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Institut National de la Recherche Agronomique, Physiologie de la Reproduction des Mammiferes Domestiques, Laboratoire de Neuroendocrinologie Sexuelle (C.V., J.-C.T., Y.T., B.M.), Nouzilly; and Collège de France, Laboratoire de Biochimie Cellulaire (J.T.), Paris, France
Address all correspondence and requests for reprints to: Dr. Benoît Malpaux, Institut National de la Recherche Agronomique, Physiologie de la Reproduction des Mammiferes Domestiques, 37380 Nouzilly, France. E-mail: malpaux{at}tours.inra.fr
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Introduction |
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Indeed, the effects of photoperiod and, therefore, melatonin on pulsatile LH secretion are mediated by central nervous system mechanisms that regulate the activity of the LHRH pulse generator system (8, 9). In particular, catecholamines seem to be heavily implicated in the photoperiodic inhibition of gonadotropic secretion in both male and female sheep (10, 11, 12, 13, 14). Furthermore, catecholamines appear to regulate PRL secretion in sheep as in many other species (15, 16, 17, 18). Interestingly, photoperiod can regulate dopaminergic activity at the level of the hypothalamus (14, 19), such that a short day treatment decreases both the activity of the rate-limiting enzyme of catecholamine biosynthesis, tyrosine hydroxylase (TH), and dopamine tissue content at the level of the median eminence (ME) (14). These changes in dopaminergic activity have been observed in conjunction with both a stimulation of LH and an inhibition of PRL secretion. Therefore, it is not clear whether these changes in dopaminergic activity are linked to the photoperiodic regulation of LH or PRL secretion. Moreover, if the photoperiodic regulation of the ME dopaminergic activity is involved in the regulation of LH and/or PRL secretion, then it must be mediated by melatonin. Although it has been shown that a dopaminergic antagonist decreases the stimulatory effect of a melatonin implant placed in the mediobasal hypothalamus on FSH secretion in the ram (20), a direct relationship between a hypothalamic catecholaminergic pathway and melatonin has never been established in sheep.
The purpose of this study was to identify the functional significance of the photoperiod-induced changes in hypothalamic dopaminergic activity with respect to the photoperiodic regulation of LH pulsatile secretion. In the first experiment, we studied the timing of these changes in relation to the short day-induced inhibition of PRL and stimulation of LH secretion to determine whether a temporal link exists with either one of these two neuroendocrine regulations. In the second experiment, we examined the effect on ME dopaminergic activity of melatonin treatment that induced short day-like effects on both LH and PRL secretion to determine whether melatonin mimics the effect of photoperiod on ME dopaminergic activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Photoperiodic and melatonin treatments
In both experiments, a preparatory photoperiodic treatment was
designed to allow all animals from one experiment to be killed at the
same time (Figs. 1
and 5), thus standardizing the conditions of
collection and storage of brain tissue samples and minimizing the
impact of environmental factors other than photoperiod, such as stress
or temperature, on PRL secretion. All animals were submitted to
alternations between constant long (LD; 16 h of light/24 h) and
short (SD; 8 h of light/24h) day lengths (Figs. 1 and 5). The
initial period of treatment was different depending on the type of
photoperiod (LD or SD) and its duration for each group. However, the
last SD/LD cycle preceding the critical SD (Exp 1) or melatonin (Exp 2)
period was similar in all groups (mean ± SD, 82
± 1 SD/85 ± 3 LD and 80 ± 1 SD/88 ± 3 LD in Exp 1
and 2, respectively).
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). In this group, LH
secretion was expected to be inhibited, whereas PRL secretion should
have been stimulated. Two other groups (5 SD and 25 SD) were killed
after 5 and 25 SD, respectively, during the establishment of inhibition
of PRL secretion but before any changes in pulsatile LH secretion. The
last group (76 SD) was submitted to 76 SD; thus, LH secretion was
expected to be maximal in this group.
Exp 2: chronology of melatonin-induced changes in TH activity
Twenty-one ewes were allocated to three groups (n = 7),
which were balanced for age and body weight. In this experiment, sc
implants of melatonin (Regulin, Hoescht, Hauxton, UK) were given to
LD-maintained ewes to stimulate LH secretion and inhibit that of PRL.
The duration of the melatonin treatment was different in each group
according to the timing of the expected melatonin SD-like effects on LH
and PRL secretion. One group was killed after 25 (25 MEL) days of
melatonin treatment, which should be sufficient to induce inhibition of
PRL but not to cause stimulation of LH secretion. Another group was
submitted to 77 days (77 MEL) of melatonin treatment, which should be
associated with stimulated LH secretion. The last group did not receive
any melatonin treatment (0 MEL) and acted as a control (Fig. 5); LH
secretion was expected to be low and that of PRL high in these
animals.
The ability of the melatonin sc implants to maintain permanently high blood concentrations of melatonin was monitored by determining diurnal (three blood samples obtained at 2-h intervals) and nocturnal (three hourly samples) mean plasma melatonin concentrations.
Determination of endocrine states
The effects of photoperiodic or melatonin treatments on LH
secretion were determined by assaying LH in biweekly blood samples
collected for 4.5 months before animals were killed (in Exp 1, weekly
samples were also taken for 1.5 months before the 4.5 month twice
weekly collection period). Secretory profiles of LH were determined by
assaying LH concentrations in serial blood samples obtained every 10
min for 6 h on two occasions: first, during LD, 7 days before the
onset of last short day (Exp 1) or melatonin (Exp 2) treatment period,
and second, 3–4 days (Exp 1) or 1–2 days (Exp 2) before animals were
killed (Figs. 1 and 5). The effects of the treatments on PRL secretion
were determined by assaying PRL in every third sample from the last
3 h of the second sampling period.
Hormonal assays
LH was assayed in duplicate 100-µl aliquots of plasma using
the RIA of Pelletier et al. (22) as modified by Montgomery
et al. (23). Assay sensitivity was 0.15 ± 0.0 ng/ml
1051-CY-LH (0.31 ng/ml NIH LH-S1). The intraassay coefficient of
variation (CV) for four plasma pools averaged 8.9%, and the interassay
CV (two assays) for these plasma pools averaged 9.4%.
PRL was assayed in duplicate 10-µl aliquots of plasma using the RIA of Kann (24). The assay sensitivity was 8.5 ng/ml NIDDK ovine PRL. The intraassay CV for two plasma pools averaged 25% (one assay).
Melatonin was assayed in duplicate 100-µl aliquots of plasma using the RIA method of Fraser (25) with an antibody first raised by Tillet et al. (26). The sensitivity was 4 pg/ml, and the intraassay (one assay) CV for two plasma pools averaged 6%.
TH activity and catecholamine assays
In Exp 1 and 2, TH activity and catecholamine content were
determined in the stalk-ME, as previously described (14). In addition,
in Exp 1, to confirm the lack of photoperiodic effects observed
previously in those hypothalamic areas, TH activity and catecholamine
content were also determined in the preoptic area (POA) and the
laterobasal (LBH), mediobasal (MBH), and mediodorsal (MDH) parts of the
hypothalamus, which were punched on thick slices (2–3 mm) of frozen
brain as previously described (14). The confirmation of the lack of
effect of photoperiod on the dopaminergic activity of the MBH
(containing the A12 nucleus) and LBH (containing the A15 nucleus) parts
of the hypothalamus (Exp 1) led us to increase the precision in the
collection procedure for Exp 2, focusing on these specific structures
rather than collecting the entire surrounding areas (27). In this
experiment, TH activity and monoamine contents were determined in the
A15 and A12 dopaminergic nuclei punched on frozen brain using a
horizontal Leitz microtome fitted with a cooling device (Leitz,
Rockleigh, NJ). The A12 and A15 nuclei were sampled at 2- to 3-mm
thickness on the anteroposterior axis in the part of the hypothalamus
located between the retrochiasmatic area (19) and the back of the
dorsal limit of the infundibular recess. The A12 nuclei were obtained
by bilateral dissection of the mediobasal part of the periventricular
area. The A15 nuclei were obtained by bilateral dissection of the
laterobasal areas bordering to the optic tracts.
For both experiments, TH activity was assayed in caudate nucleus samples as a control to determine the ability of the method to assess high levels of TH activity, whereas the optic chiasm was assayed as a negative control for TH activity.
Brain samples were individually weighed and crushed in 20 mM Na/K phosphate buffer (pH 6.5), and the tissue extract was used to determine TH activity and monoamine contents as previously described (14). TH activity was estimated by measuring the amount of tritiated water produced during the incubation of total tissue extract with tritiated L-tyrosine using the method described by Levitt et al. (28) and modified by Mueller et al. (29). For a given structure, samples of all animals were either assayed in two different assays (Exp 1) or regrouped into a single assay (Exp 2). Assay sensitivity (activity corresponding to a level of radioactivity equal to 2 times the background measured after incubation of 100 µl buffer) was 0.14 ± 0.01 U TH. The interassay CVs determined for a standard 10% ovine adrenal gland extract were 20% and 22% for Exp 1 and 2, respectively. After an extraction in an aqueous acid solution, contents of catecholamines and their metabolites were assayed by HPLC coupled to electrochemical detection as previously described (14, 19). The limits of detection were 14.5 ± 2.4 pg for noradrenaline, 33.2 ± 5.3 pg for dopamine, and 25.0 ± 4.4 pg for 3,4-dihydroxy-phenyl acetic acid (DOPAC).
Analysis of data
For each ewe, the time when circulating LH concentrations
started to rise in response to SD or melatonin treatments was defined
by the first of at least three consecutive samples in which a LH
concentration exceeded 1 ng/ml.
LH pulses were identified by means of a modification of the methods of Wallace and McNeilly (30), as described previously (9). The changes in LH pulse frequency in the same group were analyzed by a Wilcoxon test, and differences between groups were analyzed by Kruskall-Wallis tests followed by Mann-Whitney tests for comparisons by pairs.
Mean concentrations of PRL, tissue TH activity, and contents of catecholamines and their metabolites were analyzed by a one-factor ANOVA (duration of SD or melatonin exposure for Exp 1 and 2, respectively).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In contrast, LH levels remained at the basal
level (<1 ng/ml) in the other groups (Fig. 1).
Seven days before the end of the last LD period, LH pulse frequency was
low, and there was no difference between groups (0.9 ± 0.8,
0.1 ± 0.0, 0.0 ± 0.1, and 2.0 ± 0.8 pulses/6 h for
the 0 SD control, 5 SD, 25 SD, and 76 SD groups, respectively;
mean ± SEM). LH pulse frequencies 48–72 h before the
death of the animals remained low in the 0 SD control group (0.6
± 0.4 pulses/6 h) and the 5 and 25 SD groups (0.0 ± 0.0 and
0.0 ± 0.0 pulses/6 h, respectively; Fig. 2). In
contrast, in the 76 SD group, LH pulse frequency was dramatically
increased (7.1 ± 1.0 pulses/6 h) compared to that during the LD
period in the same group (P < 0.01). As a consequence,
LH pulse frequency at that time was much higher in the 76 SD group than
in any other group (P < 0.01).
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). The
inhibitory effect of SD on PRL secretion was already expressed in the 5
SD group (P < 0.01 compared to the 0 SD control
group). Most importantly, the inhibition of PRL secretion seemed to be
maximal after only 25 SD, as there was no difference in mean PRL levels
between the 25 and 76 SD groups.
Effect of SD treatment on hypothalamic TH activity and
catecholamine contents. ME: The SD treatment was
associated with a decrease in TH activity in the ME (P
< 0.001), and this effect was expressed only in the 76 SD group. In
this group, the TH activity of the ME was 33% lower than that in the 0
SD control group and 40% and 42% lower than those in 5 and 25 groups,
respectively (P < 0.01 for all comparisons; Fig. 3). The SD exposure had no significant effect on the
content of noradrenaline, dopamine, or DOPAC.
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).
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Exp 2
Melatonin implants caused a dramatic increase in the diurnal
plasma levels of this hormone, which reached values similar to the
physiological nocturnal concentrations (diurnal levels, 7.1 ± 1
before vs. 262.7 ± 14.5 pg/ml after implants;
nocturnal levels, 276.0 ± 27.0 before vs. 415.7
± 19.4 pg/ml after implants).
Effect of melatonin treatment on LH and PRL secretion. In the
77 MEL group, mean LH concentrations, measured in twice weekly samples,
were increased after 55.7 ± 4.3 days of treatment. In the 0 MEL
control and 25 MEL groups, mean LH levels remained low until the end of
the experiment (Fig. 5).
Seven days before the beginning of melatonin treatment (LD control
period), LH pulse frequency was low and did not show any difference
between groups (0.0 ± 0.0, 0.0 ± 0.0, and 0.6 ± 0.6
pulses/6 h for the 0 MEL control, 25 MEL, and 77 MEL groups,
respectively). LH pulse frequencies 24–48 h before the death of the
animals remained low in the 0 MEL control group (0.0 ± 0.0
pulses/6 h) as well as in the 25 MEL group (0.0 ± 0.0 pulses/6
h). In contrast, in the 77 MEL group, LH pulse frequency was
dramatically increased (5.4 ± 1.1 pulses/6 h) compared to that
during the LD period in the same group (P < 0.01). As
a result, at the end of the experiment, LH pulse frequency was higher
in the 77 MEL group than in the two other groups (P <
0.01; Fig. 6).
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).
Effect of melatonin treatment on hypothalamic TH activity and
catecholamine content. ME: ME TH activity in the 77 MEL
group was 50% and 41% lower than those in the 0 MEL control and 25
MEL groups, respectively (P < 0.01 for both
comparisons), and there was no significant difference between the 0 MEL
control group and the 25 MEL group (Fig. 7).
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). The dopamine and DOPAC contents in
the 25 MEL group were not different from the contents in the 0 MEL and
75 MEL groups. Melatonin implants had no effect on noradrenaline
content.
A12 and A15 nuclei: Melatonin implants had no effect on TH
activity (Fig. 8) or the dopamine, DOPAC, and
homovanillic acid contents of these two structures. In the same way,
noradrenaline content was not affected by melatonin treatment in the
area of the A15 nucleus. In the area of the A12 nucleus, there was an
effect of melatonin implants on noradrenaline content
(P < 0.05). The noradrenaline concentration in the 25
MEL group was 2-fold greater than that in the 77 MEL or the 0 MEL
control groups (9.8 ± 2.0 µg/g tissue in the 25 MEL group
vs. 5.0 ± 0.6 and 5.3 ± 0.5 µg/g tissue in the
77 MEL and 0 MEL, respectively; P < 0.05 for both
comparisons).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our second experiment suggests that melatonin can mimic the effect of short days on ME dopaminergic activity, indicating a potential implication of the ME dopaminergic pathway in the photoperiodic regulation of LH secretion. In the present study, however, short day-like effects of melatonin on gonadotropic secretion were obtained by melatonin implants maintaining permanently high levels of this hormone (Exp 2). It has been suggested that this type of melatonin treatment produces an absence of photoperiodic information, rather than an active signal (31). It is noteworthy that within the limits of our experiments, the kinetics of the changes in TH activity at the level of the ME were similar between short day exposure and melatonin implants, which suggests that melatonin implants act in the same way as short days on ME catecholaminergic synthesis. It could also be argued that the effects observed in the 77 MEL group result from a longer exposure to long days in this group compared to the other groups, because in that experiment, the total duration of LD exposure changed according to the duration of melatonin treatment. However, it is now recognized that in LD-maintained ewes the effect of melatonin implants on gonadotropin secretion is a specific action of melatonin and does not result from a prolonged exposure to LD (32). Furthermore, in our previous experiment, investigating the effect of photoperiod on hypothalamic catecholaminergic activity (14), TH activity in the ME was measured after 140 LD and 65 SD, and the difference observed between these two groups was very similar to that observed between the control groups exposed to 80 to 85 LD and either the 75 SD (Exp 1) group or the 77 MEL groups (Exp 2; i.e. 157 LD, including 77 days of melatonin treatment). It, therefore, appears that the changes in TH activity observed in the present study reflect a response to the melatonin treatment rather than a difference in the duration of exposure to LD.
Exp 2 constitutes the first demonstration in sheep that melatonin can control dopamine synthesis of the dopaminergic pathway, which is probably implicated in the inhibition of pulsatile LH secretion (14, 33). It is noteworthy that in the ram, dopamine has been proposed to mediate the effect of melatonin on FSH secretion (20). An inhibitory effect of melatonin on TH activity of the neurointermediate lobe of the pituitary gland has been previously described in the hamster. However, this melatonin-induced change in TH activity seemed to be related to noradrenergic rather than dopaminergic regulation and did not show any temporal link with the melatonin-induced changes in testis weight (34). In the hamster, SD exposure was reported to reduce dopamine and noradrenaline turnover in the ME (35). It has also been demonstrated that 10 weeks of daily afternoon melatonin injection in LD-maintained female hamsters induce a decrease in the ME dopamine content, probably resulting from a decrease in dopamine synthesis (36). Suppression of LH and FSH release in this species is presumably related to reduced noradrenaline activity, whereas reduced dopamine turnover may represent a consequence of suppressed PRL release (37, 38). In the ewe, our results suggest that the melatonin-induced decrease in TH activity at the level of the ME is involved in the stimulation of LH pulsatile secretion; however, they do not allow exclusion of the possibility that they are the consequence of suppressed PRL release. Indeed, PRL could act on dopaminergic neurons to stimulate dopamine synthesis, so that a SD-induced decrease in PRL secretion would lead to a decrease in dopamine turnover. However, this positive feedback effect of PRL on ME dopaminergic activity is usually described as an acute mechanism involved in the short term regulation of PRL secretion (preovulatory surge) (39, 40). Thus, the long latency observed between the decrease in PRL secretion (detectable after 5 SD) and the inhibition of dopaminergic activity (occurring between 25 and 77 SD) makes this hypothesis unlikely.
Our present results strongly suggest that the dopaminergic pathway of the ME constitutes a step in the photoperiodic regulation of LH secretion that mediates the effects of melatonin on LHRH neurons. The photoperiod, through melatonin, could regulate the activity of the dopaminergic neurons reaching the LHRH terminals in the ME (41). These neurons should, therefore, inhibit LHRH release in the portal vessels. Such a hypothesis is strengthened by in vitro studies showing that dopamine can inhibit LHRH secretion from the ovine ME in perifusion (41). A direct inhibitory action of dopamine on LHRH release assumes that changes in dopaminergic activity at the level of the ME occur at the same time as changes in pulsatile LH secretion. Our experiments do not allow us to address this point because we have no data for these two parameters between 25 and 76 SD or 25 and 77 days of melatonin treatment. Moreover, although dopamine contents in the ME tended to decrease in parallel with TH activity, this decrease was not significant in Exp 1. It is, therefore, not clear whether the SD-induced changes in TH activity are related to the regulation of a dopaminergic pathway. It is noteworthy, however, that in our previous studies (14, 19) as well as in Exp 2 of the present study, the SD- or melatonin-induced decrease in TH activity was associated with a significant decrease in the dopamine, but not noradrenaline, content in the ME. The reason for this discrepancy in the changes in dopamine content between Exp 1 and our other experiments is not clear; the failure to detect a reduction in dopamine content in Exp 1 may be due to a higher variability between animals, but this point needs to be clarified. Indeed, it has been shown in the male hamster, that SD induce an inhibition of noradrenaline turnover in the ME (37). In this species, however, SD are associated with an inhibition of LH secretion that is temporally linked to this decrease in noradrenaline turnover. Thus, noradrenaline appears to stimulate LH secretion in the male hamster. Such a stimulatory function of noradrenaline in the ME is not consistent with the decrease in TH activity, and potentially noradrenaline synthesis, observed during the activation of LH secretion in the ewe.
The catecholaminergic cell bodies of the neurons in which TH activity is controlled by photoperiod are not identified in these experiments. Indeed, the ME in the ewe contains dopaminergic terminals, but no cell bodies (42). Furthermore, our present experiment did not show any photoperiod-induced changes in TH activity in the A15 dopaminergic nuclei implicated in estradiol negative feedback on the gonadotropic axis (43, 44). Yet, changes in gonadotropic axis responsiveness to estradiol negative feedback constitute one of the main mechanisms of the photoperiodic regulation of pulsatile LH secretion. Photoperiodic changes in the activity of dopaminergic structures involved in the expression of the estradiol negative feedback, such as the A15 nucleus, were, therefore, expected. Relative to this, it has been shown that estradiol increases TH activity in LD-treated ewes (45) and induces c-fos gene expression in the TH-immunoreactive cells of this structure in a season-dependent manner (46). It is noteworthy that the lack of changes in TH activity in the areas of both A12 and A15 persisted in Exp 2 despite the improvement in our brain-sampling method to focus on a restricted area according to the location of these nuclei.
It appears that the photoperiodic regulation of ME dopaminergic activity does not participate in the regulation of PRL secretion. In contrast, the parallel kinetic in the changes in TH activity and LH pulsatile secretion in response to SD treatment strongly suggest that the regulation of ME dopaminergic activity can be a key component of the photoperiodic regulation of pulsatile LH secretion. Importantly, our second experiment clearly demonstrated that melatonin can mimic the effects of photoperiod on ME dopaminergic activity. This is a required condition for the consideration of dopamine as a step in the photoperiodic regulation of pulsatile LH secretion. We, therefore, can hypothesize that photoperiod regulates LH secretion at least in part via a melatonin-dependent modulation of the activity of dopaminergic inhibitory inputs on LHRH terminals in the ME. Further investigations, however, are required to study such a functional link.
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Acknowledgments |
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Footnotes |
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2 Present address: Reproductive Sciences Program, 11th Floor, 300
North Ingalls Building, Ann Arbor, Michigan 48109-0404. Supported by a
Ph.D. grant from Région Centre and Institut National de la
Recherche Agronomique.
Received July 30, 1996.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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