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Differential Proteomic Profiling of Spermatozoal Proteins of Infertile

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Differential Proteomic Profiling of Spermatozoal Proteins of Infertile Infertility Differential Proteomic Profiling of Spermatozoal Proteins of Infertile Men With Unilateral or Bilateral Varicocele Ashok Agarwal, Rakesh Sharma, Damayanthi Durairajanayagam, Zhihong Cui, Ahmet Ayaz, Sajal Gupta, Belinda Willard, Banu Gopalan, and Edmund Sabanegh OBJECTIVE To compare the sperm protein profile between infertile men with unilateral varicocele and infertile men with bilateral varicocele. METHODS This prospective study investigated 50 infertile patients with clinical varicocele (33 unilateral and 17 bilateral) seeking fertility workup between March 2012 and April 2014. Routine sperm parameters, reactive oxygen species, total antioxidant capacity, and sperm deoxyribonucleic acid fragmentation were assessed in their semen. Sperm protein profile was characterized only in pooled samples of 5 unilateral and 3 bilateral varicocele samples, respectively, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an Linear Trap Quadrupole-Orbitrap Elite hybrid mass spectrophotometer system. Differences in protein expression were analyzed using gel analysis software, followed by protein identification using mass spectroscopy analysis. Differentially expressed proteins and their abundance were quantified by comparing spectral counts, followed by bioinformatics analysis. RESULTS Unique expression of 64 proteins in the bilateral group and 31 proteins in the unilateral group was obtained. Core functions of the top protein interaction networks were post-translational modi- fication (w122 proteins associated with acetylation), protein folding, free-radical scavenging, cell death, and survival. The top molecular and cellular functions were protein degradation, free radical scavenging, and post-translational modifications, whereas the top pathways were protein ubiquitination and mitochondrial dysfunction. Major biological pathways for the 253 differen- tially expressed proteins were metabolism, apoptosis, and signal transduction. CONCLUSION Functional proteomic profiling helps identify the differential processes or pathways that are affected based on the nature of varicocele (bilateral or unilateral) and provide insights into the mechanistic implications of varicocele-associated male infertility. UROLOGY 85: 580e588, 2015. Ó 2015 Elsevier Inc. aricoceles are found in 35%-50% of men with Proteomics is emerging as a new frontier in male infertility primary infertility and in w80% of men with research and is being extensively used to investigate protein V secondary infertility, suggesting a progressive alterations in different disease states.3 Proteomic analysis has decline in fertility over time. Factors that may contribute previously been used to screen for differentially expressed to the pathogenesis of varicocele include hypoperfusion sperm proteins in infertile men afflicted with various etiol- leading to hypoxia, heat stress, oxidative stress, hormonal ogies including varicocele.4-7 Two-dimensional electro- imbalances, and exogenous toxicants.1 However, the phoresis and MALDI-TOF/TOF-MS was used to compare exact mechanism underlying varicocele-associated testic- the proteomic profile of oligozoospermic, grade 3 varicocele ular dysfunction and infertility remains unclear.2 patients with that of healthy fertile men,8 and differentially expressed proteins in grade 2 varicocele patients.9 Ashok Agarwal and Rakesh Sharma contributed equally to this study. However, no proteomics-based study has as yet, to Financial Disclosure: The authors declare that they have no relevant financial interests. our knowledge, looked into the potential alteration in Funding Support: This study was supported by funds from the Center for Reproductive sperm protein expression within the varicocele patient Medicine, Cleveland Clinic. From the Department of Urology, the Center for Reproductive Medicine, Glickman pool, mainly between unilateral and bilateral varicocele Urological & Kidney Institute, Cleveland Clinic, Cleveland, OH; Department of patients. Unilateral varicoceles are more commonly Physiology, MARA University of Technology, Selangor, Malaysia; Research Core present on the left side (35%-40%) compared with Services, Lerner Research Institute, Cleveland Clinic, Cleveland, OH; and Bioinfor- matics Consulting Services, Yorg Corporation, Plano, Texas. bilateral varicoceles (10%-15%). Thus, the aim of the Address correspondence to: Ashok Agarwal, Ph.D., HCLD, Department of Urology, present study was to examine and compare potential Center for Reproductive Medicine, Glickman Urological & Kidney Institute, Cleveland differences between the sperm protein profile within a Clinic, 10681 Carnegie Avenue, Mail Code X-11, Cleveland, OH 44195. E-mail: [email protected] subset of varicocele patients, that is, men with unilat- Submitted: November 4, 2014, accepted (with revisions): November 27, 2014 eral varicocele with that of bilateral varicocele patients. 580 ª 2015 Elsevier Inc. http://dx.doi.org/10.1016/j.urology.2014.11.030 All Rights Reserved 0090-4295/15 METHODS All MS/MS samples were analyzed using Mascot (version 2.3.02; Matrix Science, London, United Kingdom), SEQUEST (version Patients and Semen Samples 1.4.0.288; Thermo Fisher Scientific, San Jose, CA) and X! Tandem On study approval by the institutional review board, semen samples (version CYCLONE; 2010.12.01.1; The GPM; thegpm.org). were collected from 50 patients with infertility as a primary diagnosis To validate MS/MSebased peptide and protein identifications, and clinical varicocele (unilateral, n ¼ 33 and bilateral, n ¼ 17) as a Scaffold (version 4.0.6.1; Proteome Software Inc., Portland, OR) secondary diagnosis. All participating patients were examined, and was used. Peptide and protein identifications were accepted as 10 their varicocele(s) were diagnosed. Azoospermic men and men previously described.14 Protein probabilities were assigned by the with a sperm concentration of <10 million sperm/mL were excluded ProteinProphet (Systems Biology, Seattle, WA) algorithm.15 from the present study. After liquefaction, manual semen analysis Proteins were annotated with Gene Ontology (GO) terms.16 was performed to determine sperm concentration and motility, and smears were prepared for assessment of sperm morphology according to the World Health Organization criteria.11 Samples with a round Quantitative Proteomics cell concentration of >1 Â 106/mL were tested for leukocytospermia For proteomic analysis, the relative quantity of the proteins was (>1 Â 106 white blood cells/mL) and confirmed by the Endtz test.6 determined by comparing the number of spectra used to identify fi Samples positive for leukocytospermia were excluded from the final each protein. The total number of mass spectra that identi ed a proteomic pool. Reactive oxygen species (ROS) formation was particular protein (spectral counts [SpCs]) was used to measure measured by chemiluminescence assay using a Berthold lumin- the relative abundance of proteins in the samples being ometer (Autolumat Plus 953, Berthold Technologies, Oakland, compared. Normalization of SpCs using the normalized spectral TN), as described previously.12 Total antioxidant capacity was abundance factor (NSAF) approach was applied to each group fi 17 measured in the seminal plasma using the antioxidant assay kit.6 separately before relative protein quanti cation. Differentially Sperm deoxyribonucleic acid (DNA) fragmentation was evalu- expressed proteins (DEP) were obtained by applying different fi ated using a terminal deoxynucleotidyl transferaseemediated fluo- constraints for signi cance tests and/or fold-change cutoffs based rescein end labeling assay with an APO-DIRECT kit (BD on the average SpCs of the protein from multiple runs. fi Biosciences Pharmingen, San Diego, CA).13 Appropriate lters were used to identify DEP that were dependent on the overall abundance of the proteins.18 The fi Sperm Protein Extraction and In-gel Digestion abundance of the proteins was classi ed as high, medium, low, or very low based on their average SpCs among the 3 replicate Only samples with sufficient sperm concentration after running runs. Different constraints for significance tests (P value) and/or the ROS and sperm DNA fragmentation tests could be fold-change cutoffs (or NSAF ratio) were applied for these 4 considered for pooling. We therefore selected samples for abundance categories, as shown in the following: pooling from 5 of the 33 unilateral and 3 of the 17 bilateral varicocele patients who met the criteria. After complete lysis, Very Low. SpC range, 1.7-7; P .001; and NSAF ratio (2.5 protein concentration was determined using a bicinchoninic for upregulated and 0.4 for downregulated proteins). acid (BCA) protein assay kit (Thermo, Rockford, IL). Equal amounts of protein were fractionated using sodium dodecyl Low. SpC range, 8-19; P .01; and NSAF ratio (2.5 for sulfate-polyacrylamide gel electrophoresis 1-dimensional gel elec- upregulated and 0.4 for downregulated proteins). trophoresis. Samples were assayed in triplicate for global proteomic analysis and quantified using the label-free spectral counting Medium. SpC range, between 20 and 79; P .05; and NSAF method. The bands were reduced with dithiothreitol and alkylated ratio (2.0 for upregulated and 0.5 for downregulated proteins). with iodoacetamide before the in-gel digestion using trypsin. High. SpC, >80; P .05; and NSAF ratio (1.5 for upregu- Liquid ChromatographyeMass Spectrometer lated and 0.67 for downregulated proteins). After complete digestion, they were extracted from the poly- acrylamide in 2 aliquots of 30-mL
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