VoL 29, No.6, June 1978 Printed in U.8A.
FERTILITY AND STERILITY Copyright
~
1978 The American Fertility Society
PITUITARY GONADOTROPIN RESPONSIVENESS WITH DANAZOL*t
JOHN M. SHANE, M.D.:j: RICHARD KATES, M.D. ROBERT L. BARBIERI, M.D. ROBERTA B. TODD, M.S. I. J. DAVIES, M.D.
Department of Obstetrics and Gynecology, Beth Israel Hospital and Boston Hospital for Women, Harvard Medical School, Boston, Massachusetts 02115
Danazol (17a-pregn-4-en-20-yno-[2,3-dlisoxazol-I7-oV was administered daily for 4 days to castrated female rats. As previously demonstrated, danazollowered serum levels of luteinizing hormone (LH) in an apparent dose-dependent fashion. Animals which received danazol in a dose sufficient to lower serum LH responded to administered LH-releasing hormone (LHRH) with increases in serum LH levels which were not diminished as compared with those of control animals. Although these experiments do not preclude an effect of danazol directly on the pituitary, the results indicate that this agent probably lowers serum LH primarily by inhibition of hypothalamic LHRH secretion.
Danazol, a new and potentially valuable pharmacologic agent, is the isoxazol derivative of 17aethinyl testosterone (17a-pregn-4-en-20-yno-[2,3dlisoxazol-17-oD. This steroid has had human and animal trials which have demonstrated its ability to lower luteinizing hormone (LH) and folliclestimulating hormone (FSH) levels; it has therefore been described as an "antigonadotropin." This effect has been utilized in the clinical management of endometriosis, 1 precocious puberty,2 and breastdisorders.2 Any knowledge regarding its mechanism of action should enable us to administer the agent to our patients with more confidence and intelligence. In human studies, danazol, at varying dosages, depressed plasma levels of testosterone and androstenedione as well as the gonadotropins. 3 In rats, rabbits, and mice, studies have confirmed the ability of danazol to lower serum LH with little or no biologic activity as a progestin, antipro-
gestin, estrogen,or antiestrogen. Mild androgenic activity has been demonstrated at higher doses. 2. 4 The mechanism by which danazol lowers circulating LH and FSH levels has not been elucidated. This action could be postulated to result' from inhibition of pituitary responsiveness to hypothalamic LH-releasing hormone (LHRH) or to a diminution of hypothalamic stimulation of the pituitary. In the present experiment, castrated female rats were chronically treated with danazol and then challenged intraperitoneally with LHRH. The subsequent LH responses. were measured by radioimmunoassay and compared with those of appropriate controls.
MATERIALS AND METHODS
Sixty-day-old female Sprague-Dawley"derived rats from Charles River Laboratories were ovariectomized under sodium pentobarbital anesthesia 6 weeks before experimentation. The animals were maintained in a controlled environment (22 C, 14 hours of light, 10 hours of darkness) on Purina laboratory chow and tap water ad libitum; they were housed three per cage.
Received January 30, 1978; accepted February 15, 1978. *Supported by Rockefeller Foundation Grant RF72077 and United States Public Health Service Grant HD07923-03. tThis paper is the recipient of fIrst prize in. the Annual Boston Obstetrical Society Prize Paper Competition. :j:Reprintrequests: Dr. John M. Shane, 6465 South Yale, Tulsa, Okla. 74136.
0
637
June 1978
SHANE ET AL.
638
At the time of the experimentation, the rats weighed between 300 and 420 gm. They were placed in weight-matched groups, seven animals in each group. The danazol was suspended in a 10% ethanol-corn oil mixture. This mixture, in dosages of 200 mg of danazol/kg or 20 mg of danazollkg, or vehicle alone, was administered in a 3-ml volume, by gavage, once daily for 4 consecutive days. Four hours after the last feeding, each rat was given 100 mg of LHRH intraperitoneally, dissolved in 0.2 ml of 0.9% saline or the vehicle alone. This dose of LHRH as been shown to be below the dose that elicits the maximal (plateau) response to that hormone. 5 One hour after LHRH administration, under 50% CO 2-50% O2 anesthesia, blood was obtained by a single cardiac puncture. All animals were killed between 2:30 and 3:30 P.M. Blood was allowed to remain at room temperature for 0.5 hour and then was stored for 1 hour at 4 0 C to allow clot retraction, at which time the serum was removed and stored at -20 C. All determinations of luteinizing hormone were performed in duplicate in a single assay by the ovine-ovine double-antibody radioimmunoassay. 6, 7 Ovine LH was labeled with 1251. The reference standard was 'NIH LH-S16. The intra-assay coefficient of variation was less than 5%. Statistical analysis was performed by using a t-test with Dunnett's tables. The animals were separated into experimental groups as follows: group 1--control, no treatment; group 2--control, treated with corn oil gavage and intraperitoneal saline; group 3--control, corn oil gavage and intraperitoneal LHRH; group 4control, treated with low-dose'(20 mg/kg) danazol and intraperitoneal saline; group 5-control, treated with high-dose (200 mglkg) danazol and intraperitoneal saline; group 6-treatment group, low-dose danazol gavage and intraperitoneal LHRH; group 7-treatment group, high-dose danazol gavage and intraperitoneal LHRH. 0
RESULTS
The results are summarized in Figure 1. Danazol lowered serum LH levels in female castrated rats in an apparent dose-dependent fashion. In the low-dose danazol treatment group (20 mg/kg) the mean serum LH level was lowered by 41% as compared with that of the control groups. However, the absolute difference between control and low-dose groups was not statistically significant rp > 0.05). At high-dosage treatment
40
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FIG. 1. LH response by castrated female rats in indicated treatment groups.
(200 mglkg), LH was lowered by 90% as compared with control groups. The absolute difference between control and high-dosage groups was statistically significant rp < 0.01). After challenge with LHRH, there was no significant difference in mean LH levels among the three groups. Of the three treatment groups, the high-dose danazol group showed the greatest absolute increase in LH levels after LHRH stimulation. DISCUSSION
Our results support previous clinical and laboratory findings that danazol is capable of lowering serum LH. Moreover, under the conditions of these experiments, the decrease in serum LH induced by danazol was not associated with a diminished pituitary responsiveness to LHRH. Although these experiments do not preclude an effect of danazol directly on the pituitary, the results support the hypothesis that this agent lowers serum LH primarily by inhibition of hypothalamic secretion of LHRH. Acknowledgments. We would like to thank Dr. Leo E. Reichert, Jr., for the ovine LH used for radioiodination; Dr. Gordon Niswender for the anti-ovine LH #15; and the National Institute of Arthritis, Metabolism and Digestive
Vol. 29, No.6
PITUITARY GONADOTROPIN RESPONSIVENESS WITH DANAZOL
Diseases for the ovine Nlli-LH-16 standard. Appreciation is also extended to the Sterling-Winthrop Research Institute for their contribution of danazol. REFERENCES 1. Dmowski WP, Cohen MR: 'rreatment of endometriosis
with an antigonadotropin, danazol: a laparoscopic and histologic evaluation. Obstet Gynecol 46:147, 1975 2. Greenblatt RB, Dmowski WP, Mahesh VB, Scholer HFL: Clinical studies with an antigonadotropin-danazol. Fertil Steril 22:102, 1971 3. Sherins RJ, Gandy HM, Thorslund TW, Paulsen CA: Pituitary and testicular function studies: experience with a new gonadal inhibitor, 17a-pregn-4-en-20-yno-(2,3-d)isoxazol-17-01 (danazol). J Clin Endocrinol Metab 32:522, 1971
639
4. Dmowski SP, Scholar HFL, Mahesh VB, Greenblatt RB: Danazol-a synthetic steroid derivative with interesting physiologic properties. Fertil Steril 22:9, 1971 5. Mueh J, Shane JM, Naftolin F: The effects of methallibure (lCI 33,828) on LH release in castrate male rats challenged with LHRH. Endocrinology 97:493,1975 6. Niswender GD, Midgely AR Jr, Monroe SE, Reichert LE Jr: Radioimmunoassay for rat luteinizing hormone with antiovine LH serum and ovine LH'3'1. Proc Soc Exp BioI Med 128:807, 1968 7. Naftolin F, Feder HH: Suppression ofluteinizing hormone secretion in male rats by 5a-androstan-17,6-01-3-one (dihydrotestosterone) propionate. J Endocrinol 56:155, 1973
FERTILITY AND STERILITY Copyright
~
1978 The American Fertility Society
PITUITARY GONADOTROPIN RESPONSIVENESS WITH DANAZOL*t
JOHN M. SHANE, M.D.:j: RICHARD KATES, M.D. ROBERT L. BARBIERI, M.D. ROBERTA B. TODD, M.S. I. J. DAVIES, M.D.
Department of Obstetrics and Gynecology, Beth Israel Hospital and Boston Hospital for Women, Harvard Medical School, Boston, Massachusetts 02115
Danazol (17a-pregn-4-en-20-yno-[2,3-dlisoxazol-I7-oV was administered daily for 4 days to castrated female rats. As previously demonstrated, danazollowered serum levels of luteinizing hormone (LH) in an apparent dose-dependent fashion. Animals which received danazol in a dose sufficient to lower serum LH responded to administered LH-releasing hormone (LHRH) with increases in serum LH levels which were not diminished as compared with those of control animals. Although these experiments do not preclude an effect of danazol directly on the pituitary, the results indicate that this agent probably lowers serum LH primarily by inhibition of hypothalamic LHRH secretion.
Danazol, a new and potentially valuable pharmacologic agent, is the isoxazol derivative of 17aethinyl testosterone (17a-pregn-4-en-20-yno-[2,3dlisoxazol-17-oD. This steroid has had human and animal trials which have demonstrated its ability to lower luteinizing hormone (LH) and folliclestimulating hormone (FSH) levels; it has therefore been described as an "antigonadotropin." This effect has been utilized in the clinical management of endometriosis, 1 precocious puberty,2 and breastdisorders.2 Any knowledge regarding its mechanism of action should enable us to administer the agent to our patients with more confidence and intelligence. In human studies, danazol, at varying dosages, depressed plasma levels of testosterone and androstenedione as well as the gonadotropins. 3 In rats, rabbits, and mice, studies have confirmed the ability of danazol to lower serum LH with little or no biologic activity as a progestin, antipro-
gestin, estrogen,or antiestrogen. Mild androgenic activity has been demonstrated at higher doses. 2. 4 The mechanism by which danazol lowers circulating LH and FSH levels has not been elucidated. This action could be postulated to result' from inhibition of pituitary responsiveness to hypothalamic LH-releasing hormone (LHRH) or to a diminution of hypothalamic stimulation of the pituitary. In the present experiment, castrated female rats were chronically treated with danazol and then challenged intraperitoneally with LHRH. The subsequent LH responses. were measured by radioimmunoassay and compared with those of appropriate controls.
MATERIALS AND METHODS
Sixty-day-old female Sprague-Dawley"derived rats from Charles River Laboratories were ovariectomized under sodium pentobarbital anesthesia 6 weeks before experimentation. The animals were maintained in a controlled environment (22 C, 14 hours of light, 10 hours of darkness) on Purina laboratory chow and tap water ad libitum; they were housed three per cage.
Received January 30, 1978; accepted February 15, 1978. *Supported by Rockefeller Foundation Grant RF72077 and United States Public Health Service Grant HD07923-03. tThis paper is the recipient of fIrst prize in. the Annual Boston Obstetrical Society Prize Paper Competition. :j:Reprintrequests: Dr. John M. Shane, 6465 South Yale, Tulsa, Okla. 74136.
0
637
June 1978
SHANE ET AL.
638
At the time of the experimentation, the rats weighed between 300 and 420 gm. They were placed in weight-matched groups, seven animals in each group. The danazol was suspended in a 10% ethanol-corn oil mixture. This mixture, in dosages of 200 mg of danazol/kg or 20 mg of danazollkg, or vehicle alone, was administered in a 3-ml volume, by gavage, once daily for 4 consecutive days. Four hours after the last feeding, each rat was given 100 mg of LHRH intraperitoneally, dissolved in 0.2 ml of 0.9% saline or the vehicle alone. This dose of LHRH as been shown to be below the dose that elicits the maximal (plateau) response to that hormone. 5 One hour after LHRH administration, under 50% CO 2-50% O2 anesthesia, blood was obtained by a single cardiac puncture. All animals were killed between 2:30 and 3:30 P.M. Blood was allowed to remain at room temperature for 0.5 hour and then was stored for 1 hour at 4 0 C to allow clot retraction, at which time the serum was removed and stored at -20 C. All determinations of luteinizing hormone were performed in duplicate in a single assay by the ovine-ovine double-antibody radioimmunoassay. 6, 7 Ovine LH was labeled with 1251. The reference standard was 'NIH LH-S16. The intra-assay coefficient of variation was less than 5%. Statistical analysis was performed by using a t-test with Dunnett's tables. The animals were separated into experimental groups as follows: group 1--control, no treatment; group 2--control, treated with corn oil gavage and intraperitoneal saline; group 3--control, corn oil gavage and intraperitoneal LHRH; group 4control, treated with low-dose'(20 mg/kg) danazol and intraperitoneal saline; group 5-control, treated with high-dose (200 mglkg) danazol and intraperitoneal saline; group 6-treatment group, low-dose danazol gavage and intraperitoneal LHRH; group 7-treatment group, high-dose danazol gavage and intraperitoneal LHRH. 0
RESULTS
The results are summarized in Figure 1. Danazol lowered serum LH levels in female castrated rats in an apparent dose-dependent fashion. In the low-dose danazol treatment group (20 mg/kg) the mean serum LH level was lowered by 41% as compared with that of the control groups. However, the absolute difference between control and low-dose groups was not statistically significant rp > 0.05). At high-dosage treatment
40
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FIG. 1. LH response by castrated female rats in indicated treatment groups.
(200 mglkg), LH was lowered by 90% as compared with control groups. The absolute difference between control and high-dosage groups was statistically significant rp < 0.01). After challenge with LHRH, there was no significant difference in mean LH levels among the three groups. Of the three treatment groups, the high-dose danazol group showed the greatest absolute increase in LH levels after LHRH stimulation. DISCUSSION
Our results support previous clinical and laboratory findings that danazol is capable of lowering serum LH. Moreover, under the conditions of these experiments, the decrease in serum LH induced by danazol was not associated with a diminished pituitary responsiveness to LHRH. Although these experiments do not preclude an effect of danazol directly on the pituitary, the results support the hypothesis that this agent lowers serum LH primarily by inhibition of hypothalamic secretion of LHRH. Acknowledgments. We would like to thank Dr. Leo E. Reichert, Jr., for the ovine LH used for radioiodination; Dr. Gordon Niswender for the anti-ovine LH #15; and the National Institute of Arthritis, Metabolism and Digestive
Vol. 29, No.6
PITUITARY GONADOTROPIN RESPONSIVENESS WITH DANAZOL
Diseases for the ovine Nlli-LH-16 standard. Appreciation is also extended to the Sterling-Winthrop Research Institute for their contribution of danazol. REFERENCES 1. Dmowski WP, Cohen MR: 'rreatment of endometriosis
with an antigonadotropin, danazol: a laparoscopic and histologic evaluation. Obstet Gynecol 46:147, 1975 2. Greenblatt RB, Dmowski WP, Mahesh VB, Scholer HFL: Clinical studies with an antigonadotropin-danazol. Fertil Steril 22:102, 1971 3. Sherins RJ, Gandy HM, Thorslund TW, Paulsen CA: Pituitary and testicular function studies: experience with a new gonadal inhibitor, 17a-pregn-4-en-20-yno-(2,3-d)isoxazol-17-01 (danazol). J Clin Endocrinol Metab 32:522, 1971
639
4. Dmowski SP, Scholar HFL, Mahesh VB, Greenblatt RB: Danazol-a synthetic steroid derivative with interesting physiologic properties. Fertil Steril 22:9, 1971 5. Mueh J, Shane JM, Naftolin F: The effects of methallibure (lCI 33,828) on LH release in castrate male rats challenged with LHRH. Endocrinology 97:493,1975 6. Niswender GD, Midgely AR Jr, Monroe SE, Reichert LE Jr: Radioimmunoassay for rat luteinizing hormone with antiovine LH serum and ovine LH'3'1. Proc Soc Exp BioI Med 128:807, 1968 7. Naftolin F, Feder HH: Suppression ofluteinizing hormone secretion in male rats by 5a-androstan-17,6-01-3-one (dihydrotestosterone) propionate. J Endocrinol 56:155, 1973
