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Endocrinology Vol. 148, No. 9 4385-4392
Copyright © 2007 by The Endocrine Society

Temporal Dynamics of Type 2 Deiodinase Expression after Melatonin Injections in Syrian Hamsters

Shinobu Yasuo, Takashi Yoshimura, Shizufumi Ebihara and Horst-Werner Korf

Dr. Senckenbergische Anatomie (S.Y., H.-W.K.), Institute of Anatomie II, Johann Wolfgang Goethe-University Frankfurt, 60590 Frankfurt am Main, Germany; and Division of Biomodeling (T.Y., S.E.), Graduate School of Bioagricultural Sciences, and Institute for Advanced Research (T.Y.), Nagoya University, Nagoya 464-8601, Japan

Address all correspondence and requests for reprints to: Horst-Werner Korf, M.D., Ph.D., Dr. Senckenbergische Anatomie, Institute of Anatomy II, Johann Wolfgang Goethe-University Frankfurt, Theodor-Stern-kai 7, 60590 Frankfurt am Main, Germany. E-mail: korf{at}em.uni-frankfurt.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In many species living in temperate zones, reproduction is controlled by the photoperiod. Recent findings have clarified that type 2 iodothyronine deiodinase (Dio2) plays a significant role in the photoperiodic response of gonads in the mediobasal hypothalamus, converting the prohormone T4 into bioactive T3. In mammals, Dio2 expression is suppressed by long-term melatonin injections, although the signal transduction pathways that link the melatonin signal to Dio2 expression are unknown. As a first step to approach the problem, we have here investigated the temporal dynamics of the melatonin effect on Dio2 expression using male Syrian hamsters. Dio2 mRNA levels were found to show diurnal rhythms under long-day conditions in an area adjacent to the tuberoinfundibular sulcus and in the ependymal cell layer lining the ventrobasal walls of the third ventricle. Daily sc melatonin injections given in the late afternoon under long-day condition suppressed the Dio2 mRNA levels already at the first day after the onset of the treatment in the ependymal cell layer lining the ventrobasal walls of the third ventricle, and 1 d later in an area adjacent to the tuberoinfundibular sulcus. These suppressive effects were sustained for at least 2 d after a single injection. Furthermore, we examined the temporal changes of the Dio2 expression after the onset of the treatment, showing that the suppression did not occur until midday of the next day. These data suggest that melatonin is involved in the signal transduction mechanisms controlling the photoperiodic response of gonads by acting on Dio2 expression rather rapidly through indirect pathways.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FOR MANY SPECIES living in temperate zones, annual changes in photoperiod are the primary factor that regulates the timing of reproduction. In mammals, photoperiodic information is translated into a melatonin signal conveyed from the pineal gland night by night (1, 2). The duration of the melatonin signal lasts longer under short-day conditions and shorter under long-day conditions in many species (3, 4). These changes in the duration of the nocturnal melatonin peak are believed to alter the level of GnRH secretion from the hypothalamus and thereby the gonadal growth (5, 6). Although the target sites at which melatonin contributes to the seasonal regulation of reproduction are not fully understood, the primary target is thought to be located in the mediobasal hypothalamus (MBH). In Syrian hamsters, lesions of the dorsomedial nucleus and/or the dorsomedial region of the ventromedial nucleus, which contain [125I]iodomelatonin-binding sites (7, 8, 9), suppress the gonadal response to short photoperiod and melatonin in males (8, 9) and block the photoperiodic mediation of the estrous cycle in females (10). In sheep, melatonin microimplants positioned in the MBH or the premammillary hypothalamic area mimic a short-day effect on the gonadal axis (11, 12). In mammals, two high-affinity G protein-coupled melatonin receptor subtypes (MT1 and MT2) have been identified (13, 14), and MT1 is suggested to be involved in the photoperiodic gonadal regulation (15). The expression of mt1, but not that of mt2, is found in the premammillary hypothalamic area of the ewe where there is a day-night rhythm in expression (16).

Recent studies have clarified the molecular mechanisms for the photoperiodic response of gonads in birds. The exposure to long day length elicits the up-regulation of the expression of type 2 iodothyronine deiodinase (Dio2) in the MBH of Japanese quail (17). Dio2 catalyzes the conversion of prohormone T4 into bioactive T3 and controls local thyroid hormone concentration (17, 18). Furthermore, T3 administration to the MBH mimics the long-day effects on gonadal regulation in Japanese quail (17, 19), indicating that the Dio2 expression induced by long days would be critical for the photoperiodic response of gonads. This mechanism appears to be conserved in mammals, because Dio2 expression in the MBH has been shown to be regulated by the photoperiod in Djungarian (20, 21) and Syrian hamsters (22), Saanen goats (23), and Fischer 344 rats (24). The functional importance of T3 in mammals is underlined by a recent paper showing that exogenous T3 administration mimics the effect of long-day exposure in Djungarian hamsters; i.e. T3 treatment for 4 wk stimulates the gonadal development under short-day conditions and delays the beginning of the photorefractory phase after a long-term exposure to short days (25). In addition, in Djungarian and Syrian hamsters and rats, Dio2 mRNA levels are suppressed by melatonin injected daily 1–2 h before the lights were turned off under long-day conditions after 8, 1, and 3 wk of treatment, respectively (20, 22, 24). However, the signal transduction pathways that link the melatonin signal to the expression of Dio2 are totally unknown.

To address this issue, in this study, we tried to identify the exact timing at which melatonin suppresses the Dio2 mRNA levels of the MBH using male Syrian hamsters. Surprisingly, the Dio2 expression was suppressed already on the next day after the first injection. This suppressive effect was maintained for at least 2 d after a single injection. To determine whether melatonin acts on the transcription of Dio2 directly or indirectly, we examined the temporal changes of Dio2 expression after the onset of the treatment, showing that the suppression did not occur until midday of the next day. Although the single melatonin injection given in the morning caused acute suppression of the Dio2 mRNA levels, it did not elicit chronic suppression. Taken together with the earlier physiological observations, we discuss the involvement of the indirect effects of melatonin on Dio2 expression in the photoperiodic response of gonads.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All animal experimentation reported here was conducted in accordance with a protocol approved by the Committee on Animal Experiments of the Graduate School of Bioagricultural Sciences, Nagoya University; the Policy on the Use of Animals in Neuroscience Research; and the Policy on Ethics, as approved by the Society for Neuroscience, and were consistent with Federal guidelines and the European Communities Council Directive. Male Syrian hamsters 8 to 9 wk old were obtained from Charles River Laboratories (Sulzfeld, Germany, or Yokohama, Japan) and kept in light-tight boxes under long-day conditions [16 h of light, 8 h of darkness (16L:8D)] for at least 3 wk before the experiment. The boxes were placed in a room at a temperature of 22 ± 1 C. Food and water were available ad libitum and were replenished at least twice a week.

Temporal expression of Dio2 throughout the day
When hamsters were 12 wk of age, they were separated into two groups. One group was transferred to short-day conditions (8L:16D) for 3 wk. This group is named SD-hamsters. The other group was kept under long-day conditions (16L:8D) for 3 wk. This group is named LD-hamsters. Thereafter, SD- and LD-hamsters were killed at zeitgeber time (ZT; ZT0 corresponds to the light onset) 3, 9, 15, 21 (n = 3–4). The testicular weight was measured in animals killed at ZT9 to confirm the photoperiodic response of the gonads (n = 4).

Melatonin injections
When hamsters were 12 wk of age, sc injections of either melatonin (20 µg melatonin dissolved in 0.1 ml 5% ethanol and 0.9% NaCl) or vehicle (0.1 ml 5% ethanol and 0.9% NaCl) were started. Animals were kept under long-day conditions (16L:8D) throughout the experiment. The experimental design is shown in Fig. 1Go. To analyze the effects of single melatonin injections or melatonin injections daily repeated for 10 d, hamsters were divided into three groups: the first group was injected with melatonin at ZT14 every day for 10 d (10dMel), in the second group, vehicle was injected for 10 days, and the third group received only a single melatonin injection at ZT14 on d 0 (1dMel). Hamsters of each group were killed at ZT9 on d 0 (6 h before the first injection), and on d 1, 2, 4, and 10 after the onset of the treatment (n = 3) (Fig. 1AGo). To investigate the temporal changes in Dio2 mRNA levels during the first 2 d of treatment, melatonin or vehicle was injected at ZT14 for 2 d, and the animals were killed every 6 h from ZT3 on d 0 to ZT9 on d 2 (n = 3–6) (Fig. 1BGo). To elucidate whether the effects of a single melatonin injection depends on the time of day, melatonin or vehicle was injected at ZT6 or ZT14. Brains were prepared 1 and 3 h after injections at ZT6 (ZT6–1 h and ZT6–3 h), at ZT14 (ZT14–1 h and ZT14–3 h), or at ZT9 on the next day (ZT6-day1 and ZT14-day1) (n = 3) (Fig. 1CGo).


Figure 1
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  		FIG. 1. Schematic drawings of the experimental design. A, Effects of daily or single melatonin injections during 10 d. Syrian hamsters kept under 16L:8D were sc injected with melatonin or vehicle 2 h before the lights were turned off for 10 d (10dMel) or only on d 0 (1dMel). Brains were collected at ZT9 on 0, 1, 2, 4, and 10 d after the onset of the injections in each treatment. B, Temporal changes in the Dio2 mRNA levels during the first 2 d of the treatment. Melatonin or vehicle was sc injected into hamsters kept under 16L:8D 2 h before the lights were turned off. Brains were collected every 6 h from ZT3 on d 0 to ZT9 on d 2 at an interval of 6 h. C, Effects of single melatonin injections given in the morning or late afternoon. Melatonin or vehicle was sc injected into hamsters kept under 16L:8D at ZT6 or -14. Brains were collected 1 and 3 h after the injection (ZT6–1h and ZT6–3h, ZT14-1h and ZT14-3h) and at ZT9 on the next day (ZT6-day1 and ZT14-day1).

 
Fluorescence in situ hybridization
A 704-bp fragment of the Dio2 gene was amplified from Syrian hamster hypothalamus cDNA with the primers designed from the sequences that are in 100% identical regions between rats and mice as follows: forward primer 5'-atgggactcctcagcgtaga-3' and reverse primer 5'-attttccgtctctgcacgat-3'. The amplified fragment was cloned into pCRII-TOPO (Invitrogen, Carlsbad, CA). The sequence of the insertion was confirmed by sequencing performed by a local dealer (GATC Biotech Ltd., Konstanz, Germany). After linearization of plasmids, antisense and sense riboprobes were labeled with digoxigenin-UTP by in vitro transcription with SP6 or T7 RNA polymerase following the manufacturer’s procedure (Roche Diagnostics GmbH, Mannheim, Germany). For the preparation of sections, hamsters (12 wk of age) kept under long-day conditions were deeply anesthetized and were fixed by vascular perfusion using 4% paraformaldehyde in 0.1 M phosphate buffer at ZT9. Dissected brains were then immersed overnight in the same fixative and cryoprotected by sucrose solutions. Coronal sections (20 µm thick) of the MBH were prepared using a cryostat. For in situ hybridization, sections were dipped consecutively in 4% paraformaldehyde in 0.1 M phosphate buffer for 10 min, 1.5% H2O2 in methanol for 15 min, and 0.1 M triethanolamine-HCl (pH 8.0) and 0.25% acetic anhydride for 10 min at room temperature. After dehydration in increasing ethanol concentrations, heat-denatured riboprobes (800 ng/ml) were applied to slides in 60 µl buffer containing 50% formamide, 10 mM Tris-HCl (pH 7.6), 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% BSA, 200 µg/ml yeast tRNA, 0.6 M NaCl, 0.25% sodium dodecyl sulfate, 1 mM EDTA, and 10% dextran sulfate. Hybridization was carried out overnight at 52 C under Parafilm. After removal of the coverslip in 5x SSC (1x SSC contains 150 mM NaCl and 15 mM sodium citrate), the slides were washed in 2x SSC for 30 min at 55 C and then dipped into ribonuclease A solution (1 µg/ml) for 30 min at 37 C. Slides were then washed consecutively by 2x SSC for 20 min, 1x SSC for 20 min, and twice by 0.2x SSC for 20 min at 55 C. Visualization of digoxigenin label was performed following the manufacturer’s procedure of TSA Plus Fluorescence System (NEN Life Science Products, Boston, MA). Briefly, slides were incubated with blocking buffer [0.5% blocking reagent in 0.1 M Tris-HCl (pH 7.5) and 0.15 M NaCl] for 30 min, antidigoxigenin antibody conjugated with horseradish peroxidase (1:600 in blocking buffer; Roche Diagnostics) for 30 min, and fluorescence-conjugated tyramide amplification reagent for 7 min at room temperature. Slides were then washed, coverslipped, and observed with a Zeiss confocal imaging system (LSM 510; Zeiss, Göttingen, Germany). Serial sections were counterstained by 0.1% cresyl violet.

Radioactive in situ hybridization
In situ hybridization was carried out according to Yoshimura et al. (26). Antisense 45-mer oligonucleotide probes for Syrian hamster Dio2 (5'-tgcttgagcagaatgaccgagtcatagagcgccaggaagaggcag-3') were labeled with [33P]dATP (NEN Life Science Products) using terminal deoxynucleotidyl transferase (Invitrogen). Coronal sections (20 µm thick) of the MBH were prepared using a cryostat. Hybridization was carried out overnight at 42 C. Two high-stringency posthybridization washes were performed at 55 C. The sections were air dried and apposed to Biomax-MR film (Kodak, Rochester, NY) for 3 wk. 14C microscales (GE Healthcare Biosciences, Piscataway, NJ) were included in each cassette, and the relative OD was measured by using NIH Image software and converted into the radioactive value (nCi) using the 14C standard measurements.

Statistical analysis
All data are given as means ± SEM. For the daily rhythm of Dio2 expression, data were analyzed by one-way ANOVA followed by Fisher’s least significant difference post hoc test. All other data were analyzed by t test. To analyze Dio2 mRNA levels in LD- and SD-hamsters, the values at each time point were compared. In the 10dMel and 1dMel groups, values obtained for each day were compared with the values at d0. The temporal changes of Dio2 mRNA levels after melatonin injections were determined by comparing values with those from the control group (vehicle) at each corresponding time point. Statistical significance was set as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distribution of Dio2 mRNA in the MBH
We analyzed the localization of Dio2 mRNA in the MBH of Syrian hamsters kept under long-day conditions by radioactive (Fig. 2AGo) or fluorescence (Fig. 2Go, B–E) in situ hybridization. In both detection methods, Dio2 mRNA was observed 1) in the ependymal cell layer lining the ventrobasal parts of the third ventricular wall (EC) (Fig. 2Go, A and B) and 2) in an area devoid of cell bodies and adjacent to the tuberoinfundibular sulcus (TIS), which is localized in the lateral portion of the median eminence (Fig. 2Go, A and C; also see G), and as previously described in rats (24, 27, 28) and Syrian (22) and Djungarian hamsters (20). No signal was detected in these regions by sense probes (Fig. 2Go, D and E). Figure 2Go, F and G, show the region in cresyl violet stain.


Figure 2
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  		FIG. 2. Localization of Dio2 mRNA in the MBH of Syrian hamsters. Results are shown in hamsters killed at ZT9 under long-day conditions. A, Dio2 mRNA detected by 33P-labeled antisense oligonucleotide probes for Dio2. The Dio2 expression was observed in the EC (arrow) and TIS (arrowheads, also see G). B–E, Dio2 mRNA localization detected by digoxigenin-labeled riboprobes for antisense (B and C) and sense (D and E) for Dio2 in the EC (B and D) and TIS (C and E). Digoxigenin signal was detected by incubation with antidigoxigenin antibody conjugated with horseradish peroxidase, followed by fluorescein-labeled tyramide amplification reagent. F and G, Photomicrographs of the EC (F, arrow) and TIS (G, arrowhead) stained with cresyl violet. Scale bars, 1 mm (A) and 50 µm (B–G). V, Third ventricle.

 
Temporal expression of Dio2 throughout the day
The temporal changes of Dio2 mRNA levels throughout the day were examined in animals kept under long- and short-day conditions (LD- or SD-hamsters, respectively) at an interval of 6 h. In the EC, Dio2 mRNA levels were expressed rhythmically in LD-hamsters (F3,11 = 5.53; P < 0.05), and peaked at ZT9 (Fig. 3Go, A and B). In contrast, Dio2 expression in SD-hamsters remained at low levels throughout the day (P > 0.05) (Fig. 3Go, A and B). When the values of each time point were compared between SD- and LD-hamsters, a significant change was found at ZT9 (P < 0.01) (Fig. 3BGo). In the TIS, Dio2 mRNA levels also showed a diurnal rhythm in LD-hamsters (F3,11 = 4.24; P < 0.05) (Fig. 3Go, A and C), whereas those in SD-hamsters did not show temporal changes (P > 0.05) and remained constant at a moderate level throughout the day (Fig. 3Go, A and C). The differences between LD- and SD-hamsters were statistically significant at ZT9 and ZT15 (ZT9, P < 0.01; ZT15, P < 0.05) (Fig. 3CGo). Testicular weight of SD-hamsters was significantly smaller than that of LD-hamsters (P < 0.05) (Fig. 3DGo).


Figure 3
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  		FIG. 3. Effects of long days (LD, 16L:8D) or short days (SD, 8L:16D) on Dio2 mRNA levels in the MBH (A–C) and testicular weight (D) of Syrian hamsters. Animals were maintained on long or short days for 3 wk. A, Representative autoradiograms showing Dio2 expression in hamsters maintained under long or short days. Results at ZT3, -9, -15, and -21 are shown. The Dio2 expression was observed in the EC (arrow) and TIS (arrowheads). Scale bar, 500 µm. B and C, Temporal changes in the Dio2 mRNA levels in the EC (B) and TIS (C) throughout the day under long and short days. The values at ZT3 are double plotted. The black and white bars below the graphs indicate the light-dark conditions. Different letters indicate significant differences in each condition (one-way ANOVA followed by Fisher’s least significant difference post hoc test, P < 0.05). Asterisks indicate significant differences between values at corresponding time points under long and short days: *, P < 0.05; **, P < 0.01 (t test). D, Testicular weight of hamsters in long- and short-day conditions. Exposure to short days for 3 wk significantly reduced the testicular weight. Data are shown for the hamsters killed at ZT9 (*, P < 0.05, t test). Similar results were obtained when hamsters were killed at ZT4 or ZT21. Values are means ± SEM (n = 3–4).

 
Long-term effects of single or daily repeated melatonin injections on Dio2 mRNA levels
To determine the temporal dynamics and the duration of the suppressive melatonin effects on Dio2 mRNA levels, we examined the changes in the Dio2 mRNA levels by melatonin injected 2 h before the lights were turned off under the long-day conditions during 10 d of daily injections (10dMel) or 10 d after a single injection (1dMel) (Fig. 1AGo). Because Dio2 mRNA levels peaked in LD-hamsters at ZT9 in both the EC and TIS (Fig. 3Go, B and C), brains were collected at this time point. In the 10dMel group, the suppression of Dio2 mRNA levels was first detected at the next day of the first treatment in the EC (P < 0.05) (Fig. 4Go, A and B) and 1 d later in the TIS (P < 0.05) (Fig. 4Go, A and C). The suppressive effects of repeated melatonin injections persisted for the total period of the investigation (10 d) (Fig. 4Go). In the 1dMel group, the suppression of Dio2 expression in the EC persisted for 2 d (P < 0.05), and normally high levels were reached again on d 4 after treatment (Fig. 4Go, A and B). In the TIS, the suppression was observed on the second day after the injection (P < 0.05), and Dio2 mRNA levels reached the original levels on d 4 after treatment (Fig. 4Go, A and C).


Figure 4
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  		FIG. 4. Effects of daily repeated or single melatonin injections on Dio2 expression in the MBH of Syrian hamsters during 10 d. Melatonin or vehicle was sc injected 2 h before the lights were turned off under 16L:8D daily for 10 d (10dMel, vehicle) or only at d 0 (1dMel). A, Representative autoradiograms showing Dio2 expression in each treatment. Signals are located in the EC (arrow) and TIS (arrowheads). Scale bar, 500 µm. B and C, Effects of the daily or single melatonin injections on the Dio2 expression in the EC (B) and TIS (C). *, P < 0.05; **, P < 0.01, t tests. Values are means ± SEM (n = 3).

 
Short-term effects of melatonin on Dio2 mRNA levels
To elucidate short-term effects of melatonin on Dio2 mRNA levels, we investigated the temporal changes in Dio2 mRNA levels within the first 2 d after the onset of the injections at an interval of 6 h (Fig. 1BGo). In the EC, the first statistically significant suppression of Dio2 mRNA levels was observed at ZT9 1 d after the injection (P < 0.05) (Fig. 5Go, A and B). In the TIS, this suppressive effect was first detected at ZT9 on the second day (P < 0.05) (Fig. 5CGo).


Figure 5
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  		FIG. 5. Temporal changes in the Dio2 mRNA levels during the first 2 d of melatonin treatment in Syrian hamsters. Melatonin or vehicle was sc injected into hamsters kept under 16L:8D 2 h before lights were turned off. A, Representative autoradiograms showing Dio2 expression in several ZTs. The Dio2 expression is located in the EC (arrow) and in TIS (arrowheads). Scale bar, 500 µm. B and C, Temporal changes in the Dio2 mRNA levels during first 2 d of treatment in the EC (B) and TIS (C). The black and white bars below the graphs show the light-dark conditions. *, P < 0.05; **, P < 0.01, t test. Values are means ± SEM (n = 3–6).

 
Time-of-day-dependent effects of a single melatonin injection
In the above-described experiment, melatonin did not appear to elicit acute effects on Dio2 mRNA levels (Fig. 5Go). However, Dio2 mRNA is expressed rhythmically, and in the above-described experiment, melatonin injections were performed within the descending phase of the Dio2 rhythm (ZT14). To elucidate whether melatonin may elicit suppressive effects at other times of day, e.g. in the ascending phase of the Dio2 expression rhythm, we injected melatonin into hamsters kept under long-day conditions at two different time points of the day and examined Dio2 mRNA levels at 1 and 3 h after the injections and at ZT9 of the following day (Fig. 1CGo). When melatonin was injected at ZT6, suppressive effects on Dio2 mRNA levels in the EC were observed 3 h after the injection (P < 0.05) (Fig. 6AGo). However, this suppressive effect did not persist on the next day after the injection (P > 0.05) (Fig. 6AGo). In contrast, when melatonin was injected at ZT14, Dio2 mRNA levels were not acutely suppressed (P > 0.05) (Fig. 6AGo), but a significant suppression of Dio2 mRNA levels was observed at ZT9 on the next day (P < 0.05) (Fig. 6AGo). No effects were detected in the Dio2 mRNA levels of the TIS throughout this experiment (all P > 0.05) (Fig. 6BGo).


Figure 6
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  		FIG. 6. Effects of single melatonin injections given in the morning or late afternoon on Dio2 expression in the EC (A) or TIS (B). Melatonin or vehicle injections were performed at ZT6 or -14, and brains were collected 1 and 3 h after the injection and at ZT9 on d 1. *, P < 0.05, t test. Values are means ± SEM (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent findings have shown that the expression of Dio2 in the MBH plays a key role for the photoperiodic response of gonads and provide the basis to analyze the molecular mechanisms underlying the decoding seasonal melatonin signals (17, 20, 22, 23, 24). In mammals, the Dio2 expression is suppressed by long-term melatonin injections (20, 22, 24), although the signal transduction pathways that link the melatonin signal to the Dio2 expression are unknown. As an initial approach to address this issue, we have here investigated the temporal dynamics of the melatonin effect on Dio2 expression using male Syrian hamsters.

As a first step, we examined the photoperiodic effects of Dio2 mRNA levels in the MBH and testicular weight to determine whether hamsters used in this study respond properly to changes in the photoperiod. Testicular weight was significantly reduced by exposure to short days for 3 wk. The expression of Dio2 showed a day-night rhythm with high amplitude under long-day conditions (16L:8D) in the MBH. The signal was located in the EC and TIS. In the EC, Dio2 expression peaked at ZT9, and in the TIS, maximal levels were found at ZT9 and ZT15. As far as we know, this is the first demonstration of a diurnal rhythm of Dio2 expression in the MBH. This result is important to unravel the molecular basis driving the day-night rhythm in Dio2 activity and concentrations of T3 and T4, which were reported in the hypothalamus of rats (29, 30, 31) and shown to be driven by the suprachiasmatic nucleus (30). The comparison between animals maintained under long- and short-day conditions revealed that Dio2 mRNA levels were significantly higher in long-day conditions at ZT9 in the EC and at ZT9 and -15 in the TIS. These data agree with a previous study, which for a single time point (midday) shows photoperiod-dependent differences in Dio2 expression in the EC and TIS of Djungarian hamsters (20).

Previous reports show that Dio2 mRNA levels are suppressed by melatonin injected daily 1–2 h before the lights were turned off under long-day conditions after 1–8 wk of the treatment in Djungarian and Syrian hamsters and rats (20, 22, 24). To determine the onset and the duration of this suppressive effect, we injected hamsters kept under long-day conditions with melatonin for up to 10 d and determined the effects on Dio2 mRNA levels at one time point, ZT9. ZT9 was chosen for this analysis because Dio2 mRNA levels peaked at this time point in both the EC and TIS. Interestingly, Dio2 mRNA levels in the EC are suppressed by melatonin already on the first day after the onset of the treatment. In the TIS, this effect was first detected on the second day after the first melatonin injection. These rather acute effects of melatonin on Dio2 expression appear to be reflected by hormonal changes; a single injection of melatonin into male Syrian hamsters kept under 14L:10D suppresses pituitary LH levels at 12 h after the injection and plasma LH levels at 18 h (32). Moreover, Dio2 expression is stimulated in the MBH by a long photoperiod already on the first day of the long photoperiod in Japanese quail (33), and this correlates with the induction of plasma LH and c-Fos expression in the MBH within 30 h after the dawn of the first long day (33, 34, 35). Thus, the first-day response of Dio2 expression to melatonin provides us with an interesting model to investigate melatonin action on the hypophyseal gonadal axis. Compared with the earlier responses of Dio2 mRNA and also LH levels (32), the testicular regression is considerably delayed (6–8 wk) (1, 2). This indicates that regulation of Dio2 is an early event that is complemented by other, more distal mechanisms that need to be clarified in future studies.

The melatonin-induced Dio2 suppression in the EC was maintained for at least 2 d after a single injection (1dMel group). Moreover, the Dio2 suppression in the TIS, which was observed on the second day in the 10dMel group, was also retained in the 1dMel group. These data clearly indicate that the suppression of the Dio2 expression by melatonin injection reflects a long-lasting effect. Similarly, a single photoperiodic stimulus elicits long-lasting impact on the gonadal axis in Japanese quail and Djungarian hamsters. In Japanese quail, the Dio2 expression induced by a long-day stimulus is sustained for at least 4 d after returning to short days (33), and plasma LH concentrations mirror the changes in Dio2 expression, maintaining at elevated levels for at least 1 wk (33, 34). In Djungarian hamsters, exposure to a single long day at weaning stimulates testicular and somatic development 16 d after the treatment (36, 37). There are two possibilities for how melatonin elicits its long-lasting effect on the expression of Dio2: 1) melatonin might entrain the circadian clock that in turn controls the synthesis and secretion of melatonin, or 2) melatonin may elicit direct effects in its target tissues via unknown mechanisms. If the former view were correct, a single melatonin injection should sustain the changes in the patterns of melatonin secretion for several days, and indeed, this has been observed in rats subjected to prolonged light stimuli (38). However, a single melatonin injection administered in the late afternoon in animals kept under long-day condition does not affect the melatonin patterns in the pineal gland and the circulation on the next day in Djungarian hamsters (39), suggesting that this possibility can be excluded. Accordingly, the long-term suppression of Dio2 appears to be mediated by unknown mechanisms in the melatonin target tissues.

To investigate the temporal dynamics of such processes, we examined the temporal changes in Dio2 mRNA levels after melatonin injections on two consequent days. The suppression was not observed until ZT9 on the next day after the first injection, and Dio2 mRNA levels were not changed at 1 and 3 h after an injection in the late afternoon (ZT14). It should, however, be kept in mind that the readout signal of our experimental paradigm, i.e. Dio2 expression, is in the descending phase at this time point, which may mask direct effects of melatonin. We therefore injected melatonin at ZT6, when Dio2 expression is in the ascending phase. Surprisingly, melatonin elicited its suppressive effects at this time point within 3 h, suggesting that mechanisms controlling Dio2 expression, in principle, may be capable of an acute response to the melatonin signal. However, this acute effect of melatonin on Dio2 expression did not cause suppression of Dio2 expression on the next day after melatonin injection. One possible explanation for this discrepancy may be that the late afternoon injections summate with endogenous melatonin and produce a long duration elevation of circulating melatonin (40, 41), which then serves as a signal that alters Dio2 mRNA levels. Accordingly, earlier reports on Djungarinan and Syrian hamsters show that melatonin does not induce the gonadal inhibition when given in the morning, but it does so when given in late afternoon (42, 43, 44, 45). Thus, the long-term suppression of Dio2 expression after melatonin injection at ZT14 would be involved in the photoperiodic regulation of the gonads.

In the present study, the timing of Dio2 suppression by melatonin was different between the EC and TIS; Dio2 suppression in the EC by daily melatonin injections was detected on d 1, and that in the TIS was on d 2. In addition, the acute effect by the morning injection of melatonin was observed only in the EC. These region-specific differences of Dio2 expression deserve some attention. The Dio2 mRNAs are presumably located in the tanycytes as reported in rats (27, 28). The perikarya of these cells are located in the ventricular wall, and their processes extend into the median eminence and also into the TIS (27, 46, 47) (Yasuo, S., unpublished observation). The delayed response of the TIS compared with the EC may reflect the fact that Dio2 mRNAs are synthesized in the perikarya (EC) and later transported into the processes (TIS). In this view, melatonin is likely to affect Dio2 transcription primarily in the cell bodies. Recently, several genes were found in the tanycytes in the EC of the third ventricle in Djungarian hamsters and shown to be down-regulated by short days. Among these are genes encoding for cellular retinoic acid-binding protein 1, a retinoic acid transport protein, GRP50, an orphan G protein-coupled receptor, and nestin, an intermediate filament protein (48). Taken together, the EC would be the important site in the pathways decoding melatonin signals into the seasonal changes of physiology.


   Acknowledgments
 
We thank Nariman Anzari and Qian Zhang for technical support.


   Footnotes
 
This work was supported by a Japan Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad to S.Y. and in part by a Grant-in-Aid for Scientific Research [Kiban Kenkyu (S)] to S.E. from the Japan Society for the Promotion of Science.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 31, 2007

Abbreviations: Dio2, Type 2 iodothyronine deiodinase; 1dMel, single melatonin injection at ZT14 on d 0; 10dMel, injected with melatonin at ZT14 every day for 10 d; EC, ependymal cell layer lining the ventrobasal parts of the third ventricular wall; 16L:8D, 16 h of light, 8 h of darkness; MBH, mediobasal hypothalamus; TIS, area adjacent to the tuberoinfundibular sulcus; ZT, zeitgeber time.

Received April 17, 2007.

Accepted for publication May 24, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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