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Department of Obstetrics and Gynaecology, Adelaide University, Medical School, Adelaide, South Australia 5005
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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There has been
relatively little research conducted on pineal melatonin production in
laboratory mice, in part, due to the lack of appropriate assays. We
studied the pineal and plasma rhythm, response to light, adrenergic
stimulation, and metabolism of melatonin in CBA mice. With the use of a
sensitive and specific melatonin RIA, melatonin was detected in the
pineal glands at all times of the day >21 fmol/gland in CBA mice but
not in C57Bl mice. Both plasma and pineal melatonin levels peaked
2 h before dawn in a 12:12-h light-dark photoperiod (162 ± 31 pM and 1,804 ± 514 fmol/gland, respectively). A brief light
pulse (200 lx/15 min), 2 h before lights on, suppressed both
plasma and pineal melatonin to near basal levels within 30 min.
Exposure to light pulses 4 h after lights off or 2 h before
lights on resulted in delays and advances, respectively, in the early
morning decline of plasma and pineal melatonin on the next cycle.
Administration of the 
-adrenergic agonist isoproterenol (20 mg/kg) 2 and 4 h after lights on in the morning resulted in a fivefold
increase in plasma and pineal melatonin 2.5 to 3 h after the first
injection. In the mouse, unlike the rat, melatonin was shown to be
metabolized almost exclusively to 6-glucuronylmelatonin rather than
6-sulphatoxymelatonin. These studies have shown that the appropriate
methodological tools are now available for studying melatonin rhythms
in mice.
pineal gland; circadian rhythm; phase shift; 6-glucuronylmelatonin; 6-sulphatoxymelatonin
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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DESPITE THEIR USEFULNESS IN many diverse areas of biomedical research, there has been surprisingly little systematic research conducted on the production of melatonin by the pineal gland in laboratory mice. The most likely reasons for this situation are the small amount of blood that can be obtained from mice, their small pineal glands, and insensitive melatonin assays. Nevertheless, it has always been presumed that mice, like all other mammals, would synthesize melatonin during darkness. This assumption was challenged in 1986 when it was shown that C57Bl mice had <10 pg melatonin per pineal gland at various times during the day and night (8). Subsequently, it was shown that only two of the commonly used mouse strains, C3H and CBA, had high-amplitude melatonin rhythms with peak pineal melatonin content occurring 2 h before lights on (13). Other commonly used mouse strains (BALB/c, DBA/2, 129/Sv, AKR/J, and CF#1) had undetectable melatonin (<20 pg/gland) at 2, 6, and 10 h after darkness onset.
Melatonin is produced in the pineal gland by N-acetylation
of serotonin by N-acetyltransferase (NAT) followed by
O-methylation by
hydroxyindole-O-methyltransferase (HIOMT). NAT is the
rate-limiting enzyme in the synthesis of melatonin and is under
-adrenergic control from the superior cervical ganglion. When the
possible causes of the impaired melatonin production in mice were
examined, it was found that C57Bl, AKR/J, and BALB/c mice had neither
NAT nor HIOMT activity in the pineal gland, whereas another strain (NZB/BLNJ) had NAT but no HIOMT activity (7). The lack of
NAT activity in the pineal gland was later confirmed in BALB/c mice (34). In the case of C57Bl/6J mice, it was found that a
point mutation in the NAT gene results in a truncated protein that has little or no enzyme activity (30), whereas different
mutations are present in BALB/c and 129/Sv strains (30).
To date, there have been no reports on the nature of the HIOMT mutations.
The aim of the present study was to evaluate pineal melatonin synthesis, secretion, metabolism, and the response to light in CBA, C57Bl, and BALB/c mice using a variety of approaches. Of particular interest was the possibility of establishing a method to monitor melatonin metabolite excretion rhythms in a similar way to that conducted in laboratory rats (16).
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Adult male CBA, C57Bl, CBAxC57Bl (F1), and BALB/c mice were obtained from the University of Adelaide Central Animal house, and wild derived mice (32) were obtained from the Animal Resources Centre (Canning Vale, Western Australia) where they had been maintained since birth on a 12:12-h light-dark photoperiod. On arrival in our facility, they continued to be on the 12:12-h light-dark photoperiod (lights off 1900, on 0700) and were fed standard laboratory mouse food ad libitum.
Pineal and plasma melatonin rhythm studies. Male CBA and C57Bl mice were killed by decapitation at 2-h intervals in dim red light, starting at 1900 and ending at 0700. Trunk blood was collected into heparinized tubes, and plasma was harvested by centrifugation and stored frozen before assay by RIA. Skull caps with attached pineal glands were cut away and frozen in 1 ml phosphate buffer containing 0.5% BSA (PBS/BSA). Before assay, pineals were removed together with the attached dura, homogenized in the buffer, and assayed for melatonin by RIA.
Isoproterenol stimulation. Groups of five CBA mice were injected with isoproterenol (20 mg/kg sc; Sigma, St. Louis, MO) or saline, 2 h after lights on and again 2 h later and killed at various times after the last injection. Pineal glands and blood were collected as above. In a separate experiment, groups of 10 CBA and 10 C57Bl male mice were injected with isoproterenol (20 mg/kg) or saline (CBA only) at 0900 and 1100, killed at 1130, and pineals and blood were collected as above.
Effect of light on pineal and plasma melatonin. Male CBA mice entrained to a 12:12-h light-dark photoperiod were killed at 0500, 0600, and 0700 (n = 14, 10, and 10, respectively). An additional group of five animals was exposed to 200-lx light for 15 min commencing at 0500 and killed at 0530. Pineal glands and blood were collected as previously described.
In two separate experiments, the effects of a 200 lx/15 min light pulse on the timing of the morning decline in pineal melatonin content and plasma melatonin concentration were determined. Male CBA mice entrained to a 12:12-h light-dark photoperiod were exposed to light pulses at either 2300 or 0500 or were left in darkness. The lights remained off for the remainder of the experiment, and groups of five animals were killed the next morning at 0500, 0600, and 0700. Pineal glands and blood were collected for melatonin analysis as above.Urine collections. Male CBA and C57Bl mice (n = 5) were acclimated in metabolism cages for several days and fed a liquid diet (Osmolite HN, Abbot Laboratories) as previously reported for rats (16). Urine was collected at 2-h intervals from 1900 to 0900 for two nights and for one night of continuous light. The mice appeared to tolerate the liquid diet, and urine volumes in excess of 200 µl/2 h were regularly obtained overnight, with the urine flow decreasing slightly before the expected time of lights on.
To investigate the nature of the metabolism and possible metabolites of melatonin in the mouse, five CBA and five C57Bl mice were housed in metabolism cages, placed on a liquid diet, and urine was collected at 2-h intervals. At 1600 (3 h before expected darkness), mice were injected subcutaneously with 100 µg melatonin, and the lights were kept illuminated. Samples (20 µl) were assayed for 6-sulphatoxymelatonin and melatonin by RIA.Assays. Melatonin was assayed in plasma from individual mice (50-100 µl) by RIA using reagents obtained from Buhlmann Laboratories (Allschwil, Switzerland). This assay uses the G280 antibody developed in our laboratory (18). Samples diluted to 1 ml with PBS/BSA were added to C18 reverse-phase columns, and melatonin was eluted according to the instructions accompanying the assay kit. Standards were reconstituted in PBS/BSA and were also extracted to ensure that both standards and samples were treated identically. Sensitivity of the assay ranged from 21 to 43 pM depending on the volume of sample used. Pineal gland melatonin was assayed in 100 µl of homogenate by direct RIA using the Buhlmann reagents. The sensitivity was 21 fmol/gland. Urinary melatonin was assayed by direct RIA of 20 µl of urine. There has been no formal attempt to prove that the immunoreactivity detected in the mouse plasma is authentic melatonin due to the very low amounts of sample available and the low levels detected. Nevertheless, the G280 antibody used has been well characterized in terms of specificity with a large number of synthetic indoles, and it has low cross-reactivity with all naturally occurring indoles (15, 18).
The urinary metabolite 6-sulphatoxymelatonin was assayed in urine by direct RIA as previously reported for rats (1, 17) using reagents obtained from Stockgrand, Guildford, Surrey, UK.Chromatography. To investigate the metabolism of melatonin, CBA, BALB/c, and wild derived mice and a Wistar albino rat were injected intraperitoneally with 4 mg (mice) or 40 mg melatonin (rat), and urine was collected overnight. For chromatography, 5 µl of undiluted urine were applied to a silica gel thin-layer plate along with standard solutions of melatonin, 6-hydroxymelatonin, 6-sulphatoxymelatonin, and 6-glucuronylmelatonin. The plates were developed in butanol: acetic acid:water (4:1:1), and indoles were detected by spraying with Ehrlich's reagent (p-dimethylaminobenzaldehyde). Melatonin and 6-hydroxymelatonin were obtained from Sigma. 6-Sulphatoxymelatonin was synthesized using the method of Fellenberg et al. (9). 6-Glucuronylmelatonin was synthesized enzymatically using a procedure originally designed for steroid glucuronylation (36). Briefly, this involved incubating UDP-glucuronic acid (68 mg; Sigma), bovine liver UDP-glucuronyl transferase (100 mg; Sigma), and 6-hydroxymelatonin [25 mg (Sigma) in 0.5 ml of dimethylformamide] in 4.5 ml of incubation buffer (0.3 M Tris · HCl buffer, 1 mM calcium chloride, pH 8) at 37°C for 16 h. The reaction was stopped by freezing the mixture. Thin-layer chromatography revealed two major indole-positive spots, one corresponding to the starting material and another consistent with 6-glucuronylmelatonin (22). Aliquots of the reaction mixture (0.5 ml) were applied to a C18 Sep-Pak cartridge (Waters), and the presumptive 6-glucuronylmelatonin was eluted with 40% methanol (11). The material has not been crystallized or subjected to any further chemical analysis at this stage.
Statistics. The pineal and plasma melatonin rhythm data for CBA and C57Bl mice were analyzed by ANOVA, and differences between times at night were tested by post hoc Student-Newman-Keuls test. Significance was set at P < 0.05. All studies were conducted according to the guidelines set out by the National Health and Medical Research Council of Australia and were approved by the Animal Ethics Committee of Adelaide University.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Pineal and plasma melatonin rhythms.
Melatonin was detected in the pineal glands of CBA mice at all times
from 1900 to 0700, increasing from 0100, peaking at 0500, and
decreasing eightfold by 0700 (Fig. 1).
The changes in melatonin content with time were significant (ANOVA;
F = 5.6, P < 0.001). Post hoc analysis
indicated that the 0500 levels (1,800 ± 514 fmol/gland) were
higher than all other times except 0300. By contrast, melatonin content
of the C57Bl mice never exceeded 55 fmol/gland (
12 pg/gland) at any
time (Fig. 1). Nevertheless, ANOVA indicated that there was a
significant effect of time (F = 3.5, P = 0.01) with the highest melatonin content (41 ± 4.5 fmol/gland)
occurring at 0500. The lowest value (20.2 ± 3.3 fmol/gland)
occurred at 2100 [the values at 0500 were significantly higher than
those at 2100 (P = 0.04) and 2300 (P = 0.047)].
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Isoproterenol.
Administration of isoproterenol (20 mg/kg) at 0900 and again at 1100 resulted in a robust and prolonged increase in pineal melatonin content
and plasma melatonin levels 30 and 60 min after the last injection in
CBA mice (Fig. 2). Saline administration to CBA mice caused no changes in pineal or plasma melatonin levels (pineal melatonin 79 ± 15 fmol/gland; plasma melatonin <30 pM). When the response of CBA and C57Bl mice to isoproterenol was compared 30 min after the last injection, the pineal melatonin levels were 413 ± 75 and <21 fmol/gland, respectively; plasma melatonin
values were 63 ± 5 and <30 pM, respectively. No obvious acute
ill effects were observed in the mice following isoproterenol
administration.
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Light effects.
Exposure to light 2 h before dawn (0500) resulted in the
suppression of pineal melatonin content and plasma melatonin levels to
basal levels within 45 min of the start of light exposure (Fig. 3). These levels were indistinguishable
from the usual 0700 values.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The CBA mouse strain has a high-amplitude pineal melatonin rhythm with a peak 2 h before lights on (13, 33). The decrease during the 2 h before dawn is profound, with near basal levels measured at zeitgeber time 0 (ZT0; ZT12 is defined as lights off), and it is highly repeatable, indicating that it is likely to be under clock control. This is to our knowledge the first report of plasma melatonin measurement in CBA mice, and we have shown that the pineal melatonin content is highly correlated with plasma melatonin levels. Masana et al. (25) reported pineal and plasma melatonin rhythms in the related C3H strain and found that the highest plasma levels coincided with the lowest pineal melatonin content 2 h after dark onset. This relationship was not observed in animals kept under constant darkness conditions. It remains to be confirmed whether this finding is a special feature of the C3H strain. We also confirmed the greatly impaired melatonin synthesis in the commonly used C57Bl strain. The slight, but statistically significant, elevation in pineal melatonin content (but not plasma melatonin) in C57Bl mice toward light onset is difficult to explain considering that the mutation in NAT responsible for the low melatonin production has been shown to be due to a truncated nonfunctional acetyltransferase (30). Little is known about the nature of the HIOMT defect in mouse strains and whether the mutant enzyme retains some enzyme activity. It is possible that small amounts of N-acetylserotonin produced by other transferases are methylated by residual pineal HIOMT enzyme. The rhythm could then come from a low-amplitude rhythm in pineal HIOMT activity similar to the rhythm reported in rats (29). It should be pointed out that very low levels of pineal melatonin synthesis have also been reported elsewhere in C57Bl mice (33, 35).
Exposure to bright light at the time of maximal pineal melatonin content and plasma melatonin levels resulted in rapid suppression of melatonin production within 30 min. This is consistent with a previous report (12) that showed that pineal melatonin content in CBA mice decreased to basal levels after 5-min exposure to 100-lx light. The rapid decrease in plasma melatonin levels following light exposure as opposed to the gradual morning decline between 0500 and 0700 suggests that the decline may be a clock-controlled event.
Although complete profiles were not obtained due to the large numbers of animals required, a light pulse in the early dark period clearly resulted in a small but significant delay of the morning decline in melatonin production and secretion, whereas a pulse 2 h before lights on resulted in what we interpret as an advance of more than 2 h. These results are consistent with light-induced phase shifts in activity rhythms induced by light pulses in this strain (Kennaway et al., unpublished results) and other strains (3-5), although the magnitude of the shifts is smaller. This may be due to the use of the Aschoff Type II protocol (2) rather than the application of pulses after a long period of constant darkness.
The apparently large phase advance in the melatonin rhythm within one cycle following light exposure at 0500 was a little unexpected because in rats, it can take up to 4 days under similar circumstances for advances of the pineal NAT rhythm to become apparent (14). A possible explanation for this difference is the species difference in the endogenous period, which for rats free running in constant darkness is greater than 24 h and for mice less than 24 h [CBA mice in our laboratory have a free-running rhythm of wheel running of ~23.4 h (Kennaway et al., unpublished results)]. Thus, in rats, morning light pulses before producing a net advance must first counteract the natural tendency to delay, whereas in mice, morning pulses assist the natural tendency for rhythms to advance.
Recent in vitro studies by von Gall et al. (35)
highlighted the similarities between rats and mice in the control
systems responsible for the induction of NAT and synthesis of
melatonin. We have shown that in vivo stimulation of the mouse pineal
gland by the -adrenergic agonist isoproterenol during the light
period increased pineal melatonin content and melatonin secretion into blood in CBA but not C57Bl mice. In preliminary studies, we observed only small transient increases in melatonin production following a
single injection of isoproterenol. By contrast, two injections, 2 h apart in the morning, provided reliable induction of melatonin synthesis, which is reminiscent of the requirements of Syrian hamsters
for prolonged adrenergic stimulation to increase melatonin synthesis
(31). Interestingly, both mice and hamsters have maximum melatonin production late in the dark period. The failure to detect increased melatonin production or synthesis in C57Bl mice in response to isoproterenol administration supports the hypothesis that the small
amounts of melatonin detected in the pineals of some animals at night
may be synthesized from N-acetylserotonin produced by a
transferase enzyme outside the pineal gland that is not induced by
-adrenergic stimulation.
Melatonin produced by the pineal gland of mammals appears in the blood and is metabolized by the liver to 6-hydroxymelatonin, conjugated, and excreted into the urine (20). The nature of the conjugation products of melatonin was reported over 40 yr ago in rats to be 6-sulphatoxymelatonin and 6-glucuronylmelatonin (20), and this was subsequently confirmed in humans (9, 22, 23, 37). With the development of a RIA for 6-sulphatoxymelatonin (1), it has been possible to monitor the excretion of 6-sulphatoxymelatonin into the urine of humans (10), rats (17), Djungarian hamsters (21), minks (26), and pigs (19). In all cases, the excretion rate of 6-sulphatoxymelatonin is rhythmic and can be used as an index of pineal gland rhythmicity (10). There is a clear advantage in such a procedure due to the noninvasive nature and simplicity of urine collection that reduces the number of animals needed for experiments. It was thus expected that mice would produce and excrete 6-sulphatoxymelatonin and that RIA procedures might be used to follow pineal rhythms in this species. It should, however, be pointed out that while Kopin et al. (20) used both rats and mice in their influential study, the nature of the melatonin metabolites in their mouse studies was not reported.
We have provided unequivocal evidence that mice do not excrete significant amounts of 6-sulphatoxymelatonin, presumably due to a lack of an appropriate sulphotransferase. Instead, melatonin is excreted as 6-glucuronylmelatonin with only very small amounts excreted unmetabolized. Our method of detection of metabolites involved both RIA and thin-layer chromatography. In the case of the RIA, the antibody has 0.5% cross-reactivity with 6-glucuronylmelatonin, and this may be the cause of the increase in 6-sulphatoxymelatonin immunoreactivity observed in CBA mice between 0600 and 1000 and following melatonin administration. In the case of the thin-layer chromatography identification of metabolites, because Ehrlich's reagent only detects intact indole rings, we can neither exclude nor quantitate other nonindolic melatonin metabolites in the mouse. It is also important to point out that the inability to produce 6-sulphatoxymelatonin is not confined to laboratory mouse strains because the wild mouse strain also excreted only 6-glucuronylmelatonin.
The very low levels of unmetabolized melatonin excreted are in contradiction to a study by Perissin et al. (28), who not only reported melatonin in mouse urine but also found a more than fivefold increase in unmetabolized melatonin in urine at night. These researchers used a strain of mouse (BD2F1) that is a cross of two strains (DBA2 and C57Bl) both known to be melatonin deficient (13). A more recent study describing atypical plasma and pineal melatonin rhythms in this strain is remarkable for the extremely low levels of melatonin measured (24). We cannot provide an explanation for this discrepancy other than the possibility that the assay used was not specific for melatonin. Similar conclusions may be warranted in the case of the two recent publications that have challenged the idea that BALB/c, C57Bl, and AKR mice are melatonin deficient. In the first study, in each strain, melatonin was reported to be elevated for 15 min around ZT18; at all other times, the pineal melatonin content was very low (6). In the second study, melatonin was detected in BALB/c, C57Bl/6, and OF1 Swiss mice pineal glands, but the levels were very low and the pattern was very different from that observed for CBA and C3H mice (6).
Perspectives
The results of this study indicate that the CBA mouse strain has a robust melatonin rhythm that is under-adrenergic control and that
melatonin production can be suppressed by brief light pulses. There is
also compelling preliminary evidence that light pulses presented at
different times of the night phase shift melatonin rhythms. The need to
kill animals for pineals or blood may restrict the usefulness of mice
in studies of melatonin rhythmicity, although the knowledge that
6-glucuronylmelatonin is the major urinary melatonin metabolite
provides an incentive to develop assays for this compound.
Apparently, all knockout and transgenic mouse lines are on genetic backgrounds that make the animals melatonin deficient (27). This is not acceptable for researchers studying circadian rhythms or reproduction. If strains of interest (i.e., knockout) are crossed with CBA mice, an isoproterenol stimulation test based on results presented in this study would provide a simple method of monitoring the inheritance of melatonin production capacity at least until the mutations in NAT and HIOMT are defined and PCR-based genotyping strategies are developed.
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ACKNOWLEDGEMENTS |
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The authors thank S. Palnok for assistance in the early stages of the study.
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FOOTNOTES |
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This study was supported by a grant to D. J. Kennaway from the National Health and Medical Research Council of Australia.
Address for reprint requests and other correspondence: D. J. Kennaway, Dept. of Obstetrics and Gynaecology, Adelaide Univ., Medical School, Frome Road, Adelaide, South Australia 5005 (E-mail: david.kennaway{at}adelaide.edu.au).
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. Section 1734 solely to indicate this fact.
10.1152/ajpregu.00360.2001
Received 28 June 2001; accepted in final form 14 September 2001.
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TOP
ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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and plasma melatonin with 
. The
relationship between pineal and plasma melatonin in these animals is
shown in C and D. Note the scale difference for
C57Bl pineal levels in B and D. Where no SE bar
is shown, the line is obscured by the symbol.
)
and for plasma melatonin (pM; isoproterenol, 
). 
Time of administration of isoproterenol with 0 being 2 h after lights on. Where no SE bar is shown, the line is
obscured by the symbol.
