Seasonal Changes in the Circadian Plasma Melatonin Profile of the Tammar, Macropus eugenii

The circadian plasma melatonin profile of a marsupial, the tammar, was determined at various stages of the annual reproductive cycle. At 6-14 days after each of the solstices and equinoxes, six females were exposed to a photoperiod equivalent to the natural day length at these times. Serial blood samples were taken 8 days later at 2-4-hourly intervals, and plasma melatonin concentrations were measured by radioimmunoassay. Melatonin concentrations were elevated during the dark phase of each photoperiod, and there were significant changes between the profiles in each season. The amplitude of the nocturnal rise was significantly higher (P less than 0.05) during the breeding season after the summer solstice (peak 259.5 +/- 26.8 pg/ml, mean +/- SEM) and autumnal equinox (287 +/- 53.2 pg/ml) compared to those during the nonbreeding season after the winter solstice (111.5 +/- 10.5 pg/ml) and vernal equinox (154.5 +/- 10.4 pg/ml). The duration of the nocturnal rise was significantly correlated (r=0.996, P less than 0.01) with the length of the dark phase and so was shortest after the summer solstice and longest after the winter solstice. Either of these changes in amplitude or duration might provide the photoperiodic information that regulates the annual reproductive cycle of the tammar.     

Melatonin rhythms and pineal structure in a tropical bat, Anoura geoffroyi, that does not use photoperiod to regulate seasonal reproduction

Abstract: It has been hypothesized that pineal structure and function might differ between temperate zone and tropical species of mammals because of lower amplitudes of seasonal change in photoperiod and, in some areas, less seasonal climatic variation. Anoura geoffroyi produce a single offspring in November or December of each year on the Caribbean island of Trinidad, at 10°N latitude in the deep tropics. Previous work has shown that this population lacks reproductive responses to photoperiod, and must be enforcing seasonal breeding using a non-photoperiodic cue. Anoura geoffroyi have a minute, thin, and rod-like pineal gland. Throughout much of its length, the pineal courses irregularly within the ventrolateral wall of the great cerebral vein. This intimate relationship may have functional implications. Despite having a very small pineal gland, this species produced a nocturnal rise in serum melatonin. Serum melatonin levels in most individuals were below or near undetectable levels during the light period and rose to a peak averaging 100 pg/ml in the last third of the dark period. Our results indicate that, although the pineal gland of A. geoffroyi is extremely small, serum melatonin levels are comparable to those of other mammals.     

Melatonin receptors in neonatal pig brain and pituitary gland

Summer infertility remains a major problem in domestic pigs. It has been proposed that sows which display this trait are inherent seasonal breeders. The influence of photoperiod on domestic pigs has been difficult to ascertain as significant diurnal fluctuations in blood levels of the pineal hormone, melatonin, which provide a direct neuroendocrine transduction of the ambient photoperiod in other species, remain questionable in adult pigs. To investigate whether the pig is potentially receptive to melatonin, central sites of action for this hormone were localized and characterized within the brain and pituitary of the neonatal pig by in vitro autoradiography using 2-((125)I)iodomelatonin. Specific binding was distributed over a number of discrete regions of the brain including the cerebral cortex, hypothalamus, thalamus, brainstem, and cerebellum. The choroid plexus, and the pars tuberalis and pars distalis of the pituitary were also specifically labeled. Specific binding was completely abolished in the presence of 10(-7) M melatonin, and inhibited in the presence of 10(-4) M GTPgammaS (guanosine-5-0-(3-thiotriphosphate)), a non-hydrolysable analogue of GTP, in all regions examined, indicating that binding is representative of a G-protein coupled receptor. Characterization studies showed that 2-((125)I)iodomelatonin binding was saturable with a dissociation constant (Kd) in the low picomolar range (approximately 30 pM). Competition studies with iodomelatonin, melatonin, N-acetylserotonin and serotonin (5-HT) gave IC50 values similar to those previously characterized for the melatonin receptor in the ovine pars tuberalis.     

Seasonal Variation in the Daily Pattern of Plasma Melatonin in a Wild Mammal: The Mountain Hare (Lepus timidus)

Seasonal differences in daily patterns of plasma melatonin concentration were investigated in both free-living and captive mountain hares, in relation to reproductive activity. There was a marked increase in plasma melatonin concentrations at night at all times of the year. The period of elevation of plasma melatonin above the daytime values was longer in winter than in summer, correlating with the longer duration of darkness. The magnitude of the nighttime rise in melatonin concentrations did not differ significantly between seasons. There was no change in the plasma melatonin profile under similar photoperiods before the summer solstice when hares are sexually active or after the summer solstice when gonadal regression occurs, indicating that melatonin is not directly pro- or antigonadal. These field and laboratory observations support the view that the daily rhythm of melatonin secretion plays a role in the transduction of photoperiodic information in the mountain hare. Furthermore, they favor the hypotheses that the circadian melatonin rhythm transduces photoperiod information by means of the duration of nocturnal melatonin secretion or by the coincidence of elevated melatonin with a particular sensitive period, rather than by the amplitude of the nocturnal rise.     

Effect of Melatonin Administration and Long Day-Length on Endocrine Cycles in the Hedgehog Erinaceus europaeus

The effects of exogenous melatonin (subcutaneous implants containing 0.031 +/- 0.006 mg/gm body mass melatonin) or long photoperiod (18L:6D) on wild-caught adult male hedgehogs were studied. Hedgehogs were implanted with melatonin-filled or empty capsules in May, August, or September, or maintained under long photoperiod from August. Blood samples collected at monthly intervals were assayed for testosterone, melatonin, and thyroxin. Melatonin-filled capsules elevated plasma melatonin concentrations for 4-6 months. Although melatonin administration in May depressed plasma testosterone levels, testicular reactivation was advanced by 1 month the following year, and the characteristic prehibernal gain in body mass was abolished. Melatonin administration in August had no effect on plasma testosterone concentrations but reduced body mass fluctuations before and during hibernation. Hedgehogs receiving melatonin in September recovered early from hibernal body mass loss and showed a 2 month advance in testicular reactivation the following year. Maintaining hedgehogs at 18L:6D photoperiod, however, elevated plasma melatonin concentrations. Testicular reactivation the following spring was delayed by 1 month, ended 3 months early, and testosterone concentrations were depressed. All treatments depressed plasma thyroxin levels. These results suggest that elevated melatonin levels during winter are important in the regulation of endogenous endocrine cycles in the hedgehog. Hedgehogs do not respond positively to melatonin at the end of the breeding season, but are again responsive to melatonin as early as September.     

Effect of Melatonin Administration and Long Day-Length on Endocrine Cycles in the Hedgehog Erinaceus europaeus

The effects of exogenous melatonin (subcutaneous implants containing 0.031 +/- 0.006 mg/gm body mass melatonin) or long photoperiod (18L:6D) on wild-caught adult male hedgehogs were studied. Hedgehogs were implanted with melatonin-filled or empty capsules in May, August, or September, or maintained under long photoperiod from August. Blood samples collected at monthly intervals were assayed for testosterone, melatonin, and thyroxin. Melatonin-filled capsules elevated plasma melatonin concentrations for 4-6 months. Although melatonin administration in May depressed plasma testosterone levels, testicular reactivation was advanced by 1 month the following year, and the characteristic prehibernal gain in body mass was abolished. Melatonin administration in August had no effect on plasma testosterone concentrations but reduced body mass fluctuations before and during hibernation. Hedgehogs receiving melatonin in September recovered early from hibernal body mass loss and showed a 2 month advance in testicular reactivation the following year. Maintaining hedgehogs at 18L:6D photoperiod, however, elevated plasma melatonin concentrations. Testicular reactivation the following spring was delayed by 1 month, ended 3 months early, and testosterone concentrations were depressed. All treatments depressed plasma thyroxin levels. These results suggest that elevated melatonin levels during winter are important in the regulation of endogenous endocrine cycles in the hedgehog. Hedgehogs do not respond positively to melatonin at the end of the breeding season, but are again responsive to melatonin as early as September.     

Melatonin receptors in human fetal brain: 2-[(125)I]iodomelatonin binding and MT1 gene expression

The purpose of this study was to identify sites of action of melatonin in the human fetal brain by in vitro autoradiography and in situ hybridization. Specific, guanosine triphosphate (GTP) sensitive, binding of 2-[(125)I]iodomelatonin was localized to the leptomeninges, cerebellum, thalamus, hypothalamus, and brainstem. In the hypothalalmus, specific binding was present in the suprachiasmatic nuclei (SCN) as well as the arcuate, ventromedial and mammillary nuclei. In the brainstem specific binding was present in the cranial nerve nuclei including the oculomotor nuclei, the trochlear nuclei, the motor and sensory trigeminal nuclei, the facial nuclei, and the cochlear nuclei. The localization of MT1 receptor subtype gene expression as determined by in situ hybridization matched the localization of 2-[(125)I]iodomelatonin binding. No MT2 receptor subtype gene expression was detected using this technique. Thus, melatonin may act on the human fetus via the MT1 receptor subtype at a number of discrete brain sites. A major site of action of melatonin in both fetal and adult mammals is the pars tuberalis of the pituitary gland. However, no 2-[(125)I]iodomelatonin binding or melatonin receptor gene expression was detected in the pituitary gland in the present study, indicating that the pituitary, particularly the pars tuberalis, is not a site of action of melatonin in the human fetus.     

Melatonin induces tyrosine hydroxylase mRNA expression in the ventral mesencephalon but not in the hypothalamus

We have evaluated the effect of chronic administration of melatonin in terms of mRNA expression for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, and in the terms of dopamine (DA) transporter (DAT) by means of in situ hybridization. Experimental rats received daily late afternoon injections of 1.5 mg/kg melatonin for 30 days and analysis were performed in the ventral mesencephalon including the substantia nigra (SN) and ventral tegmental area (VTA), and hypothalamus. In the ventral mesencephalon, melatonin treatment significantly induced TH mRNA levels in individual dopaminergic neurons in SN and VTA. In contrast, DAT mRNA levels remained at control levels. Striatal synaptosomal DA uptake was not modified by melatonin treatment as compared with controls. Analysis of glutamic acid decarboxylase (GAD) mRNA in SN, the biosynthetic enzyme for GABAergic neurons, revealed no effect of melatonin treatment on mRNA levels for this marker. In the hypothalamus, we performed mRNA quantitation for TH in arcuate nucleus (Arc) and supraoptic nucleus (SO). Melatonin treatment failed to alter mRNA levels in either area. We detected weak but significant mRNA levels for DAT in Arc, SO, zona incerta (ZI) and periventricular hypothalamic nucleus (Pe). However, because of the low levels of mRNA in hypothalamic areas we were unable to perform a reliable measurement of DAT mRNA levels in response to melatonin treatment. We conclude that melatonin administration, that combines antioxidant capacity and a tissue-specific TH inducing effect, may be useful as a pharmacological agent to protect dopaminergic neurons from degeneration.     

Diurnal Variations of Plasma Melatonin Concentrations in Pregnant or Pseudopregnant Mink (Mustela vison) Maintained Under Different Photoperiods

Removal of the superior cervical ganglia suppresses the inhibitory role of short days in prolactin secretion and luteal activity in pregnant or pseudopregnant mink. Alternatively, timed injections of melatonin replicate the inhibitory role of short days in females maintained under long days. To understand if the diurnal variation of melatonin secretion is part of the mechanism by which the mink measure day length, diurnal variations in plasma melatonin concentrations have been measured in intact and ganglionectomized females maintained under long or short days after mating. Melatonin was measured by radioimmunoassay according to Brun et al. [Adv. Biosci. 53:41-45]. In intact females, plasma concentrations ranged from nondetectable levels to 40 pg/ml during the day, increased shortly after the onset of the dark phase, and reached peak values during the middle of the night. The duration of the elevated levels was roughly proportional to the length of the night. No diurnal variations could be detected in ganglionectomized females; melatonin levels never exceeded the day values observed in intact females.     

Melatonin and ischemia-reperfusion injury of the brain

Abstract:  This review summarizes the reports that have documented the neuroprotective effects of melatonin against ischemia/reperfusion brain injury. The studies were carried out on several species, using models of acute focal or global cerebral ischemia under different treatment schedules. The neuroprotective actions of melatonin were observed during critical evolving periods for cell processes of immediate or delayed neuronal death and brain injury, early after the ischemia/reperfusion episode. Late neural phenomena accounting either for brain damage or neuronal repair, plasticity and functional recovery taking place after ischemia/reperfusion have been rarely examined for the protective actions of melatonin. Special attention has been paid to the advantageous characteristics of melatonin as a neuroprotective drug: bioavailability into brain cells and cellular organelles targeted by morpho-functional derangement; effectiveness in exerting several neuroprotective actions, which can be amplified and prolonged by its metabolites, through direct and indirect antioxidant activity; prevention and reversal of mitochondrial malfunction, reducing inflammation, derangement of cytoskeleton organization, and pro-apoptotic cell signaling; lack of interference with thrombolytic and neuroprotective actions of other drugs; and an adequate safety profile. Thus, the immediate results of melatonin actions in reducing infarct volume, necrotic and apoptotic neuronal death, neurologic deficits, and in increasing the number of surviving neurons, may improve brain tissue preservation. The potential use of melatonin as a neuroprotective drug in clinical trials aimed to improve the outcome of patients suffering acute focal or global cerebral ischemia should be seriously considered.     

Propranolol Blockade of Short Photoperiod-Induced Gonadal Regression: Modification by Melatonin Injections or Implants

Male Syrian hamsters were exposed to short photoperiods (10L: 14D) for ten weeks and received daily ethanolic saline (1:10), propranolol (125 μg, sc) or melatonin (25 μg, sc) injections at 16: 45. Some hamsters also received melatonin implants (1 mg in 24 mg beeswax, sc) every two weeks in addition to the above treatments. Testicular weights were determined at the beginning, every three weeks, and at the end of the experiment. Serum hormone levels, hypothalamic amine levels and cortical amine levels were also determined at the conclusion of the experiment. Propranolol prevented the decline in testes weight produced by short photoperiod ( P <0.001 versus control). Propranolol was not able to prevent daily injections of melatonin from producing gonadal regression. Melatonin implants blocked testicular regression produced by both short photoperiod and melatonin injections. No differences in hypothalamic or cortical amine levels were observed between the treatment groups. These results add further support to the suggestion that propranolol is acting at the pineal gland preventing melatonin synthesis and not at a post-pineal central site which would affect melatonin binding and action.     

Melatonin induces apoptosis in human neuroblastoma cancer cells

Low concentrations (nanomolar) of melatonin had been previously shown to inhibit cell proliferation in several cancer cell lines as well as in experimental animal models. Additionally, cell growth inhibition and differentiation of prostate cancer cell lines by high concentrations (micromolar to millimolar) of melatonin have been recently reported. In the present paper, we show the induction of apoptosis by high doses of melatonin in the human neuroblastoma cell line SK-N-MC. We found accumulation of cells in the G2/M cell cycle phase and induction of cellular death, measured as lactate dehydrogenase (LDH) released into the culture medium, under millimolar concentration of melatonin. Apoptosis was evaluated using 4,6-diamidino-2-phenylindole staining, DNA gel electrophoresis, electron microscopy, and annexin V binding. Apoptosis progressed through the classical pathway, which involves caspase-3 activation. Cell death was dose and time-dependent; the lowest effective concentration of melatonin was 100 microm. Treatment with 1 mm melatonin for 6 days induced cell death in 75% of the cells. This novel finding shows that a nontoxic natural indoleamine may be potential therapy for some types of human neuroblastomas.     

Effects of Timed Melatonin Infusion on Prolactin Secretion in Pineal Denervated Goat

The effects of timed melatonin infusion on prolactin secretion were examined in the pineal denervated goat. Ovariectomized Shiba goats (n = 5) were subjected to bilateral superior cervical ganglionectomy (SCGX); this procedure resulted in complete abolition of endogenous melatonin release during the dark phase. SCGX goats failed to coordinate their prolactin secretion with the prevailing photoperiod. Melatonin was infused (20 micrograms/h, s.c.) daily into these goats either for 8 h (the long-day-type infusion) or for 16 h (the short-day-type infusion) to mimic the nocturnal profile of plasma melatonin under long days or short days, respectively. The long-day-type melatonin infusion for 9 days, in comparison with control saline infusion, accelerated prolactin secretion, inducing a nocturnal rise in plasma prolactin; this was comparable to that seen in the pineal intact goats under long photoperiods. On the other hand, the short-day-type melatonin infusion suppressed prolactin secretion throughout the day as the short-day treatment did in intact goats. The prevailing photoperiod appeared to have no distinct effect on these prolactin responses to exogenous melatonin, which were indistinguishable under 16L8D and 8L16D conditions. The results indicate that the information about external light-dark cycles is converted by the pineal gland into the endocrine signal as a daily pattern of melatonin secretion, which eventually regulates prolactin secretion from the pituitary gland of the goat.     

Melatonin receptors in the rat brain and pituitary

Abstract: 2-(¹²⁵I)iodomelatonin binding has been mapped and characterized in the brain and pituitary of the male laboratory rat using quantitative in vitro autoradiography. Specific binding was defined as that completely displaced in the presence of 1 μM melatonin. In the brain high levels of binding were localized over the suprachiasmatic nucleus (SCN), the area postrema (AP), and the spinal tract of the trigeminal nerve (Sp5). Lower densities of binding were found over the medial preoptic area (MPA), the septohypothalamic nuclei (SHy), the anterior hypothalamic area (AHA), the nuclei of the lateral olfactory tract (LOT), the paraventricular (PV), anteroventral (AV) and intermediodorsal (IMD) nuclei of the thalamus, the medial region of the lateral habenular (Lhb), the nuclei of the stria medullaris (SM), the basolateral (BL) and medial (ME) amygdaloid nuclei, the ventromedial nuclei (VMH), the arcuate nuclei (Arc), the subiculum of the hippocampus (S) and the lateral mammillary nuclei (LM). High levels of binding were also present over the pars tuberalis of the pituitary (PT) and the anterior and posterior cerebral arteries (CA). In both neuronal and non-neuronal areas, specific binding was time dependent and partially reversible in the presence of 1 μM melatonin. Binding was also saturable and of high affinity with dissociation constants (Kd) in the low picomolar range and was significantly inhibited in the presence of 104M guanosine 5'-0-(3-thiotriphosphate) (GTPγS) and 150 mM NaCl in all regions examined, indicating the presence of high affinity G-protein coupled melatonin receptors.     

Melatonin Response to Exercise Training in Women

Previous human studies have indicated that daytime melatonin levels increase when the organism is subjected to the stress of fasting and exercise. Melatonin, epinephrine, and norepinephrine levels were measured during a mock run and in the course of treadmill exercise performed before (T-1), during (T-2), and following (T-3) a progressive conditioning (running) program. Hormonal responses to the training program were determined by comparing values at T-1 and T-3. Plasma melatonin, epinephrine, and norepinephrine levels rose significantly (P less than .01) from baseline values for each exercise intensity during all three treadmill runs. While a dose-response trend was observed in each of the norepinephrine and epinephrine trials, there appeared to be a progressive diminution of this relationship in melatonin between intensities. Further, as training progressed, the peak melatonin concentration was decreased by 52% from T-1 to T-3, while peak epinephrine and norepinephrine values diminished only 19% and 8%, respectively. These results suggest that vigorous exercise training may attenuate rather than augment the secretion of pineal melatonin. Development of a human model of pineal responsiveness to exercise may contribute to the elucidation of exercise-associated reproductive disorders.     

Melatonin Rhythms in Quail: Regulation by Photoperiod and Circadian Pacemakers

The profile of melatonin in the eyes, pineal, and blood of Japanese quail was assessed in birds held under LD 16:8 and LD 6: 18 photoperiods. Melatonin levels in all three tissues showed a robust daily rhythm with higher levels occurring at night. The amplitude of the rhythm was depressed and its duration lengthened on LD 6: 18 relative to LD 16:8. The blood melatonin rhythm precisely reflected the rhythms shown by the pineal and eyes, supporting the idea that the blood rhythm is a result of melatonin secretion by both the eyes and pineal.
The ocular melatonin rhythm continued after sectioning of the optic nerve, was reentrainable to a shift in the phase of the LD cycle, and persisted for at least 2 days in constant darkness. It was concluded that either (1) an intraocular circadian clock drives the ocular melatonin rhythm, or (2) an extraocular clock drives the ocular melatonin rhythm via a route other than the efferent innervation (which enters the eye via the optic tract).     

Annual pattern of plasma melatonin and progesterone concentrations in hair and wool ewe lambs kept under natural photoperiod at lower latitudes in the southern hemisphere

To study the annual pattern of plasma melatonin and progesterone concentrations in hair [Santa Inês (SI)] and wool [Romney Marsh (RM) and Suffolk (SU)] ewe lambs kept under natural photoperiods at 21 degrees 59'S, 12 ewe lambs (four/breed) were used. For melatonin, blood samples were collected monthly throughout the year at the onset (17:00, 19:00 and 21:00 hr) and end (04:00, 06:00 and 08:00 hr) of the night, and for progesterone the samples were collected in the morning, two to three times a week throughout the year. Plasma melatonin concentrations at different times of the day changed according to the season. In diurnal periods (17:00 and 8:00 hr) no seasonal differences were observed but they became evident in the nocturnal intervals (21:00 and 4:00 hr) and transitional night-day (6:00 hr) times. The patterns of melatonin secretion were higher in winter and autumn than in spring and summer. The patterns of plasma progesterone secretion were affected by interaction between breed and season. There was no seasonal variation in plasma progesterone concentrations for SI females. The progesterone pattern for RM and SU females varied with season. The plasma levels were higher in autumn and winter than in spring and summer. At 21 degrees 59'S hair and wool ewe lambs showed the same annual pattern of plasma melatonin concentration while the annual progesterone profiles were quite different. For SI females this pattern was constant along all seasons and for RM and SU females this pattern was higher during autumn and winter than spring and summer.     

Melatonin duration gates photoperiodic vocal state change in a songbird

Seasonally breeding animals concentrate courtship to a particular time of year such that their offspring will be reared in a favorable environment. In house sparrows, Passer domesticus, primary (gonads) and secondary (song, plumage, beak color, etc) sexual characteristics are expressed differentially depending on the photoperiod. Removal of the pineal gland (PINX) has no effect on seasonal rhythms in gonad size but alters the photostimulated increase in vocal rate and complexity. Administration of long durations of melatonin, indicative of short days of winter, prevents seasonal recrudescence of song control nuclei in photostimulated house sparrows. In this study, male PINX house sparrows were exposed to three durations of melatonin, while vocalization and locomotor behavior were recorded as they were transitioned from short photoperiod to equinoctial photoperiods of spring. Birds receiving short duration melatonin or vehicle control increased dawn and dusk choruses as well as call complexity. Long durations of melatonin prevented this expansion to a spring-like vocal state observed in birds receiving the short duration of melatonin or vehicle control. The daily distribution of locomotor activity, beak color, and testis size was unaffected by treatment. Vocal state change was defined by our measures in two capacities: (i) increased dawn and dusk choruses, and (ii) an increase in calls associated with territory and mate attraction compared to the winter-like “social song.” We conclude that house sparrows use the calendar information provided by melatonin duration to control seasonal vocalization behavior, independent of effects on and of the gonads.