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Behavior |
Biological Institute, Department of Animal Physiology, University of Stuttgart, 70550 Stuttgart, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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behavior, seasonal reproduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Annual rhythms in body weight [1, 3] and reproductive state [9] persist in many European hamsters for more than one cycle, despite the fact that the animals are kept under constant photoperiods. In addition, the majority of pinealectomized hamsters show at least two consecutive cycles in body weight and reproduction [9]. In this respect, European hamsters share more similarities with true circannual species [10], such as ground squirrels [11–13], marmots [14, 15], or sheep [16] than with photoperiodic species, such as the Syrian or Siberian hamster [17–20].
Under natural conditions, both circannual and photoperiodic species are entrained with the geophysical year by the regular changes in day length [10, 20]. As has been demonstrated in ewes [21, 22] and ground squirrels [10], synchronization does not require the entire annual cycle. Instead, seasonal rhythms of reproductive activity can be entrained by exposing the animals to only one or two discrete blocks of photoperiodic information as long as they fall into a sensitive phase. For the European hamster, such an annual phase of sensitivity to short photoperiods has already been demonstrated. Under natural conditions European hamsters are not able to perceive the short photoperiod before mid-May. The sensitive phase to short photoperiods starts at that time and extends to at least the middle of July when natural photoperiods of less than 15.5 h induce gonadal regression [23].
Furthermore, there is experimental evidence for a complementary phase of sensitivity to long photoperiods during fall or winter. When animals were transferred from a natural photoperiod to a long photoperiod in either August or October (after complete gonadal atrophy), regrowth of gonads was accelerated by about 2–5 months, compared with animals kept under natural conditions [9]. In addition, the subsequent spontaneous regression of the gonads was also phase advanced, compared with control animals. These results suggest that the annual reproductive rhythm of European hamsters can be phase advanced by exposure to long photoperiods in winter. The aim of the present studies was to further characterize the complementary phase of sensitivity to long photoperiods during fall or winter, which may play an additional role in the synchronization of seasonal rhythms in the European hamster.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male and female European hamsters used for this study were obtained from our own breeding colony. All animals were born and raised at constant temperature (18 ± 2°C) and humidity (55 ± 5%), but under conditions of natural light (LDnat) at Stuttgart, Germany (latitude 48.46°N), yielding a maximum long day of 16 h 11 min at the summer solstice and a minimum short day of 8 h 15 min at the winter solstice. Food and water were given ad libitum. Body weight and gonadal status of the animals were checked once every 2 wk (experiment 2) or once every 3 wk (experiments 1 and 3) under a short enflurane anesthesia (Ethrane, Abbott, Abbott Park, IL). The studies were performed in accordance with the National Research Council publication Guide for Care and Use of Laboratory Animals (copyright 1996, National Academy of Science), the European Communities Council Directive of 24 November 1986 (86/609/EEC), and the German law.
In our hands, breeding of European hamsters is limited to approximately six to eight litters a year with an average of four to eight pups each. Therefore, both males and females were used for the present study to obtain a sufficient number of animals in each experimental group. In males the beginning of gonadal regrowth was defined by the descent of the testes into the scrotum, after which testis size was measured externally with a caliper-square. Palpation of testes and/or caliper-square measurement of testis size are standard procedures to determine the reproductive status of male hamsters [24]. It has been demonstrated for the European hamster that testis size correlates well with testis weight [9] and plasma testosterone levels [4]. In female animals the beginning of gonadal regrowth was defined by the first opening of the vagina verified by visual inspection. It has been demonstrated for Djungarian hamsters that there is a good correlation between ovarian cyclicity and the opening of the vagina [25]. In addition, preliminary studies in our own laboratory revealed that European hamsters show regular estrous cycles with a cycle length of 4– 5 days. In most animals, ovarian cyclicity started immediately after the opening of the vagina, and ended 1–2 wk before its closure. Thus, it seemed safe to assume that the first opening of the vagina is a sufficiently good indicator for the beginning of reproductive activity.
Experiment 1: Timing of Gonadal Regrowth under LDnat
Between 1998 and 2001, a total of 40 animals (12 males, 28 females) were born in our breeding colony under a natural light:dark cycle (LDnat) between mid-April and the end of July (Table 1). These animals were kept under natural lighting conditions and served as a control group to verify the timing of the first gonadal regrowth in the following spring (i.e., in the years 1999–2002). Body weight and gonadal status of the animals were checked regularly every 3 wk.
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Experiment 2: Effect of Long Photoperiods in Winter
A total of 28 animals (16 males, 12 females), born under LDnat between April and July 2001, were kept under LDnat until September 2001 (Table 1). At that time, all animals had regressed gonads. Five groups of four animals each, randomly chosen from different litters, were transferred to a 16L:8D cycle (lights on from 0400 h to 2000 h) every 4 wk from 19 September 2001 to 9 January 2002. A 16L:8D photoperiod is similar to the day length in Stuttgart during the summer solstice. One group stayed under LDnat as a control. Additionally, one group of four animals was transferred to an intermediate photoperiod (12L:12D, lights on from 0600 h to 1800 h) on 8 November 2001. At that time the natural photoperiod was approximately 9L:15D. Thus, the 12L:12D group experienced a 3-h increase of the photoperiod. Body weight and gonadal status of the animals were checked regularly every 2 wk.
Experiment 3: Gonadal Regrowth under Accelerated and Seminatural Photoperiods
A total of 22 animals (12 males, 10 females), born and raised in July 2000 under LDnat, were kept under LDnat until the middle of August 2000 (Table 1). Two groups of eight animals each were then transferred to a seminatural photoperiod (LDseminat) mimicking the natural lighting conditions of Stuttgart (i.e., light onset and offset of the artificial LD cycle were adjusted to the natural sunrise and sunset of Stuttgart once a week). Starting December 27, one group was subjected to an accelerated seminatural photoperiod (LDshift; i.e., the timing of lights on and off was changed twice as fast). As a consequence, the seasonal photoperiodic cycle of 1 yr was finished within 6 mo (Fig. 1). Six animals were maintained in natural conditions (LDnat) as controls. Body weight and gonadal status of the animals were checked regularly every 3 wk.
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Statistics
If not stated otherwise, data were analyzed using parametric and nonparametric ANOVA (STATISTICA software, StatSoft, Inc., Tulsa, OK). Where appropriate, the Nemenyi or least significant difference test was used for post hoc comparisons [26]. If not indicated otherwise, means ± SEM (
± SEM) are given in text and figures.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals born in our breeding colony during 4 consecutive years (1998–2001) under LDnat between mid-April and the end of July and kept under LDnat showed a rather high variability in the timing of gonadal regrowth in the following spring (Fig. 2). The first animal became reproductive in late December (a male) and the last in early May (a female). Kruskal-Wallis ANOVA by ranks revealed a highly significant difference between males and females (P < 0.001) but not between different years (P = 0.31). In general, regrowth of gonads occurred earlier in males than females. Gonadal regrowth of females showed two distinct peaks, a first one together with the males around 10L:14D and a second one around 13L:11D.
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Experiment 2: Effect of Long Photoperiods in Winter
Transfer to a long photoperiod (16L:8D) accelerated the timing of gonadal regrowth in all but one experimental group (Fig. 3). In groups I–III, gonads developed around the end of November. Animals in group II and III became reproductive almost simultaneously, whereas the timing of gonadal regrowth was more variable in group I (i.e., the total range was 56 days, compared with 0 and 14 days in groups II and III, respectively). In groups III–V, gonadal regrowth occurred within 14–28 days after exposure to 16L:8D. In contrast, transfer to 12L:12D did not stimulate reproductive development. Instead, animals of group VI became reproductive in the middle of March, which is at about the same time as the last two animals from the control group C. Control animals (C) kept permanently in LDnat showed a rather variable timing of gonadal regrowth, ranging from early January to the end of March. A two-way ANOVA revealed a highly significant difference between experimental groups (F6,14 = 39.9; P < 0.001) but no sex difference (F1,14 = 2.45; ns) in the timing of gonadal regrowth (i.e., the number of days between the start of the experiment on September 1 and the first appearance of testes or the opening of the vagina).
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A two-way ANOVA revealed a highly significant difference between experimental groups (F5,12 = 49.0; P < 0.001) but no sex difference (F1,12 = 0.09; ns) in the time until gonadal regrowth (i.e., the number of days between transfer to a long photoperiod and the first appearance of testes or the opening of the vagina [Fig. 4]). The shortest times (21 ± 4.0 days) were observed in groups III–V, indicating that gonadal regrowth can be stimulated within 3 wk. Animals in group II needed approximately twice as long. The longest times (67 ± 10.5 and 129 ± 6.7 days) were observed in groups I and VI, respectively. Duration of gonadal regrowth of males (22 ± 2.8 days) and females (20 ± 3.4 days) did not differ significantly.
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Experiment 3: Gonadal Regrowth under Accelerated and Seminatural Photoperiods
There was no difference in the timing of reproductive development between animals kept in LDnat and LDseminat. Both groups became reproductive at nearly the same time of the year, namely after 77 ± 5.9 days and 78 ± 6.2 days of increasing photoperiod (i.e., around the middle of March [Fig. 5]). Animals kept under LDshift showed a slightly advanced gonadal regrowth, but this effect proved to be significant only for females (Kruskal-Wallis ANOVA, H(2,11) = 8.14, P = 0.017). In addition, post hoc comparisons by the Nemenyi test revealed that females of the LDshift group were clearly advanced, compared with those kept under LDnat and LDseminat. In contrast, the males in each of the three groups developed their gonads at approximately the same time.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The experiments also demonstrate the existence of a phase of sensitivity to long photoperiods that begins in mid- November. During this phase of sensitivity, transfer to a long photoperiod induced the first signs of gonadal regrowth within 3 wk. One has to be aware, however, that estimates for both the duration of gonadal regrowth and the timing of the sensitive phase are affected by the sampling frequency. Because gonadal status was checked only once every 2 wk, animals with palpable testes or an open vagina after 14 days could have reached this stage as early as 1 day after exposure to a long photoperiod, whereas gonadal regrowth may have started already after 15 days in animals that were considered reproductive after 28 days. This experimental procedure created an artificial separation of at least 14 days between groups and also reduced the variability within groups. Nevertheless, it is safe to assume that gonadal regrowth can be induced within 14–28 days. The sensitive phase lasts at least until early January, as observed in group V. However, the end of this sensitive phase cannot be determined more precisely because the spontaneous recrudescence of gonads occurs in early spring. Indeed, some animals became reproductive even before the end of January (LDnat less than 9 h of light) without being exposed to a long photoperiod (Figs. 2 and 3). However, data obtained under LDnat also showed that quite a few females became reproductive between 17 March and 20 April (Fig. 2). At least for those animals, it seems safe to assume that gonadal regrowth was induced by the increasing natural photoperiod 2–4 wk earlier. The sensitive phase to long photoperiods may therefore last until the end of March if it is not terminated earlier by a spontaneous regrowth of the gonads.
Our data also shed some light on the length of daylight necessary to stimulate reproductive activity in spring. The second peak in the number of reproductive females in April (Fig. 2) suggests that these animals received a stimulatory signal 3 wk earlier, which corresponds to a natural photoperiod of almost 13 h at the end of March (difference between sunrise and sunset at 48.46°N latitude = 12 h 50 min). In addition, all animals of the LDshift group (Fig. 5) showed first signs of gonadal regrowth on March 7 after 70 days of increasing photoperiod, suggesting that gonadal regrowth was induced by the photoperiod perceived 3 wk earlier (i.e., around 14 February, when the experimental photoperiod was 13 h and 10 min). In contrast, transfer to 12L:12D (Fig. 3; group VI) failed to stimulate reproductive activity. Therefore, a photoperiod of approximately 13 h light seems to be both necessary and sufficient to stimulate the reproductive system of the European hamster. These results demonstrate that in European hamsters, like in Siberian hamsters, the stimulatory or inhibitory effect of a given photoperiod depends on the photoperiodic history of the animals. More than 13 h of light stimulate gonadal regrowth in animals previously exposed to short photoperiods. In contrast, as shown previously, less than 15.5 h of light induce gonadal regression in animals exposed to a long photoperiod [29].
The history-dependent effect of intermediate photoperiods may actually explain the biological significance of the finding that European hamsters show two transient periods of insensitivity during which they do not respond to either long photoperiods (present study) or short photoperiods [23]. In locations such as Stuttgart or Strasbourg, gonadal regression is induced in the middle of July by photoperiods of less than 15.5 h [29]. Gonadal atrophy is observed 4 wk later, namely in mid-August [23], when the natural photoperiod has shortened to approximately 14.5 h of light. This is longer than the critical photoperiod for inducing gonadal regrowth. However, the animals are insensitive to the stimulatory long photoperiod until mid-November, when the natural photoperiod has shortened to less than 9 h of light per day. The transient period of insensitivity thus prevents the animals from receiving a photo stimulatory signal during fall. The same logic can be applied to the opposite phase of the annual cycle. Under natural conditions, most of the animals are reproductive by the end of April, which corresponds to a natural photoperiod of 14 h. However, the phase of sensitivity to short photoperiods begins only in mid-May [23], when the natural photoperiod is longer than 15 h. Again, the transient phase of insensitivity to short photoperiods prevents the animals from receiving a short- day signal in spring.
An alternative explanation for the present findings could be that European hamsters do not respond to the long photoperiods before mid-November because exposure to photoperiods as short as those of the late fall is indeed necessary to elicit the fast response to long photoperiod observed in groups III–V (and absent in groups I and II). This possibility should be tested in future experiments by analyzing the timing of gonadal development in animals exposed to short photoperiod mimicking late fall (10L:14D or 9L:15D) before transfer to long photoperiods in September or October. Our findings could also be interpreted that European hamsters become refractory to long photoperiods by the end of summer, and that a 1- to 20-month period of exposure to short days is necessary to break this refractoriness and reinstate the sensitivity to long photoperiods. This interpretation, however, can be ruled out because it has already been shown that European hamsters kept under long photoperiods for several months will show not only spontaneous regression but also spontaneous regrowth of their gonads after approximately 3–5 months of gonadal regression [9]. The same has been observed in European hamsters kept under short photoperiods for several months (i.e., the animals show a spontaneous recrudescence of the gonads, followed by a second phase of gonadal regression [9]). Thus, European hamsters exhibit both short-day and long- day photorefractoriness. In contrast to other hamster species [30, 31], however, they do not need exposure to an opposite photoperiod to break photorefractoriness. Instead, they avoid the ill-timed perception of stimulatory or inhibitory photoperiods by establishing two periods of insensitivity during which they do not respond to long- or short-day signals, respectively.
The European hamster is a true hibernator. For such species, it is a suitable strategy to control gonadal regrowth in spring by an endogenous timer rather than a direct photoperiodic effect because at that time the animals are still in their hibernating burrows and have no information about photoperiodic changes in the outside world. The biological significance of a sensitive phase to long photoperiod may nevertheless be that of a backup mechanism for those animals, whose endogenous timing mechanisms is delayed. In addition, the sensitive phase may also serve as an entrainment mechanism by which the seasonal rhythm can be synchronized to the natural year. It has already been demonstrated for a number of species with true circannual rhythms that these circannual rhythms can be entrained or synchronized by exposing the animals to photoperiodic information only once or twice a year [10]. For example, providing photoperiodic information by means of a circadian melatonin pattern for as little as 70 days each year can maintain entrainment of the reproductive cycle in sheep [21, 22].
Data from both Masson-Pévet et al. [9] and our laboratory indeed suggest that transfer to a long photoperiod will not only induce gonadal regrowth but also phase advance the whole reproductive cycle. In male European hamsters exposed to long photoperiods by mid-October, not only gonadal regrowth but also the subsequent gonadal regression and recrudescence were advanced by 2–3 months, compared with control animals [Fig. 12 in Ref. 9]. In our study, most of the LDshift animals started gonadal regression almost simultaneously at the end of May (Fig. 5), which was most likely caused by the gradual decrease in photoperiod, effective in this group as of 4 April. Previous studies by Saboureau et al. [23] demonstrated that male hamsters initiate gonadal regression 4 wk after a short-day signal and that the perception of a short-day signal requires a phase of sensitivity for photoperiodic information. Therefore, the animals must have been sensitive at the end of April (i.e., 2 wk before sensitivity to short photoperiods occurs under natural conditions [23]). This suggests that the phase of sensitivity to short photoperiods was also advanced in the LDshift group.
The results of the present study can well be incorporated into a working model of the photoperiodic regulation of seasonal reproductive cycles in the European hamster. According to the present state of knowledge, illustrated in Figure 6, an endogenous circannual rhythm (
) provides the primary drive for both the transition from gonadal quiescence during hibernation to reproductive activity during the mating season and the transition from reproductive activity back to gonadal quiescence. The ambient photoperiod synchronizes this circannual rhythm by means of two phases of sensitivity. One phase of sensitivity to short photoperiod starts in mid-May and lasts until approximately the end of July [23]. During this phase exposure to a photoperiod of less than 15.5–15.0 h of light [critical photoperiod according to Ref. 29] will induce gonadal atrophy within 4 wk (–). Without exposure to a short photoperiod, nevertheless, most of the animals show gonadal regression, although a few weeks later than under natural conditions [9]. The short photoperiod thus serves to synchronize the endogenous circannual rhythm.
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A complementary phase of sensitivity to long photoperiods starts in mid-November and extends until March or early April. Exposure to a photoperiod longer than 13 h of light (critical photoperiod as determined in the present study) will induce gonadal regrowth within 2–4 wk (+). However, most of the animals will undergo gonadal regrowth even without exposure to a long photoperiod because they become refractory to the short photoperiods in early spring. The transition to a long photoperiod in spring is thus not as important for controlling the seasonal reproduction rhythm as the complementary transition from long to short photoperiods in summer. It remains to be determined why some animals initiate gonadal regrowth without any photoperiodic information, whereas others require a long photoperiod. However, a similar high variability has also been observed in the timing of arousal from hibernation in free-living European hamsters [32], ranging from early February for the oldest males to late April for females and juvenile males. Because hibernation is terminated by the regrowth of the gonads [33, 34], it can be assumed that the high variability in the timing of arousal is also a good indicator for a large variability in the timing of gonadal regrowth in free-living European hamsters.
In summary, the demonstration of an annual phase of sensitivity to long days provides further proof for a hypothesis proposed by Masson-Pévet et al. [9] that in European hamsters annual rhythms of body weight and reproduction are controlled by an endogenous circannual system entrained to the natural year by photoperiodic information.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Franziska Wollnik, Biological Institute, Department of Animal Physiology, University of Stuttgart, Pfaffenwaldring 57, 70550 Stuttgart, Germany. FAX: 49 711 685 5090; franziska.wollnik{at}po.uni-stuttgart.de
Received: 24 September 2003.
First decision: 17 October 2003.
Accepted: 9 January 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  S. Monecke, A. Malan, and F. Wollnik Asymmetric Control of Short Day Response in European Hamsters J Biol Rhythms, August 1, 2006; 21(4): 290 - 300. [Abstract] [PDF]
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