Molecular Human Reproduction, pp. 1–14, 2019 doi:10.1093/molehr/gaz003

ORIGINAL ARTICLE Combined proteomic and miRNome

analyses of mouse testis exposed to an Downloaded from https://academic.oup.com/molehr/advance-article-abstract/doi/10.1093/molehr/gaz003/5304489 by guest on 26 February 2019 endocrine disruptors chemicals mixture reveals altered toxicological pathways involved in male infertility

Julio Buñay 1, Eduardo Larriba3, Daniel Patiño-Garcia1, † Paulina Urriola-Muñoz1,2, Ricardo D. Moreno1,*, , and Jesús del † Mazo3,*, 1Department of Physiology, Faculty of Biological Sciences, Pontificia Universidad Catolica de Chile (PUC), Santiago 8331150, Chile 2Chemistry Institute, Pontificia Universidad Católica de Valparaíso, Valparaíso 2340000, Chile 3Department of Cellular and Molecular Biology, Centro de Investigaciones Biologicas (CIB-CSIC), 28040 Madrid, Spain

*Correspondence address. Department of Physiology, Faculty of Biological Science, Pontificia Universidad Católica de Chile, Alameda 340, 8331150 Santiago, Chile. Tel: +562-2354-2885; E-mail: [email protected] (R.D.M.)/Department of Cellular and Molecular Biology, CIB (CSIC), Ramiro de Maeztu, 9, 28040 Madrid, Spain. Tel: +34-918373112x4324; E-mail: [email protected] (J.d.M.) Submitted on May 6, 2018; resubmitted on December 23, 2018; editorial decision on January 23, 2019; accepted on January 24, 2019

The increase in male idiopathic infertility has been associated with daily exposure to endocrine disruptors chemicals (EDCs). Nevertheless, the mechanisms of action in relation to dysregulating and regulatory microRNAs are unknown. We combined proteomic and miRNome analyses of mouse testis chronically exposed to low doses of a define mixture of EDCs [phthalates: bis(2-ethylhexyl), dibutyl and benzyl-butyl; 4-nonylphenol and 4-tert-octylphenol], administered in the drinking water from conception until adulthood (post-natal Day 60/ 75) and compared them with no-exposed control mice. We analysed fertility parameters and global changes in the patterns of mice testis proteome by 2D electrophoresis/mass spectrometry, along with bioinformatic analyses of dysregulated microRNAs, and their association with published data in human infertile patients. We detected a decrease in the potential fertility of exposed mice associated with changes in the expression of 18 proteins (10 up-regulated, 8 down-regulated). Functional analysis showed that 89% were involved in cell death. Furthermore, we found a group of 23 microRNAs/isomiRs (down-regulated) correlated with six of the up-regulated target proteins (DIABLO, PGAM1, RTRAF, EIF4E, IVD and CNDP2). Regarding this, PGAM1 up-regulation was validated by Western blot and mainly detected in Sertoli cells. Some of these microRNA/ dysregulations were reported in human testis with spermatogenic failure. Overall, a chronic exposure to EDCs mixture in human males could potentially lead to spermatogenic failure through changes in microRNA expres- sion, which could post-transcriptionally dysregulate mRNA targets that encode proteins participating in cell death in testicular cells. Finally, these microRNA/protein dysregulations need to be validated with other EDCs mixtures and concentrations.

Key words: endocrine disruptors / PGAM1 / miRNAs / isomiRs / proteome / spermatogenic failure / infertility

Introduction been documented by epidemiological studies, which report correla- tions between idiopathic male infertility (specially deterioration in Endocrine disruptors chemicals (EDCs) such as phthalates and alkyl- sperm quality and quantity) and high levels of phthalates such as di-(2- phenols are environmentally widespread man-made chemicals to ethylhexyl)phthalate (DEHP), dibutyl phthalate (DBP) and benzyl butyl which humans and wildlife are chronically exposed from foetal life to phthalate (BBP or BzBP); and alkylphenols such as nonylphenol (NP) adulthood (Bergman et al., 2012). At a toxicological level, the effects and octylphenol (OP) detected in both urine and semen (Pant et al., on male fertility are a hallmark of the exposure to EDCs. This has 2008; Chen et al., 2013). Furthermore, some reports from human

† Equal contribution authorship.

© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected] 2 Buñay et al. foetal testis and experimental murine models of single exposure to approved all the experimental protocols in this work. C57BL/6J mice these compounds demonstrate that they can act as trigger factors of were bred at the CSIC or PUC animal facilities under specific, pathogen- death in testicular cells (de Jager et al., 1999; Yoshida et al., 2001; free (SPF), temperature- and humidity-controlled conditions in 12-h light/ Boekelheide et al., 2009; Lambrot et al., 2009). However, the molecu- dark cycles with ad libitum access to food and water. lar mechanisms involved have not been fully disclosed. Changes in the global proteomic profile have been detected in eja- EDCs mixture exposure

culated spermatozoa of idiopathic infertile patients, and are mainly To emulate chronic human exposure to an environmental EDCs mixture, Downloaded from https://academic.oup.com/molehr/advance-article-abstract/doi/10.1093/molehr/gaz003/5304489 by guest on 26 February 2019 associated with the folding and degradation of proteins, cytoskeleton we designed a murine model of chronic exposure to a defined mixture of and energy metabolism (Jodar et al., 2017; Bracke et al., 2018; Cao 0.3 mg/kg-bw/day of each of the following phthalates: bis(2-ethylhexyl) et al., 2018). In relation to the changes in spermatogenesis, recently a phthalate (DEHP), dibutyl phthalate (DBP), benzyl butyl phthalate (BBP) quantitative proteomic analyses in testis of infertile patients revealed (Sigma-Aldrich, USA) and 0.05 mg/kg-bw/day of each of the following alkylphenols: 4-nonylphenol (NP), 4-tert-octylphenol (OP) (Sigma-Aldrich, that over 520 proteins are dysregulated (Alikhani et al., 2017). Overall USA). The total concentration of the EDCs mixture was 1 mg/kg-bw/day. this data lead to the conclusion that a complex dysregulation of protein Phthalates were diluted in DMSO (dimethyl sulfoxide) (Sigma-Aldrich, expression occurs in testis of patients with idiopathic infertility. USA) and alkylphenols were diluted in ethanol. For the control group, we However, the causes that originate it remain unsolved. used a mixture of DMSO and ethanol (vehicles) with equivalent intake of Inside the regulatory proteins expression mechanisms, the regula- 0.25 and 0.06 g/kg-bw/day, respectively. In this toxicological approach tion of mRNA expression mediated by microRNAs (miRNAs) and (Buñay et al., 2017, 2018; Patiño-García et al., 2018), the dose of each theirs sequence variants or isomiRs have taken relevance. The EDCs was chosen based on the non-occupational exposure in human, miRNAs/isomiRs are small non-coding endogenous RNA, which are which is estimated to range between 0.3 and 143 mg/kg-bw/day (Jonsson, evolutionarily well-conserved between species and implicated in the 2006; National Toxicology Program, 2003a, 2003b, 2006; Ademollo et al., negative post-transcriptional regulatory systems in a sequence-specific 2008; Hines et al., 2011). We ensured that the chosen doses were at least manner (Guo et al., 2010; Neilsen et al., 2012). In fact, several dysre- ~1000-fold lower than the LOAEL values for reproductive traits effects in experimental male animals while still being environmentally relevant doses gulated miRNAs in testes of infertile patients have been reported by (Chapin et al., 1999; Nagao et al., 2001; Rider et al., 2010). multiple studies (Abu-Halima et al., 2014; Munoz et al., 2015; Noveski The EDCs mixture or control (vehicles) were dissolved in the drinking ‘ et al., 2016), allowing the hypothesis that an aberrant expression of water of C57BL/6 J mice in independent bottles covered with foil. The … miRNAs could promote certain alterations in spermatogenesis and final dose was calculated according to the volume of water ingested by the be a cause of infertility in males’ (Smorag et al., 2012). Additionally, mice and the (bw) recorded in a pilot study and in agreement with data in integrative reports of differentially expressed and miRNAs in the literature referring to these parameters. Water intake was controlled testis of infertile patients have also emerged, which help in exploring each day. The global water intake was not affected by the exposure to miRNA–mRNA interactions and uncovering the molecular regulatory EDCs or control. Then, the EDCs mixture or control was administered network and therapeutic targets in male infertility (Zhuang et al., 2015; with ad libitum access, to pregnant mice randomly selected at post-coital Li et al., 2016). Day 0.5 (conception) and throughout gestation and lactation. At weaning, Our previous reports showed for first time that exposure to EDCs only male offspring were selected and maintained at a maximum of four individuals per cage. The administration of the EDCs mixture or control (phthalates and alkylphenols) mixture alters specific miRNAs/isomiRs was continued in these mice until adulthood (endpoint: post-natal Day 60 involved in the control of hormonal status in mice testis (Buñay et al., and post-natal Day 75 for fertility assays) (Buñay et al., 2017, 2018). We 2017). However, the consequent effects on protein networks have choose this model in order to mimic the human where the problems of not been fully addressed. Therefore, the aim of this work was to evalu- idiopathic fertility appear from the beginning of their reproductive life. ate a functional association between dysregulated proteins and Thus, taking account that the fecundity of male mice declines with age, we miRNAs in mouse testis with impaired fertility due to the chronic consider that the choice of Day 75 of age for male mice allows us to make exposure to low-doses of mixtures of phthalates and alkylphenols. sure that the male mice have good reproductive performance. The results showed that some toxicological proteins/miRNAs path- All male litter of one pregnant mouse exposed was considered as a stat- ways involved in cell death were altered in mouse testis exposed to an istical unit (n) and we used a minimum of three distinct male litters per EDCs mixture and have potential relationship with idiopathic human group. Within a single litter, the male offspring exposed to EDCs mixture male infertility. and/or control were considered biological replicates and used in the differ- ent test.

Materials and Methods Protein extraction At the specified endpoints, animals were sacrificed, and testes were Animals and ethical statement removed, decapsulated and mechanically homogenized in radioimmuno- All procedures relating to the care and handling of animals were carried precipitation assay buffer (RIPA), along with a protease inhibitor cocktail out in accordance with the regulations of the Consejo Superior de with 2 mM AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochlor- Investigaciones Cientificas (CSIC) and the Pontifical Catholic University of ide), 0.3 μM aprotinin, 130 μM bestatin hydrochloride, 14 μM E-64, 1 mM Chile (PUC), following the European Commission (EC) guidelines (direct- EDTA and 1 μM leupeptin hemisulfate (Sigma-Aldrich, USA). Proteins ive 86/609/EEC), and the guides for the Care and Use of Agricultural were purified by centrifugation at 12 000 g at 4°C for 10 min and subse- Animals in Agricultural Research and Teaching by the National Research quently quantified by the method described by Bradford (1976). Council of Chile, respectively. The General Direction of Environment of For 2D protein electrophoresis, 150 μg of total protein was isolated CAM in Spain (Ref. PROEX 054/15) and the National Fund of Science and from testis of adult mice. Sample pooling of three biological replicates from Technology (FONDECYT) (No. 1150 532) in Chile reviewed and the control groups and the exposed groups were used, each one Proteomics/miRNAs in testis exposed to EDCs 3 contributing equally with 50 μg of total protein. Protein purification is covering the m/z 700–4000 region. The typical error observed in mass described above. These protein extracts were precipitated using a metha- accuracy for calibration was usually below 50 ppm. MALDI–MS and MS/ nol/chloroform protocol (Wessel and Flügge, 1984). Briefly, cold metha- MS data were combined through the BioTools 3.0 program (Bruker- nol and chloroform were added to the sample tubes previously incubated Daltonics, USA) to interrogate the NCBI non-redundant protein database at 4°C and then centrifuged at 13 000 g at 4°C for 15 min. Protein- SwissProt 2014_03 (542 782 sequences; 193 019 802 residues) using interfaces were washed and centrifuged twice with cold methanol whereas MASCOT software 2.3 (Matrix Science, UK). Relevant search parameters protein pellets were dried and resuspended in 2× buffer (7 M urea, 2 M were set as follows: enzyme, trypsin; fixed modifications, carbamidomethyl thiourea, 4% [w/v] CHAPS and 0.0003% [w/v] bromophenol blue). (C); oxidation (M); one missed cleavage allowed; peptide tolerance, Downloaded from https://academic.oup.com/molehr/advance-article-abstract/doi/10.1093/molehr/gaz003/5304489 by guest on 26 February 2019 Protein concentration quantification was made with the RC-DC Protein 50 ppm; MS/MS tolerance, 0.5 Da. Assay (Bio-Rad, USA). Peptide mass fingerprinting and fragmentation by MS–MALDI-TOF was carried out in the Proteomics and Genomics Facility (CIB-CSIC), a mem- ber of ProteoRed-ISCIII network. 2D electrophoresis, image acquisition and identification of the peptide footprint Western blotting For 2D electrophoresis, aliquots of 150 μg of protein from both control μ and exposed groups were diluted in a total volume of 350 μl with 2× buffer Samples of 20 g of protein homogenizing were retrieved from the testes = containing 18.2 M DTT and 0.5% of IPG buffer solution of ampholytes (pH of the control groups and the exposed to EDCs groups (n 3) and sepa- 3–10) (Bio-Rad, USA) as final concentrations. The first dimension was run rated by electrophoresis on a 12% polyacrylamide gel (sodium dodecyl sul- – on IPG strips (pH 3–10 NL, 17 cm) (Bio-Rad, USA) in a Protean IEF Cell phate polyacrylamide gel electrophoresis) under denaturing and reducing system (Bio-Rad, USA) at 20°C. Active rehydration was performed at 50 V conditions and then transferred to a nitrocellulose membrane (Thermo fi for at least 16 h. The IEF steps program was: 300 V for 45 min, linear ramp; Scienti c, USA) at 350 mA for 2 h. Next, membranes were blocked with a 3500 V for 22 h 45 min, rapid ramp; 5000 V for 30 min, rapid ramp, and solution of 3% (w/v) bovine serum albumin 0.1% (v/v) Tween in Tris- 100 V, rapid ramp, until total voltage time reached 80 000 V h; maximum buffered saline, pH 7.4 and incubated overnight with the respective pri- μ current limits: 99 μA/strip. Each gel strip was equilibrated in 5 ml of equili- mary antibodies for PGAM1 (0.4 ng/ l) (Abbexa, UK) and ß-ACTIN μ μ bration buffer (50 mM Tris:HCl pH 8.8, 2% [w/v] SDS; 6 M urea; 30% [v/ (0.3 g/ l) (Sigma-Aldrich, USA) as a loading control. Finally, the second v] glycerol) containing 52 mM DTT for 15 min and then in 5 ml of equilibra- incubation took place with their respective secondary antibodies conju- tion buffer containing 130 mM iodoacetamide for 15 min. gated with horseradish peroxidase (KPL, Gaithersburg, UK) diluted 1:5000 The second dimension was run in 1.5 mm-thick, 18 × 20 cm2 SDS- in blocking solution for 1 h at room temperature. Peroxidase activity was PAGE gels (12% acrylamide). Gels were cast according to the manufac- detected by enhanced chemiluminescence (Pierce Biotechnology, tures protocol (Bio-Rad, USA), prepared the day before, and kept at 4°C Rockford, IL). before use. Second dimension gels were run at 5 watts/gel for 30 min and then at 17 watts/gel until the BPB front reached the bottom edge, using a Immunohistochemistry cooled Protean II xi Cell (Bio-Rad, USA) and 10 μl of Precision Plus Protein PGAM1 was detected in paraffin-embedded cross-sections of testes (n = Unstained Standards solution (Bio-Rad, USA) were used as protein mar- 3) fixed in Bouin’s solution for each group and treated with sodium citrate kers. Finally, gels were stained with SYPRO Ruby protein gel stain (Bio- 0.01 M, pH 6, in heat until it boiled and then kept for 10 min to expose the Rad, USA) according to the manufacturer instructions. EXQuest Spot antigens. After, the samples were first treated with 3% H O for 10 min. Cutter (Bio-Rad, USA) was used to image the gels at different excitation/ 2 2 Then, to prevent unspecific binding, a standard protein block system (Ultra emission times and to pick the selected spots, which were then subjected V block, Thermo Scientific, USA) was applied for 10 min. Primary anti- to manual tryptic digestion. For digestion, gel pieces were washed first bodies were applied at a concentration of (4 ng/μl); samples were incu- with 50 mM ammonium bicarbonate (Sigma-Aldrich, USA) and then with bated overnight at 4°C in a humidified chamber after being washed three acetonitrile (ACN) (Scharlau, Spain). Trypsin (Promega, USA) at final con- times for 5 min in a Tris–HCl buffer, pH 7.6, with 0.3 M NaCl and 0.1% centration of 12.5 ng/μl in 50 mM ammonium bicarbonate solution was Tween-20. Biotinylated secondary antibody, streptavidin–biotinylated– added to the gel pieces for 8 h at 37°C. Finally, 100% ACN containing peroxidase complex, amplification reagent (biotinyl tyramide) and 0.5% trifluoroacetic acid (TFA) (Sigma-Aldrich, USA) was added for pep- peroxidase-conjugated streptavidin were applied step-by-step for 10 min tide extraction. Tryptic eluted peptides were dried by speed-vacuum cen- each (Thermo Scientific, USA). Afterwards, slides were washed three trifugation and resuspended in 6 μl of 30% ACN- 0.1% TFA. Then 1 μlof times for 5 min in a Tris–HCl buffer, pH 7.6, with 0.3 M NaCl and 0.1% each peptide mixture was deposited onto an 800 μm AnchorChip Tween-20. Finally, DAB (3,3-diaminobenzidine tetrahydrochloride) Plus (Bruker-Daltonics, USA) and dried at room temperature, and 1 μlof Substrate and DAB Plus Chromogen (Thermo Scientific, USA) were matrix solution (3 mg/ml α-cyano-4-hydroxycinnamic acid) in 33% ACN applied for 1 min and washed in distilled water. Samples were stained with 0.1% TFA was then deposited onto the digest and allowed to dry at room hematoxylin and observed under a phase contrast microscope (Optiphot- temperature. 2, Nikon, Tokyo, Japan) and photographed with a digital camera (CoolPix 4500, Nikon, Tokyo, Japan). Mass spectrometry analysis Samples were analysed with an Autoflex III TOF/TOF mass spectrometer Protein functional analyses, expression of fi (Bruker-Daltonics, USA). Typically, 1000 scans for peptide mass nger- miRNA/isomiRs and identification of printing (PMF) and 2000 scans for MS/MS were collected. Automated ana- – – lysis of mass data was performed using FlexAnalysis software (Bruker- miRNA mRNA protein targets Daltonics, USA). Internal calibration of MALDI-TOF mass spectra was per- Protein ontology analyses were performed firstly at the Mouse Genome formed using two trypsin autolysis ions with m/z 842.510 and m/z Informatics (MGI) and Uniprot databases. Protein–protein interactions 2211.105; as for MALDI–MS/MS, calibrations were performed with a frag- were analysed by STRING (https://string-db.org/), then functional ana- ment ion spectrum obtained from the proton adducts of a peptide mixture lysis of GO domains was performed using a Cytoscape plugging ClueGO 4 Buñay et al.

(Bindea et al., 2009) by a hypergeometric test with a P-value threshold Results ≤0.05. Small-RNAseq data of testis exposedtoEDCsmixtureorcontrolwere Chronic exposure to a EDCs mixture and obtained from NCBI Expression Omnibus (GEO) from our previous work: GSE8469 (Buñay et al.,2017). Small-RNAseq data were processed as fertility in male mice described. Briefly, MicroRNA identification and quantification were performed In previous works, we showed that exposure to a single EDC or mix- by aligned trimmed reads to the mouse genome (mm10) using Bowtie aligner ture of EDCs induces an increase in the body weight, a decrease in tes- Downloaded from https://academic.oup.com/molehr/advance-article-abstract/doi/10.1093/molehr/gaz003/5304489 by guest on 26 February 2019 (Langmead et al.,2009) and then using HTSeq script (Anders et al.,2015)with tis relative weight, testis lesions and hormonal status changes (Buñay fi GFF le from miRBase v21. Further, isomiRage software were used for isomiRs et al., 2017, 2018). In the present work, while assessing the fertility of detection and quantification (Muller et al.,2014). We carried out data normal- males exposed to a specific EDCs mixture, we found that there were ization and differential expression analysis using the DeSeq tool of the R/ no significant differences in several reproductive parameters such as Bioconductor software package (Anders et al.,2013). Correlations between miRNAs and proteins were found using the gestational rate, fertility index and potential fertility, when compared miRWalk database, combining the searches of three or more databases, to control groups (Supplementary Table SI). Interestingly, in the along with the validated mRNA targets reporter in miRWalk and a valid- exposed groups, not all males seem to be equally affected by the treat- ated miRNA:mRNA database, DIANA-TARbase v7.0 (Vlachos et al., ment. We identified four males from different litters which, once ana- 2015). The interaction map was performed using a Cytoscape. lysed by ANOVA, presented a decrease in potential fertility. Furthermore, when mating resulted in pregnancy, the number of reab- Fertility assay sorptions and the rates of pre-implantation loss and foetal mortality Gestational rate, fertility index and potential fertility were calculated were increased (Fig. 1 and Supplementary Table SI). according to Garcia et al. (2012). Representative animals of each ‘n’ per group were selected randomly and treated (control and EDCs mixture) until post-natal Day 75 to ensure their potential fertility. After this period, Changes in the proteome profile of mouse males were placed in individual cages and cohabited with two unexposed testis exposed to EDCs mixture adult females for 8 days (to ensure at least two complete female estrous First, to detect changes of testicularproteinslevelsinmiceexposedto cycles). Vaginal plugs were checked every day early in the morning (08:- 00–09:00 h). All females with vaginal plug were set apart and placed in indi- EDCs, we performed a global approach and evaluated the mouse testis vidual cages. An effective pregnancy was confirmed by the increase of the proteome using 2D electrophoresis comparing exposed mice with con- body weight. trols. We analysed a total of 246 spots/proteins matched by PDQuest Between the Days 18 and 20 post-detection of the vaginal plug, a group softwareandnormalizedbyLOESS.Basedonthedifferentialspotinten- of females were sacrificed to quantify the number of implantations and sities observed, only those proteins that change more than 1.2-fold in reabsorptions in each uterine horn and the foetal mortality rate (number exposed versus control groups were taken into account. This approach of reabsorptions/number of implantations × 100). Then, ovaries were allowed us to select 21 spots of unequal signals, plus one spot (8102) ran- fi extracted, xed in Bouin solution and processed for histological analysis. domly selected among those of equal relative abundance in all gels – Serial sections of each ovary were stained with hematoxylin eosin, and the (Supplementary Fig. S1). These 22 spots/proteins were excised from the number of corpora lutea per ovary was quantified. Another group of preg- gels for further identification of peptide mass fingerprint by MS–MALDI- nant females was maintained until the delivery. Between the post-natal TOF. Three spots (904, 1302 and 9104) could not be identified mainly due Day 1 or 3, the number of newborns was quantified. to low signals. But this experimental approach allowed us to correctly iden- fi fi Statistical analysis tify 19 spots/proteins. Of these identi ed proteins, 10 were classi ed as up-regulated and 8 as down-regulated in testis exposed to EDCs mixture For the selection and quantification of spots of interest in 2D gels, (Fig. 2AandB,TableI); the spot/protein named as 8102 was identified as PDQuest™ V8.0 (Bio-Rad) was used. First, a reference gel was created peptidyl-prolyl cis–trans isomerase A (PPIA) and no significant changes from images of the analysed gels. Then, each one of these gels were aligned with the reference gel. Spot quantification in relation to the protein levels were detected (Fig. 2C, Table I). It is worthy to note that in our previous was calculated considering the intensities of all pixels within the defined work, with the same exposure model, we did not detect changes in the boundary previously normalized. Finally, the quantitative data of each spot levels of Ppia mRNA (Buñay et al.,2017), and therefore used this transcript were normalized using a non-parametric linear regression analysis (LOESS) as an endogenous reference gene (Radonić et al.,2004). A second normal- to compare each expression level. This approach combined with MS, ization using PPIA found that the up-regulated/down-regulated proteins enabled us to identify the PPIA protein levels with no significant changes previously identified in the EDCs group had a minimum of 1.2-fold change between the two groups (control and exposed to EDCs). As others stud- (Supplementary Figure 2). Then we decided to validate our 2D electro- ies suggest, PPIA is suitable to be used as an internal control (Kim et al., phoresis/MS results by using western blot and immunohistochemistry. To 2014). Consequently, PPIA was used as an endogenous reference protein this end we chose PGAM1 as it was identified among those proteins up- (housekeeping) in order to perform a second normalization/comparison regulated by 2D electrophoresis/MS. Through Western blot, we found a of the proteins identified by MS. significant increase in the protein levels of PGAM1 in testes of mice In mass spectrometry (MS) analysis, mascot total scores >75 were con- sidered significant (P < 0.05). exposed to EDCs mixture. This result was similar to the one observed with Reproductive outcome in male mice exposed to EDCs mixture was ana- 2D electrophoresis/MS. In addition, by immunohistochemistry, PGAM1 lysed by Chi-square test, umpaired t-test and one-way ANOVA along with localized preferably in Sertoli and interstitial cells both in control and EDCs Dunnett’s post hoc test, using GraphPad Prism version 5.0. (GraphPad treated mouse testes. No obvious change in localization was detected Software, USA). between these populations (Fig. 3B). Proteomics/miRNAs in testis exposed to EDCs 5 Downloaded from https://academic.oup.com/molehr/advance-article-abstract/doi/10.1093/molehr/gaz003/5304489 by guest on 26 February 2019

Figure 1 Populations of mice exposed to EDCs mixture have compromised fertility. White diamonds and gray circles indicate male population of control mice (n = 9) and mice exposed to EDCs mixture, (n = 12), respectively. Gray circles inside white background indicate popula- tions of mice with a normal potential fertility and without induced pre-implantation losses. Green and red backgrounds indicate the populations of ani- mals in which the parameters were detected as decreased or increased, respectively.

Figure 2 Proteins that changes its expression in mouse testis exposed to EDCs.(A) Up-regulated proteins, (B) down-regulated proteins and (C) protein showing uniform expression in mouse testis exposed to EDCs compared with control. Each chart represents the relative levels by regression lineal test (LOESS) for PDQuest, significant changes by 1.2-fold. Images are representative picture of 2D electrophoresis gels in each condition. 6 Buñay et al.

Table I Changes in the proteome of mice testis exposed to EDCs mixture.

Spot Ac. Symbol Protein name MM (Da)/pI Mat’db Total Sequence Molecular Biological processh No.a codeb theorc,d pepse scoref coverage functionh (%)g ...... A. Proteins up-regulated by the exposure to EDCs mixture and involved in germ cell death Downloaded from https://academic.oup.com/molehr/advance-article-abstract/doi/10.1093/molehr/gaz003/5304489 by guest on 26 February 2019 Q9JIQ3 DIABLO Diablo homolog, 26 975/6.3 7 305 32 Activating Intrinsic apoptotic signalling 203 mitochondrial caspases. pathway Inhibitory activity (IAP) P70349 HINT1 Histidine triad 13 882/6.36 5 157 58 Hydrolase activity Intrinsic apoptotic signalling 5101 nucleotide- pathway by p53 class mediator, binding protein 1 positive regulation of calcium- mediated signalling P63073 EIF4E Eukaryotic 25 266/5.79 4 236 18 Eukaryotic Regulation of translation, positive 4301 translation initiation factor regulation of mitotic cell cycle, initiation factor 4E 4 G binding, behavioural fear response P99026 PSMB4 Proteasome 29 211/5.47 7 162 37 Threonine-type Proteasome-mediated ubiquitin- 3202 subunit beta type- endopeptidase dependent protein catabolic 4 activity process Q9D1A2 CNDP2 Cytosolic non- 53 190/5.43 6 131 12 Dipeptidase Proteolysis 3702 specific activity dipeptidase Q01768 NME2 Nucleoside 17 466/6.97 9 505 51 Nucleoside Mitophagy in response to 7101 diphosphate diphosphate mitochondrial depolarization, kinase B kinase activity, cellular response to fatty acid, fatty acid binding glucose stimulus and oxidative stress Q9DBJ1 PGAM1 Phosphoglycerate 28 928/6.67 7 365 40 Phosphoglycerate Glycolytic process 6401 mutase 1 mutase activity, protein kinase binding Q9JHI5 IVD Isovaleryl-CoA 46 695/8.53 5 200 12 Isovaleryl-CoA Lipid homoeostasis 5605 dehydrogenase, dehydrogenase mitochondrial activity, fatty-acyl- CoA binding B. Proteins down-regulated by the exposure to EDCs mixture and involved in germ cell death 0 Q8CHP8 PGP phosphoglycolate 34 975/5.21 6 170 42 Magnesium ion Carbohydrate metabolic process, phosphatase binding, dephosphorylation, peptidyl- phosphoglycolate tyrosine dephosphorylation phosphatase activity, P63017 HSPA8 Heat shock 71 055/5.37 8 362 18 ATPase activity, ATP metabolic process, chaperone- 2901 cognate 71 kDa poly(A) RNA mediated protein folding requiring protein binding, unfolded cofactor protein binding P30416 FKBP4 Peptidyl-prolyl 51 939/5.54 14 367 38 ATP binding, Androgen receptor signalling 3804 cis–trans glucocorticoid pathway, embryo implantation, isomerase FKBP4 receptor binding, male sex differentiation, poly(A) RNA reproductive structure binding development P27773 PDIA3 Protein 57 099/5.88 14 400 30 Poly(A) RNA Cell redox homoeostasis, positive 3807 disulphide- binding, protein regulation of extrinsic apoptotic isomerase A3 disulphide signalling pathway, protein folding isomerase activity P15626 GSTM2 Glutathione S- 25 871/6.9 12 455 65 Glutathione Glutathione metabolic process, 6302 transferase Mu 2 transferase xenobiotic catabolic process activity Q80W21 GSTM7 25 864/6.34 4 71 20 Continued Proteomics/miRNAs in testis exposed to EDCs 7

Table I Continued

Spot Ac. Symbol Protein name MM (Da)/pI Mat’db Total Sequence Molecular Biological processh No.a codeb theorc,d pepse scoref coverage functionh (%)g ...... Glutathione S- Glutathione Glutathione metabolic process, Downloaded from https://academic.oup.com/molehr/advance-article-abstract/doi/10.1093/molehr/gaz003/5304489 by guest on 26 February 2019 5308 transferase Mu 7 transferase regulation of release of sequestered activity, receptor calcium ion into cytosol by binding sarcoplasmic reticulum, xenobiotic catabolic process P80314 CCT2 T-complex 57 783/5.97 12 541 28 ATP binding, Binding of sperm to zona pellucida, 5705 protein 1 subunit ubiquitin protein chaperone-mediated protein beta ligase binding complex assembly, toxin transport Q60930 VDAC2 Voltage- 32 340/7.44 6 180 31 Voltage-gated Negative regulation of intrinsic 7401 dependent anion- anion channel apoptotic signalling pathway, selective channel activity, negative regulation of protein protein 2 nucleotide binding polymerization C. Other up-regulated proteins identified Q924M7 MPI Mannose-6- 47 229/5.62 7 211 23 Mannose-6- Mannose to fructose-6-phosphate 3601 phosphate phosphate metabolic process isomerase isomerase activity, Q9CQE8 RTRAF RNA 28 249/6.4 5 215 26 Poly(A) RNA Positive regulation of transcription 6303 transcription, binding, identical from RNA polymerase II promoter, translation and protein binding tRNA splicing, negative regulation transport factor of protein kinase activity protein D. Other proteins identified P17742 Ppia Peptidyl-prolyl 18 131/7.74 6 320 53 Poly(A) RNA Positive regulation of protein 8102 cis–trans binding, peptide secretion, lipid particle organization, isomerase A binding protein folding

Data depicted correspond to the most statistically significant candidates encoded in Mus musculus. aSpot numbering as shown in the 2D gels depicted in Supplementary Fig. S1. bProtein accession code from Mus musculus database. cTheoretical molecular weight (Da). dTheoretical isoelectric point (pI). eNumber of matched peptides. fMascot Total score is −10*Log(P), where, P is the probability that the observed match is a random event. gSequence coverage is the amino acids ratio (no. identified in peptides/no. in theoretical peptides from sequence data) expressed as a percentage. hOntology classification according Mouse Genome Informatics (M.G.I.) and UniprotKB.

A bioinformatic analysis of dysregulated proteins detected by 2D Association between proteins and miRNAs electrophoresis/MS was performed, disclosing some protein–protein differentially expressed in mouse testis interaction networks (Supplementary Fig. S3). annota- exposed to EDCs mixture tion of differentially expressed proteins showed an enrichment in the process of cellular response to stress, in metabolic processes and in Integrative in-silico analyses revealed that 23 miRNAs/isomiRs (differen- protein folding pathways (Fig. 4A). Furthermore, we found that eight tially down-regulated in our previous studies; Buñay et al.,2017) may be up-regulated proteins (DIABLO, HINT1, EIF4E, PSMB4, CNDP2, associated with six up-regulated proteins (33.3% of total dysregulated NME2, PGAM1 and IVD) and all down-regulated proteins (VDAC2, proteins), considering the post-transcriptional regulatory roles of identi- fi PGP, HSPA8, FKBP4, PDIA3, GSTM2, GSTM7 and CCT2), which ed miRNAs/isomiRs over the corresponding target transcripts encod- fi represent 89% of all dysregulated proteins (16 out of 18) were ing identi ed proteins (Fig. 5A). The analyses did not show correlation involved in different steps of cell death (Fig. 4B). As for the other two between down-regulated proteins and up-regulated miRNAs. An inte- up-regulated proteins, MP1 is involved in metabolic processes and grative map showed that transcripts for DIABLO, PGAM1, RTRAF, RTRAF in the regulation of transcription as an RNA binding protein EIF4E, IVD and CNDP2 were targets of more than one down-regulated fi (Table I). Therefore, cell death seems to be the major dysregulated miRNA/isomiRs, which suggested higher potential ef ciency in their pathway at the protein level. expected post-transcriptional regulation (Fig. 5B and Supplementary 8 Buñay et al. Downloaded from https://academic.oup.com/molehr/advance-article-abstract/doi/10.1093/molehr/gaz003/5304489 by guest on 26 February 2019

Figure 3 Immunolocalization and up-regulation of PGAM1 in mice testis by the exposure to EDCs mixture.(A) PGAM1 protein levels in mice testis exposed to EDCs mixture and/or control were determined by Western blot and normalized with β-ACTIN. All graphics represent the mean ± SEM, n = 4. Unpaired t test, *P < 0.05. Abbreviation: AU, arbitrary units. (B) Representative pictures of PGAM1 in mouse testes exposed to EDCs mixture and controls, bar = 50 μm.

Table SII). For example, the overexpression of DIABLO and EIF4E could miRNA/isomiRs could be also dysregulated in human testis of infertile be induced as consequence of down-regulation of five and eight man. miRNAs/isomiRs, respectively (Fig. 5B). In addition, we have identified To assess this comparative analysis, we selected a recent study of two isomiRs (miR-30c-5p_trim2 and miR-497a-5p_UA_3prime)whose label-free quantitative shotgun proteomics on testicular tissue from canonical miRNAs are reported in the DIANA-TARbase v7.0 as valid- patients with non-obstructive azoospermia, including maturation ated for specific transcripts: miR-30c-5p: Diablo and miR-497a-5p: Ivd arrest (MA) and Sertoli cell only syndrome (SCOS) (Alikhani et al., mRNAs. As the detected isomiRs have only different 3′ ends (the corre- 2017). The results revealed eight common dysregulated proteins sponding 5′ seed regions sequence of these isomiRs are the same that (Fig. 6A) in testes from azoospermic men and those of mice exposed the canonical miRNAs). Consequently, it can be expected that the tar- to the EDCs mixture. These eight common proteins represented 44% get should be the same for each dysregulated miRNA/isomiR. of proteins dysregulated by EDCs in mice, all of them involved in cell Furthermore, we showed that the 3′ UTR of Eif4e mRNA can be tar- death (EIF4E, PGAM1, DIABLO, PSMB4, PDIA3, CCT, HSPA and geted by both the miR-15b-5p (canonical miRNA) and its isomiR miR- GST). 15b-5p_AA_3prime, and this interaction is conserved in humans Then, we selected from the literature three similar reports of signifi- (Fig. 5C). This finding might explain the correlation between six identi- cant changes in the expression of miRNAs (at least 2-fold changes) in fied proteins and the loss of miRNAs/isomiRs with targets in the corre- testicular biopsies of patients with MA and SCOS syndromes (Abu- sponding mRNAs encoding such proteins, as was detected in testis Halima et al., 2014; Munoz et al., 2015; Noveski et al., 2016). These from mice exposed to the EDCs mixture. data were contrasted with the list of miRNAs that were differentially expressed by the exposure to EDCs mixture, as previously detected Changes in the expression of proteins and (Buñay et al., 2017). We found two miRNAs (miRNA-15b and miRNA- miRNAs in mouse testis exposed to EDCs 34b) that were reported dysregulated in every work on human testis mixture are linked with idiopathic male infertility with spermatogenic failure and in our previous work in mice exposed Since some animals exposed to the EDCs mixture showed a decrease to EDCs. Moreover, three miRNAs were coincidently found only in fertility, we hypothesized that some of the deregulated protein and patients with MA and SCOS reported by Abu-Halima et al., 2014 Proteomics/miRNAs in testis exposed to EDCs 9 Downloaded from https://academic.oup.com/molehr/advance-article-abstract/doi/10.1093/molehr/gaz003/5304489 by guest on 26 February 2019

Figure 4 Cell death is the main molecular function related with dysregulated proteins in testis of mice exposed to EDCs mixture. (A) GO enrichment analysis of dysregulated proteins was performed using GlueGO. Enrichment was obtained using hypergeometric distribution test, with a P-value threshold >0.05. Each colour represents a different metabolic pathway. (B) Rate of identified proteins that are involved in cell death. Molecular function was obtained according to the information available at the MGI and UniProt database.

(Abu-Halima et al., 2014) and mice exposed to EDCs mixture (miR- exposed animals which could be due to: (i) differences in the relative 382, miR-18a and miR-378) (Fig. 6B). These observations suggested individual susceptibility at the exposure level, from early periods of that proteins and miRNAs differently expressed by the exposure to gonadal development, (ii) maternal physiology, (iii) differences in the EDCs mixture might be related to spermatogenic failure and asso- amount of milk intake during lactation and (iv) differences in metabolic ciated with the risk of exposure to EDCs of human populations. rate, accumulation, elimination and/or global detoxification rates of EDCs between animals. These observations might mirror the discrep- ancy in epidemiological data reports in human adult males, pregnant Discussion women and children and the association of adverse effects due to the This work shows a global integrative profile of dysregulated proteins/ single and mixed exposure to EDCs (Hou et al., 2015; Birks et al., miRNAs in testes of mice exposed to a mixture of phthalates and alkyl- 2016; Sifakis et al., 2017). Therefore, future fertility trials should be phenols, suggesting that chronic exposure to an EDCs mixture during performed in greater number of mice covering longer exposure times their development might be a plausible cause for idiopathic male and different aging to validate the tendency shown in this work. infertility. Furthermore, we indicate that new specific epidemiological studies are Male mice are sexually mature when they are around 42–49 days necessary in the human population with different grades of exposure old. At this age, each spermatogenesis cycle takes ~35 days to be to EDCs in order to evaluate the impact on male reproductive health. completed (Griswold, 2016). Although protein/miRNAs expression By 2D/MS proteomic analysis, we detected deregulation of 18 pro- analyses and fertility tests were performed in 60 and 75 days old ani- teins in the testes of mice exposed to the EDCs mixture. In this prote- mals, respectively, we believe that this 15-day difference should not omic approach, we used a sample pooling, which is a validated and affect the correlation of results since both are in the adult fertile commonly used option (Weinkauf et al., 2006; Diz et al., 2009; Karp period. and Lilley, 2009). However, it is important to state that this proteomic As a whole, mice exposed to EDC mixtures did not present signifi- study should be seen as a biological averaging (Karp and Lilley, 2009) cant changes in global fertility. According to this preliminary study, we and that the expression of several proteins observed as dysregulated can not be certain that subfertility is more likely present in the exposed must be validated through different approaches in future studies. group than in the control group. However, the potential fertility based Our previous reports and other studies have indicated that expo- on the increase of the number of pre-implantation losses and reab- sures to EDCs mixtures induce apoptosis of germ cells which is also sorptions was significantly compromised within a sub-group of well correlated with the decrease in fertility reported in this work 10 Buñay et al. Downloaded from https://academic.oup.com/molehr/advance-article-abstract/doi/10.1093/molehr/gaz003/5304489 by guest on 26 February 2019

Figure 5 Integrative analyses of dysregulated proteins and miRNAs in mouse testis exposed to EDCs mixture.(A) Hierarchical clus- ter analysis of differentially expressed miRNAs/isomiRs involved in the post-transcriptional control of dysregulated proteins. Expression levels corres- pond to log 2 normalized read counts using DeSeq tool of the R/Bioconductor software package, in testes of mice exposed to EDCs mixture comparing with control mouse testes. (B) Integrative map of interactions between down-regulated miRNAs/isomiRs and overexpressed proteins in mouse testis exposed to EDCs mixture; red colour indicates overexpression and gray to green scale under-expression. (C) Prediction of Eif4E as target of miR-15b-5p/miR-15b-5p_AA_3 prime in human and mouse. IsomiRs nomenclature according to Muller et al. (2014).

Figure 6 Common proteins and miRNAs differentially expressed by the exposure of EDCs on mouse testis and in idiopathic infertile human males.(A) Comparative analyses of dysregulated proteins in humans with spermatogenic failure (Alikhani et al., 2017) and in mouse testis exposed to EDCs. (B) Comparative analyses of dysregulated microRNAs in humans with spermatogenic failure (Abu-Halima et al., 2014; Munoz et al., 2015; Noveski et al., 2016) and in mouse testis exposed to EDCs (Buñay et al., 2017). The name in parenthesis refers to the reference of the study. Proteomics/miRNAs in testis exposed to EDCs 11

(Manikkam et al., 2013; Buñay et al., 2017, 2018). We found, through DIABLO (SMAC) is a relevant pro-apoptotic protein with ubiqui- developmental analysis, that some forms of GST, e.g. GSTM2, are tous expression in testicular cells that showed, by 2D electrophoresis/ down-regulated in normal adults compared to prepuberal mice (Paz MS, an increased expression in the testes of mice exposed to EDCs et al., 2006), which was in agreement with the higher expression of mixture. Interestingly, preliminary reports by immunohistochemistry this GST isoform in Sertoli and spermatogonia cells than in spermato- have not showed differences in the expression of DIABLO in azoo- cytes and spermatids (Yu et al., 2003). The decrease in the expression spermic patients compared to subjects with normal testicular histology

of two glutathione S-transferases (GSTM2 and GSTM7) detected in (Bozec et al., 2008). However, Jaiswal et al. (2015) demonstrated that Downloaded from https://academic.oup.com/molehr/advance-article-abstract/doi/10.1093/molehr/gaz003/5304489 by guest on 26 February 2019 our 2D proteomic analysis, suggests that the EDCs mixture is affecting protein expression of DIABLO increased in infertile human testes with the testis during development since the early differentiation stages. In hypospermatogenesis, MA and SCOS. And recently, the study of glo- fact, the depletion of antioxidant enzymes is a known mechanism of bal proteome profile in human testis affected by MA or SCOS have oxidative stress and cell death. Thus, the decrease in these GSTs levels corroborated that DIABLO expression is dysregulated (Alikhani et al., in testis after exposure to the EDCs mixture would be in opposition to 2017). Thus, the increase in DIABLO is directly related with the dis- its cytoprotective role and directly involved in mechanism of oxidative order of regulation of apoptosis in its pathogenesis (Takagi et al., stress to induce sperm DNA damage and male infertility. 2001). In addition, it has been detected that a lack of gonadotropins New in-vitro evidence show that endoplasmic reticulum (ER) stress and androgens causes the increase of DIABLO expression and its is an early driver of the EDC-mediated perturbations (Rajamani et al., translocation to the cytoplasm in spermatocytes (Vera et al., 2006). 2017). In this way, our in-vivo approach in mouse testis exposed to Moreover, exposure to phthalates metabolites with antiandrogenic the EDCs mixture identified a group of dysregulated proteins effect such as MEHP induces (via decrease of PI3K/AKT and increase involved in ER stress. Firstly, the overexpression of the proteasome of NF-Kβ) germ cell apoptosis by increasing the levels of DIABLO subunit beta type-4 (PSMB4) responsible of proteasome assembly (Rogers et al., 2008). Here, we suggest that the increase of DIABLO is and protein degradation (Hirano et al., 2008) was correlated with the linked to the exposure to EDCs mixture in a new mechanism that decrease in at least three proteins expression. One of them, PDIA3 involves the down-regulation of miRNA/isomiRs. is a chaperone located at the ER that modulates the thiol-disulphide Another up-regulated protein was EIF4E, whose overexpression is status of proteins and is considered a survival factor. In-vitro assays associated with oncogenesis and metastatic progression in humans foundthatsomeEDCsaffectthePDIAactivity(Klett et al.,2010), (Hsieh and Ruggero, 2010; Siddiqui and Sonenberg, 2015). and further evidence shows that PDIA3 has the capacity of binding Furthermore, some studies have reported that somatic cell viability, with 17β-estradiol (Primm and Gilbert, 2001; Wong et al., 2017), a condensation, cytokinesis during the meiotic division hormone that was previously reported as decreased in mouse testis and normal production of functional sperm in spermatogenesis exposed to EDCs mixture (Buñay et al., 2017). Another down- requires EIF4E activity (Amiri et al., 2001; Ghosh and Lasko, 2015). In regulated protein was the cytosolic chaperone CCT2, a protein par- addition, in a previous study, we found that changes in the expression ticularly involved in the folding of cytoskeletal and cell cycle proteins of EIF4F complex including Eif4E depend on the type of EDCs and the (actin, tubulin and cyclin-E), which is related with PSMB4 (up-regu- time of exposure (López-Casas et al., 2012). The increase of the level lated). Consequently, altered folding of these proteins may stop the of EIF4E could be a consequence of the loss of post-transcriptional cell cycle, cause an ER stress response, disrupt the mitochondria and control due to a decrease in the level of one canonical miRNA (miR- induce apoptosis in somatic or germ cells. In addition, CCT2 15b-5p) and seven doubly adenylated 3′ end isomiRs, targeting its 3′- increases its expression from prepuberal to adult mice, suggesting an UTR. This miRNA regulation is also preserved in the human EIF4E 3′ association with late spermatogenesis (Paz et al., 2006). Finally, the UTR. At the same time, both EIF4E and miR-15b are recurrently dysre- down-regulation of heat-shock cognate protein HSPA8 was also pre- gulated in testicular biopsies of patients with spermatogenic failure, sent. HSPA8 participates in the response to ER stress with the pivotal which suggests a novel mechanism whereby the exposure to EDCs role of correcting the folding of nascent polypeptides and then coop- might decreases fertility and should be studied in depth in infertile erates in the re-folding of misfolded proteins. At the same time male patients. PSMB4, PDIA3, CCT and HSP are found dysregulated in testicular Interestingly, the mitochondrial protein Isovaleryl-CoA dehydrogen- biopsies from patients with SCOS and MA (Alikhani et al., 2017). ase (IVD) was found overexpressed and it increases in response to Therefore, the interplay of these proteins of ER stress could be impli- lipid overload, which is correlated with the obesogenic effect and cho- cated in germ-cell death induced by EDCs. lesterol increase reported by the exposure to EDCs (Heindel et al., Many efforts have been made to identify miRNAs involved in sper- 2015). Our in-silico data show that the Ivd transcript is targeted by one matogenic failure (Abu-Halima et al., 2014; Munoz et al., 2015; canonical miRNA (miR-15b-5p) and the isomiRs miR-15b- Noveski et al., 2016). Currently, new approaches with small non- 5p_AA_3prime along to three more down-regulated isomiRs. coding RNA sequencing (sncRNA-Seq) suggest that changes of Therefore, an overexpression of IVD due to the loss of the post- miRNA variants or isomiRs (more than canonical miRNAs) could bet- transcriptional control by miRNAs/isomiRs might promote high β-oxi- ter describe a pathological state (Guo et al., 2011; Kozlowska et al., dation of free fatty acids and catabolism of proteins that are related to 2013; Telonis et al., 2015). In this sense, at the reproductive level, the stress in the ER and increased mitochondrial ROS (Tumova et al., changes in isomiRs expression due to the EDCs exposure could have a 2016), thus generating mitochondrial dysfunctions commonly detected more relevant role than canonical miRNAs. Thus, we suggest that in spermatozoa of infertile patients. future studies focused on changes in the miRNome of infertile subjects Similarly, an important enzyme in the glycolytic pathway, PGAM1, (male and female) should include analyses of isomiRs. was found to be up-regulated in mouse testis exposed to EDCs. In 12 Buñay et al. addition, by immunohistochemistry we found that PGAM1 was prefer- the possibility of establishing miRNAs pathways as a specific and effect- entially expressed in Sertoli and interstitial cells. However, in relation ive pharmacological therapy for certain types of primary testicular to the protein levels, IHQ analysis could not validate the data obtained failure. by WB and 2D/MS, possibly due to differences in the treatments of the samples related to the fixation method or the epitope retrieval Supplementary data (Uhlen et al., 2016). The increase in PGAM1 suggests that the expos- fi fi Supplementary data are available at Molecular Human Reproduction ure to EDCs modi ed the glycolytic pro le in somatic cells, particularly Downloaded from https://academic.oup.com/molehr/advance-article-abstract/doi/10.1093/molehr/gaz003/5304489 by guest on 26 February 2019 in Sertoli cells to subsequently alter the energetic metabolic role sup- online. plying germ cells and induce germ cell death. In testicular biopsies from infertile men dysregulated levels of PGAM1 were found (Alikhani et al., Acknowledgements 2017), specifically down-regulated in severe hypospermatogenesis and We would like to thank MSc. Leonor Cruz Fernandes for the English overexpressed in SCOS, which is associated with an inhibition of pro- editing. liferation and apoptosis (Zhang et al., 2015). Here, we presented the first evidence of post-transcriptional control of PGAM1 mediated by ’ isomiRs, suggesting that these interactions could also be used as bio- Author s roles markers in male infertility. J.B., R.D.M. and J.d.M. designed the research; J.B., D.P-G. and P.U-M. Another up-regulated protein was the cytosolic nonspecific dipepti- performed the experiments; J.B. and E.L. contributed new analytic dase 2 (CNDP2). However, its function and implication in human fer- tools; J.B., E.L., R.D.M. and J.d.M. analysed data; and J.B., E.L., R.D.M. tility is not clear. Apparently, CNDP2 has more than one enzymatic and J.d.M. wrote the article. activity and when increased, it activates p38 and JNK/MAPK pathways to induce cell apoptosis (Zhang et al., 2014). Moreover, recent Funding research on the enzymatic activity of CNDP2 in the cellular metabo- lome has indicated that this protein can degrade lactate to its metabo- Grants from FONDECYT 1150352 to R.D.M., FONDECYT 3160273 lites N-lactoil-amino acids (N-lac-Phe) (Jansen et al., 2015). In previous to P.U.-M. and CONICYT 21120505 to J.B., Chile, and MINECO work, we indicated that lactate is a survival factor of spermatocytes BFU2013-42164-R and BFU2017-87095-R to J.d.M., Spain. (Bustamante-Marín et al., 2012). Although the function of lactate meta- bolites is unknown, it suggests that the degradation of lactate via an Conflicts of interest increase in the expression and enzymatic activity of CNDP2 could be a None declared. new mechanism that modulates germ cell apoptosis. Our analyses indi- cated that the increase in CNDP2 expression would be associated with the loss of the post-transcriptional control mediated by miR-23a- References 3p_AA_3prime, miR-320-3p_AAA_3prime, and the joint action of miR- Abu-Halima M, Backes C, Leidinger P, Keller A, Lubbad AM, Hammadeh 3085-3p/miR-3085-3p_UA_3prime. This supports our hypothesis that M, Meese E. MicroRNA expression profiles in human testicular tissues isomiRs could participate along with the corresponding canonical of infertile men with different histopathologic patterns. Fertil Steril 2014; miRNAs in the regulation of mRNA targets during spermatogenesis, 101:78–86. which can be altered by the exposure to EDCs, triggering changes in Ademollo N, Ferrara F, Delise M, Fabietti F, Funari E. Nonylphenol and metabolic and cell stress pathways. octylphenol in human breast milk. Environ Int 2008;34:984–987. 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