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m e SPERMATOZOA PROFILES IN CRYOBANKED SEMEN SAMPLES A

C e 3 9 n 9 tr 1 um t. E Es FROM TESTICULAR CANCER PATIENTS BEFORE TREATMENT xcellentiae Ashok Agarwal, PhD.,1 Eva Tvrda, PhD.,1,2 Rakesh Sharma, PhD.,1 Sajal Gupta, MD.,1 Gulfam Ahmad, PhD.,1,3 and Edmund S. Sabanegh, MD.4

1American Center For Reproductive Medicine, Cleveland Clinic, Cleveland, OH, 2Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Nitra, Slovakia, 3Physiology and Cell Biology, University of Health Sciences, Lahore, Pakistan, 4Department of Urology, Cleveland Clinic, Cleveland, OH

INTRODUCTION RESULTS Table 1: Differentially expressed compared to the infertile control group that are involved in reproductive functions Testicular cancer (TC) is the most common malignant diagnosis Proteomic analysis and Liquid chromotography mass Seminomas, mixed germ cell tumors, embryonal carcinomas and Sertoli Uniprot name Protein name Expression Description in the reproductive age group. Current attention focuses on spectrometer analysis (LC-MS) cell tumors were the most prevalent TC diagnoses. Spermatozoa Global proteomic analysis was done in triplicate and No. improving quality of life, including preservation of their fertility. concentration in cancer was significantly lower (20.40±2.77) compared 250 Decreased male fertility may be present even before the start of quantified using the label-free spectral counting method. P07288 KLK3 Prostate-specific OE/H Organ-specific glycoprotein present in normal prostatic tissue, benign prostatic H Figure 1A: Abundance of proteins in in testicular cancer patients with infertile patients (63.51±30.41; P=0.424). antigen hypertrophy and most prostatic carcinomas. Hydrolyzes semenogelin-1 leading M Following in-gel digestion and trypsinization, peptides were 200 Of the DEPs unique to the cancer patients, 5 displayed very low abundance cancer treatment. Endocrine imbalance and disturbed to the liquefaction of the seminal coagulum. L extracted The extracts were combined and evaporated to <10 P04279 SEMG1 Semenogelin-1 OE/H Produced by seminal vesicles and is a predominant protein in semen. (41.7%), 6 (50%) showed low abundance, while 1 (8.3%) displayed spermatogenesis leading to azoospermia or oligozoospermia, as Proteomic profiling 150 VL µL in Speedvac and subsequently resuspended in 1% acetic Participates in the formation of a gel matrix entrapping the accessory gland medium abundance. 194 DEPs were underexpressed while 51 were well as decreased spermatozoa motility and viability may be 1. Global proteomic analysis identified a total of 723 proteins in the secretions and ejaculated spermatozoa. Blocks capacitation mainly via overexpressed in TC. From the proteins underexpressed in TC, 131 exhibited 100 observed in pre-treatment oncological patients. Presence of a acid to make a final volume of ~30 µL for LC-MS analysis. testicular cancer group and 982 proteins in the infertile group. inhibition of reactive oxygen species generation. very low abundance, 26 low abundance, 34 medium abundance and The LC-MS system was a Finnigan LTQ-Orbitrap Elite hybrid P15309 ACPP Prostatic acid OE/H Highly expressed in the prostate, restricted to glandular and ductal epithelial 3 high abundance. Of the proteins overexpressed in TC, 5 showed very low malignancy may lead to gonadal dysfunction through a variety phosphatase cells. Highly expressed in prostate cancer and associated metastases. 50 abundance, 20 low abundance, 17 medium and 9 high abundance. of mechanisms, which may be commonly observed in males mass spectrometer system. The digest was analyzed to 2. 398 DEPs were identified between the infertile group and cancer Dephosphorylates a diverse number of substrates under acidic conditions (pH determine peptide molecular weights and tandem mass 4-6) including alkyl, aryl and acyl orthophosphate monoesters and 0 with general infertility as well. patients.141 DEPs were unique to the infertile patient group. 12 phosphorylated proteins. Uniquely expressed Overexpressed Underexpressed spectra (MS/MS) to determine amino acid sequence in were unique to the cancer group. Q02383 SEMG2 Semenogelin-2 OE/H Produced by seminal vesicles, and to a much lesser extent, epididymis. in cancer group in cancer group in cancer group successive instrument scans. Tandem mass spectra were Participates in the formation of a gel matrix (sperm coagulum) entrapping the (n=12) (n=51) (n=194) Cancer is also believed to be one of the most important accessory gland secretions and ejaculated spermatozoa. extracted by Proteome Discoverer version 1.4.1.288. All P54107 CRISP1 Cysteine-rich OE/M Expressed in the caput, corpus, and cauda regions of the epididymis, the underlying conditions of idiopathic male infertility. Numerous 3. 194 DEPs were underexpressed while 51 were overexpressed in TC secretory protein 1 ductus deferens, sperm and seminal plasma. May have a role in sperm-egg MS/MS samples were assessed using Mascot, SEQUEST, and fusion and maturation. 250 reports also suggests that patients with a decreased fertility of compared to the infertile group. H Figure 1B: Abundance of proteins in in infertile patients X!Tandem. P05154 SERPINA5 Plasma serine OE/M Predominantly expressed in the epithelium of seminal vesicles. Heparin- unknown origin may be at a higher risk for developing TC. M protease inhibitor dependent serine protease inhibitor. Inactivates serine proteases by binding 200 Of the DEPs unique to the infertile patient group, 47 (33.3%) showed L Both cancer and idiopathic infertile patients often exhibit 4. Protein abundance in cancer and infertile men is shown in Figure 1A-B. (Acrosomal serine irreversibly to their serine activation site. Involved in the regulation of very low abundance, 68 (48.2%) displayed low abundance, 23 (16.3%) common features of poor semen quality, including low sperm Criteria for protein identification and Quantitative protease inhibitor) intravascular and extravascular proteolytic processes. Alterations in its 150 VL showed medium abundance and 3 (2.12%) presented with high proteomics expression may result in defective spermatogenesis and infertility. abundance. From the proteins overexpressed in the IF group, 52 showed count, leukocytospermia and a high percentage of 5. The majority of the DEP were involved in the cellular nitrogen P25311 AZGP1 Zinc-alpha-2- OE/L Identified in semen and seminal plasma. High expression in cancer cachexia, 100 low abundance, 81 medium abundance and 61 were present high morphologically abnormal spermatozoa. It is therefore Scaffold was used to validate MS/MS-based peptide and compound metabolic processes (134/389; 34.45%), small glycoprotein causing extensive fat losses associated with some advanced cancers. abundance. Of the proteins that were underexpressed in the IF group, protein identifications. Proteins were annotated with gene Considered as a tumor biomarker for various carcinomas. 50 22 displayed very low abundance, 14 low abundance, 7 medium proposed that specific gene mutations, oxidative insult, molecule metabolic processes (132/389; 33.93%), biosynthetic Q9Y230 RUVBL2 RuvB-like 2 (48 kDa UE/H Highly expressed in testis. Possesses single-stranded DNA-stimulated ATPase abundance and 8 showed high abundance. endocrine disruption or environmental factors may play a ontology (GO) terms from National Center for Biotechnology processes (118/389; 30.33%), response to stress (108/389; TATA box-binding and ATP-dependent DNA helicase (5' to 3') activity; hexamerization is thought 0 Information (NCBI). Normalization of spectral counts using protein-interacting to be critical for ATP hydrolysis and adjacent subunits in the ring-like structure Uniquely expressed Overexpressed Underexpressed significant role in the manifestation of these two health 29.55%), anatomical and structural development (97/389; protein) contribute to the ATPase activity. Functions in decondensation at the in infertile in infertile in infertile the NSAF (normalized spectral abundance factor) approach end of mitosis. (n=141) (n=194) (n=51) disorders. 24.94%) (Figure 2). O14556 GAPDHS Glyceraldehyde-3- UE/H Testis specific protein. May play an important role in regulating the switch was applied prior to relative protein quantification. phosphate between different pathways for energy production during spermiogenesis and in Sperm cryobanking has proven to be the most reliable method Differentially expressed proteins (DEP) were separated into Reactome database dehydrogenase. the spermatozoon. Required for sperm motility and male fertility. 4 categories, as shown below: testis-specific in preserving male fertility prior to cancer therapy. Pre-freeze Mitochondrial metabolism: The major pathways included the citric acid Q9Y265 RUVBL1 RuvB-like 1 (49 kDa UE/H Ubiquitously expressed with high expression testis. Possesses single-stranded Cellular nitrogen compound metabolic process Figure 2: annotations for DEP for major cellular quality of semen is the single most important factor predicting (TCA) cycle, respiratory electron transport (31 proteins) as well as ATP TATA box-binding DNA-stimulated ATPase and ATP-dependent DNA helicase (3' to 5') activity; Transport functions Very Low (VL): spectral count range 1.7-7; p≤0.001 and protein-interacting hexamerization is thought to be critical for ATP hydrolysis and adjacent and biological processes a successful cryopreservation. The ever-increasing evidence synthesis (19 proteins). protein) subunits in the ring-like structure contribute to the ATPase activity. Functions in Response to stress linking cancer to idiopathic infertility is too significant to be (NSAF ratio ≥2.5 for overexpressed, ≤0.4 for chromatin decondensation at the end of mitosis. Anatomical structure development GO Term Mapper was used to classify the DEPs in order to identify their underexpressed proteins), Q5JQC9 AKAP4 A-kinase anchor UE/H Testis specific protein, only expressed in round spermatids. Major structural Cellular component assembly involvement in major cellular, molecular functions as well as biological protein 4 component of sperm fibrous sheath. Plays a role in sperm motility. ignored, and deserves further scrutiny. Protein Metabolism: 48 proteins were found to be associated with Cellular protein modification process processes. Among the biological processes, the majority of the DEP were Q9UFH2 DNAH17 Dynein heavy chain UE/H Expressed in testis. Force generating protein of respiratory cilia. Produces force involved in the cellular nitrogen compound metabolic processes metabolism of proteins, involving the metabolism of amino acids and Cell death Low (L): spectral count range 8-19; p≤ 0.01 and (NSAF 17, axonemal towards the minus ends of microtubules. Dynein has ATPase activity; the force- (134/389; 34.45%), small molecule metabolic processes (132/389; Thus, in this pilot study, proteomic tools were utilized to derivates (14 proteins) and post-translational protein modifications (10 producing power stroke is thought to occur on release of ADP. Involved in Macromolecular complex assembly 33.93%), biosynthetic processes (118/389; 30.33%), response to stress ratio≥2.5 for overexpressed, ≤0.4 for underexpressed sperm motility. identify protein profiles of the spermatozoon, to search for proteins). Generation of precursor metabolites and energy (108/389; 29.55%), anatomical and structural development (97/389; proteins), Q96JB1 DNAH8 Dynein heavy chain UE/H Expressed in testis. Force generating protein of respiratory cilia. Produces force 24.94%). potential clinical biomarkers of the male reproductive system 8, axonemal towards the minus ends of microtubules. Dynein has ATPase activity; the force- transmembrane transport affected by cancer or infertility. Notable transport functions were transmembrane transport of small producing power stroke is thought to occur on release of ADP. Involved in Reproduction Medium (M): spectral count range between 20-79; p≤0.05 sperm motility. Homestatic process molecules proteins and mitochondrial protein import. 28 proteins were Q96A08 HIST1H2BA H2B type 1-A UE/M Mainly expressed in testis and mature sperm (at protein level). Variant histone and (NSAF ratio ≥2.0 for overexpressed, ≤0.5 for (Histone H2B, testis) specifically required to direct the transformation of dissociating to linked to the cell cycle control, including DNA replication, regulation of 0 5 10 15 20 25 30 35 40 MATERIAL AND METHODS underexpressed proteins), protamine in male germ cells. Entirely replaces classical histone H2B prior apoptosis and maintenance. to protamine transition. Percent Distribution P0C5Z0 H2AFB2; -Bbd UE/M Present in mature sperm. A typical histone H2A which can replace conventional Patient enrollment and sample collection High (H): spectral counts >80; p≤0.05 and (NSAF ratio ≥ H2AFB3 type 2/3 (H2A Barr H2A in some nucleosomes and is associated with active transcription and After a written consent, discarded semen samples were Overall, 75 proteins were associated with disease state, 36 proteins with body-deficient) mRNA processing. Functionallly and structurally involved in male 1.5 for overexpressed, ≤0.67 for underexpressed proteins). gametogenesis. obtained from 16 patients diagnosed with testicular cancer prior immune function and 2 proteins with reproduction. Q96QH8 SPACA5; Sperm acrosome- UE/M Involved in the binding of capacitated spermatozoa to the egg's extracellular to start of any cancer therapy regardless of the extent of the SPACA5B associated protein 5 coat and induction of acrosome reaction. Bioinformatics analysis Participation of DEP in the top networks and pathways Q13733 ATP1A4 Sodium/potassium- UE/M Specifically expressed in testis and mature sperm. A catalytic component of the disease. In addition, 9 semen samples were obtained Functional annotation and enrichment analysis were transporting ATPase active enzyme. which catalyzes the hydrolysis of ATP coupled with the male-factor infertility patients without any history of malignant 35 of the DEPs were involved in key functions associated with subunit alpha-4 exchange of sodium and potassium ions across the plasma membrane. performed using publicly available bioinformatic annotation developmental disorder, hereditary disorder and metabolic disease, while (Na(+)/K(+) ATPase CONCLUSIONS disease and served as control. Semen samples were tools and databases such as GO Term Finder, GO Term alpha-4 subunit) 33 were directly related to cancer, organismal injury and abnormalities, as Q13508 ART3 Ecto-ADP- UE/M Testis specific. Alterations in its expression are linked to non-obstructive cryopreserved using the TEST-Yolk Buffer (TYB, Irvine Scientific, 1. Protein profiles of the reproductive cells affected by testicular cancer vary quite significantly than Mapper, UniProt, Software Tools for Researching Annotations well as renal and urological diseases. ribosyltransferase 3 azoospermia. Santa Ana, CA). of Proteins (STRAP) and Database for Annotation, Q8IUA0 WFDC8 WAP four-disulfide UE/M Expressed ubiquitously with the highest levels in the epididymis and testis. infertile men. core domain protein 8 Suggested to be involved in the key functions or pathways in spermatogenesis Visualization and Integrated Discovery (DAVID). Proprietary The majority of the DEPs were localized in the mitochondrion, extracellular (Putative protease and sperm maturation. Sample pooling and protein extraction software package such as IPA (Ingenuity Pathway Analysis) inhibitor WAP8) 2. Overexpression and underexpression of DEP in the testicular cancer group only may be a Once the cryoprotectant was removed, samples were pooled region and cytosol. O14967 CLGN Calmegin UE/M Detected in testis. Functions during spermatogenesis as a chaperone for a was also used to obtain consensus-based, comprehensive range of client proteins that are important for sperm adhesion onto the egg contributary factor in the severity of the disease and sperm dysfunction in these patients. after normalizng for spermatozoa and protein concentration. zona pellucida and for subsequent penetration of the zona pellucida. functional context for the large list of proteins derived from Enriched functional categories were activated processes/functions, proteins Spermatozoa were solubilized in radio-immunoprecipitation OE = overexpressed; UE = underexpressed; H = high abundance; M = medium abundance;. 3. Alterations in the sperm proteome of testicular cancer patients may provide an insight into the this proteomic study. associated with acetylation, mitochondrial functions, nucleoside binding, assay (RIPA) lysis buffer (Sigma-Aldrich, St. Louis, MO) unsatisfactory semen quality and/or compromised fertility in cancer patients prior to treatment. containing a proteinase inhibitor cocktail (Roche, Indianapolis, oxidation-reduction, as well as downregulated processes/functions. IN). After a complete lysis of spermatozoa, protein concentration 4. DEPs identified may serve as useful biomarkers in the diagnosis of testicular cancer and useful Identification of differentially expressed proteins relevant to was determined using the bicinchoninic acid (BCA) kit (Thermo, non-invasive tool to assist andrology specialists in a better identification and management of spermatogenesis Rockford, IL). Equal amounts of protein were fractionated using fertility in patients suffering from testicular cancer. 73 DEPs protein were associated with spermatogenic function in the SDS PAGE 1D gel electrophoresis. testicular cancer group compared to the infertile group. Some of the overexpressed and underexpressd DEP in testicular cancer patients are shown in Table 1.