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Sperm proteins and cancer‐testis antigens are released by the seminiferous tubules in mice and men

2021; Wiley; Volume: 35; Issue: 3 Linguagem: Inglês

10.1096/fj.202002484r

1530-6860

Liza O’Donnell, Diane Rebourcet, Laura F. Dagley, Raouda Sgaier, Giuseppe Infusini, Peter J. O’Shaughnessy, Frédéric Chalmel, Daniela Fietz, W. Weidner, Julien M. D. Legrand, Robin M. Hobbs, Robert I. McLachlan, Andrew I. Webb, Adrian Pilatz, Thorsten Diemer, Lee B. Smith, Peter G. Stanton,

Xenotransplantation and immune response

The FASEB JournalVolume 35, Issue 3 e21397 RESEARCH ARTICLEOpen Access Sperm proteins and cancer-testis antigens are released by the seminiferous tubules in mice and men Correction(s) for this article Erratum Volume 35Issue 7The FASEB Journal First Published online: June 17, 2021 Liza O'Donnell, Corresponding Author Liza O'Donnell Liza.ODonnell@hudson.org.au orcid.org/0000-0001-5848-6136 Hudson Institute of Medical Research, Clayton, VIC, Australia Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, Australia Faculty of Science, The University of Newcastle, Callaghan, NSW, Australia Correspondence Liza O'Donnell, Hudson Institute of Medical Research, 27-31 Wright Street, Clayton, VIC 3168, Australia. Email: Liza.ODonnell@hudson.org.auSearch for more papers by this authorDiane Rebourcet, Diane Rebourcet Faculty of Science, The University of Newcastle, Callaghan, NSW, AustraliaSearch for more papers by this authorLaura F. Dagley, Laura F. Dagley Walter and Eliza Hall Institute, Parkville, VIC, Australia Department of Medical Biology, University of Melbourne, Parkville, VIC, AustraliaSearch for more papers by this authorRaouda Sgaier, Raouda Sgaier Hudson Institute of Medical Research, Clayton, VIC, Australia Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, Australia Department of Urology, Pediatric Urology and Andrology, Medical Faculty, Justus-Liebig-University Giessen, UKGM GmbH, Giessen, GermanySearch for more papers by this authorGiuseppe Infusini, Giuseppe Infusini Walter and Eliza Hall Institute, Parkville, VIC, Australia Department of Medical Biology, University of Melbourne, Parkville, VIC, AustraliaSearch for more papers by this authorPeter J. O'Shaughnessy, Peter J. O'Shaughnessy Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) - UMR_S 1085, Rennes, FranceSearch for more papers by this authorFrederic Chalmel, Frederic Chalmel Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail), UMR_S 1085, University Rennes, Rennes, FranceSearch for more papers by this authorDaniela Fietz, Daniela Fietz Institute for Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University Giessen, Giessen, GermanySearch for more papers by this authorWolfgang Weidner, Wolfgang Weidner Department of Urology, Pediatric Urology and Andrology, Medical Faculty, Justus-Liebig-University Giessen, UKGM GmbH, Giessen, GermanySearch for more papers by this authorJulien M. D. Legrand, Julien M. D. Legrand Hudson Institute of Medical Research, Clayton, VIC, Australia Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, AustraliaSearch for more papers by this authorRobin M. Hobbs, Robin M. Hobbs Hudson Institute of Medical Research, Clayton, VIC, Australia Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, AustraliaSearch for more papers by this authorRobert I. McLachlan, Robert I. McLachlan Hudson Institute of Medical Research, Clayton, VIC, Australia Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, AustraliaSearch for more papers by this authorAndrew I. Webb, Andrew I. Webb Walter and Eliza Hall Institute, Parkville, VIC, Australia Department of Medical Biology, University of Melbourne, Parkville, VIC, AustraliaSearch for more papers by this authorAdrian Pilatz, Adrian Pilatz Department of Urology, Pediatric Urology and Andrology, Medical Faculty, Justus-Liebig-University Giessen, UKGM GmbH, Giessen, GermanySearch for more papers by this authorThorsten Diemer, Thorsten Diemer Department of Urology, Pediatric Urology and Andrology, Medical Faculty, Justus-Liebig-University Giessen, UKGM GmbH, Giessen, GermanySearch for more papers by this authorLee B. Smith, Lee B. Smith orcid.org/0000-0002-4103-3074 Faculty of Science, The University of Newcastle, Callaghan, NSW, Australia MRC Centre for Reproductive Health, The Queen's Medical Research Institute, University of Edinburgh, Edinburgh, UKSearch for more papers by this authorPeter G. Stanton, Peter G. Stanton orcid.org/0000-0002-6104-1565 Hudson Institute of Medical Research, Clayton, VIC, Australia Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, AustraliaSearch for more papers by this author Liza O'Donnell, Corresponding Author Liza O'Donnell Liza.ODonnell@hudson.org.au orcid.org/0000-0001-5848-6136 Hudson Institute of Medical Research, Clayton, VIC, Australia Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, Australia Faculty of Science, The University of Newcastle, Callaghan, NSW, Australia Correspondence Liza O'Donnell, Hudson Institute of Medical Research, 27-31 Wright Street, Clayton, VIC 3168, Australia. Email: Liza.ODonnell@hudson.org.auSearch for more papers by this authorDiane Rebourcet, Diane Rebourcet Faculty of Science, The University of Newcastle, Callaghan, NSW, AustraliaSearch for more papers by this authorLaura F. Dagley, Laura F. Dagley Walter and Eliza Hall Institute, Parkville, VIC, Australia Department of Medical Biology, University of Melbourne, Parkville, VIC, AustraliaSearch for more papers by this authorRaouda Sgaier, Raouda Sgaier Hudson Institute of Medical Research, Clayton, VIC, Australia Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, Australia Department of Urology, Pediatric Urology and Andrology, Medical Faculty, Justus-Liebig-University Giessen, UKGM GmbH, Giessen, GermanySearch for more papers by this authorGiuseppe Infusini, Giuseppe Infusini Walter and Eliza Hall Institute, Parkville, VIC, Australia Department of Medical Biology, University of Melbourne, Parkville, VIC, AustraliaSearch for more papers by this authorPeter J. O'Shaughnessy, Peter J. O'Shaughnessy Univ Rennes, Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail) - UMR_S 1085, Rennes, FranceSearch for more papers by this authorFrederic Chalmel, Frederic Chalmel Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail), UMR_S 1085, University Rennes, Rennes, FranceSearch for more papers by this authorDaniela Fietz, Daniela Fietz Institute for Veterinary Anatomy, Histology and Embryology, Justus-Liebig-University Giessen, Giessen, GermanySearch for more papers by this authorWolfgang Weidner, Wolfgang Weidner Department of Urology, Pediatric Urology and Andrology, Medical Faculty, Justus-Liebig-University Giessen, UKGM GmbH, Giessen, GermanySearch for more papers by this authorJulien M. D. Legrand, Julien M. D. Legrand Hudson Institute of Medical Research, Clayton, VIC, Australia Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, AustraliaSearch for more papers by this authorRobin M. Hobbs, Robin M. Hobbs Hudson Institute of Medical Research, Clayton, VIC, Australia Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, AustraliaSearch for more papers by this authorRobert I. McLachlan, Robert I. McLachlan Hudson Institute of Medical Research, Clayton, VIC, Australia Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, AustraliaSearch for more papers by this authorAndrew I. Webb, Andrew I. Webb Walter and Eliza Hall Institute, Parkville, VIC, Australia Department of Medical Biology, University of Melbourne, Parkville, VIC, AustraliaSearch for more papers by this authorAdrian Pilatz, Adrian Pilatz Department of Urology, Pediatric Urology and Andrology, Medical Faculty, Justus-Liebig-University Giessen, UKGM GmbH, Giessen, GermanySearch for more papers by this authorThorsten Diemer, Thorsten Diemer Department of Urology, Pediatric Urology and Andrology, Medical Faculty, Justus-Liebig-University Giessen, UKGM GmbH, Giessen, GermanySearch for more papers by this authorLee B. Smith, Lee B. Smith orcid.org/0000-0002-4103-3074 Faculty of Science, The University of Newcastle, Callaghan, NSW, Australia MRC Centre for Reproductive Health, The Queen's Medical Research Institute, University of Edinburgh, Edinburgh, UKSearch for more papers by this authorPeter G. Stanton, Peter G. Stanton orcid.org/0000-0002-6104-1565 Hudson Institute of Medical Research, Clayton, VIC, Australia Department of Molecular and Translational Sciences, Monash University, Clayton, VIC, AustraliaSearch for more papers by this author First published: 10 February 2021 https://doi.org/10.1096/fj.202002484RCitations: 4 Liza O'Donnell, Diane Rebourcet, and Laura F. Dagley are equal first author; Lee B. Smith and Peter G. Stanton are equal senior author. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Sperm develop from puberty in the seminiferous tubules, inside the blood-testis barrier to prevent their recognition as "non-self" by the immune system, and it is widely assumed that human sperm-specific proteins cannot access the circulatory or immune systems. Sperm-specific proteins aberrantly expressed in cancer, known as cancer-testis antigens (CTAs), are often pursued as cancer biomarkers and therapeutic targets based on the assumption they are neoantigens absent from the circulation in healthy men. Here, we identify a wide range of germ cell-derived and sperm-specific proteins, including multiple CTAs, that are selectively deposited by the Sertoli cells of the adult mouse and human seminiferous tubules into testicular interstitial fluid (TIF) that is "outside" the blood-testis barrier. From TIF, the proteins can access the circulatory- and immune systems. Disruption of spermatogenesis decreases the abundance of these proteins in mouse TIF, and a sperm-specific CTA is significantly decreased in TIF from infertile men, suggesting that exposure of certain CTAs to the immune system could depend on fertility status. The results provide a rationale for the development of blood-based tests useful in the management of male infertility and indicate CTA candidates for cancer immunotherapy and biomarker development that could show sex-specific and male-fertility-related responses. Abbreviations ACN acetonitrile CID collision-induced dissociation CTA cancer-testis antigen DTR diphtheria toxin receptor DTX diphtheria toxin FA formic acid FDR False discovery rate iBAQ intensity-based absolute quantification LDHC lactate dehydrogenase C MS mass spectrometry M-TESE microsurgical-assisted testicular sperm extraction OA obstructive azoospermia PBS phosphate-buffered saline PCA principle component analysis PSC pachytene spermatocytes PSMs peptide-spectrum matches PTMCs peritubular myoid cells rST round spermatids SCO sertoli cell-only TIF testicular interstitial fluid ZAN zonadhesin 1 INTRODUCTION Sperms form inside the seminiferous tubules of the testes at puberty, well after the establishment of the immune system in early neonatal life, and developing sperm must be protected from the immune system to prevent their recognition as foreign. Outside the seminiferous tubules, the testicular interstitium contains abundant immune cells, including macrophages, mast cells, and dendritic cells, yet it is a unique immunosuppressed microenvironment by virtue of local immunoregulatory mechanisms.1-3 Developing sperm are physically sequestered from the interstitium and resident immune cells by the blood-testis barrier.1-4 Meiotic spermatocytes and post-meiotic spermatids develop inside the blood-testis barrier, in a specialized milieu known as the adluminal compartment.4, 5 Tight junctions between the somatic Sertoli cells of the seminiferous tubules restrict the free passage of proteins into the adluminal compartment and entry of immune cells and antibodies into the tubules.4 Immune privilege in the testis is considered to be a combination of the physical sequestration of developing sperm inside the blood-testis barrier, and local immunomodulatory factors that promote an immune-suppressed environment.1, 3, 5 A widely held assumption is that proteins specifically expressed by sperm remain inside the blood-testis barrier,5, 6 protected from immune system recognition, and prevented from entering the circulation. This assumption has led to an interest in cancer-testis antigens (CTAs) as potential therapeutic targets and biomarkers of, various cancers.7-10 CTAs are proteins normally only expressed in male germ cells but aberrantly expressed in cancer.8, 9 Since sperm-specific CTAs are widely assumed to be restricted within the blood-testis barrier in healthy men, they are assumed to be neoantigens that will provoke a large immune response and thus considered excellent targets for cancer immunotherapy.7, 11-14 Because they are also assumed to be absent from the circulation in healthy men but can be aberrantly expressed in cancer, CTAs are being explored for their utility as circulating cancer biomarkers.15, 16 Although these assumptions have become accepted wisdom in the wider literature, reproductive biologists have long speculated that not all sperm-specific proteins remain inside the seminiferous tubules.6, 17-20 Vasectomy causes the leakage of sperm from the inflamed epididymis and thus should result in a massive immune response against a wide range of sperm antigens as sperm are recognized by the immune system for the first time. Yet, vasectomy is followed by the generation of an unexpectedly narrow repertoire of sperm autoantibodies, pointing to the existence of immune tolerance to at least some sperm antigens.20-22 Proof of this concept was achieved in mice, where the sperm-specific protein and CTA, lactate dehydrogenase 3 (LDH3, also known as LDHC), was shown to promote T regulatory cell (Treg)-mediated peripheral tolerance but another sperm protein, zonadhesin, was non-tolerogenic.23 These observations suggest that certain sperm proteins are not sequestered inside the seminiferous tubules by the blood-testis barrier and could encounter the immune system. However, there has been no in-depth analysis of germ cell proteins in the fluid outside of the seminiferous tubules in humans or mice. Given the interest in CTAs for immunotherapy and cancer biomarker development, it is imperative to identify whether, and which, germ cell proteins can be deposited by the seminiferous tubules into the surrounding interstitial fluid, particularly in humans. A comprehensive survey of the proteins released by human seminiferous tubules may also provide new opportunities for non-invasive monitoring of spermatogenic function. To address these issues, we completed the first in-depth characterization of the mouse and human interstitial fluid proteomes. The interstitial space between the tubules contains testicular interstitial fluid (TIF), comprised of secretions and products from the seminiferous tubules, the interstitial cells, and the circulation.24 We identify a wide range of germ cell proteins, including proteins expressed only in sperm and CTAs that are deposited by the seminiferous tubules into the TIF in mice and humans. 2 METHODS 2.1 Study approval Mice were housed and bred under standard conditions of care. Experiments were conducted with licensed permission under the UK Animal Scientific Procedures Act (1986), Home Office license number PPL 60/4200. All human procedures performed were in accordance with the ethical standards of the Institutional and/or National Research Committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. All patients were counseled preoperatively and gave written informed consent to perform testicular surgery. This study was approved by the local institutional review board (Ethik-Kommission am FB 11 "Humanmedizin," Justus-Liebig-Universität Giessen; Ref. No. 26/11). 2.2 Model of seminiferous tubule cell ablation The seminiferous epithelium was disrupted in adult mice using a model of acute (1 week) Sertoli cell ablation.25, 26 This model utilizes transgenic mice expressing diphtheria toxin receptor (DTR) specifically in Sertoli cells driven by Amh-cre.26 After one week of diphtheria toxin (DTX) administration, very few Sertoli cells are present in the seminiferous tubules (Supplemental Figure S1). Although spermatogonia, spermatocytes, and elongated spermatids are visible, many are clearly undergoing apoptosis and the mRNA expression of germ cell markers (Pouf5a1 for spermatogonia, Spo11 for spermatocytes, and Tp1, also known as Tnp1, for spermatids) are markedly reduced (Supplemental Figure S1).25 At this time, peritubular myoid cells (PTMCs) remain around the tubules but show reduced expression of the PTMC functional marker calponin.25 Leydig cell number is unaffected after one week; however, some of these cells eventually undergo apoptosis.25 There were minor but significant changes in the expression of inflammatory markers during one week of DTX treatment, but macrophage infiltration was relatively minor (Supplemental Figure S2).25, 26 2.3 Isolation of mouse TIF Adult male animals (>70 days) Amh-Cre+/+;iDTR+/+ were used for this study.25 Mice were injected with a single dose of 100ng DTX (DTX group, n = 11) or vehicle (control group, n = 12)25 and were culled 1 week later using CO2 asphyxiation and cervical dislocation. Testes were collected, weighed and TIF was collected as described27: briefly, samples were cleaned in cold PBS containing protease inhibitors (cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail, Roche, UK) and dried on filter tissue. A small incision through the tunica albuginea was made prior to centrifuging the tissues (1000 g, 1 minute, 4°C). Each testis was then suspended using sutures, decapsulated, and dipped quickly in three sequential 1.5 mL Eppendorf tubes containing PBS and protease inhibitors. TIF was collected by centrifugation (10 000 g, 15 minutes, 4°C) of the pooled sequential collection tubes and the supernatant was stored at −80°C. 2.4 Isolation of human TIF For proteomic analyses, TIF was taken from three men diagnosed as azoospermic due to distal reproductive tract obstruction (obstructive azoospermia, OA); however, all data suggested that their testicular function was normal (Supplemental Dataset 9). Normal histology of their testis was determined by morphological evaluation. Specimens from each testicular incision site (Supplemental Dataset 9) were immediately fixed in Bouin's solution and processed according to routine protocols. The semi-quantitative score count evaluation of spermatogenesis was performed according to Bergmann and Kliesch.28 For each individual retrieval site, the number of tubules containing elongated spermatids is divided by the total number of tubules examined × 10; hence, score values range from 0 to 10. This histologic diagnosis procedure allows patients to be classified into four groups: normal spermatogenesis (score 8-10), hypospermatogenesis,1-7 predominant tubular atrophy (0.1-0.9), and Sertoli cell-only tubules (SCO) (0).28 TIF was collected by experienced microsurgeons (TD, WW, AP) from patients undergoing M-TESE (microsurgical-assisted testicular sperm extraction) for sperm retrieval, as described.29 This procedure uses a midline incision and a microscope with ×15 magnification to incise the tunica albuginea and connective tissue to access the seminiferous tubules. Prior to the dissection of tubules, TIF was recovered adjacent to the tubules by applying gentle pressure on the tissue and collected using a micro-syringe (1 mL) fitted with a plastic tip. An average of 200-500 µL TIF collected per testis was immediately snap-frozen in Eppendorf tubes over dry ice in the operating theater and subsequently stored at −80°. For Western blotting, TIF was taken from n = 8 men with OA defined as above and from eight men with presumed Sertoli cell-only phenotype.28 Clinical data from the OA and SCO groups, including hormones and testis volumes, are shown in Supplemental Dataset 10A. SCO men had significantly higher levels of FSH than OA men. Spermatogenesis score28 for the biopsies taken from each patient is shown in Supplemental Dataset 10B; all SCO patients had biopsy scores of zero and were unable to have sperm retrieved from their testes during surgery. 2.5 Proteomics of mouse and human TIF 2.5.1 Trypsin digestion Protein concentrations were determined by the BCA method (Pierce, Rockford). For mouse TIF, equal amounts of mouse TIF lysate (60 μg) from DTX (n = 11) and control (vehicle-treated) mice (n = 12) were prepared for mass spectrometry analysis using the FASP protein digestion method30 with the following modifications. Protein material was reduced with Tris-(2-carboxyethyl)-phosphine (TCEP, 10 mM final concentration). Eluates were digested with sequence-grade modified trypsin Gold (Promega, V5280) (1 μg) in 50 mM ammonium bicarbonate (NH4HCO3) and incubated overnight at 37°C. Peptides were eluted with 50 mM NH4HCO3 in two 40 μL sequential washes and acidified in 1% formic acid (final concentration). For the human TIF, 200 μg of protein from three individual OA patients was prepared for mass spectrometry analysis using the USP3 protocol.31 We used a 1:1 combination mix of the two types of commercially available carboxylate beads (Sera-Mag Speed beads, #65152105050250, #45152105050250, Thermo Fisher Scientific). Beads were prepared freshly each time by rinsing with water three times prior to use and stored at 4°C at a stock concentration of 20 μg/μL. Samples were transferred to a 2 mL LoBind deepwell plate (Eppendorf, Hamburg, Germany) and reduced with 2 M dithiothreitol (DTT, 50 mM final conc.) for 1 hour at 37°C. Samples were then alkylated with 1M iodoacetamide (100 mM final conc.) for 30 mins in the dark at room temperature (RT). Samples were quenched with 2M DTT (250 mM final conc.) and 4 μL of the concentrated bead stock carboxylate beads (20 μg/μL) was added to each sample followed by the addition of acetonitrile (ACN) to a final concentration of 70% (v/v). Mixtures were left to incubate upright at RT for 20 mins to allow proteins to precipitate onto the beads. The beads were placed on a magnetic rack and washed twice with 70% ethanol and once with ACN (500 μL washes). ACN was completely evaporated from the plate using a CentriVap (Labconco, Kansas City, MO, USA) prior to the addition of 40 μL of digestion buffer (10% 2-2-2-trifluoroethanol /100 mM NH4HCO3) containing 4 μg Trypsin-gold (Promega, V5280) and 4 μg Lys-C (Wako). The plate was briefly sonicated in a water bath to disperse the beads, and the plate was transferred to a ThermoMixer C instrument (Eppendorf) for enzymatic digestion at 37°C for 1 hour (1200 rpm). The supernatant comprising of peptides was then collected from the beads using a magnetic rack (Ambion, Thermo Fisher Scientific) and an additional elution (50 μL of 2% dimethyl sulfoxide, Sigma) was performed on the beads. The eluates were pooled together then equally split across pre-equilibrated C18 stage tips for sample clean-up. Briefly, six plugs of C18 resin (3M Empore, 66883-U) were prepared in 200 μL unfiltered tips, pre-wetted with 100 μL of methanol followed by sequential washes with 100 μL of 80% acetonitrile (ACN)/5% formic acid (FA), 50% ACN/5% FA and 5% FA. The pooled peptides were then added to the spin tip and the eluate collected into a fresh lo-bind Eppendorf tube. Bound peptides were washed twice with 5% FA. Elutions (50 μL) were performed sequentially with 50% ACN/5% FA followed by 80% ACN/5% FA and collected into fresh Eppendorf tubes. All spins were performed on a benchtop centrifuge at 500 g (1000-2000 rpm) speeds. The eluates were lyophilized to dryness in MS vials (CentriVap) prior to reconstituting in 40 μL of 5 mM ammonium formate buffer, pH 10 ready for offline peptide fractionation on an HPLC. 2.5.2 Offline HPLC fractionation Tryptic peptides from each of the three human TIF samples were subjected to high pH reverse-phase analysis on an Agilent 1100 Series HPLC system equipped with a variable wavelength detector (280 nm). Fractionation was performed on XBridge Shield C18 column (10 × 100 mm, 3.5 μm bead size, Waters). Peptides were separated by their hydrophobicity at a high pH at a flow rate of 0.1 mL/min using a gradient of mobile phase A (5 mM ammonium formate, pH 10) and a mobile phase B (100% ACN), from 3% to 35% over 60 mins. Fractions were collected every minute across the gradient length and concatenated into 24 fractions. Eluted peptides were dried in a SpeedVac centrifuge and reconstituted in MS loading buffer (2% ACN/0.1% FA) prior to MS analysis. 2.5.3 Mass spectrometry and data analysis Peptides were separated by reverse-phase chromatography on a 1.6 μm C18 fused silica column (ID 75 μm, OD 360 μm × 25 cm length) packed into an emitter tip (IonOpticks, Australia), using a nano-flow HPLC (M-class, Waters). The HPLC was coupled with an Impact II UHR-QqTOF mass spectrometer (Bruker, Bremen, Germany) using a CaptiveSpray source and nanoBooster at 0.20 Bar using acetonitrile. Peptides were loaded directly onto the column at a constant flow rate of 400 nL/min with buffer A (99.9% Milli-Q water, 0.1% formic acid) and eluted with a 90 minutes linear gradient from 2% to 34% buffer B (99.9% ACN, 0.1% FA). Mass spectra were acquired in a data-dependent manner including an automatic switch between MS and MS/MS scans using a 1.5-second duty cycle and 4 Hz MS1 spectra rate followed by MS/MS scans at 8-20 Hz dependent on precursor intensity for the remainder of the cycle. MS spectra were acquired between a mass range of 200-2000 m/z. Peptide fragmentation was performed using collision-induced dissociation (CID). Raw files consisting of high-resolution MS/MS spectra were processed with MaxQuant (version 1.5.8.3) for feature detection and protein identification using the Andromeda search engine.32 Extracted peak lists were searched against the UniProtKB/Swiss-Prot Mus musculus or Homo sapiens databases (October 2016) and a separate reverse decoy database to empirically assess the false discovery rate (FDR) using strict trypsin specificity allowing up to two missed cleavages. The minimum required peptide length was set to seven amino acids. In the main search, precursor mass tolerance was 0.006 Da and fragment mass tolerance was 40 ppm. The search included variable modifications of oxidation (methionine), amino-terminal acetylation, the addition of pyroglutamate (at N-termini of glutamate and glutamine), and a fixed modification of carbamidomethyl (cysteine). The "match between runs" option in MaxQuant was used to transfer identifications made between runs on the basis of matching precursors with high mass accuracy.33 Protein abundance was calculated using the intensity-based absolute quantification (iBAQ) metric.34 Peptide-spectrum matches (PSMs) and protein identifications were filtered using a target-decoy approach at an FDR of 1%. Protein identification was based on a minimum of one unique peptide. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository35 with the dataset identifier PXD014333 and the following Username: reviewer23562@ebi.ac.uk and Password: A7eE5hnQ. 2.5.4 Label-free quantitative proteomics pipeline Statistically relevant protein expression changes between the DTX and control mouse TIF samples were identified using the default workflow in the R package Proteus (version 0.2.10) where quantitation was performed at the peptide level, with some minor differences.36 Only unique and razor peptides were considered for quantification with intensity values present in at least two out of three replicates per group. Missing values were replaced by values drawn from a normal distribution of 1.8 standard deviations and a width of 0.3 for each sample (Perseus-type). Peptides were assigned to their leading razor protein and peptide intensities were aggregated to protein intensities using the aggregateHifly function based on the high-flyer method. Peptides were assigned to their leading razor protein and peptide intensities were aggregated to protein intensities using the aggregateHifly function based on the high-flyer method.37 Protein intensities were normalized according to the normalize Quantiles function from the limma Bioconductor package.38 Differential protein expression was performed using the limmaDE function which uses the empirical Bayes moderated t tests using the limma package. Protein intensities were log2 transformed. Proteus corrects for multiple testing using the Benjamini-Hochberg FDR procedure. 2.6 Analysis of mouse TIF protein localization The gene symbol of each TIF protein was interrogated in an RNAseq dataset from normal mice and from those with adult germ cell ablation using the germ cell-specific toxicant busulfan.39 Proteins were deemed to be predominantly expressed in germ cells when their mRNA levels were decreased in whole testes by >70% after busulfan treatment. The genes corresponding to TIF proteins were also interrogated in a microarray dataset of isolated seminiferous tubule cells (Sertoli cells, spermatogonia, pachytene spermatocytes, or round spermatids), whole testes, and 18 other normal mouse tissues.40 Proteins were deemed likely to be contributed to TIF by adluminal germ cells when: (a) the protein was significantly reduced (P < .