Characterization of melatonin binding sites in the brain and retina of the frog Rana perezi

The aim of this study was to characterize 2-[125I]iodomelatonin binding sites in the neural retina and central nervous system (telencephalon, diencephalon, and optic tectum) of the anuran amphibian Rana perezi. Saturation and kinetic studies and pharmacological characterization revealed the existence of a unique melatonin-binding site that belongs to the Mel 1 receptor subtype. The affinity of this site is similar in all tissues studied (Kd, 10.5-12.8 pM), but the density varied from diencephalon and optic tectum, which exhibit the highest density, to telencephalon with the lowest. Neural retina showed an intermediate receptor density. This melatonin-binding site fulfills the requirements of a real hormone receptor; the binding is saturable, reversible, and inhibited by different melatonin agonists and antagonists. The affinity order of ligands is: 2-phenyl-melatonin = 2-I-melatonin > 6-Cl-melatonin = melatoninz > luzindole. Additionally, specific binding is decreased by non-hydrolysable GTP analogue, sodium, and by pretreatment of membranes with pertussis toxin. All these results suggest the existence of a widely distributed and pharmacologically homogeneous melatonin receptor of the subfamily Mel 1 in the nervous system of Rana perezi coupled to a Gi/o protein.     

Seasonal cycles in testicular activity in the frog, Rana perezi

Studies of seasonal testicular cycle based on spermatogenetic activity and direct measurement of plasma testosterone were made in male frog Rana perezi obtained from its natural biotope in the Iberian Peninsula. Testosterone plasma level was determined by radioimmunoassay and exhibited notable differences according to season: plasma testosterone was lowest (less than 0.5 ng/ml) in summer and then increased progressively to reach a peak in spring (3-4 ng/ml), coincident with mating. After spermiation, when an increase in temperature and photoperiod in the natural habitat occurs, levels decline. Fat bodies also show a pronounced seasonal cycle with total regression following breeding and maximal development in winter. However, testicular weight was independent of seasons, and no significant change was observed throughout the year. Histological evidence indicates that although cell nests of different types are present every month of the year, the most important spermatogenetic activity is initiated in summer. The possible relationship between spermatogenetic activity and testosterone production and the importance of environmental factors as synchronizers of seasonal reproduction are discussed.     

Effects of temperature on 2-[125I]-iodomelatonin binding to melatonin receptors in the neural retina of the frog Rana perezi

The present study analyzes the effect of temperature-dependent modifications on the binding of the analog 2-[125I]-melatonin to melatonin receptors in isolated neural retina membranes from the greenfrog Rana perezi. Association and dissociation rate constants (K+1, K-1) were exponentially increased by the assay temperature. At 15 degrees C, association and dissociation required several hours; meanwhile, at 35 degrees C, rate constants were 100- and 34-fold faster, respectively. However, the Kd constant calculated as K-1/K+1 was unmodified by the assay temperature. When frogs were acclimated at either 5 or 22 degrees C for 1 month, K+1, and K-1 constants determined at 15 and 25 degrees C were identical in both cold- and warm-acclimated groups. Thus, the binding kinetics of melatonin receptors in frog retinas did not shown any thermal compensation. Results from saturation curves and pharmacological profiles of melatonin binding sites support a lack of effect of assay temperature on the affinity of melatonin receptors in the frog retina. The inhibition of [125I]Mel binding by GTPgammaS showed clearly that the coupling of melatonin receptors to G proteins is temperature-dependent. Higher concentrations of the GTP analog were needed to inhibit specific binding when temperature decreased. The temperature effect on binding kinetics and on the G protein coupling to melatonin receptors suggests that the melatonin signal could be transduced distinctly depending on the temperature. Thus, temperature plays a major role, not only on melatonin synthesis, but also in the transduction of melatonin signal in ectotherms.     

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.     

Influence of temperature and photoperiod on plasma melatonin in the mudpuppy, Necturus maculosus.

A melatonin (MEL) radioimmunoassay employed previously only in mammals was used to estimate plasma MEL in a salamander, Necturus maculosus. Validation procedures included thin-layer chromatography of plasma extracts, parallel inhibition curves of authentic MEL and serially diluted plasma and plasma extracts and quantitative recovery of authentic MEL added to pooled Necturus plasma. A diel cycle of plasma MEL was demonstrated in mudpuppies acclimatized for a minimum of 3 weeks under a 12L:12D photoperiod and 15 +/- 1 degrees and sampled in late March. The MEL cycle persisted under a reversed photoperiod (lights on, 1800 hr), but the amplitude of the MEL peak was diminished, and the peak was more than 180 degrees out of phase with the corresponding peak under a normal photoperiod. In animals acclimated to 5 degrees in mid-June under a 12L:12D photoperiod, the diel cycle of plasma MEL continued, but both midphotophase and midscotophase concentrations were reduced compared with 15 degrees controls. The diel cycle was also present in animals acclimated to 25 degrees, but the decrease in MEL concentrations was less marked than that in 5 degrees animals. Photoperiod is apparently the primary cue for cycles in plasma MEL in Necturus, but the cycle can be influenced by temperature.     

Effects of season, temperature, and photoperiod on plasma melatonin rhythms in the goldfish, Carassius auratus

Abstract: Effects of season, environmental temperature, and photoperiod on plasma melatonin concentrations were studied in the goldfish, Carassius auratus. When goldfish were reared under natural conditions, melatonin levels at mid-dark exhibited seasonal changes, with higher levels obtained in June and September than in December and March. When fish were kept under light: dark (LD) cycle of 12: 12 at 5, 15, or 25°C during March-April, temperature-dependent increases in melatonin levels at mid-dark were observed. When animals were maintained under LD 16: 8 or LD 8: 16 in combination with temperature changes (5, 15, and 25°C) during January-February, the duration of nocturnal elevation in melatonin was controlled by the length of the scotophase while the amplitude was influenced by environmental temperature. These results indicate that plasma melatonin profiles in the goldfish exhibit seasonal changes that are regulated by both photoperiod and temperature.     

Effect of melatonin on oocyte growth and recruitment, hypophyseal gonadotrophs, and oviduct of the frog Rana cyanophlyctis maintained under natural photoperiod during the prebreeding phase

The effect of 25 and 50 micrograms melatonin on oocyte growth and recruitment, hypophyseal gonadotrophs, and oviduct was studied in Rana cyanophlyctis maintained under natural photoperiod (12.36 L/11.24 D) during the prebreeding phase (April). During the experiment minimum and maximum temperature was 20 +/- 0.2 degrees and 37.2 +/- 0.3 degrees, respectively; the daily fluctuation between these was 17 +/- 0.4 degrees. Melatonin was injected (ip) 6 days a week for 31 days and frogs were killed on the 32nd day. Treatment with 25 or 50 micrograms melatonin 2 hr after the beginning of natural photophase had no effect on the gonadosomatic index (GSI) or on the percentage and number of oocytes at each stage of development. Injections 7 hr after the beginning of the natural photophase caused a marked decrease in the percentage of medium second growth phase (MSGP) and large second growth phase (LSGP) oocytes, while the percentage of the first growth phase (FGP) oocytes increased. However, in 50% of the 25 micrograms and 62.5% of the 50 micrograms melatonin-injected frogs, there was significant reduction in the number of both MSGP and LSGP oocytes. Frogs which received melatonin (25 and 50 micrograms) 11 hr after the onset of natural photophase exhibited a significant (P less than 0.05) decrease in the number of MSGP and LSGP oocytes and GSI. Atretic follicles (AF) increased in all the melatonin-treated groups, but a significant increase was seen only in evening-injected frogs receiving 50 micrograms melatonin. The mean diameter of the largest oocytes decreased significantly in 50 micrograms melatonin-injected frogs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Minireview The influence of season, photoperiod, and pineal melatonin on immune function

Abstract: In addition to the well-documented seasonal cycles of mating and birth, there are also significant seasonal cycles of illness and death among many animal populations. Challenging winter conditions (i.e., low ambient temperature and decreased food availability) can directly induce death via hypothermia, starvation, or shock. Coping with these challenges can also indirectly increase morbidity and mortality by increasing glucocorticoid secretion, which can compromise immune function. Many environmental challenges are recurrent and thus predictable; animals could enhance survival, and presumably increase fitness, if they could anticipate immunologically challenging conditions in order to cope with these seasonal threats to health. The annual cycle of changing photoperiod provides an accurate indicator of time of year and thus allows immunological adjustments prior to the deterioration of conditions. Pineal melatonin codes day length information. Short day lengths enhance several aspects of immune function in laboratory studies, and melatonin appears to mediate many of the enhanced immunological effects of photoperiod. Generally, field studies report compromised immune function during the short days of autumn and winter. The conflict between laboratory and field data is addressed with a multifactor approach. The evidence for seasonal fluctuations in lymphatic tissue size and structure, as well as immune function and disease processes, is reviewed. The role of pineal melatonin and the hormones regulated by melatonin is discussed from an evolutionary and adaptive functional perspective. Finally, the clinical significance of seasonal fluctuations in immune function is presented. Taken together, it appears that seasonal fluctuations in immune parameters, mediated by melatonin, could have profound effects on the etiology and progression of diseases in humans and nonhuman animals. An adaptive functional perspective is critical to gain insights into the interaction among melatonin, immune function, and disease processes.     

Developmental and diel changes in plasma thyroxine and plasma and ocular melatonin in the larval and juvenile bullfrog,Rana catesbeiana

Diel variation in plasma thyroxine (T(4)), and plasma and ocular melatonin was studied in Rana catesbeiana tadpoles and postmetamorphic froglets on 12:12 and 6:18 light/dark (LD) regimens. A progressive rise in plasma T(4) initiates metamorphosis while melatonin can modulate metamorphic progress. Changes in the phase of the rhythms of these two hormones during development might influence the hormonal regulation of metamorphosis. The hormones studied exhibited LD cycle-specific diel fluctuations except in froglet plasma T(4) and all hormones at prometamorphosis on 6L:18D. On 12L:12D, plasma T(4) and ocular melatonin peaked during the scotophase at prometamorphosis and early climax, whereas the plasma melatonin acrophase shifted from the light to the dark at climax. A nocturnal peak of plasma melatonin closely correlated with the onset and offset of dark appeared in the froglet, while the peak of ocular melatonin shifted to the light. Compared to 12L:12D, the peaks of the diel fluctuations on 6L:18D occurred later than on 12L:12D in synchrony with an earlier onset, and increase in length, of the scotophase. The phase of the hormone rhythms changed during metamorphosis in such a way that the peaks of melatonin had a different relationship to the T(4) peaks as development proceeded. On both LD cycles, the 24-h mean of plasma T(4) rose at climax and fell in the froglet whereas plasma melatonin decreased at climax and then rose to a high level in the froglet. After only minor changes during metamorphosis, froglet ocular melatonin levels decreased on 12L:12D and increased on 6L:18D. The findings indicate that the hormonal flux during metamorphosis has circadian aspects, which might explain variations in the response to exogenous hormone treatment at different times of the day and LD cycle-specific timing of development. A fall in plasma melatonin at climax appears to be as much a part of the hormonal changes of metamorphosis as a rise in plasma T(4).     

Effect of estradiol on plasma melatonin levels

Melatonin levels were determined in plasma samples obtained at 15 minute intervals during a 4-hour period (08:00 - 12:00 hours) from a normal adult male on 5 consecutive days. On days 1, 2, and 3, the subject was given estradiol valerate (E2) 10 micrograms/kg at the end of each sampling period. An episodic pattern of melatonin secretion was found. Post E2 mean estradiol levels per 4 hours increased and mean melatonin and testosterone levels per 4 hours decreased significantly. A decrease in melatonin levels post E2 is the reverse of the response expected based on nonhuman animal experimental data.     

Effect of asymmetrical reductions of photoperiod on pineal melatonin, locomotor activity and gonadal condition of male Syrian hamsters

This study investigated the relationship of two overt circadian rhythms, locomotor activity and melatonin synthesis in the pineal gland, by comparing their responses to asymmetrical reductions in photoperiod. Transfer of male Syrian hamsters from long to short daylengths led to an increase in the duration of both locomotor activity and the period of melatonin synthesis. Over the course of re-entrainment, the two rhythms were held in a stable phase relationship, and the direction of the switch did not influence the rate of decompression or the final phase relationships established after 8 weeks in short daylengths. Decompression of the activity rhythm was not influenced by pinealectomy. Exposure to short photoperiods caused gonadal regression and a consequent decline in serum testosterone levels from 10 to less than 1 nmol/l. The direction of the photoperiodic switch did not affect the time-course of gonadal regression. These data demonstrate the important influence of photoperiod upon the duration of the nocturnal peak of melatonin production by the pineal and also demonstrate that this effect is one example of a more widespread response of the circadian system. A qualitatively similar signal controls both locomotor activity and melatonin synthesis, although the neural basis of this common mechanism is unclear.     

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.     

Daily cycles in plasma melatonin levels under long or short photoperiod in the common carp, Cyprinus carpio

A radioimmunoassay (RIA) for plasma melatonin (MLT) was simplified for use with the common carp (Cyprinus carpio). Plasma was partially purified with Sep-Pak C18 cartridge before RIA. The inhibition curves for the Sep-Pak C18 fraction from the plasma of carp, goldfish (Carassius auratus), yellow tail (Seriola quinqueradiata), Japanese eel (Anguilla japonica), bora (Mugil cephalus cephalus), kisu (Sillago japonica), ishigarei (Kareius bicoloratus), and shimaisaki (Rhyncopelates oxyrhynchus) were parallel with the MLT standard curve. There was a highly significant correlation between MLT added to carp plasma and that which was recovered (r = 0.997, P less than 0.01). Intraassay coefficients of variation at low, medium, and high levels were 4.7, 5.2, and 6.4, respectively. Interassay coefficients of variation at low, medium, and high levels were 11.8, 8.2, and 24.1%, respectively. The lower limit of detection was 11 pg/tube. Plasma MLT levels were investigated every 2 hr in carp under 16L-8D and 8L-16D at 24 degrees. Under both photoperiods, MLT levels showed marked daily patterns, i.e., the levels were high (220-540 pg/ml) during the dark phase and low (23-104 pg/ml) during the light phase. Tricaine methanesulfonate markedly interfered with the MLT measurements, while ethylcarbamate did not show any significant influence. Sexual difference in plasma MLT levels in carp was not observed. These observations suggest that MLT is an important hormone in photoperiodism and/or a circadian rhythm in fish.     

Distribution of adrenomedullin and proadrenomedullin N-terminal 20 peptide immunoreactivity in the pituitary gland of the frog Rana perezi

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are two multifunctional peptides processed from a common precursor which have been described in numerous mammalian organs, including the pituitary gland. Previous studies have found AM immunoreactivity in neurohypophysis nerve fibers of amphibian pituitary. In the present study, immunocytochemical and Western blot analysis in the pituitary gland of the amphibian Rana perezi demonstrated in the adenohypophysis both AM and PAMP. AM-like immunoreactivity was found in a moderate number of endocrine cells of the pars distalis. In the neurohypophysis, AM was observed not only in nerve fibers of pars nervosa and axonal projections innervating the pars intermedia, but also in the outer zone of the median eminence. PAMP staining was observed in numerous endocrine cells scattered all over the pars distalis and in some cells of the pars tuberalis, but not in the neurohypophysis. In order to compare the quantity of AM and PAMP immunoreactivity between pars distalis of female and male specimens, an image analysis study was done. Significant differences for AM immunoreactivity (p<0.001) between sexes was found, the males showing higher immunostained area percentage. Differences of PAMP immunoreactivity were not significant (p=0.599). Western blot analysis detected bands presumably corresponding to precursor and/or intermediate species in the propeptide processing.     

Effects of photoperiod on pineal melatonin in the marsh rice rat (Oryzomys palustris)

Abstract: Pineal melatonin content was examined under four different photoperi-ods (10L: 14D, 12L: 12D, 14L: 10D, and 16L: 8D) in adult female rice rats (Experiment 1). Pineal melatonin was basal during the light and increased beginning 1 hr after lights off. Within 2 hr after lights off, melatonin increased to levels that were maintained throughout the dark period. In all but one photoperiod (10L: 14D), melatonin remained elevated prior to light onset and decreased markedly within one hour after lights on. In addition, the duration of pineal melatonin was inversely related to the length of the photoperiod. In Experiment 2, the time course of pineal melatonin content on 16L: 8D was examined every 20 min during the first hour after lights off and the first hour after lights on. Melatonin content increased gradually during the first hour and decreased markedly within 20 min after lights on. These data show that pineal melatonin in female rice rats is regulated by photoperiod.     

Melatonin rhythms in European sea bass plasma and eye: influence of seasonal photoperiod and water temperature

The transduction of seasonal information from the environment (i.e., photoperiod and water temperature) into melatonin rhythms was studied in sea bass. Plasma and ocular melatonin (N-acetyl-5-methoxytryptamine) was determined in autumn, winter, spring and summer (experiment 1) under natural culture conditions, and in the summer and winter solstices under both natural and "6-month out-of-phase" photoperiods (experiment 2). At each sampling, 48 sea bass were sacrificed at a rate of 6 fish every 3 hr and the level of melatonin was determined in plasma and eye cup samples by ELISA. In experiment 1, significant diel changes were observed in plasma melatonin, with nocturnal melatonin varying from 144 pg/mL (summer) to 23 pg/mL (autumn), while diurnal melatonin remained low, around 8 pg/mL throughout the year. In experiment 2, the photoperiod length was shown to control the duration of the nocturnal melatonin rise, while the water temperature determined the amplitude of the melatonin rhythm. Ocular melatonin peaked during daytime in autumn and winter, but no significant changes were detected in summer and spring. In conclusion, plasma melatonin rhythms in sea bass reflect the pineal capacity to integrate seasonal information and supply precise calendar information, which may synchronize different physiological processes such as annual reproduction and feeding rhythms.     

Social influences on androgen levels in the southern leopard frog, Rana sphenocephala

Social cues play an important role in modulating neuroendocrine and hormonal states in many vertebrates. Social cues in the form of conspecific advertisement calls have been demonstrated to modulate plasma androgen levels in male Rana sphenocephala, the Southern leopard frog. Male R. sphenocephala were captured and brought to the laboratory for testing. Animals were exposed to one of two acoustical environments: a recording of an active conspecific breeding chorus or a recording of the same chorus in which all the spectral frequencies had been digitally shifted 500 Hz higher. Following 12 nights of exposure to either stimulus, plasma androgen levels were assessed by radioimmunoassay and gonadal morphology was examined to compare group differences. Plasma levels of dihydrotestosterone were significantly higher in males exposed to the conspecific chorus, compared with males exposed to the altered version of the chorus. Gonadal morphology did not differ between the two groups. These results suggest that in addition to environmental cues such as rainfall and other seasonal changes, social cues may also serve as environmental synchronizers among anurans to coordinate reproductive effort.     

Effects of photoperiod and temperature on rhythmic melatonin secretion from the pineal organ of the white sucker (Catostomus commersoni) in vitro.

The secretion rate of melatonin from cultured pineal organs of the white sucker was examined for several days under either a 12:12-hr light:dark (LD) cycle or continuous darkness (DD) at either 10 degrees or 20 degrees. The incubation medium was changed at 3-hr intervals and secreted melatonin was measured by RIA. Under a 12:12-hr LD cycle (0800 light on, 2000 light off) melatonin secretion was suppressed during the day and highly active at night, with larger amplitudes at 20 than at 10 degrees. In DD at 10 degrees no circadian rhythmicity in secretion was found in October or January, whereas at 20 degrees a circadian-like pattern was detected in pineals which were derived from animals reared at either 10 degrees or 20 degrees for 1 week prior to the experiment in October or January. The pineals in the DD experiment still responded to an additional 24-hr LD cycle at both temperatures even after 6 or more days. These results clearly reveal the influence of photoperiod and temperature on melatonin secretion of organ-cultured pineal glands. The existence of a circadian oscillator for melatonin secretion in the pineal gland of the white sucker is suggested.     

2-[125I]-melatonin binding sites in the central nervous system and neural retina of the frog Rana perezi: regulation by light and temperature

The objective of the present study is to test daily and seasonal changes in 2-[125I]-Melatonin ([125I]-Mel) binding in different brain areas and the retina of the frog Rana perezi as well as the possible effect of light and temperature on melatonin receptors. During the day-night cycle, binding of [125I]-Mel showed a clear rhythm in the optic tectum, diencephalon, telencephalon, and neural retina, the binding being higher in the light phase than in the dark phase. By contrast, melatonin receptors did not show any significant summer-winter differences in any of the four tissues studied. In the neural retina, but not in the brain, exposure of frogs to 24 h darkness for one week leads to significantly less [125I]-Mel binding than 24 h light exposure. This darkness-induced reduction of [125I]-Mel binding is not due to a desensitisation of binding sites by high melatonin levels. Thermal acclimation to either 5 or 22 degrees C for one month did not change the affinity (Kd) and density (Bmax) of [125I]-Mel binding sites either in the brain or the retina. All these results indicate that there is a daily rhythm in melatonin receptors in the frog brain and retina, and that the light/dark cycle can drive this rhythm in [125I]-Mel binding in the retina. Temperature apparently did not modify [125I]-Mel binding in frogs.