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a Department of Biology, College of William and Mary, Williamsburg, Virginia 23187-8795
ABSTRACT
Photoperiod is the major regulator of reproduction in temperate-zone mammals. Laboratory rats are generally considered to be nonphotoresponsive, but young male Fischer 344 (F344) rats have a uniquely robust response to short photoperiods of 8 h of light. Rats transferred at weaning from a photoperiod of 16 h to photoperiods of < 14 h of light slowed in both reproductive development and somatic growth rate. Those in photoperiods < 13 h of light underwent the strongest responses. The critical photoperiod of F344 rats can be defined as 13.5 h of light, but photoperiods of
12.5 h are required to fully suppress reproduction and somatic growth. This demonstrates that the 12-h photoperiod that is standard in some laboratory colonies would have significant effects on reproductive maturation and growth rate of this common rat strain. Young F344 rats in decreasing photoperiods that mimic natural change experienced delayed reproductive development and decreased growth rate to a greater extent and for a longer duration than those transferred at birth to a short photoperiod. The effects of gradual changes in photoperiod persisted for at least 12 wk after weaning. This indicates that young male F344 rats possess responses to photoperiod that would result in functional photoperiodism in a wild mammal.
male reproductive tract, pineal, puberty, seasonal reproduction, testes
INTRODUCTION
Detecting seasonal changes in photoperiod is critical to mammals in the temperate zone. Temperate-zone mammals use seasonal changes in photoperiod to time reproduction to avoid birth and lactation during the harsh winter months [1]. Individuals of photoperiodic species of rodents often vary in their responses to photoperiod [2]. In these species, low probability of survival through the winter months may produce selection pressure favoring winter reproductive attempts at times or places when conditions are mild or food is abundant, even if those attempts are only rarely successful [1]. In some rodent species, individuals in short winter photoperiods may be more strongly inhibited by a short photoperiod if this is combined with other environmental factors, especially food restriction, that may provide short-term cues about conditions for reproduction [3–8]. Some species of temperate-zone rodents do not inhibit reproduction in response to mild food restriction alone, but they may have a strong response to food restriction when it is combined with a short photoperiod. There is evidence that typical temperate-zone species of rodents 1) use photoperiod to monitor changes in season; 2) evaluate a suite of environmental signals, including energetic/nutritional status and direction or rate of change in photoperiod, to modify their response to a short photoperiod [2, 8–10]; 3) vary genetically in the extent to which they inhibit reproduction using these cues [11–14]; and 4) inherit reproductive photoresponsiveness as a polygenic trait that is at least partially additive [15–17].
A typical temperate-zone population of rodents may include some individuals that can be defined as being strongly photoperiodic—that is, as undergoing strong reproductive inhibition in response to photoperiods of 12–13 h of light, with inhibition persisting for 20 wk or more before the animals become refractory to the inhibitory effects of the short photoperiod. Other individuals in the same population may respond to a short photoperiod alone weakly, if at all, but they may become reproductively responsive to the photoperiod under additional conditions, such as mild food restriction, that do not by themselves inhibit reproduction. Individuals at the extreme of nonphotoresponsiveness may not respond at all to a short photoperiod, even when food is restricted, although starvation would ultimately inhibit reproduction.
Different laboratory strains of Rattus norvegicus also differ in their responses to photoperiod, with most strains tested having either no response or slight to moderate reproductive inhibition and/or reduced growth rate [18–22]. One strain, the Fischer 344 (F344), has stronger responses than other strains to short photoperiods of L8:D16 [23–25]. The modest responses of most strains that have been tested occur only when unmasked by seminatural treatments, such as food restriction [26, 27], or unnatural treatments, such as olfactory bulbectomy or sex-steroid treatment [28–30]. Wallen et al. [27] demonstrated that photoperiodic responses induced by food restriction in Harlan Sprague-Dawley rats have a critical photoperiod (below which reproduction is inhibited) of approximately 9 h of light. Other studies that have tested a range of photoperiods on rats have found similarly short critical photoperiods [19, 31]. In fact, these photoperiods are so short that they would be reached only briefly, at midwinter, in nature, and they probably would be biologically irrelevant in wild rats. The weak photoperiodic responses of most strains, coupled with the very short critical photoperiods for those effects, have lead to the characterization of rats as being functionally nonphotoperiodic [1, 26, 32, 33]. Some wild rat populations undergo seasonal changes in reproductive status [34], however, which suggests that photoperiod could play a role in reproductive regulation in wild rats. If wild rats are variable for photoresponsiveness, then domestication and inbreeding may have resulted in the fixation of different alleles for this variable trait in different strains of laboratory rats.
Results of previous studies have shown that F344 rats are responsive to short photoperiods [23, 24], but what is unknown is the critical photoperiod of these rats and the effects of a gradually reducing photoperiod, which may be more important than a response to a constant short photoperiod [35]. If F344 rats have a critical photoperiod similar to that of typical wild populations of photoresponsive rodents, and if they respond to a gradually changing photoperiod, then this strain might be considered to be functionally photoresponsive. In this study, we tested two hypotheses: that F344 rats have a critical photoperiod (
12 h of light) that could be relevant in the wild or in some laboratory settings, and that F344 rats would respond more strongly to gradual changes in photoperiod that mimic natural changes than to an abrupt switch to a single, short photoperiod.
MATERIALS AND METHODS
Experiment 1: Critical Photoperiod of F344 Rats
The F334/NHsd rats were obtained for breeding from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). Male rats were gestated and raised until weaning for 21 days in a L16:D8 photoperiod, with lights-off at 2100 h. After weaning, rats were housed individually in polypropylene cages (36 x 24 x 19 cm) with stainless-steel wire tops and provided with food (RMH 3000; Southern States Cooperative, Williamsburg, VA) and tap water ad libitum. Relative humidity was between 30%–70%, and the temperature was 23 ± 2°C.
In experiment 1a, rats were placed at weaning in one of five photoperiods (L10:D14, L12:D12, L14:D10, L16:D8, and L24:D0; hereafter referred to as L10, L12, L14, L16, and L24, respectively) in ventilated, photoperiod-controlled chambers. Sample sizes were seven to eight rats in the L10, L12, L14, and L16 groups and three rats in the L24 group. The L24 group was caused by a timer error. These results are included, however, because they add useful information. Lighting was provided by two 20-W fluorescent bulbs (F20T12 Cool White; General Electric Corp, Stamford, CT) dimmed to an intensity of 100–300 lux as measured 5 cm above the cage floor. After 4 wk of photoperiod treatment, a duration for which L8 has consistently inhibited reproductive maturation and growth in male F344 rats [23, 24], rats were killed with CO2 gas. Body weight, wet weight of the paired testes, and wet weight of the paired seminal vesicles (with the seminal vesicles emptied of their fluid contents) were measured. The experiment was run in two replicates balanced across treatments, except that the L24 group was included only in the first run.
In experiment 1b, the design was similar, except that a narrower range of photoperiods was tested (L12, L12.5:D11.5, L13:D11, L13.5D:10.5, and L14; hereafter referred to as L12, L12.5, L13, L13.5, and L14, respectively). Sample sizes were 8 to 10 rats per group.
In experiments 1a and 1b, data were collected in two runs that were balanced across treatments, and data from the two runs were combined for the analyses. We did not adjust the testis or seminal vesicle measures for differences in body weight, because previous results indicate that the faster reproductive development of F344 rats in long days is not caused by their greater food intake or body mass [24]. In that study, reproductive development was unaffected by food restriction in a long photoperiod, even when body weight had been reduced to below that of rats in a short photoperiod. Data were analyzed using ANOVA with pairwise comparisons using Fisher's probable least-squares difference (PLSD; Statview+ Graphics, v4.5; Abacus Concepts, Berkeley, CA).
Experiment 2: Effects of Simulated Natural Photoperiod Changes
Experiment 2 tested the effects of gradual decreases in photoperiod on reproductive maturation and growth. We were interested in two questions: whether this rate of change would suppress reproductive development and growth within the first few weeks after weaning, and whether this rate of change would extend the period of inhibition for a longer time than abrupt photoperiod changes. We chose a rate of change in photoperiod of 4 min shorter each day (2 min later for light onset and 2 min earlier for light offset), which is typical of September and October at temperate latitudes of approximately 40° to 50°N and slightly more rapid than the changes from August to November [36]. The experiment continued for 12 wk to allow the duration of inhibition to be assessed. Because the photoperiod decreased from L14 at a rate of 4 min total change per day, the final photoperiod became L7.1:D16.9 for the gradual change group and for an abrupt change control group.
Husbandry conditions and photoperiod chambers were as described for experiments 1a and 1b. Rats were gestated and born in L16 and then transferred for the 1–2 days after birth to L14. Some pups were cross-fostered to mothers with same-age litters to balance the sample sizes across treatments. Mothers with litters were then assigned to one of three treatments, which were 1) maintenance in the noninhibitory constant photoperiod of L14 (L14-constant; n = 12 pups from six litters), 2) abrupt change to an inhibitory short photoperiod of L7.1:D16.9 (L7-abrupt; n = 13 pups from six litters), and 3) provision of a photoperiod that gradually decreased to L7.1:D16.9 during a period of 105 days (L7-gradual; n = 14 pups from seven litters). One rat was removed from the third group midway through the study because of the sudden regression of one testis and compensatory hypertrophy of the other testis. The photoperiod of the L7-gradual group was shortened by 4 min each day by delaying light onset by 2 min and advancing light offset by 2 min. On a few days (<10 days), the photoperiod was not changed, and in these cases, the photoperiod change was doubled on the subsequent day. At 22 ± 1 days of age, pups were weaned from their dams and moved to individual cages.
Four weeks after weaning, at 50 ± 1 days of age, and at approximately 2-wk intervals thereafter (age = 64 ±1 days, 78 ± 1 days, 92 ± 1 days, and 106 ± 1 days), rats were anesthetized (or, on Day 106, overdosed) with methoxyflurane (Pitman-Moore, Inc., Mundelein, IL) and/or isoflurane (Ohmeda Caribe, Inc., Guayama, Puerto Rico) and weighed, and the external length and width of their left testis was measured with calipers by one author (PDH). A combination of methoxyflurane and isoflurane was used on the older rats to hasten the induction of anesthesia. Data collection was blind with respect to photoperiod treatment. To assess the accuracy and precision of testis measurements, some testis measurements were repeated by a second person, who was also blind with respect to treatment and to the measurements by PDH. Testis volume was estimated using the formula for a prolate spheroid (volume = width2 x length x 0.523). Data were analyzed using repeated measures ANOVA followed by one-way ANOVA for each week, with pairwise comparisons using Fisher's PLSD (Statview+ Graphics, v4.5, Abacus Concepts, Berkeley, CA).
All experiments were conducted in accordance with the Guide for Care and Use of Laboratory Animals.
RESULTS
Experiment 1: Critical Photoperiod
In experiment 1a, significant differences were found among groups in paired testis weight (F = 13.99, P < 0.0001; Fig. 1a), paired seminal vesicle weight (F = 31.55, P < 0.0001; Fig. 1b), and body weight (F = 3.10; P = 0.03; Fig. 1c). By all three measures, rats in L10 and L12 were smaller than those in L14, L16, and L24. For paired testis weight and paired seminal vesicle weight, all pairwise comparisons of groups in L10 or L12 with groups in L14, L16, and L24 were statistically significant (P < 0.05 for all comparisons). Groups in L10 and L12, however, did not differ from each other (P > 0.10 for all comparisons), nor did groups in L14, L16, and L24 differ from each other (P > 0.10 for all comparisons). For body weight, only the L12 group was significantly lighter than the L14, L16, and L24 groups in the pairwise comparison (P < 0.05 for all comparisons), whereas the L10 group did not differ significantly from any other group in the pairwise comparisons (P > 0.10 for all comparisons).
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For the narrower range of photoperiods in experiment 1b, significant differences were found in paired testis weight (F = 10.391, P < 0.0001; Fig. 2a), paired seminal vesicle weight (F = 21.158, P < 0.0001; Fig. 2b), and body weight (F = 6.978, P = 0.002; Fig. 2c) among photoperiod groups. All rats exposed to photoperiods of 13.5 h of light or less had significantly lower testis, seminal vesicle, and body weights than rats in L14 (P < 0.05 for all comparisons; Fig. 2). The data suggest that progressively shorter photoperiods from L13.5 to L12.5 cause progressively greater reproductive inhibition, and this interpretation is supported by results of the statistical analysis (Fig. 2, a and b). This is not the case for photoperiodic effects on body weight, for which all photoperiods 13.5 h had similar effects (Figs. 1c and 2c).
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Experiment 2: Effect of Gradual Photoperiod Changes
As in previous studies [23, 24], treatment with an abrupt change to a short photoperiod inhibited testicular development and reduced body weight relative to a long photoperiod (Fig. 3). However, the effects of the abrupt change from L14 to L7.1:D16.9 were smaller and shorter in duration, continuing only through Week 6 (Fisher's PLSD, P < 0.05), than those observed in previous studies [23, 24].
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Treatment with gradual changes in photoperiod affected testis size and body mass. Significant differences were found among groups in both left testis volume (F = 15.427, P < 0.001; Fig. 3a) and body weight (F = 6.297, P = 0.0045; Fig. 3b). Testis size and body weight of the L7-gradual group were significantly lower than those of the L14-constant group for the entire measurement period (Fig. 3). In addition, both measures were significantly lower in the L7-gradual group than in the L7-abrupt group for most of the measurement period (Fig. 3).
DISCUSSION
Our results suggest that F344 rats are functionally photoperiodic animals, in that they have a robust innate response to natural photoperiod changes that can extend for at least 12 wks and have a critical photoperiod similar to that of typical photoperiodic rodents. From the results of experiment 1, we found that transferring F344 rats at weaning from 16 to 13.5 h of light or less inhibits reproductive maturation and growth. In experiment 1, photoperiods of 12.5 h or less produced maximal reproductive inhibition, whereas photoperiods of 13 and 13.5 h caused intermediate responses and those of 14 h and longer were noninhibitory (Figs. 1 and 2). Results of the statistical tests indicate that maximal effects on body weight were induced by photoperiods 13.5 h, but the slightly greater mean body weights in L13 and L13.5 suggest that rats in these treatments may have undergone intermediate responses that the statistical power of our tests could not detect. Under our experimental conditions, the critical photoperiod of F344 rats was much longer than that of other strains of laboratory rats (i.e., adult Harlan Sprague-Dawley [27] and young male Wistar [31]) and similar to that of classical photoperiodic rodents under similar photoperiods (i.e., Syrian hamsters [37], Siberian hamsters [38–40], Turkish hamsters [41], white-footed mice [42, 43], and marsh rice rats [44]).
Our finding that constant photoperiods of 13.5 h of light or less delay maturation and slow growth is potentially important for researchers in other fields using the F344 rat as a model. Photoperiods for laboratory animals are typically L12, L14, or L16. Because we now have evidence from this study that young F344 rats can respond differently to L12 than to L14 or L16 in two important ways (i.e., reproductive maturation and somatic growth), studies in different laboratories that are identical in design except for photoperiods that are greater or less than L13.5 might produce different results. The F344 rats are reported to be the most commonly used strain of rats in research (M.F.W. Festing, http://www.informatics.jax.org/external/festing/rat/docs/F344.shtml) and have been the predominant strain used in studies on aging in the United States [45]. To our knowledge, no reports of photoperiodic effects on adult F344 rats have been published, but preliminary data suggest that adults may also undergo changes in food intake, body weight, and testis size in response to short photoperiod (unpublished data). We suggest that researchers should consider routinely holding F344 rats in photoperiods of L14 or L16 to remove the potential effects of a short photoperiod.
Because the critical photoperiod in rodents can vary depending on the photoperiodic history [40, 46, 47] and on the temporal pattern of change in photoperiod (i.e., abrupt or gradual) [35, 48, 49], it is valuable to test the relevance of photoperiodic responses using changes in photoperiod that are as realistic as possible. In our second experiment, we found that F344 rats are reproductively inhibited by gradually changing photoperiods. The duration of photoperiods and rate of change in this experiment were similar to a late-summer-to-winter transition at temperate latitudes of approximately 40° to 50°N. Rats in the L7-gradual group responded by having lower body mass and testis size within the first 4 wk after weaning, when the photoperiod had changed from L14 to approximately L10.5:D13.5. This indicates that gradual changes in photoperiod similar to those of the late summer and early fall months, which would be relevant to wild populations of rats, are sufficient to inhibit reproduction and growth in F344 rats. In addition, we found that reproductive inhibition and reduction in growth rate were longer lasting, and that the degree of inhibition was slightly greater in response to gradual changes in photoperiod than responses to a constant, short photoperiod in either this study (Fig. 3) or previous studies [23, 24].
In combination with those of previous studies [23, 24], our results suggest that in a temperate-zone winter, F344 rats would inhibit reproductive development and reduce food intake and growth for 12 wk or longer before becoming photorefractory. If these rats were also food restricted, this photoperiod-dependent reproductive inhibition might extend even longer before photrefractoriness and compensatory growth occurred [24]. Our measure of the critical photoperiod in F344 rats indicates that these effects could occur in rats born from the late summer through at least the midwinter months. Thus, it is appropriate to consider F344 rats as being functionally photoperiodic, with the only qualification being that their reproductive responses to photoperiod are not as dramatic as those of many, but not all, individuals from typical species of photoperiodic rodents.
Why might strains of laboratory rats differ in both photoresponsiveness and critical photoperiod? In terms of their neuroendocrine physiology, F344 rats have been suggested to be more strongly photoperiodic than other strains because of a higher sensitivity to sex steroids [50], differences in D2-dopamine-receptor densities [50], or differences in iodomelatonin binding in the thalamus [25]. Genetic differences that cause these variations in photoresponsiveness and critical photoperiod may exist for at least two reasons. First, strains might differ because mutations causing photoresponsiveness have occurred since domestication and become fixed in some laboratory strains of rats. Second, strains might differ because natural genetic variation for this trait existed in the wild source populations of laboratory rats, and different combinations of alleles for photoresponsiveness have become fixed in the different strains of laboratory rats. There is reason to suggest that the second hypothesis might be correct. In wild populations of photoperiodic species of temperate-zone rodents, laboratory experiments have often found a broad range of variation for photoresponsiveness [1, 2, 6] and a genetic basis for this variability [11, 12, 16, 17]. Several studies have provided evidence that photoresponsiveness is probably controlled by multiple loci [15, 16]. In several species of rodents, breeding lines that differ in photoresponsiveness have been created after only a few generations of selection on stock from wild populations [11, 12, 16, 17]. In addition, different inbred strains of golden hamsters, which is a classically photoperiodic species, differ in their photoperiodic responses, indicating that different alleles for photoresponsiveness have become fixed in the different strains [51].
If wild populations of rats are variable for photoresponsiveness, then variability would be expected among laboratory strains of rats, because different alleles would have been fixed in different inbred strains (and, perhaps, in some outbred strains as well). However, we would predict that most strains of laboratory rats would be relatively nonphotoresponsive because of artificial selection against photoresponsiveness during domestication. Some of the probable conditions of domestication of rats (e.g., short photoperiods during some periods of the year coupled with strong artificial selection for fertility at all times) might be expected to have selected against reproductive photoresponsiveness. Selection against nonreproductive responses to photoperiod, such as changes in food intake and body weight, probably would have been weaker, however, so these aspects of photoresponsiveness might persist in more strains. Extant variability in photoresponsiveness among laboratory rat strains should be based in part on the strength of artificial selection during domestication, in part on which alleles happen to have been fixed within any given strain, and in part on chance mutations in the laboratory that may have recreated alleles existing in the wild or produced mutations that affect photoresponsiveness but that do not persist in the wild. At present, it is not possible to exclude the hypothesis that the latter source of variation is the sole cause of photoresponsiveness in laboratory rats—that is, that photoresponsiveness in laboratory rat strains is entirely the result of mutations that would not persist in wild populations of rats. Tests of the photoresponsiveness of individuals from wild populations of rats would help to address this question.
Our results suggest that the F344 rat could be a useful new model in the study of photoperiodism. These rats would be an excellent model system because 1) their reproductive physiology and neuroendocrinology is much better known than those of common model species for the study of photoperiodism (e.g., species of hamsters and field mice), 2) equipment and reagents designed and tested for use on rats are readily available, 3) information on rat genetics is available across a broad array of outbred and inbred strains, and 4) gene-sequence information on rats is being accumulated rapidly. Two serious weaknesses of F344 rats as a model for the study of photoperiodism are 1) their relatively weaker reproductive inhibition by short photoperiods compared with standard animal models for photoperiodism and 2) the possibility that their photoresponsiveness results from mutations accumulated during and after domestication that have little relevance to natural populations. Finally, if strains of rats are fixed for different alleles that contribute quantitatively to photoresponsiveness, then quantitative-trait locus analyses and other genetic methods may allow isolation and identification of the contributions of individual genes to this polygenic, quantitative trait. We do not believe that laboratory rats could replace the traditional photoperiodic model species, but F344 laboratory rats might prove to be useful adjuncts in identifying the mechanisms and genes that contribute to photoperiodism.
ACKNOWLEDGMENTS
We thank T. Elsass, F.R. Jennings, V. Tummala, S. Majoy, S. Joiner, K. Schubert, M. Cicchetti, C.J. Sylvester, and B. Shoemaker for assistance with data collection and animal care.
FOOTNOTES
First decision: 10 April 2000.
1 Supported by NIH Grant R15 DK51334 and Jeffress Research Grant J-356 to P.D.H. and by a Minor Research Grant to M.E.G. from a Howard Hughes Medical Institute Undergraduate Biological Sciences Education Program grant to the College of William and Mary. 
2 Correspondence: Paul D. Heideman, Department of Biology, College of William and Mary, P.O. Box 8795, Williamsburg, VA 23187-8795. FAX: 757 221 6483; pdheid{at}wm.edu
Accepted: July 5, 2000.
Received: March 8, 2000.
REFERENCES
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