M. Masson-Pévet

Melatonin affects nuclear orphan receptors mrna in the rat suprachiasmatic nuclei

The pineal hormone melatonin nocturnal synthesis feeds back on the suprachiasmatic nuclei (SCN), the central circadian clock. Indeed, daily melatonin injections in free-running rats resynchronize their locomotor activity to 24 h. However, the molecular mechanisms underlying this chronobiotic effect of the hormone are poorly understood. The endogenous circadian machinery involves positive and negative transcriptional feedback loops implicating different genes (particularly period (Per) 1-3, Clock, Bmal1, cryptochrome (Cry) 1-2). While CLOCK:BMAL1 heterodimer activates the rhythmic transcription of per and cry genes, the PER and CRY proteins inhibit the CLOCK:BMAL1 complex. In previous studies, we observed that the immediate resetting effect of a melatonin injection at the end of the subjective day on the SCN circadian activity did not directly involve the above-mentioned clock genes. Recently, nuclear orphan receptors (NORs) have been presented as functional links between the regulatory loops of the molecular clock. These NORs bind to a retinoic acid receptor-related orphan receptor response element (RORE) domain and activate (RORalpha) or repress (REV-ERBalpha) bmal1 expression. In this study, we investigated whether melatonin exerts its chronobiotic effects through transcriptional regulation of these transcription factors. We monitored roralpha, rorbeta and rev-erbalpha messenger RNA (mRNA) expression levels by quantitative in situ hybridization, up to 36 h following a melatonin injection at circadian time (CT) 11.5. Results clearly showed that, while roralpha was not affected by melatonin, the hormone partially prevented the decrease of the rorbeta mRNA expression observed in control animals during the first hours following the injection. The major result is that the rev-erbalpha mRNA expression rhythm was 1.3+/-0.8-h phase-advanced in melatonin-treated animals during the first subjective night following the melatonin administration. Moreover, the bmal1 mRNA expression was 1.9+/-0.9-h phase-shifted in the second subjective night following the melatonin injection. These results clearly suggest that the NOR genes could be the link between the chronobiotic action of melatonin and the core of the molecular circadian clock.

Seasonal variations in the daily rhythm of pineal gland and/or circulating melatonin and 5-methoxytryptophol concentrations in the European hamster, Cricetus cricetus.

Day-night variations in pineal and/or circulating melatonin and 5-methoxytryptophol (5-ML) concentrations were measured monthly throughout the year in female European hamsters, Cricetus cricetus, maintained under natural conditions. Pronounced seasonal variations in the day-night rhythm of both melatonin and 5-ML were observed. As previously reported for melatonin, the daily rhythm of both methoxyindoles disappeared in spring and early summer, while a clear day-night rhythm occurred in autumn, winter, and early spring. The amplitude of the day-night variations appeared to be maximum from October until January. An inverse relationship existed between the rhythms of melatonin and 5-ML.

Are the annual reproductive and body weight rhythms in the male European hamster (Cricetus cricetus) dependent upon a photoperiodically entrained circannual clock

Masson‐Pévet M, Naimi F, Canguilhem B, Saboureau M, Bonn D, Pévet P. Are the annual reproductive and body weight rhythms in the male European hamster (Cricetus cricetus) dependent upon a photoperodically entrained circannual clock? J. Pineal Res. 1994; 17:151–163.

Effects of different doses and durations of melatonin infusions on plasma melatonin concentrations in pinealectomized Syrian hamsters: Consequences at the level of sexual activity

Abstract: The effect of different doses and durations of melatonin infusions on plasma melatonin concentrations has been studied in pinealectomized Syrian hamsters maintained under short photoperiod at either 7°C or 18°C. The effects of the infusions on plasma melatonin concentrations and on gonadal activity were compared. The results show that the minimal effective quantity of infused melatonin that induced gonadal atrophy was 40 ng/h at 7°C and 20 ng/h at 18°C. An infusion of 8 hr duration per day is necessary to inhibit sexual activity, while an infusion of 6 hr duration was ineffective. This finding suggests that the critical duration of melatonin infusion is between 6 and 8 hr. Despite the various doses of melatonin infused, plasma melatonin concentrations measured in the middle of the infusion period did not differ significantly from concentrations measured in intact animals. This finding suggests that the metabolism of infused melatonin increases as the dose of melatonin increases. Moreover, the different physiological effects observed after the various melatonin infusions cannot be explained by variations in plasma melatonin concentrations.

Circannual reproductive rhythm in the European hamster (Cricetus cricetus): Demonstration of the existence of an annual phase of sensitivity to short photoperiod

Abstract: In the European hamster (Cricetus cricetus) short photoperiod (SP) is responsible for the transition between the breeding and the resting season and data obtained previously suggest that a circannual “clock” drives the annual rhythm of reproduction. This hypothesis implies the existence of a SP‐sensitive phase of the circannual system that occurs independently of the photoperiodic regime perceived by the animals after their arousal from hibernation at the end of March. In control animals kept outside, testicular atrophy occurs in August. When the animals were transferred from outdoors to controlled SP conditions (LD 10:14 and ambient temperature Ta = 18 ± 2°C), immediately (Group II) or 2, 4, 6 wk after capture (Groups IV, V, VI, respectively), sexual arrest occurs at the same time between mid‐June and mid‐July. In the other groups, transfer from outdoors to SP either after 6, 8, 10, 12 or 14 wk (Groups VI, VII, IX, X, XI, respectively) after capture, is followed directly within 4 wk by the gonadal atrophy. When SP was applied from the beginning of August (Group XII) gonadal atrophy was observed after only 2 wk. In this last group, however, the rapid involution is the consequence of the already initiated decline in sexual activity induced by the short daylengths from July. When comparing the effect of SP in two different ambient temperatures (Ta: 18 ± 2°C vs 7 ± 2°C), immediately (Groups II vs III), 8 (Groups VII vs VIII) or 16 (Groups XII vs XIII) wk after capture, it appears that low temperature does not affect the physiological process described above. In the European hamster, after the gonadal regrowth at the end of hibernation, the animals do not need to experience increasing long days to react against SP. Gonadal inhibition is induced when, following our hypothesis, SP coincides with an endogenous period of sensitivity that extends from mid‐May to at least July‐August. The present findings complement and extend earlier evidence to support the existence of an endogenous circannual control of seasonal reproduction in the European hamster.

Seasonal regulation of melatonin receptors in rodent pars tuberalis: correlation with reproductive state

Using quantitative autoradiography, we have studied the relationship between melatonin receptor density in the pars tuberalis (PT) and photoperiodic changes in sexual activity in a photoperiodic (Syrian hamster), and a non photoperiodic (rat) rodent. Syrian hamsters exposed to short photoperiod (SP) for 13 weeks or kept in long photoperiod (LP) with daily melatonin injections for 8 weeks, had both gonadal regression and a significant decrease in 2-125I-melatonin binding site density in the PT when compared to controls. In contrast, when the animals were sexually active, photorefractory Syrian hamsters exposed to SP for 27 weeks, or rats kept for 13 weeks in SP, the PT melatonin receptor density was similar to that of control animals kept in LP. These results show clearly that a correlation exists between gonadal status and density of melatonin receptors in the PT and suggest that the PT could be the site where melatonin mediates its effects on seasonal function.

Low temperature in the golden hamster accelerates the gonadal atrophy induced by short photoperiod but does not affect the daily pattern of melatonin secretion.

Male golden hamsters were exposed to short photoperiod at either 20‡C or 5‡C. After 4 weeks a complete gonadal inhibition was observed in animals kept at 5‡C while in animals kept at 20‡C such an inhibition was much less. No significant difference in the pattern of pineal and plasma melatonin concentrations was observed between hamsters kept at 20‡C and 5‡C. If in the golden hamster pineal melatonin secretion is implicated in the transduction of the photoperiodic information, it is probably not implicated in the transduction of thermal information.

Brain and pituitary melatonin receptors in male rat during post-natal and pubertal development and the effect of pinealectomy and testosterone manipulation.

Using quantitative autoradiography, melatonin receptors have been studied during post‐natal and pubertal development of the rat in 2 brain and 2 pituitary structures. In the pars distalis of anterior pituitary, melatonin receptors decrease gradually in density after birth and disappear in 30 day‐old animals. In contrast melatonin binding is only expressed in the paraventricular nuclei of the thalamus at the age of 21–23 days and is always present in adult animals. In the suprachiasmatic nuclei and in the pars tuberalis of the pituitary, melatonin receptor density decreases after birth, remains stable for approximately 1 month and increases again at puberty to reach the birth values in the adult. This increase was absent in pinealectomized and in castrated animals but present in castrated animals receiving testosterone suggesting that it depends upon circulating testosterone and melatonin levels. These results show that melatonin receptors are differentially regulated during post‐natal development in each of the 4 structures studied, and that melatonin and testosterone are 2 factors which could be involved in the regulation of melatonin receptor density in the suprachiasmatic nuclei and pars tuberalis.

Influence of maternal melatonin on melatonin receptors in rat offspring

Using quantitative autoradiography, we have studied the influence of maternal plasma melatonin on the expression and density of melatonin receptors in the brain and pituitary of rat offspring. At birth, the same structures displayed melatonin receptors whether the rats were born to and reared by intact or pinealectomized dams. The receptor density was, however, about 20% lower in the group born to pinealectomized dams. At postnatal day 9, when the pups of both groups synthetize rhythmically their own melatonin, this difference was suppressed. These results indicate that melatonin does not appear to be a requirement for the expression of its receptors, but seems to play a stimulatory role in their synthesis.

Effect of pinealectomy and a constant high level of circulating melatonin or of 5-methoxytryptamine on the vasopressinergic innervation in the brain of the European hamster (Cricetus cricetus, L)

In the European hamster (Cricetus cricetus, L.) pinealectomy as well as a constant high level of either circulating melatonin or 5-methoxytryptamine induce marked changes in the immunocytochemically demonstrable central vasopressinergic innervation. When compared to control animals, a drastic decrease of AVP-immunoreaction was observed in the diagonal band of Broca, the lateral septum, the medial amygdala and the ventral hypothalamus. The results obtained suggest that part of the central vasopressinergic innervation is involved in pineal dependent seasonal functioning of the animal.