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DOI 10.1002/pmic.201300225

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RESEARCH ARTICLE

Proteomic characteristics of human sperm cryopreservation Shangqian Wang1∗ , Wei Wang1∗ , Yang Xu1∗ , Min Tang1 , Jianzheng Fang1 , Hongyong Sun2 , Yangyang Sun2 , Meijuan Gu2 , Zhili Liu2 , Zhaoxia Zhang2 , Faxi Lin2 , Ting Wu1 , Ninghong Song1 , Zengjun Wang1,2 , Wei Zhang1 and Changjun Yin1,2 1

State Key Laboratory of Reproductive Medicine, Department of Urology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, P. R. China 2 Human Sperm Bank, Department of Urology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, P. R. China

Human sperm cryopreservation in assisted reproductive technology is the only proven method that enables infertile men to father their own children. However, freezing and thawing reduces spermatozoon motility, viability, and fertilizing ability. An association between dysfunctional spermatozoa due to cryoinjury and protein changes has not been established. We investigated through proteomic analysis the differential protein characteristics between freeze-thawed and fresh sperm samples obtained from nine normozoospermic donors. Twenty-seven proteins differed in abundance between the two groups, and results were verified for four proteins via Western blot and immunofluorescent staining. These proteins are putatively involved in sperm motility, viability, acrosomal integrity, ATP and isocitrate content, mitochondrial membrane potential, capacitation, acrosome reaction, and intracellular calcium concentration. These marked differences suggest that dysfunctional spermatozoon after cryopreservation may be due to protein degradation and protein phosphorylation.

Received: June 8, 2013 Revised: October 16, 2013 Accepted: November 7, 2013

Keywords: Biomedicine / Mechanism / Sperm cryopreservation

 1

Additional supporting information may be found in the online version of this article at the publisher’s web-site

Introduction

Over the past decades, advances in assisted reproduction and cell manipulation techniques have increased the popular acceptance of infertility treatments. Such treatments often

Correspondence: Professor Wei Zhang, State Key Laboratory of Reproductive Medicine, Department of Urology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, 210029 Nanjing, P. R. China E-mail: [email protected] Fax: +86-25-8378-0079 Abbreviations: ATP, adenosine triphosphate/adenosine-5triphosphate; CTC, chlortetracycline; HTF, human tubal fluid; ICSI, intracytoplasmic sperm injection; MMP, mitochondrial membrane potential

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rely on the convenience of sperm storage by cryopreservation, which stabilizes cells at cryogenic temperatures for in vitro fertilization or intracytoplasmic sperm injection later. Men with azoospermia or oligozoospermia (no or low concentrations of sperm, respectively) now have the opportunity to father children even if only a single spermatozoon is harvested—cryopreservation allows storage of sperm retrieved from such patients after testicular sperm extraction or percutaneous epididymal sperm aspiration, avoiding the need for repeat biopsies or aspiration [1]. In other cases, men who must undergo procedures that entail loss of fertility or sexual function may wish to preserve the ability to have ∗ These

authors contributed equally to this work. Colour Online: See the article online to view Figs. 1, 2, and 4–6 in colour.

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children later. Thus, cryopreservation is widely used in many assisted conception units. While semen cryopreservation is the only proven method that provides many couples a chance of having children, during freezing and thawing, spermatozoa are exposed to physical and chemical stressors that result in adverse changes in membrane lipid composition, sperm motility, viability, and acrosome status [2–4]. There are many probable causes for cryodamage. Some authors have reported direct physical damage to sperm structure or function during cell freezing, related to ice formation and high osmotic pressure [5]. Sperm cryopreservation is also associated with oxidative stress and ROS-induced damage leading to lipid peroxidation and DNA damage [6]. Little is known about changes in protein levels in sperm that occur due to cryostorage. Recent advances in proteomic technology, particularly MS, have produced valuable tools for studying sperm and sperm proteins [7]. An in-depth understanding of the sperm proteome would greatly help elucidate the roles of sperm proteins in the regulation of motility, capacitation, acrosome reaction, and fertilization, and may establish biomarkers for cryodamage. In this study, we evaluated the protein content of freeze-thawed sperm samples relative to that of fresh sperm samples from the same normozoospermic donors.

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Materials and methods

2.1 Sample collection The Ethics Committee of Nanjing Medical University approved this study. The study was performed in accordance with national and international guidelines. This study included 20 sperm samples from nine normozoospermic donors obtained at Human Sperm Bank, The First Affiliated Hospital of Nanjing Medical University. All the donors gave written consent to participate in the studies after they received a thorough explanation of the study’s purpose, benefit, and possible risks. The age of the sperm donors ranged from 22 to 29 years, with a mean age of 24 years. The sperm donors were instructed to collect semen samples through masturbation after 3–5 days sexual abstinence. Semen was harvested in sterile containers. All the semen samples were given clinical and laboratory evaluations in our sperm bank in accordance with the WHO laboratory manual for the examination and processing of human semen, fifth edition [8]. The semen had the following characteristics: liquefaction time 2 mL; sperm concentration >60 × 106 /mL; motility (“a + b,” progressive) >60%; and normal morphology >5%. Liquefied semen samples were divided into two aliquots. One of the aliquots was immediately mixed with cryopreservation medium and frozen for 7 days before further investigation. The other aliquot was processed for fresh-related experiments.  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2.2 Semen cryopreservation and thawing The liquefied semen samples were mixed with an equal volume of 10% glycerol-yolk freezing medium. The equilibrated samples were then transferred to cryovials (Greiner Bio-One, Germany) and a programmable freezer to obtain cooling from 20 to −80⬚C. Then the cryovials were removed and stored in liquid nitrogen at −196⬚C. After 7 days, thawing was accomplished in a thermostat and water bath at 37⬚C for 7 min.

2.3 Sample preparation for proteomic experiments For proteomic analysis, nine donors’ ejaculates (each one divided into fresh and postthawing vials) were divided into three groups comprising three donors. For the fresh group, the liquefied ejaculate mixed with freezing medium was washed on a 60% Percoll gradient (GE Healthcare, Piscataway, NJ, USA). After centrifugation at 800 × g for 7 min, the supernatant was discarded and the pellet was washed twice in Biggers–Whitten–Whittingham (BWW) medium (GenMed Scientifics, USA). Microscopic inspection was performed to ensure the reliability and reproducibility of this washing process. The sperm pellet was solubilized in a lysis buffer (8 M urea; 2 M thiourea; 4% w/v CHAPS; 2% w/v DTT; 2% v/v IPG buffer, pH 3–10) in the presence of 1% v/w protease inhibitors-cocktail kit (Thermo Scientific, Rockford, IL, USA). Sperms were disrupted by sonication (Fisher Sonic Dismembrator, Model 300) at 20 joules for 2 s × 10 at intervals of 15 s. The samples were maintained on ice for at least 1 h and kept shaking at intervals of 15 min. Then the mixture was centrifuged at 20 000 × g for 1 h at 4⬚C to remove insoluble material. The protein concentration in each sample was determined by the Bradford method using BSA as the standard. All the sample protein was stored in cryovials at −196⬚C (liquid nitrogen) until used in the proteomic analysis.

2.4 2DE and image analysis IPG strips (24 cm, pH 3–10 NL,GE Healthcare, San Francisco, CA, USA) were rehydrated with 150 ␮g sperm protein, with three independent replications, equilibrated after IEF, run in an electrophoresis system (Ettan-Dalttwelve; GE Healthcare), and visualized by silver staining as described previously [9]. Six stained gels from the two groups were scanned. ImageMaster 2D platinum software (Version 5.0, GE Healthcare) was used to detect, quantify, and match spots as well as to perform comparative and statistical analyses of the six simultaneously run gels from the two groups. The expression level was determined using the relative volume of each spot in the gel and was expressed as % volume (spot volume,% = [spot volume/total volume of all spots resolved in the gel] × 100). Only the spots with same changing trend in all three gels from one group were considered for differential analysis. The protein expression profiles of www.proteomics-journal.com

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the postthawed sperm were compared to those of the fresh sperm. Means and SDs were calculated from the data of three gels in each group, and analyzed using ImageMaster 2D platinum software. Each paired spot was manually verified to ensure a high level of reproducibility between normalized spot volumes gels produced in triplicate data. The overlapping measures ratio was used to calculate protein expression changes, and proteins with ≥1.5-fold overlap ratio were considered statistically differentially expressed.

2.5 Protein identification As previously reported [9], selected proteins were cut from the gels, dehydrated in ACN, and dried at room temperature. Proteins were reduced with 10 mM DTT/25 mM NH4 HCO3 at 56⬚C for 1 h and alkylated with 55 mM iodoacetamide/25 mM NH4 HCO3 in the dark at room temperature for 45 min in situ. Gel pieces were thoroughly washed with 25 mM NH4 HCO3 , 50% ACN, and 100% ACN, and dried in a Speedvac. Dried gel pieces were treated with 3–4 mL of trypsin (Promega, Madison, WI, USA) solution (10 ng/mL in 25 mM ammonium bicarbonate) at 4⬚C for 30 min. Excess liquid was discarded and gel plugs were incubated at 37⬚C for 12 h. Trifluoracetic acid was added to a final concentration of 0.1% to stop the digestive reaction. Digests were immediately spotted onto 600 mM Anchorchips (Bruker Daltonics, Bremen, Germany). Spotting was achieved by pipetting 1.8 ␮L of analyte onto the MALDI target plate in duplicate three times and then adding 0.1 ␮L of 2 mg/mL a-cyano-4-hydroxycinammic acid in 0.1% trifluoracetic/33% ACN, which contained 2 mM ammonium phosphate. Bruker peptide calibration mixture was spotted down for external calibration. All samples were allowed to air dry at room temperature, and 0.1% trifluoracetic was used for on-target washing. All samples were analyzed in the positiveion, reflectron mode, on a TOF Ultraflex II mass spectrometer (Bruker Daltonics). Each acquired mass spectra (m/z range 700–4000, resolution 15 000–20 000) was processed using FlexAnalysis v2.4 software (Bruker Daltonics) with the following settings: peak detection algorithm set at SNAP (Sort Neaten Assign and Place), S/N threshold at 3, and Quality Factor Threshold at 50. The tryptic auto-digestion ion picks (842.51, 1045.56, 2211.10, and 2225.12 Da) were used as internal standards to validate the external calibration procedure. Matrix and autoproteolytic trypsin fragments, and known contaminants (e.g., keratins) were removed. The resulting lists of peptide masses were first used to search the IPI human database (67 922 sequences) and NCBInr human database (142 906 sequences) with MASCOT (v2.1.03) in an automated mode. The following parameters were used as criteria in the search: significant protein MOWSE score at p < 0.05, minimum mass accuracy at 120 ppm, trypsin as enzyme, one missed cleavage site allowed, cysteine carbamidomethylation, acrylamide-modified  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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cysteine, methionine oxidation and similarity of pI, and relative molecular mass specified, the minimum sequence coverage at 15%.

2.6 Western blot analysis Samples containing 80–100 ␮g of protein from fresh, freezethawed, precapacitated, or capacitated sperm were electrophoresed on a 12% SDS polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (GE Healthcare). The membranes were blocked in TBS containing 5% nonfat milk powder for 1 h and then incubated overnight in monoclonal antiaconitase 2, polyclonal anti-ENO1 (both 1:1000, Abcam, Cambridge, MA, USA), monoclonal antivimentin (1:200, Santa Cruz, CA, USA), polyclonal anti-TEKT1 (1:1000, Abcam), monoclonal antiphosphotyrosine (1:1000, Millipore, MA, USA), anti-␤-tubulin (1:2000, Abcam), and anti-␤-actin (1:1000, Abcam) diluted in TBS/5% nonfat milk powder. ␤-Tubulin and ␤-actin expressions were used as loading controls. Membranes were washed three times (10 min each) with TBS and then incubated for 1 h with HRP-conjugated goat-anti-rabbit IgG or goat-anti-mouse IgG (1:2000; Thermo Scientific). Lastly, membranes were washed three times with TBS, and images were captured with a Molecular Imager ChemiDoc XRS+ (Bio-Rad). Densitometry was performed using Image Lab Software (Bio-Rad).

2.7 Sperm immunofluorescence The spermatozoa were washed three times by centrifugation for 5 min at 300 × g resuspended in PBS, and air-dried onto poly-L-lysine-coated coverslips. The sperm cells were fixed with 4% paraformaldehyde for 1 h. After washing, the slides were treated with PBS containing 0.5% Triton X-100 at room temperature for 15 min. The slides were then treated with a 5% BSA blocking solution at room temperature and incubated overnight with primary antibody at 4⬚C. Sperm were incubated again with the secondary anti-goat IgG labeled with Alexa Fluor 594 (Jackson, USA) or labeled with FITC (Santa Cruz) at 1:200 dilution for 1 h at room temperature. Immunofluorescent staining was visualized using a confocal microscope (LSM 710, Zeiss).

2.8 Assessment of spermatozoa motility and concentration Motility of spermatozoa was evaluated using a Makler counting chamber. The motility of each spermatozoa was graded “rapid (a),” “progressive (a + b),” “total motility (a + b + c)” or “static (d)” according to the WHO laboratory manual [8]. Spermatozoa concentrations were also evaluated using a Makler chamber after sample collection and thawing. The evaluation www.proteomics-journal.com

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was performed again after adding cryomedium to eliminate dilution bias.

2.9 Assessment of spermatozoa viability Spermatozoa viability was determined using a one-step eosin–nigrosin staining technique [10]. Spermatozoa that appeared white or unstained were classified as live, while those that were pink or red in the head region were considered dead. At least 200 spermatozoa were assessed for each preparation.

2.10 Assessment of spermatozoa acrosome integrity An aliquot of sperm suspension was incubated with an equal volume of 2% trypan blue (Sigma-Aldrich, MO, USA) for 10 min at 37⬚C. Then, aliquots were washed two or three times with PBS medium. At least 200 cells were assessed according to the staining patterns, that is, either spermatozoa with intact acrosomes, showing a pink or purple acrosomal region; or spermatozoa with detached acrosomes, showing a white acrosomal region.

2.11 Measurement of intracellular adenosine-5 -triphosphate (ATP) content The intracellular ATP content was determined using the bioluminescent method with an ATP bioluminescent somatic cell assay kit (Sigma-Aldrich) in accordance with the manufacturer’s instructions. Spermatozoa collected from the fresh and freeze-thawed groups were washed and resuspended in BWW. The concentration of the sperm sample was determined using a hemocytometer. One-tenth of a milliliter mixture of 1×Somatic Cell ATP Releasing Reagent, ultrapure water, and the cell sample were transferred to the reaction vial (96-well plate). A standard mixture was prepared and also transferred to the reaction vial. Relative light units were measured with a Microplate Luminometer (SynergyMx, Biotek, USA) in accordance with the manufacturer’s instructions after automatic instillation with 0.1 mL ATP Assay Mix Solution. The amount (moles) of ATP in the cell sample was calculated as: ATP(SAM) =

ATP(IS) × L (SAM) , L (SAM)+IS − L (SAM)

where ATP(IS) is the ATP in moles in the added internal standard, L(SAM) is the light emitted by the cell sample, and L(SAM+IS) is the light emitted by the combined cell sample and internal standard.

2.12 Measurement of intracellular isocitrate content The change of intracellular isocitrate between fresh and freeze-thawed sperm was determined with an Isocitrate Assay  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Kit (Sigma-Aldrich). In accordance with the manufacturer’s procedure, 2 × 106 spermatozoa of each of the groups were separately homogenized in 100 ␮L of Isocitrate Assay Buffer. Reaction Mixes that included isocitrate enzyme and substrate mix were added to the samples, as well as isocitrate standards. The amount of isocitrate present in the samples was determined from the standard curve according to the A450 absorption.

2.13 Measurement of mitochondrial membrane potential (MMP) The cationic dye JC-1 (5,5 ,6 6 -tetrachloro-1,1 ,3,3 tetraethylbenzimadazolylcarbocyanine iodide) was used for MMP measurement. Fresh and freeze-thawed ejaculates were purified on a two-layer (90 and 45%) discontinuous Percoll gradient as described above. Samples were analyzed by flow cytometry (BD FACSCalibur) with a minimum of 10 000 cells to measure JC-1 fluorescence at excitation wavelength 488 nm, and the emission wavelengths 529 and 590 nm for J-monomeric (green fluorescence) and J-aggregate (red fluorescence) forms, respectively, with a flow rate of 400– 450 cells/s. The ratio of red fluorescence to green fluorescence was calculated to determine the MMP. A higher ratio indicates better mitochondrial functioning.

2.14 Evaluation of capacitation and the acrosome reaction monitored by chlortetracycline (CTC) fluorescent staining Fresh and freeze-thawed ejaculates were purified on a twolayer (90 and 45%) discontinuous Percoll gradient. After centrifugation at 300 × g for 20 min, the cell pellet was washed twice in BWW medium and resuspended in human tubal fluid (InvitroCare, Frederik, USA) to a final concentration of 3 × 107 cells/mL for capacitation and acrosome reaction. After incubation for 150 min at 37⬚C in a 5% CO2 /95% O2 incubator to permit capacitation, one-half of the sperm were then immediately stained with the vital dye Hoechst 33258 (Sigma-Aldrich) for 2 min in the dark at 37⬚C to discriminate between living (negative staining) and dead cells (positive staining). Hoechst 33258-negative staining spermatozoa were counted to assess capacitation and the acrosome reaction by acrosomal status. The sperm samples were washed with human tubal fluid medium before staining with CTC for 1 h at 37⬚C. Sperm were smeared on slides and observed under a Zeiss Axioskop plus2 fluorescent microscope at an excitation wavelength of 488 nm. The degree of spermatozoa capacitation was evaluated by the incidence of including “B” pattern or “B” plus “AR” patterns (Fig. 6A). For the evaluation of the acrosome reaction, the remaining sperm were incubated for another 150 min and then stimulated for 15 min with 15 ␮M progesterone (Sigma-Aldrich). The samples were then stained with CTC as www.proteomics-journal.com

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described above. The percentages of capacitated and acrosome-reacted sperm were obtained by subtracting values obtained after 1 h of incubation, to exclude cells destabilized during the freeze-thawing process and spontaneous acrosome reaction.

2.15 Measurement of intracellular calcium concentration [Ca2+ ]i The concentration of intracellular [Ca2+ ] was determined with an FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Briefly, after 1 h, 4 h, and thereafter 15 min stimulation of incubation as described above, sperm suspensions were loaded with 5 ␮M Fluo-3 AM (Invitrogen, Carlsbad, CA, USA) at 37⬚C and 5% CO2 for 30 min and washed twice with BWW at 800 × g for 5 min to remove free Fluo-3 AM. The fluorescence of Fluo-3 was excited at 488 nm and measured with a 530-nm filter. Photomultiplier tube voltages and gains were set to optimize the dynamic range of the signal. The fluorescence intensity was quantified for 10 000 individual spermatozoa.

3

Results

3.1 Detection of protein differences One hundred and fifty micrograms of protein were loaded on each gel. Representative 2D maps of the fresh and freezethawed groups are shown in Fig. 1 and the remaining are

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shown in Supporting Information File 1; all the differentiated spots are marked on them. Comparison of the protein amounts in the two groups revealed that 30 spots for the freeze-thawed group had increased or decreased amounts relative to fresh sperm samples. We chose 27 of these, with the minimum fold changes >1.5, for further protein identification (Table 1). We found that 22 proteins were at higher levels and five were at lower levels in the freeze-thawed samples relative to the fresh samples.

3.2 Functional analysis of identified proteins One of the purposes of this study was to understand the function of the proteins that had changed as the result of cryogenic temperatures and thawing. To gain a better understanding of these 27 proteins, we performed a functional analysis. The proteins were grouped and named using the Gene Ontology annotation in the bioinformatic Database for Annotation, Visualization and Integrated Discovery (DAVID). We then classified the proteins as cellular component, molecular function, and biological process. More detailed analysis of the cellular processes influenced by these 27 proteins was performed using PathwayStudio software, an automated text-mining tool that generates pathways from entries in the PubMed and other related databases. The results are expressed graphically in Fig. 2: proteins involved in processes such as oxidative stress, regulation of cell shape, spermatogenesis, and metabolism are indicated. We determined that 20 proteins have been well documented in the Human Sperm Proteome Database (http://reprod.njmu.edu.cn/hspd/index.py) [11].

Figure 1. A representative 2DE map of total proteins extracted from purified human spermatozoa. (A) Proteins extracted from the fresh group. (B) Proteins extracted from the freeze-thawed group. Numbered spots were significantly increased or decreased in the freeze-thawed group compared with the fresh samples and were identified by MALDI-TOF (Table 1).

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Proteomics 2014, 14, 298–310 Table 1. Proteins with a significantly higher or lower expression in the frozen-thawed samples compared to the fresh samples

Spot no.

Protein (gene)

NCBI no.

MASCOT score

Sequence coverage (%)

Function

Change in levels

56

Vimentin (VIM)

gi|62414289

153

49

Decrease

67

Tektin-1 (TEKT1)

gi|16753231

158

47

93

Aconitate hydratase, mitochondrial (ACO2)

gi|4501867

133

29

104

PREDICTED:UPF0562 protein C7orf55-like isoform 2 (C7orf55) Succinyl-CoA:3-ketoacid CoA transferase (OXCT1) Protein 4.1 isoform 3 (EPB41)

gi|114616276

77

26

Protective function against oxidative and genetic cell damage. Structural component of ciliary and flagellar microtubules. A potential sensitive and early biomarker for mitochondrial oxidant stress. Not yet clear.

gi|118505335

80

34

gi|260436834

67

19

105 135

192

Alanyl-tRNA editing protein Aarsd1 (AARSD1)

gi|387528025

87

36

209

Chain A, crystal structure of human thymidylate kinase with Fltmp And Appnhp (DTYMK) Ubiquitin carboxyl-terminal esterase L3 (UCHL3)

gi|29726715

160

50

gi|119600949

75

28

gi|11545873

99

31

gi|47778923

77

28

233

242

244

SPARC-related modular calcium-binding protein 1 isoform 2 precursor (SMOC1) NADH-cytochrome b5 reductase 2 (CYB5R2)

Key enzyme for ketone body catabolism. Contribute to a stabilization of sperm membranes in association with spectrin and actin. Functions in trans to edit the amino acid moiety from incorrectly charged tRNA(Ala). Catalyzes the conversion of dTMP to dTDP.

Involved in capacitation, energy into tubules, regulates activity of enzymes. Multifunctional roles during embryogenesis.

A key enzyme involved in the mediation of NADH-induced redox activity in human sperm. May be involved in inhibiting testis spermatogenesis apoptosis; affects sperm motility. Not yet clear. A multicatalytic proteinase complex, cleaves peptides with Arg, Phe, Tyr, Leu, and Glu; sperm-ZP penetration during mammalian fertilization. DNA-binding transcription factor activity.

Decrease

Decrease

Increase

Decrease Increase

Increase

Increase

Increase

Increase

Decrease

Increase

246

DnaJ homolog subfamily B member 13 (DNAJB13)

gi|39204547

84

34

290 330

hCG2021076 Proteasome (prosome, macropain) subunit, alpha type, 1, isoform CRA_b (PSMA1)

gi|119598574 gi|119588883

230 88

65 31

333

POU domain transcription factor OCT4-pg4 (POU5F1P4) hCG1642777 Ankyrin repeat and LEM domain-containing protein 2 (ANKLE2) Migration-inducing gene 14

gi|321173247

81

18

gi|119585591 gi|148664230

222 72

83 17

Not yet clear. Involved in mitotic nuclear envelope reassembly.

Increase Increase

gi|38570361

85

31

Not yet clear.

Increase

337 340

423

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Increase Increase

Increase

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Table 1. Continued

Spot no.

Protein (gene)

NCBI no.

MASCOT score

Sequence coverage (%)

Function

Change in levels

440

Protein FAM166A (FAM166A) Alpha-enolase isoform 1 (ENO1)

gi|48717426

192

46

Spermatogenesis.

Increase

gi|4503571

78

28

Associated with posttranslational modifications during sperm maturation and capacitation. Produce energy in microtubules and protect male gametes against oxidative stress. ATP binding. Structural component of ciliary and flagellar microtubules. Forms filamentous polymers in the walls of ciliary and flagellar microtubules. Same as TEKT4 Not yet clear. The 26S protease is involved in the ATP-dependent degradation of ubiquitinated proteins. May be involved in the sperm tail fibrous sheath, a major sperm tail structure. Glycolytic enzyme that catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to ADP. Protective function against oxidative stress.

Increase

460

477 500

GLUL protein (GLUL) Tektin-4 (TEKT4)

gi|22749655 gi|21389613

163 259

37 66

536 539 554

Tektin-3 (TEKT3) Unnamed protein product PSMC3(26S protease regulatory subunit 6A) (PSMC3)

gi|13994250 gi|16553074 gi|48145579

202 78 128

45 27 40

616

Testis specific, 10 (NYD-SP7) (TSGA10)

gi|22382177

108

29

631

Pyruvate kinase isozymes M1/M2 isoform c (PKM2)

gi|332164775

147

35

3.3 Western blot analysis of four representative proteins and phosphoproteins Western blot analysis of four candidate proteins: aconitate hydratase, mitochondrial (ACO2), alpha-enolase (ENO1), tektin1 (TEKT1), and vimentin (VIM) were performed to validate the 2DE results. Tubulin and actin were used as internal controls. The results confirmed the differential protein levels observed via 2DE (Fig. 3A and B). The freeze-thawed group had a higher level of tyrosine phosphorylation compared with the fresh (Fig. 3C).

3.4 Immunofluorescence analysis of four different proteins To verify the identified 2DE results as well as the associated functions, four candidate proteins were selected for immunofluorescence studies. The results showed diverse localization in sperm (Fig. 4A–J). According to immunolocalization, we identified that ACO2 localized in  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Increase Increase

Increase Increase Increase

Increase

Increase

the sperm midpiece, ENO1 in the principal piece, TEKT1 in the whole tail and acrosome, and VIM in the head and neck. These results confirmed the validity of the protein identification.

3.5 Sperm parameters, viability, and acrosomal integrity assessment Cryopreservation and thawing resulted in a significant reduction in spermatozoa motility parameters (Fig. 5A, Supporting Information Table 1). Progressive spermatozoa (group “a + b”) had a mean value of 67% before freezing and a mean value of 50% after thawing (p < 0.01). All the remaining parameters decreased significantly. Sperm viability, which was determined using the eosin–nigrosin staining method, revealed a mean value of 84% before freezing (range from 77 to 91%). This value diminished to 72% (range from 64 to 80%) after thawing (p < 0.01, Supporting Information Table 2). The percentage of spermatozoa with intact acrosomes after thawing was lower than this percentage in the fresh sample www.proteomics-journal.com

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Figure 2. Ontology and pathway analysis. (A) Ontology analysis of 27 identified differentially expressed proteins in the freeze-thawed samples compared to the fresh group. The classification of the protein set was performed according to the GO terms: “cellular component” (pie inside), and “biological process” (pie outside). (B) Regulated pathways involving cryoinjury mechanisms as predicted by PathwayStudio software. The regulated processes are represented by squares. Regulation events are displayed using arrows and documented by literature citations. The references linked to each protein mined by this software are listed in Supporting Information Table 8.

group (fresh: 76%, freeze-thawed: 58%; p < 0.05, Supporting Information Table 3).

less-damaged mitochondrial function. The mean ratio of the fresh sperm group (1.42) was higher than that of the freezethawed (0.82; p < 0.01; Fig. 5F).

3.6 Content assessment of intracellular ATP Nine fresh samples were compared with nine matched freezethawed samples using the bioluminescent method. The number of sperm in the reaction vial was determined based on the sample’s sperm concentration. The ATP content of the fresh samples was higher than that of the freeze-thawed (p < 0.01; Fig. 5B, Supporting Information Table 4).

3.7 Content assessment of intracellular isocitrate Six samples from the two groups were collected to measure the change in isocitrate content, using a colorimetric detection method. The concentration of isocitrate in fresh sperm was higher than that of the freeze-thawed (p = 0.002; Fig. 5C, Supporting Information Table 5)

3.9 Measurement of sperm capacitation and acrosomal reaction We stained precapacitated, capacitated, and progesteronechallenged sperm with CTC to monitor the capacitation and acrosome reaction process for sperm from both the fresh and freeze-thawed groups. We chose three time points during the sperm capacitation culture (1 h, 4 h, and thereafter 15 min following progesterone challenge) to calculate the proportion of three patterns (F, B, AR) in 200 Hoe33258-negative sperms. The results showed that there was no significant differences in either the capacitation rate or spontaneous acrosome reaction rate between the fresh and freeze-thawed groups at 1 h. There was also no significant difference in capacitation rate at 4 h between these two groups. However, after the progesterone challenge, the capacitation rate of both the fresh and freeze-thawed groups was significantly increased (p < 0.01, Supporting Information Table 6).

3.8 Analysis for mitochondrial function of spermatozoa 3.10 Indirect determination of [Ca2+ ]i The result of MMP was reported as a ratio of the mean red fluorescence intensity to the mean green fluorescence intensity. The higher ratio represents a higher MMP and also means a  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The mean fluorescence intensity of Fluo-3 was used to reflect the [Ca2+ ]i . The [Ca2+ ]i of the freeze-thawed group was www.proteomics-journal.com

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Figure 3. Validation of the 2DE results by Western blot. (A) Western blot analyses with anti-ACO2, anti-ENO1, anti-TEKT1, antiVIM, anti-␤-tubulin, and anti-␤-actin antibodies were performed on 50–100 ␮g of total protein extracts prepared from sperm proteins from three pairs of fresh (Pre) and freeze-thawed (Post) samples. (B) Bars represent the density of gel bands determined from three samples. Quantifications of ACO2, ENO1, and TEKT1/tubulin as well as VIM/actin protein ratios are shown and the data were expressed as mean ± SD. (C) Western blot was stained for tyrosine phosphorylation, and stripped and reprobed for detection of tubulin as a loading control.

significantly lower than that of the fresh sperm group at the different time points. Similar to the CTC stain, the [Ca2+ ]i after the progesterone challenge increased significantly in both groups, but with a higher level in the fresh sperm group (Fig. 6, Supporting Information Table 7).

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Discussion

The method of choice to separate and visualize proteins for the last 30 years has been 2D-PAGE [7, 12]. Although certain proteins cannot be represented due to a range of technical limitations (e.g., molecular weight and proteins either too acidic, too basic, or too hydrophobic) and also due to the relatively low abundance of proteins, 2D-PAGE delivers a map of intact proteins, which reflects changes in protein abundance, isoforms, and actual molecular mass (Mr) and pI [12]. Although work by Li et al. [13] has provided the first proteomic analysis for the sperm of common carp, which has helped an understanding of the mechanisms of cryodamage, no proteomic analysis has previously been completed with regard to human sperm cryodamage. Using the 2D-PAGE technique, we studied changes in proteins and protein levels in cryopreserved-thawed sperm samples from normal donors relative to fresh sperm samples. We found 27 proteins whose levels were either increased or decreased in the frozen-thawed sperm (Table 1). We summarize that two main mechanisms could be responsible for reduction of freeze-thawed sperm  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 4. Immunofluorescence analysis of four proteins whose abundance differed between the fresh and freeze-thawed groups. A, C, E, and G represent the locations of ACO2, ENO1, TEKT1, and VIM, respectively. B, D, F, and H each represent a merged immunofluorescence double staining of Hoechst and DIC. I and J represent the negative controls mouse-IgG, and rabbit-IgG, respectively.

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Figure 5. (A) Comparisons of sperm parameters between fresh and freeze-thawed groups. *p < 0.05, **p < 0.01, data are presented as mean ± SEM. (B) The ATP content from the fresh group was higher than frozen-thawed group, p < 0.01. (C) The concentration of isocitrate in freeze-thawed sperm was lower than that of the fresh group, p < 0.01. (D and E) Results from fresh and freeze-thawed group. (F) Ratio of the mean red fluorescence intensity (R1) to mean green fluorescence intensity (R2) of JC-1 staining revealed that mitochondrial function was damaged after cryopreservation, p < 0.01.

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Figure 6. Three patterns of chlortetracycline fluorescence staining observed on human spermatozoa cultured in a human tubal fluid medium. (Aa) “F” with uniform fluorescence over the whole head; (Ab) “B” with a fluorescence-free band in the postacrosomal region; (Ac) “AR” with dull fluorescence over the whole head; (Ad) dead spermatozoa with a Hoechst 33258 staining. (B) The bar reveals proportions of the three patterns of CTC at 1 h, and cap+ means the capacitated plus acrosome-reacted sperm at 4 h; ARP means the acrosome reaction rate after 15 min of progesterone stimulation. (C and D) [Ca2+ ]i of freeze-thawed group was significantly lower than that of the fresh group at different time points. [Ca2+ ]i after progesterone challenge increased significantly in both groups, nevertheless with a higher level in fresh sperm. (Da) Fresh group; (Db) frozen-thawed group. The small square at the upper left of each fluo-3 result is 7AAD staining. UL, the vitality rate; X, mean fluorescence intensity of Fluo-3.

concentration, velocity, motility, and intact acrosome rate (Fig. 5A)—protein degradation and protein phosphorylation. Three of five degraded proteins (ACO2, TEKT-1, VIM, succinyl-CoA:3-ketoacid CoA transferase, and NADHcytochrome b5 reductase 2) were identified by Western blot (Fig. 3A) and immunofluorescence (Fig. 4B, F, H). ACO2 is involved in the tricarboxylic acid cycle and catalyzed the isomerization of citrate to isocitrate via cis-aconitate, which associates with ATP-dependent sperm motility [14, 15]. Our results showed that the isocitrate content was significantly decreased in freeze-thawed sperm, which indicated that the degraded ACO2 lowered the production of isocitrate needed in the tricarboxylic acid cycle. The immunofluorescence result showed that ACO2 was localized to the sperm midpiece, also called the mitochondrial sheath.  C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The ATP content and the MMP of mitochondria in freezethawed sperm were significantly lower than those were in fresh sperm (Fig. 5B, D, E), which may show that ACO2 was one of the mitochondrial proteins and that cryopreservation degraded the mitochondrial proteins, leading to the injury of sperm mitochondria and decreasing ATP production. As a result, the motility of sperm after thawing was decreased. Previously, a study was reported that a decreased abundance of ACO2 may be associated with oxidative injury in mitochondria [16]. Several authors have reported the presence of vimentin in the head of human spermatozoa [17, 18], which is confirmed by our results (Fig. 4H). Vimentin may have an important role in the assembly and stabilization of the specialized cell surface domains of the spermatozoa and,

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hence, in the surface-associated events of fertilization [17]. It might also contribute to the plasticity of the nuclear and mitochondrial genome by mediating recombination events whose products are transferred to succeeding cell generations. In this way, vimentin may enable mammals and their progeny to better cope with stressful health- and life-threatening environmental changes and thus give them a higher chance of survival [19]. We found that the decreasing intact acrosome rate in freeze-thawed sperm (Fig. 5A) may be due to the degradation of cytoskeletal components such as the intermediate filament protein vimentin. Tektin-1 (TEKT1), belongs to the tektin family, is structural component of ciliary and flagellar microtubules. Tektins in sperm motility have been well shown by several studies [20, 21] and a low level of TEKT1 in an asthenozoospermic subject was found [22]. The degradation of movementrelated proteins may be responsible for the reduced motility in freeze-thawed sperm. Succinyl-CoA:3-ketoacid CoA transferase (OXCT1) enzyme is involved in ketone metabolism, which produces two acetyl CoA molecules capable of entering the tricarboxylic acid cycle [23]. Another effect of OXCT1 deficiency in sperm could be a consequential compromised glycolysis [24]. Decreased OXCT1 might be associated with the ATP deficiency in freeze-thawed sperm. NADH-cytochrome b5 reductase 2 (CYB5R2) as a key enzyme involved in the mediation of NADH-induced redox activity in human sperm [25]. This may demonstrate the production of excessive ROS in the freeze-thawed group. Interestingly, we found that the majority of altered proteins in freeze-thawed sperm were more abundant relative to fresh sperm (Table 1), which made us speculate that there must be another cryoinjury mechanism besides protein degradation. In this regard, protein posttranslational modification might be responsible for this interesting phenomenon. Protein phosphorylation, a posttranslational modification that allows the cell to control various cellular processes, is an important means of modifying sperm functions [26]. Our Western blot results showed that the level of tyrosine phosphorylated proteins in the freeze-thawed group was higher than those in the fresh group (Fig. 3C), suggesting that the cryopreservation and thawing process could lead to a high level of tyrosine phosphorylation. It has been well documented that capacitation is associated with tyrosine phosphorylation of sperm proteins, including that of humans, mice, cattle, and swine [27–30]. Moreover, it was shown that cooling sperm resulted in changes that were similar to those for sperm incubated in a capacitation-supporting medium, indicating capacitationlike changes during cryopreservation [31]. Several studies have reported higher [Ca2+ ]i , capacitation, and acrosome reaction rate in freeze-thawed sperm compared with fresh sperm [26, 32]. The researchers concluded that these changes in the capacitation-like state were detrimental to fertility and sperm were rendered unstable and had a short lifespan. Yet other studies revealed a different picture [33], in which [Ca2+ ]i decreased significantly in freeze C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

thawed sperm. We found lower levels of [Ca2+ ]i in live freezethawed sperm (7-AAD-negative) compared with fresh at three different time points. However, the [Ca2+ ]i increased after progesterone stimulated. The increase in [Ca2+ ]i in live freezethawed sperm (7-AAD-negative) was lower than that of fresh sperm after progesterone stimulation (Fig. 6C and D), which was consistent with CTC results. We failed to find any significant difference in capacitation or acrosome reaction rate between the freeze-thawed and fresh sperm at 1-h or 4-h incubation by three patterns of the CTC test (Fig. 6A, Supporting Information Table 6). However, we found that the proportion of live acrosome-reacted sperm in the fresh group was higher than that of the freeze-thawed after progesterone stimulation (Fig. 6B, Supporting Information Table 6), indicating that the fertility potential decreased after cryopreservation. Other studies have reported that high levels of protein phosphorylation may be related with sperm motility, hyperactivated motility, and capacitation [34], but this appears contradicted by our results. The high level of protein phosphorylation caused by cryopreservation failed to increase the capacitation rate or hyperactivated motility, indicating that cryopreservation stimulated a capacitation-like change other than real capacitation. In summary, this is the first comprehensive description of the human spermatozoa proteome after cryopreservation. We observed marked differences in protein degradation and protein phosphorylation between the fresh and freeze-thawed groups that are associated with changes in sperm motility, viability, acrosomal integrity, ATP content, MMP, capacitation, acrosome reaction, and [Ca2+ ]i . The authors have declared no conflict of interest.

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References

[1] Donnelly, E. T., McClure, N., Lewis, S. E., Cryopreservation of human semen and prepared sperm: effects on motility parameters and DNA integrity. Fertil. Steril. 2001, 76, 892–900. [2] Alvarez, J. G., Storey, B. T., Evidence that membrane stress contributes more than lipid peroxidation to sublethal cryodamage in cryopreserved human sperm: glycerol and other polyols as sole cryoprotectant. J. Androl. 1993, 14, 199–209. [3] Sinan, O., Esra, E., Ayca, I., Derya, O., Sercin, K., Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa. J. Assist. Reprod. Genet. 2008, 25, 403–411. [4] O’Connell, M., McClure, N., Lewis, S. E., The effects of cryopreservation on sperm morphology, motility and mitochondrial function. Hum. Reprod. 2002, 17, 704–709. [5] Brotherton, J., Cryopreservation of human semen. Arch. Androl. 1990, 25, 181–195. [6] Agarwal, A., Makker, K., Sharma, R., Clinical relevance of oxidative stress in male factor infertility: an update. Am. J. Reprod. Immunol. 2008, 59, 2–11.

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310

S. Wang et al.

[7] Xu, W., Hu, H., Wang, Z., Chen, X. et al., Proteomic characteristics of spermatozoa in normozoospermic patients with infertility. J. Proteomics 2012, 75, 5426–5436. [8] World Health Organization, Department of Reproductive Health and Research. WHO laboratory manual for the examination and processing of human semen. (Fifth edition). http://www.who.int/reproductivehealth/publications/infertili ty/9789241547789/en/ [9] Huo, R., Zhu, Y. F., Ma, X., Lin, M. et al., Differential expression of glucose-regulated protein 78 during spermatogenesis. Cell Tissue Res. 2004, 316, 359–367. ¨ [10] Bjorndahl, L., Soderlund, I., Kvist, U., Evaluation of the onestep Eosin–Nigrosin staining technique for human sperm vitality assessment. Hum. Reprod. 2003, 4, 813–816. [11] Zhou, T., Zhou, Z. M., Guo, X. J., Bioinformatics for spermatogenesis: annotation of male reproduction based on proteomics. Asian J. Androl. 2013, 15, 594–602. [12] Zhao, C., Guo, X. J., Shi, Z. H., Wang, F. Q. et al., Role of translation by mitochondrial-type ribosomes during sperm capacitation: an analysis based on a proteomic approach. Proteomics 2009, 9, 1385–1399. [13] Li, P., Humak, M., Koubek, P., Sulc, M. et al., Ice-age endurance: the effects of cryopreservation on proteins of sperm of common carp, Cyprinus carpio L. Theriogenology 2010, 74, 413–423. [14] Beinert, H., Kennedy, M. C., Aconitase, a two-faced protein: enzyme and iron regulatory factor. FASEB J. 1993, 7, 1442–1449. [15] Turner, R. M., Moving to the beat: a review of mammalian sperm motility regulation. Reprod. Fertil. Dev. 2006, 18, 25–38. [16] Lee, Y. H., Chung, M. C., Lin, Q., Boelsterli, U. A., Troglitazoneinduced hepatic mitochondrial proteome expression dynamics in heterozygous Sod2(+/-) mice: two-stage oxidative injury. Toxicol. Appl. Pharmacol. 2008, 15, 231, 43–51. [17] Virtanen, I., Badley, R. A., Paasivuo, R., Lehto, V. P., Distinct cytoskeletal domains revealed in sprem cells. J. Cell Biol. 1984, 99, 1083–1091. [18] Palecek, J., Peknicova, J., Vitu, M., Changes in immunochemical localization of cytoskeletal proteins in human and boar spermatozoa before and after acrosome reaction. Folia Biol. 1999, 45, 13–20. [19] Tolstonog, G. V., Shoeman, R. L., Traub, U., Traub, P., Role of the intermediate filament protein vimentin in delaying senescence and in the spontaneous immortalization of mouse embryo fibroblasts. DNA Cell Biol. 2001, 20, 509–520. [20] Tanaka, H., Iguchi, N., Toyama, Y., Kitamura, K. et al., Mice deficient in the axonemal protein Tektin-t exhibit male infertility and immotile-cilium syndrome due to impaired inner arm dynein function. Mol. Cell Biol. 2004, 24, 7958–7964. [21] Tanaka, H., Takahashi, T., Iguchi, N., Kitamura, K. et al., Ke-

 C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics 2014, 14, 298–310 tone bodies could support the motility but not the acrosome reaction of mouse sperm. Int. J. Androl. 2004, 27, 172–177. [22] Siva, A. B., Kameshwari, D. B., Singh, V., Pavani, K. et al., Proteomics-based study on asthenozoospermia: differential expression of proteasome alpha complex. Mol. Hum. Reprod. 2010, 16, 452–462. [23] Solomon, F., Jencks, W. P., Identification of an enzymegamma-glutamyl coenzyme A intermediate from coenzyme A transferase. J. Biol. Chem. 1969, 244, 1079–1081. [24] Cotter, D. G., Schugar, R. C., Wentz, A. E., d’Avignon, D. A. et al., Successful adaptation to ketosis by mice with tissuespecific deficiency of ketone body oxidation. Am. J. Physiol. Endocrinol. Metab. 2013, 304, E363–374. [25] Baker, M. A., Krutskikh, A., Curry, B. J., Hetherington, L., Aitken, R. J., Identification of cytochrome-b5 reductase as the enzyme responsible for NADH-dependent lucigenin chemiluminescence in human spermatozoa. Biol. Reprod. 2005, 73, 334–342. [26] Satorre, M. M., Breininger, E., Beconi, M. T., Beorlegui, N. B., Alpha-tocopherol modifies tyrosine phosphorylation and capacitation-like state of cryopreserved porcine sperm. Theriogenology 2007, 68, 958–965. [27] Luconi, M., Kraustz, C., Forti, G., Baldi, E., Extracellular calcium negatively modulates tyrosine phosphorylation and tyrosine kinase activity during capacitation of human spermatozoa. Biol. Reprod. 1996, 55, 207–216. [28] Visconti, P. E., Moore, G. D., Bailey, J. L., Leclerc, P. et al., Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP dependent pathway. Development 1995, 121, 1139–1159. [29] Galantino-Homer, H. L., Visconti, P. E., Kopf, G. S., Regulation of protein tyrosine phosphorylation during bovine sperm capacitation by a cAMP-dependent pathway. Biol. Reprod. 1997, 56, 707–719. [30] Flesch, F. M., Colembrander, B., van Golde, L. M., Gadella, B. M., Capacitation induces tyrosine phosphorylation of proteins in the boar sperm plasma membrane. Biochem. Biophys. Res. Commun. 1999, 262, 787–792. [31] Green, C. E., Watson, P. F., Comparison of the capacitationlike state of cooled boar spermatozoa with true capacitation. Reproduction 2001, 122, 889–898. [32] Collin, S, Bailey, J. L. Assessment of intracellular calcium levels in cryopreserved bovine sperm by flow cytometry. Theriogenology 1999, 51, 341. [33] Kumaresan, A., Siqueira, A. P., Hossain, M. S., Johannisson, A. et al., Quantification of kinetic changes in protein tyrosine phosphorylation and cytosolic Ca2 + concentration in boar spermatozoa during cryopreservation. Reprod. Fertil. Dev. 2012, 24, 531–542. [34] Chan, C. C., Shui, H. A., Wu, C. H., Wang, C. Y. et al., Motility and protein phosphorylation in healthy and asthenozoospermic sperm. J. Proteome Res. 2009, 8, 5382–5386.

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