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Porseryd, T., Reyhanian Caspillo, N., Volkova, K., Elabbas, L., Källman, T. et al. (2018)Testis transcriptome alterations in zebrafish (Danio rerio) with reduced fertility due todevelopmental exposure to 17α-ethinyl estradiolGeneral and Comparative Endocrinology, 262: 44-58https://doi.org/10.1016/j.ygcen.2018.03.011

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General and Comparative Endocrinology 262 (2018) 44–58

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General and Comparative Endocrinology

journal homepage: www.elsevier .com/locate /ygcen

Research paper

Testis transcriptome alterations in zebrafish (Danio rerio) with reducedfertility due to developmental exposure to 17a-ethinyl estradiol

https://doi.org/10.1016/j.ygcen.2018.03.0110016-6480/� 2018 The Authors. Published by Elsevier Inc.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

⇑ Corresponding author.E-mail address: [email protected] (T. Porseryd).

1 These authors have contributed equally to this work.

T. Porseryd a,1,⇑, N. Reyhanian Caspillo a,b,1, K. Volkova a,b, L. Elabbas a, T. Källman c,d, P. Dinnétz a,P-E. Olsson b, I. Porsch-Hällström a

a School of Natural Sciences, Technology and Environmental Studies, Södertörn University, SE-141 89 Huddinge, SwedenbÖrebro Life Science Center, School of Science and Technology, Örebro University, SE-701 82 Örebro, SwedencNational Bioinformatics Infrastructure Sweden, Uppsala University, 75124 Uppsala, Swedend Science for Life Laboratory and Department of Medical Biochemistry and Microbiology, Uppsala University, 75123 Uppsala, Sweden

a r t i c l e i n f o a b s t r a c t

Article history:Received 15 November 2017Revised 22 February 2018Accepted 7 March 2018Available online 8 March 2018

17a-Ethinylestradiol (EE2) is a ubiquitous aquatic contaminant shown to decrease fish fertility at lowconcentrations, especially in fish exposed during development. The mechanisms of the decreased fertilityare not fully understood. In this study, we perform transcriptome analysis by RNA sequencing of testesfrom zebrafish with previously reported lowered fertility due to exposure to low concentrations of EE2during development. Fish were exposed to 1.2 and 1.6 ng/L (measured concentration; nominal concentra-tions 3 and 10 ng/L) of EE2 from fertilization to 80 days of age, followed by 82 days of remediation inclean water. RNA sequencing analysis revealed 249 and 16 genes to be differentially expressed afterexposure to 1.2 and 1.6 ng/L, respectively; a larger inter-sample variation was noted in the latter.Expression of 11 genes were altered by both exposures and in the same direction. The coding sequencesmost affected could be categorized to the putative functions cell signalling, proteolysis, protein metabolictransport and lipid metabolic process. Several homeobox transcription factors involved in developmentand differentiation showed increased expression in response to EE2 and differential expression of genesrelated to cell death, differentiation and proliferation was observed. In addition, several genes related tosteroid synthesis, testis development and function were differentially expressed. A number of genes asso-ciated with spermatogenesis in zebrafish and/or mouse were also found to be differentially expressed.Further, differences in non-coding sequences were observed, among them several differentially expressedmiRNA that might contribute to testis gene regulation at post-transcriptional level. This study has gen-erated insights of changes in gene expression that accompany fertility alterations in zebrafish males thatpersist after developmental exposure to environmental relevant concentrations of EE2 that persist fol-lowed by clean water to adulthood. Hopefully, this will generate hypotheses to test in search for mech-anistic explanations.� 2018 The Authors. Published by Elsevier Inc. This is anopenaccess article under theCCBY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

The synthetic 17a-etinylestradiol (EE2) is a potent endocrinedisrupting chemical (EDC) frequently found in effluents and sur-face waters from sewage treatment plants, inevitably affectingthe aquatic wildlife (Laurenson et al., 2014, Aris et al., 2014). Inaddition to being persistent in the environment and showing signsof biomagnification (Aris et al., 2014), EE2 is also tenfold morepotent in fish than its natural counterpart, 17b-estradiol (Dennyet al., 2005). The Predicted No Effect Concentration (PNEC) of EE2

for aquatic animals is as low as 0.1 ng/L (Desbrow et al., 1998,Caldwell et al., 2012). Unfortunately, concentrations up to 300ng/L have been found in effluents from sewage treatment plants(Kolpin et al., 2002, Sun et al., 2013, Hannah et al., 2009,Laurenson et al., 2014). Reproductive organs are often targets forestrogenic EDCs in animals, causing alterations in reproductivecapability and physiology. In adult animals, the effects of EDCson reproduction are essentially reversible, while exposure duringnarrow windows in early development seems to be irreversible,manifested at the level of tissue organization (McLachlan, 2001)representing the basis for the theory of ‘‘fetal origins of adult dis-ease” (Calkins and Devaskar, 2011). Effects of EDCs on fertilityand reproduction as well as reproductive tract malformation havebeen identified in rodents, and the developing foetus seems

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particularly sensitive (McLachlan, 2001, Newbold et al., 2006,McLachlan et al., 2012, Salian et al., 2009). Transgenerational trans-mission of decreased fertility has been observed and concomitantalterations in the testis transcriptome has been demonstrated.(Anway et al., 2005). Estrogens are important actors in epigeneticmechanisms, and the persistent changes seem to be mediated bysuchmechanisms (Prados et al., 2015, Klinge, 2015b, Skinner, 2014).

Field studies and laboratory experiments in fish have demon-strated reproductive alterations caused by EDC exposure (Joblinget al., 1998, Ankley et al., 2009, Arukwe, 2001, Weber et al., 2003,Fenske et al., 2005). Environmentally relevant concentrations ofEE2 have been shown to affect fertility and reproduction in fish(Corcoran et al., 2010) and has been shown to promote feminiza-tion in adult male fish and induce the female yolk precursor vitel-logenin (VTG) in several species including zebrafish, medakarainbow trout, fathead minnow, roach and three-spined stickle-back (Scholz et al., 2004, Seki et al., 2002, Rose et al., 2002, Langeet al., 2012). Ongoing EE2 exposure in male zebrafish has beenshown to inhibit spermatogenesis and reduce testis size (Van denBelt et al., 2002) and reduce fecundity and fertilization success(Santos et al., 2007). Earlier findings when examining geneexpression using quantitative PCR or microarray-based studies ofEE2-exposed fish gonads during ongoing exposure, revealingalterations in expression e.g. in genes involved in the HPG axis,development and male sex differentiation (Santos et al., 2007,Filby et al., 2007, Miller et al., 2012, Reyhanian Caspillo et al.,2014, Garriz et al., 2017).

In line with studies from rodents, effects from adult exposureseems reversible to a large extent (Baumann et al., 2014, Schäferset al., 2007) while zebrafish exposed to EE2 during developmentexhibit a decrease in fertility, still discernible after long remedia-tion in clean water (Fenske et al., 2005, Hill and Janz, 2003, Linand Janz, 2006, Van den Belt et al., 2002, Volkova et al., 2015).Transgenerational effects transmitting a phenotype of reduced fer-tility over several generations has been demonstrated in medaka(Oryzias latipes) after exposure to EE2 (Bhandari et al., 2015). Suchlong lasting modifications of fertility could negatively affect indi-vidual and species fitness to a much higher degree than short timetemporal effects of adult exposure particularly as many marinespecies return to shallow coastal regions and rivers to reproduce.Considering the long term effects of estrogens on the reproductiveaxis, little is known regarding the mechanisms of the induced mal-functions of the gonads. Genome-wide searches including non-coding RNAs will be essential to further understand the underlyingmolecular pathways. More in depth analyses using RNA sequenceanalysis (RNAseq) in fish has so far been showing alterations ofthe transcriptome in guppy brain (Saaristo et al., 2017) and in rareminnow gonads (Gao et al., 2017) during ongoing adult exposureto EE2. We have previously found gene expression changes persist-ing after developmental exposure to EE2 in the zebrafish brain andlong-time remediation to adulthood (Porseryd et al., 2017). Tran-scriptome studies of fish testes after developmental EDC exposureto identify transcriptomic alterations persisting after remediationare however to the best of our knowledge non-existing. Since EE2affects several reproductive parameters and the effects on fertilityof exposure during development seem to be irreversible (Volkovaet al., 2015, Larsen et al., 2008, Maunder et al., 2007, Bhandariet al., 2015), it is highly relevant to examine transcriptome alter-ations in the gonads after developmental exposure. In a previousstudy, where zebrafish were developmentally exposed to 1.2 and1.6 ng/L (nominal 3 and 10 ng/L) of EE2 and raised to adulthoodin clean water, lasting decreases in fertility in both sexes werefound (Volkova et al., 2015). The objective of this study was tofurther understand the reasons for the fertility reduction in malezebrafish by performing a transcriptome analysis followed by afunctional classification of predicted biological function of the

genes found differentially expressed in the testes in these males.In search for relevant biological functions, GO-terms were searchedfor both zebrafish genes and the corresponding mouse orthologues.

2. Methods

2.1. Experimental design

This study is an extension of a previously described experiment(Volkova et al., 2015) where fertilized zebrafish (Danio rerio)embryos (AB strain, Karolinska Institute Core Facility, Huddinge,Sweden) were exposed to the nominal concentrations 0, 3 and10 ng/L of EE2 (Sigma Aldrich, USA) in 5 ppm acetone. Fertilizedeggs from each parental pair (8 pairs) were divided into batches,each assigned to one of the three exposure groups, and exposedfrom fertilization until 80 days post fertilization. During the first6 weeks, fish larvae were raised in 1L glass tanks with 60% solutionexchange every second day, fed daily with Paramecia, and fromweek 5 Artemia twice a day. Sera Dry Flakes (Vipan, Germany)were added to the diet twice a day from week 6, when the larvaefrom each parental pair and treatment were transferred to separatenet cages in 20 L glass tanks in a flow-through system exchanging1/3 of the volume per day. After 80 days, the fish were transferredto 2L tanks, still separated by parental pair and treatment, andreared in clean water under standard zebrafishmaintenance condi-tions (25–27 �C, pH 7.0, 12/12 h light/dark cycle) until adulthood,resulting in an 82-day remediation period. From 40 days, the sexeswere separated based on visual secondary sex characteristics andrechecked weekly for sex changes. All handling of the fish wereperformed according to the Swedish Animal care legislation, andapproved by the Southern Stockholm Animal Research Ethics Com-mittee (DNR S130-09).

EE2 levels were analyzed by LC-MS/MS resulting in measuredconcentrations of 0, 1.2 and 1.6 ng/L. For further details aboutchemical analyses see Volkova et al. (2015). A possible explanationto the deviation from the nominal concentrations could be that theraising of zebrafish larvae requires intense feeding resulting in aformation of a lipid-rich layer on the aquaria walls trapping thelipophilic EE2.

2.2. Dissection and sex verification

At the completion of the experiment, fish were anaesthetized in0.5‰ 2-phenoxyethanol (Sigma-Aldrich) followed by immediatedecapitation. Testes were dissected and stored individually inRNA later (Sigma-Aldrich) at �80 �C.

2.3. Ion proton RNA sequencing

In the RNAseq analysis, four individual testes from differentfamily groups were used from each exposure (0, 1.2 and 1.6 ng/LEE2). Testes were homogenized and total RNA extracted with TriR-eagent (Sigma-Aldrich, Germany) according to the instructionsfrom the manufacturer. Total RNA samples went through qualitychecking with R6K Screen Tape at Bea Core Facility (KarolinskaInstitute, Huddinge, Sweden).

IonProton RNA sequencing was performed by SciLifeLab(Uppsala University, Sweden). The total RNA libraries weresubjected to treatment with Ribo-ZeroTM rRNA Removal Kit(Epicentre/Illumina), purified using Agencourt RNAClean XP Kit(Beckman Coulter), treated with RNaseIII according to the Ion TotalRNA-Seq protocol (Life Technologies) and subsequently purifiedwith Magnetic Bead Cleanup Module (Life Technologies). Size andquantity of RNA fragments were assessed on the Agilent 2100Bioanalyzer system (RNA 6000 Pico kit, Agilent) before proceeding

46 T. Porseryd et al. / General and Comparative Endocrinology 262 (2018) 44–58

to library preparation, using the Ion Total RNA-Seq kit (Life Tech-nologies). The libraries were amplified according to the protocoland purified with Magnetic Bead Cleanup Module. Samples werequantified using the Agilent 2100 Bioanalyzer system and Frag-ment Analyzer (Advanced Analytical) and pooled followed byemulsion PCR on the Ion OneTouchTM 2 systemwith the Ion ProtonTM

Template OT2 200 v3 Kit (Life Technologies) chemistry. Sampleswere enriched using the Ion OneTouchTM ES (Life Technologies),loaded on an Ion PI v2 Chip (2 samples per chip) and sequencedon the Ion ProtonTM System using Ion ProtonTM Sequencing 200 v3Kit (read length 200 base pairs, 40 million reads per sample, LifeTechnologies) chemistry.

2.4. Bioinformatics and biostatistics

Quality control of the reads was done with the software FastQCversion 0.11.2 (Andrews 2010). The short reads were mapped toversion 9 of the Danio rerio genome using the splice aware alignerStar version 2.3.1o (Dobin et al., 2013) using the ensemble genemodels to facilitate efficient mapping of spliced reads.

The mapped reads were converted to count data with HTSeqversion 0.7.1 (Anders et al., 2014). The R package edgeR(Robinson et al., 2010) that estimate expected variance in geneexpression from a negative binomial distribution and uses a gener-alized linear model to identify differentially expressed genes wereemployed to detect genes that have a different level of geneexpression in testes from fish subjected to 1.2 ng/L or 1.6 ng/L ofEE2 compared with unexposed fish. To control for family effects,family was used as block in the analyses. Only genes that had anexpression level higher than 1 count per million in at least threeof the sequence libraries were retained for test of differential geneexpression. Finally, estimated p-values were corrected for multipletesting with false discovery rate (FDR) correction, and genes show-ing FDR-corrected p-values <0.05 were regarded as significantlydifferentially expressed.

2.5. Analysis of putative function of differentially expressed genes

Classification of genes and predictions of biological gene func-tion were performed manually with GO-terms from zebrafishand mouse (Mus musculus). Zebrafish GO-terms were found in Zfin(Howe et al., 2013). Orthologue search was done in Ensembl (Yateset al., 2016) and GO-terms for mouse were found in MGI (Blakeet al., 2017).

2.6. Quantitative Real-Time PCR

The expression of six selected differentially expressed geneswere determined with qPCR to verify the differential expressionobserved in the RNAseq analysis. Oligonucleotide primers(Table S1) were designed using the Primer-Blast primer designingtool (Ye et al., 2012). RNA was isolated from individual testes from5 control males and 5 males exposed to 1.2 ng/L EE2, homogenizedin 0.8 ml/sample of TriReagent (Sigma-Aldrich, Germany) andquantified using NanoDrop ND-1000 spectrophotometer, andRNA quality was determined as the 260/280 nm absorption ratio.Reverse transcription was performed using HotStarTaq Plus MasterMix Kit (Qiagen) from 0.5 mg RNA per sample. Bio-Rad C1000 TouchThermal cyclerTM, CFX96TM Real-Time System was used for quantita-tive Real-Time PCR (qPCR). Each sample was run in triplicates andcontained 5mL cDNA template (350 ng RNA), 1 mL H2O, 10 mL iTaqTM

Universal SYBR� Green Supermix (BIO-RAD) and 2 mL each offorward- and reverse primer. Amplification was conducted withan initial denaturation step at 95� C for 3 min followed by 40 cyclesof 30 s at 94� C, 30 s at Tm �C and 60 s at 72 �C. Melt dissociationcurve was performed after amplification at 65 �C + 0.5 �C/s in total

of 60 repeats. All samples were run in triplicates, including a nega-tive control, and a mean Ct value was obtained from the triplicates,whichwas used for quantification of normalized expression accord-ing to the 2�D Ct method (Schmittgen and Livak, 2008). Elongationfactor 1A, (Elf1a) was used as reference gene. The data wasexpressed as relativemRNA expression after normalization for eachsample in the EE2-exposed and the control group. The efficiency ofeach primer pair was evaluated by standard curves from a dilutionseries to ensure a satisfactory efficiency (<90%).

Data from qPCR were analyzed in the statistical software R(R Core Team, 2017) with Welch Two Sample t-test, assumingun-equal variances, testing the upregulation with a one tailedt-test to avoid a type II error.

3. Results

3.1. Differential gene expression analysis

Of the 33737 genes annotated, 21651 had gene expression highenough for differential gene expression analysis. In total, 254 geneswere found to be significantly altered in expression by the devel-opmental exposure, 249 in the 1.2 ng/L exposure and 16 in the1.6 ng/L EE2, respectively. Of the 16 differentially expressed genesin testes from the 1.6 ng/L EE2 exposure, 11 were also differentiallyexpressed in the 1.2 ng/L group, and the directions of the alter-ations were the same. The full list of differential expressed genesand their statistical values are shown in Table S2. The markedlylower number of differentially expressed genes in testis from thegroup exposed to 1.6 ng/L may in part be explained by the largerintra-individual variation observed in this group. A majority ofthe differentially expressed genes, 91% (2 3 1) showed an increasedexpression due to the EE2 exposure, only 9% (23) of the genes weredownregulated.

3.2. Analysis of putative function of differentially expressed genes

Functional analysis was performed manually based on informa-tion from rodent orthologue data in addition to the zebrafish data-base zfin. The categories and number of genes categorized in eachbiological pathways (BP) are shown in Fig. 1. The top putative BPsassigned in the testis from the 1.2 ng/L exposure group are prote-olysis (28 differently expressed genes), cell signalling (20), trans-port (16), lipid metabolic transport (15), regulation oftranscription (15), protein metabolic process (11), cytoskeleton(10), and cell adhesion (10). 17 transcripts were found under thecategory non-coding RNA. 35 genes did not have any GO-termsor further information and were categorized unknown (Table S2).In the 1.6 ng/L group proteolysis was identified as top putative bio-logical pathway with two differentially expressed genes. 5 geneswere put under the category non-coding RNA and the rest of the12 genes differentially expressed were assigned as single genesinto categories or categorized as unknown.

The description of the genes below are differently expressed intestes exposed to 1.2 ng/L EE2 compared to the control unlessotherwise stated. When differently expressed in the 1.6 ng/L expo-sure, or in both exposures, this is denoted in the text and referredto Table 8 where the differential genes in this exposure areexhibited.

3.3. Lipid, carbohydrate, protein and nucleic acid metabolic process

The genes under these categories are shown in Table 1. Amongthe 15 genes upregulated by EE2 annotated under lipid metabolicprocesses several encoded lipases pla2glb, cel1, cel2 and mgll, butalso apoeb involved in cholesterol biosynthesis and rbp2a and rpb

Fig. 1. The expressed biological functions (categories) from the manual analysis of genes differentially expressed in zebra fish testes after developmental exposure to A) 1.2ng/L EE2 and B) 1.6 ng/L EE2 after remediation in clean water for 82 days.


4 involved in retinoic acid metabolic process. The latter is in mouseimplied in male gonad development and spermatogenesis. In addi-tion, srd5a2a, encoding the steroid 5a reductase, in mice shown tobe involved in male gonad development and sex differentiation, isupregulated, together with ptgs2a, a prostaglandin biosynthesisenzyme and ptgdsb1. After exposure to 1.6 ng/L cyp17a2, with agene product involved in progesterone metabolism, showedenhanced transcript level (Table 8).

The carbohydrate metabolic process group contained upregula-tion by EE2 in testis of genes encoding enzymes involved in carbo-hydrate synthesis and catabolism, such as zgc:922137 (upregulatedby both exposure levels), pck1 and amy2a, but also ins, encoding

pre-proinsulin. Two upregulated genes encoding cytochrome c oxi-dases cox6a2 and cox7a1 involved in electron transport were alsodenoted under carbohydrate metabolism.

Eleven upregulated genes were found under the categoryprotein metabolic process. These genes encoded the spermine/spermidine acyltransferase sat1a2, an amine oxidase, aoc2, andthe fibrinogen-like fgl2a. Also showing increased expression werebhlmt, gamt and mat1a, with gene products engaged inS-adenosyl methionine biosynthesis; gamt has in mouse also theGO-term spermatogenesis.

Three EE2-upregulated genes in the category of nucleic acidmetabolic process coded for nucleoside biosynthesis and

Table 1Genes assigned to the biological functions lipid metabolic process, carbohydrate metabolic process, protein metabolic process and nucleic acid metabolic process from thefunctional analyses of the transcripts significantly differentially expressed in testes of zebrafish developmentally exposed to 1.2 ng/L and remediated in clean water (FDR-corrected p values < 0.05). Categories were assigned from the information of GO-terms found in Zfin and MGI.

Ensembl ID Gene name Gene ID logFC

Lipid metabolic processENSDARG00000004539 prostaglandin-endoperoxide synthase 2a ptgs2a 2.78ENSDARG00000006427 fatty acid binding protein 2 fabp2 5.96ENSDARG00000009153 phospholipase A2, group IB pla2g1b 5.55ENSDARG00000017490 carboxyl ester lipase, tandem duplicate 1 cel.1 5.73ENSDARG00000027088 prostaglandin D2 synthase b, tandem duplicate 1 Ptgdsb.1 3.15ENSDARG00000029822 carboxyl ester lipase, tandem duplicate 2 cel.2 6.07ENSDARG00000036820 monoglyceride lipase mgll 1.73ENSDARG00000040295 apolipoprotein Eb apoeb 2.84ENSDARG00000043587 steroid-5-alpha-reductase, alpha polypeptide 2a srd5a2a 3.95ENSDARG00000044802 arylacetamide deacetylase-like 4 aadacl4 3.16ENSDARG00000077505 retinol binding protein 4, plasma rbp4 4.51ENSDARG00000070038 retinol binding protein 2a, cellular rbp2a 4.14ENSDARG00000073820 zgc:174917 zgc:174917 4.50ENSDARG00000075958 si:dkey-266f7.9 si:dkey-266f7.9 2.58ENSDARG00000076015 si:ch73-139e5.2 PLBD1 (2 of 2) -3.77

Carbohydrate metabolic processENSDARG00000009443 zgc:92137 zgc:92137 6.08ENSDARG00000013522 phosphoenolpyruvate carboxykinase 1 pck1 3.62ENSDARG00000013856 Amylase, alpha 2A amy2a 5.62ENSDARG00000035350 preproinsulin ins 7.16ENSDARG00000054588 cytochrome c oxidase subunit VIa polypeptide 2 cox6a2 2.44ENSDARG00000056874 lysozyme g-like 1 lygl1 1.85ENSDARG00000060471 glucosaminyl (N-acetyl) transferase 3, mucin type gcnt3 2.18ENSDARG00000069464 cytochrome c oxidase subunit VIIa polypeptide 1 cox7a1 1.76

Nucleic acid metabolic processENSDARG00000005565 ectonucleoside triphosphate diphosphohydrolase 8 entpd8 2.20ENSDARG00000012539 deoxyribonuclease I dnase1 5.11ENSDARG00000039429 adenosine kinase a adka 2.24ENSDARG00000068644 urate (5-hydroxyiso-) hydrolase urah 2.88ENSDARG00000078069 ribonucleotide reductase M2 polypeptide rrm2 -7.38

Protein metabolic processENSDARG00000013430 betaine-homocysteine methyltransferase bhmt 6.03ENSDARG00000014646 amine oxidase, copper containing 2 aoc2 1.69ENSDARG00000019861 fibrinogen-like 2a fgl2a 1.77ENSDARG00000021154 zgc:92040 PRODH2 4.22ENSDARG00000035652 spermidine/spermine N1-acetyltransferase 1a, duplicate 2 sat1a.2 2.04ENSDARG00000039605 methionine adenosyltransferase I, alpha mat1a 7.41ENSDARG00000054543 SAM domain, SH3 domain and nuclear localisation signals 1a samsn1a 2.23ENSDARG00000058053 serpin peptidase inhibitor, clade G (C1 inhibitor), member 1 serping1 3.64ENSDARG00000070844 guanidinoacetate N-methyltransferase gamt 2.30ENSDARG00000091231 BX572619.7 BX572619.7 2.61ENSDARG00000091609 serine peptidase inhibitor, Kazal type 4 SPINK4 4.87


catabolism, entpd8, adka and urah. The deoxyribonuclease dnase1was also upregulated. A more than sevenfold reduction wasobserved in the ribonucleotide reductase rrm2. Further, expressionof the msh3 gene, involved in mismatch repair, was decreasedmore than 7-fold by 1.6 ng/L.

3.4. Response to chemical, estrogen, hormone and stress, and circadianrhythm

The genes under these categories are found under Table 2. Thegenes under the category response to chemical consisted mainly ofupregulated genes encoding enzymes involved in xenobiotic meta-bolism, such as two cytochrome P450 isozymes, cyp1a andcyp2aa12, the metallothionein mt2, the glutathione transferasegst1a, the carboxypeptidase cpb1 and the alcohol dehydrogenaseadh8a. Also, the uncharacterized xenobiotic-responsible gene si:ch211-17 m20.5 was upregulated. Response to estrogen consistedof 3 upregulated genes, agxtb, involved in glyoxylate biosynthesisand fbp1b involved in sucrose biosynthesis, and the aquaporinaqp12. In response to hormone two upregulated genes, adma,encoding adrenomedullin a, which in mouse is involved in andro-gen metabolism, and dio2, encoding iodothyronine deiodinase 2,

were observed. Genes assigned to response to stress included theupregulated genes encoding cahz, pdia2 and bco21, encodingenzymes engaged in response to oxidative stress, hypotonic salin-ity and redox homeostasis, and two hemoglobin-encoding genesinvolved in hypoxia, ba1 and ba2. Two genes involved in theresponse to starvation were upregulated by EE2 in testis, gip,encoding gastric inhibitory polypeptide and gcga, encoding gluca-gon. Two genes encoding proteins involved in circadian regulationof gene expression, bhlhe40 and prok2 were upregulated in testesfrom EE2-exposed male zebrafish.

3.5. Cell proliferation, cell differentiation and cell death

The genes categorized under cell proliferation, differentiation,and death are shown in Table 3. The expression of the pimr191and ckap5 genes, encoding a mitotic cell cycle regulator and acytoskeleton-associated protein, involved in spindle organizationand centrosome duplication, respectively, were upregulated byEE2. Increased expression of ckap5 was observed also after expo-sure to 1.6 ng/L EE2. Increased expression was observed of theppdpfa, tm4sf4, agr1 and agr2 genes involved in cell differentiation.Also, the uncharacterized NBL1 and VSIR showed increased

Table 2Genes assigned to the biological functions response to chemicals, response to estrogen, response to hormone, response to stress and circadian rhythm from the functionalanalyses of the transcripts significantly differentially expressed in testes of zebrafish developmentally exposed to 1.2 ng/L and remediated in clean water (FDR-corrected p values< 0.05). Categories were assigned from the information of GO-terms found in Zfin and MGI.


Response to chemicalsENSDARG00000026039 cytochrome P450, family 1, subfamily A cyp1a 6.98ENSDARG00000028367 sulfotransferase family 2, cytosolic sulfotransferase 3 sult2st3 5.02ENSDARG00000041623 metallothionein 2 mt2 1.83ENSDARG00000042428 glutathione S-transferase theta 1a gstt1a 2.86ENSDARG00000045442 carboxypeptidase B1 cpb1 5.97ENSDARG00000091156 cytochrome P450, family 2, subfamily AA, polypeptide 12 cyp2aa12 2.79ENSDARG00000091211 alcohol dehydrogenase 8a adh8a 4.55ENSDARG00000091996 si:ch211-117 m20.5 si:ch211-117 m20.5 3.81

Response to estrogenENSDARG00000018478 alanine-glyoxylate aminotransferase b agxtb 5.67ENSDARG00000020364 fructose-1,6-bisphosphatase 1b fbp1b 7.64ENSDARG00000043279 aquaporin 12 aqp12 1.62

Response to hormoneENSDARG00000015263 adrenomedullin a adma 1.92ENSDARG00000094857 iodothyronine deiodinase 2 dio2 4.40

Response to stressENSDARG00000011166 carbonic anhydrase cahz 1.81ENSDARG00000018263 protein disulfide isomerase family A, member 2 pdia2 4.31ENSDARG00000027620 beta-carotene 15, 15-dioxygenase 2, like bco2l 3.26ENSDARG00000071306 gastric inhibitory polypeptide gip 8.37ENSDARG00000079296 glucagon a gcga 2.72ENSDARG00000089087 BA1 globin ba1 6.19ENSDARG00000097238 hemoglobin, beta adult 1 ba2 4.72

Circadian rythmENSDARG00000004060 basic helix-loop-helix family, member e40 bhlhe40 2.59ENSDARG00000091616 prokineticin 2 prok2 1.58

Table 3Genes assigned to the biological functions cell death, cell differentiation, cell proliferation, cell adhesion, cell communication from the functional analyses of the transcriptssignificantly differentially expressed in testes of zebrafish developmentally exposed to 1.2 ng/L and remediated in clean water (FDR-corrected p values < 0.05). Categories wereassigned from the information of GO-terms found in Zfin and MGI.


Cell deathENSDARG00000040178 hepatitis A virus cellular receptor 1 havcr1 2.56ENSDARG00000040930 DEP domain containing MTOR-interacting protein deptor 3.04ENSDARG00000060682 anterior gradient 1 agr1 3.71ENSDARG00000070480 anterior gradient 2 agr2 2.65ENSDARG00000078859 G0/G1 switch 2 g0s2 3.04

Cell differentiationENSDARG00000007682 pancreatic progenitor cell differentiation and proliferation factor a ppdpfa 1.84ENSDARG00000031898 neuroblastoma 1, DAN family BMP antagonist NBL1 3.12ENSDARG00000040747 transmembrane 4 L six family member 4 tm4sf4 4.61ENSDARG00000052004 caveolin 1 cav1 1.87ENSDARG00000068784 zgc:153073 VSIR 1.65

Cell proliferationENSDARG00000055683 Pim proto-oncogene, serine/threonine kinase, related 191 pimr191 2.96ENSDARG00000073898 cytoskeleton associated protein 5 ckap5 3.94

Cell adhesionENSDARG00000009544 claudin b cldnb 5.76ENSDARG00000015955 claudin c cldnc 1.93ENSDARG00000018923 FAT atypical cadherin 2 fat2 2.15ENSDARG00000045835 si:dkey-14d8.6 si:dkey-14d8.6 6.49ENSDARG00000056767 integrin beta 3a itgb3a 3.20ENSDARG00000060715 procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 plod3 5.42ENSDARG00000061186 integrin alpha 4 itga4 1.61ENSDARG00000069503 claudin h cldnh 4.33ENSDARG00000076386 ependymin-like 1 epdl1 2.85ENSDARG00000045834 si:dkey-14d8.7 si:dkey-14d8.7 6.70

Cell communicationENSDARG00000040799 somatostatin 1, tandem duplicate 1 sst1.1 5.30ENSDARG00000041787 connexin 32.3 cx32.3 5.59ENSDARG00000041797 connexin 28.9 cx28.9 5.97ENSDARG00000041799 connexin 43 cx43 2.13ENSDARG00000056134 C1q and TNF related 5 c1qtnf5 2.32ENSDARG00000058733 Indian hedgehog homolog a ihha 3.51ENSDARG00000078366 roundabout, axon guidance receptor, homolog 2 robo2 1.66



expression. Further, some genes involved in apoptotic signalling,deptor, havcr1 and g0s2 showed increased expression. The classifi-cations under these three categories are, however, somewhat arbi-trary, as many genes received more than one of the three GO-terms.

3.6. Cell communication, migration and adhesion

In the cell communication category (Table 3), EE2-dependentupregulation of three genes encoding connexins, cx32.3, cx28.9and cx43, were discerned. In this group were also the genes robo2,roundabout axon guidance receptor homolog 2, inha, Indian hedge-hog homolog a and sst1, somatostatin 1, tandem duplicate 1, foundto have increased expression. Two genes with function in cellmotility (Table 6), dnaaf2 and mylka, were upregulated; the formerby both 1.2 and 1.6 ng/L EE2. Cell adhesion-associated genes(Table 3) showing increased expression includes FAT atypical cad-herin, fat2, one integrin a (itga4) and one integrin b, (itgb3a). Also,epdl1, involved in cell-matrix adhesion showed higher expressionin response to EE2 together with si:dkey-14d8.6 and si:dkey-14d8.7 (increased by both exposures), the gene products of whichare involved in collagen fibril organization.

3.7. Cell signalling and regulation of transcription

The genes under the category cell signalling and regulation oftranscription are shown in Table 4. Differential expression of 20genes involved in cell signalling were observed in testis in response

Table 4Genes assigned to the biological functions cell signalling and regulation of transcription frtestes of zebrafish developmentally exposed to 1.2 ng/L and remediated in clean water (Fterms found in Zfin and MGI.

Ensemble ID Gene name

Cell signalingENSDARG00000009978 ictacalcinENSDARG00000012071 angiopoietin-like 1aENSDARG00000012968 ras homolog family member UbENSDARG00000024759 inhibin, beta AbENSDARG00000027807 fyn-related Src family tyrosine kinaseENSDARG00000037116 chemokine (C-X-C motif) ligand 12aENSDARG00000041110 DnaJ (Hsp40) homolog, subfamily C, mENSDARG00000042189 tetraspanin 33bENSDARG00000052000 caveolin 2ENSDARG00000052470 insulin-like growth factor binding proENSDARG00000056045 si:ch73-139e5.4ENSDARG00000058115 fibroblast growth factor receptor 2ENSDARG00000058991 CU571081.1ENSDARG00000059348 relaxin/insulin-like family peptide recENSDARG00000060627 huntingtin interacting protein 1 relatENSDARG00000069388 transmembrane protein 88bENSDARG00000070863 chondroitin sulfate proteoglycan 5bENSDARG00000070903 MET proto-oncogene, receptor tyrosinENSDARG00000089544 alanyl (membrane) aminopeptidaseENSDARG00000090106 solute carrier family 17

Regulation of transcriptionENSDARG00000011956 distal-less homeobox 4aENSDARG00000012078 Meis homeobox 1bENSDARG00000017400 Kruppel-like factor 1ENSDARG00000019150 forkhead box A1ENSDARG00000020354 LIM homeobox transcription factor 1,ENSDARG00000036074 CCAAT/enhancer binding protein (C/EENSDARG00000042725 CCAAT/enhancer binding protein (C/EENSDARG00000055398 forkhead box C1bENSDARG00000056030 homeobox B7aENSDARG00000061391 grainyhead-like transcription factor 1ENSDARG00000074397 basonuclin 1ENSDARG00000077982 E74-like factor 3ENSDARG00000079078 si:ch211-5 k11.8ENSDARG00000086881 immediate early response 2ENSDARG00000091127 Kruppel-like factor 15

to developmental EE2 exposure. Among them were the ras familymember gene rhoub, met coding for MET receptor tyrosine kinaseand fynrk, encoding a Src kinase. Two genes with potential rele-vance for fertility were also upregulated, inhbab encoding inhibinb Ab which in mouse is involved in male gonad development andcxcl12a, which gene product is a chemokine ligand involved ingerm cell migration in zebrafish. Further, the genes fgfr2 and rfx-p3.3b encoding a fibroblast growth factor receptor and a relaxin/insulin-like family peptide receptor, respectively were also upreg-ulated as well as igfbp2a, an insulin-like growth factor bindingprotein.

Many of the 15 differentially expressed genes in the group reg-ulation of transcription encoded homeobox transcription factorsinvolved in development, such as dlx4a, kflf1. klfl15, hoxb7a, foxa1and foxc1b were upregulated while lmx1a was downregulated. Inaddition, upregulation of two genes encoding CAAT/enhancerbinding transcription factors were observed, cebpa and cebpb. Also,the expression of BNC1 encoding basonuclin, which has noGO-identification in zebrafish but in mouse is involved inspermatogenesis and chromosome organization, is enhanced bydevelopmental EE2 exposure.

3.8. Proteolysis

This category, shown in Table 5, showed the largest number ofgenes, 28, all having increased expression, demonstrating a mas-sive increased proteolytic function in testis in response to EE2exposure. Among them are 6 of 7 members of the cela1 family, four

om the functional analyses of the transcripts significantly differentially expressed inDR-corrected p values < 0.05). Categories were assigned from the information of GO-

Gene ID logFC

icn 1.95angptl1a 2.35rhoub 1.41inhbab 1.93fynrk 4.85cxcl12a 1.96

ember 3a dnajc3a 1.69tspan33b -3.58cav2 1.83

tein 2a igfbp2a 3.30GUCY2C -1.29fgfr2 1.58CU571081.1 2.46

eptor 3.3b rxfp3.3b 2.47ed a hip1Ra -1.50

tmem88b 2.03cspg5b -1.55

e kinase met 1.72ANPEP (4 of 7) 6.45slc17a7b 1.70

dlx4a 2.76meis1b 1.51klf1 3.52foxa1 3.28

alpha lmx1a -1.75BP), alpha cebpa 2.12BP), beta cebpb 2.29

foxc1b 2.46hoxb7a 2.20grhl1 4.25BNC1 1.96elf3 7.73si:ch211-5 k11.8 4.51IER2 1.91klf15 1.80

Table 5Genes assigned to the biological functions Immune system process, Proteolysis and Other from the functional analyses of the transcripts significantly differentially expressed intestes of zebrafish developmentally exposed to 1.2 ng/L and remediated in clean water (FDR-corrected p values < 0.05). Categories were assigned from the information of GO-terms found in Zfin and MGI.


Immune system processENSDARG00000015626 peptidoglycan recognition protein 6 pglyrp6 5.24ENSDARG00000062788 immunoresponsive gene 1, like irg1l 5.56ENSDARG00000075163 chemokine (C-X-C motif) ligand 20 cxcl20 2.87

ProteolysisENSDARG00000030915 carboxypeptidase A1 cpa1 5.90ENSDARG00000010146 carboxypeptidase A2 cpa2 4.07ENSDARG00000043722 carboxypeptidase A4 cpa4 5.29ENSDARG00000021339 carboxypeptidase A5 cpa5 5.89ENSDARG00000024503 six-cysteine containing astacin protease 3 c6ast3 5.79ENSDARG00000052578 six-cysteine containing astacin protease 4 c6ast4 5.35ENSDARG00000034862 coagulation factor VII f7 6.24ENSDARG00000088581 coagulation factor X f10 2.05ENSDARG00000039579 complement factor D cfd 2.79ENSDARG00000039730 zgc:112160 zgc:112160 6.49ENSDARG00000040282 zgc:92590 zgc:92590 5.36ENSDARG00000041644 transmembrane protein 27 TMEM27 2.86ENSDARG00000042993 protease, serine 1 prss1 6.38ENSDARG00000017314 zgc:92041 cela1(1 of many) 6.04ENSDARG00000043171 zgc:112302 cela1 (3 of many) 4.55ENSDARG00000043173 zgc:92745 cela1 (4 of many) 5.59ENSDARG00000043175 zgc:112368 cela1 (5 of many) 6.03ENSDARG00000077058 zgc:92511 cela1 (6 of many) 5.85ENSDARG00000095462 si:dkey-57c15.4 cela1 (7 of many) 5.87ENSDARG00000044204 endonuclease, polyU-specific endou 5.33ENSDARG00000056744 elastase 2 ela2 5.43ENSDARG00000056765 elastase 2 like ela2l 5.92ENSDARG00000007276 elastase 3 like ela3l 5.99ENSDARG00000068680 chymotrypsin-like ctrl 6.46ENSDARG00000090428 chymotrypsinogen B1 ctrb1 6.43ENSDARG00000079274 prss59.1 prss59.1 6.21ENSDARG00000073742 protease, serine, 59, tandem duplicate 2 prss59.2 6.66ENSDARG00000093844 zgc:136461 zgc:136461 7.26

OtherENSDARG00000015337 catechol-O-methyltransferase a Comta 4.81ENSDARG00000038424 si:dkey-8 k3.2 Si:key-83 k3.2 2.97ENSDARG00000054239 ghrelin/obestatin prepropeptide Ghrl 6.75ENSDARG00000095590 secreted phosphoprotein 2 Spp2 2.89


members of carboxypeptidase A family (cpa4 by both concentra-tions) and 3 in the elastase family.

3.9. Immune system processes

Altered expression of three genes (Table 5) involved in inflam-matory response and defence response to bacterium were found,pglyrp6 encoding a peptidoglycan-recognising protein, irg1l,immune responsive gene1, like and cxcl20, encoding chemokineligand 20. It should be noted that many genes in other categorieswere associated to function in immune response in zebrafishand/or mouse.

3.10. Structural components, cytoskeleton and transport

Genes coding for structural components, cytoskeleton andtransport are found in Table 6. Six structural genes encoding ker-atins (krt1-19d, 4, 5, 17, 18 and 92), in mouse involved in interme-diate filament organization, showed increased transcriptabundance in EE2 exposed testis. In the category cytoskeleton therewas increased expression of genes involved in actin filament poly-merization and organization, such as scin, tagln2, fhl3b, pls3 andzgz:86896 as well as RGS14, with a function in mouse in spindleorganization and chromosome segregation. Enhanced expressionof genes coding for proteins involved in ion transport wasobserved, including grid1b, slc4a4b and slco2b1, and three aquapor-

ins. Also, clc16a7 and -a9a in the solute carrier family 16, engagedin monocarboxylic acid trans-membrane transport, and the cacnagene, encoding a calcium channel protein which in mouse isinvolved in sperm motility, showed increased expression.

3.11. Non-coding RNA and post-transcriptional process

Non-coding RNA and post-transcriptional process are categoriesshown in Table 7. Increased abundance of several miRNAs wasobserved in response to EE2 exposure during development:mir462 and mir122 by 1.2 ng/L, mir16b by 1.6 ng/L and mir214 byboth exposure concentrations. The unknown miRNA AL935186.3was markedly decreased in abundance in the group exposed to1.2 ng/L. The category post-transcriptional processes containedaltered abundance of unknown linc-, sno- sn- and misc RNAs,unknown antisense RNAs and a mitochondrial tRNA. In addition,zpf3611, involved in mRNA destabilization and, in mouse, in nega-tive regulation of mitotic phase transition and si:ch211-153b23.4engaged in regulation of translation fidelity were all increasedwhile pdcb, involved in queuosine biosynthetic process and si:ch211-153b23.4 was decreased.

3.12. Verification of differential expression by qPCR

Analysis of gene expression performed by qPCR in isolatedtestes samples from control fish and fish exposed to 1.2 ng/L EE2

Table 7Genes assigned to the biological functions Post-transcriptional process and Non-coding RNA from the functional analyses of the transcripts significantly differentially expressed intestes of zebrafish developmentally exposed to 1.2 ng/L and remediated in clean water (FDR-corrected p values < 0.05). Categories were assigned from the information of GO-terms found in Zfin and MGI.


Post-transcriptional processENSDARG00000016154 zinc finger protein 36, C3H type-like 1a zfp36l1a 1.32ENSDARG00000017634 phosducin b pdcb -6.37ENSDARG00000077169 si:ch211-153b23.4 si:ch211-153b23.4 4.51

Non-coding RNAENSDARG00000081077 U1 spliceosomal RNA U1* -2.64ENSDARG00000081580 Small nucleolar RNA SNORD99 SNORD99 1.93ENSDARG00000082123 si:dkey-262 k9.3 si:dkey-262 k9.3 2.28ENSDARG00000082377 microRNA 462 mir462 3.59ENSDARG00000083042 microRNA 214a mir214a* 4.44ENSDARG00000083634 micrRNA122 mir122 5.99ENSDARG00000085168 AL935186.3 AL935186.3 -7.44ENSDARG00000086157 Small nucleolar RNA SNORD60 SNORD60 -1.55ENSDARG00000086676 Metazoan signal recognition particle RNA Metazoa_SRP 2.08ENSDARG00000086852 Metazoan signal recognition particle RNA Metazoa_SRP 2.90ENSDARG00000087368 Metazoan signal recognition particle RNA Metazoa_SRP 2.93ENSDARG00000088158 Metazoan signal recognition particle RNA Metazoa_SRP 4.46ENSDARG00000097712 CR848667.1 CR848667.1 -1.68ENSDARG00000097926 si:dkey-262 k9.3 si:dkey-262 k9.3 2.39ENSDARG00000097996 CR848784.5 CR848784.5* 6.97ENSDARG00000090559 Metazoan signal recognition particle RNA Metazoa_SRP 2.78ENSDARG00000083266 Small nucleolar SNORD12/SNORD106 SNORD12 -2.36

Table 6Genes assigned to the biological functions Cytoskeleton, Structural components and Transport from the functional analyses of the transcripts significantly differentially expressedin testes of zebrafish developmentally exposed to 1.2 ng/L and remediated in clean water (FDR-corrected p values < 0.05). Categories were assigned from the information of GO-terms found in Zfin and MGI.


CytoskeletonENSDARG00000004017 sperm associated antigen 1a spag1a 2.60ENSDARG00000010728 scinderin scin 2.21ENSDARG00000032405 zgc:85975 zgc:85975 1.41ENSDARG00000033466 transgelin 2 tagln2 1.86ENSDARG00000037655 plastin 3 (T isoform) pls3 1.93ENSDARG00000058348 scinderin like b scinlb 1.49ENSDARG00000059158 four and a half LIM domains 3b fhl3b 1.77ENSDARG00000060200 regulator of G protein signalling 14 RGS14 (2 of 2) 2.59ENSDARG00000061862 myosin XVIIIAb myo18ab 1.77ENSDARG00000090369 zgc:86896 zgc:86896 2.49

Structural componentsENSDARG00000017624 keratin 4 krt4 2.98ENSDARG00000023082 keratin, type 1, gene 19d krt1-19d 3.42ENSDARG00000036830 keratin 17 krt17 4.14ENSDARG00000036834 keratin 92 krt92 2.90ENSDARG00000041339 krt18 krt18 7.77ENSDARG00000058371 keratin 5 krt5 5.54

TransportENSDARG00000003808 aquaporin 3a aqp3a 3.98ENSDARG00000007275 si:ch211-251b21.1 si:ch211-251b21.1 1.94ENSDARG00000013926 solute carrier family 16, member 9a slc16a9a 2.41ENSDARG00000021573 solute carrier family 16, member 7 (monocarboxylic acid transporter 2) slc16a7 1.60ENSDARG00000023290 fatty acid binding protein 3 fabp3 2.29ENSDARG00000023713 aquaporin 1a (colton blood group) tandem duplicate 1 aqp1a.1 3.21ENSDARG00000029832 solute carrier family 26 (anion exchanger), member 1 slc26a1 5.31ENSDARG00000042617 aquaporin 11 aqp11 1.90ENSDARG00000044161 glutamate receptor, ionotropic, delta 1b grid1b -2.35ENSDARG00000044808 solute carrier family 4 (sodium bicarbonate cotransporter), member 4b slc4a4b 1.42ENSDARG00000054609 solute carrier organic anion transporter family, member 2B1 slco2b1 2.56ENSDARG00000060350 apolipoprotein Da, duplicate 2 apoda.2 4.12ENSDARG00000061990 potassium voltage-gated channel, subfamily H (eag-related), member 4b kcnh4b -2.97ENSDARG00000062346 calcium channel, voltage-dependent, R type, alpha 1E subunit a cacna1ea -1.45ENSDARG00000088079 transmembrane p24 trafficking protein 6 tmed6 3.16


(n = 5 in both groups) aimed to verify the differential expressionidentified by RNAseq. (Fig. 2) There was a significant upregulationof bhle40 (t = 2.24, df = 7.6, p = 0.028), cxcl20 (t = 4.51, df = 7.2, p =0.001), bhmt (t = 2.39, df = 4.15, p = 0.036), ptgs2a (t = 2.62, df = 6.9,

p-= 0.017) and ba1 (t = 2.43, df = 8.0, p = 0.021), in agreement to thetranscriptome analysis while no significant alteration wasobserved in cxcl12 (t = 0.99, df = 6.3, p = 0.179) expression com-pared to relative expression in control testes.

Fig. 2. Relative mRNA expression of bhle40 (log fold change in RNAseq 2.59), cxcl20 (2.87), bhmt (6.03), ptgs2a (2.78), ba1 (6.19) and cxcl12 (1.96) in the testes of fishdevelopmentally exposed to 0 or 1.2 ng/L of EE2 and remediated in clean water for 82 days. The mRNA was quantified with qPCR and normalized against the expression ofElf1a. Data represent mean ± SEM of 5 samples per exposure group, *p < 0.05, **p < 0.01.


4. Discussion

In search for transcriptome alterations accompanying thedecreased fertilization success after a long remediation to adult-hood in zebrafish males developmentally exposed to low concen-trations of EE2 (Volkova et al., 2015), we performed analysis ofthe testis transcriptome in these males. RNA sequencing is a pow-erful way to identify exposure-generated alterations without á pri-ori target selection, resulting in a bias-free experimental approach.In this analysis, 249 and 16 genes showed significantly differentialexpression in testes after exposure during development to 1.2 and1.6 ng/L, respectively, and after long remediation in clean water,compared with testes from unexposed fish. The lower number ofdifferentially expressed genes in the 1.6 exposure group wasaccompanied by a larger intra-sample variation in this group,which decreases the possibility to significantly validate differencesbetween exposure groups. Most of the genes (11 of 16, Table 8) dif-ferentially expressed in the 1.6 ng/L exposure group testes werealso differentially expressed in testes in the 1.2 ng/L exposuregroup. This indicates that the difference in number of significantlydifferently expressed genes between exposure levels can be due todifferences in experimental variation, and not necessarily to bio-logical differences. However, EDCs often show non-monotonicdose–response curves at very low doses (Vandenberg et al.,2012) and it cannot be excluded that the variation betweenexposure groups reflect a true biological variation due to aninverted U-shaped dose–response curve. Transcriptome analysesof zebrafish are still sparse, and this study is the first documentinglong lasting gonadal transcriptome changes accompanying thedecreased fertility after developmental exposure to EE2 and a longremediation period, with several findings of relevance regardingthe lowered fertility.

It is well established in fish that effects seen on sex ratio, gonadmaturity, brain aromatase and vtg after adult and developmental

exposure to EE2 are reversible after remediation (Hill and Janz,2003, Baumann et al., 2014, Larsen et al., 2009). There is no gonadhistology data from the fish in this study but several studies haveshown that also gonad morphology is restored so that no morpho-logical discrepancies could be found after long remediation periodswhen exposure levels are low (Maack and Segner, 2004, Weberet al., 2003, Xu et al., 2008), suggesting, but not excluding, that his-tological changes were not present in the testis of the developmen-tally exposed zebrafish in this study.

Estrogens have a role in the developing testis and estrogenreceptors and aromatase are found at all stages of testicular devel-opment (O’Donnell et al., 2001). Estrogen is also important forspermatogenesis (Carreau et al., 2011, O’Donnell et al., 2001) andestrogen receptors are abundantly present in fish gonads in bothmales and females (Filby And Tyler, 2005). It has been suggestedthat exposure to exogenous estrogens during development leadingto reduction in testis size and sperm counts in rodents is caused byestrogen interfering with functional maturation of Sertoli cells(Sharpe, 1998). Exposure to EE2 during adulthood has previouslybeen shown to affect genes related to development, and male sexdetermination and differentiation in fish testis (ReyhanianCaspillo et al., 2014, Filby et al., 2007, Feswick et al., 2016,Nikoleris et al., 2016) and a few of these genes, such as ptgs2aand srd5a2a, where found to be differentially expressed in thisstudy. Exposure to EE2 during adulthood also lead to alterationsin gene expression in the HPG axis in both brain and testis in pej-jerey fish (Garriz et al., 2017). DES, another synthetic estrogen hasshown to impair spermatogenesis in zebrafish by disputing keygenes in the HPG axis as well as meiosis and apoptosis pathways(Yin et al., 2017) and also juvenile catfish show alterations in theHPG axis under ongoing exposure to DES (Liu et al., 2018). Recentstudies with ER-knockout fish and aromatase deficient zebrafishhave shown that fertility in males seems not to be directly depen-dent on estrogen signalling (Tang et al., 2017, Lu et al., 2017) but

Table 8Genes from significantly differentially expressed in the testes of zebrafish developmentally exposed to the 1.6 ng/L EE2 and remediated in clean water (FDR-corrected p values <0.05). The genes are displayed under the categories assigned to them in the functional analysis from information of GO-terms found in Zfin and MGI. Gene IDs marked* are alsofound differentially expressed in the 1.2 ng/L EE2 exposure.

Ensembl ID Gene name Gene logFC

Carbohydrate metabolic processENSDARG00000009443 zgc:92137 zgc:92137* 2.68

Cell migrationENSDARG00000041134 dynein, axonemal, assembly factor 2 dnaaf2* 1.97

Cell communicationENSDARG00000045834 si:dkey-14d8.7 si:dkey-14d8.7* 3.61

ProteolysisENSDARG00000043722 carboxypeptidase A4 cpa4* 2.85ENSDARG00000093844 zgc:136461 zgc:136461* 3.90Lipid metabolic processENSDARG00000053966 cytochrome P450, family 17, subfamily A, polypeptide 2 cyp17a2 1.54

DNA repairENSDARG00000063276 mutS homolog 3 msh3 -7.31Cell proliferationENSDARG00000073898 cytoskeleton associated protein 5 ckap5* 3.99

Non-coding RNAENSDARG00000081252 microRNA16b mir16b 2.34ENSDARG00000083042 microRNA214a mir214a* 4.06ENSDARG00000082628 U1 spliceosomal RNA U1* 5.35ENSDARG00000080404 U6 spliceosomal RNA U6 -2.18ENSDARG00000097996 CR848784.5 CR848784.5* 7.17

UnknownENSDARG00000040553 transmembrane protein 97 tmem97 3.21ENSDARG00000074613 si:ch211-240 l19.6 si:ch211-240 l19.6* 3.17ENSDARG00000088811 BX248496.1 BX248496.1* 2.64


were mediated via influence of the HPG axis. In the aromatase defi-cient zebra fish LH and FSH protein levels were also increased inthe pituitary (Tang et al., 2017). A previous transcriptome studyof the brain of zebra fish developmentally exposed to EE2 in similarconcentrations showed no differential expression of genes con-nected to the HPG axes after a long recovery period in clean water(Porseryd et al., 2017). However, in the present study we find somegenes and pathways previously shown to be upregulated in fishtestis by the gonadotropins FSH and LH (Sambroni et al., 2013).For example genes coding for inhibin subunits, cytochrome c oxi-dase subunits, keratin, and insulin growth factor binding proteinwere shown to be upregulated by FSH and/or LH in concordancewith the present study. This support that the HPG axis is involvedin the organizational effects of EE2 exposure during developmentin the testis, although no longer affected in the brain after termina-tion of exposure.

A low number of genes were connected to the GO-termresponse to estrogen in this study, 3 in the 1.2 ng/L exposure groupand none in the 1.6 ng/L exposure group, supporting that directeffects on estrogen target genes is to some extent remediated afterrecovery of exposure. In addition, upregulation was observed ofmeis1b, recently shown to be regulated by estrogen, but not classi-fied under the response to estrogen GO-term. The encoded proteinis a Hox cofactor, contributing to Hox activity during development(Moens and Selleri, 2006). The low number of traditional estrogentargets among the differently expressed genes in this study are inagreement with that developmental EE2 exposure in these malesdid not lead to organizational alterations in expression of vtg. qPCRanalysis of the expression of vtg2 in the livers of the males did notshow any significant difference in relative expression between thecontrol group and the two exposure groups (Volkova et al., 2015).Vtg expression is among the consequences of estrogen exposurethat has been shown to be fully reversed after cessation of develop-mental exposure.

The highest number of affected genes were found in the cate-gory proteolysis (23). Together with 11 genes differently regulated

in protein metabolic process, this indicates that EE2 exposure havelarge impact on the cellular and extracellular protein state in testis.GO-terms related to protein modification and protein metabolismhas been shown to be the most affected biological processes in zeb-rafish males exposed to 5 ng/L EE2 for 21 days during adulthood(Santos et al., 2007). In addition, the EE2 exposure induced alter-ations in sperm quality in that study.

16 gene transcripts with differential expression were catego-rized under the pathway lipid metabolic processes. Among themupregulation of the gene encoding prostaglandin-endoperoxidasesynthase 2a, ptgs2a was observed in the 1.2 ng/L EE2 exposuregroup. Ptgs2a, also named COX 2, is involved in embryogenesisand suggested to take an active part in sex determination in zebra-fish. High expression of ptgs2a support ovary development, andinhibition result in male-biased sex ratios (Pradhan et al., 2012)and ptgs2 has been shown to be upregulated by adult exposureof EE2 in a study of male fathead minnow testis gene expression(Garcia-Reyero et al., 2009). Possibly, the increased expressionobserved in the present study is a sign of de-masculinization.Upregulation of prostaglandin D2 synthase b, tandem duplicate1, ptgdsb.1, was also observed. Other members of the prostaglandinD2 synthase family mediate the conversion of prostaglandin H2 toprostaglandin D2, which is associated with testis differentiation(Pradhan and Olsson, 2014) but the present form b1 does not seemto have prostaglandin D-synthase activity (Fujimori et al., 2006).More studies are needed to assess the biological significance ofthese findings.

Two genes upregulated by EE2 in the category lipid metabolicprocess encode enzymes in steroid hormone biosynthesis,cytochrome P-450 17a2, cyp17a2, in the 1.6 ng/L exposure groupand steroid 5a-reductase a2a (srd5a2a) in the 1.2 ng/L exposuregroup. The cyp17a2 protein product converts progesterone to17a-hydroxyprogesterone, and further to androstenedione orDHEA. The protein product of cyp17a2 in zebrafish does notperform the second, 17,20-lyase reaction associated with thisenzyme in mammals (Pallan et al., 2015). This might lead to an


accumulation of 17a-progesterone and an unbalance amongsteroidogenic substrates. The srd5a2a gene product convertstestosterone to dihydrotestosterone, an androgen more effectivelythan testosterone, which has been regarded as less importantin the gonads as opposed to the brain. It is synthesized in fishtestes (Margiotta-Casaluci et al., 2013), but the biologicalfunction of dihydrotestosterone in fish gonads is poorly known(Martyniuk et al., 2013). The SRD5A enzyme can also convert4-androstenedione to 5a-androstenedione, a pathway towards lessactive androgens (Martyniuk et al., 2013). Further, in the categoryresponse to hormones, upregulation of adrenomedullin a, adma,which in mouse is involved in androgen metabolism, wasupregulated. The physiological implications of these findings areat present unclear, but it cannot be excluded that testicularhormone homeostasis is affected by EE2.

In the category cell signalling EE2 upregulated many genesinvolved in cell surface receptor signalling and in many signaltransduction pathways, such as MAPK, SMAD, NFkB, Src, Notch,Wnt, FGF and TGFb. The biological relevance of these changes isdifficult to interpret, but clearly EE2 altered cell signalling in a lotof ways capable of affecting testicular function. Exposure of EE2also affected several genes in the category regulation of transcrip-tion. Among them are many transcription factors involved indevelopment and differentiation, such as distal-less homeobox4a, dlx4a, Kruppel-like factor, klf, 1 and 15, forkhead box A1 andC1b, foxa and foxc1b, homeobox b7a, hoxb7a, and grainyhead-liketranscription factor, ghrl1. In this category is also the above-mentioned estrogen regulated Hox coactivator Meis homeobox1b, meis1b, and basonuclin 1, BNC1 which in mouse is involved inchromosome organization during spermatogenesis.

Three genes found upregulated in the testis were categorizedunder cell proliferation, six genes under cell differentiation andfour under apoptosis (Table 3). Spermatogenesis is crucial for tes-ticular function and dependent on a balance between cell prolifer-ation and apoptosis and the differentiation of the zebrafish testisfrom the juvenile ovary has been suggested to be regulated byapoptosis (Liew and Orban, 2014). Regulator of G protein signalling14, RGS14, and cytoskeleton-associated protein 5, ckap5, the latterupregulated in both exposure groups, have functions associatedwith spindle organization and centrosome duplication in mouseand zebrafish. In fish, adult exposure to EE2 result in decrease inearly spermatogenic stages and increase in apoptotic germ cells(Miller et al., 2012, Balch et al., 2004) and life-cycle EE2 exposurein Medaka resulted in male-specific apoptosis in the gonads.(Weber et al., 2004). Also a study of zebrafish gonads during differ-entiation revealed that expression of genes in apoptotic pathwayswere altered by ongoing EE2 exposure (Luzio et al., 2016). A genefound to strongly promote apoptosis, g0s2, were one of the genesupregulated by exposure to 1.2 ng/L EE2. g0s2was also upregulatedin a microarray study of medaka testis after 14 days adult EE2exposure, where altered expression of genes involved in apoptosis,cell cycle regulation and proliferation was found (Miller et al.,2012). Two additional genes in common with the present studywas observed, inha involved in cell communication and gamt cate-gorized under protein metabolic process. In a knockout mousemodel for GAMT deficiency, impaired fertility was demonstratedand morphological examinations revealed distinct changes in theseminiferous tubules to be the factor causing disturbed spermato-genesis (Schmidt et al., 2004).

In this study several genes in different categories were found tobe associated to germ cell function in zebrafish or mouse. Amongthem are inhibin beta Ab, inhbab, involved in male gonad develop-ment in mouse, chemokine (C-X-C motif) ligand 12a, cxcl12a,involved in germ cell migration in zebrafish and germ cell develop-ment in mouse, and forkhead box C1b, foxc1b, also denoted to theGO-term germ cell migration in mouse. The zinc-finger protein

basonuclin 2, encoded by the upregulated BNC1 gene, is involvedin regulation of meiosis initiation and progression in mouse malegerm cells (Vanhoutteghem et al., 2014), and has, together withguanidinoacetate N-methyltransferase, gamt, mentioned above,prokineticin 2, prok2, and retinoic binding protein 4, rbp4, theGO-term spermatogenesis in mouse. The similarities in the sper-matogenic process in teleost fish and mammals (Leal et al., 2009)support that the genes have a similar function in the zebrafish tes-tis. Further, the mouse orthologue to six-cysteine containing asta-cin protease, C6ast 3 and C6ast 4 is a negative regulator offertilization (Sachdev et al., 2012). Calcium channel, voltage-dependent, R type, alpha 1E subunit a, cacna1ea (Sakata et al.,2002) and MET proto-oncogene, met, (Catizone et al., 2002) areinvolved in sperm motility. Although the connections are superfi-cial, these results imply that testes that have been EE2-exposedduring development have several changes with relevance forfertility.

The expression of DNA mismatch recognition protein MutShomolog 3, msh3, was more than 7-fold lower in testis from theexposure group 1.6 ng/L compared to the control group. Mismatchrepair is strongly associated with recombination and repair duringmeiosis, and with fertility in eukaryotes (Surtees and Alani, 2006,Surtees et al., 2004). Deficiency in one of the mismatch repair pro-teins, Mlh1, results in zebrafish male sterility (Feitsma et al., 2007),and cadmium down-regulates the MutS homologs msh2 and msh6in developing zebrafish (Hsu et al., 2013). The lack of significanteffect on msh3 in testis after exposure to 1.2 ng/L EE2 makes, theconnection weaker.

Epigenetic alterations are tightly associated with male sper-matogenesis and infertility in mammals (Rajender et al., 2011),and EDCs have been shown to induce transgenerational effects inrats and mice on male fertility, with concomitant testis transcrip-tome and methylome alterations (Wolstenholme et al., 2011,Salian et al., 2009, Anway et al., 2008, Manikkam et al., 2012,Guerrero-Bosagna et al., 2012, Prados et al., 2015). Female preg-nant rats exposed to vinclozin, an androgen antagonist, had maleoffspring with reduced spermatogenic capacity passed on to gener-ation F4. The male offspring had cell defects in form of enhancedapoptosis and reduced sperm count and motility (Anway et al.,2005). A follow up study conducting transcriptome and epigenomeanalysis on sertoli cells from the F3 generation of the vinclozinmothers showed alterations in both the transcriptome and epigen-ome as a result of exposure. In the transcriptome over 400 geneswas differentially expressed and over 100 promoter differentialDNA methylation regions were found modified (Guerrero-Bosagna and Skinner, 2012). Similar transgenerational phenotypesof reduced fertility has been demonstrated in medaka (Oryziaslatipes) induced by EE2 and bisphenol A (Bhandari et al., 2015)and by dioxin in zebrafish (Baker et al., 2014), but the epigeneticshave so far not been investigated. Altered promoter methylationhas however been observed in the 5flanking region of the vtg 1gene in liver and brain in zebrafish exposed to EE2 (Stromqvistet al., 2010) and in several genes in zebrafish embryos exposedto benzo(a)pyrene (Corrales et al., 2014) suggesting that alteredmethylation pattern might be involved also in the transcriptomealterations in this study however, studies of the contribution ofhistone modifications and DNA methylation to the observed differ-ences at transcriptional level should be further analyzed.

In addition to DNA methylation and histone modifications alsomiRNAs can give rise to epigenetic alterations. miRNA regulationby estrogens and EDCs have been demonstrated (Klinge, 2012,Klinge, 2015c, Klinge, 2015a), affecting gene expression at post-transcriptional level. Nonylphenol changes miRNA-expression inmouse Sertoli cells, leading to cytotoxicity (Choi et al., 2011). Fourof the genes in the 1.2 ng/L exposure group and 2 in the 1.6 ng/Lexposure group were miRNAs with GO-terms epigenetic regulation


of gene expression and gene silencing (Table 7). They have previ-ously been shown to be expressed during zebrafish development(Chen et al., 2005). In this study the expression of mir214 wasincreased in both EE2 groups, mir122, mir462 and the novel mi-RNA Al935186.3 enhanced in the 1.2 ng/L exposure group andmir16b in the 1.6 ng/L exposure group (was just above significancelevel in 1.2 ng/L). Differential expression of mir214 have beenshown to alter the expression of genes in the Hedgehog and dis-patched 2 signalling pathways during zebrafish development(Flynt et al., 2007, Li et al., 2008). Estrogen exposure during adult-hood caused changes in miRNA expression in zebrafish liver andvitellogenin 3, vtg3, has been identified as a target gene formir122 and estrogen receptor 1, esr1, is a target gene for mir214(Cohen and Smith, 2014). It is possible that these alterations inmiRNAs might affect fertility by post-transcriptional effects on tes-tis gene expression. However, these observations warrant furtherexperiments at the post transcriptional level of protein expressionof miRNA target genes to clarify if miRNA contributes to the lowerfertility.

In conclusion, we used RNA sequencing to identify gene expres-sion modification in the testis transcriptome of zebrafish showingreduced fertility due to exposure to EE2 during development thatpersist as adults after a long period in clean water. We found alarge number of genes that were differentially expressed in testisas a result of the developmental EE2 exposure still discernible aftera long remediation in clean water. Differential expression of sev-eral genes involved in steroid metabolism, in testis developmentand function, and in spermatogenesis was observed that mightbe involved in the reduced fertility. Furthermore, differentialexpression of several miRNAs that might affect testes gene expres-sion on a post-transcriptional level, were identified.

Acknowledgments

This study was supported by the Swedish Baltic Sea Foundation(Grant no: 1556/42/2011) and the Stockholm County Council(Grant no:806/3.1.1/2014). The authors would like to acknowledgesupport of the National Genomics Infrastructure (NGI) hosted bySciLifeLab/Uppsala Genome Center and UPPMAX for providingassistance in massive parallel sequencing and computationalinfrastructure. Work performed at NGI / Uppsala Genome Centerhas been funded by RFI/VR and Science for Life Laboratory, Sweden.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at https://doi.org/10.1016/j.ygcen.2018.03.011.

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Testis transcriptome alterations in zebrafish General and...

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