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Regular Article |
a Reproductive Sciences Program, Department of Physiology, University of Michigan, Ann Arbor, Michigan 48109-0404
ABSTRACT
An endogenous circannual rhythm drives the seasonal reproductive cycle of a broad spectrum of species. This rhythm is synchronized to the seasons (i.e., entrained) by photoperiod, which acts by regulating the circadian pattern of melatonin secretion from the pineal gland. Prior work has revealed that melatonin patterns secreted in spring/summer entrain the circannual rhythm of reproductive neuroendocrine activity in sheep, whereas secretions in winter do not. The goal of this study was to determine if inability of the winter-melatonin pattern to entrain the rhythm is due to the specific melatonin pattern secreted in winter or to the stage of the circannual rhythm at that time of year. Either a summer- or a winter-melatonin pattern was infused for 70 days into pinealectomized ewes, centered around the summer solstice, when an effective stimulus readily entrains the rhythm. The ewes were ovariectomized and treated with constant-release estradiol implants, and circannual cycles of reproductive neuroendocrine activity were monitored by serum LH concentrations. Only the summer-melatonin pattern entrained the circannual reproductive rhythm. The inability of the winter pattern to do so indicates that the mere presence of a circadian melatonin pattern, in itself, is insufficient for entrainment. Rather, the characteristics of the melatonin pattern, in particular a pattern that mimics the photoperiodic signals of summer, determines entrainment of the circannual rhythm of reproductive neuroendocrine activity in the ewe.
anterior pituitary, circadian rhythm, estradiol, GnRH, hypothalamus, LH, melatonin, pineal, seasonal reproduction
INTRODUCTION
Optimal propagation and survival of many species requires coordination of reproductive processes with changes in the external environment. In a broad range of species, seasonal reproductive transitions are timed by photoperiodic cues that synchronize an internally generated rhythm, often termed a circannual rhythm, because its period approximates 1 yr [1]. This synchronization process, which ensures that various rhythm stages coincide among individuals and align appropriately to the seasons, is referred to as entrainment and is achieved via melatonin. Changes in photoperiod induce corresponding alterations in the circadian pattern of melatonin secretion from the pineal gland [2]. Melatonin, in turn, acts as a physiological timekeeping hormone; its nocturnal secretory pattern provides an endocrine signal for day length [2].
Prior work in sheep, ground squirrels, and other mammalian species expressing circannual rhythms indicates that elimination of the circadian secretion of melatonin by removal of the pineal gland disrupts photoperiodic responsiveness and causes circannual rhythms to free run [3–5]. In sheep, provision of a circadian melatonin pattern for as little as 70 days each year can maintain entrainment of the circannual rhythm of reproductive neuroendocrine activity following pinealectomy [6]. Nevertheless, not all seasonal melatonin patterns are equally effective. Replacement with melatonin patterns mimicking those secreted in spring/summer is more effective than an autumnal pattern, and replacement of the pattern secreted in winter is ineffective [5]. Those studies, however, matched the season-specific melatonin patterns to their corresponding season (i.e., summer pattern delivered in summer, winter pattern in winter). Research in the field of circadian rhythms indicates that circadian entrainment requires exposure to the entraining agent (i.e., light) during a specific stage of the rhythm [7]. Time of exposure to a photoperiodic signal may also be critical for circannual rhythm entrainment [8]. Thus, the differential efficacy of the season-specific melatonin replacements in entraining the circannual reproductive rhythm of sheep, as mentioned earlier, could have reflected either quality of the simulated photoperiodic signal (i.e., characteristics of the melatonin pattern) or stage of the rhythm when the signal was applied.
In this study we assessed the importance of photoperiodic signal quality to entrainment of the circannual reproductive rhythm by determining if any circadian melatonin pattern would be efficacious if applied during a sensitive rhythm stage. Specifically, we tested whether a winter-melatonin pattern, applied in spring/summer when the rhythm is normally synchronized [5], can entrain the circannual rhythm of reproductive neuroendocrine activity in pinealectomized ewes.
MATERIALS AND METHODS
Animals and Experimental Design
The study was conducted on 27 adult Suffolk ewes maintained at the Sheep Research Facility near Ann Arbor, MI (42°18'N). The ewes were ovariectomized in August 1994 and treated s.c. with a 3-cm Silastic capsule containing estradiol (Sigma Chemical Co., St. Louis, MO) to maintain a luteal phase serum estradiol concentration [9]. Serum LH was used as an indicator of seasonal reproductive neuroendocrine status [9]. In this model, a high LH concentration reflects high frequency pulses of GnRH and LH secretion, which is indicative of the breeding season, whereas low LH reflects infrequent GnRH and LH pulses typical of anestrus [10]. In December 1994 (late breeding season), the ewes were pinealectomized as described elsewhere [11]. Pinealectomy at this time has little effect on the ongoing seasonal breeding cycle but causes subsequent cycles to become desynchronized (i.e., free run) [12]. After pinealectomy, the ewes were moved to light-proof rooms where lighting (35 lux at eye level) was adjusted twice a week to simulate natural photoperiod, including 60 min for civil twilight. Temperature was not regulated. All procedures were approved by the University of Michigan Committee for the Use and Care of Animals.
The experimental design is illustrated in Figure 1. After pinealectomy, the ewes were randomly allocated to the following three groups (n = 9/group): 1) noninfused controls, 2) summer-melatonin pattern (8 h infusion each 24 h to simulate long days), and 3) winter-melatonin pattern (14 h infusion each 24 h to simulate short days). The infusions were designed to reproduce naturally occurring circulating melatonin profiles on either the summer or winter solstice. Infusions were restricted to 70 consecutive nights each year, centered around June 21 for 2 yr after pinealectomy (Fig. 1, entrainment portion of the study). After Year 2, melatonin was discontinued and the ewes were monitored through Year 4 to document development of free-running circannual cycles (Fig. 1, free-run portion). For infusion, melatonin (Sigma Chemical Co.) in physiological saline solution was i.v. delivered (44 µg/h) via jugular cannula using battery-powered backpack pumps (Model AS-6MP; Autosyringe, Hooksett, NH), as previously described [13]. This allowed nightly infusion without the need to restrain the ewes. During each 24-h period, the midpoint of the infusion coincided with the middle of the dark phase of the light-dark cycle.
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Blood Sampling and Hormone Assays
Blood was sampled by jugular venipuncture. To monitor circannual LH cycles, samples were obtained twice a week and LH was measured in duplicate in 10- to 200-µl aliquots of serum using a modification [14] of a previously described radioimmunoassay [15, 16]. LH values are expressed in terms of NIH-LH-S12. Assay sensitivity averaged 0.65 ± 0.19 ng/ml (51 assays). Within and between assay coefficients of variation (CV) averaged 6% and 10%, respectively. Blood for melatonin assay was sampled at night under a dim red light after pinealectomy to document completeness of pineal removal, during the first week of melatonin infusion to confirm appropriate dosing, and serially across 24 h to verify desired circadian patterns were achieved. Melatonin was assayed in duplicate in 200-µl aliquots of serum using a radioimmunoassay described elsewhere [17]. Sensitivity averaged 9 pg/ml and within and between assay CV averaged 7% and 9%, respectively (four assays).
Data Analysis
Serum LH concentrations were subjected to logarithmic transformation to normalize variability across a wide range of values. Circannual LH cycles in each ewe were then identified by a cluster cycle detection algorithm [18, 19]. LH cycles were divided into high and low stages (i.e., neuroendocrine breeding and anestrus seasons, respectively) using a probability level of 
5% to discriminate between contiguous clusters of high and low LH values. Transition times between high and low clusters were taken as the midpoints of any intermediate clusters when such intermediates existed. Period of LH cycles was calculated as the interval between midpoints of successive high LH stages.
RESULTS
Melatonin
Completeness of pinealectomy was confirmed by absence of a nocturnal increase in serum melatonin concentrations in all ewes. Figure 2 illustrates diurnal patterns of circulating melatonin for each of the three experimental groups during the final week of the 70-day infusion period in Year 1 and in pineal-intact ewes maintained outdoors and sampled at the same time. Noninfused pinealectomized controls had virtually undetectable melatonin values (Fig. 2B). Melatonin infusion restored the expected summer or winter diurnal pattern of circulating melatonin (Fig. 2A). Mean values while the pumps were turned on (200–300 pg/ml) were comparable to those in the pineal-intact ewes (Fig. 2, A and B). As expected, the duration of elevated melatonin levels in the summer-melatonin group was ~6 h shorter than that in the winter-melatonin group.
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Circannual LH Cycles
Figure 3 depicts serum LH patterns across the entire 4-yr study for one representative ewe in each group. The horizontal bars above each hormonal profile indicate high and low stages of LH cycles as determined by the cycle detection algorithm. Figure 4 summarizes timing of high and low stages of LH cycles in each ewe for all 4 yr. Before pinealectomy, LH increased in September/October, signifying onset of the natural breeding season (Year 1). Thereafter, LH cycles in noninfused controls became desynchronized and appeared to free run (Figs. 3A and 4A). High/low cycle stages in these controls did not coincide among ewes and did not consistently align to the natural breeding season (September–February). This documents photoperiodic nonresponsiveness of pinealectomized ewes.
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The summer-melatonin pattern entrained 12-mo LH cycles (Figs. 3B and 4B). Variability in onset of the LH elevation in these entrained cycles was no different from that in this group before pinealectomy. Standard deviation for date of onset of the LH rise prior to pinealectomy (11 days, Year 1) was comparable to that thereafter, when melatonin was infused (15 and 10 days in Years 2 and 3). The period of these entrained cycles equaled 1 yr (364 ± 3 days between midpoints of LH rises in Years 2 and 3). These LH elevations, however, were shorter than those before pinealectomy (84 ± 6 vs. 111 ± 6 days; P < 0.05 by paired t-test), and they began 1–2 wk early. Discontinuing melatonin in Year 3 caused LH cycles to become desynchronized, confirming entrainment by the summer-melatonin pattern.
In striking contrast to efficacy of the summer-melatonin pattern, the winter pattern failed to entrain the rhythm (Figs. 3C and 4C). During the first year after pinealectomy, LH rises were less evident than in the other groups (December 1994–December 1995). LH rises the following year tended to be clustered in the spring and summer (1996) but their timing was extremely variable among ewes. Overall, ewes receiving the winter-melatonin pattern did not exhibit tightly synchronized LH cycles, and times of high LH were not confined to the natural breeding season.
DISCUSSION
The backdrop for this study was the prior finding that infusion of a spring- or summer-melatonin pattern during the spring or summer seasons entrained circannual cycles of LH secretion in pinealectomized ewes, whereas infusion of a winter-melatonin pattern during the winter did not [5]. It was not determined in the prior work, however, if inability of the winter melatonin pattern to entrain the rhythm reflected quality of the simulated photoperiodic signal (i.e., characteristics of the melatonin pattern) or the stage of the underlying rhythm during which the signal was applied. The goal of this study was to distinguish between these two possibilities. By infusing either a summer- or a winter-melatonin pattern into pinealectomized ewes centered around the summer solstice when melatonin can readily synchronize the rhythm [5], we have gathered strong evidence that the mere presence of a circadian melatonin pattern, in itself, is not sufficient for entrainment. Rather, the characteristic of the pattern, particularly its long-day nature, is crucial.
One potential caveat to this conclusion is that the melatonin delivery occurred entirely at night in the summer-melatonin group, whereas delivery overlapped into the daytime in the winter-melatonin group (Fig. 2). Thus, it could be argued that, if there were a circadian rhythm of sensitivity to melatonin, the winter pattern might have been ineffective because the infusion mismatched the photoperiodic cycle and did not properly align with a circadian-sensitive period. This, however, seems unlikely because prior work does not support the existence of a circadian rhythm of sensitivity to melatonin in pinealectomized sheep [13]. Further, earlier work has demonstrated that both the winter- and summer-melatonin patterns can produce appropriate reproductive responses in pinealectomized ewes when mismatched with the photoperiod (i.e., short-day melatonin infused in long days and vice versa) [20, 21]. These considerations are in keeping with the conclusion that the melatonin patterns, and therefore the photoperiods of summer, provide an effective entraining signal, whereas those of winter do not.
This conclusion prompts intriguing questions relative to photoperiodic regulation of circannual rhythms. Is there a role for short days, photoperiodic history, and rhythm stage? Although the short-day signal failed to entrain the rhythm, our findings do not discount a role for short days in regulating seasonal reproduction. Shortening days after the summer solstice are needed to maintain a full duration breeding season [22, 23]. This most likely explains the abbreviated LH elevation in our ewes receiving long-day melatonin, because the infusions were of constant duration. Further, short days in winter sensitize the photo-neuroendocrine axis of pineal-intact ewes to long days that will be experienced in spring [24]. Nonetheless, in terms of entraining the circannual rhythm, the short-day signal alone was ineffective.
With regard to photoperiodic history, prior work in birds and mammals indicates photoperiods experienced in the recent past, as well as directional change in day length, influence photoperiodic responses [25–27]. The fixed long-day melatonin pattern in our study, however, entrained the circannual reproductive rhythm, and this signal was just as effective as a gradually changing melatonin pattern used previously [5] to simulate natural secretory profiles of either spring or summer. Thus, absolute photoperiod, rather than change itself, appears critical for circannual rhythm entrainment. Related to this, the efficacy of a fixed melatonin pattern in entraining the rhythm in pinealectomized ewes suggests either the pineal gland is not necessary to compile a photoperiodic history or an extremely long-term memory exists for photoperiods experienced before pinealectomy (a memory lasting longer than 1 yr in this study). Alternatively, photoperiodic history may not be crucial to circannual rhythm entrainment. The latter possibility does not discount the importance of photoperiodic history to seasonal regulation, but it would suggest that this is not obligatory for rhythm entrainment.
Regarding rhythm stage, two theoretical models may be considered for photoperiodic entrainment. In the first, quality of the photoperiodic signal is critical, with entrainment occurring only when a specific photoperiod is experienced. In the second, stage of the circannual rhythm is crucial. Namely, as for circadian rhythms [7], photic cues may advance or delay circannual rhythms, or have no effect at all, depending on when the cue is perceived [8]. These models are not mutually exclusive; entrainment may require a specific photoperiod during a particular rhythm stage. Consistent with a role for stage are findings that a given photoperiod can differentially affect seasonal cycles of fish, birds, and mammals (including sheep) depending on the season of exposure [8, 28–30]. However, those studies, unlike the present one, utilized animals that had intact photoperiodic response systems. Therefore, photoperiod existing at the start of those experiments could have influenced the response, precluding definitive conclusions regarding a role for stage.
Although the present findings, in themselves, do not allow definitive conclusions regarding rhythm stage (because the differing melatonin patterns were provided at only one rhythm stage), they do permit a strong inference when considered together with prior work using the same animal model. The collective results, summarized in Table 1, consistently suggest that the summer-melatonin pattern entrains the rhythm, whereas the winter pattern does not. In one study [31], the summer pattern, infused into pinealectomized ewes at random stages of the free-running rhythm synchronized reproductive onset equally well, regardless of rhythm stage. Although discouraging a role for stage, that study monitored reproductive response through only one circannual cycle, which is insufficient for definitive assessment of circannual entrainment. In another study, a summer-melatonin pattern infused in winter did entrain the rhythm of pinealectomized ewes [6]. However, the relevance of those findings to discrimination between photoperiodic signal quality and rhythm stage is limited because the rhythm had previously been phase-shifted by 6 mo so that the summer signal was actually given during the summer stage of the rhythm. The present findings add important new insight into this issue by demonstrating that the winter-melatonin pattern fails to entrain the rhythm when delivered during the summer stage of the rhythm. If stage alone were critical, this treatment should have produced entrainment. Collectively, these findings favor the importance of photoperiodic signal quality, rather than rhythm stage, for entraining the circannual reproductive rhythm of sheep. It does remain possible, however, that an interaction exists between photoperiod and rhythm stage, which optimizes photoperiodic entrainment of this circannual rhythm.
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Finally, it is important to emphasize that circannual rhythms are widespread throughout the animal kingdom. They occur in both long- and short-day breeders, and they generate a host of seasonal processes beyond reproduction, such as hibernation, migration, metabolism, and changes in pelage [1]. Our findings in the ewe may lend themselves to a broader understanding for timing of seasonal rhythms. For example, in hibernating circannual species such as ground squirrels [4, 32] and woodchucks [33], photoperiodic exposure in spring/summer could maintain precisely timed seasonal cycles despite the disruption of photoperiodic signaling during periods of hibernation in autumn/winter. In long distance migratory species experiencing "nonsymmetrical" annual cycles of geophysical cues, the ability of a relatively brief photoperiodic exposure to entrain an entire yearly cycle could provide a protective mechanism for maintaining precise timing of seasonal rhythms. For transequatorial migrants, a stage of photoperiodic insensitivity may also be needed to buffer against a bimodal photoperiodic cycle that these species would typically encounter. These considerations are in keeping with the general concept that entrainment by exposure to a critical photoperiod during only part of the year may provide a mechanism for coordinating annual timekeeping functions that regulate reproduction and other seasonal processes in a broad spectrum of species.
ACKNOWLEDGMENTS
We thank Dr. Deborah Battaglia, Dr. Jennifer Bowen, Douglas Doop, Holly Krasa, Gary McCalla, and Edmund Tanhehco for their excellent assistance with the animal experimentation and hormone assays; Drs. Josephine Arendt, Gordon Niswender, Leo Reichert, Jr., and Al Parlow for supplying assay reagents; and Drs. Theresa Lee, Arthur Vander, and Deborah Battaglia for critiquing the manuscript.
FOOTNOTES
First decision: 30 March 2000.
1 Supported by NSF-IBN grant 92-06510, the Sheep Research and Assays and Reagents Core Facilities of the P30 Center for the Study of Reproduction, HD-18258, and the Office of the Vice President for Research at the University of Michigan. 
2 Correspondence: Fred J. Karsch, Reproductive Sciences Program, 300 N. Ingalls Building, University of Michigan, Ann Arbor, MI 48109-0404. FAX: 734 936 8620; fjkarsch{at}umich.edu
3 Current address: Animal & Food Sciences Division, P.O. Box 84, Lincoln University, Canterbury, New Zealand.
Accepted: April 18, 2000.
Received: March 7, 2000.
REFERENCES
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