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Neuroendocrinology |
Medical Research Council, Human Reproductive Sciences Unit,2 Centre for Reproductive Biology, Edinburgh EH16 4SB, United Kingdom
Prince Henry's Institute of Medical Research,3 Clayton, Victoria 3168, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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anterior pituitary, environment, hypothalamus, melatonin, pituitary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Decoding of the melatonin signal appears to occur in the PT, but evidence suggests that long-term, cyclical changes in PRL secretion may also be generated within the pituitary gland [11, 12]. For example, transfer of HPD rams from short days (8L:16D) to constant long days (16L:8D) for 48 wk induces a clearly defined, long-term cycle of activation, involution (after 12 wk), and reactivation (after 36 wk), with a remarkably similar profile for individual HPD animals. The reverse light treatment, using constant short days, produces a partially dampened and more variable, cyclical response [12]. Because the long-term changes under constant long days occur without any alteration in the daily pattern of melatonin secretion (this faithfully reflects the period of darkness), it appears that prolonged exposure to a static melatonin signal induces a spontaneous change in the output response, which is termed photorefractoriness. This is presumed to occur at the level of the pituitary gland, and the full PRL cycle has a period of more than 10 mo, which is indicative of a type of circannual rhythm [13]. Whether the timing system resides within the PT cells that decode the melatonin signal or is a special feature of the lactotropes that cycle in synchrony from the active to the inactive state, however, remains unknown.
To further investigate these pituitary mechanisms, the present study tested the hypothesis that pharmacological manipulation of PRL secretion in HPD sheep under a constant photoperiod would not affect the timing of the cycle of activation, involution, and reactivation of the PRL axis. This was thought to be likely if the long-term timing was dictated at the level of the PT independently of the output signal. In one experiment, HPD Soay rams exposed to constant long days were treated with bromocriptine to chronically suppress blood PRL concentrations for 12 wk, and the subsequent effect on the timing of the spontaneous PRL cycle of photorefractoriness and reactivation was assessed by comparison with a control group. In a second experiment, HPD Soay rams exposed to constant long days were treated with sulpiride (a specific dopamine D2-receptor antagonist [14]) during midphotorefractoriness to test whether an intrinsic dopaminergic mechanism might be involved in this long-term photoperiodic response. This was thought to be unlikely, because an acute injection of sulpiride elicits a minimal increase in PRL secretion in HPD sheep, in contrast to the marked response in hypothalamic-intact controls, in which the homeostatic dopamine system regulates PRL secretion [1, 2]. Blood PRL concentrations were measured twice weekly to follow the long-term changes in the PRL axis, and the melatonin signal was measured in serial, hourly samples collected at selected times. Other parameters (e.g., blood FSH concentrations, testis diameter, and pelage molt) were measured to correlate with PRL or to confirm the effectiveness of the HPD surgery.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The experiments were conducted in accordance with the Animals (Scientific Procedures) Act of 1986 and were authorized by the Home Office (Dundee, U.K.). Adult rams of the Soay breed of feral sheep, which show pronounced photoperiodically induced seasonal cycles in PRL secretion and other seasonal characteristics, were used in the present study [15]. The animals were housed permanently in light-controlled rooms and routinely exposed to alternating 16-wk periods of long days (16L:8D) and short days (8L:16D) to entrain the long-term endocrine cycles. Light intensity was approximately 160 lux at the animals' eye level, and dim red light (<5 lux) was used during the dark phase to a provide minimal illumination within the room. The time of lights-on was constant (zeitgeber time [ZT] 0), and adjustments in photoperiod were achieved by abruptly changing the time of lights-out by 8 h (between ZT 8 and ZT 16). The animals received a maintenance diet of commercial grass nuts (Vitagrass; Vitagrass Farms Ltd., Cumbria, U.K.), with hay and water ad libitum. The temperature in the animal rooms was controlled using a regulated airflow and heating system. This maintained the ambient temperature between 8 and 20°C, and the daily maximum and minimum temperatures were recorded.
HPD Operations
All rams received the standardized HPD operation at least 12 mo before the start of the present study. This involved surgical ablation of the median eminence and placement of an aluminum foil barrier between the hypothalamus and pituitary gland in a single, 2-h operation [2, 16]. The animals developed the expected clinical signs of permanent hypothalamopituitary disconnection, including polyurea, gonadal regression, and increasing obesity [17]. No hormonal replacement therapy was given to the HPD rams, and they remained in good health.
Experimental Manipulations
Experiment 1: Chronic treatment of HPD rams with bromocriptine This experiment utilized two groups of similar HPD rams (mean body weight at start of experiment, 45.3 kg; age, 4–5 yr) living in adjacent rooms. The animals, preconditioned to the alternating 16-wk long-day/short-day regimen for more than 12 mo, were switched from short days (8L:16D, end of 16-wk cycle) to long days (16L:8D) for 48 wk (Experimental Week 0 = start of long days). From Experimental Weeks 12–24, the rams received either daily s.c. injections of vehicle (1.0 ml ethanol:0.9% saline, 50:50 by volume) to act as controls (control group, n = 6) or daily s.c. injections of the dopamine-receptor agonist, bromocriptine (0.01–0.04 mg/kg dissolved in 1.0 ml of ethanol:0.9% saline, 50:50 by volume) to chronically suppress PRL secretion (bromocriptine group, n = 6). All injections were given in the early light phase (ZT 1–3) on the side of the neck, and the injection site was varied to minimize inflammation. The bromocriptine was obtained from Sigma-Aldrich (Poole, Dorset, U.K.), and a new solution was prepared weekly. The dose range was selected based on previous studies in sheep [1, 18]. The initial dose of bromocriptine of 0.04 mg kg-1 day-1 was reduced to 0.01 mg kg-1 day-1 after the first 2 wk, because the higher dose caused a notable decrease in food intake in some of the HPD rams (evidenced by food rations remaining after 24 h). The time of treatment (12–24 wk under constant long days) was selected to start before the blood PRL concentrations declined (i.e. before the onset of photorefractoriness) based on a previous study in HPD rams exposed to constant long days. The treatment duration of 12 wk was selected to allow detection of an alteration in the phase of the endogenous PRL cycle that varies between individuals by up to 6 weeks [12].
Experiment 2: Chronic treatment of HPD rams with sulpiride This experiment utilized two young groups of HPD rams (mean body weight at start of experiment, 32.8 kg; age, 3–4 yr) living in adjacent rooms. The animals, entrained to the alternating long-day/short-day regimen for more than 12 mo, were switched from short days (8L:16D, end of 16-wk cycle) to long days (16L:8D) for 48 wk, as in experiment 1 (Experimental Week 0 = start of long days). From Experimental Weeks 22–30, the animals received either twice-daily s.c. injections of vehicle (1.0 ml, see below) to act as controls (control group, n = 6) or twice-daily s.c. injections of the dopamine-receptor antagonist, sulpiride (0.6 mg/kg dissolved in 1.0 ml of 0.1 M tartaric acid in 0.9% saline with the pH adjusted to near neutrality with 1 M NaOH) to chronically block dopamine D2 receptors expressed by the lactotropes (sulpiride group, n = 6). The injections were given in the early and late light phase (ZT 1–3 and 12–14, respectively) on the side of the neck, and the injection site was varied to minimize inflammation. The sulpiride was obtained from Sigma-Aldrich, and a new solution was prepared weekly. The dose was selected based on previous studies in sheep, in which a single injection (0.6 mg/kg) produced a sustained increase in PRL secretion lasting more than 8 h in hypothalamic-intact Soay rams [1, 19]. This dose produced no behavioral effects in the HPD rams in the current experiment. The timing of treatment (from 22 to 30 wk under long days) was designed to commence during the declining phase of the PRL cycle (midphotorefractoriness), and the treatment duration of 8 wk was selected as a chronic treatment period that would produce a detectable change in the timing of the long-term photorefractory response if this was dependent on control through a dopamine D2-receptor mechanism.
Data Collection
To measure the changes in PRL and FSH secretion throughout the experiments, blood samples were collected twice weekly by venipuncture from the jugular vein of each animal (sampling time, ZT 2–4). The samples were heparinized and the plasma separated by centrifugation (2000 rpm for 20 min) within 30 min and stored at -20°C. Every 2 wk, the pattern of wool growth on the scrotum (short, <0.6 cm; medium, 0.7–1.4 cm; long, >1.5 cm) and molting of the pelage (% molt/ease of plucking) was recorded as a biological response to PRL [20]. The diameter of the testis was also measured through the scrotum using calipers, and the intensity of the androgen-dependent skin coloration in the inguinal region was assessed on a 0–5 scale, both as indexes of gonadal activity [21]. The data for testis size and sex skin color were not collected for a 14-wk period (Experimental Weeks 4–18) in experiment 1 and for a 10-wk period (Experimental Weeks 36–46) in experiment 2 because of a U.K. Foot and Mouth Disease Restriction Order that limited the movement of research personnel.
To measure the daily rhythm in melatonin and PRL secretion, sequential blood samples were collected hourly for 24 h (starting at ZT 4) from all animals. This was carried out on selected occasions before and after the drug treatments (experiment 1: Weeks 18, 36, and 48 of long days; experiment 2: Weeks 8 and 48 of long days). For the repeated sampling, a polythene cannula was inserted into the jugular vein on the day before each study, and blood samples were collected via a three-way tap using heparinized saline to maintain patency of the indwelling cannula. The heparinized blood samples were collected onto ice and the plasma separated within 30 min and stored at -20°C.
Radioimmunoassays
The concentrations of PRL and FSH were measured using routine RIAs validated for sheep plasma [22, 23]. The PRL assay had a lower limit of detection (10% decrease in binding relative to binding without ligand [Bo]) of 0.5 µg NIH-PRL-S13/L plasma and intra- and interassay coefficients of variation (CVs) of 7.8% and 9.0% respectively, based on low-, medium-, and high-quality control samples measured in 12 assays. The corresponding values for the FSH assay were 0.2 µg NIDDK-FSH-RP2/L plasma, 6.5%, and 13%, respectively. The concentrations of melatonin in the hourly plasma samples were measured using a direct RIA [24]. The assay was performed using a commercial rat antibody (Stockgrand, Guildford, Surrey, U.K.) at a working dilution of 1:3000, iodinated melatonin (Amersham plc, Buckinghamshire, U.K.), and melatonin (Genzyme, Haverhill, Suffolk, U.K.) as standard. The sensitivity of the assay was 40–60 pM/L, and the mean intra- and interassay CVs were <10%. All sequential samples from one animal for the hourly series were included in the same assay.
Statistical Analysis
The long-term PRL profiles measured under the constant long days were analyzed for significant changes with respect to time (Week of Experiment) and treatment (control/drug treatment) by two-way analysis of variance (ANOVA) with repeated measures. For the analysis, the PRL data in experiment 1 were divided into four 12-wk periods, which were defined as pretreatment (Weeks 0–12), treatment (Weeks 12.5–24), early posttreatment (Weeks 24.5–36), and late posttreatment (Weeks 36.5–48). In experiment 2, the PRL data were assessed for an effect of time and a time x treatment interaction for three 8- to 22-wk periods, which were defined as pretreatment (Weeks 0–22), treatment (Weeks 22.5–30), and posttreatment (Weeks 30.5–48). These periods were preselected based on the predicted effects of the drug treatments and the PRL profiles in HPD rams exposed to a change from short days to constant long days [12]. The time of the minimum plasma PRL concentrations during the posttreatment period (long-term PRL minimum) was used as an index of the time of photorefractoriness. This was calculated for each animal using a three-point moving average based on the half-weekly samples, and the values were expressed as weeks relative to the start of constant long days and compared between groups by ANOVA. Week 36 was the expected PRL nadir/maximum refractoriness under long days [12]. The slope and intercept for the PRL data during spontaneous reactivation at Weeks 36–48 were also compared by linear regression analysis using PRISM 3.0a for Macintosh (GraphPad Prism, San Diego, CA). The correlation coefficients between mean weekly ambient temperature and plasma PRL concentrations were calculated as well.
For the analysis of the melatonin profiles, the nocturnal melatonin peak was defined as two or more consecutive blood samples with melatonin concentrations greater than the mean basal concentrations plus 2-fold the intraassay CV. The duration of the melatonin peak was taken as the total number of consecutive blood samples that constituted the peak (not disrupted by more than a single basal sample). The dark-phase melatonin concentration was taken as the mean of all values for all blood samples collected during darkness. In all cases, the values were calculated for the individuals and then for the group (mean ± SEM). The statistical significance of the changes in the melatonin parameters under the constant photoperiod was assessed by ANOVA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Prolactin The effect of the chronic treatment with the dopamine agonist, bromocriptine, on the long-term cycle in PRL secretion in the HPD rams is illustrated in Figure 1. In the control group, as expected, the blood PRL concentrations increased during the pretreatment phase (Weeks 0–12) because of the initial change from short to long days and decreased during the treatment and early posttreatment phases (Weeks 12–24 and 24–36, respectively) because of the development of refractoriness. Spontaneous reactivation in PRL secretion occurred during the late posttreatment phase (Weeks 36–48). The statistical analysis revealed a significant (P < 0.001) effect of time on PRL concentrations during all four phases of the experiment, with the direction of change altering from increasing to decreasing and then back to increasing according to the phase of the cycle under constant long days. The mean weekly changes in PRL concentrations were not correlated with the changes in ambient temperature in the animal rooms for the full experiment (HPD controls 0–48 wk of long days, PRL vs. temperature: r2 = 0.002, not significant) or for full period minus the first 4 wk, when the initial effect of the change in photoperiod predominated (HPD controls 4–48 wk of long days, PRL vs. temperature: r2 = 0.203, not significant).
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Bromocriptine caused a marked suppression in the blood PRL concentrations throughout the 12-wk treatment phase and a notable rebound increase extending for 6–8 wk during the early posttreatment phase. These drug effects were evident as a significant (P < 0.001) time x treatment interaction for the PRL values during these two phases of the experiment (treatment and early posttreatment) but not at other times (pretreatment and late posttreatment), when the PRL secretion changed in parallel to the control group.
The time of the minimum blood PRL concentrations during the posttreatment period (index of photorefractoriness) was similar in the control and bromocriptine groups (PRL minimum, 35.3 ± 0.6 and 37.0 ± 0.4 wk for the bromocriptine and control groups, respectively; mean ± SEM, n = 6, not significant). The linear regression analysis of the blood PRL concentrations after 36 wk under long days also revealed no significant difference between the two groups in either the calculated slope or intercept, indicating that the bromocriptine treatment had not affected the rate of spontaneous increase in PRL secretion or the timing of the long-term PRL cycle.
Pelage molt Molting of the pelage correlated with the increases in PRL secretion observed in the HPD rams (Fig. 1). In both control and bromocriptine groups, a molt occurred at Weeks 6–14, following the initial photoperiod-induced increase in PRL concentrations (pretreatment), and another molt occurred beginning at Week 46, following the spontaneous increase in PRL concentrations under long days (late posttreatment). In the bromocriptine group, an additional conspicuous molt was observed after the end of the treatment with bromocriptine at Weeks 28–33 that was related to the rebound increase in blood PRL concentrations (early posttreatment) (Fig. 1). This was a more extensive molt than normally seen in HPD rams and resulted in the shedding of wool across the entire body.
Reproductive parameters The blood concentrations of FSH in the HPD rams were close to the minimum detection limit of the RIA throughout the experiment, and no significant changes were observed with time or between control and bromocriptine treatment groups for the four phases of the experiment. The testes were markedly regressed, and no androgen-dependent sexual skin coloration was seen at any stage. In the control HPD rams, testis diameter increased marginally to a maximum at Week 20 under long days and then declined. In the bromocriptine-treated animals, the increase in testis diameter occurred at a later time, with a maximum at Week 34 under long days; theses changes were evident as a significant (P < 0.01) time x treatment interaction for testis diameter during the posttreatment phase (Fig. 1).
Melatonin and PRL rhythm The 24-h blood melatonin and PRL profiles in the HPD rams at Weeks 18, 36, and 48 under long days are illustrated in Figure 2. At all times, the blood melatonin concentrations were low during the light phase and increased during the dark phase. The duration of the nocturnal melatonin peak closely reflected the duration of darkness. During the treatment phase (Week 18), the duration and amplitude of the melatonin peak was similar between groups (melatonin peak duration: 9.3 ± 0.2 and 9.0 ± 0 h for the control and bromocriptine-treated rams, respectively; dark-phase mean melatonin concentrations: 368.4 ± 83.6 and 329.0 ± 92.9 pM/L for the control and bromocriptine-treated rams, respectively; not significant). These melatonin parameters were similar at Weeks 36 and 48 (posttreatment), with no significant differences between groups (Fig. 2).
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The blood PRL concentrations in the HPD rams did not vary consistently across the 24-h cycle, but the mean values changed markedly according to the stage of the long-term PRL cycle and the drug treatment regimen. At Week 18, during the treatment phase, PRL concentrations were relatively high in the controls but fully suppressed throughout the 24-h cycle in the bromocriptine group. At Week 36, the profiles were reversed; the controls became photorefractory, and the bromocriptine group showed a rebound response after the end of the treatment (Figs. 1 and 2). At Week 48, PRL concentrations were again increased, with a similar 24-h pattern in both groups.
Experiment 2: Effects of Sulpiride
Prolactin The effect of the chronic treatment with the dopamine-receptor antagonist, sulpiride, given to HPD rams during photorefractoriness is illustrated in Figure 3. In the control group, a very well-defined cycle of activation, involution, and reactivation in PRL axis was observed under the constant photoperiod, as in experiment 1. Again, the mean weekly changes in PRL concentrations were not correlated with the changes in ambient temperature (HPD controls 0–48 wk of long days, PRL vs. temperature: r2 = 0.009, not significant). Sulpiride caused a minor, nonsignificant increase in the blood plasma concentrations of PRL during the initial 4 wk of treatment but otherwise had no effect. The statistical analysis revealed a significant (P < 0.001) effect of time on the PRL concentrations during the treatment (Weeks 22–30, decreased PRL) and posttreatment phases of the experiment, but no significant time x treatment interaction was found in the comparison between the control and sulpiride groups.
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The time of the minimum blood PRL concentrations under long days (index of photorefractoriness) was similar in the two groups (PRL minimum, 37.9 ± 0.4 and 37.6 ± 0.9 wk for the sulpiride and control groups, respectively; mean ± SEM, n = 6, not significant). The timing and rate of increase in the blood PRL concentrations after 36 wk under long days was again similar for the two groups (Fig. 3).
Pelage molt Two molting periods were associated with the early and late increases in PRL secretion, as with the control animals in experiment 1. The treatment with sulpiride had no effect on the sequence of molts.
Reproductive parameters The blood concentrations of FSH were consistently low throughout the experiment, and no significant changes with time or related to treatment were observed. The testes were markedly regressed, and no androgen-dependent sexual skin coloration was seen. Testis diameter increased marginally but significantly (P < 0.01) during the pretreatment phase in both groups to a maximum at Week 18, but no effect of the sulpiride treatment on testis diameter was noted at any phase of the experiment.
Melatonin and PRL rhythm The 24-h melatonin and PRL profiles measured at Weeks 8 and 48 under constant long days are illustrated in Figure 4. At both times, the blood melatonin concentrations were low during the light phase and increased during the dark phase. At Week 8 (pretreatment), the duration and amplitude of the melatonin peak was similar between the two groups (melatonin peak duration: 8.8 ± 0.1h and 8.7 ± 0.2 h for the control and sulpiride-treated rams, respectively; dark-phase mean melatonin concentrations: 372.6 ± 86.6 and 309.6 ± 70.0 pmol/L for the control and sulpiride-treated rams, respectively; not significant). These parameters were also similar at Week 48 (posttreatment), with no significant differences between groups (Fig. 3).
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The 24-h PRL profiles for the HPD rams again revealed no consistent diurnal rhythmicity. The hourly mean PRL values were high at Week 8 and lower at Week 48, consistent with the long-term PRL cycle, but no significant difference was found between the two groups.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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No evidence in the present study suggested that the 24-h melatonin signal encoding the photoperiod changed during the prolonged exposure to long days or following the different drug treatments. The long-term photorefractory cycle in PRL secretion was therefore generated in the presence of a constant-long-day melatonin signal (short duration signal of 
8 h/day). The photorefractory phase began after some 16–20 wk of long days, and the recovery phase commenced after 34–38 wk, with a remarkably similar long-term profile for the individual animals. This is very similar to the PRL profiles described previously in HPD rams exposed to the long-day regimen [12]. The nature of the ambient photoperiod, albeit constant, is clearly important in dictating this high-amplitude, long-term PRL cycle, because a very different endocrine profile is expressed under constant short days and because a change to short days at any time during prolonged long days results in an immediate decline in PRL secretion [2, 12, 28]. Changes in ambient temperature in the animal rooms are unlikely to account for the long-term PRL cycle, because the pattern was essentially the same in the control animals in both experiments of the present study (although these were run 6 mo out of phase) and because the weekly variations in blood PRL concentrations were not correlated with changes in ambient temperature for the full experiment or when the data for the first 4 wk were deleted to avoid the inductive effect of the acute change in photoperiod. Overall, the results support the view that under long days, the short-duration melatonin signal is permanently inductive to the PRL axis, "driving" the sequential stages of activation, decline (photorefractoriness), and reactivation with precise timing over the period of a year. Likewise, in hamster models, photorefractoriness to short days results from prolonged exposure to a long-duration melatonin signal, and pinealectomy abruptly interrupts the long-term PRL response [29, 30]. Because the current data derive from an HPD sheep model in which the pituitary is isolated from the control of the hypothalamus, we conclude that the intrinsic timekeeping mechanism resides within the pituitary gland.
The cellular mechanisms that dictate the long-term PRL cycle must account for the coordinated secretion by lactotropes, the development of refractoriness to the constant photoperiod, and the spontaneous reactivation after many months. The cells of the PT are the logical site of control in the pituitary gland, because they express a high density of melatonin receptors, secrete factors (tuberalins) that modulate PRL release, and are strategically placed in the rostrodorsal region of the pituitary gland, close to the median eminence and portal vasculature supplying the pars distalis. The development of photorefractoriness does not appear to result from a loss of melatonin receptors in the PT, a change in receptor-G protein coupling, or a change in the inhibitory cAMP response to melatonin, at least based on the results of early studies in Siberian hamsters rendered refractory to short days [31]. More detailed work has revealed that melatonin inhibits MT1-receptor expression in the PT and that a 24-h cycle exists in receptor expression inverse to the blood melatonin rhythm, with increased amplitude under long days compared with short days [32, 33], which also occurs in sheep [34]. Still, no evidence suggests that photorefractoriness results from melatonin receptor-related down-regulation or desensitization in the target cells of the PT. The possibility that changes in clock gene expression in the PT may underlie the timing response has been proposed, most recently based on the measurement of 24-h patterns of expression of the core clock genes in sheep exposed to long and short days [8, 13]. Notably, the beginning of the light phase (melatonin offset) was closely associated with the activation of period gene expression (Per1 and Per2), whereas the beginning of the dark phase (melatonin onset) was linked with the activation cryptochrome gene expression (Cry1 and Cry2) under both photoperiods. The temporal phase relationship between Per and Cry expression thus varied with daylength, providing a potential molecular mechanism for decoding the melatonin signal at the level of the PT. A speculative, but testable, extension of this concept is that photorefractoriness results from the uncoupling of the melatonin control of the clock genes such that the Per/Cry interval no longer reflects the photoperiod [13].
The HPD rams expressed a normal diurnal rhythm in melatonin secretion, but no clear evidence of diurnal rhythmicity was found in the blood concentrations of PRL, which is a well-defined feature in the hypothalamic-intact animal [2]. This confirms that HPD surgery blocks the circadian control of PRL release. In addition, this indicates that the hypothalamic dopaminergic and adrenergic mechanisms that regulate PRL secretion may relay input from the circadian pacemaker system located in the suprachiasmatic nuclei to control the diurnal PRL rhythm as well as provide for the homeostatic control through the positive-feedback effect of PRL [1, 35]. The lack of a short-term, 1:1 relationship between blood PRL and melatonin concentrations throughout the 24-h cycle in the HPD rams illustrates that melatonin does not act directly to control the photoperiod-induced changes in PRL secretion. Instead, the melatonin-target tissues are responsive to changes in signal duration over a 24-h period, and decoding this signal is regarded as a special property of the PT cells [13].
In the present study, the blockade of PRL secretion with bromocriptine had no effect on the long-term PRL cycle, but it did cause a prolonged change in PRL release lasting for many weeks. This was evident as a posttreatment rebound increase in blood PRL concentrations, occurring when the control animals were becoming photorefractory to long days. This increase in PRL secretion is unlikely to result from a homeostatic response governed by the hypothalamus [35], because the HPD lesion disrupts the hypothalamic control, as confirmed by the absence of a PRL response to the dopamine-receptor antagonist, sulpiride, in experiment 2. The rebound response is also unlikely to result from parallel changes in other endocrine systems induced by the chronic bromocriptine (e.g., gonadal steroid secretion), because FSH secretion was blocked (as expected in the HPD rams) and because no evidence of androgen secretion was found (sex skin coloration was absent throughout). Other pituitary mechanisms are also markedly compromised in the HPD animal [17]. More probably, this effect on PRL release is generated within the pituitary gland because of the effect of the intrinsic circannual rhythm-generating mechanism and/or the way in which bromocriptine causes a temporal dissociation between the function of the lactotropes and the secretion of paracrine factors from cells in the pituitary gland. At the end of the bromocriptine treatment, at 24 wk into long days, it might be predicted that the PT would still be secreting tuberalins at half-maximum value, based on the PRL profiles in the control animals (blood PRL, 50% of maximum) (Fig. 1), and this might stimulate PRL secretion in lactotropes as they recover from the chronic inhibition. There would be no hypothalamic inhibitory feedback mechanism to terminate such a response. The long period of recovery fits well with the view that many paracrine and autocrine mechanisms operate within the pituitary to govern the function of the lactotrope [36].
A final point of interest relates to the observed minor changes in testis size and the occurrence of periodic pelage molts in the HPD animals. Testis diameter increased marginally to a maximum at 18–20 wk under constant long days, and the timing was delayed by the chronic treatment with bromocriptine. This is consistent with the view that PRL acts in the testis to affect gonadal activity, albeit with minimal effect in the absence of gonadotropins [28]. The changes in testis size lag many weeks behind the changes in PRL secretion. Previous studies have shown that PRL receptors are expressed in the Leydig cells in the testicular interstitium and in germ cells in the seminiferous tubules, and PRL activates second-messenger signaling in the ovine testis [37]. In the present study, molting of the pelage on the scrotum occurred following the initial increase in PRL secretion induced by the switch from short to long days and following the second increase in PRL secretion toward the end of 48 wk of long days, as described previously [12]. This relates to the known action of PRL in the dermal papilla [38, 39], stimulating the resumption of the hair follicle cycle and affecting the hair phenotype (summer/winter). The result nicely illustrates how the molt cycle provides a convenient index for the activity of the PRL axis. The molt triggered by the treatment with bromocriptine was of special interest, because it extended across the whole body and was more complete than is normally evident in HPD sheep following switches in photoperiod or treatments with melatonin [26]. This fits with the view that very low circulating PRL concentrations are required to induce all hair follicles to enter telogen (quiescence), and once in this state, an increase in PRL levels can reactivate the hair follicle cycle and trigger the synchronous shedding of old hair fibers, as occurs during spring molt in the normal animal. In untreated HPD sheep, baseline blood PRL concentrations are always increased compared to intact animals, even under short days, presumably because of the absence of the inhibitory control by the hypothalamus, and this favors continued hair follicle activity in some areas of the body (back and sides) and, thereby, prevents the full molt [2].
In conclusion, the present study demonstrates that the chronic blockade of PRL secretion, or the blockade of dopamine receptors using a receptor antagonist, in HPD sheep living under prolonged, constant long days has no effect on the development of photorefractoriness or the timing of the spontaneous reactivation in PRL secretion. The 24-h melatonin rhythm faithfully reflected the photoperiod; thus, the photorefractory response was not caused by a change in the melatonin signal. Because these observations were made in HPD animals with an isolated pituitary gland and other evidence suggests that melatonin receptors are heavily expressed by cells in the PT and not by lactotropes, the results are consistent with the working hypothesis that the photoperiod/melatonin timer is in the PT cell, not in the lactotrope. Secretion of PRL per se is not required for development of the long-term photorefractory response.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received: 27 March 2003.
First decision: 16 April 2003.
Accepted: 23 May 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 G. A. Lincoln, I. J. Clarke, R. A. Hut, and D. G. Hazlerigg Characterizing a Mammalian Circannual Pacemaker Science, December 22, 2006; 314(5807): 1941 - 1944. [Abstract] [Full Text] [PDF]
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