Revealing genes associated with vitellogenesis in the liver of the zebrafish (Danio rerio) by transcriptome profiling
© Levi et al. 2009
Received: 05 September 2008
Accepted: 31 March 2009
Published: 31 March 2009
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© Levi et al. 2009
Received: 05 September 2008
Accepted: 31 March 2009
Published: 31 March 2009
In oviparous vertebrates, including fish, vitellogenesis consists of highly regulated pathways involving 17β-estradiol (E2). Previous studies focused on a relatively small number of hepatic expressed genes during vitellogenesis. This study aims to identify hepatic genes involved in vitellogenesis and regulated by E2, by using zebrafish microarray gene expression profiling, and to provide information on functional distinctive genes expressed in the liver of a vitellogenic female, using zebrafish as a model fish.
Genes associated with vitellogenesis were revealed by the following paired t-tests (SAM) comparisons: a) two-month old vitellogenic (Vit2) females were compared with non-vitellogenic (NV) females, showing 825 differentially expressed transcripts during early stages of vitellogenesis, b) four-month old vitellogenic (Vit4) females were compared with NV females, showing 1,046 differentially expressed transcripts during vitellogenesis and c) E2-treated males were compared with control males, showing 1,828 differentially expressed transcripts regulated by E2. A Venn diagram revealed 822 common transcripts in the three groups, indicating that these transcripts were involved in vitellogenesis and putatively regulated by E2. In addition, 431 transcripts were differentially expressed in Vit2 and Vit4 females but not in E2-treated males, indicating that they were putatively not up-regulated by E2. Correspondence analysis showed high similarity in expression profiles of Vit2 with Vit4 and of NV females with control males. The E2-treated males differed from the other groups. The repertoire of genes putatively regulated by E2 in vitellogenic females included genes associated with protein synthesis and reproduction. Genes associated with the immune system processes and biological adhesion, were among the genes that were putatively not regulated by E2. E2-treated males expressed a large array of transcripts that were not associated with vitellogenesis.
The study revealed several genes that were not reported before as being regulated by E2. Also, the hepatic expression of several genes was reported here for the first time.
Gene expression profiling of liver samples revealed 1,046 differentially expressed transcripts during vitellogenesis of which at least ~64% were regulated by E2. The results raise the question on the regulation pattern and temporal pleiotropic expression of hepatic genes in vitellogenic females.
The accumulation of yolk in oocytes of oviparous animals during oocyte development is essential for proper embryonic development after fertilization and is therefore, a key process in successful reproduction. In fish, the egg yolk protein precursors (vitellogenins) are synthesized in the liver, secreted to the plasma and transported to the oocytes for uptake in a process known as vitellogenesis. Several metabolic changes occur during vitellogenesis in the maturing female fish as reflected in the pronounced increase in liver weight, RNA content, lipid deposition, glycogen depletion, plasma proteins, calcium and magnesium and phosphoprotein content [1, 3].
The most dominant trigger of vitellogenin (vtg) expression is the ovarian steroid hormone 17β-estradiol (E2) that is synthesized under the regulation of the hypothalamic–pituitary–gonad axis [reviewed in ]. Most data to-date supports the premise that the action of estrogens is mediated principally through specific nuclear Estrogen receptors (ERs). In the "classical" or "genomic" mechanism of E2 action, estrogens diffuse into the cell and bind to ERs, which are located in the cytosol or the nucleus of target cells. After ligand binding, the ERs form homo- or hetero dimers that bind to specific palindromic estrogen response elements (ERE) sequences  in the promoter region of estrogen-responsive genes, resulting in recruitment of coactivators or corepressors to the promoter. Subsequently this leads to increased or decreased mRNA levels and associated protein synthesis, resulting in the physiological response . Two main ERs (ERa and ERb) were characterized in mammals, birds and fish. Three ER subtypes were described so far for fish and include the Estrogen receptor 1, Estrogen receptor 2b and Estrogen receptor 2a [with the gene names of estrogen receptor 1 (esr1), estrogen receptor 2b (esr2b) and estrogen receptor 2a (esr2a), respectively) [7, 9]. Some of the effects of estrogens are so rapid that they cannot depend on RNA and protein synthesis and are known as non-genomic actions. They involve activating protein-kinase cascades, leading eventually to regulation of gene expression through phosphorylation and activation of transcription factors (TFs) within the nucleus [10, 12].
Hepatic expression of vtg is tightly coupled to E2-dependent up-regulation of esr1 expression [13, 15]. Vtg is specific to maturing females and therefore assessment of vtg expression or Vtg plasma levels is considered a useful approach in evaluating female maturity related with peripheral gonadal steroid changes . This protein is normally not detected in males or juveniles, but yolk precursor proteins can be detected in males or juveniles exposed to estrogens. Hepatocytes synthesize yolk precursor proteins when stimulated with exogenous estrogens or substances that mimic estrogens. Several changes in hepatic morphology such as proliferation of the endoplasmic reticulum and the Golgi apparatus also accompany estrogen stimulation. These aspects were investigated in several oviparous species [4, 17–23].
Vtg is a large (MW; 250–600 kDa) and complex calcium-binding phospholipoglycoprotein and in order to reach the end product found in the plasma, substantial post-translational modification must occur within liver cells. First, the protein backbone of Vtg is synthesized on membrane-bound ribosomes and subsequently the Vtg molecule is lipidated, glycosylated and phosphorylated [Reviewed in ]. In addition, Vtg may carry additional compounds such as retinal that are also transported to the developing oocytes . The genes involved in these processes have not been fully elucidated. In zebrafish, seven vtg genes were previously identified  but recent proteome profiling data from maturing ovarian follicles indicates the occurrence of eight vtg's . The proteins fall into three main families represented by Vitellogenin 1 or VtgAo1 (with five corresponding genes, vtg 1, 4, 5, 6 and 7), Vitellogenin 2 or VtgAo2 (with two vtg2 genes) and Vitellogenin 3 or Vtg C (encoded by vtg3). Many more genes appear in the genome of zebrafish and fourteen of these genes were tightly linked to chromosome 22, while the phosvitinless gene (vtg3) was located on chromosome 11 .
In recent years, the study of hepatic expressed genes involved in fish vitellogenesis focused on few genes such as vtg, esr1, insulin-like growth factor 1 (igf1), zona pellucida glycoproteins (zp's), choriogenin H, cytochrome p450, family 1, subfamily a (cyp1a; also known as cyp1a1) and peroxisome proliferator-activated receptors (ppar's) [3, 15, 28–32] that are known to be regulated by estrogen. It is also well known that teleost apolipoproteins such as Apolipoprotein A-I (Apoa1), Apolipoprotein A-II (Apoa2) and Apolipoprotein E (Apoe) are regulated by E2 and presumably contribute to changes in the lipoprotein classes during vitellogenesis in fish [33, 35]. A high-throughput expression genomics approach would provide complementary information to the single-gene approaches used so far. Large-scale microarrays, available for model fish species including zebrafish, provide the opportunity to simultaneously monitor the expression of thousands of genes in different physiological stages during vitellogenesis. This approach has already been used with success to elucidate the zebrafish embryonic transcriptome [36, 37], to understand the molecular pathways defining gender specifiCity in zebrafish [38, 39] and to explore hepatic gene expression after exposure to different estrogens [40, 44].
The present study aims to identify genes involved in vitellogenesis and putatively regulated by E2 in the liver of zebrafish as a model fish, by using zebrafish oligonucleotide microarrays. Comparison of the hepatic expression profiles of vitellogenic and non-vitellogenic females provides information on the genes associated with vitellogenesis. In order to reveal E2-regulated genes, E2 treatment was administered to males for 48 hr at levels detected in the plasma of vitellogenic female. Genes suggested to be regulated by E2 were revealed by comparing the gene expression profiles of E2-treated males with those of vitellogenic females. The results also provide comparative information on the hepatic transcriptome profiles of 2- and 4- month old vitellogenic and non-vitellogenic females, E2-treated and control males and of the resemblance in gene expression profiles of these five groups. Distinctive putative pathways for the liver of vitellogenic females were found by analyses of the molecular functions and biological processes of the different treatment groups.
A list of 20 most regulated annotated genes during vitellogenesis and after E2 treatment of males.
Function according to http://www.ncbi.nlm.nih.gov/sites/entrez gene
Vitellogenin 3, phosvitinless
Egg yolk precursor, phosvitinless.
Similar to reticulon 1
Associates with the endoplasmic reticulum.
Nothepsin, cathepsin e
Eukaryotic aspartyl (acid) protease.
Similar to low density lipoprotein receptor
Plays a central role in cholesterol metabolism.
Moderately similar to spectrin repeat containing, nuclear envelope 1
Involved in cytoskeletal structure.
Similar to ectonucleoside triphosphate diphosphohydrolase 4
Cleaving nucleotide tri- and diphosphates.
Similar to follistatin-like 1
Play an important role in tissue specific regulation.
Similar to human Prefoldin subunit 6
Binds and stabilizes newly synthesized polypeptides.
Lag1 homolog, ceramide synthase 2
May play a role in the regulation of cell growth.
Ankyrin repeat domain 6
Ankyrin repeats mediate protein-protein interactions.
Similar to nitric oxide synthase interacting protein
Promotes translocation of eNOS from the plasma membrane to intracellular sites.
Similar to heterogeneous nuclear ribonucleoprotein h1
RNA recognition/binding motif.
Similar to family with sequence similarity 46 c
The function of this gene is unknown.
Egg yolk precursor.
Similar to human guanylate binding protein1
Specifically bind guanine nucleotides (GMP, GDP, and GTP).
Similar to human protein fam20c precursor?
Has a crucial role in normal bone development.
Similar to glutamate receptor, ionotropic, kainate 1
Ligand-gated ion channel.
Similar to lectin, mannose-binding, 1 precursor
Mannose-specific lectin, a member of a Mannose-specific lectin, a member of a the secretory pathway of animal cells.
Similar to rna polymerase ii associated protein 1
The function of this gene is unknown.
Estrogen receptor 1
A ligand-activated transcription factor.
Paired t-test analysis (SAM) comparing Vit2 or Vit4 females with NV females revealed 263 or 374, respectively transcripts that were not regulated by E2 (Fig 6B; see Additional file 2). The proportion of the down-regulated transcripts was higher (60.0% and 56.4% for Vit2 and Vit4 females, respectively) than the up-regulated transcripts indicating a slightly reverse trend from the E2 regulated genes.
The number of genes regulated or not-regulated by E2 in selected GO functions.
Not regulated by E2
Lipid metabolic process
Immune system process
Transcription factor activity
Validation of the microarray results was performed by testing the relative expression of 16 genes (see Additional file 4) in the same RNA samples that were used for the chip hybridization, by real-time PCR. The 16 tested transcripts were: 1) genes highly expressed in vitellogenic females and known to be induced by E2 treatment [esr1, vtg1, vtg3, nots and cytochrome p450 2k6 (cyp2k6)], 2) genes that were highly expressed in males and known to be down-regulated by E2 (cyp1a1 and igf1), and 3) genes that showed significantly different expression levels in the tested groups [retinoic acid receptor alpha a (raraa), alcohol dehydrogenase 5 (adh5), alcohol dehydrogenase 8b (adh8b), igf1 and retinol dehydrogenase 10 (rdh10), retinol dehydrogenase 14 (rdh14), dehydrogenase/reductase (SDR family) member 10 (dhrs10), stearoyl-desaturase (sCd), fatty acid desaturase 2 (fads2) and steroidogenic acute regulatory protein (star)]. A very high correlation was found between the microarray and real-time PCR results, with regression coefficients (R2 values) ranging from 0.9102 to 0.9340 (see Additional file 5).
This study aims at identifying and characterizing genes associated with vitellogenesis and defining the role of E2 in their regulation by using five physiological groups: non-vitellogenic females, vitellogenic females (2- and 4-month old), E2-treated males and control males. The expression levels of esr1 and vtg3 corresponding with the E2 plasma levels of females and E2 treated males, confirmed the efficacy of the E2 treatment. They also depicted the physiological State of the vitellogenic females, indicating that Vit2 females were at an interim stage between NV and Vit4 females.
The results provided a novel insight into the number and scope of hepatically regulated genes during vitellogenesis and indicated that most genes were regulated during the early stages of the process as young vitellogenic females (Vit2) differed by only 33 transcripts from older females (Vit4). Moreover, only ~64% of the transcripts regulated during vitellogenesis were suggested to be also regulated by E2. The resemblance in the gene expression pattern between non-vitellogenic females and males, stresses the specific change in pattern taking place during vitellogenesis in females. This change cannot be simply attributed to E2 as E2 treatment of males at physiological concentrations, resulted in a 1.8 fold higher number of genes than those regulated during vitellogenesis. These results also emphasize that the wide effects of xenobiotics with estrogen activity [45, 47] are not confined to genes associated with oocyte development. The following discussion sections highlight selected specific genes and putative pathways that were regulated during vitellogenesis and E2 treatment of males. Some of these putative pathways, were previously shown to be regulated in E2- treated males .
The list of the 20 most differentially expressed hepatic transcripts includes genes known to be regulated by E2, genes that were not recorded previously as regulated by E2 or genes that were not previously reported to be expressed in the liver. Eight of the 20 most differentially expressed genes (Table 1), were previously reported to be regulated by E2, including: vtg1 and vtg3 [25, 48–nots [42, 51], syne1 , fst1 , nosip , grik1  and esr1 [15, 49]. Transcripts that were not associated previously with E2 regulation include reticulon1 (rtn1), entpd4 and lman1. A few transcripts (syne1, fstl1 and the fam20c) were not reported previously in hepatic cells. Proteins associated with cytoskeleton formation (syne1 and ank) showed higher up-regulation in vitellogenic females than after E2 treatment of males, supporting a putative role in the growth of the liver  and of the dramatic increase in protein synthesis and secretion by the endoplasmic reticulum  during vitellogenesis.
Zebrafish display eight different variant gene sequences [25, 27] for vtg genes but the array used here included probes only for vtg3 and vtg1. Since vtg1 shows high sequence similarity with vtg4, vtg5, vtg6 and vtg7 (all coding for VtgAo1), transcript levels for vtg1 may also reflect the expression of these genes. The higher expression levels of vtg3 compared with vtg1, may suggest their differential regulation by E2 stems from differences in the estrogen response elements (ERE's) in the promoter regions .
Numerous prominent and putatively regulated functions were revealed in this study to take place during vitellogenesis and include lipid metabolism and lipid binding, hormone and cholesterol binding, transcription factor activity, immune response, immune system processes and cell adhesion (Table 2 and see Additional file 3). The task of allocating processes and functional significance to genes that were putatively regulated by E2 and also to those that were putatively not regulated by E2, was faced with a general difficulty for zebrafish. Linking specific pathways or modes of function with genes by the general descriptors provided by Gene Ontology (GO) for zebrafish is problematic due to the incomplete annotation of the zebrafish genome and a deficiency in functional studies. Consequently, several GO terms rely on homology of putative functions described in higher vertebrate species.
Several genes with a role in lipid metabolism were reported to be regulated by E2 [57, 58]. Genes associated with lipid metabolic processes putatively regulated in vitellogenic females or putatively regulated by E2, were also identified in the present study. Transcripts indicating a change in plasma lipoproteins were identified here, supporting previous published results on higher levels of plasma lipoproteins during vitellogenesis in fish [34, 35]. Plasma lipoproteins associated with transport of lipids are mainly synthesized in the liver and intestine . The protein components of lipoproteins, the apolipoproteins, form distinct complexes and two gene clusters, one consisting of apoa1, apoa4 and apoc3 and the other of apoe (apoeb for zebrafish), apoc, apoc2 and apoa4. These apolipoproteins are known from mammals and some were also characterized in fish . In the current study, there were no significant changes in apoa1 gene expression, in contrast with previous studies reporting on the downregulation of apoa1 gene expression after E2 and EE2 treatment [34, 42, 43]. Interestingly, Apoa1 was also found to serve as an antimicrobial protein and to be associated with the immune response system in fish . However, the expression of apoa4 and apoeb was down-regulated in vitellogenic females in comparison to NV females or after E2 treatment of males (see Additional file 1 and Additional file 3).
Several genes in the lipid metabolic processes are associated with the PPARs signaling pathway [62, 63] and some of them were found to be regulated in this study. The Ppars are ligand-inducible TFs belonging to the nuclear hormone receptor superfamily and are important regulators of lipid and energy homeostasis. Three isotypes have been identified in mammals, birds and amphibians, termed Ppar alpha (Ppara), Ppar beta (Pparb) (also known as Ppar delta) and Ppar gamma (Pparg) and each isotype is a product of one gene and shows distinct tissue distribution . Ppara functions in regulating reversible induction of β-oxidation in specific tissues but mainly the liver . Unexpectedly, there was no significant change in the expression of ppara in the liver of vitellogenic females or E2-treated males. The Pparb presumably functions in global control of lipid homeostasis and cellular proliferation and differentiation in mammals, is expressed in the liver and is moderately activated by a range of unsaturated fatty acids. Multiple/isoforms genes were found for pparb in several fish species, with two genes for pparb in zebrafish [64, 67]. The pparb1 was up-regulated in Vit4 females and control males while pparb2 was down-regulated in Vit4 and Vit2 females and by E2 treatment. In mammals, Pparg is associated with fat accumulation, particularly in adipocytes and in lipid accumulation in macrophages . Here, the pparg coactivator related 1 (ppargcr1) was up-regulated in Vit4 females and after E2 treatment (see Additional file 1).
A large array of transcripts putatively associated with lipid metabolism, was found to be regulated during vitellogenesis and by E2 treatment (see Additional file 3). Among them were genes coding for enzymes associated with fatty acid elongation and metabolism, and bile acid metabolism. Also, several transcripts of putative members of the cytochrome 450 superfamily were found to be regulated during vitellogenesis. This group of monooxygenases catalyzes several reactions involved in xenobiotic and drugs metabolism, synthesis of cholesterol, steroids and lipids.
One of the explanations for the higher transcript levels of star, hsd3b7 and hsd17b3 in NV females, could be related to the variable levels of steroid hormones in the plasma of females during onset of vitellogenesis [77, 78].
The process of vitellogenesis is known to be regulated by E2 through the induction of esr1, as mentioned before. A similar putative expression pattern for genes coding for two additional hormone binding receptors was suggested to take place during vitellogenesis and regulated by E2 (Table 2, see Additional file 3). These include the progesterone receptor membrane component 2 (pgmrc) and the kappa opioid receptor 1 (oprk1) as shown by the regulation of their respective ESTs; an EST similar to pgmrc2 (AW153364) and an EST similar to oprk1 (BG883146). Transcripts of pgmrc genes were found in livers of mammals and oviparous vertebrates including zebrafish but their functions remain unknown [79, 80]. The gene pgmrc2 is known to be involved in progestin signaling in several vertebrate reproductive tissues and in the brain (reviewed in ) and Pgmrc1 (closely related to Pgmrc2) was suggested to have a role in the regulation of oocyte maturation in trout ovarian follicles . Since PGMRC1 was suggested to be regulated by testosterone in porcine hepatocytes , the high transcript levels of pgmrc2 found in vitellogenic females could be linked with the elevated testosterone levels associated with higher E2 plasma levels [77, 78]. The opioid receptors have multiple effects on reproductive, endocrine and immune functions. Transcripts of oprk were found to be widely expressed in rat tissues including in the liver, yet their function in the liver remained unknown .
Surprisingly, all transcripts related to immune system processes in this study, were down-regulated in vitellogenic females and putatively not regulated by E2 (Fig 8). Three of these transcripts are also related to the immune response GO term (Table 2, see Additional file 3). The other transcripts related to the immune response term were all up-regulated by E2 treatment and some were also up-regulated during vitellogenesis (Table 2, see Additional file 3). This is in contrast to previous studies where E2 treatment repressed the expression of immune system and immune response related transcripts [92, 93]. Down-regulation in transcript levels in vitellogenic females (but not regulated by E2), was observed for genes coding for a class of intracellular molecules that play a role in coupling T-cell antigen receptor stimulation to the activation of integrins, major histocompatibility complex class genes (mhc's), proteases mediating programmed cell death or apoptosis [caspase 8 (casp8)] and a gene coding for an adherens junction protein [catenin beta 1 (ctnnb1)], functioning in communication and adhesion between cells, and anchoring the actin cytoskeleton. The genes showing high transcript levels in vitellogenic females and E2 treated males include an interferon activated gene, a TF that regulates mhc class II genes and the oprk1 previously described to be affected by steroid hormones, including E2 [94, 95].
Microarray profiling of liver samples revealed expression patterns characteristic of vitellogenic females of which only ~64% were found to be putatively regulated by E2. The repertoire of regulated genes implicates a wide range of functions especially those associated with protein synthesis, lipid metabolism, steroid biosynthesis, hormone binding and TF. Genes associated with the immune system and biological adhesion were among the genes that were up-regulated in vitellogenic females but not in E2-treated males, indicating that they were putatively not regulated by E2. E2-treated males expressed a large array of genes that were not associated with vitellogenesis. The study revealed several genes (rtn1, entpd4, lman1) that were not reported before as being regulated by E2. Also, the hepatic expression of several genes (e.g., syne1, fstl1, fam20c, star) is reported here for the first time for fish liver. In general, these results raise the question on the identity of the factors that regulate the pleiotropic expression of hepatic genes in vitellogenic females, in addition to E2.
Zebrafish were purchased from a local fish supplier (A&H Holdings, Israel LTD). All fish were maintained in 5-liter aquaria with UV treated, recycled and dechlorinated water and at ambient temperature of 25 ± 2°C with a light/dark cycle of 14/10 hr. The fish were fed twice a day, with shrimp nauplii (PGT, Eilat, Israel) in the morning and fish eggs in the afternoon. Non-vitellogenic females were kept under a light/dark cycle of 6/18 hr and fed twice a day with dry pellet food to avoid access to steroid compounds that maybe found in live food. For histological analysis the fish body or ovaries were fixed in Bouin's. Fixation, sectioning, and histological examination were performed according to . Paraffin sections of 4–7 µm were stained with hematoxylin and eosin. The terminology used by  for the zebrafish was adopted for this study. All fish were anaesthetized with Tricaine (Sigma-Aldrich, USA) before experimental procedures  and treatment of fish adhered with institutional regulations.
Two experiments were performed, one for evaluating the effect of E2 exposure in males and a second experiment for comparing gene expression in the liver of non-vitellogenic females with that of vitellogenic females. The experiments designs were as follows: 1) Four months old zebrafish were divided into three groups consisting of 8 fish in each group: i) adult spermeating males weighing 2.45 ± 0.207 g (N = 8) were exposed to E2 (Sigma-Aldrich, USA) by immersion for 48 hr (group E2-treated males). The concentration used was 5 μg/L (18 nM), as 3–4 ng/ml was determined to be the E2 natural concentration in the plasma of adult vitellogenic female ZF . A period of 48 hr of exposure was chosen as the highest expression level of esr1 was reached after 12 hr  and the expression of vtg stabilized after 48 hr of exposure to E2 and lasted for 17 days . The hepatosomatic index (HIS) of E2-treated males was 5.6 ± 0.6 after treatment. ii) control males weighing 2.26 ± 0.246 g (N = 8;) and a HIS of 3.2 ± 0.4; iii) vitellogenic females weighing 3.95 ± 0.303 g (N = 8) and showing a HIS of 5.9 ± 0.4 (group Vit4). Four replicate samples were prepared for each group and each replicate consisted of a pooled sample from livers of two fish. The samples were pooled after RNA extraction (see below). The fish in this experiment were kept with a light/dark cycle of 14/10 hr. The fish were fed twice a day, with shrimp nauplii (PGT, Eilat, Israel) in the morning and fish eggs in the afternoon. 2) In order to reveal the differences between vitellogenic and non-vitellogenic females, a second experiment was designed. One month old zebrafish were divided into two groups consisting of 32 fish in each group. Due to the small size of the liver, pooled samples from eight fish were prepared for each of the four replicates in the expression studies. The pooling of samples was done after total RNA extraction (see below). The groups consisted of: i) fish that were kept under a light/dark cycle of 14/10 hr for 5 weeks weighing 2.56 ± 0.426 g (N = 32) and a HIS of 5.6 ± 0.4, with ovaries in vitellogenic stage (group Vit2). Fish were kept at 25 ± 2°C and fed twice a day, with shrimp nauplii (PGT, Eilat, Israel) in the morning and with fish eggs in the afternoon. ii) fish that were kept under a light/dark cycle of 6/18 hr for the same time period and weighing 1.44 ± 0.391 g (N = 32) and showing a HIS of 4.6 ± 0.3, with non-vitellogenic ovaries (group NV). This group was fed only with dry pelleted food. In order to show the reversibility of this condition, some of the non-vitellogenic females were placed in tanks under the regular 10 hr light/14 hr dark photoperiod cycle. Fish were kept at 25 ± 2°C and were fed twice a day, with shrimp nauplii (PGT, Eilat, Israel) in the morning and with fish eggs in the afternoon. Ovaries collected after three weeks from these females were in the vitellogenic stage.
Determination of sex and of developmental stages of ovaries, were done by microscopic examination of the gonads. Oocyte stages were determined according to . The livers collected from all five groups were frozen instantly in liquid nitrogen and stored in -80°C until further use.
Measurements of E2 concentration in plasma was performed in similar designed separate experiments. Blood samples were collected using Micro-Hematocrit Tubes with Heparin (VWR, USA) from all tested groups and stored at -80°C. E2 concentrations were measured using Estradiol EIA Kit (Cayman, USA) according to the manufacture's protocol.
Total RNA was extracted from whole livers of zebrafish using Trizol reagent (GIBCO, USA) according to the manufacture's protocol followed by a clean-up and DNase treatement using RNeasy MiniElut Kit (Qiagene, Germany). After clean-up, 3 μl of the RNA samples were separated on 1.2% agarose gel to evaluate their quality and concentration. According to the gel picture, pooled samples from the livers of two fish were prepared for each group in Experiment 1 and of livers from eight fish for each group in the second experiment. Each RNA pool was quantified (A260) and assessed for purity (A260:A280 ratio) using Gene Quant (Amersham, UK) and by visual inspection of the 3 μg RNA separated on a denaturing gel.
Zebrafish microarrays were prepared by the Kimmel Cancer Center at Thomas Jefferson University (TJU), Philadelphia, USA. The microarray is a single color system based on zebrafish oligonucleotide library from Compugen/Sigma Genosys and consists of 16,399 oligonucleotide probes (65 nt), representing 16,288 unique gene clusters. In order to minimize non-specific binding, CodeLink slides with a special coating were used. Also β-actin internal controls were used to monitor the labeling and hybridization quality.
Processed chips were scanned by using a Perkin Elmer ScanArray® XL 5000 Scanner, software version 3.1. Images were quantified by PerkinElmer (USA) Quant Array® Software 3.0. Quantization used the fixed circle method and outputs were total intensities. Microarray data were normalized across all arrays using quantile normalization of data in log base 2 scale . This method corrects background noise and non-specific hybridization.
Statistical analysis was performed using Significant Analysis of Microarray (SAM) software . For the multiclass analysis a false discovery rate (FDR) of 0.1% was used. For paired t-test comparisons between the different groups a FDR of 0.03% was used. Correspondence Analysis (COA) was performed using MultiExperiment Viewer (MeV) version 4.1. Clustering analysis was performed using Coupled Two Way Clustering (CTWC) algorithm [103, 105].
Real-time PCR was performed using the same RNA samples used for microarray hybridization (n = 16). For cDNA synthesis, 4 μg of total RNA were mixed with 0.1 µg of oligo-d(T) (Promega, USA), 4 µl of Bio-RT 5× buffer, 2 µM dNTP mix (Promega, USA), 200 U of Bio-RT (Bio-lab, Israel) and H2O to reach a final volume of 20µl. After an incubation of 1 h at 37°C, 80 µl of H2O were added to the reaction. The PCR mixture consisted of 0.5 ul of cDNA sample, 70 nM of each primer (see Additional file 4) and 12.5 μl of SYBR Green master mix (ABgene, UK), in a final volume of 25 μl.
Amplification was carried out in a GenAmp 5700 thermocycler (PE Applied Biosystems, USA) and according to the manufacture's protocol. Amplification was performed in triplicates and the results were analyzed with REST-384 version 2 . The relative expression of the 16 tested genes (see Additional file 4) was calculated using zebrafish elongation factor 1 alpha (ef1a) as a reference gene. The gene ef1a was recently shown to be a suitable reference gene for tissue analysis, developmental and E2 exposure studies of zebrafish [41, 107].
The microarray annotations were updated using BlastX program against nr database of the GeneBank. Blast2GO software  was used for achieving GO annotations for the 2,523 differentially expressed genes found by SAM analysis (see Additional file 1 and Additional file 3). Combinations of gene lists were performed using Gene List Venn Diagrams software  (Fig. 5).
This study was supported by the Israel Science Foundation Grant 1184/04. We would like to thank Ms. Hana Bernard for assistance in the graphics. We would also like to thank the reviewers for useful comments.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.