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• W2892462906 abstract "Proteins are essential for sperm function, including their fertilizing capacity. Pig spermatozoa, emitted in well-defined ejaculate fractions, vary in their functionality, which could be related to different sperm protein composition. This study aimed (i) to update the porcine sperm proteome and (ii) to identify proteins differentially expressed in mature spermatozoa from cauda epididymis and those delivered in separate ejaculate fractions. Ejaculates from nine mature and fertile boars were manually collected in three separate portions: the first 10 ml of the sperm-rich ejaculate fraction (SRF), the rest of the SRF and the post-SRF. The contents of cauda epididymides of the boars were collected post-mortem by retrograde duct perfusion, generating four different semen sources for each boar. Following centrifugation, the resulting pellets of each semen source were initially pooled and later split to generate two technical replicates per source. The resulting eight sperm samples (two per semen source) were subjected to iTRAQ-based 2D-LC-MS/MS for protein identification and quantification. A total of 1,723 proteins were identified (974 of Sus scrofa taxonomy) and 1,602 of them were also quantified (960 of Sus scrofa taxonomy). After an ANOVA test, 32 Sus scrofa proteins showed quantitative differences (p < 0.01) among semen sources, which was particularly relevant for sperm functionality in the post-SRF. The present study showed that the proteome of boar spermatozoa is remodeled during ejaculation involving proteins clearly implicated in sperm function. The findings provide valuable groundwork for further studies focused on identifying protein biomarkers of sperm fertility. Proteins are essential for sperm function, including their fertilizing capacity. Pig spermatozoa, emitted in well-defined ejaculate fractions, vary in their functionality, which could be related to different sperm protein composition. This study aimed (i) to update the porcine sperm proteome and (ii) to identify proteins differentially expressed in mature spermatozoa from cauda epididymis and those delivered in separate ejaculate fractions. Ejaculates from nine mature and fertile boars were manually collected in three separate portions: the first 10 ml of the sperm-rich ejaculate fraction (SRF), the rest of the SRF and the post-SRF. The contents of cauda epididymides of the boars were collected post-mortem by retrograde duct perfusion, generating four different semen sources for each boar. Following centrifugation, the resulting pellets of each semen source were initially pooled and later split to generate two technical replicates per source. The resulting eight sperm samples (two per semen source) were subjected to iTRAQ-based 2D-LC-MS/MS for protein identification and quantification. A total of 1,723 proteins were identified (974 of Sus scrofa taxonomy) and 1,602 of them were also quantified (960 of Sus scrofa taxonomy). After an ANOVA test, 32 Sus scrofa proteins showed quantitative differences (p < 0.01) among semen sources, which was particularly relevant for sperm functionality in the post-SRF. The present study showed that the proteome of boar spermatozoa is remodeled during ejaculation involving proteins clearly implicated in sperm function. The findings provide valuable groundwork for further studies focused on identifying protein biomarkers of sperm fertility. Spermatozoa are highly differentiated, structurally complex and dynamic cells that deliver the paternal genome/epigenome to mature oocytes. To achieve this goal, the spermatozoon must attain full fertilization capacity, which implies they underwent molecular and/or functional maturational changes in the epididymis to later display an active forward movement, undergo capacitation, zona binding and the acrosome reaction, and chromatin decondensation during fertilization (1Gadella B.M. Reproductive tract modifications of the boar sperm surface.Mol. Reprod. Dev. 2017; 84: 822-831Crossref PubMed Scopus (13) Google Scholar). Most of these essential functional processes involve modifications in the expression of proteins through sperm interaction with the surrounding environment, as evidenced in spermatozoa from rodents, humans and livestock species, including pigs (2Park Y.J. Kim J. You Y.A. Pang M.G. Proteomic revolution to improve tools for evaluating male fertility in animals.J. Proteome Res. 2013; 12: 4738-4747Crossref PubMed Scopus (35) Google Scholar, 3Mohanty G. Swain N. Samanta L. Sperm Proteome: What is on the horizon?.Reprod. Sci. 2015; 22: 638-653Crossref PubMed Scopus (8) Google Scholar, 4Dostàlovà Z. Calvete J.J. Sanz L. Töpfer-Petersen E. Quantitation of boar spermadhesins in accessory sex gland fluids and on the surface of epididymal, ejaculated and capacitated spermatozoa.Biochim. Biophys. Acta. 1994; 1200: 48-54Crossref PubMed Scopus (103) Google Scholar, 5Ensslin M. Vogel T. Calvete J.J. Thole H.H. Schmidtke J. Matsuda T. Töpfer-Petersen E. Molecular cloning and characterization of P47, a novel boar sperm-associated zona pellucida-binding protein homologous to a family of mammalian secretory proteins.Biol. Reprod. 1998; 58: 1057-1064Crossref PubMed Scopus (83) Google Scholar, 6Li J. Liu F. Liu X. Liu J. Zhu P. Wan F. Jin S. Wang W. Li N. Liu J. Wang H. Mapping of the human testicular proteome and its relationship with that of the epididymis and spermatozoa.Mol. Cell. Proteomics. 2011; 10M110.004630Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 7Skerget S. Rosenow M.A. Petritis K. Karr T.L. Sperm Proteome Maturation in the Mouse Epididymis.PLoS ONE. 2015; 10: e0140650Crossref PubMed Scopus (74) Google Scholar). Because the ejaculate contains a heterogeneous suspension of spermatozoa, sperm sub-populations of different quality and functionality are present (8Roca J. Parrilla I. Gil M.A. Cuello C. Martinez E.A. Rodriguez-Martinez H. Non-viable sperm in the ejaculate: Lethal escorts for contemporary viable sperm.Anim. Reprod. Sci. 2016; 169: 24-31Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). D'Amours et al. (9D'Amours O. Frenette G. Bourassa S. Calvo É Blondin P Sullivan R. Proteomic markers of functional sperm population in bovines: Comparison of low- and high-density spermatozoa following cryopreservation.J. Proteome Res. 2018; 17: 177-188Crossref PubMed Scopus (22) Google Scholar) suggested that qualitative and/or quantitative differences in protein expression among these sperm subpopulations probably resulted in an unbalanced interaction between spermatozoa and the changing surrounding environment during ejaculation. This noticeable hypothesis, derived from empirical studies of ejaculated bull sperm sub-populations artificially generated through cell separation methods, needs testing. The porcine is an excellent model for such testing because, as in human, the ejaculate is emitted in fractions, where the cauda epididymal spermatozoa are sequentially exposed to the differential secretion of the sexual accessory glands, building separate ejaculate fractions (10Rodríguez-Martínez H. Kvist U. Saravia F. Wallgren M. Johannisson A. Sanz L. Peña F.J. Martínez E.A. Roca J. Vázquez J.M. Calvete J.J. The physiological roles of the boar ejaculate.Soc. Reprod. Fertil. Suppl. 2009; 66: 1-21PubMed Google Scholar). These different fractions/environments apparently impose remarkable differences in the capability of spermatozoa to withstand technologies such as cryopreservation (11Saravia P. Wallgren M. Johannisson A. Calvete J.J. Sanz L. Peña F.J. Roca J. Rodríguez-Martínez H. Exposure to the seminal plasma of different portions of the boar ejaculate modulates the survival of spermatozoa cryopreserved in MiniFlatPacks.Theriogenology. 2009; 71: 662-675Crossref PubMed Scopus (64) Google Scholar, 12Alkmin D.V. Perez-Patiño C. Barranco I. Parrilla I. Vazquez J.M. Martinez E.A. Rodriguez-Martinez H. Roca J. Boar sperm cryosurvival is better after exposure to seminal plasma from selected fractions than to those from entire ejaculate.Cryobiology. 2014; 69: 203-210Crossref PubMed Scopus (44) Google Scholar, 13Li J. Barranco I. Tvarijonaviciute A. Molina M.F. Martinez E.A. Rodriguez-Martinez H. Parrilla I. Roca J. Seminal plasma antioxidants are directly involved in boar sperm cryotolerance.Theriogenology. 2018; 107: 27-35Crossref PubMed Scopus (45) Google Scholar). A recent study (14Li J. Roca J. Perez-Patiño C. Barranco I. Martinez E.A. Rodriguez-Martinez H. Parrilla I. Is boar sperm freezability more intrinsically linked to spermatozoa than to the surrounding seminal plasma?.Anim. Rep. Sci. 2018; Crossref Scopus (16) Google Scholar) suggested differences in the proteome of spermatozoa in the different boar ejaculate fractions. The present study aimed to update the porcine sperm proteome (15Feugang J.M. Liao S.F. Willard S.T. Ryan P.L. In-depth proteomic analysis of boar spermatozoa through shotgun and gel-based methods.BMC Genomics. 2018; 19: 62Crossref PubMed Scopus (21) Google Scholar), taking advantage of the increased annotation of protein-coding genes (16Marx H. Hahne H. Ulbrich S.E. Schnieke A. Rottmann O. Frishman D. Kuster B. Annotation of the domestic pig genome by quantitative proteogenomics.J. Proteome Res. 2017; 16: 2887-2898Crossref PubMed Scopus (22) Google Scholar). In addition, it aimed to identify and measure, using isobaric tags for relative and absolute quantification (iTRAQ) 1The abbreviations used are:iTRAQisobaric tags for relative and absolute quantificationSRFsperm-rich ejaculate fractionAIartificial inseminationVTHveterinary teaching hospitalSPseminal plasmaTEABtriethylammonium bicarbonate bufferTCEP2-carboxyethyl phosphineMMTSS-methyl methanethiosulphonateFAformic acidFDRfalse discovery ratePCAprincipal component analysisMeVmulti-experiment viewerFCfold changeGOgene ontologyDDI1DNA damage inducible 1 homolog 1CES1Carboxyl ester hydrolasepB1porcine B1BSPbovine seminal plasmaFN1Fibronectin 1TSP1Thrombospondin-1ROSreactive oxygen speciesWGAwheat germ agglutininCOL18A1type XVIII Collagen α1 chain., eventual putative differences in protein composition between mature spermatozoa retrieved from the cauda epididymis and from the most representative ejaculate fractions, specifically the sperm peak (first 10 ml of the sperm-rich ejaculate fraction, SRF), the remaining SRF and the post-SRF. This approach allowed the measurement of the exchange in proteins experienced by the spermatozoa during ejaculation and provided evidence for the relevance of the different sexual accessory glands in such exchange. isobaric tags for relative and absolute quantification sperm-rich ejaculate fraction artificial insemination veterinary teaching hospital seminal plasma triethylammonium bicarbonate buffer 2-carboxyethyl phosphine S-methyl methanethiosulphonate formic acid false discovery rate principal component analysis multi-experiment viewer fold change gene ontology DNA damage inducible 1 homolog 1 Carboxyl ester hydrolase porcine B1 bovine seminal plasma Fibronectin 1 Thrombospondin-1 reactive oxygen species wheat germ agglutinin type XVIII Collagen α1 chain. All procedures involving animals followed international guidelines (Directive 2010/63/EU), including the approval of the Bioethics Committee of Murcia University (research code: 639/2012) and of the Local Ethical Committee for Experimentation with Animals at Linköping, Sweden (permit nr ID-1400). Semen and cauda epididymal contents were retrieved from nine mature and fertile boars (Sus scrofa) of different breeds and crossbreds. The boars were housed in an artificial insemination (AI) center (Topigs Norsvin España) located in Murcia (Spain) and regularly used in conventional AI programs (two ejaculates collected per week). The boars were kept under the same management conditions, housed in individual pens under controlled regimens of temperature (15–25 °C) and light (16 h per day), with free access to water, and fed with commercial feedstuff for mature boars. One ejaculate per boar was manually collected in separate portions, specifically the first 10 ml of the SRF (P1), the remaining SRF (P2), and the post-SRF (P3) using the gloved-hand method. A proportional mixture of each ejaculate fraction was used to verify that all collected ejaculates fulfilled the standard thresholds of sperm quantity and quality needed for the preparation of AI semen doses, specifically more than 200 × 106 sperm/ml (SP-100 NucleoCounter; ChemoMetec A/S, Allerød, Denmark) with more than 70 and 75% of them motile (objectively evaluated using ISASV1® CASA; Proiser R+D, Valencia, Spain) and viable (cytometrically evaluated after staining using Hoechst 33342 and propidium iodide; BD FACSCanto II cytometer; Becton Dickinson Co, Franklin Lakes, NJ), respectively. The boars, still healthy and fit to deliver semen, were eventually removed from the AI center because of genetic replacement reasons and slaughtered (Slaughterhouse Agroalimentaria de Teruel, Teruel, Spain). Immediately post-mortem, the scrotal contents were collected and transported in insulated containers (5 °C) to the Andrology Laboratory of Veterinary Teaching Hospital of University of Murcia (VTH) within 4 h. Immediately after ejaculation, the semen samples of each ejaculate fraction were centrifuged twice (1,500 × g at rt for 10 min, Rotofix 32 A, Hettich Zentrifuge, Tuttlingen, Germany) to separate the seminal plasma (SP) from a sperm pellet. The pellets were washed twice with PBS (1,500 × g at rt, 10 min) with a final suspension in PBS (1/3, v/v). These PBS-suspended sperm pellets were transported in thermal containers to the VTH, arriving within 2 h of ejaculate collection. Once in the VTH-laboratory, the PBS-suspended pellets were again centrifuged (2,400 × g, rt, 3 min; Megafuge 1.0 R, Heraeus, Hanau, Germany) and cytometrically checked for sperm content after staining with Hoechst 33342. As expected for the species (17Ford W.C. Regulation of sperm function by reactive oxygen species.Hum. Reprod. Update. 2004; 10: 387-399Crossref PubMed Scopus (228) Google Scholar), the pellets contained >97% of spermatozoa. Finally, the sperm suspensions were diluted with PBS to a final concentration of 1,000 × 106 spermatozoa/ml, aliquoted in 1 ml volumes and stored at −80 °C (Ultra Low Freezer; Haier, Schomberg, Ontario, Canada) until use. The contents of the epididymal caudae were collected following the procedure described by Alkmin et al. (12Alkmin D.V. Perez-Patiño C. Barranco I. Parrilla I. Vazquez J.M. Martinez E.A. Rodriguez-Martinez H. Roca J. Boar sperm cryosurvival is better after exposure to seminal plasma from selected fractions than to those from entire ejaculate.Cryobiology. 2014; 69: 203-210Crossref PubMed Scopus (44) Google Scholar) with slight modifications. The cauda epididymides were carefully dissected, a needle was placed in the ductus deferens, and air was retrogradely infused. The luminal fluid was collected at a section of the ductus made at the corpus-cauda limit. The harvested fluid of the two caudae of each boar was pooled and microscopically evaluated to confirm that more than 75% of viable, mature spermatozoa were present. The cauda epididymal sperm pellets were processed and stored following the same protocol for ejaculated spermatozoa described above. A total of four sperm samples from different semen sources were generated by each of the 9 boars. The proteomic analyses were carried out in the Proteomics Unit of the University of Valencia, Valencia, Spain (member of the PRB2-ISCIII ProteoRed Proteomics Platform). The sperm pellets were thawed at rt and centrifuged at 14,000 × g at 10 °C for 10 min (Eppendorf 5424R, Eppendorf AG, Hamburg, Germany) to obtain protein-enriched fractions. Two extraction cycles, which involved dilution in 200 μl of U/T/C lysis buffer (7 m Urea, 2 m thiourea and 4% CHAPS) and constant rotation at 5 °C during 1 h, were performed. Thereafter, the results of the two extraction cycles were combined and treated with 10% (final concentration) of TCA (Fisher Scientific, Madrid, Spain) and stored overnight at 5 °C to achieve complete sperm lysis. Thereafter, samples were diluted in 200 μl of MilliQ water (Merck Millipore, Darmstadt, Germany) and centrifuged at 14,000 × g for 2 h. A TCA/Acetone protocol was used for protein precipitation; the TCA-treated samples were diluted (1:4, v/v) in a TCA-cold acetone solution, stirred and stored on ice for 15 min. The samples were then centrifuged (14,000 × g, at 4 °C for 20 min; Eppendorf 5424R) and the resulting pellets were washed twice with 1 ml ice-cold acetone and centrifuged (14,000 × g, at 4 °C for 20 min; Eppendorf 5424R). The pellets were incubated overnight at rt to volatilize the residual acetone. The dried pellets were lysed by dilution in 200 μl of protein extraction reagent (8 m urea and 0.5 m triethylammonium bicarbonate buffer [TEAB]), sonicated and constantly rotated at rt for 1 h. Protein concentration was measured using the Lowry modified RC DCTM Protein Assay Kit (Bio-Rad, Richmond, CA). A total of 100 μg of final protein extract per sample was used for iTRAQ analysis. The iTRAQ labeling was performed using the AB SCIEX kit (Framingham, MA). Cysteine residues were blocked by incubation in 4 μl of 50 mm 2-carboxyethyl phosphine (TCEP) at 37 °C for 180 min to avoid undesirable secondary urea reactions. Sulfhydryl groups were alkylated with 1 μl of 200 mm S-methyl methanethiosulfonate (MMTS) at rt for 10 min. Urea was diluted to 2 m with 0.5 m of TEAB buffer to a final volume of 100 μl. The protein samples were digested with 10 μg of sequencing-grade modified trypsin (Promega Corporation, Madison, WI) diluted in 0.5 m of TEAB buffer, and incubated at 37 °C overnight. The digested protein samples were dried in a centrifuge vacuum concentrator (ISS 110 SpeedVac System, Thermo Savant, ThermoScientific, Langenselbold, Germany), dissolved in 80 μl of TEAB buffer in ethanol solution (3:7, v/v) and sonicated for 10 min. Then, the resulting peptide mixtures were labeled with the appropriate iTRAQ reagents following the protocol of the 8-plex iTRAQ labeling kit (AB SCIEX). The iTRAQ-labeled peptides were then incubated at rt for 3 h, mixed, aliquoted in 250 μg portions and dried by vacuum centrifugation. The dried iTRAQ-labeled peptides were subjected to fractionation by IEF separation following the protocol of Krijgsveld et al. (18Krijgsveld J. Gauci S. Dormeyer W. Heck A.J. In-gel isoelectric focusing of peptides as a tool for improved protein identification.J. Proteome Res. 2006; 5: 1721-1730Crossref PubMed Scopus (94) Google Scholar) with minor modifications. Briefly, 250 μg of the peptide mixture was brought up to 8 m urea in the presence of IPG buffer, 3–11 NL (GE Healthcare Life Sciences, Little Chalfont, United Kingdom) and applied to 13 cm IPG dry strips, 3–11 NL (GE Healthcare), which were isoelectrofocused with 5,000 V up to 25,000 Vh. Thereafter, strips were washed with MilliQ-grade water and cut into 11 equal pieces. The isoelectrofocused peptides were extracted from the strips with 120 μl of the following sequential five extracted solutions (Fisher Scientific): (1Gadella B.M. Reproductive tract modifications of the boar sperm surface.Mol. Reprod. Dev. 2017; 84: 822-831Crossref PubMed Scopus (13) Google Scholar) 5% aqueous ACN 0.1% TFA, (2Park Y.J. Kim J. You Y.A. Pang M.G. Proteomic revolution to improve tools for evaluating male fertility in animals.J. Proteome Res. 2013; 12: 4738-4747Crossref PubMed Scopus (35) Google Scholar) 20% ACN 0.1% TFA, (3Mohanty G. Swain N. Samanta L. Sperm Proteome: What is on the horizon?.Reprod. Sci. 2015; 22: 638-653Crossref PubMed Scopus (8) Google Scholar) 50% ACN 0.1% TFA, (4Dostàlovà Z. Calvete J.J. Sanz L. Töpfer-Petersen E. Quantitation of boar spermadhesins in accessory sex gland fluids and on the surface of epididymal, ejaculated and capacitated spermatozoa.Biochim. Biophys. Acta. 1994; 1200: 48-54Crossref PubMed Scopus (103) Google Scholar) 70% ACN 0.1% TFA and (5Ensslin M. Vogel T. Calvete J.J. Thole H.H. Schmidtke J. Matsuda T. Töpfer-Petersen E. Molecular cloning and characterization of P47, a novel boar sperm-associated zona pellucida-binding protein homologous to a family of mammalian secretory proteins.Biol. Reprod. 1998; 58: 1057-1064Crossref PubMed Scopus (83) Google Scholar) 99.9% ACN 0.1% TFA. All peptide fractions were combined, dried by vacuum centrifugation and redissolved with 40 μl of 2% ACN 0.1% TFA. The samples were cleaned and concentrated by C18 silica homemade “in tip” columns, dried by speed vacuum and resuspended to a concentration of ca. 0.30 μg/μl in 2% ACN 0.1% TFA (the peptide concentration was determined assuming 100% performance). The labeled peptides were analyzed by LC using a NanoLC Ultra 1-D plus Eksigent (Eksigent Technologies, Dublin, CA), which was directly connected to a TripleTOF 5600 mass spectrometer (AB SCIEX). Briefly, 5 μl from each sample was loaded onto a trap column (NanoLC column, Chrom XP C18–3 μm, 350 μm × 0.5 mm; Eksigent Technologies) and desalted with 0.1% TFA at 3 μl/min for 5 min. Then, the peptides were eluted from the trap column and separated using an analytical LC-column (3 μm particle size C18-CL, 75 μm x 12 cm, Nikkyo Technos Co®, Tokyo, Japan) equilibrated in 5% ACN and 0.1% formic acid (FA) (Fisher Scientific). Peptide elution was performed by applying a linear gradient of solvents A (0.1% FA in water) and B (0.1% FA in ACN) from 5% to 35% of solvent B in A at a constant flow rate of 300 nL/min over 90 min. The eluted peptides were thereafter ionized using an ESI Nanospray III ion source (AB SCIEX) for analysis with a TripleTOF 5600 mass spectrometer coupled to the NanoLC system. The samples were ionized by applying 2.8 kV to the spray emitter and the TripleTOF was operated in an information-dependent acquisition mode, in which a TOF MS scan was made from 350 to 1250 m/z, accumulating for 250 ms TOF followed by 75 product ion scans from 100–1500 m/z; the 25 most abundant multiply charged (2+, 3+, 4+ or 5+) precursor peptide ions were automatically selected. Ions with 1+ and unassigned charge states were rejected from the MS/MS analysis. The collision energy was automatically set by the instrument rolling collision energies for iTRAQ labeled peptides. The generated SCIEX.wiff data-files were processed using the ProteinPilot v5.0 search engine (AB SCIEX) for protein identification and quantification with a peptide confidence threshold of 95% and a false discovery rate (FDR) less than 1% at the protein level. The Paragon algorithm (4.0.0.0, 4767) of ProteinPilot was used to search against the Uniprot_mammalia database (version 20180307 with 3,810,720 proteins searched) with the following parameters: iTRAQ quantitation, trypsin specificity, cys-alkylation (MMTS), no taxonomy restrictions, and the search effort set to throughout. The identified proteins were grouped by the Pro GroupTM algorithm (ProteinPilotTM Software) following the Pro Group Report recommendation (http://www3.appliedbiosystems.com/cms/groups/mcb_marketing/documents/generaldocuments/cms_040586.pdf). Protein groups were exclusively made from observed peptides, and the grouping was guided by the spectral usage. Consequently, unobserved regions of protein sequence were not considered to explain the data. Bioinformatics of all identified and differentially expressed sperm-proteins was manually performed using the comprehensive bioinformatics tool for functional annotation UniProt KB database (www.uniprot.org) downloaded 28/03/2018, containing 111,425,245 total entries with 40,710 of them encoded in the Sus scrofa taxonomy. This analysis allowed the elucidation of the different functions and processes in which the differentially expressed sperm proteins were putatively involved. Three independent sets of ontology were used in the annotation: “molecular function,” “biological processes” in which the proteins participate, and their “cellular components.” A total of 130 proteins were not considered for collation, as they showed no similarity with database entries. Spermatozoa of nine mature and fertile boars were used, obtained from four different sources per boar; (P0) cauda epididymis, (P1) the first 10 ml of the SRF, (P2) the remaining SRF and (P3) the post-SRF. Before proteomic analysis, the sperm pellets derived from the same source per boar (either cauda epididymis or fractions of the ejaculate) were mixed together to diminish individual effects. Consequently, four single sperm pools were built (P0-P3) to study the proteome of pre- and post-ejaculation mature boar spermatozoa. To validate analytical reliability, each sperm pool was in turn split into two aliquots to generate two technical replicates. Samples labeled with the appropriate iTRAQ reagents corresponded to the first and second technical replicate, respectively: spermatozoa from P0: 113 and 117; spermatozoa from P1: 114 and 118; spermatozoa from P2: 115 and 119 and spermatozoa from P3: 116 and 121. The relative quantification of sperm proteins was achieved by comparison of the relative intensities of reporter ions of different sperm samples (P0, P1, P2, and P3), dividing the iTRAQ reporter groups (114, 115, 116, 117, 118, 119, and 121) by the peak intensity of 113. Principal component analysis (PCA) was performed to evaluate the discriminative ability of sperm proteins in the four sperm samples using the Origin Software (OriginLab, Northampton, MA). Thereafter, the Multiexperiment Viewer (MeV) software (version 4.8) (http://www.tm4.org/mev.html) was used for statistical normalization following software instructions. An ANOVA test was used to identify the differentially expressed sperm proteins among the four sperm samples. Proteins were considered differentially expressed with an adjusted p value < 0.01, and those with a fold change (FC) ≥ 1.5 after log2 transformation were highlighted. The results of the hierarchical clustering analysis of the proteome profile of the different sperm samples were shown with a heat map after z-score normalization, using Euclidean distances. Quantitative analysis was done only on proteins identified in all sperm samples. Differences in the gene ontology (GO) distribution between total and differentially expressed proteins were analyzed using a Chi-square analysis. A total of 1,723 proteins were identified with a cutoff of unused prot score > 1.3 (corresponding to a confidence limit of 95% and an FDR < 1%), 974 of them belonging to the Sus scrofa taxonomy. Among the identified proteins, 1,602 were successfully quantified, 960 of them belonging to the Sus scrofa taxonomy. All the identified proteins were present in the spermatozoa of the four sperm sources. The complete list of identified and quantified proteins and the ratio of the peak area of the iTRAQ reporter ion displaying the relative abundance of each protein is shown in supplemental Data S1. The PCA analysis of all identified proteins showed proportions of variance of 24.9%, 15.1% and 14.5% for PC1, PC2 and PC3, respectively (supplemental Fig. S1). The PCA of identified proteins encoded in the Sus scrofa taxonomy showed similar distributions, explaining 29.5%, 15.7% and 13.5% of the variance in PC1, PC2 and PC3, respectively (Fig. 1). PC1 had the highest discrimination among sperm sources. Specifically, the sperm samples were grouped into three distinct branches; the first one included the P0 and P1 sources, whereas the second and third included the P2 and P3, respectively. The ANOVA test revealed a total of 43 proteins differentially expressed (p < 0.01) among the sperm sources, 32 of them belonging to the Sus scrofa taxonomy. The quantitative value of the 43 differentially expressed proteins, following data normalization per source and the FC estimation among sources after log2 transformation, is shown in supplemental Data S2. The expression pattern of the differentially expressed sperm proteins of the two technical replicates of each sperm source is graphically presented as a heat map in Fig. 2 (proteins encoded in Sus scrofa taxonomy) and supplemental Fig. S2 (all differentially expressed proteins). The dendrograms of the two heat-maps showed that the technical replicates merged into a close cluster, highlighting the robustness of the analysis carried out. The dendrograms also showed that the distance between the cluster grouping P0-P2 source and P3 source samples was large, showing that the greatest differences in protein expression were between these two clusters. Twenty-eight of the 32 differentially expressed Sus scrofa proteins showed an FC ≥ 1.5 among sources (Table I). Only three of these proteins were differentially expressed between spermatozoa of the epididymis (P0) and the P1-P2 ejaculate fractions (SRF), being these three proteins overexpressed in P0. In contrast, a larger number of proteins (n = 20), were differentially expressed between the post-SRF (P3) and the other sperm sources (epididymis -P0-, first 10 ml of SRF -P1- and rest of SRF -P2-). Notably, these 20 differentially expressed proteins were overexpressed in the spermatozoa of the post-SRF.Fig. 2Heat map with dendrograms representing the differentially expressed proteins belonging to the Sus scrofa among the four sperm samples: (P0) mature spermatozoa from the cauda epididymis, (P1) spermatozoa from the first 10 ml of the sperm-rich ejaculate fraction (SRF), (P2) the remainder of the SRF and (P3) the post-SRF. The data were obtained from two technical replicates of each sperm source. The hierarchical clustering tree of sperm sources is shown at the top. The relative expression level of each differentially expressed protein is shown on a color scale from red (highest level) to green (lowest level).View Large Image Figure ViewerDownload Hi-res image" @default.
• W2892462906 created "2018-10-05" @default.
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• W2892462906 creator A5011323287 @default.
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• W2892462906 date "2019-01-01" @default.
• W2892462906 modified "2023-10-17" @default.
• W2892462906 title "The Proteome of Pig Spermatozoa Is Remodeled During Ejaculation" @default.
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