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Background: Cryopreservation of epididymal spermatozoa is important in cases in which it is not possible to collect semen using normal methods, as the sudden death of an animal or a catastrophic injury. However, the freezing and thawing processes cause stress to spermatozoa, including cold shock, osmotic damage, and ice crystal formation, thereby reducing sperm quality. We assessed the motility (%), motion kinematics, capacitation status, and viability of spermatozoa using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining. Moreover, we identified proteins associated with cryostress using a proteomic approach and performed western blotting to validate two-dimensional electrophoresis (2-DE) results using two commercial antibodies. Results: Cryopreservation reduced viability (%), motility (%), straight-line velocity (VSL), average path velocity (VAP), amplitude of lateral head displacement (ALH), and capacitated spermatozoa, whereas straightness (STR) and the acrosome reaction increased after cryopreservation (P < 0.05). Nine proteins were differentially expressed (two proteins decreased and seven increased) (>3 fold, P < 0.05) before and after cryopreservation. The proteins differentially expressed following cryopreservation are putatively related to several signaling pathways, including the ephrinR-actin pathway, the ROS metabolism pathway, actin cytoskeleton assembly, actin cytoskeleton regulation, and the guanylate cyclase pathway. Conclusion: The results of the current study provide information on epididymal sperm proteome dynamics and possible protein markers of cryo-stress during cryopreservation. This information will further the basic understanding of cryopreservation and aid future studies aiming to identify the mechanism of cryostress responses. Keywords: Cryopreservation, Cryostress, Protein, Spermatozoa Background bovine is regarded as a model of successful cryopreser- For decades, sperm cryopreservation has been an im- vation among various species, the process causes up to portant tool for assisted reproductive techniques and 50% loss of viable spermatozoa . storing genetic resources [1, 2]. Using cryopreservation During the different stages of cryopreservation, sperm- for effective sperm storage has enabled advances in the atozoa are exposed to various types of stress, such as livestock industry and management of infertility [3, 4]. cold shock, osmotic damage, and ice crystal formation However, in cases in which it is not possible to collect [7, 9]. Furthermore, cryopreservation induces the excess spermatozoa normally such as unexpected death and production of reactive oxygen species (ROS), disruption catastrophic injury, cryopreservation of epididymal sper- of mitochondrial membrane potential, and an apoptosis- matozoa can be advantageous. Therefore, preservation like phenomenon in spermatozoa [1, 10]. These changes of epididymal spermatozoa can play an important role in are associated with alterations in sperm membrane human and animal reproduction. compounds, resulting in dramatic reductions in motility, The freezing and thawing processes of sperm cryo- viability, and fertilizing ability . preservation inevitably cause structural and functional It is well known that sperm functions and structures are alterations, thereby reducing fertility [5–7]. Although the affected by protein degradation and post-translational modifications, such as phosphorylation, during cryo- * Correspondence: email@example.com preservation [5, 10]. Research over the past decade has Department of Animal Science & Technology, Chung-Ang University, provided compelling evidence that spermatozoa are Anseong, Gyeonggi-Do 456-756, Republic of Korea © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yoon et al. Journal of Animal Science and Biotechnology (2016) 7:67 Page 2 of 12 damaged substantially during cryopreservation, thereby chamber (Makler, Haifa, Israel) using the 10× objective reducing sperm function and semen quality [3, 4]. How- in phase contrast mode. The obtained images were ana- ever, little is known about cryostress at the protein level in lyzed to assess sperm motility (%), average path velocity spermatozoa. Advances in two-dimensional electrophoresis (VAP), straight-line velocity (VSL), curvilinear velocity (2-DE) and mass spectrometry techniques have enabled (VCL), wobble (WOB), straightness (STR), linearity (LIN), the identification of proteins related to cryopreservation and amplitude of lateral head displacement (ALH). At . Therefore, these methods may facilitate the discovery least 250 sperm cells were recorded for each sample. of biomarkers of cryostress at the proteome level in spermatozoa. Assessment of capacitation status by Hoechst 33258 In the present study, bull epididymal sperm cryopreser- (H33258)/chlortetracycline fluorescence (CTC) vation was used as a model system of cryopreservation. The H33258/CTC dual staining method was performed as First, we evaluated various sperm parameters before- and previously described [16, 18]. Briefly, 15 μL of H33258 after-cryopreservation. To identify the protein markers re- solution (10 μg H33258/mL PBS) was added to 135 μL lated to cryostress, a comprehensive proteomic study of of the sample. After a 10 min incubation at room the associated signaling pathways was performed. temperature (RT), 250 μL of 2% (w/v) polyvinylpyrroli- done (Sigma-Aldrich) in PBS was added to the mixture. Methods Then, the sample was washed by centrifugation at Sample collection 700 × g for 5 min and the pellet was resuspended in Native Korean Bull (Hanwoo) testes were transferred on 150 μLof PBS and150 μL of CTC solution (750 mmol/L ice from a local slaughterhouse (Seomun Co., Hwaseong, CTC in 5 μL of buffer composed of 20 mmol/L Tris, Korea) to the laboratory within 3 h . Sperm samples 130 mmol/L NaCl, and 5 mmol/L cysteine, pH 7.4). were obtained from nine individual bull epididymides. A Capacitation status and viability were analyzed using a small cut was made at the cauda epididymis of the testis Microphot-FXA microscope (Nikon, Tokyo, Japan) under and backflushed Phosphate-buffered saline (PBS; Sigma- epifluorescent illumination. Four capacitation patterns Aldrich, St. Louis, MO, USA) into the end of the vas were observed: live acrosome-reacted (AR), live capaci- deferens using a 10-mL syringe. Each collected sample tated (B), live non-capacitated (F), or dead (D). For each was washed by centrifugation at 700 × g for 15 min . sample, at least 400 spermatozoa per slide were evaluated. To rule out individual difference in resistance to cryo- stress, the nine samples were mixed for the sperm 2DE, gel-image analysis parameter and proteome analyses. All procedures were All procedures followed the methods of Kwon et al. . performed according to the guidelines for the ethical Samples were centrifuged at 700 × g for 15 min with iso- treatment of animals by the Institutional Animal Care and tonic 45% Percoll in PBS [4, 17, 19]. After rehydrating with Use Committee of Chung-Ang University, Seoul, Korea. 250 μg solubilized protein samples for 12 h at 4 °C, 24-cm- long NL Immobiline DryStrips (pH 3–11; Amersham, Cryopreservation of spermatozoa Piscataway, NJ, USA) were focused for first-dimension Sperm cryopreservation was performed as previously electrophoresis using an IPGphor isoelectric focusing sys- described [15, 16]. Briefly, washed samples were diluted tem. After equilibrate the strips, 2-DE was performed using (100 × 10 cells/mL) in Tris-egg yolk buffer (TYB; 12.5% (w/v) SDS-polyacrylamide gel electrophoresis 250 mmol/L Tris, 88.5 mmol/L citric acid, 68.8 mmol/L (SDS-PAGE) gels. Silver staining was performed and glucose, and 20% egg yolk) and cooled to 4 °C over 2 h. the gels were scanned with a GS-800 calibrated scanner Then an equal volume of TYB containing 12% glycerol (Bio-Rad, Hercules, CA, USA). To detect, quantify, and was added to dilute the sample. After equilibration at 4 °C match spots and to perform the comparative and statis- for 2 h, samples packaged into 0.5-mL straws were frozen tical analyses, PDQuest 8.0 software (Bio-Rad, Hercules, in liquid nitrogen vapor for 15 min. Finally, the straws were CA, USA) was used. plunged into liquid nitrogen for long-term storage. Sam- ples were thawed at 37 °C for 1 min after 2 wk of In-gel digestion cryopreservation. Proteins were subjected to in-gel trypsin digestion. Ex- cised gel spots were destained with 100 μL of destaining Computer-assisted sperm analysis (CASA) solution (30 mmol/L potassium ferricyanide, 100 mmol/L Sperm motility (%) and kinematics of the samples were sodium thiosulfate) with shaking for 5 min. After removal analyzed by the method of Kown et al. using CASA of the solution, gel spots were incubated with 200 mmol/L system (Sperm Analysis Imaging System version SAIS- ammonium bicarbonate for 20 min. The gel pieces were PLUS 10.1; Medical Supply, Seoul, Korea) . Briefly, dehydrated with 100 μL of acetonitrile and dried in a vac- 10 μL of each sample was observed on 37 °C Makler uum centrifuge. The procedure was repeated three times. Yoon et al. Journal of Animal Science and Biotechnology (2016) 7:67 Page 3 of 12 The dried gel pieces were rehydrated with 20 μLof peptide fragment files were used to search the database 50 mmol/L ammonium bicarbonate containing 0.2 μg using the Mascot search engine (Matrix Science), and modified trypsin (Promega, Madison, WI, USA) for the results were limited to Sus scrofa. Oxidized methio- 45 min on ice. After removing the solution, 70 μLof nine was set as a variable modification, and carbamido- 50 mmol/L ammonium bicarbonate was added. The di- methylated cysteine was set as a fixed modification. The gestion was performed overnight at 37 °C. The peptide mass tolerance was set at ±1 and ±0.6 Da for the pep- solution was desalted using a C18 nano column (home- tides and fragments, respectively. High scores were de- made, Waters Corp., Milford, MA, USA). fined as those above the default significance threshold in MASCOT (P < 0.05, peptide score > 50). Desalting and concentration Custom-made chromatographic columns were used for Signaling pathway desalting and concentrating the peptide mixture prior to To identify signaling pathways associated with the pro- mass spectrometric analysis. A column consisting of 100– tein markers, Pathway Studio (v 9.0, Aridane Genomics, 300 nL of Poros Reversed-phase R2 material (20–30-μm Rockville, MD, USA) was used. Differentially expressed beads, PerSeptive Biosystems, Framingham, MA, USA) proteins were analyzed in Pathway Studio to determine was packed in a constricted GELoader Tip (Eppendorf, significantly matched pathways for each protein. Hamburg, Germany). A 10-mL syringe was used to force liquid through the column by applying gentle air pressure. Western blotting Thirty microliters of the peptide mixture from the diges- Western blotting was performed as described previously tion supernatant was diluted with 30 μL of 5% formic acid, , with modifications. Commercial polyclonal anti- loaded onto the column, and washed with 30 μLof5%for- SOD2 (Abcam, Cambridge, MA, USA) and polyclonal mic acid. For analyses by tandem mass spectrometry (MS/ anti-NUDFV2 (Abcam) were used, and monoclonal anti- MS) analyses, peptides were eluted with 1.5 μLof50% α-tubulin (Abcam) was used as a control. Briefly, the methanol/49% H O/1% formic acid directly into a pre- samples were washed by centrifugation in DPBS at coated borosilicate nanoelectrospray needle (EconoTip™, 10,000 × g for 5 min. The pellets were re-suspended with NewObjective,Woburn,MA,USA). lysis buffer containing 5% 2-mercaptoethanol and incu- bated for 10 min at RT. The samples were electropho- ESI-MS/MS resed on a 12% SDS-polyacrylamide gel and transferred MS/MS of peptides generated by in-gel digestion was per- to polyvinylidene fluoride membranes (Amersham). The formed by nano-electrospray ionization (ESI) on a MicroQ- membranes were blocked with PBS-Tween containing TOF III mass spectrometer. The source temperature was 5% skim milk powder (blocking solution) for 3 h at RT. RT. A potential of 1 kV was applied to the precoated After washing, the membranes were incubated overnight borosilicate nanoelectrospray needles (EconoTip™,New with anti-NDUFV2 (1:3,000) and anti-SOD2 (1:5,000) di- Objective) in the ion source combined with a nitrogen luted with blocking solution. Then, the membranes were back-pressure of 0–5 psi to produce a stable flow rate incubated with horseradish peroxidase conjugated anti- (10–30 nL/min). The cone voltage was 800 V. The rabbit immunoglobulin G (1:3,000, Abcam) for 1 h. After quadrupole analyzer was used to select precursor ions the membranes were washed, proteins were detected by for fragmentation in the hexapole collision cell. The –5 enhanced chemiluminescence reagents. All bands were collision gas was Ar at a pressure of 6–7×10 mbar scanned with a GS-800 Calibrated Imaging Densitometer and the collision energy was 15–40 V. Product ions (Bio-Rad) and analyzed with Quantity One (v.4.6, Bio-Rad). were analyzed using an orthogonal time-of-flight The signal intensity ratios of the bands were calculated for (TOF) mass analyzer, fitted with a reflector, a micro- SOD and NUDFV2/α-tubulin. channel plate detector, and a time-to-digital converter. The data were processed using a peptide sequence system. Statistical analysis Data were analyzed with SPSS v.21.0 (SPSS Inc., Chicago, Database search IL, USA). The Student’s two-tailed t-test was used to com- An MS/MS ion search was assigned as the ion search pare the values from before and after cryopreservation option in MASCOT software (MASCOT version 2.3, after performing normality and variance homogeneity Matrix Science, Boston, MA, USA). Peptide fragment tests. In Pathway Studio, Fisher’s Exact Test was used to files were obtained from the peptide peaks in ESI-MS by determine if the pathways were statistically correlated ESI-MS/MS. Trypsin was selected as the enzyme, with with differentially expressed proteins. P <0.05 was con- one potentially missed cleavage site. An ESI-QTOF in- sidered statistically significant. Data are expressed as strument was used for protein mass determination. The the mean ± SEM. Fisher’s exact test was used to determine Yoon et al. Journal of Animal Science and Biotechnology (2016) 7:67 Page 4 of 12 the probability that a protein is involved in a particular and NDUFV2 were detected at 25 and 27 kDa, res- signaling pathway (P <0.05). pectively. The densities of SOD2 and NDUFV2 expres- sion were higher after cryopreservation than before (Fig. 5, Results P < 0.05), consistent with the 2-DE results. Sperm parameters The motility and motion kinematics of spermatozoa be- Informatics fore and after cryopreservation were measured by the Nine proteins were differentially expressed before and CASA technique, as described previously . Motility after cryopreservation. They were analyzed using Pathway (%), VCL, VAP, and ALH significantly decreased during Studio 9. Five signaling pathways were significantly corre- cryopreservation (Figs. 1 and 2, P < 0.05), whereas STR lated with four proteins (Fig. 6 and Table 2, P <0.05). increased significantly (Fig. 2, P < 0.05). However, there CAPZB and ODF2 were correlated with the ephrinR-actin were no significant differences in VSL, WOB, BCF, or signaling pathway and CAPZB, TPI, and ODF2 were cor- LIN (Fig. 2). To evaluate sperm capacitation status and related with the Notch pathway (Table 2, P <0.05). The viability, CTC/H33258 dual staining was performed. The actin cytoskeleton assembly pathway was correlated with AR pattern increased significantly, whereas the F pattern CAPZB and actin cytoskeleton regulation was correlated significantly decreased during cryopreservation (Fig. 3, with CAPZB and ODF2 (Table 2, P < 0.05). Moreover, the P < 0.05). However, there was no difference in the B guanylate cyclase pathway was correlated with CAPZB, pattern (Fig. 3). The viability of frozen-thawed sperma- NDPK, and ODF2 (Table 2, P <0.05). tozoa was significantly lower than that of spermatozoa before cryopreservation (Fig. 1, P <0.05). Discussion Numerous studies have prviously reported that ejacu- 2-DE lated and epididymal spermatozoa have differences in Spermatozoa samples were analyzed by 2-DE before sperm maturity, fertility, and sensitivity to freezing. and after cryopreservation. A total of 285 proteins were However, in certain situations, it is not possible to col- detected, and nine proteins were found to be differen- lect semen using normal methods, and the cryopreserva- tially expressed (>3 fold, Table 1 and Fig. 4). The tion of epididymal spermatozoa can be an acceptable expression of major fibrous sheath protein (AKAP), F1- alternative. Epididymal spermatozoa can be stored for ATPase complexed with aurovertin B (F1-ATPase), more than 24 h without loss of fertility . Moreover, a triosephosphate isomerase (TPI), nucleoside diphos- recent study reported that epididymal spermatozoa had phatekinase7(NDPK7), NADH dehydrogenaseflavo- a similar fertilizing-capacity as fresh ejaculated sperm- protein 2 (NDUFV2), F-actin-capping protein subunit atozoa using an in vitro fertilization system . There- beta (CAPZB), and superoxide dismutase 2 (SOD2) fore, the cryopreservation of epididymal sperm can be a were higher after cryopreservation. However, expres- useful assisted reproductive technology for the livestock sion of outer dense fiber protein 2 (ODF2) and unchar- industry and preserving genetic resources. However, the acterized protein LOC616410 were higher in before effects of cryo-stress at the proteome level, especially in cryopreservation (Table 1 and Fig. 4). epididymal spermatozoa, have not yet fully understood. During cryopreservation, spermatozoa are exposed to Western blot various types of stress such as cold shock, osmotic stress, Western blotting analysis was performed to confirm and ice crystal formation [1, 10]. These environmental the 2-DE results. Two differentially expressed proteins changes cause physical and chemical damage to spermato- were examined using commercial antibodies. SOD2 zoa, resulting in changes of various parameters, including Fig. 1 Effect of cryopreservation on sperm. a Viability b Motility before and after cryopreservation. Data are presented as mean ± SEM. (*P <0.05, n =9) Yoon et al. Journal of Animal Science and Biotechnology (2016) 7:67 Page 5 of 12 Fig. 2 Effect of cryopreservation on sperm motion kinematics. a Curvilinear velocity b Straight-line velocity c Average path velocity d Straightness e Wobble f Amplitude of lateral head g Beat-cross frequency h Linearity before and after cryopreservation. Data are presented as mean ± SEM. (*P < 0.05, n =9) viability, motility, motion kinematics, and capacitation representation of sperm movement and its relationship status . with sperm viability . Our result showed that viability Motility is one of the most important sperm fertility was significantly reduced during cryopreservation. Mo- parameters, and numerous studies have described its es- tility (%), velocity parameters (VCL, VSL, and VAP), sential role in sperm transport to the site of fertilization and ALH were also significantly reduced. These parame- in the female reproductive tract [12, 23]. Moreover, mo- ters are closely related to sperm semen quality and tion kinematics assessed by CASA provide an accurate fertility . Yoon et al. Journal of Animal Science and Biotechnology (2016) 7:67 Page 6 of 12 Fig. 3 Effect of cryopreservation on live sperm capacitation status. a AR pattern. b B pattern. c F pattern assessed by combined CTC/H33258 staining. Data are presented as mean ± SEM. (*P < 0.05, n =9) Capacitation refers to the structural and metabolic alter- which cause damage to sperm surface proteins [5, 10]. ations that allow spermatozoa to fertilize an oocyte [17, 26]. Moreover, cryoprotectant toxicity can induce alterations Without capacitation, spermatozoa lack fertilization ability, in sperm membrane components . For these reasons, although they are motile and morphologically normal . the mechanisms of cryopreservation and the physiological Cryopreservation also results in nonfunctional capacitation, alterations of spermatozoa should be examined at the i.e., premature capacitated spermatozoa. Premature capaci- protein level using sperm proteomic studies. 2-DE is a tation causes an aberrant acrosome reaction in spermato- typical proteomic technique for purifying individual pro- zoa [5, 28]. In our study, the AR pattern significantly teins from various samples . Therefore, we studied the increased and the F pattern significantly decreased after mechanisms of cryostress using the 2-DE technique and cryopreservation. confirmed the results using western blotting. It has been reported that membrane cytoskeletal com- It is generally accepted that transcription, translation, ponents are sensitive to temperature and cryoprotectants, and protein synthesis do not occur in mature Table 1 Differentially expressed (>3-fold) proteins of spermatozoa at different steps of cryopreservation a b Spot no. Protein NCBI no. MASCOT score Expression ratio Search 228 Major fibrous sheath protein chain D, bovine mitochondrial gi|4588120 88.0 3.87 NCBI 1304 F1-ATPase complexed with aurovertin B gi|1827812 183.0 12.57 NCBI 2311 PREDICTED: Uncharacterized protein LOC616410 gi|119914715 299.0 0.25 NCBI 4208 Triosephosphate isomerase gi|61888856 174.0 6.48 NCBI 4418 Nucleoside diphosphate kinase 7 gi|62751773 64.0 6.13 SwissProt 4607 Outer dense fiber protein 2 gi|84000345 290.0 0.46 NCBI 5204 NADH dehydrogenase flavoprotein 2 gi|72004 67.0 5.30 SwissProt 5305 F-actin-capping protein subunit beta gi|28603770 53.0 5.73 NCBI 7206 Superoxide dismutase, mitochondrial gi|88853816 74.0 3.92 NCBI MASCOT scores are −10×log(P), where P is the probability that the observed match is a random event. Individual scores > 40 indicate identity or extensive homology (P < 0.05) Expression ratio is the ratio of relative volume of protein spots with after cryopreservation value to over before Yoon et al. Journal of Animal Science and Biotechnology (2016) 7:67 Page 7 of 12 Fig. 4 2-DE Separation of proteins by 2-DE. 2-DE gels were stained with silver nitrate and analyzed using PDQuest 8.0 software. Protein spots from (a) before cryopreservation and after cryopreservation. b The expression of seven proteins increased significantly after cryopreservation. c The expression of two proteins decreased significantly after cryopreservation. Differentially expressed (>3-fold) proteins were determined by comparing samples before and after cryopreservation (P <0.05, n = 9). The line indicates the landmark of equal levels for before cryopreservation spermatozoa [30, 31]. However, several studies report infertility . The absence of ODF also could result in that mature spermatozoa have the ability to synthesize nonfunctional tails and affect sperm movement . proteins [17, 32, 33]. These studies suggest that an- ODF2 expression decreased after cryopreservation, and other possible mechanism, such as post translational as a result, motility and viability were also found to modifications is involved in cryostress . We decrease. found nine proteins that were differentially expressed SOD2, a member of the superoxide dismutase family, before and after cryopreservation (Table 1). Among is an isozyme of superoxide dismutase and an antioxi- these nine, two proteins (ODF2 and LOC616410) dant . SOD2 can improve cell survival by reducing decreased and seven proteins (SOD2, CAPZB, NDUFV2, the level of ROS [38, 39]. In the testis, it reacts with NDPK, TPI, F1-ATPase, and AKAP) increased after ROS directly and reduces toxicity . During cryo- cryopreservation. preservation, oxidative stress damages spermatozoa . ODF2 is a cytoskeletal structure protein localized in In our study, SOD2 was extremely highly expressed after sperm flagella . It functions in the maintenance of cryopreservation. Therefore, it is tempting to speculate sperm structures and movement . ODF could be that this increased expression reflects a defensive re- consider as a marker of male infertility factor and de- sponse to protect the spermatozoa against oxidative fects of ODF can lead to abnormal morphology and stress during cryopreservation . Yoon et al. Journal of Animal Science and Biotechnology (2016) 7:67 Page 8 of 12 Fig. 5 Expression of SOD and NDUFV2 before and after cryopreservation. a Ratio of SOD2 to α-tubulin expression before and after cryopreservation. b Ratio of NDUFV2 to α-tubulin expression before and after cryopreservation. c Expression of SOD, NDUFV2 and before and α-tubulin after cryopreservation. Data are presented as mean ± SEM. (*P < 0.05, n =9) CAPZB is a member of the F-actin capping protein . This enzyme plays an important role in sperm me- family and a major cytoskeletal protein . CAPZB is tabolism, such as capacitation and the acrosome reaction. involved in F-actin function. F-actin depolymerization Bone et al.  have shown that the inhibition of TPI pre- inhibits capacitation and acrosome reactions . Capping vents capacitation in rat spermatozoa. The decreased F proteins assemble and disassemble filaments in the outer pattern and increased AR pattern in our capacitation acrosomal membrane during capacitation, and these status results might be associated with the high expression mechanisms are important for the induction of the of TPI. TPI also induces early capacitation after cryo- acrosome reaction . The higher expression of CAPZB preservation, resulting in cell death. This protein is after cryopreservation may be related to capacitation-like highly expressed in spermatozoa that have low motility changes during cryopreservation. Therefore, the capacita- parameters or poor freezability . In the current tion status results from our study suggest that the increase study, TPI was highly expressed after cryopreservation in CAPZB is related to changes in the AR and F patterns. (Fig. 4), which may indicate that spermatozoa are dam- NDPK localizes in mitochondria and catalyzes revers- aged during cryopreservation. In addition, our results ible reactions involving a nucleoside triphosphate at the show that viability and motility decreased (Fig. 1). expense of ATP . NDPK is also widely known to Moreover, in human sperm, the levels of TPI were regulate transcription and cell proliferation as well as higher in asthenozoospermic samples than in normos- energy metabolism . Sperm motility is closely associ- permic samples . ated with energy metabolism and excessive metabolic ac- Four of the nine proteins that were differentially tivity could reduce the life span of spermatozoa . We expressed before and after cryopreservation were signifi- believe that it might be associated with the decreased cantly correlated with five signaling pathways (Table 2). viability and motility in our study (Fig. 1). Moreover, These signaling pathways are also associated with sperm NDPK has protective effects against oxidative stress . functions. The ROS metabolism pathway is associated Cryostresses such as oxidative stress and cold shock with SOD2 (Table 2). ROS is necessary for sperm me- cause metabolic and functional changes in spermatozoa tabolismand varioussperm functionssuchasviability, . In our study, NDPK7 increased after cryopreservation capacitation and fertility, however high levels of ROS (Fig. 4). This suggests that oxidative stress and changes in induce oxidative damages to spermatozoa . Oxida- energy metabolism occur to protect spermatozoa during tive damage causes lower motility, DNA damages, and cryopreservation. lipid peroxidation . Membrane phospholipids are TPI is an enzyme that promotes the conversion of dihy- related to cell freezability, which affects sperm motility droxyacetone phosphate to D-glyceraldehyde 3-phosphate and mitochondrial potential [16, 56]. SOD2 is a major Yoon et al. Journal of Animal Science and Biotechnology (2016) 7:67 Page 9 of 12 Fig. 6 Signaling pathways associated with differentially expressed proteins as identified by Pathway Studio. a ROS metabolism is associated with SOD2. b Guanylate cyclase pathway is associated with CAPZB, NDPK, and ODF2. c EphrinR-actin signaling pathway is associated with CAPZB and ODF2. d Actin cytoskeleton regulation associated with CAPZB and ODF2. e Actin cytoskeleton assembly is associated with CAPZB Yoon et al. Journal of Animal Science and Biotechnology (2016) 7:67 Page 10 of 12 Table 2 Signaling pathways associated with differentially biomarkers could be used in future studies to identify the expressed proteins as identified by Pathway Studio mechanism of cryostress responses. Signaling pathways Overlapping entities P-value Abbreviations EphrinR - > Actin Signaling CAPZB, ODF2 0.005 2-DE: Two-dimensional electrophoresis; AKAP: Major fibrous sheath protein; ALH: Amplitude of lateral head displacement; AR: Live acrosome-reacted; ROS metabolism SOD2 0.041 B: Live capacitated; CAPZB: F-actin-capping protein subunit beta; Actin Cytoskeleton Assembly CAPZB 0.016 CASA: Computer-assisted sperm analysis; CTC: Chlortetracycline fluorescence; D: Dead; ESI: Nano-electrospray ionization; F: Live non-capacitated; F1-ATPase: Actin Cytoskeleton Regulation CAPZB, ODF2 0.020 F1-ATPase complexed with aurovertin B; H33258: Hoechst 33258; LIN: Linearity; Guanylate Cyclase Pathway CAPZB, NDPK, ODF2 0.034 MS/MS: Tandem mass spectrometry; NDPK7: Nucleoside diphosphate kinase 7; NDUFV2: NADH dehydrogenase flavoprotein 2; ODF2: Outer dense fiber protein 2; PBS: Phosphate-buffered saline; ROS: Reactive oxygen species; RT: Room temperature; SOD2: Superoxide dismutase 2; STR: Straightness; TOF: Time-of- flight; TPI: Triosephosphate isomerase; TYB: Tris–egg yolk buffer; VAP: Verage path antioxidant, and it reacts with ROS directly to protect velocity; VCL: Curvilinear velocity; VSL: Straight-line velocity; WOB: Wobble against oxidative stress . ROS metabolism is also re- lated the the guanylate cyclase pathway and energy me- Acknowledgments This work was carried out with the support of “Cooperative Research Program tabolism [57, 58]. for Agriculture Science & Technology Development (Project No. PJ01106101)” The guanylate cyclase pathway plays an important role in Rural Development Administration, Republic of Korea. 2+ 2+ Ca influx and ROS metabolism . The uptake of Ca Funding is a key factor in sperm capacitation and accelerates the ac- Funding for this study was provided by “Cooperative Research Program for rosome reaction . In the current study, the alterations Agriculture Science & Technology Development (Project No. PJ01106101)” in capacitation status after cryopreservation may be closely Rural Development Administration (RDA). RDA had no role in the study design, collection, analysis or interpretation of the data, writing of the manuscript, or related to this pathway (Fig. 3). Moreover, guanylate cyclase the decision to submit the paper for publication. is associated with sperm motility and energy metabolism . Our results show that CAPZB, NDPK, and ODF2 are Availability of data and materials The data will not be shared. We provided all data and information on the significantly associated with the guanylate cyclase pathway software, database, and equipment that we used in the present study. In (Table 2). NDPK has protective effects against oxidative addition, we did not use any new software. stress and a strong relationship with energy metabolism Authors’ contributions . The increase in NDPK in our study may have been in- S.J.Y., M.S.R., W.S.K., D.Y.R., and Y.J.P. performed the experiments, analyzed the duced by ROS or excessive energy activity resulting in re- data, and drafted the manuscript. M.G.P. supervised the design of the study duced motility and viability (Fig. 1). Moreover, the increase and data analysis and revised the manuscript. All authors critically reviewed the manuscript for intellectual content and gave final approval for the version in SOD2 also can be explained by excessive ROS caused by to be published. cryopreservation (Fig. 1, Table 1). CAPZB was also associated with actin cytoskeleton as- Competing interests The authors have declared that there are no conflicts of interests that could sembly and actin regulation pathways (Table 2). These be perceived as prejudicing the impartiality of this paper. pathways are involved in sperm capacitation and the acro- some reaction . Phosphatidylinositol (4, 5)-bisphosphate Consent for publication Not applicable. and phosphatidylinositol (3, 4, 5)-trisphosphate in these pathways play important roles in the regulation of actin Ethics approval and consent to participate polymerization . Sperm capacitation and the acrosome All procedures were performed according to the guidelines for the ethical reaction are closely associated with actin polymerization treatment of animals by the Institutional Animal Care and Use Committee of Chung-Ang University, Seoul, Korea. . F-actin in the actin-related pathways plays a major role in the remodeling of actin structure, the acrosome reaction, Received: 29 June 2016 Accepted: 1 November 2016 and male fertility . It is possible that the significant alterations of capacitation status in our results are related References to these pathways (Fig. 3). 1. Olaciregui M, Gil L, Monton A, Luno V, Jerez RA, Marti JI. Cryopreservation of epididymal stallion sperm. Cryobiology. 2014;68(1):91–5. 2. Mota Filho AC, Silva HV, Nunes TG, de Souza MB, de Freitas LA, de Araujo AA, Conclusions et al. Cryopreservation of canine epididymal sperm using ACP-106c and TRIS. 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Journal of Animal Science and Biotechnology – Springer Journals
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