The Effect of Constant Illumination on the Circadian Rhythms of Plasma Thyrotropin and Corticosterone and on the Estrous Cycle in the Rat

Division of Endocrinology, Department of Medicine, University of Oregon Health Science Center, Portland, Oregon 97201 ABSTRACT. Young adult female rats were kept on a normal light/dark (LD) schedule (12 h light beginning at 0600 h) for 10 days. Eighteen rats were then kept in constant light (LL) for 110 days; 9 controls were maintained on LD. All rats were then kept on LD for a further 30 days. Vaginal smears were made 5 days per week. Plasma TSH and corticosterone were

plasma hormone concentration were present in individual LL rats. Within 1 week of the return to LD, normal estrous, TSH, and corticosterone rhythms had resumed. There was a significant negative correlation of plasma TSH and corticosterone (P < .02) during LD but not during LL (P > 0.1). From these and other data, we conclude that a free-running circadian rhythm of plasma corticosterone, and probably of TSH, occurs within a few days of transfer from LD to LL. These rhythms are independent of each other, indicating different pacemakers for each. Both "free-run" in LL before the estrous cycle is lost. In spite of prolonged free-running, entrainment of all 3 cycles resumes promptly when LD cues are restored. (Endocrinology 101: 1304, 1977)

measured on consecutive days at 1100—1130 h and

1745-1815 h. LD controls maintained normal rhythms of plasma TSH and corticosterone and of estrus throughout the experiment. After beginning LL, plasma corticosterone and TSH AM-PM differences in group means disappeared within one week, while constant estrus did not appear until approximately 4 weeks. Marked AM-PM differences in

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HYTHMIC fluctuations in the secretion of several hormones have been demonstrated in a variety of species. In the rat, a strong nyctohemeral rhythm of adrenal glucocorticoid secretion has been known for many years. Recently, several laboratories have shown that there is also a definite nyctohemeral rhythm for TSH secretion with a zenith in plasma TSH at mid-day (1-3). The rat is a species primarily active at night, whereas man is primarily active during the day. Thus, it is not surprising that the zenith of secretory activity of various hormones is approximately 180° out of phase between the two species. In both species there is a negative correlation between plasma TSH and glucocorticoid concentration. Since glucocorticoids will suppress TSH secretion under certain conditions, it Received January 17, 1977. Supported by grants from the National Institute of Arthritis, Metabolism and Digestive Diseases, NIH, Bethesda, Maryland. Presented in part at the 52nd meeting of the American Thyroid Association, September 1976.

has been postulated that the secretion of TSH might be under negative feedback control of glucocorticoids (4). However, recent evidence (3) suggests that this is unlikely since the TSH nyctohemeral rhythm is maintained in adrenalectomized animals which do not have a plasma corticosterone rhythm. Conversely, the nyctohemeral rhythm of TSH disappears but that of corticosterone is unaffected in hypothyroid rats. It is unknown whether there is one common pacemaker for both these rhythms or whether each is under the control of a separate pacemaker. They are dependent on neural regulation since hypothalamic deafferentation abolishes both (5). To probe further into this area, we have examined the effect of constant illumination on the reproductive, adrenal, and TSH rhythms. In the rat, constant light disrupts normal female reproductive cycles after a few weeks. The animals go into a state of persistent estrus in which ovulation does not occur, there is no LH surge, and the animals have a relatively constant secretion

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HITOSHI FUKUDA, MONTE A. GREER, LESLIE ROBERTS, SUSAN E. GREER, AND PATRICIA PANTON

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Materials and Methods Young adult 250 g female Sprague-Dawley rats were maintained three per cage on a normal light/dark schedule with 12 h light beginning at 0600 h. After 10 days, 18 animals were moved to an adjoining room and kept in constant light for 110 days. Nine controls were maintained on the normal light/dark schedule. At the end of the 110 days of constant illumination, all rats were maintained for a further 30 days on the standard light/dark cycle. All groups were fed Purina Laboratory Chow and tap water ad lib. Room temperatures were maintained at 24 ± 1 C. Vaginal smears were made 5 days a week in all animals except as indicated. Blood (0.6 ml) was removed percutaneously from the subclavian vein under light ether anesthesia within 2 min after removing the rats from their home cage. Plasma TSH and corticosterone concentrations are not altered during this procedure (10). One blood sample was obtained on each of 2 consecutive days at 1100-1130 h on one day and at 1745-1815 h on the other, at approximately weekly intervals. In our laboratory, these times correspond to the nyctohemeral TSH and corticosterone peak concentration in plasma, respectively (3). After the experimental animals were placed in constant light, they were divided into 3 groups of 6 each. Sampling on 2 consecutive days was rotated each week among the 3 groups to minimize blood loss. During the middle of the LL period, sometimes only one group was sampled per week. Each experimental rat thus had 2 consecutive AM and PM blood samples obtained at least every 3 weeks and no more often than weekly. During a 2-month period in the center of the experiment, blood was not taken from the control animals which were maintained under normal lighting conditions. Blood samples were immediately placed in ice and centrifuged at 4 C within 30 min. Plasma was separated and frozen at - 2 0 C until assayed. TSH was measured by radioimmunoassay employing the rat TSH kit supplied by the

Rat Pituitary Hormone Distribution Program of the NIAMDD. The stated potency of 0.22 U/mg of the supplied purified rat TSH was used in making calculations of plasma TSH concentration. Plasma corticosterone was measured by a semi-automated microfluorescence method (11). All experimental samples were measured in the same assay at the end of the experiment to avoid interassay variation. Statistical analysis of AM-PM differences was made with the paired t test. The relation between plasma concentration of TSH and corticosterone was evaluated by calculating the correlation coefficient.

Results

The pattern of estrous cycles is shown in Fig. 1. Normal estrous cycles were maintained in all except one of the control rats maintained on a normal lighting cycle. The exceptional animal went into spontaneous persistent estrus during the 17th week. The experimental rats had normal cycles before being placed in constant light. The majority of animals exhibited prolonged periods of estrus, sometimes interspersed with prolonged periods of diestrus, after the 4th week of constant light. Four animals showed some evidence of prolonged estrus before the 4th week. Within 1 week after returning to the normal light-dark cycle, all but one of the surviving rats had a resumption of relatively normal estrous cycles. The effect of constant light on plasma corticosterone fluctuations is shown in Fig. 2. During the initial control light/dark period, a significant PM rise in plasma corticosterone was shown by all animals. After 1 week in constant light, there was no significant circadian rhythm in group mean plasma corticosterone (paired t). However, pronounced AM-PM fluctuations occurred in individual animals. This same pattern persisted throughout the exposure to constant light as shown by the illustrated examples at 2, 4, 8, and 12 weeks. A normal nyctohemeral plasma corticosterone rhythm was restored within 1 week after returning the rats to the normal light/dark environment.

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of estrogen which maintains an estrous vaginal smear. Some studies have also been done on the effect of constant light on adrenal rhythms, but the data are not in complete accord (6-9). In general, there is agreement that the normal "group mean" circadian adrenal rhythm is abolished.

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FIG. 1. Diagrammatic representation of vaginal smears. Each rat is shown on an individual horizontal line which is solid during days with a predominantly proestrous or estrous smear. The period of constant light for the experimental rats is enclosed in heavy black lines. Smears were not made in the control rats between 3 and 13 weeks. Animal death is indicated by +.

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The effect of constant light on plasma marked temporalfluctuationsin plasma TSH TSH fluctuations is shown in Fig. 3 for the in individual rats in constant light. This same animals. The group mean plasma TSH suggests that rhythmic fluctuations were had lost its circadian rhythm by the end of persisting with plasma TSH as well as with the first week of constant light, but a corticosterone, although there could now be significant rhythm of group means reap- no entrainment to normal light/dark cues. peared during the first week of returning Although the nyctohemeral differences the animals to a normal light/dark environ- shown in Fig. 3 at 8 weeks of constant ment. As with plasma corticosterone, al- light were statistically significant by the though there was no significant difference paired t test, it is probable that this was between AM-PM group means, there were due to coincidence. The differences were

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FIG. 2. AM-PM differences in plasma corticosterone in the experimental group. The lighting conditions are indicated in the upper left of each panel and the number of weeks on that particular schedule is in the upper right. Values for individual rats are shown with lines and group means with bars. Statistical analysis was by paired t. LL is constant light.

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not significant at other sampling periods. Discussion The controls maintained in the normal light/ Our data indicate that the normal temdark environment throughout the period of poral rhythms for plasma corticosterone and observation exhibited a normal nyctoTSH are lost within one week of placing hemeral rhythm of plasma TSH and coranimals in a constant light environment. ticosterone at each sampling period. These rhythms are restored within one week Correlation of plasma TSH and corof reinstituting normal light/dark cycles. ticosterone concentrations is shown in Table Group data, based on samples from different 1. There was a significant negative corrats at each time point, have previously demrelation of plasma corticosterone and TSH onstrated a loss of normal adrenal rhythmicin the control animals maintained throughity in rats in constant light (6-9). However, out on a normal light/dark cycle and in the experimental animals during the time they group data cannot distinguish between loss were on a normal light/dark cycle. How- of synchronization of rhythms and loss of ever, there was no significant correlation rhythmicity. In our studies, samples were of plasma TSH and corticosterone during obtained throughout in the same individual rats. Marked temporalfluctuationsin plasma the constant light period. concentration of corticosterone and TSH were observed in the majority of animals, TABLE 1. Correlation of plasma TSH and corticosterone indicating that a circadian rhythmicity might Group be maintained but that this was free-running, since it could not be entrained to Lighting Experimental Control light/dark cues which did not exist. The LD r = - 0.238 r = - 0.212 animals were fed and watered and their P < 0.02 P < 0.05 cages cleaned at the same time in the conn = 88 n = 114 stant light room as in the adjoining animal rooms with normal light/dark cycles. ApparLL r = 0.058 P>0.1 ently, such physical activity was insufficient n = 237 to induce a normal temporal entrainment of the measured hormonal rhythms. LD, light-dark cycle; LL, constant light.

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FIG. 3. AM-PM differences in plasma TSH for the same samples shown in Fig. 2.

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cocorticoids (4). However, there is evidence against this hypothesis. The nyctohemeral rhythm of plasma TSH concentration persists in adrenalectomized rats where there is no rhythm of plasma corticosterone (3). TSH rhythmicity is lost under conditions where there is a high rate of TSH secretion, such as in severe iodine deficiency or chronic antithyroid treatment while normal nyctohemeral plasma corticosterone rhythmicity is maintained (3). This suggests that there may be separate pacemakers for adrenal and thyroid rhythms. A similar conclusion has been reached from data obtained in man (15). Our present data are in accord with there being independent pacemakers controlling ACTH and TSH secretion. A significant negative correlation was seen between plasma corticosterone and TSH during normal light/dark periods, but this correlation was lost in constant light even though in individual rats marked AM-PM fluctuations occurred in plasma concentration of both hormones and a free-running circadian rhythm for plasma corticosterone was demonstrated in the majority of rats in constant light that were adequately examined (12). Although only plasma corticosterone was measured in these studies, we assume that the fluctuations in plasma corticosterone are due to corresponding fluctuations in plasma ACTH secretion. Unfortunately, ACTH could not be measured directly because of the marked rise that would occur in plasma ACTH due solely to the stress of the ether anesthesia. Plasma corticosterone is unaffected by ether anesthesia of the duration employed in these studies and returns to basal levels within 90 min after each of repeated stresses similar to those of the present study (16). Ether anesthesia has also been shown not to affect plasma TSH concentration under our conditions (10). The loss of normal estrous cycles and the appearance of persistent vaginal estrus did not occur in the majority of animals placed in constant light until the fourth week. Normal temporal plasma corticosterone and

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Our data were not obtained at sufficiently frequent intervals to establish whether the observed fluctuations in hormone concentration in the constant light rats were random or were following a free-running circadian rhythm. This was examined in more detail in a separate study using these same animals (12). Nine rats which demonstrated persistent estrus after 15 weeks in constant light and 9 control rats which had been maintained on the normal light/dark cycle had blood samples taken from a tail vein at 4-h intervals over a 44-h period. Eight of the 9 control rats demonstrated a 24-h periodicity in plasma corticosterone concentration with peaks entrained to the onset of dark. Five of the 9 constant light rats demonstrated an unentrained circadian periodicity of approximately 24 h. The other 4 constant light rats had apparently arhythmic fluctuations. Unfortunately, since only 0.3 ml blood samples were taken to minimize blood loss over this 2-day period, we had insufficient plasma to determine whether a similar circadian rhythmicity in plasma TSH could be demonstrated. The individual patterns of the constant light rats in our study (12) differed from the arhythmic fluctuations in plasma corticosterone found by Krieger (9) in 80-dayold rats reared from birth in constant light. The discrepancy between these findings suggests that the age at which a rat is placed in constant light may be critical. A circadian periodicity in corticosterone does not appear until approximately 30 days of age (13) and a demonstrable retinohypothalamic tract appears at about the same time (14). Light-dark cycles in early life may be necessary to establish a circadian rhythmicity in neural control of pituitary secretion. Nyctohemeral rhythms for plasma ACTH and corticosterone and for TSH have been observed in both man and the rat. Since glucocorticoids have at least a transient effect to depress plasma TSH secretion, it has been suggested that TSH secretion may be regulated by the fluctuations in plasma glu-

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7. 8. 9. 10. 11. 12. 13. 14.

1. Leppaluoto, J., T. Ranta, and J. Toumisto, Ada Physiol Scand 90: 699, 1974. 2. Azukizawa, M., H. V. Roohk, J. J. DiStefano III, and J. M. Hershman, Clin Res 23: 92A, 1975 (Abstract). 3. Fukuda, H., M. A. Greer, L. Roberts, C. F. Allen,

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V. Critchlow, and M. Wilson, Endocrinology 97: 1424, 1975. Nicoloff, J. T., D. A. Fisher, and H. D. Appelman, Jr.,7 Clin Invest 49: 1922, 1970. Fukuda, H., and M. A. Greer, Endocrinology 97: 749, 1975. Critchlow, V., R. A. Liebelt, M. Bar-Sela, W. Mountcastle, and H. S. Lipscomb, Am J Physiol 205: 807, 1963. Scheving, L. E., and J. E. Pauly, Am J Physiol 210: 1112, 1966. Cheifetz, P., N. Gaffud, and J. F. Dingman, Endocrinology 82: 1117, 1968. Krieger, D. T., Endocrinology 93: 1077, 1973. Fukuda, H., N. Yasuda, and M. A. Greer, Endocrinology 97: 924, 1975. Greer, M. A., C. F. Allen, P. Panton, and J. P. Allen, Endocrinology 96: 718, 1975. Wilson, M. M., and M. A. Greer, Proc Soc Exp Biol Med 154: 69, 1977. Allen, C , and J. W. Kendall, Endocrinology 80: 926, 1967. Campbell, C. B. G., and J. A. Ramaley, Endocrinology 94: 1201, 1974. Van Cauter, E., R. Leclercq, L. VanHaelst, and J. Golstein, ; Clin Endocrinol Metab 39: 645, 1974. Cook, D. M., J. P. Allen, M. A. Greer, and C. F. Allen, Endocr Res Commun 1: 347, 1974.

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TSH rhythms were lost much earlier. Although we do not have direct data from these animals, we assume that the persistent vaginal estrus was due to loss of a normal LH surge resulting in failure of ovulation and normal luteinization. The loss of correlation of plasma concentrations of corticosterone and TSH and the relatively delayed onset of persistent estrus compared to the onset of loss of normal temporal rhythmicity of the other 2 hormones suggest that separate pacemakers are involved in the control of each of the organ systems through regulation of secretion of ACTH, TSH, and gonadotropins, respectively.

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The effect of constant illumination on the circadian rhythms of plasma thyrotropin and corticosterone and on the estrous cycle in the rat.

The Effect of Constant Illumination on the Circadian Rhythms of Plasma Thyrotropin and Corticosterone and on the Estrous Cycle in the Rat Division of...
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