| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
a Biology Department, College of William and Mary, Williamsburg, Virginia 23187–8795
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Laboratory rats are derived from temperate zone ancestors, but adult laboratory rats are not traditionally considered reproductively photoperiodic [4–6]. However, a reproductive response to short days (SD) and/or melatonin treatment can be induced in adult male Sprague-Dawley, Wistar, and some other strains of rats by several procedures: food deprivation, neonatal androgen treatment, chronic exposure to exogenous testosterone (T), or olfactory bulbectomy ([7–11], but see [12]).
In contrast, puberty can be delayed in some rats by treatment with photoperiod alone. Constant dark [13], blinding [11], or very short days (light 
2 h) [14, 15] can delay puberty in some strains of rats, but the effects reported have been slight, on the order of a 1- to 2-wk delay in vaginal opening or a 0–20% smaller testis size relative to controls. In Wistar rats, testicular development is inhibited by constant dark [16] (testes 30% smaller than those of controls after 8 wk). In the more biologically reasonable condition of SD (6–8 h light), testicular development of Wistar or Sprague-Dawley rats is either uninhibited (Wistar: no difference after 7 wk [17]; Sprague-Dawley: no difference from 2 to 10 wk [18]) or moderately inhibited (Wistar: 30% smaller after 4 wk [19]). In Wistar rats, daily injections of melatonin 1–3 h before lights-out on a 12L:12D cycle induce greater testicular inhibition than SD, with melatonin-injected rats having testes ca. 50% smaller than those of controls [20]. Young Fischer 344 (F344) rats are more strongly inhibited than other strains by blinding [21] or SD [18]. For example, males in SD for 2–6 wk have testes averaging up to 50% smaller than males in long days (LD) [18]. Collectively, these data show that strains of laboratory rats have the neuroanatomical connections that mediate reproductive sensitivity to photoperiod [10, 22] but that these connections normally range from nonfunctional in adults to functional in young rats of some strains [18, 20].
In theory, peripubertal rodents should be more strongly affected by inhibitory photoperiods than are adults [23, 24], because young rodents are less experienced and have lower reserves of energy, and thus are more likely to fail to raise offspring or even to die in a reproductive attempt. Higher sensitivity of younger animals to inhibitory photoperiods has been reported in rats and some other rodents [19, 23, 25, 26], including the strongly photoperiodic Siberian hamster [26, 27]. Because a typical rodent can expect only one or two reproductive opportunities in its life, the regulation of puberty by photoperiod is probably more important than adult photoresponsiveness for many species. For most rodents, the correct timing of puberty may be their single most important reproductive decision.
While pubertal inhibition in response to SD or similar treatments is robust in F344 rats, reproduction is not inhibited as strongly as in the classically photoresponsive rodents—species of hamsters and voles, or species of Peromyscus (deer mice and white-footed mice). We hypothesize that F344 rats have two components to photoresponsiveness—one an obligate inhibition by SD, and the other a facultative inhibition in response to biologically relevant challenges. Thus, it is possible that the combined obligate and facultative responses to SD might induce reproductive inhibition in some rats to a degree similar to that in highly photoresponsive rodents such as hamsters, white-footed mice, or voles. In the young of nonphotoperiodic strains of rats, photoperiod-dependent reproductive suppression can be induced by neonatal T injection [16] or food restriction [9, 28]. However, the degree of suppression of the reproductive system is similar to that of young F344 rats treated with SD alone, suggesting that stronger seasonal inhibition is not possible in these strains.
This study tested the hypothesis that F344 rats, which clearly have an obligate inhibition of reproduction in SD, also have an additional, facultative inhibition of reproduction in SD. Two treatments were chosen. The first, 30% food restriction (FR), was chosen to mimic the biologically reasonable condition of low food availability during maturation. The second, delivery of exogenous testosterone propionate (TP) to neonatal males, is a biologically unrealistic pharmacological treatment that we presume alters organizational events in the development of the reproductive axis. The latter treatment does not mimic any biologically realistic challenge, but it was chosen to test whether F344 rats had the potential for stronger responses to inhibitory photoperiod than occur in SD alone.
We predicted that, if F344 rats have the capacity to be fully reproductively inhibited in SD by a combination of obligate and facultative responses to SD, then both FR and TP would enhance the effects of SD. Alternatively, if F344 rats have the capacity for only modest inhibition of the reproductive axis, then SD alone would fully inhibit the reproductive axis, and other treatments would have no additional effect.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This experiment tested whether F344 rats have the capacity for stronger responses to SD than that induced by SD alone. At age 3 days, 20 male F344 rats gestated and born in LD (16L:8D; lights-on at 0500 h) received s.c. injections of 1 mg of TP in 0.1 ml of corn oil. The dose was chosen because, in a pilot study, this dose inhibited reproductive development in SD but not LD. Twenty control rats received injections of oil vehicle alone. Rats were kept in LD until aged 21 ± 2 days, when both the TP and control groups were divided into weight-matched halves, with one half of each group placed into a second room in SD (8L:16D; lights-on at 0900 h) or kept in LD (n = 10 rats/group in the four treatments). The experiment was run in three replicates balanced across treatments; the data from the three replicates were combined for the analyses.
Rats were housed individually in polypropylene cages (36 x 24 x 19 cm) and provided with food (RMH 3000; Southern States Cooperative; Williamsburg, VA) and tap water ad libidum (ad lib). Temperature was 23 ± 2°C, and relative humidity was between 30% and 70%. Experiments were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.
Rats were weighed, and the length and width (to nearest 0.1 mm) of the right testis was measured at 2-wk intervals until 12 wk of treatment, and thereafter at longer intervals. During testis measurements, the rats were lightly anesthetized with isofluorane (Ohmeda PPD Inc., Liberty Corner, NJ) maintained as necessary with methoxyflurane (Pitman-Moore, Inc., Mundelein, IL). Testis volume was estimated using the formula for a prolate spheroid (width2 x length x 0.523), which is highly correlated with testis weight in our hands [18]. Because testes are somewhat compressible in a live animal, producing a potential subjective component to testis measurements, all measurements presented for this experiment were taken by a single person (LMY) blind with respect to treatment.
Experiment 2
This experiment tested whether the biologically reasonable challenge of mild food restriction could trigger facultative enhancement of reproductive inhibition by SD. Forty male Fischer 344 rats (Charles River Laboratories, Raleigh, NC; gestated and born in 12L:12D), aged 22 ± 2 days, were assigned to one of four weight-matched treatment groups: LD and ad lib food (RMH 3000) (LD-ad lib), SD and ad lib food (SD-ad lib), LD and FR (LD-FR), and SD and FR (SD-FR). All rats were held in a single room in fan-ventilated photoperiod chambers (86 x 58 x 49 or 160 x 69 x 40 cm) lit by 20-W fluorescent bulbs (illuminance of 200–250 lux 5 cm above the floor of the cage). Temperature within the boxes was 23 ± 2°C.
The level of FR (30%) was chosen after a pilot experiment that showed a very slight inhibition of testicular growth in LD by 30% food reduction, and little or no effect of 20% or 10% food reduction. Rats held in SD voluntarily reduced their food intake by approximately 10–15% from Week 3 until the preliminary experiment was terminated at 10 wk (p < 0.05; Fig. 1). Thus, the FR treatments were 30% of the amount of food eaten by same-age ad lib-fed controls in the same photoperiod. FR rats were fed daily between 1400 and 1700 h. Testes and body weight were measured at 2-wk intervals, beginning after 1 wk of photoperiod and FR treatment, according to the procedures of experiment 1. As in experiment 1, all measurements presented were taken by a single person (RWD) blind with respect to treatment.
|
Originally all treatments had 10 subjects. Two animals in the LD-FR group developed testicular abnormalities in one or both testes. All three of these testes became extremely flaccid and cyanotic (as seen through the skin of the scrotum), and on autopsy at Week 9 appeared dark blue-gray and had an unusually prominent vasculature with possible varicosities. Both animals were removed from the study. Finally, one rat in the SD-FR group died under anesthesia. Thus, final cell sizes were 8–10 rats/treatment group.
Statistical Analysis
Testis volumes were not standardized according to body weight, as we believe that absolute testis size is a better indicator of reproductive maturation than relative testis size. A number of previous studies have shown body weight to be a poor predictor of sperm count [29], and photoperiod can affect body weight independently of testis size in rodents (e.g., [30, 31]). Our previous work on F344 rats [18] showed similar qualitative results using adjusted or unadjusted measurements of testis size. In experiment 2, in particular, we were interested in the effect of FR on absolute measures of reproductive development, not on reproductive development relative to somatic growth, and so adjustment by body weight was inappropriate.
Data on food intake were analyzed by repeated measures ANOVA. In experiments 1 and 2, data were analyzed by repeated measures two-way ANOVA for each experiment, followed by two-way ANOVA for each week with post-hoc contrast comparisons of all pairs of means. Attained significance levels < 0.05 were considered significant. Analyses were performed using Superanova (v 1.11) or Statview+ Graphics (v 1.04 A; both by Abacus Concepts, Berkeley, CA) on a Power Macintosh 6100 computer (Apple Computer, Cupertino, CA). All means are presented with their standard errors.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Both SD and TP inhibited testicular growth (F = 220.6, p < 0.0001, and F = 122.3, p < 0.0001, respectively; Fig. 2). The effect of both photoperiod and TP were significant at Week 2 and persisted through Week 12 (Fig. 2), but by Week 21, the effects of both TP and photoperiod were no longer apparent, and testis size was similar in all four treatments (Fig. 2). There was a significant interaction between SD and TP treatment (F = 8.3, p < 0.005), showing that TP treatment enhanced the inhibitory effect of SD (Fig. 2). At the time of their greatest differences, at Weeks 4 and 6, the combined effect of SD and TP resulted in testes approximately 10% of the size of LD controls. Before Week 8, the combination of SD and TP almost completely halted testicular growth.
|
TP treatment did not affect body weight (F = 3.2, p > 0.05; Fig. 3). In contrast, SD significantly inhibited increases in body weight (F = 204.1, p < 0.0001), and SD rats in both treatments weighed approximately 20% less than LD rats in most weeks, confirming previous results [18]. The effect of SD on body weight was significant from Week 2 through Week 16, but at Week 21 the effect of SD was no longer apparent (Fig. 3). There was no interaction between photoperiod and TP treatment (F = 0.1, p > 0.05).
|
Experiment 2
Both SD and FR inhibited testicular growth (F = 153.9, p < 0.0001, and F = 56.1, p < 0.0001, respectively; Fig. 4). The effects of photoperiod and of food restriction were significant at Week 1, and both persisted through all 13 wk of the study (Fig. 4). There was a significant interaction between SD and FR treatment (F = 19.2; p < 0.001), showing that FR treatment enhanced the inhibitory effect of SD (Fig. 4). At the time of their greatest differences, at Weeks 3 and 5, the combined effect of SD and FR resulted in testes approximately 70% smaller than the LD controls.
|
Both SD and FR treatment affected increases in body weight (F = 153.1, p < 0.0001, and F = 576.4, p < 0.0001, respectively; Fig. 5). The effect of food was significant after 1 wk, and the effect of SD was significant after 3 wk (Fig. 5). There was a significant interaction between SD and FR treatment (F = 9.2; p < 0.005). The interaction is difficult to interpret, but it appears that FR enhanced the inhibitory effect of SD on body weight in the second half of the experiment, while in early weeks the effects of FR and SD were essentially additive. Control SD rats weighed 10–25% less than control LD rats in most weeks, while FR rats weighed about 20% less than ad lib controls in the same photoperiod (Fig. 5).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The effect of neonatal TP and SD on F344 rats was greater than the effect of neonatal TP and either blinding or SD on Sprague-Dawley [11], Wistar [16], or Wistar-Konarovice strains of rats [17]. Similarly, the effect of FR with SD on F344 rats appears to be greater than in other strains (e.g., [6, 9]). Thus, SD alone or in combination with FR or neonatal TP induces greater reproductive inhibition in F344 rats than in other strains.
Interestingly, the effect of neonatal TP treatment was clearly restricted to reproductive responses to SD. Neonatal TP strongly inhibited testicular growth in SD but had no effect on body weight. This suggests that SD may act independently on the reproductive axis and body weight, such that neonatal TP can enhance one effect but not the other. If so, then neonatal TP may act directly on photoresponsive elements of the reproductive axis. Alternatively, the mechanism by which neonatal TP enhances reproductive photoresponsiveness may be through a nonspecific enhancement of reproductive inhibition from any source (e.g., photoperiod, stress, or food). Our results do not allow us to distinguish between these possibilities.
Neonatal androgens have powerful organizational effects on neural control of reproductive behavior and function. The enhancement of photoresponsiveness by exogenous androgen given neonatally is likely to be due to these organizational effects, but the mechanism is unknown. Neonatal androgens may enhance the sensitivity of the reproductive axis to feedback inhibition by androgens in adult life. F344 rats are known to be unusually sensitive to the effects of steroids and steroid negative feedback on the reproductive system [21, 32, 33]. This suggests that the robust photoresponsiveness of F344 rats may be due to photoperiodic activation of a steroid negative feedback pathway acting on GnRH secretion.
The 2- to 4-wk added delay in reproductive maturation induced by the combination of SD and FR could be highly significant to young rats in the wild. However, the combined effect of SD and FR together was considerably weaker than the reproductive inhibition produced by SD alone in many obligately photoresponsive species. The level of food restriction we chose was relatively modest, and it is possible that greater food restriction, which would be likely for some animals in the wild, might produce a more dramatic effect.
While SD inhibits food intake and body weight gain as well as testicular development (similar inhibition of weight gain by SD was reported by Boon et al. [34] for Wistar rats), the effects of photoperiod on reproduction are not due to inhibition of feeding and body weight. In experiment 2, we found that the effects of SD on reproduction were independent of the effects of food intake or of body weight. Food-restricted LD rats had lower body weights (Fig. 5) but much larger testes (Fig. 4) than SD-ad lib controls. Thus, reduction in food intake and lowered body weight are not the cause of reproductive inhibition by SD. Lower body weight in SD may hold adaptive significance unrelated to reproduction. In wild rodents, it may reduce daily energy requirements in winter (reviewed in [35]), a response to photoperiod that can be independent of changes in testis size [1].
Truly opportunistic breeders, such as the tropical cane mouse, show neither photoperiodic inhibition of reproduction [36] nor interactions between photoperiod and neonatal androgen treatment or FR [37]. In contrast, seasonal breeders such as the deer mouse (Peromyscus maniculatus) show strongly inhibited reproduction in SD, with greater inhibition when FR and SD are combined [3, 29, 38, 39]. Deer mice have both phenotypic and genetic variation in the magnitude of their response to SD alone [1, 35, 40]. In F344 rats, the pathway regulating reproduction is intermediate to that of purely opportunistic breeders such as the cane mouse and classic seasonal breeders such as deer mice or hamsters. FR alone has almost no effect on reproductive development in F344 rats, while SD produces a delay of approximately 4 wk in testicular growth. The combination of SD and FR extends the delay in testicular growth, such that FR animals in SD had reproductive growth delayed by an additional 2–4 wk; this total of 6–8 wk represents a large delay for an animal whose wild relatives have a life expectancy that is generally on the order of a few months.
It is probably significant that the response to photoperiod is present to varying degrees in different strains of rats. The implication is that some of the wild ancestors of laboratory rats had the capacity to undergo both obligate and facultative inhibition of reproduction in SD, with longer reproductive delays in SD when food was scarce but faster maturation when food was sufficiently abundant to permit successful reproduction. The process of domestication of the wild Norway rat may have carried naturally occurring individual variation in seasonal reproductive responses into the laboratory. In the creation of relatively inbred laboratory strains (such as Sprague-Dawley and Wistar) and highly inbred lines (F344), different strains and lines were fixed for varying combinations of alleles that contribute to the regulation of seasonal reproduction. While this strain variation may not permit us to reconstruct or infer mechanisms of seasonal reproduction in wild rats, it may prove useful in elucidation of the contribution to seasonality of specific genes and the neuroendocrine phenotypes they control.
Our results should alter the interpretation of the significance of "unmasking" of photoresponsiveness in laboratory rats. Far from being a laboratory phenomenon of little relevance, our results suggest that these responses are important components of a seasonal response integrating multiple environmental inputs. In previous studies on other strains of rats, the effects of photoperiod have been modest enough to suggest that they might be of little or no biological importance in a wild rat (e.g., [6]). This study lends support to the hypothesis that seasonal reproductive regulation in temperate zone rodents, including Rattus norvegicus, typically involves the integration of multiple cues. Ultimately, in order to fully understand seasonality, it will be necessary to understand the effects of these environmental cues both alone and in combination.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Paul D. Heideman, Biology Department, College of William and Mary, PO Box 8795, Williamsburg, VA 23187–8795. FAX: (757) 221–6483; pdheid{at}facstaff.wm.edu
3 Current address: Program in Cell and Molecular Biology, Baylor College of Medicine, Houston, TX 77030.
4 Current address: University of Virginia Medical School, Charlottesville, VA 22908.
Accepted: March 25, 1998.
Received: January 9, 1998.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  S. Yasuo, M. Watanabe, M. Iigo, T. J. Nakamura, T. Watanabe, T. Takagi, H. Ono, S. Ebihara, and T. Yoshimura Differential response of type 2 deiodinase gene expression to photoperiod between photoperiodic Fischer 344 and nonphotoperiodic Wistar rats Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2007; 292(3): R1315 - R1319. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. R Francisco, C. M Raymond, and P. D Heideman Short photoperiod inhibition of growth in body mass and reproduction in ACI, BUF, and PVG inbred rats Reproduction, December 1, 2004; 128(6): 857 - 862. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Lorincz, M. B. Shoemaker, and P. D. Heideman Genetic variation in photoperiodism among naturally photoperiodic rat strains Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2001; 281(6): R1817 - R1824. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. D. Heideman, C. K. Bierl, and M. E. Galvez Inhibition of Reproductive Maturation and Somatic Growth of Fischer 344 Rats by Photoperiods Shorter than L14:D10 and by Gradually Decreasing Photoperiod Biol Reprod, November 1, 2000; 63(5): 1525 - 1530. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
