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Functional proteomic analysis of seminal plasma proteins in men with various semen parameters

Functional proteomic analysis of seminal plasma proteins in men with various semen parameters Background: Alterations at the molecular level in spermatozoa and seminal plasma can affect male fertility. The objective of this study was to determine if analysis of differential expression of proteins in varying semen parameters can serve as potential biomarkers for male infertility. Methods: The differential expression of proteins in the seminal plasma of men based on sperm count and morphology were examined utilizing proteomic tools. Subjects were categorized based on sperm concentration and morphology into 4 groups: 1) normal sperm count and normal morphology (NN); 2) normal sperm count and abnormal morphology (NA); 3) oligozoospermia and normal morphology (ON); and 4) oligozoospermia and abnormal morphology (OA). Proteomic analysis was performed by LC-MS/MS followed by functional bioinformatics analysis. Protein distribution in the NA, ON and OA groups was compared with that of the NN group. Results: Twenty proteins were differentially expressed among the 4 groups. Among the unique proteins identified, 3 were downregulated in the NA group, 1 in the ON group and 1 in the OA group while 2 were upregulated in the ON and OA groups. The functional analysis 1) identified biological regulation as the major processes affected and 2) determined that most of the identified proteins were of extracellular origin. Conclusions: We have identified proteins that are over-or underexpressed in the seminal plasma of men with poor sperm quality. The distinct presence of some of the proteins may serve as potential biomarkers and provide insight into the mechanistic role played by these proteins in male infertility. Further studies using Western Blot analysis are required to validate these findings. Background morphology have been documented [5]. Idiopathic and un- Infertility is a major problem in 15% of couples world- explained infertility cannot be diagnosed by routine sperm wide. Male factors may play a role in half of these cases function tests [6]. Similarly, oligozoospermic men may have [1]. Most causes of male infertility are idiopathic. Semen other underlying pathologies that may contribute to infer- analysis remains the cornerstone in the evaluation of male tility. Evaluation solely based on semen analysis is insuffi- infertility. However, the data generated from this routine cient to determine the fertility status of the male partner. testing do not provide any insight into the underlying Spermatogenesis is a complex process that involves de- problems associated with developing spermatozoa. Sperm velopment of the undifferentiated germ cells into a highly morphology plays an important role in conception, and specialized spermatozoon capable of fertilizing an oocyte both fertilization and pregnancy rates are affected when [7]. Fertilization requires physical proximity of the sperm- morphologically normal sperms are below 5%. It is also a atozoa and the oocytes. Seminal plasma composed of reflection of poor testicular physiology and is an important secretions from the testis, epididymis and male accessory factor in male infertility [2-4]. However, a significant over- glands [8] provides a favorable environment and serves as lap of semen parameters such as sperm count, motility and a vehicle for the spermatozoa as it travels to meet the oocyte. * Correspondence: Agarwaa@ccf.org Seminal plasma contains unique proteins necessary for Center for Reproductive Medicine, Glickman Urological and Kidney Institute, sperm function and survival [9,10]. Seminal plasma pro- Cleveland Clinic, Cleveland, OH, USA teins play a variety of roles—they help protect the sperm Full list of author information is available at the end of the article © 2013 Sharma et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 2 of 20 http://www.rbej.com/content/11/1/38 by binding to the sperm surface during ejaculation and Semen analysis play a key role in capacitation, acrosome reaction, and Following complete liquefaction (average time: 20 minutes sperm-egg fusion [11,12]. They can also modulate im- and no more than 60 min.), manual semen analysis was mune response in male and female reproductive tracts, performed using a MicroCell counting chamber (Vitrolife, ensuring that the most competent spermatozoa meet the San Diego, CA) to determine sperm concentration and oocyte during fertilization [13]. Thus, seminal plasma percentage motility according to WHO guidelines [22]. proteins can serve as important biomarkers for male Viability was determined with Eosin - Nigrosin stain. infertility [14]. Smears of the raw semen were stained with a Diff-Quik Conventional 1-Dimensional gel electrophoresis studies kit (Baxter Healthcare Corporation, Inc., McGaw Park, IL) have provided information in relation to sperm proteins for assessment of sperm morphology according to WHO and their function in normal and abnormal spermato- criteria [22]. zoa [15,16]. Advancements in mass- spectrometry and After analysis of semen parameters, aliquots of the proteomic-basedtechniqueshavemadeitpossibleto samples were frozen at −80°C for proteomic analysis. analyze the complex protein mixtures found in tissues and body fluids. Several attempts have been made to identify these proteins using high-throughput techniques Preparation of samples for proteomic analysis such as matrix assisted laser desorption ionization – Samples were divided into 4 groups based only on time of flight (MALDI-TOF) mass spectrometry (MS) normal sperm concentration and normal morphology and liquid chromatography – tandem mass spectrometry parameters according to WHO criteria [22]. The groups (LC-MS/MS) and linear ion trap (LTQ-Orbitrap) mass were as follows: Group 1: normal sperm count and spectrometry [17-21]. normal morphology (NN = 26); Group 2: normal sperm Alterations at the molecular level in spermatozoa and count and abnormal morphology (NA = 22); Group 3: the seminal plasma may contribute to male infertility. oligozoospermia and normal morphology (ON = 6) and However, even after accounting for all the advances in group 4: oligozoospermia and abnormal morphology proteomics, there has been a great lack of detailed data (OA = 10). in the area of comparative analysis of seminal plasma To prepare the samples for proteomic analysis, they proteins associated with male infertility. were thawed, and clear seminal plasma was separated The objective of the present study was 1) to compare from the sperm pellet by centrifugation at 3,000 g for the differential expression of proteins in the seminal 30 minutes to ensure complete removal of the cellular plasma from subjects with normal or abnormal sperm components. Seminal plasma samples were pooled into concentration and sperm morphology utilizing proteomic replicates (NN = 5; NA = 4; ON = 1; OA = 2). Each sam- tools such as LC-MS/MS and 2) utilize the functional bio- ple was dissolved in 98% acetonitrile containing 0.1% informatics analysis to identify the cellular origin and the trifluoroacetic acid followed by lyophilization at −80°C differentially affected processes and/or pathways of these under vacuum for 2 days. The lyophilized sample was proteins to gain insights into the mechanistic roles played used to estimate the protein content. The samples were by these proteins in effecting the observed phenotypes. first precipitated in cold acetone and centrifuged at These analyses could possibly identify potential bio- 10,000 g for 15 minutes. The acetone was poured off, markers for male infertility. and the protein pellet was allowed to dry at room temperature. The protein pellet was solubilized in a buf- fer of 6 M urea, 100 mM Tris, pH 8.0. The proteins were Methods then reduced by the addition of DTT (200 mM in After obtaining Institutional Review Board approval, 100 mM Tris) for 15 minutes at room temperature and written consent was obtained from all subjects. Semen then alkylated by the addition of 200 mM iodoacetamide samples were obtained from 64 subjects who were (200 mM in 100 mM Tris) for 20 minutes at room healthy male volunteers of unproven fertility (n = 21) temperature. The urea concentration was then reduced and men presenting to our infertility clinic for evaluation to approximately 1.2 M, and trypsin was added at a (n = 43). Semen samples were collected by masturbation ratio of 1:50. Digestion was carried out overnight at after 2–3 days of sexual abstinence. Samples with room temperature. The digestion was stopped the next leukocytospermia--a high concentration of white blood morning by adding acetic acid to lower the pH to <6, cells (>1 × 10 WBC/mL)–were examined for the pres- and the samples were centrifuged to remove insoluble ence of granulocytes by the peroxidase or the Endtz test. material. The digests were then prepared for LC-MS/MS The patients with a positive Endtz test were excluded analysis by using PepClean C-18 spin columns to desalt from the study. Semen analysis was conducted according the samples, which were then brought up in 50 μLof 1% to WHO criteria as described below [22]. acetic acid. Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 3 of 20 http://www.rbej.com/content/11/1/38 Liquid chromatography – mass spectrometer analysis database index, molecular weight, peptide coverage and (LC-MS/MS) Mascot score is shown in Table 1. A protein was consid- The LC-MS system is a Finnigan LTQ linear ion trap ered significant if the SC cut off value was ≥ 10 in at mass spectrometer system. The high performance liquid least one sample and present in at least 50% of the chromatography (HPLC) column was a self-packed samples in a group. They were considered ‘low abun- 9cm×75 μm (internal diameter) Phenomenex Jupiter dant’ if the SC cut-off value was ≤10 in all the samples. C18 reversed-phase capillary chromatography column. The differentially expressed proteins (DEP) in the NA, Ten μL volumes of the extract were injected, and the pep- OA, and ON groups were categorized based on the NSC tides that were eluted from the column by an acetonitrile/ ratio cut-off values of > 2 (for over-expressed) or < 0.5 0.1% formic acid gradient at a flow rate of 0.25 μL/min (for under-expressed) in comparison to the NN group. were introduced into the source of the mass spectrometer We identified a total of 35 proteins; of these, 10 were on-line. The microelectrospray ion source was set at classified as low abundant. Amongst the remaining 25 2.5 kV. The digest was analyzed using the data-dependent significantly abundant proteins (24 in NN, 23 in NA, 20 multitask capability of the instrument acquiring full scan in OA, and 16 in ON), 11 were present in all the samples, mass spectra to determine peptide molecular weights and and 13 proteins were identified as unique to one or two or product ion spectra to determine the amino acid sequence three of the four samples. 20 proteins were identified as in successive instrument scans [23]. This mode of analysis differentially expressed in the NA, OA, and ON groups as produces approximately 2500 collision-induced dissoci- compared to NN group, with 2 proteins differentially ation (CID) spectra of ions ranging in abundance over sev- expressed in all three groups (Figure 1). The remaining eral orders of magnitude. The spectral count (SC) for each 18 were present in either of the groups (Figure 2). A protein was determined. Normalized spectral count (NSC) detailed list of the proteins classified under these cat- was obtained by dividing the spectral count for each pro- egories (Common, Unique, Significant, Low Abundant, tein and the total number of spectral counts identified in and Differentially Expressed) is shown in Table 2. the sample. The spectral counts were quantitated by tak- ing the normalized spectral count ratio for two sets of Identification of the common proteins samples. A protein was considered to be differentially Our analyses revealed a set of 11 proteins that were expressed if there was at least a two-fold difference in the common to all the samples in the 4 groups (Table 2). spectral count ratios between the two samples. Prolactin induced protein (PIP), semenogelin II (SgII) precursor, albumin preprotein, lactotransferrin, epididy- Data analysis mal secretory protein E1 precursor, extracellular matrix All CID spectra collected in the experiment were used to protein 1 isoform 1 precursor, prosaposin isoform A search the National Center for Biotechnology Information preprotein, cathepsin D preprotein, prostate specific (NCBI) human reference sequence database with the antigen isoform 1 preprotein, zinc alpha-2 glycoprotein search engine MASCOT (Matrix Science, Boston, MA, 1, and clusterin isoform 1 were the common proteins www.matrixscience.com). After identification, a database identified. consisting of all proteins identified in these searches was created and used for a second set of searches. These Identification of differentially expressed proteins searches were performed with a program called As shown in Figure 1, the DEP list encompassed pro- SEQUEST, and the results from these SEQUEST searches teins that overlapped with other categories (common, were used to determine the spectral counts. Furthermore, unique, low abundant and significant). The common functional bioinformatics analysis was done using publicly proteins that were also differentially expressed included available software packages such as Gene Ontology anno- prostate specific antigen isoform I preprotein; zinc tations from GO Term Finder [24] and GO Term Mapper alpha-2-glycoprotein 1 and clusterin isoform 1. Five low [25], UniProt [26], STRAP [27], and BioGPS [28]) as well abundant proteins (transferrin; secretory leukocyte as proprietary software packages (Ingenuity Pathway Ana- peptidase inhibitor precursor; ubiquitin and ribosomal lysis (IPA) from Ingenuity® Systems [29], and Metacore™ protein S27a precursor; protein tyrosine phosphatase from GeneGo Inc. [30]) to identify the differentially receptor type, sigma isoform 1 precursor, and acidic affected processes, pathways, interactions, and cellular epididymal glycoprotein-like 1 isoform 1 precursor) distribution of the proteins in the four study groups. were included as differentially expressed because their NSC ratio comparison met the 2-fold cutoff criteria. Results Of the 20 differentially expressed proteins, mucin 6, Analysis of the proteins identified by LC-MS/MS gastric; orosomucoid 1 precursor and acidic epididy- The proteins identified in the 12 replicates from NN, mal glycoprotein-like isoform 1 precursor were unique NA, ON and OA group showing protein name, NCBI proteins that were down regulated in the NA group. Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 4 of 20 http://www.rbej.com/content/11/1/38 Table 1 Identification of proteins in the 12 replicates from NN, NA, ON and OA group showing protein name, NCBI database index, molecular weight, peptide coverage and Mascot score Identification LTQ No. Protein name NCBI database index Calculated MW in PI Peptides Mascot number kDa (%coverage) score Sample NN1 semenogelin II precursor 4506885 65 9 8(10%) 9536 prolactin induced protein 4505821 16 8.2 3(20%) 5335 albumin preproprotein 4502027 71 5.9 9(22%) 4805 epididymal secretory protein E1 precursor 5453678 16 7.5 3 (21%) 794 prosaposin isoform a preproprotein 11386147 59 5 1 (2%) 658 mucin 6, gastric 151301154 263 7.2 4 (3%) 461 prostate specific antigen isoform 4 preproprotein 71834855 24 7 3 (15%) 460 armadillo repeat protein 4502247 105 6.3 3(5%) 413 cathepsin D preproprotein 4503143 45 6.1 1 (4%) 285 zinc alpha-2-glycoprotein 1 4502337 34 5.7 2 (12%) 260 cystatin S precursor 4503109 16 4.9 2 (31%) 178 ubiquitin and ribosomal protein S27a precursor 4506713 18 9.6 1 (10%) 173 clusterin isoform 1 42716297 58 6.2 3 (10%) 142 Sample NN2 prolactin-induced protein 4505821 16 8.2 10 (69%) 32886 semenogelin II precursor 4506885 65 9 17 (33%) 22468 semenogelin I isoform b preproprotein 38049014 45 9.2 13 (33%) 5388 albumin preproprotein 4502027 71 5.9 24 (54%) 15508 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 11 (61%) 5087 lactotransferrin precursor 54607120 80 8.5 22 (42%) 4281 zinc alpha-2-glycoprotein 1 4502337 34 5.7 11 (44%) 3424 cystatin S precursor 4503109 16 4.9 5 (44%) 1809 prosaposin isoform a preproprotein 11386147 59 5 9 (33%) 880 epididymal secretory protein E1 precursor 5453678 16 7.5 4 (52%) 786 serine proteinase inhibitor, clade A, member 1 50363217 46 5.3 11 (48%) 737 mucin 6, gastric 151301154 263 7.2 11 (9%) 524 extracellular matrix protein 1 isoform 1 precursor 221316614 62 6.2 7 (31%) 504 cystatin C precursor 4503107 16 9 6 (68%) 432 tissue inhibitor of metalloproteinase 1 precursor 4507509 23 8.4 4 (40%) 411 fibronectin 1 isoform 3 preproprotein 16933542 262 5.4 8(11%) 353 cathepsin D preproprotein 4503143 45 6.1 3 (11%) 266 acid phosphatase, prostate short isoform precursor 6382064 44 5.8 2 (12%) 166 carboxypeptidase E preproprotein 4503009 53 5 5 (20%) 153 clusterin isoform 1 42716297 58 6.2 2 (7%) 106 Sample NN3 prolactin-induced protein 4505821 16 8.2 10 (66%) 24697 semenogelin II precursor 4506885 65 9 19 (35%) 13533 semenogelin I isoform b preproprotein 38049014 45 9.2 15 (39%) 2633 albumin preproprotein 4502027 71 5.9 30 (60%) 8842 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 16 (70%) 8634 lactotransferrin precursor 54607120 80 8.5 23 (49%) 4607 Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 5 of 20 http://www.rbej.com/content/11/1/38 Table 1 Identification of proteins in the 12 replicates from NN, NA, ON and OA group showing protein name, NCBI database index, molecular weight, peptide coverage and Mascot score (Continued) zinc alpha-2-glycoprotein 1 4502337 34 5.7 9 (42%) 3935 epididymal secretory protein E1 precursor 5453678 16 7.5 6 (52%) 1214 tissue inhibitor of metalloproteinase 1 precursor 4507509 23 8.4 3 (29%) 987 prosaposin isoform a preproprotein 11386147 59 5 5 (21%) 833 extracellular matrix protein 1 isoform 1 precursor 221316614 62 6.2 6 (22%) 766 cystatin S precursor 4503109 16 4.9 5 (49%) 621 beta 2 microglobulin precursor 4757826 13 6 2 (21%) 523 fibronectin 1 isoform 3 preproprotein 16933542 262 5.4 4 (2%) 410 orosomucoid 1 precursor 167857790 23 5 3 (16%) 397 cystatin C precursor 4503107 16 9 3 (26%) 301 mucin 6, gastric 151301154 263 7.2 7 (7%) 288 cathepsin D preproprotein 4503143 45 6.1 2 (11%) 228 carboxypeptidase E preproprotein 4503009 53 5 3 (16%) 193 acidic epididymal glycoprotein-like 1 isoform 1 precursor 25121982 29 5.5 2 (10%) 166 galectin 3 binding protein 5031863 66 5.1 4 (11%) 165 clusterin isoform 1 42716297 58 6.2 2 (7%) 158 acid phosphatase, prostate short isoform precursor 6382064 44 5.8 5 (16%) 128 prostaglandin (H2) D-isomerase −1 peptide 32171249 21 7.6 1 (8%) 115 Sample NN4 prolactin-induced protein 4505821 16 8.2 12 (76%) 36052 semenogelin II precursor 4506885 65 9 16 (32%) 17746 semenogelin I isoform b preproprotein 38049014 45 9.2 14 (34%) 2723 lactotransferrin precursor 54607120 80 8.5 39 (61%) 8695 albumin preproprotein 4502027 71 5.9 23 (48%) 8361 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 15 (67%) 7417 zinc alpha-2-glycoprotein 1 4502337 34 5.7 11 (38%) 3979 mucin 6, gastric 151301154 263 7.2 14 (12%) 1685 epididymal secretory protein E1 precursor 5453678 16 7.5 7 (70%) 1428 acid phosphatase, prostate short isoform precursor 6382064 44 5.8 9 (26%) 1384 clusterin isoform 1 42716297 58 6.2 6 (16%) 1148 orosomucoid 1 precursor 167857790 23 5 4 (32%) 942 orosomucoid 2 4505529 23 5 2 (12%) 207 prosaposin isoform a preproprotein 11386147 59 5 6 (27%) 941 extracellular matrix protein 1 isoform 1 precursor 221316614 62 6.2 7 (25%) 731 tissue inhibitor of metalloproteinase 1 precursor 4507509 23 8.4 3 (29%) 635 beta 2 microglobulin precursor 4757826 13 6 2 (21%) 489 fibronectin 1 isoform 3 preproprotein 16933542 262 5.4 4 (3%) 447 cystatin C precursor 4503107 16 9 3 (26%) 391 carboxypeptidase E preproprotein 4503009 53 5 4 (14%) 358 galectin 3 binding protein 5031863 66 5.1 5 (14%) 341 transferrin 4557871 79 6.8 2 (4%) 275 acidic epididymal glycoprotein-like 1 isoform 1 precursor 25121982 29 5.5 2 (10%) 238 cystatin S precursor 4503109 16 4.9 2 (20%) 205 serine proteinase inhibitor, clade A, member 1 50363217 46 5.3 3 (12%) 187 Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 6 of 20 http://www.rbej.com/content/11/1/38 Table 1 Identification of proteins in the 12 replicates from NN, NA, ON and OA group showing protein name, NCBI database index, molecular weight, peptide coverage and Mascot score (Continued) cathepsin D preproprotein 4503143 45 6.1 2 (11%) 185 prostaglandin (H2) D-isomerase −1 peptide 32171249 21 7.6 1 (8%) 133 Sample NN5 prolactin-induced protein 4505821 16 8.2 8 (44%) 15001 semenogelin II precursor 4506885 65 9 10 (14%) 7235 albumin preproprotein 4502027 71 5.9 17 (39%) 2621 epididymal secretory protein E1 precursor 5453678 16 7.5 3 (23%) 552 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 5 (22%) 539 cystatin S precursor 4503109 16 4.9 2 (23%) 332 zinc alpha-2-glycoprotein 1 4502337 34 5.7 4 (19%) 250 prosaposin isoform a preproprotein 11386147 59 5 1 (2%) 239 prostatic acid phosphatase precursor 6382064 44 5.8 2 (4%) 180 lactotransferrin 54607120 80 8.5 4 (10%) 173 clusterin isoform 1 42716297 58 6.2 2 (7%) 157 galectin 3 binding protein 5031863 66 5.1 2 (4%) 152 extracellular matrix protein 1 isoform 1 precursor 4758236 62 6.2 1 (5%) 119 Sample NA1 prolactin-induced protein 4505821 16 8.2 10 (62%) 17127 albumin preproprotein 4502027 71 5.9 36 (61%) 9157 semenogelin II precursor 4506885 65 9 22 (39%) 7469 semenogelin I isoform b preproprotein 38049014 45 9.2 19 (42%) 2622 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 14 (67%) 5287 lactotransferrin 54607120 80 8.5 19 (40%) 2696 zinc alpha-2-glycoprotein 1 4502337 34 5.7 9 (38%) 1904 epididymal secretory protein E1 precursor 5453678 16 7.5 6 (68%) 1228 extracellular matrix protein 1 isoform 1 precursor 221316614 62 6.2 6 (22%) 635 serine proteinase inhibitor, clade A, member 1 50363217 46 5.3 8 (27%) 616 tissue inhibitor of metalloproteinase 1 precursor 4507509 23 8.4 2 (19%) 487 prosaposin isoform a preproprotein 11386147 59 5 4 (15%) 475 beta 2 microglobulin precursor 4757826 13 6 2 (21%) 400 fibronectin 1 isoform 3 preproprotein 16933542 262 5.4 7 (5%) 398 cystatin C precursor 4503107 16 9 3 (26%) 340 cystatin S precursor 4503109 16 4.9 2 (20%) 261 mucin 6, gastric isoform 1 89033736 185 6.3 4 (5%) 238 prostaglandin (H2) D-isomerase 32171249 21 7.6 1 (8%) 227 prostatic acid phosphatase precursor 6382064 44 5.8 4 (8%) 189 protein tyrosine phosphatase, receptor type, sigma isoform 1 104487006 218 6.1 5 (5%) 180 precursor transferrin 4557871 79 6.8 3 (17%) 174 clusterin isoform 1 42716297 58 6.2 4 (11%) 171 cathepsin D preproprotein 4503143 45 6.1 1 (4%) 149 galectin 3 binding protein 5031863 66 5.1 5 (14%) 101 Sample NA2 prolactin-induced protein 4505821 16 8.2 12 (76%) 21197 Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 7 of 20 http://www.rbej.com/content/11/1/38 Table 1 Identification of proteins in the 12 replicates from NN, NA, ON and OA group showing protein name, NCBI database index, molecular weight, peptide coverage and Mascot score (Continued) semenogelin II precursor 4506885 65 9 18 (36%) 9137 semenogelin I isoform b preproprotein 38049014 45 9.2 13 (33%) 1777 albumin preproprotein 4502027 71 5.9 26 (55%) 7695 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 16 (67%) 4963 zinc alpha-2-glycoprotein 1 4502337 34 5.7 13 (44%) 3134 lactotransferrin 54607120 80 8.5 24 (46%) 3105 epididymal secretory protein E1 precursor 5453678 16 7.5 5 (68%) 1276 prosaposin isoform a preproprotein 11386147 59 5 7 (26%) 662 tissue inhibitor of metalloproteinase 1 precursor 4507509 23 8.4 3 (29%) 660 serine proteinase inhibitor, clade A, member 1 50363217 46 5.3 5 (20%) 612 extracellular matrix protein 1 isoform 1 precursor 4758236 62 6.2 7 (22%) 577 cystatin C precursor 4503107 16 9 3 (26%) 548 beta 2 microglobulin precursor 4757826 13 6 2 (21%) 443 prostatic acid phosphatase precursor 6382064 44 5.8 8 (18%) 410 cathepsin D preproprotein 4503143 45 6.1 5 (25%) 375 clusterin isoform 1 42716297 58 6.2 2 (7%) 308 DJ-1 protein 31543380 20 6.3 2 (26%) 279 carboxypeptidase E preproprotein 4503009 53 5 4 (19%) 278 galectin 3 binding protein 5031863 66 5.1 5 (14%) 266 cystatin S precursor 4503109 16 4.9 6 (64%) 212 CD177 molecule 110735433 47 5.6 2 (9%) 199 mucin 6, gastric isoform 1 89033736 185 6.3 3 (4%) 196 fibronectin 1 isoform 3 preproprotein 16933542 262 5.4 4 (4%) 172 secretory leukocyte peptidase inhibitor precursor 4507065 15 9.1 2 (28%) 162 cathepsin B preproprotein 4503139 38 5.8 3 (12%) 127 Sample NA3 prolactin-induced protein 4505821 16 8.2 5 (37%) 6642 semenogelin II precursor 4506885 65 9 6 (9%) 5126 albumin preproprotein 4502027 71 5.9 7 (17%) 2077 prosaposin isoform a preproprotein 11386147 59 5 2 (8%) 438 mucin 6, gastric isoform 1 89033736 185 6.3 3 (2%) 269 epididymal secretory protein E1 precursor 5453678 16 7.5 2 (21%) 210 zinc alpha-2-glycoprotein 1 4502337 34 5.7 3 (24%) 203 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 3 (13%) 141 clusterin isoform 1 42716297 58 6.2 3 (10%) 117 Sample NA4 prolactin-induced protein 4505821 16 8.2 10(76%) 22296 semenogelin II precursor 4506885 65 9 16 (30%) 11486 semenogelin I isoform b preproprotein 38049014 45 9.2 13 (33%) 3038 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 14 (67%) 3631 zinc alpha-2-glycoprotein 1 4502337 34 5.7 11 (38%) 3260 lactotransferrin 54607120 80 8.5 16 (45%) 2926 prosaposin isoform a preproprotein 11386147 59 5 5 (20%) 773 epididymal secretory protein E1 precursor 5453678 16 7.5 3 (39%) 533 Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 8 of 20 http://www.rbej.com/content/11/1/38 Table 1 Identification of proteins in the 12 replicates from NN, NA, ON and OA group showing protein name, NCBI database index, molecular weight, peptide coverage and Mascot score (Continued) serine proteinase inhibitor, clade A, member 1 50363217 46 5.3 4 (12%) 506 extracellular matrix protein 1 isoform 1 precursor 4758236 62 6.2 4 (17%) 454 beta 2 microglobulin precursor 4757826 13 6 2 (21%) 388 tissue inhibitor of metalloproteinase 1 precursor 4507509 23 8.4 3 (29%) 351 cystatin S precursor 4503109 16 4.9 5 (48%) 321 cystatin C precursor 4503107 16 9 2 (26%) 280 cathepsin D preproprotein 4503143 45 6.1 2 (11%) 200 cathepsin B preproprotein 4503139 38 5.8 2 (10%) 186 carboxypeptidase E preproprotein 4503009 53 5 3 (11%) 162 Sample OA1 prolactin-induced protein 4505821 16 8.2 12 (77%) 22670 semenogelin II precursor 4506885 65 9 19(32%) 13764 semenogelin I isoform b preproprotein 38049014 45 9.2 15 (27%) 6586 albumin preproprotein 4502027 71 5.9 26 (51%) 7715 lactotransferrin 54607120 80 8.5 28 (54%) 5063 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 11 (64%) 3309 zinc alpha-2-glycoprotein 1 4502337 34 5.7 9 (40%) 3298 ankyrin repeat domain 11 56676397 299 6.7 3 (1%) 1056 PREDICTED: mucin 6, gastric isoform 1 89033736 185 6.3 10 (14%) 808 epididymal secretory protein E1 precursor 5453678 16 7.5 7 (70%) 635 clusterin isoform 1 42716297 58 6.2 4 (13%) 512 fibronectin 1 isoform 2 preproprotein 47132551 269 5.3 10 (7%) 503 extracellular matrix protein 1 isoform 1 precursor 4758236 62 6.2 5 (28%) 420 prosaposin isoform a preproprotein 11386147 59 5 4 (18%) 407 prostatic acid phosphatase precursor 6382064 44 5.8 9 (28%) 365 tissue inhibitor of metalloproteinase 1 precursor 4507509 23 8.4 4 (35%) 337 beta 2 microglobulin precursor – 1 peptide 4757826 13 6 1 (18%) 262 serine proteinase inhibitor, clade A, member 1 50363217 46 5.3 4 (12%) 242 transferrin 4557871 79 6.8 3 (4%) 211 cathepsin D preproprotein 4503143 45 6.1 2 (11%) 206 cystatin C precursor 4503107 16 9 5 (52%) 199 cystatin S precursor 4503109 16 4.9 6 (68%) 198 secretory leukocyte peptidase inhibitor precursor −1 peptide 4507065 15 9.1 1 (18%) 175 carboxypeptidase E preproprotein 4503009 53 5 2 (8%) 123 galectin 3 binding protein 5031863 66 5.1 3 (8%) 105 cathepsin B preproprotein 4503139 38 5.8 2 (12%) 95 expressed in prostate and testis 19923082 14 8.2 1 (10%) 86 macrophage migration inhibitory factor – 1 peptide 4505185 12 7.7 1 (9%) 84 prostaglandin (H2) D-isomerase −1 peptide 32171249 21 7.6 1 (8%) 75 Sample OA2 prolactin-induced protein 4505821 16 8.2 8 (51%) 13511 semenogelin II precursor 4506885 65 9 11 (18%) 6235 albumin preproprotein 4502027 71 5.9 10 (23%) 1848 cystatin S precursor 4503109 16 4.9 3 (28%) 522 Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 9 of 20 http://www.rbej.com/content/11/1/38 Table 1 Identification of proteins in the 12 replicates from NN, NA, ON and OA group showing protein name, NCBI database index, molecular weight, peptide coverage and Mascot score (Continued) prostate specific antigen isoform 4 preproprotein 71834855 24 7 4 (25%) 354 prosaposin isoform a preproprotein 11386147 59 5 1 (2%) 324 clusterin isoform 1 42716297 58 6.2 3 (10%) 308 mucin 6, gastric isoform 1 89033736 185 6.3 2 (1%) 306 zinc alpha-2-glycoprotein 1 4502337 34 5.7 4 (17%) 291 epididymal secretory protein E1 precursor 5453678 16 7.5 3 (38%) 184 ankyrin repeat domain 11 56676397 299 6.7 3 (1%) 133 carboxypeptidase E preproprotein 4503009 53 5 2 (8%) 121 lactotransferrin precursor 54607120 80 8.5 3 (9%) 115 extracellular matrix protein 1 isoform 1 precursor 4758236 62 6.2 1 (5%) 108 galectin 3 binding protein 5031863 66 5.1 1 (2%) 98 cathepsin D preproprotein 4503143 45 6.1 2 (13%) 95 lactamase, beta isoform a 26051231 61 8.7 2 (10%) 90 prostatic acid phosphatase precursor 6382064 44 5.8 1 (4%) 78 Sample ON prolactin-induced protein 4505821 16 8.2 9 (69%) 33212 semenogelin II precursor 4506885 65 9 20 (35%) 10703 semenogelin I isoform b preproprotein 38049014 45 9.2 17 ((38%) 2722 albumin preproprotein 4502027 71 5.9 25 (52%) 9519 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 14 (62%) 6487 zinc alpha-2-glycoprotein 1 4502337 34 5.7 9 (38%) 5967 lactotransferrin precursor 54607120 80 8.5 18(40%) 3770 serine proteinase inhibitor, clade A, member 1 50363217 46 5.3 4 (15%) 1138 prosaposin isoform a preproprotein 11386147 59 5 6 (26%) 1014 epididymal secretory protein E1 precursor 5453678 16 7.5 3 (39%) 937 tissue inhibitor of metalloproteinase 1 precursor 4507509 23 8.4 2 (19%) 866 extracellular matrix protein 1 isoform 1 precursor 221316614 62 6.2 3 (12%) 637 beta 2 microglobulin precursor 4757826 13 6 2 (21%) 633 mucin 6, gastric 151301154 263 7.2 5 (5%) 407 fibronectin 1 isoform 3 preproprotein 16933542 262 5.4 2 (1%) 352 cathepsin D preproprotein 4503143 45 6.1 2 (11%) 334 cystatin C precursor 4503107 16 9 3 (26%) 320 carboxypeptidase E preproprotein 4503009 53 5 3 (11%) 172 clusterin isoform 1 42716297 58 6.2 2 (7%) 147 acid phosphatase, prostate short isoform precursor 6382064 44 5.8 3 (8%) 132 secretory leukocyte peptidase inhibitor precursor 4507065 15 9.1 2 (42%) 123 protein tyrosine phosphatase, receptor type, sigma isoform 1 104487006 218 6.1 4 (3%) 112 precursor The proteins identified as unique and upregulated in 1 precursor); while clusterin 1 was downregulated in the OA group were: prostate specific antigen isoform this group. 1 preprotein; semenogelin I isoform b preprotein. Some unique proteins were absent in some of the Cystatin C precursor was found to be downregulated groups but were differentially expressed in other groups. in the OA group. The ON group showed an These proteins included the DJ-1 protein, which was upregulation of two unique proteins (zinc alpha-2 absent in the OA groups, whereas the ankyrin repeat glycoprotein 1 and tissue inhibitor of metalloproteinase domain 11 was absent in the NN group. Also included Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 10 of 20 http://www.rbej.com/content/11/1/38 Figure 1 Broad categorical analysis of proteomics data. Differentially expressed proteins list encompasses proteins that overlap with other categories (common, unique, low abundant and significant) of proteins. in this category was orosomucoid 1 precursor, which while orosomucoid 2 was present only in the NN. Pro- was found in significant abundance in the NN samples tein tyrosine phosphatase, receptor type, sigma isoform but in low abundance in NA sample. 1 precursor and acidic epididymal glycoprotein - like 1 isoform 1 precursor protein were absent in the OA Identification of the low abundant proteins group. Many of the identified proteins were low abundant proteins (SC ≤10) (Table 2). Some of these proteins were Cellular distribution and significant biological processes restricted to a particular group while they were absent in for proteins in four groups other groups. Transferrin, secretory leukocyte peptidase The functional analysis revealed that most of the signifi- inhibitor precursor, ubiquitin and ribosomal protein cant proteins in each of the four groups (NN, NA, OA, S27 a precursor, prostaglandin H2 D isomerase were and ON) had a predominant cellular distribution in the some of theproteinsthatwereabsentinON group. extracellular region followed by their presence in the The CD177 molecule was absent only in the NN group; intracellular organelles (Figure 3). The distribution of Figure 2 Venn diagram showing distribution of 20 differentially expressed proteins. This was based on the NSC ratio cut-off >2 across 3 samples, NA, OA, and ON in comparison to the baseline NN sample. Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 11 of 20 http://www.rbej.com/content/11/1/38 Table 2 Detailed list of classification of 35 proteins based on their presence, abundance, and differential expression No. Protein Names NCBI UniProt No. of Proteins Low Differentially Expressed Significant Accession Accession samples present in abundant Proteins (in NA, OA, and ON) proteins in No. No. groups proteins compared to NN groups (SC ≤ 10) 1 prolactin-induced protein 4505821 P12273 12 NN(5), NA NN, NA, OA, (4), OA(2), ON ON(1) 2 semenogelin II precursor 4506885 Q02383 12 NN(5), NA NN, NA, OA, (4), OA(2), ON ON(1) 3 albumin preproprotein 4502027 P02768 12 NN(5), NA NN, NA, OA, (4), OA(2), ON ON(1) 4 lactotransferrin 54607120 P02788 12 NN(5), NA NN, NA, OA, (4), OA(2), ON ON(1) 5 epididymal secretory protein 5453678 P61916 12 NN(5), NA NN, NA, OA, E1 precursor (4), OA(2), ON ON(1) 6 extracellular matrix protein 1 221316614 Q16610 12 NN(5), NA NN, NA, OA, isoform 1 precursor (4), OA(2), ON ON(1) 7 prosaposin isoform a 11386147 P07602 12 NN(5), NA NN, NA, OA, preproprotein (4), OA(2), ON ON(1) 8 cathepsin D preproprotein 4503143 P07339 12 NN(5), NA NN, NA, OA, (4), OA(2), ON ON(1) 9 prostate specific antigen 4502173 Q546G3 12 NN(5), NA ↑ in OA NN, NA, OA, isoform 1 preproprotein (4), OA(2), ON ON(1) 10 zinc alpha-2-glycoprotein 1 4502337 P25311 12 NN(5), NA ↑ in ON NN, NA, OA, (4), OA(2), ON ON(1) 11 clusterin isoform 1 42716297 P10909 12 NN(5), NA Low in ON ↓ in ON NN, NA, OA (4), OA(2), ON(1) 12 mucin 6, gastric 151301154 Q6W4X9 11 NN(4), NA ↓ in NA NN, NA, OA, (4), OA(2), ON ON(1) 13 cystatin S precursor 4503109 P01036 11 NN(4), NA Low in ON ↓ in NA, OA, ON NN, NA, OA (4), OA(2), ON(1) 14 galectin 3 binding protein 5031863 Q08380 11 NN(5), NA Low in OA ↓ in OA, ON NN, NA (3), OA(2), and ON ON(1) 15 semenogelin I isoform b 38049014 P04279 10 NN(3), NA ↑ in OA NN, NA, OA, preproprotein (4), OA(2), ON ON(1) 16 prostatic acid phosphatase 6382064 P15309 10 NN(4), NA Low in ON ↓in NA, ON NN, NA, OA precursor (3), OA(2), ON(1) 17 cystatin C precursor 4503107 P01034 9 NN(4), NA ↓in OA NN, NA, OA, (3), OA(1), ON ON(1) 18 tissue inhibitor of 4507509 Q6FGX5 8 NN(3), NA ↑ in ON NN, NA, OA, metalloproteinase 1 (3), OA(1), ON precursor ON(1) Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 12 of 20 http://www.rbej.com/content/11/1/38 Table 2 Detailed list of classification of 35 proteins based on their presence, abundance, and differential expression (Continued) 19 beta 2 microglobulin 4757826 P61769 8 NN(3), NA Low in OA ↓ in OA; Up in ON NN, NA, ON precursor (3), OA(1), ON(1) 20 DJ-1 protein 31543380 Q99497 6 NN(2), NA ↑ in NA, ON NN, NA, ON (3), ON(1) 21 ankyrin repeat domain 11 56676397 Q6UB99 4 NA(1), OA Low in NA ↓ in OA, ON OA (2), ON(1) and ON 22 orosomucoid 1 precursor 167857790 P02763 2 NN(2), NA Low in NA ↓in NA NN (1) 23 serine proteinase inhibitor, 50363217 P01009 8 NN(3), NA ↑ in NA,ON; ↓ in OA NA, OA, ON clade A, member 1 (3), OA(1), ON(1) 24 transferrin 4557871 Q06AH7 6 NN(2), NA Low ↑ in NA, OA NONE (3), OA(1) abundant 25 secretory leukocyte 4507065 P03973 5 NN(1), NA Low ↑ in NA, OA NONE peptidase inhibitor precursor (3), OA(1) abundant 26 ubiquitin and ribosomal 4506713 P62979 4 NN(2), NA Low ↓ in NA, OA NONE protein S27a precursor (1), OA(1) abundant 27 protein tyrosine 104487006 Q13332 4 NN(1), NA Low ↑ in NA, ON NONE phosphatase, receptor type, (2), ON(1) abundant sigma isoform 1 precursor 28 acidic epididymal 25121982 P54107 3 NN(2), NA Low ↓in NA NONE glycoprotein-like 1 isoform 1 (1) abundant precursor 29 prostaglandin H2 D- 32171249 P41222 5 NN(2), NA Low NONE isomerase (2), OA(1) abundant 30 cathepsin B preproprotein 4503139 P07858 6 NN(3), NA Low NONE (3) abundant 31 expressed in prostate and 19923082 Q8WXA2 4 NN(2), NA Low NONE testis (1), OA(1) abundant 32 orosomucoid 2 4505529 P19652 1 NN(1) Low NONE abundant 33 CD177 molecule 110735433 Q8N6Q3 3 NA(1), OA Low NONE (1), ON(1n abundant with (1)) 34 carboxypeptidase E 4503009 P16870 9 NN(3), NA Low in OA NN, NA preproprotein (3), OA(2), and ON ON(1) 35 fibronectin 1 isoform 2 47132551 P02751 10 NN(4), NA Low in ON NN, NA, OA preproprotein (4), OA(1), ON(1) NN = normal sperm count and normal morphology; NA = normal sperm count and abnormal morphology; ON = oligozoospermia and normal morphology; OA = oligozoospermia and abnormal morphology; number in the parenthesis indicates the number of samples. proteins in the NA group was comparable in most of the involved in the biological process were regulatory in cases to the NN group but was different from the ON function (Figure 4). Based on the distribution pattern of and OA groups. The OA group showed only ~15% of the regulatory proteins, the OA groups showed the least the proteins localized in the plasma membrane region involvement of proteins (60%) in regulation compared compared to the other groups, with the maximum num- with 70% - 75% seen in the NN, NA and ON groups. A ber of proteins (~20%) localized in the lysosomal and smaller number of proteins were involved in other func- vacuolar regions. The ON group showed the least distri- tional processes such as protein complex assembly, cell bution of proteins in the nuclear region compared to the morphogenesis, membrane organization, protein matur- extracellular region. ation and trafficking in all the 4 groups. Interestingly, The functional analysis of the significant proteins in none of the proteins in the ON group were involved in each of the groups revealed that most of the proteins any of these processes. Importantly, of all the major Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 13 of 20 http://www.rbej.com/content/11/1/38 Figure 3 Functional annotations from consolidated findings using publicly available software tools (GO term mapper, GO term finder, UniProtKB, STRAP, BioGPS) and proprietary pathway software packages (Ingenuity Pathway Analysis and Metacore™) showing cellular distribution of significant proteins in NN, NA, OA, and ON groups. processes represented, the OA groups showed the lowest compared to DEP and low abundance proteins. The distribution of signal transduction proteins (15%) and extracellular region showed the highest distribution had little or no role in neurological system processing, (91%) of the common proteins whereas they were membrane organization and protein maturation. absent in the ribosomal and endosomal regions. Higher distribution of differentially expressed proteins was seen Comparison of cellular distribution and biological in the cytosolic and Golgi regions compared to the processes amongst the common, DEP, and low abundant common or low abundance proteins. The low abundant proteins proteins were absent in the protein complex and A detailed evaluation of the cellular localization of the secretory granular region but their localization was common, DEP and low abundant proteins is shown in found to be high in the nuclear and endosomal regions. Figure 5. A higher distribution of the common proteins A comparative analysis of the proteins involved in was seen in the majority of cellular compartments various biological processes in the three groups are Figure 4 Functional annotations from consolidated findings using publicly available software tools (GO term mapper, GO term finder, UniProtKB, STRAP, BioGPS) and proprietary pathway software packages (Ingenuity Pathway Analysis and Metacore™) showing biological processes of significant proteins in NN, NA, OA and ON group. Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 14 of 20 http://www.rbej.com/content/11/1/38 Figure 5 Functional annotations from consolidated findings using publicly available software tools (GO term mapper, GO term finder, UniProtKB, STRAP, BioGPS) and proprietary pathway software packages (Ingenuity Pathway Analysis and Metacore™) showing comparison of cellular distribution amongst the proteins in Common, DEP, and Low abundant category. shown in Figure 6. The proteins commonly expressed in metabolism, vesicle-mediated transport and defense all 4 groups played a significant role in many of the bio- response. logical processes such as cellular development, molecu- lar transport, and stress response, interactions with cells Pathways and network analysis using IPA and metacore™ and organisms, cellular processes and in processes relat- Based on the dataset derived from common, DEP and ing to the immune system. Compared with the common low abundance proteins, pathways, biological functions proteins, DEP were comparable for the regulatory pro- and networks of interactions were derived utilizing the cesses, but were reduced in all biological processes. two proprietary pathway packages, IPA and Metacore™. Higher distribution was seen in protein metabolic The important processes affected by common proteins process, vesicle mediated transport, defense response were lipid metabolism (epididymal secretory protein E1 and cellular protein modification process. The low abun- precursor, prosaposin isoform A preprotein, clusterin dant proteins were seen to be involved mainly in protein isoform 1, lactotransferrin and cathepsin D preprotein), Figure 6 Functional annotations from consolidated findings using publicly available software tools (GO term mapper, GO term finder, UniProtKB, STRAP, BioGPS) and proprietary pathway software packages (Ingenuity Pathway Analysis and Metacore™) showing Comparison of Biological Processes amongst the proteins in Common, DEP, and Low abundant category. Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 15 of 20 http://www.rbej.com/content/11/1/38 cell death and survival (cathepsin D preprotein, lacto- contributor to seminal plasma. It is a very rich source of transferrin, clusterin isoform 1, prosaposin isoform A protein with concentration ranges from 35 to 55 g/l preprotein and prostate specific antigen isoform 1 [9,10]. It provides a safe environment for spermatozoa to preprotein), and cellular development (clusterin isoform carry out their physiological functions. Understanding 1, prostate specific antigen isoform 1 preprotein, lacto- the protein profile of human seminal plasma is import- transferrin, extracellular matrix protein 1 isoform 1 pre- ant because it has a profound impact on sperm physi- cursor, cathepsin D preprotein and epididymal secretory ology and thus may affect sperm functioning [31]. proteinE1precursor). In this novel study, we identified 35 proteins based on The important processes affected by DEP showed a SEQUEST scoring in the seminal plasma of men with higher involvement in carbohydrate metabolism and varying semen parameters and categorized them into nephrosis (ankyrin repeat domain 11, beta 2 microglobulin common, differentially expressed, and low abundant (B2MG) precursor, clusterin isoform 1, cystatin C pre- proteins. The large variation in the number of proteins cursor, prostate specific antigen isoform 1 preprotein, identified by any given technique depends mainly on the DJ-1 protein, protein tyrosine phosphatase, receptor sample preparation and mass spectrometry technology type, sigma isoform 1 precursor, transferrin). The major available [32-36]. Recently, the LTQ-Orbitrap mass pathways involved are proteolysis (extracellular matrix; spectrometer has become the cutting-edge instrument ECM), remodeling and connective tissue degradation, for LC/MDS/MS based approaches to characterize the immune response, clathrin-mediated endocytosis sig- seminal proteome. In our study, we used in-solution naling, lipid antigen presentation by CD1, and intrinsic digestion of proteins with the online LC-MS system. prothrombin activation pathway. Similarly, in the low Seminal plasma from different subsets was pooled to abundant proteins the top biological functions included form 4 distinct study groups. There are numerous stud- the cellular development, growth proliferation, DNA ies in the proteomic literature that refer to the benefits replication, recombination, and repair (prostaglandin (H2) of pooling samples where it may not be feasible to D-isomerase, protein tyrosine phosphatase, receptor type, analyze individual samples due to limitations of the sample sigma isoform 1 precursor and transferrin). or the study design [8,17,37,38]. We also studied the major biological functions of the Of the 11 proteins found in all the samples, 9 were DEP in each of the 3 groups and found that free radical associated with sperm function, and the common pro- scavenging was the topmost function in the NA group, teins comprised the majority of the secretions of the while cell-to-cell signaling and interaction were seen in prostate gland, the seminal vesicles and epididymis. all 3 groups. Genes that encode for 7 differentially Some of the proteins or their isoforms detected in the expressed proteins (cysteine-rich secretory protein 1, seminal plasma were zinc alpha-2-glycoprotein 1, clusterin, clusterin, prostatic acid phosphatase (PAP), mucin 6, pros- lactotransferrin, prostate specific antigen. These were simi- tate specific antigen (PSA), zinc alpha-2-glycoprotein 1, lar to those reported by Utleg et al. [39]. and DJ-1) are known to be regulated by androgen recep- Prostate specific antigen is a serine protease that tor. The transcriptional network showed the activation cleaves semenogelin by hydrolysis and thus liquefies the of prostate induction by the androgen receptor signaling semen coagulum and facilitates sperm motility and cap- pathway. DJ-1 protein, protein tyrosine phosphatase, acitation [40,41]. Our study showed that prostate specific receptor type, sigma isoform 1 precursor and transferrin antigen isoform I preprotein was one of the common were observed to interact with other proteins in the proteins, thus indicating its importance in all the 4 pathway database and affect processes related to cellular groups. Prolactin-induced protein (PIP) and Sg II are function and maintenance. Similarly, in the OA group, important common proteins that have a profound im- prostate specific antigen isoform 1 preprotein and pact on sperm physiology. PIP has specificity to fibro- transferrin formed the topmost network, encompassing nectin and constitutes about 1% of seminal coagulum key processes such as gene expression, tissue morphology [42]. It may play a vital role in fibronectin breakdown and cell cycle. The ON group showed immunological dis- during liquefaction. Viscous samples have been reported ease, antigen presentation, cell-to-cell signaling and inter- to show reduced amounts of PIP and PIP precursor, action (B2MG precursor, DJ-1 protein, receptor type, which may also be a contributory factor towards incom- sigma isoform 1 precursor) as the key processes affected plete liquefaction [43]. Both Sg I and II are the major in its topmost network proteins of the coagulum. They represent 20-40% of the seminal plasma proteins. Studies have shown increased Discussion Sg concentrations in asthenozoospermic men [44,45]. Seminal plasma is a mixture of secretions of several male Our study showed that both prolactin and semenogelin accessory glands, including prostate, seminal vesicles, II were in all samples but they were not differentially epididymis and Cowper’s gland. Prostate gland is a major expressed, indicating that men with low sperm count or Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 16 of 20 http://www.rbej.com/content/11/1/38 abnormal morphology may not be affected by these pro- [47-49]. Patients with severe oligozoospermia (<1 × 10 ) teins—a conclusion also made by Milardi et al. [18]. were also shown to have increased levels of seminal PAP Epididymal secretory protein E I precursor, albumin [50]. PAP is produced in the prostate gland and is present preprotein, lactotransferrin, extracellular matrix protein in the seminal fluid at a concentration of 1 mg/mL. It is E1 precursor, prosaposin isoform a preprotein, cathepsin an important tumor marker and acts as a negative growth D preprotein were some of the commonly expressed regulator in prostate cancer [51]. proteins that were not differentially expressed in any of A proteomic analysis of seminal fluid, rather than the groups, suggesting that these proteins may not play blood, has been proposed as as a better starting point to a significant role in sperm concentration or morphology. identify changes that may serve as specific and sensitive The highest percentage of the common proteins was markers of prostate dysfunction [17]. These authors iden- seen in the extracellular region (Figure 5). Human sem- tified more than 100 proteins such as PSA, semenogelin I inal plasma proteins can bind to sperm surface proteins and II, clusterin etc. which have been shown to affect in the ejaculate and form a protective layer around the sperm quality. spermatozoon [12] as well as a sperm reservoir in the The increased expression of semenogelin I in the OA oviduct [46]. It is likely that the extracellular origin of group suggests that the accessory gland secretions have most of the common proteins may play a key role in the a profound impact on oligozoospermic men with abnor- binding activity of the proteins. The absence of common mal morphology. Wang et al. reported semenogelin I proteins in the ribosomal and endosomal region (Figure 5) and mucin were not differentially expressed and there- suggests a relatively low involvement in protein metab- fore had no effect in asthenozoospermic men [21]. Our olism, as also seen by the low distribution of common study also included the differentially expressed proteins, proteins in the ‘protein metabolic process’ category, especially DJ-1 secreted from the testis, epididymis and (Figure 6). Zinc alpha-2 glycoprotein and clusterin play prostate [39,52]. DJ-1 has a high level of expression in a role in signal transduction while lactotransferrin is a the testis. We found DJ-1 to be overexpressed in the NA transport and structural protein [39]. Prostate specific and ON groups. We also documented that orosomucoid 1 antigen has been shown to have enzymatic activity. Our precursor was downregulated in the NA group whereas results show a high distribution of signal transducing expression of orosomucoid 2 was comparable in all protein among the common proteins, suggesting the groups, though it was present in low abundance. However, importance of clusterin, isoform 1 and zinc alpha-2 Wang et al. reported the overexpression of orosomucoid 1 glycoprotein 1 (Figure 6). This was also confirmed from and orosomucoid 2 in asthenozoospermic patients [21]. the pathway and network analysis, which also These proteins have also been found in high abundance in highlighted the role these proteins play in molecular post vasectomy patients [8]. transport, cell death and survival and lipid metabolism. B2MG was found to be under-expressed in OA while A clear overlap was observed for some of the differen- over-expressed in the ON samples. B2MG is present in tially expressed proteins. These included the prostate spe- all nucleated cells. It is one of the two polypeptide cific antigen isoform 1 preprotein, zinc alpha 2 glycoprotein chains of the major histocompatiblity complex (MHC) 1, and clusterin isoform 1. While semenogelin II precursor class I molecule. In humans B2MG is coded by the B2M was seen in common proteins, the semenogelin I isoform b gene. It is a marker of cellular immune system. It is a preprotein was found to be upregulated in the OA group. naturally occurring protein and can detect certain types This suggests that it may contribute to the low motility of tumor cells in the blood and kidney, and some inflam- seen in this group compared to other groups. matory and autoimmune disorders [53,54]. In our study Clusterin isoform was downregulated in the ON as well as that reported by Fung et al. [17], many of the group. This is an interesting finding, given that clusterin proteins in the seminal plasma were identified to be fore- isoform 1 has been shown to be downregulated in pros- runners of prostate disease, suggesting a pathogenic role tate cancer. The proteome of the seminal fluid is largely for B2MG [55,56]. Similarly high levels of B2MG were attributed to secretions from the prostate gland, and reported in the seminal plasma of azoospermic men com- approximately 10% is contributed by the testis and the pared to controls [10,57,58]. An inverse correlation was epididymis [8]. reported between B2MG levels in seminal fluid and sperm Prostate gland is a major contributor to seminal plasma. count [10]. Furthermore, one of the proteins - prostatic acid phos- Similary, we reported the underexpression of galectin phatase (PAP) was significantly increased in azoospermic 3 binding protein in oligozoospermic group. Galectin-3 men compared to oligozoospermic men and asthenozoo- is a carbohydrate-binding protein whose expression level spermic men [10]. In our study, PAP levels were down has been shown to correlate with metastatic potential in regulated in the NA and ON groups. PAP levels have also a number of different types of tumors. Galectin-3 is been reported to correlate with sperm concentration downregulated in prostate cancer. The altered Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 17 of 20 http://www.rbej.com/content/11/1/38 downregulation pattern of galectin-3 observed between response and that low levels of this protein are seen in tumor stages suggests different roles for galectin-3 in the stress conditions. Low distribution of B2MG in DEP progression of prostate cancer [59]. compared to normal and low abundant proteins further TIMP 1 can inhibit tumor growth, invasion and me- suggests that in addition to B2MG, there are other pro- tastasis through their matrix metalloproteinases (MMP) teins that also play a key role in immune system processes. inhibitory activity [60,61]. TIMPs play an inhibitory role, We further validated this observation from the pathway and any imbalance between the two may result in pro- analysis, which showed that that the common protein gression of the disease [62]. TIMP was found to be over- prosaposin isoform, a preprotein, is involved in lipid anti- expressed in the ON group. gen presentation by CD1. Amongst the proteins that were uniquely under- We observed overlapping of differentially expressed expressed in the OA group was ankyrin repeat domain proteins with the low abundant proteins in our study. 11 (ANKRD11). Ankyrins function as adaptor proteins These included transferrin, secretory leukocyte peptidase [63,64] and play a vital role in membrane skeleton inhibitor precursor, ubiquitin and ribosomal protein organization, ionic transport, maintenance of cell polar- S27a precursor, protein tyrosine phosphatase, receptor ity as well as cell-cell adhesion regulation. Data suggest type, sigma isoform 1 precursor and acidic epidiymal that ANKRD11 may act as a tumor suppressor [65]. It’s glycoprotein- like 1 isoform 1 precursor. Of these pro- presence in the semen samples has not been well docu- teins, protein tyrosine phosphatase, receptor type, sigma mented, but it is likely that its overexpression in isoform 1 precursor, acidic epididymal glycoprotein-like oligozoospermic men may play a role in apoptosis. isoform 1 precursor were amongst the low abundant Clusterin isoform 1 can be both pro- and antiapoptotic proteins. Protein tyrosine phosphatase, receptor type, depending upon the isoform expressed [66,67]. It plays a sigma isoform 1 precursor were upregulated while acidic key role in signal transduction and is involved in apop- epididymal glycoprotein-like isoform 1 precursor was tosis of spermatocytes, sperm maturation, and down regulated in the NA group. The acidic epididymal spermiation. Low signal transducing proteins were seen glycoprotein like I isoform I precursor is a member of in the OA group compared with the other groups, as the cysteine - rich secretory protein (CRISP) family and shown in Figure 4. is encoded by the gene CRISP-I. It is expressed in the Compared with the proteins involved in stress response, epididymis and is secreted into the epididymal lumen. It as elucidated from GO annotations (Figure 4), the NA and binds to the post acrosomal region of the head, where it ON groups had a higher distribution of stress proteins may play a role in sperm-egg fusion. The reported low such as DJ-1 whereas a lower distribution of this protein abundance of acidic epididymal glycoprotein like I iso- was seen in OA group. DJ-1 protein is a multifunctional, form I precursor protein is concordant with the findings highly conserved antioxidant protein and is upregulated in of Batruch et al. who also reported its low abundance in hyperglycemia [68,69]. It is mainly involved in the control the seminal plasma of post vasectomy patients compared of oxidative stress and is downregulated in the seminal to controls [8]. plasma of asthenozoospermic men [21]. DJ-1 is activated Transferrin is one of the serum proteins that has been in stress conditions, but with higher levels of stress (as characterized in the seminal plasma, but its role in male seen in OA), there is depletion of antioxidant levels and infertility is unclear [20]. Our study showed this protein low expression of DJ-1 protein. Furthermore, the major was present in low levels in the NN group but was up biological function that comes into focus through IPA in regulated in NA and OA groups. Prostaglandin H2 D the NA group is free-radical scavenging activity. This fur- isomerase, cathepsin B preprotein, orosomucoid 2 and ther supports the fact that DJ-1 was activated in the NA the CD177 molecule were the low abundant proteins that group. Significant proteins were distributed in the lyso- were not differentially expressed in any of the groups. In our study, CD177 was absent in the NN group but present somal and vacuolar regions in higher amounts in the OA group (Figure 3), suggesting that proteins with phagocytic in all the other groups although they were not activity may be activated in stress conditions. Previous differentially expressed. Our findings are consistent with previous publications that reported low concentrations of studies have reported reduced levels of DJ-1 in sperm in response to toxic exposure of male rats to ornidazole and CD177 in fertile men (control samples) compared to epichlorhydrin [70]. postvasectomy men [8] but are contrary to the findings of Wang et al. who reported upregulation of CD177 in Utleg et al. categorized prostatic acid phosphatase (PAP) precursor as an enzymatic protein, and the pres- asthenozoospermic patients [21]. Cathepsin B preprotein ence of this protein in our study suggests that it plays a was present in the NN and NA groups, showing its speci- similar role [39]. The distribution pattern of immune ficity to the normal sperm count while prostaglandin H2 system response in all the groups is shown in Figure 4. D isomerase was absent in the ON group. Cathepsin B It is likely that B2MG is the major protein in immune preprotein found in the lysosomal region is a thiol Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 18 of 20 http://www.rbej.com/content/11/1/38 protease believed to participate in intracellular degrad- Author details Center for Reproductive Medicine, Glickman Urological and Kidney Institute, ation. It has also been implicated in tumor invasion and Cleveland Clinic, Cleveland, OH, USA. Center for Cellular and Molecular metastasis. The prostaglandin (H2) D-isomerase is Biology, Hyderabad, Andhra Pradesh, India. Bioinformatics Core Services, expressed in the testis, epididymis and prostate and is se- Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA. Proteomics Core Services, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA. creted into the seminal fluid. It binds small non-substrate Molecular Biotechnology Core lab, Lerner Research Institute, Cleveland lipophilic molecules and may act as a scavenger for harm- Clinic, Cleveland, OH, USA. Permanent Address: Ravenshaw University, ful hydrophobic molecules. The prostaglandin (H2) D- Cuttack, Odisha, India. isomerase and cathepsin B protein are potentially involved Received: 15 January 2013 Accepted: 22 March 2013 in biological processes such as vesicle-mediated transport Published: 11 May 2013 and defense response. The low abundant proteins showed a marked distribution in the extracellular region and may References play a regulatory role. The major pathways in which the 1. The Practice Committee of the American Society for Reproductive Medicine: low abundant proteins were involved were prostanoid bio- Diagnostic evaluation of the infertile male: a committee opinion. Fertil Steril 2012, 98:294–301. synthesis and eicosanoid signaling and acute phase re- 2. Kruger TF, Menkveld R, Stander FS, Lombard CJ, Van der M, van Zyle JA, sponse signaling, but the distribution of signal Smith K: Sperm morphologic features as a prognostic factor in in vitro transduction proteins was decreased in low abundant fertilization. Fertil Steril 1986, 46:1118–1123. 3. Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Veeck LL, proteins. Morshedi M, Brugo S: New method of evaluating sperm morphology with predictive value for human in vitro fertilization. Urology 1987, 30:248–251. 4. 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Batruch I, Lecker I, Kagedan D, Smith CR, Mullen BJ, Grober E, Lo KC, Our findings from the bioinformatic analysis shows that Diamandis EP, Jarvi KA: Proteomic analysis of seminal plasma from stress proteins such as DJ-1 are differentially regulated normal volunteers and post-vasectomy patients identifies over 2000 and expressed in the different study groups, suggesting proteins and candidate biomarkers of the urogenital system. J Proteome Res 2011, 10:941–953. that some of these proteins may serve as a potential bio- 9. Pilch B, Mann M: Large-scale and high-confidence proteomic analysis of markers in identifying the mechanistic role in men with human seminal plasma. Genome Biol 2006, 7(5):R40. poor sperm quality. 10. Davalieva K, Kiprijanovska S, Noveski P, Plaseski T, Kocevska B, Broussard C, Plaseska-Karanfilska D: Proteomic analysis of seminal plasma in men with different spermatogenic impairment. Andrologia 2012, 44:256–264. Competing interests 11. 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RJ participated in organizing the 15. Mengual L, Ballescá JL, Ascaso C, Oliva R: Marked differences in protamine subject information and review of the study findings. ES provided the content and P1/P2 ratios in sperm cells from percoll fractions between subjects and participated in the study design. All authors read and approved patients and controls. J Androl 2003, 24:438–447. the final manuscript. 16. Aoki VW, Moskovtsev SI, Willis J, Liu L, Mullen JB, Carrell DT: DNA integrity is compromised in protamine-deficient human sperm. J Androl 2005, 26:741–748. Acknowledgements 17. Fung KY, Glode LM, Green S, Duncan MW: A comprehensive This study was supported by Cleveland Clinic, Research Program Committee. characterization of the peptide and protein constituents of human Authors wish to thank the Cleveland Clinic Andrology laboratory personnel seminal fluid. Prostate 2004, 61:171–181. for their help with scheduling of subjects used in this study. 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Biol Pharm Bull 2002, 25:853–856. doi:10.1186/1477-7827-11-38 Cite this article as: Sharma et al.: Functional proteomic analysis of seminal plasma proteins in men with various semen parameters. Reproductive Biology and Endocrinology 2013 11:38. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Reproductive Biology and Endocrinology Springer Journals

Functional proteomic analysis of seminal plasma proteins in men with various semen parameters

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Springer Journals
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Copyright © 2013 by Sharma et al.; licensee BioMed Central Ltd.
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Medicine & Public Health; Reproductive Medicine; Endocrinology
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1477-7827
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Abstract

Background: Alterations at the molecular level in spermatozoa and seminal plasma can affect male fertility. The objective of this study was to determine if analysis of differential expression of proteins in varying semen parameters can serve as potential biomarkers for male infertility. Methods: The differential expression of proteins in the seminal plasma of men based on sperm count and morphology were examined utilizing proteomic tools. Subjects were categorized based on sperm concentration and morphology into 4 groups: 1) normal sperm count and normal morphology (NN); 2) normal sperm count and abnormal morphology (NA); 3) oligozoospermia and normal morphology (ON); and 4) oligozoospermia and abnormal morphology (OA). Proteomic analysis was performed by LC-MS/MS followed by functional bioinformatics analysis. Protein distribution in the NA, ON and OA groups was compared with that of the NN group. Results: Twenty proteins were differentially expressed among the 4 groups. Among the unique proteins identified, 3 were downregulated in the NA group, 1 in the ON group and 1 in the OA group while 2 were upregulated in the ON and OA groups. The functional analysis 1) identified biological regulation as the major processes affected and 2) determined that most of the identified proteins were of extracellular origin. Conclusions: We have identified proteins that are over-or underexpressed in the seminal plasma of men with poor sperm quality. The distinct presence of some of the proteins may serve as potential biomarkers and provide insight into the mechanistic role played by these proteins in male infertility. Further studies using Western Blot analysis are required to validate these findings. Background morphology have been documented [5]. Idiopathic and un- Infertility is a major problem in 15% of couples world- explained infertility cannot be diagnosed by routine sperm wide. Male factors may play a role in half of these cases function tests [6]. Similarly, oligozoospermic men may have [1]. Most causes of male infertility are idiopathic. Semen other underlying pathologies that may contribute to infer- analysis remains the cornerstone in the evaluation of male tility. Evaluation solely based on semen analysis is insuffi- infertility. However, the data generated from this routine cient to determine the fertility status of the male partner. testing do not provide any insight into the underlying Spermatogenesis is a complex process that involves de- problems associated with developing spermatozoa. Sperm velopment of the undifferentiated germ cells into a highly morphology plays an important role in conception, and specialized spermatozoon capable of fertilizing an oocyte both fertilization and pregnancy rates are affected when [7]. Fertilization requires physical proximity of the sperm- morphologically normal sperms are below 5%. It is also a atozoa and the oocytes. Seminal plasma composed of reflection of poor testicular physiology and is an important secretions from the testis, epididymis and male accessory factor in male infertility [2-4]. However, a significant over- glands [8] provides a favorable environment and serves as lap of semen parameters such as sperm count, motility and a vehicle for the spermatozoa as it travels to meet the oocyte. * Correspondence: Agarwaa@ccf.org Seminal plasma contains unique proteins necessary for Center for Reproductive Medicine, Glickman Urological and Kidney Institute, sperm function and survival [9,10]. Seminal plasma pro- Cleveland Clinic, Cleveland, OH, USA teins play a variety of roles—they help protect the sperm Full list of author information is available at the end of the article © 2013 Sharma et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 2 of 20 http://www.rbej.com/content/11/1/38 by binding to the sperm surface during ejaculation and Semen analysis play a key role in capacitation, acrosome reaction, and Following complete liquefaction (average time: 20 minutes sperm-egg fusion [11,12]. They can also modulate im- and no more than 60 min.), manual semen analysis was mune response in male and female reproductive tracts, performed using a MicroCell counting chamber (Vitrolife, ensuring that the most competent spermatozoa meet the San Diego, CA) to determine sperm concentration and oocyte during fertilization [13]. Thus, seminal plasma percentage motility according to WHO guidelines [22]. proteins can serve as important biomarkers for male Viability was determined with Eosin - Nigrosin stain. infertility [14]. Smears of the raw semen were stained with a Diff-Quik Conventional 1-Dimensional gel electrophoresis studies kit (Baxter Healthcare Corporation, Inc., McGaw Park, IL) have provided information in relation to sperm proteins for assessment of sperm morphology according to WHO and their function in normal and abnormal spermato- criteria [22]. zoa [15,16]. Advancements in mass- spectrometry and After analysis of semen parameters, aliquots of the proteomic-basedtechniqueshavemadeitpossibleto samples were frozen at −80°C for proteomic analysis. analyze the complex protein mixtures found in tissues and body fluids. Several attempts have been made to identify these proteins using high-throughput techniques Preparation of samples for proteomic analysis such as matrix assisted laser desorption ionization – Samples were divided into 4 groups based only on time of flight (MALDI-TOF) mass spectrometry (MS) normal sperm concentration and normal morphology and liquid chromatography – tandem mass spectrometry parameters according to WHO criteria [22]. The groups (LC-MS/MS) and linear ion trap (LTQ-Orbitrap) mass were as follows: Group 1: normal sperm count and spectrometry [17-21]. normal morphology (NN = 26); Group 2: normal sperm Alterations at the molecular level in spermatozoa and count and abnormal morphology (NA = 22); Group 3: the seminal plasma may contribute to male infertility. oligozoospermia and normal morphology (ON = 6) and However, even after accounting for all the advances in group 4: oligozoospermia and abnormal morphology proteomics, there has been a great lack of detailed data (OA = 10). in the area of comparative analysis of seminal plasma To prepare the samples for proteomic analysis, they proteins associated with male infertility. were thawed, and clear seminal plasma was separated The objective of the present study was 1) to compare from the sperm pellet by centrifugation at 3,000 g for the differential expression of proteins in the seminal 30 minutes to ensure complete removal of the cellular plasma from subjects with normal or abnormal sperm components. Seminal plasma samples were pooled into concentration and sperm morphology utilizing proteomic replicates (NN = 5; NA = 4; ON = 1; OA = 2). Each sam- tools such as LC-MS/MS and 2) utilize the functional bio- ple was dissolved in 98% acetonitrile containing 0.1% informatics analysis to identify the cellular origin and the trifluoroacetic acid followed by lyophilization at −80°C differentially affected processes and/or pathways of these under vacuum for 2 days. The lyophilized sample was proteins to gain insights into the mechanistic roles played used to estimate the protein content. The samples were by these proteins in effecting the observed phenotypes. first precipitated in cold acetone and centrifuged at These analyses could possibly identify potential bio- 10,000 g for 15 minutes. The acetone was poured off, markers for male infertility. and the protein pellet was allowed to dry at room temperature. The protein pellet was solubilized in a buf- fer of 6 M urea, 100 mM Tris, pH 8.0. The proteins were Methods then reduced by the addition of DTT (200 mM in After obtaining Institutional Review Board approval, 100 mM Tris) for 15 minutes at room temperature and written consent was obtained from all subjects. Semen then alkylated by the addition of 200 mM iodoacetamide samples were obtained from 64 subjects who were (200 mM in 100 mM Tris) for 20 minutes at room healthy male volunteers of unproven fertility (n = 21) temperature. The urea concentration was then reduced and men presenting to our infertility clinic for evaluation to approximately 1.2 M, and trypsin was added at a (n = 43). Semen samples were collected by masturbation ratio of 1:50. Digestion was carried out overnight at after 2–3 days of sexual abstinence. Samples with room temperature. The digestion was stopped the next leukocytospermia--a high concentration of white blood morning by adding acetic acid to lower the pH to <6, cells (>1 × 10 WBC/mL)–were examined for the pres- and the samples were centrifuged to remove insoluble ence of granulocytes by the peroxidase or the Endtz test. material. The digests were then prepared for LC-MS/MS The patients with a positive Endtz test were excluded analysis by using PepClean C-18 spin columns to desalt from the study. Semen analysis was conducted according the samples, which were then brought up in 50 μLof 1% to WHO criteria as described below [22]. acetic acid. Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 3 of 20 http://www.rbej.com/content/11/1/38 Liquid chromatography – mass spectrometer analysis database index, molecular weight, peptide coverage and (LC-MS/MS) Mascot score is shown in Table 1. A protein was consid- The LC-MS system is a Finnigan LTQ linear ion trap ered significant if the SC cut off value was ≥ 10 in at mass spectrometer system. The high performance liquid least one sample and present in at least 50% of the chromatography (HPLC) column was a self-packed samples in a group. They were considered ‘low abun- 9cm×75 μm (internal diameter) Phenomenex Jupiter dant’ if the SC cut-off value was ≤10 in all the samples. C18 reversed-phase capillary chromatography column. The differentially expressed proteins (DEP) in the NA, Ten μL volumes of the extract were injected, and the pep- OA, and ON groups were categorized based on the NSC tides that were eluted from the column by an acetonitrile/ ratio cut-off values of > 2 (for over-expressed) or < 0.5 0.1% formic acid gradient at a flow rate of 0.25 μL/min (for under-expressed) in comparison to the NN group. were introduced into the source of the mass spectrometer We identified a total of 35 proteins; of these, 10 were on-line. The microelectrospray ion source was set at classified as low abundant. Amongst the remaining 25 2.5 kV. The digest was analyzed using the data-dependent significantly abundant proteins (24 in NN, 23 in NA, 20 multitask capability of the instrument acquiring full scan in OA, and 16 in ON), 11 were present in all the samples, mass spectra to determine peptide molecular weights and and 13 proteins were identified as unique to one or two or product ion spectra to determine the amino acid sequence three of the four samples. 20 proteins were identified as in successive instrument scans [23]. This mode of analysis differentially expressed in the NA, OA, and ON groups as produces approximately 2500 collision-induced dissoci- compared to NN group, with 2 proteins differentially ation (CID) spectra of ions ranging in abundance over sev- expressed in all three groups (Figure 1). The remaining eral orders of magnitude. The spectral count (SC) for each 18 were present in either of the groups (Figure 2). A protein was determined. Normalized spectral count (NSC) detailed list of the proteins classified under these cat- was obtained by dividing the spectral count for each pro- egories (Common, Unique, Significant, Low Abundant, tein and the total number of spectral counts identified in and Differentially Expressed) is shown in Table 2. the sample. The spectral counts were quantitated by tak- ing the normalized spectral count ratio for two sets of Identification of the common proteins samples. A protein was considered to be differentially Our analyses revealed a set of 11 proteins that were expressed if there was at least a two-fold difference in the common to all the samples in the 4 groups (Table 2). spectral count ratios between the two samples. Prolactin induced protein (PIP), semenogelin II (SgII) precursor, albumin preprotein, lactotransferrin, epididy- Data analysis mal secretory protein E1 precursor, extracellular matrix All CID spectra collected in the experiment were used to protein 1 isoform 1 precursor, prosaposin isoform A search the National Center for Biotechnology Information preprotein, cathepsin D preprotein, prostate specific (NCBI) human reference sequence database with the antigen isoform 1 preprotein, zinc alpha-2 glycoprotein search engine MASCOT (Matrix Science, Boston, MA, 1, and clusterin isoform 1 were the common proteins www.matrixscience.com). After identification, a database identified. consisting of all proteins identified in these searches was created and used for a second set of searches. These Identification of differentially expressed proteins searches were performed with a program called As shown in Figure 1, the DEP list encompassed pro- SEQUEST, and the results from these SEQUEST searches teins that overlapped with other categories (common, were used to determine the spectral counts. Furthermore, unique, low abundant and significant). The common functional bioinformatics analysis was done using publicly proteins that were also differentially expressed included available software packages such as Gene Ontology anno- prostate specific antigen isoform I preprotein; zinc tations from GO Term Finder [24] and GO Term Mapper alpha-2-glycoprotein 1 and clusterin isoform 1. Five low [25], UniProt [26], STRAP [27], and BioGPS [28]) as well abundant proteins (transferrin; secretory leukocyte as proprietary software packages (Ingenuity Pathway Ana- peptidase inhibitor precursor; ubiquitin and ribosomal lysis (IPA) from Ingenuity® Systems [29], and Metacore™ protein S27a precursor; protein tyrosine phosphatase from GeneGo Inc. [30]) to identify the differentially receptor type, sigma isoform 1 precursor, and acidic affected processes, pathways, interactions, and cellular epididymal glycoprotein-like 1 isoform 1 precursor) distribution of the proteins in the four study groups. were included as differentially expressed because their NSC ratio comparison met the 2-fold cutoff criteria. Results Of the 20 differentially expressed proteins, mucin 6, Analysis of the proteins identified by LC-MS/MS gastric; orosomucoid 1 precursor and acidic epididy- The proteins identified in the 12 replicates from NN, mal glycoprotein-like isoform 1 precursor were unique NA, ON and OA group showing protein name, NCBI proteins that were down regulated in the NA group. Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 4 of 20 http://www.rbej.com/content/11/1/38 Table 1 Identification of proteins in the 12 replicates from NN, NA, ON and OA group showing protein name, NCBI database index, molecular weight, peptide coverage and Mascot score Identification LTQ No. Protein name NCBI database index Calculated MW in PI Peptides Mascot number kDa (%coverage) score Sample NN1 semenogelin II precursor 4506885 65 9 8(10%) 9536 prolactin induced protein 4505821 16 8.2 3(20%) 5335 albumin preproprotein 4502027 71 5.9 9(22%) 4805 epididymal secretory protein E1 precursor 5453678 16 7.5 3 (21%) 794 prosaposin isoform a preproprotein 11386147 59 5 1 (2%) 658 mucin 6, gastric 151301154 263 7.2 4 (3%) 461 prostate specific antigen isoform 4 preproprotein 71834855 24 7 3 (15%) 460 armadillo repeat protein 4502247 105 6.3 3(5%) 413 cathepsin D preproprotein 4503143 45 6.1 1 (4%) 285 zinc alpha-2-glycoprotein 1 4502337 34 5.7 2 (12%) 260 cystatin S precursor 4503109 16 4.9 2 (31%) 178 ubiquitin and ribosomal protein S27a precursor 4506713 18 9.6 1 (10%) 173 clusterin isoform 1 42716297 58 6.2 3 (10%) 142 Sample NN2 prolactin-induced protein 4505821 16 8.2 10 (69%) 32886 semenogelin II precursor 4506885 65 9 17 (33%) 22468 semenogelin I isoform b preproprotein 38049014 45 9.2 13 (33%) 5388 albumin preproprotein 4502027 71 5.9 24 (54%) 15508 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 11 (61%) 5087 lactotransferrin precursor 54607120 80 8.5 22 (42%) 4281 zinc alpha-2-glycoprotein 1 4502337 34 5.7 11 (44%) 3424 cystatin S precursor 4503109 16 4.9 5 (44%) 1809 prosaposin isoform a preproprotein 11386147 59 5 9 (33%) 880 epididymal secretory protein E1 precursor 5453678 16 7.5 4 (52%) 786 serine proteinase inhibitor, clade A, member 1 50363217 46 5.3 11 (48%) 737 mucin 6, gastric 151301154 263 7.2 11 (9%) 524 extracellular matrix protein 1 isoform 1 precursor 221316614 62 6.2 7 (31%) 504 cystatin C precursor 4503107 16 9 6 (68%) 432 tissue inhibitor of metalloproteinase 1 precursor 4507509 23 8.4 4 (40%) 411 fibronectin 1 isoform 3 preproprotein 16933542 262 5.4 8(11%) 353 cathepsin D preproprotein 4503143 45 6.1 3 (11%) 266 acid phosphatase, prostate short isoform precursor 6382064 44 5.8 2 (12%) 166 carboxypeptidase E preproprotein 4503009 53 5 5 (20%) 153 clusterin isoform 1 42716297 58 6.2 2 (7%) 106 Sample NN3 prolactin-induced protein 4505821 16 8.2 10 (66%) 24697 semenogelin II precursor 4506885 65 9 19 (35%) 13533 semenogelin I isoform b preproprotein 38049014 45 9.2 15 (39%) 2633 albumin preproprotein 4502027 71 5.9 30 (60%) 8842 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 16 (70%) 8634 lactotransferrin precursor 54607120 80 8.5 23 (49%) 4607 Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 5 of 20 http://www.rbej.com/content/11/1/38 Table 1 Identification of proteins in the 12 replicates from NN, NA, ON and OA group showing protein name, NCBI database index, molecular weight, peptide coverage and Mascot score (Continued) zinc alpha-2-glycoprotein 1 4502337 34 5.7 9 (42%) 3935 epididymal secretory protein E1 precursor 5453678 16 7.5 6 (52%) 1214 tissue inhibitor of metalloproteinase 1 precursor 4507509 23 8.4 3 (29%) 987 prosaposin isoform a preproprotein 11386147 59 5 5 (21%) 833 extracellular matrix protein 1 isoform 1 precursor 221316614 62 6.2 6 (22%) 766 cystatin S precursor 4503109 16 4.9 5 (49%) 621 beta 2 microglobulin precursor 4757826 13 6 2 (21%) 523 fibronectin 1 isoform 3 preproprotein 16933542 262 5.4 4 (2%) 410 orosomucoid 1 precursor 167857790 23 5 3 (16%) 397 cystatin C precursor 4503107 16 9 3 (26%) 301 mucin 6, gastric 151301154 263 7.2 7 (7%) 288 cathepsin D preproprotein 4503143 45 6.1 2 (11%) 228 carboxypeptidase E preproprotein 4503009 53 5 3 (16%) 193 acidic epididymal glycoprotein-like 1 isoform 1 precursor 25121982 29 5.5 2 (10%) 166 galectin 3 binding protein 5031863 66 5.1 4 (11%) 165 clusterin isoform 1 42716297 58 6.2 2 (7%) 158 acid phosphatase, prostate short isoform precursor 6382064 44 5.8 5 (16%) 128 prostaglandin (H2) D-isomerase −1 peptide 32171249 21 7.6 1 (8%) 115 Sample NN4 prolactin-induced protein 4505821 16 8.2 12 (76%) 36052 semenogelin II precursor 4506885 65 9 16 (32%) 17746 semenogelin I isoform b preproprotein 38049014 45 9.2 14 (34%) 2723 lactotransferrin precursor 54607120 80 8.5 39 (61%) 8695 albumin preproprotein 4502027 71 5.9 23 (48%) 8361 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 15 (67%) 7417 zinc alpha-2-glycoprotein 1 4502337 34 5.7 11 (38%) 3979 mucin 6, gastric 151301154 263 7.2 14 (12%) 1685 epididymal secretory protein E1 precursor 5453678 16 7.5 7 (70%) 1428 acid phosphatase, prostate short isoform precursor 6382064 44 5.8 9 (26%) 1384 clusterin isoform 1 42716297 58 6.2 6 (16%) 1148 orosomucoid 1 precursor 167857790 23 5 4 (32%) 942 orosomucoid 2 4505529 23 5 2 (12%) 207 prosaposin isoform a preproprotein 11386147 59 5 6 (27%) 941 extracellular matrix protein 1 isoform 1 precursor 221316614 62 6.2 7 (25%) 731 tissue inhibitor of metalloproteinase 1 precursor 4507509 23 8.4 3 (29%) 635 beta 2 microglobulin precursor 4757826 13 6 2 (21%) 489 fibronectin 1 isoform 3 preproprotein 16933542 262 5.4 4 (3%) 447 cystatin C precursor 4503107 16 9 3 (26%) 391 carboxypeptidase E preproprotein 4503009 53 5 4 (14%) 358 galectin 3 binding protein 5031863 66 5.1 5 (14%) 341 transferrin 4557871 79 6.8 2 (4%) 275 acidic epididymal glycoprotein-like 1 isoform 1 precursor 25121982 29 5.5 2 (10%) 238 cystatin S precursor 4503109 16 4.9 2 (20%) 205 serine proteinase inhibitor, clade A, member 1 50363217 46 5.3 3 (12%) 187 Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 6 of 20 http://www.rbej.com/content/11/1/38 Table 1 Identification of proteins in the 12 replicates from NN, NA, ON and OA group showing protein name, NCBI database index, molecular weight, peptide coverage and Mascot score (Continued) cathepsin D preproprotein 4503143 45 6.1 2 (11%) 185 prostaglandin (H2) D-isomerase −1 peptide 32171249 21 7.6 1 (8%) 133 Sample NN5 prolactin-induced protein 4505821 16 8.2 8 (44%) 15001 semenogelin II precursor 4506885 65 9 10 (14%) 7235 albumin preproprotein 4502027 71 5.9 17 (39%) 2621 epididymal secretory protein E1 precursor 5453678 16 7.5 3 (23%) 552 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 5 (22%) 539 cystatin S precursor 4503109 16 4.9 2 (23%) 332 zinc alpha-2-glycoprotein 1 4502337 34 5.7 4 (19%) 250 prosaposin isoform a preproprotein 11386147 59 5 1 (2%) 239 prostatic acid phosphatase precursor 6382064 44 5.8 2 (4%) 180 lactotransferrin 54607120 80 8.5 4 (10%) 173 clusterin isoform 1 42716297 58 6.2 2 (7%) 157 galectin 3 binding protein 5031863 66 5.1 2 (4%) 152 extracellular matrix protein 1 isoform 1 precursor 4758236 62 6.2 1 (5%) 119 Sample NA1 prolactin-induced protein 4505821 16 8.2 10 (62%) 17127 albumin preproprotein 4502027 71 5.9 36 (61%) 9157 semenogelin II precursor 4506885 65 9 22 (39%) 7469 semenogelin I isoform b preproprotein 38049014 45 9.2 19 (42%) 2622 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 14 (67%) 5287 lactotransferrin 54607120 80 8.5 19 (40%) 2696 zinc alpha-2-glycoprotein 1 4502337 34 5.7 9 (38%) 1904 epididymal secretory protein E1 precursor 5453678 16 7.5 6 (68%) 1228 extracellular matrix protein 1 isoform 1 precursor 221316614 62 6.2 6 (22%) 635 serine proteinase inhibitor, clade A, member 1 50363217 46 5.3 8 (27%) 616 tissue inhibitor of metalloproteinase 1 precursor 4507509 23 8.4 2 (19%) 487 prosaposin isoform a preproprotein 11386147 59 5 4 (15%) 475 beta 2 microglobulin precursor 4757826 13 6 2 (21%) 400 fibronectin 1 isoform 3 preproprotein 16933542 262 5.4 7 (5%) 398 cystatin C precursor 4503107 16 9 3 (26%) 340 cystatin S precursor 4503109 16 4.9 2 (20%) 261 mucin 6, gastric isoform 1 89033736 185 6.3 4 (5%) 238 prostaglandin (H2) D-isomerase 32171249 21 7.6 1 (8%) 227 prostatic acid phosphatase precursor 6382064 44 5.8 4 (8%) 189 protein tyrosine phosphatase, receptor type, sigma isoform 1 104487006 218 6.1 5 (5%) 180 precursor transferrin 4557871 79 6.8 3 (17%) 174 clusterin isoform 1 42716297 58 6.2 4 (11%) 171 cathepsin D preproprotein 4503143 45 6.1 1 (4%) 149 galectin 3 binding protein 5031863 66 5.1 5 (14%) 101 Sample NA2 prolactin-induced protein 4505821 16 8.2 12 (76%) 21197 Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 7 of 20 http://www.rbej.com/content/11/1/38 Table 1 Identification of proteins in the 12 replicates from NN, NA, ON and OA group showing protein name, NCBI database index, molecular weight, peptide coverage and Mascot score (Continued) semenogelin II precursor 4506885 65 9 18 (36%) 9137 semenogelin I isoform b preproprotein 38049014 45 9.2 13 (33%) 1777 albumin preproprotein 4502027 71 5.9 26 (55%) 7695 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 16 (67%) 4963 zinc alpha-2-glycoprotein 1 4502337 34 5.7 13 (44%) 3134 lactotransferrin 54607120 80 8.5 24 (46%) 3105 epididymal secretory protein E1 precursor 5453678 16 7.5 5 (68%) 1276 prosaposin isoform a preproprotein 11386147 59 5 7 (26%) 662 tissue inhibitor of metalloproteinase 1 precursor 4507509 23 8.4 3 (29%) 660 serine proteinase inhibitor, clade A, member 1 50363217 46 5.3 5 (20%) 612 extracellular matrix protein 1 isoform 1 precursor 4758236 62 6.2 7 (22%) 577 cystatin C precursor 4503107 16 9 3 (26%) 548 beta 2 microglobulin precursor 4757826 13 6 2 (21%) 443 prostatic acid phosphatase precursor 6382064 44 5.8 8 (18%) 410 cathepsin D preproprotein 4503143 45 6.1 5 (25%) 375 clusterin isoform 1 42716297 58 6.2 2 (7%) 308 DJ-1 protein 31543380 20 6.3 2 (26%) 279 carboxypeptidase E preproprotein 4503009 53 5 4 (19%) 278 galectin 3 binding protein 5031863 66 5.1 5 (14%) 266 cystatin S precursor 4503109 16 4.9 6 (64%) 212 CD177 molecule 110735433 47 5.6 2 (9%) 199 mucin 6, gastric isoform 1 89033736 185 6.3 3 (4%) 196 fibronectin 1 isoform 3 preproprotein 16933542 262 5.4 4 (4%) 172 secretory leukocyte peptidase inhibitor precursor 4507065 15 9.1 2 (28%) 162 cathepsin B preproprotein 4503139 38 5.8 3 (12%) 127 Sample NA3 prolactin-induced protein 4505821 16 8.2 5 (37%) 6642 semenogelin II precursor 4506885 65 9 6 (9%) 5126 albumin preproprotein 4502027 71 5.9 7 (17%) 2077 prosaposin isoform a preproprotein 11386147 59 5 2 (8%) 438 mucin 6, gastric isoform 1 89033736 185 6.3 3 (2%) 269 epididymal secretory protein E1 precursor 5453678 16 7.5 2 (21%) 210 zinc alpha-2-glycoprotein 1 4502337 34 5.7 3 (24%) 203 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 3 (13%) 141 clusterin isoform 1 42716297 58 6.2 3 (10%) 117 Sample NA4 prolactin-induced protein 4505821 16 8.2 10(76%) 22296 semenogelin II precursor 4506885 65 9 16 (30%) 11486 semenogelin I isoform b preproprotein 38049014 45 9.2 13 (33%) 3038 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 14 (67%) 3631 zinc alpha-2-glycoprotein 1 4502337 34 5.7 11 (38%) 3260 lactotransferrin 54607120 80 8.5 16 (45%) 2926 prosaposin isoform a preproprotein 11386147 59 5 5 (20%) 773 epididymal secretory protein E1 precursor 5453678 16 7.5 3 (39%) 533 Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 8 of 20 http://www.rbej.com/content/11/1/38 Table 1 Identification of proteins in the 12 replicates from NN, NA, ON and OA group showing protein name, NCBI database index, molecular weight, peptide coverage and Mascot score (Continued) serine proteinase inhibitor, clade A, member 1 50363217 46 5.3 4 (12%) 506 extracellular matrix protein 1 isoform 1 precursor 4758236 62 6.2 4 (17%) 454 beta 2 microglobulin precursor 4757826 13 6 2 (21%) 388 tissue inhibitor of metalloproteinase 1 precursor 4507509 23 8.4 3 (29%) 351 cystatin S precursor 4503109 16 4.9 5 (48%) 321 cystatin C precursor 4503107 16 9 2 (26%) 280 cathepsin D preproprotein 4503143 45 6.1 2 (11%) 200 cathepsin B preproprotein 4503139 38 5.8 2 (10%) 186 carboxypeptidase E preproprotein 4503009 53 5 3 (11%) 162 Sample OA1 prolactin-induced protein 4505821 16 8.2 12 (77%) 22670 semenogelin II precursor 4506885 65 9 19(32%) 13764 semenogelin I isoform b preproprotein 38049014 45 9.2 15 (27%) 6586 albumin preproprotein 4502027 71 5.9 26 (51%) 7715 lactotransferrin 54607120 80 8.5 28 (54%) 5063 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 11 (64%) 3309 zinc alpha-2-glycoprotein 1 4502337 34 5.7 9 (40%) 3298 ankyrin repeat domain 11 56676397 299 6.7 3 (1%) 1056 PREDICTED: mucin 6, gastric isoform 1 89033736 185 6.3 10 (14%) 808 epididymal secretory protein E1 precursor 5453678 16 7.5 7 (70%) 635 clusterin isoform 1 42716297 58 6.2 4 (13%) 512 fibronectin 1 isoform 2 preproprotein 47132551 269 5.3 10 (7%) 503 extracellular matrix protein 1 isoform 1 precursor 4758236 62 6.2 5 (28%) 420 prosaposin isoform a preproprotein 11386147 59 5 4 (18%) 407 prostatic acid phosphatase precursor 6382064 44 5.8 9 (28%) 365 tissue inhibitor of metalloproteinase 1 precursor 4507509 23 8.4 4 (35%) 337 beta 2 microglobulin precursor – 1 peptide 4757826 13 6 1 (18%) 262 serine proteinase inhibitor, clade A, member 1 50363217 46 5.3 4 (12%) 242 transferrin 4557871 79 6.8 3 (4%) 211 cathepsin D preproprotein 4503143 45 6.1 2 (11%) 206 cystatin C precursor 4503107 16 9 5 (52%) 199 cystatin S precursor 4503109 16 4.9 6 (68%) 198 secretory leukocyte peptidase inhibitor precursor −1 peptide 4507065 15 9.1 1 (18%) 175 carboxypeptidase E preproprotein 4503009 53 5 2 (8%) 123 galectin 3 binding protein 5031863 66 5.1 3 (8%) 105 cathepsin B preproprotein 4503139 38 5.8 2 (12%) 95 expressed in prostate and testis 19923082 14 8.2 1 (10%) 86 macrophage migration inhibitory factor – 1 peptide 4505185 12 7.7 1 (9%) 84 prostaglandin (H2) D-isomerase −1 peptide 32171249 21 7.6 1 (8%) 75 Sample OA2 prolactin-induced protein 4505821 16 8.2 8 (51%) 13511 semenogelin II precursor 4506885 65 9 11 (18%) 6235 albumin preproprotein 4502027 71 5.9 10 (23%) 1848 cystatin S precursor 4503109 16 4.9 3 (28%) 522 Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 9 of 20 http://www.rbej.com/content/11/1/38 Table 1 Identification of proteins in the 12 replicates from NN, NA, ON and OA group showing protein name, NCBI database index, molecular weight, peptide coverage and Mascot score (Continued) prostate specific antigen isoform 4 preproprotein 71834855 24 7 4 (25%) 354 prosaposin isoform a preproprotein 11386147 59 5 1 (2%) 324 clusterin isoform 1 42716297 58 6.2 3 (10%) 308 mucin 6, gastric isoform 1 89033736 185 6.3 2 (1%) 306 zinc alpha-2-glycoprotein 1 4502337 34 5.7 4 (17%) 291 epididymal secretory protein E1 precursor 5453678 16 7.5 3 (38%) 184 ankyrin repeat domain 11 56676397 299 6.7 3 (1%) 133 carboxypeptidase E preproprotein 4503009 53 5 2 (8%) 121 lactotransferrin precursor 54607120 80 8.5 3 (9%) 115 extracellular matrix protein 1 isoform 1 precursor 4758236 62 6.2 1 (5%) 108 galectin 3 binding protein 5031863 66 5.1 1 (2%) 98 cathepsin D preproprotein 4503143 45 6.1 2 (13%) 95 lactamase, beta isoform a 26051231 61 8.7 2 (10%) 90 prostatic acid phosphatase precursor 6382064 44 5.8 1 (4%) 78 Sample ON prolactin-induced protein 4505821 16 8.2 9 (69%) 33212 semenogelin II precursor 4506885 65 9 20 (35%) 10703 semenogelin I isoform b preproprotein 38049014 45 9.2 17 ((38%) 2722 albumin preproprotein 4502027 71 5.9 25 (52%) 9519 prostate specific antigen isoform 1 preproprotein 4502173 29 7.6 14 (62%) 6487 zinc alpha-2-glycoprotein 1 4502337 34 5.7 9 (38%) 5967 lactotransferrin precursor 54607120 80 8.5 18(40%) 3770 serine proteinase inhibitor, clade A, member 1 50363217 46 5.3 4 (15%) 1138 prosaposin isoform a preproprotein 11386147 59 5 6 (26%) 1014 epididymal secretory protein E1 precursor 5453678 16 7.5 3 (39%) 937 tissue inhibitor of metalloproteinase 1 precursor 4507509 23 8.4 2 (19%) 866 extracellular matrix protein 1 isoform 1 precursor 221316614 62 6.2 3 (12%) 637 beta 2 microglobulin precursor 4757826 13 6 2 (21%) 633 mucin 6, gastric 151301154 263 7.2 5 (5%) 407 fibronectin 1 isoform 3 preproprotein 16933542 262 5.4 2 (1%) 352 cathepsin D preproprotein 4503143 45 6.1 2 (11%) 334 cystatin C precursor 4503107 16 9 3 (26%) 320 carboxypeptidase E preproprotein 4503009 53 5 3 (11%) 172 clusterin isoform 1 42716297 58 6.2 2 (7%) 147 acid phosphatase, prostate short isoform precursor 6382064 44 5.8 3 (8%) 132 secretory leukocyte peptidase inhibitor precursor 4507065 15 9.1 2 (42%) 123 protein tyrosine phosphatase, receptor type, sigma isoform 1 104487006 218 6.1 4 (3%) 112 precursor The proteins identified as unique and upregulated in 1 precursor); while clusterin 1 was downregulated in the OA group were: prostate specific antigen isoform this group. 1 preprotein; semenogelin I isoform b preprotein. Some unique proteins were absent in some of the Cystatin C precursor was found to be downregulated groups but were differentially expressed in other groups. in the OA group. The ON group showed an These proteins included the DJ-1 protein, which was upregulation of two unique proteins (zinc alpha-2 absent in the OA groups, whereas the ankyrin repeat glycoprotein 1 and tissue inhibitor of metalloproteinase domain 11 was absent in the NN group. Also included Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 10 of 20 http://www.rbej.com/content/11/1/38 Figure 1 Broad categorical analysis of proteomics data. Differentially expressed proteins list encompasses proteins that overlap with other categories (common, unique, low abundant and significant) of proteins. in this category was orosomucoid 1 precursor, which while orosomucoid 2 was present only in the NN. Pro- was found in significant abundance in the NN samples tein tyrosine phosphatase, receptor type, sigma isoform but in low abundance in NA sample. 1 precursor and acidic epididymal glycoprotein - like 1 isoform 1 precursor protein were absent in the OA Identification of the low abundant proteins group. Many of the identified proteins were low abundant proteins (SC ≤10) (Table 2). Some of these proteins were Cellular distribution and significant biological processes restricted to a particular group while they were absent in for proteins in four groups other groups. Transferrin, secretory leukocyte peptidase The functional analysis revealed that most of the signifi- inhibitor precursor, ubiquitin and ribosomal protein cant proteins in each of the four groups (NN, NA, OA, S27 a precursor, prostaglandin H2 D isomerase were and ON) had a predominant cellular distribution in the some of theproteinsthatwereabsentinON group. extracellular region followed by their presence in the The CD177 molecule was absent only in the NN group; intracellular organelles (Figure 3). The distribution of Figure 2 Venn diagram showing distribution of 20 differentially expressed proteins. This was based on the NSC ratio cut-off >2 across 3 samples, NA, OA, and ON in comparison to the baseline NN sample. Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 11 of 20 http://www.rbej.com/content/11/1/38 Table 2 Detailed list of classification of 35 proteins based on their presence, abundance, and differential expression No. Protein Names NCBI UniProt No. of Proteins Low Differentially Expressed Significant Accession Accession samples present in abundant Proteins (in NA, OA, and ON) proteins in No. No. groups proteins compared to NN groups (SC ≤ 10) 1 prolactin-induced protein 4505821 P12273 12 NN(5), NA NN, NA, OA, (4), OA(2), ON ON(1) 2 semenogelin II precursor 4506885 Q02383 12 NN(5), NA NN, NA, OA, (4), OA(2), ON ON(1) 3 albumin preproprotein 4502027 P02768 12 NN(5), NA NN, NA, OA, (4), OA(2), ON ON(1) 4 lactotransferrin 54607120 P02788 12 NN(5), NA NN, NA, OA, (4), OA(2), ON ON(1) 5 epididymal secretory protein 5453678 P61916 12 NN(5), NA NN, NA, OA, E1 precursor (4), OA(2), ON ON(1) 6 extracellular matrix protein 1 221316614 Q16610 12 NN(5), NA NN, NA, OA, isoform 1 precursor (4), OA(2), ON ON(1) 7 prosaposin isoform a 11386147 P07602 12 NN(5), NA NN, NA, OA, preproprotein (4), OA(2), ON ON(1) 8 cathepsin D preproprotein 4503143 P07339 12 NN(5), NA NN, NA, OA, (4), OA(2), ON ON(1) 9 prostate specific antigen 4502173 Q546G3 12 NN(5), NA ↑ in OA NN, NA, OA, isoform 1 preproprotein (4), OA(2), ON ON(1) 10 zinc alpha-2-glycoprotein 1 4502337 P25311 12 NN(5), NA ↑ in ON NN, NA, OA, (4), OA(2), ON ON(1) 11 clusterin isoform 1 42716297 P10909 12 NN(5), NA Low in ON ↓ in ON NN, NA, OA (4), OA(2), ON(1) 12 mucin 6, gastric 151301154 Q6W4X9 11 NN(4), NA ↓ in NA NN, NA, OA, (4), OA(2), ON ON(1) 13 cystatin S precursor 4503109 P01036 11 NN(4), NA Low in ON ↓ in NA, OA, ON NN, NA, OA (4), OA(2), ON(1) 14 galectin 3 binding protein 5031863 Q08380 11 NN(5), NA Low in OA ↓ in OA, ON NN, NA (3), OA(2), and ON ON(1) 15 semenogelin I isoform b 38049014 P04279 10 NN(3), NA ↑ in OA NN, NA, OA, preproprotein (4), OA(2), ON ON(1) 16 prostatic acid phosphatase 6382064 P15309 10 NN(4), NA Low in ON ↓in NA, ON NN, NA, OA precursor (3), OA(2), ON(1) 17 cystatin C precursor 4503107 P01034 9 NN(4), NA ↓in OA NN, NA, OA, (3), OA(1), ON ON(1) 18 tissue inhibitor of 4507509 Q6FGX5 8 NN(3), NA ↑ in ON NN, NA, OA, metalloproteinase 1 (3), OA(1), ON precursor ON(1) Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 12 of 20 http://www.rbej.com/content/11/1/38 Table 2 Detailed list of classification of 35 proteins based on their presence, abundance, and differential expression (Continued) 19 beta 2 microglobulin 4757826 P61769 8 NN(3), NA Low in OA ↓ in OA; Up in ON NN, NA, ON precursor (3), OA(1), ON(1) 20 DJ-1 protein 31543380 Q99497 6 NN(2), NA ↑ in NA, ON NN, NA, ON (3), ON(1) 21 ankyrin repeat domain 11 56676397 Q6UB99 4 NA(1), OA Low in NA ↓ in OA, ON OA (2), ON(1) and ON 22 orosomucoid 1 precursor 167857790 P02763 2 NN(2), NA Low in NA ↓in NA NN (1) 23 serine proteinase inhibitor, 50363217 P01009 8 NN(3), NA ↑ in NA,ON; ↓ in OA NA, OA, ON clade A, member 1 (3), OA(1), ON(1) 24 transferrin 4557871 Q06AH7 6 NN(2), NA Low ↑ in NA, OA NONE (3), OA(1) abundant 25 secretory leukocyte 4507065 P03973 5 NN(1), NA Low ↑ in NA, OA NONE peptidase inhibitor precursor (3), OA(1) abundant 26 ubiquitin and ribosomal 4506713 P62979 4 NN(2), NA Low ↓ in NA, OA NONE protein S27a precursor (1), OA(1) abundant 27 protein tyrosine 104487006 Q13332 4 NN(1), NA Low ↑ in NA, ON NONE phosphatase, receptor type, (2), ON(1) abundant sigma isoform 1 precursor 28 acidic epididymal 25121982 P54107 3 NN(2), NA Low ↓in NA NONE glycoprotein-like 1 isoform 1 (1) abundant precursor 29 prostaglandin H2 D- 32171249 P41222 5 NN(2), NA Low NONE isomerase (2), OA(1) abundant 30 cathepsin B preproprotein 4503139 P07858 6 NN(3), NA Low NONE (3) abundant 31 expressed in prostate and 19923082 Q8WXA2 4 NN(2), NA Low NONE testis (1), OA(1) abundant 32 orosomucoid 2 4505529 P19652 1 NN(1) Low NONE abundant 33 CD177 molecule 110735433 Q8N6Q3 3 NA(1), OA Low NONE (1), ON(1n abundant with (1)) 34 carboxypeptidase E 4503009 P16870 9 NN(3), NA Low in OA NN, NA preproprotein (3), OA(2), and ON ON(1) 35 fibronectin 1 isoform 2 47132551 P02751 10 NN(4), NA Low in ON NN, NA, OA preproprotein (4), OA(1), ON(1) NN = normal sperm count and normal morphology; NA = normal sperm count and abnormal morphology; ON = oligozoospermia and normal morphology; OA = oligozoospermia and abnormal morphology; number in the parenthesis indicates the number of samples. proteins in the NA group was comparable in most of the involved in the biological process were regulatory in cases to the NN group but was different from the ON function (Figure 4). Based on the distribution pattern of and OA groups. The OA group showed only ~15% of the regulatory proteins, the OA groups showed the least the proteins localized in the plasma membrane region involvement of proteins (60%) in regulation compared compared to the other groups, with the maximum num- with 70% - 75% seen in the NN, NA and ON groups. A ber of proteins (~20%) localized in the lysosomal and smaller number of proteins were involved in other func- vacuolar regions. The ON group showed the least distri- tional processes such as protein complex assembly, cell bution of proteins in the nuclear region compared to the morphogenesis, membrane organization, protein matur- extracellular region. ation and trafficking in all the 4 groups. Interestingly, The functional analysis of the significant proteins in none of the proteins in the ON group were involved in each of the groups revealed that most of the proteins any of these processes. Importantly, of all the major Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 13 of 20 http://www.rbej.com/content/11/1/38 Figure 3 Functional annotations from consolidated findings using publicly available software tools (GO term mapper, GO term finder, UniProtKB, STRAP, BioGPS) and proprietary pathway software packages (Ingenuity Pathway Analysis and Metacore™) showing cellular distribution of significant proteins in NN, NA, OA, and ON groups. processes represented, the OA groups showed the lowest compared to DEP and low abundance proteins. The distribution of signal transduction proteins (15%) and extracellular region showed the highest distribution had little or no role in neurological system processing, (91%) of the common proteins whereas they were membrane organization and protein maturation. absent in the ribosomal and endosomal regions. Higher distribution of differentially expressed proteins was seen Comparison of cellular distribution and biological in the cytosolic and Golgi regions compared to the processes amongst the common, DEP, and low abundant common or low abundance proteins. The low abundant proteins proteins were absent in the protein complex and A detailed evaluation of the cellular localization of the secretory granular region but their localization was common, DEP and low abundant proteins is shown in found to be high in the nuclear and endosomal regions. Figure 5. A higher distribution of the common proteins A comparative analysis of the proteins involved in was seen in the majority of cellular compartments various biological processes in the three groups are Figure 4 Functional annotations from consolidated findings using publicly available software tools (GO term mapper, GO term finder, UniProtKB, STRAP, BioGPS) and proprietary pathway software packages (Ingenuity Pathway Analysis and Metacore™) showing biological processes of significant proteins in NN, NA, OA and ON group. Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 14 of 20 http://www.rbej.com/content/11/1/38 Figure 5 Functional annotations from consolidated findings using publicly available software tools (GO term mapper, GO term finder, UniProtKB, STRAP, BioGPS) and proprietary pathway software packages (Ingenuity Pathway Analysis and Metacore™) showing comparison of cellular distribution amongst the proteins in Common, DEP, and Low abundant category. shown in Figure 6. The proteins commonly expressed in metabolism, vesicle-mediated transport and defense all 4 groups played a significant role in many of the bio- response. logical processes such as cellular development, molecu- lar transport, and stress response, interactions with cells Pathways and network analysis using IPA and metacore™ and organisms, cellular processes and in processes relat- Based on the dataset derived from common, DEP and ing to the immune system. Compared with the common low abundance proteins, pathways, biological functions proteins, DEP were comparable for the regulatory pro- and networks of interactions were derived utilizing the cesses, but were reduced in all biological processes. two proprietary pathway packages, IPA and Metacore™. Higher distribution was seen in protein metabolic The important processes affected by common proteins process, vesicle mediated transport, defense response were lipid metabolism (epididymal secretory protein E1 and cellular protein modification process. The low abun- precursor, prosaposin isoform A preprotein, clusterin dant proteins were seen to be involved mainly in protein isoform 1, lactotransferrin and cathepsin D preprotein), Figure 6 Functional annotations from consolidated findings using publicly available software tools (GO term mapper, GO term finder, UniProtKB, STRAP, BioGPS) and proprietary pathway software packages (Ingenuity Pathway Analysis and Metacore™) showing Comparison of Biological Processes amongst the proteins in Common, DEP, and Low abundant category. Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 15 of 20 http://www.rbej.com/content/11/1/38 cell death and survival (cathepsin D preprotein, lacto- contributor to seminal plasma. It is a very rich source of transferrin, clusterin isoform 1, prosaposin isoform A protein with concentration ranges from 35 to 55 g/l preprotein and prostate specific antigen isoform 1 [9,10]. It provides a safe environment for spermatozoa to preprotein), and cellular development (clusterin isoform carry out their physiological functions. Understanding 1, prostate specific antigen isoform 1 preprotein, lacto- the protein profile of human seminal plasma is import- transferrin, extracellular matrix protein 1 isoform 1 pre- ant because it has a profound impact on sperm physi- cursor, cathepsin D preprotein and epididymal secretory ology and thus may affect sperm functioning [31]. proteinE1precursor). In this novel study, we identified 35 proteins based on The important processes affected by DEP showed a SEQUEST scoring in the seminal plasma of men with higher involvement in carbohydrate metabolism and varying semen parameters and categorized them into nephrosis (ankyrin repeat domain 11, beta 2 microglobulin common, differentially expressed, and low abundant (B2MG) precursor, clusterin isoform 1, cystatin C pre- proteins. The large variation in the number of proteins cursor, prostate specific antigen isoform 1 preprotein, identified by any given technique depends mainly on the DJ-1 protein, protein tyrosine phosphatase, receptor sample preparation and mass spectrometry technology type, sigma isoform 1 precursor, transferrin). The major available [32-36]. Recently, the LTQ-Orbitrap mass pathways involved are proteolysis (extracellular matrix; spectrometer has become the cutting-edge instrument ECM), remodeling and connective tissue degradation, for LC/MDS/MS based approaches to characterize the immune response, clathrin-mediated endocytosis sig- seminal proteome. In our study, we used in-solution naling, lipid antigen presentation by CD1, and intrinsic digestion of proteins with the online LC-MS system. prothrombin activation pathway. Similarly, in the low Seminal plasma from different subsets was pooled to abundant proteins the top biological functions included form 4 distinct study groups. There are numerous stud- the cellular development, growth proliferation, DNA ies in the proteomic literature that refer to the benefits replication, recombination, and repair (prostaglandin (H2) of pooling samples where it may not be feasible to D-isomerase, protein tyrosine phosphatase, receptor type, analyze individual samples due to limitations of the sample sigma isoform 1 precursor and transferrin). or the study design [8,17,37,38]. We also studied the major biological functions of the Of the 11 proteins found in all the samples, 9 were DEP in each of the 3 groups and found that free radical associated with sperm function, and the common pro- scavenging was the topmost function in the NA group, teins comprised the majority of the secretions of the while cell-to-cell signaling and interaction were seen in prostate gland, the seminal vesicles and epididymis. all 3 groups. Genes that encode for 7 differentially Some of the proteins or their isoforms detected in the expressed proteins (cysteine-rich secretory protein 1, seminal plasma were zinc alpha-2-glycoprotein 1, clusterin, clusterin, prostatic acid phosphatase (PAP), mucin 6, pros- lactotransferrin, prostate specific antigen. These were simi- tate specific antigen (PSA), zinc alpha-2-glycoprotein 1, lar to those reported by Utleg et al. [39]. and DJ-1) are known to be regulated by androgen recep- Prostate specific antigen is a serine protease that tor. The transcriptional network showed the activation cleaves semenogelin by hydrolysis and thus liquefies the of prostate induction by the androgen receptor signaling semen coagulum and facilitates sperm motility and cap- pathway. DJ-1 protein, protein tyrosine phosphatase, acitation [40,41]. Our study showed that prostate specific receptor type, sigma isoform 1 precursor and transferrin antigen isoform I preprotein was one of the common were observed to interact with other proteins in the proteins, thus indicating its importance in all the 4 pathway database and affect processes related to cellular groups. Prolactin-induced protein (PIP) and Sg II are function and maintenance. Similarly, in the OA group, important common proteins that have a profound im- prostate specific antigen isoform 1 preprotein and pact on sperm physiology. PIP has specificity to fibro- transferrin formed the topmost network, encompassing nectin and constitutes about 1% of seminal coagulum key processes such as gene expression, tissue morphology [42]. It may play a vital role in fibronectin breakdown and cell cycle. The ON group showed immunological dis- during liquefaction. Viscous samples have been reported ease, antigen presentation, cell-to-cell signaling and inter- to show reduced amounts of PIP and PIP precursor, action (B2MG precursor, DJ-1 protein, receptor type, which may also be a contributory factor towards incom- sigma isoform 1 precursor) as the key processes affected plete liquefaction [43]. Both Sg I and II are the major in its topmost network proteins of the coagulum. They represent 20-40% of the seminal plasma proteins. Studies have shown increased Discussion Sg concentrations in asthenozoospermic men [44,45]. Seminal plasma is a mixture of secretions of several male Our study showed that both prolactin and semenogelin accessory glands, including prostate, seminal vesicles, II were in all samples but they were not differentially epididymis and Cowper’s gland. Prostate gland is a major expressed, indicating that men with low sperm count or Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 16 of 20 http://www.rbej.com/content/11/1/38 abnormal morphology may not be affected by these pro- [47-49]. Patients with severe oligozoospermia (<1 × 10 ) teins—a conclusion also made by Milardi et al. [18]. were also shown to have increased levels of seminal PAP Epididymal secretory protein E I precursor, albumin [50]. PAP is produced in the prostate gland and is present preprotein, lactotransferrin, extracellular matrix protein in the seminal fluid at a concentration of 1 mg/mL. It is E1 precursor, prosaposin isoform a preprotein, cathepsin an important tumor marker and acts as a negative growth D preprotein were some of the commonly expressed regulator in prostate cancer [51]. proteins that were not differentially expressed in any of A proteomic analysis of seminal fluid, rather than the groups, suggesting that these proteins may not play blood, has been proposed as as a better starting point to a significant role in sperm concentration or morphology. identify changes that may serve as specific and sensitive The highest percentage of the common proteins was markers of prostate dysfunction [17]. These authors iden- seen in the extracellular region (Figure 5). Human sem- tified more than 100 proteins such as PSA, semenogelin I inal plasma proteins can bind to sperm surface proteins and II, clusterin etc. which have been shown to affect in the ejaculate and form a protective layer around the sperm quality. spermatozoon [12] as well as a sperm reservoir in the The increased expression of semenogelin I in the OA oviduct [46]. It is likely that the extracellular origin of group suggests that the accessory gland secretions have most of the common proteins may play a key role in the a profound impact on oligozoospermic men with abnor- binding activity of the proteins. The absence of common mal morphology. Wang et al. reported semenogelin I proteins in the ribosomal and endosomal region (Figure 5) and mucin were not differentially expressed and there- suggests a relatively low involvement in protein metab- fore had no effect in asthenozoospermic men [21]. Our olism, as also seen by the low distribution of common study also included the differentially expressed proteins, proteins in the ‘protein metabolic process’ category, especially DJ-1 secreted from the testis, epididymis and (Figure 6). Zinc alpha-2 glycoprotein and clusterin play prostate [39,52]. DJ-1 has a high level of expression in a role in signal transduction while lactotransferrin is a the testis. We found DJ-1 to be overexpressed in the NA transport and structural protein [39]. Prostate specific and ON groups. We also documented that orosomucoid 1 antigen has been shown to have enzymatic activity. Our precursor was downregulated in the NA group whereas results show a high distribution of signal transducing expression of orosomucoid 2 was comparable in all protein among the common proteins, suggesting the groups, though it was present in low abundance. However, importance of clusterin, isoform 1 and zinc alpha-2 Wang et al. reported the overexpression of orosomucoid 1 glycoprotein 1 (Figure 6). This was also confirmed from and orosomucoid 2 in asthenozoospermic patients [21]. the pathway and network analysis, which also These proteins have also been found in high abundance in highlighted the role these proteins play in molecular post vasectomy patients [8]. transport, cell death and survival and lipid metabolism. B2MG was found to be under-expressed in OA while A clear overlap was observed for some of the differen- over-expressed in the ON samples. B2MG is present in tially expressed proteins. These included the prostate spe- all nucleated cells. It is one of the two polypeptide cific antigen isoform 1 preprotein, zinc alpha 2 glycoprotein chains of the major histocompatiblity complex (MHC) 1, and clusterin isoform 1. While semenogelin II precursor class I molecule. In humans B2MG is coded by the B2M was seen in common proteins, the semenogelin I isoform b gene. It is a marker of cellular immune system. It is a preprotein was found to be upregulated in the OA group. naturally occurring protein and can detect certain types This suggests that it may contribute to the low motility of tumor cells in the blood and kidney, and some inflam- seen in this group compared to other groups. matory and autoimmune disorders [53,54]. In our study Clusterin isoform was downregulated in the ON as well as that reported by Fung et al. [17], many of the group. This is an interesting finding, given that clusterin proteins in the seminal plasma were identified to be fore- isoform 1 has been shown to be downregulated in pros- runners of prostate disease, suggesting a pathogenic role tate cancer. The proteome of the seminal fluid is largely for B2MG [55,56]. Similarly high levels of B2MG were attributed to secretions from the prostate gland, and reported in the seminal plasma of azoospermic men com- approximately 10% is contributed by the testis and the pared to controls [10,57,58]. An inverse correlation was epididymis [8]. reported between B2MG levels in seminal fluid and sperm Prostate gland is a major contributor to seminal plasma. count [10]. Furthermore, one of the proteins - prostatic acid phos- Similary, we reported the underexpression of galectin phatase (PAP) was significantly increased in azoospermic 3 binding protein in oligozoospermic group. Galectin-3 men compared to oligozoospermic men and asthenozoo- is a carbohydrate-binding protein whose expression level spermic men [10]. In our study, PAP levels were down has been shown to correlate with metastatic potential in regulated in the NA and ON groups. PAP levels have also a number of different types of tumors. Galectin-3 is been reported to correlate with sperm concentration downregulated in prostate cancer. The altered Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 17 of 20 http://www.rbej.com/content/11/1/38 downregulation pattern of galectin-3 observed between response and that low levels of this protein are seen in tumor stages suggests different roles for galectin-3 in the stress conditions. Low distribution of B2MG in DEP progression of prostate cancer [59]. compared to normal and low abundant proteins further TIMP 1 can inhibit tumor growth, invasion and me- suggests that in addition to B2MG, there are other pro- tastasis through their matrix metalloproteinases (MMP) teins that also play a key role in immune system processes. inhibitory activity [60,61]. TIMPs play an inhibitory role, We further validated this observation from the pathway and any imbalance between the two may result in pro- analysis, which showed that that the common protein gression of the disease [62]. TIMP was found to be over- prosaposin isoform, a preprotein, is involved in lipid anti- expressed in the ON group. gen presentation by CD1. Amongst the proteins that were uniquely under- We observed overlapping of differentially expressed expressed in the OA group was ankyrin repeat domain proteins with the low abundant proteins in our study. 11 (ANKRD11). Ankyrins function as adaptor proteins These included transferrin, secretory leukocyte peptidase [63,64] and play a vital role in membrane skeleton inhibitor precursor, ubiquitin and ribosomal protein organization, ionic transport, maintenance of cell polar- S27a precursor, protein tyrosine phosphatase, receptor ity as well as cell-cell adhesion regulation. Data suggest type, sigma isoform 1 precursor and acidic epidiymal that ANKRD11 may act as a tumor suppressor [65]. It’s glycoprotein- like 1 isoform 1 precursor. Of these pro- presence in the semen samples has not been well docu- teins, protein tyrosine phosphatase, receptor type, sigma mented, but it is likely that its overexpression in isoform 1 precursor, acidic epididymal glycoprotein-like oligozoospermic men may play a role in apoptosis. isoform 1 precursor were amongst the low abundant Clusterin isoform 1 can be both pro- and antiapoptotic proteins. Protein tyrosine phosphatase, receptor type, depending upon the isoform expressed [66,67]. It plays a sigma isoform 1 precursor were upregulated while acidic key role in signal transduction and is involved in apop- epididymal glycoprotein-like isoform 1 precursor was tosis of spermatocytes, sperm maturation, and down regulated in the NA group. The acidic epididymal spermiation. Low signal transducing proteins were seen glycoprotein like I isoform I precursor is a member of in the OA group compared with the other groups, as the cysteine - rich secretory protein (CRISP) family and shown in Figure 4. is encoded by the gene CRISP-I. It is expressed in the Compared with the proteins involved in stress response, epididymis and is secreted into the epididymal lumen. It as elucidated from GO annotations (Figure 4), the NA and binds to the post acrosomal region of the head, where it ON groups had a higher distribution of stress proteins may play a role in sperm-egg fusion. The reported low such as DJ-1 whereas a lower distribution of this protein abundance of acidic epididymal glycoprotein like I iso- was seen in OA group. DJ-1 protein is a multifunctional, form I precursor protein is concordant with the findings highly conserved antioxidant protein and is upregulated in of Batruch et al. who also reported its low abundance in hyperglycemia [68,69]. It is mainly involved in the control the seminal plasma of post vasectomy patients compared of oxidative stress and is downregulated in the seminal to controls [8]. plasma of asthenozoospermic men [21]. DJ-1 is activated Transferrin is one of the serum proteins that has been in stress conditions, but with higher levels of stress (as characterized in the seminal plasma, but its role in male seen in OA), there is depletion of antioxidant levels and infertility is unclear [20]. Our study showed this protein low expression of DJ-1 protein. Furthermore, the major was present in low levels in the NN group but was up biological function that comes into focus through IPA in regulated in NA and OA groups. Prostaglandin H2 D the NA group is free-radical scavenging activity. This fur- isomerase, cathepsin B preprotein, orosomucoid 2 and ther supports the fact that DJ-1 was activated in the NA the CD177 molecule were the low abundant proteins that group. Significant proteins were distributed in the lyso- were not differentially expressed in any of the groups. In our study, CD177 was absent in the NN group but present somal and vacuolar regions in higher amounts in the OA group (Figure 3), suggesting that proteins with phagocytic in all the other groups although they were not activity may be activated in stress conditions. Previous differentially expressed. Our findings are consistent with previous publications that reported low concentrations of studies have reported reduced levels of DJ-1 in sperm in response to toxic exposure of male rats to ornidazole and CD177 in fertile men (control samples) compared to epichlorhydrin [70]. postvasectomy men [8] but are contrary to the findings of Wang et al. who reported upregulation of CD177 in Utleg et al. categorized prostatic acid phosphatase (PAP) precursor as an enzymatic protein, and the pres- asthenozoospermic patients [21]. Cathepsin B preprotein ence of this protein in our study suggests that it plays a was present in the NN and NA groups, showing its speci- similar role [39]. The distribution pattern of immune ficity to the normal sperm count while prostaglandin H2 system response in all the groups is shown in Figure 4. D isomerase was absent in the ON group. Cathepsin B It is likely that B2MG is the major protein in immune preprotein found in the lysosomal region is a thiol Sharma et al. Reproductive Biology and Endocrinology 2013, 11:38 Page 18 of 20 http://www.rbej.com/content/11/1/38 protease believed to participate in intracellular degrad- Author details Center for Reproductive Medicine, Glickman Urological and Kidney Institute, ation. It has also been implicated in tumor invasion and Cleveland Clinic, Cleveland, OH, USA. Center for Cellular and Molecular metastasis. The prostaglandin (H2) D-isomerase is Biology, Hyderabad, Andhra Pradesh, India. Bioinformatics Core Services, expressed in the testis, epididymis and prostate and is se- Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA. Proteomics Core Services, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA. creted into the seminal fluid. It binds small non-substrate Molecular Biotechnology Core lab, Lerner Research Institute, Cleveland lipophilic molecules and may act as a scavenger for harm- Clinic, Cleveland, OH, USA. Permanent Address: Ravenshaw University, ful hydrophobic molecules. The prostaglandin (H2) D- Cuttack, Odisha, India. isomerase and cathepsin B protein are potentially involved Received: 15 January 2013 Accepted: 22 March 2013 in biological processes such as vesicle-mediated transport Published: 11 May 2013 and defense response. The low abundant proteins showed a marked distribution in the extracellular region and may References play a regulatory role. The major pathways in which the 1. The Practice Committee of the American Society for Reproductive Medicine: low abundant proteins were involved were prostanoid bio- Diagnostic evaluation of the infertile male: a committee opinion. Fertil Steril 2012, 98:294–301. synthesis and eicosanoid signaling and acute phase re- 2. Kruger TF, Menkveld R, Stander FS, Lombard CJ, Van der M, van Zyle JA, sponse signaling, but the distribution of signal Smith K: Sperm morphologic features as a prognostic factor in in vitro transduction proteins was decreased in low abundant fertilization. Fertil Steril 1986, 46:1118–1123. 3. Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Veeck LL, proteins. Morshedi M, Brugo S: New method of evaluating sperm morphology with predictive value for human in vitro fertilization. Urology 1987, 30:248–251. 4. 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Saleh RA, Agarwal A, Nada EA, El-Tonsy MH, Sharma RK, Meyer A, Nelson have highlighted the distribution pattern of these pro- DR, Thomas AJ: Negative effects of increased sperm DNA damage in teins in cell organelles and described their biological relation to seminal oxidative stress in men with idiopathic and male processes. We have also illustrated the high involvement factor infertility. Fertil Steril 2003, 79:1597–1605. 7. Sharma R, Agarwal A: Spermatogenesis: an overview.In Human sperm of proteins in cellular development signal transduction. chromatin: structure and function. Sperm chromatin. Biological and clinical The overexpression or underexpression of these proteins applications in male infertility and assisted reproduction. Edited by Zini A, in the 4 groups illustrates their role in male infertility. Agarwal A. Springer Science + Business Media, LLC; 2012:19–44. 8. 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Biol Pharm Bull 2002, 25:853–856. doi:10.1186/1477-7827-11-38 Cite this article as: Sharma et al.: Functional proteomic analysis of seminal plasma proteins in men with various semen parameters. Reproductive Biology and Endocrinology 2013 11:38. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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