Aquatic Toxicology 149 (2014) 1–7
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Distribution of vitellogenin in zebrafish (Danio rerio) tissues for biomarker analysis Liqiao Zhong a,b,1 , Li Yuan a,∗,1 , Yu Rao a,2 , Zhouquan Li a,b , Xiaohua Zhang a , Tao Liao a,3 , Ying Xu a , Heping Dai a,∗ a State Key Laboratory of Fresh Water Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, 7 Southern East Lake Road, Wuhan 430072, PR China b University of Chinese Academy of Sciences, Yuquan Road 19A, Beijing 100039, PR China
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Article history: Received 22 August 2013 Received in revised form 25 January 2014 Accepted 27 January 2014 Available online 2 February 2014 Keywords: Vitellogenin Biomarker Zebrafish Distribution
a b s t r a c t Vitellogenin (VTG), the precursor of yolk proteins, is a sensitive biomarker of estrogenic contamination in aquatic environments. Traditionally, VTG was believed to be synthesized under the control of estrogen in the livers of mature females and then secreted into the blood, before being taken up by the ovaries and cleaved into lipovitellin and phosvitin, which provide nutrition for developing embryos. However, recent studies have reported that the liver is not the only tissue to express VTG and this has led to questions over the precise tissue distribution of VTG in zebrafish. Moreover, studies in zebrafish on the expression of the VTG protein are rare. Using Western blotting and reverse-transcriptase polymerase chain reaction, this present study reports that the VTG protein and VTG1 mRNA were detected not only in the liver, but also in various extrahepatic tissues, including the heart, spleen, kidney, skin, muscle, gill, eye and brain tissues, of female and 17␣-ethinylestradiol (EE2 )-treated male zebrafish. Due to the high expression levels of VTG and the ease of taking samples, skin and eye tissues were chosen to evaluate the effects of varying doses and exposure times of EE2 on male zebrafish. The VTG gene and protein were induced by EE2 exposure in liver, skin and eye tissues of male zebrafish in dose- and time-dependent patterns. Therefore, we suggest that zebrafish skin and eye tissues may be alternatives to plasma and liver tissues for VTG biomarker analysis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In recent years, there has been increasing concern for the adverse effects of endocrine disrupting chemicals (EDCs), as these can interfere with the actions of hormones (Zoeller et al., 2012). Exposure to EDCs can be harmful to human health and wildlife, as these chemicals can disrupt the endocrine, reproductive and immune systems (Colborn et al., 1993), which will render organisms more susceptible to infections (Milla et al., 2011; Nakamura and Kariyazono, 2010; Schug et al., 2011) and many other problems, ranging from behavioral deficits to infertility to chronic disease
∗ Corresponding authors. Tel.: +86 27 68780716; fax: +86 27 68780123. E-mail addresses: [email protected] (L. Yuan), [email protected] (H. Dai). 1 These authors contributed equally to this work and should be considered co-first authors. 2 Current address: School of Bioengineering, Xihua University, 999 Jinzhou Road, Chengdu 610039, PR China. 3 Current address: Institute for Farm Products Processing and NuclearAgricultural Technology, Hubei Academy of Agricultural Sciences, 8 Nanhu YaoYuan Road, Wuhan 430064, PR China. 0166-445X/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquatox.2014.01.022
(Colborn et al., 1993; Diamanti-Kandarakis et al., 2009; Roig et al., 2013). Various reports have demonstrated that vitellogenin (VTG) is a sensitive biomarker of estrogenic pollution (Denslow et al., 1999; Heppell et al., 1995; Jones et al., 2000; Marin and Matozzo, 2004; Matozzo et al., 2008; Sumpter and Jobling, 1995). VTG, the precursor of the major egg yolk protein in oviparous animals, is a large serum glycolipophosphoprotein (300–600 kDa depending on species) (Wallace, 1985). It was generally thought that VTG was produced in the livers of females in response to estradiol-17 (E2), and then secreted to the bloodstream for transport to the developing oocytes (Byrne et al., 1989; Copeland et al., 1986; Wallace, 1985). Levels of VTG are lower in juvenile females and undetectable in males (Denslow et al., 1999; Heppell et al., 1995; Liao et al., 2006; Matozzo et al., 2008); however, when exposed to exogenous estrogens or estrogen mimics, immature females and males can synthesize and secrete VTG (Denslow et al., 1999; Heppell et al., 1995; Jones et al., 2000; Liao et al., 2006; Marin and Matozzo, 2004; Matozzo et al., 2008; Sumpter and Jobling, 1995; Wallace, 1985). Recent studies in zebrafish (Danio rerio) suggested that VTG is expressed not only in the liver, but also in extrahepatic tissues, including the heart, brain (Yin et al., 2009), skin (Jin et al.,
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2008), gill (Islinger et al., 2003), white adipose tissue (TingaudSequeira et al., 2012), intestine, ovary, muscle and estrogen-treated testes of male fish (Wang et al., 2005). VTG expression has also been detected in the gill, ovary and testes of white cloud mountain minnow (Tanichthys albonube) (Wang et al., 2010), the testes and kidney of spotted ray (Torpedo marmoratas) (Del Giudice et al., 2011), and the heart and brain of Chinese rare minnow (Gobiocypris rarus) (Ma et al., 2009). In addition, juvenile salmon were induced to express VTG in the skin when exposed to nonylphenol (Arukwe and Roe, 2008). Until now, the distribution of VTG in zebrafish has been reported incompletely and most studies have relied only on mRNA detection. Moreover, the biological functions of VTG synthesized in extrahepatic tissues in response to estrogens have remained unknown. In addition, it is unclear whether zebrafish tissues other than blood and liver are better suited for detecting VTG as a biomarker of estrogenic contamination. Therefore, in this present study, we aimed to investigate the expression and distribution of VTG mRNA and protein in thirteen kinds of zebrafish tissue, and find new alternative tissues which can be easily sampled for VTG biomarker analyses. 2. Materials and methods 2.1. Fish and treatment Adult zebrafish were bred and maintained in 10-L or 20-L glass aquaria with 4 L or 8 L dechlorinated water respectively, operating with a 14:10 h light:dark cycle at 23–26 ◦ C. Fish were acclimated for 1 week prior to use and fed with Tubifex worms once per day. Stock solution of 10 mg/L and 1 mg/L was prepared by dissolving 17␣-ethinylestradiol (EE2 ; TCI chemicals; E0037) in dimethyl sulfoxide (DMSO; Sigma; D2650). Two groups of adult zebrafish (9 male together with 9 female) in 10-L glass aquaria were used to analyze the tissue distribution of VTG with or without EE2 treatment. One group was exposed for 7 days to 50 ng/L (168.7 pmol/L) of EE2 , and the control group was exposed to DMSO (20 l in 4 L water). To investigate the effects of different concentrations of EE2 on VTG expression in male adult zebrafish, groups of 12–13 fish per group were exposed to 0, 3.125, 6.25, 12.5, 25 and 50 ng EE2/L (0, 10.55, 21.09, 42.18, 84.35 and 168.7 pmol EE2 /L) for 7 days in 10-L glass aquaria, and DMSO (25 l) only was added to the water of control tanks. Two groups containing 48 male adult zebrafish in 20-L glass aquaria were applied to study the effects of EE2 on VTG expression after different exposure times. One group was exposed to 50 ng/L (168.7 pmol/L) of EE2 , and the control group was exposed to DMSO (40 l in 8 L water). Samples of 12 fish were taken after 0, 2, 4 and 7 days of exposure. The final concentration of DMSO was within 0.01% in each group. Exposure solutions were completely renewed once per day. 2.2. Sampling After exposure, fish body lengths and weights were measured, and then blood was collected from the caudal peduncle according to the method described by Liao et al. (2006). And then the blood was put into TRIZOL reagent (Invitrogen; 15596-026) or 100 l icecold phosphate-buffered saline (PBS; pH 7.5) for RNA analysis or protein analysis, respectively. Scales were scraped off from the tail to the head of fish using a blunt tip tweezers. The residual scales were washed out with ice-cold PBS, and skin was separated from muscle with a slender tweezers. Then, the liver, brain, eyes, gills, heart, spleen, kidney, intestines, ovaries, testes, muscle and skin of each fish were separated. Each sample was divided into two parts: for RNA analysis, tissues from three individuals with the same sex were pooled, resulting in 3 pooled samples analyzed per treatment
and stored in TRIZOL reagent at −80 ◦ C; for protein assays, the tissues from three or four fish with the same sex were pooled as one sample and all samples except blood were washed with ice-cold PBS (pH 7.5) three times to avoid contamination by the blood, and then homogenized using a glass homogenizer in 10 volumes of icecold PBS containing a protease inhibition cocktail (Amresco; M222). The homogenates were centrifuged at 13,000 × g for 15 min at 4 ◦ C. For blood samples for protein analysis, the plasma was isolated by centrifugation for 3 min at 15,000 × g. Total protein was extracted from the supernatant of each sample, quantified by Bradford assay (Bradford, 1976), and then frozen immediately at −80 ◦ C. 2.3. Western blot analysis of VTG Western blot analysis was performed as described previously with minor modifications (Sambrook and Russell, 2001). A total of 25 g protein from each fish sample was separated using 5% stacking and 10% separating sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were subsequently transferred to a nitrocellulose membrane (Millipore; HATF00010). The membrane was cut into two pieces according to different molecular weights, which allowed for the separate detection of VTG and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The membrane was blocked with 4% skimmed milk in PBS. For VTG analysis, the blocked membrane was incubated for 1 h at room temperature (RT) with antiserum (diluted to 1:5000) against zebrafish VTG, which was kindly provided by Liao et al. (2006). After washing three times in 0.1% Tween-20 in PBS for 10 min each time, followed by three similar washes with PBS, the membrane was incubated for 1 h at RT with peroxidaseconjugated goat anti-rabbit IgG (diluted to 1:5000; Pierce; 31466). After this, the membrane was visualized using an enhanced chemiluminescence (ECL) assay (Boster; AR1171) according to the manufacturer’s procedure. Similar procedures were performed to detect GAPDH except that a mouse monoclonal antibody against GAPDH (diluted to 1:2000; Santa Cruz; sc-47724) and peroxidaseconjugated goat anti-mouse IgG (diluted to 1:5000; Pierce; 31431) were used as primary and secondary antibodies, respectively. VTG levels were quantified using ImageJ software (National Institutes of Health), and the relative level of the VTG protein in the control group was set as 1. 2.4. RNA extraction, reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) The tissue samples in TRIZOL reagent were homogenized using an RNase-free glass homogenizer on ice. The sample volumes should not exceed 10% of the volume of TRIZOL reagent. Total RNA was extracted from the homogenized tissue samples in TRIZOL reagent according to the protocol recommended by the manufacturer (Invitrogen). The quality and integrity of the total RNA were determined by electrophoresis in agarose gel. The concentration of RNA in each sample was measured with a NanoDrop ND-1000 UV-visible spectrophotometer. The RNA samples were digested with DNases (RNase free; Fermentas; EN0521) to remove the genomic DNA. cDNA was reverse transcribed from 2 g of RNA of each sample using the RevertAidTM First Strand cDNA Synthesis Kit (Fermentas; K1622) according to the manufacturer’s procedure. For RT-PCR and qPCR, a 130 bp fragment of zebrafish VTG1 was amplified with VTG1 forward (5 -ACGAACAGCGAGAAAGAGATTG3 ) and reverse primers (5 -GATGGGAACAGCGACAGGA-3 ). ˇ-actin was amplified as an internal control for calibration purposes using forward (5 -ATGGATGAGGAAATCGCTGCC-3 ) and reverse primers (5 -CTCCCTGATGTCTGGGTCGTC-3 ). These primers were designed as described by Jin et al. (2008) and used to amplify a 127 bp region of the zebrafish ˇ-actin gene. The PCRs were performed on a
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Fig. 1. Tissue distribution of the VTG in adult zebrafish with or without 50 ng (168.7 pmol) EE2 /L treatment. (A) Expression of VTG protein in various tissues of adult zebrafish as detected by Western blotting after application of polyclonal rabbit anti-zebrafish VTG antibody. A total of 25 g protein pooled from three fish was applied per well. Analysis of glyceraldehyde phosphate dehydrogenase (GAPDH) served as a constitutive control to show that equal amounts of protein were used per well. (B) VTG1 mRNA expression as measured by RT-PCR in adult zebrafish. Total RNA samples were collected from tissues of three adult zebrafish. ˇ-actin mRNA was used as an internal control. M, male zebrafish; F, female zebrafish; Std, molecular weight standard; −, without EE2 treatment; +, exposure to 50 ng (168.7 pmol) EE2 /L for 7 days.
thermal cycler (Bio-Rad T100TM ) at 95 ◦ C for 2 min, followed by 40 cycles of denaturation at 95 ◦ C for 15 s, annealing at 60 ◦ C for 15 s and extension at 72 ◦ C for 45 s, before a final termination step of 72 ◦ C for 5 min. qPCR was carried out on a real-time PCR detection system (CFX ConnectTM Real-Time PCR Detection System Bio-Rad) running the same program as for the RT-PCR. Each 20-L reaction mixture contained 1 L cDNA, 10 L SYBR Green Mix (TOYOBO; QPK-201), and 125 nM of forward and reverse primers. Standard plots for each target gene were produced by plotting cycle threshold (Ct) values against log-transformed 10-fold dilutions of cDNA sample from controlled female liver. The corresponding standard curve was used to evaluate the amplification efficiency of each primer pair. The amplification efficiency values for VTG1 and ˇactin were 100.9% and 96.9%, respectively. The relative expression level of VTG1 in each sample was calibrated by comparison to ˇ-actin transcript level in the same sample using the equation: (Ct) = Ct(VTG1) − Ct(ˇ-actin) . The relative level of the VTG1 transcript in the control group was set as 1, so the fold-induction change of relative VTG1 expression in each exposed group was calculated as 2−(Ct(exposed) − Ct(control)) . Each sample was run in triplicate.
2.5. Statistical analyses Data of relative VTG1 expression were presented as mean ± standard deviation (SD) and were log-transformed before statistical analysis to achieve variance homogeneity. All statistical analysis was performed using SPSS 13.0 software (SPSS, Chicago, IL, USA), and p < 0.05 was considered to be a statistically significant difference. Statistical differences among treatment groups as well as tissues in the same treatment were assessed by one-way analysis of variance (ANOVA) followed by a Tukey’s post hoc tests.
3. Results 3.1. Distribution of VTG in zebrafish Western blot analysis with rabbit polyclonal antibodies against zebrafish VTG was used to detect the distribution of VTG protein in adult zebrafish. GAPDH was used as an internal reference to confirm that equal amounts of protein were used in the analyses. As shown in Fig. 1A, prior to EE2 treatment, there was no positive signal for VTG in any of the 13 tissues and organs from male fish; however, in female fish VTG was detectable in all tissues and organs examined, except the intestine and ovaries, and the strongest signal came from the plasma. After exposure to 50 ng (168.7 pmol) EE2 /L for 7 days, male fish showed detectable VTG signals in all tissues except the intestine, while levels of VTG were greater in female tissues after EE2 exposure except for in the ovaries and intestine. It was difficult to detect VTG in the intestine of male and female fish and only low molecular protein bands were detected that may be degradation products of VTG due to the abundance of enzymes found in the intestine. Since the anti-VTG antibody can also recognize yolk protein, the mature protein of VTG, it also could be detected by Western blotting. In the ovary tissues, yolk protein signals were detected rather than signals for VTG. EE2 had no apparent effect on levels of yolk protein compared with the control group. In gill and kidney tissues, besides VTG, weak signals for yolk protein were detected in control females while strong signals were detected in males and females exposed to EE2 . The VTG mRNA expression levels in various tissues of adult zebrafish were investigated using RT-PCR with specific primers for VTG1. In male fish, VTG1 expression was detected in the heart, liver, spleen, skin, muscle, eye, fin, and brain tissues prior to EE2 treatment; while after EE2 exposure, the expression of VTG1 was detected in all tissues and organs except the blood (Fig. 1B). In
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Fig. 2. Western blot analysis of VTG protein in liver, plasma, skin and eye samples of adult male zebrafish exposed to 0, 3.125, 6.25, 12.5, 25 or 50 ng (0, 10.55, 21.09, 42.18, 84.35 or 168.7 pmol) EE2 /L for 7 days. Polyclonal rabbit anti-zebrafish VTG antibody was used in the analysis. A total of 25 g protein pooled from three fish was applied per well. Analysis of glyceraldehyde phosphate dehydrogenase (GAPDH) served as a constitutive control to show that equal amounts of protein were used per well. The bar charts below show the VTG protein levels quantified using ImageJ software according to the Western blot results. The values represent the fold changes, and the relative level of the VTG protein in the control group was set as 1.
female fish, RT-PCR revealed that VTG1 mRNA was detected in all tissues and organs except the blood with or without EE2 treatment (Fig. 1B). Furthermore, after EE2 treatment, VTG1 was up-regulated in all samples except the brain and blood in male fish, while in female fish there was no obvious up-regulation of VTG1 in all samples except the liver and skin. 3.2. Effect of different concentrations of EE2 on VTG expression in male fish Owing to ease of sampling, the skin and eyes of male fish were selected for further study to assess the effects of different concentrations of EE2 on VTG expression. Results were compared to VTG levels in the liver and plasma as these tissues are traditionally used
for VTG analysis. Western blotting showed that the amount of VTG in each sample containing 25 g of total protein was greatest in the plasma, lower in the skin and eyes, and lowest in the liver tissue (Fig. 2). Concentration-dependent increases of VTG expression were shown during EE2 exposure for 7 days in the plasma, skin, eyes and liver tissues of male fish. A slight change of VTG expression was observed in plasma at 6.25 ng (21.09 pmol) EE2 /L, while VTG expression was increased obviously at 12.5 ng (42.18 pmol) EE2 /L in the liver, skin and eyes. Greatest VTG levels were observed at 50 ng (168.7 pmol) EE2 /L in all four tissue types.qPCR was carried out to confirm the effects of different doses of EE2 on the expression of VTG1 mRNA (Fig. 3). Since there is no expression of VTG1 mRNA in the blood (Fig. 1B), only the liver, skin and eye tissues were used in this investigation. As shown in Fig. 3, significant dose-dependent
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4. Discussion
Fig. 3. Real-time quantitative PCR analysis of the expression levels of VTG1 mRNA in liver, skin and eye samples of adult male zebrafish exposed to 0, 3.125, 6.25, 12.5, 25 or 50 ng (0, 10.55, 21.09, 42.18, 84.35 or 168.7 pmol) EE2 /L for 7 days. Values are means ± SD (n = 3), and were normalized against ˇ-actin as a housekeeping gene. Data were analyzed by one-way ANOVA followed by a Tukey’s multiple comparison test. Different uppercase letters denote a significant difference among treatment groups within tissue; different lowercase letters denote a significant difference among tissues within treatment (P < 0.05). The relative mRNA expression levels of VTG1 are shown with a logarithmic scale. The products of real-time quantitative PCR were analyzed on 1.5% agarose gel.
increases of VTG1 mRNA expression were observed during EE2 exposure in all three tissues examined. It could be seen that liver was the most sensitive tissue to EE2 and 3.125 ng EE2 /L (10.55 pmol EE2 /L) was enough to induce significant increase of VTG1 mRNA. While significant increases of VTG1 mRNA in skin and eye were apparent at 6.25 and 12.5 ng EE2/L (21.09 and 42.18 pmol EE2 /L), respectively, and levels peaked in all tissues at 50 ng (168.7 pmol) EE2 /L. The induced expression of VTG1 mRNA in the eye was much lower than observed for the skin and liver samples after exposure to EE2 .
3.3. Effect of exposure to 50 ng (168.7 pmol) EE2 /L for different times on VTG expression in male fish Western blotting and qPCR analysis were used to investigate the effect of EE2 on VTG expression in liver, plasma, skin and eye tissues of male zebrafish at different times after exposure to 50 ng (168.7 pmol) EE2 /L. Western blot analysis showed significant increases of VTG expression in a time-dependent manner at 0, 2, 4 and 7 days after exposure to EE2 in all four tissues examined. Earliest induction of VTG was detected in the plasma and a strong signal was detected at 2 days after EE2 exposure, while in liver, skin and eye tissues a strong signal was detected from 4 days onwards. Greatest VTG expression was observed at 7 days after exposure in all four tissue types (Fig. 4). The qPCR results revealed that EE2 induced VTG1 mRNA expression in a time-dependent manner in liver, skin and eye tissues. Significant increases of VTG1 mRNA were detected in all three tissues at days 2. Meanwhile, greatest expression of VTG1 mRNA was observed after 7 days of EE2 exposure in all three tissues. Among the three tissues, the expression of VTG1 mRNA induced by EE2 in the eye was much lower than observed for the skin and liver samples (Fig. 5).
The short generation time of zebrafish has meant that it has been widely exploited as a model organism for investigating chemical toxicity (Hill et al., 2005; McGrath and Li, 2008; Scholz et al., 2008; Segner, 2009). VTG has been proven to be an ideal biomarker for measuring exposure to xenoestrogen in oviparous fish (Denslow et al., 1999; Heppell et al., 1995); however, recently the distribution of VTG in different tissues has been questioned. Previous studies of zebrafish have reported that VTG transcripts were detected not only in the liver, but also in extrahepatic tissues (Islinger et al., 2003; Jin et al., 2008; Tingaud-Sequeira et al., 2012; Wang et al., 2005; Yin et al., 2009), even though expression of the VTG protein has been rarely reported in extrahepatic tissues. Therefore, in this present study, the distribution of VTG (protein and mRNA transcript levels) in zebrafish was investigated. VTG was not detected in all the tissues tested for male fish prior to exposure to EE2 , while after treatment with 50 ng (168.7 pmol) EE2 /L VTG was detected in all tissues except for the intestine. In female fish, the VTG protein was present in all tissues examined except for the intestine and ovaries whether EE2 treatment was applied or not. Interestingly, VTG was induced in male fish by EE2 treatment to a greater extent in the eye, skin, heart and plasma tissues compared to the liver which is used conventionally for VTG detection. No VTG signals were detected in the ovaries but there were signals for yolk protein, probably because VTG is taken up by the ovaries and cleaved into yolk protein (Wahli, 1988; Wallace, 1985). It is interesting to note that besides VTG, the yolk protein and several other protein bands of low molecular weights were present in gill and kidney samples, which may be because these are important excretory organs in fish, and it is known that VTG is eliminated from the blood through the kidneys and gills (Del Giudice et al., 2011). Therefore, we suppose that VTG degradation occurred by enzymolysis during VTG elimination. RT-PCR was used to confirm the RNA expression of VTG1 in 13 tissue types. There are seven VTG genes in zebrafish and, among these, VTG1 is the most highly expressed (Tong et al., 2004; Wang et al., 2005). VTG1 mRNA is used as a biomarker for EDCs in zebrafish (Muncke and Eggen, 2006; Tong et al., 2004). The RT-PCR results showed that VTG1 was expressed in all tissues except the blood in female fish whether or not they had been treated with EE2 and in male fish that had been treated with EE2 (Fig. 1B). Even without EE2 treatment, VTG1 mRNA was detected in the liver and several extrahepatic tissues (e.g. heart, spleen, skin, muscle, eye, fin, and brain) of male fish. Compared with other tissues, the liver has relatively greater mRNA levels of VTG1 and lower VTG protein levels in response to EE2 , which suggests that the liver may be a major organ synthesizing VTG and transporting it by blood to the ovaries (Byrne et al., 1989; Copeland et al., 1986; Wallace, 1985). On the other hand, some extrahepatic tissues, such as heart, spleen, skin and eyes, have both relatively high mRNA levels and protein levels in response to EE2 , which suggests that these organs can synthesize VTG, but do not or only rarely transport it away. Conversely, the intestine has relative higher mRNA VTG1 levels and undetectable levels of VTG protein, which suggests that VTG may be digested rapidly by the abundant proteinases found in the intestine. VTG expression in many extrahepatic tissues may be due to the extremely wide distribution of white adipose tissue, which has been identified previously to express the VTG1 gene in zebrafish (Tingaud-Sequeira et al., 2012). Further studies need to be performed to understand the biological functions of the induced VTG in these extrahepatic tissues. Taking samples of liver and blood plasma from small fish like zebrafish is very difficult and, due to the limited amount of sample obtained, liver and plasma tissues from individual fish often have to be combined to create enough sample for accurate analyses. For example, livers and plasma samples from more than three fish
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Fig. 4. Western blot analysis of VTG protein in liver, plasma, skin and eye samples of adult male zebrafish exposed to 50 ng (168.7 pmol) EE2 /L for 0, 2, 4 and 7 days. Polyclonal rabbit anti-zebrafish VTG antibody was used in the analysis. A total of 25 g protein pooled from three fish was applied per well. Analysis of glyceraldehyde phosphate dehydrogenase (GAPDH) served as a constitutive control to show that equal amounts of protein were used per well. The bar charts below show the VTG protein levels quantified using ImageJ software according to the Western blot results. The values represent the fold changes, and the relative level of the VTG protein in the control group was set as 1.
were pooled for Western blot analysis in this present study. However, samples of skin and eyes are much easier to collect and these can be obtained in sufficient amounts to allow individual fish to be studied. Owing to the high expression of VTG and the ease with which the tissues can be sampled, the skin and eyes of zebrafish were chosen to assess the levels of VTG that were induced by EE2 treatment at various doses and at different times after exposure. Then, these data were compared with liver and plasma samples that are used traditionally for biomarker studies. Interestingly, the plasma, liver, skin and eye tissues from male zebrafish all showed concentration- and time-dependent patterns of VTG
protein expression after EE2 treatment (Figs. 2 and 4). The subsequent qPCR analyses also revealed that EE2 induced significant dose- and time-dependent increases of VTG1 mRNA expression in skin and eye tissues as well as in the liver (Figs. 3 and 5). Thus, skin and eye tissues are suggested to be alternative choices for VTG biomarker detection studies in zebrafish. Skin and eye tissues are suitable as alternative tissues for analysis of VTG protein detection, while the skin is more suited than the eye to replace the liver for VTG mRNA analysis. In conclusion, this present study demonstrates that VTG protein and mRNA was detected not only in the liver but also in various
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Fig. 5. Real-time quantitative PCR analysis the of the expression levels of VTG1 mRNA in liver, skin and eye samples of adult male zebrafish exposed to 50 ng (168.7 pmol) EE2 /L for 0, 2, 4 and 7 days. Values are means ± SD (n = 3), and were normalized against ˇ-actin as a housekeeping gene. Data were analyzed by oneway ANOVA followed by a Tukey’s multiple comparison test. Different uppercase letters denote a significant difference among treatment groups within tissue; different lowercase letters denote a significant difference among tissues within treatment (P < 0.05). The relative mRNA expression levels of VTG1 are shown with a logarithmic scale. The products of real-time quantitative PCR were analyzed on 1.5% agarose gel.
extrahepatic tissues. Due to the ease of collecting samples, skin and eye tissues are suggested as new alternatives to plasma and liver for VTG biomarker analyses. Acknowledgments This work was supported by grants from the Natural Science Foundation of China (Nos. 21037004 and 20777091), and the State Key Laboratory of Fresh water Ecology and Biotechnology (2011FBZ10). References Arukwe, A., Roe, K., 2008. Molecular and cellular detection of expression of vitellogenin and zona radiata protein in liver and skin of juvenile salmon (Salmo salar) exposed to nonylphenol. Cell Tissue Res. 331, 701–712. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Byrne, B.M., Gruber, M., Ab, G., 1989. The evolution of egg yolk proteins. Prog. Biophys. Mol. Biol. 53, 33–69. Colborn, T., Saal, F.S.V., Soto, A.M., 1993. Developmental effects of endocrinedisrupting chemicals in wildlife and humans. Environ. Health Perspect. 101, 378–384. Copeland, P.A., Sumpter, J.P., Walker, T.K., Croft, M., 1986. Vitellogenin levels in male and female rainbow trout (Salmo gairdneri Richardson) at various stages of the reproductive cycle. Comp. Biochem. Physiol. B 83, 487–493. Del Giudice, G., Prisco, M., Agnese, M., Verderame, M., Limatola, E., Andreuccetti, P., 2011. Expression of vitellogenin in the testis and kidney of the spotted ray Torpedo marmorata exposed to 17beta-estradiol. Gen. Comp. Endocrinol. 174, 318–325. Denslow, N.D., Chow, M.C., Kroll, K.J., Green, L., 1999. Vitellogenin as a biomarker of exposure for estrogen or estrogen mimics. Ecotoxicology 8, 385–398.
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Bull. 48, 835–839. Matozzo, V., Gagne, F., Marin, M.G., Ricciardi, F., Blaise, C., 2008. Vitellogenin as a biomarker of exposure to estrogenic compounds in aquatic invertebrates: a review. Environ. Int. 34, 531–545. McGrath, P., Li, C.Q., 2008. Zebrafish: a predictive model for assessing drug-induced toxicity. Drug Discov. Today 13, 394–401. Milla, S., Depiereux, S., Kestemont, P., 2011. The effects of estrogenic and androgenic endocrine disruptors on the immune system of fish: a review. Ecotoxicology 20, 305–319. Muncke, J., Eggen, R.I.L., 2006. Vitellogenin 1 mRNA as an early molecular biomarker for endocrine disruption in developing zebrafish (Danio rerio). Environ. Toxicol. Chem. 25, 2734–2741. Nakamura, K., Kariyazono, H., 2010. Influence of endocrine-disrupting chemicals on the immune system. J. Health Sci. 56, 361–373. Roig, B., Mnif, W., Hassine, A.I.H., Zidi, I., Bayle, S., Bartegi, A., Thomas, O., 2013. Endocrine disrupting chemicals and human health risk assessment: a critical review. Crit. Rev. Environ Sci. Technol. 43, 2297–2351. Sambrook, J., Russell, D.V., 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York. Scholz, S., Fischer, S., Gundel, U., Kuster, E., Luckenbach, T., Voelker, D., 2008. The zebrafish embryo model in environmental risk assessment—applications beyond acute toxicity testing. Environ. Sci. Pollut. Res. Int. 15, 394–404. Schug, T.T., Janesick, A., Blumberg, B., Heindel, J.J., 2011. Endocrine disrupting chemicals and disease susceptibility. J. Steroid Biochem. 127, 204–215. Segner, H., 2009. Zebrafish (Danio rerio) as a model organism for investigating endocrine disruption. Comp. Biochem. Physiol. C: Toxicol. Pharmacol. 149, 187–195. Sumpter, J.P., Jobling, S., 1995. Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment. Environ. Health Perspect. 103, 173–178. Tingaud-Sequeira, A., Knoll-Gellida, A., Andre, M., Babin, P.J., 2012. Vitellogenin expression in white adipose tissue in female teleost fish. Biol. Reprod. 86, 38. Tong, Y., Shan, T., Poh, Y.K., Yan, T., Wang, H., Lam, S.H., Gong, Z., 2004. Molecular cloning of zebrafish and medaka vitellogenin genes and comparison of their expression in response to 17beta-estradiol. Gene 328, 25–36. Wahli, W., 1988. Evolution and expression of vitellogenin genes. Trends Genet. 4, 227–232. Wallace, R.A., 1985. Vitellogenesis and oocyte growth in nonmammalian vertebrates. Dev. Biol. 1, 127–177. Wang, H., Tan, J.T.T., Emelyanov, A., Korzh, V., Gong, Z.Y., 2005. Hepatic and extrahepatic expression of vitellogenin genes in the zebrafish, Danio rerio. Gene 356, 91–100. Wang, R.L., Gao, Y., Zhang, L.H., Zhang, Y.K., Fang, Z.Q., He, J.G., Zhang, W.M., Ma, G.Z., 2010. Cloning, expression, and induction by 17-beta estradiol (E-2) of a vitellogenin gene in the white cloud mountain minnow Tanichthys albonubes. 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Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox
Distribution of vitellogenin in zebrafish (Danio rerio) tissues for biomarker analysis Liqiao Zhong a,b,1 , Li Yuan a,∗,1 , Yu Rao a,2 , Zhouquan Li a,b , Xiaohua Zhang a , Tao Liao a,3 , Ying Xu a , Heping Dai a,∗ a State Key Laboratory of Fresh Water Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, 7 Southern East Lake Road, Wuhan 430072, PR China b University of Chinese Academy of Sciences, Yuquan Road 19A, Beijing 100039, PR China
a r t i c l e
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Article history: Received 22 August 2013 Received in revised form 25 January 2014 Accepted 27 January 2014 Available online 2 February 2014 Keywords: Vitellogenin Biomarker Zebrafish Distribution
a b s t r a c t Vitellogenin (VTG), the precursor of yolk proteins, is a sensitive biomarker of estrogenic contamination in aquatic environments. Traditionally, VTG was believed to be synthesized under the control of estrogen in the livers of mature females and then secreted into the blood, before being taken up by the ovaries and cleaved into lipovitellin and phosvitin, which provide nutrition for developing embryos. However, recent studies have reported that the liver is not the only tissue to express VTG and this has led to questions over the precise tissue distribution of VTG in zebrafish. Moreover, studies in zebrafish on the expression of the VTG protein are rare. Using Western blotting and reverse-transcriptase polymerase chain reaction, this present study reports that the VTG protein and VTG1 mRNA were detected not only in the liver, but also in various extrahepatic tissues, including the heart, spleen, kidney, skin, muscle, gill, eye and brain tissues, of female and 17␣-ethinylestradiol (EE2 )-treated male zebrafish. Due to the high expression levels of VTG and the ease of taking samples, skin and eye tissues were chosen to evaluate the effects of varying doses and exposure times of EE2 on male zebrafish. The VTG gene and protein were induced by EE2 exposure in liver, skin and eye tissues of male zebrafish in dose- and time-dependent patterns. Therefore, we suggest that zebrafish skin and eye tissues may be alternatives to plasma and liver tissues for VTG biomarker analysis. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In recent years, there has been increasing concern for the adverse effects of endocrine disrupting chemicals (EDCs), as these can interfere with the actions of hormones (Zoeller et al., 2012). Exposure to EDCs can be harmful to human health and wildlife, as these chemicals can disrupt the endocrine, reproductive and immune systems (Colborn et al., 1993), which will render organisms more susceptible to infections (Milla et al., 2011; Nakamura and Kariyazono, 2010; Schug et al., 2011) and many other problems, ranging from behavioral deficits to infertility to chronic disease
∗ Corresponding authors. Tel.: +86 27 68780716; fax: +86 27 68780123. E-mail addresses: [email protected] (L. Yuan), [email protected] (H. Dai). 1 These authors contributed equally to this work and should be considered co-first authors. 2 Current address: School of Bioengineering, Xihua University, 999 Jinzhou Road, Chengdu 610039, PR China. 3 Current address: Institute for Farm Products Processing and NuclearAgricultural Technology, Hubei Academy of Agricultural Sciences, 8 Nanhu YaoYuan Road, Wuhan 430064, PR China. 0166-445X/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquatox.2014.01.022
(Colborn et al., 1993; Diamanti-Kandarakis et al., 2009; Roig et al., 2013). Various reports have demonstrated that vitellogenin (VTG) is a sensitive biomarker of estrogenic pollution (Denslow et al., 1999; Heppell et al., 1995; Jones et al., 2000; Marin and Matozzo, 2004; Matozzo et al., 2008; Sumpter and Jobling, 1995). VTG, the precursor of the major egg yolk protein in oviparous animals, is a large serum glycolipophosphoprotein (300–600 kDa depending on species) (Wallace, 1985). It was generally thought that VTG was produced in the livers of females in response to estradiol-17 (E2), and then secreted to the bloodstream for transport to the developing oocytes (Byrne et al., 1989; Copeland et al., 1986; Wallace, 1985). Levels of VTG are lower in juvenile females and undetectable in males (Denslow et al., 1999; Heppell et al., 1995; Liao et al., 2006; Matozzo et al., 2008); however, when exposed to exogenous estrogens or estrogen mimics, immature females and males can synthesize and secrete VTG (Denslow et al., 1999; Heppell et al., 1995; Jones et al., 2000; Liao et al., 2006; Marin and Matozzo, 2004; Matozzo et al., 2008; Sumpter and Jobling, 1995; Wallace, 1985). Recent studies in zebrafish (Danio rerio) suggested that VTG is expressed not only in the liver, but also in extrahepatic tissues, including the heart, brain (Yin et al., 2009), skin (Jin et al.,
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2008), gill (Islinger et al., 2003), white adipose tissue (TingaudSequeira et al., 2012), intestine, ovary, muscle and estrogen-treated testes of male fish (Wang et al., 2005). VTG expression has also been detected in the gill, ovary and testes of white cloud mountain minnow (Tanichthys albonube) (Wang et al., 2010), the testes and kidney of spotted ray (Torpedo marmoratas) (Del Giudice et al., 2011), and the heart and brain of Chinese rare minnow (Gobiocypris rarus) (Ma et al., 2009). In addition, juvenile salmon were induced to express VTG in the skin when exposed to nonylphenol (Arukwe and Roe, 2008). Until now, the distribution of VTG in zebrafish has been reported incompletely and most studies have relied only on mRNA detection. Moreover, the biological functions of VTG synthesized in extrahepatic tissues in response to estrogens have remained unknown. In addition, it is unclear whether zebrafish tissues other than blood and liver are better suited for detecting VTG as a biomarker of estrogenic contamination. Therefore, in this present study, we aimed to investigate the expression and distribution of VTG mRNA and protein in thirteen kinds of zebrafish tissue, and find new alternative tissues which can be easily sampled for VTG biomarker analyses. 2. Materials and methods 2.1. Fish and treatment Adult zebrafish were bred and maintained in 10-L or 20-L glass aquaria with 4 L or 8 L dechlorinated water respectively, operating with a 14:10 h light:dark cycle at 23–26 ◦ C. Fish were acclimated for 1 week prior to use and fed with Tubifex worms once per day. Stock solution of 10 mg/L and 1 mg/L was prepared by dissolving 17␣-ethinylestradiol (EE2 ; TCI chemicals; E0037) in dimethyl sulfoxide (DMSO; Sigma; D2650). Two groups of adult zebrafish (9 male together with 9 female) in 10-L glass aquaria were used to analyze the tissue distribution of VTG with or without EE2 treatment. One group was exposed for 7 days to 50 ng/L (168.7 pmol/L) of EE2 , and the control group was exposed to DMSO (20 l in 4 L water). To investigate the effects of different concentrations of EE2 on VTG expression in male adult zebrafish, groups of 12–13 fish per group were exposed to 0, 3.125, 6.25, 12.5, 25 and 50 ng EE2/L (0, 10.55, 21.09, 42.18, 84.35 and 168.7 pmol EE2 /L) for 7 days in 10-L glass aquaria, and DMSO (25 l) only was added to the water of control tanks. Two groups containing 48 male adult zebrafish in 20-L glass aquaria were applied to study the effects of EE2 on VTG expression after different exposure times. One group was exposed to 50 ng/L (168.7 pmol/L) of EE2 , and the control group was exposed to DMSO (40 l in 8 L water). Samples of 12 fish were taken after 0, 2, 4 and 7 days of exposure. The final concentration of DMSO was within 0.01% in each group. Exposure solutions were completely renewed once per day. 2.2. Sampling After exposure, fish body lengths and weights were measured, and then blood was collected from the caudal peduncle according to the method described by Liao et al. (2006). And then the blood was put into TRIZOL reagent (Invitrogen; 15596-026) or 100 l icecold phosphate-buffered saline (PBS; pH 7.5) for RNA analysis or protein analysis, respectively. Scales were scraped off from the tail to the head of fish using a blunt tip tweezers. The residual scales were washed out with ice-cold PBS, and skin was separated from muscle with a slender tweezers. Then, the liver, brain, eyes, gills, heart, spleen, kidney, intestines, ovaries, testes, muscle and skin of each fish were separated. Each sample was divided into two parts: for RNA analysis, tissues from three individuals with the same sex were pooled, resulting in 3 pooled samples analyzed per treatment
and stored in TRIZOL reagent at −80 ◦ C; for protein assays, the tissues from three or four fish with the same sex were pooled as one sample and all samples except blood were washed with ice-cold PBS (pH 7.5) three times to avoid contamination by the blood, and then homogenized using a glass homogenizer in 10 volumes of icecold PBS containing a protease inhibition cocktail (Amresco; M222). The homogenates were centrifuged at 13,000 × g for 15 min at 4 ◦ C. For blood samples for protein analysis, the plasma was isolated by centrifugation for 3 min at 15,000 × g. Total protein was extracted from the supernatant of each sample, quantified by Bradford assay (Bradford, 1976), and then frozen immediately at −80 ◦ C. 2.3. Western blot analysis of VTG Western blot analysis was performed as described previously with minor modifications (Sambrook and Russell, 2001). A total of 25 g protein from each fish sample was separated using 5% stacking and 10% separating sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were subsequently transferred to a nitrocellulose membrane (Millipore; HATF00010). The membrane was cut into two pieces according to different molecular weights, which allowed for the separate detection of VTG and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The membrane was blocked with 4% skimmed milk in PBS. For VTG analysis, the blocked membrane was incubated for 1 h at room temperature (RT) with antiserum (diluted to 1:5000) against zebrafish VTG, which was kindly provided by Liao et al. (2006). After washing three times in 0.1% Tween-20 in PBS for 10 min each time, followed by three similar washes with PBS, the membrane was incubated for 1 h at RT with peroxidaseconjugated goat anti-rabbit IgG (diluted to 1:5000; Pierce; 31466). After this, the membrane was visualized using an enhanced chemiluminescence (ECL) assay (Boster; AR1171) according to the manufacturer’s procedure. Similar procedures were performed to detect GAPDH except that a mouse monoclonal antibody against GAPDH (diluted to 1:2000; Santa Cruz; sc-47724) and peroxidaseconjugated goat anti-mouse IgG (diluted to 1:5000; Pierce; 31431) were used as primary and secondary antibodies, respectively. VTG levels were quantified using ImageJ software (National Institutes of Health), and the relative level of the VTG protein in the control group was set as 1. 2.4. RNA extraction, reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) The tissue samples in TRIZOL reagent were homogenized using an RNase-free glass homogenizer on ice. The sample volumes should not exceed 10% of the volume of TRIZOL reagent. Total RNA was extracted from the homogenized tissue samples in TRIZOL reagent according to the protocol recommended by the manufacturer (Invitrogen). The quality and integrity of the total RNA were determined by electrophoresis in agarose gel. The concentration of RNA in each sample was measured with a NanoDrop ND-1000 UV-visible spectrophotometer. The RNA samples were digested with DNases (RNase free; Fermentas; EN0521) to remove the genomic DNA. cDNA was reverse transcribed from 2 g of RNA of each sample using the RevertAidTM First Strand cDNA Synthesis Kit (Fermentas; K1622) according to the manufacturer’s procedure. For RT-PCR and qPCR, a 130 bp fragment of zebrafish VTG1 was amplified with VTG1 forward (5 -ACGAACAGCGAGAAAGAGATTG3 ) and reverse primers (5 -GATGGGAACAGCGACAGGA-3 ). ˇ-actin was amplified as an internal control for calibration purposes using forward (5 -ATGGATGAGGAAATCGCTGCC-3 ) and reverse primers (5 -CTCCCTGATGTCTGGGTCGTC-3 ). These primers were designed as described by Jin et al. (2008) and used to amplify a 127 bp region of the zebrafish ˇ-actin gene. The PCRs were performed on a
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Fig. 1. Tissue distribution of the VTG in adult zebrafish with or without 50 ng (168.7 pmol) EE2 /L treatment. (A) Expression of VTG protein in various tissues of adult zebrafish as detected by Western blotting after application of polyclonal rabbit anti-zebrafish VTG antibody. A total of 25 g protein pooled from three fish was applied per well. Analysis of glyceraldehyde phosphate dehydrogenase (GAPDH) served as a constitutive control to show that equal amounts of protein were used per well. (B) VTG1 mRNA expression as measured by RT-PCR in adult zebrafish. Total RNA samples were collected from tissues of three adult zebrafish. ˇ-actin mRNA was used as an internal control. M, male zebrafish; F, female zebrafish; Std, molecular weight standard; −, without EE2 treatment; +, exposure to 50 ng (168.7 pmol) EE2 /L for 7 days.
thermal cycler (Bio-Rad T100TM ) at 95 ◦ C for 2 min, followed by 40 cycles of denaturation at 95 ◦ C for 15 s, annealing at 60 ◦ C for 15 s and extension at 72 ◦ C for 45 s, before a final termination step of 72 ◦ C for 5 min. qPCR was carried out on a real-time PCR detection system (CFX ConnectTM Real-Time PCR Detection System Bio-Rad) running the same program as for the RT-PCR. Each 20-L reaction mixture contained 1 L cDNA, 10 L SYBR Green Mix (TOYOBO; QPK-201), and 125 nM of forward and reverse primers. Standard plots for each target gene were produced by plotting cycle threshold (Ct) values against log-transformed 10-fold dilutions of cDNA sample from controlled female liver. The corresponding standard curve was used to evaluate the amplification efficiency of each primer pair. The amplification efficiency values for VTG1 and ˇactin were 100.9% and 96.9%, respectively. The relative expression level of VTG1 in each sample was calibrated by comparison to ˇ-actin transcript level in the same sample using the equation: (Ct) = Ct(VTG1) − Ct(ˇ-actin) . The relative level of the VTG1 transcript in the control group was set as 1, so the fold-induction change of relative VTG1 expression in each exposed group was calculated as 2−(Ct(exposed) − Ct(control)) . Each sample was run in triplicate.
2.5. Statistical analyses Data of relative VTG1 expression were presented as mean ± standard deviation (SD) and were log-transformed before statistical analysis to achieve variance homogeneity. All statistical analysis was performed using SPSS 13.0 software (SPSS, Chicago, IL, USA), and p < 0.05 was considered to be a statistically significant difference. Statistical differences among treatment groups as well as tissues in the same treatment were assessed by one-way analysis of variance (ANOVA) followed by a Tukey’s post hoc tests.
3. Results 3.1. Distribution of VTG in zebrafish Western blot analysis with rabbit polyclonal antibodies against zebrafish VTG was used to detect the distribution of VTG protein in adult zebrafish. GAPDH was used as an internal reference to confirm that equal amounts of protein were used in the analyses. As shown in Fig. 1A, prior to EE2 treatment, there was no positive signal for VTG in any of the 13 tissues and organs from male fish; however, in female fish VTG was detectable in all tissues and organs examined, except the intestine and ovaries, and the strongest signal came from the plasma. After exposure to 50 ng (168.7 pmol) EE2 /L for 7 days, male fish showed detectable VTG signals in all tissues except the intestine, while levels of VTG were greater in female tissues after EE2 exposure except for in the ovaries and intestine. It was difficult to detect VTG in the intestine of male and female fish and only low molecular protein bands were detected that may be degradation products of VTG due to the abundance of enzymes found in the intestine. Since the anti-VTG antibody can also recognize yolk protein, the mature protein of VTG, it also could be detected by Western blotting. In the ovary tissues, yolk protein signals were detected rather than signals for VTG. EE2 had no apparent effect on levels of yolk protein compared with the control group. In gill and kidney tissues, besides VTG, weak signals for yolk protein were detected in control females while strong signals were detected in males and females exposed to EE2 . The VTG mRNA expression levels in various tissues of adult zebrafish were investigated using RT-PCR with specific primers for VTG1. In male fish, VTG1 expression was detected in the heart, liver, spleen, skin, muscle, eye, fin, and brain tissues prior to EE2 treatment; while after EE2 exposure, the expression of VTG1 was detected in all tissues and organs except the blood (Fig. 1B). In
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Fig. 2. Western blot analysis of VTG protein in liver, plasma, skin and eye samples of adult male zebrafish exposed to 0, 3.125, 6.25, 12.5, 25 or 50 ng (0, 10.55, 21.09, 42.18, 84.35 or 168.7 pmol) EE2 /L for 7 days. Polyclonal rabbit anti-zebrafish VTG antibody was used in the analysis. A total of 25 g protein pooled from three fish was applied per well. Analysis of glyceraldehyde phosphate dehydrogenase (GAPDH) served as a constitutive control to show that equal amounts of protein were used per well. The bar charts below show the VTG protein levels quantified using ImageJ software according to the Western blot results. The values represent the fold changes, and the relative level of the VTG protein in the control group was set as 1.
female fish, RT-PCR revealed that VTG1 mRNA was detected in all tissues and organs except the blood with or without EE2 treatment (Fig. 1B). Furthermore, after EE2 treatment, VTG1 was up-regulated in all samples except the brain and blood in male fish, while in female fish there was no obvious up-regulation of VTG1 in all samples except the liver and skin. 3.2. Effect of different concentrations of EE2 on VTG expression in male fish Owing to ease of sampling, the skin and eyes of male fish were selected for further study to assess the effects of different concentrations of EE2 on VTG expression. Results were compared to VTG levels in the liver and plasma as these tissues are traditionally used
for VTG analysis. Western blotting showed that the amount of VTG in each sample containing 25 g of total protein was greatest in the plasma, lower in the skin and eyes, and lowest in the liver tissue (Fig. 2). Concentration-dependent increases of VTG expression were shown during EE2 exposure for 7 days in the plasma, skin, eyes and liver tissues of male fish. A slight change of VTG expression was observed in plasma at 6.25 ng (21.09 pmol) EE2 /L, while VTG expression was increased obviously at 12.5 ng (42.18 pmol) EE2 /L in the liver, skin and eyes. Greatest VTG levels were observed at 50 ng (168.7 pmol) EE2 /L in all four tissue types.qPCR was carried out to confirm the effects of different doses of EE2 on the expression of VTG1 mRNA (Fig. 3). Since there is no expression of VTG1 mRNA in the blood (Fig. 1B), only the liver, skin and eye tissues were used in this investigation. As shown in Fig. 3, significant dose-dependent
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4. Discussion
Fig. 3. Real-time quantitative PCR analysis of the expression levels of VTG1 mRNA in liver, skin and eye samples of adult male zebrafish exposed to 0, 3.125, 6.25, 12.5, 25 or 50 ng (0, 10.55, 21.09, 42.18, 84.35 or 168.7 pmol) EE2 /L for 7 days. Values are means ± SD (n = 3), and were normalized against ˇ-actin as a housekeeping gene. Data were analyzed by one-way ANOVA followed by a Tukey’s multiple comparison test. Different uppercase letters denote a significant difference among treatment groups within tissue; different lowercase letters denote a significant difference among tissues within treatment (P < 0.05). The relative mRNA expression levels of VTG1 are shown with a logarithmic scale. The products of real-time quantitative PCR were analyzed on 1.5% agarose gel.
increases of VTG1 mRNA expression were observed during EE2 exposure in all three tissues examined. It could be seen that liver was the most sensitive tissue to EE2 and 3.125 ng EE2 /L (10.55 pmol EE2 /L) was enough to induce significant increase of VTG1 mRNA. While significant increases of VTG1 mRNA in skin and eye were apparent at 6.25 and 12.5 ng EE2/L (21.09 and 42.18 pmol EE2 /L), respectively, and levels peaked in all tissues at 50 ng (168.7 pmol) EE2 /L. The induced expression of VTG1 mRNA in the eye was much lower than observed for the skin and liver samples after exposure to EE2 .
3.3. Effect of exposure to 50 ng (168.7 pmol) EE2 /L for different times on VTG expression in male fish Western blotting and qPCR analysis were used to investigate the effect of EE2 on VTG expression in liver, plasma, skin and eye tissues of male zebrafish at different times after exposure to 50 ng (168.7 pmol) EE2 /L. Western blot analysis showed significant increases of VTG expression in a time-dependent manner at 0, 2, 4 and 7 days after exposure to EE2 in all four tissues examined. Earliest induction of VTG was detected in the plasma and a strong signal was detected at 2 days after EE2 exposure, while in liver, skin and eye tissues a strong signal was detected from 4 days onwards. Greatest VTG expression was observed at 7 days after exposure in all four tissue types (Fig. 4). The qPCR results revealed that EE2 induced VTG1 mRNA expression in a time-dependent manner in liver, skin and eye tissues. Significant increases of VTG1 mRNA were detected in all three tissues at days 2. Meanwhile, greatest expression of VTG1 mRNA was observed after 7 days of EE2 exposure in all three tissues. Among the three tissues, the expression of VTG1 mRNA induced by EE2 in the eye was much lower than observed for the skin and liver samples (Fig. 5).
The short generation time of zebrafish has meant that it has been widely exploited as a model organism for investigating chemical toxicity (Hill et al., 2005; McGrath and Li, 2008; Scholz et al., 2008; Segner, 2009). VTG has been proven to be an ideal biomarker for measuring exposure to xenoestrogen in oviparous fish (Denslow et al., 1999; Heppell et al., 1995); however, recently the distribution of VTG in different tissues has been questioned. Previous studies of zebrafish have reported that VTG transcripts were detected not only in the liver, but also in extrahepatic tissues (Islinger et al., 2003; Jin et al., 2008; Tingaud-Sequeira et al., 2012; Wang et al., 2005; Yin et al., 2009), even though expression of the VTG protein has been rarely reported in extrahepatic tissues. Therefore, in this present study, the distribution of VTG (protein and mRNA transcript levels) in zebrafish was investigated. VTG was not detected in all the tissues tested for male fish prior to exposure to EE2 , while after treatment with 50 ng (168.7 pmol) EE2 /L VTG was detected in all tissues except for the intestine. In female fish, the VTG protein was present in all tissues examined except for the intestine and ovaries whether EE2 treatment was applied or not. Interestingly, VTG was induced in male fish by EE2 treatment to a greater extent in the eye, skin, heart and plasma tissues compared to the liver which is used conventionally for VTG detection. No VTG signals were detected in the ovaries but there were signals for yolk protein, probably because VTG is taken up by the ovaries and cleaved into yolk protein (Wahli, 1988; Wallace, 1985). It is interesting to note that besides VTG, the yolk protein and several other protein bands of low molecular weights were present in gill and kidney samples, which may be because these are important excretory organs in fish, and it is known that VTG is eliminated from the blood through the kidneys and gills (Del Giudice et al., 2011). Therefore, we suppose that VTG degradation occurred by enzymolysis during VTG elimination. RT-PCR was used to confirm the RNA expression of VTG1 in 13 tissue types. There are seven VTG genes in zebrafish and, among these, VTG1 is the most highly expressed (Tong et al., 2004; Wang et al., 2005). VTG1 mRNA is used as a biomarker for EDCs in zebrafish (Muncke and Eggen, 2006; Tong et al., 2004). The RT-PCR results showed that VTG1 was expressed in all tissues except the blood in female fish whether or not they had been treated with EE2 and in male fish that had been treated with EE2 (Fig. 1B). Even without EE2 treatment, VTG1 mRNA was detected in the liver and several extrahepatic tissues (e.g. heart, spleen, skin, muscle, eye, fin, and brain) of male fish. Compared with other tissues, the liver has relatively greater mRNA levels of VTG1 and lower VTG protein levels in response to EE2 , which suggests that the liver may be a major organ synthesizing VTG and transporting it by blood to the ovaries (Byrne et al., 1989; Copeland et al., 1986; Wallace, 1985). On the other hand, some extrahepatic tissues, such as heart, spleen, skin and eyes, have both relatively high mRNA levels and protein levels in response to EE2 , which suggests that these organs can synthesize VTG, but do not or only rarely transport it away. Conversely, the intestine has relative higher mRNA VTG1 levels and undetectable levels of VTG protein, which suggests that VTG may be digested rapidly by the abundant proteinases found in the intestine. VTG expression in many extrahepatic tissues may be due to the extremely wide distribution of white adipose tissue, which has been identified previously to express the VTG1 gene in zebrafish (Tingaud-Sequeira et al., 2012). Further studies need to be performed to understand the biological functions of the induced VTG in these extrahepatic tissues. Taking samples of liver and blood plasma from small fish like zebrafish is very difficult and, due to the limited amount of sample obtained, liver and plasma tissues from individual fish often have to be combined to create enough sample for accurate analyses. For example, livers and plasma samples from more than three fish
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Fig. 4. Western blot analysis of VTG protein in liver, plasma, skin and eye samples of adult male zebrafish exposed to 50 ng (168.7 pmol) EE2 /L for 0, 2, 4 and 7 days. Polyclonal rabbit anti-zebrafish VTG antibody was used in the analysis. A total of 25 g protein pooled from three fish was applied per well. Analysis of glyceraldehyde phosphate dehydrogenase (GAPDH) served as a constitutive control to show that equal amounts of protein were used per well. The bar charts below show the VTG protein levels quantified using ImageJ software according to the Western blot results. The values represent the fold changes, and the relative level of the VTG protein in the control group was set as 1.
were pooled for Western blot analysis in this present study. However, samples of skin and eyes are much easier to collect and these can be obtained in sufficient amounts to allow individual fish to be studied. Owing to the high expression of VTG and the ease with which the tissues can be sampled, the skin and eyes of zebrafish were chosen to assess the levels of VTG that were induced by EE2 treatment at various doses and at different times after exposure. Then, these data were compared with liver and plasma samples that are used traditionally for biomarker studies. Interestingly, the plasma, liver, skin and eye tissues from male zebrafish all showed concentration- and time-dependent patterns of VTG
protein expression after EE2 treatment (Figs. 2 and 4). The subsequent qPCR analyses also revealed that EE2 induced significant dose- and time-dependent increases of VTG1 mRNA expression in skin and eye tissues as well as in the liver (Figs. 3 and 5). Thus, skin and eye tissues are suggested to be alternative choices for VTG biomarker detection studies in zebrafish. Skin and eye tissues are suitable as alternative tissues for analysis of VTG protein detection, while the skin is more suited than the eye to replace the liver for VTG mRNA analysis. In conclusion, this present study demonstrates that VTG protein and mRNA was detected not only in the liver but also in various
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Fig. 5. Real-time quantitative PCR analysis the of the expression levels of VTG1 mRNA in liver, skin and eye samples of adult male zebrafish exposed to 50 ng (168.7 pmol) EE2 /L for 0, 2, 4 and 7 days. Values are means ± SD (n = 3), and were normalized against ˇ-actin as a housekeeping gene. Data were analyzed by oneway ANOVA followed by a Tukey’s multiple comparison test. Different uppercase letters denote a significant difference among treatment groups within tissue; different lowercase letters denote a significant difference among tissues within treatment (P < 0.05). The relative mRNA expression levels of VTG1 are shown with a logarithmic scale. The products of real-time quantitative PCR were analyzed on 1.5% agarose gel.
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