A novel melatonin antagonist, N-(2,4-dinitrophenyl)-5-methoxytryptamine neutralizes some effects of melatonin in the female Syrian hamster

In this present study we evaluated the ability of a recently synthesized melatonin antagonist, N-(2,4-dinitrophenyl)-5-methoxytryptamine (ML-23), to antagonize the effects of afternoon injections of melatonin on the reproductive and thyroid axes in the female Syrian hamster. Thirty-six animals were divided into four groups and treated daily for 13 weeks with an afternoon injection of melatonin (25 micrograms/injection) or saline diluent. ML-23 was given via the drinking water to both melatonin- and saline-treated groups. The experiment was continued until 78% of melatonin-treated animals exhibited acyclicity. The results show that ML-23 partially reversed the effects of melatonin on pituitary follicle-stimulating hormone concentrations but was without effect on the decreased pituitary and plasma prolactin concentrations induced by melatonin treatment. Furthermore, ML-23 antagonized the effects of melatonin on plasma thyroxine levels and significantly increased plasma triiodothyronine concentrations and the free triiodothyronine index when used in combination with melatonin. The decrease in ovarian weight and plasma estradiol, but not progesterone, obtained with melatonin treatment also was reversed by ML-23. Our data suggest that ML-23 prevents the effects of melatonin treatment on ovarian weight, pituitary follicle-stimulating hormone levels, plasma estradiol, and thyroxine concentrations in the female Syrian hamster. Since ML-23 did not prevent the effects of melatonin on pituitary weight, plasma luteinizing hormone and prolactin, and pituitary prolactin concentrations, the actions of ML-23 may involve only peripheral sites of action of melatonin. Alternatively, the dose of ML-23 may not have been optimal to prevent all of the central effects of the indoleamine.     

Geographic differences for delay of sexual maturation in Peromyscus leucopus: effects of photoperiod, pinealectomy, and melatonin

Effects of short-day photoperiod, pinealectomy, and melatonin on sexual maturation were tested in Peromyscus leucopus from either Connecticut (CT) or Georgia (GA). Laboratory reared-stocks from CT and GA were exposed to short daylength (photoperiod) from birth or 25 days of age. At 12 wk of age, delay in sexual maturation was indicated in most CT mice by decreased testis length, combined testes weight, and seminal vesicle weight. Conversely, GA animals did not delay sexual maturation when exposed to short-day photoperiod from either birth or 25 days of age. These results indicate that responses to short daylengths differ for juvenile CT and GA populations. In a second experiment, pinealectomized or sham-operated CT males were exposed to short-day (9L:15D) or long-day (16L:8D) photoperiod from birth. Pinealectomy blocked the effect of short daylength on reproduction. Therefore, the pineal must be involved in the delay of sexual maturation observed for short-day CT mice. The effects of melatonin, a pineal gland hormone, were tested with chronic s.c. implants or daily injections. In CT mice given either melatonin implants or afternoon injections, sexual maturation was delayed. GA mice were insensitive to all melatonin treatments. Further, no differences in circadian organization (phase angle, duration of activity, period under constant dark) between GA and CT animals were apparent. Collectively, these studies indicate that melatonin is involved in the mechanism responsible for delay of sexual maturation in CT mice. Short-day insensitivity of GA Peromyscus leucopus probably results from a deficiency in the melatonin effector pathway and is not due to a disruption of circadian organization.     

Hormonal modulation of pineal melatonin synthesis in rats and Syrian hamsters: effects of streptozotocin-induced diabetes and insulin injections

Pineal levels of tryptophan, 5-hydroxytryptophan, serotonin, N-acetylserotonin, melatonin, 5-hydroxyindoleacetic acid and the enzyme activities of N-acetyltransferase and hydroxyindole-O-methyltransferase were determined in male albino rats and Syrian hamsters that were injected with insulin twice daily for three days, or injected with streptozotocin to induce diabetes. Neither insulin injections nor streptozotocin diabetes had any effect on pineal melatonin production in rats. In hamsters, diabetes reduced the nocturnal peak of pineal melatonin content by approximately one half, while insulin injections had no effect on pineal melatonin levels; however, insulin injections did cause a slight increase in pineal N-acetyltransferase activity. These findings indicate that the pineal gland of the hamster may be more sensitive to alterations in plasma insulin levels than the same organ in rats.     

Differential effects of isoproterenol injections on the levels of pineal N-acetyltransferase, serum N-acetylserotonin and melatonin

Rats housed under diurnal lighting conditions were either injected with isoproterenol (ISO), 0.5 mg/kg subcutaneous (SC) and sacrificed at different times up to 180 minutes afterwards, or injected with different doses of ISO (0.2 mg/kg to 5.0 mg/kg intraperitoneally (IP] and sacrificed 120 minutes later. Pineal N-acetyltransferase (NATase), serum N-acetylserotonin (NAS) and serum melatonin (MT) levels were determined. It was found that both pineal NATase and serum MT responded to the injection with peak increase at 120 minutes after the injection. This increase in pineal NATase and serum MT levels were also found to be dose-dependent. It was also observed that at 30 minutes after ISO injection, the serum MT level already demonstrated a significant increase which preceeded any increase in the pineal NATase activity. The underlying mechanism for this observation remains undetermined. Unlike serum MT and pineal NATase, there were no changes in serum NAS levels after injections of ISO at all the doses tested or up to 180 minutes after injection of the drug at 0.5 mg/kg dose SC. This suggests that serum NAS level is neither regulated by pineal NATase activity nor is the pineal gland the major source of NAS in circulation. This also indicates that serum NAS level is not influenced by beta-adrenergic stimulation.     

Pineal N-acetyltransferase, pineal and serum melatonin response to isoproterenol stimulation in underfed male rats

Adult male rats were subjected to 4 weeks of 50% food restriction under lighting regimen of 14 h light and 10 h dark. The pineal response to isoproterenol (ISO) was determined. In the time-course study, animals were injected with 0.5 mg/Kg ISO subcutaneously (SC) and killed at different times up to 180 min post injection. In the dose-response study, various doses of ISO (0.2 mg/Kg to 5.0 mg/Kg) were injected intraperitoneally (IP) and animals were killed 120 min post injection. Body weight, pineal N-acetyltransferase (NATase), pineal and serum melatonin (MT) were determined. After 4 weeks of restricted feeding, body weight was reduced by 40%. In the time-course study, peak pineal NATase occurred 120 min post injection in the ad libitum fed animals. By contrast, the food restricted animals showed a gradual increase of pineal NATase up to 180 min post injection. In the dose-response study, the ad libitum fed animals demonstrated a dose dependent increase of pineal NATase up to 5 mg/kg dose. The food restricted animals, however, achieved their maximal pineal NATase at 1 mg/Kg dose with no further increment at 5 mg/Kg dose. These differences in responsiveness were also reflected in pineal and serum MT levels. These results indicate that underfed animals have abnormal pineal NATase, pineal and serum MT responses to ISO stimulation.     

Age-related characteristics of the effects of epithalamin on serotonin metabolism in the pineal gland of rats]

The biosynthesis of serotonin into melatonin was decreased in old (18-20-month) in comparison to young (4-5-month) male Wistar rats. 5-day morning injections to young and old rats with polypeptide pineal preparation (epithalamin) in a dose of 2.5 mg/kg of body weight induced the increase in the night peak of serotonin, N-acetylserotonin and melatonin in young and melatonin alone in old rats and did not influence 5-methoxytryptamine, 5-oxy- and 5-methoxyindoleacetic acids level. These data support suggestion of ultrashort loop between pineal peptides and indoles and that epithalamin increases the metabolism of serotonin into melatonin.     

Melatonin reverses pinealectomy-induced decrease of benzodiazepine binding in rat cerebral cortex

Pinealectomy of rats resulted in significant depression of benzodiazepine receptors (assessed by [³H]flunitrazepam binding) in cerebral cortex 3–14 days after surgery without affecting their affinity significantly. A single s.c. injection of melatonin (800 μg/kg body wt) restored the depressed brain benzodiazepine receptor sites. Single melatonin injections (up to 1600 μg/kg) to intact rats did not affect brain benzodiazepine binding when injected at either morning or evening hours. Daily melatonin treatment to intact rats for 5 days augmented benzodiazepine receptor density in brain (morning injections) or its dissociation constant (evening injections). Melatonin added in vitro to rat cerebral cortex membranes only slightly depressed [³H]flunitrazepam binding at 100 μM concentrations. These results point out a link between pineal activity and benzodiazepine receptor function in rats. They also indicate that pharmacological doses of melatonin affect benzodiazepine binding sites in rat cerebral cortex.     

Age-related changes in 24-hour rhythms of norepinephrine content and serotonin turnover in rat pineal gland: effect of melatonin treatment

The 24-hour rhythms of pineal norepinephrine (NE) content and serotonin (5-HT) turnover [estimated from the ratio of 5-hydroxyindoleacetic acid (5-HIAA) to 5-HT] were studied in young (2 months) and aged (18-20 months) Wistar rats killed at 6 different time points throughout a 24-hour cycle. In the first study, significant changes dependent on the time of day were identified, with acrophases in the first half of the activity span for both parameters. Old rats showed significantly smaller mesor and amplitude of the 24-hour rhythm of pineal NE content. They also showed decreased amplitude of the pineal 5-HT turnover rhythm, in the absence of changes in mesor. In old rats, pineal 5-HT and 5-HIAA concentrations were 41-47% of those found in young rats. In a second study, young and old rats received daily intraperitoneal injections of melatonin (30 microg) or vehicle for 11 days at 19.00 h (i.e. 11 h after light on). Analyzed as a main factor in a factorial analysis of variance, both pineal NE content and 5-HT turnover decreased in old rats while pineal 5-HT turnover increased after melatonin treatment. Melatonin treatment augmented the amplitude of the 24-hour rhythm of pineal NE content by 120 and 52% in young and old rats, respectively. The amplitude of the 24-hour rhythm of pineal 5-HT turnover almost doubled after melatonin treatment in young rats and did not change in old rats. Melatonin injection did not modify the rhythm's acrophase. The results indicate that old rats had lower amplitude and lower mesor values of 24-hour variations in pineal NE content and 5-HT turnover. Melatonin treatment only partly restored pineal NE content and was devoid of activity on pineal 5-HT turnover and 5-HT and 5-HIAA concentration in old rats. Impairment of pineal melatonin synthesizing capacity and intrapineal responses to melatonin may underlie pineal aging in rats.     

Characterization of the protective effects of melatonin and related indoles against alpha-naphthylisothiocyanate-induced liver injury in rats

The protective effect of melatonin, 6-hydroxymelatonin and N-acetylserotonin against alpha-naphthylisothiocyanate (ANIT)-induced liver injury was investigated and compared in rats injected once with the hepatotoxicant (75 mg/kg body weight). In rats injected with ANIT alone, liver injury with cholestasis developed within 24 h, as indicated by both serum levels of alanine aminotransferase (SGPT) and aspartic acid aminotransferase (SGOT) activities and serum total bilirubin concentration. The administration of melatonin or 6-hydroxymelatonin (10 mg/kg body weight) to ANIT-injected rats reduced significantly the serum levels of both SGPT and SGOT and the serum total bilirubin concentration. For all hepatic biochemical markers, melatonin was more effective that 6-hydroxymelatonin. By comparison, the administration of N-acetylserotonin (10 mg/kg body weight) to ANIT-injected rats did not reduce the serum levels of either hepatic enzymes or the serum total bilirubin concentration. In ANIT-injected rats, hepatic lipid peroxidation (LPO) was significantly higher than in control animals and this increase was significantly reduced by either melatonin, 6-hydroxymelatonin or N-acetylserotonin. Furthermore, ANIT treatment caused a significant reduction in liver microsomal membrane fluidity and this reduction was completely reversed by the three indoles. The liver from ANIT-injected rats showed several histopathological alterations; above all there was an acute infiltration of polymorphonuclear neutrophils and an increase in the number of apparent apoptotic hepatocytes. The concurrent administration of melatonin reduced the severity of all morphological alterations, specially the neutrophil infiltration and the number of presumed apoptotic cells. On the contrary, the administration of 6-hydroxymelatonin or N-acetylserotonin did not provide any protective effect in terms of the histopathological alterations. These results indicate that melatonin protects against ANIT-induced liver injury with cholestasis in rats, and suggests that this protective effect is likely due to its antioxidant properties and above all to its capacity to inhibit liver neutrophil infiltration, a critical factor in the pathogenesis of ANIT-induced liver injury. 6-hydroxymelatonin, although able to provide partial protection against the ANIT-induced hepatic injury, probably through its antioxidant properties by mechanisms that are unclear, was unable to reduce neutrophil infiltration. Finally, N-acetylserotonin in the experimental conditions of this study, only exhibited some antioxidant protection but had no protective effect against ANIT-induced hepatic damage.     

A 15-minute light pulse during darkness prevents the antigonadotrophic action of afternoon melatonin injections in male hamsters

When adult male Syrian hamsters were maintained under 14 h light and 10 h darkness daily (lights on from 0600-2000 h), peak pineal melatonin levels (705 pg/gland) were attained at 0500 h. When the dark phase of the light:dark cycle was interrupted with a 15 min pulse of light from 2300–2315 h (3 h after lights out), the highest melatonin levels achieved was roughly 400 pg/gland. Finally, if the 15 min pulse of light was given at 0200–0215 h (6 h after lights out) the nocturnal rise in pineal melatonin was completely abolished. Having made these observations, a second experiment was designed to determine the ability of afternoon melatonin injections to inhibit reproduction in hamsters kept under an uninterrupted 1410 cycle or under the same lighting regimen where the dark phase was interrupted with a 15 min pulse of light (0200–0215 h). In the uninterrupted light:dark schedule the daily afternoon injection of 25 g melatonin caused the testes and the accessory sex organs to atrophy within 11 weeks. Conversely, if the dark phase was interrupted with light between 0200–0215 h, afternoon melatonin injections were incapable of inhibiting the growth of the reproductive organs. The findings suggest that exogenously administered melatonin normally synergizes with endogenously produced melatonin to cause gonadal involution in hamsters.     

Diurnal Sensitivity of the Neuroendocrine-Reproductive Axis to the Antigonadotrophic Influence of Melatonin in Male Syrian Hamsters with Experimentally Altered Cortisol Rhythms

Two different experimental models were used to test if a temporal relationship exists between the rhythm of adrenal steroid secretion and the vulnerability of the hamster reproductive system to short photoperiod exposure or to the daily afternoon injection of melatonin. In the first experiment adrenalectomized hamsters were implanted with a Cortisol pellet to provide a sustained, rather than rhythmic, level of the hormone. The animals were either placed in short photoperiod or given a daily afternoon melatonin injection. In both cases the gonads underwent atrophy. In the second experiment adrenalectomized hamsters were given a Cortisol injection either in the morning (approx. 8 hr before the subsequent afternoon injection of melatonin) or in the afternoon (approx. 1 hr before the subsequent melatonin injection). Measurements of testicular and accessory organ weights 7 weeks later indicated regression of the reproductive system in both the groups when compared with their appropriate controls. Depressed levels of plasma LH. PRL, testosterone and thyroxine (T4) in these animals confirmed the melatonin induced gonadal collapse. The results suggest that apparently there is no temporal correlation between the rhythm of secretion of the adrenal steroids and the responsiveness of the reproductive system to late afternoon injection of melatonin. Interestingly, all the adrenalectomized Cortisol injected control animals (not receiving melatonin) had depressed plasma LH and PRL while the testicular weights and plasma testosterone titers remain unaffected.     

A novel melatonin agonist Neu-P11 facilitates memory performance and improves cognitive impairment in a rat model of Alzheimer' disease

Previous studies have shown that melatonin is implicated in modulating learning and memory processing. Melatonin also exerts neuroprotective activities against Aβ-induced injury in vitro and in vivo. Neu-P11 (piromelatine, N-(2-(5-methoxy-1H-indol-3-yl)ethyl)-4-oxo-4H-pyran-2-carboxamide) is a novel melatonin (MT1/MT2) receptor agonist and a serotonin 5-HT1A/1D receptor agonist recently developed for the treatment of insomnia. In the present study we firstly investigated whether Neu-P11 and melatonin enhance memory performance in the novel object recognition (NOR) task in rats, and then assessed whether Neu-P11 and melatonin improve neuronal and cognitive impairment in a rat model of Alzheimer' disease (AD) induced by intrahippocampal Aβ_(1–42) injection. The results showed that a single morning or afternoon administration of Neu-P11 enhanced object recognition memory measured at 4 or 24 h after training. Melatonin was effective in the memory facilitating effects only when administered in the afternoon. Further results showed that intrahippocampal Aβ_(1–42) injection resulted in hippocampal cellular loss, as well as decreased learning ability and memory in the Y maze and NOR tasks in rats. Neu-P11 but not melatonin attenuated cellular loss and cognitive impairment in the rat AD model. The current data suggest that Neu-P11 may serve as a novel agent for the treatment of AD.     

Adrenal-mediated depression of N-acetyltransferase activity and melatonin levels in the rat pineal gland

N-acetyltransferase (NAT) is believed to be the rate-limiting enzyme in the synthesis of melatonin from serotonin in the pineal gland. Norepinephrine released from sympathetic nerve endings within the pineal gland stimulates NAT activity and, therefore, melatonin synthesis. When an animal is subjected to a stressful stimulus, it would be expected that the increase in plasma stimulus, it would be expected that the increase in plasma catecholamines originating from the adrenal medulla and/or the sympathetic nervous system would result in a stimulation of pineal NAT activity. Adult male rats were given a 1.5cc injection of physiological saline subcutaneously into the back leg. Compared to non-injected controls, animals stressed in this manner were shown to have significantly lower pineal melatonin content 10 min after the saline injection late in the light phase of the light/dark cycle (at 18.30 h-lights on at 07.00 h). To test this more thoroughly, a time course study was conducted during the dark phase (at 02.00 h-5 hours after lights out) when pineal NAT activity and melatonin levels are either increasing or elevated. NAT activity and melatonin levels in the pineal were significantly depressed in stressed animals as compared to controls by 10 min after the saline injection, and remained so until 60 min after injection. By 90 min they had returned to control values. In the next study the nighttime response of the pineal to stress was compared in intact and adrenalectomized rats. Adrenalectomy prevented the changes in NAT activity and melatonin content associated with the saline injection. Some factor, such as a catecholamine or corticosterone from the adrenal, seems to be eliciting the response in the pineal to the saline injection. It is not known if the factor is acting centrally or directly on the pineal gland.     

Dose-dependent entrainment of rat circadian rhythms by daily injection of melatonin

Previous work in our laboratory has shown that daily injection of large doses of the pineal hormone melatonin entrains the free-running locomotor rhythms of rats held in constant darkness and synchronizes the disrupted patterns of rats maintained in constant bright light. The present experiments determined the dose-response characteristics of entrainment to daily melatonin injections and made preliminary biochemical estimates of blood melatonin levels and half-lives after two critical doses of the hormone. The data indicated that the median effective dose for melatonin as an entraining agent in free-running rats was 5.45 +/- 1.33 micrograms/kg, considerably lower than doses previously employed and lower than doses employed in reproductive and metabolic studies in rats and hamsters. The data further indicated that the response to melatonin was quantal; rats either entrained to melatonin or they did not. No "partial entrainment" was evident, nor were there differences in phase angle, activity, or period among all effective doses. Biochemical estimates of blood melatonin after either 1 mg/kg or 1 microgram/kg of melatonin indicated that all effective doses resulted in supraphysiological levels of blood melatonin, although doses of 1 microgram/kg resulted in blood levels that were within one order of magnitude of normal nighttime values. Together, the data suggest that the rat circadian system is sensitive to the pineal hormone melatonin at or below doses required to effect rodent reproduction. Whether this sensitivity reflects a role for the pineal gland in rat circadian organization, however, still remains to be determined.     

Locally synthesized angiotensin modulates pineal melatonin generation

We aimed to study the mechanisms and the significance of the influence exerted by the renin-angiotensin system (RAS) on the pineal melatonin production. Pineal melatonin and other indoles were determined by HPLC with electrochemical detection after angiotensin AT1-receptor blockade with Losartan in vivo or in cultured glands. N-acetyltransferase (NAT) activity was radiometricaly measured. To test the in vivo relevance of the local RAS, pineal melatonin and its indole precursors were determined in transgenic rats with inhibited production of angiotensinogen exclusively in astrocytes, TGR(ASrAOGEN). Tryptophan hydroxylase (TPH) and NAT mRNA levels were determined by real-time RT-PCR. Pineal melatonin content was significantly decreased by AT1-receptor blockade in vivo, in cultured glands and in TGR(ASrAOGEN) (35%, 32.4% and 17.5% from control, respectively). Losartan produced a significant decrease of pineal 5-hydroxytryptophan, serotonin, 5-hydroxyindole acetic acid and N-acetylserotonin in pineal cultures. Also, the pineal content of the precursor indoles in TGR(ASrAOGEN) rats was significantly lowered. The reduction of 5-hydroxytryptophan levels by 33-75% in both in vivo and in vitro studies suggests a decreased activity of TPH. Moreover, the TPH mRNA levels in TGR(ASrAOGEN) rats were significantly lower than control rats. On the other hand, NAT activity was unaffected by Losartan in pineal culture and its expression was not significantly different from control in TGR(ASrAOGEN) rats. Our results demonstrate that a local pineal RAS exerts a tonic modulation of indole synthesis by influencing the activity of TPH via AT1-receptors.     

Determination of rat pineal gland melatonin content by a radioimmunoassay.

Melatonin content in individual rat pineal glands was measured by radioimmunoassay (RIA). The RIA used can very reliably detect as little as 50 pg of melatonin. The various precursors, analogues, and the metabolite of melatonin (6-hydroxymelatonin) which were tested for cross-reactivity were not recognized by the antibody. The effects on melatonin levels in rat pineal glands following the administration of L-tryptophan, 5-hydroxy-L-tryptophan, serotonin, N-acetylserotonin, melatonin and pargyline are also presented.     

Beta-adrenergic blockers prevent short photoperiod-induced gonadal regression, but not melatonin-induced regression in male Syrian hamsters

Pineal melatonin synthesis is regulated by norepinephrine at beta-adrenergic receptors on pinealocytes. Melatonin released from the pineal is believed to be responsible for short photoperiod-induced gonadal regression. By blocking melatonin production at the beta-adrenergic receptor with beta-adrenergic antagonists, short photoperiod-induced gonadal regression was prevented. Propranolol or nadolol injected daily in the late afternoon prohibited short photoperiod-produced testicular regression in male Syrian hamsters. Likewise, propranolol and nadolol pellets were able to block short photoperiod-induced gonadal regression. Interestingly, in the presence of beta-adrenergic antagonists, gonadal regression was still produced by daily afternoon injections of melatonin. These results indicate that propranolol or nadolol can be used to "pharmacologically pinealectomize" hamsters and block the physiological action of the pineal.     

The pineal and melatonin in the regulation of pituitary-thyroid axis

Studies of thyroid physiology in rats and hamsters support the view that the pineal gland has an anti-thyrotropic action. While chronic exposure of hamsters to short photoperiod, darkness, or blindness results in a depression of plasma thyroxin and plasma TSH, removal of the pineal gland, which synthesizes melatonin, prevents these effects. Melatonin administration, in the form of daily injections given late in the photoperiod, also results in inhibition of plasma thyroxin and plasma TSH. These anti-thyrotropic effects are similar to the anti-gonadotropic effects of melatonin. The results of a variety of experiments are consistent with the view that melatonin acts on a neuroendocrine control mechanism influencing synthesis or release of hypothalamic thyrotropin releasing hormone (TRH).     

Increase in testosterone sensitivity induced by constant light in relation to melatonin injections in rats.

In this experiment we investigated whether the lack of the nocturnal melatonin peak under constant light would cause an increase in testosterone sensitivity. Castrated rats were kept under periodic or constant light for one week. They received a daily injection of vehicle, testosterone propionate (125 micrograms), melatonin (50 micrograms) or testosterone plus melatonin (125 micrograms + 50 micrograms). Serum and pituitary gonadotrophins and pineal melatonin were measured at the end of the experiment. Under constant light, testosterone injections reduced the serum luteinizing hormone concentration in castrated rats to that in intact rats, but, under periodic light, the decrease was smaller. Melatonin did not reverse the stronger effect of testosterone under constant light. The serum melatonin peak produced by the exogenous melatonin injection had a higher amplitude, shorter duration and earlier appearance than the physiological melatonin peak. Exogenous melatonin did not modify the physiological melatonin secretion, measured either as serum melatonin concentration or pineal melatonin content on the consecutive day. We conclude that the increase in testosterone negative feedback sensitivity of castrated rats under constant light was not due to the absence of the nocturnal melatonin pulse.     

Time and reproductive phase-dependent effects of exogenous melatonin on the pineal gland and ovary of a nocturnal bird, the Indian spotted owlet, Athene brama

In a tropical nocturnal bird, the Indian spotted owlet, Athene brama, the intraperitonial injection of an identical amount (20 mg/100 g b. wt/day) of exogenous melatonin (MEL) for 15 consecutive days increased the pineal weight and plasma MEL level in sexually active birds while it decreased them in inactive birds more potently when injected in the evening (18.30-19.30 h) rather than the morning (0500-0600 h). On the other hand, more efficiently than the morning hour treatment, the evening hour MEL injection decreased the ovary weight and plasma estradiol and progesterone levels both in sexually active and inactive birds, but more potently in active than inactive birds. Thus, the exogenous MEL showed the time and reproductive phase dependent effects on the pineal gland and the ovary of this nocturnal bird.