Edited by: Allan V. Kalueff, St. Petersburg State University, Russia
Reviewed by: Matthew O. Parker, University of Portsmouth, UK; Denis Broock Rosemberg, Universidade Federal de Santa Maria, Brazil
*Correspondence: Tove Porseryd
†These authors have contributed equally to this work.
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The synthetic estrogen 17α-ethinylestradiol (EE2) is an endocrine disrupting compound of concern due to its persistence and widespread presence in the aquatic environment. Effects of developmental exposure to low concentrations of EE2 in fish on reproduction and behavior not only persisted to adulthood, but have also been observed to be transmitted to several generations of unexposed progeny. To investigate the possible biological mechanisms of the persistent anxiogenic phenotype, we exposed zebrafish embryos for 80 days post fertilization to 0, 3, and 10 ng/L EE2 (measured concentrations 2.14 and 7.34 ng/L). After discontinued exposure, the animals were allowed to recover for 120 days in clean water. Adult males and females were later tested for changes in stress response and shoal cohesion, and whole-brain gene expression was analyzed with RNA sequencing. The results show increased anxiety in the novel tank and scototaxis tests, and increased shoal cohesion in fish exposed during development to EE2. RNA sequencing revealed 34 coding genes differentially expressed in male brains and 62 in female brains as a result of EE2 exposure. Several differences were observed between males and females in differential gene expression, with only one gene,
17α-ethinylestradiol (EE2) is the most potent endocrine disrupting compound (EDC) pollutant contaminating the aquatic environment (Aris et al.,
In addition to the effects of EDCs on the reproductive axis in fish we and others have reported a significant influence also on non-reproductive behaviors in adult fish of importance for fitness, such as risky behavior, aggression, anxiety, and shoaling (Espmark Wibe et al.,
In this study, we analyze if persistent effects on gene expression in the zebrafish brain could be discerned in the adult fish after developmental EE2 exposure, accompanying an anxiogenic phenotype. We postulated that effects of anxiety should affect the regulation of genes related to the stress axis in the brain, but presumably also several other functions. To achieve this, we repeated the study mentioned above (Volkova et al.,
Fertilized zebrafish eggs from eight separate parental pairs of the wild type strain AB were obtained from the Karolinska Institute Zebrafish Core Facility, Solna, Sweden. Eggs from each parental pair were divided into three lots and assigned to treatment groups of 0, 3, and 10 ng EE2/L, respectively. EE2 (17α-ethinylestradiol, Sigma-Aldrich) was dissolved in acetone and stock solutions were mixed with pre-heated fish maintenance system water to final nominal concentrations of 0, 3, and 10 ng EE2/L. The nominal concentrations were considered relevant as they are within the range of concentrations measured in effluents and surface waters (Ternes,
Water samples were collected from different tanks at nine separate occasions during the exposure and stored in darkness at −20°C until analysis. EE2 concentrations were analyzed in single or duplicate samples as previously described in Volkova et al. (
The behaviors were analyzed in the novel tank test (NT; Egan et al.,
In the scototaxis test, the test tank (20 × 20 × 40 cm) was divided into one black and one white half and filled with pre-heated tap water up to 10 cm. The tank had two transparent central sliding doors, creating a compartment of 5 × 20 cm. The test fish was introduced into the central compartment, and after a 5 min habituation period the sliding doors were raised and the behavior of the fish was video recorded from above for 5 min. Latency to first entrance into the white half, number of transitions to white half and total time spent in white half was recorded.
All tests were video recorded and manually analyzed. Behavior analyses were not blinded for treatment due to logistical reasons. Three tanks were operating in parallel, and the behavior tests were performed between 9:00 a.m. and 1:00 p.m.
Fish previously macroscopically determined as males or females were re-examined based on secondary sexual characteristics, and separated before behavior testing. After behavior testing, two fish of each sex from each family group and treatment group were weighed and then sacrificed by anaesthetization in 0.5‰ 2-phenoxyethanol (Sigma-Aldrich) followed by immediate decapitation. During dissection, gonads and livers were removed and weighed and Gonadosomatic index (GSI) and hepatosomatic index (HSI) were calculated by the formula: organ weight/fish weight × 100. Brains were removed and stored at −80°C in RNA later (Sigma-Aldrich).
Based on the results in the novel tank test, RNA sequencing analysis was selected to be performed on control male brains and brains from males exposed to 3 ng/L EE2, while for females, brains were from unexposed females and females exposed to 10 ng/L EE2. Three biological replicates were used in the RNA sequencing analysis for each treatment group and sex. In the female analysis, fish from the same family groups were represented in both the control and treated samples; meaning that the control females and exposed females were siblings. Male samples were however taken from different family groups and the exposed males were therefore not the siblings of the control males. Brains were homogenized and total RNA extracted with TriReagent (Sigma-Aldrich) according to the manufacturer's protocol. Total RNA from selected samples was submitted to Bea Core Facility (Karolinska Institute, Huddinge, Sweden) for quality checking with R6K Screen Tape. IonProton RNA sequencing was performed by SciLifeLab (Uppsala University, Sweden) collecting ~40 million reads per sample.
The RNA was treated with Ribo-Zero™ rRNA Removal Kit (Epicentre/Illumina) to remove ribosomal RNA, and purified using Agencourt RNAClean XP Kit (Beckman Coulter). The RNA was then treated with RNaseIII according to the Ion Total RNA-Seq protocol (Life Technologies) and purified with Magnetic Bead Cleanup Module (Life Technologies). The size and quantity of RNA fragments were assessed on the Agilent 2100 Bioanalyzer system (RNA 6000 Pico kit, Agilent) before proceeding to library preparation, using the Ion Total RNA-Seq kit (Life Technologies). The libraries were amplified according to the protocol and purified with Magnetic Bead Cleanup Module (Life Technologies). Samples were then quantified using the Agilent 2100 Bioanalyzer system (Agilent) and Fragment Analyzer (Advanced Analytical) and pooled followed by emulsion PCR on the Ion OneTouch™ 2 system using the Ion Proton™ Template OT2 200 v3 Kit (Life Technologies) chemistry. Enriching was conducted using the Ion OneTouch™ ES (Life Technologies). Samples were loaded on an Ion PI v2 Chip (2 samples per chip) and sequenced on the Ion Proton™ System using Ion Proton™ Sequencing 200 v3 Kit (200 bp read length, Life Technologies) chemistry.
The software FastQC (
Classification of genes and predictions of biological gene function were performed manually, due to the observed low detection capacity of several automated classifications tested. Manual ortholog search was done in Ensembl. GO-terms were found in Zfin (for zebrafish), Entrez gene (for human and rodent orthologs) and NGNC (human orthologs).
For verification of the RNAseq results, the expression of selected differentially expressed genes in the male brain were analyzed with qPCR. RNA isolation was performed with individual brains from 10 control males and 10 males exposed to 3 ng/L, homogenized in TriReagent (0.8 ml/sample, Sigma-Aldrich, Germany) according to the manufacturer, quantified using NanoDrop ND-1000 spectrophotometer, and RNA quality verified by the 260/280 nm absorption ratio. Reverse transcription was performed from Quantitect® Reverse Transcription Kit (Qiagen) from 1 μg RNA per sample and then diluted 1:10 for qPCR reactions. Bio-Rad C1000 Touch™ Thermal cycler, CFX96™ Real-Time System was used for quantitative Real-Time PCR (qPCR). qPCR reactions were run in triplicates and contained 5 μL cDNA template (25 ng RNA), 2.5 μL each of forward and reverse primer and 10 μL iTaq™ Universal SYBR® Green Supermix (BIO-RAD), using cycling parameters as recommended by the manufacturer. Oligonucleotide primers (Table
18S r RNA |
5′-AATGTCTGCCCTATCAACTTTC-′3 |
3′-TGGATGTGGTAGCCGTTTC-′5 | |
Elf1a |
5′-TACCCTCCTCTTGGTCGCTTT-′3 |
3′-ACCTTTGGAACGGTGTGATTG-′5 | |
Msmo1 |
5′-TCAGCATCCCTTATGACTGG-′3 |
3′-AATGGAGAAGTGAAGTCGTGA-′5 | |
Sqlea |
5′-ACAGCTACCAGAGCACTTAAA-′3 |
3′-CGCATGTTATACGCATCTCC-′5 | |
Lss |
5′-TACAAGCATTTCTTGAGGCAG-′3 |
3′-CAGTCAGCAACTATCCAACC-′5 | |
Hmgcs1 |
5′-CGCTGCTACACTTTACTCCA-′3 |
3′-TAGTTTGCTAAATGGTGGGTTTC-′5 |
Data from the behavior tests, GSI and HSI, and qPCR was analyzed in the statistical package R (R Core Team,
The actual concentration of EE2 was determined from water samples from 9 occasions during the 80 days exposure period. The determined actual concentrations of EE2 (mean ± SEM) in samples taken from the treatment tanks were 2.14 ± 0.33 and 7.34 ± 1.42 ng/L for 3 and 10 ng/L, respectively. This represents 71 and 73% of the nominal concentrations. Control water samples contained no detectable levels (<0.5 ng/L) of EE2.
Of the dissected animals all fish phenotypically classified as females contained ovaries, and all fish phenotypically classified as males had testes (
Bodyweight (log) | 4.852 | 0.0884 | |||||
Controls | 16 | 0.393 | 0.018 | ||||
3 ng/L | 15 | 0.367 | 0.013 | − | |||
10 ng/L | 16 | 0.421 | 0.018 | − | |||
HSI (log) | 9.096 | 0.0106 |
|||||
Controls | 14 | 0.709 | 0.111 | ||||
3 ng/L | 15 | 0.875 | 0.129 | 0.221 | |||
10 ng/L | 16 | 1.005 | 0.101 | 0.0071 |
|||
GSI (log) | 10.05 | 0.0066 |
|||||
Controls | 16 | 1.558 | 0.222 | ||||
3 ng/L | 15 | 1.028 | 0.122 | 0.0117 |
|||
10 ng/L | 16 | 1.51 | 0.224 | 0.9750 | |||
Bodyweight (log) | 1.433 | 0.4886 | |||||
Controls | 16 | 0.616 | 0.036 | ||||
3 ng/L | 16 | 0.619 | 0.038 | − | |||
10 ng/L | 14 | 0.731 | 0.095 | − | |||
HSI (log) | 0.489 | 0.783 | − | ||||
Controls | 15 | 2.01 | 0.298 | − | |||
3 ng/L | 16 | 1.818 | 0.201 | − | |||
10 ng/L | 14 | 2.115 | 0.234 | − | |||
GSI (log) | − | ||||||
Controls | 16 | 12.08 | 1.168 | 2.871 | 0.238 | − | |
3 ng/L | 16 | 19.86 | 6.163 | − | |||
10 ng/L | 14 | 14.60 | 1.866 | − |
There were significant interactions between treatment and sex for latency to upper half (Chisq = 7.48,
Latency to upper half | 7.78 | 0.021 |
45.05 | <0.001 |
7.48 | 0.024 |
|||
Controls | 78 | ||||||||
3 ng/L | 79 | 0.139 | |||||||
10 ng/L | 78 | 0.022 |
|||||||
Total transitions (log) | 1.505 | 0.471 | 48.29 | <0.001 |
14.17 | <0.001 |
|||
Controls | 78 | ||||||||
3 ng/L | 79 | − | |||||||
10 ng/L | 78 | − | |||||||
Total time in upper half | 13.77 | 0.001 |
12.77 | <0.001 |
3.39 | 0.183 | |||
Controls | 78 | ||||||||
3 ng/L | 79 | 0.008 |
|||||||
10 ng/L | 78 | <0.001 |
|||||||
Latency to opposite half | 0.482 | 0.79 | 16.35 | <0.001 |
1.84 | 0.40 | |||
Controls | 57 | ||||||||
3 ng/L | 58 | − | |||||||
10 ng/L | 51 | − | |||||||
Total transitions | 24.80 | <0.001 |
202.48 | <0.001 |
1.94 | 0.40 | |||
Controls | 57 | ||||||||
3 ng/L | 58 | <0.001 |
|||||||
10 ng/L | 51 | <0.001 |
|||||||
Time in opposite half | 1.45 | 0.49 | 45.38 | <0.001 |
2.70 | 0.26 | |||
Controls | 57 | ||||||||
3 ng/L | 58 | − | |||||||
10 ng/L | 51 | − | |||||||
Latency to cross (log) | 5.74 | 0.057 | 21.32 | <0.001 |
0.77 | 0.68 | |||
Controls | 69 | ||||||||
3 ng/L | 68 | − | |||||||
10 ng/L | 79 | − | |||||||
Total transitions (log) | 13.03 | 0.0015 |
77.14 | <0.001 |
46.23 | <0.001 |
|||
Controls | 69 | ||||||||
3 ng/L | 68 | <0.001 |
|||||||
10 ng/L | 79 | 0.662 | |||||||
Time in white zone (log) | 6.16 | 0.046 |
7.0 | 0.0008 |
9.28 | 0.0097 |
|||
Controls | 69 | ||||||||
3 ng/L | 68 | 0.02 |
|||||||
10 ng/L | 79 | 0.88 |
To further evaluate the effects, we analyzed males (
Swimming activity measured during the last minute of the novel tank test revealed average ±
Fish that failed to make contact with the group within 5 min were excluded from the analyses which slightly lowered the number of observations (NControl = 57, N3 ng = 58, N10 ng = 51). Twenty-one (8 female and 13 male fish), 21 (13 female and 9 male fish), and 27 (24 female and 3 male fish) fishes where excluded for this reason in the 0, 3, and 10 ng/L treatment groups, respectively. EE2 exposure significantly decreased the number of transitions made away from shoal (Chisq = 24.8,
The models including both males and females (Ncontrol = 69, N3 ng = 68, N10 ng = 79) reveal significant interactions between sex and treatment in number of entries into white half (Chisq = 46.2,
Analyzing males (NControl = 39, N3 ng = 38, N10 ng = 40) and females (NControl = 30, N3 ng = 29, N10 ng = 39) separately (Figure
Of the 33,373 gene features that were annotated in genome sequence, 18,792 in the females, and 18,445 in males had gene expression levels high enough to be retained for analysis. One hundred and forty-six sequences, 95 protein-encoding, and 51 non-coding sequences, were observed to be differentially expressed in brains from developmentally EE2-exposed zebrafish after recovery, compared to brains from unexposed controls. In the brain samples from males, 34 coding genes showed significantly different expression levels between control males and males exposed to 3 ng/L EE2. For the female brains, 62 coding genes were differentially expressed due to the developmental exposure to 10 ng/L EE2. Only one gene was affected by developmental EE2 exposure in both male and female brains:
ENSDARG00000057652 | dbpb | 0.92 | 6.82 | <0.001 | <0.001 | Regulation of transcription (DNA templated), sequence-specific DNA binding | DBP |
ENSDARG00000033160 | nr1d1 | 2.21 | 5.98 | <0.001 | <0.001 | Regulation of transcription (DNA templated), Sequence-specific DNA binding, steroid and thyroid hormone receptor activity | NR1D1 |
ENSDARG00000056885 | per1a | 1.83 | 6.24 | <0.001 | <0.001 | Photoperiodism, signal transduction, response to hydrogen peroxide | PER1 |
ENSDARG00000058094 | CIART (1 of 2) | 1.99 | 5.54 | <0.001 | 0.003 | Not available | CIART |
ENSDARG00000088171 | CIART (2 of 2) | 0.81 | 6.21 | <0.001 | 0.03 | Not available | CIART |
ENSDARG00000075397 | cipca | 1.65 | 3.91 | <0.001 | 0.004 | Not available | CIPC |
ENSDARG00000041691 | bhlhe41 | 1.01 | 7.83 | <0.001 | 0.028 | Negative regulation of transcription (DNA templated), protein dimerization, DNA binding | BHLHE41 |
ENSDARG00000068468 | DNAH3 | −1.51 | 5.26 | <0.001 | <0.001 | Not available | DNAH3 |
ENSDARG00000056888 | DNAH8 | −1.86 | 6.06 | <0.001 | <0.001 | Not available | Unknown |
ENSDARG00000041723 | zgc:55461 | −1.48 | 4.72 | <0.001 | 0.002 | Protein polymerization, microtubule-based process, GTP binding, nucleotide binding | TUBB4A/TUBB4B |
ENSDARG00000042708 | tuba8l | −0.90 | 4.58 | <0.001 | 0.036 | Microtubule-based process, protein polymerization, GTP binding, nucleotide binding | TUBA8 |
ENSDARG00000087352 | AL935046.1 | −1.38 | 5.07 | <0.001 | 0.037 | Not available | DNAH2 |
ENSDARG00000001993 | myhb | 1.67 | 3.53 | <0.001 | 0.039 | Motor activity, ATP binding, actin binding, Myosin complex | MYH1/13/2/8/4 |
ENSDARG00000059987 | dnah12 | −1.10 | 3.88 | <0.001 | 0.045 | Microtubule-based movement, microtubule motor activity | DNAH12 |
ENSDARG00000040528 | Igals3bpb | 1.17 | 5.37 | <0.001 | <0.001 | Cell adhesion, scavenger receptor activity | LGALS3BP |
ENSDARG00000026611 | socs3b |
−1.21 | 4.43 | <0.001 | <0.001 | JAK-STAT cascade, protein kinase inhibitor activity | SOCS3 |
ENSDARG00000053136 | b2m | −0.80 | 6.69 | <0.001 | 0.001 | Immune system process, MHCI protein complex | B2M |
ENSDARG00000079412 | ftr02 | −3.20 | 1.39 | <0.001 | 0.004 | Metal ion binding, zinc ion binding | TRIM29 |
ENSDARG00000060711 | SV2B | −1.21 | 4.89 | <0.001 | 0.013 | Not available | SV2B |
ENSDARG00000074749 | abca12 | −3.08 | 2.93 | <0.001 | <0.001 | Lipid transport, cholesterol efflux, phospholipid transporter activity | ABCA12 |
ENSDARG00000039406 | prom2 | −1.96 | 4.03 | <0.001 | 0.003 | Integral component of membrane | PROM2 |
ENSDARG00000040295 | apoeb | −1.12 | 7.78 | <0.001 | 0.03 | Lipid transport, cholesterol biosynthetic process, negative regulation of neuron apoptotic process | APOE |
ENSDARG00000003462 | fech | −0.94 | 5.67 | <0.001 | <0.001 | Erythrocyte maturation, heme biosynthetic process, ferrochelatase activity | FECH |
ENSDARG00000031952 | mb | 3.13 | 3.31 | <0.001 | 0.002 | Vasculogenesis, oxygen transport, response to hypoxia, oxygen binding, heme binding | MB |
ENSDARG00000055101 | hmox2a | −0.72 | 5.41 | <0.001 | 0.008 | Hemeoxygenase (decyclizing) activity, oxidation-reduction process, heme oxidation | HMOX2 |
ENSDARG00000078529 | Bai1b | 0.82 | 8.61 | <0.001 | 0.04 | Regulation of angiogenesis | BAI1 |
ENSDARG00000089195 | pcdh2g20 | −1.90 | 2.10 | <0.001 | 0.02 | Cell adhesion, calcium ion binding | Unknown |
ENSDARG00000014047 | cldn7b | −2.32 | 2.87 | <0.001 | 0.04 | Structural molecular activity | CLDN7 |
ENSDARG00000092933 | cbx8a | −4.59 | 1.32 | <0.001 | <0.001 | Nucleus | CBX8 |
ENSDARG00000060645 | sirt7 | −1.34 | 4.49 | <0.001 | 0.004 | NAD+ binding | SIRT7 |
ENSDARG00000051970 | smu1b | 1.21 | 6.44 | <0.001 | 0.010 | Not available | SMU1 |
ENSDARG00000063572 | perp | −0.91 | 5.07 | <0.001 | 0.015 | Regulation of apoptotic process, response to UV | PERP |
ENSDARG00000020084 | tg | −2.62 | 1.93 | <0.001 | 0.004 | Carboxylic ester hydrolase activity | TG |
ENSDARG00000002494 | itgb6 | −2.29 | 1.70 | <0.001 | 0.018 | Integrin-mediated signaling pathway, cell adhesion, receptor activity | ITGB6 |
ENSDARG00000016470 | anxa5b | −2.15 | 3.06 | <0.001 | 0.02 | Negative regulation of coagulation, regulation of cell motility, calcium-dependent phospholipid binding | ANXA5 |
ENSDARG00000026611 | socs3b |
−1.21 | 4.43 | <0.001 | <0.001 | JAK-STAT cascade, protein kinase inhibitor activity | SOCS3 |
ENSDARG00000092115 | eif4a1a | −1.18 | 8.96 | <0.001 | <0.01 | Translational initiation, nucleotide binding, ATP binding, helicase activity, hydrolase activity | SENP3, EIF4A1 |
ENSDARG00000091245 | dnajc7 | −1.22 | 4.84 | <0.001 | <0.001 | Not available | DNAJC7 |
ENSDARG00000009978 | icn | −1.91 | 4.54 | <0.001 | 0.03 | Calcium ion binding | S100A2/3/4/5/6 |
ENSDARG00000069476 | spint2 | −3.43 | 3.45 | <0.001 | 0.01 | Serine-type endopeptidase inhibitor activity | SPINT2 |
ENSDARG00000024195 | znf395b | 0.95 | 5.74 | <0.001 | 0.01 | Metal ion binding | ZNF395 |
ENSDARG00000068923 | unmodl1 | −2.20 | 2.49 | <0.001 | 0.019 | Peptidase inhibitor activity, calcium ion binding | UMODL1 |
ENSDARG00000075984 | hsbp1l1 | −2.53 | 1.34 | <0.001 | 0.025 | Not available | HSBP1 |
ENSDARG00000038729 | s100z | −1.53 | 2.97 | <0.001 | 0.038 | Metal ion binding, calcium binding | S100Z |
ENSDARG00000074807 | tbrg4 | −0.75 | 4.65 | <0.001 | 0.048 | Protein kinase activity | TBRG4 |
ENSDARG00000052012 | rtn4rl2a | 1.09 | 5.51 | <0.001 | 0.042 | Not available | RTN4RL2 |
ENSDARG00000089382 | zgc:158463 | −3.63 | 7.93 | <0.001 | <0.001 | Not available | Unknown |
ENSDARG00000088436 | CT956064.3 | −3.58 | 5.84 | <0.001 | <0.001 | Not available | Unknown |
ENSDARG00000091744 | BX296557.7 | −2.97 | 7.17 | <0.001 | <0.001 | Not available | Unknown |
ENSDARG00000031588 | si:dkey-239b22.1 | −4.22 | 3.44 | <0.001 | <0.001 | Cell-matrix adhesion | Unknown |
ENSDARG00000068374 | si:ch211-132b12.7 | 1.50 | 6.56 | <0.001 | <0.001 | Not available | Unknown |
ENSDARG00000091234 | CU019646.2 | −2.25 | 2.30 | <0.001 | 0.001 | Not available | Unknown |
ENSDARG00000067703 | NLRP | 1.32 | 4.62 | <0.001 | 0.0037 | Not available | Unknown |
ENSDARG00000079175 | si:ch211-79k12.1 | −2.44 | 2.44 | <0.001 | 0.004 | Not available | Unknown |
ENSDARG00000088142 | Unknown | 1.72 | 4.74 | <0.001 | 0.004 | Not available | Unknown |
ENSDARG00000087067 | CABZ01073069.1 | 3.73 | 0.84 | <0.001 | 0.007 | Not available | Unknown |
ENSDARG00000088938 | BX548011.4 | −3.12 | 1.34 | <0.001 | 0.008 | Not available | Unknown |
ENSDARG00000036700 | Unknown | −1.71 | 5.00 | <0.001 | <0.01 | Not available | Unknown |
ENSDARG00000093780 | si:ch211-212c13.6 | −2.42 | 3.63 | <0.001 | <0.01 | Not available | Unknown |
ENSDARG00000042829 | si:dkey-30j22.1 | −2.46 | 3.73 | <0.001 | 0.019 | Calcium ion binding, extracellular matrix structural constituent | Unknown |
ENSDARG00000070212 | zgc:158463 | −1.69 | 9.23 | <0.001 | 0.03 | Not available | Unknown |
ENSDARG00000080675 | si:dkey-71b5.7 | −2.71 | 3.70 | <0.001 | 0.019 | Not available | Unknown |
ENSDARG00000090930 | si:ch211-120g10.1 | −1.93 | 4.54 | <0.001 | <0.001 | Not available | Unknown |
ENSDARG00000052738 | hmgcs1 | 1.01 | 5.75 | <0.001 | 0.01 | Oligodendrocyte development, isoprenoid biosynthetic process | HMGCS1 |
ENSDARG00000079946 | sqlea | 1.84 | 4.21 | <0.001 | 0.01 | Fatty acid biosynthesis process, metabolic process, oxidation reduction process, 3-hydroxyacyl-CoA dehydrogenase activity, flavin adenine dinucleotide binding, oxidoreductase activity, squalene monooxygenase activity | SQLE |
ENSDARG00000061274 | lss | 1.34 | 3.95 | <0.001 | 0.02 | Baruol synthase activity | LSS |
ENSDARG00000055876 | msmo1 | 0.98 | 5.08 | <0.001 | 0.01 | Fatty acid biosynthesis process, endoplasmic reticulum, C-4 methylsterol oxidase activity | MSMO1 |
ENSDARG00000060711 | SV2B | 0.86 | 5.25 | <0.001 | 0.01 | Not available | SV2B |
ENSDARG00000090540 | Svbb | −0.68 | 5.47 | <0.001 | 0.02 | Transmembrane transport, integral component of membrane, transmembrane transporter activity | SV2B |
ENSDARG00000078624 | arhgef9b |
−1.05 | 5.27 | <0.001 | 0.005 | Regulation of Rho protein signal transduction, Rho guanyl-nucleotide exchange factor activity | ARHGEF9 |
ENSDARG00000079015 | brca2 | 1.20 | 4.02 | <0.001 | 0.01 | DNA repair, regulation of transcription, female gonad development, spermatogenesis, centrosome, γ-tubulin binding | BRCA2 |
ENSDARG00000055754 | smc1a | 1.01 | 6.22 | <0.001 | 0.01 | DNA repair, chromosome organization, ATP binding, chromatin binding | SMC1A |
ENSDARG00000024877 | ptgr1 | −2.32 | 4.24 | <0.001 | 0.015 | Oxidation-reduction process, oxidoreductase activity, zinc ion binding | PTGR1 |
ENSDARG00000070396 | serpinb1l2 | 5.71 | 1.11 | <0.001 | <0.001 | Negative regulation of endopeptidase activity, extracellular space, serine-type endopeptidase activity | SERPINB1 |
ENSDARG00000070449 | tspan5b | −2.02 | 2.88 | <0.001 | 0.002 | Integral component of membrane | TSPAN5 |
ENSDARG00000097011 | hbaa1 | 1.48 | 6.56 | <0.001 | 0.005 | Response to hypoxia, hemoglobin complex, heme binding | HBA1 |
ENSDARG00000069735 | si:ch211-5k11.6 | 2.79 | 2.01 | <0.001 | 0.008 | Oxygen transport, hemoglobin complex, heme binding | Unknown |
ENSDARG00000078322 | col12a1a | 0.94 | 5.00 | <0.001 | 0.019 | Collagen trimer | COL12A1 |
ENSDARG00000033760 | pmelb | 1.90 | 2.62 | <0.001 | 0.005 | Not available | PMEL |
ENSDARG00000087678 | rbm22 | 2.46 | 1.52 | <0.001 | 0.04 | RNA splicing, multicellular organismal development | RBM22 |
ENSDARG00000022185 | RGL3 | −1.58 | 2.51 | <0.001 | 0.03 | Not available | RGL3 |
ENSDARG00000086838 | ITGA2 | −3.48 | 1.33 | <0.001 | 0.007 | Not available | ITGA2 |
ENSDARG00000078624 | arhgef9b |
−1.05 | 5.27 | <0.001 | 0.005 | Regulation of Rho protein signal transduction, Rho guanyl-nucleotide exchange factor activity | ARHGEF9 |
ENSDARG00000092281 | flnb | −0.93 | 4.35 | <0.001 | 0.04 | Not available | FLNB |
ENSDARG00000009524 | rnf150b | 0.63 | 7.20 | <0.001 | 0.01 | Metal ion binding, zinc ion binding | RNF150 |
ENSDARG00000077799 | EGR4 | 1.22 | 5.50 | <0.001 | 0.015 | Not available | EGR4 |
ENSDARG00000041294 | noxo1a | −2.65 | 1.36 | <0.001 | 0.002 | Phosphatidylinositol binding | NOXO1 |
ENSDARG00000042988 | SLC24A2 (1 of 2) | −3.50 | 2.95 | <0.001 | 0.009 | Not available | SLC24A2 |
ENSDARG00000058508 | CFAP70 | −2.48 | 4.29 | <0.001 | 0.01 | Not available | CFAP70 |
ENSDARG00000094587 | si:dkey-35m8.1 | −0.87 | 4.67 | <0.001 | 0.017 | Not available | Unknown |
ENSDARG00000070516 | si:dkeyp-52c3.2 | 4.74 | 3.86 | <0.001 | <0.001 | GTP binding | Unknown |
ENSDARG00000070506 | CR450842.1 | 6.58 | 1.17 | <0.001 | <0.001 | Not available | Unknown |
ENSDARG00000093998 | si:ch73-7i4.2 | −7.47 | 1.90 | <0.001 | <0.001 | Not available | Unknown |
ENSDARG00000068621 | si:ch211-181d7.3 | 0.99 | 6.44 | <0.001 | <0.001 | Not available | Unknown |
ENSDARG00000089831 | NLRP6 | −1.21 | 4.66 | <0.001 | <0.001 | Not available | Unknown |
ENSDARG00000068621 | si:ch211-181d7.3 | 0.99 | 6.44 | <0.001 | <0.001 | Not available | Unknown |
ENSDARG00000089582 | si:dkey-265e15.2 | 2.75 | 1.19 | <0.001 | <0.001 | Not available | Unknown |
ENSDARG00000091847 | si:ch211-181d7.1 | 0.84 | 5.85 | <0.001 | 0.01 | ATP binding, nucleotide binding | Unknown |
Functional analyses were performed for males and females separately. As several automated classifications tested yielded low percentage of genes classified to function, a manual search was performed based on information from human and rodent ortholog data offered through Entrez gene and NGNC databases in addition to the zebrafish database Zfin. This search identified 73% of all differentially expressed genes (45 out of 62 genes for females and 25 out of 34 genes for males). Differentially expressed genes in Tables
A manual homolog search in Ensembl identified a total of seven differentially expressed genes encoding proteins presumably involved in the circadian rhythm in brains from developmentally EE2-exposed compared with unexposed female brains (Table
Cholesterol biosynthesis was identified as the top putative pathway affected by developmental EE2-exposure in zebrafish male brains after 120 days of remediation (Table
Several genes involved in synaptogenesis and synapse function were affected by developmental EE2 exposure in both male and female zebrafish brains. In male brains, in addition to the altered expression of the two synaptic vesicle genes mentioned above,
Developmental EE2 exposure affected seven genes related to the cytoskeleton and motor proteins in zebrafish female brains (Table
Several genes coding for heme-binding proteins were affected by developmental EE2 treatment in both male and female zebrafish. In male brains, EE2 exposure during early life resulted in an upregulation of
The brain expression of several genes in different ways involved in the immune response and inflammation were found to be affected by developmental exposure to EE2. The macrophage associated lectin
The brain expression of two genes involved in DNA repair and recombination,
Of the differentially expressed sequences 48 of the 110 in the females and 3 of 34 in males were annotated as non-coding RNA (ncRNA). A significant fraction, 19 of the 48 ncRNA in the female brain were novel microRNAs (miRNAs) whilst none of the 3 in males were miRNA (Table
ENSDARG00000088865 | −3.62 | 5.46 | <0.001 | <0.001 | 5 | Unknown |
ENSDARG00000088533 | −3.87 | 5.16 | <0.001 | <0.001 | 5 | Unknown |
ENSDARG00000088976 | −3.54 | 3.17 | <0.001 | <0.001 | 5 | Unknown |
ENSDARG00000087432 | −3.41 | 6.30 | <0.001 | <0.001 | 5 | Unknown |
ENSDARG00000088430 | −3.34 | 4.26 | <0.001 | <0.001 | 5 | Unknown |
ENSDARG00000087068 | −4.47 | 3.42 | <0.001 | <0.001 | 5 | Unknown |
ENSDARG00000089384 | −3.42 | 2.91 | <0.001 | <0.001 | 20 | Unknown |
ENSDARG00000086686 | −3.77 | 2.17 | <0.001 | <0.001 | 20 | Unknown |
ENSDARG00000088510 | −2.61 | 4.08 | <0.001 | <0.001 | 5 | Unknown |
ENSDARG00000084533 | −3.39 | 2.86 | <0.001 | <0.001 | 20 | Unknown |
ENSDARG00000084962 | −2.78 | 2.46 | <0.001 | <0.001 | 20 | Unknown |
ENSDARG00000090280 | −2.58 | 2.30 | <0.001 | <0.001 | 20 | Unknown |
ENSDARG00000090175 | −3.33 | 4.74 | <0.001 | <0.001 | 5 | Unknown |
ENSDARG00000091738 | −3.15 | 2.00 | <0.001 | <0.001 | 20 | Unknown |
ENSDARG00000090733 | −3.66 | 2.16 | <0.001 | <0.001 | 5 | Unknown |
ENSDARG00000088313 | −2.99 | 2.40 | <0.001 | 0.0035 | 20 | Unknown |
ENSDARG00000088673 | −3.04 | 1.82 | <0.001 | 0.0190 | 20 | Unknown |
ENSDARG00000087315 | −2.87 | 2.43 | <0.001 | 0.0425 | 20 | Unknown |
ENSDARG00000088865 | −3.62 | 5.46 | <0.001 | <0.001 | 5 | Unknown |
ENSDARG00000082087 | −1.18 | 5.75 | <0.001 | <0.001 | 7 | SNORD31 |
ENSDARG00000082611 | −1.15 | 6.17 | <0.001 | <0.001 | 7 | SNORA23 |
ENSDARG00000083108 | −1.25 | 5.64 | <0.001 | <0.001 | 20 | Yes, several |
ENSDARG00000083400 | −0.81 | 6.17 | <0.001 | <0.001 | 20 | Yes, several |
ENSDARG00000083784 | −1.34 | 4.65 | <0.001 | 0.0013 | 7 | SNORD22 |
ENSDARG00000082008 | −2.22 | 5.61 | <0.001 | 0.0013 | 10 | SNORD14C |
ENSDARG00000081115 | 1.11 | 8.63 | <0.001 | 0.0059 | 23 | Yes. several |
ENSDARG00000080942 | −1.80 | 4.31 | <0.001 | 0.0071 | 2 | SNORD66 |
ENSDARG00000081931 | −0.71 | 6.32 | <0.001 | 0.0086 | 19 | Yes, several |
ENSDARG00000084828 | −0.91 | 7.09 | <0.001 | 0.0107 | 4 | Yes, several |
ENSDARG00000081849 | −1.04 | 4.22 | <0.001 | 0.0112 | 1 | Yes, several |
ENSDARG00000082928 | −1.16 | 4.38 | <0.001 | 0.0137 | 5 | Yes, several |
ENSDARG00000084628 | −1.54 | 5.55 | <0.001 | 0.0251 | 20 | SNORA27 |
ENSDARG00000083378 | −1.11 | 4.51 | <0.001 | 0.0327 | 5 | Yes, several |
ENSDARG00000081235 | −0.77 | 6.73 | <0.001 | 0.0398 | 10 | SNORD14D |
ENSDARG00000083063 | −1.29 | 6.19 | <0.001 | <0.001 | 8 | Unknown |
ENSDARG00000083455 | −3.34 | 0.52 | <0.001 | 0.0024 | 4 | Yes, several |
ENSDARG00000082472 | 4.33 | 8.56 | <0.001 | 0.0046 | 13 | Unkown |
ENSDARG00000091828 | −6.47 | 5.23 | <0.001 | <0.001 | 5 | Yes, several |
ENSDARG00000091776 | −5.69 | 4.92 | <0.001 | <0.001 | 5 | Yes, several |
ENSDARG00000090705 | −5.03 | 5.21 | <0.001 | 0.0086 | 4 | Yes, 2 |
ENSDARG00000083480 | −5.15 | 7.24 | <0.001 | <0.001 | MT | Unknown |
ENSDARG00000082084 | −2.39 | 4.73 | <0.001 | 0.0135 | MT | Unknown |
ENSDARG00000082753 | −1.68 | 11.74 | <0.001 | 0.0142 | MT | Unknown |
ENSDARG00000091988 | −3.25 | 0.27 | <0.001 | 0.0196 | 19 | Unknown |
ENSDARG00000096403 | −2.20 | 3.92 | <0.001 | <0.001 | 20 | Unknown |
ENSDARG00000095598 | −1.39 | 2.68 | <0.001 | 0.0260 | 9 | Unknown |
ENSDARG00000084661 | −1.54 | 4.36 | <0.001 | <0.001 | 2 | Unknown |
ENSDARG00000080300 | 6.56 | 9.75 | <0.001 | 0.018 | 7 | Yes, several |
ENSDARG00000084622 | 0.92 | 8.14 | <0.001 | 0.046 | 15 | Yes, several |
Verification of the differential gene expression observed by RNA sequencing analysis was performed by qPCR for four selected target genes in the cholesterol biosynthesis pathway that was differentially upregulated by 3 ng/L EE2 in male brains. Ten brain samples each in exposure and control groups were analyzed. As shown in Figure
The present study verifies and extends our previous findings (Volkova et al.,
We used the same nominal concentrations as in Volkova et al. (
The current study verified that developmental EE2 exposure of zebrafish increases anxiety as adults, after a long remediation period in clean water. Increased anxiety is well-established as an EDC effect in mammals (Dugard et al.,
In the shoaling test, developmentally exposed fish ventured away from the shoal less in agreement with previous results (Volkova et al.,
To learn more about what changes in the brain could be discerned to accompany the behavior phenotype, we performed RNA sequencing of the brain transcriptome. We acknowledge that this transcriptome analysis is hampered by the few biological replicates, and conclusions from the data have to be drawn with caution. Also, the use of whole brains might hide region-specific alterations. Transcriptome data are challenging to interpret, and we can only speculate on connections between identified pathways and imprinting of increased stress sensitivity due to developmental EE2 exposure. However, RNA sequencing analyses of zebrafish brain are still few, and this study represents, to the best of our knowledge, the first study of estrogenic effects induced during development and persisting as a behavior phenotype in the adult fish. Further studies are needed to support, or contradict, the current findings.
No effects were observed in genes involved in the stress axis, neither on CRH, related genes in the hypothalamo-pituitary-interrenal (HPI) axis nor the monoamine system shown to be involved in their regulation. We did, however, identify some possible candidate pathways that might indirectly affect anxiety behavior. It is thus possible that the alterations in anxiety behavior induced during development are not mediated via the most obvious target genes in the brain. The connection between estrogens, stress and the regulation of HPI axis and monoaminergic system in fish is well-established (Winberg and Nilsson,
Only one gene,
The most significantly affected pathway in females was the circadian rhythm (Table
In males, the top putative pathway identified was cholesterol biosynthesis with four genes upregulated (Table
In females, three genes with the ability to bind lipids were found to be down-regulated by developmental EE2 exposure (Table
The current study identified six differentially expressed genes with recognized functions in the synapse. One of these genes,
In the female brain, a surprisingly high number of differentially expressed novel miRNA was observed (Table
This study has verified our previous findings of persistent effects on stress behavior in response to developmental exposure to low doses of EE2. RNA sequencing of male and female brain transcriptome revealed differences in differential expression between the sexes. No effects on genes belonging to the stress axis was observed in any of the sexes, but the expression of several genes in the regulation of circadian rhythm in females and cholesterol biosynthesis in males were found to be affected. Both pathways have previously been implied in anxiety regulation. Also, altered expression of several genes associated with synaptic function was observed, which might turn out to be important for the developmental modulations resulting in an anxiety phenotype. Further studies are needed to evaluate the significance of these findings, representing an initial survey of the effects of developmental exposure to the ubiquitous environmental contaminant EE2 on the brain transcriptome in the adult zebrafish.
TP planned, designed, and conducted experiment, performed qPCR, prepared figures and tables and completed the manuscript. KV planned, designed, and conducted experiment, performed manual functional classifications of coding genes and prepared tables, and wrote first draft of the paper and thereafter reviewed drafts of the paper. NR planned, designed, and conducted experiment and reviewed drafts of the paper. TK performed bioinformatics and biostatistics and reviewed drafts of the paper. PD performed statistical analyses and prepared figures and reviewed drafts of the paper. IP participated in and held main responsibility for the project from planning of experiment to the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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). We thank Victoria Barclay and Alexandros Christakopoulos, department of Laboratory Medicine, Karolinska Institute, Sweden, for chemical analyses. The authors would like to acknowledge support of the National Genomics Infrastructure (NGI) hosted by SciLifeLab/Uppsala Genome Center and UPPMAX for providing assistance in massive parallel sequencing and computational infrastructure. Work performed at NGI/Uppsala Genome Center has been funded by RFI/VR and Science for Life Laboratory, Sweden.
The Supplementary Material for this article can be found online at:
Endocrine disrupting compound
17α-ethinyl estradiol
novel tank
RNA sequencing
quantitative Real Time polymerase chain reaction
non-coding RNA
micro RNA.