Plasma TSH Levels, by Radioimmunoassay, During the Estrous Cycle of the Rat J. DAVID KIEFFER, HEIDI MOVER, AND FARAHE MALOOF Thyroid Unit and Departments of Medicine at the Massachusetts General Hospital and Harvard MedicalSchool, Boston, Massachusetts 02114 ABSTRACT. Between 1000 and 1200 hr on any given morning, female rats displaying regular 4-day estrous cycles were bled by decapitation. Plasma TSH concentrations were determined by homologous radioimmunoassay (RIA), in terms of NIAMDD-Rat-TSH-I-1 standards. Mean plasma TSH concentrations ± SE were 1.2 ± 0.1, 1.5 ± 0.2, 2.3 ± 0.6, and 1.1 ± 0.1 ng/ml, for rats killed at proestrus, estrus, diestrus-1, and diestrus-2, respec-
tively. Statistical analysis revealed no significant differences among the TSH levels, except for the elevation at diestrus-1, which is of borderline significance at the 0.05 probability level. Our findings, based on RIA, are in contrast to previous reports, based on bioassays, of an elevation of plasma TSH at estrus. (Endocrinology 96: 535, 1975)
REVIOUS studies of circulating levels of TSH during the estrous cycle of the rat have indicated that the serum TSH concentration at estrus is greater than the concentration on any other day of the cycle (1,2). These findings, as well as the report that the pituitary content of TSH, on the morning of estrus, is lower than the content at 1200 or 1900 hr on proestrus (3), supported the hypothesis (4) that there may be a discharge of TSH from the pituitary during late proestrus. The TSH values reported in these previous studies (1-3) were determined by bioassay, utilizing either the stasis tadpole method (5) or the McKenzie method (6). Our report presents plasma TSH values during the rat estrous cycle, as measured by a sensitive and specific homologous radioimmunoassay (RIA). (7).
All rats killed at estrus had a full complement (13.6 ± 0.6) of fresh tubal ova. The uteri of all rats killed at proestrus showed moderate to marked distention with fluid ("ballooning"); uteri were not distended on any other day of the cycle.
P
Materials and Methods Animals. Adult female rats (224 ± 2 g) of the Charles River CD strain were housed with lights on only between 0700 and 1900 hr. Tap water and Purina Lab Chow were always available. Vaginal smears were taken daily in the morning, including the morning of autopsy for any given animal. Rats were used only after they had displayed at least two consecutive 4-day estrous cycles immediately before the cycle in which they were autopsied. One to 1.5 hr after vaginal smears were taken, rats selected as cyclic were decapitated without anesthesia, between 1000 and 1200 hr. The blood was collected into heparinized tubes. After separation, the plasmas were stored at -20 C until assayed. Received May 10, 1974.
Assay. Plasma TSH was determined by a homologous rat RIA, using the NIAMDD materials according to methods developed in this laboratory (7). Additional experience with these methods has revealed coefficients of variation between assays ranging from 10.0% to 19.1%, for four plasmas measured in each of five assays. Coefficients of variation within assays have ranged from 0.1% to 4.9%, for 25 plasmas measured in duplicate; these were selected randomly, five from each offiveassays. A somewhat higher range for the intra.-assay coefficient—2.6% to 10.1%—was
found for five samples assessed in quadruplicate in one assay. Plasma TSH levels are expressed in terms of NIAMDD-Rat-TSH-I-1, which has a biological potency of about 35 IU/mg in the McKenzie assay, according to information supplied by the NIAMDD. All of the TSH values reported in the present paper were determined in the same assay; and all were well above the minimum detectable plasma TSH level, which was 0.2 ng/ml. Statistics. Data were assessed by analysis of variance and the Studentized range test.
Results The data are summarized in Table 1. Statistical analysis shows that the differences in the plasma TSH concentrations of proestrous, estrous, and diestrous-2 rats do not approach significance at the 0.05 level. The significance of the slight elevation of plasma TSH observed at
535
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NOTES AND COMMENTS
536
TABLE 1. Plasma TSH concentrations during the estrous cycle of the rat Stage of estrous cycle
Number of rats
Plasma TSH ng/ml (mean ± SE)
Proestrus Estrus Diestrus-1 Diestrus-2
13 11 11 12
1.2 ± 0.1 1.5 ± 0.2 2.3 ± 0.6 1.1 ± 0.1
The only significant difference at the 0.05 level is between Diestrus-1 and Diestrus-2. See text. diestrus-1 is borderline. The difference between diestrus-1 and diestrus-2 barely attains significance (0.01 < p < 0.05), while that between diestrus-1 and proestrus barely fails of significance at the 0.05 level. The difference between diestrus-1 and estrus is clearly not significant (p > 0.05). Discussion Brown-Grant (4) has hypothesized that the increased accumulation of iodide by the thyroid gland at estrus (2,4) is due to a nearly concomitant elevation of plasma TSH. A further hypothesis (4) was that such an elevation of plasma TSH might be due to the possible effectiveness of luteinizing hormone-releasing hormone (LRH) in stimulating the secretion of TSH as well as that of LH. Our findings indicate that plasma TSH is not elevated on the morning of estrus. Moreover, as our study neared completion, a report (8) appeared which provides important complementary evidence. The plasma TSH of rats was measured by homologous RIA at six times, from 1230 to 1700 hr on the afternoon of proestrus; no significant changes were found. This result reveals no surge of plasma TSH concomitant with the ovulatory surge of LH on the afternoon of proestrus. Current information does not permit a definitive interpretation of the apparent differences between the earlier determinations of serum TSH during the estrous cycle of the rat (1,2) and the more recent data reported in the present paper and that by Blake (8). At least the following three possibilities merit consideration: 1) The discordant findings may be due to intrinsic differences between the bioassay methods used to measure TSH in the earlier studies and the homologous RIA methods
Endo • 1975 Vol 96 • No 2
employed in the more recent work. 2) Differences in the procedures used for maintaining rats, for assigning them to various stages of the estrous cycle, and for collecting blood, may have contributed to the dissimilarity of the findings. For instance, in the present study and that of Blake (8), the light-dark schedule was controlled, and the times at which blood was taken were specified. Neither of these matters is discussed in the earlier reports (1,2). Additionally, the varying procedures probably involved different degrees of nonspecific stress, which has been reported to lower the plasma TSH of male rats (9). Thus, such factors as the particular techniques used for obtaining vaginal smears, and the length of time between taking smears and collecting blood, might possibly have affected TSH levels. However, it seems unlikely that stress is of major importance in the present context, since one of the investigations (2) which reported an elevated serum TSH level at estrus included the use of ether anesthesia immediately before taking blood. Such use of ether was reported (9) to be more "stressful" — that is, to result in lower plasma TSH levels— than decapitation without anesthesia, as in the present study, in which no elevation of TSH at estrus was found. 3) There may be differences among various strains of rats with respect to circulating TSH levels during the estrous cycle. Further research, including determination of TSH levels at times in the cycle not yet examined, and an analysis of the same sets of plasmas by both bioassay and RIA, will be needed to resolve the uncertainties discussed above. Nevertheless, the convergent evidence of our results and those of Blake (8) indicates that re-examination of hypotheses invoking elevated plasma TSH in proestrus or estrus is in order.
Acknowledgments This work was supported by USPHS Grant AM16791-03A1. We thank Dr. Albert F. Parlow and the NIAMDD Rat Pituitary Hormone Distribution Program for rat TSH and anti-TSH. The expert secretarial assistance of Mrs. Ruth DiBlasi is gratefully acknowledged.
References 1. Soliman, F. A., and H. M. Badawi, Nature 177: 235, 1956.
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NOTES AND COMMENTS 2. Boccabella, A. V., and E. A. Alger, Endocrinology 81: 121, 1967. 3. Newcomer, W. S., and K. Brown-Grant, / Endocrinol 50: 699, 1971. 4. Brown-Grant, K.J Physiol (Lond) 184: 402, 1966. 5. D'Angelo, S. A., and A. S. Gordon, Endocrinology 46: 39, 1950.
537
6. McKenzie, J. M., Endocrinology 63: 372, 1958. 7. Kieffer, J. D., B. D. Weintraub, W. Baigelman, S. Leeman, and F. Maloof, Acta Endocrinol (Khh) 76: 495, 1974. 8. Blake, C. A., Endocrinology 94: 503, 1974. 9. Ducommun, P., E. Sakiz, and R. Guillemin, Proc Soc Exp Biol Med 121: 921, 1966.
1975 Laurentian Hormone Conference Mt. Tremblant Lodge, Quebec, Canada, August 24-29, 1975 SPEAKER
TITLE (Tentative)
Gordon M. Tomkins
Genetic Studies on Hormone Action
Joseph Chayen
The Cytochemical Bioassay of Hormones
William L. Duax
Crystal Structures of Steroids: Molecular Conformation and Biological Function
Wilfrid F. White and John F. Wilber
The Gonadotropin Releasing Hormone and Thyrotropin Releasing Hormone. Distribution and Effects in Central Nervous System
Selna L. Kaplan and
Ontogenesis of Human Fetal Hormones
Melvin G. Grumbach William D. Odell
Etiology of Sexual Maturation
Judith Vaitukaitis
The Gonadotropin Subunits: Clinical Investigations
Peter H. Bennett and Max Miller
Epidemiological Studies of Diabetes in Pima Indians
Jacques Genest
The Adrenal Cortex and Essential Hypertension
Bryan Hudson
Clinical and Basic Study of Male Reproductive
Endocrinology Anthony R. Means Jack H. Oppenheimer
Follicle Stimulating Hormone; The Sertoli Cell and Spermatogenesis Studies of Mechanism of Action of the Thyroid Hormone Symposium on (3 Receptors
Jerry D. Gardner and Gerald D. Aurbach
Receptor Function and Ion Transport in Turkey Erythrocytes
Robert Lefkowitz
Receptor Regulation of Adenylate Cyclase
Alfred G. Gilman
Agonist Interaction in Hybrid Cells
Henry Bourne and Kenneth Melman
Cyclic AMP Responsiveness in Mutant Cells
Investigators interested in attending this meeting may request application forms from the Conference Office, 45 Shattuck Street, Boston, Massachusetts, 02115. A limited number of travel fellowships will be available to help young investigators who have a special interest in the program and who could not otherwise attend the Conference. Applications are due April 30, 1975.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 19:06 For personal use only. No other uses without permission. . All rights reserved.
tively. Statistical analysis revealed no significant differences among the TSH levels, except for the elevation at diestrus-1, which is of borderline significance at the 0.05 probability level. Our findings, based on RIA, are in contrast to previous reports, based on bioassays, of an elevation of plasma TSH at estrus. (Endocrinology 96: 535, 1975)
REVIOUS studies of circulating levels of TSH during the estrous cycle of the rat have indicated that the serum TSH concentration at estrus is greater than the concentration on any other day of the cycle (1,2). These findings, as well as the report that the pituitary content of TSH, on the morning of estrus, is lower than the content at 1200 or 1900 hr on proestrus (3), supported the hypothesis (4) that there may be a discharge of TSH from the pituitary during late proestrus. The TSH values reported in these previous studies (1-3) were determined by bioassay, utilizing either the stasis tadpole method (5) or the McKenzie method (6). Our report presents plasma TSH values during the rat estrous cycle, as measured by a sensitive and specific homologous radioimmunoassay (RIA). (7).
All rats killed at estrus had a full complement (13.6 ± 0.6) of fresh tubal ova. The uteri of all rats killed at proestrus showed moderate to marked distention with fluid ("ballooning"); uteri were not distended on any other day of the cycle.
P
Materials and Methods Animals. Adult female rats (224 ± 2 g) of the Charles River CD strain were housed with lights on only between 0700 and 1900 hr. Tap water and Purina Lab Chow were always available. Vaginal smears were taken daily in the morning, including the morning of autopsy for any given animal. Rats were used only after they had displayed at least two consecutive 4-day estrous cycles immediately before the cycle in which they were autopsied. One to 1.5 hr after vaginal smears were taken, rats selected as cyclic were decapitated without anesthesia, between 1000 and 1200 hr. The blood was collected into heparinized tubes. After separation, the plasmas were stored at -20 C until assayed. Received May 10, 1974.
Assay. Plasma TSH was determined by a homologous rat RIA, using the NIAMDD materials according to methods developed in this laboratory (7). Additional experience with these methods has revealed coefficients of variation between assays ranging from 10.0% to 19.1%, for four plasmas measured in each of five assays. Coefficients of variation within assays have ranged from 0.1% to 4.9%, for 25 plasmas measured in duplicate; these were selected randomly, five from each offiveassays. A somewhat higher range for the intra.-assay coefficient—2.6% to 10.1%—was
found for five samples assessed in quadruplicate in one assay. Plasma TSH levels are expressed in terms of NIAMDD-Rat-TSH-I-1, which has a biological potency of about 35 IU/mg in the McKenzie assay, according to information supplied by the NIAMDD. All of the TSH values reported in the present paper were determined in the same assay; and all were well above the minimum detectable plasma TSH level, which was 0.2 ng/ml. Statistics. Data were assessed by analysis of variance and the Studentized range test.
Results The data are summarized in Table 1. Statistical analysis shows that the differences in the plasma TSH concentrations of proestrous, estrous, and diestrous-2 rats do not approach significance at the 0.05 level. The significance of the slight elevation of plasma TSH observed at
535
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NOTES AND COMMENTS
536
TABLE 1. Plasma TSH concentrations during the estrous cycle of the rat Stage of estrous cycle
Number of rats
Plasma TSH ng/ml (mean ± SE)
Proestrus Estrus Diestrus-1 Diestrus-2
13 11 11 12
1.2 ± 0.1 1.5 ± 0.2 2.3 ± 0.6 1.1 ± 0.1
The only significant difference at the 0.05 level is between Diestrus-1 and Diestrus-2. See text. diestrus-1 is borderline. The difference between diestrus-1 and diestrus-2 barely attains significance (0.01 < p < 0.05), while that between diestrus-1 and proestrus barely fails of significance at the 0.05 level. The difference between diestrus-1 and estrus is clearly not significant (p > 0.05). Discussion Brown-Grant (4) has hypothesized that the increased accumulation of iodide by the thyroid gland at estrus (2,4) is due to a nearly concomitant elevation of plasma TSH. A further hypothesis (4) was that such an elevation of plasma TSH might be due to the possible effectiveness of luteinizing hormone-releasing hormone (LRH) in stimulating the secretion of TSH as well as that of LH. Our findings indicate that plasma TSH is not elevated on the morning of estrus. Moreover, as our study neared completion, a report (8) appeared which provides important complementary evidence. The plasma TSH of rats was measured by homologous RIA at six times, from 1230 to 1700 hr on the afternoon of proestrus; no significant changes were found. This result reveals no surge of plasma TSH concomitant with the ovulatory surge of LH on the afternoon of proestrus. Current information does not permit a definitive interpretation of the apparent differences between the earlier determinations of serum TSH during the estrous cycle of the rat (1,2) and the more recent data reported in the present paper and that by Blake (8). At least the following three possibilities merit consideration: 1) The discordant findings may be due to intrinsic differences between the bioassay methods used to measure TSH in the earlier studies and the homologous RIA methods
Endo • 1975 Vol 96 • No 2
employed in the more recent work. 2) Differences in the procedures used for maintaining rats, for assigning them to various stages of the estrous cycle, and for collecting blood, may have contributed to the dissimilarity of the findings. For instance, in the present study and that of Blake (8), the light-dark schedule was controlled, and the times at which blood was taken were specified. Neither of these matters is discussed in the earlier reports (1,2). Additionally, the varying procedures probably involved different degrees of nonspecific stress, which has been reported to lower the plasma TSH of male rats (9). Thus, such factors as the particular techniques used for obtaining vaginal smears, and the length of time between taking smears and collecting blood, might possibly have affected TSH levels. However, it seems unlikely that stress is of major importance in the present context, since one of the investigations (2) which reported an elevated serum TSH level at estrus included the use of ether anesthesia immediately before taking blood. Such use of ether was reported (9) to be more "stressful" — that is, to result in lower plasma TSH levels— than decapitation without anesthesia, as in the present study, in which no elevation of TSH at estrus was found. 3) There may be differences among various strains of rats with respect to circulating TSH levels during the estrous cycle. Further research, including determination of TSH levels at times in the cycle not yet examined, and an analysis of the same sets of plasmas by both bioassay and RIA, will be needed to resolve the uncertainties discussed above. Nevertheless, the convergent evidence of our results and those of Blake (8) indicates that re-examination of hypotheses invoking elevated plasma TSH in proestrus or estrus is in order.
Acknowledgments This work was supported by USPHS Grant AM16791-03A1. We thank Dr. Albert F. Parlow and the NIAMDD Rat Pituitary Hormone Distribution Program for rat TSH and anti-TSH. The expert secretarial assistance of Mrs. Ruth DiBlasi is gratefully acknowledged.
References 1. Soliman, F. A., and H. M. Badawi, Nature 177: 235, 1956.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 19:06 For personal use only. No other uses without permission. . All rights reserved.
NOTES AND COMMENTS 2. Boccabella, A. V., and E. A. Alger, Endocrinology 81: 121, 1967. 3. Newcomer, W. S., and K. Brown-Grant, / Endocrinol 50: 699, 1971. 4. Brown-Grant, K.J Physiol (Lond) 184: 402, 1966. 5. D'Angelo, S. A., and A. S. Gordon, Endocrinology 46: 39, 1950.
537
6. McKenzie, J. M., Endocrinology 63: 372, 1958. 7. Kieffer, J. D., B. D. Weintraub, W. Baigelman, S. Leeman, and F. Maloof, Acta Endocrinol (Khh) 76: 495, 1974. 8. Blake, C. A., Endocrinology 94: 503, 1974. 9. Ducommun, P., E. Sakiz, and R. Guillemin, Proc Soc Exp Biol Med 121: 921, 1966.
1975 Laurentian Hormone Conference Mt. Tremblant Lodge, Quebec, Canada, August 24-29, 1975 SPEAKER
TITLE (Tentative)
Gordon M. Tomkins
Genetic Studies on Hormone Action
Joseph Chayen
The Cytochemical Bioassay of Hormones
William L. Duax
Crystal Structures of Steroids: Molecular Conformation and Biological Function
Wilfrid F. White and John F. Wilber
The Gonadotropin Releasing Hormone and Thyrotropin Releasing Hormone. Distribution and Effects in Central Nervous System
Selna L. Kaplan and
Ontogenesis of Human Fetal Hormones
Melvin G. Grumbach William D. Odell
Etiology of Sexual Maturation
Judith Vaitukaitis
The Gonadotropin Subunits: Clinical Investigations
Peter H. Bennett and Max Miller
Epidemiological Studies of Diabetes in Pima Indians
Jacques Genest
The Adrenal Cortex and Essential Hypertension
Bryan Hudson
Clinical and Basic Study of Male Reproductive
Endocrinology Anthony R. Means Jack H. Oppenheimer
Follicle Stimulating Hormone; The Sertoli Cell and Spermatogenesis Studies of Mechanism of Action of the Thyroid Hormone Symposium on (3 Receptors
Jerry D. Gardner and Gerald D. Aurbach
Receptor Function and Ion Transport in Turkey Erythrocytes
Robert Lefkowitz
Receptor Regulation of Adenylate Cyclase
Alfred G. Gilman
Agonist Interaction in Hybrid Cells
Henry Bourne and Kenneth Melman
Cyclic AMP Responsiveness in Mutant Cells
Investigators interested in attending this meeting may request application forms from the Conference Office, 45 Shattuck Street, Boston, Massachusetts, 02115. A limited number of travel fellowships will be available to help young investigators who have a special interest in the program and who could not otherwise attend the Conference. Applications are due April 30, 1975.
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