From gestational chronodisruption to noncommunicable diseases: Pathophysiological mechanisms of programming of adult diseases, and the potential therapeutic role of melatonin.

During gestation, the developing fetus relies on precise maternal circadian signals for optimal growth and preparation for extrauterine life. These signals regulate the daily delivery of oxygen, nutrients, hormones, and other biophysical factors while synchronizing fetal rhythms with the external photoperiod. However, modern lifestyle factors such as light pollution and shift work can induce gestational chronodisruption, leading to the desynchronization of maternal and fetal circadian rhythms. Such disruptions have been associated with adverse effects on cardiovascular, neurodevelopmental, metabolic, and endocrine functions in the fetus, increasing the susceptibility to noncommunicable diseases (NCDs) in adult life. This aligns with the Developmental Origins of Health and Disease theory, suggesting that early-life exposures can significantly influence health outcomes later in life. The consequences of gestational chronodisruption also extend into adulthood. Environmental factors like diet and stress can exacerbate the adverse effects of these disruptions, underscoring the importance of maintaining a healthy circadian rhythm across the lifespan to prevent NCDs and mitigate the impact of gestational chronodisruption on aging. Research efforts are currently aimed at identifying potential interventions to prevent or mitigate the effects of gestational chronodisruption. Melatonin supplementation during pregnancy emerges as a promising intervention, although further investigation is required to fully understand the precise mechanisms involved and to develop effective strategies for promoting health and preventing NCDs in individuals affected by gestational chronodisruption.

Maternal melatonin treatment rescues endocrine, inflammatory, and transcriptional deregulation in the adult rat female offspring from gestational chronodisruption.

Introduction: Gestational chronodisruption impact maternal circadian rhythms, inhibiting the nocturnal increase of melatonin, a critical hormone that contributes to maternal changes adaptation, entrains circadian rhythms, and prepares the fetus for birth and successful health in adulthood. In rats, we know that gestational chronodisruption by maternal chronic photoperiod shifting (CPS) impaired maternal melatonin levels and resulted in long-term metabolic and cardiovascular effects in adult male offspring. Here, we investigated the consequences of CPS on mother and adult female offspring and explored the effects of melatonin maternal supplementation. Also, we tested whether maternal melatonin administration during gestational chronodisruption rescues maternal circadian rhythms, pregnancy outcomes, and transcriptional functions in adult female offspring. Methods: Female rats raised and maintained in photoperiod 12:12 light: dark were mated and separated into three groups: (a) Control photoperiod 12:12 (LD); (b) CPS photoperiod; and (c) CPS+Mel mothers supplemented with melatonin in the drinking water throughout gestation. In the mother, we evaluated maternal circadian rhythms by telemetry and pregnancy outcomes, in the long-term, we study adult female offspring by evaluating endocrine and inflammatory markers and the mRNA expression of functional genes involved in adrenal, cardiac, and renal function. Results: In the mothers, CPS disrupted circadian rhythms of locomotor activity, body temperature, and heart rate and increased gestational length by almost 12-h and birth weight by 12%, all of which were rescued by maternal melatonin administration. In the female offspring, we found blunted day/night differences in circulating levels of melatonin and corticosterone, abnormal patterns of pro-inflammatory cytokines Interleukin-1a (IL1a), Interleukin-6 (IL6), and Interleukin-10 (IL10); and differential expression in 18 out of 24 adrenal, cardiac, and renal mRNAs evaluated. Conclusion: Maternal melatonin contributed to maintaining the maternal circadian rhythms in mothers exposed to CPS, and the re-establishing the expression of 60% of the altered mRNAs to control levels in the female offspring. Although we did not analyze the effects on kidney, adrenal, and heart physiology, our results reinforce the idea that altered maternal circadian rhythms, resulting from exposure to light at night, should be a mechanism involved in the programming of Non-Communicable Diseases.

New integrative approaches to discovery of pathophysiological mechanisms triggered by night shift work.

Synchronization to periodic cues such as food/water availability and light/dark cycles is crucial for living organisms' homeostasis. Both factors have been heavily influenced by human activity, with artificial light at night (ALAN) being an evolutionary challenge imposed over roughly the last century. Evidence from studies in humans and animal models shows that overt circadian misalignment, such as that imposed to about 20% of the workforce by night shift work (NSW), negatively impinges on the internal temporal order of endocrinology, physiology, metabolism, and behavior. Moreover, NSW is often associated to mistimed feeding, with both unnatural behaviors being known to increase the risk of chronic diseases, such as eating disorders, overweight, obesity, cardiovascular, metabolic (particularly type 2 diabetes mellitus) and gastrointestinal disorders, some types of cancer, as well as mental disease including sleep disturbances, cognitive disorders, and depression. Regarding deleterious effects of ALAN on reproduction, increased risk of miscarriage, preterm delivery and low birth weight have been reported in shift-worker women. These mounting lines of evidence prompt further efforts to advance our understanding of the effects of long-term NSW on health. Emerging data suggest that NSW with or without mistimed feeding modify gene expression and functional readouts in different tissues/organs, which seem to translate into persistent cardiometabolic and endocrine dysfunction. However, this research avenue still faces multiple challenges, such as functional characterization of new experimental models more closely resembling human long-term NSW and mistimed feeding in males versus females; studying further target organs; identifying molecular changes by means of deep multi-omics analyses; and exploring biomarkers of NSW with translational medicine potential. Using high-throughput and systems biology is a relatively new approach to study NSW, aimed to generate experiments addressing new biological factors, pathways, and mechanisms, going beyond the boundaries of the circadian clock molecular machinery.

Maternal Chronodisruption Throughout Pregnancy Impairs Glucose Homeostasis and Adipose Tissue Physiology in the Male Rat Offspring.

Compelling evidence in rats support the idea that gestational chronodisruption induces major changes in maternal circadian rhythms and fetal development and that these changes impact adult life at many physiological levels. Using a model of chronic photoperiod shifting throughout gestation (CPS), in which pregnant female rats (Sprague-Dawley strain; n = 16 per group) were exposed to lighting schedule manipulation every 3-4 days reversing the photoperiod completely or light/dark photoperiod (12/12; LD), we explored in the adult rat male offspring body weight gain, glucose homeostasis, adipose tissue content, adipose tissue response to norepinephrine (NE), and adipose tissue proteomic in the basal condition with standard diet (SD) and in response to high-fat diet (HFD). In adult CPS male (100-200 days old; n = 8 per group), we found increasing body weight, under SD and adiposity. Also, we found an increased response to intraperitoneal glucose (IGTT). After 12 weeks of HFD, white adipose tissue depots in CPS offspring were increased further, and higher IGTT and lower intraperitoneal insulin tolerance response were found, despite the lack of changes in food intake. In in vitro experiments, we observed that adipose tissue (WAT and BAT) glycerol response to NE from CPS offspring was decreased, and it was completely abolished by HFD. At the proteomic level, in CPS adipose tissue, 275 proteins displayed differential expression, compared with LD animals fed with a standard diet. Interestingly, CPS offspring and LD fed with HFD showed 20 proteins in common (2 upregulated and 18 downregulated). Based on these common proteins, the IPA analysis found that two functional pathways were significantly altered by CPS: network 1 (AKT/ERK) and network 2 (TNF/IL4; data are available via ProteomeXchange with identifier PXD026315). The present data show that gestational chronodisruption induced deleterious effects in adipose tissue recruitment and function, supporting the idea that adipose tissue function was programmed in utero by gestational chronodisruption, inducing deficient metabolic responses that persist into adulthood.

Long-Term Effects of Altered Photoperiod During Pregnancy on Liver Gene Expression of the Progeny.

Experimental and epidemiological studies have revealed a relationship between an adverse intrauterine environment and chronic non-communicable disease (NCD) like cardiovascular disease (CVD) in adulthood. An important risk factor for CVD is the deregulation of the fibrinolytic system particularly high levels of expression of plasminogen activator inhibitor 1 (Pai-1). Chronic exposure to altered photoperiod disrupts the circadian organization of physiology in the pregnant female, known as gestational chronodisruption, and cause long-term effects on the adult offspring's circadian physiology. The Pai-1 expression is regulated by the molecular components of the circadian system, termed clock genes. The present study aimed to evaluate the long-term effects of chronic photoperiod shifts (CPS) during pregnancy on the expression of the clock genes and the fibrinolytic system in the liver of adult male offspring. Our results using an animal model demonstrated statistically significant differences at the transcriptional level in males gestated under CPS. At 90 days of postnatal age, the liver transcript levels of the clock gene Bmal1 were downregulated, whereas Rorα, Rorγ, Nfil3, and Pai-1 were upregulated. Our data indicate that CPS during pregnancy affects gene expression in the liver of male adult progeny, showing that alteration of the photoperiod in the mother's environment leads to persistent effects in the offspring. In conclusion, these results reveal for the first time the long-term effects of gestational chronodisruption on the transcriptional activity of one well-established risk factor associated with CVD in the adult male offspring.

Fetal Programming of Renal Dysfunction and High Blood Pressure by Chronodisruption.

Adverse prenatal conditions are known to impose significant trade-offs impinging on health and disease balance during adult life. Among several deleterious factors associated with complicated pregnancy, alteration of the gestational photoperiod remains largely unknown. Previously, we reported that prenatal manipulation of the photoperiod has adverse effects on the mother, fetus, and adult offspring; including cardiac hypertrophy. Here, we investigated whether chronic photoperiod shifting (CPS) during gestation may program adult renal function and blood pressure regulation. To this end, pregnant rats were subjected to CPS throughout pregnancy to evaluate the renal effects on the fetus and adult offspring. In the kidney at 18 days of gestation, both clock and clock-controlled gene expression did not display a daily pattern, although there were recurrent weaves of transcriptional activity along the 24 h in the control group. Using DNA microarray, significant differential expression was found for 1,703 transcripts in CPS relative to control fetal kidney (835 up-regulated and 868 down-regulated). Functional genomics assessment revealed alteration of diverse gene networks in the CPS fetal kidney, including regulation of transcription, aldosterone-regulated Na+ reabsorption and connective tissue differentiation. In adult offspring at 90 days of age, circulating proinflammatory cytokines IL-1ß and IL-6 were increased under CPS conditions. In these individuals, CPS did not modify kidney clock gene expression but had effects on different genes with specific functions in the nephron. Next, we evaluated several renal markers and the response of blood pressure to 4%NaCl in the diet for 4 weeks (i.e., at 150 days of age). CPS animals displayed elevated systolic blood pressure in basal conditions that remained elevated in response to 4%NaCl, relative to control conditions. At this age, CPS modified the expression of Nhe3, Ncc, Atp1a1, Nr3c1 (glucocorticoid receptor), and Nr3c2 (mineralocorticoid receptor); while Nkcc, Col3A1, and Opn were modified in the CPS 4%+NaCl group. Furthermore, CPS decreased protein expression of Kallikrein and COX-2, both involved in sodium handling. In conclusion, gestational chronodisruption programs kidney dysfunction at different levels, conceivably underlying the prehypertensive phenotype observed in the adult CPS offspring.

Developmental Programming of Capuchin Monkey Adrenal Dysfunction by Gestational Chronodisruption.

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 3ß-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 3ß-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.

Deciphering the Function of the Blunt Circadian Rhythm of Melatonin in the Newborn Lamb: Impact on Adrenal and Heart.

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.

Gestational Chronodisruption Impairs Circadian Physiology in Rat Male Offspring, Increasing the Risk of Chronic Disease.

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.

Gestation under chronic constant light leads to extensive gene expression changes in the fetal rat liver.

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 (r(2) = 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.

Gestational chronodisruption impairs hippocampal expression of NMDA receptor subunits Grin1b/Grin3a and spatial memory in the adult offspring.

Epidemiological and experimental evidence correlates adverse intrauterine conditions with the onset of disease later in life. For a fetus to achieve a successful transition to extrauterine life, a myriad of temporally integrated humoral/biophysical signals must be accurately provided by the mother. We and others have shown the existence of daily rhythms in the fetus, with peripheral clocks being entrained by maternal cues, such as transplacental melatonin signaling. Among developing tissues, the fetal hippocampus is a key structure for learning and memory processing that may be anticipated as a sensitive target of gestational chronodisruption. Here, we used pregnant rats exposed to constant light treated with or without melatonin as a model of gestational chronodisruption, to investigate effects on the putative fetal hippocampus clock, as well as on adult offspring's rhythms, endocrine and spatial memory outcomes. The hippocampus of fetuses gestated under light:dark photoperiod (12:12 LD) displayed daily oscillatory expression of the clock genes Bmal1 and Per2, clock-controlled genes Mtnr1b, Slc2a4, Nr3c1 and NMDA receptor subunits 1B-3A-3B. In contrast, in the hippocampus of fetuses gestated under constant light (LL), these oscillations were suppressed. In the adult LL offspring (reared in LD during postpartum), we observed complete lack of day/night differences in plasma melatonin and decreased day/night differences in plasma corticosterone. In the adult LL offspring, overall hippocampal day/night difference of gene expression was decreased, which was accompanied by a significant deficit of spatial memory. Notably, maternal melatonin replacement to dams subjected to gestational chronodisruption prevented the effects observed in both, LL fetuses and adult LL offspring. Collectively, the present data point to adverse effects of gestational chronodisruption on long-term cognitive function; raising challenging questions about the consequences of shift work during pregnancy. The present study also supports that developmental plasticity in response to photoperiodic cues may be modulated by maternal melatonin.

Impact of gestational chronodisruption on fetal cardiac genomics.

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 photoperiod (LD). RNA isolated from E18 heart was subjected to microarray analysis (Affymetrix platform for 28,000 genes). Integrated transcriptional changes were assessed by gene ontology and pathway analysis. Significant 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 steroidogenesis 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 relevant 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.

Impact of Maternal Melatonin Suppression on Amount and Functionality of Brown Adipose Tissue (BAT) in the Newborn Sheep.

In human and sheep newborns, brown adipose tissue (BAT) accrued during fetal development is used for newborn thermogenesis. Here, we explored the role of maternal melatonin during gestation on the amount and functionality of BAT in the neonate. We studied BAT from six lambs gestated by ewes exposed to constant light from 63% gestation until delivery to suppress melatonin (LL), six lambs gestated by ewes exposed to LL but receiving daily oral melatonin (12 mg at 1700 h, LL + Mel) and another six control lambs gestated by ewes maintained in 12 h light:12 h dark (LD). Lambs were instrumented at 2 days of age. At 4-6 days of age, they were exposed to 24°C (thermal neutrality conditions) for 1 h, 4°C for 1 h, and 24°C for 1 h. Afterward, lambs were euthanized and BAT was dissected for mRNA measurement, histology, and ex vivo experiments. LL newborns had lower central BAT and skin temperature under thermal neutrality and at 4°C, and higher plasma norepinephrine concentration than LD newborns. In response to 4°C, they had a pronounced decrease in skin temperature and did not increase plasma glycerol. BAT weight in LL newborns was about half of that of LD newborns. Ex vivo, BAT from LL newborns showed increased basal lipolysis and did not respond to NE. In addition, expression of adipogenic/thermogenic genes (UCP1, ADBR3, PPARγ, PPARα, PGC1α, C/EBPß, and perilipin) and of the clock genes Bmal1, Clock, and Per2 was increased. Remarkably, the effects observed in LL newborns were absent in LL + Mel newborns. Thus, our results support that maternal melatonin during gestation is important in determining amount and normal functionality of BAT in the neonate.

Impact of chronodisruption during primate pregnancy on the maternal and newborn temperature rhythms.

Disruption of the maternal environment during pregnancy is a key contributor to offspring diseases that develop in adult life. To explore the impact of chronodisruption during pregnancy in primates, we exposed pregnant capuchin monkeys to constant light (eliminating the maternal melatonin rhythm) from the last third of gestation to term. Maternal temperature and activity circadian rhythms were assessed as well as the newborn temperature rhythm. Additionally we studied the effect of daily maternal melatonin replacement during pregnancy on these rhythms. Ten pregnant capuchin monkeys were exposed to constant light from 60% of gestation to term. Five received a daily oral dose of melatonin (250 µg kg/body weight) at 1800 h (LL+Mel) and the other five a placebo (LL). Six additional pregnant females were maintained in a 14∶10 light:dark cycles and their newborns were used as controls (LD). Rhythms were recorded 96 h before delivery in the mother and at 4-6 days of age in the newborn. Exposure to constant light had no effect on the maternal body temperature rhythm however it delayed the acrophase of the activity rhythm. Neither rhythm was affected by melatonin replacement. In contrast, maternal exposure to constant light affected the newborn body temperature rhythm. This rhythm was entrained in control newborns whereas LL newborns showed a random distribution of the acrophases over 24-h. In addition, mean temperature was decreased (34.0±0.6 vs 36.1±0.2°C, in LL and control, respectively P<0.05). Maternal melatonin replacement during pregnancy re-synchronized the acrophases and restored mean temperature to the values in control newborns. Our findings demonstrate that prenatal melatonin is a Zeitgeber for the newborn temperature rhythm and supports normal body temperature maintenance. Altogether these prenatal melatonin effects highlight the physiological importance of the maternal melatonin rhythm during pregnancy for the newborn primate.

Timed maternal melatonin treatment reverses circadian disruption of the fetal adrenal clock imposed by exposure to constant light.

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.

Melatonina inhibe la respuesta de cortisol a ACTH: posible mediación a través del gen reloj Per1 / Melatonin inhibition of the cortisol response to ACTH may be exerted through period circadian protein homolog 1 (Per1)

Background: Circadian cortisol production results from the interaction of the circadian production of ACTH, the autonomic nervous system and intrinsic factors within the gland. An additional regulator is the neuro-hormone melatonin. In human adrenal gland cultures, melatonin inhibited ACTH stimulated cortisol production and Per1 mRNA expression. ACTH actions on the adrenal involve early and late responses. Aim: To investigate the effects of melatonin on the time course of ACTH stimulated cortisol production and of Per1 expression in the lamb adrenal gland. Material and Methods: Adrenal glands and plasma of five newborn lambs were obtained. Adrenal glands were cut in 15 mg explants. Three of these explants were stored for RNA extraction. The rest of explants were using in different culture protocols with ACTH and melatonin. Results: Lambs had an in vivo a circadian variation in plasma cortisol and in adrenal Per1 expression. In vitro, ACTH stimulated an early and late increase in cortisol production and an early increase in Per1 expression reaching a maximum at 3 hours of treatment. Melatonin inhibited the early Per1 response to ACTH without affecting the early ACTH stimulated cortisol production. However, melatonin inhibited the late response of cortisol production to ACTH. Conclusions: The inhibitory actions of melatonin on Per1 response to ACTH may contribute to the inhibitory effects of melatonin on adrenal steroidogenic response to ACTH.

Circadian rhythms in the fetus.

Throughout gestation, the close relationship between mothers and their progeny ensures adequate development and a successful transition to postnatal life. By living inside the maternal compartment, the fetus is inevitably exposed to rhythms of the maternal internal milieu such as temperature; rhythms originated by maternal food intake and maternal melatonin, one of the few maternal hormones that cross the placenta unaltered. The fetus, immature by adult standards, is however perfectly fit to accomplish the dual functions of living in the uterine environment and developing the necessary tools to "mature" for the next step, i.e. to be a competent newborn. In the fetal physiological context, organ function differs from the same organ's function in the newborn and adult. This may also extend to the developing circadian system. The information reviewed here suggests that the fetal circadian system is organized differently from that of the adult. Moreover, the fetal circadian rhythm is not just present simply as the initial immature expression of a mechanism that has function in the postnatal animal only. We propose that the fetal suprachiasmatic nucleus (SCN) of the hypothalamus and fetal organs are peripheral maternal circadian oscillators, entrained by different maternal signals. Conceptually, the arrangement produces internal temporal order during fetal life, inside the maternal compartment. Following birth, it will allow for postnatal integration of the scattered fetal circadian clocks into an adult-like circadian system commanded by the SCN.

La melatonina reduce la respuesta de cortisol al ACTH en humanos / Melatonin reduces cortisol response to ACTH in humans

Background: Melatonin receptors are widely distributed in human tissues but they have not been reported in human adrenal gland. Aim: To assess if the human adrenal gland expresses melatonin receptors and if melatonin affeets cortisol response to ACTH in dexamethasone suppressed volunteers. Material and methods: Adrenal glands were obtained from 4 patients undergoing unilateral nephrectomy-adrenalectomy for renal cáncer. Expression of mRNA MT1 and MT2 melatonin receptors was measured by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). The effect of melatonin on the response to intravenous (i.v.) ACTH was tested (randomized cross-over, double-blind, placebo-controlled tríal) in eight young healthy males pretreated with dexamethasone (1 mg) at 23:00 h. On the next day at 08:00 h, an i.v. Une was inserted, at 08:30 h, and after a blood sample, subjeets ingested 6 mg melatonin or placebo. At 09:00 h, 1-24 ACTH (Cortrosyn, 1µg/1.73 m² body surface área) was injected, drawing samples at 0, 15, 30, 45 and 60 minutes after. Melatonin, cortisol, cortisone, progesterone, aldosterone, DHEA-S, testosterone and prolactin were measured by immunoassay. Results: The four adrenal glands expressed only MT1 receptor mRNA. Melatonin ingestión reduced the cortisol response to ACTH from 14.6+1.45µg/dl at 60 min in the placebo group to 10.8+1.2µg/dl in the melatonin group (p <0.01 mixed model test). It did not affect other steroid hormone levels and abolished the morningphysiological decline of prolactin. Conclusions: The expression ofMTl melatonin receptor in the human adrenal, and the melatonin reduction of ACTH-stimulated cortisol production suggest a direct melatonin action on the adrenal gland .

Evidence of a role for melatonin in fetal sheep physiology: direct actions of melatonin on fetal cerebral artery, brown adipose tissue and adrenal gland.

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.