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Towards Understanding Male Infertility After Spinal Cord Injury Using Quantitative Proteomics
2016; Elsevier BV; Volume: 15; Issue: 4 Linguagem: Inglês
10.1074/mcp.m115.052175
ISSN1535-9484
AutoresBarbara Ferreira da Silva, Chen Meng, Dominic Helm, Fiona Pachl, Jürgen Schiller, Emad Ibrahim, Charles M. Lynne, Nancy L. Brackett, Ricardo Pimenta Bertolla, Bernhard Küster,
Tópico(s)Urologic and reproductive health conditions
ResumoThe study of male infertility after spinal cord injury (SCI) has enhanced the understanding of seminal plasma (SP) as an important regulator of spermatozoa function. However, the most important factors leading to the diminished sperm motility and viability observed in semen of men with SCI remained unknown. Thus, to explore SP related molecular mechanisms underlying infertility after SCI, we used mass spectrometry-based quantitative proteomics to compare SP retrieved from SCI patients to normal controls. As a result, we present an in-depth characterization of the human SP proteome, identifying ∼2,800 individual proteins, and describe, in detail, the differential proteome observed in SCI. Our analysis demonstrates that a hyper-activation of the immune system may influence some seminal processes, which likely are not triggered by microbial infection. Moreover, we show evidence of an important prostate gland functional failure, i.e. diminished abundance of metabolic enzymes related to ATP turnover and those secreted via prostasomes. Further we identify the main outcome related to this fact and that it is intrinsically linked to the low sperm motility in SCI. Together, our data highlights the molecular pathways hindering fertility in SCI and shed new light on other causes of male infertility. The study of male infertility after spinal cord injury (SCI) has enhanced the understanding of seminal plasma (SP) as an important regulator of spermatozoa function. However, the most important factors leading to the diminished sperm motility and viability observed in semen of men with SCI remained unknown. Thus, to explore SP related molecular mechanisms underlying infertility after SCI, we used mass spectrometry-based quantitative proteomics to compare SP retrieved from SCI patients to normal controls. As a result, we present an in-depth characterization of the human SP proteome, identifying ∼2,800 individual proteins, and describe, in detail, the differential proteome observed in SCI. Our analysis demonstrates that a hyper-activation of the immune system may influence some seminal processes, which likely are not triggered by microbial infection. Moreover, we show evidence of an important prostate gland functional failure, i.e. diminished abundance of metabolic enzymes related to ATP turnover and those secreted via prostasomes. Further we identify the main outcome related to this fact and that it is intrinsically linked to the low sperm motility in SCI. Together, our data highlights the molecular pathways hindering fertility in SCI and shed new light on other causes of male infertility. For many years, seminal plasma (SP) 1The abbreviations used are:SPSeminal plasmaCASAComputer-aided semen analysisCIDCollision induced dissociationDAVIDDatabase for Annotation, Visualization and Integrated DiscoveryDDAData dependent acquisitionDMSODimethyl sulfoxideDTTDithiothreitolESIElectrospray ionizationFDRProtein false discovery rateHCDHigher energy collisional dissociationHPLCHigh-performance liquid chromatographyhSAXHydrophilic Strong Anion ExchangeKEEGKyoto Encyclopedia of Genes and GenomesLC-MS/MSLiquid chromatography-tandem mass spectrometryLTQ-OrbitrapLinear trap quadrupole-OrbitrapPVSPenile vibratory stimulationSCISpinal cord injurySTRStraightnessTEABTriethylammonium bicarbonateVAPAverage path velocityWBCWhite blood cellWHOWorld Health Organization., the liquid component of semen, was believed to have a single and simple physiological significance as the carrier of spermatozoa through both male and female reproductive tracts. It was around 50 years ago when the compositional complexity of this fluid started to be investigated, demonstrating that SP not only aids in cellular transport but also provides energy and metabolic support to the transiting spermatozoa (1..Mann, T., (1964) Protein constituents and enzymes of the seminal plasma. The Biochemistry of Semen and of the Male Reproductive, 2nd Ed., pp. 161–192, Methuen, London.Google Scholar, 2.Marshburn P.B. Giddings A. Causby S. Matthews M.L. Usadi R.S. Steuerwald N. Hurst B.S. Influence of ejaculatory abstinence on seminal total antioxidant capacity and sperm membrane lipid peroxidation.Fertil. Steril. 2014; 102: 705-710Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Today, growing evidence indicates that SP plays a role far beyond what was once envisioned, including acting as an essential regulator of spermatozoa function contributing to (enabling/hindering) the cellular ability of fertilization (3.Rodríguez-Martínez H. Kvist U. Ernerudh J. Sanz L. Calvete J.J. Seminal plasma proteins: what role do they play?.Am. J. Reprod. Immunol. Suppl. 2011; 66: 11-22Crossref PubMed Scopus (275) Google Scholar). Composed of secretions derived from the testis, epididymis and male accessory glands (prostate, seminal vesicles and bulbourethral glands), SP is a mixture of sugars, inorganic ions, organic salts, (phospho)lipids and proteins (4.Druart X. Rickard J.P. Mactier S. Kohnke P.L. Kershaw-Young C.M. Bathgate R. Gibb Z. Crossett B. Tsikis G. Labas V. Harichaux G. Grupen C.G. de Graaf S.P. Proteomic characterization and cross species comparison of mammalian seminal plasma.J. Proteomics. 2013; 91: 13-22Crossref PubMed Scopus (124) Google Scholar). Such a heterogeneous composition emphasizes the complex biochemical cascades triggered within SP during, and immediately after, ejaculation and defines the beneficial and/or detrimental nature of SP in the overall reproductive process (5.Maxwell W.M. de Graaf S.P. Ghaoui Rel-H. Evans G. Seminal plasma effects on sperm handling and female fertility.Soc. Reprod. Fertil. Suppl. 2007; 64: 13-38PubMed Google Scholar). From a clinical point of view, studies have confirmed the participation of SP in the etiology of male infertility. By studying semen of men with spinal cord injury (SCI), who become infertile after a traumatic injury and often present with an unusual seminal profile characterized by normal sperm concentration but extremely impaired sperm motility and viability, Brackett et al. demonstrated how SP can impair sperm function leading to infertility (6.Brackett N.L. Davi R.C. Padron O.F. Lynne C.M. Seminal plasma of spinal cord injured men inhibits sperm motility of normal men.J. Urol. 1996; 155: 1632-1635Crossref PubMed Scopus (86) Google Scholar). Specifically, the authors mixed SP obtained from SCI patients with spermatozoa from normal donors and vice versa. Defining sperm motility as the main evaluation parameter, it was found that, after 5 min, SP from SCI men inhibited motility of spermatozoa from normal controls. Conversely, SP from controls improved cellular motility of spermatozoa from SCI patients, clearly indicating that the SP of these patients present abnormalities that are somehow deleterious to sperm. In order to strengthen these findings, the same authors compared vas deferens aspirated to ejaculated spermatozoa in SCI patients and controls (7.Brackett N.L. Lynne C.M. Aballa T.C. Ferrell S.M. Sperm motility from the vas deferens of spinal cord injured men is higher than from the ejaculate.J. Urol. 2000; 164: 712-715Crossref PubMed Google Scholar). Interestingly, sperm motility and viability were significantly higher when spermatozoa were directly aspirated from the vas deferens before any contact with the glandular fractions of the ejaculate in SCI patients. Although aspirated cells from patients presented somewhat lower motility and viability compared with controls, implying that epididymal or testicular factors may also be responsible, by far the greatest decrease in the measured sperm parameters was observed after contact with SP. Seminal plasma Computer-aided semen analysis Collision induced dissociation Database for Annotation, Visualization and Integrated Discovery Data dependent acquisition Dimethyl sulfoxide Dithiothreitol Electrospray ionization Protein false discovery rate Higher energy collisional dissociation High-performance liquid chromatography Hydrophilic Strong Anion Exchange Kyoto Encyclopedia of Genes and Genomes Liquid chromatography-tandem mass spectrometry Linear trap quadrupole-Orbitrap Penile vibratory stimulation Spinal cord injury Straightness Triethylammonium bicarbonate Average path velocity White blood cell World Health Organization. Proteins are highly abundant molecules in human SP. The average protein concentration ranges from 35 to 55 mg/ml (8.Pilch B. Mann M. Large-scale and high-confidence proteomic analysis of human seminal plasma.Genome Biol. 2006; 7: R40Crossref PubMed Scopus (301) Google Scholar). Proteins also constitute the main level of functional interaction with spermatozoa. Some SP proteins are known to be specific for key cellular processes such as sperm capacitation (9.De Jonge C. Biological basis for human capacitation.Hum. Reprod. Update. 2005; 11: 205-214Crossref PubMed Scopus (137) Google Scholar), sperm-zona pellucida interaction, and sperm-oocyte fusion (10.Primakoff P. Myles D.G. Penetration, adhesion, and fusion in mammalian sperm-egg interaction.Science. 2002; 296: 2183-2185Crossref PubMed Scopus (273) Google Scholar, 11.Evans J.P. Kopf G.S. Molecular mechanisms of sperm-egg interactions and egg activation.Andrologia. 1998; 30: 297-307Crossref PubMed Google Scholar). We previously presented an initial qualitative report of the SP proteome from SCI patients and control individuals (12.da Silva B.F. Souza G.H. Lo Turco E.G. Del Giudice P.T. Soler T.B. Spaine D.M. Borrelli Junior M. Gozzo F.C. Pilau E.J. Garcia J.S. Ferreira C.R. Eberlin M.N. Bertolla R.P. Differential seminal plasma proteome according to semen retrieval in men with spinal cord injury.Fertil. Steril. 2013; 100: 959-969Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). In that study, a total of 638 individual proteins were identified and 119 proteins showed differential expression. At that time, we observed that prostatic proteins such as prostatic specific acid phosphatase (PSAP) and other enzymes like carboxypeptidases (e.g. CPE) were absent in patients. At the same time, a variety of proteins including apolipoproteins (e.g. APOB) and immunoglobulins (e.g. IGHG2) were found exclusively in samples from SCI patients. These findings suggested a deviation from homeostasis occurring in the SP of SCI patients, presumably altering its function and accounting for the poor seminal quality, which is typical of these individuals (12.da Silva B.F. Souza G.H. Lo Turco E.G. Del Giudice P.T. Soler T.B. Spaine D.M. Borrelli Junior M. Gozzo F.C. Pilau E.J. Garcia J.S. Ferreira C.R. Eberlin M.N. Bertolla R.P. Differential seminal plasma proteome according to semen retrieval in men with spinal cord injury.Fertil. Steril. 2013; 100: 959-969Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Nevertheless, how a SCI post-traumatic scenario influences the actions of SP proteins leading to infertility and which pathways hinder sperm function remained unclear. In the present study, we used MS based proteomics to qualitatively and quantitatively assess the SP proteome of SCI patients and controls. We investigated SP obtained from SCI patients both on pooled samples as well as individual patients. As a result, we obtained the most extensive list of human SP proteins reported to date (2,820 identified proteins). We also report on the possible molecular mechanisms underlying SCI related infertility. Thus, our results not only describe the SCI related infertility in a molecular level but also improve the general knowledge about the relation between SP and spermatozoa functionality, constituting a valuable data set for future studies. All SCI patients and control volunteers included in the study presented good general health and were participating in the Male Fertility Research Program of the Miami Project to Cure Paralysis, University of Miami Miller School of Medicine. The study was approved by the University of Miami Institutional Review Board, and informed consent was obtained from each of the subjects. In total, seminal samples from 12 SCI patients and 11 controls were collected, analyzed and further prepared for proteomic analysis. The mean age ± standard error of SCI subjects was 38 ± 10 years and the level of injury ranged from C4 to T6. All patients had already passed the period of spinal shock (≥12 months after injury) by the time of semen collection, which was obtained using the standard method of penile vibratory stimulation (PVS) as described elsewhere (13.Brackett N.L. Ibrahim E. Iremashvili V. Aballa T.C. Lynne C.M. Treatment of ejaculatory dysfunction in men with spinal cord injury: a single center experience of more than 18 years.J. Urol. 2010; 183: 2304-2308Crossref PubMed Scopus (94) Google Scholar); only antegrade specimens were collected. All control volunteers were noninjured and normospermic men with no known history of infertility. Fertility status and/or proved paternity were not considered for including a donor in the control group. Controls collected semen by masturbation in specific sterile containers after at least 3 days, but not longer than 7 days, of ejaculatory abstinence. After liquefaction at room temperature, semen analysis was performed for all samples. Sperm concentration (millions of sperm/ml ejaculate), total sperm count, sperm motility (% with forward progression) were evaluated by computer-aided semen analysis (CASA) using the IVOS II Clinical system version 12.2 (Hamilton Thorne, MA). The progressive motility settings were 250 μ/s for the path velocity (VAP) and 80% for the straightness (STR). The cut-off parameter for the slow cells was 5.0 μ/s for the VAP and 11.0 μ/s for the progressive velocity (VSL). The seminal white blood cell (WBC) concentration (millions of WBC/ml ejaculate) was measured by unstained wet smear. All reference values were used according to the guidelines from the World Health Organization (WHO) (14.World Health Organization WHO manual for the examination of human semen and sperm-cervical mucus interaction.4th Ed. Cambridge University Press, Cambridge, UK1999Google Scholar). Detailed information about the individual patients are provided in supplemental Table S1. Immediately after semen analysis, all samples were centrifuged at 1,000 × g for 15 min. The pellet was discarded and the supernatant (i.e. seminal plasma) recovered and stored at −80 °C until use. To perform the proteomics assays, all samples were thawed at room temperature and centrifuged at 1,000 × g for 30 min at 4 °C to eliminate remaining cellular debris or occasional spermatozoa. Supernatants were collected and total protein concentration was assessed for each SCI and control subject by the Bradford assay (15.Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216068) Google Scholar). Initially, samples from 10 SCI patients were pooled together to form a SCI representative pool. Similarly, the 11 control donors were pooled together to form a representative control pool. SCI and control pools were prepared containing 1 mg of total protein. Both groups were further analyzed in triplicate. In a second step, SCI individual samples were prepared to contain 1 mg of protein each. SCI and control pools and the 12 individual SCI samples were resolved on 50 mm triethylammonium bicarbonate (TEAB), 8 m urea buffer and vortexed thoroughly. The disulfide bonds of SP proteins were reduced with 10 mm dithiothreitol (DTT) at 37 °C for 1 h and then alkylated with 55 mm chloroacetamide at room temperature for 30 min in the dark. A urea concentration of 6 m was adjusted with 50 mm TEAB and 20 μg of Lys-C (Wako, Osaka, Japan) was added for the first digestion step (enzyme to protein ratio 1:50). Samples were then diluted to ≤ 2 m urea with 50 mm TEAB and 20 μg of trypsin (modified sequencing grade; Promega, Wisconsin, USA) was added to the samples (enzyme to protein ratio 1:50). For both digestion conditions proteolysis were carried out at 37 °C for 4 h (Lys-C) and overnight (trypsin). The digested samples were dried down by vacuum centrifugation (1,500 r.p.m. at room temperature) and the resulting peptides were suspended in 5% (v/v) formic acid. The on-column stable isotope dimethyl labeling was described elsewhere (16.Boersema P.J. Raijmakers R. Lemeer S. Mohammed S. Heck A.J. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics.Nat. Protoc. 2009; 4: 484-494Crossref PubMed Scopus (1062) Google Scholar) and performed using SepPak C18 cartridges (Waters, MA). The SepPak columns were washed with 100% acetonitrile and conditioned with 0.1% (v/v) formic acid. Each sample (pool SCI, pool control and individuals SCI) was loaded on a separate SepPak column. The columns were washed with 0.1% (v/v) formic acid and then flushed with the respective labeling reagent (light for SCI samples or intermediate for control sample). Labeling reagents were prepared by mixing 50 mm sodium phosphate buffer (pH 7.5) with 4% (v/v) formaldehyde in water (CH2O for light reagent or CD2O for intermediate) and 0.6 m sodium cyanoborohydride in water (NaBH3CN). Columns were again washed with 0.1% (v/v) formic acid and labeled peptides eluted from the column with a 0.1% (v/v) formic acid, 60% acetonitrile (v/v) solution. The labeling efficiency was estimated by measuring a fraction of each sample by LC-MS. Differentially labeled samples were finally mixed 1:1 (pool SCI: pool control; each individual SCI: pool control). Mixed samples were dried down by vacuum centrifugation (1,500 r.p.m. at room temperature) for a further off-line fractionation step. The hydrophilic Strong Anion Exchange (hSAX) chromatography was adapted from Ritorto et al. (17.Ritorto M.S. Cook K. Tyagi K. Pedrioli P.G. Trost M. Hydrophilic strong anion exchange (hSAX) chromatography for highly orthogonal peptide separation of complex proteomes.J. Proteome Res. 2013; 12: 2449-2457Crossref PubMed Scopus (52) Google Scholar) and performed using a Dionex Ultimate 3000 HPLC system (Dionex Corp., CA) equipped with an IonPac AG24 guard column (2 × 50 mm, Thermo Fisher Scientific, MA) and an IonPac AS24 SAX-column (2 × 250 mm, Thermo Fisher Scientific). The mixed samples were suspended in 20 mm Tris-HCl (pH 8.0) and injected in the system. Peptide separation was achieved with a flow rate of 0.25 ml/min (buffer A: 20 mm Tris-HCl pH 8.0 and buffer B: 20 mm Tris-HCl pH 8.0, 1 m NaCl). An initial 3 min equilibration step with 100% buffer A followed by elution with a linear 17 min gradient up to 40% buffer B was used. Buffer B was increased to 100% in 10 min and held constant for another 10 min. A subsequent switch to 100% buffer A in 3 min was followed by column re-equilibration with 100% buffer A for 10 min. A total of 48 fractions were collected for the pool experiment whereas a total of 36 fractions (combined into 30 final fractions) were collected for each sample from the individual experiment. All chromatograms were recorded by a UV detector and fractions dried down for subsequent analysis. For the pool experiment Nano-flow LC-MS/MS was performed by coupling an Eksigent nanoLC-Ultra 1D (Eksigent Technologies, CA) to an Orbitrap Elite (Thermo Scientific, Bremen, Germany). The collected 48 fractions were dissolved in 20 μl 0.1% formic acid and 10 μl were injected for each analysis. Peptides were delivered to a trap column (100 μm i.d. x 2 cm, packed in-house with Reprosil PUR C18-AQ, 5 μm resin, Dr. Maisch, Ammerbuch, Germany) at a flow rate of 5 μl/min in 100% buffer A (0.1% formic acid in HPLC grade water). After 20 min of loading and washing, peptides were transferred to an analytical column (75 μm x 40 cm, packed in-house with ReprosilGOLD C18, 3 μm resin, Dr. Maisch, Ammerbuch, Germany) and separated using a 60 min gradient from 4% to 32% of solvent B (0.1% formic acid, 5% DMSO in acetonitrile; solvent A: 0.1% formic acid, 5% DMSO in water) at 300 nL/minute flow rate. The Orbitrap Elite was operated in data dependent mode (DDA), automatically switching between MS and MS2. Full scan MS spectra were acquired in the Orbitrap at 30,000 (m/z 400) resolution after accumulation to a target value of 1,000,000. Internal calibration was performed using a DMSO derivate at m/z 401.92272. Tandem mass spectra were generated for up to 15 peptide precursors in the orbitrap for fragmention using higher energy collisional dissociation (HCD) at normalized collision energy of 30% and a resolution of 15,000 with a target value of 20,000 charges after accumulation for a maximum of 100 ms. For the individual experiment measurements using the LTQ-Orbitrap XL ETD (Thermo Scientific) employed the same LC conditions as described for the pool experiment and similar data acquisition parameters. Full scan MS spectra were acquired in the Orbitrap at 60,000 resolution. Tandem mass spectra were generated for up to ten peptide precursors in the linear ion trap for fragmentation using collision-induced dissociation (CID) at normalized collision energy of 35% after accumulation to a target value of 5,000 for a maximum of 100 ms. Raw MS spectra were processed by MaxQuant (version 1.5.2.8) for peak detection and quantification. MS/MS spectra were searched against the Uniprot human reference proteome database (70,076 sequences, downloaded on November 6th, 2015) by Andromeda search engine enabling contaminants and the reversed versions of all sequences with the following search parameters: Carbamidomethylation of cysteine residues as fixed modification and Acetyl (Protein N-term), Oxidation (M) as variable modifications. Trypsin/P was specified as the proteolytic enzyme with up to 2 missed cleavages allowed. The mass accuracy of the precursor ions was decided by the time-dependent recalibration algorithm of MaxQuant, fragment ion mass tolerance was set to of 0.6 Da. The maximum false discovery rate for proteins and peptides was 0.01 and a minimum peptide length of six amino acids was required. Quantification mode with the dimethyl Lys 0 and N-term 0 as light labels and dimethyl Lys 4 and N-term 4 as heavy labels was selected. All other parameters are the default setting in MaxQuant. Quantitative ratios were calculated by MaxQuant based on two light and medium label partners for each protein and normalized by shifting the median of the total ratio population to 1. Normalized ratios were used for the differential expression analysis and statistical significance was assessed using paired t test on proteins that are quantified all replicates of the pool experiment and in at least two samples of the individual experiment. Statistical analyses were performed using the R software (version 3.0.0). Classification and functional enrichment analysis of the identified and differential expressed proteins (adjusted p < 0.05) were performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) (http://david.abcc.ncifcrf.gov), a Bioinformatics Database for the biological process (BP), molecular function (MF), and cellular component (CC). Tissue origin analysis was also performed through the use of DAVID. The Kyoto Encyclopedia of Genes and Genomes (KEEG) (http://www.genome.jp/kegg/) database was used to map the differential expressed proteins to KEGG pathways for biological interpretation. For the creation and investigation of protein-protein interaction maps Cytoscape 2.8.2 (http://www.cytoscape.org) with Bisogenet 1.41 (http://apps.cytoscape.org/apps/bisogenet) plugin was used. This plugin integrates data from well-known interaction databases including DIP, BIOGRID, HPRD, BIND, MINT, and INTACT and displays the result as an interaction network within Cytoscape. For our purposes only the physical interactions were considered. The original mass spectrometric raw data files along with the MaxQuant search result files are available on proteomeXchange (18.Vizcaino J.A. Cote R.G. Csordas A. Dianes J.A. Fabregat A. Foster J.M. Griss J. Alpi E. Birim M. Contell J. O'Kelly G. Schoenegger A. Ovelleiro D. Perez-Riverol Y. Reisinger F. Rios D. Wang R. Hermjakob H. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013.Nucleic Acids Res. 2013; 41: D1063-1069Crossref PubMed Scopus (1596) Google Scholar), http://www.proteomexchange.org, under the accession number PXD002145). There is a growing body of evidence pointing to SP as a key regulator of spermatozoa homeostasis and as an important effector of male infertility. It has been described that the success of the fertilization process is intrinsically related to the complex protein content present in SP (19.Mogielnicka-Brzozowska M. Kordan W. Characteristics of selected seminal plasma proteins and their application in the improvement of the reproductive processes in mammals.Pol. J. Vet. Sci. 2011; 14: 489-499Crossref PubMed Scopus (32) Google Scholar), which serves functions in the different steps such as sperm capacitation (9.De Jonge C. Biological basis for human capacitation.Hum. Reprod. Update. 2005; 11: 205-214Crossref PubMed Scopus (137) Google Scholar), immune response inside the uterus, formation of the tubal sperm reservoir, sperm-zona pellucida interaction, and sperm-oocyte fusion (20.Kim B.J. Choi Y.M. Rah S.Y. Park D.R. Park S.A. Chung Y.J. Park S.M. Park J.K. Jang K.Y. Kim U.H. Seminal CD38 is a pivotal regulator for fetomaternal tolerance.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 1559-1564Crossref PubMed Scopus (32) Google Scholar, 21.Evans J.P. Kopf G.S. Molecular mechanisms of sperm-egg interactions and egg activation.Andrologia. 1998; 30: 297-307Crossref PubMed Scopus (57) Google Scholar, 22.Yi Y.J. Manandhar G. Oko R.J. Breed W.G. Sutovsky P. Mechanism of sperm–zona pellucida penetration during mammalian fertilization: 26S proteasome as a candidate egg coat lysin.Soc. Reprod. Fertil. Suppl. 2007; 63: 385-408PubMed Google Scholar). Additionally, Brackett et al. demonstrated that SP is capable of impairing and/or restoring sperm motility (6.Brackett N.L. Davi R.C. Padron O.F. Lynne C.M. Seminal plasma of spinal cord injured men inhibits sperm motility of normal men.J. Urol. 1996; 155: 1632-1635Crossref PubMed Scopus (86) Google Scholar). In the present study, we used state-of-the-art proteomics to extend the coverage of the human SP proteome and to perform an in depth analysis (as described in Fig. 1) of proteins present in the SP of SCI patients. As a result, we have obtained the most extensive proteomic analysis of SP to date, extending coverage by about three times over the most cited study on the human SP proteome (8.Pilch B. Mann M. Large-scale and high-confidence proteomic analysis of human seminal plasma.Genome Biol. 2006; 7: R40Crossref PubMed Scopus (301) Google Scholar) (Fig. 2A). In total 2,550 proteins were identified in the pool experiment (Fig. 2B, supplemental Table S2 and S3) at a protein false discovery rate (FDR) of 1%. On average, 2,279 proteins were identified per technical replicate (supplemental Fig. S1A) and the overlap among the experiments are shown in supplemental Figs. S1B and S1C. For the individual experiment (i.e. proteome measurements of individual patients), a total of 1,534 proteins were identified (Fig. 1C, supplemental Table S4 and S5, protein FDR 1%) and ∼1,000 proteins were identified per individual patient (supplemental Fig. S1C). When combining both data sets, a total of 2,820 nonredundant proteins were identified (Fig. 2D, supplemental Table S6). Our data set ranges from high abundance proteins such as the semenogelins, kallikreins, the main serine proteases in SP, lactotransferrin and fibronectin to very low abundance enzymes such as phosphatases and dehydrogenases. Moreover, GO enrichment analysis showed that the majority of proteins is composed of extracellular and intracellular proteins (Fig. 2E). Interestingly, we identified proteins from almost all cellular compartments including the endoplasmic reticulum, mitochondria, nucleus, membrane, cytosol, and cytoplasm. These proteins likely originate from shedded epithelial cells and/or from secretory cells. Furthermore, the identification of high amounts of certain proteins likes acrosin (ACR), a serine protease with trypsin-like specificity stored specifically in the acrosome of mature sperm cells (23.Marí S.I. Rawe V. Biancotti J.C. Charreau E.H. Dain L. Vazquez-Levin M.H. Biochemical and molecular studies of the proacrosin/acrosin system in patients with unexplained infertility.Fertil. Steril. 2003; 79: 1676-16169Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), shows that cellular disruption or leakages of spermatozoa also contribute to the contamination of SP with cellular proteins. In addition, as previously described, SP bears large amounts of immune system proteins, many of which may be produced and released by leukocytes. Quantitative ratios of proteins, between SCI and control samples, were obtained by dimethyl labeling and calculated using the software package MaxQuant (24.MaxQuant Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p. p. b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9214) Google Scholar) and requiring a minimum of two pairs of labeled peptides per protein. For the pool experiment, three technical replicates were analyzed and the good reproducibility among replicates (supplemental Figs. S2A and S2B) enabled statistical analysis within and across groups. Similarly, the 12 individual analyses showed good reproducibility, enabling statistical analysis to consider each single patient as one biological replicate in order to assess differences within the SCI group. The data was normalized by shifting the median of all protein ratios to 1 (i.e. no change between SCI and control,
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