Cyclic Variations in the Increased Responsiveness of the Pituitary to Luteinizing Hormone-Releasing Hormone (LHRH) Induced by LHRH A. CASTRO-VAZQUEZ1 AND S. M. M C C A N N 2 Department of Physiology, The University of Texas Health Science Center at Dallas, Southwestern Medical School, Dallas, Texas 75235 Only small increases in FSH followed these injections and there was no increase in responsiveness to the second injection in any of the groups. It is postulated that rising estrogen titers can bring about a priming action of LHRH which enhances the response of the gland to a second injection on proestrus. The priming action of LHRH may magnify the response of the pituitary to endogenous decapeptide release to further increase the release of LH on the afternoon of proestrus. (Endocrinology 97: 13, 1975)
ABSTRACT. Following a single iv injection of 50 ng of LH-releasing hormone (LHRH), a small increase in plasma LH titers was observed and this response was greater on proestrus and estrus than in other stages of the cycle. When a second injection of a similar dose of the neurohormone was given one hour later, the response to the second injection was markedly enhanced on proestrus but only slightly increased at other stages of the cycle. The response to LHRH in androgenized females was similar to that observed on proestrus, and, as on proestrus, an enhanced response to a second injection occurred.
P
REVIOUS work from this laboratory (1-3) and from those of others (4-7) has demonstrated cyclic changes in the response of the pituitary to a single injection of LHRH. The release of both FSH and LH increased during proestrus to reach a maximum during the preovulatory release of LH and then declined to a minimum during diestrus. The time course of the increased responsiveness followed, with a lag period, the time of heightened estrogen release which begins on diestrus II and reaches a maximum in early proestrus (8,9). The association of maximal responsiveness to LHRH with the preovulatory discharge of LH thought to be brought about by increased release of LHRH, plus the fact that both the preovulatory discharge and the heightened responsiveness could be blocked by administration of Nembutal at 1 PM on proestrus, suggested that the Received January 23, 1975. 1 Postdoctoral Fellow of the World Health Organization, on leave from ILAFIR, Universidad del Salvador, San Miguel, Argentina. 2 Supported by grants from NIH (AM 10073 and HD 05151), the Ford Foundation and the Texas Population Research Institute.
release of endogenous LHRH might in some manner enhance the pituitary's responsiveness to the subsequently released neurohormone (4,7). Previous attempts to demonstrate an ability of LHRH to increase responsiveness to subsequent injections of LHRH were unsuccessful in ovariectomized, estrogen-primed animals (Libertun, unpublished data); however, we were able to show that median eminence lesions blocked the increased responsiveness to LHRH which occurred during the LH release induced by progesterone in the ovariectomized, estrogen-primed rat (10). In the meantime, Aiyer et at. (11) reported in an abstract that injection of a priming dose of LHRH at 1 PM on proestrus enhanced the responsiveness of the gland to a second injection of LHRH given 1 h later. In the present experiments, the potentiation of the response to LHRH produced by a previous injection of the neurohormone has been studied during various stages of the estrous cycle and in neonatally androgenized females. A pronounced priming action of LHRH was demonstrated on proestrus and in androgenized females but not at other stages of the estrous cycle. 13
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 06:04 For personal use only. No other uses without permission. . All rights reserved.
14
CASTRO-VAZQUEZ AND McCANN
Materials and Methods Animals. Adult female rats of the SpragueDawley strain (Simonsen Laboratories, Gilroy, California) were kept under conditions of controlled light (lights on between 5:00 and 19:00 h) and temperature (21 C) and given free access to Purina rat chow and water. After arrival they were allowed to become accustomed to the new environment for at least one week. After this period, vaginal smears were taken at least 6 days a week and rats displaying at least two consecutive cycles of 4 or 5 days duration were used. Only 7 out of 41 animals had 5-day cycles. Since the results obtained with these rats were similar to those obtained with rats with 4-day cycles, the results were pooled for presentation. Results are reported of only those animals whose uteri fulfilled the criteria of Long and Evans (12) for the expected stage of the cycle, i.e., 1) a ballooned uterus at proestrus, 2) either a slightly distended or an empty uterus at estrus, and 3) a slender and empty uterus at diestrus. Androgenized rats were obtained by injecting females on the fourth day of life (birth = day 0) with 50 fig of testosterone propionate sc (13). They were used when adult if they exhibited at least 6 consecutive days of vaginal cornification. At autopsy, no corpora lutea were seen macroscopically in the ovaries. Injections and blood withdrawal. Synthetic LHRH (50 ng in 0.2 ml of 0.9% NaCl)3 was injected into the external jugular vein which was also used for obtaining heparinized blood samples (0.8 ml) while the animals were anesthetized with tribromoethanol (14). An initial dose of 25 mg/100 g body weight of tribromoethanol was injected ip between 1300-1330 h and additional doses were administered thereafter when necessary. Blood samples were obtained just prior to (3-5 min after injection of tribromoethanol) and at 20 and 60 min postinjection of LHRH. Then the second injection of LHRH was given and additional blood samples were removed at 20 and 60 min following this injection. Plasma was separated by centrifugation at low speed at 4 C and stored frozen until the day of 3
The synthetic LHRH was generously provided by Dr. R. Deghenghi, Ayerst, Ltd., Montreal.
Endo • 1975 Vol 97 • No 1
assay. LH was measured by the ovine-ovine radioimmunoassay of Niswender et al. (15).4 RP-1 rat pituitary LH was used as reference standard and results were expressed in terms of NIH-LH-S1 reference preparation. FSH was measured using the kits supplied by NIAMDD.4 The RP-1 rat pituitary FSH reference preparation was used. The paired t test was used to evaluate the significance of increases or decreases in plasma LH which took place in each group of rats. Comparisons between values in two groups were made with Student's t test.
Results
Plasma luteinizing hormone concentration before injection of LHRH. Plasma LH levels at 13:00 h showed minor fluctuations during the cycle, being minimal during diestrus II and rising slightly but significantly on proestrus (P < 0.001) and diestrus I (P < 0.01) (Figs. 1-5). Androgenized females showed LH levels at 13:00 h (0.9 ± O.I5 ng/ml) that were significantly lower (P < 0.05) than those on proestrus (1.3 ± 0.25 ng/ml) (Figs. 6, 7). Response to a single injection of LHRH. The iv injection of 50 ng of the neurohormone produced significant increases in LH titers at all stages of the cycle (Figs. 1-5). However, only small changes in the response at different stages could be observed with this dose of LHRH (Fig. 5). The response was minimal on diestrus II, rose significantly at proestrus (P < 0.001), remained high during estrus and fell at diestrus I to values that were not significantly different from the response during diestrus II (Fig. 5). These results are in agreement with previous experiments that detected no clear cyclic differences in the 4
Antiovine LH serum and purified rat LH for radioiodination were provided through the courtesy of Dr. G. Niswender (Colorado State University) and Dr. L. Reichert (Emory University). Kits for the determination of FSH were obtained through the NIAMD NIH Pituitary Hormone Program. 5 Mean ± SEM.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 06:04 For personal use only. No other uses without permission. . All rights reserved.
INCREASED RESPONSE TO LHRH INDUCED BY LHRH response to low doses of LHRH (2,16). Increases in plasma FSH levels were inconsistently found after the injection of 50 ng of LHRH, and on proestrus and diestrus I they reached significance (P < 0.01) (Table 1). Androgenized females responded to the first LHRH injection with elevated LH titers similar to those obtained in proestrus (Figs. 2, 6) in agreement with a recent report (17). No increases in FSH were observed. Response to a second injection of LHRH given 1 h later. The second injection of LHRH again produced a significant rise in LH levels on all days of the cycle (Figs. 1-5). The magnitude of this second response showed remarkable cyclic differences if compared with the response to the first injection (Fig. 5). At proestrus, the increase in plasma LH was 4.4 times greater than the increase following the first injection of LHRH (P < 0.001). Appropriate controls injected with saline instead of the first injection of LHRH did not show any potentiation of the response to the second injection; the increment was 3.0 ± 1.3 ng/ml5 which was similar to the
15
3O-|
20-
10-
oJ
20
60 80 TIME (min after 1st injection)
120
FIG. 2. Effect of two consectuive injections (50 ng) of LHRH (arrows) on plasma titers of LH in proestrous rats (individual values).
increment of 3.0 ± 0.3 ng/ml5 in response to the first injection in the other group of proestrous rats. On the contrary, no significant differences between both responses to LHRH were observed on estrus or on diestrus II; a small but significant (P < 0.01) potentiation of the second response was observed on diestrus I (Fig. 5). 30-i
20-
20-
10-
10-
20
60
80
120
TIME (min ofter lit injection)
FIG. 1. Effect of two consecutive injections (50 ng) of LHRH (arrows) on plasma titers of LH in diestrous II rats (individual values).
oJ
20
60 80 TIME (min after 1st injection)
120
FIG. 3. Effect of two consectuive injections (50 ng) of LHRH (arrows) on plasma titers of LH in estrous rats (individual values).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 06:04 For personal use only. No other uses without permission. . All rights reserved.
16
CASTRO-VAZQUEZ AND McCANN 30-,
Endo • 1975 Vol 97 • No 1
significant increases were observed in FSH levels. A control group injected with saline instead of the first injection of LHRH showed no potentiation of the response to LHRH, the increment in plasma LH was 3.8 ± 0.6 ng/ml5, an increase similar to that obtained in response to the first injection of LRH in the group which was primed with LHRH (3.6 ± 0.3 ng/ml).
20-
10-
Discussion 0
J
1
1
60
eo
120
TIME (min after 1st injection)
FIG. 4. Effect of two consecutive injections (50 ng) of LHRH (arrows) on plasma titers of LH in diestrous I rats (individual values).
Again, FSH changes after the second injection were not as consistent as the LH changes (Table 1); The increments were only significant in the groups on proestrus (P < 0.01) and on estrus (P < 0.05). No significant potentiation of the second response was observed at any stage of the cycle. Androgenized females showed a potentiation of the second LH response similar to that of proestrous rats (Figs. 6 and 7). No
The dose of LHRH used in the present experiments produced only small increases in plasma LH and did not reveal the conspicuous changes in responsiveness of the pituitary during the estrous cycle that have been reported with larger doses (1-7), although the response was higher on proestrus and estrus. When this small dose was followed by the injection of an equal amount of the neurohormone 1 h later, the response to the second injection was markedly increased on proestrus. Since a priming injection of the diluent did not augment responsiveness of the gland, this would appear to be a specific response to the priming injection of LHRH. In sharp contrast, there was only a slight increase in responsiveness to the second injection of LHRH at the other stages of the cycle. The
10-
FIG. 5. Comparison of the initial LH values at 1300 h (triangles) and the increments after the first (open circles) and second injection (black dots) of LHRH during the estrous cycle (means ± SEM).
5-
0J DIESTRUS I I
PROESTRUS
ESTRUS
DIESTRUS I
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 06:04 For personal use only. No other uses without permission. . All rights reserved.
17
INCREASED RESPONSE TO LHRH INDUCED BY LHRH 45-1
40-
15-. 20-
10-
10-
0
c
J
20
60
80
120
TIME (min after 1st injection)
FIG. 6. Effect of two consecutive injections (50 ng) of LHRH (arrows) on plasma concentrations of LH in androgenized females (individual values).
5-
present results thus confirm the findings of Aiyer et al. (11) that priming with LHRH on proestrus can increase responsiveness of 0the gland and further show that this re1st 2nd INITIAL INCREMENT INCREMENT LEVEL sponse is maximal on proestrus. Since the priming effect of LHRH was FIG. 7. Comparison of the initial LH values and the only apparent on proestrus, it may be increments after the first and second injection of induced by the prior elevation of estrogen LHRH in androgenized females (means ± SEM). titers in early proestrus (8,9). This concept is supported by the fact that androgenized not clear. Since estrogen can increase the females with constant vaginal cornification responsiveness to LHRH in vivo and in as a result of estrogen secretion from their vitro (18-21), it is possible that the action ovaries also showed an increased response of estrogen is directly on the gland to bring to the second injection of LHRH which out the priming action of LHRH. Another was similar in magnitude to that of proes- possibility is that estrogen, by an action on trous rats. the CNS, induces enhanced release of The mechanism by which the priming endogenous LHRH on the afternoon of action of LHRH is enhanced by estrogen is proestrus which brings on the priming TABLE 1. Effect of two consecutive doses of LHRH (1 h apart) on plasma levels of FSH (ng/ml ± SEM) Diestrus II (7)1
Proestrus (8)
Estrus (7)
Diestrus I (6)
Androgenized (7)
Maximal increment after first injection
5.6±4.6 2
37.5 ± 9.73
9.6 ± 6.3
16.4 ± 3.73
24.7 ± 20.5
Maximal increment after second injection
4.3 ± 2.1
49.1 ± 14.03
13.0 ± 4.93
8.5 ± 3.7
30.1 ± 13.2
Type of rat
1
Figures in parentheses indicate number of animals per group. Mean ± SEM. 3 The increment is significant. 2
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 06:04 For personal use only. No other uses without permission. . All rights reserved.
18
CASTRO-VAZQUEZ AND McCANN
E n d o < 1975 Vol 97 i No 1
effect. The inability of LHRH to prime the decapeptide will need to be used to detergland on the day of estrus could be related mine if a similar priming of the response of to the long interval since the occurrence of the FSH secreting cells can be detected. In other experiments, it has been possihigh estrogen titers, or it could be related to the prior LH release on proestrus with ble to show that the increased responsivepartial depletion of glandular content (2,22). ness induced by a prior injection of LHRH The mechanism by which LHRH en- can also be demonstrated even if LHRH hances the response to the second injection priming follows ovariectomy on proestrus, of the neurohormone is unknown. Several which indicates that this response does not possibilities suggest themselves. First, require altered release of ovarian steroids LHRH may increase the synthesis of a in, response to the initial injection of readily releaseable pool of LH so that the LHRH (23). Following the completion of response to the second injection is en- these experiments, Aiyer et al. (24) rehanced. Since the fraction of the glandular ported results similar to those which we content of LH which is released in re- have obtained. They also observed that the sponse to LHRH is quite small (2), detec- priming action of LHRH was particularly tion of this change would be difficult if not prominent on proestrus and that it was not impossible by direct assay of glandular abolished by ovariectomy. They also subcontent. Second, it is conceivable that jected the animals to adrenalectomy and LHRH may induce the formation of addi- adrenalectomy plus ovariectomy. These tional LHRH receptors on the gonado- procedures also failed to block the introphs. This also would enhance the re- creased responsiveness. sponse to a second injection. Further studies are required to elucidate the Ackno wl edgments mechanism by which the priming action We gratefully acknowledge the technical assistance develops. of Mrs. Suzan Raymond and Miss Teresa Fornieles. Even though the mechanism by which LHRH exerts its priming action is unReferences known, it probably plays an important physiological role in magnifying the re1. Cooper, K. J., C. P. Fawcett, and S. M. McCann, y lease of LH on proestrus. One can specuEndocrinol 57: 187, 1973. late that there are two components to the 2. , , and , Endocrinology 95: increased responsiveness of the gland to 1293, 1974. 3. Zeballos, G., and S. M. McCann, Endocrinology LHRH which occurs during the preovula(In press). tory surge of LH. The first component is the 4. Aiyer, M. S., G. Fink, and F. Greig, / Endocrinol increased responsiveness to LHRH in60: 47, 1974. duced by prior estrogen secretion and the 5. Martin, J. E., L. Tyrey, J. W. Everett, and R. E. second component is increased responFellows, Endocrinology 94: 556, 1974. siveness induced by the priming effect of 6. Rippel, R. H., E. S. Johnson, and W. F. White, Proc Soc Exp Biol Med 143: 55, 1973. small amounts of endogenous LHRH. Then 7. Gordon, J. H., and S. Reichlin, Endocrinology 94: during the proestrous discharge, increased 974, 1974. release of LHRH can act on a maximally 8. Brown-Grant, K., D. Exley, and F. Naftolin, J responsive gland to produce the precipiEndocrinol 48: 295, 1970. tous preovulatory discharge of LH. 9. Kalra, S. P., and P. S. Kalra, Endocrinology 95: 1711, 1974. The small doses of the decapeptide used 10. Libertun, C., and S. M. McCann, Fed Proc 33: in the present study induced very small 212, 1974 (Abstract). releases of FSH and no clear priming 11. Aiyer, M. S., S. A. Chiappa, G. Fink, and F. Greig, action was observed. Higher doses of the J Physiol (Lond) 234: 81P, 1973.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 06:04 For personal use only. No other uses without permission. . All rights reserved.
INCREASED RESPONSE TO LHRH INDUCED BY LHRH 12. Long, J. A., and H. M. Evans, Mem Univ Calif 6: 1, 1922. 13. Gorski, R. A., In Martini, L., and W. F. Ganong (eds.), Frontiers in Neuroendocrinology, Oxford University Press, New York, 1971, p. 237. 14. Zeballos, G., and S. M. McCann, Proc Soc Exp Biol Med 145: 415, 1974. 15. Niswender, G. D., A. R. Midgley, S. E. Monroe, and L. E. Reichert, Proc Soc Exp Biol Med 128: 807, 1968. 16. Blake, C. A., R. L. Norman, and C. H. Sawyer, In Gual, C., and E. Rosemberg (eds.), Hypothalamic Hypophysiotropic Hormones. Physiological and Clinical Studies, Excerpta Medica, Amsterdam, 1973, p. 33. 17. Mennin, S. P., K. Kubo, and R. A. Gorski, Endocrinology 95: 412, 1974.
19
18. Arimura, A., and A. V. Schally, Proc Soc Exp Biol Med 136: 290, 1971. 19. Libertun, C., K. J. Cooper, C. P. Fawcett, and S. M. McCann, Endocrinology 94: 518, 1974. 20. , R. Orias, and S. M. McCann, Endocrinology 94: 1094, 1974. 21. Cooper, K. J., C. P. Fawcett, and S. M. McCann, Proc Soc Exp Biol Med 145: 422, 1974. 22. Schwartz, N. B., Recent Progr Horm Res 25: 1, 1969. 23. Cooper, K. J., and S. M. McCann, Proc Symposium on Hypothalamic Hormones: Chemistry, Physiology, Pharmacology and Clinical Uses, Milan, 1974 (In press). 24. Aiyer, M. S., S. A. Chiappa, and G. Fink, J Endocrinol 62: 573, 1974.
International Symposium on the Hypothalamus and Endocrine Functions A symposium on neuroendocrinology co-sponsored by the International Society for Neuroendocrinology, the Canadian Society of Endocrinology and Metabolism and Laval University will be held at the Hotel Concorde,
Quebec City, on September 22-25, 1975. The main program will consist of twenty-six 35-minute lectures divided under the following topics: chemistry and assay of hypothalamic hormones, localization of hypothalamic hormones, control of PRL and GH secretion and action, steroid feedback, clinical applications of hypothalamic hormones, control of hypothalamic hormone secretion and somatostatin. Poster session(s) will also be held. The deadline for submission of abstracts is August 1. For additional information, please contact: Dr. Fernand Labrie Chairman, Organizing Committee Laboratory of Molecular Endocrinology Centre Hospitalier de l'Universite Laval 2705 Boul. Laurier Quebec G1V 4G6, Canada
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 06:04 For personal use only. No other uses without permission. . All rights reserved.
ABSTRACT. Following a single iv injection of 50 ng of LH-releasing hormone (LHRH), a small increase in plasma LH titers was observed and this response was greater on proestrus and estrus than in other stages of the cycle. When a second injection of a similar dose of the neurohormone was given one hour later, the response to the second injection was markedly enhanced on proestrus but only slightly increased at other stages of the cycle. The response to LHRH in androgenized females was similar to that observed on proestrus, and, as on proestrus, an enhanced response to a second injection occurred.
P
REVIOUS work from this laboratory (1-3) and from those of others (4-7) has demonstrated cyclic changes in the response of the pituitary to a single injection of LHRH. The release of both FSH and LH increased during proestrus to reach a maximum during the preovulatory release of LH and then declined to a minimum during diestrus. The time course of the increased responsiveness followed, with a lag period, the time of heightened estrogen release which begins on diestrus II and reaches a maximum in early proestrus (8,9). The association of maximal responsiveness to LHRH with the preovulatory discharge of LH thought to be brought about by increased release of LHRH, plus the fact that both the preovulatory discharge and the heightened responsiveness could be blocked by administration of Nembutal at 1 PM on proestrus, suggested that the Received January 23, 1975. 1 Postdoctoral Fellow of the World Health Organization, on leave from ILAFIR, Universidad del Salvador, San Miguel, Argentina. 2 Supported by grants from NIH (AM 10073 and HD 05151), the Ford Foundation and the Texas Population Research Institute.
release of endogenous LHRH might in some manner enhance the pituitary's responsiveness to the subsequently released neurohormone (4,7). Previous attempts to demonstrate an ability of LHRH to increase responsiveness to subsequent injections of LHRH were unsuccessful in ovariectomized, estrogen-primed animals (Libertun, unpublished data); however, we were able to show that median eminence lesions blocked the increased responsiveness to LHRH which occurred during the LH release induced by progesterone in the ovariectomized, estrogen-primed rat (10). In the meantime, Aiyer et at. (11) reported in an abstract that injection of a priming dose of LHRH at 1 PM on proestrus enhanced the responsiveness of the gland to a second injection of LHRH given 1 h later. In the present experiments, the potentiation of the response to LHRH produced by a previous injection of the neurohormone has been studied during various stages of the estrous cycle and in neonatally androgenized females. A pronounced priming action of LHRH was demonstrated on proestrus and in androgenized females but not at other stages of the estrous cycle. 13
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 06:04 For personal use only. No other uses without permission. . All rights reserved.
14
CASTRO-VAZQUEZ AND McCANN
Materials and Methods Animals. Adult female rats of the SpragueDawley strain (Simonsen Laboratories, Gilroy, California) were kept under conditions of controlled light (lights on between 5:00 and 19:00 h) and temperature (21 C) and given free access to Purina rat chow and water. After arrival they were allowed to become accustomed to the new environment for at least one week. After this period, vaginal smears were taken at least 6 days a week and rats displaying at least two consecutive cycles of 4 or 5 days duration were used. Only 7 out of 41 animals had 5-day cycles. Since the results obtained with these rats were similar to those obtained with rats with 4-day cycles, the results were pooled for presentation. Results are reported of only those animals whose uteri fulfilled the criteria of Long and Evans (12) for the expected stage of the cycle, i.e., 1) a ballooned uterus at proestrus, 2) either a slightly distended or an empty uterus at estrus, and 3) a slender and empty uterus at diestrus. Androgenized rats were obtained by injecting females on the fourth day of life (birth = day 0) with 50 fig of testosterone propionate sc (13). They were used when adult if they exhibited at least 6 consecutive days of vaginal cornification. At autopsy, no corpora lutea were seen macroscopically in the ovaries. Injections and blood withdrawal. Synthetic LHRH (50 ng in 0.2 ml of 0.9% NaCl)3 was injected into the external jugular vein which was also used for obtaining heparinized blood samples (0.8 ml) while the animals were anesthetized with tribromoethanol (14). An initial dose of 25 mg/100 g body weight of tribromoethanol was injected ip between 1300-1330 h and additional doses were administered thereafter when necessary. Blood samples were obtained just prior to (3-5 min after injection of tribromoethanol) and at 20 and 60 min postinjection of LHRH. Then the second injection of LHRH was given and additional blood samples were removed at 20 and 60 min following this injection. Plasma was separated by centrifugation at low speed at 4 C and stored frozen until the day of 3
The synthetic LHRH was generously provided by Dr. R. Deghenghi, Ayerst, Ltd., Montreal.
Endo • 1975 Vol 97 • No 1
assay. LH was measured by the ovine-ovine radioimmunoassay of Niswender et al. (15).4 RP-1 rat pituitary LH was used as reference standard and results were expressed in terms of NIH-LH-S1 reference preparation. FSH was measured using the kits supplied by NIAMDD.4 The RP-1 rat pituitary FSH reference preparation was used. The paired t test was used to evaluate the significance of increases or decreases in plasma LH which took place in each group of rats. Comparisons between values in two groups were made with Student's t test.
Results
Plasma luteinizing hormone concentration before injection of LHRH. Plasma LH levels at 13:00 h showed minor fluctuations during the cycle, being minimal during diestrus II and rising slightly but significantly on proestrus (P < 0.001) and diestrus I (P < 0.01) (Figs. 1-5). Androgenized females showed LH levels at 13:00 h (0.9 ± O.I5 ng/ml) that were significantly lower (P < 0.05) than those on proestrus (1.3 ± 0.25 ng/ml) (Figs. 6, 7). Response to a single injection of LHRH. The iv injection of 50 ng of the neurohormone produced significant increases in LH titers at all stages of the cycle (Figs. 1-5). However, only small changes in the response at different stages could be observed with this dose of LHRH (Fig. 5). The response was minimal on diestrus II, rose significantly at proestrus (P < 0.001), remained high during estrus and fell at diestrus I to values that were not significantly different from the response during diestrus II (Fig. 5). These results are in agreement with previous experiments that detected no clear cyclic differences in the 4
Antiovine LH serum and purified rat LH for radioiodination were provided through the courtesy of Dr. G. Niswender (Colorado State University) and Dr. L. Reichert (Emory University). Kits for the determination of FSH were obtained through the NIAMD NIH Pituitary Hormone Program. 5 Mean ± SEM.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 06:04 For personal use only. No other uses without permission. . All rights reserved.
INCREASED RESPONSE TO LHRH INDUCED BY LHRH response to low doses of LHRH (2,16). Increases in plasma FSH levels were inconsistently found after the injection of 50 ng of LHRH, and on proestrus and diestrus I they reached significance (P < 0.01) (Table 1). Androgenized females responded to the first LHRH injection with elevated LH titers similar to those obtained in proestrus (Figs. 2, 6) in agreement with a recent report (17). No increases in FSH were observed. Response to a second injection of LHRH given 1 h later. The second injection of LHRH again produced a significant rise in LH levels on all days of the cycle (Figs. 1-5). The magnitude of this second response showed remarkable cyclic differences if compared with the response to the first injection (Fig. 5). At proestrus, the increase in plasma LH was 4.4 times greater than the increase following the first injection of LHRH (P < 0.001). Appropriate controls injected with saline instead of the first injection of LHRH did not show any potentiation of the response to the second injection; the increment was 3.0 ± 1.3 ng/ml5 which was similar to the
15
3O-|
20-
10-
oJ
20
60 80 TIME (min after 1st injection)
120
FIG. 2. Effect of two consectuive injections (50 ng) of LHRH (arrows) on plasma titers of LH in proestrous rats (individual values).
increment of 3.0 ± 0.3 ng/ml5 in response to the first injection in the other group of proestrous rats. On the contrary, no significant differences between both responses to LHRH were observed on estrus or on diestrus II; a small but significant (P < 0.01) potentiation of the second response was observed on diestrus I (Fig. 5). 30-i
20-
20-
10-
10-
20
60
80
120
TIME (min ofter lit injection)
FIG. 1. Effect of two consecutive injections (50 ng) of LHRH (arrows) on plasma titers of LH in diestrous II rats (individual values).
oJ
20
60 80 TIME (min after 1st injection)
120
FIG. 3. Effect of two consectuive injections (50 ng) of LHRH (arrows) on plasma titers of LH in estrous rats (individual values).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 06:04 For personal use only. No other uses without permission. . All rights reserved.
16
CASTRO-VAZQUEZ AND McCANN 30-,
Endo • 1975 Vol 97 • No 1
significant increases were observed in FSH levels. A control group injected with saline instead of the first injection of LHRH showed no potentiation of the response to LHRH, the increment in plasma LH was 3.8 ± 0.6 ng/ml5, an increase similar to that obtained in response to the first injection of LRH in the group which was primed with LHRH (3.6 ± 0.3 ng/ml).
20-
10-
Discussion 0
J
1
1
60
eo
120
TIME (min after 1st injection)
FIG. 4. Effect of two consecutive injections (50 ng) of LHRH (arrows) on plasma titers of LH in diestrous I rats (individual values).
Again, FSH changes after the second injection were not as consistent as the LH changes (Table 1); The increments were only significant in the groups on proestrus (P < 0.01) and on estrus (P < 0.05). No significant potentiation of the second response was observed at any stage of the cycle. Androgenized females showed a potentiation of the second LH response similar to that of proestrous rats (Figs. 6 and 7). No
The dose of LHRH used in the present experiments produced only small increases in plasma LH and did not reveal the conspicuous changes in responsiveness of the pituitary during the estrous cycle that have been reported with larger doses (1-7), although the response was higher on proestrus and estrus. When this small dose was followed by the injection of an equal amount of the neurohormone 1 h later, the response to the second injection was markedly increased on proestrus. Since a priming injection of the diluent did not augment responsiveness of the gland, this would appear to be a specific response to the priming injection of LHRH. In sharp contrast, there was only a slight increase in responsiveness to the second injection of LHRH at the other stages of the cycle. The
10-
FIG. 5. Comparison of the initial LH values at 1300 h (triangles) and the increments after the first (open circles) and second injection (black dots) of LHRH during the estrous cycle (means ± SEM).
5-
0J DIESTRUS I I
PROESTRUS
ESTRUS
DIESTRUS I
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 06:04 For personal use only. No other uses without permission. . All rights reserved.
17
INCREASED RESPONSE TO LHRH INDUCED BY LHRH 45-1
40-
15-. 20-
10-
10-
0
c
J
20
60
80
120
TIME (min after 1st injection)
FIG. 6. Effect of two consecutive injections (50 ng) of LHRH (arrows) on plasma concentrations of LH in androgenized females (individual values).
5-
present results thus confirm the findings of Aiyer et al. (11) that priming with LHRH on proestrus can increase responsiveness of 0the gland and further show that this re1st 2nd INITIAL INCREMENT INCREMENT LEVEL sponse is maximal on proestrus. Since the priming effect of LHRH was FIG. 7. Comparison of the initial LH values and the only apparent on proestrus, it may be increments after the first and second injection of induced by the prior elevation of estrogen LHRH in androgenized females (means ± SEM). titers in early proestrus (8,9). This concept is supported by the fact that androgenized not clear. Since estrogen can increase the females with constant vaginal cornification responsiveness to LHRH in vivo and in as a result of estrogen secretion from their vitro (18-21), it is possible that the action ovaries also showed an increased response of estrogen is directly on the gland to bring to the second injection of LHRH which out the priming action of LHRH. Another was similar in magnitude to that of proes- possibility is that estrogen, by an action on trous rats. the CNS, induces enhanced release of The mechanism by which the priming endogenous LHRH on the afternoon of action of LHRH is enhanced by estrogen is proestrus which brings on the priming TABLE 1. Effect of two consecutive doses of LHRH (1 h apart) on plasma levels of FSH (ng/ml ± SEM) Diestrus II (7)1
Proestrus (8)
Estrus (7)
Diestrus I (6)
Androgenized (7)
Maximal increment after first injection
5.6±4.6 2
37.5 ± 9.73
9.6 ± 6.3
16.4 ± 3.73
24.7 ± 20.5
Maximal increment after second injection
4.3 ± 2.1
49.1 ± 14.03
13.0 ± 4.93
8.5 ± 3.7
30.1 ± 13.2
Type of rat
1
Figures in parentheses indicate number of animals per group. Mean ± SEM. 3 The increment is significant. 2
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 06:04 For personal use only. No other uses without permission. . All rights reserved.
18
CASTRO-VAZQUEZ AND McCANN
E n d o < 1975 Vol 97 i No 1
effect. The inability of LHRH to prime the decapeptide will need to be used to detergland on the day of estrus could be related mine if a similar priming of the response of to the long interval since the occurrence of the FSH secreting cells can be detected. In other experiments, it has been possihigh estrogen titers, or it could be related to the prior LH release on proestrus with ble to show that the increased responsivepartial depletion of glandular content (2,22). ness induced by a prior injection of LHRH The mechanism by which LHRH en- can also be demonstrated even if LHRH hances the response to the second injection priming follows ovariectomy on proestrus, of the neurohormone is unknown. Several which indicates that this response does not possibilities suggest themselves. First, require altered release of ovarian steroids LHRH may increase the synthesis of a in, response to the initial injection of readily releaseable pool of LH so that the LHRH (23). Following the completion of response to the second injection is en- these experiments, Aiyer et al. (24) rehanced. Since the fraction of the glandular ported results similar to those which we content of LH which is released in re- have obtained. They also observed that the sponse to LHRH is quite small (2), detec- priming action of LHRH was particularly tion of this change would be difficult if not prominent on proestrus and that it was not impossible by direct assay of glandular abolished by ovariectomy. They also subcontent. Second, it is conceivable that jected the animals to adrenalectomy and LHRH may induce the formation of addi- adrenalectomy plus ovariectomy. These tional LHRH receptors on the gonado- procedures also failed to block the introphs. This also would enhance the re- creased responsiveness. sponse to a second injection. Further studies are required to elucidate the Ackno wl edgments mechanism by which the priming action We gratefully acknowledge the technical assistance develops. of Mrs. Suzan Raymond and Miss Teresa Fornieles. Even though the mechanism by which LHRH exerts its priming action is unReferences known, it probably plays an important physiological role in magnifying the re1. Cooper, K. J., C. P. Fawcett, and S. M. McCann, y lease of LH on proestrus. One can specuEndocrinol 57: 187, 1973. late that there are two components to the 2. , , and , Endocrinology 95: increased responsiveness of the gland to 1293, 1974. 3. Zeballos, G., and S. M. McCann, Endocrinology LHRH which occurs during the preovula(In press). tory surge of LH. The first component is the 4. Aiyer, M. S., G. Fink, and F. Greig, / Endocrinol increased responsiveness to LHRH in60: 47, 1974. duced by prior estrogen secretion and the 5. Martin, J. E., L. Tyrey, J. W. Everett, and R. E. second component is increased responFellows, Endocrinology 94: 556, 1974. siveness induced by the priming effect of 6. Rippel, R. H., E. S. Johnson, and W. F. White, Proc Soc Exp Biol Med 143: 55, 1973. small amounts of endogenous LHRH. Then 7. Gordon, J. H., and S. Reichlin, Endocrinology 94: during the proestrous discharge, increased 974, 1974. release of LHRH can act on a maximally 8. Brown-Grant, K., D. Exley, and F. Naftolin, J responsive gland to produce the precipiEndocrinol 48: 295, 1970. tous preovulatory discharge of LH. 9. Kalra, S. P., and P. S. Kalra, Endocrinology 95: 1711, 1974. The small doses of the decapeptide used 10. Libertun, C., and S. M. McCann, Fed Proc 33: in the present study induced very small 212, 1974 (Abstract). releases of FSH and no clear priming 11. Aiyer, M. S., S. A. Chiappa, G. Fink, and F. Greig, action was observed. Higher doses of the J Physiol (Lond) 234: 81P, 1973.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 06:04 For personal use only. No other uses without permission. . All rights reserved.
INCREASED RESPONSE TO LHRH INDUCED BY LHRH 12. Long, J. A., and H. M. Evans, Mem Univ Calif 6: 1, 1922. 13. Gorski, R. A., In Martini, L., and W. F. Ganong (eds.), Frontiers in Neuroendocrinology, Oxford University Press, New York, 1971, p. 237. 14. Zeballos, G., and S. M. McCann, Proc Soc Exp Biol Med 145: 415, 1974. 15. Niswender, G. D., A. R. Midgley, S. E. Monroe, and L. E. Reichert, Proc Soc Exp Biol Med 128: 807, 1968. 16. Blake, C. A., R. L. Norman, and C. H. Sawyer, In Gual, C., and E. Rosemberg (eds.), Hypothalamic Hypophysiotropic Hormones. Physiological and Clinical Studies, Excerpta Medica, Amsterdam, 1973, p. 33. 17. Mennin, S. P., K. Kubo, and R. A. Gorski, Endocrinology 95: 412, 1974.
19
18. Arimura, A., and A. V. Schally, Proc Soc Exp Biol Med 136: 290, 1971. 19. Libertun, C., K. J. Cooper, C. P. Fawcett, and S. M. McCann, Endocrinology 94: 518, 1974. 20. , R. Orias, and S. M. McCann, Endocrinology 94: 1094, 1974. 21. Cooper, K. J., C. P. Fawcett, and S. M. McCann, Proc Soc Exp Biol Med 145: 422, 1974. 22. Schwartz, N. B., Recent Progr Horm Res 25: 1, 1969. 23. Cooper, K. J., and S. M. McCann, Proc Symposium on Hypothalamic Hormones: Chemistry, Physiology, Pharmacology and Clinical Uses, Milan, 1974 (In press). 24. Aiyer, M. S., S. A. Chiappa, and G. Fink, J Endocrinol 62: 573, 1974.
International Symposium on the Hypothalamus and Endocrine Functions A symposium on neuroendocrinology co-sponsored by the International Society for Neuroendocrinology, the Canadian Society of Endocrinology and Metabolism and Laval University will be held at the Hotel Concorde,
Quebec City, on September 22-25, 1975. The main program will consist of twenty-six 35-minute lectures divided under the following topics: chemistry and assay of hypothalamic hormones, localization of hypothalamic hormones, control of PRL and GH secretion and action, steroid feedback, clinical applications of hypothalamic hormones, control of hypothalamic hormone secretion and somatostatin. Poster session(s) will also be held. The deadline for submission of abstracts is August 1. For additional information, please contact: Dr. Fernand Labrie Chairman, Organizing Committee Laboratory of Molecular Endocrinology Centre Hospitalier de l'Universite Laval 2705 Boul. Laurier Quebec G1V 4G6, Canada
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 06:04 For personal use only. No other uses without permission. . All rights reserved.
