Effects of Melatonin on Liver Estrogen Receptor and Vitellogenin Expression in Rainbow Trout: An in Vitro and in Vivo Study

General and Comparative Endocrinology 118, 344 –353 (2000) doi:10.1006/gcen.2000.7472, available online at http://www.idealibrary.com on

Effects of Melatonin on Liver Estrogen Receptor and Vitellogenin Expression in Rainbow Trout: An in Vitro and in Vivo Study David Mazurais,* Mark Porter,† Christe`le Lethimonier,* Gwenola Le Dre´an,* Pascale Le Goff,* Clive Randall,† Farzad Pakdel,* Niall Bromage,† and Olivier Kah* *Endocrinologie Mole´culaire de la Reproduction, UPRES-A CNRS 6026, Campus de Beaulieu, 35042 Rennes Cedex, France; and †Institute of Aquaculture, The University of Stirling, Stirling FK9 4LA, United Kingdom Accepted February 10, 2000

length are commonly used by the trout farming industry to meet the demand for eggs at any time of the year (Bromage et al., 1984, 1992, 1993). However, the mechanisms underlying the effects of photoperiod on seasonal reproduction are far from being fully understood. Although it is not clearly established in fish (Mayer et al., 1997), it is believed that, in vertebrates, the pineal hormone melatonin mediates many circadian and seasonal rhythmic activities (Cardinali et al., 1997) acting primarily through central receptors which were characterized using the specific ligand [2- 125I]iodomelatonin. Binding studies and autoradiography have shown that these receptors belong to the superfamily of G protein-coupled receptors and have allowed their distribution within the brain to be mapped. In fish, one class of high-affinity melatoninbinding sites has been characterized in the brain of several species, with a prominent distribution in the pretectal region, the optic tectum, and the cerebellum, although other regions exhibited binding to a lower extent (Martinoli et al., 1991; Vernadakis et al., 1998; Davies et al., 1994; Ekstro¨m and Vanecek, 1992; Pang et al., 1994). More recently, two mammalian melatonin receptor subtypes, Mel1a and Mel1b, have been identified by molecular cloning studies (Reppert, 1997). Recent studies in rainbow trout cloned two Mel1a and one Mel1b receptors and demonstrated that these re-

Although melatonin is believed to mediate many seasonal and circadian effects of photoperiod on reproduction in salmonids, the precise mechanisms underlying such effects are still largely unknown. Recent data of the literature indicate a relationship between melatonin and expression of estrogen receptors (ER) in various tissues. In this study, the effects of melatonin on estrogen receptor and/or vitellogenin expression were studied by a combination of in vivo and in vitro experiments. In yeast stably expressing ER and transfected with an estrogen-responsive element-␤-galactosidase reporter gene, melatonin had no effect on basal or E2-stimulated ER expression. Incubation of hepatocyte aggregates with melatonin (10 ⴚ8 to 10 ⴚ4) for 16 or 48 h did not modify the E2stimulated ER and vitellogenin mRNA, as measured by dot blots. Finally, neither pinealectomy nor melatonin implants caused any effect on basal or E2-stimulated ER and vitellogenin mRNA contents in the liver. Altogether, these results suggest that, athough we cannot exclude potential effects at the brain or pituitary levels, melatonin has no or little effects on estrogen receptor in the liver. © 2000 Academic Press

In rainbow trout, as in many other salmonids, the timing of gonadal recrudescence, maturation, and spawning is synchronized by the seasonal changes in photoperiod. Consequently, manipulations of day 344

0016-6480/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

345

Effects of Melatonin in Rainbow Trout

ceptors are expressed mainly in components of the visual systems (Mazurais et al., 1999), confirming the data obtained by radioautography. However, the low expression of melatonin receptor messengers within the neuroendocrine hypothalamus and the pituitary makes the identification of the precise targets of melatonin difficult. There are many reports in the literature indicating that melatonin influences estradiol receptor (ER) expression. For example, it has been shown that brain estrogen receptor expression varies during the light/ dark cycle of female ovariectomized rats, an effect partially mimicked by melatonin treatment (Roy and Wilson, 1981). In golden hamster, significant decrease in ER immunoreactivity was noted in the medial preoptic area and bed nucleus of the stria terminalis in response to melatonin, whereas other hypothalamic areas which express ER, e.g., the anterior hypothalamus, showed less dramatic, but still significant, changes (Lawson et al., 1992). In another study, hypothalamic ER mRNA measured by ribonuclease protection assay was decreased by 25% in response to melatonin in both intact and ovariectomized animals (Hill et al., 1996). Similarly, the number of ER-immunoreactive cells detected within the preoptic area, but not the hypothalamus, was approximately 20% higher in ewes exposed to long days compared to ewes exposed to short days (Skinner and Herbison, 1997). Such effects of melatonin on ER expression have also been reported in peripheral tissues. For example, melatonin has also been shown to increase cytoplasmic ER activity in hamster uteri in vivo and in vitro (Danforth et al., 1983a). The molecular mechanisms underlying these effects of photoperiod or melatonin on ER expression are still largely unknown due to the lack of an appropriate model and have been studied only in the breast cancer cells MCF-7 (Danforth et al., 1983b). In these cells, it has been shown that melatonin has antiproliferative effects at concentrations as low as 1 nM (Hill and Blask, 1988), an effect that appears to involve ER, as ER-negative cell lines were unresponsive to melatonin (Hill et al., 1992). Further studies have demonstrated that melatonin significantly suppressed both ER protein and ER mRNA expression in MCF-7 cells in a time- and dose-dependent manner but that this effect is serum dependent (Molis et al., 1993, 1994). More recently, in MCF-7 cells transfected with an estrogen-responsive element (ERE)-luciferase reporter

construct, it was shown that EGF (epidermal growth factor) or insulin cooperates with melatonin to cause an estrogen-independent transactivation of the ER (Ram et al., 1998). Altogether, these results indicate a relationship between melatonin and ER expression in a number of tissues and species. In oviparous species, the liver is a major target of estradiol, which triggers the expression of its own receptor and subsequently that of vitellogenin. The mechanisms underlying the effects of estradiol in upregulating the expression of its own receptor have been thoroughly studied in rainbow trout and involve a well-defined ERE located in the promoter region of the rainbow trout ER (rtER) gene (Pakdel et al., 1989, 1991; Le Dre´an et al., 1995; Flouriot et al., 1996; Pakdel et al., 1997). In addition, hepatocytes maintained in aggregates have proved useful for studying the effects of hormones or xenobiotics on rtER expression (Flouriot et al., 1993; Petit et al., 1997, 1999). For these reasons, we have examined the potential effects of melatonin on liver rtER and vitellogenin expression in vivo following pinealectomy or melatonin implantation and in vitro on hepatocyte aggegrates. In addition, we have looked at the potential direct effects of melatonin on the expression of a reporter gene placed under the control of estrogen-responsive elements in yeast stably expressing rtER (Petit et al., 1995, 1997).

MATERIAL AND METHODS Yeast Strain and ␤-Galactosidase Assays The yeast strain used in this study was BJ-ECZ (a leu2 trp1 ura3-52 prb1-1122 pep4-3prc1-407 gal2::URA32ER E-CYC 1-lacZ) containing the ␤-galactosidase reporter gene under the control of two estrogen-responsive elements. Yeast cells were also transformed by the rtER yeast expression vector using a lithium acetate method and selected by growth on complete minimal medium (0.13% dropout powder lacking uracil and tryptophan, 0.67% yeast nitrogen base, 0.5% (NH 4) 2SO 4, and 1% dextrose; Petit et al., 1995). The ␤-galactosidase assays were performed as described previously (Petit et al., 1997) in the absence or presence of 17␤-estradiol and increasing concentrations of melatonin (for 4 h at 30°C) for the transacti-

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

346

vation assays. ␤-Galactosidase activity was measured at 420 nm using the o-nitrophenyl ␤-d-galactopyranoside (NpGal) substrate. The formation of colored product was quantified with a spectrophotometer. The ␤-galactosidase activity was expressed in Miller units.

In Vitro Experiments Animals. Male rainbow trout (Oncorhynchus mykiss), weighing approx 500 g, were supplied by a trout farm (Le Drennec, France) and kept in recycled running water at 15°C under a natural photoperiod until use. Hepatocyte isolation and culture. Fish were anesthetized with phenoxyethanol (1:20,000) and the liver was dissociated by perfusion of collagenase A (Boerhinger, Meylan, France), as described by Seglen (1973) and adapted to trout (Maitre et al., 1986). The cell suspension was filtered through a 70-mesh sieve and the pellet was collected by centrifugation (50g for 5 min) at 18°C. The nonparenchymal and damaged cells were removed by centrifugation. The hepatocyte pellet was resuspended in Dulbecco’s modified Eagle’s medium/Ham’s F-12 nutrient mixture (1:1 mixture, with l-glutamine and 15 mM Hepes, without phenol red) supplemented with 15 mM TES (N-tris [hydroxymethyl methyl-2 amino-ethanesulfonic acid]; 2-([2-hydroxy-1,1 bis(hydroxymethyl)-ethyl] amino) ethanesulfonic acid), 12 mM NaHCO 3, 1% (v/v) antibiotics (penicillin, streptomycin, and amphotericin B; Sigma), and 2% (v/v) Ultroser SF (Biosepra, Villeneuve la Garenne, France). The cells were plated in 60-mm untreated plastic petri dishes (Falcon; 1–2 ⫻ 10 7 cells/5 ml medium/dish). Aggregates were obtained by constant gyratory shaking at 55 rpm at 18°C (Novotron; INFORS AG, Massy, France). The culture medium was changed every 2 days. After hormonal treatment, cells were harvested and pelleted by centrifugation (at 50g for 5 min) and stored at ⫺70°C until use. Estradiol-17␤ (Sigma) and melatonin (Sigma), dissolved in ethanol at the appropriate doses, were added to the culture medium in the following ratio: 1/1000 e (v/v). Cells were treated for 16 or 48 h without renewing the medium. At the end of the experiment, media were collected for melatonin radioimmunoassay (Randall et al., 1995) to check potential melatonin degradation.

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

Mazurais et al.

Dot blot analysis. Total RNA was prepared using Trizol reagent (Gibco BRL, Germany). Total RNA samples (5 ␮g) were spotted onto a nylon Biodyne A membrane (Pall, St Germain en Laye, France), using a Bio-Rad dot blot apparatus, as described by Cheley and Anderson (1984). The membrane was prehybridized (50% formamide, 5⫻ SSC, 5⫻ Denhart’s solution, 5 mM NaH 2PO 4, pH 6.5, 0.1 mg/ml Thymus calf DNA, and 0.1% SDS) at 42°C for 6 h and hydridized under stringent conditions (50% formamide, 5⫻ SSC, 1⫻ Denhart’s solution, 20 mM NaH 2PO 4, pH 6.5, 0.05 mg/ml Thymus calf DNA, 0.1% SDS, and 2 ⫻ 10 6 cpm/ml of a radiolabeled probe). Rainbow trout vitellogenin (rtVg) and actin cDNA were radiolabeled by the random priming technique, as previously described (Pakdel et al., 1989). The estradiol receptor (rtER) single-strand probe was labeled using Dynabeads M-280 (Dynal, Oslo, Norway). After 16 h of hybridization, blots were washed four times with L1 buffer (2⫻ SSC, 0.1% SDS) for 5 min at room temperature and then three times for 15 min at 55°C with L2 buffer (0.1⫻ SSC, 0.1% SDS). The washed blots were autoradiographed at ⫺70°C. Radioactivity was quantified by instantimager (Packard) or densitometry.

In Vivo Experiments Animals. Female rainbow trout (mean weight ⫾ SEM: 665.8 g ⫾ 9.8; GSI: 0.5%) of South African origin were maintained at the University of Stirling’s freshwater facility at Buckieburn (56°N). The tanks (square, 1000 L) were in a flow-through system (5 L/min) supplied from a reservoir 1 km from the site and at ambient seasonal temperatures (1–17°C). All fish were maintained under a natural photoperiod. Weights of all experimental fish were regularly recorded and used to provide the recommended daily ration of commercially available trout diets (Trouw Aquaculture UK Ltd., Witham, UK). Fish manipulation. A 1:20,000 concentration of 2-phenoxyethanol (Sigma, St Louis, MO) was used as an anesthetic whenever fish were handled to reduce stress and damage to the fish. Fish were placed in aerated freshwater for recovery, which was usually observed within 5 min. Blood samples were taken via the caudal dorsal aorta of anesthetised fish using 2-ml heparinized syringes (4 mg/ml; Sigma). The plasma was then transferred to new polystyrene tubes (LP3;

347

Effects of Melatonin in Rainbow Trout

FIG. 1. Experimental design of the in vivo experiments. In Experiment 1, four groups of fish were either left intact (Control), sham-operated (Sham), pinealectomized (PinX), or implanted with melatonin (Mel-implanted) on August 7 and were sacrificed on August 10. In Experiment 2, five groups of fish were either left intact (Control and E2), sham-operated (Sham), pinealectomized (PinX), or implanted with melatonin (Mel-implanted) on August 7. On September 4, groups E2, PinX, and Mel-implanted received an E2 injection. All groups were sacrificed on September 7.

Luckhams Ltd., Burgess Hill, Sussex, UK) on ice prior to centrifugation at 2500 rpm for 15 min at 4°C. The supernatant was transferred to polystyrene tubes (LP3; Luckhams Ltd.) and stored at ⫺70°C until analysis. Pinealectomy was performed as described in Porter et al. (1996). The melatonin implants (“Regulin;” Schering Agrochemicals, Alexandria, Australia) contained 18 mg of melatonin and utilized a polymer coating to allow a slow and constant release. An implanter (Schering) was used to administer the implants intramuscularly 1 cm below the dorsal fin. Estradiol-17␤ (E2; Sigma) was dissolved in alcohol, then made up to a 4 mg/ml solution in 0.85% saline, and administered intraperitoneally at a dose of 1 mg/kg. On the day of sacrifice, blood samples were taken, and the brain and liver were dissected before being frozen in isopentane at ⫺50°C. Brain and liver samples were then sent by airfreight (on dry ice) to the laboratory in Rennes. Melatonin radioimmunoassays were performed as described in Randall et al. (1995). Pilot experiment. In a pilot experiment, two groups of fish were sacrificed during the middle of the dark phase or the middle of the light phase to check whether differences in rtER and Vg mRNA could be detected in the liver. As no differences were observed, animals were sacrificed at 12:00 h in the subsequent experiments. Experimental design. Two experiments were performed and the experimental design is shown in Fig.

1. In Experiment 1, four groups of fish were either left intact (Control), sham-operated (Sham), pinealectomized (PinX), or implanted with melatonin (Mel-implanted) on August 7 and were sacrificed on August 10. In Experiment 2, four groups of fish were either left intact (Control), sham-operated (Sham), pinealectomized (PinX), or implanted with melatonin (Mel-implanted) on August 7, received an E2 injection on September 4, and were sacrificed on September 7.

Statistical Analysis Data are expressed as mean ⫾ standard error of the mean (SEM). Differences between means were tested by one-way analysis of variance (ANOVA), followed by the Fisher test. In all cases, significance was accepted at P ⬍ 0.05.

RESULTS Effects of Melatonin on rtER Expression in a Yeast Transcriptional Assay Figure 2A shows that, in yeast cotransfected with the rtER cDNA and with an ERE-␤-galactosidase reporter construct, melatonin was unable to affect the basal ␤-galactosidase activity, whereas E2 (10 ⫺8 M) was highly effective, demonstrating that the as-

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

348

Mazurais et al.

FIG. 2. Effects of melatonin on basal (A) and E2-stimulated (B) rtER expression in the yeast transcriptional ␤-galactosidase assay (mean ⫾ SE; n ⫽ 4). Melatonin concentrations are given in M.

say was functional. Figure 2B shows that melatonin was also unable to affect the E2-stimulated ␤-galactosidase activity. A similar experiment performed with the human ER (hER) gave identical results (data not shown).

Effects of Melatonin on rtER and Vitellogenin Expression in Hepatocyte Aggregates Three independent experiments were performed and gave similar results. Melatonin at doses ranging from 10 ⫺4 to 10 ⫺8 M was unable to affect the E2stimulated rtER and vitellogenin expression. The effects of melatonin alone were not tested because of the

usually low and highly variable basal levels. Figure 3 shows an experiment in which melatonin was given for 16 h at 10 ⫺4, 10 ⫺5, and 10 ⫺6 M. Although both ER and vitellogenin mRNA were strongly increased by E2, no effect of melatonin was detected. In another experiment, hepatocyte aggregates were exposed to melatonin for 48 h. Figure 4 shows the results on vitellogenin expression which was, as expected, strongly stimulated by E2 but not affected by melatonin. In this latter experiment, melatonin levels were assayed in the culture medium at the end of the experiment and were found to be approximatively half of the theoretical concentration used for the incubation.

FIG. 3. In vitro effects of melatonin (16 h) on E2-stimulated rtER (A) and vitellogenin (B) mRNA contents of hepatocyte aggregates (mean ⫾ SE; n ⫽ 4).

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

349

Effects of Melatonin in Rainbow Trout

FIG. 4. In vitro effects of melatonin (48 h) on E2-stimulated vitellogenin mRNA contents of hepatocyte aggregates (mean ⫾ SE; n ⫽ 4).

noted that mean light phase levels of plasma melatonin in the implanted individuals was significantly lower (P ⬍ 0.05) than the dark phase levels. The results of these experiments are shown in Figs. 5 and 6. It can be seen that neither pinealectomy nor melatonin implants caused any clear short-term effects on rtER or vitellogenin mRNA contents in the liver. However, in the case of vitellogenin, interpretation was difficult due to the rather large individual variations. As expected, in Experiments 1 and 2, E2 stimulated both rtER and vitellogenin mRNA contents. In Experiment 2, a slight but significant stimulatory effect of melatonin implants was detected on E2-stimulated rtER expression, but this effect had no impact on vitellogenin expression.

Effects of Pinealectomy and Melatonin Implants on rtER and Vitellogenin Expression in Vivo

DISCUSSION

Experiment 1 was designed to look at the short-term effects of pinealectomy and melatonin implants on basal rtER and vitellogenin expression. In Experiment 2, the long-term effects of pinealectomy and melatonin implants were studied in combination with a shortterm E2 treatment. The melatonin levels in the different groups (Experiment 1) are given in Table 1. In the sham-operated group, the dark phase melatonin level was significantly greater than the basal light phase level (P ⬍ 0.05); however, this was not the case in the pinealectomized group in which neither light nor dark phase levels were significantly different from the sham operation light phase level. The melatonin-implanted group had significantly elevated (P ⬍ 0.05) light and dark phase plasma melatonin levels compared to the sham operation dark phase level. It should also be

Although modifications of photoperiod clearly affect the seasonality of reproduction in a number of teleost species and although melatonin is believed to underlie these effects, there is to date no precise indication of how, when, and where melatonin affects the reproductive axis in fish. Several studies have attempted to identify reproductive parameters influenced by photoperiod manipulations in salmonids, and accelerating effects have been reported with respect to salmon GnRH (sGnRH) brain expression (Amano et al., 1994, 1995), sGnRH and pituitary gonadotrophin contents (Amano et al., 1994), and estradiol, testosterone, and vitellogenin plasma levels (Campbell, 1995; Bon et al., 1997). However, whether such effects were a cause or a consequence of the advanced maturation is not known. Studies in the stickleback failed to show any effects of melatonin treatments

TABLE 1 Mean (⫾SEM, n ⫽ 10) Mid-Light and Mid-Dark Phase Plasma Melatonin Levels Collected from Rainbow Trout Maintained under Ambient Photoperiod after Either Sham Operations, Pinealectomy, or Melatonin Implantation

Mid-light Phase Mid-dark Phase

Sham operation

Pinealectomy

Melatonin implantation

48.4 ⫾ 12.9 pg/ml 204.6 ⫾ 15.5 pg/ml*

26.0 ⫾ 2.5 pg/ml 28.4 ⫾ 3.4 pg/ml

866.0 ⫾ 53.7 pg/ml# 1184.2 ⫾ 38.2 pg/ml*#

* Significantly different from the respective mid-light phase group (P ⬍ 0.05). # Significantly different from the mid-dark phase sham-operated group (P ⬍ 0.05).

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

350

Mazurais et al.

FIG. 5. Effects of melatonin implants (Mel) and pinealectomy (Pin-X) on rtER (A) and vitellogenin (B) expression in the liver of trout. Animals were sacrificed 3 days after surgery (Experiment 1; mean ⫾ SE; n ⫽ 5).

under long photoperiod on the development of secondary sexual characters (Mayer et al., 1997). In the catfish Heteropneustes fossilis, effects of intraperitoneal administration of melatonin and 5-methoxytryptophol on ovarian activity were investigated during different seasons of the annual reproductive cycle under natural photothermal conditions. The results showed that both compounds were effective in inhibiting ovarian vitellogenesis and inducing atresia in the catfish (Joy and Agha, 1991). In this study, we tested the hypothesis that melatonin could interfere with the expression of estrogen receptors

which are essential in the process of vitellogenesis at all levels of the reproductive axis (Kah et al., 1997; Pakdel et al., 1997). Because estrogen receptors are ligand-dependent transcription factors, any impact on rtER expression would ultimately alter that of estrogen-dependent genes. For this reason, we looked at the effects of melatonin on the hepatic expression of rtER and vitellogenin, two estrogen-dependent genes (Flouriot et al., 1996). As rtER levels vary considerably from one animal to another, this hypothesis was tested both in vivo and in vitro. However, the present results show that melatonin had no significant effects on rtER and vitellogenin expression in hepa-

FIG. 6. Effects of melatonin implants (Mel) and pinealectomy (Pin-X) on rtER (A) and vitellogenin (B) expression in the liver of trout. Animals were sacrificed 1 month after surgery and received an E2 injection 3 days before sacrifice (Experiment 2; mean ⫾ SE; n ⫽ 5). (A) *Significantly different from Contr. and Sham groups, P ⬍ 0.05; **significantly different from E2 and PinX groups, P ⬍ 0.05). (B) *Significantly different from Contr. and Sham groups, P ⬍ 0.05.

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

Effects of Melatonin in Rainbow Trout

tocyte aggregates maintained in vitro. In addition, neither pinealectomy nor melatonin implants had any significant effects on basal or E2-stimulated rtER and vitellogenin expression in the liver. Furthermore, no direct effect of melatonin on rtER expression was detected in a yeast transcriptional assay. As mentioned in the introduction, there are different lines of evidence showing that melatonin has the capacity to influence the effects of several steroids, including estrogens, on target organs (Bergstrom and Hakanson, 1998). For this reason, we were willing to test the hypothesis of potential direct estrogen-mimetic or anti-estrogenic effects of melatonin on rtER in the yeast transcriptional assay developed in our laboratory. In this assay, estradiol or estrogen-mimetic compounds are able to transactivate the rtER, resulting in activation of the transcription rate of the ERE-␤-galactosidase construct (Petit et al., 1995, 1997). Accordingly, E2 caused a marked increase of ␤-galactosidase activity, whereas melatonin alone had no effect and did not modify the effect of E2, whatever the dose. This indicates that melatonin by itself is unable to transactivate the receptor and does not inhibit the transcriptional regulatory activity of the fully activated (estradiol-bound) rtER. These results are in agreement with previous studies on the human ER (Ram et al., 1998) and rule out the possibility that melatonin directly interacts with any particular domain of the rtER. However, there was still the possibility that melatonin acts via other mechanisms, including via its own receptor, to alter rtER mRNA expression and subsequently that of estrogen-dependent genes. The trout liver was a good potential target tissue to study, as it strongly expresses rtER in response to E2 and because the transcriptional activity of the activated rtER can be evaluated by monitoring rtER itself and vitellogenin (Flouriot et al., 1993, 1996; Pakdel et al., 1997). Indeed, extensive studies have shown that the rtER gene in the liver is upregulated by its own product, a mechanism that relies on the presence of an ERE on the proximal promoter of the rtER gene (Le Dre´an et al., 1995). Furthermore, hepatocyte aggregates have already proved useful for studying the estrogenic capacities of xenobiotics (Petit et al., 1997). At the present time, it is not known whether the trout liver expresses melatonin receptors, but specific iodo-melatonin-binding sites have been reported in liver membranes of another oviparous species, the quail (Wan and Pang, 1995). Our own RT-PCR experiments indicate that the

351

trout Mel1a and Mel1b receptors are not expressed in the liver (D. Mazurais and O. Kah, unpublished results), but, at the present stage, we cannot rule out the possibility that other subtypes, such as a Mel1c detected in chicken and zebrafish, are present in the liver (Reppert et al., 1995). In addition, high-affinity melatonin-binding sites have been reported in liver nuclei in the rat (Acuna-Castroviejo et al., 1993, 1994). In any case, our in vitro experiments indicate that exposure of hepatocyte aggregates to melatonin has no effect on the E2-stimulated rtER or vitellogenin mRNA contents, as assessed by dot blots. As an inhibitory effect of melatonin was expected, and because the basal expression is low and variable, we did not study the effect of melatonin on basal rtER mRNA levels. To verify that melatonin was not degraded during the incubation period, the melatonin levels in the incubation media were measured by radioimmunoassay at the end of the longest incubation period (48 h). In addition, the fact that the cells were highly responsive to E2 indicates that the transcriptional machinery had remained functional under the culture conditions. This suggests that any effects of melatonin on rtER or vitellogenin expression are likely to be minor. The in vivo experiments were originally designed to look at the potential effect of melatonin on brain rtER expression as monitored by an RNase protection assay developed in our laboratory (G. Le Dre´an, unpublished results). However, because of the low central expression of rtER, this method proved inappropriate. Measurements of liver rtER and vitellogenin mRNA by dot blots showed that neither pinealectomy nor melatonin implants significantly affected basal rtER expression. However, in this experiment, the important individual variations in the levels of rtER, which are commonly observed among females, may have hidden possible significant responses. In another experiment, we looked at the long-term (4 weeks) effects of melatonin implants or pinealectomy on E2-stimulated rtER expression. No clear effects could be detected, although a tendency for increased rtER expression was noticeable in the fish that were simultaneously implanted with E2 and melatonin. This increase in rtER expression was minor and not accompanied by an increase in vitellogenin expression. At this stage, the possibility that melatonin affects the central expression of rtER cannot be excluded, but this question will have to be addressed using in situ hybridization. Studies in our laboratory have shown

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

352

that rtER are expressed mainly in the ventral telencephalon, anteroventral preoptic region, and mediobasal hypothalamus (Salbert et al., 1991; Anglade et al., 1994), but none of these regions heavily expresses melatonin receptors (Mel1a or Mel1b) as detected by in situ hybridization or [ 125I]iodomelatonin radioautography (Mazurais et al., 1999). However, we cannot exclude indirect effects since, in mammals, the distributions of melatonin receptors and estrogen receptors do not significantly overlap, although significant effects of melatonin on estrogen receptor expression have been reported (Roy and Wilson, 1981; Lawson et al., 1992; Skinner and Herbison, 1997). Altogether, these data indicate that melatonin does not significantly affect expression of rtER or that of vitellogenin, two estrogen-dependent genes in the liver. This rules out the possibility that melatonin directly influences the initiation of vitellogenesis in the liver through estrogen receptor expression.

ACKNOWLEDGMENTS This study was supported by the European Union (Fair PL96.1410), CNRS, INRA, and the “Fondation Langlois.”

REFERENCES Acuna-Castroviejo, D., Pablos, M. I., Menendez-Pelaez, A., and Reiter, R. J. (1993). Melatonin receptors in purified cell nuclei of liver. Res. Commun. Chem. Pathol. Pharmacol. 82, 253–256. Acuna-Castroviejo, D., Reiter, R. J., Menendez-Pelaez, A., Pablos, M. I., and Burgos, A. (1994). Characterization of high-affinity melatonin binding sites in purified cell nuclei of rat liver. J. Pineal Res. 16, 100 –112. Amano, M., Okumoto, N., Kitamura, S., Ikuta, K., Suzuki, Y., and Aida, K. (1994). Salmon gonadotropin-releasing hormone and gonadotropin are involved in precocious maturation induced by photoperiod manipulation in underyearling male masu salmon, Oncorhynchus masou. Gen. Comp. Endocrinol. 95, 368 –373. Amano, M., Hyodo, S., Kitamura, S., Ikuta, K., Suzuki, Y., Urano, A., and Aida, K. (1995). Short photoperiod accelerates preoptic and ventral telencephalic salmon GnRH synthesis and precocious maturation in underyearling male masu salmon. Gen. Comp. Endocrinol. 99, 22–27. Anglade, I., Pakdel, F., Bailhache, T., Petit, F., Valotaire, Y., Je´go, P., and Kah, O. (1994). Distribution of estrogen receptor-immunore-

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

Mazurais et al.

active cells in the brain of the rainbow trout (Oncorhynchus mykiss). J. Neuroendocrinol. 6, 573–583. Bergstrom, W. H., and Hakanson, D. O. (1998). Melatonin: The dark force. Adv. Pediatr. 45, 91–106. Bon, E., Corraze, G., Kaushik, S., and Le Menn, F. (1997). Effects of accelerated photoperiod regimes on the reproductive cycle of the female rainbow trout: I-Seasonal variations of plasma lipids correlated with vitellogenesis. Comp. Biochem. Physiol. 118A, 183–190. Bromage, N., Elliot, J., Springate, J. R., and Whitehead, C. (1984). The effects of constant photoperiods on the timing of spawning in the rainbow trout. Aquaculture 43, 213–223. Bromage, N., Jones, J., Randall, C., Thrush, M., Davies, B., Springate, J., Duston, J., and Barker, G. (1992). Broodstock management, fecundity, egg quality and timing of egg production in the rainbow trout (Oncorhynchus mykiss). Aquaculture 100, 141–166. Bromage, N., Randall, C., Davies, B., Thrush, M., Duston, J., Carillo, M., and Zanuy, S. (1993). Photoperiodisrn and the control of reproduction and development in farmed fish. In “Aquaculture: Fundamental and Applied Research” (B. Lahlou and P. Vitiello, Eds.), pp. 81–102, Am. Geophys. Union, Washington, DC. Campbell, B. (1995). “Environmental and Hormonal Control of Reproduction in the Female Rainbow Trout, Oncorhynchus mykiss.” PhD thesis, Univ. of Stirling, Stirling, UK. Cardinali, D. P., Golombek, D. A., Rosenstein, R. E., Cutrera, R. A., and Esquifino, A. I. (1997). Melatonin site and mechanism of action: Single or multiple? J. Pineal Res. 23, 32–39. Cheley, S., and Anderson, R. (1984). A reproducible micoranalytical method for the detection of specific RNA sequences by dot blot hyrbidization. Anal. Biochem. 137, 15–19. Danforth, D. N., Jr., Tamarkin, L., Do, R., and Lippman, M. E. (1983a). Melatonin-induced increase in cytoplasmic estrogen receptor activity in hamster uteri. Endocrinology 113, 81– 85. Danforth, D. N., Jr., Tamarkin, L., and Lippman, M. E. (1983b). Melatonin increases oestrogen receptor binding activity of human breast cancer cells. Nature 305, 323–325. Davies, B., Hannah, L. T., Randall, C. F., Bromage, N., and Williams, L. M. (1994). Central melatonin binding sites in rainbow trout (Onchorynchus mykiss). Gen. Comp. Endocrinol. 96, 19 –26. Ekstro¨m, P., and Vanecek, J. (1992). Localization of 2-[ 125I]Iodomelatonin binding sites in the brain of the Atlantic salmon. Neuroendocrinology 55, 529 –537. Flouriot, G., Vaillant, C., Salbert, G., Pelissero, C., Guiraud, J. M., and Valotaire, Y. (1993). Monolayer and aggregate cultures of rainbow trout hepatocytes: Long-term and stable liver-specific expression in aggregates. J. Cell Sci. 105, 407– 416. Flouriot, G., Pakdel, F., and Valotaire, Y. (1996). Transcriptional and post-transcriptional regulation of rainbow trout estrogen receptor and vitellogenin gene expression. Mol. Cell. Endocrinol. 124, 173– 183. Hill, S. M., and Blask, D. E. (1988). Effects of the pineal hormone melatonin on the proliferation and morphological characteristics of human breast cancer cells (MCF-7) in culture. Cancer Res. 48, 6121–3126. Hill, S. M., Spriggs, L. L., Simon, M. A., Muraoka, H., and Blask, D. E. (1992). The growth inhibitory action of melatonin on human

Effects of Melatonin in Rainbow Trout

breast cancer cells is linked to the estrogen response system. Cancer Lett. 64, 249 –256. Hill, S. M., Spriggs, L. L., Lawson, N. O., and Harlan, R. E. (1996). Effects of melatonin on estrogen receptor expression in the forebrain of outbred (Lak.LVG) golden hamsters. Brain Res. 742, 107– 114. Joy, K. P., and Agha, A. K. (1991). Seasonal effects of administration of melatonin and 5-methoxytryptophol on ovarian activity in the catfish Heteropneustes fossilis (Bloch). J. Pineal Res. 10, 65–70. Kah, O., Anglade, I., Linard, B., Pakdel, F., Salbert, G., Bailhache, T., Ducouret, B., Saligaut, C., Le Goff, P., Valotaire, Y., and Je´go, P. (1997). Estrogen receptors in the brain–pituitary complex and the neuroendocrine control of gonadotrophin release in rainbow trout. Fish Physiol. Biochem. 17, 53– 62. Lawson, N. O., Wee, B. E., Blask, D. E., Castles, C. G., Spriggs, L. L., and Hill, S. M. (1992). Melatonin decreases estrogen receptor expression in the medial preoptic area of inbred (LSH/SsLak) golden hamsters. Biol. Reprod. 47, 1082–1090. Le Drean, Y., Lazennec, G., Kern, L., Saligaut, D., Pakdel, F., and Valotaire, Y. (1995). Characterization of an estrogen-responsive element implicated in regulation of the rainbow trout estrogen receptor gene. J. Mol. Endocrinol. 15, 37– 47. Maitre, J. L., Valotaire, Y., and Guguen-Guillouzeau, C. (1986). Estradiol 17␤ stimulation of vitellogenin synthesis in primary culture of male rainbow trout hepatocytes. In Vitro 22, 337–343. Martinoli, M. G., Williams, L. M., Kah, O., Titchener, L. T., and Pelletier, G. (1991). Localization and characterization of melatonin binding sites in the brain of the goldfish. Mol. Cell. Neurosci. 2, 78 – 85. Mayer, I., Bornestaf, C., Wetterberg, L., and Borg, B. (1997). Melatonin does not prevent long photoperiod stimulation of secondary sexual characters in the male three-spined stickleback, Gasterosteus aculeatus. Gen. Comp. Endocrinol. 108, 386 –394. Mazurais, D., Brierley, I., Anglade, I., Drew, J., Randall, C., Bromage, N., Michel, D., Kah, O., and Williams, L. M. (1999) Central melatonin receptors in the rainbow trout: Comparative distribution of ligand binding and gene expression. J. Comp. Neurol. 409, 313–324. Molis, T. M., Walters, M. R., and Hill, S. M. (1993). Melatonin modulation of estrogen receptor expression in MCF-7 human breast cancer cells. Int. J. Oncol. 3, 687– 694. Molis, T. M., Spriggs, L. L., and Hill, S. M. (1994). Modulation of estrogen receptor mRNA expression by melatonin in MCF-7 human breast cancer cells. Mol. Endocrinol. 8, 1681–1690. Pakdel, F., Le Guellec, C., Vaillant, C., Le Roux, M. G., and Valotaire, Y. (1989). Identification and estrogen induction of two estrogen receptors (ER) messenger ribonucleic acids in the rainbow trout liver: Sequence homology with other ERs. Mol. Endocrinol. 3, 44 –51. Pakdel, F., Feon, S., Le Gac, F., Le Menn, F., and Valotaire, Y. (1991). In vivo estrogen induction of hepatic estrogen receptor mRNA and correlation with vitellogenin mRNA in rainbow trout. Mol. Cell. Endocrinol. 75, 205–212. Pakdel, F., Delaunay, F., Ducouret, B., Flouriot, G., Kern, L., Lazennec, G., Le Dre´an, Y., Petit, F., Salbert, G., Saligaut, D., Tujague,

353 M., and Valotaire Y. (1997). Regulation of gene expression and biological activity of rainbow trout estrogen receptor. Fish Physiol. Biochem. 17, 123–133. Pang, C. S., Ali, M. A., Reddy, P. K., Leatherland, J. F., Brown, G. M., and Pang, S. F. (1994). 2-Iodo melatonin binding in the brain of four salmonids. Biol. Signals 3, 230 –238. Petit, F., Valotaire, Y., and Pakdel, F. (1995). Differential functional activities of rainbow trout and human estrogen receptors expressed in the yeast Saccharomyces cerevisiae. Eur. J. Biochem. 233, 584 –592. Petit, F., Le Goff, P., Cravedi, J. P., Valotaire, Y., and Pakdel, F. (1997). Two complementary bioassays for screening the estrogenic potency of xenobiotics: Recombinant yeast for trout estrogen receptor and trout hepatocyte cultures. J. Mol. Endocrinol. 19, 321–335. Petit, F., Le Goff, P., Crave´di, J. P., Kah, O., Valotaire, Y., and Pakdel, F. (1999). Trout estrogen receptor sensitivity to xenobiotics as tested by different bioassays. Aquaculture, in press. Porter, M. J. R., Randall, C. F., and Bromage, N. R. (1996). The effect of pineal removal on circulating melatonin levels in Atlantic salmon parr. J. Fish. Biol. 48, 1011–1013. Ram, P. T., Kiefer, T., Silverman, M., Song, Y., Brown, G. M., and Hill, S. M. (1998). Estrogen receptor transactivation in MCF-7 breast cancer cells by melatonin and growth factors. Mol. Cell. Endocrinol. 141, 53– 64. Randall, C. F., Bromage, N. R., Thorpe, J. E., Miles, M. S., and Muir, J. S. (1995). Melatonin rhythms in Atlantic salmon (Salmo salar) maintained under natural and out-of-phase photoperiods. Gen. Comp. Endocrinol. 98, 73– 86. Reppert, S. M. (1997). Melatonin receptors: Molecular biology of a new family of G protein-coupled receptors. J. Biol. Rhythms 12, 528 –531. Reppert, S. M., Weaver, D. R., Cassone, V. M., Godson, C., and Kolakowski, L. F. (1995). Melatonin receptors are for the birds: Molecular analysis of two receptor subtypes differentially expressed in chick brain. Neuron 15, 1003–1015. Roy, E. J., and Wilson, M. A. (1981). Diurnal rhythm of cytoplasmic estrogen receptors in the rat brain in the absence of circulating estrogens. Science 213, 1525–1527. Salbert, G., Bonnec, G., Le Goff, P., Boujard, D., Valotaire, Y., and Je´go, P. (1991). Localization of the estradiol mRNA in the forebrain of the rainbow trout. Mol. Cell. Endocrinol. 78, 173–180. Seglen, P. O. (1973). Preparation of rat liver cells II. Effect of ions and chelators on tissue dispersion. Exp. Cell Res 76, 25–30. Skinner, D. C., and Herbison, A. E. (1997). Effects of photoperiod on estrogen receptor, tyrosine hydroxylase, neuropeptide Y, and beta-endorphin immunoreactivity in the ewe hypothalamus. Endocrinology 138, 2585–2595. Vernadakis, A. J., Bemis, W. E., and Bittman, E. L. (1998). Localization and partial characterization of melatonin receptors in amphioxus, hagfish, lamprey, and skate. Gen. Comp. Endocrinol. 110, 67–78. Wan, Q., and Pang, S. F. (1995). 2-[125I]iodomelatonin binding sites in the quail liver: Characterization and the effect of guanosine 5⬘-O-(3-thiotriphosphate). Biol. Signals 4, 24 –31.

Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.