Sensitive gene-expression changes

Both male and female juvenile fish exposed to 0.87 ng EE₂/L were analyzed with microarray. The microarray analysis of female fish suggested that only three out of four females had an induced expression of the known estrogen-responsive gene ZP3. In contrast, an induction was present in all eight males. This observation suggested that some juvenile females may have sufficient endogenous estrogen to induce sensitive estrogen-responsive genes. Thus, in our search for genes responding to low concentrations of estrogens only the microarray results from male fish were used.

Thirty-six sets of cDNAs (presumably corresponding to 29 genes) were regulated in male fish both by the low and the high concentration of EE₂ (Table 1). All of the cDNAs responded in a dose-dependent manner. ZP3 was the most differentially expressed gene in fish exposed to both high and low concentrations with a fold change of 84 and 3.5 respectively. VTG was not affected by the low concentration while it was up-regulated 537 times by 10 ng EE₂/L as measured by quantitative RT-PCR (qPCR) (Figure 1).

cGRASP ID	M-value 0.87 ng/L	M-value 10 ng/L	Annotation
CK991165	1.40	5.60	[GO] [P10761] Zona pellucida sperm-binding protein 3 precursor (Zona pellucida glycoprotein ZP3) (Sperm receptor) (Zona pellucida protein C),
CB492227	-0.23	-0.34	[GO] [P23506] Protein-L-isoaspartate(D-aspartate) O-methyltransferase (EC 2,1,1,77) (Protein-beta-aspartate methyltransferase) (PIMT) (Protein L- isoaspartyl/D-aspartyl methyltransferase)
CA054422	0.22	0.43	UNKNOWN
CB496664	0.30	0.50	[GO] [Q9D0E1] Heterogeneous nuclear ribonucleoprotein M (hnRNP M),
CB497378	0.27	0.69	[GO] [P15532] Nucleoside diphosphate kinase A (EC 2,7,4,6) (NDK A) (NDP kinase A) (Tumor metastatic process-associated protein) (Metastasis inhibition factor NM23) (NDPK-A) (nm23-M1),
CB511030	0.27	0.47	[GO] [P15532] Nucleoside diphosphate kinase A (EC 2,7,4,6) (NDK A) (NDP kinase A) (Tumor metastatic process-associated protein) (Metastasis inhibition factor NM23) (NDPK-A) (nm23-M1)
CK991305	0.27	0.59	[GO] [Q01768] Nucleoside diphosphate kinase B (EC 2,7,4,6) (NDK B) (NDP kinase B) (nm23-M2) (P18),
CA037915	0.30	0.38	[GO] [P35505] Fumarylacetoacetase (EC 3,7,1,2) (Fumarylacetoacetate hydrolase) (Beta-diketonase) (FAA),
CA060608	0.21	0.46	[GO] [P56384] ATP synthase lipid-binding protein, mitochondrial precursor (EC 3,6,3,14) (ATP synthase proteolipid P3) (ATPase protein 9) (ATPase subunit C),
CB496562	0.16	0.32	[GO] [Q9CY58] Plasminogen activator inhibitor 1 RNA-binding protein (PAI1 RNA- binding protein 1) (PAI-RBP1),
CB516182	0.46	0.56	[GO] [O08709] Peroxiredoxin 6 (EC 1,11,1,15) (Antioxidant protein 2) (1-Cys peroxiredoxin) (1-Cys PRX) (Acidic calcium-independent phospholipase A2) (EC 3,1,1,-) (aiPLA2) (Non-selenium glutathione peroxidase) (EC 1,11,1,7) (NSGPx),
CB511422	0.25	0.50	[GO] [P15532] Nucleoside diphosphate kinase A (EC 2,7,4,6) (NDK A) (NDP kinase A) (Tumor metastatic process-associated protein) (Metastasis inhibition factor NM23) (NDPK-A) (nm23-M1),
CB496931	0.80	2.58	[GO] [P11404] Fatty acid-binding protein, heart (H-FABP) (Heart-type fatty acid- binding protein) (Mammary-derived growth inhibitor) (MDGI),
CB497374	0.60	2.08	[GO] [P11404] Fatty acid-binding protein, heart (H-FABP) (Heart-type fatty acid- binding protein) (Mammary-derived growth inhibitor) (MDGI),
CB505692	-0.33	-0.42	UNKNOWN
CB497174	0.53	1.71	[NR] [XP_423045] PREDICTED: similar to nudix (nucleoside diphosphate linked moiety X)-type motif 7; coenzyme A diphosphatase [Gallus gallus]
CB497649	0.21	0.52	[GO] [Q01768] Nucleoside diphosphate kinase B (EC 2,7,4,6) (NDK B) (NDP kinase B) (nm23-M2) (P18),
CA037988	0.23	0.41	[NT] [AJ488155] Pachymedusa dacnicolor partial mRNA for ribosomal protein S16 (rps16 gene)
CB499596	0.14	0.49	[NR] [NP_077217] hydroxysteroid dehydrogenase like 2 [Mus musculus]
CB489314	-1.01	-1.11	UNKNOWN
CA769854	0.64	2.17	[GO] [P11404] Fatty acid-binding protein, heart (H-FABP) (Heart-type fatty acid- binding protein) (Mammary-derived growth inhibitor) (MDGI),
CB509453	-0.32	-0.48	[GO] [O16797] 60S ribosomal protein L3,
CB500821	-0.26	-0.36	[GO] [P62918] 60S ribosomal protein L8,
CB492885	-0.15	-0.31	UNKNOWN
CA061403	0.42	0.72	UNKNOWN
CB498219	0.24	0.51	[NR] [XP_613218] PREDICTED: similar to 24-dehydrocholesterol reductase precursor, partial [Bos taurus]
CA054168	-0.39	-0.58	UNKNOWN
CB515449	0.34	-0.60	[GO] [P50247] Adenosylhomocysteinase (EC 3,3,1,1) (S-adenosyl-L-homocysteine hydrolase) (AdoHcyase) (Liver copper binding protein) (CUBP),
CB515945	-0.36	-0.49	[NR] [NP_683732] RNA binding motif protein 5 [Mus musculus]
CA057448	-0.30	-0.33	UNKNOWN
CA036745	-0.62	-0.74	[NT] [XM_532501] PREDICTED: Canis familiaris similar to Chimerin (chimaerin) 2 (LOC475267), mRNA
CB509472	-0.29	-0.42	UNKNOWN
CB494192	0.23	0.33	[GO] [P09411] Phosphoglycerate kinase 1 (EC 2,7,2,3),
CB496589	-0.49	-0.82	UNKNOWN
CB513882	-0.48	-0.60	[NR] [XP_413822] PREDICTED: similar to normal mucosa of esophagus specific 1 [Gallus gallus]
CK990857	-0.64	-1.11	UNKNOWN

cDNAs or sets of cDNA putatively sensitive to estrogen exposure as judged by the presence on the top 250-lists ranked by moderated t-statistics on both 0.87 and 10 ng EE2/L exposure experiments in male juvenile rainbow trout. cDNAs corresponding to genes that were selected for qPCR analysis are marked with bold text. Note that several cDNAs may likely correspond to the same gene.

Robust gene-expression changes

A meta-analysis was performed with the aim to identify robust estrogen-responsive genes. The microarray data from fish (both sexes) exposed to 10 ng/L from the present study and available microarray data from three other exposure studies with fish and estradiol (E₂) or EE₂ were used in the meta-analysis [16, 20, 22]. Information about the different studies is shown in Table 2. Transcripts (360) presumably corresponding to 55 genes or groups of paralog genes were identified as differentially expressed in at least two of the four different studies (see Additional file 1). VTG and ZP3 were differentially expressed in all four studies and nine genes had an altered expression in at least three studies (Figure 2). It should be noted that ZP1 and the estrogen receptor-α, which are well-know estrogen-responsive genes in fish, have poor sequence representation on the cGRASP microarrays and are therefore not present in Figure 2.

	Present study	Hook et al. 2006	Tilton et al. 2006	Kishi et al. 2006
Species	O.mykiss	O.mykiss	O.mykiss	O.latipes
Sex	juvenile	male	juvenile	male
Estrogenic substance	EE2	EE2	E2	E2
Exposure	Water, 10 ng/L	Water, 50 ng/L	Dietary, 5 ppm	Water, 100 ng/L
Water temperature (C°)	10	12	12	24
Duration (days)	14	7	12	21
Platform	Two-channel spotted cDNA (GRASP 16 k v.1)	Two-channel spotted cDNA (GRASP 16 k v.1)	Two-channel spotted cDNA (GRASP 3.7 k v.1)	One-channel oligonucleotide (60 mer) (Kishi et al. 2006)
Experimental Setup	Direct comparison, 8 biological replicates	Direct comparison, 3 biological × 3 technical replicates	Reference design, 2 biological × 2 technical replicates	6 control and 3 exposed biological replicates
Number of cDNAs/probes	16006	16006	3700	22587
Pre-processing	Loess, no background correction	Loess	Loess	Robust Multichip
Statistical method used for ranking	Moderated t-statistic	Student t-statistic	fold change	Student t-statistic
Selected cDNA/probes	250 (167 induced, 83 suppressed)	189 (48 induced, 141 suppressed)	366 (127 induced, 239 suppressed)	381 (242 induced, 139 suppressed)
Source for sequences	cDNA, cGRASP	cDNA, cGRASP	cDNA, cGRASP	Transcripts, TIGR (OLGI release 4.0)
Number of matches (a in D.rerio	91	89	190	184

a) A match is defined as a tblastx hit with a E-value less than 10^-25.

Confirmation of microarray data with quantitative RT-PCR

Genes that were likely to be both sensitive (Table 1) and robust (Figure 2) were chosen for subsequent qPCR analysis. Three genes fulfilled these criteria: ZP3, a nucleoside diphosphate kinase (nm23) and fatty acid binding protein 3 (fabp3 or H-FABP). In addition, VTG was subjected to the qPCR analysis as well as the reference gene ubiquitin. In accordance with the microarray results the expression of VTG, ZP3 and nm23 were significantly induced in fish exposed to the high concentration. Also, as suggested by the microarrays, ZP3 and nm23 were significantly induced by the low concentration as well, whereas VTG expression was not induced (Figure 1). In stark contrast to the microarray results fabp3 had no tendency to any regulation caused by the treatment but showed a large variation within each treatment group (data not shown). The fabp3 and nm23 qPCR products were sequenced in order to confirm the amplification of the right products and they were identical to fabp3 and nm23 [EMBL:U95296, AF350241] in rainbow trout (data not shown).

Experimental animals, exposure and preparation of hepatic total RNA

Fish from a previously published study were analysed [29]. The experimental setup was in short: 15, 14 and 14 juvenile rainbow trout (weighing around 100 g) were divided into three aquaria and exposed for two weeks to measured concentrations of 0, 0.87 and 10 ng/L respectively of EE₂ in a flow-through system. Water samples were taken from the low and high EE₂ concentration aquaria, before the transfer of the fish, on day 8 and on day 13. One sample was collected from the control aquaria on day 8. Solid phase columns were used to extract and purify EE₂ from the water followed by derivatization (pentafluorobenzoylester) and further purification. EE₂-concentrations were determined using GC/MS. The limit of detection (signal-to-noise set to 5) was 0.01 ng/L. Samuelsson et al showed that the fish exposed to 10 ng/L EE₂ had significantly increased plasma levels of VTG, increased hepatosomatic index and the plasma metabolite profile were affected by the treatment. However, in the fish exposed to 0.87 ng/L neither induction of plasma VTG protein nor an altered metabolite pattern could be demonstrated using a specific VTG-ELISA and NMR respectively [29]. Gene responses in liver are widely used as biomarkers for environmental pollutants, i.e. estrogens, and the hepatic responses to estrogens are not restricted to a short developmental period. A prerequisite for our meta-analysis was availability of additional array-data from the same tissue in estrogen-exposed fish. Only hepatic microarray data was available in the literature, which therefore also contributed to our choice of tissue. Livers were collected and snap frozen in liquid nitrogen. Total hepatic RNA was isolated from individual trout liver using TRI reagent (Sigma chemicals Co, St Louis, MO, USA). RNA quality and quantity were assessed by agarose gel electrophoresis and spectrophotometric measurements (Nanodrop 1000, NanoDrop Technologies, USA and Spectra MAXplus, Molecular Devices, CA, USA).

Microarray chip, hybridisation and wash

Salmonid cDNA microarrays (GRASP16k v1) were purchased from cGRASP, Univerity of Victoria, BC, Canada [21]. Microarray fabrication and quality control have previously been described in von Schalburg et al. [30]. The array contains 13,421 Atlantic salmon (Salmon salar) cDNAs and 2,576 rainbow trout cDNAs that together with a few more expressed sequence tags (ESTs) from other salmonid fish results in 16 006 spotted cDNAs in total. It has previously been shown that the sequence similarity between the Atlantic salmon and rainbow trout is sufficiently high for cross species use of the array [31].

Several cDNAs on the array correspond to the same gene and to reduce redundancy, a sequence based clustering was made as follows. Each cDNA sequence was compared to all other sequences on the array using BLAST [32]. A stringent cut-off value of at least 98% sequence similarity over 250 base-pairs or more was used to define equality. Single-link clustering was then applied which resulted in 13853 sets of cDNAs.

Slide preparation have been described in detail in von Schalburg et al. [30]. Briefly, 8 μg of total RNA was reverse transcribed and labelled using SuperScript Indirect cDNA labelling System kit (Invitrogen, Carlsbad, CA, USA) and fluorescent dyes Cy5 and Cy3 (GE Healthcare, Buckinghamshire, UK). cDNA from one control fish and one exposed fish were labelled and hybridised to the same array. Every other pair was dye swapped to compensate for cyanine flour effects. Eight male control fish were paired with male fish exposed to 0.87 ng EE2/L matching individual weights and lengths as closely as possible. Four of the same male control fish were also paired to fish exposed to 10 ng EE2/L. In the same way, four female control fish were paired both to females exposed to the low dose and the high dose. Hybridisation and wash were performed as described before by von Schalburg et al. [30] with the exception of the prehybridization that was preformed for 1.5 h in 5xSSC, 0.1% SDS, 0.2% BSA at 49°C. In total 20 microarrays were analysed.

Microarray analyses

Fluorescent images of hybridized arrays were acquired using an Agilent MicroArray Scanner (Agilent Technologies, Palo Alto, CA, USA). Intensity data were extracted from TIFF images using Imagene version 6.0 (BioDiscovery, CA, USA). The statistical analysis was performed using the R package [33] LIMMA [34], which is available at the Bioconductor repository [35, 36]. For each cDNA on the chip, M-values (log₂ fold change) and A-values (average log₂ intensity) were calculated. Loess normalization was applied to each array to remove intensity dependent trends [37]. For each set of cDNAs (defined above), an M-value was calculated by taking the average of the M-values of all the cDNAs in the set. Next, each set was annotated based on the cDNA with highest A-value (i.e. the spot with best hybridization). Finally, the sets of cDNAs were ranked by moderated t-statistic [34] to reduce the proportion of false positives. Data from the complete microarray experiment is available according to the MIAME guidelines at Array Express [38].

Meta-analysis

Microarray data from four different studies on estrogen-exposed fish were subjected to a meta-analysis with the aim to identify robust estrogen responsive genes [16, 20, 22]. Another study with estrogen-exposed adult female zebrafish was excluded since the control fish presumable had high levels of endogenuous estrogen (neither VTG nor ZP3 was regulated in this study) [39]. To our knowledge, no other relevant microarray studies covering more than 3000 cDNAs/transcripts were publicly available (i.e. open access to transcript sequences) in October 2006 when we performed the meta-analyses. The studies included are summarized in Table 2.

The meta-analysis was done as follows. For each study, a list of the reported estrogen-regulated genes and the corresponding transcripts/cDNA-sequences was created. Note that the studies used different statistical methods to find the regulated genes (Table 2).

From the present study, the topmost 250 sets of cDNA from fish (both female and male) exposed to 10 ng EE2/L were chosen. To compare the platforms, zebrafish was used as a reference species. It was chosen since it is almost fully sequenced and well annotated compared to the other species involved. All transcripts/cDNAs were compared to the zebrafish transcriptome available through Ensembl release 40 (26,679 in total) using tblastx [32]. A cut-off E-value of 10^-25 was used to define a match. This resulted in a list of 360 zebrafish transcripts that had a match to transcripts/cDNAs from at least two studies (the transcripts should be regulated in the same direction). The list of zebrafish transcripts contained both multiple transcripts from the same gene (different splice variants) and paralogs and therefore the list was clustered into groups of transcripts. A similarity indicator matrix was created by comparing all transcripts in the list to each other using tblastx. Pairs of transcripts with an E-value of 10^-50 or less were defined to be equal. Otherwise the distance was set to zero. Single link clustering was then applied to create the groups of transcripts. Finally, all transcripts were annotated using Ensembl. The complete list of transcripts is available in Additional file 1.

Quantitative RT-PCR

To confirm regulation of four selected genes, the abundances of the mRNAs were analysed with qPCR. The qPCR was performed on isolated total RNA from the same fish used in the microarray analysis. Total RNA (0.5 μg) was reverse transcribed in duplicate with a mixture of random hexamers and oligo(dT) primers, using the iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). The cDNA synthesis was performed according to the manufacturer's instructions, except for a scale-down of the reaction volume to 10 μl. Pooled RNA samples were used as no reverse transcriptase controls to control for genomic contamination. It was discovered that three samples might have been contaminated with DNA. These samples were treated with DNase and new qPCR analyses were done. PCR primers for ZP3 [EMBL:AF231708], nm23 [EMBL:AF350241], fabp3 [EMBL:U95296], VTG [EMBL:X92804], β-actin [EMBL:AJ438158] and ubiquitin [EMBL:AB036060] were designed using Primer3 software [40]. Primer sequences were as follows: 5'-ccctgcgtatctttgtgga-3' and 5'-gtgggaacctgtcattttgg-3' for ZP3; 5'-ccttcttccctggtctcgt-3' and 5'-gatgatgttcctgcccactt-3' for nm23 ; 5'-ctttccctgtttcccctcct-3' and 5'-tgctgtgtgcttcttgctactc-3' for VTG; 5'-ggggcagtatggcttgtatg-3' and 5'-ctggcaccctaatcacctct-3' for beta actin; 5'-cgatagacggtggtaagatgg-3' and 5'-aggtgtggcaaagggtagtg-3' for fabp3 ; 5'- atgtcaaggccaagatccag -3' and 5'-ataatgcctccacgaagacg -3' for ubiquitin. For all genes except the reference gene, ubiquitin (for which the qPCR was performed according to a previously published protocol [41]), the qPCR reactions contained 1× Real Time PCR Buffer, 3 mM MgCl₂, 400 μM dNTP, 300 nM of each primer, 1 U TaKaRa Ex Taq™ R-PCR Version 2.1 (TaKaRa Bio Inc., Shiga, Japan), 0.25× SYBR Green I (Molecular Probes Eugene, OR, USA) and cDNA corresponding to 20 ng total RNA, in a final reaction volume of 20 μl. Real-time qPCR was performed on a Stratagene Mx3005p with 30 sec initial denaturation at 95°C, followed by 45 cycles of 95°C for 20s, 60°C for 20s and 72°C for 20s. A melting curve analysis was performed after each run to verify specific amplification. In addition the qPCR products were subjected to an agarose gel electrophoresis to confirm the expected size of the product. Both beta actin and ubiquitin were chosen as potential reference genes. Beta actin had a high variance and also a tendency to be regulated in the high dose group and therefore only ubiquitin was used. All signals were normalized against ubiquitin and ratios were calculated for exposed fish compared to control fish paired in the same manner as in the microarray analysis. Paired single-sided student's t-test were performed to test for significantly regulated genes. Since all samples could not be run at the same occasion, two standard samples were run at all occasions in order to enable a compensation for a possible run to run variation. Applying a run to run factor made little difference and the differently expressed genes VTG, ZP3 and nm23 were significant up-regulated both with and without applying the factor. The presented qPCR results are calculated without this factor.