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a Faculty of Veterinary Medicine and
b Science, University College Dublin, Dublin 4, Ireland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6 h increase in day length) failed to influence prolactin secretion, all other long-day treatments (> 6 h increase in day length) initiated a rise in prolactin in at least 75% of ewes in each group (p < 0.05). The aim of experiment 2 was to determine whether ewes failed to secrete prolactin during a 35 LD photoperiod from September 21 because they did not recognize a 6-h increase in day length at any time of year as a stimulatory photoperiod signal or because hypothalamic/pituitary regulation of prolactin synthesis or secretion is compromised in September. The results demonstrated that while hypothalamic regulation of prolactin secretion and pituitary stores of prolactin were normal at all times of year examined, the ability of ewes to secrete prolactin in response to a long-day photoperiod signal appears to be dependent on photoperiodic history rather than the time of year of the photoperiodic challenge.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is not known whether circannual rhythms are regulated by one signal or by a number of signals that function independently of one another [7]. Several aspects of the regulation of prolactin and gonadotropin circannual cycles suggest that they may be regulated by independent signals. First, disassociation of prolactin and LH secretion was demonstrated in ewes exposed to specific photoperiod signals [8–10] and in ewes maintained in constant photoperiods, in which annual rhythms in prolactin and LH secretion were observed [1, 2]. Second, recent data suggest that while melatonin acts at the level of the hypothalamus to regulate GnRH/LH secretion, melatonin acts primarily at the level of the pituitary gland to regulate prolactin secretion [11–15]. Third, in the Syrian hamster, photoperiodic history can influence the ability of a specific photoperiod signal to initiate LH secretion, while prolactin is secreted according to the photoperiod signal regardless of the photoperiodic history [16, 17]. Thus, it was hypothesized that the differential responses of the LH and prolactin axes to photoperiod in the hamster represented the expression of different time-measuring systems and that the role of photoperiodic history varied among different neuroendocrine rhythms [16].
The relationship between gonadotropin and prolactin rhythm regulation in ewes is less clear than in hamsters, although LH secretion in response to photoperiod challenges is well documented. When ewes are removed from natural photoperiod after the winter solstice and are immediately exposed to 35 long days followed by short days, reproductive activity is initiated approximately 100 days later [18]. However, exposure to long days followed by short days between the autumn equinox and the winter solstice fails to initiate reproductive activity in ewes at a later time [18, 19]. Seasonal differences in the ability of this photoperiodic challenge to initiate reproductive activity was attributed to photoperiodic history before the long-day photoperiodic challenge [20]. Thus, on natural photoperiod, the endogenous rhythm of LH secretion in ewes is considered to be photorefractory between the autumn equinox and winter solstice. The objective of experiment 1 was to determine whether the prolactin secretory system in ewes, like the LH system, is also unresponsive to long- followed by short-day stimulation at certain times of the year, and particularly between the autumn equinox and winter solstice. The results of experiment 1 identified a specific time period during which the prolactin system was unresponsive to a long-day photoperiod signal. Therefore, the objective of experiment 2 was to determine the mechanism by which prolactin secretion was disrupted in this model at this time of year. Could releasable pools of prolactin or dopaminergic regulation of prolactin vary with time of year? Our hypothesis was that the prolactin axis of ewes fails to respond to a long-day signal close to the autumn equinox simply because the change in day length after transfer from natural photoperiod to a long-day photoperiod signal of 18L:6D is not a sufficient change at our latitude to be interpreted as a long-day signal at this time of year.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Two experiments were carried out using sexually mature Suffolk cross ewes in Dublin, Ireland (53°18'N). Ewes were maintained on natural photoperiod until transferred indoors into photochambers. Photoperiod was regulated by electronic timers operating fluorescent bulbs that provided approximately 350 lux lateral to the head of the sheep during periods of illumination. A dim red light producing less than 2 lux at 1 m distance, mounted 2 m above the sheep, remained on continuously to facilitate stock inspection during periods of darkness. The housed animals had access to hay and water ad libitum and received 0.5 kg/head per day of supplementary concentrates to maintain body weight.
Experiment 1a: What Is the Prolactin Response to a 35-Long-Day Photoperiod between the Autumn Equinox and the Winter Solstice?
Ewes (8 per group) were transferred from outdoor natural photoperiod to the photochamber and immediately given a photoperiod treatment of 35 long days (35 LD; 18L:6D) followed by continuous exposure to a fixed short day length (8.5L:15.5D) at the following times of year (Fig. 1a): 35 LD from 1) September 21, 2) October 26, and 3) November 30. As positive controls, ewes were exposed to 35 LD from 4) January 4 and 5) February 8. Negative controls (6) were maintained on continuous exposure to a fixed short day length (8.5L:15.5D) from the winter solstice. Blood samples were collected once weekly and assayed for prolactin. LH data, as a measure of reproductive activity, have previously been presented [18].
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Experiment 1b: Are There Differences in the Melatonin Profile of Ewes Exposed to 35 LD in September vs. February?
In a separate group of ewes (5 per group), blood samples were collected once per hour for 24 h in animals exposed to 35 LD from September and February (controls) on Day 34 of the long-day exposure. Lights were changed on the hour, and blood samples were collected on the half hour; therefore, we could determine melatonin concentrations within 30 min of lights-on and -off. Serum was assayed for melatonin as described below.
Experiment 2a: Can the Prolactin Axis Interpret a 6-Hour Increase in Day Length?
Ewes (8 per group) were taken from natural photoperiod and immediately exposed to a photoperiod treatment as follows (Fig. 1b): 1) 35 LD (18L:6D; 6-h increase in day length from natural photoperiod) from September 21; 2) 35 LD (18L:6D) a 9-h increase in day length from November 30; 3) 35 days of a 6-h increase in day length from natural photoperiod from November 30 (15L:9D); and 4) 12L:12D from September 21 followed by 35 days of a 6-h increase in day length from November 30. Controls (5) were maintained on simulated natural photoperiod (SNP) from September 21.
Experiment 2b: Is Hypothalamic/Pituitary Gland Regulation of Prolactin Secretion Compromised in Ewes in September/October?
On Days 34 and 35 of a 35-LD photoperiod exposure from September 21 (samples collected on October 27–28) and November 30 (samples collected on January 4–5), ewes were challenged with thyrotropin-releasing hormone (TRH; 100 µg i.v.; Sigma, P-1319, Poole, Dorset, UK) and sulpiride (42 mg i.m.; Sigma S-7771) 24 h apart (Fig. 1b). These doses were similar to doses used by Lincoln and Clarke [13]. Sulpiride is a specific dopamine D2 receptor antagonist [21]. The D2 receptor mediates the hypothalamic inhibitory effect of dopamine in the pituitary gland [22]. TRH provides an alternative way to elicit prolactin secretion and provides an index of releasable pools of prolactin. Blood samples were collected every 15 min for 1 h before and 3 h after each challenge.
RIAs
Blood samples were collected and stored at room temperature for 1 h and at 4°C for 24 h. Samples were then centrifuged at 700 x g for 20 min. Serum was collected and stored at -20°C until assayed.
Serum prolactin concentrations were determined using the RIA method of McNeilly and Andrews [23]. Sensitivity of the assay, as defined as 95% binding, was 0.5 ng/ml. Mean inter- and intraassay coefficients of variation were 6.6% and 7.5%. Melatonin concentrations were determined in a single assay as described by Ronayne et al. [24]. Sensitivity of the assay, as defined as 95% binding, was 18 pg/ml. Intraassay CV for two serum pools with mean values of 36 and 324 pg/ml were 6.6% and 7.5%, respectively.
Mean Air Temperature
In experiment 1, temperature was recorded daily in the photoperiod rooms using maximum/minimum thermometers. The mean air temperature per week and per month was determined by averaging the daily mean of the maximum and minimum recordings.
Statistical Analyses
In experiment 1, data were analyzed using a nonlinear least-squares analysis using the NLIN procedure from SAS/STAT [25]. Using this procedure, data from each animal were plotted, and a line was fitted to the data after transfer from long to short days. A second line was drawn on the baseline concentrations under short days. Animals were defined as having elevated concentrations of prolactin during exposure to long days if the line fitted to the prolactin concentrations after transfer to short days had a significantly negative slope, demonstrating that there was a decline after transfer to short days. This indicated elevated levels during exposure to long days. The date of intersection of the two lines was determined by the program and was taken as the date when prolactin concentrations had returned to baseline. The Jacknife resampling method was used to determine standard errors at this intersection point [26]. Differences in monthly temperatures were analyzed using ANOVA. Melatonin data were log-transformed and analyzed using two-way ANOVA (treatment vs. time).
In experiment 2, differences in the prolactin profile between animals in different treatment groups during the period of long-day stimulation was analyzed using a general linear mixed model using SAS. Sulpiride and TRH data were analyzed using ANOVA for repeated measures.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rise in prolactin concentrations during exposure to long days Prolactin concentrations remained at baseline in all ewes exposed to 35 LD from September 21. A greater number of ewes (6 or more per treatment group, n = 8; p < 0.05) had a rise in circulating concentrations of prolactin during exposure to 35 LD from October 26, November 30, January 4, and February 8 (Fig. 2). Over the time period of the experiment, prolactin concentrations remained at baseline in the negative control ewes maintained on short days from the winter solstice.
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Decline in prolactin concentrations after transfer to short days Because ewes exposed to 35 LD from September did not have a rise in prolactin during exposure to long days, they did not express a subsequent decrease in prolactin after exposure to short days. Prolactin concentrations in ewes exposed to 35 LD from November 30, January 4, and February 8 returned to baseline within 7 wk of exposure to short days. In contrast, prolactin concentrations in ewes exposed to 35 LD from October 26 returned to baseline levels within a shorter time (p < 0.05; Fig. 2).
Temperature Temperature fluctuated in the photoperiod rooms over the period of the experiment, with highest average room temperatures in May (18.7 ± 0.6°C) and lower temperatures in December (12.1 ± 0.5°C) and January (12.0 ± 0.3°C). However, there was no association between the changes in temperature and the plasma concentrations of prolactin in controls maintained on short days from the winter solstice over the time period of the experiment (Fig. 2).
Experiment 1b: Were There Differences in the Melatonin Profile of Ewes Exposed to 35 LD in September vs. February?
Serum melatonin concentrations were similar in both treatment groups (p > 0.05). Melatonin concentrations increased from baseline values (below sensitivity of the assay, 18 pg/ml) within 30 min of lights-off. Melatonin concentrations reached maximum concentrations of 100 ± 25 pg/ml in animals exposed to 35 LD from September and 115 ± 35 pg/ml in animals exposed to 35 LD from March, and subsequently returned to baseline concentrations within 30 min of lights-on in ewes in both treatments. The duration of elevated concentrations of melatonin was the same in both treatment groups (p > 0.05).
Experiment 2a: Can the Prolactin Axis Interpret a 6-Hour Increase in Day Length?
Ewes transferred from SNP to a 9-h increase in day length for 35 days from November 30 had elevated concentrations of prolactin within 2 wk of exposure to long days (72.8 ± 13.8 ng/ml; p < 0.05; Fig. 3), confirming the results of experiment 1a. Similarly, prolactin concentrations increased in ewes transferred from SNP to a 6-h increase in day length for 35 days from November 30 (99.5 ± 22.2; Fig. 3; p < 0.05).
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In contrast, weekly prolactin concentrations did not change in ewes before and during exposure to a 6-h increase in day length for 35 days from September 21 (p > 0.05; Fig. 3). Similarly, ewes failed to secrete prolactin in elevated concentrations after a 6-h increase in day length from a higher baseline photoperiod of 12L:12D in November in comparison to pre-treatment values or SNP controls (p > 0.05; Fig. 3).
Experiment 2b: Was Hypothalamic/Pituitary Gland Regulation of Prolactin Secretion Compromised in September/October?
Interestingly, ewes exposed to 35 LD from both September and November elicited similar prolactin secretory profiles after stimulation with TRH and sulpiride (Fig. 4). Prolactin concentrations rose above baseline for the 3-h sampling period, with peak values of 612 ± 159 (September) and 653 ± 81 (November) ng/ml for TRH and 1311 ± 246 (September) and 673 ± 143 (November) ng/ml for sulpiride (p > 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An interesting question that arose from the observation that prolactin was not secreted during exposure to 35 LD from September 21 was what mechanism regulated the lack of a cellular response to a signal that at other times of the year was stimulatory. There were four possible answers. First, the pineal gland may not have been secreting a melatonin profile reflecting ambient photoperiod during this period. Second, some hypothalamic neuromediator was inhibiting prolactin secretion. Third, pituitary stores of prolactin were reduced. Fourth, the prolactin axis of ewes may simply not be able to interpret a 6-h increase in day length at our latitude as a long-day signal. The results of experiment 1b demonstrated that circulating concentrations of melatonin reflect ambient photoperiod during exposure to 35 LD from September 21. Thus, the lack of prolactin secretion in response to a long-day photoperiod signal is not caused by a change in the melatonin signal, but can be attributed to the post-pineal processing of the photoperiodic message. The results of experiment 2b demonstrated that the prolactin response to a sulpiride challenge was similar during exposure to 35 LD from both September 21 and November 30. This demonstrated that hypothalamic regulation of prolactin secretion is not different at this time of year. There was no difference in the pattern of prolactin secretion from the pituitary gland after a TRH challenge during long-day stimulation from either September or November, demonstrating that releasable pools of prolactin were normal. Thus, these experiments discount the possibility that either melatonin secretion, dopaminergic regulation of prolactin secretion, or releasable pools of prolactin could account for the inability of a 35 LD signal to initiate prolactin secretion from September 21. Could it simply be that animals could not interpret a 6-h increase in day length at this time of year?
When ewes were taken from natural photoperiod and exposed to a 35 LD photoperiod signal from September 21, they were exposed to a 6-h increase in day length, from 12L:12D to 18L:6D. When animals were taken from natural photoperiod and exposed to a 35 LD signal from November 30, they were exposed to a 9-h increase in day length, from 9L:15D to 18L:6D. In experiment 2, animals received a 6-h increase in day length from either 12L:12D to 18L:6D or 9L:15D to 15L:9D from November 30. The prolactin axis of ewes could not interpret a 6-h increase from 12L as a long-day signal, but could interpret a 6-h increase in day length from 9L as a long-day signal at this time of year. These data strongly suggest that it is the photoperiodic history of the ewe that determines the ability of an increase in day length to stimulate prolactin secretion.
When the responses of ewes to a 35 LD photoperiod signal followed by short days are compared for prolactin (experiment 1a) and LH [18], it appears that these two endocrine systems respond differently to the same photoperiod challenge between the autumn equinox and the winter solstice. When the temporal relationship between LH and prolactin secretion is examined, there are two separate relationships to consider. The first is the relationship between the immediate secretion of LH and prolactin at the time of the long-day signal. Ewes were reproductively active from September to January and thus were secreting elevated concentrations of LH during this time period. Exposure to 35 LD at this time of year initiated a rise in prolactin concentrations during exposure to long days. Thus, elevated concentrations of LH did not exclude the secretion of prolactin. When ewes were exposed to 35 LD from February, they expressed a rise in prolactin while they were in an anestrous condition with undetectable levels of LH. Thus, the immediate LH profile does not influence the response of the prolactin axis to long days.
The second relationship is the relationship between long-term LH and prolactin secretion in response to the long-day signal. When ewes are exposed to 35 LD after the winter solstice, LH is secreted in elevated concentrations after approximately 100 days from the end of the long-day signal, and prolactin is secreted in elevated concentrations during exposure to long days. Thus, after the winter solstice, long days synchronize endogenous rhythms of both LH and prolactin secretion. Exposure to long days before the winter solstice, however, does not produce such a predictable response. Exposure to long days of 35–90 days duration before the winter solstice fails to initiate a rise in LH secretion 100 days from the end of the long-day signal. In contrast, prolactin was secreted during exposure to 35 LD at any time period between October 26 and the spring equinox. Thus, there is variation in the times at which the two rhythms are sensitive to long days. The results of our experiment provide clear evidence that the secretion of prolactin and LH are regulated independently and that concentrations of one hormone do not regulate the levels of the other hormone.
In conclusion, the results of these experiments are evidence that there is a seasonal variation in the ability of long days to influence prolactin secretion. We have identified a time period of approximately 35 days after the autumn equinox during which the prolactin axis fails to respond to a long-day photoperiod signal. The ability of ewes to secrete prolactin in response to a long-day photoperiod signal appears to be dependent on photoperiodic history rather than the time of year of the photoperiodic challenge.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: T. Sweeney, Faculty of Veterinary Medicine, University College Dublin, Ballsbridge, Dublin 4, Ireland. FAX: 353 1 6600883; tsweeney{at}vetmed.ucd.ie
Accepted: September 2, 1998.
Received: April 15, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
