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Crowding-induced changes in basal and stress levels of thyrotropin and somatotropin in male rats
BEHAVIORAL AND NEURAL BIOLOGY
48, 334-343 (1987)
Crowding-Induced Changes in Basal and Stress Levels of Thyrotropin and Somatotropin in Male Rats A. ARMARIO,*'l C. GARCIA-MARQUEZ,* AND T. JOLIN~*Departamento de Biologfa Celular y Fisiologia, Universidad Aut6noma de Barcelona, Barcelona, y Plnstituto de Investigaciones Biomddicas, CSIC, Universidad Aut6noma de Madrid, Madrid, Spain
The effects of crowding on thyrotropin (TSH) and somatotropin (GH) secretion were studied in two-month-old male rats. Crowded rats (9-10 per cage) showed lower serum GH levels than controls (3 per cage). Likewise, serum GH was lower in crowded rats after acute exposure to stress. However, percentage inhibition of GH secretion induced by acute stress was similar in crowded and control rats. Crowding reduced the TSH response to acute stress. The results found with the administration of hypothalamic regulatory factors suggest that the impaired GH and TSH secretion observed in crowded rats was not likely to he at the pituitary level. Therefore, altered neuroendocrine control of GH and TSH secretion appears to exist in crowded rats. Preliminary results obtained in rats crowded from weaning to adulthood suggest that food restriction only partially accounts for the changes observed in crowded rats. © 1987AcademicPress, Inc. It is well-established that c r o w d i n g induces s o m e i m p o r t a n t alterations in the p h y s i o l o g y and b e h a v i o r o f animals (Archer, 1970; C a l h o u n , 1962; Christian & Davis, 1964). It has b e e n r e p e a t e d l y o b s e r v e d that c r o w d i n g r e d u c e d f o o d intake, b o d y weight gain, and r e p r o d u c t i v e capacities in animals maintained in l a b o r a t o r y conditions w i t h o u t the possibility to migrate (Armario, Ortiz, & Balasch, 1984a; Christian, L l o y d , & Davis 1965; Ortiz, A r m a r i o , Castellanos, & Balasch, 1985). H o w e v e r , the endocrine alterations underlying m o s t o f the o b s e r v e d c h a n g e s are not k n o w n b e c a u s e no studies h a v e b e e n u n d e r t a k e n to elucidate the effects o f c r o w d i n g on h o r m o n e s o t h e r than t h o s e o f the p i t u i t a r y - a d r e n a l and pituitary-gonadal axes (Armario, Castellanos, & Balasch, 1984b; Armario & L o p e z - C a l d e r o n , 1986; Daniels-Severs, G o o d w i n , Keil, & V e r n i k o s Danelis, 1973; M c C a r t h y , G r e e n , & Sohal, 1976; Nowell, 1980; Ortiz et al., 1985). To whom requests for reprints should be addressed at Departamento de Biologfa Celular y Fisiologfa, Facultad de Ciencias, Universidad Aut6noma de Barcelona, Bellaterra, Barcelona, Spain. 334 0163-1047/87 $3.00 Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
CROWDING AND TSH AND GH LEVELS
335
Among the hormones of the anterior pituitary, thyrotropin (TSH) and somatotropin (GH) appear to play critical roles in the control of growth and adjustment of metabolic processes to altered food intake (Armario, Montero, & Jolin, 1987; Ortiz-Caro, Gonzalez, & Jolin, 1984). Therefore it seemed of interest to study the influence of crowding on the circulating levels of these hormones. In addition, we studied the effect of crowding on the response of these hormones to an acute stress for two main reasons: First, this type of stimuli might contribute to make evident changes which are not detected by measuring basal hormone levels only. Second, crowding could alter the response of these hormones to an acute superimposed stress by altering the sensitivity of these neuroendocrine axes to stressful stimuli.
MATERIAL AND METHODS
Animals and general procedures. Male Sprague-Dawley rats were used. They were maintained in a controlled environment (lights on from 0700 to 1900 h, temperature 23°C) in cages of 48 × 23 × 14 cm for at least 10 days before starting experiments to allow acclimation to our laboratory. Food and water were freely available. The animals were killed by decapitation between 9 and 11 AM in a room adjacent to the animal house. The time elapsed between touching the cages and decapitation was less than 30 s. The trunk blood was collected in plastic tubes maintained in ice-cold water. The serum obtained was frozen at -20°C. Experiment 1. Rats approximately 70 days old at the beginning of the experimental phase were used. They were randomly assigned to control (3 rats per cage) and crowding (10 rats per cage) groups. Fourteen days later, four animals from each group were killed without stress to obtain basal hormone values. Others (6 per group) were subjected to forced swimming stress in a water tank (50 cm diameter and 60 cm high, temperature of water 18°C) for 30 min (except between the 15th and the 30th min when the animals were allowed to rest to avoid exhaustion). Experiment 2. The aim of the second experiment was to study the influence of the period of crowding on the variables under investigation. In addition, corticosterone response to stress was also assessed to study the possible effects of an altered adrenocortical responsiveness to acute stress on TSH and GH secretion. Rats with the same characteristics as those in the Experiment 1 were used, but they were approximately two months old at the beginning of the experimental phase. They were housed in groups of three (control) or nine (crowding) per cage. The periods of crowding were 10 and 30 days. Those assigned to 10 days of crowding were maintained three per cage until 10 days before the sacrifice of the animals. At that time they were rehoused in groups of nine per cage. Four rats from each group were killed without stress. The others were
336
ARMARIO, GARCIA-MARQUEZ, AND JOLIN
subjected to 10 (6 rats per group) or 30 (8 rats per group) min of forced swimming as detailed above. Experiment 3. This experiment was designed to study the mechanisms underlying the changes in GH and TSH secretion observed in the two previous experiments. Rats of the same characteristics as those in the Experiment 2 were used. They were housed in groups of 3 (control) or 10 (crowding) per cage for 30 days. Animals from the two groups were divided into subgroups and injected ip with saline, thyrotropin releasing hormone (TRH), human growth hormone-releasing hormone (GH-RH) fragment 1-40, or somatostatin at doses of 2 ~g/kg body wt. Thirteen minutes later they were killed. All hypothalamic factors were purchased from Sigma. Hormone assays. All hormones were determined by radioimmunoassay. Corticosterone was assayed as previously described (Armario & Castellanos, 1984). GH and TSH were analyzed by double-antibody radioimmunoassays using the reagents kindly provided by Dr. Parlow (NIADDK, NIH, Bethesda, MD). The results of GH and TSH were expressed in terms of their respective standards. All samples to be statistically compared were processed within the same assay to avoid interassay variability. Intraassay coefficients of variation were always below 8%. Statistical analysis. The results were analyzed with a two-way ANOVA (first and second experiments) or with the Student's t test. Comparisons of more than two means under the same acute treatment were carried out with the Duncan's test (p < .05). Where necessary data were log. transformed to achieve homogeneity of variances. RESULTS
Experiment 1 Figure 1 shows the effect of crowding on TSH and GH. The ANOVA revealed significant effects of both crowding (p < .02) and acute stress (p < .001) on TSH secretion. Although the interaction between the two main factors was not statistically significant, the effect of crowding was apparently more marked after stress. GH levels were altered by crowding (p < .03) and acute stress (p < .001); the interaction between the two factors was nonsignificant. The differences in serum GH levels between crowded and control rats were more evident in the basal state.
Experiment 2 Crowding significantly reduced body weight gain. Thus, body weight at the end of the experiment were (means ___ SD, n = 27) 397 +_ 34, 371 _ 35, and 364 _+ 52 in controls, rats crowded for 10 days, and rats crowded for 30 days, respectively. The effects of the two periods of crowding on serum corticosterone are depicted in Fig. 2. A significant effect of the period of acute stress
CROWDING AND TSH AND GH LEVELS
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FIG. 1. Serum TSH and GH levels in control and crowded rats. Means and SEM are represented. Open bars indicate control and closed bars crowded rats. The number of animals per group is give.n inside the bar. p < .05 between the two experimental groups, *p at least .05 vs respective basal values. For other details of the statistical analysis see text.
(p < .001), but not of previous chronic treatment was found. Figure 3 shows serum TSH and GH levels in crowded and control rats. The ANOVA revealed significant effects of previous chronic treatment (p < .001) and period of acute stress (p < .001) on TSH levels. The interaction between the two factors was also significant (p < .006). It is apparent from the results that the effect of crowding was similar regardless of the period of crowding. Both crowding (p < .001) and acute stress (p < .001) resulted in impaired GH release. No significant interaction between the two factors was found. The effect of crowding on GH secretion was independent of the period of crowding.
Experiment 3 Serum levels of TSH and GH were lower in crowded than in control rats after saline administration (p < .01). For this reason the response
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FI6. 3. Serum TSH and GH levels in control and crowded rats. Means and SEM are represented. Open bars indicate controls, lined bars rats crowded for 10 days, and closed bars rats crowded for 30 days. The number of animals per group is given inside the bars. The numbers under bars indicate the period (min) of exposure to acute stress. Differences between stress and basal levels were always significant and they are not indicated. Bars representing rats subjected to the same acute treatment labeled with different letters are statistically different (Duncan's test, p < .05).
to the hypothalamic factors was expressed in both absolute and relative values. Stimulation with TRH resulted in high TSH levels in both control and crowded rats, with no difference between the two experimental groups being observed (Table 1). However, the percentage stimulation was greater (p < .01) in crowded than in control rats. After somatostatin administration serum TSH levels were similar in control and crowded rats. However, percentage changes differed in the two groups (p < .05) because somatostatin administration significantly inhibited TSH secretion in control rats but not in crowded rats. The response of GH to hypothalamic factors is depicted in Table 2. Serum GH levels were significantly lower in crowded than in control rats (p < .05) after GH-RH administration. In contrast, the percentage increase was higher (p < .01) in the former animals. Somatostatin caused the same percentage inhibition of GH secretion in the two experimental groups, but absolute serum GH levels were lower (p < .05) in crowded than in control rats.
DISCUSSION The response of GH and TSH to forced swimming stress is in agreement with the pattern of response of the two hormones to acute stress in the rat. This is characterized by a decrease in GH and an increase in TSH (Armario, Castellanos, & Balasch, 1984c, 1984d; Kokka, Garcia, George & Elliott, 1972; Gartner, Buttner, Dohler, Friedel, Lindena, & Trautschold, 1980; Schalch & Reichlin, 1966). Although inhibition of TSH secretion has been reported after exposure to various acute stressors (Armario, Hidalgo, & Restrepo, 1985; Simpkins, Hudson, & Meites, 1978), in the
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present experiment TSH release was expected as rats swam in cold water, and cold is a potent stimulus for TSH release (Hefco, Krulich, Illner, & Larsen, 1975). The present results demonstrate that crowding can inhibit basal and stress levels of TSH and GH in the rat. While the response of TSH to stress was consistently depressed by crowding, its basal levels were significantly depressed only in one of three experiments. As the experiments were done at different times of the year, it seems that circannual changes in the sensitivity of the pituitary-thyroid axis to some environmental stimuli might exist. However, other unknown factors to explain the differences between experiments may also be relevant. The inhibitory action of crowding on TSH and GH was the same after 10 or 30 days of crowding, so that the changes were not dependent on the length of the crowding period. Crowded rats showed lower body weight than control rats in the three experiments. This is in accordance with previous reports in which reduced food intake and body weight gain were found in crowded rats (Armario et al., 1984a, 1984b). Impaired TSH and GH secretion might be, at least partially, a consequence of the reduction of food intake since proteincalorie deficit depressed basal GH and TSH secretion in rats (Armario et al., 1987; Ortiz-Caro et al., 1984). In addition, rats crowded after weaning and killed two months later showed lower body weight gain and lower basal serum TSH levels, but similar serum GH levels, than pairfed rats (unpublished data). To our knowledge it is not possible to design an adequate experimental approach to the problem of the reduced food intake caused by crowding, but these data suggest that the decrease in food intake always accompanying crowding only partially accounts for the changes observed in crowded rats. The finding in the second experiment that basal TSH levels were normal whereas the activation induced by acute stress was significantly lower in crowded than in control rats indicates that stress levels of TSH might be more sensitive to abnormalities caused by crowding than its basal levels. Serum TSH levels achieved after pituitary stimulation with TRH were similar in the two experimental groups. However, the percentage increase was higher in crowded rats which suggests enhanced responsiveness of crowded rats to TRH. In addition, somatostatin administration reduced TSH secretion in accordance with a previous report (Drouin, De Lean, Rainville, Lachance, & Labrie, 1976)--in control but not in crowded rats. This suggests either that thyrotropin-producing cells of crowded rats were less responsive to somatostatin or that crowding resulted in increased somatostatinergic activity and no effect of additional somatostatin on TSH secretion would be evident. In any case, it appears that impaired TSH secretion observed in crowded rats might be located at the hypo-
342
ARMARIO, GARCIA-MARQUEZ, AND JOLIN
thalamic rather than at the pituitary level. Although absolute GH levels were lower in crowded than in control rats after GH-RH administration, the percentage increase was greater in the former animals. Likewise, the percentage response to somatostatin was the same in the two groups, but absolute values were lower in crowded rats. Taken together, these data indicate that changes in GH in response to its hypothalamic regulatory factors could not play a major role in the inhibition of GH secretion observed in crowded rats. The specific contribution of the hypothalamus remains uncertain, but increased somatostatinergic activity might be involved since somatostatin appears to mediate the inhibition of GH caused by food deprivation (Tannenbaum, Epelbaum, Colle, Brazeau, & Martin, 1978). The contribution of the pituitary-adrenal axis to crowding-induced changes in GH and TSH secretion was not directly assessed but a major role appears unlikely because crowding did not alter adrenal weight (Armario et al., 1984a, 984b) and no differences in adrenocortical response to acute stress were observed between control and crowded rats. Impaired GH and TSH secretion observed in crowded animals might be the endocrine basis of the reduction of growth repeatedly observed in crowded animals. Although the reduction of food intake might partially account for the changes in GH and TSH secretion caused by crowding, its precise contribution to the observed changes remains to be established. REFERENCES Archer, J. (1970). Effects of population density on behaviour in rodents. In J. H. Crook (Ed.), Social behaviour in birds and mammals (pp. 169-210). London: Academic Press. Armario, A., & Castellanos, J. M. (1984). A simple procedure for direct corticosterone radioimmunoassay in the rat. Revista Espanola de Fisiologia, 40, 437-442. Armario, A., Ortiz, R., Balasch, J. (1984a). Effect of crowding on some physiological and behavioral variables in adult male rats. Physiology and Behavior, 32, 35-37. Armario, A., Castellanos, J. M., & Balasch, J. (1984b). Effect of crowding on emotional reactivity in male rats. Neuroendocrinology, 39, 330-333. Armario, A., Castellanos, J. M., & Balasch, J. (1984c). Effect of acute and chromc psychogenic stress on corticoadrenal and pituitary-thyroid hormones in male rats. Hormone Research, 20, 241-245. Armano, A., Castellanos, J. M., & Balasch, J. (1984d). Adaptation of anterior pituitary hormones to chronic noise in male rats. Behavioral and Neural Biology, 41, 71-76. Armario, A., Hidalgo, J., & Restrepo, C. (1985). Thyrotropin response to stress and other stimuli in peripuberal and adult male rats. Neuroendocrinology Letters, 6, 121-126. Armario, A., & Lopez-Calderon, A. (1986). Pituitary-gonadal function in adult male rats subjected to crowding. Endocrine Research. 12, 115-122. Armario, A., Montero, J. L., & Jolin, T. (1987). Chronic food restriction and the circadian rhythms of pituitary-adrenal hormones, growth hormone and thyroid-stimulating hormone. Annals of Nutrition and Metabolism, 31, 81-87. Calhoun, J. B. (1962). Population density and social pathology. Scientific American 206, 139-148. Christian, J. J., & Davis, D. E. (1964). Endocrines, behavior and population. Science, 146, 1550-1560.
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Christian, J. J., Lloyd, J. A., & Davis, D. E. (1965). The role of endocrines in the selfregulation of mammalian populations. Recent Progress in Hormone Research, 21,501578. Daniels-Severs, A., Goodwin, A., Keil, L. C., & Vernikos-Danelis, J. (1973). Effect of crowding and cold on the pituitary-adrenal system: Responsiveness to an acute stimulus during chronic stress. Pharmacology, 9, 348-356. Drouin, J., De Lean, A., Rainville, D., Lachance, R., & Labrie, F. (1976). Characteristics of the interaction between thyrotropin-releasing hormone and somatostatin for thyrotropin and prolactin release. Endocrinology, 98, 514-521. Gartner, K., Buttner, D., Dohler, K., Friedel, R., Lindena, J., & Trauschold, I. (1980). Stress response of rats to handling and experimental procedures. Laboratory Animals, 14, 267-274. Hefco, E., Krulich, L., Illner, P., & Larsen, P. R. (1975). Effect of acute exposure to cold on the activity of the hypothalamic-pituitary-thyroid system. Endocrinology, 97, 1185-1195. Kokka, N., Garcia, J. F., George, R., & Elliott, H. W. (1972). Growth hormone and ACTH secretion: Evidence for an inverse relationship in rats. Endocrinology, 90, 735743. McCarthy, J. L., Green, W., & Sohal, R. S. (1976). Crowding stress and adrenal mitochondrial 11-fl-hydroxylation in vitro. Proceedings of the Society for Experimental Biology and Medicine, 153, 528-531. Nowell, N. W. (1980). Adrenocortical function in relation to mammalian population densities and hierarchies. In I. C. Jones & I. W. Henderson (Eds.), General, comparative and clinical endocrinology of the adrenal cortex (Vol. 3, pp. 350-393). London: Academic Press. Ortiz, R., Armario, A., Castellanos, J. M., & Balasch, J. (1985). Post-weaning crowding induces corticoadrenal hyperreactivity in male mice. Physiology and Behavior, 34, 857-860. Ortiz-Caro, J., Gonzalez, C., & Jolin, T. (1984). Diurnal variations of plasma growth hormone, thyrotropin, thyroxine, and trliodothyronine in streptozotocin-diabetic and food-restricted rats. Endocrinology, 115, 2227-2232. Schalch, D. S., & Reichlin, S. (1966). Plasma growth hormone concentration in the rat determined by radioimmunoassay: Influence of sex, pregnancy, lactation, anesthesia, hypophysectomy and extrasellar pituitary transplants. Endocrinology 79, 275-290. Simpkins, J. W., Hudson, C. A., & Meites, J. (1978). Differential effects of stress on release of thyroid-stimulating hormone in young and old male rats. Proceedings of the Society for Experimental Biology and Medicine, 157, 144-147. Tannenbaum, G. S., Epelbaum, J., Colle, E., Brazeau, P., & Martin, J. B. (1978). Antiserum to somatostatin reverses starvation-induced inhibition of growth hormone but not insulin secretion. Endocrinology, 102, 1909-1914.
48, 334-343 (1987)
Crowding-Induced Changes in Basal and Stress Levels of Thyrotropin and Somatotropin in Male Rats A. ARMARIO,*'l C. GARCIA-MARQUEZ,* AND T. JOLIN~*Departamento de Biologfa Celular y Fisiologia, Universidad Aut6noma de Barcelona, Barcelona, y Plnstituto de Investigaciones Biomddicas, CSIC, Universidad Aut6noma de Madrid, Madrid, Spain
The effects of crowding on thyrotropin (TSH) and somatotropin (GH) secretion were studied in two-month-old male rats. Crowded rats (9-10 per cage) showed lower serum GH levels than controls (3 per cage). Likewise, serum GH was lower in crowded rats after acute exposure to stress. However, percentage inhibition of GH secretion induced by acute stress was similar in crowded and control rats. Crowding reduced the TSH response to acute stress. The results found with the administration of hypothalamic regulatory factors suggest that the impaired GH and TSH secretion observed in crowded rats was not likely to he at the pituitary level. Therefore, altered neuroendocrine control of GH and TSH secretion appears to exist in crowded rats. Preliminary results obtained in rats crowded from weaning to adulthood suggest that food restriction only partially accounts for the changes observed in crowded rats. © 1987AcademicPress, Inc. It is well-established that c r o w d i n g induces s o m e i m p o r t a n t alterations in the p h y s i o l o g y and b e h a v i o r o f animals (Archer, 1970; C a l h o u n , 1962; Christian & Davis, 1964). It has b e e n r e p e a t e d l y o b s e r v e d that c r o w d i n g r e d u c e d f o o d intake, b o d y weight gain, and r e p r o d u c t i v e capacities in animals maintained in l a b o r a t o r y conditions w i t h o u t the possibility to migrate (Armario, Ortiz, & Balasch, 1984a; Christian, L l o y d , & Davis 1965; Ortiz, A r m a r i o , Castellanos, & Balasch, 1985). H o w e v e r , the endocrine alterations underlying m o s t o f the o b s e r v e d c h a n g e s are not k n o w n b e c a u s e no studies h a v e b e e n u n d e r t a k e n to elucidate the effects o f c r o w d i n g on h o r m o n e s o t h e r than t h o s e o f the p i t u i t a r y - a d r e n a l and pituitary-gonadal axes (Armario, Castellanos, & Balasch, 1984b; Armario & L o p e z - C a l d e r o n , 1986; Daniels-Severs, G o o d w i n , Keil, & V e r n i k o s Danelis, 1973; M c C a r t h y , G r e e n , & Sohal, 1976; Nowell, 1980; Ortiz et al., 1985). To whom requests for reprints should be addressed at Departamento de Biologfa Celular y Fisiologfa, Facultad de Ciencias, Universidad Aut6noma de Barcelona, Bellaterra, Barcelona, Spain. 334 0163-1047/87 $3.00 Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
CROWDING AND TSH AND GH LEVELS
335
Among the hormones of the anterior pituitary, thyrotropin (TSH) and somatotropin (GH) appear to play critical roles in the control of growth and adjustment of metabolic processes to altered food intake (Armario, Montero, & Jolin, 1987; Ortiz-Caro, Gonzalez, & Jolin, 1984). Therefore it seemed of interest to study the influence of crowding on the circulating levels of these hormones. In addition, we studied the effect of crowding on the response of these hormones to an acute stress for two main reasons: First, this type of stimuli might contribute to make evident changes which are not detected by measuring basal hormone levels only. Second, crowding could alter the response of these hormones to an acute superimposed stress by altering the sensitivity of these neuroendocrine axes to stressful stimuli.
MATERIAL AND METHODS
Animals and general procedures. Male Sprague-Dawley rats were used. They were maintained in a controlled environment (lights on from 0700 to 1900 h, temperature 23°C) in cages of 48 × 23 × 14 cm for at least 10 days before starting experiments to allow acclimation to our laboratory. Food and water were freely available. The animals were killed by decapitation between 9 and 11 AM in a room adjacent to the animal house. The time elapsed between touching the cages and decapitation was less than 30 s. The trunk blood was collected in plastic tubes maintained in ice-cold water. The serum obtained was frozen at -20°C. Experiment 1. Rats approximately 70 days old at the beginning of the experimental phase were used. They were randomly assigned to control (3 rats per cage) and crowding (10 rats per cage) groups. Fourteen days later, four animals from each group were killed without stress to obtain basal hormone values. Others (6 per group) were subjected to forced swimming stress in a water tank (50 cm diameter and 60 cm high, temperature of water 18°C) for 30 min (except between the 15th and the 30th min when the animals were allowed to rest to avoid exhaustion). Experiment 2. The aim of the second experiment was to study the influence of the period of crowding on the variables under investigation. In addition, corticosterone response to stress was also assessed to study the possible effects of an altered adrenocortical responsiveness to acute stress on TSH and GH secretion. Rats with the same characteristics as those in the Experiment 1 were used, but they were approximately two months old at the beginning of the experimental phase. They were housed in groups of three (control) or nine (crowding) per cage. The periods of crowding were 10 and 30 days. Those assigned to 10 days of crowding were maintained three per cage until 10 days before the sacrifice of the animals. At that time they were rehoused in groups of nine per cage. Four rats from each group were killed without stress. The others were
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ARMARIO, GARCIA-MARQUEZ, AND JOLIN
subjected to 10 (6 rats per group) or 30 (8 rats per group) min of forced swimming as detailed above. Experiment 3. This experiment was designed to study the mechanisms underlying the changes in GH and TSH secretion observed in the two previous experiments. Rats of the same characteristics as those in the Experiment 2 were used. They were housed in groups of 3 (control) or 10 (crowding) per cage for 30 days. Animals from the two groups were divided into subgroups and injected ip with saline, thyrotropin releasing hormone (TRH), human growth hormone-releasing hormone (GH-RH) fragment 1-40, or somatostatin at doses of 2 ~g/kg body wt. Thirteen minutes later they were killed. All hypothalamic factors were purchased from Sigma. Hormone assays. All hormones were determined by radioimmunoassay. Corticosterone was assayed as previously described (Armario & Castellanos, 1984). GH and TSH were analyzed by double-antibody radioimmunoassays using the reagents kindly provided by Dr. Parlow (NIADDK, NIH, Bethesda, MD). The results of GH and TSH were expressed in terms of their respective standards. All samples to be statistically compared were processed within the same assay to avoid interassay variability. Intraassay coefficients of variation were always below 8%. Statistical analysis. The results were analyzed with a two-way ANOVA (first and second experiments) or with the Student's t test. Comparisons of more than two means under the same acute treatment were carried out with the Duncan's test (p < .05). Where necessary data were log. transformed to achieve homogeneity of variances. RESULTS
Experiment 1 Figure 1 shows the effect of crowding on TSH and GH. The ANOVA revealed significant effects of both crowding (p < .02) and acute stress (p < .001) on TSH secretion. Although the interaction between the two main factors was not statistically significant, the effect of crowding was apparently more marked after stress. GH levels were altered by crowding (p < .03) and acute stress (p < .001); the interaction between the two factors was nonsignificant. The differences in serum GH levels between crowded and control rats were more evident in the basal state.
Experiment 2 Crowding significantly reduced body weight gain. Thus, body weight at the end of the experiment were (means ___ SD, n = 27) 397 +_ 34, 371 _ 35, and 364 _+ 52 in controls, rats crowded for 10 days, and rats crowded for 30 days, respectively. The effects of the two periods of crowding on serum corticosterone are depicted in Fig. 2. A significant effect of the period of acute stress
CROWDING AND TSH AND GH LEVELS
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FIG. 1. Serum TSH and GH levels in control and crowded rats. Means and SEM are represented. Open bars indicate control and closed bars crowded rats. The number of animals per group is give.n inside the bar. p < .05 between the two experimental groups, *p at least .05 vs respective basal values. For other details of the statistical analysis see text.
(p < .001), but not of previous chronic treatment was found. Figure 3 shows serum TSH and GH levels in crowded and control rats. The ANOVA revealed significant effects of previous chronic treatment (p < .001) and period of acute stress (p < .001) on TSH levels. The interaction between the two factors was also significant (p < .006). It is apparent from the results that the effect of crowding was similar regardless of the period of crowding. Both crowding (p < .001) and acute stress (p < .001) resulted in impaired GH release. No significant interaction between the two factors was found. The effect of crowding on GH secretion was independent of the period of crowding.
Experiment 3 Serum levels of TSH and GH were lower in crowded than in control rats after saline administration (p < .01). For this reason the response
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Fie. 2. Serum corticosterone levels in control and crowded rats. Means and SEM are represented. Open bars indicate controls, lined bars rats crowded for 10 days, and closed bars rats crowded for 30 days. The number of animals per group is given inside the bars. The numbers under the bars indicate the period of exposure (min) to acute stress. No significant differences between the three housing conditions were observed. The differences between basal and stress values were always significant and they are not indicated.
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FI6. 3. Serum TSH and GH levels in control and crowded rats. Means and SEM are represented. Open bars indicate controls, lined bars rats crowded for 10 days, and closed bars rats crowded for 30 days. The number of animals per group is given inside the bars. The numbers under bars indicate the period (min) of exposure to acute stress. Differences between stress and basal levels were always significant and they are not indicated. Bars representing rats subjected to the same acute treatment labeled with different letters are statistically different (Duncan's test, p < .05).
to the hypothalamic factors was expressed in both absolute and relative values. Stimulation with TRH resulted in high TSH levels in both control and crowded rats, with no difference between the two experimental groups being observed (Table 1). However, the percentage stimulation was greater (p < .01) in crowded than in control rats. After somatostatin administration serum TSH levels were similar in control and crowded rats. However, percentage changes differed in the two groups (p < .05) because somatostatin administration significantly inhibited TSH secretion in control rats but not in crowded rats. The response of GH to hypothalamic factors is depicted in Table 2. Serum GH levels were significantly lower in crowded than in control rats (p < .05) after GH-RH administration. In contrast, the percentage increase was higher (p < .01) in the former animals. Somatostatin caused the same percentage inhibition of GH secretion in the two experimental groups, but absolute serum GH levels were lower (p < .05) in crowded than in control rats.
DISCUSSION The response of GH and TSH to forced swimming stress is in agreement with the pattern of response of the two hormones to acute stress in the rat. This is characterized by a decrease in GH and an increase in TSH (Armario, Castellanos, & Balasch, 1984c, 1984d; Kokka, Garcia, George & Elliott, 1972; Gartner, Buttner, Dohler, Friedel, Lindena, & Trautschold, 1980; Schalch & Reichlin, 1966). Although inhibition of TSH secretion has been reported after exposure to various acute stressors (Armario, Hidalgo, & Restrepo, 1985; Simpkins, Hudson, & Meites, 1978), in the
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present experiment TSH release was expected as rats swam in cold water, and cold is a potent stimulus for TSH release (Hefco, Krulich, Illner, & Larsen, 1975). The present results demonstrate that crowding can inhibit basal and stress levels of TSH and GH in the rat. While the response of TSH to stress was consistently depressed by crowding, its basal levels were significantly depressed only in one of three experiments. As the experiments were done at different times of the year, it seems that circannual changes in the sensitivity of the pituitary-thyroid axis to some environmental stimuli might exist. However, other unknown factors to explain the differences between experiments may also be relevant. The inhibitory action of crowding on TSH and GH was the same after 10 or 30 days of crowding, so that the changes were not dependent on the length of the crowding period. Crowded rats showed lower body weight than control rats in the three experiments. This is in accordance with previous reports in which reduced food intake and body weight gain were found in crowded rats (Armario et al., 1984a, 1984b). Impaired TSH and GH secretion might be, at least partially, a consequence of the reduction of food intake since proteincalorie deficit depressed basal GH and TSH secretion in rats (Armario et al., 1987; Ortiz-Caro et al., 1984). In addition, rats crowded after weaning and killed two months later showed lower body weight gain and lower basal serum TSH levels, but similar serum GH levels, than pairfed rats (unpublished data). To our knowledge it is not possible to design an adequate experimental approach to the problem of the reduced food intake caused by crowding, but these data suggest that the decrease in food intake always accompanying crowding only partially accounts for the changes observed in crowded rats. The finding in the second experiment that basal TSH levels were normal whereas the activation induced by acute stress was significantly lower in crowded than in control rats indicates that stress levels of TSH might be more sensitive to abnormalities caused by crowding than its basal levels. Serum TSH levels achieved after pituitary stimulation with TRH were similar in the two experimental groups. However, the percentage increase was higher in crowded rats which suggests enhanced responsiveness of crowded rats to TRH. In addition, somatostatin administration reduced TSH secretion in accordance with a previous report (Drouin, De Lean, Rainville, Lachance, & Labrie, 1976)--in control but not in crowded rats. This suggests either that thyrotropin-producing cells of crowded rats were less responsive to somatostatin or that crowding resulted in increased somatostatinergic activity and no effect of additional somatostatin on TSH secretion would be evident. In any case, it appears that impaired TSH secretion observed in crowded rats might be located at the hypo-
342
ARMARIO, GARCIA-MARQUEZ, AND JOLIN
thalamic rather than at the pituitary level. Although absolute GH levels were lower in crowded than in control rats after GH-RH administration, the percentage increase was greater in the former animals. Likewise, the percentage response to somatostatin was the same in the two groups, but absolute values were lower in crowded rats. Taken together, these data indicate that changes in GH in response to its hypothalamic regulatory factors could not play a major role in the inhibition of GH secretion observed in crowded rats. The specific contribution of the hypothalamus remains uncertain, but increased somatostatinergic activity might be involved since somatostatin appears to mediate the inhibition of GH caused by food deprivation (Tannenbaum, Epelbaum, Colle, Brazeau, & Martin, 1978). The contribution of the pituitary-adrenal axis to crowding-induced changes in GH and TSH secretion was not directly assessed but a major role appears unlikely because crowding did not alter adrenal weight (Armario et al., 1984a, 984b) and no differences in adrenocortical response to acute stress were observed between control and crowded rats. Impaired GH and TSH secretion observed in crowded animals might be the endocrine basis of the reduction of growth repeatedly observed in crowded animals. Although the reduction of food intake might partially account for the changes in GH and TSH secretion caused by crowding, its precise contribution to the observed changes remains to be established. REFERENCES Archer, J. (1970). Effects of population density on behaviour in rodents. In J. H. Crook (Ed.), Social behaviour in birds and mammals (pp. 169-210). London: Academic Press. Armario, A., & Castellanos, J. M. (1984). A simple procedure for direct corticosterone radioimmunoassay in the rat. Revista Espanola de Fisiologia, 40, 437-442. Armario, A., Ortiz, R., Balasch, J. (1984a). Effect of crowding on some physiological and behavioral variables in adult male rats. Physiology and Behavior, 32, 35-37. Armario, A., Castellanos, J. M., & Balasch, J. (1984b). Effect of crowding on emotional reactivity in male rats. Neuroendocrinology, 39, 330-333. Armario, A., Castellanos, J. M., & Balasch, J. (1984c). Effect of acute and chromc psychogenic stress on corticoadrenal and pituitary-thyroid hormones in male rats. Hormone Research, 20, 241-245. Armano, A., Castellanos, J. M., & Balasch, J. (1984d). Adaptation of anterior pituitary hormones to chronic noise in male rats. Behavioral and Neural Biology, 41, 71-76. Armario, A., Hidalgo, J., & Restrepo, C. (1985). Thyrotropin response to stress and other stimuli in peripuberal and adult male rats. Neuroendocrinology Letters, 6, 121-126. Armario, A., & Lopez-Calderon, A. (1986). Pituitary-gonadal function in adult male rats subjected to crowding. Endocrine Research. 12, 115-122. Armario, A., Montero, J. L., & Jolin, T. (1987). Chronic food restriction and the circadian rhythms of pituitary-adrenal hormones, growth hormone and thyroid-stimulating hormone. Annals of Nutrition and Metabolism, 31, 81-87. Calhoun, J. B. (1962). Population density and social pathology. Scientific American 206, 139-148. Christian, J. J., & Davis, D. E. (1964). Endocrines, behavior and population. Science, 146, 1550-1560.
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Christian, J. J., Lloyd, J. A., & Davis, D. E. (1965). The role of endocrines in the selfregulation of mammalian populations. Recent Progress in Hormone Research, 21,501578. Daniels-Severs, A., Goodwin, A., Keil, L. C., & Vernikos-Danelis, J. (1973). Effect of crowding and cold on the pituitary-adrenal system: Responsiveness to an acute stimulus during chronic stress. Pharmacology, 9, 348-356. Drouin, J., De Lean, A., Rainville, D., Lachance, R., & Labrie, F. (1976). Characteristics of the interaction between thyrotropin-releasing hormone and somatostatin for thyrotropin and prolactin release. Endocrinology, 98, 514-521. Gartner, K., Buttner, D., Dohler, K., Friedel, R., Lindena, J., & Trauschold, I. (1980). Stress response of rats to handling and experimental procedures. Laboratory Animals, 14, 267-274. Hefco, E., Krulich, L., Illner, P., & Larsen, P. R. (1975). Effect of acute exposure to cold on the activity of the hypothalamic-pituitary-thyroid system. Endocrinology, 97, 1185-1195. Kokka, N., Garcia, J. F., George, R., & Elliott, H. W. (1972). Growth hormone and ACTH secretion: Evidence for an inverse relationship in rats. Endocrinology, 90, 735743. McCarthy, J. L., Green, W., & Sohal, R. S. (1976). Crowding stress and adrenal mitochondrial 11-fl-hydroxylation in vitro. Proceedings of the Society for Experimental Biology and Medicine, 153, 528-531. Nowell, N. W. (1980). Adrenocortical function in relation to mammalian population densities and hierarchies. In I. C. Jones & I. W. Henderson (Eds.), General, comparative and clinical endocrinology of the adrenal cortex (Vol. 3, pp. 350-393). London: Academic Press. Ortiz, R., Armario, A., Castellanos, J. M., & Balasch, J. (1985). Post-weaning crowding induces corticoadrenal hyperreactivity in male mice. Physiology and Behavior, 34, 857-860. Ortiz-Caro, J., Gonzalez, C., & Jolin, T. (1984). Diurnal variations of plasma growth hormone, thyrotropin, thyroxine, and trliodothyronine in streptozotocin-diabetic and food-restricted rats. Endocrinology, 115, 2227-2232. Schalch, D. S., & Reichlin, S. (1966). Plasma growth hormone concentration in the rat determined by radioimmunoassay: Influence of sex, pregnancy, lactation, anesthesia, hypophysectomy and extrasellar pituitary transplants. Endocrinology 79, 275-290. Simpkins, J. W., Hudson, C. A., & Meites, J. (1978). Differential effects of stress on release of thyroid-stimulating hormone in young and old male rats. Proceedings of the Society for Experimental Biology and Medicine, 157, 144-147. Tannenbaum, G. S., Epelbaum, J., Colle, E., Brazeau, P., & Martin, J. B. (1978). Antiserum to somatostatin reverses starvation-induced inhibition of growth hormone but not insulin secretion. Endocrinology, 102, 1909-1914.