1. Introduction
Growth hormone, prolactin and somatolactin belong to the same hormone family and have a similar structure in teleost fishes [1]. Each of these three hormones appear to have opposite or specific functions in electrolyte balance in teleosts [2]. Teleost fish in freshwater environment face two primary challenges: preventing the loss of ions to the external hypoosmotic environment and preventing the influx of water. Prolactin plays a central role in these activities during the adaptation of fish to fresh water, as evidenced by its ability to increase plasma ion concentrations (primarily Na+ and Cl-) and decrease the permeability of osmoregulatory organs, such as gill, kidney and intestine [3]. In seawater environment, in contrast, diffusive water loss is counteracted by drinking seawater and actively taking up Na+, Cl- and water across the gastrointestinal tract, and the gill actively secretes Na+ and Cl- through chloride cells. Growth hormone activates these gill chloride cells with ion transporters (e.g., Na+,K+-ATPase and the Na+,K+, 2Cl- cotransporter) involved in secretion of Na+ and Cl- across the branchial epithelium [3], and appears to stimulate the intestinal absorption [4]. On the other hand, somatolactin is proposed to be involved in regulation of acid-base, calcium and phosphate levels in several species [2, 3, 5-7], but the role of somatolactin in the intestine is unknown. Although the presence of receptors for these hormones in ion-transporting organs has been reported in a variety of teleost species [8-14] and several studies have investigated the direct effects of the teleost hormones on osmoregulatory organs [5, 15-18], the modes of action of the hormones are still unclear. In particular, the opposite
In our
2. Materials and methods
2.1. Animals
Adult medaka (
2.2. Tissue culture
As previously described [23], esophagi were gently sliced open along the long axis and cut into halves. Each explant was placed in a individual well of 96-well culture plates containing preincubation medium (MEM with Hanks' salts, 25 mM HEPES, 5 mg/ml BSA, 250 U/ml penicillin G, and 250 µg/ml streptomycin sulfate, adjusted to pH 7.8 at 25°C). After several hours, the medium was replaced with MEM containing Earle's salts, 4 mg/ml BSA, 292 µg/ml L-glutamine, 50 U/ml penicillin G and 50 µg/ml streptomycin sulfate adjusted to pH 7.8 when saturated with 99% O2 / 1% CO2. The medium osmolarity was adjusted to 300 mOsm/kg with NaCl. Explants were randomly assigned to control and treatment groups (
2.3. Cell proliferation assay
At given time points, cultured explants were pulsed with an oxidation-reduction indicator WST-1 (10% vol/vol, Roche, Tokyo, Japan) for 4 h and color development (A450 nm-A600 nm) was quantified to measure the activity of mitochondrial dehydrogenases. This activity is proportional to the number of viable cultured cells and is expressed as the cell proliferating index [35]. The result after pre-incubation (on day 0) was used to correct for differences in initial tissue content per esophageal slice, and also for quantification of apoptosis.
2.4. Quantification of apoptosis
DNA internucleosomal fragmentation in the esophagus was assessed using a cell death detection ELISA kit (Roche, Tokyo, Japan) according to the manufacturer’s instructions. This kit uses a quantitative sandwich ELISA that specifically measures the histone region (H1, H2A, H2B, H3, and H4) of mono- and oligonucleosomes [36] in teleosts [37] that are released during apoptosis, but not during necrosis [38]. After a 10-minute reaction, color development (A405 nm-A492 nm) was quantified using an MTP-300 microplate reader (Corona, Ibaragi, Japan).
2.5. Proliferating cell nuclear antigen (PCNA) immunohistochemistry
To label proliferating cells in the esophagus, we used a mouse monoclonal antibody (clone PC10; Sigma, Tokyo, Japan) against proliferating cell nuclear antigen (PCNA), as described previously for teleosts [14, 19, 33]. In our previous study on the teleost esophagus [19], the level of PCNA immunoreactivity was in agreement with the uptake of [3H]-thymidine. Slides were immersed in 0.3% H2O2 in methanol at 20°C for 30 min to inactivate endogenous peroxidase activity. After washing in PBS, the sections were placed in 5% normal goat serum in PBS at room temperature for 1 h to block non-specific binding. Sections were subsequently incubated at 4°C overnight with the primary antibody diluted 1:100 in a solution containing 0.5% Triton X-100 and 1% BSA (Sigma, Tokyo, Japan) in PBS. Sections were then washed 3 times in PBS, incubated with peroxidase-labeled goat anti-mouse secondary antibody (Sigma, Tokyo, Japan) diluted 1:70 in PBS containing 0.5% Triton X-100 and 1% BSA at room temperature for 1 h, and then developed for 5 min with DAB substrate solution (Roche, Tokyo, Japan). Controls omitting the PCNA primary antibody were performed and yielded no immunoreactivity.
2.6. In situ 3'-end labeling of DNA (TUNEL)
Nuclei of apoptotic cells were detected by the TUNEL method [39] using an
2.7. Statistical analysis
The significance of differences between the means for cell proliferation were analyzed using two-way repeated measures analysis of variance (ANOVA), with time within groups (after application of treatment) as one factor and treatment among or between groups as the other factor. Since there was a significant interaction between treatment and time, each time was analyzed separately to identify differences among the treatments using the appropriate post-hoc test. The significance of differences among means for concentration-response relationships for apoptosis was also tested using ANOVA followed by a post-hoc test. All data were checked for normality and equal variances. Where assumptions of normality or equal variances were not satisfied, equivalent non-parametric tests were used. Results were considered significant for
3. Results
The effects of growth hormone, prolactin and somatolactin (1, 10 and 100 ng/ml) on esophageal cell proliferation for 8 days in culture are shown in Figures 1 and 2. Addition of prolactin at 10 ng/ml to the culture medium significantly (
We examined the effects of growth hormone, prolactin and somatolactin (1, 10 and 100 ng/ml) on esophageal apoptosis after 8 days of culture, since esophageal apoptosis has been shown to be induced significantly 5-10 days after salinity acclimation and hormonal treatment of fish [14, 19, 33]. Addition of growth hormone (10 ng/ml) to the culture medium significantly induced esophageal apoptosis (
4. Discussion
Prolactin is an important hormone for freshwater adaptation in teleost species, whereas growth hormone is involved in seawater adaptation in several euryhaline fishes [3]. In accord with the greater permeability of the esophagus in seawater-acclimated euryhaline fish than in freshwater-acclimated fish [20], we have previously shown that apoptosis throughout the esophageal epithelium occurs for the simple columnar epithelium in seawater and that cell proliferation is induced for the stratified epithelium, which is composed of numerous mucus cells, in fresh water [14, 19]. We have also shown that injection of prolactin stimulates cell proliferation in the esophageal epithelium [33], but neither the mode nor specificity of the action of prolactin is clear. The present study shows that the esophagus of medaka is responsive to prolactin and growth hormone after several days in culture, with induction of cell proliferation and apoptosis, respectively. This is the first demonstration of opposite
The
There are few reports on the
At 100 ng/ml prolactin or growth hormone, the above significant effects on esophageal cell turnover were disappeared. Very high doses of these hormones may also activate receptors for the other hormones even in homologous systems. It is reported that prolactin can bind to growth hormone receptor in tilapia (
In our previous study using this esophagus culture system, low levels of cortisol stimulate epithelial apoptosis through glucocorticoid receptors in seawater, whereas high levels of cortisol induce epithelial cell proliferation also via glucocorticoid receptors in freshwater [23]. Interactions of prolactin/growth hormone with glucocorticoid may play an important role in the cell turnover in osmoregulatory esophageal epithelia during acclimation to different salinities. In the gill, cortisol is suggested to promote the differentiation of the ion-secretory chloride cell (seawater-form) with growth hormone, and also expedite the differentiation of ion-uptake chloride cell (freshwater-form) by interacting with prolactin [3, 58]. In the amphibian metamorphosis, on the other hand, thyroid hormones with glucocorticoid signaling induce apoptosis in the regression of tadpole tail, whereas prolactin prevents this apoptosis [22](Fig. 4B). Although apoptosis by glucocorticoid appear to be conserved throughout the vertebrates, thyroid hormone has no significant effect on esophageal cell proliferation or apoptosis in euryhaline fish [33]. Therefore, we hypothesize that thyroid hormones are involved only in irreversible metamorphosis and/or developmental processes.
5. Conclusions
Our
Acknowledgement
This study was supported in part by grants to T.S (Grants-in-Aid for Scientific Research (C) Nos. 17570049, 19570057 and from JSPS) and to H.T (JSPS Research Fellowships for Young Scientists Nos. 192156 and 214892).
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