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Medical Research Council, Reproductive Biology Unit, Centre for Reproductive Biology, Edinburgh EH3 9EW, Scotland, United Kingdom
Address all correspondence and requests for reprints to: Dr. Domingo J. Tortonese, Department of Anatomy, University of Bristol, Southwell Street, Bristol BS2 8EJ, England, United Kingdom.
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Abstract |
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Introduction |
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The participation of EOP in the inhibitory regulation of GnRH secretion is also well documented. In sheep, opioid-binding sites were detected in the hypothalamus (11) and ß-endorphin immunoreactive neurons were identified in the arcuate nucleus with fiber tracts extending to the preoptic area and median eminence (12, 13). Moreover, central blockade of EOP systems with opioid receptor antagonists placed in the mediobasal hypothalamus or in the preoptic area stimulated pulsatile secretion of LH in sexually active animals (14, 15). The activity of inhibitory EOP systems also appears to be affected by photoperiod but, in contrast to DA, the EOP tone seems to be enhanced under SD during the sexually active phase. This concept is supported by studies showing that treatment with an opioid antagonist stimulated pulsatile LH secretion in sexually active rams under SD but had little or no effect when given to sexually inactive animals exposed to LD (16, 17).
The experimental evidence therefore indicates that the activity of DA systems is enhanced in photoinhibited animals exposed to LD when the EOP tone is reduced and GnRH secretion suppressed, whereas the activity of DA systems is reduced in photostimulated animals exposed to SD when the EOP tone is increased and GnRH secretion is at maximum. Based on these observations, in this study we propose that inhibitory DA pathways interact with EOP networks to regulate GnRH/gonadotropin secretion in sheep and that photoperiod modulates this interplay between DA and EOP systems as part of the mechanisms involved in the photoperiodic control of the seasonal reproductive cycle. The hypothesis was tested in vivo by evaluating the ability of a specific DA-D2 receptor agonist (bromocriptine) or antagonist (sulpiride) to modify the pulsatile LH response to an opioid antagonist (naloxone) in rams exposed to SD (sexually active) or exposed to LD (sexually inactive). DA-D2 receptors have been shown to be specifically implicated in the inhibitory regulation of GnRH/LH secretion in sheep (4). Based on the preceding hypothesis, the predictions were that the effects of naloxone would be greater under SD compared with LD and that this response to the EOP antagonist would be prevented by pretreatment with the DA agonist. Conversely, it was anticipated that blockade of DA systems with the DA antagonist would enhance the response to the EOP antagonist only under LD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Drugs
Bromocriptine (DA-D2 receptor agonist) was
generously provided by Sandoz Products Ltd.
(Feltham, Middlesex, UK); it was dissolved, immediately before
administration, in a solution containing 0.1 M tartaric
acid, 15% ethanol, and 0.9% NaCl. Sulpiride (DA-D2 receptor
antagonist) was purchased from Sigma Chemical Co. Ltd.
(Poole, Dorset, UK); it was dissolved, within 24 h of
injection, in 0.1 M tartaric acid, 0.01 M NaOH,
and 0.9% NaCl. Naloxone (opioid receptor antagonist) was purchased
from Sigma Chemical Co. Ltd. and dissolved in saline on
the day of injection.
Experimental strategy and design
The acute effects of the DA-D2 receptor agonist
bromocriptine or of the DA-D2 receptor antagonist
sulpiride, on the pulsatile LH response to the EOP receptor antagonist
naloxone were investigated in sexually active Soay rams exposed to SD,
and then reassessed in the same animals when sexually inactive under
LD.
Exp 1. At 12 weeks into SD, sexually active rams were randomly assigned to the following treatments: 1) control (Control), 2) bromocriptine (Brom), 3) sulpiride (Sulp), 4) naloxone (Nal), 5) pretreatment with bromocriptine followed by naloxone (Brom+Nal), and 6) pretreatment with sulpiride followed by naloxone (Sulp+Nal). There were eight animals in each experimental group. In the Control group, rams received (s.c.) an injection of vehicle (0.1 M tartaric acid in saline). In groups Brom and Brom+Nal, bromocriptine was injected sc at a dose of 0.06 mg/kg; this dose was chosen on the basis of its suppressive effects on pulsatile LH secretion in a previous study in Soay rams (5). In groups Sulp and Sulp+Nal, animals received sc 0.59 mg/kg sulpiride; this dose was previously shown to be effective in stimulating pulsatile LH secretion in this breed of sheep, with no adverse side effects (5). In groups Nal, Brom+Nal, and Sulp+Nal, naloxone was administered iv at a dose of 1.6 mg/kg; the selection of this dose was based on its reported effectiveness in stimulating pulsatile secretion of LH in Soay rams (16, 17). In groups Brom+Nal and Sulp+Nal, bromocriptine or sulpiride was injected 1 h before (-1 h) the administration of naloxone, which was given at 0 h concomitantly with the single treatments in the remaining four groups (i.e. Control, Brom, Sulp, and Nal). The acute effects of treatments on the pulsatile secretion of LH were evaluated by measuring the concentrations of LH in serial blood samples taken at 10-min intervals over a 6-h period starting at the time of vehicle/drug injections (0 h). On the previous day blood samples were also taken, every 10 min for 6 h, after an injection of saline, to assess concentrations of LH as a double control within animal. Samples were collected from indwelling jugular cannulae inserted 48 h before the experiment, transferred into heparinized tubes, and centrifuged within 30 min; the plasma was stored at -20 C until concentrations of LH were determined by RIA.
Exp 2. All animals of Exp 1 remained under SD for a further 4-week period and were then exposed to LD; this change in photoperiod is known to induce a sexually inactive state (18). After exposure to LD for 8 weeks, during the peak of sexual inactivity, each animal was assigned to the same treatment as in Exp 1. The effects of treatments on the pulsatile secretion of LH were monitored in serial blood samples collected as described previously for SD.
Hormone assays
Concentrations of LH in plasma were measured in 200-µl
aliquots by RIA (19). The limit of detection (90% B/Bo), expressed as
NIH-LHs 18 (National Institute of Diabetes and Digestive and Kidney
Diseases, Bethesda, MD), was 0.2 ng/ml. The antibody (ASMN R 29) was
used at a working dilution of 1:120,000. The mean intra- and interassay
coefficients of variation (CV) for a total of 17 assays were 7.5 and
9.8%, respectively, based on low, medium, and high quality control
samples.
Statistical analyzes
In both experiments, the effects of individual treatments on the
secretion of LH were first examined by ANOVA for repeated measurements
over time and then, for all treatments, by one-factor ANOVA followed by
Dunnett’s test using the General Linear Model Procedure of the
Statistical Analysis System. LH pulses were identified following
the previously reported criteria (20) of two consecutive high values of
which at least one must exceed the mean of the preceding two values by
at least twice the intraassay CV. The amplitude of each pulse (peak
value minus the mean of the two values preceding the pulse) was
calculated, and the differences between treatments were examined by
ANOVA followed by Fisher’s test. Due to the nonparametric nature of LH
pulse frequency, Kruskal Wallis test and Wilcoxon and match pairs
signed and rank test were used to determine treatment effects on this
variable.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(temporal patterns) and
illustrated for all animals in both Fig. 2 (mean concentrations) and Table 1 (LH pulse frequency and amplitude).
Control rams showed a typical pattern of LH secretion during the
sexually active state with approximately one pulse every 2 h
(2.9 ± 0.3 pulses/6 h). The DA agonist bromocriptine
significantly suppressed this episodic release of LH (1.1 ± 0.4
pulses/6 h; P < 0.01), while blockade of DA receptors
with sulpiride had no effect (2.7 ± 0.2 pulses/6 h). In contrast,
under this photoperiod, the EOP antagonist naloxone evoked a
significant stimulation of pulsatile secretion of LH (5.1 ± 0.5
pulses/6 h; P < 0.01). This stimulatory effect was
completely prevented by the pretreatment with the DA agonist in
Brom+Nal rams (1.6 ± 0.4 pulses/6 h), but not affected by the
preadministration of the DA antagonist in Sulp+Nal animals (5.1 ±
0.4 pulses/6 h).
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) were clearly reflected
on the mean concentrations of this hormone in plasma (Control,
0.95 ± 0.08; Brom, 0.45 ± 0.06; Sulp, 0.96 ± 0.14;
Nal, 1.96 ± 0.35; Brom+Nal, 0.51 ± 0.08; and Sulp+Nal,
1.85 ± 0.29 ng/ml; P < 0.01; Fig. 2). These
effects were mainly accounted for by changes in LH pulse frequency
(Table 1). However, the administration of the DA agonist, in Brom and
Brom+Nal groups, also induced a significant reduction in LH pulse
amplitude (P < 0.01; Table 1). It is important to note that vehicle administration did not affect the pattern of LH secretion in Control rams when compared with an injection of saline on the previous day (within-animal control, 0.93 ± 0.08 vs. 0.95 ± 0.08 ng/ml for saline and vehicle, respectively). Similarly, there were no differences across groups on the day before the experiment when all animals received saline (Control, 0.93 ± 0.08; Brom, 0.85 ± 0.07; Sulp, 0.83 ± 0.08; Nal, 0.82 ± 0.09; Brom+Nal, 0.94 ± 0.06; and Sulp+Nal, 0.78 ± 0.08 ng/ml; P > 0.01).
Effects of treatments on pulsatile LH secretion in sexually
inactive animals exposed to LD (Exp 2)
The temporal LH response to treatments under LD is illustrated for
individual representative animals in Fig. 1, while the overall response
on mean concentrations of LH and the effects of treatments on LH pulse
frequency and amplitude are presented in Fig. 2 and Table 1,
respectively. Control rams showed a characteristic pattern of LH
secretion for sexually inactive animals, with low basal concentrations
(0.24 ± 0.03 ng/ml) and sporadic low-amplitude LH episodes
(0.9 ± 0.3 pulses/6 h). Whereas treatment with
bromocriptine had no significant effect on this pattern of
LH release (0.24 ± 0.04 ng/ml; 0.4 ± 0.3 pulses/6 h), the
administration of sulpiride resulted in a marginal increase in the
frequency of LH pulses (1.4 ± 0.5 pulses/6 h) but not in mean LH
concentrations (0.25 ± 0.03 ng/ml). Indeed, sulpiride
statistically (P < 0.05) increased the frequency of LH
pulses only within group (i.e. when compared with the
effects of saline treatment in the same animals on the previous day:
0.7 ± 0.3 vs. 1.4 ± 0.5 pulses/6 h for saline
and sulpiride, respectively). Treatment with naloxone induced a rapid
and transient increase in the secretion of LH (0.48 ± 0.09 ng/ml;
P < 0.01), which was normally observed as a single
pulse immediately after injection (1.5 ± 0.2 pulses/6 h). This
response was blocked by pretreatment with the DA agonist in Brom+Nal
rams (0.23 ± 0.03 ng/ml; 0.6 ± 0.3 pulses/6 h), resulting
in a pattern of LH release that did not differ from those observed for
Control or Brom animals. Under this photoperiod, however, the
pretreatment with the DA antagonist (which on its own statistically
increased the frequency of LH pulses only within animal), significantly
enhanced the response to the EOP antagonist in Sulp+Nal rams (1.32
± 0.41 ng/ml), leading to an increase in LH pulse frequency (2.0
± 0.2 pulses/6 h; P < 0.01) and high-amplitude LH
pulses throughout the duration of the sampling period.
With the exception of sulpiride, the effects of treatments on the
secretory pattern of LH were reflected on the mean concentrations of
this hormone in plasma and accounted for by reciprocal changes
in LH pulse frequency. However, the stimulatory effects of Sulp+Nal
also resulted in a significant increase in LH pulse amplitude
(P < 0.01; Table 1).
As for Exp 1, vehicle administration in Control rams had no effect on LH secretion when compared with an injection of saline on the previous day (within-animal control; 0.26 ± 0.02 vs. 0.24 ± 0.03 ng/ml for saline and vehicle, respectively). Moreover, all groups showed similar LH values on the day before the experiment when the animals were treated with saline (Control, 0.26 ± 0.02; Brom, 0.24 ± 0.03; Sulp, 0.21 ± 0.02; Nal, 0.23 ± 0.03; Brom+Nal, 0.21 ± 0.02; Sulp+Nal, 0.29 ± 0.03 ng/ml; P > 0.01).
The administration of bromocriptine, sulpiride, or naloxone alone or in combination did not result in adverse side effects under either photoperiod.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous pharmacological studies have shown that inhibitory DA systems participate in the photoperiodic control of GnRH/gonadotropin secretion in both rams and ewes (3, 5, 8, 21, 22, 23, 24). Moreover, in the ewe, disruption of DA pathways by radiofrequency lesions increased pulsatile LH release (25), while frontal hypothalamic deafferentation blocked the photoperiod-induced changes in LH secretion and caused some animals to cycle continuously (26, 27). In agreement with those reports, the suppressive effects of DA on GnRH/LH and the modulatory role of photoperiod in this mechanism of control were corroborated in the current study. Bromocriptine suppressed pulsatile secretion of LH in rams exposed to SD, but was without effect under LD. In turn, sulpiride did not affect the secretory pattern of LH under SD, but increased the frequency of LH pulses under LD. It should be noted, however, that the magnitude of the LH response to sulpiride under LD was smaller than anticipated and that it reached statistical significance only when compared with the effects of vehicle in the same animals. This is in contrast to previous work where a robust increase, both in mean concentrations of LH and in LH pulse frequency, had been observed in response to this antagonist under LD (5). It must be brought into consideration, however, that in the current study the DA antagonist was administered at an earlier stage in the reproductive cycle (8 vs. 10 weeks after a shift from SD), i.e. at the peak of sexual inactivity and when the DA tone is thought to be highest; therefore, a larger dose may have been required to counteract the suppressive effects of DA at this stage. Nonetheless, the concept that an increase in DA tone is induced by LD is indeed further supported by recent observations showing that the content of DA (9, 10) and the synthesis activity of catecholaminergic neurons (10, 28) in the ewe median eminence are higher under LD than under SD. The involvement of DA in the photoperiodic control of LH release is likely to imply direct actions on GnRH neurons; DA axons were shown to synapse with GnRH terminals in the ewe median eminence (2) and the placement of microimplants of sulpiride or bromocriptine in this region of the brain modified the stimulatory effects of microimplants of melatonin on the GnRH/gonadotrophic axis in sexually inactive rams exposed to LD (29). Taken together, the results of the current study and those of previous reports support the notion that DA systems are involved in the photoperiodic control of GnRH/gonadotropin secretion in sheep and that a full activation of these inhibitory neural systems is induced by LD.
Blockade of opioid receptors with naloxone evoked an immediate and robust increase in the pulsatile release of LH in sexually active rams exposed to SD; the response to naloxone was smaller under LD when the animals were sexually inactive. These results support the hypothesis proposing that, in contrast to DA, the activity of inhibitory EOP systems is increased under SD. The results are also in agreement with previous reports in gonadal-intact animals showing potent stimulatory effects of naloxone on LH secretion during the sexually active phase under SD but not during reproductive quiescence under LD (16, 17, 30). The opioid inhibition of LH release under SD reflects central inhibitory effects of hypothalamic EOP systems on GnRH. Naloxone stimulated GnRH secretion in ewes and rams (31, 32, 33), and the placement of microimplants of naloxone in the hypothalamus or the infusion of the antagonist into the lateral cerebral ventricles increased pulsatile LH release in the ewe (14, 15). Taken together, these observations and the results of the present study support the concept that the activity of inhibitory EOP systems is modulated by photoperiod and that these systems become fully active under SD.
The hypothesis tested in this study was that DA and EOP systems
interact to regulate GnRH/gonadotropin secretion in sheep and that
photoperiod modulates this interaction to relay its effects on the
seasonal reproductive cycle. Specifically, we proposed that DA pathways
are inhibitory not only to GnRH neurons but also to EOP networks (Fig. 3). Based on this hypothesis, the
photoperiod-induced changes in DA tone would result in reversed changes
in EOP tone. This is based on the aforementioned observations that the
increase in DA activity induced by LD, which leads to suppression of
GnRH/LH release and to reproductive quiescence, is concomitant with a
reduction in EOP tone, whereas the decrease in DA activity induced by
SD that results in activation of the GnRH/LH/gonadal axis is
concomitant with an increase in EOP tone. The results of the current
study support this hypothesis. The potent stimulation of LH release
induced by naloxone under SD was negated by the pretreatment with
bromocriptine. This implies that activation of DA
receptors at this stage leads to suppression of EOP pathways, so that
blockade of inhibitory EOP systems no longer results in stimulation of
GnRH/LH release. Conversely, the preadministration of sulpiride
significantly enhanced the stimulatory effects of naloxone observed
under LD. Furthermore, the preadministration of sulpiride did not
modify the response to naloxone under SD. The interpretation is that
blockade of DA receptors under LD, when the DA tone is increased, leads
to derepression of inhibitory EOP systems so that subsequent blockade
of these systems with naloxone induces a potent stimulation of GnRH/LH
release as observed under SD. It is unlikely that, as recently proposed
for the rat (34), the interaction between these two neuronal systems
occurs in the reverse order, i.e. that EOP pathways are
inhibitory to DA networks, because in this species DA antagonists were
shown to increase ß-endorphin immunoreactivity in the hypothalamus
(35), and DA agonists or DA itself inhibited ß-endorphin hypothalamic
content and release through DA-D2 receptors (36, 37). Moreover,
previous studies from this laboratory have shown that in the ram,
blockade of DA systems with sulpiride provoked a marked increase in
ß-endorphin release which, in addition, was significantly higher
under LD than SD (38). This implies that inhibitory DA pathways play an
important role in the photoperiodic regulation of GnRH/LH secretion in
sheep, both directly and indirectly through an interaction with EOP
systems. In turn, the EOP are more likely to be associated to the
modulation of the dynamics of GnRH release and the synchronization of
the activity of GnRH neurons. This latter theory is supported by recent
studies showing that treatments with naloxone affected the shape and
duration of GnRH pulses in portal blood of breeding season ewes (33)
and the GnRH pulse amplitude in push-pull perfusates of the ewe median
eminence during the follicular phase of the estrous cycle (39).
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The pituitary gland itself may represent another target for the control of gonadotropin secretion by DA and EOP. Even though in the current study the effects of single or combined treatments on mean concentrations of LH primarily reflected similar effects on LH pulse frequency, there were also significant effects on LH pulse amplitude. Specifically, bromocriptine suppressed LH pulse amplitude under SD, both when given on its own and in combination with naloxone, while a significant increase in the amplitude of LH pulses was observed in animals receiving the preadministration of sulpiride followed by naloxone under LD. The suppressive effects of bromocriptine on LH pulse amplitude may reflect indeed a pituitary effect; not only have DA-binding sites been identified in the pituitary gland (50, 51, 52), but a subpopulation of gonadotrophs were reported to contain DA receptors and treatment with DA was shown to reduce the LH response to exogenous GnRH in pituitary stalk-transected ewes (53). In contrast, in view of the aforementioned effects of naloxone on GnRH pulse size (33), the increase in LH pulse amplitude observed under LD in response to the combined treatment is more likely to reflect central actions within the hypothalamus, although a possible pituitary effect cannot be completely ruled out. Nevertheless, in view of the fact that LH pulses are highly correlated to GnRH pulses in both ewes (54, 55) and rams (56), the possibility that the pituitary may represent another target for the actions of DA and EOP in the photoperiodic regulation of gonadotropin secretion in sheep would be of relatively minor physiological significance.
In conclusion, the results of this study are consistent with the hypothesis proposing the existence of an interaction between DA and EOP systems in the photoperiodic regulation of gonadotrophin secretion in sheep. To our knowledge, this is the first report showing that while DA and EOP both act to inhibit GnRH/gonadotropin release, inhibitory DA pathways also interact with EOP networks and that changes in day length modulate this neuronal interplay as part of the mechanisms involved in the photoperiodic control of the seasonal reproductive cycle. The results help to explain the paradox that the EOP tone is apparently reduced in photoinhibited animals.
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Acknowledgments |
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Received May 29, 1998.
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References |
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opiate receptor subtypes. J
Neuroendocrinol 8:771–776[CrossRef][Medline]
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,
Statistically significant pulses.
