Stress suppresses testicular and body weight in young Syrian hamsters under short photoperiod

Physiology&Behavior,Vol. 53, pp. 917-922, 1993

0031-9384/93 $6.00 + .00 Copyright © 1993 PergamonPress Ltd.

Printed in the USA.

Stress Suppresses Testicular and Body Weight in Young Syrian Hamsters Under Short Photoperiod NOBUO

IBUKA, l SHUUICHI

ICHIKAWA

AND HIDEE NISHIOKA

Department of Psychology, Shiga University, Otsu-shi, Shiga-ken, 520, Japan R e c e i v e d 19 J u n e 1992 IBUKA, N., S. ICHIKAWA AND H. NISHIOKA. Stress suppresses testicularand body weight in young Syrian hamsters under short photoperiod. PHYSIOL BEHAV 53(5) 917-922, 1993.--Two experiments were performed to investigate the effect of stress on testicular weight and body mass in young adult male Syrian hamsters under long or short photoperiods. We hypothesized that water or food deprivation causes stress and that the amount of stress depends on unpredictable timing of deprivation. More specifically, water or food deprivation on unpredictable days was considered more stressful for animals than regular deprivation on fixed days even if the total lengths of deprivation were the same for the two treatments. Experiment 1 showed that water deprivation on unpredictable days caused more suppressive effects on testicular weight and body mass than that on fixed days under short photoperiod. Experiment 2 indicated that unpredictable food deprivation on a quarter or half of the days throughout the 12-week testing period under short photoperiod also induced more detrimental effects on testes and body growth than predictable deprivation once every 4 days or every other day. These findings clearly suggested that the stress associated with water or food caused more suppressive effects on testicular weight and body mass than the shortage of water or food per se. Stress

Photoperiod

Syrian hamster

Testicular weight

THE Syrian hamster (Mesocricetus auratus) is a long-day species whose reproductive activity as well as body weight is affected by a few environmental factors. First, gonadal activity is largely regulated by photoperiod or daylength. In male Syrian hamsters that are transferred from a long to a short photoperiod of less than 12.5 h light/day, testicular regression occurs within several weeks (5,16). The Syrian hamster, unlike the Djungarian hamster (Phodopus sungorus), increases its body weight under a short photoperiod (1). There is good evidence that the pineal gland is involved in mediating photoperiodic information on reproductive function and body weight in Syrian hamsters (1,12). Second, low ambient temperature of 5 - 6 ° C for 5-10 weeks is known to induce testicular regression accompanied by cessation of spermatogenesis (4,6). Nutrition is a third environmental factor that influences seasonal reproductive function in some rodent species. Acute food deprivation has antigonadal effects in laboratory rats (7,8) and Syrian hamsters (3,11). Underfeeding, if prolonged, can decrease testicular weight in Syrian hamsters (9). Refeeding reverses the effect of starvation on testicular function (3). Water deprivation also produces suppression of reproduction in Mongolian gerbils (17). Recently, Rusak (14) reported that daily injection of an oil vehicle, i.e., a stressful factor, caused testicular regression in Syr-

Body mass

ian hamsters when they were transferred from LD 16:8 to LD 14:10 photoperiod. This study suggests that the decreasing photoperiod has a larger antigonadal effect, when combined with stress. Thus, environmental stressors such as reduced food or water availability may combine with decreasing photoperiod to trigger endocrine changes and induce testicular regression. Jansky et al. (9) similarly showed that water deprivation and a short photoperiod worked additively and induced testicular regression most effectively. Reduced food or water availability is apparently the most c o m m o n stressor in natural e n v i r o n m e n t s (9). The problem here is that reduced food or water availability can be interpreted as either stress or lack of nutrition (3,9, l 1). The studies thus far reported have not elucidated the relative importance of n u t r i t i o n and stress in testicular function and body growth. The present study was designed to separate effects of stress from those of nutrition. We hypothesized that food or water deprivation causes stress, a n d that the degree of unpredictability of food or water availability determines the m a g n i t u d e of stress. Stronger stress m a y be induced by uncertainty of food or water availability. That is, food or water deprivation on unpredictable days m a y be more stressful for animals t h a n that on fixed days even if the total lengths of deprivation days are the same.

i Requests for reprints should be addressed to Dr. Nobuo Ibuka.

917

918

IBUKA, ICHIKAWA AND NISHIOKA EXPERIMENT

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Animals and procedure. Sixty male Syrian hamsters at 3 weeks of age were purchased from SLC Breeding Colonies in Shizuoka. They were housed in a long photoperiod (LP, LD 16:8, lights on from 0500 to 2100 h) for 10 weeks from the day of arrival, and were given ad lib access to food (Oriental Rat Mouse Chow) and water. Then they were randomly divided into five groups and maintained for another 13 weeks under one of the following conditions. We employed long or short photoperiods to investigate the interactive effect of stress from water deprivation and photoperiod in the testing period. In condition 1, animals (n = 12) were given water every day under LP (the L control group). In condition 2, animals (n = 12) were regularly deprived of water every other day under LP (the LR group). In condition 3, animals (n = 12) were given water every day under a short photoperiod (SP, LD 8:16, lights on from 0900 to 1700 h, the S control group). In condition 4, animals (n = 12) were regularly deprived of water every other day under SP (the SR group). In condition 5, animals (n = 12) were deprived of water irregularly on 50% of the days under SP until the end of the experiment (the SIR group). Water deprivation was started at noon as a rule and lasted for 24 h every other day for the LR and the SR groups. The length of deprivation, however, varied depending on the predetermined schedule and lasted for 3 days (maximum) in rare cases for the SIR group. Food was always available for all animals throughout testing. The animals were housed in groups of four in opaque polycarbonate cages (39 × 34 × 18 cm) with wire mesh tops. Wood chips were used as cage bedding. Three cages were placed in a black light-tight chamber (130 × 50 × 40 cm) illuminated by two 10-W white fluorescent lights that provided about 400 lux at the cage floor level. Ventilation of the chamber was provided by a fan in continuous operation. The room temperature was kept at 22 + 2°C. Body weight of each individual was recorded in the light period between 1100 and 1300 h at weekly intervals throughout testing. After the 13-week exposure to each experimental conditions, the animals were sacrificed under deep sodium pentobarbital anesthesia (100 mg/kg). Then their testes were removed and immediately weighed to the nearest 1 mg. The experimental schedule is summarized in the lower part of Fig. 1. Results Testes. Figure 2 shows average testes weights in the five groups of hamsters. An analysis of variance revealed that there were significant differences in paired testes weight among the five groups, F(4, 55) = 10.58, p < 0.01, after the 13-week exposure to each condition. Pairwise comparisons of the group means showed that testes weight in the SIR group was lighter than that in any of the other four groups (p < 0.05). The latter were not

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AGE IN WEEKS FIG. I. The experimental schedules of Experiment l (lower half) and Experiment 2 (upper halt). LP indicates a long photoperiod with LD 16: 8 (lights on from 0500 to 2100 h), and SPa short photoperiod with LD 8:16 (lights on from 0900 to 1700 h). R means regular water- or fooddeprivation and IR irregular water- or food-deprivation. For example, R 50% indicates that complete deprivation occurs every other day while IR 50% means that deprivation occurs randomly on 50% of the days throughout the testing period. Asterisks denote the day of sacrifice of the animals. significantly different from each other. That is, only the hamsters deprived of water irregularly on 50% of the days under SP had smaller testes than those of the other four groups. The testes of the hamsters deprived of water every other day did not differ in mass from those of the control animals given water daily not only under the long photoperiodic condition (L vs. LR) but also under the short photoperiodic condition (S vs. SR). Body weight. Although the five groups did not differ significantly from each other in body weight at the start of the experiment, individual weight variations became rather large at the end of the 10-week exposure to the long photoperiod. Accordingly, instead of absolute body weight, weight gain or loss was calculated as a percent change relative to the final value immediately before the animals were transferred to each experimental condition. Figure 3 shows average weight change in the five groups over the 13 weeks. The five groups increased their body weight at different rates after transfer to the different experimental treatments. Two-way analyses of variance were used to evaluate the effects of the treatments and the weeks on body growth rate. Main effects of both the experimental treatments, F(4, 55) = 42.29, p < 0.001, and the weeks, F( 12, 660) = 182.24, p < 0.001, were highly significant. In addition, the interaction between the treatments and the weeks were also highly significant, F(48,660) = 31.56, p < 0.001. An analysis of variance of the group means at the end of testing (week 13) showed that the growth rate was smaller in the SIR group than in three other groups (L, S, SR; p < 0.05). Each of the three groups transferred to the short photoperiod showed different rates of growth after water deprivation. That is, unpredictable deprivation (the SIR group) of water on 50% of the days caused a more detrimental effect on growth than predictable water deprivation (the SR group) every other day (p < 0.05) or no deprivation (the S group; p < 0.05). Predictable deprivation, however (the LR group), under the long photoperiod also pro-

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919 They were housed in a long photoperiod (LD 16:8, lights on from 0500 to 2100 h, LP) for 6 weeks from the day of arrival, and were given free access to food (Oriental Rat Mouse Chow) and water. Then they were transferred to a short photoperiod (LD 8:16, lights on from 0900 to 1700 h), and were maintained in one of the following five conditions for another 12 weeks. In condition 1, animals (n = 11) were fed every day (control) until the end of testing (12th week). In condition 2, animals (n = 12) were deprived of food irregularly on 25% of the days (IR 25) until the end of the 10th week. In condition 3, animals (n = 12) were deprived of food irregularly on 50% of the days (IR 50) until the end of the 10th week. In condition 4, animals (n = 12) were deprived of food regularly on 25% of the days, i.e., every 4 days (R 25) until the end of the 10th week. In condition 5, animals (n = 13) were deprived of food regularly on 50% of the days (R 50), i.e., every other day until the end of the 10th week. Food deprivation was started at noon as a rule on the deprivation days. The length of deprivation varied depending on the predetermined schedule and lasted for 3 or 4 days in rare cases for the IR 50 and the IR 25 groups. Water was always available throughout testing. The animals in the four experimental groups were refed daily in the last 2 (1 lth and 12th) weeks of the experiment. The rearing conditions (illumination, temperature, etc.) were the same as in Experiment I. Body weight of each individual was recorded in the light period between 1100 and 1300 h at weekly intervals throughout the experiment. After the 12-week exposure to each experimental condition, the animals were sacrificed under deep sodium pentobarbital anesthesia. Their testes were removed and immediately weighed to the nearest 1 mg.

FIG. 2. Means (black circles) and standard deviations (vertical bars) of paired testes weightsafter 13 weeks ofexposure to fivewater-deprivation conditions. Abscissaindicates groups. L; control group with a long photoperiod (LP). S; control group with a short photoperiod (SP). LR; group deprived of water every other day under LP. SR; group deprived of water every other day under SP. SIR; group deprived of water randomly on 50% of the days under SP.

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EXPERIMENT 2 We found in Experiment 1 that testicular regression occurred only when the animals (the SIR group) were deprived of water unpredictively on 50% of the days under the short photoperiod (SP). The animals (the SR group) deprived of water regularly every other day under SP did not show any testicular regression. These findings suggest that uncertainty of water availability is more detrimental to testicular function than water deprivation per se. In Experiment 2 we deprived animals of food in place of water under SP, and introduced more systematically uncertainty of food availability as a parameter of stress.

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Testes. Figure 4 shows average testes weight in the five groups measured at the end of the experiment. The data indicate that food deprivation induced a decrease in testes weight regardless of its severity or predictability. Furthermore, unpredictable food deprivation induced a more suppressive effect on testicular function than predictable food deprivation even if the animals were exposed to the same amount of deprivation. An analysis of variance was run to verify this impression. It showed that there were significant differences in paired testes weight among the five groups after the 12-week exposure to each treatment, F(4, 55) = 16.16, p < 0.01. Pairwise comparison of the group means showed that testes weight differed significantly at 5% level between the following group pairs; control vs. R50, control vs. IR50, control vs. IR25, R50 vs. IR50, R25 vs. 1R25, and IR50 vs. R25. No significant differences were seen between the remaining group pairs (control vs. R25, R50 vs. IR25, IR25 vs. IR50, and R25 vs. R50). Body weight. Figure 5 shows average changes in body weight over the 12th week after the animals were transferred to the five experimental conditions. A change of body weight was calculated as a percentage of the final body weight immediately before each animal was exposed to its treatment. Values for each group are expressed relative to its own baseline values in week 0 as in Experiment I.

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As seen from Fig. 5, the growth rates after transfer to the experimental treatments differed significantly among the five groups, F(4, 55) = 185.33, p < 0.01. The control animals, fed daily throughout testing, gradually and consistently increased their body weight after transfer to the short photoperiod,/7(11, 110) = 298.3, p < 0.001. In contrast, the animals (the R50 and IR50 groups) deprived of food on 50% of the days throughout testing, whether on predictable days or not, lost markedly their body weight, F(11, 132) = 66.77, p < 0.00 l, for the R50 group, F(l l, 121) = 111.80, p < 0.001, for the IR50 group, although the animals (the R25 and IR25 groups) deprived of food on 25% of the days did not alter their body weight largely. Pairwise analysis of the group means at the 10th week showed that the growth rate significantly differed between the control group and each of the other four groups (p < 0.05), between the IR50 group and the R50 group (p < 0.05) but not between the IR25 and the R25 group. Refeeding in the I lth and 12th weeks rapidly restored the animals' body weight in both the IR50 group and the R50 group. GENERAL DISCUSSION

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The results of Experiment 1 indicated that unpredictable water deprivation on 50% of the testing days under the short photoperiod (SP) had the most suppressive effect on testicular function. But the testes did not regress at all when the animals were deprived of water every other day under SP. It seems, therefore, that uncertainty or unpredictability of water deprivation, rather than the scarcity of water per se, caused the regression of testes.

SUPPRESSION OF TESTICULAR AND BODY WEIGHT

921

It has been reported that, when Syrian hamsters were transferred from a long photoperiod (LP) to SP, their testes began to regress within several weeks (16). However, prepuberal hamsters were known to be insensitive to SP (15,18). Thus, Syrian hamsters, unlike Djungarian hamsters or white-footed mice, reach puberty totally independent of photoperiod. We expected in our experiments that the testes would regress in 12 or 13 weeks after exposure to SP because the animals of the control groups had already reached adulthood at the beginning of SP exposure. But we could not obtain the regression of testes in spite of the exposure to SP in Experiment 2 as well as in Experiment 1. The SP insensitive period in our Syrian strain seemed to extend into young adulthood. In some cases, adult Syrian hamsters raised in SP failed to regress their testes in 15 or more consecutive weeks (18). But our results are not in general agreement with the earlier studies ( 13,15). A recent study (10) reported that Djungarian hamsters from American and German stocks differed both in the percentage of the animals responding to SP and in the degree and timing of their response. German stock hamsters showed a higher percentage of photoresponsive individuals, lost body weight more rapidly, and molted earlier than the American stock. Their study and our present data suggest a possibility that different stocks of Syrian hamsters may also show variations in photoresponsiveness. Now an experiment to clarify this problem is in progress. The results of Experiment 2 showed that, whether the animals were deprived of food unpredictably or predictably, intermittent food deprivation for 12 weeks caused regression of testes. More importantly, as in Experiment l, unpredictable food deprivation induced a more suppressive effect on testes than predictable deprivation. Here, again, the stress on uncertainty of food deprivation or availability caused the more detrimental effect on testes than the scarcity of food per se. In a recent study (14) the unnatural stress of control oil injection induced testicular regression. The suppressive effect on testes was obtained when this stress was given under an LD 14" 10 light regime after exposure to an LD 16:8 light regime. In natural habitats reduced food availability may interact with decreasing photoperiod. Thus, stress from food or water deprivation was expected to be potentiated by exposure to short photoperiod. It was apparent from Experiment 2 that stress created by uncertainty of food availability caused the more suppressive effect on testicular weight and body mass under short photoperiod. But it remains to be determined whether the similar suppressive effect on testicular and body weight is obtained under long photoperiod.

Our present findings suggest that stress may be more important than nutritional factors associated with water or food shortage in testicular regression under a short photoperiod. Not only in animals but also in humans various kinds of chronic stress may have suppressive influences on the reproductive system (2). In severe cases stress may induce cessation of menstrous cycles or regression of testes as reported here (2,8). It is very difficult to separate completely the stress component from the nutritional aspect of food or water deprivation. A experimental paradigm in the present experiments was an approach to solve this problem. However, a question remains whether the irregular and regular schedules were physiologically identical to each other. A solution to the question might be to measure the actual amounts of eating or drinking of both groups during the deprivation period. The present study (Experiment l) indicated that weight gains in Syrian hamsters were larger under the short photoperiod than the long photoperiod (see Fig. 3). Weight gains were particularly remarkable when the hamsters were maintained in SP after LP. These findings confirmed and extended earlier observations (1,9). Experiment 1 showed that the animals deprived of water every other day under SP continued to grow more slowly than the control animals given water every day. But the animals deprived of water unpredictably on 50% of the days did not show any weight gain at all over the 13 weeks. Here, again, the stress of unpredictable water deprivation caused the most detrimental effect on body growth. The findings of Experiment 2 on body growth indicated that food deprivation, regardless of whether the hamsters were deprived of food every other day or unpredictably on 50% of the days, caused the remarkable weight loss. Furthermore, the unpredictable food deprivation tended to suppress more largely body growth. This trend was obvious in the later weeks of testing for the 50% irregular deprivation group. The suppressive effect of food deprivation on body growth was larger than that of water deprivation. This might be related to the fact that in laboratory environments as well as in natural habitats Syrian hamsters can survive for prolonged time without drinking water (9). To summarize, the present study clearly suggested that unpredictability or uncertainty of food or water deprivation induced more suppressive effects than shortage of food or water per se on testicular function and body growth. ACKNOWLEDGEMENTS The authors wish to express their thanks to Dr. Shinya Suzuki for polishing up the English manuscript and for helpful comments.

REFERENCES 1. Bartness, T. J.; Wade, G. N. Photoperiodic control of body weight and energy metabolism in Syrian hamsters (Mesocricetus auratus): Role of pineal gland, melatonin, gonads, and diet. Endocrinology 114:492-498; 1984. 2. Campbell,C. S.; Turek, F. W. Cyclicfunction of the mmalian ovary. In: Aschoff, J., ed. Handbook of behavioral neurobiology, vol. 4. Biological rhythms. New York: Plenum Press; 1981:523-545. 3. Eskes, G. Gonadal responses to food restriction in intact and pinealectomized male golden hamsters. J. Reprod. Fertil. 68:85-90; 1983. 4. Frehn, J. L.; Liu, C. C. Effects of temperature, photoperiod and hibernation on the testes of golden hamsters. J. Exp. Zool. 174:317323; 1970. 5. Gaston, S.; Menaker M. Photoperiodic control of hamster testis. Science 158:925-928; 1967.

6. Hoffman, R. A.; Hester, R. J.; Towns, C. Effect of light and temperature on the endocrinesystemof the goldenhamster (Mesocricetus auratus Waterhouse). Comp. Biochem. Physiol. 15:525-533; 1965. 7. Howland, B. E. The influenceof feed restriction and subsequent refeeding on gonadotrophin secretion and serum testosterone levels in male rats. J. Reprod. Fertil. 44:429-436; 1975. 8. Howland, B. E.; Skinner, K. R. Effect of starvation on LH levelsin male and female hamsters. J. Reprod. Fertil. 32:505-507; 1973. 9. Jansky, L.; Haddad, G.; Pospisilova, D.; Dvorak, P. Effect of external factors on gonadal activity and body mass of male golden hamsters (Mesocricetus auratus). J. Comp. Physiol. [B] 156:717725; 1986. 10. Lynch, G. R.; Lynch, C. B.; Kliman, R. M. Genetic analyses of photoresponsivenessin the Djungarian hamster Phodopus sungorus. J. Comp. Physiol. [A] 164:475-481; 1989.

922 I 1. Printz, R. H.; Greenwald, G. S. Effects of starvation on follicular development in the cyclic hamster. Endocrinology 86:290-295; 1970. 12. Reiter, R. J. Circannual reproductive rhythms in mammals related to photoperiod and pineal function: A review. Chronobiologia I: 365-395; 1974. 13. Rollag, M. D.; Dipinto, M. N.; Stetson, M. H. Ontogeny of the gonadal response of golden hamsters to short photoperiod, blinding, and melatonin. Biol. Reprod. 27:895-902; 1982. 14. Rusak, B. Interactive effects of stress and photoperiod history on gonadal condition in male Syrian hamsters. J. Pineal Res. 5:41-50; 1988. 15. Sisk, C. L.; Turek, F. W. Reproductive responsiveness to short pho-

IBUKA, I C H I K A W A A N D NISHIOKA

toperiod develops postnatally in male golden hamsters. J. Androl. 8:91-96; 1987. 16. "l-urek, F. W.; Elliott, J. A.; Alvis, J. D.; Menaker, M. Efl~ct of prolonged exposure to nonstimulatory photoperiods on the activity of the neuroendocrine-testicular axis of golden hamsters. Biol. Reprod. 13:475-481; 1975. 17. Yahr, P.; Kessler, S. Suppression of reproduction in water-deprived Mongolian gerbils (Meriones unguicu/atus). Biol. Reprod. 12:249254; 1975. 18. Zucker~ 1.: Johnston, P. G.; Frost, D. Comparative, physiological and biochronometric analyses of rodent seasonal reproductive cycles. In: Hubinont, P. O., ed. Progress in reproductive biology. Basel: Karger; 1980:102-133.