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Key points Light at night is essential to a 24/7 society, but it has negative consequences on health. Basically, light at night induces an alteration of our biological clocks, known as chronodisruption, with effects even when this occurs during pregnancy. Here we explored the developmental impact of gestational chronodisruption (chronic photoperiod shift, CPS) on adult and fetal adrenal biorhythms and function. We found that gestational chronodisruption altered fetal and adult adrenal function, at the molecular, morphological and physiological levels. The differences between control and CPS offspring suggest desynchronization of the adrenal circadian clock and steroidogenic pathway, leading to abnormal stress responses and metabolic adaptation, potentially increasing the risk of developing chronic diseases. Abstract Light at night is essential to a 24/7 society, but it has negative consequences on health. Basically, light at night induces an alteration of our biological clocks, known as chronodisruption, with effects even when this occurs during pregnancy. Indeed, an abnormal photoperiod during gestation alters fetal development, inducing long-term effects on the offspring. Accordingly, we carried out a longitudinal study in rats, exploring the impact of gestational chronodisruption on the adrenal biorhythms and function of the offspring. Adult rats (90 days old) gestated under chronic photoperiod shift (CPS) decrease the time spent in the open arm zone of an elevated plus maze to 62% and increase the rearing time to 170%. CPS adults maintained individual daily changes in corticosterone, but their acrophases were distributed from 12.00 h to 06.00 h. CPS offspring maintained clock gene expression and oscillation, nevertheless no daily rhythm was observed in genes involved in the regulation and synthesis of steroids. Consistent with adult adrenal gland being programmed during fetal life, blunted daily rhythms of corticosterone, core clock gene machinery, and steroidogenic genes were observed in CPS fetal adrenal glands. Comparisons of the global transcriptome of CPS versus control fetal adrenal gland revealed that 1078 genes were differentially expressed (641 down-regulated and 437 up-regulated). In silico analysis revealed significant changes in Lipid Metabolism, Small Molecule Biochemistry, Cellular Development and the Inflammatory Response pathway (z score: 48-20). Altogether, the present results demonstrate that gestational chronodisruption changed fetal and adult adrenal function. This could translate to long-term abnormal stress responses and metabolic adaptation, increasing the risk of developing chronic diseases.

Recent reports account for altered metabolism in adult offspring from pregnancy subjected to abnormal photoperiod, suggesting fetal programming of liver physiology. To generate a pipeline of subsequent mechanistic experiments addressing strong candidate genes, here we investigated the effects of constant gestational light on the fetal liver transcriptome. At 10 days of gestation, dams were randomized in two groups ( n = 7 each): constant light (LL) and normal photoperiod (12 h light/12 h dark; LD). At 18 days of gestation, RNA was isolated from the fetal liver and subjected to DNA microarray (Affymetrix platform for 28,000 genes). Selected differential mRNAs were validated by quantitative PCR (qPCR), while integrated transcriptional changes were analyzed with Ingenuity Pathway Analysis and other bioinformatics tools. Comparison of LL relative to LD fetal liver led to the following findings. Significant differential expression was found for 3,431 transcripts (1,960 upregulated and 1,471 downregulated), with 393 of them displaying ≥ 1.5-fold change. We validated 27 selected transcripts by qPCR, which displayed fold-change values highly correlated with microarray ( r2= 0.91). Different markers of nonalcoholic fatty liver disease were either upregulated (e.g., Ndn and Pnpla3) or downregulated (e.g., Gnmt, Bhmt1/2, Sult1a1, Mpo, and Mat1a). Diverse pathways were altered, including hematopoiesis, coagulation cascade, complement system, and carbohydrate and lipid metabolism. The microRNAs 7a-1, 431, 146a, and 153 were upregulated, while the abundant hepatic miRNA 122 was downregulated. Constant gestational light induced extensive modification of the fetal liver transcriptome. A number of differentially expressed transcripts belong to fundamental functional pathways, potentially contributing to long-term liver disease.

In the capuchin monkey (Cebus apella), a new-world nonhuman primate, maternal exposure to constant light during last third of gestation induces precocious maturation of the fetal adrenal and increased plasma cortisol in the newborn. Here, we further explored the effects of this challenge on the developmental programming of adrenal function in newborn and infant capuchin monkeys. We measured (i) plasma dehydroepiandrosterone sulphate (DHAS) and cortisol response to ACTH in infants with suppressed endogenous ACTH, (ii) plasma DHAS and cortisol response to ACTH in vitro, and (iii) adrenal weight and expression level of key factors in steroid synthesis (StAR and 3β-HSD). In one-month-old infants from mothers subjected to constant light, plasma levels of cortisol and cortisol response to ACTH were twofold higher, whereas plasma levels of DHAS and DHAS response to ACTH were markedly reduced, compared to control conditions. At 10 months of age, DHAS levels were still lower but closer to control animals, whereas cortisol response to ACTH was similar in both experimental groups. A compensatory response was detected at the adrenal level, consisting of a 30% increase in adrenal weight and about 50% reduction of both StAR and 3β-HSD mRNA and protein expression and the magnitude of DHAS and cortisol response to ACTH in vitro. Hence, at birth and at 10 months of age, there were differential effects in DHAS, cortisol production, and their response to ACTH. However, by 10 months of age, these subsided, leading to a normal cortisol response to ACTH. These compensatory mechanisms may help to overcome the adrenal alterations induced during pregnancy to restore normal cortisol concentrations in the growing infant.

Neonatal lambs, as with human and other neonates, have low arrhythmic endogenous levels of melatonin for several weeks until they start their own pineal rhythm of melatonin production at approximately 2 weeks of life. During pregnancy, daily rhythmic transfer of maternal melatonin to the fetus has important physiological roles in sheep, nonhuman primates, and rats. This melatonin rhythm provides a circadian signal and also participates in adjusting the physiology of several organs in preparation for extrauterine life. We propose that the ensuing absence of a melatonin rhythm plays a role in neonatal adaptation. To test this hypothesis, we studied the effects of imposing a high-amplitude melatonin rhythm in the newborn lamb on (1) clock time-related changes in cortisol and plasma variables and (2) clock time-related changes of gene expression of clock genes and selected functional genes in the adrenal gland and heart. We treated newborn lambs with a daily oral dose of melatonin (0.25 mg/kg) from birth to 5 days of age, recreating a high-amplitude melatonin rhythm. This treatment suppressed clock time-related changes of plasma adrenocorticotropic hormone, cortisol, clock gene expression, and functional genes in the newborn adrenal gland. In the heart, it decreased heart/body weight ratio, increased expression of Anp and Bnp, and resulted in different heart gene expression from control newborns. The interference of this postnatal melatonin treatment with the normal postnatal pattern of adrenocortical function and heart development support a physiological role for the window of flat postnatal melatonin levels during the neonatal transition.

Chronic exposure to light at night, as in shift work, alters biological clocks (chronodisruption), negatively impacting pregnancy outcome in humans. Actually the interaction of maternal and fetal circadian systems could be a key factor determining a fitting health in adults. We propose that chronic photoperiod shift (CPS) during pregnancy alter maternal circadian rhythms and impair circadian physiology in the adult offspring, increasing health risks. Pregnant rats were exposed to normal photoperiod (12 h light, 12 h dark) or to CPS until 85% of gestation. The effects of gestational CPS were evaluated on the mother and adult offspring. In the mother we measured rhythms of heart rate, body temperature, and activity through gestation and daily rhythms of plasma variables (melatonin, corticosterone, aldosterone, and markers of renal function) at 18 days of gestation. In adult offspring, we measured rhythms of the clock gene expression in the suprachiasmatic nucleus (SCN), locomotor activity, body temperature, heart rate, blood pressure, plasma variables, glucose tolerance, and corticosterone response to ACTH. CPS altered all maternal circadian rhythms, lengthened gestation, and increased newborn weight. The adult CPS offspring presented normal rhythms of clock gene expression in the SCN, locomotor activity, and body temperature. However, the daily rhythm of plasma melatonin was absent, and corticosterone, aldosterone, renal markers, blood pressure, and heart rate rhythms were altered. Moreover, CPS offspring presented decreased glucose tolerance and an abnormal corticosterone response to ACTH. Altogether these data show that gestational CPS induced long-term effects on the offspring circadian system, wherein a normal SCN coexists with altered endocrine, cardiovascular, and metabolic function.

Although the fetal pineal gland does not secrete melatonin, the fetus is exposed to melatonin of maternal origin. In the non-human primate fetus, melatonin acts as a trophic hormone for the adrenal gland, stimulating growth while restraining cortisol production. This latter physiological activity led us to hypothesize that melatonin may influence some fetal functions critical for neonatal adaptation to extrauterine life. To test this hypothesis we explored (i) the presence of G-protein-coupled melatonin binding sites and (ii) the direct modulatory effects of melatonin on noradrenaline (norepinephrine)-induced middle cerebral artery (MCA) contraction, brown adipose tissue (BAT) lypolysis and ACTH-induced adrenal cortisol production in fetal sheep. We found that melatonin directly inhibits the response to noradrenaline in the MCA and BAT, and also inhibits the response to ACTH in the adrenal gland. Melatonin inhibition was reversed by the melatonin antagonist luzindole only in the fetal adrenal. MCA, BAT and adrenal tissue displayed specific high-affinity melatonin binding sites coupled to G-protein (Kd values: MCA 64 ± 1 pm, BAT 98.44 ± 2.12 pm and adrenal 4.123 ± 3.22 pm). Melatonin binding was displaced by luzindole only in the adrenal gland, supporting the idea that action in the MCA and BAT is mediated by different melatonin receptors. These direct inhibitory responses to melatonin support a role for melatonin in fetal physiology, which we propose prevents major contraction of cerebral vessels, restrains cortisol release and restricts BAT lypolysis during fetal life.

The circadian production of glucocorticoids involves the concerted action of several factors that eventually allow an adequate adaptation to the environment. Circadian rhythms are controlled by the circadian timing system that comprises peripheral oscillators and a central rhythm generator located in the suprachiasmatic nucleus (SCN) of the hypothalamus, driven by the self-regulatory interaction of a set of proteins encoded by genes named clock genes. Here we describe the phase relationship between the SCN and adrenal gland for the expression of selected core clock transcripts (Per-2, Bmal-1) in the adult capuchin monkey, a New World, diurnal nonhuman primate. In the SCN we found a higher expression of Bmal-1 during the h of darkness (2000–0200 h) and Per-2 during daytime h (1400 h). The adrenal gland expressed clock genes in oscillatory fashion, with higher values for Bmal-1 during the day (1400–2000 h), whereas Per-2 was higher at nighttime (about 0200 h), resulting in a 9- to 12-h antiphase pattern. In the adrenal gland, the oscillation of clock genes was accompanied by rhythmic expression of a functional output, the steroidogenic enzyme 3β-hydroxysteroid dehydrogenase. Furthermore, we show that adrenal explants maintained oscillatory expression of Per-2 and Bmal-1 for at least 36 h in culture. The acrophase of both transcripts, but not its overall expression along the incubation, was blunted by 100 nm melatonin. Altogether, these results demonstrate oscillation of clock genes in the SCN and adrenal gland of a diurnal primate and support an oscillation of clock genes in the adrenal gland that may be modulated by the neurohormone melatonin.

We tested the hypothesis that in primates, maternal melatonin restrains fetal and newborn adrenal cortisol production. A functional G-protein-coupled MT1 membrane-bound melatonin receptor was detected in 90% gestation capuchin monkey fetal adrenals by (a) 2-[125I] iodomelatonin binding (Kd, 75.7 ± 6.9 pm; Bmax, 2.6 ± 0.4 fmol (mg protein)−1), (b) cDNA identification, and (c) melatonin inhibition of adrenocorticotrophic hormone (ACTH)- and corticotrophin-releasing hormone (CRH)-stimulated cortisol but not of dehydroepiandrosterone sulphate (DHAS) production in vitro. Melatonin also inhibited ACTH-induced 3β-hydroxysteroid dehydrogenase mRNA expression. To assess the physiological relevance of these findings, we next studied the effect of chronic maternal melatonin suppression (induced by exposure to constant light during the last third of gestation) on maternal plasma oestradiol during gestation and on plasma cortisol concentration in the 4- to 6-day-old newborn. Constant light suppressed maternal melatonin without affecting maternal plasma oestradiol concentration, consistent with no effect on fetal DHAS, the precursor of maternal oestradiol. However, newborns from mothers under constant light condition had twice as much plasma cortisol as newborns from mothers maintained under a normal light–dark schedule. Newborns from mothers exposed to chronic constant light and daily melatonin replacement had normal plasma cortisol concentration. Our results support a role of maternal melatonin in fetal and neonatal primate cortisol regulation.

The pineal hormone melatonin participates in circadian, seasonal, and reproductive physiology. The presence of melatonin binding sites in human brain and peripheral tissues is well documented. However, in the mammalian adrenal gland, low-affinity melatonin binding sites have been detected only in the rat by some but not all authors. Conflicting evidence for a regulatory role of melatonin on adrenal cortisol production, prompted us to investigate this possibility in a New World primate, the capuchin monkey. Expression of melatonin receptors in the adrenal cortex was demonstrated through pharmacological characterization and autoradiographic localization of 2-[125I]iodomelatonin binding sites (dissociation constant = 96.9 +/- 15 pM; maximal binding capacity = 3.8 +/- 0.4 fmol/mg protein). The mt1 identity of these receptors was established by cDNA sequencing. Melatonin treatment of dispersed cells and explants from adrenal gland did not affect basal cortisol production. However, cortisol production stimulated by 100 nM ACTH was significantly inhibited by low melatonin concentrations (0.1-100 nM); this inhibitory effect was reversed by the mt1/MT2 melatonin antagonist luzindole. Melatonin also inhibited dibutyril-cAMP-stimulated cortisol production, suggesting that melatonin acts through a cAMP-independent signaling pathway. The present data demonstrate that the primate adrenal gland cortex expresses functional mt1 melatonin receptors and shows that melatonin inhibits ACTH-stimulated cortisol production.

article i nfo Cardiac gene networks Whole transcriptome Gestational chronodisruption Fetal programming of adult disease Left ventricle hypertrophy We recently reported that gestational chronodisruption induces fetal growth restriction and marked effects on fetal adrenal physiology. Here, whole-transcriptome profiling was used to test whether gestational chronodisruption modifies gene expression in the fetal heart, potentially altering cardiac development. At day 10 of gestation (E10), pregnant rats were randomized in two groups: constant light (LL) and control 12 h light/12 h dark photope- riod (LD). RNAisolated from E18 heartwas subjectedto microarray analysis(Affymetrix platform for28,000 genes). Integrated transcriptional changes were assesse db y gene ontology and pathway analysis. Signi ficant differential expression was found for 383 transcripts in LL relative to LD fetal heart (280 up-regulated and 103 down- regulated); with 42 of them displaying a 1.5-fold or greater change in gene expression. Deregulated markers of cardiovascular disease accounted for alteration of diverse gene networks in LL fetal heart, including local steroido- genesis and vascular calcification, as well as cardiac hypertrophy, stenosis and necrosis/cell death. DNA integrity was also overrepresented, including a 2.1-fold increase of Hmga1 mRNA, which encodes for a profuse architectural transcription factor. microRNA analysis revealed up-regulation of miRNAs 218-1 and 501 and concurrent down- regulation of their validated target genes. In addition, persistent down-regulation of Kcnip2 mRNA and hypertrophy of the left ventricle were found in the heart from 90 days-old offspring from LL mothers. The dysregulation of a rel- evant fraction of the fetal cardiac transcriptome, together with the diversity and complexity of the gene networks altered by gestational chronodisruption, suggest enduring molecular changes which may shape the hypertrophy observed in the left ventricle of adult LL offspring.

Surprisingly, in our modern 24/7 society, there is scant information on the impact of developmental chronodisruption like the one experienced by shift worker pregnant women on fetal and postnatal physiology. There are important differences between the maternal and fetal circadian systems; for instance, the suprachiasmatic nucleus is the master clock in the mother but not in the fetus. Despite this, several tissues/organs display circadian oscillations in the fetus. Our hypothesis is that the maternal plasma melatonin rhythm drives the fetal circadian system, which in turn relies this information to other fetal tissues through corticosterone rhythmic signaling. The present data show that suppression of the maternal plasma melatonin circadian rhythm, secondary to exposure of pregnant rats to constant light along the second half of gestation, had several effects on fetal development. First, it induced intrauterine growth retardation. Second, in the fetal adrenal in vivo it markedly affected the mRNA expression level of clock genes and clock-controlled genes as well as it lowered the content and precluded the rhythm of corticosterone. Third, an altered in vitro fetal adrenal response to ACTH of both, corticosterone production and relative expression of clock genes and steroidogenic genes was observed. All these changes were reversed when the mother received a daily dose of melatonin during the subjective night; supporting a role of melatonin on overall fetal development and pointing to it as a 'time giver' for the fetal adrenal gland. Thus, the present results collectively support that the maternal circadian rhythm of melatonin is a key signal for the generation and/or synchronization of the circadian rhythms in the fetal adrenal gland. In turn, low levels and lack of a circadian rhythm of fetal corticosterone may be responsible of fetal growth restriction; potentially inducing long term effects in the offspring, possibility that warrants further research.

In nonhuman primates and rodents, melatonin acting directly on the adrenal gland, inhibits glucocorticoid response to ACTH. In these species, an intrinsic adrenal circadian clock is involved in ACTH-stimulated glucocorticoid production. We investigated whether these findings apply to the human adrenal gland by determining i) expression of clock genes in vivo and ii) direct effects of melatonin in ACTH-stimulated adrenal explants over a) expression of the clock genes PER1 (Period 1) mRNA and BMAL1 [Brain-Muscle (ARNT)-like] protein, ACTH-induced steroidogenic acute regulatory protein (StAR), and 3β-hydroxysteroid dehydrogenase (3β-HSD) and b) over cortisol and progesterone production. Adrenal tissue was obtained from 6 renal cancer patients undergoing unilateral nephrectomy-adrenalectomy. Expression of the clock genes PER1, PER2, CRY2 (Cryptochrome 2), CLOCK (Circadian Locomotor Output Cycles Kaput) and BMAL1, was investigated by RT-PCR in a normal adrenal and in an adenoma. In independent experiments, explants from 4 normal adrenals were preincubated in culture medium (6 h) followed by 12 h in: medium alone; ACTH (100 nM); ACTH plus melatonin (100 nM); and melatonin alone. The explants' content of PER1 mRNA (real-time PCR) and StAR, 3β-HSD, BMAL1 (immuno slot-blot), and their cortisol and progesterone production (RIA) were measured. The human adrenal gland expresses the clock genes PER1, PER2, CRY2, CLOCK, and BMAL1. ACTH increased PER1 mRNA, BMAL1, StAR, and 3β-HSD protein levels, and cortisol and progesterone production. Melatonin inhibited these ACTH effects. Our study demonstrates, for the first time, direct inhibitory effects of melatonin upon several ACTH responses in the human adrenal gland.

The adrenal gland in the adult is a peripheral circadian clock involved in the coordination of energy intake and expenditure, required for adaptation to the external environment. During fetal life, a peripheral circadian clock is present in the nonhuman primate adrenal gland. Whether this extends to the fetal adrenal gland like the rat is unknown. Here we explored in vivo and in vitro whether the rat fetal adrenal is a peripheral circadian clock entrained by melatonin. We measured the 24-h changes in adrenal content of corticosterone and in the expression of clock genes Per-2 and Bmal-1 and of steroidogenic acute regulatory protein (StAR), Mt1 melatonin receptor, and early growth response protein 1 (Egr-1) expression. In culture, we explored whether oscillatory expression of these genes persisted during 48 h and the effect of a 4-h melatonin pulse on their expression. In vivo, the rat fetal adrenal gland showed circadian expression of Bmal-1 and Per-2 in antiphase (acrophases at 2200 and 1300 h, respectively) as well as of Mt1 and Egr-1. This was accompanied by circadian rhythms of corticosterone content and of StAR expression both peaking at 0600 h. The 24-h oscillatory expression of Bmal-1, Per-2, StAR, Mt1, and Egr-1 persisted during 48 h in culture; however, the antiphase between Per-2 and Bmal-1 was lost. The pulse of melatonin shifted the acrophases of all the genes studied and restored the antiphase between Per-2 and Bmal-1. Thus, in the rat, the fetal adrenal is a strong peripheral clock potentially amenable to regulation by maternal melatonin.

Timely production of glucocorticoid hormones in response to ACTH is essential for survival by coordinating energy intake and expenditure and acting as homeostatic regulators against stress. Adrenal cortisol response to ACTH is clock time dependent, suggesting that an intrinsic circadian oscillator in the adrenal cortex contributes to modulate the response to ACTH. Circadian clock gene expression has been reported in the adrenal cortex of several species. However, there are no reports accounting for potential involvement of adrenal clock proteins on cortisol response to ACTH. Here we explored whether the clock protein cryptochrome 2 (CRY2) knockdown modifies the adrenal response to ACTH in a primate. Adrenal gland explants from adult capuchin monkey (n = 5) were preincubated for 6 h with transfection vehicle (control) or with two different Cry2 antisense and sense probes followed by 48 h incubation in medium alone (no ACTH) or with 100 nm ACTH. Under control and sense conditions, ACTH increased cortisol production, whereas CRY2 suppression inhibited ACTH-stimulated cortisol production. Expression of the steroidogenic enzymes steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase at 48 h of incubation was increased by ACTH in control explants and suppressed by Cry2 knockdown. Additionally, we found that Cry2 knockdown decreased the expression of the clock gene brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (Bmal1) at the mRNA and protein levels. Altogether these results strongly support that the clock protein CRY2 is involved in the mechanism by which ACTH increases the expression of steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase. Thus, adequate expression levels of components of the adrenal circadian clock are required for an appropriate cortisol response to ACTH.

Although the fetal pineal gland does not secrete melatonin, the fetus is exposed to melatonin of maternal origin. In the non-human primate fetus, melatonin acts as a trophic hormone for the adrenal gland, stimulating growth while restraining cortisol production. This latter physiological activity led us to hypothesize that melatonin may influence some fetal functions critical for neonatal adaptation to extrauterine life. To test this hypothesis we explored (i) the presence of G-protein-coupled melatonin binding sites and (ii) the direct modulatory effects of melatonin on noradrenaline (norepinephrine)-induced middle cerebral artery (MCA) contraction, brown adipose tissue (BAT) lypolysis and ACTH-induced adrenal cortisol production in fetal sheep. We found that melatonin directly inhibits the response to noradrenaline in the MCA and BAT, and also inhibits the response to ACTH in the adrenal gland. Melatonin inhibition was reversed by the melatonin antagonist luzindole only in the fetal adrenal. MCA, BAT and adrenal tissue displayed specific high-affinity melatonin binding sites coupled to G-protein (K(d) values: MCA 64 +/- 1 pm, BAT 98.44 +/- 2.12 pm and adrenal 4.123 +/- 3.22 pm). Melatonin binding was displaced by luzindole only in the adrenal gland, supporting the idea that action in the MCA and BAT is mediated by different melatonin receptors. These direct inhibitory responses to melatonin support a role for melatonin in fetal physiology, which we propose prevents major contraction of cerebral vessels, restrains cortisol release and restricts BAT lypolysis during fetal life.

We previously demonstrated that melatonin is involved in the regulation of adrenal glucocorticoid production in diurnal primates through activation of MT1 membrane-bound melatonin receptors. However, whether melatonin has a similar role in nocturnal rodents remains unclear. Using an integrative approach, here we show that the adult rat adrenal gland expresses a functional MT1 melatonin receptor in a rhythmic fashion. We found that: 1) expression of the cognate mRNA encoding for the MT1 membrane-bound melatonin receptor, displaying higher levels in the day/night transition (1800–2200 h); 2) expression of the predicted 37-kDa MT1 polypeptide in immunoblots from adrenals collected at 2200 h but not 1000 h; 3) no expression of the MT2 melatonin receptor mRNA and protein; 4) specific high-affinity 2-[125I]iodomelatonin binding in membrane fractions and frozen sections from adrenals collected at 2200 h but not 0800 h (dissociation constant = 14.22 ± 1.23 pm; maximal binding capacity = 0.88 ± 0.02 fmol/mg protein); and 5) in vitro clock time-dependent inhibition of ACTH-stimulated corticosterone production by 1–100 nm melatonin, which was reversed by 1 μm luzindole (a melatonin membrane receptor antagonist). Our findings indicate not only expression but also high amplitude diurnal variation of functional MT1 melatonin receptors in the rat adrenal gland. It is conceivable that plasma melatonin may play a role to fine-tune corticosterone production in nocturnal rodents, probably contributing to the down slope of the corticosterone rhythm.

We tested the hypothesis that the capuchin monkey adrenal (Cebus apella) gland has oscillatory properties that are independent of adrenocorticotropic hormone (ACTH) by exploring under ACTH suppression by dexamethasone: (i) maintenance of a circadian rhythm of plasma cortisol and (ii) clock time dependency of plasma cortisol response to exogenous ACTH. The capuchin monkey had a clear ACTH and plasma cortisol rhythm. Dexamethasone treatment resulted in low non-rhythmic ACTH levels and decreased cortisol to 1/10 of control values; nevertheless, the circadian rhythm of plasma cortisol persisted. We found that cortisol response to exogenous ACTH was clock time-dependent. The maximal response to ACTH occurred at the acrophase of the cortisol rhythm (0800 h). These results suggest that the capuchin monkey adrenal cortex may possess intrinsic oscillatory properties that participate in the circadian rhythm of adrenal cortisol secretion and in the circadian cortisol response to ACTH.

In the adult mammal the circadian system, which allows predictive adaptation to daily environmental changes, comprises peripheral oscillators in most tissues, commanded by the suprachiasmatic nucleus (SCN) of the hypothalamus. The external environment of the fetus is provided by its mother. In primates, maternal melatonin is a candidate to entrain fetal circadian rhythms, including the SCN rhythms of metabolic activity. We found in the 90% of gestation capuchin monkey fetus expression of the clock genes Bmal-1, Per-2, Cry-2, and Clock in the SCN, adrenal, pituitary, brown fat, and pineal. Bmal-1, Per-2, and the melatonin 1 receptor (MT1) showed a robust oscillatory expression in SCN and adrenal gland, whereas a circadian rhythm of dehydroepiandrosterone sulphate was found in plasma. Maternal melatonin suppression changed the expression of Bmal-1, Per-2, and MT1 in the fetal SCN. These effects were reversed by maternal melatonin replacement. In contrast, neither maternal melatonin suppression nor its replacement had effects on the expression of Per-2 and Bmal-1 or MT1 in the fetal adrenal gland or the circadian rhythm of fetal plasma dehydroepiandrosterone sulphate. Our data suggest that maternal melatonin is a Zeitgeber for the fetal SCN but probably not for the adrenal gland.

The primate fetal adrenal reaches a large size relative to body weight followed by a rapid decrease in size in the postnatal period. We tested the hypothesis that maternal melatonin stimulates growth and prevents maturation of the primate fetal adrenal gland. We suppressed maternal melatonin by exposing eight pregnant capuchin monkeys to constant light (LL) from 63% to 90% gestation (term 155 days). Three of these received daily oral melatonin replacement (LL + Mel). Five mothers remaining in light:dark cycle were used as controls. Fetuses were delivered at 90% gestation. The absence of maternal melatonin selectively decreased fetal adrenal weight (Control: 488.8 +/- 51.5; LL: 363.2 +/- 27.7 and LL + Mel 519 +/- 46 mg; P < 0.05 ANOVA) without effecting fetal weight, placental weight or the weight of other fetal tissues. Changes in fetal adrenal size were accompanied by an increase in the levels of Delta5-3beta-hydroxysteroid dehydrogenase (3beta-HSD) mRNA (Control: 0.8 +/- 0.2; LL: 5.2 +/- 0.6 and LL + Mel 0.8 +/- 0.1; 3beta-HSD/18S-rRNA; P < 0.05 ANOVA). In vitro we found that maternal melatonin suppression increased basal progesterone production to levels similar to those of the adult adrenal gland (Control: 0.36 +/- 0.09; LL 0.99 +/- 0.13; LL + Mel 0.18 +/- 0.06 and adult: 0.88 +/- 0.10 ng/mg of tissue; P < 0.05 ANOVA) but no change in cortisol production. We found an increased production of cortisone (Control: 1.65 +/- 0.60; LL: 5.44 +/- 0.63; LL + Mel: 2.90 +/- 0.38 and adult: 1.70 +/- 0.45 ng/mg of tissue; P < 0.05 ANOVA). Collectively, the effects of maternal melatonin suppression and their reversion by maternal melatonin replacement suggest that maternal melatonin stimulates growth and prevents maturation of the capuchin monkey fetal adrenal gland.

The circadian time-keeping system ensures predictive adaptation of individuals to the reproducible 24-h day/night alternations of our planet by generating the 24-h (circadian) rhythms found in hormone release and cardiovascular, biophysical and behavioral functions, and others. In mammals, the master clock resides in the suprachiasmatic nucleus (SCN) of the hypothalamus. The molecular events determining the functional oscillation of the SCN neurons with a period of 24-h involve recurrent expression of several clock proteins that interact in complex transcription/translation feedback loops. In mammals, a glutamatergic monosynaptic pathway originating from the retina regulaltes the clock gene expression pattern in the SCN neurons, synchronizing them to the light:dark cycle. The emerging concept is that neural/humoral output signals from the SCN impinge upon peripheral clocks located in other areas of the brain, heart, lung, gastrointestinal tract, liver, kidney, fibroblasts, and most of the cell phenotypes, resulting in overt circadian rhythms in integrated physiological functions. Here we review the impact of day/night alternation on integrated physiology; the molecular mechanisms and input/output signaling pathways involved in SCN circadian function; the current concept of peripheral clocks; and the potential role of melatonin as a circadian neuroendocrine transducer.

The circadian time-keeping system is the neural system that allows predictive adaptation of individuals to the reproducible 24-hour day/night alternations of our planet. A biological clock, the suprachiasmatic nucleus, receives environmental information and imposes a circadian pattern to physiological functions. Since the suprachiasmatic nucleus develops early in gestation and circadian rhythms are present in the fetus and newborn, the circadian system seems to be functional in fetal life and can receive circadian inputs through the mother. The neonate moves to an environment in which the main time giving signal is the light:dark cycle. Teleologically, a term newborn should be fit to face this challenge. But this may be quite different for a preterm infant that trades the circadian environment to which it was previously exposed for the timeless environment of the Neonatal Intensive Care Nursery. Scientists and physicians should seek new experimental and clinical approaches to answer the challenging questions of perinatal chronomedicine.