There has been relatively little research conducted on pineal melatonin production in laboratory mice, in part, due to the lack of appropriate assays. We studied the pineal and plasma rhythm, response to light, adrenergic stimulation, and metabolism of melatonin in CBA mice. With the use of a sensitive and specific melatonin RIA, melatonin was detected in the pineal glands at all times of the day >21 fmol/gland in CBA mice but not in C57Bl mice. Both plasma and pineal melatonin levels peaked 2 h before dawn in a 12:12-h light-dark photoperiod (162 ± 31 pM and 1,804 ± 514 fmol/gland, respectively). A brief light pulse (200 lx/15 min), 2 h before lights on, suppressed both plasma and pineal melatonin to near basal levels within 30 min. Exposure to light pulses 4 h after lights off or 2 h before lights on resulted in delays and advances, respectively, in the early morning decline of plasma and pineal melatonin on the next cycle. Administration of the β-adrenergic agonist isoproterenol (20 mg/kg) 2 and 4 h after lights on in the morning resulted in a fivefold increase in plasma and pineal melatonin 2.5 to 3 h after the first injection. In the mouse, unlike the rat, melatonin was shown to be metabolized almost exclusively to 6-glucuronylmelatonin rather than 6-sulphatoxymelatonin. These studies have shown that the appropriate methodological tools are now available for studying melatonin rhythms in mice.

A specific practical radioimmunoassay suited to determinations of melatonin in both tissues and body fluids is described. The rabbit antibody employed was raised to an antigen formed by condensation between N-acetylserotonin and the Mannich adduct of bovine serum albumin and formaldehyde. Substitution was shown by protonmagnetic resonance spectroscopy to occur exclusively at die 4 position of the indole nucleus. The antibody reacted with a variety of N-acetylated indoles, and absolute specificity was dependent upon the extraction procedure and column (Lipidex 5000) chromatography.In addition to the usual reliability criteria, the validity of the assay was checked by gas chromatography-mass spectrometry using [ 2 H 3 ]melatonin as an internal standard, the preparation of which is described. The occurrence of melatonin in the plasma of man, sheep, rat and chicken was confirmed, and its presence in the plasma of the pig (22-76 pg/ml), donkey (24-128 pg/ml), cow (20-320 pg/ml), camel (29-221 pg/ml) and a scincid lizard (20-500 pg/ml) established. A nocturnal rise in plasma melatonin content occurred in all species.Melatonin was found in the plasma of ewes 2-12 weeks after pinealectomy, but the nocturnal rise was abolished. The results establish a nyctohemeral variation in plasma melatonin in a wide variety of species, and indicate that sources of melatonin other than the pineal may assume precedence following pinalectomy. (Endocrinology 101: 119, 1977)

The geographic isolation and the prolonged absence of sunlight during winter make Antarctica an interesting environment for studying circadian rhythms. This study explored the effects of wintering on sleep, hormonal, and electrolyte rhythms in four human subjects living in a small Antarctic base. Up to the last sunset sleep, 6-sulfatoxymelatonin, cortisol, sodium, and potassium rhythms were synchronized within clock time. During the 126 days of winter, when there was no sunlight, the circadian rhythms of all measures free ran in each individual. For example, the free-running periods for the cortisol excretory rhythm were 24 h 29 min, 24 h 45 min, 25 h 7 min, and 25 h 14 min for subjects C, J, K, and G, respectively. The period lengths of C, J, and K were significantly different, whereas there was no significant difference between K and G. The phase relationships between each rhythm remained constant in three out of the four subjects. Total daily output and rhythm amplitude for 6-sulfatoxymelatonin, potassium, and sodium remained constant during the entrained and free-running stages of the study. Significant changes in total daily cortisol excretion were observed during the year with one subject producing less and two subjects more while the rhythms were free running. When the sun reappeared during spring, all rhythms again synchronized and entrained to the daylight. These results show that 1) circadian rhythms can free run, even when the subjects have knowledge of time; and 2) within a small communal group, individuals can maintain unique free-running periods.

Melatonin and wheel-running rhythmicity and the effects of acute and chronic light pulses on these rhythms were studied in Clock(Delta19) mutant mice selectively bred to synthesize melatonin. Homozygous melatonin-proficient Clock(Delta19) mutant mice (Clock(Delta19/Delta19)-MEL) produced melatonin rhythmically, with peak production 2 h later than the wild-type controls (i.e., just before lights on). By contrast, the time of onset of wheel-running activity occurred within a 20-min period around lights off, irrespective of the genotype. Melatonin production in the mutants spontaneously decreased within 1 h of the expected time of lights on. On placement of the mice in continuous darkness, the melatonin rhythm persisted, and the peak occurred 2 h later in each cycle over the first two cycles, consistent with the endogenous period of the mutant. This contrasted with the onset of wheel-running activity, which did not shift for several days in constant darkness. A light pulse around the time of expected lights on followed by constant darkness reduced the expected 2-h delay of the melatonin peak of the mutants to approximately 1 h and advanced the time of the melatonin peak in the wild-type mice. When the Clock(Delta19/Delta19)-MEL mice were maintained in a skeleton photoperiod of daily 15-min light pulses, a higher proportion entrained to the schedule (57%) than melatonin-deficient mutants (9%). These results provide compelling evidence that mice with the Clock(Delta19) mutation express essentially normal rhythmicity, albeit with an underlying endogenous period of 26-27 h, and they can be entrained by brief exposure to light. They also raise important questions about the role of Clock in rhythmicity and the usefulness of monitoring behavioral rhythms compared with hormonal rhythms.

The emergence of melatonin rhythmicity was studied in 163 infants between 46-55 weeks postconception by monitoring the excretion of the urinary melatonin metabolite 6-sulfatoxymelatonin (aMT.6S). From this population, we examined the effects of gender, season, multiple birth, home birth, previous sudden infant death syndrome in the family, premature labor, spontaneous rupture of membranes, preeclampsia, intrauterine growth restriction, and nursery lighting on pineal rhythmicity. As previously reported, rhythmic excretion of aMT.6S appeared between 49-55 weeks postconception (9-15 weeks of age) in singleton babies born at term in the hospital. Full-term infants who had a sibling die of sudden infant death syndrome had a pattern of melatonin rhythm development no different from that of the control full-term infants. In contrast, full-term infants born at home and full-term twins born in the hospital had significantly lower aMT.6S excretion than hospital-born singleton infants at the same ages despite similar body weights (e.g. at 52 weeks postconception; 1.8 +/- 0.4, 1.1 +/- 0.3, and 3.6 +/ -0.5 nmol/day, respectively). In full-term infants, there was no difference in the development of melatonin rhythmicity between the sexes, with season or method of delivery (vaginal vs. caesarean). The premature infants were divided into 5 groups (babies born after premature labor, premature rupture of membranes, preeclampsia, intrauterine growth restriction, and fetal distress). All premature infants had a delay in the appearance of aMT.6S rhythms in the urine in relation to chronological age. When the infants were compared on the basis of weeks since conception, those infants born after spontaneous premature labor excreted amounts of aMT.6S no different from those of full-term singleton infants during the period of study. In contrast, the premature rupture of membranes, preeclampsia, and fetal distressed infants excreted 50% less aMT.6S, and intrauterine growth restricted infants excreted 67% less at the same postconceptional ages. These differences were due to reduced nocturnal excretion of the metabolite. In an attempt to accelerate the development of melatonin rhythmicity, premature labor and premature rupture of membranes infants were randomly assigned to be totally deprived of light (using phototherapy eye shields) or partially deprived of light by moving them to a dimly lit room each night for the last 3-8 weeks of their stay in the hospital nursery. Babies born after premature labor produced normal amounts of aMT.6S between 46-52 weeks postconception, and this pattern was not affected by the nocturnal light deprivation. Infants born after premature rupture of membranes and totally deprived of light at night had aMT.6S excretion rhythms at 52 weeks postconception no different from those of full-term hospital-born infants or premature labor infants, whereas those in infants placed in dim light were similar to those in untreated premature rupture of membranes infants. These results suggest that premature birth alone is not...

The rhythm of melatonin in rat milk and the capacity of pups to synthesize and metabolize melatonin were studied. Melatonin was undetectable in milk in the light (< 21 pM), but increased rapidly 2-4 h after dark to peak at 357 +/- 66 pM at mid-dark. Oral or subcutaneous administration of melatonin to 5- and 10-day-old pups resulted in peak plasma melatonin levels 30 min after administration and rapid metabolism. Increases in pineal and plasma melatonin levels at night were detected at 5 and 6 days of age, respectively. Isoproterenol administration (2 microg/g body wt) at mid-light to day 10 pups increased plasma melatonin from 312 +/- 40 pM to 1,298 +/- 160 pM, whereas propranolol (2 microg/g body wt) suppressed nocturnal melatonin secretion from 1,270 +/- 128 pM to 395 +/- 66 pM. The rise of pineal and plasma melatonin in day 10 pups occurred 1 and 2 h after dark onset, respectively, preceding the onset in dams by 3 and 4 h, respectively. Propranolol administration to 2- and 5-day lactating dams inhibited plasma and milk melatonin at night but had no effect on their suckling pups. Transfer of melatonin via the milk is unlikely to provide an entraining signal for rat pups.

The relationship between circadian rhythmicity and rodent reproductive cyclicity is well established, but the impact of disrupted clock gene function on reproduction has not been investigated. This study evaluated the reproductive performance of melatonin deficient and proficient mice carrying a mutation in the core circadian gene, Clock. In natural matings, melatonin deficient Clock mutant mice took 2 to 3 days longer to mate and to subsequently deliver pups than their control line. The melatonin proficient mutants (Clock-MEL) had a smaller, but still significant delay (P < 0.05). The Clock mutation resulted in smaller median litter sizes compared to the control lines (7 v. 8 pups, P < 0.05) while melatonin proficiency reversed this difference. Survival to weaning was 84% and 80% for the melatonin deficient and proficient Clock mutant lines respectively, compared to 94 to 96% for their control lines. When immature mice were subjected to a standard PMSG/HCG superovulation protocol, Clock-MEL mice had lowered fertility and significantly fewer ovulations than their control line although embryo development appeared to be only slightly affected (Table 1). 19 ± 5 17% 48% 32% 3% When kept in constant darkness, 7 of 15 Clock-MEL mice, became arrhythmic, but still became pregnant. The 7 mice that free ran for at least 14 days in constant darkness with a period of 27.1 h also became pregnant.This study has shown that a mutation in the Clock gene that results in a protein incapable of initiating the transcription of target genes has significant, but subtle effects on reproductive performance. The capacity to produce melatonin or additional genes introduced along with the genes for the melatonin synthesising enzymes reduced the impact of the mutation further. It would appear that redundancy within the circadian timing system allows the reproductive cyclicity to persist in Clock mutant mice, albeit at a suboptimal level.

Abstract. Urinary excretion of 6-suphatoxy melatonin, cortisol, potassium and sodium was monitored at four hourly intervals for 24 h in 30 normal subjects at the summer and winter solstices. The 24 h profiles were fitted to sine curves and mean 24-h excretion, time of maximum excretion and amplitude of the curves compared. The excretion of 6-sulphatoxy melatonin was remarkably stable at the two times of the year (24-h excretion 108 ± 6.3 nmol in summer and 105 ± 6.3 nmol in winter, mean ± sem). The time of maximum excretion was significantly delayed in winter by 1 h and 40 min. Urinary cortisol excretion was significantly higher in winter, however, the amplitude was unaltered. The time of maximum excretion of cortisol was significantly delayed by 1 h and 34 min. Potassium and sodium excretion were both unaffected by seasonal influences. These results contrast with results in some animal species in which the duration of the melatonin signal is thought to be the key determinant in subsequent melatonin action. In humans it is likely that the phasing of the melatonin rhythm is of prime importance.