Test of ML23 as an antagonist to the effects of melatonin
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Test of ML23 as an antagonist to the effects of melatonin
in the ram
G. A. Lincoln and R. W. Kelly
MRC Reproductive Biology Unit, 37 Chalmers Street, Edinburgh EH3 9EW, UK
Summary. In Exp. 1, four groups of 8 yearling Soay rams were housed under long days
(16L:8D) to induce reproductive quiescence and were treated daily for 12 weeks with:
(I) vehicle (2 or 4 ml 50% ethanol/water), (II) ML23 (2 mg), (III) melatonin (2 mg)
and (IV) melatonin and ML23 (2 mg of each). All treatments were given orally in the
mid-light phase. In the rams receiving melatonin (Group III) there was an earlier in-
crease in the plasma concentrations of FSH and testosterone and regrowth of the testes
compared to the controls (time to maximum testicular diameter: 10\m=.\0\m=+-\0\m=.\5 and
15\m=.\3\m=+-\1\m=.\2weeks). These differences were reversed after the end of the 12-week treat-
ments when rapid testicular regression occurred in melatonin-treated rams but not in
the controls. In the group receiving ML23 and melatonin (Group IV), there was early
reactivation and regression of the reproductive axis as in the melatonin group (testis
max. 9\m=.\9 \m=+-\0\m=.\7and 10\m=.\0\m=+-\0\m=.\5weeks) while in the group receiving ML23 alone (Group
II) there was a slower redevelopment and regression as in the controls (testis max.
15\m=.\7\m=+-\1\m=.\1and 15\m=.\3\m=+-\1\m=.\2weeks). The comparison between the 4 groups in the changes
in the blood concentrations of prolactin, voluntary food intake and total body weight
also indicated that the treatment with ML23 failed to modify the effect of melatonin
(combined treatment vs melatonin) or the effect of the long day photoperiod (ML23
vs vehicle).
In Exp. 2, a more soluble derivative of ML23 (called ML23-lysine) was tested in two
groups of 4 adult Soay rams which were housed under long days (16L:8D) and treated
daily for 12 weeks, as above with: (I) melatonin (2 mg) and (II) melatonin (2 mg) and
ML23-lysine (4 mg). In both groups there was a similar reactivation and regression of
the testes with no significant differences between the two treatments.
The overall results illustrate that the treatments with melatonin in this protocol were
effective at influencing a wide range of reproductive and non-reproductive measures in
the ram, but that ML23 was unable to antagonize these biological effects of melatonin.
Keywords: ram; seasonal reproductive cycles; photoperiod; pineal gland; melatonin receptors
Introduction
A derivative of tryptamine ((N-2,4-dinitrophenyl)-5-methoxy tryptamine; ML23) which blocks two
different biological effects of melatonin in the rat has recently been identified: the inhibitory effect
of melatonin on the release of dopamine from rat hypothalamus in vitro (Zisapel & Laudon, 1987)
and the delaying effect of melatonin on sexual maturation in the male rat in vivo (Laudon et al,
1988) were prevented by concomitant treatment with an equal dose of ML23. The aim of the
current study was to test this putative melatonin antagonist for its ability to block the effect of
melatonin in the sheep.
The sheep is a photoperiodic species in which the annual cycle in daylength affects the timing of
the seasonal reproductive cycle through alterations in the 24-h pattern of melatonin secretion from
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via free accessthe pineal gland (Arendt, 1986). Melatonin is secreted largely at night and the duration of the
period of release codes for night length to influence the hypothalamic-pituitary-gonadal axis
(Bittman et al, 1983). A switch from long days to short days leads to an increase in the duration of
melatonin secretion and this results in a resurgence of reproductive activity and the onset of the new
breeding season. The administration of exogenous melatonin in the afternoon to sheep exposed to
long days, which extends the exposure to melatonin, causes early onset of ovarian activity in ewes
similar to that produced by transfer to short days (Kennaway et al, 1982; English et al, 1986;
Wigzell et al, 1988). The protocol of administering melatonin in the afternoon to photo-inhibited
rams was used in the current study to test the effectiveness of ML23 as a melatonin antagonist. The
prediction was that, if ML23 blocked the biological effects of melatonin, the daily treatment with
this drug would modify the reproductive responses both to the administered melatonin (effect of
exogenous melatonin) and to the prevailing photoperiod (effect of endogenous melatonin). Since
ML23 is relatively insoluble, a more soluble derivative was also tested (called ML23-lysine). The
changes in the diameter of the testes, intensity of the sexual skin colouration, concentrations of
FSH, prolactin and testosterone in the blood plasma and the voluntary food intake and total body
weight were monitored to provide a wide range in reproductive and non-reproductive indicators of
the effects of melatonin.
The results for part of this study have been presented in abstract form (Lincoln & Kelly, 1988).
The nomenclature of ML23 which was given as N-(3,5-dinitrophenyl)-5 methoxy tryptamine by
Laudon et al (1988) has been corrected to N-(2,4-dinitrophenyl)-5-methoxy tryptamine (see
J. Endocr. (1988) 118, p. 160).
Materials and Methods
Animals and routine measurements
The animals used in these experiments were rams of the Soay breed of semi-domesticated sheep which show a
marked seasonal reproductive cycle readily manipulated by changes in daylength (Lincoln & Short, 1980). They were
housed in individual pens in adjacent light-controlled rooms (8 animals/room) at the Marshall Building near
Edinburgh and given a diet of grassnuts with hay ad libitum. Every week a heparinized blood sample was collected
from the jugular vein and the plasma stored at 20°C until the hormone concentrations were measured by RIA.
Every 2 weeks the diameter of the testes and the intensity of the sexual skin colouration were recorded (Lincoln &
—
Davidson, 1977) and every 4 weeks the body weight of the rams was measured. On one occasion during Exp. 1 the
total weight of food consumed was recorded daily for 5 days.
Drugs
Melatonin was obtained from Sigma Chemicals (Sigma, Poole, Dorset, UK). ML23 (N-2,4-dinitrophenyl)-5-
methoxy tryptamine) was prepared by coupling 5-methoxy tryptamine (Sigma, Poole, Dorset, UK) with fluoro-2,4-
dinitrobenzene (Aldrich, Gillingham, Dorset, UK) as described by Laudon et a! (1988). ML23-lysine was prepared in
a similar manner but using l,5-difluoro-2,4-dinitrobenzene (Aldrich) and further reacting the product with a 1-2m
excess of L-lysine (Sigma). The product was recrystallized from methanol and was essentially pure by t.l.c.
Treatments
Experiment I. Yearling rams (N 32) were moved indoors in February 1987 and housed under long days (16 h
=
light:8 h darkness, 16L:8D) to induce full testicular regression. The treatments began after 14 weeks with 4 equal
groups: (I) solvent vehicle as control (2 or 4 ml ethanol/water (50:50), v/v), (II) ML23 (2 mg ML23 in 2 ml 50%
ethanol), (III) melatonin (2 mg melatonin in 2 ml 50% ethanol), and (IV) ML23 and melatonin combined (2 mg ML23
and 2 mg melatonin both in 2 ml 50% ethanol). All treatments were repeated daily at 16:00 h (lights on 08:00 h and off
at 24:00 h) for 12 weeks and involved placing the drug inside the mouth using a 5-ml syringe. The routine monitoring
of the reproductive changes was continued for 12 weeks after the end of the drug treatments.
Experiment 2. Adult rams (N 8) which had been living indoors for at least 2 years under alternating 16-week
=
periods of long days (16L:8D) and short days (8L:16D) were exposed to long days (16L:8D) to induce testicular
regression. The treatments began after 12 weeks as two equal groups: (I) melatonin (2 mg melatonin dissolved in 2 or
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via free access4 ml 50% ethanol), and (II) melatonin and ML23-lysine (2 mg melatonin dissolved in 2 ml 50% ethanol and 4 mg
ML23-lysine dissolved in 0.2 ml dimethyl sulphoxide and 2 ml 50% ethanol). The treatments were repeated daily
for 12 weeks and involved placing the drug inside the mouth using a 5-ml syringe. The routine monitoring of the
reproductive changes was continued for 16 weeks after the end of the drug treatment.
Radioimmunoassays
Previously validated RIAs were used for the measurement of the blood plasma concentrations of FSH (McNeiUy
eta!, 1976; using NIADDK-anti oFSH-I as first antibody, McNeiUy et a!, 1986), prolactin (McNeiUy & Andrews,
1974) and testosterone (Corker & Davidson, 1978; using an iodinated tracer, Sharpe & Bartlett, 1985). The FSH assay
had intra- and inter-assay coefficients of variation of 71% and 23-7% respectively and a lower limit of sensitivity
(twice s.d. from buffer control) of 5 ng NIH-FSH-S14/ml. The corresponding values for the prolactin assay were
4-9%, 15-9% and 0-5 ng NIH-P-S15/ml and for the testosterone assay 10-3%, 10-3% and 0-2 ng/ml.
Statistical analysis
In the two experiments, the long-term data for each parameter were divided into a 12-week period during the
treatment and a12-week period after the treatment. The significance of differences between groups during these two
periods was assessed by ANOVA and when a time versus treatment interaction was indicated, the differences were
assessed using the Newman-Keuls t test.
Results
Experiment 1: ML23 and melatonin alone and in combination
The long-term changes in the reproductive measures in the 4 groups of rams in Exp. 1 are
summarized in Fig. 1 and other characteristics of the animals at the end of the treatments are shown
in Fig. 2.
At the beginning, the rams in all groups were sexually inactive as judged by the small size of the
testes, low plasma concentrations of FSH and testosterone and high plasma concentrations of
prolactin. In the group that received melatonin for 12 weeks there was an earlier reactivation of the
reproductive axis compared to the control group. This involved a rapid increase in plasma concen¬
trations of FSH and testosterone, growth of the testes and the early appearance of the sexual skin
colouration; at the same time there was a rapid decline in the plasma concentrations of prolactin
(Figs 1 & 2). There were significant differences between the melatonin and control groups during
the 12 weeks of treatment for all the hormonal values (P < 005, ANOVA). By the end of the
treatments, the rams that received melatonin (Group III) were fully sexually active well in advance
of the controls, with peak plasma concentrations of testosterone and low values of prolactin; there
were also decreases in total body weight and voluntary food intake as expected for animals entering
the rutting condition (compare Groups I and III, Figs 1 & 2). After the end of the treatments, the
differences between Groups I and III became further accentuated as the rams which had received
melatonin showed a dramatic regression of the reproductive axis. During the 12 weeks after
treatment, there were significant differences between the melatonin and control groups in all the
recorded measures (P < 001; ANOVA, Fig. 1).
In the rams that received ML23 (Group II), there was a slow redevelopment of testicular
activity with a pattern not significantly different from that occurring in the control group for any of
the reproductive measures during the treatment or post-treatment phases of the experiment (Fig.
1). Furthermore, in Group IV rams (ML23 + melatonin), there was a rapid onset of testicular
activity as occurred in the rams treated with melatonin alone, with no significant differences
between these two groups. These results illustrate that the ML23 treatments failed to modify the
effects of the prevailing long-day photoperiod or the effects of exogenous melatonin in the rams.
Experiment 2: ML23-lysine and melatonin
In the rams that received melatonin in Exp. 2 (Group I), there was also a conspicuous increase
in the plasma concentrations of FSH and testosterone and growth of the testes during the 12-week
treatment, which was reversed during the period after treatment as the testes underwent regression.
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Group IV
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via free accessIn Group II (ML23-lysine + melatonin) there was a very similar reproductive cycle. The time from
the start of treatment to the maximum testicular diameter was 12-3 ± 0-5 weeks (mean ± s.e.m.)
for Group I and 13-0 + 0-8 weeks for Group II. The corresponding values for the other repro¬
ductive measures were as follows: plasma FSH, 80 ± 0-8 and 7-3 + 0-6 weeks, and plasma testos¬
terone, 13-7 ± 0-6 and 110+ 1-8 weeks. The plasma concentrations of prolactin decreased during
the treatment in both groups, reaching minimum values after 3-7 ± 0-6 weeks in Group I and
40 + 0-2 weeks in Group II, and there was a rapid increase in the concentrations of prolactin after
the end of the treatments in all animals. The statistical analysis revealed no significant differences
between the endocrine changes in the two groups.
- g&M III
Group
Fig. 2. Time of maximum testicular diameter and appearance of the sexual skin colouration
(sexual flush) and measurements of voluntary food intake and total body weight loss (assessed
at the end of the treatments) in groups of 8 sexually mature yearling Soay rams housed indoors
under long days (16L:8D) and treated for 12 weeks as follows: Group I, solvent vehicle as
control; Group II, ML23; Group III, melatonin; Group IV, melatonin + ML23. The values are
mean ± s.e.m. and significant differences were assessed by ANOVA and Newman-Keuls t test;
*P < 005, **P < 001 compared with control group.
Discussion
This is the first demonstration that the daily administration of melatonin in the afternoon to rams
housed under long days induces reactivation of the reproductive axis as predicted from studies in the
ewe (Kennaway et al, 1982). The treatments resulted in an increase in the plasma concentrations
Fig. 1. Changes (mean ± s.e.m.) every 2 weeks in the diameter of the testes and concentration
of FSH, testosterone and prolactin in blood plasma of groups of 8 yearling Soay rams housed
indoors under long days (16L:8D) and given one of the following treatments: Group I, solvent
vehicle as control; Group II, 2 mg ML23; Group III, 2 mg melatonin; Group IV, 2 mg
melatonin + 2 mg ML23. Each ram received the treatments orally every day for 12 weeks
(indicated by horizontal bar) and the experiment continued for a further 12 weeks.
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via free accessof FSH which was evident within 3 weeks and full testicular activity occurred after 10-14 weeks.
Other changes included a decline in the blood concentrations of prolactin and a decrease in voluntary
food intake and body weight. All these changes are a normal feature of the response to a change from
long days to short days (Lincoln & Short, 1980; Lincoln & Ebling, 1985). Further evidence to indicate
that the exogenous melatonin had elicited a short-day response was provided by the results for the
12-week period after the treatments. There was a rapid decline in the plasma concentrations of FSH
and testosterone, regression of the testes and an increase in the plasma concentrations of prolactin,
all of which are normal responses to a switch from short days to long days.
The cycle of reactivation and regression of the reproductive axis in the rams treated with mela¬
tonin was virtually identical to that in rams receiving the combination of melatonin and ML23.
This shows clearly that the ML23 did not modify the biological effects induced by the exogenous
melatonin. The more soluble derivative, ML23-lysine, was also ineffective. Furthermore, the results
for the rams which received ML23 alone were very similar to those for the animals which acted as
normal controls and received no drug treatment. This shows that the ML23 failed to modify the
effects of the endogenous melatonin which is involved in relaying the effects of the prevailing long
day photoperiod. There was no evidence therefore, from these experiments using this particular
protocol, that ML23 acted as a melatonin antagonist.
The original study which characterized the biological effect of ML23 was carried out with
pubertal male rats (Laudon et al, 1988). ML23 when administered i.p. by timed injection or orally
in the drinking water totally or partly blocked the inhibitory effects of exogenous melatonin on the
secretion of testosterone and the growth of the accessory sex glands. The dose of ML23 which
antagonized the melatonin was equivalent to (weight/weight) or 10 times less than that of the
agonist. The study also demonstrated that, after oral treatment, ML23 rapidly entered the blood
circulation and brain tissues and had a relatively long half-life (42 min).
A number of explanations can be proposed to explain the negative results for ML23 obtained in the
current study with the rams in contrast to the result with rats. The mechanism of action of melatonin
in the photoperiodic control in the ram may be different from the action of melatonin in the control of
puberty in the rat. ML23 may be ineffective because the oral method of administration was not
adequate to deliver the drug into the brain tissues of the rams at the concentration necessary to block
the actions of melatonin. The presence of the rumen may have modified absorption and prevented
intact ML23 entering the tissues. The concentrations of ML23 in the blood and tissues of the brain
were not measured in the current study so low absorption could account for the negative results.
Another explanation is that ML23 is not an effective antagonist competing with melatonin at
the receptor sites in the hypothalamus as originally proposed. In a recent competitive binding study
it has been shown that ML23 has a very low potency in inhibiting the binding of 125I-labelled
melatonin (0062%) in crude membrane preparations from the hypothalamus of male rats (English
& Arendt, 1988). This result has also been confirmed in preparations from the pars tuberalis of
sheep (Morgan et al, 1989).
Using the combined evidence that ML23 is relatively ineffective at blocking the binding of
melatonin in vitro and is ineffective in blocking the biological actions of melatonin in vivo as in the
current study, it is concluded that ML23 is not a useful melatonin antagonist.
We thank our colleagues at the Marshall Building for the care of the animals; Norah Anderson
for help with blood sampling; and Gillian Hay and Susan Maguire for expert technical assistance
with the radioimmunoassays and data analysis. NIADDK kindly supplied the ovine FSH and
prolactin standards.
References
Arendt, J. (1986) Role of the pineal gland and melatonin Bittman, E.L., Dempsey, R.J. & Karsch, F.J. ( 1983) Pineal
in seasonal reproductive function in mammals. melatonin secretion drives the reproductive response
Oxford Rev. Reprod. Biol. 8, 266-320. to daylength in theewe. Endocrinology 113,2276-2283.
Downloaded from Bioscientifica.com at 06/11/2024 11:23:06PM
via free accessCorker, C.S. & Davidson, D.W. (1978) The radio¬ Lincoln, G.A. & Short, R.V. (1980) Seasonal breeding:
immunoassay of testosterone in various biological Nature's contraceptive. Recent Prog. Horm. Res. 36,
fluids without chromatography. Steroid Biochem. 9, 1-52.
373-374. McNeiUy, A.S. & Andrews, P. (1974) Purification and
English, J. & Arendt, J. (1988) Characterisation and characterisation of caprine prolactin. /. Endocr. 60,
circadian rhythmicity of a melatonin binding site in 359-367.
the rat hypothalamus. J. Reprod. Fert., Abstract McNeiUy, A.S., Jonassen, J.A. & Fraser, H.M. (1986)
Series 1, Abstr. 8. Suppression of follicular development after chronic
English, J., Poulton, A.L., Arendt, J. & Symons, A. (1986) LHRH immunoneutralization in the ewe. J. Reprod.
A comparison of the efficency of melatonin treat¬ Fert.l6,4U-490.
ments in advancing oestrus in ewes. J. Reprod. Fert. McNeiUy, J.R., McNeiUy, A.S., Walton, J.S. &
11, 321-327. Cunningham, F.J. (1976) Development and appli¬
Kennaway, D.J., Gilmore, T.A. & Seamark, R.E. (1982) cation of a heterologous radioimmunoassay for
Effect of melatonin feeding on serum prolactin and ovine follicle-stimulating hormone. J. Endocr. 70,
gonadotropin and the onset of seasonal estrous 69-70.
cyclicity in sheep. Endocrinologv 110, 1766-1772. Morgan, P.J., Williams, L.M., Davidson, G., Lawson, W.
Laudon, M., Yaron, Z. & Zisapel, N. (1988) N-(3,5- & HoweU, E. (1989) Melatonin receptors on ovine
dinitrophenyl)-5 methoxytryptamine, a novel melato¬ pars tuberalis: characterization and autoradiographi-
nin antagonist: effects on sexual maturation of the cal localization. J. Neuroendocr. 1, 1-4.
male and female rat on oestrous cycles in female rats. Sharpe, R. M. & Bartlett, J.M.S. (1985) Intratesticular
J. Endocr. 116,43-53. distribution of testosterone in rats and the relation¬
Lincoln, G.A. & Davidson, W. (1977) The relationship ship to the concentrations of a peptide that stimulates
between sexual and aggressive behaviour and pitu¬ testosterone secretion. /. Reprod. Fert. 74, 223-236.
itary and testicular activity during the seasonal sexual Wigzell, S., Robinson, J. J., WaUace, J.M. & Aitken,
cycle of rams and the influence of photoperiod. J. R.P. (1988) Duration of melatonin treatment and
Reprod. Fert. 49, 267-276. ovarian activity in ewes. Anim. Prod. 46, 510, Abstr.
Lincoln, G.A. & Ebling, F.J.P. (1985) Effect of constant Zisapel, N. & Laudon, M. (1987) A novel melatonin
release implants of melatonin on seasonal cycles in antagonist affects melatonin mediated processes in
reproduction, prolactin secretion and moulting in vitro and in vivo. Eur. J. Pharmacol. 136, 259-260.
rams. J. Reprod. Fert. 73, 241-253.
Lincoln, G.A. & Kelly, R.W. (1988) Test of a putative
melatonin antagonist in the ram. J. Reprod. Fert.,
Abstract Series, Abstr. 19. Received 16 January 1989
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