperm

368

penetration during porcine IVF.

369

It remains to be clarified to what extent inhibition of acrosomal proteases involved in zona

370

lysis has contributed to the decrease in sperm penetration, as by our approach we could not

371

distinguish between interference with the release of acrosomal content and zona lysis. Real

372

time imaging of sperm-oocyte interaction could be a first approach to evaluate the time

373

necessary for zona penetration in medium with and without serine protease inhibitors. The

374

starting point of zona penetration is the release of acrosomal content, real time imaging should

375

thus be accompanied by fluorescent live staining of spermatozoa allowing to evaluate the

376

onset and completion of the acrosome reaction. This requires a more complex experimental

377

design and for now there are only few studies in which the release of acrosomal content and

378

the subsequent zona lysis was studied separately [16, 44].

379

Pre-incubation of oocytes with AEBSF and STI reduced sperm penetration rate in subsequent

380

IVF, which was not in line with the results of the fertilization experiments with zona-free

381

oocytes and the predominant effect of inhibitors on sperm penetration of the ZP. The

382

ambiguity between the results from the oocyte pre-incubation experiment versus the other

383

experiments cannot readily be explained and we can only speculate about possible reasons. To

384

us, the first possibility that needs to be tested is whether the viscous matrix of the cumulus

AC C

EP

TE D

M AN U

SC

RI PT

360

16

[Type here]

ACCEPTED MANUSCRIPT

oophorus might have captured protease inhibitor after which, by carry over to the fertilization

386

droplet, the inhibitor could have been present during sperm interaction with the ZP. The

387

incorporation of proteins from follicular fluid or medium into the cumulus matrix of COCs

388

has been observed before [45, 46].

389

The high incidence of polyspermic fertilization during porcine IVF is still a major problem to

390

be solved. Several modifications of the IVF protocol have been proposed to minimize

391

polyspermy rate [47-50]. The finding in the present study that serine protease inhibitors were

392

able to decrease sperm penetration raised the question whether this characteristic could be

393

used to minimize polyspermic fertilization, preferentially without affecting total fertilization

394

rate. Unfortunately, the decrease in polyspermy rate by inhibitors AEBSF and STI coincided

395

with a decrease in total fertilization. In conclusion, the results of the present in vitro study

396

confirm the involvement of a serine protease in sperm binding to the ZP and demonstrate a

397

role for trypsin-like serine protease activity in the acrosome reaction. Further research is

398

warranted to unravel the identity of the involved serine proteases in combination with their

399

exact mode of action.

SC

M AN U

TE D

EP AC C

400

RI PT

385

17

[Type here]

ACCEPTED MANUSCRIPT

401

Declaration of interest

402

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

403

the impartiality of the research reported.

RI PT

404 405

Funding

406

This study was supported by Research Foundation Flanders (grant numbers 1.1.450.09.N.00

407

and 1.1.450.11.N.01).

SC

408

Acknowledgements

410

The authors wish to thank Isabel Lemahieu and Petra Van Damme for their excellent

411

technical assistance. Frozen epididymal boar semen was kindly provided by Dr. D Rath,

412

Institute of Animal Science and Animal Behaviour, Mariensee, Germany. The authors are

413

members of the COST Action FA0702 “Maternal Interaction with Gametes and Embryos”.

TE D EP AC C

414

M AN U

409

18

[Type here]

ACCEPTED MANUSCRIPT

References

416

[1] Primakoff P, Myles DG. Penetration, adhesion, and fusion in mammalian sperm-egg

417

interaction. Science. 2002;296:2183-5.

418

[2] Yanagimachi R. Mammalian Fertilization. In: Knobil E, Neill JD, editors. The Physiology

419

of Reproduction. second ed. New York: Raven Press Ltd; 1994. p. 189-317.

420

[3] Tulsiani DR, Abou-Haila A, Loeser CR, Pereira BM. The biological and functional

421

significance of the sperm acrosome and acrosomal enzymes in mammalian fertilization. Exp

422

Cell Res. 1998;240:151-64.

423

[4] Honda A, Siruntawineti J, Baba T. Role of acrosomal matrix proteases in sperm-zona

424

pellucida interactions. Hum Reprod Update. 2002;8:405-12.

425

[5] Cesari A, Monclus MdlA, Tejón GP, Clementi M, Fornes MW. Regulated serine

426

proteinase lytic system on mammalian sperm surface: There must be a role. Theriogenology.

427

2010;74:699-711.e5.

428

[6] Iwamoto K, Ikeda K, Yonezawa N, Noguchi S, Kudo K, Hamano S, et al. Disulfide

429

formation in bovine zona pellucida glycoproteins during fertilization: evidence for the

430

involvement of cystine cross-linkages in hardening of the zona pellucida. J Reprod Fertil.

431

1999;117:395-402.

432

[7] Burkart AD, Xiong B, Baibakov B, Jiménez-Movilla M, Dean J. Ovastacin, a cortical

433

granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy. The Journal of

434

Cell Biology. 2012;197:37-44.

435

[8] Shimada M, Nishibori M, Yamashita Y, Ito J, Mori T, Richards JS. Down-regulated

436

expression of A disintegrin and metalloproteinase with thrombospondin-like repeats-1 by

437

progesterone receptor antagonist is associated with impaired expansion of porcine cumulus-

438

oocyte complexes. Endocrinology. 2004;145:4603-14.

AC C

EP

TE D

M AN U

SC

RI PT

415

19

[Type here]

ACCEPTED MANUSCRIPT

[9] Coy P, Jiménez-Movilla M, García-Vázquez FA, Mondéjar I, Grullón L, Romar R.

440

Oocytes use the plasminogen-plasmin system to remove supernumerary spermatozoa. Human

441

reproduction. 2012;27:1985-93.

442

[10] Tranter R, Read JA, Jones R, Brady RL. Effector sites in the three-dimensional structure

443

of mammalian sperm beta-acrosin. Structure. 2000;8:1179-88.

444

[11] Urch UA, Patel H. The interaction of boar sperm proacrosin with its natural substrate, the

445

zona pellucida, and with polysulfated polysaccharides. Development. 1991;111:1165-72.

446

[12] Jones R. Interaction of zona pellucida glycoproteins, sulphated carbohydrates and

447

synthetic polymers with proacrosin, the putative egg-binding protein from mammalian

448

spermatozoa. Development. 1991;111:1155-63.

449

[13] Howes E, Pascall JC, Engel W, Jones R. Interactions between mouse ZP2 glycoprotein

450

and proacrosin; a mechanism for secondary binding of sperm to the zona pellucida during

451

fertilization. Journal of Cell Science. 2001;114:4127-36.

452

[14] Leggio LL, Williams RM, Jones R. Some effects of zona pellucida glycoproteins and

453

sulfated polymers on the autoactivation of boar sperm proacrosin and activity of β-acrosin.

454

Journal of reproduction and fertility. 1994;100:177-85.

455

[15] Eberspaecher U, Gerwien J, Habenicht U-F, Schleuning W-D, Donner P. Activation and

456

subsequent degradation of proacrosin is mediated by zona pellucida glycoproteins, negatively

457

charged polysaccharides, and DNA. Molecular Reproduction and Development. 1991;30:164-

458

70.

459

[16] Yamagata K, Murayama K, Okabe M, Toshimori K, Nakanishi T, Kashiwabara S, et al.

460

Acrosin accelerates the dispersal of sperm acrosomal proteins during acrosome reaction. J

461

Biol Chem. 1998;273:10470-4.

AC C

EP

TE D

M AN U

SC

RI PT

439

20

[Type here]

ACCEPTED MANUSCRIPT

[17] Smith R, Jenkins A, Lourbakos A, Thompson P, Ramakrishnan V, Tomlinson J, et al.

463

Evidence for the activation of PAR-2 by the sperm protease, acrosin: expression of the

464

receptor on oocytes. FEBS Letters. 2000;484:285-90.

465

[18] Cherr GN, Drobnis EZ, Katz DF. Localization of cortical granule constituents before and

466

after exocytosis in the hamster egg. Journal of Experimental Zoology. 1988;246:81-93.

467

[19] Hoodbhoy T, Talbot P. Mammalian cortical granules: contents, fate, and function.

468

Molecular reproduction and development. 1994;39:439-48.

469

[20] Rekkas CA, Besenfelder U, Havlicek V, Vainas E, Brem G. Plasminogen activator

470

activity in cortical granules of bovine oocytes during in vitro maturation. Theriogenology.

471

2002;57:1897-905.

472

[21] Mondéjar I, Grullón LA, García-Vázquez FA, Romar R, Coy P. Fertilization outcome

473

could be regulated by binding of oviductal plasminogen to oocytes and by releasing of

474

plasminogen activators during interplay between gametes. Fertility and sterility. 2012;97:453-

475

61. e3.

476

[22] Van Soom A, Tanghe S, De Pauw I, Maes D, De Kruif A. Function of the cumulus

477

oophorus before and during mammalian fertilization. Reproduction in Domestic Animals.

478

2002;37:144-51.

479

[23] Kikuchi K, Nagai T, Motlik J, Shioya Y, Izaike Y. Effect of follicle cells on in vitro

480

fertilization of pig follicular oocytes. Theriogenology. 1993;39:593-9.

481

[24] Romar R, Coy P, Ruiz S, Gadea J, Rath D. Effects of oviductal and cumulus cells on in

482

vitro fertilization and embryo development of porcine oocytes fertilized with epididymal

483

spermatozoa. Theriogenology. 2003;59:975-86.

484

[25] Romar R, Coy P, Gadea J, Rath D. Effect of oviductal and cumulus cells on zona

485

pellucida and cortical granules of porcine oocytes fertilized in vitro with epididymal

486

spermatozoa. Anim Reprod Sci. 2005;85:287-300.

AC C

EP

TE D

M AN U

SC

RI PT

462

21

[Type here]

ACCEPTED MANUSCRIPT

[26] D'Alessandris C, Canipari R, Di Giacomo M, Epifano O, Camaioni A, Siracusa G, et al.

488

Control of mouse cumulus cell-oocyte complex integrity before and after ovulation:

489

Plasminogen activator synthesis and matrix degradation. Endocrinology. 2001;142:3033-40.

490

[27] Beek J, Maes D, Nauwynck H, Piepers S, Van Soom A. A critical assessment of the

491

effect of serine protease inhibitors on porcine fertilization and quality parameters of porcine

492

spermatozoa in vitro. Reproductive Biology. 2015;15:9-19.

493

[28] Petters RM, Wells KD. Culture of pig embryos. Journal of reproduction and fertility

494

Supplement. 1993;48:61-73.

495

[29] Rath D, Long CR, Dobrinsky JR, Welch GR, Schreier LL, Johnson LA. In vitro

496

production of sexed embryos for gender preselection: high-speed sorting of X-chromosome-

497

bearing sperm to produce pigs after embryo transfer. J Anim Sci. 1999;77:3346-52.

498

[30] Rath D, Niemann H. In vitro fertilization of porcine oocytes with fresh and frozen-

499

thawed ejaculated or frozen-thawed epididymal semen obtained from identical boars.

500

Theriogenology. 1997;47:785-93.

501

[31] Kim NH, Funahashi H, Abeydeera LR, Moon SJ, Prather RS, Day BN. Effects of

502

oviductal fluid on sperm penetration and cortical granule exocytosis during fertilization of pig

503

oocytes in vitro. J Reprod Fertil. 1996;107:79-86.

504

[32] Abeydeera LR. In vitro production of embryos in swine. Theriogenology. 2002;57:256-

505

73.

506

[33] Beynon RJ, Bond JS. Proteolytic enzymes : a practical approach. 2nd ed. Oxford ; New

507

York: Oxford University Press; 2001.

508

[34] Kunitz M. Isolation of a crystalline protein compound of trypsin and of soybean trypsin-

509

inhibitor. J Gen Physiol. 1947;30:311-20.

AC C

EP

TE D

M AN U

SC

RI PT

487

22

[Type here]

ACCEPTED MANUSCRIPT

[35] Beek J, Nauwynck H, Maes D, Van Soom A. Inhibitors of zinc-dependent

511

metalloproteases hinder sperm passage through the cumulus oophorus during porcine

512

fertilization in vitro. Reproduction. 2012;144:687-97.

513

[36] Stambaugh R, Buckley J. Studies on acrosomal proteinase of rabbit spermatozoa.

514

Biochim Biophys Acta. 1972;284:473-7.

515

[37] Miyamoto H, Chang MC. Effects of protease inhibitors on the fertilizing capacity of

516

hamster spermatozoa. Biol Reprod. 1973;9:533-7.

517

[38] Saling PM. Involvement of trypsin-like activity in binding of mouse spermatozoa to

518

zonae pellucidae. Proceedings of the National Academy of Sciences of the United States of

519

America-Biological Sciences. 1981;78:6231-5.

520

[39] Jones R, Brown CR. Identification of a zona-binding protein from boar spermatozoa as

521

proacrosin. Exp Cell Res. 1987;171:503-8.

522

[40] Muller-Esterl W, Wendt V, Leidl W, Dann O, Shaw E, Wagner G, et al. Intra-acrosomal

523

inhibition of boar acrosin by synthetic proteinase inhibitors. J Reprod Fertil. 1983;67:13-8.

524

[41] Honda A, Yamagata K, Sugiura S, Watanabe K, Baba T. A mouse serine protease TESP5

525

is selectively included into lipid rafts of sperm membrane presumably as a

526

glycosylphosphatidylinositol-anchored protein. J Biol Chem. 2002;277:16976-84.

527

[42] De Jonge CJ, Mack SR, Zaneveld LJD. Inhibition of the human sperm acrosome reaction

528

by proteinase inhibitors. Gamete Research. 1989;23:387-97.

529

[43] Deppe M, Morales P, Sanchez R. Effect of protease inhibitors on the acrosome reaction

530

and sperm-zona pellucida binding in bovine sperm. Reprod Domest Anim. 2008;43:713-9.

531

[44] Yamagata K, Murayama K, Kohno N, Kashiwabara S, Baba T. p-Aminobenzamidine-

532

sensitive acrosomal protease(s) other than acrosin serve the sperm penetration of the egg zona

533

pellucida in mouse. Zygote. 1998;6:311-9.

AC C

EP

TE D

M AN U

SC

RI PT

510

23

[Type here]

ACCEPTED MANUSCRIPT

[45] Fraser LR. Albumin is required to support the acrosome reaction but not capacitation in

535

mouse spermatozoa in vitro. J Reprod Fertil. 1985;74:185-96.

536

[46] Bijttebier J, Tilleman K, Dhaenens M, Deforce D, Van Soom A, Maes D. Comparative

537

proteome analysis of porcine follicular fluid and serum reveals that excessive alpha(2)-

538

macroglobulin in serum hampers successful expansion of cumulus-oocyte complexes.

539

Proteomics. 2009;9:4554-65.

540

[47] Funahashi H, Nagai T. Sperm selection by a climbing-over-a-wall IVF method reduces

541

the incidence of polyspermic penetration of porcine oocytes. Journal of Reproduction and

542

Development. 2000;46:319-24.

543

[48] Park C-H, Lee S-G, Choi D-H, Lee C-K. A modified swim-up method reduces

544

polyspermy during in vitro fertilization of porcine oocytes. Animal reproduction science.

545

2009;115:169-81.

546

[49] Almiñana C, Gil MA, Cuello C, Parrilla I, Roca J, Vazquez JM, et al. Effects of

547

ultrashort gamete co-incubation time on porcine in vitro fertilization. Animal Reproduction

548

Science. 2008;106:393-401.

549

[50] Li Y-H, Ma W, Li M, Hou Y, Jiao L-H, Wang W-H. Reduced polyspermic penetration in

550

porcine oocytes inseminated in a new in vitro fertilization (IVF) system: straw IVF. Biology

551

of reproduction. 2003;69:1580-5.

553

SC

M AN U

TE D

EP

AC C

552

RI PT

534

554

555

556

24

[Type here]

ACCEPTED MANUSCRIPT

Figure legends

558

Fig. 1: Effect of AEBSF (100 µM) and STI (5 µM) on total fertilization and polyspermy rate

559

in cumulus-intact (CI) and cumulus-free (CF) porcine oocytes. Data represent mean ± SEM of

560

3 replicates (n = 820 oocytes). *(P<0.05).

561

Fig. 2: Effect of AEBSF (100 µM) and STI (5 µM) on the sperm number per fertilized oocyte

562

Data represent mean ± SD of 3 replicates (n = 820 oocytes). CI: cumulus-intact; CF: cumulus-

563

free;

564

spermatozoa. *(P<0.05).

565

Fig. 3: Number of spermatozoa bound to the zona pellucida (ZP) after 4 h of gamete co-

566

incubation in medium without inhibitor (control) or in medium with either AEBSF (100 µM)

567

or STI (5 µM). Data represent mean difference ± SD of 3 replicates (n = 240 oocytes).

568

*(P<0.05).

569

Fig. 4: Effect of gamete pre-incubation with AEBSF (100 µM) and STI (5 µM) on total

570

fertilization and polyspermy rate. Data represent mean ± SEM (STI, 3 replicates, n = 523

571

oocytes) (AEBSF, 4 replicates, n = 756 oocytes). *(P<0.05).

572

Fig. 5: Percentage of acrosome intact spermatozoa after 1 h incubation in medium without

573

inhibitor (control), in medium with AEBSF (100 µM) or in medium with STI (5 µM), either

574

without (-) or with (+) supplementation of calcium ionophore (A23187). Data represent mean

575

± SD of 3 replicates. *(P<0.05).

pre-incubation;

COC:

cumulus-oocyte-complex;

sp:

SC

pre-inc.:

M AN U

zona-free;

AC C

EP

TE D

ZF:

RI PT

557

576

25

ACCEPTED MANUSCRIPT Total fertilization (%) 100 90 80 70

cumulus-intact

60

*

cumulus-free

40 30

*

20 10 0

50 45 40 35 30 20 15 10 5 0

TE D

25

AC C

EP

1 Control

STI3

SC

Polyspermy (%) 55

2 AEBSF

M AN U

1 Control

RI PT

50

2 AEBSF

* STI3

cumulus-intact

cumulus-free

ACCEPTED MANUSCRIPT

3

Sperm number per fertilized oocyte

RI PT

2,5 2 1,5

*

* *

*

SC

1

M AN U

0,5 0 cumulus-intact

cumulus-free

3,5 3

TE D

2,5 2 1,5

*

*

EP

1

zona-free

0,5 0

pre-inc sp

AC C

pre-inc COC

pre-inc COC

pre-inc sp

control AEBSF STI

Number of spermatozoa

M AN U

100 90

*

80 70

40 30

EP

20

TE D

60 50

SC

RI PT

ACCEPTED MANUSCRIPT

10

AC C

0

Control AEBSF

1

Control

STI

ACCEPTED MANUSCRIPT

Polyspermy (%)

Total fertilization (%) 100

90 80

70

70

60

60

50

50

40

40

30

30

20

20

10

10

0

Control AEBSF

0

1 COC Pre-inc.

2 sp Pre-inc.

1 COC Pre-inc.

2 sp Pre-inc.

Polyspermy (%)

Total fertilization (%) 100

TE D

100

90

Control

90

80

80

70

STI

*

40

AC C

*

EP

70

60 50

*

M AN U

80

RI PT

*

SC

90

100

30 20 10 0 1 COC Pre-inc.

2 sp Pre-inc.

60 50 40 30 20

*

10

*

0

Pre-inc.1 COC

Pre-inc.2sp

ACCEPTED MANUSCRIPT Acrosome reacted spermatozoa (%)

*

100 90 80

*

70

RI PT

60 50 40 30 20 0

C -

-

-

C +

AEBSF STI +

+

AC C

EP

TE D

M AN U

ca-ion

AEBSF STI

SC

10

ACCEPTED MANUSCRIPT • •

AC C

EP

TE D

M AN U

SC

RI PT

• •

We evaluated the effect of serine protease inhibitors AEBSF and STI on porcine IVF AEBSF and STI inhibit sperm penetration of cumulus-intact and cumulusfree oocytes AEBSF but not STI inhibits sperm binding to the zona pellucida Both AEBSF and STI strongly inhibit the calcium-induced acrosome reaction

ACCEPTED MANUSCRIPT

B

PSA

Hoechst

Supplemental Figure

M AN U

SC

RI PT

A

Fluorescent images of spermatozoa stained with FITC-labeled Pisum Sativum Agglutinin (A) and Hoechst (B) (original magnification X 600).

3A

AC C

EP

TE D

White arrow indicates an acrosome-intact sperm cell, red arrow indicates an acrosome-reacted sperm cell

3B