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Biology of Reproduction 59, 960-968 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Pulsatile Luteinizing Hormone Secretion in the Ovariectomized, Thyroidectomized Red Deer Hind Following Treatment with Dopaminergic and Opioidergic Agonists and Antagonists1

Greg M. Anderson3,a, , and Graham K. Barrell2,a

a Animal and Food Sciences Division, Lincoln University, Canterbury, New Zealand


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Two experiments were conducted to determine whether dopaminergic or opioidergic pathways are modulated by thyroid gland secretions for seasonal suppression of LH secretion in red deer hinds. Ovariectomized (n = 5) or ovariectomized and thyroidectomized (n = 4) hinds, treated with estradiol implants, received the dopamine agonist bromocriptine or the antagonist sulpiride during pulse bleeds in July (breeding season) and October (nonbreeding season). Comparison of July and October mean plasma LH concentration (3.5 ± 1.3, 0.7 ± 0.1 ng/ml, respectively), pulse frequency (1.9 ± 0.4, 0.7 ± 0.2 pulses/4 h), and pulse amplitude (1.3 ± 0.5, 0.7 ± 0.02 ng/ml) showed lower (p < 0.05) levels in October, and these levels were not significantly affected by thyroidectomy or drug treatment. In the absence of estradiol implants, the hinds received bromocriptine or morphine during the breeding season (July) and their antagonists, sulpiride or naloxone, respectively, in the nonbreeding season (November). In euthyroid hinds there was a seasonal decrease (p < 0.05) in mean plasma LH concentration, pulse frequency, and pulse amplitude, which did not occur in thyroidectomized hinds. There were no effects of drug treatment on LH concentration except for a small increase following sulpiride in November. Plasma prolactin concentration was significantly increased by antagonists and decreased by agonists on most occasions. We conclude that in red deer hinds, seasonal regulation of LH secretion does not involve dopamine or endogenous opioids and the thyroid gland is required specifically for LH suppression in the absence of estradiol.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The red deer (Cervus elaphus) hind exhibits a circannual cycle of reproductive activity, with periods of estrous cyclicity occurring during winter under shortened ambient photoperiod [1–3], similar to that of the ewe [4, 5]. However, recent studies indicate that differences in seasonal gonadotropin secretion patterns exist between the two species. Red deer exhibit dramatic seasonal fluctuations of LH secretion even in the absence of gonadal steroids [3], as well as in exogenous GnRH-induced LH secretion [3, 6], whereas the seasonal oscillations in these parameters in the domestic ewe are considerably smaller [7–10] or even undetectable [11, 12], and seasonal reproductive quiescence is usually attributed primarily to the dramatic increase in negative feedback of estradiol on the hypothalamic GnRH pulse generator (e.g., [12]).

Little is known about the neuronal control of GnRH secretion in deer. Since the vast majority of the GnRH neurons do not possess estradiol receptors in the ewe [13, 14] and other species [15–17], it is generally considered that the effects of this steroid in suppressing tonic LH secretion during anestrus must be relayed via other neurons. Considerable evidence has been obtained over the last 15 years to show that dopaminergic neurons fulfil this role in the ewe [18–20] and ram [21, 22]. This has been most clearly demonstrated by measuring changes in LH secretion in response to injection of receptor agonists and antagonists. In such experiments, dopaminergic D2 receptor agonists are able to suppress LH transiently during the breeding season, while in the nonbreeding season D2 receptor antagonists overcome LH suppression (e.g., [21]).

Several reports have indicated that opioid peptides are also able to suppress LH concentration in ewes during the nonbreeding season in the absence of gonadal steroids [23–25], while others disagree with this finding [26–28]. The ovariectomized red deer hind presents a unique model for studying such "steroid-independent" mechanisms of reproductive suppression due to the large seasonal fluctuations in plasma LH concentrations exhibited by this species.

The seasonal transition to the state of reproductive quiescence in the ewe [29–31] and red deer stag [32] has been shown within the last decade to be dependent on the presence of thyroid hormones. In the ewe, this role is specifically manifested as a disruption of onset of estradiol-induced GnRH suppression in animals thyroidectomized during the breeding season [33] so that LH concentrations remain elevated throughout the nonbreeding season [29, 30, 34–36]. Since the mechanism by which thyroid hormones elicit seasonal transitions is not yet understood, we wished to investigate whether the thyroid glands are required for the development of inhibitory neuronal pathways at the end of the breeding season in red deer hinds.

In the first experiment reported here, we used a receptor agonist and antagonist to determine whether thyroidectomy affects the LH secretory response to dopaminergic analogues in ovariectomized estradiol-treated red deer hinds during anestrus. The second experiment tested whether the thyroidectomy affects the response to dopaminergic or opioidergic analogues in the absence of gonadal steroids. As it is well established that prolactin secretion is modified by dopaminergic and opioidergic agonists and antagonists [23, 37–39], prolactin responses to these drugs were measured in addition to LH as an indicator of their biological effectiveness.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Management

Ten mature red deer hinds (mean live weight at the beginning of the study 86.7 ± 4.4 kg) were ovariectomized (n = 5) or ovariectomized and thyroidectomized (THX) (n = 5) early in the breeding season (May 1995). Ovariectomy was performed aseptically by midventral laparotomy under general anesthesia induced by 250 mg i.v. sodium thiopentone (Pentothal; Techvet Laboratories Ltd., Auckland, New Zealand) following recumbency induced by 114 mg i.m. xylazine hydrochloride (Thiazine 50; RWR Veterinary Products Pty Ltd., NSW, Australia). Thyroidectomy was performed as previously described [40] using sodium thiopentone and xylazine hydrochloride as above. Hinds were maintained outdoors (lat 43°39'S) on pasture of predominantly ryegrass and white clover for the following two years. Ryegrass silage was fed as a supplement during winter. To monitor thyroid status, blood samples were collected monthly by jugular venepuncture into 10-ml heparinized tubes for measurement of plasma total tri-iodothyronine (T3). During intensive collection periods, blood samples were drawn via indwelling 14-gauge jugular catheters (Terumo, Tokyo, Japan) into 10-ml syringes and heparinized within 4 min. Plasma was removed after centrifugation at 1500 x g and stored at -20°C until assayed. All procedures were approved by the Lincoln University Animal Ethics Committee, and the experiments in this paper were conducted in accordance with the Guiding Principles for Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.

Experiment 1

To determine whether the thyroid gland is required for a dopaminergic neuronal pathway to mediate estradiol-induced suppression of LH in red deer during the nonbreeding season, hinds were treated in early June 1995 with slow-release silicone rubber implants containing estradiol-17ß (Compudose 200; Elanco Animal Health, Auckland, New Zealand; cut transversely so that each hind received one third of an implant containing 8 mg estradiol-17ß s.c. in the right ear) and were challenged with a dopaminergic agonist and antagonist during the breeding and nonbreeding season as outlined below. Estradiol implants were removed between 1 August and 25 August as part of another experiment.

In July (midbreeding season), all hinds received an i.m. vehicle (3.0 ml of a 1:1 mixture of 15% ethanol and 0.1 M tartaric acid) injection followed 4 h later by either a single i.m. injection of the dopamine-D2 receptor agonist 2-bromo-{alpha}-ergocryptine methanesulfonate (bromocriptine, 0.06 mg/kg) or the dopamine-D2 receptor antagonist (-)-sulpiride (0.60 mg/kg). The ethanol/tartaric acid mixture was the vehicle for both drugs; both vehicle components have been shown separately not to affect LH concentrations in sheep [18, 27]. Blood samples were taken every 10 min for plasma LH analysis from the time of vehicle injection until 5 h after drug injection. Plasma prolactin concentration was measured at -240, -120, 0, 40, 80, 160, and 300 min relative to drug injections. Four days later, hinds received the same treatment but were injected with the other drug, using the same treatment regimen so that all animals received both agonist and antagonist. This procedure was repeated during late October (nonbreeding season). Drugs and dosages were selected for their ability to elicit changes in LH and prolactin concentrations and for absence of clinical side effects in similar studies in sheep and red deer [21, 41–44]. All drugs were purchased from Sigma Chemical Co. (St. Louis, MO).

Experiment 2

To determine whether thyroidectomy affects the LH secretory response to dopaminergic or opioidergic analogues in the absence of gonadal steroids, hinds were treated at least 3 mo after estradiol implant removal with receptor agonists during the breeding season and receptor antagonists during the nonbreeding season. In July 1996, all hinds were treated with 0.06 mg/kg bromocriptine i.m., and blood was sampled for determination of plasma LH and prolactin concentrations as for experiment 1, except that the vehicle was 3 ml of 15% ethanol. One week later, hinds were treated with a saline vehicle injection followed 4 h later by a single injection of 1 mg/kg morphine sulphate i.v. in 3.0 ml of 0.9% saline solution (David Bull Laboratories, Melbourne, Vic., Australia). Blood samples were taken at 10-min intervals from the time of vehicle injection until 4 h after morphine injection for measurement of plasma LH concentration and for measurement of plasma prolactin concentration at -240, -120, 0, 10, 20, 40, 80, and 160 min relative to morphine injection. Morphine was administered as described in other published experiments [45, 46], none of which reported any unwanted side-effects at this dose.

In early November (the time of maximal LH suppression in ovariectomized red deer hinds), receptor antagonists were injected according to exactly the same protocol and sampling regime as for the agonists during the breeding season. Antagonists for dopamine and opioid peptides were, respectively, sulpiride (0.60 mg/kg i.m.) and naloxone hydrochloride (2.5 mg/kg i.v.) (Sigma Chemical Co.). The vehicle for sulpiride was 3 ml 0.1 M tartaric acid, and for naloxone, 2 ml 0.9% saline solution. The dose of naloxone was based on previous studies in sheep [25, 45, 46].

Hormone Analysis

Plasma LH concentrations were measured in duplicate 100-µl aliquots by heterologous RIA, using the method described previously for sheep plasma [47] and validated for red deer plasma [48]. Values are expressed in terms of the ovine standard, NIAMDD-LH-S20; iodinated ovine LH (NIADDK-oLH-1–2) was used as tracer, and primary antiserum was NIADDK-anti-oLH-1 (AFP-192279) (all obtained through the National Hormone and Pituitary Program). The sensitivity (95% confidence limit at 0 ng/ml) averaged 0.4 ng/ml over the 18 assays used for these results. Intraassay coefficients of variation (CV) averaged 16.9% and 12.3%, respectively, for plasma pools displacing radiolabeled LH to approximately 84% and 64% of the total bound, and interassay CV were 26.6% and 17.5% for the same plasma pools. Serially diluted deer plasma produced a binding curve parallel to that of the ovine standard.

Total T3 was assayed using a commercially available kit (Coat-A-Count Total T3; Diagnostic Products Corp., Los Angeles, CA) in duplicate 100-µl aliquots. Serial dilution of deer plasma produced a binding curve in the assay that was parallel to the standard curve. Sensitivity of the assay averaged 0.04 nM over 5 assays. The average intra- and interassay CV were 10.1% and 10.7%, respectively, for plasma pools that displaced radiolabeled T3 to 62% and 24% of the total bound.

Plasma prolactin concentration was measured in triplicate 50-µl aliquots by ELISA as has been described previously [49]. NIADDK-o-PRL-19 was the ovine antigen for conjugation and the reference standard, in terms of which results are expressed. Rabbit anti-ovine prolactin primary antiserum was provided by Dr. D.F.M. van de Wiele. Assay sensitivity averaged 7.2 ng/ml (3 separate assays involving 21 ELISA microtiter plates), and intraassay CV averaged 14.1% for plasma pools displacing thyroglobulin-conjugated prolactin to 65%, 46%, and 27% of the total bound. Interassay CV averaged 9.9% for a plasma pool displacing thyroglobulin-conjugated prolactin to 27% of the total bound. The binding curve produced by serial dilution of deer plasma was parallel to that of the ovine standard.

Data Analysis

One THX hind became debilitated and died during 1995, and was not replaced until June the following year; data from this hind are excluded from the nonbreeding season of experiment 1.

Hormone concentrations below the average assay sensitivity were assigned a value equal to the sensitivity. LH pulse amplitude was calculated as the peak LH concentration minus that of the preceding nadir. A pulse of LH during intensive sampling periods was defined as any increase in concentration in which 1) concentrations were elevated relative to pre- and post-nadirs for at least 2 consecutive samples, 2) the pulse peaked within 2 sampling intervals, 3) the increment between peak and nadir concentrations exceeded by at least 2 standard deviations both the pre- and post-nadirs, and 4) the peak amplitude exceeded the sensitivity of the assay [50].

Responses to drug injections were identified using paired Student's t-tests to compare average post-drug plasma LH concentration with the average pre-drug concentration, or, in the case of plasma prolactin concentrations, with the concentration immediately before drug injection since a large diurnal variation was observed for this hormone. As thyroidectomy did not affect plasma concentration of prolactin or the prolactin response to drug challenges, these data were pooled for euthyroid and THX hinds. Pre-drug injection data from the two intensive sampling dates in each season were pooled before analysis of variance or paired t-tests to examine the effects of thyroidectomy and season, respectively, on mean plasma LH concentration, LH pulse frequency, LH pulse amplitude, and prolactin concentration. Hormone concentrations were transformed to their logarithms (base 10) before statistical analysis. Mean values are presented ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiments 1 and 2: Plasma T3 Concentration and Effects of Thyroidectomy

Plasma concentrations of total T3 were similar in experiments 1 and 2. In euthyroid hinds, a seasonal pattern of plasma total T3 concentration was observed, with a nadir concentration of 0.9 ± 0.1 nM in winter (June) and a peak concentration of 1.9 ± 0.2 nM in summer (p < 0.001). In contrast, mean plasma concentrations of total T3 for THX hinds were low (< 0.2 nM) at all times and often undetectable (average concentration over both experiments was 0.1 ± 0.1 nM).

Live weight did not differ significantly (p > 0.05) between THX and euthyroid hinds throughout both experiments. Thyroidectomy generally prevented molting of the pelage in spring and growth of the summer coat. There were no other obvious side-effects of hypothyroidism.

Experiment 1

Mean plasma prolactin concentration before drug injections in all hinds was lower during the breeding season (July) than during the nonbreeding season (October) (73.1 ± 17.5 ng/ml and 146.9 ± 32.8 ng/ml, respectively; p < 0.01). In both seasons, sulpiride caused an increase (p < 0.01) in mean plasma prolactin concentration, and bromocriptine decreased mean plasma prolactin concentration in the nonbreeding season (p < 0.001) but not in the breeding season, when concentrations were already low (Fig. 1).



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  		FIG. 1. Mean (± SEM) concentrations of plasma prolactin in response to bromocriptine (open circles) or sulpiride (solid circles) during July (breeding season; upper panel) or October (nonbreeding season; lower panel) in ovariectomized estradiol-implanted hinds in experiment 1. Open arrows, vehicle injection times; filled arrows, drug injection times. Data are pooled from 5 euthyroid and 4 THX hinds. Note the different scales on the graphs.

Nine-hour profiles of plasma LH concentrations from representative hinds are shown in Figure 2. Mean plasma LH concentration and pulse frequency during the period before drug injections were lower in the nonbreeding season than in the breeding season (p < 0.05) in both euthyroid and THX hinds, but pulse amplitude declined significantly (p < 0.05) in euthyroid hinds only (Table 1). In contrast to prolactin, mean plasma LH concentration, and pulse frequency and amplitude in all hinds were unaffected by bromocriptine or sulpiride in either season (p > 0.05; Fig. 3).



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  		FIG. 2. Individual plasma LH profiles representative of ovariectomized estradiol-implanted hinds in experiment 1 during bromocriptine (a) or sulpiride (b) challenges in the breeding season (upper panels) and nonbreeding season (lower panels). Profiles are representative of both euthyroid and THX hinds. Closed circles, peak of each pulse; open arrows, vehicle injections; filled arrows, drug injections.


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  		TABLE 1. LH pulsatility in ovariectomized estradiol-implanted hinds in Experiment 1 before drug injections.



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  		FIG. 3. Mean (± SEM) change in plasma LH concentrations (open bars), LH pulse frequency (shaded bars), and LH pulse amplitude (solid bars) in ovariectomized estradiol-implanted hinds in experiment 1 following bromocriptine or sulpiride during the breeding or nonbreeding season. There were no significant responses (p > 0.05; paired t-test). Data are pooled from 5 euthyroid and 4 THX hinds.

Experiment 2

In contrast to experiment 1, mean plasma prolactin concentration before drug injections was not significantly lower during the breeding season than during the nonbreeding season (271.8 ± 53.0 ng/ml and 253.4 ± 24.5 ng/ml, respectively; p > 0.05). Bromocriptine and morphine both caused a decrease (p < 0.01), whereas sulpiride and naloxone caused an increase (p < 0.01) in plasma prolactin concentration (Fig. 4).



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  		FIG. 4. Mean (± SEM) concentrations of plasma prolactin in response to bromocriptine (open circles) or morphine (open triangles) injections during July (breeding season; upper panel) and sulpiride (solid circles) or naloxone (solid triangles) injections during November (nonbreeding season; lower panel) in ovariectomized hinds in experiment 2. Open arrows, vehicle injection times; filled arrows, drug injection times. Data are pooled from 5 euthyroid and 5 THX hinds. Note the different scales on the graphs.

Profiles of plasma LH concentrations from representative hinds are shown in Figure 5. Mean plasma LH concentration, pulse frequency, and pulse amplitude during the period before drug injections were lower in the nonbreeding season than during the breeding season in euthyroid hinds (p < 0.05). In THX hinds, however, mean plasma LH concentration and pulse amplitude increased in the nonbreeding season (p < 0.05), whereas pulse frequency did not change (Table 2). Mean plasma LH concentration, and pulse frequency and amplitude in all hinds were unaffected by any of the drug treatments in either season (p > 0.05) except for a small increase in concentration (p < 0.05) following sulpiride in November (Fig. 6).



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  		FIG. 5. Individual plasma LH profiles representative of ovariectomized hinds in experiment 2 receiving bromocriptine (a) or sulpiride (b and c) and morphine (d) or naloxone (e and f) challenges in the breeding or nonbreeding season. Upper panels) Plasma LH profiles from the breeding season, representative of both euthyroid and THX hinds; middle panels) euthyroid profiles; lower panels) THX profiles during the nonbreeding season. Closed circles, peak of each pulse; open arrows, vehicle injection times; filled arrows, drug injection times.


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  		TABLE 2. LH pulsatility in ovariectomized hinds in Experiment 2 before drug injections.



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  		FIG. 6. Mean (± SEM) change in plasma LH concentrations (open bars), LH pulse frequency (shaded bars), and LH pulse amplitude (solid bars) in ovariectomized hinds in experiment 2 (n = 10) following drug challenges during the breeding or nonbreeding season. * Significant response (p < 0.05; paired t-test). Data are pooled from 5 euthyroid and 5 THX hinds.

Morphine caused panting and signs of anxiety (e.g., pacing behavior) in 3 of the 10 hinds, beginning approximately 30 min after injection and lasting up to 4 h.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To our knowledge, this study is the first of its kind to be reported for deer. Most of our present understanding of the neural pathways regulating seasonal reproductive cycles comes from experiments using the ewe [18]. Our results strongly suggest that the neural mechanisms that modify GnRH secretion may differ between the two species, since treatment with dopaminergic and opioidergic agonists and antagonists that previously have been shown to evoke clear LH responses in sheep failed to do so in deer.

In experiments 1 and 2, the dopamine agonist bromocriptine and antagonist sulpiride were administered at doses that have been shown to be effective in evoking LH responses lasting for over 4 h in rams [21]. Since bromocriptine suppressed LH concentration and pulse frequency only under short days, and sulpiride increased LH concentration and pulse frequency only under long days, it was concluded in that study, as in most similar studies using ovariectomized estradiol-treated ewes [18, 51, 52] or intact ewes [18, 53, 54], that an inhibitory dopaminergic neural system is activated during the nonbreeding season to suppress LH and bring about the sexually inactive state. In two studies using ovariectomized estradiol-treated ewes [55, 56] and one using intact or testosterone-treated castrated rams [56], there was no LH response to the dopamine antagonist pimozide. Although the results of these studies may initially appear to be comparable to the results of experiment 1, it must be noted that whereas sulpiride is a specific dopaminergic-D2 antagonist [57, 58], pimozide is less specific for these receptors and binds also to dopaminergic-D1 receptors in the brain [59]. In a comparative study of the two drugs, sulpiride elicited an LH response under long days in intact rams while pimozide was ineffective in this regard [21], and it was surmised that activation of dopaminergic-D1 receptor-mediated responses may possibly negate effects of dopamine on GnRH/LH secretion.

Since the lack of LH responses to bromocriptine and sulpiride in red deer hinds in experiment 1 is not in agreement with most comparable studies in intact or castrated steroid-treated sheep, our results may indicate that the neuromodulation of GnRH pulse generation differs between the two species. Although we cannot rule out a mere quantitative species difference in the dose of bromocriptine or sulpiride required to elicit an LH response in red deer hinds, our results suggest that dopaminergic-D2 receptor-mediated neural pathways are not important modulators of LH suppression in the presence of gonadal steroids in hinds. This conclusion is based on the following three reasons. First, the doses of bromocriptine and sulpiride were sufficient to generate robust changes in prolactin secretion, a response known to be activated by dopaminergic-D2 receptor agonists and antagonists in sheep [44] and red deer hinds [42, 43]. Although prolactin responses do not necessarily prove that the dose of drug used was sufficient to affect dopaminergic-D2 receptor-mediated pathways that mediate GnRH secretion, they do at least show that the drug treatments were effective in binding to dopaminergic-D2 receptors and evoking an appropriate biological response. Second, we have given bromocriptine at five times the dose used in the current experiment to ovariectomized estradiol-implanted hinds during the nonbreeding season, and LH secretion remained unchanged, even though we recorded similar prolactin responses to those shown here (unpublished). This indicates that the lack of an effect of bromocriptine in experiment 1 on LH secretion was unlikely to be due to an inadequate dose. Third, differences in neuromodulation of seasonal reproduction between sheep and other species are known to exist; for example dopaminergic agonists and antagonists do not evoke LH responses in the anestrous mare [60].

Most studies of dopaminergic neural pathways using the ovariectomized sheep without steroid replacement have concluded that the seasonal reproductive changes exhibited by this animal model are not mediated by dopamine [27, 51, 54]. This is in agreement with results from experiment 2, in which bromocriptine and sulpiride evoked prolactin but not pulsatile LH responses in ovariectomized hinds that had not received estradiol. Neural pathways that are not activated by gonadal steroids are of particular significance to red deer reproduction [3] since they appear to make a much larger contribution to seasonal breeding in this species than in the ewe. Therefore, in experiment 2 we examined the role of endogenous opioid peptides, as these appear to have an inhibitory role that is independent of estradiol in some [23–25] but not all [26–28] studies of seasonal LH secretion in ovariectomized sheep. As was the case for bromocriptine and sulpiride, pulsatile LH secretion did not change in response to morphine in the breeding season, or to naloxone, its antagonist, in the nonbreeding season, although plasma prolactin concentrations were significantly increased by morphine and decreased by naloxone. One possible explanation for the lack of LH response to these drugs is that endogenous opioid peptides suppress LH secretion during puberty in red deer hinds, but non-opioidergic systems progressively take over this role during post-pubertal maturation, as has been demonstrated in some studies in the ewe [25, 61], cow [62], and human [63]. Our results also do not preclude a role for opioids in inhibiting GnRH pulse size, as has been demonstrated by Goodman et al. [64] in ovariectomized ewes irrespective of estradiol treatment, since the effects of naloxone on episodic GnRH secretion in that study were not clearly reflected in episodic LH secretion and would be unlikely to be detected in studies, such as the current experiment, in which GnRH was not measured directly. Notwithstanding these considerations, our results indicate that endogenous opioid peptides do not play a major role in seasonal suppression of LH concentrations in the ovariectomized red deer hind.

As was noted previously in relation to bromocriptine and sulpiride, the prolactin responses to morphine and naloxone in the current experiment confirm that the dosages used were sufficient to elicit biological responses in this species. However, the response observed in our hinds was not the same as has been reported in studies of sheep [23, 38], in which morphine has stimulatory effects on circulating prolactin concentrations. Endogenous opioid peptides are thought to stimulate prolactin secretion by reducing dopamine release into the hypothalamo-pituitary portal circulation [65–68]; our results suggest that opioids do not act this way in the red deer hind. On all sampling dates, a decline in mean plasma prolactin concentration occurred during the first 4 h of sampling. As the mean plasma prolactin concentrations were considerably higher in these experiments than in many other reported studies using red deer (e.g., [42, 43]), it is possible that this progressive decline reflects a stress period at the start of sampling. The lower mean concentration of plasma prolactin in hinds in experiment 1 in the presence of estradiol, compared with hinds in experiment 2, and the absence of a seasonal decrease in plasma prolactin concentration during the breeding season in ovariectomized hinds in experiment 2 may indicate an effect of estradiol on prolactin secretion in this species.

In this study, neither dopamine nor endogenous opioid peptides were implicated in seasonal LH suppression in either euthyroid or THX red deer hinds; thus the pathways on which thyroid hormones act to bring about the anestrous state remain to be discovered. It is, however, extremely interesting to note that thyroidectomy was able to overcome the suppression of episodic LH secretion in our hinds when gonadal steroids were absent (experiment 2) but could not overcome estradiol-induced LH suppression (experiment 1). This is in contrast to the growing body of evidence for the ewe, in which the thyroid gland has been shown to be required for the profound increase in estradiol negative feedback that occurs at the end of the breeding season [29–31, 33–36]. Episodic LH secretion characteristics in ovariectomized estradiol-treated hinds in experiment 1 differed from those previously described for ovariectomized estradiol-treated ewes in two ways. Firstly, in the deer, both LH pulse frequency and pulse amplitude were suppressed in the presence of estradiol during the nonbreeding season, whereas in the ewe, estradiol primarily suppresses pulse frequency at this time [69]. Since estradiol greatly magnifies the seasonal fluctuations in pituitary responsiveness to GnRH that occur in red deer hinds [3], the decrease in LH pulse amplitude during the nonbreeding season is likely to reflect a reduction in the amount of LH released from the anterior pituitary gland in response to GnRH pulses, rather than a decrease in GnRH pulse size. Secondly, thyroidectomy during the breeding season was unable to overcome these effects on LH secretion, since LH pulse frequency and amplitude were as much suppressed by estradiol in THX hinds as in euthyroid hinds during the nonbreeding season. Taken together with the absence of LH responses to dopaminergic-D2 receptor agonists and antagonists in experiment 1, our results indicate that the mechanisms contributing to seasonal breeding in the ovariectomized, estradiol-treated red deer hind operate differently from those in the ewe. A limitation of this conclusion, however, is our current inability to measure estradiol reliably in cervine plasma and, hence, verify that the concentration produced by the implant was within the normal physiological range.

Pulsatile LH secretion in euthyroid ovariectomized hinds (experiment 2) during the breeding season had characteristics similar to those recorded by Limsirichaikul [70], but during the nonbreeding season, pulse frequency and amplitude were much lower in the current experiment. This difference can be explained by the time of year in which sampling was conducted, since pulses were measured close to the breeding season in February and March in the study of Limsirichaikul. More recent experiments have shown that LH concentrations in ovariectomized hinds at that time of year are similar to those during the breeding season ([3], unpublished results). The present results describe episodic LH secretion in ovariectomized hinds during the breeding season, and during the nonbreeding season at the time of maximal reproductive neuroendocrine suppression (i.e., just before the summer solstice). In euthyroid hinds, pulse frequency was low at this time compared with the breeding season. This finding is consistent with results from ovariectomized ewes [9, 34, 69, 71], although the magnitude of the seasonal change appears to be much greater in hinds. Ovariectomized ewes show an increase in pulse amplitude during the nonbreeding season [34, 69, 71], but in contrast pulse amplitude declined in the current experiment. The role of thyroid hormones in seasonal LH secretion in the ewe in the absence of gonadal steroids has received no direct attention; however, the results of Moenter et al. [34] suggest that pulse frequency and amplitude may remain at breeding season levels in thyroidectomized, ovariectomized ewes during the nonbreeding season. Thyroidectomy prevented the seasonal decline in LH pulse frequency in the ovariectomized hinds in experiment 2 but, in addition, caused pulse amplitude to increase to more than twice the breeding season amplitudes. One possible explanation for this is that high-amplitude pulses of GnRH occur in hinds during the nonbreeding season as in ewes [72], but correspondingly large LH pulses are prevented due to the seasonal decline in responsiveness of the anterior pituitary gland to GnRH [3]. Since this seasonal decline in pituitary responsiveness is greatly diminished in the absence of the thyroid gland (unpublished results), high-amplitude LH pulses can be expressed in thyroidectomized hinds. Since methods for collecting portal blood and measuring GnRH have not been developed in our laboratory, it is not currently possible to test this hypothesis directly.

In conclusion, the general absence of LH responses to bromocriptine or sulpiride in the presence or absence of estradiol, despite the fact that these drugs appeared to bind effectively to dopaminergic-D2 receptors as evidenced by appropriate changes in prolactin secretion, indicates that dopaminergic-D2 receptor-mediated neural pathways are not important modulators of LH regulation in this species. Furthermore, endogenous opioid peptides do not appear to modulate LH suppression in the absence of gonadal steroids, as morphine and naloxone elicited prolactin but not LH responses in ovariectomized hinds. Since thyroidectomy in these experiments was able to overcome suppression of episodic LH secretion in the absence, but not in the presence, of estradiol, we are currently investigating the role of another neural system that has been associated with LH regulation in ovariectomized ewes, the serotonergic system [27, 73], to determine whether thyroid hormones influence seasonal reproduction in red deer hinds by modifying this pathway.


   ACKNOWLEDGMENTS
 
Grateful thanks are extended to Mr. M.J. Keeley for assistance with animal handling and sample collection, and to Elanco Animal Health for donation of the Compudose implants. LH assay reagents were provided by Dr. A.F. Parlow, Pituitary Hormones and Antisera Center, Torrance, California, and prolactin antiserum was provided by Dr. D.F.M. van de Wiele.


   FOOTNOTES
 
1 Financial assistance was provided by the Lottery Science Research Committee of the New Zealand Lottery Grants Board and by the Canterbury Branch of the New Zealand Deer Farmers Association. Back

2 Correspondence: Graham K. Barrell, Animal and Food Sciences Division, P.O. Box 84, Lincoln University, Canterbury, New Zealand. FAX: 64 3 3253851; barrell{at}whio.lincoln.ac.nz Back

3 Current address: Department of Physiology, West Virginia University, Morgantown, WV 26506–9229. Back

Accepted: June 4, 1998.

Received: March 5, 1998.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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