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a MRC Reproductive Biology Unit, Centre for Reproductive Biology, Edinburgh EH3 9EW, Scotland, United Kingdom
b Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To further investigate the role of the pituitary gland in this photoperiodic relay, we have now measured the patterns of PRL secretion in HPD rams exposed to a prolonged constant photoperiod of either long (16L:8D) or short days (8L:16D). The purpose was to establish whether the lesioned animals become refractory to the initial response to a change in photoperiod and express long-term changes in PRL secretion under constant photoperiod, as seen in intact sheep [5–7]. The persistence of such temporal changes in the absence of hypothalamic control would indicate that, besides activating a response to photoperiod, the target cell-lactotroph relay within the pituitary plays an important role in the overall timing of the annual PRL cycle. In this study, the blood concentrations of melatonin were measured throughout 24 h on selected occasions to check the fidelity of the nocturnal melatonin peak as an index of night length. In addition, the long-term changes in the blood concentrations of FSH were measured to illustrate the effect of the HPD lesion on an endocrine system that is known to be dependent on control by the hypothalamus [8]. The overt changes in pelage, testis size, and sexual skin coloration were recorded as overt indices of the biological effects of PRL and gonadotropins [9–11].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult rams of the Soay breed of feral sheep, which show pronounced photoperiodically induced seasonal cycles in testicular activity, coat growth, and other seasonal characteristics, were used in the study [12,13]. The animals were adult (4 yr old), with a mean body weight of 45.4 kg at the start of the experiment. They were housed permanently in light-controlled rooms and routinely exposed to alternating 16-week periods of long days (16L:8D, LD) and short days (8L:16D, SD) to entrain the long-term endocrine cycles. Light intensity was approximately 160 lux at the animals' eye level. The time of lights-on was constant (0800 h), and adjustments in photoperiod were achieved by abruptly changing the time of lights-out by 8 h. The animals received a maintenance diet of commercial grass "nuts" (Vitagrass, Vitagrass Farms Ltd., Cumbria, UK), 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 10 and 20°C. The constant photoperiod was initiated in August, and the overall mean temperatures during the 4 phases of the experiment (see below and Fig. 1) were 17.0°C, 11.6°C, 11.9°C, and 16.1°C.
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Experimental Manipulations
The rams were housed in two adjacent rooms (n = 6/room) and received the HPD operation at 8 wk into long days. The HPD operation involved the surgical ablation of the median eminence and the placement of an aluminum foil barrier between the hypothalamus and pituitary gland [1,8]. The animals developed the expected clinical signs of permanent hypothalamo-pituitary disconnection including polyurea, gonadal regression, and increasing obesity [1,9]. No hormonal replacement therapy was given to the HPD rams, and they remained in good health. Immediately after the HPD operations, the photoperiod was changed to short days in one group but remained unchanged in the other group. After 8 wk, and subsequently every 16 wk for 48 wk, the photoperiod was switched between long and short days such that the animals were on a reversed lighting schedule. This was designed to produce two adjacent HPD groups in different photoperiod-induced physiological states (high and low PRL secretion) for the current experiment. The two experimental groups were treated as follows: 1) one group of HPD rams, preconditioned to short days, was exposed to long days for 48 wk (LD group), and 2) the other group of HPD rams, preconditioned to long days, was exposed to short days for 48 wk (SD group). At the end of the treatment, the photoperiod was switched back to the pretreatment schedule for 12 wk in both groups to measure the response to an abrupt change in photoperiod after the prolonged experiment. The total experimental period was 60 wk (48 wk constant photoperiod plus 12 wk reversed photoperiod; see Fig 1).
Data Collection
To record the endocrine changes throughout the experiment, blood samples were collected twice weekly from the jugular vein by venipuncture from each animal during the light phase (between 1000 and 1200 h). The samples were heparinized, and the plasma was separated by centrifugation within 30 min and stored at -20°C until PRL and FSH concentrations were measured by RIA. Every 2 wk, the pattern of wool growth and molting on the scrotum, and the diameter of the testes were measured to correlate with the changes in PRL and FSH secretion [9,11]. The intensity of the androgen-dependent sexual skin coloration in the inguinal region was also visually scored [14].
On three occasions—at weeks 10, 24, and 44 under the constant photoperiod—sequential blood samples were collected hourly for 24 h from all animals to measure the daily rhythm in melatonin secretion. 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 3-way tap using heparinized saline to maintain patency of the indwelling cannula. The heparinized blood samples were collected onto ice, and the plasma was separated within 30 min and stored at -20°C.
RIAs
The concentrations of PRL and FSH were measured in weekly blood samples collected from the rams using routine RIAs validated for sheep plasma for PRL [15] and for FSH [16]. The PRL assay had a lower limit of detection (10% decrease in binding relative to Bo) of 0.5 µg/l NIH-PRL-S13 plasma, and intra- and interassay coefficients of variation (CV) of 6.4% and 8.5%, respectively, based on low-, medium-, and high-quality control samples measured in 6 assays. The corresponding values for the FSH assay were 0.2 µg NIDDK-FSH-RP2/l, 9.2%, and 12.0%. The concentrations of melatonin in the hourly plasma samples were measured using a direct RIA [17] as previously validated for sheep plasma [18]. The assay was performed using the antibody (Stockgrand, Guildford, Surrey, UK) at a working dilution of 1:3000, tritiated melatonin (Amersham, Amersham plc, Buckinghamshire, UK), and melatonin as standard (Genzyme, Haverhill, Suffolk, UK). The sensitivity of the assay was 40–60 pM, and the mean intra- and interassay CVs were 6.8% and 9.2%, respectively. All sequential samples from one animal for the hourly series were included in the same assay.
Statistical Analysis
The half-weekly profiles of PRL during the 48 wk under constant photoperiod were analyzed for significant changes with respect to time (weeks of experiment) by ANOVA. For this, the data were divided into four consecutive, 12-weekly periods (24 half-weekly sample points per animal) to assess the significance of the time-dependent changes at different phases of the study. The periods were as follows: phase 1—experimental weeks 1–12; phase 2—experimental weeks 13–24; phase 3—experimental weeks 25–36; phase 4—experimental weeks 37–48. These periods were preselected on the basis of the latency of the different phases of the PRL response to treatment with a constant-release implant of melatonin that mimics the effect of exposure to constant short days [4,19]. The mean hormonal concentrations for all four phases were compared between LD and SD groups by ANOVA. Linear correlations between PRL concentrations and time for each 12-wk period were calculated using a Cricket Graph Program (Cricket Graph Inc., Philadelphia, PA). The times of maximum and minimum plasma PRL concentrations were assessed for each animal using a 3-point moving average, and the values were expressed in weeks relative to the start of the period of constant photoperiod as a group mean ± SEM.
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 2x 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. The maximum melatonin concentration was taken as the mean of 3 adjacent values that were maximum for each 24-h complete profile. In all cases, the values were calculated for the individuals and then for the group (mean ± SEM, Table 1). The statistical significance of the changes in the melatonin parameters from weeks 10, 24, and 44 under the constant photoperiod was assessed by ANOVA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The long-term effects of the experimental manipulations on PRL secretion and the timing of the pelage molt in the two groups of HPD rams are illustrated in Figures 1 and 2. In the LD group, the blood plasma concentrations of PRL were decreased at the start of the experiment because of prior exposure to short days. Transfer to long days caused a marked increase in the concentrations of PRL beginning within 1 wk, followed by a biphasic pattern extending throughout the 48 wk of treatment (Fig. 1). Maximum PRL values initially occurred at 8–12 wk into long days (mean PRL concentration: 143.3 ± 8.4 µg/l; mean time of maximum: 8.8 ± 1.2 wk). The concentrations then declined to a minimum at 34–38 wk (mean PRL concentration: 15.6 ± 1.6 µg/l; mean time of minimum: 34.5 ± 1.5 wk), before increasing again until the end of the 48 wk of long days. The switch back to short days at this time induced an immediate decline in the plasma concentrations of PRL (Fig. 1). The long-term PRL profiles were very similar for all animals within the LD group (Fig. 2). Molting of the pelage in the HPD rams was associated with the initial increase in PRL concentrations at the start of long days (normal spring molt). There was also a less complete molt after the secondary increase in PRL concentrations towards the end of long days (extending into short days, Fig. 1).
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In the SD group, the blood plasma concentrations of PRL were increased at the start of the experiment because of prior exposure to long days. The transfer to short days caused an immediate decrease in the concentrations of PRL (mean PRL concentration: 8.6 ± 2.6 µg/l; mean time of minimum: 17.1 ± 2.0 wk). PRL values subsequently increased to an attenuated maximum at 22–48 wk under short days, with notable variability between animals (mean PRL concentration: 46.4 ± 5.5 µg/l; mean time of maximum: 30.2 ± 3.2 wk). The return to long days after 48 wk induced a rapid increase in the concentrations of PRL, with the exception of one animal in which PRL secretion was already enhanced (Fig. 2). A partial molt of the pelage occurred after the minor increase in PRL under short days, and a full molt followed the major increase induced by long days (Fig. 1).
The statistical analysis revealed that there was a significant (P < 0.001) time-dependent change in plasma concentrations of PRL during the 48-wk experiment under the constant photoperiods, and a significant (P < 0.001) time-by-treatment (LD/SD) interaction. In the LD group, concentrations of PRL were increased compared with those of the SD group during phases 1, 2, and 4 (PRL mean ± SEM, LD group: 101.1 ± 6.6, 90.1 ± 5.1, 32.4 ± 3.6 and 49.0 ± 4.3; SD group: 16.3 ± 2.0, 15.2 ± 2.0, 23.9 ± 4.5 and 15.8 ± 3.3 µg/l plasma; phases 1–4 respectively). In the LD group, the long-term PRL profile was characterized by a significant (P < 0.01) positive slope during phase 1, a negative slope during phases 2 and 3, and a positive slope during phase 4. In the SD group, the long-term PRL profile was characterized by a significant negative slope during phase 1, a positive slope during phase 2, and no consistent pattern in phases 3 and 4 (data not shown).
FSH Secretion and Testicular Activity
The HPD rams in both the LD and SD groups had very low blood plasma concentrations of FSH, close to the lower limit of sensitivity of the RIA. There was no detectable change in FSH values during the prolonged photoperiods and no difference between the LD and SD groups (FSH mean ± SEM, LD group: 0.21 ± 0.04, 0.21 ± 0.04, 0.24 ± 0.06, and 0.19 ± 0.06; SD group: 0.18 ± 0.01, 0.18 ± 0.01, 0.16 ± 0.01, and 0.15 ± 0.01 µg/l plasma; phases 1–4 respectively). The testes of all animals were markedly regressed, and there was no androgen-dependent sexual skin coloration at any stage. In the LD group, the diameter of the testis was marginally increased (P < 0.05), compared with that of the SD group during phase 2, but was not different at other times (testis diameter mean ± SEM, LD group: 35.1 ± 1.0, 37.0 ± 1.3, 35.4 ± 1.8, and 32.3 ± 2.3; SD group: 34.4 ±0.9, 31.7 ± 0.7, 30.9 ± 0.7 and 30.1 ± 1.0 mm; phases 1–4 respectively).
Melatonin Profiles Throughout 24 Hours Under Constant Photoperiod
The changes in the blood plasma concentrations of melatonin throughout 24 h in the HPD rams at the 3 sampling periods under the constant photoperiod are illustrated in Figure 3 and summarized in Table 1. In the LD group, the melatonin concentrations were low during the light phase and increased during the dark phase. The duration of the nocturnal melatonin peak was close to 8 h at weeks 10, 24, and 44, and there was no significant change in any of the melatonin parameters during the prolonged exposure to long days (Table 1). In the SD group, there was also a clearly defined diurnal rhythm in melatonin concentrations at all stages. The duration of the nocturnal melatonin peak was close to 14 h at week 10 (associated with the long dark phase under short days). The peak duration, mean dark-phase melatonin concentrations, and mean maximum melatonin concentrations decreased significantly from 10 to 44 wk under short days (Fig. 3, Table 1). There was no clear association between the characteristics of the daily melatonin profile at weeks 24 and 44 and the individual variation in the long-term changes in PRL secretion under short days.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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If the HPD model represents an isolated pituitary preparation, the important conclusion that can be drawn from the current results is that the melatonin signal that encodes the photoperiod may act directly at the level of the pituitary gland to control PRL secretion. Moreover, both the acute inductive effects and chronic refractory effects of photoperiod may be generated within the pituitary. This is inferred from the way the blood concentrations of PRL changed progressively in the HPD rams throughout the 48 wk under the constant light regimens in a photoperiod-specific manner. The differential responses to the two photoperiods are likely to be due to the observed differences in the duration of nocturnal melatonin secretion, since a change in this parameter has been shown to mediate effects of photoperiod in sheep [13,22]. The animals exposed to long days had a predictable short-duration melatonin peak associated with the short night and initially expressed high prolactin secretion as expected under long days, while the animals on short days showed the converse association. Under the prolonged photoperiod, however, PRL secretion continued to change as a spontaneous cycle, with a decline and reactivation under long days and an essentially opposite pattern under short days. These long-term changes cannot be readily explained by spontaneous changes in the encoding melatonin signal, at least under long days, when the melatonin peak accurately reflected night length during the experiment. The differences in PRL secretion between the two groups are also unlikely to be due to other environmental influences, since the animals were housed under similar conditions in adjacent light-controlled rooms. The animals experienced a relatively constant environment with a standardized diet, and the ambient temperature was partially regulated. The recorded cycle of growth and molting of the pelage was closely correlated with the variation in PRL secretion, consistent with a role of PRL in the control of hair follicle activity [10], and was not associated with the changes in temperature in the animal rooms. There was also no confounding effect of a photoperiod-induced change in the reproductive axis, because the HPD rams were permanently hypogonadal, with no cyclical changes in androgen secretion (absence of the sexual skin coloration) or in overt sexual and aggressive behavior. Overall, the data support the view that continuous exposure to a static melatonin signal results in a time-dependent change in the PRL response, an effect apparently regulated within the pituitary gland.
The only previous comparable study involved the treatment of intact and HPD Soay rams with peripheral constant-release implants of melatonin for 48 wk, while the animals were maintained under a long day photoperiod [4]. In this experiment, the chronic treatment with melatonin caused an initial inhibition in blood PRL concentrations followed by partially recovery after 8–12 wk. The addition of a second melatonin implant after 20 wk failed to suppress PRL secretion, demonstrating a loss of responsiveness to melatonin, i.e., refractoriness [4]. The long-term profile in blood PRL concentrations was similar for both intact and HPD rams, and it was notably similar to that induced by exposure to prolonged short days in the current study. The results are reproduced for the two types of experiments in Figure 4. These data provide support for the view that a pharmacological, continuous melatonin signal from an implant induces biological effects indistinguishable from those induced by short days [23,24] and this applies in HPD animals. The results also indicate that the observed partial breakdown of the melatonin signal under prolonged short days did not disrupt the short-day response. No groups of intact rams were included in the current experiment because of the limitation of space, but published data in other sheep breeds demonstrate that long-term, cyclical changes in PRL secretion occur in rams maintained under a constant photoperiod for periods up to 3 years. The oscillations are of high amplitude under long days but are attenuated or absent under short days [5,6]. This is a situation similar to that observed in the HPD Soay rams and indicates that photoperiod persistently modulates PRL secretion despite the development of refractoriness and the generation of a cyclical pattern. The current treatment of 48 wk was not long enough to establish whether HPD rams are able to express circannual cycles in PRL secretion under constant photoperiod as occurs in normal intact rams. This remains a possibility since PRL secretion increased spontaneously after 40 wk under long days in the HPD animals, while the timing of the pattern of PRL secretion was variable between individuals under short days, consistent with an intrinsic control mechanism.
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The cellular mechanisms that mediate the time-dependent effects of melatonin on PRL secretion are largely unresolved. Such mechanisms must account for the coordinated secretion by lactotrophs, the response to photoperiod, and the development and recovery of refractoriness, all operating within the tissues of the pituitary gland. High-affinity melatonin receptors (ML1a subtype) [25] are abundantly expressed by cells in the ovine pars tuberalis/zona tuberalis (PT) but not by lactotrophs in the pars distalis [26]. Thus, it is logical to presume that the melatonin signal acts indirectly through the PT to control PRL secretion. Melatonin inhibits cAMP-induced activation of protein synthesis and secretion by ovine PT cells [27,28], and there is evidence that PT cells in primary culture secrete factors that promote PRL secretion [29]. PT cells also vary their cellular response depending on the timing or duration of exposure to melatonin [30,31]. This includes the activation of the early response gene oPer1 [32]; thus the PT may have clock-like properties. Accordingly, the chronic effects of photoperiod on PRL secretion may be due to the time-dependent responses of PT cells reacting to the daily melatonin signal. Long days (short duration signal) would predictably permit maximum activity of the PT cells and hence promote the activity of the lactotrophs, while short days (long duration signal) would be inhibitory. The refractory response expressed under the fixed photoperiods could be due to a change in the responsiveness of the PT target cells or any component of the intercellular relay. The involvement of a paracrine mechanism within the pituitary gland may explain the coordinated secretion by lactotrophs, and the time-dependent responsiveness may explain the temporal variation in PRL secretion under a fixed melatonin stimulus.
In conclusion, this study demonstrates for the first time that robust, long-term, cyclical changes in PRL secretion persist in HPD rams in which the neural-hormonal link between the hypothalamus and pituitary gland has been surgically disrupted. HPD animals express an apparently normal response to an acute switch in photoperiod, become refractory to a fixed photoperiod, and express intrinsically generated long-term patterns of PRL secretion. If these effects are not mediated through the hypothalamus, they must be generated within the multicellular systems of the pituitary gland. The pituitary gland may be a more important center for integration and timing than previously recognized.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Correspondence: G.A. Lincoln, MRC Reproductive Biology Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, Scotland, UK. FAX: 0131-228-5571; g.lincoln{at}ed-rbu.mrc.ac.uk 
Accepted: October 6, 1999.
Received: July 13, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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