María Luisa Forcelledo

Maternal melatonin selectively inhibits cortisol production in the primate fetal adrenal gland

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.

Perinatal neuroendocrine regulation. Development of the circadian time-keeping system

During gestation, the perinatal neuroendocrine axis keeps clock time. In primates, the suprachiasmatic nucleus (biological clock in mammals), shows oscillatory function by midgestation. There is evidence in rodents that the mother, during pregnancy, entrains the fetal suprachiasmatic nucleus (SCN) and newborn circadian rhythms. We are investigating the role of maternal melatonin as an entraining signal for the newborn circadian time-keeping system in the Cebus apella (New World non-human primate). Twenty-four hour rhythms of temperature and cortisol are present in the 4 days old C. apella newborn. Preliminary data suggests that inhibition of maternal melatonin by exposing pregnant females to constant light alters these rhythms. We have found binding sites for melatonin and expression of mRNA for Mel 1A receptor in hypothalamus, kidney and testis. These preliminary results suggest that maternal melatonin may play a role in relating the perinatal circadian time-keeping system to environmental signals.

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.

Additive effect of RU 486 and anordrin on pregnancy interruption in the mouse

The effect of various doses of anordrin and RU 486, alone or combined, on serum progesterone (P) levels, fetal resorption, and recovery of ovulation was studied in mice. Each drug was given as a single sc injection on day 7 of pregnancy and autopsy was performed on days 8, 9, or 11. Serum P was normal at 24 h but fell significantly 48 h after treatment with anordrin (0.05 mg). Doses of 0.05 or 0.2 mg anordrin were effective in interrupting pregnancy in 30% and 70% of pregnant mice, respectively. RU 486, 0.01 mg per mouse, induced a pronounced decrease of P levels 24 h after treatment and interrupted pregnancy in 50% of pregnant mice. The combined treatment with submaximal doses of anordrin plus RU 486 did not further decrease P levels, but increased the proportion of mice with fetal resorptions to 90%. The combination of small doses of anordrin with RU 486 had an additive effect on pregnancy termination. The additive effect required a dose of RU 486 above the threshold level. Direct observation of aborted fetuses indicated that the resorptive process occurred earlier with RU 486 than with anordrin. Recovery of ovulation was associated with pregnancy termination in a high proportion of mice treated with either drug or their combination.

Subcellular hypothalamic fractions active on ovulation and luteinizing hormone release in the rat.

Nuclear, microsomal and cytosol fractions were obtained from hypothalamic cells by differential centrifugation in sucrose solutions of different molarities. The nuclear fraction (NF) from immature female rats had an inhibitory effect on ovulation induced with pregnant mares serum (PMS) in immature rats and with luteinizing hormone (LH) in chlorpromazine (CPZ)-treated proestrous rats. The microsomal fraction from the same rats increased both types of ovulation. Nuclear and microsomal fractions obtained from immature male rats were inactive on ovulation. Cytosol fractions were inactive. NFs active to inhibit ovulation significantly reduced the release of LH induced with synthetic LH-RH in immature male rats and in chronically castrated male rats primed with testosterone (T).