Ontogenetic expression and 17β-estradiol regulation of immune-related genes in early life stages of Japanese medaka (Oryzias latipes)

Fish & Shellfish Immunology 30 (2011) 1131e1137

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Ontogenetic expression and 17b-estradiol regulation of immune-related genes in early life stages of Japanese medaka (Oryzias latipes) Liwei Sun, Xiaolu Shao, Yudan Wu, Jingming Li, Qinfang Zhou, Bo Lin, Shenyuan Bao, Zhengwei Fu* College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 August 2010 Received in revised form 14 February 2011 Accepted 20 February 2011 Available online 6 March 2011

Accumulating evidence suggests that environmental endocrine disrupting chemicals (EDCs) may exert adverse effects on aquatic organisms via the modulation of immune competence in addition to the endocrine system. However, to date, most studies have been undertaken only on biochemical and histopathological endpoints, and few studies have addressed the role of immune response gene transcript abundance in response to estrogen. In the present study, the ontogenetic expression of immune-related genes, including three complement components (C3-1, C3-2 and Bf/C2), two cytokines (IL-21 and type I IFN [IFN]), lysozyme (LZM), novel immune-type receptor (NITR-18), Ikaros (IK) and ceruloplasmin (CP) were characterized during different developmental periods (from 0 to 28 d post-hatch [dph]) in Japanese medaka. Furthermore, the responses of these genes to natural estrogen (i.e., 17b-estradiol [E2]) were evaluated. E2 exposure at sublethal concentrations (0.1e10 mg/L) down-regulated the gene expression of C3-1, C3-2, Bf/C2, LZM and CP, while up-regulating the expression of IL-21, IFN, NITR-18 and IK. The results demonstrate a very different trend in gene expression in fish larvae exposed to E2 when compared with the ontogenetic changes in control, suggesting that exposure to environmental chemicals with estrogenic activities may interfere with immune-related genes and thus potentially influence the susceptibility of fish to opportunistic infections. These findings confirm the ability of exogenous estrogens to elicit changes in immune-related gene expression, and broaden our understanding about the mechanisms underlying the actions of EDCs. In addition, the expression profiles of immune-related genes can be developed for use as biomarkers for future immunotoxicological studies. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Endocrine disrupting chemicals Innate immune system Gene expression Estrogen

1. Introduction The aquatic environment represents the ultimate sink for a broad group of natural and synthetic chemicals. There is now powerful evidence that aquatic animals are endangered by the effects of these chemical substances, including environmental endocrine disrupting chemicals (EDCs) [1]. In fish, endocrine disruption has been postulated as the cause of various adverse effects such as reduced fertility, abnormal sexual differentiation and developmental abnormalities [2]. These phenomena have attracted significant scientific and public attention during the last two decades, and stimulated considerable research efforts on the mechanism of action of various EDCs [1,3]. Recent field and laboratory studies suggest that the modulation of the reproductive tissues by sex steroids or EDCs may also affect the immune system [4]. For example, it is well documented that estrogen, xenoestrogens, and other steroid hormones could disrupt * Corresponding author. Tel.: þ86 571 8832 0599. E-mail address: [email protected] (Z. Fu). 1050-4648/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2011.02.020

various aspects of the immune response of fish in vivo, such as phagocytosis, immunoglobulin levels, bactericidal activity, and nitric oxide production [4e8]. These immunosuppressive effects could also be observed in vitro, primarily on the primary cultured leukocytes derived from different tissues and their corresponding cell lines [8e14]. Additionally, exposure to wastewater treatment work (WwTW) effluents has been associated with wide adverse health effects in fish, including inhibition of the immune response [15e17], and the environmental estrogens present in effluents are thought to be partly responsible for this immunosuppression. In fact, whereas once the endocrine and the immune system were considered as two separate entities, today there is increasing insight into their mutual interaction [4]. However, the bi-directional communication between the endocrine and immune systems is not completely understood in teleost fish, even if it has been widely demonstrated and accepted in mammals [4]. It is well known that the innate immune system in fish acts as the first line of host defense against pathogenic organisms and foreign materials, and it is thought to be of key importance in primary defense and in instructing the adaptive immunity to

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mount a response by providing the biological context e the “danger signal” [18]. During the early life stages of fish, it is believed that the innate immune system is of vital importance for disease resistance [19] because the immune system is still developing and specific immunity is non-functional [20]. However, only a limited number of studies have focused on the different maturation states of the immune system during fish development. Moreover, since fish in the early life stages are generally considered to be relatively sensitive to the effects of environmental stressors [21], more consideration should be given to the potential hazards to the fish population concerning the impairment of the immune system after EDCs exposure in embryos or larvae. Research into the effects of sex steroids and EDCs on the fish immune system has been characterized by a heavy reliance on biochemical and histopathological endpoints, but the molecular mechanisms of action are largely unknown. More recently, researchers have begun examining gene expression in response to chemical exposure, mostly as an approach to provide greater mechanistic understanding of the chemical effects as well as to develop biomarkers of exposure and effects [21,22]. Since an increasing number of immune-related gene sequences from different teleost species have become available, the impact of environmental toxicants (e.g., arsenic [23,24] and insecticides [25,26]), and natural and synthetic steroid estrogens [26e28] on the mRNA expression of genes involved in the innate immune system has been investigated. The results of these studies suggest that these chemical substances, including EDCs, interfere with the expression of these genes, thus potentially impairing immune competence. However, the discrepancies and scarcities addressing the immune-reproductive system interactions at the level of gene expression still remain. The objective of this study was to characterize and quantify ontogenetic changes in immune-related gene expression during larval and larvaeejuvenile transition development in Japanese medaka. These genes included three complement components (i.e., C3-1, C3-2 and Bf/C2), two cytokines (i.e., IL-21 and type I IFN [IFN]), lysozyme (LZM), novel immune-type receptor (NITR-18), Ikaros (IK) and ceruloplasmin (CP). It is well known that the complement system is one of the first lines of immune defense as well as a modifier of acquired immunity [18,29]. The complement system is activated though three different but partially overlapping routes: the classical pathway, the alternative pathway and the lectin pathway [19,29]. Complement C3 is the central protein of all three activation pathways and the major opsonin of the complement system, which is also essential for the generation of the membrane attack complex [29]. Besides complement C3, other components, such as complement C2 and factor B, also play important roles in the innate immune response. Moreover, inflammation typifies the innate immune response and an inflammatory insult will result in a cytokine cascade [30]. To date, many cytokines have been cloned from fish, including tumor necrosis factors, interleukins and several chemokines [18]. Therefore, the IL-21 and type I IFN genes were included in this study. Lysozyme is also an important defense molecule of fish innate immune system, which possesses lytic activity against microbial invasion and is expressed in a wide variety of tissues [31]. Additionally, the Ikaros (IK) gene encodes a transcription factor which is used as an early lymphoid marker [20]. With respect to NITR genes, it is suggested that they are probably involved in regulating the function of cytotoxic and likely other types of immune reactive cells [32]. It has been suggested that CP acts as a nonspecific factor of viral immunity. Therefore, the detection of these genes will extend our investigation on the maturation of the medaka immune system. Additionally, we evaluated the responses of these genes to the endogenous estrogen 17b-estradiol (E2) at sublethal concentrations. The results obtained from this study will also enable a better understanding of influence

of sex steroidal hormone on the immune system during the early life stages of teleost fish. 2. Materials and methods 2.1. Fish husbandry Japanese medaka (d-rR) stock used in this study originated from the Laboratory of Freshwater Fish at the Bioscience Center of Nagoya University, Japan. Breeding pairs (approximately 6 months old) were maintained in charcoal-dechlorinated tap water at a constant temperature (25  1  C) with a photoperiod of 16:8 h (light:dark). The eggs spawned from stock females were collected within 4 h after fertilization. Egg clutches were pooled and separated. Eggs were then disinfected by placing them in a 0.9% solution of hydrogen peroxide for 10 min and checked for fertilization using a dissecting microscope. The embryos were counted, and every thirty embryos were transferred into a glass dish containing 150 mL dechlorinated tap water with 2 mg/L methylene blue and incubated under the same conditions described above for adult fish. In order to minimize variations caused by the different developmental stages, only larvae that hatched on the 9th day post-fertilization (within 8 h) were selected for the following test procedures. 2.2. Exposure conditions Exposure studies were conducted in glass beakers containing 150 mL of test solution, under the same conditions described above. At the beginning of exposure, approximately thirty newly hatched larvae were placed in one beaker. During the development period, the numbers of larvae were adapted in order to maintain the appropriate loading rate. Based on the exposure concentrations of E2 employed in previous reports [6,26] and our preliminary test, larvae were exposed to E2 (SigmaeAldrich, St Louis, MO) at concentrations of 0.1, 1 and 10 mg/L; the concentration of acetone (solvent carrier) never exceeded 0.01% of the final concentration. The solvent control group was exposed to 0.01% acetone alone. There were at least three separate tanks of fish used for each treatment level. The test solutions were renewed daily. At regular intervals (0, 3, 6, 10, 14, 21 and 28 days post-hatch [dph]), the larvae were sampled at about 18:00, flash-frozen in dry ice and then stored at 80  C for gene expression analyses. The larvae were observed twice daily and any dead larvae were removed. The larvae were fed twice per day with Paramecium until 4 dph followed by newly hatched brine shrimp (Artemia nauplii). 2.3. Quantitative real-time PCR assay Total RNA of whole fish was extracted with TRIzol reagent (Invitrogen, USA), following the manufacturer’s protocol. Due to vast differences in size, different numbers of larvae (ranging from about 12 larvae for those aged 0 and 3 dph to 5 larvae for those aged 28 dph) were pooled into one sample. There were five replicate samples at each time point per age group. The quality of total RNA was evaluated by agarose gel electrophoresis and optical density (260/280 ratio 1.8e2.0). First-strand cDNA synthesis was performed using the ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. Real-time PCR with SYBR green detection was performed on the Mastercycler ep realplex (Eppendorf, Hamburg, Germany) according to protocols established by the manufacturer (SYBRÒ Green Real-time PCR Master Mix, Toyobo). Samples were amplified under the following conditions: denaturation for 1 min at 95  C, followed by 40 cycles of 15 s at 95  C, and 1 min at 60  C. Nine target genes were chosen for quantification. The oligonucleotide primers specific for these genes were designed with

L. Sun et al. / Fish & Shellfish Immunology 30 (2011) 1131e1137

PrimerExpress software (Applied Biosystems, Foster City, CA) and are summarized in Table 1. The relative gene quantitation was expressed in relation to the expression of a reference gene, ribosomal protein L7 (RPL-7), because this gene showed little variation in its expression during the early stages of development or following EDC treatment in Japanese medaka [33]. 2.4. Data analysis Throughout this study, data are presented as means  standard error of the mean. Prior to conducting statistical comparisons, data were assessed for the normality and homogeneity of variances using the KolmogoroveSmirnov one-sample test and Levene’s test, respectively. When necessary, data were transformed for normalization and to reduce heterogeneity of variance. Intergroup differences were assessed by analysis of variance (ANOVA) followed by Dunnett’s or Student-NewmaneKeuls post-hoc test. The critical value for statistical significance was p < 0.05. All statistical analyses were conducted with the SPSS 13.0 software (SPSS, Chicago, IL). 3. Results 3.1. Expression profiles of immune-related genes during the first 28 days post-hatch of Japanese medaka Overall survival of larvae was greater than 90% in all clutches studied. The specificity of the quantitative real-time PCR products was confirmed by melting curve analyses as well as by agarose gel electrophoresis (data not shown). The expression levels of the immune-related genes in Japanese medaka aged 3, 6, 10, 14, 21 and 28 dph in relation to 0 dph larvae are presented in Fig. 1. The complement component C3 genes, termed C3-1 and C3-2, showed similar trends of increasing expression after hatching (Fig. 1A and B). Compared with 0 dph larvae, significant up-regulation of both genes was first observed at 10 dph (w5.5- and 4.8fold, respectively). At 28 dph, both genes were expressed at their maximal levels (w12.9- and 7.8-fold, respectively) during the study period. The expression level of Bf/C2 (Fig. 1C) stayed relatively constant during the first 21 dph and then increased significantly at 28 dph (w2.1-fold). After hatching, the expression of IL-21 increased and reached a maximum at 10 dph and then gradually declined, returning to levels similar to that observed at 0 dph (Fig. 1D). The expression of

Table 1 Summary of primers used in this study. Target gene Accession # Primer sequences (50 e30 )

Size (bp)

C3-1

AB025575

118

C3-2

AB025576

Bf/C2

D84063

IL-21

EF513162

LZM

AU179932

IFN

BN001095

CP

BM309822

NITR-18

EU419369

IK

AB274723

RPL-7

DQ118296

F-CCT AAA CAG CAA GCA CAG ACT CAC R-CCC AGC ATC AAA GAA CAC ACT C F-GAA AAC GGA GAG GGA AAA GTA GTG R-GCA CGC TAA CAG AAA CAA AGA TG F-ATC GCC TTG GAC ATT TCA GAG A R-GAC ACA GTG AAG GCT GCA ATC T F-CTG CTG CAG CCT CAA AGC GT R-TGA GGT GCA GTT TTG GCA CGA F-ACC AAT GCC ATC AAC CAC AA R-GTT GAC TCT TCC GGT TTT TCC A F-GGA AGT GTC TGC CCT GTT TGA R-GAG TGA AGC TCA TCA GCC TGT C F-TTG GCT CGC ATA CCA TTC C R-CGT TTT CCC TCA CAT CTC ACA C F-CAT CCC CAC CTC ATT TAC ACA A R-CCG AAC AGG ATT TCT CCA CAC F-TGA CCA AGC AAT CAA CAG CG R-GCT TGT GGA GGC CAT ACA AA F-CGC CAG ATC TTC AAC GGT GTA T R-AGG CTC AGC AAT CCT CAG CAT

106 101 166 102 108 108 149

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another cytokine, IFN, did not change during the first 10 days followed by a gradual decrease to the level which was only 1/5 of 0 dph value by 28 dph (Fig. 1E). The relative expression of LZM appeared to increase over the developmental period studied (Fig. 1F). Significant up-regulation (w18.4-fold) of this gene was observed at 14 dph. By 28 dph, the expression level had increased more than 25-fold above the initial expression level. In terms of expression changes for NITR-18, up-regulation was observed between 3 and 6 dph but expression dropped markedly thereafter (Fig. 1G). Although not statistically significant, the expression of NITR-18 between 14 and 28 dph decreased to a level below that observed on 0 dph. No changes in the expression of IK were found through the first four weeks after medaka hatching (Fig. 1H). An up-regulation in CP expression started at 3 dph (w7.2-fold) compared with that on 0 dph and was maintained through the study period (Fig. 1I). 3.2. Effects of E2 on the immune-related genes of Japanese medaka during the first 28 days post-hatch Throughout the exposure period, no differences were found in mortality between the E2-treated fish and controls (data not shown), indicating that the chemical tested was not acutely toxic at the concentrations used. The changes in immune-related gene expression in response to E2 exposure are presented in Fig. 2. The mRNA expression of complement component C3 genes C31 and C3-2 demonstrated similar trends after E2 treatment, decreasing in a concentration- and time-dependent manner (Fig. 2A and B). It was observed that 1 mg/L E2 exposure caused significantly reduced expression of both genes after 6 days. At 14 or 21 days of exposure, the expression level had declined markedly by more than 5-fold compared to that of the control group. Exposure to E2 also resulted in down-regulation in Bf/C2 expression levels when compared to the control group (Fig. 2C). After only 6 days of E2 exposure, a significant decrease was observed even at the lowest exposure concentration (0.1 mg/L). However, after prolonged exposure, this effect on expression was less severe (Fig. 2C). In contrast, with respect to two cytokines, i.e. IL-21 and IFN, fish exposed to E2 demonstrated increases in gene expression (Fig. 2D and E). Discrepancies were noted at specific sample points, but the up-regulation trend caused by E2 persisted over the period studied. The expression level reached a maximum at 28 dph in the highest concentration treatment group (10 mg/L E2), with a 4.3-fold induction for IL-21 and a 14.1-fold induction for IFN. Fish exposed to E2 demonstrated marked decreases in LZM expression levels (Fig. 2F). Excluding 3 and 10 dph, significant down-regulation was observed at every concentration tested throughout most of the study period, with expression decreasing to less than 5% of the control level. In contrast, E2 up-regulated NITR18 mRNA expression, which peaked at 28 dph in the highest exposure group with an 8.1-fold induction compared to unexposed controls (Fig. 2G). The expression of IK followed a similar pattern of increased expression in response to E2 exposure, reaching maximum induction (w15.0-fold) at 28 dph in the highest exposure group (10 mg/L E2) (Fig. 2H). For CP, a significant decrease in expression was found in the 1 mg/L E2 treatment group as early as 3 dph (Fig. 2I). A trend of dose-dependent decline was then observed in the period that followed. However, expression returned to levels similar to that observed in the unexposed controls by 28 dph. 4. Discussion

119 72

In this study, we characterized temporal expression profiles of immune-related genes during the early life stages of Japanese medaka. Furthermore, abundance of these gene products in

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Fig. 1. Quantitative expression profiles of immune-related genes, including (A) C3-1, (B) C3-2, (C) Bf/C2, (D) IL-21, (E) IFN, (F) LZM, (G) NITR-18, (H) IK and (I) CP, from Japanese medaka during the first 28 dph in relation to those observed at 0 dph. The results are represented as means  S.E.M. (n ¼ 5) and expressed as the ratio of target gene mRNA/RPL-7 mRNA. Statistically significant differences are denoted by different letters (p < 0.05, one-way ANOVA followed by Student-NewmaneKeuls post-hoc test).

response to the endogenous estrogen, 17b-estradiol was also evaluated. The results demonstrated that sublethal E2 exposure resulted in significant modulation of several immune response gene mRNA concentrations. This suggests that defense functions of the immune system in Japanese medaka may be influenced by estrogen.

Employing fish in early life stages are generally considered as a promising alternative to utilizing juvenile and adult fish in toxicological studies [21]. Because the developmental process is still ongoing during this period, the knowledge of ontogenetic changes in gene expression is vital for interpreting toxic effects and selecting suitable biomarkers for toxicological evaluation. However,

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Fig. 2. Transcript level of the mRNA of immune-related genes, including (A) C3-1, (B) C3-2, (C) Bf/C2, (D) IL-21, (E) IFN, (F) LZM, (G) NITR-18, (H) IK and (I) CP, in Japanese medaka larvae exposed to 0.1, 1 and 10 mg/L E2. The results are represented as means  S.E.M. (n ¼ 5) and expressed as the ratio of target gene mRNA/RPL-7 mRNA. Asterisks denote statistical significance in relation to the control (*p < 0.05 and **p < 0.01, one-way ANOVA followed by Dunnett’s post-hoc test).

similar to other genes, the literature regarding the ontogenetic changes in genes involved in immune system is relatively scarce. Lam et al. [20] monitored the expression of six immune-related genes in zebrafish (Danio rerio) until 105 days post-fertilization (dpf), and correlated these findings with the in situ detection of cells expressing these genes in lymphoid tissues. Also using

zebrafish, Wang et al. [19] investigated the ontogenetic expression of key complement component genes during the first 24 dpf and then examined the responses of these genes to lipopolysaccharide challenge. However, with respect to Japanese medaka, while cloning and characterization of some immune-related genes have been performed, little is known about the temporal changes in gene

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expression in early life stages. In this study, nine critical target genes of the immune system were chosen for quantification during early development because their genomic sequences were available in the GenBank database and the corresponding primers for quantitative real-time PCR could be designed properly. In fish, the complement systems are well developed [29] and the transcription of most complement components begins early in development [19]. As mentioned above, C3 is the central complement component of all activation pathways and interacts with many proteins [18]. In this study, two C3 genes were monitored since it was not clear whether these multiple C3 sequences represented multiple C3 genes or alleles of a single gene [34]. Compared with 0 dph, steady increases in C3-1 and C3-2 expression were observed, both reaching peak induction at 28 dph (Fig. 1A and B). Because C3 is predominantly synthesized in the liver in fish [35], the ontogenetic appearance of C3 transcripts was probably due to liver development [36]. Similar increasing trends were also found during the development of zebrafish [19] and Atlantic salmon (Salmo salar) [37], although in rainbow trout (Oncorhynchus mykiss) the increasing expression of C3 before hatch followed by a drop [38]. When fish were exposed to E2, expression of both C3-1 and C3-2 declined in a concentration- and time-dependent manner (Fig. 2A and B). This suppression effect, in contrast to their significant up-regulation during larval development (Fig. 1A and B), implied that the expression of these genes was modulated by E2, which thus potentially interfered with the complement system. Similarly in gilthead seabream (Sparus aurata), Cuesta et al. [4] found that treatment with E2 decreased complement activity at 3 and 7 days post-injection. In addition to complement C3, complement C2 and factor B were also monitored in this study. Kuroda et al. [39] isolated medaka cDNA clones corresponding to those of the mammalian Bf or C2 and named the gene medaka Bf/C2 due to the difficulty in assigning the sequence to Bf or C2. Similar to C3, a gradual increase in the expression of Bf/C2 was found, although the level of induction was relatively lower than that observed for C3 (Fig. 1C). This increase may also be attributed to development of the liver. E2 exposure significantly suppressed the expression of Bf/C2 (Fig. 2C), suggesting the potential impact of E2 on complement system functioning. Cytokines encompass a large and diverse family of polypeptide regulators that are secreted by specific cells of the immune system and play important roles in initiating and regulating the inflammatory processes essential for innate immunity [40]. In this study, the expression of IL-21 and type I IFN was detected. It has been reported that IL-2, IL-15 and IL-21 are closely related, signal through a common receptor and have similar biological activities [40]. With respect to IFNs, these proteins induce vertebrate cells into a state of antiviral activity and activate transcriptional regulation of several hundred IFN-stimulated genes [41]. In this study, a bell-shaped curve in expression for IL-21, which increased during the first 10 dph and then declined, was observed (Fig. 1D). In terms of IFN, the expression level stayed relatively constant during the first 10 dph and declined thereafter (Fig. 1E). However, because very little research has been conducted on the expression of IL-21 and IFN in teleost larvae, few data are available for comparison and interpretation of the gene expression changes. On the other hand, the presence of E2 in the water resulted in significant up-regulation of both IL-21 and IFN expression (Fig. 2D and E). The different trends in gene expression between exposed and unexposed fish revealed the modulation of E2 on the activities of cytokines. In the previous report, it was demonstrated that IL-2 is linked to the reproductive axis of mammalian [42] and IL-21 acts through a common receptor as IL-2 which could be the cause of results in this study. Recently, Jin et al. [26] examined the expression of several other cytokine genes (i.e., TNFa, IFN, IL-1b, IL-8, CXCL-Clc,

and CC-chemokine) in newly hatched zebrafish exposed to chemical substances (including E2 and other EDCs) and also found that these genes were regulated significantly. The level or activity of lysozyme is an important index of innate immunity in fish. It is well documented that fish lysozyme possesses lytic activity against pathogenic bacteria and activates the complement system and phagocytes [31]. The continuous increase in LZM expression, peaking at 28 dph at about 25-fold induction (Fig. 1F), is probably a consequence of the role of lysozyme in defense against bacteria because it was reported that bacterial growth occurs in fish eggs as early as 2 h post-hatch [35]. Moreover, because fish lysozyme is mainly distributed in the head kidney [31], the development and maturation of this organ coincides with the concomitant increase in LZM expression. However, a sharp decrease in LZM expression levels was observed when following E2 exposure (Fig. 2F). The result concurred with a previous report that showed that exposure to estrogen caused a significant decrease in lysozyme activity in both fingerling and juvenile Japanese sea bass (Lateolabrax japonicus) [6]. The IK gene encodes a transcription factor that has been shown to be essential for the correct differentiation of B and T lymphocytes in mice [43]. In zebrafish, IK has been shown to be a suitable marker for lymphoid progenitors during early development [20]. In this study, no changes in the expression levels of IK were found through the first 28 dph (Fig. 1H). This result agrees with the results of Lam et al. [20] that reported that IK expression was moderate and only a relatively small change occurred over 105 dpf. It was suggested that because IK encoded a transcription factor, its expression is well-regulated and transient in a limited pool of lymphoid progenitors. When IKexpressing cells begin to express Rag-1, which encodes a protein involved in genomic rearrangement of T-cell receptor and immunoglobulin loci, IK expression was rapidly down-regulated and limited any further increase [20]. However, after exposure to a high concentration of E2 for a relatively long time, the expression of IK in medaka was up-regulated significantly (Fig. 2H). With respect to NITR and CP, the available findings are relatively preliminary. It was reported that NITR genes found in bony fish are members of diversified multigene families and encode type I transmembrane proteins [44]. It is also known that CP synthesis could be induced by several cytokines and other factors, suggesting a link between this protein and immune function [45]. In this study, the expression of NITR-18 was significantly up-regulated at 3 and 6 dph and then returned to levels similar to that observed at 0 dph (Fig. 1G). However, treatment with E2 tended to up-regulate the expression of NITR-18 (Fig. 2G). On the other hand, up-regulation of CP expression was found in the unexposed larvae (Fig. 1I). In contrast, E2 had significant inhibitory effects on CP expression in Japanese medaka larvae (Fig. 2I). Interestingly, at 28 dph, the expression level of CP returned to near-control levels, implying the possibility of fish adaptation to E2 during prolonged exposure. The results of the present study, coupled with previous researches, clearly demonstrate that sex steroids or EDCs, can significantly interfere with the immune system and suggest the possible cross-talk between reproductive and immune systems. Although the concentrations of E2 used in this laboratory study were relatively higher than the existing environmental concentrations, such levels in animals are possible as a result of joint effects of the combination of various chemicals with estrogenic activities. Furthermore, it should be noted that for some genes, significant changes in expression were observed in the lowest E2 exposure group (Fig. 2), which implied that lower concentrations of E2 (<0.1 mg/L) may also exert effects on immune function. However, although we characterized and quantified changes in expression for these immune-related genes, the mechanisms of temporal patterns or responses to E2 could not be completely interpreted because the immune functions of some genes were largely

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unknown and little research was available for comparison. Despite its preliminary nature, the present study did focus attention on the ontogenetic changes in the expression of immune-related genes and the effects of environmental chemicals with estrogenic activities on the immune system in the teleost. In addition, the immune-related gene expression profiles can also be developed for use as biomarkers in future immunotoxicological studies. Acknowledgments We gratefully acknowledge the National Natural Science Foundation of China (No. 20907044, No. 20837002), the Natural Science Foundation of Zhejiang Province (No. Y5090265), and the program from the Department of Education of Zhejiang Province (No. Y200806131) for supporting this research.

[20]

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