Aberrant Upregulation of Compensatory Redox Molecular Machines May Contribute to Sperm Dysfunction in Infertile Men with Unilateral Varicocele: a Proteomic Insight

ANTIOXIDANTS & REDOX SIGNALING Volume 00, Number 00, 2019 ª Mary Ann Liebert, Inc. DOI: 10.1089/ars.2019.7828

Aberrant Upregulation of Compensatory Redox Molecular Machines May Contribute to Sperm Dysfunction in Infertile Men with Unilateral Varicocele: A Proteomic Insight

Nirlipta Swain,1,2,* Luna Samanta,1–3,* Ashok Agarwal,2 Sugandh Kumar,4,5 Anshuman Dixit,4 Banu Gopalan,6 Damayanthi Durairajanayagam,7 Rakesh Sharma,2 Peter N. Pushparaj,8,9 and Saradha Baskaran2

Abstract

Aims: To understand the molecular pathways involved in oxidative stress (OS)-mediated sperm dysfunction against a hypoxic and hyperthermic microenvironment backdrop of varicocele through a proteomic approach. Results: Protein selection (261) based on their role in redox homeostasis and/or oxidative/hyperthermic/hypoxic stress response from the sperm proteome data set of unilateral varicocele (UV) in comparison with fertile control displayed 85 to be differentially expressed. Upregulation of cellular oxidant detoxification and glutathione and reduced nicotinamide adenine dinucleotide (NADH) metabolism accompanied with downregulation of protein folding, energy metabolism, and heat stress responses were observed in the UV group. Ingenuity pathway analysis (IPA) predicted suppression of oxidative phosphorylation (OXPHOS) (validated by Western blotting [WB]) along with augmentation in OS and mitochondrial dysfunction in UV. The top affected networks indicated by IPA involved heat shock proteins (HSPs: HSPA2 and HSP90B1). Their expression profile was corroborated by immunocytochemistry and WB. Hypoxia-inducible factor 1A as an upstream regulator of HSPs was predicted by MetaCore. Occurrence of reductive stress in UV spermatozoa was corroborated by thiol redox status. Innovation: This is the first evidence of a novel pathway showing aberrant redox homeostasis against chronic hypoxic insult in varicocele leading to sperm dysfunction. Conclusions: Upregulation of antioxidant system and dysfunctional OXPHOS would have shifted the redox balance of biological redox couples (GSH/GSSG, NAD+/NADH, and NADP+/NADPH) to a more reducing state leading to reductive stress. Chronic reductive stress-induced OS may be involved in sperm dysfunction in infertile men with UV, where the role of HSPs cannot be ignored. Intervention with antioxidant therapy warrants proper prior investigation. Antioxid. Redox Signal. XX, XXX–XXX.

Keywords: reductive stress, oxidative stress, HSPA2, HSP90B1, spermatozoa, varicocele

1Redox Biology Laboratory, Department of Zoology, School of Life Sciences, Ravenshaw University, Odisha, India. 2American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, Ohio. 3Centre for Excellence in Environment and Public Health, Ravenshaw University, Odisha, India. 4Computational Biology and Bioinformatics Laboratory, Institute of Life Sciences, Bhubaneswar, Odisha, India. 5School of Biotechnology, KIIT University, Bhubaneswar, Odisha, India. 6Yorg Corporation, Plano, Texas. 7Department of Physiology, Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh, Malaysia. 8Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah, Saudi Arabia. 9Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah, Saudi Arabia. *These authors contributed equally to this work.

1 2 SWAIN ET AL.

Results Innovation Semen analysis and assessment of OS in sperm Oxidative stress has been advocated as the common consequence to a diverse etiology of infertility, including Semen results showed that infertile men with UV had a sperm concentration of 22.55 (12.21, 35.53) · 106/mL com- varicocele. Nevertheless, there remains a paucity of infor- 6 mation regarding the mechanism involved. This novel study pared with FD having 64.90 (46.46, 75.58) · 10 /mL. Per- demonstrates the distinct role of chronic reductive stress in centage motility and normal morphology were significantly reactive oxygen species generation in spermatozoa from lower in the infertile UV group [40.23 (35.6, 47)%; 4.4 (2.39, infertile men who presented with unilateral varicocele 6.38)%] in comparison with the FD group [60.2 (55.5, (Fig. 9). Our findings have for the first time demonstrated 63.28)%; 8.4 (6.26, 18.03)%], respectively. Evaluation of reductive stress as an adaptive response, which when not seminal oxidation/reduction potential (ORP) revealed sig- reconciled, results in sperm damage where the role of heat nificantly higher levels in the UV group [2.2 (1.76, 3.3) mV/ shock protein downregulation cannot be undermined. million sperm/mL] when compared with the FD group [1.23 Therefore, special caution should be taken before advising (0.8, 1.53) mV/million sperm/mL], which indicated an im- antioxidant therapy for sperm quality enhancement. balance in seminal redox homeostasis in UV subjects. ROS levels were 1436.22 (416.25, 2791.2) RLU/Sec/million sperm in UV, and 66.26 (24.1, 112.1) RLU/Sec/million Introduction sperm in FD, specifying significant increment in ROS levels in spermatozoa of UV patients. aricocele is the abnormal dilation and tortuosity of the Vpampiniform venous plexus within the spermatic cord Selection of DEPs from the data set based (5). Decreased blood supply in a varicocele testis may lead to on their involvement in redox homeostasis hypoxia-mediated oxidative stress (OS) and subsequently and/or oxidative/heat/hypoxic stress response impaired sperm function. Moreover, distinct increment in the Using the gene ontology (GO) analysis platform, 261 levels of reactive oxygen species (ROS) in chronic hyper- proteins were selected based on their involvement in (1) re- thermic conditions has been demonstrated in cellular models dox homeostasis and/or (2) response to stress (OS/heat stress/ (26). However, fertile men with varicocele might have cy- hypoxic stress), of which, 85 were DEPs, with 28 DEPs being toprotective mechanisms to combat the aforementioned ox- overexpressed and 33 DEPs underexpressed in UV (Fig. 1a idative damage (9). It could be hypothesized that ischemic/ and Supplementary Table S1). Seventeen DEPs were uniquely hypoxia and hyperthermia combined with induced OS is the detected in FD and seven DEPs were uniquely detected in UV mediating machinery for testicular tissue damage in infertile sperm. Clustering of DEPs between FD and UV groups was men with varicocele (9, 30, 33, 49). distinctly represented by principal component analysis (PCA)— Despite being principally a unilateral disease, Wang et al. for biplot, heatmap, and scatterplot (Fig. 1b–d). the first time reported the bilateral damage caused by unilateral varicoceles (UVs) (69). Left-sided varicocele is reported to induce bilateral testicular hypoxia that could eventually lead to GO enrichment analysis of selected DEPs bilateral germ cell apoptosis. In a pioneering study by our Molecular functions and biological processes associated with group, we had reported the differentially expressed proteins the selected proteins as investigated using the bioinformatic tool (DEPs) in infertile men with unilateral varicocele (UV) in suggest that redox homeostasis is severely impaired. The pro- comparison with healthy fertile donor (FD) men (7). teins found in the respiratory chain complex (GO:0098803), Recently, we demonstrated impairment of the mitochondrial mitochondrial protein complex (GO:0098798), catalytic com- structure and function, including hypoxia sensing, in infertile plex (GO:1902494), transferase complex (GO:1990234), men with varicocele and hypothesized that this in turn might and oxidoreductase complex (GO:1990204) were mostly have altered the mitochondrial redox circuitry of sperm, result- underexpressed in the UV group. DEPs enriched in bio- ing in sperm dysfunction. We validated the hypothesis using a logical processes such as cellular oxidant detoxification combination of wet laboratory and in silico techniques (59). (GO:0098869); regulation of cell redox homeostasis (GO: The present study aims to further explore the DEPs de- 0045454); glutathione (GSH) metabolism (GO:0006749); tected in UV men compared with fertile men and to charac- and reduced nicotinamide adenine dinucleotide (NADH) terize the proteins into those related or responsive to (1) redox (GO:0006734) metabolic process were mostly overexpressed homeostasis and/or (2) stress (OS/heat stress/hypoxic stress). in UV group and enriched by overexpressed proteins. On the The aberrant redox homeostasis on exposure to chronic contrary, a higher percentage of underexpressed proteins in hypoxic and hyperthermic conditions would explain the UV group were associated with protein folding (GO:0035966, molecular mechanisms leading to sperm OS in varicocele. GO:0006986, GO:0042026, and GO:0051131); energy me- Furthermore, with the etiology of varicocele being multi- tabolism (GO:0045333, GO:0006120, GO:0022904, GO: factorial, researchers have also explored the exposure of 0042773, GO:0042775, GO:0006119, and GO:0043462); and exogenous toxicants in mediating oxidative testicular dam- cellular response to heat stress (GO:1900034, GO:0034605, age in the testis of varicocele patients (62). A recent report GO:0009408), resulting in downregulation of the above pro- determined the impact of metal toxicity in inducing oxidative cesses. Similarly, an overview of enriched functions demon- and nitrosative stresses (27). The present work also targeted strated that proteins associated with oxidoreductase activity the major toxicity functions enriched by selected DEPs to with NAD(P) as an H acceptor (GO:0016668), peroxidase postulate the contribution of the latter in causing varicocele- activity (GO:0004601), and flavin adenine dinucleotide (FAD) associated infertility. binding (GO:0050660) were mostly overexpressed in UV 3

FIG. 1. Characterization of DEPs based on their involvement in redox homeostasis and/or oxidative/heat/hypoxic stress response in spermatozoa of UV men in comparison with FD. (a) Venn diagram showing the distribution of 261 global proteins and 85 DEPs. (b) 2D-PCA Biplot taking NSAF values of 85 selected DEPs demonstrated a distinct clustering of data points between the two groups. The biplot showed a complete separation of FD and UV proteome expression data (scores) as dots, with loading of overexpressed proteins being more towards UV group and loading for underexpressed proteins being more towards FD group. Principal component PC2 separated 3 sample replicates of FD (FD1, FD2, FD3) from the UV sample replicates (UV1, UV2, UV3). (c) Heat map representation of a hierarchical cluster of 85 DEPs. A color scale representation of data matrix alongside dendrograms for rows and columns was displayed. The dendrogram for sample replicates (column clustering) separated the samples according to their clinical diagnosis into FD and UV. Hierarchical clustering analysis between protein expression profiles of DEPs (row clustering) separated overexpressed DEPs in UV from underexpressed DEPs in UV. The blue and red color denotes low and high expression levels, respectively. (d) Scatter plot showing the log2 fold change difference values of common 61 selected DEPs. Red dots denote overexpressed proteins and blue stars denote underexpressed proteins. DEP, differentially expressed protein; FD, fertile donor; NSAF, normalized spectral abundance factor; PCA, principal component analysis; UV, unilateral varicocele. Redrawn by the Cleveland Clinic Center for Medical Art & Photography. Image used with permission. Color images are available online. 4 SWAIN ET AL. group; whereas proteins with NADH dehydrogenase (ubiqui- heat shock proteins (HSPs) downregulated in UV, HSP90B1 none) activity (GO:0008137), unfolded protein binding (HSP90b) and HSPA2 were selected for validation based on (GO:0051082), isomerase activity (GO:0003756), and oxido- their involvement with top two affected disease and function reductase activity (GO:0015037) of protein disulfide were networks as revealed by IPA network analysis (Fig. 4a, b). underexpressed in UV group compared with FD group. The most enriched pathophysiological functions affected by HSP90B1 was ‘‘posttranslational modification, protein Pathway enrichment analysis of selected DEPs folding, cancer’’ (IPA score = 53), while HSPA2 was found to be associated with ‘‘cell death and survival, cellular develop- ClueGO pathway enrichment analysis of DEPs demon- ment, cellular growth and proliferation’’ (IPA score = 18) strated enrichment of 233 pathways curated from KEGG, (Supplementary Table S2). Transcriptional regulation of 12 REACTOME, and Wiki pathway databases (Supplementary key proteins, including HSPA2 and HSP90B1 by hypoxia- Fig. S1). The metabolic pathways mostly affected included inducible factor 1A (HIF1A), was probed using MetaCore glycolysis (KEGG:00010 and R-HSA:70171), TCA cycle/ (Fig. 4c). Furthermore, the top five Tox Lists and Tox Func- ETC (KEGG:00020, R-HSA:71403, R-HSA:1428517, R- tions determined by IPA-Tox showed that OS and mitochon- HSA:71406, WP:78, WP:2453, KEGG:00190, R-HSA:611105, drial dysfunction are the most affected toxicity functions R-HSA:163200, and WP:111), and fatty acid metabolism (R- (Supplementary Table S3). Protein–protein interaction analy- HSA:611105 and R-HSA:163200). In addition, attenuation of sis from the STRING database showed a strong association the following pathways (KEGG: 04714, R-HSA: 3371453, and among the DEPs with a very high combined interaction score R-HSA: 3371556) indicated poor heat shock response (HSR). >0.9, thereby offering additional information about the com- Furthermore, a disturbed regulatory and metabolic signaling in plex interactive links between these DEPs (Fig. 5). an imbalanced redox state would be apparent from the dysre- gulated expression of proteins associated with OS (R-HSA: Expression and localization pattern 3299685 and WP: 408) and glutathione metabolism (KEGG: of the HSPs in sperm 00480, R-HSA: 174403, R-HSA: 156590, and WP: 100). To corroborate the alteration in the redox environment of sperm in Low expression of HSPA2 and HSP90B1 in spermatozoa of the UV patients, GSH/glutathione disulfide (GSSG) concen- UV patients compared with FD was validated by both immu- trations as a representative redox couple were measured. An nocytochemistry (ICC) and Western blot results (Fig. 6). Im- upsurge in the absolute concentrations in GSH indicated an munocytochemically, both HSPA2 and HDP90B1 were enhanced reducing capacity (Fig. 2a), while the increased GSH/ observed to be localized throughout the sperm (Fig. 6a, e). GSSG ratio further testified the occurrence of reductive stress in HDP90B1 had a predominant expression at the posterior por- spermatozoa of UV group (Fig. 2b). Correspondingly, a lower tion of head, in most of the sperm of FD. In contrast, HSPA2 was seen to be uniformly distributed at the head and neck steady-state reduction potential, that is, a more negative Ehc value in UV patient’s sperm compared with FD men (Fig. 2c), region, with a punctate expression at the tail in both the groups. demonstrated a reductive state in the spermatozoa of UV men. No change in the distribution/localization pattern of the The possible activation or suppression states of targeted aforementioned proteins was detected in the sperm of both pathways were analyzed using the ingenuity pathway anal- groups, however, decreased expression of these proteins could ysis (IPA). The IPA canonical pathway clearly predicted the be evidently inferred from the intensity spectrum images and suppressed activity of oxidative phosphorylation (OXPHOS) integrated density measurements (Fig. 6a, b, e, and f). The pathway with a negative activation z score of -2.0, con- Western blot results (Fig. 6c, d, g, and h) further corroborated ceivably due to downregulation of associated DEPs. The an obliterated expression of HSPs in the sperm of varicocele customized OXPHOS pathway generated by IPA also high- patients, implying poor protection to hypoxia- and hyperthermia- lighted the presumed sites of ROS production in mitochon- mediated OS in the UV group. dria (Fig. 3a). Diminished expression of OXPHOS proteins as Discussion corroborated by Western blot (Fig. 3b) and band intensities (Fig. 3c) further indicated the inhibition of proper OXPHOS Augmented levels of ROS despite the unusual over- in spermatozoa from the UV group and subsequent accu- expression profile of antioxidants in spermatozoa of UV pa- mulation of unused reducing equivalents. Out of the eight tients implicated the possible occurrence of reductive stress FIG. 2. Comparison of glutathione redox status in the spermatozoa of FDs and infertile UV patients. (a) Levels of reduced (GSH) and oxidized glutathione (GSSG); (b) thiol redox status; (c) half-cell reduction potential (Ehc). *p < 0.05. GSH, glutathione; GSSG, glutathione disulfide. Re- drawn by the Cleveland Clinic Center for Medical Art & Photography. Image used with permission. Color images are available online. 5

FIG. 3. Underexpression of proteins involved in oxidative phosphorylation (OXPHOS) pathway. (a) Pathway generated by ICP analysis using down regulated DEPs (green), implying suppression of OXPHOS in spermatozoa of unilateral varicocele patients. The network was overlayed with potential sites of ROS release indicated by pink dotted lines. Lines: interactions, arrowheads: directionality, Dotted line: inferred or indirect interaction, and absence of arrowheads: a binding interaction. (b) Western blot showing expression of mitochondrial OXPHOS proteins in FD and UV sperm samples. PC: positive control (human heart tissue lysate - mitochondrial extract). (c) Corre- sponding densitometry analysis of OXPHOS proteins in UV sperm with respect to FD sperm, with total protein normalization (in arbitrary unit). *p < 0.05. ICP, ingenuity canonical pathway; OXPHOS, oxidative phosphorylation; PC, positive control; ROS, reactive oxygen species. Redrawn by the Cleveland Clinic Center for Medical Art & Photography. Image used with permission. Color images are available online. 6

FIG. 4. Functional analysis of the top disease and function networks. (a) Network 1: Posttranslational modiﬁcation, protein folding, and cancer, centered around HSP90B1. (b) Network 2: cell death and survival, cellular development, cellular growth and proliferation centered around HSPA2. Red: overexpressed, green: underexpressed proteins in UV sperm in comparison with FD, and white: predicted protein not detected in our data set, lines: interactions and arrowheads: directionality. (c) Predicted upstream transcriptional regulator HIF1A of the 12 DEPs, including HSP90B1 and HSPA2, by MetaCore analysis. Blue circles: downregulated and red circles: upregulated proteins in UV spermatozoa in comparison with FD. The intensity of the color reﬂects the log2 expression levels of the proteins. HIF1A, hypoxia-inducible factor 1A. Redrawn by the Cleveland Clinic Center for Medical Art & Photography. Image used with permission. Color images are available online. 7

FIG. 5. STRING protein-protein interactome map of DEPs. Network contained 85 nodes with 142 edges (vs. 22 expected edges); clustering coefficient: 0.533; enrichment p-value <0.001. Confidence score threshold was set at 0.7 (high) for analyses. DEPs were subjected to MCL-clustering taking MCL-inflation parameter of 1.8. The eight clusters obtained were color coded, and the solid and dotted lines indicated connection within the same and different clusters, respectively. MCL, Markov clustering. Redrawn by the Cleveland Clinic Center for Medical Art & Photography. Image used with permission. Color images are available online. 8

FIG. 6. Expression profile of heat shock proteins HSP90B1 and HSPA2 in FDs and infertile UV patients. (a, e) Confocal photomicrograph showing immunocytochemical localization and expression profile of the respective proteins; (b, f) corresponding box-whisker plot of fluorescence intensity/integrated density of ICC (in arbitrary units). *p < 0.01. (c, g) Representative WB image of the respective proteins; (d, h) corresponding densitometry analysis with total protein normalization (in arbitrary unit). *p < 0.01. ICC, immunocytochemistry; WB, Western blot. Redrawn by the Cleveland Clinic Center for Medical Art & Photography. Image used with permission. Color images are available online. VARICOCELE-INDUCED REDUCTIVE STRESS IN SPERMATOZOA 9 in this patient group. Although the term reductive stress was Moreover, accumulation of cytosolic NADH inhibits first introduced by Albrecht Wendel in 1987 (70), it remains glyceraldehyde-3-phosphate dehydrogenase favoring alternate less explored. Reductive stress is characterized by an imbal- utilization of glycolysis-derived triose phosphates (72). As the anced redox state associated with an aberrant increment in levels glycolytic intermediaries are directed to the advance glycation of reducing equivalents, such as NADH/reduced NAH phos- end-product pathway, methylglyoxal is generated, which is a phate, along with increased activation of antioxidant enzymes potent intracellular glycating agent and ROS producer (58). and reduced pro-oxidant capacity (21). Deleterious effects of The conversion of methylglyoxal to lactate in varicocele sperm reductive stress with overproduction of ROS were reported to be could be mediated by overexpression of lactoylglutathione ly- prevalent in hypoxic conditions where the availability of ter- ase (nSC ratio = 4.53) and hydroxyacylglutathione hydrolase minal electron acceptor (oxygen) is compromised (28, 51, 53). (nSC ratio = 1.53) (22) (Fig. 5b). Accumulation of lactate with This makes the sperm samples in the UV patient group an ideal an underexpressed pyruvate dehydrogenase (nSC ra- model to study the incidence of reductive stress in augmenting tio = 0.37 for subunit a, 0.26 for subunit b)wouldleadto ROS production with respect to sperm biology. reductive stress in varicocele patients. Switching from Due to reduced venous outflow from the testis and epi- glycolysis to the pentose phosphate pathway enhances the didymis, a state of relative hypoxia develops in varicocele formation of NADPH as shown in Figure 7a (31). In UV sper- patients (12). Cellular hypoxia is characterized by genetic matozoa, a significant overexpression of 6-phosphogluconate and metabolic reprogramming that is facilitated by stabili- dehydrogenase (nSC ratio = 2.01) would have further aug- zation of a transcription factor such as HIF1A. Studies have mented reductive stress. Thus, in an environment where re- reported HIF1A to induce testicular hypofunction in varico- dox homeostasis is severely impaired, mitochondria become cele condition (38, 41, 69). In a hypoxic milieu, HIF1A en- both a source and a target of ROS. Moreover, reductive stress hances the expression of glycolytic genes (42), especially can lead to generation of ROS due to autoxidation of one and glucose transporters, glycolytic enzymes, lactate dehydro- more mitochondrial complex(es) (23, 65). genase A (LDHA), and pyruvate dehydrogenase kinase (60). Nuclear factor erythroid 2-related factor 2 (NRF2) is a In our study, we have detected overexpression of LDHA redox-sensitive transcription factor that is activated in re- (nSC ratio = 1.09) and several glycolytic enzymes such as sponse to hypoxia (67). The activated NRF2 then binds to the triose phosphate (nSC ratio = 1.53), glucose-6-phosphate antioxidant responsive element and enhances the expression isomerase (nSC ratio = 5.87), testis-specific glyceraldehyde- of several antioxidants (50). Unrestricted activation of NRF2 3-phosphate dehydrogenase (nSC ratio = 1.49), and a-enolase would lead to reductive stress, due to augmented production (nSC ratio = 1.42) in spermatozoa from UV group. However, and accumulation of antioxidant enzymes and reducing the glycolytic kinases were underexpressed in varicocele equivalents (17, 54). The entire array of antioxidant defence sperm, namely hexokinase (nSC ratio = 0.69), phosphofruc- work in cascade and such a scenario would eventually result tokinase (nSC ratio = 0.69), and pyruvate kinase isoenzyme in increased ROS generation (40). A disturbed feedback M1/M2 (nSC ratio = 0.21), implying a disturbed glycolysis. regulating the entire redox homeostasis was corroborated by HIF-1A is a ubiquitous, constitutively synthesized nuclear high-level static ORP (sORP) in the semen of UV group in factor that, in normal conditions, is hydroxylated by prolyl comparison with the FD group. Figure 7b schematically hydroxylase domain (PHD) enzyme and subsequently de- represents the de novo synthesis of GSH, which is increased graded by the ubiquitin/proteasome pathway (74). In UV in varicocele sperm due to the exclusive presence of GSH spermatozoa, upregulation of components of oxoglutarate synthetase. Recycling of Cys-Gly of the c-glutamyl cycle (36) dehydrogenase complex (nSc ratio = 2.09) and malate de- and synthesis of cysteine by the trans-sulfuration pathway (45) hydrogenase (nSC ratio = 2.45) could be presumed to con- are mediated by an upregulated level of cytosolic nonspecific vert 2-oxoglutarate to succinyl-CoA (11). Lower levels of dipeptidase isoform 2 (nSC ratio = 2.22) and adenosylhomo- 2-oxoglutarate and higher levels of succinate are reported cysteinase isoform 1 (nSC ratio = 5.10), respectively, in vari- to inactivate PHD enzymes and stabilize HIF1 A (1). cocele sperm. The higher levels of absolute GSH and a NADH is formed mainly by conventional glucose meta- subsequent decrease in reduction potential in sperm of UV pa- bolic pathways. As schematically illustrated in Figure 7a, it is tients have further substantiated the above findings (Fig. 2). plausible that overproduction of NADH can be predicted due The GSH (GSH/GPx) and thioredoxin/peroxiredoxin to upregulation of NADH producing glycolytic and TCA (TRDX-PRDX) are the primary regulatory antioxidant sys- cycle enzymes in UV spermatozoa. However, a major cohort tems. However, when the supply of their natural electron of sperm in a varicocele patient has been reported to have acceptors such as GSSG or oxidized TRDX is limited, these inactive mitochondria (15) and poor mitochondrial respiratory antioxidant systems convert to oxidant generators. Under re- efficiency (25). In congruence with the above results, we found ductive stress, NADPH could be presumed to supply electrons a downregulated expression of all OXPHOS proteins in UV to the FAD unit of glutathione reductase (GR) and thioredoxin - sperm sample (Fig. 3a). Incompetent oxidation of reductive reductase (TXNRD) subsequently to oxygen to produce O2 respiratory equivalents, such as NADH, hydroquinone (fully (Fig. 8a) (39). In UV group, we found high amounts of GR reduced) form of FAD, ubiquinone, and respiratory cyto- (nSC ratio = 5.77) and absence of glutathione peroxidase 1 chromes, creates an electron pressure in the mitochondria (66). (GPX1). It has been demonstrated that absence of GPX1 with The major sites of an electron leakage occur at mitochondrial high amounts of GSH would deteriorate the reductive stress complex I and III, where monoelectronic reduction of O2 is condition (10). Likewise, NADPH-dependent augmented thermodynamically and kinetically feasible (19, 47, 68). Due to ROS generation may result from high amounts of TXNRD damage to the electron transport chain (ETC) or low adenosine (nSc ratio = 2.98) as its preferred endogenous oxidized thior- triphosphate synthesis, electrons can be speculated to prema- edoxin 1 (Trx) substrate is in limited supply in sperm from UV turely escape from ETC and produce ROS (43, 44) (Fig. 3b). group (nSc ratio of TRDX = 0.60) (20). The thioredoxin 10

FIG. 7. Schematic representation showing the proposed mechanism for over production of NADH/NADPH and GSH in spermatozoa of unilateral varicocele patients. (a) Production of NADH/NADPH. (b) Production of GSH. Red: upregulated, blue: downregulated and purple: exclusive presence of enzymes in unilateral varicocele spermatozoa in comparison to fertile donor spermatozoa. Redrawn by the Cleveland Clinic Center for Medical Art & Photography. Image used with permission. Color images are available online. 11

FIG. 8. Predicted dysfunsctional glutathione (GSH/GPx) and TRDX-PRDX system in spermatozoa of UV patients. (a) Schematic representation of the flow of electrons in the GSH (GSH/GPx) and TRDX-PRDX regulatory antioxidant systems in expected normal state, that is, in FD sperm samples (FD), and presumed abnormal state, that is, UV sperm samples. When the supply of their natural electron acceptors such as GSSG or oxidized TRDX is limited, NADPH could be presumed to supply electrons to the FAD unit - of GR and TXNRD subsequently to oxygen to produce O2 ; (b) Schematic representation of possible generation of many species of ROS from superoxide due to overexpression of SOD, QSOX1, and MPO in UV sperm samples. FAD, flavin adenine dinucleotide; GR, glutathione reductase; MPO, myeloperoxidase; QSOX1, quiescin-sulfhydryl oxidase; SOD, superoxide dismutase; TRDX-PRDX, thioredoxin/peroxiredoxin; TXNRD, thioredoxin reductase. Redrawn by the Cleveland Clinic Center for Medical Art & Photo- graphy. Image used with permission. Color images are available online. 12 SWAIN ET AL. domain-containing proteins, such as TXNDC2, TXNDC3, tivation of genes associated with OS and hypoxia (52). In fact, TXNDC12, and TXNDC17 required for proper disulfide hyperthermia may contribute to cellular hypoxia-related OS bridge formation in the sperm tail proteins, are also found at injury and vice versa. Heat stress has been found to elevate undetectable levels in the UV sperm (46). Moreover, GSH hypoxia-induced free radical generation (29). The rise in tes- could be postulated to increase mitochondrial membrane ticular temperature is reported to aggravate oxidoreductive potential, thereby resulting in redox recycling of ubiquinone stress, especially in chronic hypoxia (57). Shiraishi et al. (64) or reverse electron flow from succinate to NAD+ and thereby demonstrated a strong correlation between testicular apoptosis increasing mitochondrial ROS production (63). The most and OS with increased scrotal temperature in testis with vari- abundant redox couple in a cell is the GSSG/2GSH couple. cocele. In such a scenario, hypoxic tolerance is mediated by The biological status of the cell is correlated with the half-cell stimulationofHSR,aschaperoneactivityofHSPmighthelpto reduction potential (Ehc) of the GSSG/2GSH couple. In fact, combat hypoxia-induced OS (34, 35). However, lower expres- the results of the present study showed a perturbed GSH to sion of HSPs detected in our study would have undermined the GSSG ratio and increased Ehc. Glutathionylation of mito- multifaceted prosurvival effects of HSPs in varicocele sperm. chondrial target(s) would also drive ETC toward superoxide In silico analysis along with validation of annihilated ex- - (O2 ) generation (75). As shown in Figure 8b, the generated pression of nodal proteins, dysfunctional OXPHOS pathway, - O2 is rapidly converted to hydrogen peroxide (H2O2) with and a more negative reduction potential with the disturbed high levels of superoxide dismutase as detected in sperm from balance in the pivotal redox couple (GSH/GSSG) in varicocele the UV group (nSc ratio = 2.48 of SOD1, 2.94 of SOD2) (75). patients support our hypothesis (Fig. 9). A further detailed In addition, protein folding enzymes such as protein disulfide- measurement of other redox couples, particularly NAD+/NADH isomerase (PDI) were downregulated (nSC ratio = 0.56 of and NADP+/NADPH, and expression profile of few more key PDIA1, 0.24 of PDIA3, 0.01 of PDIA4, and 0.35 of PDIA6), proteins of the redox regulatory pathways in a larger cohort of while quiescin-sulfhydryl oxidase (QSOX1) was upregulated patients will further strengthen our findings and help designing (nSc ratio = 12.73). With QSOX1 being the major enzyme for special treatment modalities for these patients. protein folding in sperm from the UV group, large amounts of H2O2 would be produced as a by-product (32). In such a Materials and Methods scenario, overexpression of myeloperoxidase (nSC ra- Selection of patients tio = 8.20) in sperm from UV would catalyze the formation of hypochlorous acid from H2O2, which will subsequently yield The study was carried out in 2015–2016, after obtaining many species of ROS as shown in Figure 8b (48). approval from the Institutional Review Board of Cleveland Several studies have demonstrated lower sperm concentra- Clinic, USA. Signed written consent was obtained from all tion, motility, sperm function, higher sperm DNA damage, as subjects before enrollment. Thirty-three infertile patients well as increased levels of ROS after scrotal hyperthermia in diagnosed with UV and 10 fertile normozoospermic men varicocele animal models (55, 56). Transient mild testicular without clinical varicocele (categorized as FD), all with an hyperthermia instigates complex stress response, including ac- age limit of 20–40 years, were included in the study. All

FIG. 9. Annihilated expression of nodal proteins, dysfunctional OXPHOS pathway. Schematic representation of the proposed hypothesis on reductive stress-induced oxidative stress-mediated sperm dysfunction in UV. Redrawn by the Cleveland Clinic Center for Medical Art & Photography. Image used with permission. Color images are available online. VARICOCELE-INDUCED REDUCTIVE STRESS IN SPERMATOZOA 13 fertile subjects had no clinical history of varicocele and had the luminol-dependant chemiluminescence with the Berthold fathered at least one healthy child naturally within the last 2 luminometer (Model: AutoLumat plus LB 953, Oakridge, years. Infertile patients were diagnosed with clinical varicocele TN). ROS of the test sample were calculated by subtracting basedonscrotalpalpationinatemperature-controlled room the negative control average from their average. The sample (23.8C) with adequate illumination, and the gradation of var- ROS are then corrected by dividing them with ‘‘sperm con- icocele was done based on criteria by Dubin and Amelar (24). centration/mL.’’ Results were expressed as relative light Both infertile men and control subjects with a sperm concen- units, RLU/s/million sperm/mL. tration <10 · 106/mL, leukocyte concentration ‡1 · 106/mL, history of any systemic illnesses and chronic fever before en- Test Sample ROS average }RLU=s} for test sample rollment, and female factor infertility were excluded. ¼ average }RLU=s} for negative control Sample collection and processing Corrected Test sample ROS ¼ Calculated sample ROS=sperm Semen samples were collected by masturbation after 2– concentration ðmillion sperm=mLÞ; i:e:; RLU=s= 3 days of ejaculatory abstinence. Following complete lique- million sperm=mL faction for 15–30 min at 37C, routine semen analysis was performed according to the WHO guidelines (71) as depicted earlier (4). Sperm concentration and motility were deter- Assessment of reductive stress and calculation mined by manual semen analysis using a MicroCell counting of redox potential chamber (Vitrolife, San Diego, CA). Percent viability was Total GSH equivalents (GSH + GSSG) were measured determined by eosin/nigrosin staining. Sperm morphology spectrophotometrically by GR recycling assay at the expense of was assessed by staining semen smears with a Diff-Quik kit oxidation of NADPH using 5-5¢-dithiobis 2-nitrobenzoic acid (Baxter Healthcare Corporation, Inc., McGaw Park, IL). (DTNB; Ellman’s reagent); and GSSG was measured under Sperm morphology was evaluated according to Kruger’s similar conditions after masking GSH with 2-vinylpyridine as strict criteria as described in the WHO, 2010 guidelines (71). 6 described elsewhere (18). For measuring the total GSH Semen samples having a round cell concentration >1 · 10 / equivalents (GSH + GSSG), GR was added to the assay for mL were tested for leukocytospermia, by the peroxidase or reduction of GSSG to GSH at the expense of oxidation of the Endtz test. NADPH. The reduction potential (Ehc)ofGSH/GSSGcouple was calculated using the Nernst equation. Measurement of ORP in semen Postseparation from seminal plasma, spermatozoa were ORP is the measurement of oxidative or reductive stress washed twice with chilled PBS, and then sonicated in 6 re- (redox balance) in semen. The ORP was measured in the peated cycles of 10 s sonic pulses at high intensity with 1-min semen using the MiOXSYS System (Aytu Bioscience, intervals between pulses on ice. Then, the sperm lysates were Englewood, CO), as described earlier (3, 6, 8). Normalized centrifuged at 10,000 g for 15 min at 4C. The proteins in the ORP was expressed as mV/million sperm/mL. Using the supernatant were estimated by bicinchoninic acid assay MiOXSYS System, a 30 lL sample was loaded and the test (Pierce BCA Protein assay kit; Thermo Fisher Scientific) as starts when the sample fills the reference electrode. The per the manufacturer’s protocol. Then, they were precipitated electrochemical circuit is completed and sORP is displayed with ice-cold 5% trichloroacetic acid containing 0.01 N HCl, on the MiOXSYS Analyzer display screen. ORP provides a and cleared by centrifugation. The deproteinized supernatants snapshot of current redox balance. A higher sORP reading is were used for the assay. In brief, the assay mixture (final indicative of OS. Normalized ORP is expressed as mV/mil- volume 200 lL) contained 3 mM NADPH in 125 mM phos- lion sperm/mL. phate buffer containing 6.3 mM EDTA (pH 7.5), DTNB (0.6 mM), and sperm lysate (30–40 lg protein). The reaction was started with the addition of 2 lLGR(*1 U; Sigma- Measurement of ROS in sperm Aldrich, St. Louis, MO). Subsequently, the yellow chro- ROS was measured by chemiluminescence assay using matophore (2-nitro-5-thiobenzoate: TNB2–) formed by the luminol (5-amino-2, 3-dihydro-1, 4-phthalazinedione) as the interaction of contributing SH groups from GSH and GSSG probe (2, 61). Luminol is an extremely sensitive dye that (after conversion by GR) with DTNB was monitored at detects both extracellular and intracellular ROS at neutral pH. 405 nm in an ELx800 Absorbance Microplate Reader (Bio- Luminol generates light signal after combining with oxida- Tek Instruments, Inc., Winooski, VT) at 1-min intervals for tive end-products, which is measured with a luminometer. 6 min. All the determinations were normalized to protein For measuring ROS, a working solution of luminol content. The absolute GSH amount was quantified from dif- (Sigma-Aldrich, United Kingdom) (5 mM) was prepared ference between the total GSH equivalent and the obtained from a stock solution (100m) of luminol in dimethyl sulf- GSSG value. Reductive stress index was calculated as ratio oxide. For positive control, 50 lL of 30% H2O2 was used. between GSH and GSSG. The glutathione redox potential was The assay mixture consisted of only phosphate-buffered calculated by Nernst equation for half-reaction: Ehc =-240– saline (PBS) (400 lL) for the blank; PBS (400 lL) +5mM 61.5/2 ln{[GSH]2/GSSG} mV; where -240 mV is the stan- luminol (10 lL) for the negative controls; PBS (395 lL) dard redox potential (Eo) of GSH at pH 0, -61.5/2 denotes RT/ +30% H2O2 (5 lL) + of 5 mM luminol (10 lL) for the positive zF, that is, R = gas constant (8.314 J/K/mol), T = absolute controls; and sperm suspension (400 lL) +5mM luminol temperature of 37C or 310 K, F = Faraday constant (10 lL) for test sample. All samples were vortexed gently (9.64853 · 104 C/mol); z = number of electrons exchanged in immediately, and levels of ROS were assessed by measuring the chemical reaction GSSG +2e- + 2H à 2GSH. 14 SWAIN ET AL.

Identification of differentially expressed sperm proteins Western blot validation The DEPs reported in our earlier publication (7) were se- Expression of the OXPHOS mitochondrial complexes and lected for secondary in silico analysis based on the following HSPs was verified in sperm samples of seven fertile men, and criteria: (1) those related to redox homeostasis, and/or (2) five infertile UV patients using Western blotting (WB). those responsive to stress (OS/heat stress/hypoxic stress). Washed spermatozoa were lysed in RIPA lysis buffer DEPs were identified by the spectral count (SC) method, (Sigma-Aldrich, St. Louis, MO) containing proteinase in- considering their normalized spectral abundance factor hibitor cocktail (Roche, Indianapolis, IN). The supernatants (NSAF). NSAF of a particular protein ‘‘j’’ with total ‘‘n’’ no. were collected and treated with the required volume of of proteins was calculated from the following formula: Laemmli sample application buffer (BioRad, CA) containing n [(NSAF)j = (SC/protein length)j/+ (SC protein length)]. The 1% b-mercaptoethanol. The mixture (*20–30 lg of protein) average value of NSAF for each identified protein taking into was boiled for 5 min; 15 lL of each sample was applied to account all the replicates of sample is used for further each well of a 4%–15% Mini-PROTEAN TGX Precast gel quantitative analysis. This approach takes into account the (Biorad, CA) and electrophoresed for 2 h at 90 V along with a sample-to-sample variation that is obtained when performing set of molecular weight markers (Sigma Chemical Co., St. replicate analyses of a sample. The abundance of the proteins Louis, MO). The resolved protein bands were transferred was classified as high (H), medium (M), low (L), or very low onto polyvinylidene difluoride (PVDF) membranes at 20 V (VL) based on their average SC among the three replicate for 30 min using a transfer buffer of 25 mmol/LTris base, runs. Different constraints for significance tests ( p value) 192 mmol/L glycine, and 20% methanol. The blots were and/or fold change cutoffs (or NSAF ratio) were applied for blocked for 2 h at room temperature with SuperBlock these four abundance categories for selection of DEPs, as blocking buffer (Thermo Scientific, Rockford, IL). The explained in the aforementioned published study (7). blocking buffer was decanted and blots were incubated Categorization and selection of DEPs were performed using overnight at 4C, with a primary antibody diluted at 1:1000 the GO analysis platform, which comprised publicly available concentration in 3% bovine serum albumin (BSA) in tris- bioinformatic annotation tools and databases such as GO Term buffered saline with Triton X (TBST). The respective Finder (16), GO Term Mapper, UniProt (73), and Software for primary antibodies used were total OXPHOS Human WB Researching Annotations of Proteins [STRAP] V_1.5.0.0 (13). Antibody Cocktail (mouse IgG; ab110411; Abcam), anti- Quantitative patterns across the selected DEPs and samples HSP90B1 (rabbit IgG; AB3468; Millipore), and anti-HSPA2 were organized in a matrix format using the heatmap (37). (rabbit IgG;ab154374; Abcam). Blots were then washed us- To visualize hierarchical clustering, a heatmap was con- ing TBST and incubated with horseradish peroxidase- structed in R.3.4.4 package using the ComplexHeatmap li- conjugated respective IgG (anti-mouse IgG; ab6728; Abcam; brary. Hierarchical clustering was performed taking the anti-rabbit IgG; ab97051; Abcam) in 1:10,000 dilution for 1 h Euclidean distance correlation matrix for complete linkage at room temperature following washes in TBST. Antibody/ to design the heatmap dendrogram. protein complexes were visualized using an enhanced chemiluminescence kit-Pierce ECL Western Blotting Sub- strate (Thermo Scientific, Rockford, IL) using ChemiDoc Bioinformatic analysis MP Imaging System (BioRad, Hercules, CA). Post-Western blot detection, the PVDF membranes were stripped. After GO enrichment analysis was conducted using the washing with TBST, the membranes were treated with col- ClueGO plugin V_2.5.2 (75) using Cytoscape software loidal gold total protein stain (#170-6527; BioRad, CA ) and V_3.6.1. A two-sided hypergeometric test was performed imaged. The developed blots were subjected to densitometric using this platform for the enrichment of GO terms, fol- analysis using Image Lab software (BioRad) according to p lowed by the Bonferroni -value correction method for standard methods normalizing with total protein stained with multiple testing. Functionally grouped networks with GO colloidal gold. In addition to individual (n = 4) WB analyses, terms as nodes were linked based on their kappa score pooled specimens (n = 3) from both UV and FD group were level (‡0.3). Enriched GO terms were classified accord- run against each other to maintain biological variability. ing to their enrichment score, for ‘‘biological processes,’’ Normalization of sample pooling was performed by both ‘‘cellular component,’’ and ‘‘molecular function’’ using sperm number (each sample contributed with an equal functional annotations. The gene ontological relationship number of spermatozoa) and protein content. was retrieved from EBI-UNIPROT_GOA (September 4, 2018). ICC validation of HSPs Key biological pathways affected by selected DEPs were queried using both open source tools such as ClueGO plugin Semen was collected from seven fertile men, and five in- V_2.5.2 (14) in Cytoscape software V_3.6.1, and commercial fertile UV patients for validation. For ICC analysis of the two pathway packages such as IPA as provided by Ingenuity selected HSPs, spermatozoa were isolated from seminal Systems, V_ 47547484, release date: February 8, 2019; and plasma postliquefaction by centrifugation and fixed with MetaCore Pathway Maps and Disease Marker Sets (as paraformaldehyde (PFA) (final concentration 2%). After provided by GeneGo, Inc., V_19.1, release date: May 2019). fixing the cells with PFA, the cells were permeabilized with IPA-Tox was used to determine the top toxicity functions in 0.1% Triton X-100 in PBS (5 min). Subsequently, the cells combination with toxicity lists and link experimental data to were blocked with 5% BSA for 2 h. The blocking buffer was clinical pathology endpoints. The known and predicted pro- removed, and cells were incubated overnight at 4C, with the tein interactions of DEPs were explored from STRING da- rabbit primary antibody specifically recognizing HSPA2 tabase using STRING web tool V_10.5. (ab154374; Abcam) and HSP90b (AB3468; Millipore). Both VARICOCELE-INDUCED REDUCTIVE STRESS IN SPERMATOZOA 15 the antibodies were used at a dilution of 1:100 in 1% BSA in miluminescence assay. J Assist Reprod Genet 32: 1721– PBST buffer (PBS supplemented with 0.1% Tween-20). 1729, 2015. After extensive washings, detection was carried out using 3. Agarwal A and Bui AD. Oxidation-reduction potential as a appropriate AlexaFluor-488-labeled anti-rabbit (ab150077; new marker for oxidative stress: correlation to male infer- Abcam) at 1:1000 dilutions. Cells were counterstained with tility. Investig Clin Urol 58: 385–399, 2017. Hoechst (bisbenzimide; H 33258; Sigma ). All images were 4. Agarwal A, Gupta S, and Sharma R. Andrological Eva- taken on a confocal laser-scanning microscope (Leica Mi- luation of Male Infertility. Switzerland: Springer Interna- crosystems) with a 63 · -objective and analyzed with the tional Publishing, 2016. Leica Application Suite X image examiner software. After 5. 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Reprod Biol Endocrinol 13: 8, 2015. presented as median (25th, 75th percentile) and compared 8. Agarwal A, Sharma R, Roychoudhury S, Du Plessis S, and between infertile UV men and FD control men by the non- Sabanegh E. MiOXSYS: a novel method of measuring parametric Mann–Whitney test for semen analysis and ICC oxidation reduction potential in semen and seminal plasma. results. A two-tailed Student’s t-test was used to compare Fertil Steril 106: 566–573 e10, 2016. 9. Agarwal A, Sharma RK, Desai NR, Prabakaran S, Tavares intensity readings of WB and biochemical data on GSH/ A, and Sabanegh E. Role of oxidative stress in patho- GSSG redox couple. All analyses were done using IBM- genesis of varicocele and infertility. Urology 73: 461–469, SPSS software V_21 and p-value <0.05 was considered sig- 2009. nificant. A PCA of quantified NSAF of selected DEPs was 10. 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Abbreviations Used (Cont.) PVDF ¼ polyvinylidene diﬂuoride QSOX1 quiescin-sulfhydryl oxidase NSAF ¼ normalized spectral abundance factor ¼ ORP ¼ oxidation/reduction potential ROS ¼ reactive oxygen species SC spectral count OS ¼ oxidative stress ¼ PBS ¼ phosphate-buffered saline sORP ¼ static ORP TBST tris-buffered saline with Triton X PCA ¼ principal component analysis ¼ PDI ¼ protein disulﬁde-isomerase TRDX ¼ thioredoxin TXNRD thioredoxin reductase PFA ¼ paraformaldehyde ¼ PHD ¼ prolyl hydroxylase domain UV ¼ unilateral varicocele WB Western blotting PRDX ¼ peroxiredoxin ¼