Seasonal changes in pineal melatonin content and hydroxyindole-O-methyltransferase activity in juvenile chinook salmon, Oncorhynchus tshawytscha

Juvenile fall and spring chinook salmon were examined for seasonal changes in pineal hydroxyindole-O-methyltransferase (HIOMT) activity. Numerous small peaks in pineal HIOMT specific activity were found in fall chinook salmon and HIOMT activity per pineal gland increased linearly with age and/or size. In spring chinook salmon, cyclic changes in pineal HIOMT specific activity occurred in midsummer and midwinter, independent of the photoperiod under which the fish were reared. HIOMT activity per pineal gland showed a peak in midwinter. Activity increased with age and/or size of the fish during the following spring. Melatonin content of the pineal gland of spring chinook salmon did not change seasonally in fish reared under a normal photoperiod. Fish reared under constant illumination had about 50% higher pineal melatonin content than those under a normal photoperiod until November. At that time, pineal melatonin content decreased to the same level as that of fish reared under a normal photoperiod.     

Effect of photoperiod on the fish innate immune system: a link between fish pineal gland and the immune system

The pineal gland via its secretory product, melatonin, influences the light-dark rhythm in most vertebrates including fish. Apart from the information concerning this circadian rhythm, the interrelation of the melatonin with other physiological processes has not been considered in fish. Thus, we evaluated the changes in the humoral innate immune system of seabream (Sparus aurata L.) and sea bass (Dicentrarchus labrax L.) specimens exposed to a constant light-dark photoperiod (12 hr L:12 hr D). Serum was obtained from blood samples collected at 02:00, 08:00 hr (light-on), 14:00, 20:00 hr (light-off) and at 08:00 hr again. Among the humoral innate immune responses, complement, lysozyme and peroxidase activities were determined. Complement activity was higher during the day than during the night in both fish species. Seabream lysozyme activity reached its maximum at 20:00 and 02:00 hr but was hardly affected in sea bass. Finally, the peroxidase activity of seabream was significantly higher at 08:00 hr than during the rest of the cycle while, in sea bass, it showed little variation. The present results demonstrate that the humoral innate immune system has a circadian rhythm based on the light-dark cycle and that this cycle might be affected by the pineal gland.     

Vasoactive intestinal peptide stimulates N-acetyltransferase and hydroxyindole-O-methyltransferase activities and melatonin production in cultured rat but not in Syrian hamster pineal glands

The purpose of this study was to compare the responses of the Syrian hamster and rat pineal glands in organ culture to vasoactive intestinal peptide (VIP). The endpoints in these studies were the activities of pineal N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), as well as pineal and medium melatonin levels. When rat pineal glands were incubated with either VIP (1 microM) or isoproterenol (1 microM), a beta-adrenergic agonist, a significant increase in NAT and HIOMT activities and melatonin levels were observed within 3 hr. Conversely, during the day, VIP (1 microM) was ineffective in stimulating these parameters in hamster pineal gland after incubation times of either 2, 4, 6, or 8 hr. In another experiment, hamster pineal glands were collected from animals killed in the late dark period (after 30 min light exposure). In these glands, isoproterenol promoted NAT activity and melatonin production; however, VIP was ineffective in stimulating either NAT or HIOMT activities; likewise, VIP had no stimulatory effect on pineal melatonin levels at night. Finally, when hamster pineal glands at night were incubated with either 0, 10 nM, 100 nM, 10 microM, or 100 microM VIP, no changes in any parameter of melatonin synthesis were measured. The results indicate that the hamster pineal gland, unlike that of the rat, may not respond to VIP with an increased melatonin production.     

Hydroxyindole-O-methyltransferase (HIOMT) and melatonin in the pineal gland of the antarctic penguins (Pygoscelis adeliae and P. papua)

Pineal melatonin content of the antarctic penguins, Pygoscelis adeliae and Pygoscelis papua, and pineal hydroxyindole-O-methyltransferase (HIOMT) activity in P. adeliae was studied in the polar summer. Both melatonin and HIOMT were assayed when the environmental light was maximal and minimal (12.00 and 24.00). Neither the melatonin content was different between the 2 hr in the species studied, nor the HIOMT activity in P. adeliae pineals. These results suggest that either melatonin biosynthesis does not occur rhythmically in the organ during Antarctic summer, or melatonin biosynthesis is independent of the daily variation in light intensity.     

Effects of time, photoperiod, and pinealectomy on ocular and plasma melatonin concentrations in the chick

Pinealectomized and intact chicks raised under cyclic (14L:10D) and noncyclic (OL;24L) conditions of environmental lighting were sampled 60 hr and 6 weeks after pinealectomy to evaluate the effects of pineal ablation on plasma and ocular melatonin concentrations. Little or no immunoreactive melatonin was present in plasma 60 hr after pinealectomy, while appreciable amounts (38-70% of control values) were measurable 6 weeks later. We also found photoperiod to be an important variable in determining the relative amounts of melatonin present in the eyes and the pineal gland. At night or in continuous darkness, the eyes contain proportionately larger amounts of melatonin; by day, or in continuous light, they contain less. Finally, it was determined that pinealectomy significantly increases ocular melatonin concentrations (+ 62-80%) in chicks raised in the 14L:10D and 24L environments. These results show that compensatory mechanisms exist for maintaining circulating levels of melatonin in the absence of the pineal, and that they require some time to develop. Furthermore, there appears to be a dynamic interrelationship between the eyes and the pineal gland in terms of melatonin production. This is evidenced by the differential effects of environmental lighting on melatonin concentrations in these tissues and by the increased ocular concentrations of melatonin in pinealectomized animals.     

A direct influence of moonlight intensity on changes in melatonin production by cultured pineal glands of the golden rabbitfish, Siganus guttatus

Rabbitfish are a restricted lunar-synchronized spawner that spawns around a species-specific lunar phase. It is not known how the fish perceive changes in cues from the moon. One possible explanation is that rabbitfish utilize changes in moonlight intensity to establish synchrony. The purpose of the present study was to examine whether or not the pineal gland of the golden rabbitfish can directly perceive changes in moonlight intensity. Isolated pineal glands were statically cultured under natural or artificial light conditions and melatonin secreted into the culture medium was measured using a time-resolved fluoroimmunoassay. Under an artificial light/dark cycle, melatonin secretion significantly increased during the dark phase. Under continuous light conditions, melatonin secretion was suppressed, while culture under continuous dark conditions seemed to duplicate melatonin secretion corresponding to the light/dark cycle in which the fish were acclimated. When cultured pineal glands were kept under natural light conditions on the dates of the full and the new moon, small amounts of melatonin were secreted at night. Moreover, exposure of cultured pineal glands to artificial and natural light conditions resulted in a significant decrease of melatonin secretion within 2 hr. These results suggest that the isolated pineal gland of golden rabbitfish responds to environmental light cycles and that 'brightness' of the night moon has an influence on melatonin secretion from the isolated pineal gland.     

Estrogen modulates the nocturnal synthesis of melatonin in peripubertal female rats

Abstract: Our objective was to evaluate the effects of estrogen deficit and of estrogen stimulation on the synthesis of pineal melatonin in female rats during the peripubertal period. The levels of melatonin and N-acetylserotonin (NAS) and the activities of N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) were determined in homogenates of pineal glands obtained from peripubertal female Sprague- Dawley rats 4 to 12 weeks of age in the mid-dark during the daily light/dark cycle. Animals were ovariectomized at 4 weeks of age; daily administration of estradiol benzoate (E₂B, 1.0 μg/d, s.c.) was initiated at 4 weeks of age. A peak in the pineal levels of melatonin and NAS and in NAT activity was observed in untreated (control) rats with intact ovaries at 6 weeks. HIOMT activity increased from Week 4 to 6 and remained unchanged thereafter. Ovariectomy at Week 4 led to significant increases in the levels of melatonin and of NAS and NAT in activity at Week 8. NAT activity Week 10 resembled that of control animals, but levels of melatonin and NAS were slightly elevated. Ovariectomy did not affect HIOMT activity. Subcutaneous injection of E₂B significantly decreased the levels of melatonin and NAS and of NAT activity at Week 4, as compared with those in control rats. E₂B suppressed the ovariectomy-induced elevation of levels of melatonin and NAS and of NAT activity, similar to the effect in control animals. E₂B did not affect HIOMT activity. Our results suggest that estrogen modulates the nocturnal synthesis of melatonin in the pineal gland in peripubertal female rats. The effects of estrogen on melatonin synthesis appeared to be mediated by the modulation of NAT activity.     

Pineal and circulating melatonin rhythms in the box turtle, Terrapene carolina triunguis: effect of photoperiod, light pulse, and environmental temperature

Pineal and circulating melatonin concentrations have been measured throughout the 24-hr cycle in the box turtle, Terrapene carolina triunguis, under different conditions of photoperiod and temperature. An obvious effect of photoperiod on the duration of the night rise of pineal and circulating melatonin is observed; the period of elevated melatonin is 4.30 hr in long photoperiod (18L:6D) and 11.00 hr in short photoperiod (8L:16D). A single pulse of 1 hr illumination beginning 1.30 hr after the onset of darkness, in a 16L:8D cycle, has no effect on pineal or circulating melatonin levels. A clear effect of environmental temperature on the amplitude of the day-night rhythm of melatonin production is observed. A possible role of the pineal of poikilotherms in the transduction of several environmental factors, via the daily pattern of melatonin secretion, is hypothesized.     

Diurnal and circadian rhythms in melatonin synthesis in the turkey pineal gland and retina

The pineal gland and retina of the turkey rhythmically produce melatonin. In birds kept under a daily light-dark (LD) illumination cycle melatonin concentrations in the pineal gland and retina were low during the light phase and high during the dark phase. A similar melatonin rhythm with high night-time values was also observed in the plasma. The pineal and retinal melatonin rhythms mirror oscillations in the activity of serotonin N-acetyltransferase (AANAT; the penultimate enzyme in the melatonin biosynthetic pathway). In contrast, in both the pineal gland and retina the activity of the enzyme hydroxyindole-O-methyltransferase (HIOMT) did not exhibit significant changes throughout the 24-h period. Acute exposure of turkeys to light at night dramatically decreased melatonin levels in the pineal gland, retina and plasma. The rhythms in AANAT activity and melatonin concentrations in the turkey pineal gland and retina were circadian in nature as they persisted under conditions of constant darkness (DD). Under DD, however, the amplitudes of AANAT and melatonin rhythms were significantly lower (by 50-80%) than those found under the LD cycle. The findings indicate that melatonin rhythmicity in the turkey pineal gland and retina is regulated both by light and the endogenous circadian clock. The rapid dampening of the rhythms under DD suggests that of these two regulatory factors, environmental light may be the primary stimulus in the maintenance of the high amplitude melatonin rhythms in the turkey.     

Hydroxyindole-O-methyltransferase mRNA expression in a subpopulation of photoreceptors in the chicken retina

Abstract: Hydroxyindole O -methyltransferase (HIOMT, EC 2.1.1.4) catalyzes the final step in the synthesis of melatonin in the pineal gland and retina. HIOMT mRNA was localized by in situ hybridization in the chicken retina to some, but clearly not all, photoreceptors, while in the pineal gland, most pinealocytes displayed a positive hybridization signal. The in situ hybridization localization was confirmed by immunocytochemistry, using an antibody directed against a synthetic chicken HIOMT peptide. Western blot analysis demonstrated an immunoreactive protein of about 40 kilodaltons in the pineal, but the HIOMT protein was below detectable levels in the retina. However, the HIOMT-peptide antibody did identify a modestly immunoreactive subpopulation of retinal photoreceptors. These observations suggest that, in the chicken, melatonin biosynthetic activity is located mainly in a subpopulation of retinal photoreceptors and in most pinealocytes.     

Changes in nocturnal pineal melatonin synthesis during the perimenopausal period: Relation to estrogen levels in female rats

To evaluate changes in melatonin synthesis during the perimenopausal period in the female rat and to determine the effects of estrogen on melatonin synthesis, pineal levels of tryptophan, melatonin and norepinephrine and activities of N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT) were determined. Homogenates for assay were prepared from the pineal glands of female virgin Sprague-Dawley rats between 4 and 24 months of age in the middle of the dark period of a daily light/dark cycle. Serum 17 beta-estradiol (E2) concentrations were also determined. Pineal melatonin levels significantly decreased from month 4 12 and significantly increased from month 12 16, decreasing thereafter. Serum E2 concentrations significantly decreased from month 12-16, and remained low thereafter. No significant changes in tryptophan or norepinephrine were seen. NAT activities paralleled the time course of changes in melatonin. HIOMT activities decreased gradually from month 4 24. Subcutaneous implantation of an E2 capsule between months 12 and 16 resulted in significant decreases in levels of melatonin and NAT activity at month 16. Ovariectomy at month 4 or 12 led to significant increases in the levels of melatonin and NAT activity. These findings represent a temporal increase in pineal melatonin synthesis during the perimenopausal period, and suggest that the increase in melatonin synthesis activity at that time might result from decreasing levels of endogenous estrogen. The effect of estrogen on melatonin synthesis appeared to involve modulation of NAT activity.     

Seasonal relationship between pineal hydroxyindole-O-methyltransferase (HIOMT) activity and reproductive status in the pony.

Pony pineal glands and female reproductive tracts were collected monthly for 1 year from a local slaughterhouse. Pineal gland weights did not change significantly throughout the year. Pineal gland tissue homogenates were assayed for hydroxyindole-O-methyltransferase (HIOMT) activity with N-acetylserotonin as the primary substrate. The greatest HIOMT activity was obtained with N-acetylserotonin as substrate, but three other related 5-OH indole substrates (5-hydroxytryptophol, serotonin, and 5-hydroxy-2-methylindole) were also methylated. HIOMT activity with all substrates was highest during the late fall and winter (November–February) and lowest during the spring, summer, and early fall (April–October). Percentage of mares ovulatory was inversely related to pineal HIOMT activity, except that there was a 1- to 2-month lag phase. These findings are consistent with a hypothesized antigonadal activity of both melatonin and the pineal in mammals.     

A rhythm in hydroxyindole-O-methyltransferase (HIOMT) activity in the scincid lizard, Lampropholas guichenoti

Hydroxyindole-O-methyltransferase (HIOMT) activity was observed in the pineal organ and the retina of the lateral eyes of Lampropholas guichenoti. No activity was recorded from the parietal eye of this species. There was a distinct diurnal pattern to the changes in HIOMT activity in both the pineal organ and the retina, there being a peak of activity 2 hr after the onset of darkness and another greater burst of activity commencing 1 hr before the onset of light. When lizards were exposed to an artificial lighting regime which was different from their normal environmental lighting the peak of HIOMT activity associated with the onset of darkness was established after 5 days, whereas that associated with the start of the light period took approximately 12 days to appear. It is concluded from these results that the pineal of Lampropholas is capable of setting itself to a particular photoperiod and is capable of measuring a lapse of time in order to do so.     

Circadian rhythms of plasma melatonin in the Adelie penguin (Pygoscelis adeliae) in constant dim light and artificial photoperiods

The response of plasma melatonin in Adelie penguins (Pygoscelis adeliae) to constant dim light and to light/dark cycles was measured to determine the capacity of the pineal gland to secrete melatonin after exposure to continuous daylight for 2 months. Penguins were moved in mid-summer from the natural photoperiod to either constant dim light (n = 10), to a 12L:12D light/dark cycle (n = 5), or to a 12L:12D light/dark cycle with a 30 min light pulse (50-155 lux) on the third (n = 4) or sixth (n = 5) "night." Blood samples were collected regularly through cannulae for up to 33 h. The birds in dim light were sampled after 2 days, with samples obtained over at least 24 h from 7 birds. Three of these birds had melatonin rhythms (peak levels 66.7-130.2 pg/ml) whereas the other 4 birds had constant low levels (less than 44 pg/ml). The phase of the rhythm was similar for all 3 birds. This is consistent with the pacemaker that regulates the circadian rhythm of melatonin secretion being entrained to a period of 24 h when the penguins were exposed to the natural photoperiod. Mean melatonin levels (42.7 +/- 2.5 pg/ml) were elevated compared to those previously reported in penguins under natural daylight. All penguins held under a 12L:12D light/dark cycle had melatonin rhythms. The phase and form of these rhythms were similar to those reported for other birds, and they appeared to be circadian rhythms entrained by the light/dark cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term daily melatonin infusion induces a large increase in N-acetyltransferase activity, hydroxyindole-O-methyltransferase activity, and melatonin content in the Harderian gland and eye of pinealectomized male Siberian hamsters (Phodopus sungorus)

The effects of long-term daily melatonin infusions on the melatonin synthetic pathway in the Harderian glands and eyes of male Siberian hamsters were studied. Hamsters were pinealectomized (PX) and infused daily for 8 hr with either melatonin (6 microg/hr) or vehicle for 7 days in short photoperiod (SP, 10L:14D), followed by 14 wk in either SP (SP group) or in constant darkness (DD group). After the infusion period (15 wk), the infusion was stopped and animals were transferred into SP for 3 wk. The hamsters were then killed at midday or midnight. Exogenous melatonin infusion caused an increase in the Harderian gland weight, which was still evident 3 wk after the end of the treatment. In addition, exogenous melatonin increased endogenous melatonin concentrations (4-fold) and hydroxyindole-O-methyltransferase (HIOMT) activity (2-fold). N-acetyltransferase (NAT) activity, however, was not increased, and no day/night difference in melatonin content and HIOMT activity was observed in the Harderian glands. In the eye, melatonin infusions significantly increased day and night-time melatonin levels (up to 3-fold) and both NAT and HIOMT activities (up to 3.5-fold). This effect of melatonin treatment was observed in both SP and DD groups. These observations demonstrate that exogenously-infused melatonin at relatively high doses activates the synthesis of endogenous melatonin in the Harderian gland and eye of the Siberian hamster. Circulating levels of melatonin were also markedly increased, indicating that in these conditions melatonin may be released from extra-pineal sites.     

Cloning and expression of arylalkylamine N-acetyltranferase-2 during early development and metamorphosis in the sole Solea senegalensis

The arylalkylamine N-acetyltransferase (AANAT) is a key enzyme in the rhythmic production of melatonin. Two Aanats are expressed in teleost fish, one retinal specific, Aanat1, and the other one pineal specific, Aanat2, being the latter the main enzyme responsible of the plasma nocturnal melatonin increase in fish. In anurans melatonin has been involved in metamorphosis through antagonizing thyroid hormone function; however, no available data reports a relationship between melatonin system and metamorphosis in fish. In this study, we have cloned the AANAT2 (SsAanat2) in a flatfish, Solea senegalensis, and studied its sites of expression and developmental expression pattern by in situ hybridization and Real Time PCR. These studies allowed us to demonstrate a specific signal in the pineal gland of sole larvae from 2 days post-fertilization (dpf), which was evident until post-metamorphosis. Immunohistochemical analysis on the hybridized slides showed that the sole pineal Aanat2 expressing cells corresponded to pineal photoreceptor cells. Real Time PCR was performed in animals kept under natural photoperiod and sampled at different stages from 0 to 21 dpf (including pre-, early-, middle- and late-metamorphic stages) and at midlight (ML) and middark (MD) daytimes. Sole Aanat2 expression was higher at MD than at ML from 2 dpf and at most developmental stages analyzed. The highest AANAT2 mRNA abundance was observed at 2 and 4 dpf. A significant 60-fold reduction in Aanat2 expression was seen just before metamorphosis demonstrating, for the first time in a vertebrate species, that the expression of pineal AANAT and thyroid hormones levels exhibit an inverse pattern during metamorphosis.     

Plasma melatonin in the neotenic tiger salamander (Ambystoma tigrinum): Effects of photoperiod and pinealectomy

A nonequilibrium, double-antibody radioimmunoassay was validated for melatonin extracted from tiger salamander plasma. Radioimmunoassay of plasma samples that were collected from salamanders killed at 6-hr intervals, starting at 0600 hr and ending at 2400 hr (12:12 L:D; lights on at 0600 hr), indicated a significant increase in plasma melatonin at 2400 hr and 0600 hr compared to 1200 hr, with the 1800 hr sampling time value being intermediate. Animals which had been pinealectomized, sham pinealectomized, or anesthetized only, and then killed and sampled at either midphotophase or midscotophase, demonstrated that this operation reduced circulating melatonin only during the scotophase. These results indicate the presence of other, non-pineal sources for circulating melatonin and that the pineal body is responsible for the increased titers of melatonin measured during the scotophase.     

Pineal melatonin concentrations during day and night in the adult hedgehog: Effect of a light pulse at night and superior cervical ganglionectomy

The European hedgehog (Erinaceus europaeus L.) is a hibernating mammal and seasonal breeder in which numerous circadian and circannual rhythms are entrained and synchronized by photoperiod. The present study was undertaken in order to establish the involvement of the pineal gland in transducing the photoperiodic message in this species. Pineal melatonin concentrations were determined by radioimmunoassay in female hedgehogs kept under natural climatic conditions and killed during the light:dark (L:D) cycle in spring and autumn, after the interruption of darkness by a 45 min light pulse, and after bilateral superior cervical ganglionectomy (SCGx). Absolute melatonin concentrations were low (less than 100 pg/pineal) in the pineal gland. Under natural climatic conditions, in spring and in autumn, pineal melatonin concentrations exhibited a marked diurnal rhythmicity, with very low levels in the day (1200: less than 10 pg/pineal) and high levels during the night (2200: 71.9 +/- 18.6 pg/pineal; 0200: 42.5 +/- 15.6 pg/pineal). The 45 min light pulse during darkness depressed rapidly and significantly the melatonin content (dark + light [D + L]: less than 10 pg/pineal), but a subsequent return to darkness restored high melatonin content after approximately 2 h (D + L + D: 65.4 +/- 20.2 pg/pineal). After bilateral SCGx, melatonin concentrations were reduced and no increase during night could be observed, either in animals sacrificed 42 days after SCGx or in animals killed 2 years after SCGx. In the hedgehog, as in other mammals, pineal melatonin concentrations are related to the light:dark cycle. Such data indicate that during the year, in this species, the effects of light on seasonal endocrine rhythms may be mediated by the pineal gland.