Genetic variability in plasma melatonin in sheep is due to pineal weight, not to variations in enzyme activities

Steven L. Coon1, Luis A. Zarazaga2, Benoît Malpaux2, Jean-Paul Ravault2, Loys Bodin3, Pierre Voisin4, Joan L. Weller1, David C. Klein1, and Philippe Chemineau2

1 Section on Neuroendocrinology, Laboratory of Developmental Biology, National Institutes of Health, Bethesda, Maryland 20892; 2 Neuroendocrinologie Sexuelle, Physiologie de la Reproduction, Institut National de la Recherche Agronomique, Centre National de la Recherche Scientifique Unité de Recherche Associée 1291, 37380 Nouzilly; 3 Station d'Amélioration Génétique des Animaux, Institut National de la Recherche Agronomique, 31326 Castanet Tolosan; and 4 Faculté Sciences, Neurobiologie et Neuroendocrinologie Cellulaire, Centre National de la Recherche Scientifique UMR 1869, 86002 Poitiers, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study was conducted to determine the origin of the high variability in the mean nocturnal plasma melatonin concentration (MC) in sheep. Two extreme groups of 25 lambs each [low (L) and high (H)] were obtained by calculating their genetic value on the basis of the MC of their parents. The MC of lambs was significantly higher in the H group than in the L group (L: 189.7 ± 24.4 vs. H: 344.1 ± 33.0 pg/ml, P < 0.001). Within each group, 13 lambs were slaughtered during the day (D) and 12 lambs during the night (N). Pineal weight was significantly higher in the H group than in the L group (L: 83.5 ± 6.7 vs. H: 119.1 ± 9.2 mg, P < 0.01) but did not differ between D and N. The amount of melatonin released in vitro per milligram of pineal gland, the arylalkylamine N-acetyltransferase (AANAT) activity, the AANAT protein content, and the level of AANAT mRNA differed significantly between D and N but not with genetic group. Hydroxyindole O-methyltransferase activity did not differ significantly between D and N or between genetic groups. Therefore, the genetic difference in MC between the two groups of lambs was attributed to a difference in pineal size, not in enzymatic activity of the pinealocytes.

pineal gland; arylalkylamine N-acetyltransferase; hydroxyindole O-methyltransferase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MELATONIN IS THE PINEAL PRODUCT that transduces the neural photoperiodic information received by the retina into a hormonal message, which is read by a large variety of tissues in the whole organism (1). In most animals, melatonin is secreted only during the nocturnal phase of the nycthemeral rhythm, and the duration of nocturnal melatonin secretion is considered the mechanism by which photoperiodic changes are perceived for the control of reproduction (2). On the one hand, a large variability in the dates of onset and dates of offset of the annual breeding season between ewes has been described (12), but the physiological reasons for this variability are unknown. On the other hand, a large variability in the nocturnal plasma melatonin concentrations between individuals has been described in various mammalian species, including humans (1) and sheep (1, 3). This between-individual variability in melatonin secretion may partly explain the differences in dates of the breeding season. In sheep, some ewes had a mean nocturnal plasma melatonin concentration ~50 pg/ml, whereas others had levels exceeding 500 pg/ml; estimation of the heritability coefficient of this trait showed that this variability is under a strong genetic control (17). Similar observations were made in humans regarding melatonin in the urine (15). In sheep, such variability originates from melatonin production, predominantly occurring in the pineal gland but not from hepatic melatonin catabolism (18).

Melatonin is synthesized in the pineal gland from serotonin by the sequential action of two enzymes, arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) and hydroxyindole O-methyltransferase (HIOMT; EC 2.1.1.4) (8). Daily rhythms in melatonin production are controlled by rhythms of AANAT activity, but the level of production may be limited by HIOMT activity.

Thus the genetic differences in plasma melatonin concentration between animals may be due to a difference in either pineal size or the ability of the pineal to synthesize and/or secrete melatonin. In the present study we used two extreme groups of lambs genetically selected on the basis of their parent's plasma melatonin levels to investigate whether this genetic variability was due to 1) the size of the pineal gland; 2) a difference in the secretory activity of the pineal gland; or 3) a difference in the activity, quantity, or mRNA level of AANAT and HIOMT.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals, Blood Sampling and Slaughtering

This experiment was carried out at the Institut National de la Recherche Agronomique, Research Center of Nouzilly, France (45°N). The whole Ile-de-France flock from which the experimental lambs were obtained is a large flock of ~2,500 animals that is divided into six different families. At regular intervals, sires are purchased from various private external flocks and are introduced to prevent inbreeding and maintain genetic connections with the national French scheme of genetic improvement of the Ile-de-France breed.

The genetic value, defined as the sum of the average effects of the genes an individual carries (6), of ~400 lambs born at the same time of the year (mid-March, dawn at 0730, dusk at 1930) was calculated on the basis of the endogenous nocturnal plasma melatonin concentration of their parents, which had been determined at the June and December solstices (17, 18). Two extreme groups of 25 male lambs each were chosen on the basis of differences in their genetic value [low (L) group and high (H) group].

Blood was collected 24 h before slaughter (16th of July, dawn at 0600, dusk at 2145), twice during the day (1100 and 1200), and four times during the night (2300, 2400, 0100, and 0200). Blood samples were obtained by venipuncture of jugular veins and were collected under dim-red light (<1 lux at 20 cm) to avoid any direct illumination of the eyes. Plasma was immediately separated by centrifugation and stored at -20°C until assayed for melatonin. Lambs were slaughtered at 4 mo of age, on the 17th of July, by a licensed butcher and in accordance with the authorization for animal experimentation no. A37801 of the French Ministry of Agriculture. Lambs were weighed 24 h before slaughter, and carcass weight was measured 24 h after slaughter. Within each group, 13 lambs were slaughtered during the day (D, mean slaughtering time 1029, range 0930-1135) and 12 lambs during the night under dim-red light (N, mean slaughtering time 0057, range 2400-0215). The pineal gland was removed and weighed immediately. Approximately a quarter of each pineal gland was weighed and used for in vitro melatonin secretion (expressed per milligram of fresh pineal gland) for 30 h, whereas two other parts of the gland were immediately deep-frozen for enzymatic activity measurements (see Determinations of enzymatic activities, protein, and mRNA contents).

In Vitro Melatonin Release

To compare the two genetic groups of lambs in the ability of the pineal glands, per se, to synthetize and release melatonin in the absence of any adrenergic stimulation, we used portions of glands that were maintained in vitro during 30 h and for which we measured melatonin release. The method used was adapted from that described in Maronde et al. (9). For each lamb, immediately after slaughter and removal of the pineal gland, about a quarter of the gland was dissected and weighed. On average, portions of glands used for determination of in vitro release weighed 21.6 ± 1.5 (SE) mg. Each quarter was then placed immediately in culture wells with 1 ml of DMEM and F-12 media at 37°C under 5% CO2. The culture medium was collected at regular intervals after the onset of the culture up to 30 h and replaced immediately by fresh medium. The samples were frozen immediately until assayed for melatonin (see Radioimmunoassay of Melatonin).

Determinations of Enzymatic Activities, Protein, and mRNA Contents

AANAT. A second portion of each pineal gland was used to determine AANAT activity, AANAT protein content, and AANAT mRNA levels. The tissues were homogenized in 10 volumes of 100 mM sodium phosphate buffer, pH 6.8, containing 2 mM dithiothreitol (DTT), protease inhibitors [0.5 mM 4-(2-aminoethyl)benzenesulfonylfluoride; 1 µM leupeptin; 40 µg/ml bestatin], and 200 units/ml RNAsin and 200 units/ml RNAsin Ribonuclease inhibitor. A 100-µl sample of each homogenate was immediately added to 1 ml of Trizol (GIBCO-BRL) for extraction of mRNA (see below). The remainder of the homogenates were centrifuged at 14,000 g for 20 min, and aliquots of the supernatant were removed for determination of N-acetyltransferase activity and AANAT protein.

N-Acetyltransferase activity was assayed, as previously described (10), by incubating an aliquot of supernatant with (final concentrations are given) [acetyl-3H]acetylcoenzyme A (0.5 mM; 4 µCi/µmol) and tryptamine (1 mM) in 100 mM phosphate buffer, pH 6.8, for 25 min at 37°C. N-acetyltryptamine was extracted with chloroform, dried, and counted by liquid scintillation.

The level of AANAT protein was measured by SDS-PAGE. The proteins were separated on a 13.5% acrylamide gel (30.8% T acrylamide, 2.6% C bis-acrylamide) run for 16 h at 60 V. The proteins were transferred onto 0.45-µm Immobilon-P membranes with a graphite semidry blotter. The membranes were dried and then blocked with 10% milk in PBS containing 0.2% Tween-20 (TPBS) and 0.5% thimerosal for 2 h and with 10% fish gelatin in TPBS and 0.5% thimerosal for 2 h. The AANAT protein was detected using Anti-2345XVI (1:350; 16 h). The blots were then incubated with goat anti-rabbit peroxidase-labeled secondary antibody (0.0083 µg/ml; 1 h), which was imaged using the LumiGLO Substrate kit (Kirkegarrd and Perry Labs) according to the company's protocol. The film images were analyzed using Adobe 3.0.5 software and quantitated using National Institutes of Health Image VI.57 software. The antiserum used for detecting and quantitating AANAT, Anti-2345, was raised in a rabbit against peptide AANAT-1 (ovine sequence PGRQRRHTLPANEFRC; Hazelton Labs, Vienna, VA). This 16-mer corresponds to amino acid residues 24-39 of ovine AANAT. Anti-2345 was immunopurified using membrane-bound sheep GST-AANAT fusion protein and was designated Anti-2345XVI. This antibody detects recombinant ovine AANAT and a 25-kDa band in ovine pineal glands that comigrates with the recombinant AANAT. Detection of the recombinant and native bands is completely blocked by preincubation of Anti-2345XVI with peptide AANAT-1. This same band in ovine tissue is also detected with other antibodies directed against distinct regions of ovine AANAT. This antibody has previously been shown to detect a putative AANAT band, which varies with AANAT activity in ovine pineal homogenates on a 24-h cycle and during experimental manipulations (8).

Northern blot analysis was conducted using total RNA extracted by the guanidine HCl/phenol procedure (5). For each lane, 20 µg of total RNA were separated on a 1.5% agarose-0.7 M formaldehyde gel, transferred to a charged nylon membrane by passive capillary transfer, and cross-linked to the membrane with ultraviolet. The hybridization probe was 32P-labeled by random priming of a full-length ovine AANAT cDNA clone (clone 87A). Blots were hybridized at 68°C for 1.5 h in QuikHyb buffer (Stratagene, La Jolla, CA). The final wash was in 0.1× standard sodium citrate-0.1% SDS at 60°C for 15 min. After being stripped, the blots were hybridized with a glyceraldehyde-3-phosphate dehydrogenase probe to normalize for RNA loading. Blots were quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

HIOMT. A third portion of each pineal gland was used to determine HIOMT activity, which was assayed as previously described (11) by incubating an aliquot of supernatant with (final concentrations are given) S-[methyl-3H]adenosyl-L-methionine (0.1 mM; 25 µCi/µmol) and N-acetylserotonin (0.1 mM) in 50 mM phosphate buffer, pH 7.9, for 30 min at 37°C. The [3H]melatonin formed was chloroform-extracted directly from the assay mixture, as previously described (11). This method is valid for HIOMT measurements in pineal homogenates: because of the high level of HIOMT activity in this tissue, [3H]melatonin accounts for >90% of the chloroform-extractable radioactivity, as can be verified in blanks without acceptor substrate.

Radioimmunoassay of Melatonin

Melatonin concentrations were estimated in duplicate aliquots of 100 µl of blood plasma or culture medium from tissue culture by radioimmunoassay by use of the technique of Fraser et al. (7) with an antibody raised by Tillet et al. (13). The sensitivity of the assay was 4 pg/ml of plasma or culture medium. The inter- and intra-assay coefficients of variation, estimated from plasma pools of every 50 unknown samples, were 2.2 and 13.6%, respectively.

Statistical Analysis

Plasma samples that were below the limit of detection of the radioimmunoassay were arbitrarily assigned the limit of detection (4 pg/ml of plasma) for the statistical analysis. Statistical analyses were performed using SUPERANOVA (Abacus Concepts, Berkeley, CA), with an effect of the genetic group (L vs. H) and an effect of the conditions of slaughtering (D vs. N); alternatively, daytime values of plasma melatonin concentrations were analyzed by a Mann-Whitney nonparametric test (Statview; Abacus Concepts, Berkeley, CA) because some values fall below the limit of detection of the assay. Unless otherwise stated, data are presented as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

As expected, mean plasma melatonin concentrations 24 h before slaughter were significantly higher in the H group than in the L group, both during the day and during the night (L: 5.8 ± 0.7 vs. H: 12.7 ± 1.8 pg/ml and L: 189.7 ± 24.4 vs. H: 344.1 ± 33.0 pg/ml, respectively; P < 0.001). No significant effects of genetic group and day or night slaughtering were detected on live weight or on carcass weight of lambs (37.2 ± 0.9 and 19.3 ± 0.4 kg, respectively). Mean pineal weight was significantly higher in the H group than in the L group (L: 83.5 ± 6.7 vs. H: 119.1 ± 9.2 mg; P < 0.01) but did not differ with day and night (Fig. 1). A highly significant correlation was detected between mean nocturnal plasma melatonin concentrations 24 h before slaughter and pineal weight ( y = 2.8535x + 4.1877; r2 = 0.628; P < 0.01).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1.   Pineal weight of genetically extreme lambs [low (L) and high (H) producers of melatonin] slaughtered during the day or during the night (D vs. N). Data are presented as means ± SE (in each genetic group: n = 13 for D, 12 for N). Statistical effects: slaughtering condition (D vs. N), not significant (NS); genetic group (L vs. H), P < 0.01.

Although in vitro melatonin release was higher during the night than during the day (D: 1,405 ± 81 vs. N: 2,106 ± 84 pg/mg of gland; P < 0.0004), there was no difference in the amount of melatonin released per milligram of pineal gland between the two genetic groups (Fig. 2). In vitro melatonin release was affected by time (release decreased with time, P < 0.0001) and by the interaction between time and day/night (P < 0.01). The largest decrease in melatonin release was observed during the first 4 h of secretion, whereas melatonin release was stabilized or slightly increased from 6 to 10 h and then decreased progressively from 10 to 30 h after the onset of the culture. A significant interaction between time and slaughtering condition was detected, with a more pronounced decrease for the pineal fragments sampled from lambs slaughtered at night. There was no interaction between genetic group and time, which means that the temporal dynamics of melatonin release were identical in the pineal fragments collected from the two extreme groups of lambs.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   In vitro melatonin production by pineal fragments (pg/mg of gland) from genetically extreme lambs (L and H producers of melatonin) slaughtered during the day or during the night (D vs. N). Data are presented as means ± SE (in each genetic group: n = 13 for D, 12 for N). Statistical effects: slaughtering condition (D vs. N), P < 0.0004; genetic group (L vs. H), NS; time, P < 0.0001; interactions: slaughtering condition × time, P < 0.01; genetic group × time, NS.

For all three AANAT parameters measured, there was no difference between the L and H groups, although a significant difference was found between day and night for all these parameters (Fig. 3; activity, D: 125.6 ± 7.1 vs. N: 303.4 ± 18.9 nmol · h-1 · mg protein-1, P < 0.0001; protein content, D: 36.0 ± 0.3 vs. N: 250.7 ± 9.9 optical density units, P < 0.0001; mRNA, D: 1,264 ± 55 vs. N: 1,481 ± 48 optical density units, P < 0.01). There was also no interaction detected between the genetic group and time of slaughter for any parameter. Figure 4 shows examples of data from each of the four groups of lambs.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 3.   Arylalkylamine N-acetyltransferase (AANAT) enzymatic activity, AANAT protein content, and AANAT mRNA level in pineal extracts from genetically extreme lambs (L and H producers of melatonin) slaughtered during the day or during the night (D vs. N). Data are presented as means ± SE (n = 8). Statistical effects: slaughtering condition (D vs. N), P < 0.0001 for enzymatic activity and protein content and P < 0.01 for mRNA level; genetic group (L vs. H), NS; interaction, NS. O.D., optical density.



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 4.   Representative examples of AANAT enzymatic activity, AANAT protein content, and AANAT mRNA level in pineal extracts from 8 genetically extreme lambs (L and H producers of melatonin) slaughtered during the day or during the night (D vs. N).

HIOMT activity did not differ significantly between day and night or between genetic groups, and there was no interaction between factors (8.89 ± 0.43 nmol · h-1 · mg protein-1).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The differences in plasma melatonin concentration between the two genetically extreme groups of lambs were dramatic, both during daytime and during nighttime. For both sampling periods, plasma melatonin concentration of lambs from the high group was about twice as high as that of the lambs from the low group. Because the lambs for this study were chosen on the basis of the nocturnal plasma melatonin concentration of their parents, this result confirms the genetic control of the variability of this trait and shows that these two sets of lambs were suitable for investigating the mechanisms of this heritability.

Among the parameters studied, the only significant difference between the two genetic groups was pineal weight. This indicates that the differences in plasma melatonin levels are due to an increased production of melatonin as a result of a larger pineal gland rather than an enhanced pineal weight-specific capability for melatonin synthesis or secretion. In vitro melatonin secretion per milligram of pineal gland did not differ between the two genetic groups but was significantly higher after slaughtering at night than after slaughtering during the day. Even if the time of slaughtering influenced the pattern of secretion (demonstrated by the existence of an interaction between time of culture and day/night), in vitro melatonin secretion was higher at most time points after slaughtering at night. This indicates clearly that the mechanisms involved in this in vitro secretion were identical between genetic groups but that they were dependent on the state (active/inactive) of the pineal glands at time of collection.

AANAT activity was significantly higher when lambs were slaughtered during the night than when lambs were slaughtered during the day. This result confirms previous results obtained in adult sheep (5) and rats (8). Such a difference in AANAT activity between day and night explains the nocturnal increase in melatonin plasma concentration observed 24 h before slaughter in the lambs of the present study. This difference in AANAT activity reflects the day vs. night levels of AANAT protein in the pineal glands, which in turn are partly controlled by AANAT gene expression, as evidenced by a small but significant nocturnal rise in AANAT mRNA. This indicates that both pre- and posttranslational mechanisms are operating to regulate the daily rhythm of AANAT activity, which is consistent with published results regarding adult sheep (5). In contrast, however, no significant effect of the genetic group was detected on AANAT activity, AANAT protein content, or AANAT mRNA. Similarly, the last biochemical parameter studied here, HIOMT activity, did not differ between the two extreme groups of lambs. This evidence indicates that genetic control of the melatonin synthetic enzymes is not a likely explanation for the observed differences in plasma melatonin concentration.

The most obvious explanation is that these two groups differed widely in their pineal weight. Mean pineal weight from lambs of the H group was 43% higher than mean pineal weight from lambs of the L group. Thus these two sets of lambs, at 4 mo of age, simply differed by their pineal size, and not by the enzymatic characteristics of their pineal gland. This between-individual difference in pineal weight has been previously observed, both in sheep (12) and in humans (1, 14); however, to our knowledge, the relationship with the plasma melatonin concentration has not been measured, and we demonstrated here that a strong correlation exists between plasma melatonin concentration and pineal weight. We have shown that pineal weight is under a strong genetic influence. The variations in pineal weight are probably proportional to the total quantity of secreting tissue, because 1) the sheep pineal gland is composed primarily of pinealocytes (>80%; Ref. 1); and 2) the plasma melatonin concentration of the two groups of lambs used in this study differed in proportion to the size of their pineal gland. It remains to be determined, however, whether the larger pineal glands of the H group are due to an increase in the number, or an increase in size, of pinealocytes relative to the L group. It is probable that a difference in pineal size already exists at birth, because a difference in plasma melatonin concentration has previously been observed in similar extreme groups of young lambs (A. Gómez-Brunet, B. Malpaux, and P. Chemineau, unpublished results). This suggests that the alleles of the gene(s) affecting pineal size probably act in utero; such genes remain to be identified.

The question of the existence of a relationship between the genetic variability in pineal weight and/or melatonin secretion and reproductive performances, such as dates of onset and offset of the annual breeding season, fertility, and litter size, should also be addressed. In a limited number of animals, it was suggested that the relative melatonin ratio of day to night could be related to the date of onset of the ovulatory activity (4, 16), but this relationship needs to be confirmed on a larger set of ewes. Regarding any relationship with fertility or litter size, unfortunately all ewes in the flock from which the animals used in the present experiment came are hormonally synchronized, a process that always induces a high and nonvariable fertility rate. To our knowledge, no other result about these questions has been published in the literature.


    ACKNOWLEDGEMENTS

The authors thank Agnès Daveau and Françoise Maurice-Mandon for advice in the technical procedures, Didier Chesneau and Mathieu Ouvray for technical help, Jean Voisin and Christian Moussu for slaughtering the lambs, and the breeders of the Institut National de la Recherche Agronomique Research Center of Nouzilly for the supply and care of the experimental animals.


    FOOTNOTES

L. A. Zarazaga was supported by a postdoctoral grant from Diputación General de Aragón and Institut National de la Recherche Agronomique-Direction Scientifique des Productions Animales.

Present address for L. A. Zarazaga: Universidad de Huelva, Dpto. de Ciencias Agroforestales, Torre de Aldebarán no. 5, E.P.S. La Rábida, Ctra. Palos de la Frontera s/n, 21819 La Rábida, Huelva, Spain.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for correspondence and reprint requests: P. Chemineau, INRA-PRMD, 37380 Nouzilly, France (E-mail: Philippe.Chemineau{at}tours.inra.fr).

Received 15 March 1999; accepted in final form 10 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Arendt, J. Melatonin and the Mammalian Pineal Gland, edited by J. Arendt. London: Chapman & Hall, 1995, p. 331.

2.   Bittman, E. L., A. H. Kaynard, D. H. Olster, J. E. Robinson, S. M. Yellon, and F. J. Karsch. Pineal melatonin mediates photoperiodic control of pulsatile luteinizing hormone secretion in the ewe. Neuroendocrinology 40: 409-418, 1985[Medline].

3.   Chemineau, P., I. Beltrán de Heredia, A. Daveau, and L. Bodin. High repeatability of the amplitude and duration of the nycthemeral rhythm of the plasma melatonin concentrations in the Ile-de-France ewes. J. Pineal Res. 21: 1-6, 1996[Medline].

4.   Chemineau, P., F. Maurice, and A. Daveau. Re-initiation of ovulatory activity by melatonin given as a constant-release implant in long-day treated Ile-de-France ewes, depends on endogenous secretion of melatonin. In: Melatonin and the Pineal Gland---From Basic Science to Clinical Application, edited by Y. Touitou, J. Arendt, and P. Pévet. Paris: Elsevier, 1993, p. 247-250.

5.   Coon, S. L., P. H. Roseboom, R. Baler, J. L. Weller, M. A. A. Namboodiri, E. V. Koonin, and D. C. Klein. Pineal serotonin N-acetyltransferase: expression cloning and molecular analysis. Science 70: 1681-1683, 1995.

6.   Falconer, D. S. Introduction to Quantitative Genetics (3rd ed.). Harlow, UK: Longman, 1989, p. 438.

7.   Fraser, S., P. Cowen, M. Franklin, C. Franey, and J. Arendt. Direct radio-immunoassay for melatonin in plasma. Clin. Chem. 20: 396-397, 1983[Free Full Text].

8.   Klein, D. C., S. L. Coon, P. H. Roseboom, J. L. Weller, M. Bernard, J. A. Gastel, M. Zatz, P. M. Iuvone, I. R. Rodriguez, V. Bégay, J. Falcón, G. M. Cahill, V. M. Cassone, and R. Baler. The melatonin rhythm-generating enzyme: molecular regulation of serotonin N-acetyltransferase in the pineal gland. Rec. Progr. Hormone Res. 52: 307-358, 1997[Medline].

9.   Maronde, E., R. Middendorff, B. Mayer, and J. Olcese. The effect of NO-Donors in bovine and rat pineal cells: stimulation of cGMP and cGMP independent inhibition of melatonin synthesis. J. Neuroendocrinol. 7: 207-214, 1995[Medline].

10.   Parfitt, A., J. L. Weller, and D. C. Klein. Beta adrenergic-blockers decrease adrenergically stimulated N-acetyltransferase activity in pineal glands in organ culture. Neuropharmacology 15: 353-358, 1976[Medline].

11.   Sugden, D., V. Ceña, and D. Klein. Hydroxyindole-O-methyltransferase. Methods Enzymol. 142: 590-596, 1986.

12.   Thimonier, J., and P. Mauléon. Variations saisonnières du comportement d'oestrus et des activités ovarienne et hypophysaire chez les ovins. Ann. Biol. Anim. Biochim. Biophys. 9: 233-250, 1969.

13.   Tillet, Y., J. P. Ravault, C. Selve, G. Evin, B. Castro, and M. P. Dubois. Conditions d'utilisation d'anticorps spécifiques pour la visualisatin immunohistochimique de la sérotonine et de la mélatonine dans la glande pinéale deu mouton. Compte-Rendus Acad. Sci., Paris 303: 77-82, 1986.

14.   Wetterberg, L. Melatonin in humans: physiological and clinical studies. J. Neural Transm. Suppl. 13: 290-291, 1978.

15.   Wetterberg, L., L. Iselius, and J. Lindsten. Genetic regulation of melatonin excretion in urine. Clin. Genet. 24: 399-402, 1983[Medline].

16.   Zarazaga, L. A., F. Forcada, J. A. Abecia, and J. M. Lozano. Date of reinitiation of the breeding season could be related with relative changes in plasma melatonin amplitude in ewes. In: Pineal Update---From Molecular Mechanisms to Clinical Implications, edited by S. M. Webb, M. Puig-Domingo, M. Möller, and P. Pévet. New York: PJD Publications, 1996, p. 295-300.

17.   Zarazaga, L. A., B. Malpaux, L. Bodin, and P. Chemineau. The large variability in melatonin blood levels in ewes is under strong genetic influence. Am. J. Physiol. 274 (Endocrinol. Metab. 37): E607-E610, 1998[Abstract/Free Full Text].

18.   Zarazaga, L. A., B. Malpaux, D. Guillaume, L. Bodin, and P. Chemineau. Genetic variability in melatonin concentrations in ewes originates in its synthesis, not in its catabolism. Am. J. Physiol. 274 (Endocrinol. Metab. 37): E1086-E1090, 1998[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 277(5):E792-E797
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Coon, S. L.
Articles by Chemineau, P.
Articles citing this Article
PubMed
PubMed Citation
Articles by Coon, S. L.
Articles by Chemineau, P.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online