Differential Proteomic Analysis of Carbon Ion Radiation in Sheep Sperm

Journal of Integrative Agriculture2013, 12(9): 1629-1637 September 2013

© 2013, CAAS. All rights reserved. Published by Elsevier Ltd.doi:10.1016/S2095-3119(13)60558-4

RESEARCH ARTICLE

Differential Proteomic Analysis of Carbon Ion Radiation in Sheep Sperm

HE Yu-xuan1*, LI Hong-yan2, 3, 4, 5*, ZHANG Yong1, HE Jian-hua6, ZHANG Hong2, 3, 4 and ZHAO Xing-xu1

1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, P.R.China2 Department of Medical Physics, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, P.R.China3 Key Laboratory of Heavy Ion Radiation Medicine, Chinese Academy of Sciences, Lanzhou 730000, P.R.China4 Key Laboratory of Heavy Ion Radiation Medicine of Gansu Province, Lanzhou 730000, P.R.China5 University of Chinese Academy of Sciences, Beijing 100049, P.R.China6 Lanzhou Agricultural Science and Technology Research Promotion Center, Lanzhou 730030, P.R.China

Abstract

This study is first to investigate proteomic changes in sheep sperm induced by carbon ion radiation using two-dimensionalelectrophoresis (2-DE) analysis in the project of breeding a new variety of sheep. Differential expression proteins weredetected using the PDQuest 8.0 software after staining with Coomassie blue. Valid spots were then analyzed throughliquid chromatography tandem mass spectrometry (LC-MS/MS). Among the 480 total protein spots displayed in 2-D gels,6 specific protein spots were observed in sperm gels. A search against protein sequences in the National Center forBiotechnology Information databases (NCBI) indicated that differentially expressed proteins correspond to two proteins,identified to be enolase and transcription factor AP-2-alpha (TFAP-2α). The two proteins were up-regulated in the irradiatedsperm. To the best of our knowledge, this study is the first to identify proteomic changes induced by carbon ion radiation insheep sperm. The analysis of differential expression protein may be useful in identifying new breeding markers in sheepreproduction and in clarifying the mechanisms involved in irradiation or space breeding.

Key words: sheep, sperm protein, two-dimensional polyacrylamide gel electrophoresis analysis, proteome, carbon ion radiation, irradiation breeding

Received 26 March, 2012 Accepted 28 May, 2012HE Yu-xuan, E-mail: [email protected]; LI Hong-yan, E-mail: [email protected]; Correspondence ZHAO Xing-xu, Tel: +86-931-7632482, E-mail: [email protected]; ZHANG Hong, Tel: +86-931-4969344, E-mail: [email protected]* These authors contributed equally to this study.

INTRODUCTION

Irradiation technology has been widely used for mu-tagenic breeding of rice (Sung et al. 2005), maize (Liuet al. 2012) and wheat plants (Wang et al. 2012); how-ever reports on this technology in mammalian repro-duction are rare. Using a large sample without involv-ing a great expense for source material is possible inplants. Unlike their plant breeding colleagues, usingradiation in large animals requires large investment foreach animal. Furthermore, any misjudgment of theproper doses, which may kill the breeding materials, is

not notable in plants, but would result in large fatal lossin animals. Therefore, animals cannot be directly usedfor irradiation breeding because of the various types ofcells involved, wherein the effects after radiation wouldbe difficult to predict. Only germ cells have been cho-sen in experiments since this type of cells can transmitthe beneficial effects of mutation. The most widelyused sample of this cell is poultry egg, which has beenused for decades to study the effects of ion radiation(Verde et al. 2004; Daòová et al. 2010).

Sheep sperm was used as experimental material inthe current study. On one hand, mammalian sperm is adeceptively simple and terminally differentiated cell

1630 HE Yu-xuan et al.

© 2013, CAAS. All rights reserved. Published by Elsevier Ltd.

(Ramalho-Santos et al. 2007), which is very stable andnot susceptible to mutation. Zhang et al. (1999) re-ported that heavy ion at low doses (0.5, 1, and 2 Gy)improves human sperm motility and acrosome reaction,demonstrating that low dose of heavy ion may improvesperm vitality in vitro. On the other hand, sperms canbe easily carried to outer space to accomplish spacebreeding. Sperms can be used to conduct fertilizationin vitro (IVF) after irradiation, thus, sperm is the bestmaterial for this experiment.

Proteomics was used to elucidate the biochemicaland biophysics mechanisms, as well as to discover dif-ferentially expressed proteins that control animal pro-duction and quality (Miarelli and Signorelli 2010). Study-ing sperms at DNA/RNA level is difficult due to theirunusual structure. Therefore, it is more practical tomake some progress at its molecular level by studyingthe functions of sperm proteins using proteomic ways(Peddinti et al. 2008). The biological effects of highlinear energy transfer (LET) heavy ions show certainbiological advantages compared with low LET radiation,such as X- or γ-rays (Breakely and Kronenberg 1998;Murakami et al. 2001; Lee et al. 2005; Pathak et al.2007). However, the biological effects of radiation havebeen extensively reported to impact proteins in cells(Zhang et al. 2006). Some important players, such asDNA ligase IV/XRCC4 (X-ray cross complementationgroup 4), DNA-dependent protein kinase (DNA-PK),Ku70 and Ku80, and Rad51 protein and its paraloguesRad52 and Rad54 play crucial roles during radiation(Su et al. 2009). Furthermore, distinct changes innumbers, kinds, and activities of total soluble proteins,esterase isozyme, and peroxidase isozyme have beenobserved after low-energy ion implantation (Guijunet al. 2006).

The current experiment, as part of the project ofbreeding a new variety of sheep, represents a prelimi-nary approach to a comparative study of irradiated sheepsperm via comparative proteomics. This investigationis conducted to detect expressed proteins in irradiatedand control sperms through two-dimensional electro-phoresis (2-DE) as a platform for parallel analysis, andto identify differential proteins using an ion trap massspectrometer equipped with high performance liquidchromatography (HPLC) system. In addition, this in-vestigation is conducted to probe the mechanism of

irradiation or space breeding, evaluate the damage in-duced by radiation, and find new biomarkers of breed-ing-associated proteins for domestic animals.

RESULTS

2-DE map results of sperm protein

The match rate of the 2-DE gels was 65%. A total of480 proteins were detected in the proteome profile ofthe sperm gels in Fig. 1. The MW and pI of theseproteins ranged from 14.4 to 116 kDa (the scope ofprotein marker) and 4.00 to 6.72 kDa, respectively. Inaddition, obvious proteome difference between irradi-ated and unirradiated sperm was observed. Six differ-entially expressed proteins were found, 5 protein spotswere up-regulated, and 1 protein spot was absent in theirradiated sperm gels.

2-DE map results of seminal plasma protein

A total of 275 individual protein spots were detectedand compared in Fig. 2. The match rate of 2-DE gelswas 63%. No differentially expressed proteins werefound between the two groups.

Analysis and identification of differentiallyexpressed proteins

The six differentially expressed proteins were detectedusing the PDQuest 8.0 software. Spot numbers corre-spond to the first column of Table 1, which shows theidentified proteins. Spot 6 was absent and other pro-tein spots were up-regulated in the irradiated sperm.The LC-MS/MS system identified all six spots. Thedifferential spots were excised from the gels and tryp-tic peptides were extracted. The peptides were thenautomatically analyzed through HPLC equipped withion trap mass spectrometer. Protein identification wascarried out via peptide sequencing using the search pro-grams of SEQUEST. They were then searched againstthe National Center for Biotechnology Information da-tabase (NCBI). Fig. 1 illustractes the representativesperm protein profiles. The six differentially expressedproteins were identified to be two proteins, are listed in

Differential Proteomic Analysis of Carbon Ion Radiation in Sheep Sperm 1631


Table 1 List of up-regulated proteins in irradiated sperm

Protein spot Protein name Accession pI/MW Sequence Score Coverage (%)1 Enolase (Ovis aries) 7331113 6.18/23 056.17 HAGNKLAMQEFMILPVGASSFREAMRIGAEVYHHL 30.2 12.73

KGVIKAKYGKDATNVGDEGGFAPNILENNEALELLKTAIQAAGYPDKVVIGMDVAASEFYRNGKYDLDFKSPDDP

2 Transcription factor 57619230 9.22/53 927.00 MPPRLASVKIPYDWGRKGPFRFWRIFCQSRAVGWF 30.1 4.09AP-2-alpha (O. aries) LAAACGRAGRFRTQPAEWPTPDAVFSPLGLALFQD

RHDGASNGTARLPQLGTVGQSPYTSAPPLSHTPNADFQPPYFPPPYQPIYPQSQDPYSHVNDPYSLNPLHAQPQPQHPGWPGQRQSQESGLLHTHRGLPHQLSGLDPRRDYRRHEDLLHGPHGLGSGLGDLPIHSLPHAIEDVPHVEDPGINIPDQTVIKKGPVSLSKSNSNAVSSIPINKDNLFGGVVNPNEVFCSVPGRLSLLSSTSKYKVTVAEVQRRLSPPECLNASLLGGVLRRAKSKNGGRSLREKLDKIGLNLPAGRRKAANVTLLTSLVEGEAVHLARDFGYVCETEFPAKAVAEFLNRQHSDPNEQVTRKNMLLATKQICKEFTDLLAQDRSPLGNSRPNPILEPGIQSCLTHFNLISHGFGSPAVCAAVTALQNYLTEALKAMDKMYLSNNPNSHTDNSAKSSDKEEKHRK

The underlines stand for the partial peptides of spots matched with the amino acids. pI, isoelctric point; MW, molecular weight marker.

Fig. 1 2-DE gels of control (A) and irradiated sperm (B). Marked spot numbers refer to numbers in Table 1. MW, molecular weight marker;IEF, isoelectric focusing; SDS, sodium dodecyl sulphate polyacrylamide gel electrophoresis.

Fig. 2 2-DE gels of control (A) and irradiated seminal plasma (B). The missing protein in irradiated sperm was not found in 2-DE gel ofirradiated seminal plasma.



Table 1. Spot 1 and spot 2 correspond to enolase andTFAP-2α, respectively. Fig. 3 shows the 2-DE pro-files of enolase and TFAP-2α and the normalized spotvolume of these two proteins. Compared with the con-trol group, the volume of enolase and TFAP-2α in the0.5 Gy group significantly increased, respectively(P<0.01). Fig. 4 shows that MS/MS analysis of thedouble-charged peptide ions with an m/z ratio of726.62. The N- and C-terminal fragments of the pep-tide produced through breakage at the peptide bond inLCQ were designated b and y, respectively.

DISCUSSION

Selection of irradiation dose

It was indicated that proteins are major initial targets offree radicals in cells, and the damage to DNA and lipidsis a secondary process after radiation (Distel et al.2006). High doses of IR have been demonstrated toinhibit overall protein synthesis in highly transformedcells by acting on the cap initiation complex or by re-ducing levels of initiation factor eIF4G, a member ofthe complex. Most mRNAs are translated in mamma-

lian cells through a cap-dependent mechanism and arethought to use the cap initiation complex to recruit andassemble scanning ribosome (Braunstein et al. 2009).A radiation dose of 0.5 Gy was selected in this study toinvestigate the proteomic changes in sheep sperm in-duced by middle dose carbon ion radiation, and to en-sure the genetic stability is at maximum.

The purpose of analysis seminal plasma

Protein changes in human sperm after cryopreservationwere studied. The expression of heat-shock protein 90obviously decreased, which was not found in seminalplasma. The result showed that heat-shock protein 90cannot leak from the sperm, but is degraded or modi-fied in the process of cryopreservation. Seminal plasmawas analyzed to find whether differentially expressedproteins leaked from the sperm, which indirectly vali-dated the impact of radiation on the sperm plasmamembrane. This study showed that the absence ofprotein was due to the degradation or modification inthe process of irradiation.

Enolase

Enolase was discovered in 1934 by Lohman andMayerhof (Pancholi et al. 2001), it is a glycoltic en-zyme that catalyzes to generate two molecules of ATP(Andre et al. 2002). Dynein proteins provide move-ment for sperm tail and tail use energy from ATP hy-drolysis to slide microtubules. When glucose is con-verted to pyruvate by glycolysis, two molecules of ATPare released (Gitlitsa et al. 2000). Two isoforms inhuman sperm were designated as ENO-S and ENO-αα. The two isoforms were provided two parametersto describe different aspects of the sperm: ENO-ααcharacterizes abnormal immature sperm, whereas ENO-S characterizes normally developed sperm (Andreet al. 2002). Proteome changes in sheep sperm aftercryopreservation were studied, we found the enolasewas down-regulated in frozen-thawed sperm and con-cluded that the under-expression of enolase may re-lated with the decreased of sperm motility aftercryopreservation (Li et al. 2011). Enolase was up-regu-lated in the irradiated sheep sperm in the current study,this result was consistent with our previous results that

Fig. 3 A, 2-D profiles of enolase and TFAP-2α. B, relativeexpression level of differentially expressed proteins between enolaseand TFAP-2α. The height of the columns represented the relativeexpressions of the protein spots. **, P<0.01.



irradiated 0.5 and 3 Gy, respectively, in sheep sperm(Li et al. 2011). The over-expression of enolase lead-ing to glycolysis can generate more ATP to protect thesperm against or reduce damage in the process of irra-diation and improve sperm motility.

TFAP-2α

Activator protein 2 (TFAP-2) is a sequence-specificDNA-binding transcription factor known to regulateeither activating or repressing effect on the target genedirectly or indirectly (Choi et al. 2008). TFAP-2, firstcloned by Williams, regulates several aspects of cellproliferation, differentiation, and death (Nyormoiet al. 2001). The expression of TFAP-2α is associ-a ted wi th the embryonic d i f ferent ia t ion ofneuroectodermal, urogenital, and ectodermal tissues(Müller et al. 2004). Overexpression of TFAP-2α inN-ras-transformed teratocarcinoma cells correlates

with resistance to retinoic acid-induced cell differ-entiation (Nyormoi et al. 2001). Recently, some docu-ments have demonstrated that reduced expression ofTFAP-2α is associated with cancer progression invarious cancers (Zhao et al. 2001). The overexpressionof TFAP-2α can suppress the invasion of ovarian can-cer cells (Zhang et al. 2000). The over-expressedTFAP-2α can activate some protection mechanismsand suppress abnormal death of sheep sperm in theprocess of irradiation.

CONCLUSION

Differential expression of sheep sperm proteins wasinvestigated after irradiation in the current research.Enolase and TFAP-2α were up-regulated in the irradi-ated sperm. This study investigated for the first timethe effect of sheep sperm by heavy ion radiation at

Fig. 4 MS/MS analysis of the double-charged peptide ions with an m/z ratio of 726.62. b and y designate the N- and C-terminal fragmentsof the peptide produced by breakage at the peptide bond in LCQ, respectively. A, VVIGMDVAASEFYR was the detected peptide fragment.B, LSPPECLNASLLGGVLRRAK was the detected peptide fragment.



proteomic level. Proteomics can be used as a tool tounderstand the mechanisms of irradiated sperm andevaluate the damage induced by radiation. Moreover,protein identification may provide the basis for discov-ering potential biomarkers of irradiation or space breed-ing in domestic animal.

MATERIALS AND METHODS

Chemicals

Two-dimensional gel electrophoresis (2-DE) marker, Tris,ammonium persulfate (AP), sodium dodecyl sulfate(SDS), N,N,N ,́N -́tetramethylethylene diamine (TEMED),glycin, acrylamide N,N -́methylenebisacrylamide, bro-mophenol blue, Coomassie brilliant blue (CBB) G-250,thiourea, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), glycerol, bovine serum al-b u m i n ( B S A ) , a g a r o s e , D i t h i o t h r e i t o l ( D T T ) ,iodoacetamide, trypsin, acetonitrile and urea were pur-chased from Sigma-Aldrich Chemical Company (USA).Immobilized pH gradients (IPG) strip (170 mm×63 mm;pH 4-7; non linear), Bio-lyte (pH 4-7) and mineral oilwere purchased from Bio-Rad Laboratories Ltd. (USA).2-D Clean-Up Kit was purchased from AmershamPharmacia (GE, Sweden).

Doses collection

Our group conducted several studies on selecting doses,which were divided into six dose groups including 0.1, 0.2,0.3, 0.4, 0.5, and 1 Gy. DNA integrity was then evaluatedthrough comet assay experiments. Sperm DNA damagewas not obvious when doses were lower than 0.5 Gy; thus,0.5 Gy was used in this study.

Semen collection, dilution and examination

Semen was collected using an artificial vagina from fivemature and healthy Dorset male sheep, and was well mixed.Semen (1 mL) was mixed with 1 mL of diluents (2.422 gTris, 1.34 g citric acid, 0.5 g fructose, 500 IU benzylpeni-cillin and streptomycin in 100 mL of deionized water)(Hafez et al. 2000; Triwulanningsih et al. 2010). The mix-ture was then divided into two groups (2 mL for eachgroup), protected from light, and incubated at 37°C for 30min. Smears were prepared for motility examination. Atleast 200 spermatozoa were subsequently evaluated un-der light microscope at ×200 magnification (Nikon, Japan)(Janetta et al. 2009).

Irradiation

Heavy ion beam current was 12C6+ at 200 MeV/U and 31.3keV �m-1 of the beam entrance, which was supplied by theheavy ion research facility in Lanzhou (HIRFL) at the Insti-tute of Modern Physics, Chinese Academy of Sciences(Lanzhou, China). The dose rate was approximately 0.5 Gymin-1 with the dose at 0.5 Gy. Air isolation room was usedto detect the dose. The acquisition of dose data was auto-matically controlled by a computer. Particle fluence wasdetermined from an air-ionization chamber signal accord-ing to the calibration of the detector (PTW-UNIDOS, PTW-Freiburg Co., Wiesbaden, Germany). One group was irra-diated at room temperature. Unirradiated semen was usedas the control. Two groups were first placed in a bucket at37°C after irradiation, and were then brought back to thelaboratory immediately.

Sperm proteins extraction

Sperm swim-up technique was used to float the sperm. Bothsemen samples were centrifuged at 400×g for 15 min, andsupernatants were collected and labeled for the next step.The pellets were suspended in pre-warmed 2.5 mL of DMEMculture medium and kept at 37°C for 1 h in 5% CO (Jameelet al. 2008). The supernatant was collected, and 10 �L wasdropped on the cell counter for counting. The cells werecollected via centrifugation at 6 000×g for 5 min, and thenthe supernatant was discarded. Sediments were resuspendedwith 0.25 mol L-1 sucrose (containing 5 mmol L-1 Tris) andcentrifuged at 5 000×g for 10 min. Sperm proteins were pre-pared using the Trizol method. About 5×107 sperm cellswere mixed with 1 mL of Trizol (Kirkland et al. 2006). Spermproteins were dissolved with 10 �L of lysis solution (8 mol L-1

urea, 2 mol L-1 thiourea, 4% CHAPS, 65 mmol L-1 DTT), andtotal protein concentration was measured using the Bradfordprotein assays (Bradford et al. 1976).

Seminal plasma proteins extraction

The labeled supernatants contained seminal plasma ofboth groups. Seminal plasma proteins were prepared us-ing the acetone precipitation method (Chen et al. 2005),and then treated with the 2-D Clean-Up Kit. Briefly, thesupernatants were precipitated with four-fold cold ac-etone overnight. The sediments were rinsed three timeswith 90% cold alcohol, centrifuged at 5 000×g for 10 min,and then dried at room temperature. Finally, the sedi-ments were treated with a 2-D Clean-Up Kit. Seminalplasma proteins were dissolved with 10 �L of lysis solu-tion (8 mol L-1 urea, 4% CHAPS, 65 mmol L-1 DTT), andtotal protein concentration was measured using theBradford protein assays (Bradford et al. 1976).



2-DE, image analysis

Sperm (450 �g) and seminal proteins (600 �g) were uni-formly mixed with 350 �L of loading buffer (8 mol L-1 urea,2% CHAPS, 65 mmol L-1 DTT, 0.2% (w/v) Bio-Lyte 4-7ampholytes, and bromophenol blue (trace)). The mixtureswere then transferred to the tray for IEF using a ProteanIEF cell (Bio-Rad Laboratories, USA). After focusing, IPGstrips were equilibrated to reduce protein disulfide bondsin 10 mL equilibrating solution (6 mol L-1 urea, 30% (v/v)glycerol, 2% (w/v) SDS, 50 mmol L-1 Tris-HCl, pH 8.8, 1%(w/v) DTT) for each strip, with gentle rocking for 15 min.The free cysteine residues of the proteins were then alky-lated to prevent the reformation of disulfide bonds by rock-ing each strip for 15 min in a 10 mL solution (6 mol L-1 urea,30% (v/v) glycerol, 2% (w/v) SDS, 50 mmol L-1 Tris-HCl, pH8.8, 2.5% (w/v) iodoacetamide). Subsequently, the stripswere affixed onto homogeneous 12% polyacrylamide SDS-PAGE gels (30% acrylamide solution, 1.5 mol L-1 Tris (pH8.8), 10% SDS, 10% ammonium persulfate, TEMED andultrapure water) using a Protean II xi cell (Bio-RadLaboratories, USA) (Bartosz et al. 2006; Reinhardt et al.2009). SDS-PAGE gel was stained with Coomassie Brilliantblue G-250 solution (each 500 mL contained 50 g ammo-nium sulfate, 50 mL phosphoric acid, 100 mL methanol, anddeionized water), and then placed on a rocker and shakenfor at least 3 h (Neuhoff et al. 1988). The stained gels weredecolored three times using deionized water, and thenscanned using the molecular imager pharos FX (Bio-RadLaboratories, USA). The images were analyzed and quanti-fied using the PDQuest 8.0 software (Bio-Rad Laboratories,USA) to identify valid spots. PDQuest spot detection soft-ware was used with appropriate selection of the faintest andthe smallest spots and a large representative section of theimage containing spots, steaks, and background gradationto correct the noise. Three independent samples were re-peated three times for each pooled sample. Gel images wereprocessed to remove backgrounds and to automatically de-tect protein spots. Normalization against the total intensityof all spots present in the gel was done for comparisons ofprotein levels among gels. Hence, only different proteinspots in the gel pairs and successful analysis by mass spec-trometry are listed as differently expressed proteins. ANOVA(ver. 17.0, SAS Institute, Cary, N.C., USA) was used to testthe significance of the relative expression of identified pro-teins in all gels.

In-gel digestion, MS/MS protein identification anddatabase search

Differential spots of protein were excised manually by us-ing pipette tips which were clipped off to form an orificewith inner diameter of 2 mm. Each spot was placed into a1.5 mL microtube. The particles were washed twice with

ultrapure water for 15 min, and then twice with ultrapurewater/acetonitrile (1:1, v/v) for 15 min at room temperature.After washing gel pieces were shrunk in acetonitrile anddried, then subjected to incubation in 50 mmol L-1 NH4HCO3containing 0.01% sequence-grade trypsin (5 ng mL-1 forfaint and 10 ng mL-1 for stronger spots) at 37°C overnight.Then, the peptides were extracted twice with 50% acetoni-trile and 0.2% formic acid, applying one round of vortexingand sonication (20 min each).

Peptide mass was determined on an ion trap mass spec-trometer (LCQ Deca XP, Thermo Finnigan, USA) equippedwith a Surveyor HPLC system (Thermo). Peptides wereidentified using SEQUEST software (Bioworks 2.0, ThermoFinnigan), which used the tandem mass spectra of peptideions to search against the publicly available NCBInonredundant protein database (http://www.ncbi.nlm.nih.gov). The protein identification criteria were based onDelta CN ( 0.1) and cross-correlation scores (Xcorr, onecharge 1.9, two charges 2.2, three charges 3.75) (Yanget al. 2009).

AcknowledgementsThis work was supported by the Key Scientific Technol-ogy Research Projects of Gansu Province, China(1102NKDA022), the Major Program of Ministry of Agri-culture to Cultivate New Varieties of Genetically ModifiedOrganisms of China (2008ZX08008-003), the National Ba-sic Research Program of China, the National Natural Sci-ence Foundation of China (2010CB834202).

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(Managing editor ZHANG Juan)

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Differential Proteomic Analysis of Carbon Ion Radiation in Sheep Sperm