TY - JOUR
AU - Tsai, Pei-Shiue Jason
AB - Abstract Sulfhydryl oxidation is part of the sperm maturation process essential for the acquisition of sperm fertilization competency and its structural stabilization; however, the specific sulfhydryl oxidases that fulfill these roles have yet to be identified. In this study, we investigate the potential involvement of one atypical thiol oxidase family called quiescin Q6/sulfhydryl oxidase (QSOX) using the mouse epididymis as our model system. With multidisciplinary approaches, we show that QSOX isoform 1 and 2 exhibit complementary distribution throughout the epididymal duct, but that each variant possesses distinct subcellular localization within the epididymal principal cells. While QSOX2 was exclusively present in the Golgi apparatus of the caput and corpus epididymis, QSOX1c, the most profusely express QSOX1 variant, was abundantly present in the cauda luminal fluids. Moreover, immunohistochemistry studies together with proteomic identification in isolated epididymosomes provided evidence substantiating the release of QSOX2, but not QSOX1c, via an apocrine secretory pathway. Furthermore, we demonstrate for the first time, distinct association of QSOX1c and QSOX2 with the sperm acrosome and implantation fossa, during different stages of their epididymal maturation. In conclusion, our study provides the first comprehensive comparisons between QSOX1 and QSOX2 in the mouse epididymis, revealing their distinct epididymal distribution, cellular localization, mechanisms of secretion and sperm membrane association. Together, these data suggest that QSOX1 and QSOX2 have discrete biological functions in male germ cell development. Introduction Spermiogenesis takes place and is completed in the testis; however, fully differentiated testicular spermatozoa are not yet competent to fertilize an oocyte. Spermatozoa are required to travel through the convoluted epididymal tubule where they undergo sequential morphological and biochemical modifications known as epididymal sperm maturation [1, 2]. The epididymis is anatomically divided into three major segments, caput (head), corpus (body), and cauda (tail), and each of these segments exhibits distinct luminal environments. The regional-specific microenvironment is established as a result of variations in gene and protein expression profiles as well as different proportions of three epithelial cell types (basal, narrow, and principal cells). It is known that these distinct luminal environments contribute to regional-specific sperm maturation processes [3–5], including the addition/removal of proteins to/from the sperm membrane surface [6], the attachment of glycosylphosphatidylinositol (GPI)-anchored proteins, and the post-translational modification (PTM) of intrinsic sperm proteins, the latter of which includes phosphorylation, glycosylation, and disulfide bond formation [7–9]. Among different PTMs, disulfide bond formation is critical for correct protein folding and the stabilization of tertiary and quaternary structure of essential proteins [10]. Furthermore, disulfide bond formation is highly correlated with sperm physiological functions such as tyrosine phosphorylation [11], sperm chromatin condensation [12], as well as sperm tail structure stabilization and normal wave patterns of sperm motility [13]. Although disulfide bond formation is critical for the above-mentioned processes, the specific enzyme(s) that catalyze the oxidation of free thiols to promote disulfide bond conversion during sperm maturation have yet to be completely resolved. Some studies have revealed that free thiols labeled by monobromobimane are predominantly observed in caput spermatozoa, while the number of free thiols on the sperm membrane surface decreased drastically in the cauda sperm (84% vs 14% in caput and cauda sperm, respectively) [11, 14, 15]. However, the identity of the actual protein candidates that experience this form of PTM, and their precise role in sperm membrane stabilization or sperm physiology, remains unclear. As early as 1979, a member of the protein family termed Quiescin Q6-Sulfydryl Oxidases (QSOX) was first discovered in the rat seminal vesicle. The QSOX protein was characterized as a flavin adenine dinucleotide (FAD) responsible for sulfhydryl oxidation and catalyzing the oxidation of thiols into disulfides following the reaction 2R-SH + O2→R-S-S-R + H2O2 [16]. Two Qsox transcript variants, namely Qsox1 and Qsox2, have since been identified in mammalian cells and are located on chromosome 1 and 9, respectively in both human and mouse [17]. Moreover, these genes give rise to a variety of different protein isoforms in multiple species. For example, the mouse QSOX1 family contains isoforms a, b, c, and d (http://www.Uniport.org) and similarly, mouse QSOX2 comprises a, b, and c variants (http://www.Uniport.org). Compared to the QSOX1a/2a isoforms, which consist of an additional transmembrane domain, all other isoforms (QSOX1b-d or QSOX2b-c) are truncated due to alternative splicing events [18, 19]. Despite the fact that QSOX1 and QSOX2 share 40% identity in their primary amino acid structure and 68% in their functional domains [20], the exact biological properties and the biochemical characteristics of QSOX proteins remain to be elucidated. In this study, we have utilized the mouse model to characterize the epididymal distribution and the subcellular localization of QSOX1 and QSOX2 proteins. Moreover, we have also investigated the secretory mechanisms underlying QSOX1/2 release to the epididymal luminal compartment and their subsequent interaction with maturing spermatozoa. Materials and methods Chemicals, reagents, and antibodies Chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Rabbit polyclonal anti-QSOX2 and anti-QSOX1 that detect all splicing variants were purchased from Abcam (Cambridge, UK) and Santa Cruz (Texas, USA), respectively. To investigate specific QSOX1 variant c which showed the most abundant expression in reproductive tissues, anti-QSOX1 isoform c (QSOX1c) anti-serum was generated and kindly provided by Dr SH Li at the Mackay Memorial Hospital (Taiwan) [21]. For loading control of the immunoblot, the eukaryotic translation elongation factor 2 was used and purchased from Abcam (Cambridge, UK). All secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA, USA). Animals Eight-week-old male ICR mice were purchased from National Laboratory Animal Center (NAR Labs, Taiwan) and were housed in groups (3 mice/cage) in a certified animal facility at the School of Veterinary Medicine, National Taiwan University under a 12/12 h controlled dark-light cycle with ad libitum access to food and water, and were used throughout the study. All animal experiments were carried out under the regulation and permission of IACUC protocols at National Taiwan University (NTU-103-EL-86; NTU-104-EL-00081, Taiwan). Immunohistochemistry staining For immunohistochemistry staining, 5-μm paraffin-embedded tissue sections were deparaffinized with xylene and rehydrated with 100%–80% ethanol as described earlier [17]. Antigen retrieval was carried out with 10 mM citrate buffer (pH 9.0) at 121°C for 3 min. Endogenous peroxidase was subsequently removed by incubation of slides with 3% (v/v) hydrogen peroxide (H2O2) for 30 min at room temperature (RT), and nonspecific signals were minimized by incubation of slides with filtered 2.5% normal goat serum (NGS) diluted in Tris-buffered saline (5 mM Tris, 250 mM sucrose, pH 7.4, TBS) for 30 min at RT. Sections were subsequently incubated with primary antibodies (1:1000 for QSOX1c or 1:100 for QSOX2) overnight at 4°C. Polymer-HRP reagent (BioGenex HRP kit) was used as a secondary antibody after intensive washes of sections with TBS. Reactions were developed with 2% diaminobenzidine (DAB, Dako Real DAB + Chromogen) for 10 min, and slides were counterstained with hematoxylin (Muto Pure Chemicals Co. Ltd, Tokyo, Japan) and mounted for evaluation. Immunoblotting For protein sample preparation, freshly obtained materials were homogenized on ice with tissue homogenization buffer (250 mM sucrose, 1 mM EDTA, 20 mM Tris/Hepes, 1% Triton X-100, pH 7.4) supplemented with protease inhibitor cocktail tablet (EDTA free, Roche, Mannheim, Germany). For quantitative protein profile in the epididymosomes, purified epididymosomes from 8–12 mice were pooled to generate a sufficient quantity of material. Protein concentrations were measured and standardized according to the BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA). Equivalent amount of total protein extract was resuspended with LDS loading buffer (Invitrogen) in the presence of reducing agent (50 mM dithiothreitol [DTT]). Samples were heated at 95°C for 10 min and cooled on ice before loading on gels. Bio-Rad Mini-PROTEIN® electrophoresis system was used (Bio-Rad Laboratories Ltd, Hertfordshire, UK), and standard manufactory protocol was followed. Briefly, proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (gradient T-Pro EZ Gel Solution, T-Pro Biotechnology, NTC, Taiwan) and wet-blotted onto a polyvinylidene difluoride membrane (Immobilon-P, Millipore, Billerica, MA, USA). After blocking for 1 h with blocking buffer (TBST [5 mM Tris, 250 mM sucrose, pH 7.4 with 0.05% v/v Tween-20] supplemented with 5% milk powder) at RT, blots were incubated with primary antibody (1:5000 for QSOX1c or 1:250 for QSOX2) for overnight at 4°C. After washing in TBST, secondary antibody was subsequently added and blots were incubated at RT for an additional 1 h. Protein signals were visualized by chemiluminescence (Merck Ltd, Taiwan) and were detected with ChemiDoc™ XRS + system (Bio-Rad). When necessary, blots were stripped (Invitrogen) and re-probed for other proteins of interests. Structural analysis was used to compare the relative QSOX expressions between male reproductive organs (Figure 1B and C). Figure 1. View largeDownload slide Cellular localizations and tissue abundance of QSOX1c and QSOX2 in the male reproductive tissue. Male reproductive tissues including the testis, epididymis, vas deferens, and seminal vesicle were harvested from an 8-week-old male mouse and were subjected to immunohistochemical studies and western blotting analyses. (A) QSOX1c and QSOX2 proteins exhibit homogenous cytosolic pattern in the epithelium of testis and vas deferens. Within the epididymal epithelium, QSOX2 was strongly detected in a restricted spherical structure at the apical ridge of the epithelial cells, while QSOX1c was detected mostly in the epididymal lumen. In addition to cell-bearing signals, a large amount of QSOX1c-positive secretory vesicles were identified (marked with red arrow head) in the seminal vesicle lumen, suggesting this protein is secreted at multiple sites in the male reproductive tract. (B) Western blotting analyses on whole tissue homogenates indicated seminal vesicles contained relatively more (∼69.9%) QSOX1c protein detected at ∼65 kDa than in other tissues. (C) In contrast, QSOX2 protein was dominantly expressed in the epididymis (∼51%) as a ∼75 kDa protein. All experiments were repeated at least three times and representative images are presented. L: lumen; Epi.: epithelium; Int.: intestinal area. Figure 1. View largeDownload slide Cellular localizations and tissue abundance of QSOX1c and QSOX2 in the male reproductive tissue. Male reproductive tissues including the testis, epididymis, vas deferens, and seminal vesicle were harvested from an 8-week-old male mouse and were subjected to immunohistochemical studies and western blotting analyses. (A) QSOX1c and QSOX2 proteins exhibit homogenous cytosolic pattern in the epithelium of testis and vas deferens. Within the epididymal epithelium, QSOX2 was strongly detected in a restricted spherical structure at the apical ridge of the epithelial cells, while QSOX1c was detected mostly in the epididymal lumen. In addition to cell-bearing signals, a large amount of QSOX1c-positive secretory vesicles were identified (marked with red arrow head) in the seminal vesicle lumen, suggesting this protein is secreted at multiple sites in the male reproductive tract. (B) Western blotting analyses on whole tissue homogenates indicated seminal vesicles contained relatively more (∼69.9%) QSOX1c protein detected at ∼65 kDa than in other tissues. (C) In contrast, QSOX2 protein was dominantly expressed in the epididymis (∼51%) as a ∼75 kDa protein. All experiments were repeated at least three times and representative images are presented. L: lumen; Epi.: epithelium; Int.: intestinal area. Indirect immunofluorescent staining and image acquisition For indirect immunofluorescent staining, 5-μm paraffin-embedded tissue sections were deparaffinized, rehydrated, and underwent antigen retrieval as mentioned above. After blocking with 1% BSA for 30 min at RT, tissue sections were further permeabilized with 100% ice-cold methanol at –20°C for 10 min. Anti-QSOX1c antiserum or anti-QSOX2 antibody was used at a dilution of 1:1000 (for QSOX1c) or 1:250 (for QSOX2) for overnight incubation at 4°C. Sections were subsequently incubated with secondary antibodies for 1.5 h at RT. Stained sections were mounted with Vectashield in the presence of diamidino-2-phenylindole (DAPI, Vector Lab, Peterborough, UK). As for negative controls, each immunoreaction was accompanied by a reaction omitting the primary antibody. All samples were evaluated with either Olympus IX83 epifluorescent microscopy or Leica TCS SP5 II confocal scanning microscopy and analyzed with ImageJ (NIH; http://rsb.info.nih.gov/ij/) or CellSens software (Olympus, Tokyo, Japan). Background subtraction and contrast/brightness enhancement (up to ∼30% enhancement using the maximum slider in both softwares) were performed identically for all images (including control images) in the same experiment. Isolation and characterization of mouse epididymal fluid and epididymosomes Isolation and characterization of mouse epididymosomes were carried out as previously described [22, 23]. Briefly, epididymides from 8-week-old male ICR mice were carefully separated from fat and overlying connective tissue immediately after euthanasia. Epididymal segments corresponding to the caput, corpus, and cauda were dissected, and luminal fluid was aspirated from each segment in a modified Biggers, Whitten, and Whittingham media (BWW; pH 7.4, osmolarity 300 mOsm/kg) [23, 24]. The tissue was subjected to mild agitation, and the medium was subsequently filtered through membranes with 70 μm pore size. The resultant suspension was collected and prepared for epididymosome isolation as described earlier [22, 23]. The isolation protocol involved sequential centrifugation of the epididymal fluid suspensions with increasing velocity (500 g, 2000 g, 4000 g, 8000 g, 17,000 g) to eliminate all cellular debris before layering the supernatant onto a discontinuous OptiPrep gradient (40%, 20%, 10%, and 5%). The gradient was subjected to ultracentrifugation at 100,000 g for 18 h at 4°C, and 12 equivalent fractions were collected. Each collected fraction was further diluted in PBS and subjected to a final ultracentrifugation (100,000 g, 3 h, 4°C). Each sample was analyzed on a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) to determine mean particle size. Immunoblotting was carried out on isolated epididymosomes using specific antibodies known for the validation of extracellular vesicles, including anti- CD9, anti-FLOT1, anti-PSMD7, anti-HSP90B1, and anti-TUBB antibodies as demonstrated in our earlier studies [22, 23, 25, 26]. Tandem mass spectrometry (nanoLC-MS/MS) quantitative analyses For protein identification from the isolated epididymosomes, NanoLC–MS/MS was performed using a Dionex UltiMate 3000 nanoLC system (Dionex, Sunnyvale, CA, USA). Trypsin was used as the digestion enzyme, and digested peptides were suspended in 0.1% formic acid and directly loaded onto the analytical column packed with ReproSil Pur C18 AQ 3 μm sorbent. Peptides were eluted using a 110 min gradient from 0 to 34% buffer (95% ACN, 0.1% formic acid) at 250 nl min−1 and nanoelectrosprayed into a Q-Exactive Plus (Thermo Fisher Scientific, Bremen, Germany). Precursor scan of intact peptides was measured in the Orbitrap by scanning from m/z 350 to 1500 (with a resolution of 70,000), the 10 most intense multiply charged precursors were selected for high energy collision dissociation fragmentation with a normalized collision energy of 30.0, then measured in the Orbitrap at a resolution of 17,500. Data were converted to peak lists using Xcalibur (Thermo Fisher Scientific), and searched using Proteome Discoverer version 2.0 (Thermo Fisher Scientific) against all mouse entries in SwissProt. Mass tolerances in MS and MS/MS modes were 10 ppm and 0.02 Da, respectively, and up to two missed cleavages were allowed. Identification criteria consisted of a minimum of two uniquely matched peptides per protein, and a MASCOT score ≥35 to identify proteins of interest. Only proteins interrogated at a peptide level sequenced with two unique peptides each scoring an E-value <0.05 are reported. Indirect immunolabeling of QSOX1c and QSOX2 on epididymal spermatozoa Sperm cells from different epididymal segments were obtained by carefully separating the caput, corpus, and cauda epididymis from fat and overlying connective tissue on a temperature-controlled dissecting microscopy (Olympus, SZ61) immediately after euthanasia. Small incisions were made on these tissues and sperm cells were allowed to swim out for 10 min in a 500 μl droplet of Whitten's medium (Wm: 100 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.5 mM glucose, 1 mM pyruvic acid, 4.8 mM calcium L-lactate hydrate, 20 mM HEPES, pH 7.4). To examine QSOX–sperm association, sperm suspensions were collected and divided into two equivalent proportions for either an unwashed group or washed group (with Wm medium, centrifugation at 800 g for 5 min and for three times). A standard wet- mounted indirect sperm immunolabeling protocol without membrane permeabilization step was followed. Briefly, sperm cells were fixed with 4% (v/v) paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) + 0.2% (v/v) glutaraldehyde (GA, Sigma) for 1 h at RT. Fixed sperm cells were rinsed twice with PBS (600 g, 5 min), followed by a 15 min incubation in blocking buffer (5% NGS [Vector Lab, Burlingame, CA] diluted in PBS supplement with 100 mM glycine). After two rinses in PBS (600 g, 5 min), sperm cells were resuspended in blocking buffer for 1 h at RT. Sperm cells were subsequently incubated with either anti-QSOX1c anti-serum (1:1000 dilution in the blocking buffer) or anti-QSOX2 antibody (1:250 dilution in blocking buffer) overnight at 4°C. Unbound primary antibody was removed by two additional washes with PBS. Goat anti-rabbit Alexa-594 was used as a secondary antibody (1:100 dilution in the blocking buffer). A 1:1 ratio of the sperm suspension and the Vectashield (Vector Lab, Burlingame, CA) were mixed, mounted, and sealed on a Superfrost® plus microscope slide (Thermo Scientific; Menzel GmbH & Co KG, Braunschweig, Germany) for further microscopy evaluation. N-glycosylation of the mouse epididymal QSOX protein Epididymal fluids isolated from caput, corpus, and cauda were digested with or without peptide N-glycosidase F (PNGase F, New England Biolabs, MA, USA) according to the manufacturer's instructions. Briefly, a 10 μl total reaction mixture, containing 12.5 μg of total protein with 1 μl of Glycoprotein Denaturing Buffer (10X) and H2O, was denatured by heating at 100°C for 10 min. The denatured protein mixture was chilled on ice and centrifuged for 10 s, following which 2 μl of GlycoBuffer 2 (10X), 2 μl of 10% NP - 40 and 6 μl of H2O, with or without 1 μl of PNGase F, were added. The mixture was incubated at 37°C for 1 h and used for western blotting assay as previously described. Results Mouse QSOX1c and QSOX2 exhibit distinct cellular localizations and are enriched in male reproductive organs Based on western blotting analysis of whole tissue homogenates using anti-QSOX1 and anti-QSOX2 antibodies (detect all variants in both isoforms), we observed QSOX1 variant c was much abundant than isoform a, b, and d in the male reproductive tracts (Supplementary Figure S1). As a result, in-house production of polyclonal anti-QSOX1c anti-serum was generated as using methods previously published for the generation of antisera against CEACAM10 [21] and used in this study. As for QSOX2, commercially available QSOX2 antibody was used. First, the tissue distribution and cellular localization of QSOX1c and QSOX2 in male reproductive organs including testes, epididymides, vas deferens, and seminal vesicles were assessed. As shown in Figure 1, positive labeling for both QSOX1c and QSOX2 was detected in all tissues examined. However, apparent differences in the cellular localization of QSOX1c and QSOX2 were detected in the epididymis and the seminal vesicle. In the epididymis, QSOX1c was characterized by abundant luminal staining in addition to homogenous distribution within the cytosol of the lining epithelial cells. In contrast, QSOX2 displayed a more restricted profile of labeling, appearing to be concentrated within the apical domain of epididymal principal cells. QSOX2 was also far less abundant in the epididymal lumen compared to QSOX1c (Figure 1A). In the seminal vesicles, QSOX1c was detected in both intracellular and extracellular domains, with the latter appearing restricted to a population of large spherical vesicle-like structures in the lumen (Figure 1A, red arrows). Western blotting analyses revealed the labeling of single predominant bands of ∼65 and 75 kDa corresponding to QSOX1c and QSOX2, respectively (Figure 1B and C). Notably, QSOX1c was more highly expressed in the seminal vesicles relative to the other male reproductive tissues assessed (Figure 1B). On the contrary, QSOX2 was abundantly present in the epididymis and expressed at more modest levels in the testis and the vas deferens (Figure 1C). Semiquantification analysis of western blotting experiments demonstrated the densitometry of QSOX1c and QSOX2 in different male reproductive tissues, confirming that QSOX1c was mostly abundant (69.9%) in the seminal vesicles and QSOX2 had the highest expression in the epididymis (64.6%) (Figure 1B and C). Complementary tissue distribution of QSOX1c and QSOX2 in the epididymis The epididymis is a highly segmented male reproductive tissue with distinct, regional-specific profiles of protein and gene expression. Accordingly, we next examined whether QSOX proteins also exhibited regional-specific expression patterns within this tissue. As shown in Figure 2A, QSOX1c protein expression was first detected in the distal portion of the caput epididymis before experiencing progressive enrichment toward the downstream segments of the corpus and cauda. On the contrary, QSOX2 protein expression was first apparent in the initial segment and thereafter was consistently detected throughout the caput and proximal region of the corpus, before gradually diminishing in the distal corpus and cauda epididymis. The validity of these regional-specific expression patterns of QSOX protein isoforms was further supported by western blotting analyses on total tissue homogenates (Figure 2B and C). This analysis revealed that the ∼65 kDa protein band corresponding to QSOX1c was dominantly expressed in the corpus and cauda epididymis. By contrast, the ∼75 kDa QSOX2 protein was most abundantly expressed in the caput and corpus epididymal regions (Figure 2B). Besides distinct epididymal tissue distributions of QSOX1c and QSOX2, the unique cellular localizations of these protein isoforms were also demonstrated. QSOX1c was abundantly present in the epididymal lumen rather than in the intracellular domain, while QSOX2 was predominantly detected at the epididymal epithelium (Figures 1A and A). Figure 2. View largeDownload slide Regional-specific protein expression of QSOX1c and QSOX2 in the epididymis. Whole epididymal cross sections were subjected to indirect immunofluorescence studies. (A) QSOX1c signal (in green) appeared in the lumen of distal corpus and the intensity of staining was gradually increased toward the cauda epididymal lumen. In contrast, QSOX2 signal (in red) was associated within the epithelium starting from the initial segment (IS) and gradually diminishing toward the distal corpus. (B) Epididymal tissue homogenates from the caput (Cap), corpus (Cor), and cauda (Cau) segments were analyzed by semiquantified western blotting. In agreement with immunofluorescence data, QSOX1c protein was enriched in the cauda epididymis more than in the caput or corpus, while QSOX2 was mainly present in the caput and corpus epididymis. (C) To further validate the presence of QSOX proteins in the epididymal lumen, epididymal fluid (EF) was isolated from different segments. Both QSOX1c and QSOX2 proteins were identified in the EF, with the segment-specific enrichment of these proteins being similar to that documented in epididymal tissue. However, in contrast to a single protein band observed in tissue homogenates, additional protein bands were detected at the higher molecular weights of ∼85 and ∼100 kDa for QSOX1c and QSOX2, respectively. Figure 2. View largeDownload slide Regional-specific protein expression of QSOX1c and QSOX2 in the epididymis. Whole epididymal cross sections were subjected to indirect immunofluorescence studies. (A) QSOX1c signal (in green) appeared in the lumen of distal corpus and the intensity of staining was gradually increased toward the cauda epididymal lumen. In contrast, QSOX2 signal (in red) was associated within the epithelium starting from the initial segment (IS) and gradually diminishing toward the distal corpus. (B) Epididymal tissue homogenates from the caput (Cap), corpus (Cor), and cauda (Cau) segments were analyzed by semiquantified western blotting. In agreement with immunofluorescence data, QSOX1c protein was enriched in the cauda epididymis more than in the caput or corpus, while QSOX2 was mainly present in the caput and corpus epididymis. (C) To further validate the presence of QSOX proteins in the epididymal lumen, epididymal fluid (EF) was isolated from different segments. Both QSOX1c and QSOX2 proteins were identified in the EF, with the segment-specific enrichment of these proteins being similar to that documented in epididymal tissue. However, in contrast to a single protein band observed in tissue homogenates, additional protein bands were detected at the higher molecular weights of ∼85 and ∼100 kDa for QSOX1c and QSOX2, respectively. Considering the fact that epididymal protein–sperm interactions take place within the epididymal lumen, we also collected fluids from different epididymal regions and analyzed the presence of QSOX proteins within this milieu. As shown in Figure 2C, QSOX1c distribution in the epididymal fluid was similar to that of the protein's tissue distribution, with the protein being predominantly detected in the luminal fluids sampled from the corpus and cauda segments (Figure 2A and B). Intriguingly, however, the predominant ∼65 kDa QSOX1c-positive band detected in both epididymal tissue and fluids was accompanied by a secondary, higher molecular weight band of ∼85 kDa. This additional band was particularly evident in cauda epididymal fluid (Figure 2C), but was also present, albeit at relatively low levels in cauda epididymal tissue (Figure 2B). Notably, the labeling of QSOX2 protein also differed between epididymal tissue and fluid samples. Unlike epididymal tissue, QSOX2 was detected exclusively at a molecular weight of ∼100 kDa in the epididymal fluid isolated from caput and corpus segments (Figure 2C). In the downstream caudal epididymal fluid however, the ∼100 kDa QSOX2-positive band was accompanied by a second, far more intensely labeled band, resolving at ∼75 kDa (Figure 2C). Together, these data illustrate a complementary pattern of tissue distribution of QSOX1c and QSOX2 in the mouse epididymis, and identify distinct changes in the molecular weight of the proteins expressed in the epididymal epithelium versus those residing in the luminal environment. Epididymal QSOX2 but not QSOX1c is aggregated in the Golgi apparatus prior to its secretion In contrast to the homogenous cytosolic distribution of QSOX1c, QSOX2 was observed in a restrained structure at the apical region of the epididymal epithelium. Accordingly, we next examined whether this QSOX2 labeling co-localized with distinct cellular organelles. By dual staining of QSOX2 with organelle-specific protein markers, we demonstrated that QSOX2 (in red) was highly co-localized with Golgi 58K associated protein formiminotransferase cyclodeamidase (FTCD, in green) at the apical region of the principal cells (Figure 3B). In contrast, QSOX1c (in red) was detected homogenously throughout the cytosol of the epithelium, with only minimal co-localization with FTCD (in green, Figure 3A). These data demonstrate, for the first time, distinct cellular patterns of QSOX isoform expression and raise the prospect that QSOX2, but not QSOX1, is preferentially accumulated in the Golgi apparatus prior to its secretion to the epididymal lumen. On the basis of these data we also infer that QSOX isoforms possess different biological properties and/or fulfill different functions in the male reproductive tissues. Figure 3. View largeDownload slide Colocalization analysis demonstrated the accumulation of QSOX2 but not QSOX1c in the Golgi apparatus of epididymal epithelium. Corpus epididymal sections were double stained with QSOX proteins and the Golgi apparatus-specific marker protein Golgi 58k. (A) In line with our earlier observation, the QSOX1c (shown in red) was homogenously distributed in the cytosol of epididymal epithelium and in the extracellular lumen, whereas Golgi 58K (shown in green) was detected specifically in the Golgi apparatus with minimal co-localization with QSOX1c. (B) In contrast to QSOX1c, QSOX2 (shown in red) and Golgi 58K (shown in green) were highly colocalized demonstrating QSOX2, but not QSOX1c, accumulated in the Golgi apparatus of epididymal epithelium. L: lumen; Int.: intestinal area. Figure 3. View largeDownload slide Colocalization analysis demonstrated the accumulation of QSOX2 but not QSOX1c in the Golgi apparatus of epididymal epithelium. Corpus epididymal sections were double stained with QSOX proteins and the Golgi apparatus-specific marker protein Golgi 58k. (A) In line with our earlier observation, the QSOX1c (shown in red) was homogenously distributed in the cytosol of epididymal epithelium and in the extracellular lumen, whereas Golgi 58K (shown in green) was detected specifically in the Golgi apparatus with minimal co-localization with QSOX1c. (B) In contrast to QSOX1c, QSOX2 (shown in red) and Golgi 58K (shown in green) were highly colocalized demonstrating QSOX2, but not QSOX1c, accumulated in the Golgi apparatus of epididymal epithelium. L: lumen; Int.: intestinal area. Epididymosome isolation and protein identification revealed different secretory mechanisms for mouse epididymal QSOX1c and QSOX2 There are two known secretory mechanisms for the release of epididymal proteins to the luminal environment: merocrine and apocrine [27]. We therefore investigated whether either of these specific secretory mechanisms could account for the release of QSOX protein into the epididymal lumen. As shown in Figure 4A, we observed QSOX2, but not QSOX1c, -enriched vesicle-like structures appearing to pinch off from the apical domain of the epididymal epithelium (Figure 4A1). Higher magnification images confirmed the presence of these QSOX2-positive vesicles accumulating at the apical margin of the epididymal epithelia (Figure 4A2 marked with red arrowheads), and permitted measurement of their approximate dimensions. The average diameter of these vesicles was 1.12 ± 0.035 μm (Figure 4B), which was considerably larger than the expected size for secretory vesicles or epididymosomes (measuring in the nanometer range). We therefore conclude that these vesicles likely represent apical blebs, a highly characteristic feature of the apocrine secretory pathway. Figure 4. View largeDownload slide QSOX2, but not QSOX1c, is released from epididymal epithelium via an apocrine secretory pathway and is detected in epididymosomes. (A) Caput epididymal sections were subjected to immunohistochemistry and assessed via light microscopy. Apart from Golgi apparatus-associating QSOX2 (shown in brown), multiple QSOX2-containing vesicle-like structures (marked by red arrow heads) were observed pinching off at the apical margin of the epididymal epithelium. (B) The average size of these vesicle-like structures was measured as 1.12 ± 0.035 μm, which corresponds to the approximate size of apical blebs commonly associated with the apocrine secretory process. (C) Western blotting analysis was applied to investigate the presence of QSOX2 in isolated epididymosomes. The QSOX2 protein identified in epididymosome preparations exhibited identical segment-specific enrichment to that observed in whole epididymal fluids. Moreover, the QSOX2 protein detected in epididymosomes was of a similar molecular weight (i.e. 100 kDa) as that recorded in the epididymal fluids, but differed from that of the 75 kDa protein detected in epididymal tissue homogenates. (D) Proteomic analysis further confirmed the presence of QSOX2, but not QSOX1c, in isolated epididymosomes. L: lumen. Figure 4. View largeDownload slide QSOX2, but not QSOX1c, is released from epididymal epithelium via an apocrine secretory pathway and is detected in epididymosomes. (A) Caput epididymal sections were subjected to immunohistochemistry and assessed via light microscopy. Apart from Golgi apparatus-associating QSOX2 (shown in brown), multiple QSOX2-containing vesicle-like structures (marked by red arrow heads) were observed pinching off at the apical margin of the epididymal epithelium. (B) The average size of these vesicle-like structures was measured as 1.12 ± 0.035 μm, which corresponds to the approximate size of apical blebs commonly associated with the apocrine secretory process. (C) Western blotting analysis was applied to investigate the presence of QSOX2 in isolated epididymosomes. The QSOX2 protein identified in epididymosome preparations exhibited identical segment-specific enrichment to that observed in whole epididymal fluids. Moreover, the QSOX2 protein detected in epididymosomes was of a similar molecular weight (i.e. 100 kDa) as that recorded in the epididymal fluids, but differed from that of the 75 kDa protein detected in epididymal tissue homogenates. (D) Proteomic analysis further confirmed the presence of QSOX2, but not QSOX1c, in isolated epididymosomes. L: lumen. It is known that the apical blebs shed from the epididymal epithelium via apocrine secretory pathways eventually degrade within the epididymal lumen, releasing a population of smaller extracellular vesicle structures termed epididymosomes. To confirm the presence of QSOX proteins in epididymosomes, and thus substantiate the hypothesized apocrine secretory mechanism of QSOX2, we performed epididymosome isolation. Based on proteomic profiles of epididymosome content, QSOX2, but not QSOX1, nor QSOX1c was identified (detailed proteomic results are shown in Figure 4C). In accordance with our earlier data from isolated epididymal fluid (Figure 2C), western blotting analysis of purified epididymosomes confirmed the presence, and the substantial enrichment of the ∼100 kDa of QSOX2 in the caput and corpus epididymosomes. Although QSOX2 was also detected in epididymosomes isolated from the cauda epididymis, only the ∼75 kDa variant was present in these vesicles (Figure 4C). Moreover, the relative abundance of the QSOX2 protein was considerably reduced in cauda epididymosomes versus those sampled from more proximal epididymal segments (Figure 4C). Distinct sperm membrane surface association of QSOX1c and QSOX2 To investigate segment-specific sperm–QSOX associations, we isolated caput, corpus, and cauda spermatozoa and carried out for immunolabeling of surface antigens (i.e. spermatozoa were fixed but nonpermeabilized). As shown in Figure 5A, QSOX1c (in green) was observed over the entirety of the sperm surface, but a strong acrosome association (indicated with green arrowhead) was evident in both caput and corpus spermatozoa. By contrast, the acrosome association of QSOX1c was barely detectable in equivalent populations of cauda epididymal spermatozoa. Accordingly, quantification analysis demonstrated a striking reduction in the number of sperm with positive QSOX1c staining in the acrosomal domain in the caput (67%) and corpus epididymal segments (63%), compared to that of their downstream cauda counterparts (23%) (Figure 5B). Unlike QSOX1c, QSOX2 was detected mainly on the membrane overlying the sperm connecting piece, mid-piece, and principal piece of the tail (Figure 5A, showed in red). Moreover, an intense QSOX2 signal was observed in the vicinity of the implantation fossa (IF, indicated with red arrow) of caput and corpus spermatozoa. Quantification analysis demonstrated that a higher percentage of the caput (55%) and corpus (62%) spermatozoa exhibited QSOX2-positive signal at the IF, with comparatively few of the cauda spermatozoa (8%) exhibiting a similar staining pattern (Figure 5C). Such data demonstrate that both QSOX1c and QSOX2 exhibit regional-specific patterns of sperm association, presumably reflecting stage-wise biological functions in the context of epididymal sperm maturation and storage. Figure 5. View largeDownload slide QSOX1c and QSOX2 exhibited distinct but specific sperm surface association. To investigate the association of QSOX proteins with maturing spermatozoa, sperm cells were isolated from the caput, corpus, and cauda epididymis prior to being subjected to fixation and a nonpermeabilized immunofluorescence staining process. (A) QSOX1c (in green) was observed on the surface of the sperm head, mid-piece, and principal piece of the tail. However, in caput epididymal sperm, a strong association of QSOX1c was detected exclusively overlying the acrosomal domain (indicated by green arrow head). In contrast, QSOX2 (in red) was observed mostly on the sperm head and mid-piece with minimal labeling on the membrane overlying the principal piece of the sperm tail. An additional foci of QSOX2 labeling was detected at the implantation fossa (indicated by red arrowheads) in caput and corpus spermatozoa, but not in cauda spermatozoa. (B) A higher percentage of caput (67%) and corpus (63%) spermatozoa exhibited acrosome-specific QSOX1c staining than cauda sperm (23%). (C) Likewise, QSOX2 was predominantly detected at implantation fossa of the caput (55%) and corpus (62%) sperm, and only a small percentage of cauda spermatozoa (8%) were positively labeled for QSOX2 protein at the implantation fossa. Two hundred sperm cells were manually quantified for each QSOX staining, and representative images are presented. Figure 5. View largeDownload slide QSOX1c and QSOX2 exhibited distinct but specific sperm surface association. To investigate the association of QSOX proteins with maturing spermatozoa, sperm cells were isolated from the caput, corpus, and cauda epididymis prior to being subjected to fixation and a nonpermeabilized immunofluorescence staining process. (A) QSOX1c (in green) was observed on the surface of the sperm head, mid-piece, and principal piece of the tail. However, in caput epididymal sperm, a strong association of QSOX1c was detected exclusively overlying the acrosomal domain (indicated by green arrow head). In contrast, QSOX2 (in red) was observed mostly on the sperm head and mid-piece with minimal labeling on the membrane overlying the principal piece of the sperm tail. An additional foci of QSOX2 labeling was detected at the implantation fossa (indicated by red arrowheads) in caput and corpus spermatozoa, but not in cauda spermatozoa. (B) A higher percentage of caput (67%) and corpus (63%) spermatozoa exhibited acrosome-specific QSOX1c staining than cauda sperm (23%). (C) Likewise, QSOX2 was predominantly detected at implantation fossa of the caput (55%) and corpus (62%) sperm, and only a small percentage of cauda spermatozoa (8%) were positively labeled for QSOX2 protein at the implantation fossa. Two hundred sperm cells were manually quantified for each QSOX staining, and representative images are presented. Figure 6. View largeDownload slide Proposed models for the epididymal secretion of mouse QSOX1c and QSOX2. Based on data presented in this study, QSOX1c is likely to be secreted via a constitutive secretory pathway, and thereafter may be responsible for stabilization and/or modification of the sperm membrane overlying the acrosomal domain (left panel). QSOX2 protein, on the other hand, localizes strongly to the aggregates within the Golgi apparatus where it is likely packaged into epididymosomes awaiting the arrival of appropriate stimuli in order to trigger its release via an apocrine secretory pathway. After release into the epididymal lumen, a specific QSOX2–sperm association occurs at the implantation fossa, suggesting the involvement of this protein in the manipulation of sperm morphology required to support motility. Figure 6. View largeDownload slide Proposed models for the epididymal secretion of mouse QSOX1c and QSOX2. Based on data presented in this study, QSOX1c is likely to be secreted via a constitutive secretory pathway, and thereafter may be responsible for stabilization and/or modification of the sperm membrane overlying the acrosomal domain (left panel). QSOX2 protein, on the other hand, localizes strongly to the aggregates within the Golgi apparatus where it is likely packaged into epididymosomes awaiting the arrival of appropriate stimuli in order to trigger its release via an apocrine secretory pathway. After release into the epididymal lumen, a specific QSOX2–sperm association occurs at the implantation fossa, suggesting the involvement of this protein in the manipulation of sperm morphology required to support motility. Discussion Post-testicular spermatozoa are exposed to segment-specific luminal environments during their epididymal transit and functional maturation. Among the myriad of changes that accompany this phase of sperm development, the process of sulfhydryl oxidation has been shown to play an important role in sperm nuclear protamine condensation [12], flagellar stabilization [13], and acrosomal enzyme modification [28]. As in all cells, a delicate balance exists between the number of sulfhydryl groups and disulfide bonds, a balance that is regulated by the activity and overall abundance of sulfhydryl oxidase(s) [29, 30]. Many sulfhydryl oxidases exhibit regional-specific activities in the epididymis suggesting their involvement in different aspects of the sperm maturation process. For example, selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx), which plays a crucial role in sperm chromatin condensation, is abundantly present in the anterior region of the epididymis [13, 31, 32]. By contrast, other thiol peroxidases, such as protein-disulfide isomerase-associated 3, which has been implicated in sperm–oocyte fusion, exhibits a higher concentration in the proximal epididymal segments [33, 34]. Despite having been identified in the epididymis of multiple species, the secretory mechanisms and the exact biological functions of sulfhydryl oxidases are currently poorly understood. In previous studies, we have provided the first evidence that the epididymis expresses both QSOX1 and QSOX2 proteins, with distinct biochemical properties and isoforms having been documented [17]. In the current study, we have used the mouse as a model to extend our characterization of QSOX proteins by investigating the mechanism(s) through which they are secreted into the epididymal lumen and their association with the maturing sperm membrane thereafter. Based on the evidence presented in this study, we conclude that QSOX1c, the most abundantly expressed QSOX1 variants, and QSOX2 exhibit a complementary epididymal luminal distribution with distinct subcellular localization profiles. Furthermore, high confidence proteomic identification of QSOX2, but not QSOX1c, within isolated epididymosomes, and the distinct sperm acrosome or IF association of QSOX1c and QSOX2, respectively, suggested that these proteins engage different secretory mechanisms and fulfill discrete biological functions. It is well established that different epididymal regions exhibit distinctive functions, a fact attributed to the creation of highly specialized, segment-specific luminal microenvironments. In this context, the caput and corpus epididymis have been primarily implicated in promoting early and late sperm maturational events, while the cauda epididymis serves as the major sperm storage site attributed to the production of various proteins that stabilize the sperm architecture [5, 35, 36]. Previous proteomic studies by Ijiri et al and Kameshwari et al suggested that most of the caput-specific proteins contribute to the functional modifications on sperm structural proteins, such as actin and outer dense fiber proteins [37, 38]. Other studies have shown that the structural characteristics of sperm organelles (e.g. nucleus, centrosome, flagellum) are modulated by serial sulfhydryl oxidation during spermiogenesis and the early stages of epididymal maturation within the proximal epididymis. This agrees with evidence for high enzymatic activity among certain members of the protein disulfide isomerase and thioredoxin families within the proximal epididymal regions [13, 32, 39, 40]. These earlier findings echo current study on the abundant epididymal expression of QSOX2 within the Golgi apparatus of proximal epididymal epithelium cells (Figures 2 and 3), ideally positioning the protein for secretion into the epididymal lumen whereupon it could participate in the postspermiogenesis structural stabilization of maturing spermatozoa. Post-translational modifications are known to participate in many biological processes (e.g. the glycosylation, phosphorylation, the attachment of GPI anchors, acetyl, alkyl, phosphate or addition of peptides including SUMO or ubiquitin) [7]. Among those processes, glycosylation is a commonly seen and well-characterized PTM. Glycosylation is a ubiquitous process mainly occurs in the Golgi apparatus and participates the maturation of intrinsic proteins [7, 41]. During epididymal maturation, sperm proteins undergo either glycosylation or de-glycosylation to become functional [42]. Moreover, comparative glycomic studies of the epididymal fluid showed that the level of protein (de)-glycosylation in the epididymis exhibits region-specific and maturation-dependent manner [41]. We observed a shifted protein molecular weight of the QSOX2 in the isolated epididymal luminal fluids and epididymosomes (Figures 2C and 4C), the increase of QSOX2 molecular weight may account for QSOX2 undergoing glycosylation particular in the caput and corpus Golgi apparatus, and thus, functional QSOX2 was secreted into the lumen as glycoform. Accordingly, our N-glycosylation assay demonstrated that the shifted molecular weight of QSOX2 was indeed attributed to N-glycosylation that the expected molecular weight of nascent QSOX2 was revealed after the de-N-glycosylation process (Supplementary Figure S2B). In contrast, the higher molecular weight of QSOX1c detected at ∼85 kDa in the corpus and cauda epididymal fluids (Figure 2C) did not fall back to the expected size of nascent QSOX1c at ∼65kDa after de-N-glycosylation (supplementary Figure S2A) suggesting other yet to be characterized PTM processes may also attribute to QSOX1c protein maturation. Furthermore, QSOX2 was shown to exhibit strong association with populations of caput and corpus epididymal spermatozoa, localizing to the mid- and principal-piece of their tails and displaying a particularly intense signal foci at the IF adjacent to the sperm head (Figure 5, indicated with red arrowhead). Notably, in the mouse model the centriole pair is initially restricted in the IF of caput sperm, prior to the centrioles being destroyed as these cells continue their descent through more distal regions of the epididymis [43]. The stabilization of sperm centrosomes is critical for structural maturation of epididymal spermatozoa, such as sperm flagellogenesis [44]. Although we are not able to distinguish at this moment whether the observed QSOX2 signal at the sperm IF is originated from the testis or is secreted from the epididymal epithelium and thereafter associated with maturating sperm cells, our data suggest that QSOX2 might be critical for sperm tail elongation and imparting straightness on this structure. Further functional genetic characterizations of QSOX2 will be helpful to elucidate the exact functions of QSOX2. Distinct from the proximal epididymis, the cauda luminal environment is enriched with antioxidant enzymes that regulate and maintain the redox status of the sperm storage reservoir and thereby protect spermatozoa against oxidative damage [45]. In this context, peroxiredoxin 6 and superoxide dismutase have been reported to act as scavenging enzymes to help mitigate the impact of ROS-mediated toxic effects on spermatozoa during their prolonged storage in the cauda epididymis [38, 46]. In a similar context, it has been reported by Morel et al that QSOX1 can afford protection to somatic cells, such as those of the human breast cancer MCF-7 lineage, against apoptosis-induced oxidative stress during in vitro culture [47]. Thus, our finding that QSOX1c is abundantly present in the lumen of distal epididymis suggests this protein may form part of the antioxidant defenses needed to protect mature spermatozoa. Aside from this putative role, it is also of particular interest that QSOX1c was observed to localize mainly to the acrosomal plasma membrane of caput and corpus, but not cauda sperm cells (Figure 5). This situation stands in marked contrast to the reciprocal, increasing gradient of QSOX1c accumulation documented in epididymal luminal fluids (Figures 2 and 5). Such findings raise the prospect that QSOX1c may participate in reorganization of the structural characteristics of the sperm head plasma membrane, an event that is recognized as one of the most prominent maturational changes that accompany epididymal transit [48]. Indeed, the acquisition of sperm–oocyte fusion ability has been shown to be mediated by re-folding of membranous molecules triggered by thiol-disulfide exchanges [49]. At present, however, it remains to be determined whether QSOX1c association with the head of proximal epididymal sperm is related to the remodeling of the plasma membrane components needed for gamete fusion. Similarly, further research is needed to assess whether the abundant QSOX1c observed in the distal epididymis serves to stabilize the luminal environment required for sperm storage. Protein secretion from the epididymal epithelium is known to be mediated via either (1) the constitutive merocrine or (2) the regulated apocrine secretory pathways [27]. The former is the classical pathway for epithelium to liberate secretory vesicles, consisting of fully matured proteins, into the extracellular lumen by exocytosis [50]. The latter apocrine pathway is instead characterized by the formation of large protrusions at the apex of principal cells that are referred to as apical blebs. The pinching off the blebs and eventually degradation within the epididymal lumen sees the release of small membranous vesicles, called epididymosomes, into this extracellular environment [6, 51]. Epididymosomes are defined as exosome-like secretory vesicles, ranging in size from 50 to 500 nm, which have been documented in the epididymal lumen of numerous species, including the mouse [52]. These heterogeneous small membranous vesicles have been suggested to carry genetic information (DNA, RNA) and materials that are required for sperm fertilizing ability and plasma membrane remodeling [23, 27, 51, 53]. Additionally, compartmentalization of proteins in epididymosomes has been shown to coordinate the association of epididymal proteins with the different structures of the spermatozoon [54]. For example, microphage migration inhibitory factor is transferred as a cytosolic protein from within the epididymosomes to the intracellular dense fibers of the sperm flagellum [55]. Alternatively, the GPI-anchored protein P26h/P34H is associated with membrane raft domains in the epididymosome and subsequently transferred to the acrosomal region of maturing spermatozoa, a domain that is also enriched with membrane rafts [56]. Of interest, both QSOX1c and QSOX2 were readily detected in mouse epididymal luminal fluids (Figure 2C). However, only QSOX2 and not QSOX1c was identified in isolated epididymosomes. Based on these data, we infer that QSOX1c and QSOX2 enter the epididymal lumen via different secretory mechanisms, with QSOX1c most likely being secreted via the classical exocytotic merocrine pathway, and QSOX2 being released from the epididymal epithelium via association with epididymosomes in an apocrine secretory pathway. Notably, the regulation of apical blebbing associated with the apocrine pathway has been shown to be dependent on testicular luminal factors and/or cross-talk between epididymal cells responding to testicular factors [57]. It is therefore likely that QSOX2 secretion is regulated by specific, but as yet unidentified, luminal factors that are intimately involved in the process of sperm maturation. In conclusion, we have provided the first comprehensive comparison between two isoforms of the QSOX protein family, QSOX1c and QSOX2, in the mouse epididymis (Figure 6). The complementary epididymal tissue distributions, distinct secretory pathways, and their association with specific, but different, sperm membrane domains all suggest the involvement of epididymal QSOX proteins in different stages of sperm maturation and/or storage. Specifically, we postulate that the QSOX proteins are likely to be of fundamental importance in the regulation of key membrane surface modifications that accompany sperm transit through the epididymis. Supplementary data Supplementary data are available at BIOLRE online. Supplementary Figure S1. The profound expression of QSOX1 variant c in the male reproductive tracts. Male reproductive tissues including the testis, epididymis, vas deferens, and seminal vesicle were harvested from an 8-week-old male mouse. Standard western blotting analysis was performed to determine the relative abundancy of each splicing variants of mouse QSOX1. In the epididymis, vas deferens, and seminal vesicle, the dominant band was observed at ∼65 kDa corresponding to QSOX1 variant c. On the other hand, the relatively weak QSOX1-positive signal was also detected in the testis, epididymis, and seminal vesicle at ∼75 kDa. Supplementary Figure S2. De-glycosylation analyses revealed N-glycosylation status of secreted QSOX1c and QSOX2 proteins. The epididymal fluids (EF) collected from the caput, corpus, and cauda were treated with (+) or without (–) PNGase F to assess the extent of N-glycosylation by mobility shifts on western blotting. (A) In the corpus and cauda EF, the dominant signal of QSOX1c was detected at ∼65 kDa, whereas the additional band was weakly present at ∼85 kDa. After the PNGase treatment, QSOX1c signals with reduced molecular weight were observed at ∼80 and ∼63 kDa. (B) Prior to de-N-glycosylation process, QSOX2 was predominantly expressed at ∼100 kDa in the caput and corpus EF. 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TI - Mouse quiescin sulfhydryl oxidases exhibit distinct epididymal luminal distribution with segment-specific sperm surface associations
JF - Biology of Reproduction
DO - 10.1093/biolre/ioy125
DA - 2018-05-24
UR - https://www.deepdyve.com/lp/oxford-university-press/mouse-quiescin-sulfhydryl-oxidases-exhibit-distinct-epididymal-luminal-EGVHodIHDT
SP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -