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Endocrinology Vol. 147, No. 10 4618-4626
Copyright © 2006 by The Endocrine Society

Maternal Melatonin Effects on Clock Gene Expression in a Nonhuman Primate Fetus

C. Torres-Farfan, V. Rocco, C. Monsó, F. J. Valenzuela, C. Campino, A. Germain, F. Torrealba, G. J. Valenzuela and M. Seron-Ferre

Departamento de Ciencias Fisiológicas (C.T.F., V.R., C.M., F.J.V., F.T., M.S.-F.), Facultad de Ciencias Biológicas, Departamento de Endocrinología (C.C.), Facultad de Medicina, Pontificia Universidad Católica de Chile, Casilla 114D, Santiago, Chile; Unidad de Medicina Materno-Fetal (A.G.), Centro Especializado de Vigilancia Materno Fetal, Departamento de Obstetricia y Ginecología, Clinica Las Condes, Santiago, Chile; and Department of Women’s Health Arrowhead Regional Medical Center (G.J.V.), Colton, California 92324

Address all correspondence and requests for reprints to: María Serón-Ferré, Departamento de Ciencias Fisiológicas, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Casilla (P.O. Box) 114D, Santiago, Chile. E-mail: mseron{at}puc.cl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE ADULT, most body functions follow a rhythmic pattern adjusted to 24 h (circadian rhythms), which is controlled by the circadian timing system (1, 2). In mammals, this system comprises peripheral oscillators located in most tissues of the body and a central rhythm generator located in the suprachiasmatic nucleus (SCN) of the hypothalamus. At the cell level, circadian rhythms are driven by the self-regulatory interaction of a set of genes (Bmal-1, Per1–2, Cry1–2, and Clock; named clock genes) and their protein products. The heterodimer of the proteins CLOCK:BMAL-1 binds E-box elements (CACGTG/T) at the promoter region of Per1–2 and Cry1–2, inducing their transcription (3). Conversely, PER1–2 and CRY1–2 proteins, by interacting with the CLOCK:BMAL-1 heterodimer operate as negative regulators inhibiting their own transcription. In adult animals, oscillatory expression of clock genes has been demonstrated in the SCN and in several peripheral tissues. The circadian oscillation of clock genes expression controls the expression of genes involved in multiple cellular functions in the 24-h, and results in the overt circadian rhythms in the individual (reviewed in Refs. 1 , 4 , and 5).

The external environment of the fetus is provided by its mother. The fetal SCN shows rhythms of metabolic activity early in gestation in squirrel monkeys (6) and of c-FOS in sheep (7, 8) and of metabolic and electric activity in rodents (9, 10, 11). In addition, fetuses of precocious species like the human, rhesus, and sheep present circadian rhythms: respiratory movements, limb movements, heart rate, and production of cortisol in the human fetus (12, 13), DHAS in rhesus (14), prolactin in sheep (15, 16), suggesting a circadian organization that uses maternal signals to adapt to the maternal environment.

Development of the circadian system has been studied mostly in laboratory rodents, whose newborns show an immature nervous system at birth. In rats, mice, and hamsters, the ontogeny of overt circadian rhythms is postnatal and clock genes are present in the SCN but do not oscillate at birth (17). There are no data on the clock gene expression in the SCN or peripheral tissues of fetuses of primates or other precocious species. Similarly, the maternal signal/s (Zeitgeber) conveying temporal information to the fetus are unknown. A good candidate signal is melatonin, one of the few maternal hormones crossing the placenta without being altered. As a result, the fetus, whose pineal does not synthesize melatonin, is exposed to the maternal melatonin rhythm (18).

We hypothesize that the SCN and adrenal gland of the capuchin monkey fetus display oscillatory circadian clock gene expression and that maternal melatonin is a Zeitgeber for the fetal SCN and the adrenal gland. To test this hypothesis, we first investigated clock gene expression in the fetal SCN and the fetal adrenal gland under normal conditions. Secondly, we investigated the effect of maternal melatonin suppression and replacement on clock gene expression in the fetal SCN and the adrenal gland.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Animal handling and care was performed following the recommendations of the National Institutes of Health Guide for Animal Experimentation Care. The Commission on Bioethics and Biosafety of the Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, approved the study protocols.

Capuchin monkeys (Cebus apella) were obtained from the Animal Care Facility of the Pontificia Universidad Católica de Chile. In the colony, animals are kept in individual cages in a room with controlled temperature and humidity, water available ad libitum and food administered twice a day. The light:dark (LD) cycle in the facility was 14 h light, 10 h dark (lights on at 0700 h). Meals were given at 1200 and 1800 h.

Collection of maternal and fetal blood samples and fetal tissues
Blood samples and fetal tissues used in this report were obtained from 90% of gestation (142.2 ± 2.1; term 159.6 ± 1.7 d) fetal capuchin monkeys whose mothers were maintained in LD (14:10) and from mothers chronically exposed to constant light (LL) from 60–90% gestation as described previously (19, 20). These pregnant females were maintained with lights continuously on (2000 lux at the head level) from about 100 d gestation up to hysterotomy at about 90% of gestation. Six females received a teaspoon of fruit juice daily at 1600 h as placebo (LL group) for 40.8 ± 1.3 d and the other three (LL+ Mel group) received 250 µg/kg body weight of melatonin (Maver Ltd. Laboratory, Santiago, Chile) in fruit juice daily at 1600 h for 44.3 ± 1.2 d.

At about 90% of gestation, females were anesthetized with a mixture of 1% halothane/oxygen, and a maternal blood sample was drawn from the saphenous vein. The fetuses were delivered by hysterotomy performed under sterile conditions, a blood sample was taken from the umbilical artery and the fetus was euthanized immediately with an overdose of sodium thiopental (100 mg/kg of weight) given in the umbilical vein. Fetal weight and biparietal diameter were recorded and the fetal adrenal glands, SCN and other organs were dissected and weighed immediately after necropsy. The SCN, when feasible, and pituitary, pineal, thyroid and brown fat were dissected. To dissect out a hypothalamic block that included the SCN, we relied on our previous experience with blocking the SCN in newborn capuchin monkey (13), our work on fetuses of other mammals (22, 23) as well as on the atlas of the adult C. apella by Manocha et al. (24). We made a transverse cut just rostral to the optic chiasm and another transverse cut about 1 mm posterior to the optic chiasm. A horizontal cut 1 mm below the lateral ventricles as seen from the front and two parasagittal cuts lateral to the border of the chiasm completed the blocking of the SCN. The SCN is indicated in level 15.5 of Manocha’s atlas. The blocks varied in size depending on age, and they were, roughly, 4 mm in the antero-posterior plane, 5 mm wide, and 4 mm in the dorso-ventral plane. Tissues were stored in TRIzol (Life Technologies Inc., Rockville, MD) and kept at –20 C for further measurement of mRNA levels. Other fetal tissues were incorporated to the Colony Tissue Bank. Blood samples were centrifuged and plasma was stored at –20 C until assayed.

After suturing the surgical wound, the females were given an oral analgesic (Tramal, 1 drop/kg; Grunenthal, Santiago, Chile), and im antiinflamatory (Ketofen 1%, 0.1 ml/kg; Merial, Lyon, France) and antibiotics (Baytril 5%, 0.1 ml/kg, Bayer S.A., Brasil; and Benacillin 1.5 ml, Troy Laboratories PTY Ltd., New South Wales, Australia). Upon recovery from anesthesia, the females were returned their cages. Tramal was given daily for the next 3 d, Ketofen and Baytril were given daily for 5 d, and a second dose of Benacillin was given 48 h after surgery. The wound was cleaned daily for 5 d using sterile saline and Larvispray (Pfizer, Animal Health Division, Santiago, Chile).

Hysterotomies were performed at 0800 h in five LD fetuses and three LL fetuses and at 2000 h in three LD fetuses. Fetal adrenal tissue (n = 5) and SCN blocks (n = 7) and maternal and fetal blood samples obtained at 1400 h (n = 5) were from LD fetuses and from three LL and three LL + Mel fetuses euthanized for other studies (19, 20). The time of surgery was selected on the basis of reported patterns of clock gene oscillation in adult animals. The characteristics of the fetuses are shown in Table 1Go.


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  		TABLE 1. Fetal variables in LD, LL, and LL + Mel treatments at different clock times (mean ± SE)

 
Plasma cortisol and DHAS assays
Plasma cortisol concentration was measured by RIA using a highly specific antiserum (25); generously donated by Dr. Doris Haack (Department of Pharmacology, Heidelberg University, Germany) and [1,2,6,7-3H] cortisol (78 Ci/mmol; NEN Life Science Products, Boston, MA) as tracer. The range of the standard curve was 12.5–800 pg/500 µl. Fetal and maternal plasma samples were diluted 1:2000 or 1:5000, respectively, in buffer 0.1% gelatin PBS (pH 7.0) to fit into the standard curve. The diluted samples were incubated for 10 min at 95 C to destroy binding proteins before assayed. The inter- and intraassay coefficients were less than 10% (26).

Plasma DHAS was measured using a Kit from Diagnostic System Laboratories, Inc. (Webster, TX), following the manufacturer’s instructions.

Semiquantitative analyses by RT-PCR
The primers for Bmal-1, Per-2, Cry-2, and Clock were designed from human, rat, mouse, and sheep cDNA sequences available at GenBank using OLIGO 4.1 (Primer Analysis Software, Plymouth, MN) and BLASTN 2.2.1 tools (Ref. 27 and www.ncbi.nlm.nih.gov), and were synthesized by Invitrogen Corp. (Carlsbad, CA). The sequence of the primers, size of the expected PCR products and location between exons according to the clock genes structure reported for human, are given in Table 2Go. 18S-rRNA was amplified as housekeeping gene using the primers described by Einspanier et al. (28). SuperScript II RNaseH– reverse transcriptase and Taq DNA polymerase were purchased from Invitrogen Corp. Random hexamers, 10 mM deoxynucleotide triphosphates, and 500-bp DNA ladder were purchased from Promega Corp. (Madison, WI). cDNAs were synthesized from 3 µg of total RNA using random hexamers. The lineal range of cycles for the each gene was established using cDNA pools of adrenal and SCN. Two microliters of cDNA were mixed with a PCR solution [20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM of each deoxynucleotide triphosphates, 0.2 µM of each primer and 1.25 U Taq DNA polymerase] in a total volume of 25 µl and amplified in an Eppedorff thermocycler (AG model 22331; Hamburg, Germany). The RT-PCR products were purified by chromatography (DNA Wizard PCR Preps; Promega Corp.) and sequenced in the Ecology Department of our faculty. The homology degree of each RT-PCR product with the corresponding human clock gene sequence was determined using the BLASTN 2.2.1 tool (27). Percentage identity was 93% for Per-2, 97% for Bmal-1, 98% for Cry-2 and 97% for Clock. The GenBank accession numbers of the partial cDNA sequences obtained for capuchin monkey Bmal-1, Per-2, Cry-2, and Clock and the PCR conditions are shown in Table 2Go. Expression of MT1 receptor and of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) was determined as previously described (29, 19). PCR products were separated by electrophoresis on a 2% agarose-ethidium bromide gel, the gel image was captured with a digital camera (Olympus Camedia Master 4.1, Tokyo, Japan) using DocIt Software (UVP, Inc., Upland, CA) and the density of the band was measured using the software Scion Image (Scion Corp., Frederick, MD) and corrected by the density of 300 bp standard (12.5 ng). All adrenal and SCN samples were analyzed in triplicate and in several dilutions (150–0.5 ng of RNA/tube) and in at least two assays. Mean coefficient of variation was 20.0 ± 1.3%.


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  		TABLE 2. Summary of sequence of the PCR primers, exon location (according to the clock genes structure reported for human), size of the expected PCR product, annealing temperature and number of cycles used to amplify partial cDNAs for Bmal-1, Per-2, Clock, Cry-2, and 18S-rRNA in capuchin monkey adrenal and SCN.

 
Data analysis
Data are expressed as mean ± SE. The mean ratio gene mRNA/18S-rRNA per ng of RNA were transformed to arcsin (30) and the means of the ratios were compared by ANOVA followed by the post hoc Tukey test. Fetal and maternal plasma cortisol and DHAS concentration were analyzed by ANOVA followed by the post hoc Tukey test. Statistical analyses were performed using GraphPad Prism software (version 3.02; GraphPad Software Inc., San Diego, CA). Results were considered significant when P values were < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Clock gene expression in fetal tissues
We studied the expression of Bmal-1, Per-2, Cry-2, and Clock in fetal SCN, adrenal, pituitary, thyroid, brown fat, and pineal from three LD fetuses euthanized at 0800, 1400, and 2000 h (Fig. 1Go). These tissues were chosen because their functional status in the fetus is known. The fetal SCN shows rhythms of activity in squirrel monkey, sheep, and rat (6, 7, 8, 9, 10, 11); the primate fetal adrenal is an active steroid secreting organ (19, 31), the pituitary and the thyroid secrete their respective hormones during fetal life (32, 33), and brown adipose tissue accumulates in the fetus to serve as a thermogenic substrate right after birth (34). The fetal pineal was chosen because it is well established that melatonin synthesis is absent during fetal life (18). As shown in Fig. 1Go, expression of Bmal-1, Per-2, Cry-2, and Clock, was detected in fetal SCN, adrenal, pituitary, thyroid, and brown fat. In the pineal, Bmal-1, Cry-2, and Clock were detected but not Per-2 and the expression of Cry-2 was higher in the fetal pineal than in the other tissues. Overall, the data show that clock genes are expressed with variable abundance in different fetal tissues, requiring the use of different amounts of RNA for detection. At comparable levels of expression of 18S-rRNA (Fig. 1Go), Bmal-1 and Per-2 were more abundant in the fetal SCN and the fetal adrenal than in the other tissues tested. In addition, levels of expression may be related to the clock time at which the tissue was obtained. Bmal-1 expression tends to be higher at 2000 h in most tissues, whereas Per-2 and Cry-2 may be higher at 0800 and 1400 h. Clock expression was similar at the three time intervals studied in pituitary, thyroid, and brown fat.


Figure 1
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  		FIG. 1. Clock gene expression (Bmal-1, Per-2, Cry-2, and Clock) in capuchin monkey SCN, adrenal, pituitary, thyroid, brown fat, and pineal in LD fetuses. Fetuses were euthanized at 0800, 1400, and 2000 h. The bands shown in the gel were obtained using different amounts of RNA for gene in each tissue. Bmal-1 was detected in 30 ng of RNA in the fetal SCN, in 15 ng in the fetal adrenal, and in 300 ng in the other tissues. Per-2 was detected in 30 ng of RNA in the SCN, in 150 ng of RNA in the fetal adrenal, and in 300 ng of RNA in the other tissues. Cry-2 and Clock were measured in 150 ng of RNA in all tissues. 18S-rRNA was detected using 7.5 ng of RNA in all tissues.

 
Oscillatory expression of Bmal-1 and Per-2 in the fetal SCN and fetal adrenal gland
The data shown in Fig. 1Go indicated an abundant expression of Bmal-1 and Per-2 in the fetal SCN and adrenal gland that seem to vary with clock time. This was examined by measuring the expression of these genes by semiquantitative RT-PCR. As shown in Fig. 2Go, a time of day difference was detected in the SCN and adrenal gland for Bmal-1 and Per-2. In both tissues, the highest expression of Bmal-1 was detected at 2000 h and the lower values at 0800 h and 1400 h (P < 0.001 and P = 0.003, ANOVA and Tukey’s multiple comparison test, fetal SCN and fetal adrenal, respectively). The data suggest that the acrophase of Bmal-1 occurs at about 2300 h in both the fetal SCN and the fetal adrenal gland.


Figure 2
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  		FIG. 2. Oscillatory expression of Bmal-1 and Per-2 in the capuchin monkey SCN and adrenal gland in LD fetuses. Fetal SCN (left upper panels): mean ± SE of Bmal-1 and Per-2 expression measured by RT-PCR at 0800 h (n = 4), 1400 h (n = 7) and 2000 h (n = 3). *, Different from 0800 h and 1400 h, P = 0.001, ANOVA and Tukey’s test; {dagger}, different from 1400 h and 2000 h, P < 0.05, ANOVA and Tukey’s test. In each graph, the value at 0800 h is repeated in the next 24 h. Fetal adrenal gland (right upper panels): mean ± SE of Bmal-1 and Per-2 expression measured by RT-PCR at 0800 h (n = 5), 1400 h (n = 5), and 2000 h (n = 3). *, Different from 0800 h and 1400 h, P < 0.05, ANOVA and Tukey’s test; {dagger}, different from 1400 h and 2000 h, P < 0.001, ANOVA, and Tukey’s test. In each graph the value at 0800 h is repeated in the next 24 h. Lower panels, Representative gels of three fetal SCN and three fetal adrenal glands at each clock time. Bmal-1 and Per-2 measurements were performed in 7.5 and 30 ng of RNA, respectively. The dark bars indicate lights-off hours.

 
Expression of Per-2 was higher in the SCN than in the fetal adrenal gland, but in both tissues this gene showed an almost inverse pattern to that of Bmal-1, with a maximum at 0800 and lower values at 1400 and at 2000 h (P = 0.003 and P < 0.001, ANOVA and Tukey’s multiple comparison test, fetal SCN and fetal adrenal, respectively). The data suggest that the acrophase of Per-2 occurs at about 0700 h in both, the fetal SCN and the fetal adrenal. Thus, the highest values of Bmal-1 and Per-2 in the capuchin monkey fetal SCN and fetal adrenal were observed at beginning and at the end of the night to which the mother is exposed, respectively.

Indicators of oscillatory function in the fetal SCN and in the fetal adrenal gland
From previous data, we knew that both tissues expressed the MT1 melatonin receptor and that the fetal adrenal gland expressed the enzyme 3ß-HSD (19) involved in cortisol synthesis (31, 35) and that it secretes DHAS and cortisol (19). DHAS found in the fetal circulation is of fetal origin as maternal DHAS does not cross the placenta (31), whereas some of the cortisol present in the fetus is transferred from the mother through the placenta (36). To assess effects of the treatments on adrenal function, we measured clock time changes in the concentration of these steroids in the umbilical artery.

As shown in Fig. 3AGo, expression of the MT1 melatonin receptor in the fetal SCN was higher at 2000 than at 1400 and 0800 h (P < 0.05, ANOVA and Tukey’s test). The three markers of fetal adrenal function, MT1 receptor and 3ß-HSD expression and plasma steroid concentration showed clock time-related changes. MT1 receptor expression (Fig. 3BGo) was higher at 1400 and 2000 h than at 0800 h (P < 0.05, ANOVA and Tukey’s test) and 3ß-HSD expression (Fig. 3CGo) was higher at 2000 than at 0800 and 1400 h (P = 0.041, ANOVA and Tukey’s test). Umbilical artery DHAS concentration (Fig. 3DGo) showed a significant increase at 1400 and 2000 h (P < 0.001, ANOVA and Tukey’s test). At the three clock times, maternal DHAS concentration was about 50 times lower than the fetal and showed a maximum at 0800 h (data not shown). In contrast to the pattern of DHAS concentration, mean concentration of umbilical artery cortisol was higher at 0800 than at 1400 and 2000 h, following a pattern similar to that of maternal plasma cortisol, suggestive of maternal passage of cortisol to the fetus (Table 3Go).


Figure 3
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  		FIG. 3. Oscillatory expression of the MT1 melatonin receptor in SCN and adrenal gland, 3ß-HSD in fetal adrenal and umbilical artery plasma DHAS concentration in LD fetuses. A, Mean ± SE of MT1 expression in the fetal SCN at 0800 h (n = 3), 1400 h (n = 4) and 2000 h (n = 3). B. Mean ± SE of MT1 expression in the fetal adrenal at 0800 h (n = 3), 1400 h (n = 4) and 2000 h (n = 3). C, Mean ± SE of 3ß-HSD expression in the fetal adrenal gland at 0800 h (n = 3), 1400 h (n = 4) and 2000 h (n = 3). D, Mean ± SE umbilical artery plasma DHAS concentration measured at 0800-h (n = 5), 1400-h (n = 5) and 2000-h (n = 3). *, Different from 0800 h, P < 0.05, ANOVA and Tukey’s test.

 

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  		TABLE 3. Fetal and maternal plasma cortisol concentration (mean ± SE; µg/ml) at different clock times in control, LL, and LL + Mel treatments

 
Effects of maternal melatonin suppression on the fetal SCN and fetal adrenal gland
To assess whether maternal melatonin may be a Zeitgeber to the fetal SCN and the fetal adrenal gland, mothers were chronically maintained in constant light from 60–90% of gestation. This treatment suppresses maternal melatonin in the rhesus and capuchin monkey, without affecting the maternal rhythm of cortisol (37, 19) and temperature (Seron-Ferre, M., unpublished data). We measured Bmal-1, Per-2, and MT1 expression in the fetal SCN; Bmal-1, Per-2, MT1, and 3ß-HSD expression in the fetal adrenal gland and also umbilical artery DHAS concentration at 0800 and 1400 h.

In the fetal SCN, the absence of maternal melatonin changed the expression of Bmal-1, Per-2, and MT1 receptor. As shown in Fig. 4Go, the expression of Bmal-1 was low at 0800 and increased at 1400 h in contrast to the two low values observed in the SCN of LD fetuses. Conversely, two low values were observed for Per-2 expression at this time interval, instead of the peak at 0800 and the low value at 1400 h observed in the SCN of LD fetuses. Finally, the increase in the MT1 receptor observed at 1400 h in LD fetuses was abolished by chronic maternal melatonin suppression. The effect of chronic maternal melatonin suppression on Bmal-1 and MT1 expression in the fetal SCN at 1400 h was reversed in fetuses whose mothers were exposed to constant light but received a daily oral dose of melatonin.


Figure 4
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  		FIG. 4. Effect of maternal melatonin suppression and replacement in the fetal SCN. Expression of Bmal-1, Per-2, and MT1 melatonin receptor (RT-PCR) at 0800 h and 1400 h in the SCN of fetuses of mothers kept in 14:10 LD (n = 4 at 0800 h and 5 at 1400 h), of mothers kept in LL (n = 3 at 0800 h and 1400 h) and of mothers kept in chronic constant light receiving a daily oral dose of melatonin (LL+ mel, n = 3 at 1400). *, Different from LD at the same clock time; {dagger}, different from 0800 h (P < 0.05, ANOVA and Tukey’s test).

 
In contrast to the effects on the fetal SCN, maternal melatonin suppression did not affect Bmal-1, Per-2, or MT1 receptor expression in the fetal adrenal (Fig. 5Go) or the increase in fetal plasma DHAS concentration at 0800 and 1400 h (data not shown). As expected, maternal melatonin replacement did not affect either clock gene expression and MT1 receptor or DHAS concentration at 1400 h.


Figure 5
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  		FIG. 5. Effect of maternal melatonin suppression and replacement in the fetal adrenal. Expression of Bmal-1, Per-2, and MT1 melatonin receptor at 0800 h and 1400 h in the adrenal of fetuses of mothers kept in LD 14:10 (n = 5 at 0800 h and at 1400 h), of mothers kept in chronic constant light (LL, n = 3 at 0800 h and 1400 h) and of mothers kept in chronic constant light receiving a daily oral dose of melatonin (LL+ mel, n = 3 at 1400). {dagger}, Different from 0800 h (P < 0.05, ANOVA and Tukey’s test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data show that oscillatory circadian clock gene expression can be readily detected in the SCN and in the adrenal of the capuchin monkey fetus and that, in the fetal adrenal clock gene oscillation is time related to circadian steroid production. We found that maternal melatonin suppression shifted Bmal-1 and Per-2 expression in the fetal SCN but not in the fetal adrenal gland. The effect of maternal melatonin suppression on the SCN was reversed by maternal melatonin replacement. The lack of effect of chronic maternal melatonin suppression on the fetal adrenal gland was accompanied by maintenance of the pattern of DHAS secretion. Altogether, our data are consistent with maternal melatonin being a circadian signal for the fetal SCN but not for the fetal adrenal gland.

Clock genes are expressed in most tissues in the adult (5, 38). Similarly, the four clock genes tested: Per-2, Cry-2, Bmal-1, and Clock were detected at 90% gestation in the SCN, the adrenal and pituitary, thyroid, and brown adipose tissue of the capuchin monkey fetus. These are tissues that exert several functions in fetal life either producing hormones (adrenal, pituitary, thyroid; Refs. 19 and 31, 32, 33) or serving as thermogenic substrate (adipose tissue; Ref. 34), whereas the fetal pineal starts secreting melatonin after birth (39). Expression of Bmal-1 and Per-2 was higher in the SCN and adrenal than in the other tissues. The fetal pineal did not express Per-2 but expressed Bmal-1 and Clock and had a significantly higher expression of Cry-2 than the other tissues tested. Whether the absence of Per-2 expression relates to the lack of pineal function needs to be explored. Expression of clock genes at late gestation has been studied in the rat (40, 41), mice (42), and hamster SCN (43) and the rat fetal heart (44). Our findings suggest that fetal tissues express most of the clock genes at late gestation in the capuchin.

The presence of Bmal-1 and Per-2 in the fetal SCN and fetal adrenal gland was accompanied by daytime changes in expression. The expression of Cry-2 and Clock was not measured. In the SCN and adrenal, the peaks of Bmal-1 and Per-2 expression were in antiphase, Bmal-1 peaking at the beginning of the night and Per-2 peaking about 10 h later, at the end of the night. The timing of maximal expression of Bmal-1 at the beginning of the night in the fetal capuchin SCN is similar to that reported in adult SCN of mice, hamster, and sheep (45, 46, 47), whereas the timing of expression of Per-2 is not. In the adult, most reports show a maximum in the expression of Per-2 between middle and the end of the day in mice, rat, hamster, and sheep (45, 46, 47, 48, 49). Thus, the 10-h interval between the increases of Bmal-1 and Per-2 in the capuchin SCN would be shorter than that reported in these adult animals. Whether this reflects a functional difference between fetal and adult SCN or species differences is not known because there are no data on clock gene expression in the SCN of the capuchin or other primates. Oscillatory expression of clock genes has been reported recently in the adult rhesus monkey adrenal gland (50) and the pattern of expression of Bmal-1 and Per-2 is almost identical with what we found in the fetal capuchin monkey adrenal.

The position of the maxima of Bmal-1 and Per-2 expression in the fetal capuchin SCN and fetal adrenal gland at the beginning and end of the night led us to investigate whether the daily nocturnal increase of maternal melatonin may signal nighttime to these fetal tissues. Melatonin receptors are present in the human fetal SCN (51, 52). As shown before (19), and also as confirmed in the present study, the fetal capuchin SCN and adrenal gland express the MT1 melatonin receptors. We found that in both the fetal SCN and the adrenal, expression of the MT1 melatonin receptor showed clock time changes, as reported in adult rat SCN (53). We investigated the effect of chronic maternal melatonin suppression in the pattern of expression of Bmal-1 and Per-2 in the fetal SCN and fetal adrenal gland. Because of restrictions in the number of fetuses available, we studied expression of Bmal-1 and Per-2 in three fetuses at 0800 and three at 1400 h. Effects of the treatment were different in the SCN and adrenal. In the SCN, absence of maternal melatonin suppressed the peak of Per-2 observed at 0800 h in LD fetuses; instead, values at 0800 and 1400 h were both low. For Bmal-1, instead of the low values observed at 0800 and 1400 h in the SCN of LD fetuses, a low value was observed at 0800 h followed by an increase at 1400 h. Due to the limitation in clock time points studied, we cannot distinguish in the present experiments whether the effects observed in the SCN represent free running of Bmal-1 and Per-2 expression, a phase shift in the expression of these genes, or selective effects on Per-2 expression. Effects on clock gene expression were accompanied by a suppression of MT1 melatonin receptor expression at 1400 h. Maternal melatonin replacement restored Bmal-1 and MT1 melatonin receptor expression to the levels found in LD conditions, although as discussed before we cannot distinguish the underlying processes. The issue of maternal melatonin as an entraining signal for the fetal SCN or fetal circadian rhythms is controversial. Suppression of maternal melatonin by exposure of the mother to constant light has been shown to result in free running of the prolactin and vasopressin rhythms in fetal sheep (54, 55). In rodents, maternal pinealectomy has no effects on day/night changes in metabolic activity in the fetal rat SCN (56). On the other hand timed injection of melatonin into SCN-lesioned maternal hamsters restores synchrony in the rhythm of activity of the newborns (57). In the adult, melatonin has been shown to shift the rhythm of electrical activity of rat SCN slices (58) and to act as a chronobiotic in the human (59). The present data showing effects of maternal melatonin suppression on Bmal-1 and MT1 melatonin receptor expression that are restored by maternal melatonin replacement support a role for maternal melatonin as a Zeitgeber for the fetal primate SCN.

Neither maternal melatonin suppression nor maternal melatonin replacement had effect on expression of Bmal-1 and Per-2 or MT1 melatonin receptor in the fetal adrenal gland. MT1 melatonin receptors are active in the fetal adrenal gland as shown by inhibition of ACTH-stimulated cortisol production in vitro (19). A caveat of the present experiments is that chronic melatonin suppression induces changes in fetal adrenal morphology, increasing the size of the zone containing cortisol producing cells, effects that are reversed by maternal melatonin administration (20). Therefore, we cannot ascertain whether these morphological changes masked effects of maternal melatonin suppression on fetal adrenal clock gene expression. To assess whether chronic melatonin suppression affected oscillatory fetal adrenal function, we measured the concentrations of cortisol and DHAS in the umbilical artery. Umbilical artery cortisol concentration was about a third of the maternal concentration. However, the concordance of the time of day changes in umbilical artery cortisol with those observed in the maternal circulation, suggest a maternal contribution to fetal cortisol (36), precluding the dissection of circadian changes in fetal adrenal cortisol production. Production of fetal cortisol is rhythmic in the human term fetus, measured as difference in concentration between the umbilical artery and vein (60), not done in the present experiments. The capuchin fetal adrenal, like the adrenal gland of the human and other nonhuman primates produces DHAS, and it is known that maternal DHAS does not cross the placenta (31). In fact, we found that maternal DHAS concentration was about 20 times lower than fetal plasma concentration. A decrease in DHAS in maternal plasma during pregnancy is also found in women, as a result of the effective placental metabolism of DHAS to estradiol (31). A rhythm in umbilical artery plasma concentration of DHAS was present in LD fetuses, as shown in chronically catheterized rhesus fetuses (14). The temporal pattern of DHAS concentration was not affected by chronic maternal melatonin suppression or by daily melatonin replacement. DHAS is a precursor for placental synthesis of estradiol. In keeping with the lack of effect of chronic maternal melatonin suppression on the fetal DHAS rhythm, the circadian pattern of maternal estradiol is not changed during chronic maternal melatonin suppression in rhesus (37), or chronic maternal melatonin suppression and replacement in the capuchin (19).

Our data show that the absence of maternal melatonin changes Bmal-1 and Per-2 expression in the fetal SCN. However, these changes were not reflected in the circadian fetal adrenal steroidogenic function. The present data bring about this question: How is the fetal circadian system organized? The data suggest that circadian fetal adrenal function may not be under fetal SCN control. Are the fetal SCN and fetal adrenal gland under separated maternal circadian control, the SCN by maternal melatonin and the fetal adrenal by another maternal signal? Chronic maternal melatonin suppression does not abolish the maternal circadian rhythms of cortisol (19, 37) or of body temperature (Seron-Ferre, M., unpublished observations), and the fetus would still be subjected to maternal metabolic signals. It is tempting to speculate that both the fetal SCN and fetal adrenal are peripheral circadian clocks and that the central clock resides in the mother providing circadian information through melatonin to the SCN and through other unknown signal to the fetal adrenal gland. These possibilities need to be investigated, in the light of the emerging interest on the impact of the circadian system development in human neonatal care (21).


   Acknowledgments
 
We are very grateful to Renato Ebensperger, D.V.M. and to the personnel of the nonhuman primate colony for expert animal care, to Auristela Rojas for helping with the steroid assays, to Mauricio Mondaca for editorial support, and to Drs. Anibal Llanos and Hans G. Richter for useful discussions and comments on the manuscript


   Footnotes
 
The present work was supported by Grant 98/LABENDO/Resource Maintenance Grant-2 from the World Health Organization and Fondecyt 1030-425 from Fondo Nacional de Ciencia y Tecnologia de Chile. C.T.-F. postdoctoral fellowship was supported by a grant from San Bernardino Medical Foundation and MECESUP PUC-0211.

Author Disclosure Summary: All of the authors have nothing to declare.

First Published Online July 13, 2006

Abbreviations: DHAS, Dehydroepiandrosterone sulphate; 3ß-HSD, 3ß-hydroxysteroid dehydrogenase; LD, light-dark; LL, constant light; MT1, melatonin 1 receptor; SCN, suprachiasmatic nucleus.

Received May 10, 2006.

Accepted for publication July 6, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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