Effects of Morphine and Methadone on Serum Testosterone and Luteinizing Hormone Levels and on the Secondary Sex Organs of the Male Rat THEODORE J. CICERO,1 EDWARD R. MEYER, ROY D. BELL, AND GARY A. KOCH2 Department of Psychiatry, Washington University School of Medicine, St. Louis, Missouri 63110 hormone (FSH) were unaltered. On the basis of these observations, it seems reasonable to conclude that the narcotics inhibit the secretion of LH, by an action either in the hypothalamus (e.g., suppression of LH-releasing hormone) or directly in the pituitary gland, which leads to a reduction in serum testosterone levels and a subsequent reduction in the wet-tissue weight and secretory activity of the secondary sex organs. (Endocrinology 98: 367, 1976)
ABSTRACT. The effects of morphine and methadone on the endocrine control of the male rat's sexual function were examined. The results indicate that these narcotics markedly reduce the structural and functional integrity of the secondary sex organs by producing a pronounced reduction in serum testosterone levels. Serum levels of luteinizing hormone (LH) were not detectable in narcotic-treated animals, whereas serum levels of the follicle stimulating
T
male heroin or methadone users (12,13), in contrast to the other reports (7-11). The purpose of the studies described in this paper was to attempt to answer a number of questions raised in our earlier studies. The following issues were examined: a) What is the time course for morphine's effects on serum testosterone levels and the wet tissue weights of the secondary sex organs? b) Do narcotics other than morphine adversely affect the secondary sex organs and lower serum testosterone levels? c) Are the effects of the morphine on androgen levels and secondary sex organ functions similar to those produced by castration? d) Does testosterone reverse the effects of morphine on the secondary sex organs? e) Are the effects of the morphine on testosterone levels and secondary sex organ function influenced by the age of the rat? Two additional studies were carried out in an effort to determine at what point along the hypothalamic-pituitary-gonadal axis the narcotics might exert their principal effects. In the first study, hypophysectomized animals were maintained on human chorionic gonaReceived July 22, 1975. dotropin (hCG) until testosterone levels and This research was supported in part by USPHS grants secondary sex organ weights returned to DA-00259 and DA-01407. normal levels. The animals then were 1 Recipient of Research Scientist Development treated with morphine. The rationale for this Award MH-70180. 2 study was that if the narcotics disrupted the Research Fellow supported by grant MH-05938.
HE EFFECTS of narcotics on neuroendocrine systems have not been rigorously evaluated. However, there has been considerable recent interest in the effects of morphine, and other narcotics, on the endocrine control of sexual function (1-4). In several studies conducted in this laboratory (5-7), we have observed that chronic narcotic administration produces a marked atrophy of the secondary sex organs and a suppression of plasma testosterone levels in the male rat. In subsequent studies in the human male (7), it was found that long-term methadone administration markedly impaired the function of the secondary sex organs and depressed testosterone levels. Other investigators have reported a methadone-induced reduction in serum testosterone levels in the human (8-10) and, in addition, Martin et al. (11) have also demonstrated decreased gonadotropin secretion during methadone maintenance. It should be noted, however, that Cushman was unable to demonstrate a reduction in either luteinizing hormone or testosterone levels in
367
368
CICERO, MEYER, BELL AND KOCH
hypothalamic-pituitary-gonadal axis at the level of either the testes or the secondary sex organs, then morphine should still reduce testosterone levels and/or the secondary sex organ function in this preparation. If, on the other hand, the narcotics exerted their effects in the hypothalamus or pituitary gland, morphine should be ineffective in these animals. The final study was conducted to assess more directly the effects of morphine on pituitary function, specifically on the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Materials and Methods Animals and chemicals. Male rats of the Holtzman strain were used in all studies except those employing hypophysectomized animals. In these studies, hypophysectomized SpragueDawley rats were obtained from the Zivic-Miller Laboratories, Allisson Park, Pennsylvania. Morphine and placebo pellets suitable for implantation were formulated by the Private Formulae Company, St. Louis, Mo. (14,15). A formulated diet (16) suitable for hypophysectomized animals was purchased from General Biochemicals Inc., Chagrin Falls, Ohio. In addition, the hypophysectomized rats drank as their sole source of fluid, a solution which contained, per liter: 2030 mg NaCl, 85.75 mg KC1, 36.0 mg CaCl2, 154.0 mg MgSO4-7H2O, 1.0 mg ZnCl2, 0.062 mg FeCl2, 0.125 mg CuSo4-5H2O, 50 g sucrose, and 2.5 mg cortisone. Morphine pellet implantation. Morphine or placebo pellets were implanted subcutaneously, according to procedures described elsewhere (15). In all experiments, the animals were sacrificed 3 days later. Morphine and methadone injection regimens. In several studies a regimen of morphine or methadone injections was employed as the chronic drug delivery system. The following subcutaneous injection schedule was used for morphine: 10 mg/kg twice daily for 2 days; 20 mg/kg twice daily for 2 days; 50 mg/kg twice daily for 2 days; 75 mg/kg twice daily for 2 days; 100 mg/kg twice daily for 2 days; and 150 mg/kg twice daily for 5 days. For methadone, the injection sequence was: 5 mg/kg twice daily for 5 days; 7.5 mg/kg twice daily for 5 days, and 10 mg/kg twice
Endo c 1976 Vol 98 < No 2
daily for 10 days. Control rats were injected with saline on the same schedule. Hypophysectomy studies. Hypophysectomized rats upon arrival in the laboratory were immediately placed on the special diet and solution described above. Nine days after their arrival, rats from the hypophysectomized and sham-operated groups were sacrified and the weights of the secondary sex organs and plasma testosterone levels were determined to establish the adequacy of the hypophysectomy. On the 10th day, each rat in the hypophysectomized group was injected subcutaneously with 10 IU (3.3 fig) hCG in 0.9% saline. When normal testicular and secondary sex organ function had been restored, the rats were implanted with morphine or placebo pellets. hCG injections continued throughout the three-day pellet implantation period in the hypophysectomized groups. All animals were killed at the end of the three-day pellet implantation period. Organ extraction, testosterone, LH and FSH assays. Rats were decapitated and blood was collected from the carcasses. The blood was allowed to stand for 4 hours at 4 C, was then centrifuged at 2,500 RPM for 30 min, and the sera was stored at —20 C. Serum testosterone levels were determined by a radioimmunoassay described elsewhere (5,6). Reagents for the LH and FSH radioimmunoassays were generously provided by the NIAMDD Rat Pituitary Hormone Distribution Program. The radioimmunoassays for LH and FSH have been described elsewhere (17,18). The following organs were also obtained: brain, liver, spleen, testes, seminal vesicles, prostate gland, and the epididymis.
Results Time course of morphine's effects. Rats were implanted with morphine pellets and were then killed at various intervals. The results of these studies are presented in Table 1. Within 6 hours of pellet implantation, testosterone levels were reduced by about 70%. Testosterone levels continued to decline slowly over the next 3 days, at which time they were 77% lower than control levels. There was no decrease in the wet tissue weights of the seminal vesicles which could be correlated with the large decrease in testosterone levels observed 6 hours after
NARCOTICS AND SEXUAL ENDOCRINOLOGY TABLE 1. The
369
mean ±: SEM1 plasma testosterone levels and wet tissue weights of the seminal vesicles in rats at various intervals after morphine pellet implantation Post-implantation interval 6h
0 Testosterone ng/ml Seminal Vesicles mg
3.02 ±
.54
132.3 ±23.1
0.95* ± 155.9
.11
±12.7
24 h
48 h
0.82* ±.24
0.68* ±.19
84.1* ±9.4
72 h
81.1* ±4.4
0.71*
±.08
77.5* ±11.9
* P < .001. 1 Ten rats were examined at each time interval.
pellet implantation. However, by 24 hours, the weights of the seminal vesicles were reduced by 36% in comparison with controls. Over the next 2 days the wet tissue weights of the seminal vesicles declined only slightly. The time course of morphine's effects on the prostate and epididymis (not shown) were identical to that described above for the seminal vesicles. There was no effect of morphine on any of the other organs examined in this or subsequent studies. Comparison of morphine's effects with castration. Rats were divided into four equal groups: castrated, sham-operated, and unoperated animals implanted with either moiphine or placebo pellets, and killed 3 days later. The results of the studies are presented in Table 2. Castration, as expected, markedly reduced serum testosterone levels and also the wet tissue weights of the seminal vesicles and prostate glands. Morphine also depleted testosterone levels and reduced the weights of the secondary sex organs, although neither effect was as great as that observed in the castrates. Testosterone replacement. Morphine pellet-implanted rats were given subcutaneous injections of either sesame oil or testosterone (0.10 mg/kg, subcutaneously), suspended in sesame oil, throughout the 3-day period of pellet-implantation. Additional animals were implanted with either morphine or placebo pellets but were not given testosterone. The results of this study are shown in Table 3. In those rats implanted
with morphine pellets only, the weights of the secondary sex organs were markedly lower than the placebo levels. In contrast to these data, however, there was no reduction in the secondary sex organ weights in morphine-implanted rats treated with testosterone. Effects of age on the secondary sex organs. Rats were injected with morphine for 15 days beginning at 30,45,60, or 90 days of age. The results of this study are shown in Fig. 1. Data are presented for only the 45, 60, and 90-day-old animals since morphine was extremely toxic in the 30-day-old animals and a large number died prior to completion of the study. At the end of the 15-day injection regimen, serum testosterone levels were significantly lower (P < .001) than control levels in all age groups. In both the 60 and 90-day-old groups, testosterone levels declined to about 20% of control levels within 5 days of morphine treatment and remained at approximately the same TABLE 2. The effects of castration and morphine-pellet implantation on the mean ± SEM wet weights of the seminal vesicles and prostate glands and serum testosterone levels of the rat Croup
N
Seminal vesicles mg
Prostate mg
Testosterone ng/ml
Castrates Moiphine Placebo!
7 8 17
82.3 ± 9.2f 105.1 ± 7.61 145.6 ± 6.2
77.4 ± 4.6f 123.4 ± 6.8* 145.1 ± 4.6
0.2J ± 0.02f 0.59 ± 0.48f 4.11 ± 0.67
* P < .05 when compared with placebo. t P < .01 when compared with placebo. \ The data obtained from placebo-implanted sham-operated and unoperated animals were pooled since there were no differences between the two groups.
CICERO, MEYER, BELL AND KOCH
370
the 45-day-old animals than in the 60 and 90-day-old rats (F < .01), particularly by the end of the 15-day morphine injection regimen.
TABLE 3. The effects of testosterone (O.lOmg/kg) on the mean ± SEM wet weights of the seminal vesicles, prostate glands, and testes of rats implanted with morphine or placebo pellets for 3 days Organs
Group
Seminal vesicles
Prostate glands
Testes
mg
mg
ing
N
Placebo'
135.2 ± 3.8
16
Morphine
11
Morphine plu < testosterone.
11
86.9 ± 4.8* f 145.8 ± 6.7
136.5 ± 13.2
2,790 ± 80
110.3 ± 7.6*t
2,840 ±70
156.0 ± 14.8
2,796 ± 80
Endo • 1976 Vol 98 • No 2
Effects of methadone on the secondary sex organs and serum testosterone levels. The effects of a chronic series of methadone injections on the secondary sex organs and serum testosterone levels are shown in Table 4. Within 10 days, methadone produced a significant reduction in the weights of the secondary sex organs and serum testosterone levels. These effects were further enhanced by the 20th day of treatment.
* P < .05 when com pared with plaeelx f P < .05 w len com pared with morphine plus testos terone. 1 The place «> value< represent a pool o(the data deri ved from placeboimplanted mt.s which i eceived testosterone and those \\ hich did not since there were IK dirt'erei ces between the t vo groups.
level throughout the remainder of the study. In contrast to these findings, testosterone levels declined slowly in the 45-day-old rats during the course of morphine administration, reaching a low point of 35% of control levels by the 15th day of treatment. Regarding the weight of the secondary sex organs, the effect of morphine appeared to be inversely related to the age of the animal. That is, moiphine reduced the weight of the secondary sex organs to a greater degree in
Effects of hypophysectomy on morphineinduced secondary sex organ atrophy. The effects of moiphine on the weights of the seminal vesicles, prostate glands, and testes of hypophysectomized animals, are shown in Table 5. Morphine produced the expected reduction in the weight of the secondary sex organs in sham-operated rats. In contrast to these results, however, morphine was completely ineffective in altering the weight of
100
A
45 Day
^
60 Day
90 Day
80-
260 z o
^x
FIG. 1. The effect of a chronic series of morphine injections on serum testosterone levels and the weights of the prostate glands and seminal vesicles in male rats. Data are presented for animals (N = 10 at each time interval) killed 5, 10, and 15 days after the initiation of the injections. Control levels of testosterone (ng/ml) for the 45, 60, and 90-day-old animals were 2.6 ± 0.3, 4.3 ± 1.1, and 4.7 ± 1.2, respectively.
\
u O40Io Testosterone • Seminal Vesicle & Prostate Gland
20-
•
5
i
10
-
i
X
1
1
1
1
15 5 10 15 5 DAYS ON MORPHINE INJECTION
x
x->
1
1
10
15
NARCOTICS AND SEXUAL ENDOCRINOLOGY TABLE 4. The effects of methadone on the mean ± SEM wet tissue weights of the seminal vesicles and prostate glands and the serum testosterone levels in the rat N
Seminal vesicles
Prostate
Testosterone
10 claws Saline Methadone
10 9
101.0 ± 4.6 77.4 ± 6.7*
127.8 ± 11.6 82.6 ± 8.9*
6.4 ± 1.9 0.7 ± 0.2t
20 claws Saline Methadone
10 8
156.0 ± 6.9 94.4 ± 9.8t
205.9 ± 19.5 119.7 ± 21.5f
5.1 ± 0 . 8 1.4 ± 0.3t
P < .05.
the secondary sex organs in hypophysectomized animals which received supplemental hCG injections. Effects of morphine on LH and FSH levels. The effects of a chronic series of morphine injections on serum levels of LH and FSH are presented in Table 6. As can be seen LH was not detectable in the serum obtained from morphine-treated animals. The serum levels of FSH were unaltered by chronic morphine injections. Discussion The results of these studies indicate that morphine and methadone markedly reduce the weights of the secondary sex organs in the male rat via a ^reduction in serum testosterone levels. This conclusion is based on the following observations: a) morphine's effects on the weight, secretory activity, and cytoarchitectural features of the secondary sex organs are virtually identical to those produced by castration (19); b) after the administration of morphine, the first change observed in morphine-treated rats was a sharp drop in serum testosterone levels which was followed some time later, by a reduction in the weight of the secondary sex organs. These data suggest that a reduction in serum testosterone levels is a necessary intermediate step in morphine's subsequent effects on the weight of the secondary sex organs; c) testosterone reversed morphine's effects on the wet-tissue weights of the secondary sex organs. On the basis of these
371
three observations, it seems reasonable to conclude that morphine produces its effects via a reduction in serum testosterone levels. Although the mechanism underlying morphine's effects on serum testosterone levels cannot be ascertained from the studies described in this paper, our data are consistent with the interpretation that the narcotics depress the hypothalamic or pituitary factors associated with the release of testosterone. This conclusion is supported, at least in part, by our findings that morphine did not adversely affect the function of the secondary sex organs in hypophysectomized animals in which normal testicular function had been restored by treatment with hCG. These data suggest that a peripheral action of the narcotics probably cannot account for the observed effects, although it would be unwise at present to rule it out completely. More direct evidence for a central action of TABLE 5. The mean ± SEM wet tissue weights of the seminal vesicles, prostate glands, and testes in morphine and placebo implanted sham-operated rats, and in morphine and placebo-implanted hypophysectomized (Hypox) rats which received daily injections of hCG Seminal vesicles
Prostate
Testes
N
mg
mg
mg
S hammorphine
10
64.1 ± 3.7*
109.6 ± 6.1*
2,418 ± 38
Shamplacebo
10
90.7 ± 7.3
156.9 ± 10.9
2,278 ± 112
HypoxmorphinehCC
10
98.1 ± 12.0
155.0 ± 13.4
717 ± 30
HypoxplacebohCG
10
100.4 ± 6.7
129.5 ± 7.87
794 ± 61
*P < .01 when compared with respective control.
TABLE 6. The effects of morphine on mean ± SEM serum LH and FSH levels in rats treated with a chronic series (15 days) or morphine or saline injections Group
N
LH ng/ml
FSH ng/ml
Morphine Saline
10
N.D.f 18.2 ± 5.2
756.4 ± 50.7 808.8 ± 34.4
f N.D. = not detectable. Lower limit detectable with this assay is approximately 4.5 ng/ml.
372
CICERO, MEYER, BELL AND KOCH
morphine, on either the pituitary or hypothalamus, was provided by the findings that morphine significantly depleted serum LH levels. Consequently, it seems likely that the narcotics in some way block the release of LH which leads to a reduction in serum testosterone levels and a subsequent reduction in the weights of the secondary sex organs. Whether the narcotics inhibit LH secretion by an action in the hypothalamus (e.g., suppression of the LH-releasing factor) or in the pituitary is unknown, at present, but is currently under investigation. The observation that serum levels of only LH, and not FSH, were reduced by chronic morphine administration is somewhat puzzling, particularly if only one hypothalamic releasing factor exists for these two gonadotropins. These findings either suggest that LH-RF and FSH-RF are indeed different hormones or that the narcotics directly interfere with the secretion of LH, but not FSH, by the pituitary. On the basis of currently available information it is not possible to choose between these two alternative explanations. The effects of narcotics on serum testosterone levels and the weights of the secondary sex organs, appears to be dependent upon the age of the animal. That is, in the present studies, serum testosterone levels were reduced to a greater degree in older animals than they were in younger ones, whereas the weights of the secondary sex organs were less affected in 60 and 90-dayold animals than in the 45-day-old animals. In conclusion, the data presented in this paper indicate that the narcotics lower testosterone levels in the male rat, and subsequently the wet tissue weights of the
Endo • 1976 Vol 98 • No 2
secondary sex organs, by interfering in some way with normal hypothalamic-pituitary function. References 1. Barraclough, C , and C. Sawyer, Endocrinology 57: 329, 1955. 2. Sawyer, C , In Nalbandov, A. (ed.), Advances in Neuroendocrinology, University of Illinois Press, Urbana, 1963, p. 444. 3. Wikler, A., In Baker, A. (ed.), Clinical Neurology, vol. 2, Harper and Row, New York, 1971, p. 1. 4. George, R., In Clouet, D. (ed.), Narcotic Drugs: Biochemical Pharmacology, Plenum Press, New York, 1971, p. 283. 5. Cicero, T. J., E. R. Meyer, R. Bell, and W. G. Wiest, Res Commun Chem Pathol Pharmacol 7: 17, 1974. 6. Cicero, T. J., E. R. Meyer, W. G. Wiest, J. W. Olney, and R. D. Bell, / Pharmacol Exp Ther 192: 542, 1975. 7. Cicero, T. J., R. D. Bell, W. G. Wiest, J. H. Allison, K. Polakoski, and E. Robins, N Engl J Med 292: 882, 1975. 8. Azizi, F., A. G. Vagenakis, C. Longcope, S. H. Ingbar, and L. E. Braverman, Steroids 22: 467, 1973. 9. Mendelson, J. H., J. E. Mendelson, and V. D. Patch, Fed Proc 33: 232, 1974 (abstract). 10. Mendelson, J. H., J. E. Mendelson, and V. D. Patch, J Phannacol Exp Ther 192: 211, 1975. 11. Martin, W. R., D. R. Jasinski, C. A. Haertzen, D. C. Kay, B. E. Jones, P. A. Mansky, and R. W. Carpenter, Arch Gen Psychiat 28: 286, 1973. 12. Cushman, P., AmJ Med 55: 452, 1973. 13. Cushman, P., N Y State J Med 72: 1261, 1972. 14. Gibson, R., and J. TingstadJ Pharm Sci 59: 426, 1970. 15. Cicero, T. J., and E. R. Meyer, J Pharmacol Exp Ther 184: 404, 1973. 16. Shaw, J. H., and R. O. Greep, Endocrinology 44: 520, 1949. 17. Midgley, A., Endocrinology 79: 10, 1966. 18. Midgley, A.JClinEndocrinolMetab27: 295,1967. 19. Moore, C. R., In Allen, E., C. H. Danforth and E. A. Doisy (eds.), Sex and Internal Secretions, Williams and Wilkins, Baltimore, 1939, p. 389.
ABSTRACT. The effects of morphine and methadone on the endocrine control of the male rat's sexual function were examined. The results indicate that these narcotics markedly reduce the structural and functional integrity of the secondary sex organs by producing a pronounced reduction in serum testosterone levels. Serum levels of luteinizing hormone (LH) were not detectable in narcotic-treated animals, whereas serum levels of the follicle stimulating
T
male heroin or methadone users (12,13), in contrast to the other reports (7-11). The purpose of the studies described in this paper was to attempt to answer a number of questions raised in our earlier studies. The following issues were examined: a) What is the time course for morphine's effects on serum testosterone levels and the wet tissue weights of the secondary sex organs? b) Do narcotics other than morphine adversely affect the secondary sex organs and lower serum testosterone levels? c) Are the effects of the morphine on androgen levels and secondary sex organ functions similar to those produced by castration? d) Does testosterone reverse the effects of morphine on the secondary sex organs? e) Are the effects of the morphine on testosterone levels and secondary sex organ function influenced by the age of the rat? Two additional studies were carried out in an effort to determine at what point along the hypothalamic-pituitary-gonadal axis the narcotics might exert their principal effects. In the first study, hypophysectomized animals were maintained on human chorionic gonaReceived July 22, 1975. dotropin (hCG) until testosterone levels and This research was supported in part by USPHS grants secondary sex organ weights returned to DA-00259 and DA-01407. normal levels. The animals then were 1 Recipient of Research Scientist Development treated with morphine. The rationale for this Award MH-70180. 2 study was that if the narcotics disrupted the Research Fellow supported by grant MH-05938.
HE EFFECTS of narcotics on neuroendocrine systems have not been rigorously evaluated. However, there has been considerable recent interest in the effects of morphine, and other narcotics, on the endocrine control of sexual function (1-4). In several studies conducted in this laboratory (5-7), we have observed that chronic narcotic administration produces a marked atrophy of the secondary sex organs and a suppression of plasma testosterone levels in the male rat. In subsequent studies in the human male (7), it was found that long-term methadone administration markedly impaired the function of the secondary sex organs and depressed testosterone levels. Other investigators have reported a methadone-induced reduction in serum testosterone levels in the human (8-10) and, in addition, Martin et al. (11) have also demonstrated decreased gonadotropin secretion during methadone maintenance. It should be noted, however, that Cushman was unable to demonstrate a reduction in either luteinizing hormone or testosterone levels in
367
368
CICERO, MEYER, BELL AND KOCH
hypothalamic-pituitary-gonadal axis at the level of either the testes or the secondary sex organs, then morphine should still reduce testosterone levels and/or the secondary sex organ function in this preparation. If, on the other hand, the narcotics exerted their effects in the hypothalamus or pituitary gland, morphine should be ineffective in these animals. The final study was conducted to assess more directly the effects of morphine on pituitary function, specifically on the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Materials and Methods Animals and chemicals. Male rats of the Holtzman strain were used in all studies except those employing hypophysectomized animals. In these studies, hypophysectomized SpragueDawley rats were obtained from the Zivic-Miller Laboratories, Allisson Park, Pennsylvania. Morphine and placebo pellets suitable for implantation were formulated by the Private Formulae Company, St. Louis, Mo. (14,15). A formulated diet (16) suitable for hypophysectomized animals was purchased from General Biochemicals Inc., Chagrin Falls, Ohio. In addition, the hypophysectomized rats drank as their sole source of fluid, a solution which contained, per liter: 2030 mg NaCl, 85.75 mg KC1, 36.0 mg CaCl2, 154.0 mg MgSO4-7H2O, 1.0 mg ZnCl2, 0.062 mg FeCl2, 0.125 mg CuSo4-5H2O, 50 g sucrose, and 2.5 mg cortisone. Morphine pellet implantation. Morphine or placebo pellets were implanted subcutaneously, according to procedures described elsewhere (15). In all experiments, the animals were sacrificed 3 days later. Morphine and methadone injection regimens. In several studies a regimen of morphine or methadone injections was employed as the chronic drug delivery system. The following subcutaneous injection schedule was used for morphine: 10 mg/kg twice daily for 2 days; 20 mg/kg twice daily for 2 days; 50 mg/kg twice daily for 2 days; 75 mg/kg twice daily for 2 days; 100 mg/kg twice daily for 2 days; and 150 mg/kg twice daily for 5 days. For methadone, the injection sequence was: 5 mg/kg twice daily for 5 days; 7.5 mg/kg twice daily for 5 days, and 10 mg/kg twice
Endo c 1976 Vol 98 < No 2
daily for 10 days. Control rats were injected with saline on the same schedule. Hypophysectomy studies. Hypophysectomized rats upon arrival in the laboratory were immediately placed on the special diet and solution described above. Nine days after their arrival, rats from the hypophysectomized and sham-operated groups were sacrified and the weights of the secondary sex organs and plasma testosterone levels were determined to establish the adequacy of the hypophysectomy. On the 10th day, each rat in the hypophysectomized group was injected subcutaneously with 10 IU (3.3 fig) hCG in 0.9% saline. When normal testicular and secondary sex organ function had been restored, the rats were implanted with morphine or placebo pellets. hCG injections continued throughout the three-day pellet implantation period in the hypophysectomized groups. All animals were killed at the end of the three-day pellet implantation period. Organ extraction, testosterone, LH and FSH assays. Rats were decapitated and blood was collected from the carcasses. The blood was allowed to stand for 4 hours at 4 C, was then centrifuged at 2,500 RPM for 30 min, and the sera was stored at —20 C. Serum testosterone levels were determined by a radioimmunoassay described elsewhere (5,6). Reagents for the LH and FSH radioimmunoassays were generously provided by the NIAMDD Rat Pituitary Hormone Distribution Program. The radioimmunoassays for LH and FSH have been described elsewhere (17,18). The following organs were also obtained: brain, liver, spleen, testes, seminal vesicles, prostate gland, and the epididymis.
Results Time course of morphine's effects. Rats were implanted with morphine pellets and were then killed at various intervals. The results of these studies are presented in Table 1. Within 6 hours of pellet implantation, testosterone levels were reduced by about 70%. Testosterone levels continued to decline slowly over the next 3 days, at which time they were 77% lower than control levels. There was no decrease in the wet tissue weights of the seminal vesicles which could be correlated with the large decrease in testosterone levels observed 6 hours after
NARCOTICS AND SEXUAL ENDOCRINOLOGY TABLE 1. The
369
mean ±: SEM1 plasma testosterone levels and wet tissue weights of the seminal vesicles in rats at various intervals after morphine pellet implantation Post-implantation interval 6h
0 Testosterone ng/ml Seminal Vesicles mg
3.02 ±
.54
132.3 ±23.1
0.95* ± 155.9
.11
±12.7
24 h
48 h
0.82* ±.24
0.68* ±.19
84.1* ±9.4
72 h
81.1* ±4.4
0.71*
±.08
77.5* ±11.9
* P < .001. 1 Ten rats were examined at each time interval.
pellet implantation. However, by 24 hours, the weights of the seminal vesicles were reduced by 36% in comparison with controls. Over the next 2 days the wet tissue weights of the seminal vesicles declined only slightly. The time course of morphine's effects on the prostate and epididymis (not shown) were identical to that described above for the seminal vesicles. There was no effect of morphine on any of the other organs examined in this or subsequent studies. Comparison of morphine's effects with castration. Rats were divided into four equal groups: castrated, sham-operated, and unoperated animals implanted with either moiphine or placebo pellets, and killed 3 days later. The results of the studies are presented in Table 2. Castration, as expected, markedly reduced serum testosterone levels and also the wet tissue weights of the seminal vesicles and prostate glands. Morphine also depleted testosterone levels and reduced the weights of the secondary sex organs, although neither effect was as great as that observed in the castrates. Testosterone replacement. Morphine pellet-implanted rats were given subcutaneous injections of either sesame oil or testosterone (0.10 mg/kg, subcutaneously), suspended in sesame oil, throughout the 3-day period of pellet-implantation. Additional animals were implanted with either morphine or placebo pellets but were not given testosterone. The results of this study are shown in Table 3. In those rats implanted
with morphine pellets only, the weights of the secondary sex organs were markedly lower than the placebo levels. In contrast to these data, however, there was no reduction in the secondary sex organ weights in morphine-implanted rats treated with testosterone. Effects of age on the secondary sex organs. Rats were injected with morphine for 15 days beginning at 30,45,60, or 90 days of age. The results of this study are shown in Fig. 1. Data are presented for only the 45, 60, and 90-day-old animals since morphine was extremely toxic in the 30-day-old animals and a large number died prior to completion of the study. At the end of the 15-day injection regimen, serum testosterone levels were significantly lower (P < .001) than control levels in all age groups. In both the 60 and 90-day-old groups, testosterone levels declined to about 20% of control levels within 5 days of morphine treatment and remained at approximately the same TABLE 2. The effects of castration and morphine-pellet implantation on the mean ± SEM wet weights of the seminal vesicles and prostate glands and serum testosterone levels of the rat Croup
N
Seminal vesicles mg
Prostate mg
Testosterone ng/ml
Castrates Moiphine Placebo!
7 8 17
82.3 ± 9.2f 105.1 ± 7.61 145.6 ± 6.2
77.4 ± 4.6f 123.4 ± 6.8* 145.1 ± 4.6
0.2J ± 0.02f 0.59 ± 0.48f 4.11 ± 0.67
* P < .05 when compared with placebo. t P < .01 when compared with placebo. \ The data obtained from placebo-implanted sham-operated and unoperated animals were pooled since there were no differences between the two groups.
CICERO, MEYER, BELL AND KOCH
370
the 45-day-old animals than in the 60 and 90-day-old rats (F < .01), particularly by the end of the 15-day morphine injection regimen.
TABLE 3. The effects of testosterone (O.lOmg/kg) on the mean ± SEM wet weights of the seminal vesicles, prostate glands, and testes of rats implanted with morphine or placebo pellets for 3 days Organs
Group
Seminal vesicles
Prostate glands
Testes
mg
mg
ing
N
Placebo'
135.2 ± 3.8
16
Morphine
11
Morphine plu < testosterone.
11
86.9 ± 4.8* f 145.8 ± 6.7
136.5 ± 13.2
2,790 ± 80
110.3 ± 7.6*t
2,840 ±70
156.0 ± 14.8
2,796 ± 80
Endo • 1976 Vol 98 • No 2
Effects of methadone on the secondary sex organs and serum testosterone levels. The effects of a chronic series of methadone injections on the secondary sex organs and serum testosterone levels are shown in Table 4. Within 10 days, methadone produced a significant reduction in the weights of the secondary sex organs and serum testosterone levels. These effects were further enhanced by the 20th day of treatment.
* P < .05 when com pared with plaeelx f P < .05 w len com pared with morphine plus testos terone. 1 The place «> value< represent a pool o(the data deri ved from placeboimplanted mt.s which i eceived testosterone and those \\ hich did not since there were IK dirt'erei ces between the t vo groups.
level throughout the remainder of the study. In contrast to these findings, testosterone levels declined slowly in the 45-day-old rats during the course of morphine administration, reaching a low point of 35% of control levels by the 15th day of treatment. Regarding the weight of the secondary sex organs, the effect of morphine appeared to be inversely related to the age of the animal. That is, moiphine reduced the weight of the secondary sex organs to a greater degree in
Effects of hypophysectomy on morphineinduced secondary sex organ atrophy. The effects of moiphine on the weights of the seminal vesicles, prostate glands, and testes of hypophysectomized animals, are shown in Table 5. Morphine produced the expected reduction in the weight of the secondary sex organs in sham-operated rats. In contrast to these results, however, morphine was completely ineffective in altering the weight of
100
A
45 Day
^
60 Day
90 Day
80-
260 z o
^x
FIG. 1. The effect of a chronic series of morphine injections on serum testosterone levels and the weights of the prostate glands and seminal vesicles in male rats. Data are presented for animals (N = 10 at each time interval) killed 5, 10, and 15 days after the initiation of the injections. Control levels of testosterone (ng/ml) for the 45, 60, and 90-day-old animals were 2.6 ± 0.3, 4.3 ± 1.1, and 4.7 ± 1.2, respectively.
\
u O40Io Testosterone • Seminal Vesicle & Prostate Gland
20-
•
5
i
10
-
i
X
1
1
1
1
15 5 10 15 5 DAYS ON MORPHINE INJECTION
x
x->
1
1
10
15
NARCOTICS AND SEXUAL ENDOCRINOLOGY TABLE 4. The effects of methadone on the mean ± SEM wet tissue weights of the seminal vesicles and prostate glands and the serum testosterone levels in the rat N
Seminal vesicles
Prostate
Testosterone
10 claws Saline Methadone
10 9
101.0 ± 4.6 77.4 ± 6.7*
127.8 ± 11.6 82.6 ± 8.9*
6.4 ± 1.9 0.7 ± 0.2t
20 claws Saline Methadone
10 8
156.0 ± 6.9 94.4 ± 9.8t
205.9 ± 19.5 119.7 ± 21.5f
5.1 ± 0 . 8 1.4 ± 0.3t
P < .05.
the secondary sex organs in hypophysectomized animals which received supplemental hCG injections. Effects of morphine on LH and FSH levels. The effects of a chronic series of morphine injections on serum levels of LH and FSH are presented in Table 6. As can be seen LH was not detectable in the serum obtained from morphine-treated animals. The serum levels of FSH were unaltered by chronic morphine injections. Discussion The results of these studies indicate that morphine and methadone markedly reduce the weights of the secondary sex organs in the male rat via a ^reduction in serum testosterone levels. This conclusion is based on the following observations: a) morphine's effects on the weight, secretory activity, and cytoarchitectural features of the secondary sex organs are virtually identical to those produced by castration (19); b) after the administration of morphine, the first change observed in morphine-treated rats was a sharp drop in serum testosterone levels which was followed some time later, by a reduction in the weight of the secondary sex organs. These data suggest that a reduction in serum testosterone levels is a necessary intermediate step in morphine's subsequent effects on the weight of the secondary sex organs; c) testosterone reversed morphine's effects on the wet-tissue weights of the secondary sex organs. On the basis of these
371
three observations, it seems reasonable to conclude that morphine produces its effects via a reduction in serum testosterone levels. Although the mechanism underlying morphine's effects on serum testosterone levels cannot be ascertained from the studies described in this paper, our data are consistent with the interpretation that the narcotics depress the hypothalamic or pituitary factors associated with the release of testosterone. This conclusion is supported, at least in part, by our findings that morphine did not adversely affect the function of the secondary sex organs in hypophysectomized animals in which normal testicular function had been restored by treatment with hCG. These data suggest that a peripheral action of the narcotics probably cannot account for the observed effects, although it would be unwise at present to rule it out completely. More direct evidence for a central action of TABLE 5. The mean ± SEM wet tissue weights of the seminal vesicles, prostate glands, and testes in morphine and placebo implanted sham-operated rats, and in morphine and placebo-implanted hypophysectomized (Hypox) rats which received daily injections of hCG Seminal vesicles
Prostate
Testes
N
mg
mg
mg
S hammorphine
10
64.1 ± 3.7*
109.6 ± 6.1*
2,418 ± 38
Shamplacebo
10
90.7 ± 7.3
156.9 ± 10.9
2,278 ± 112
HypoxmorphinehCC
10
98.1 ± 12.0
155.0 ± 13.4
717 ± 30
HypoxplacebohCG
10
100.4 ± 6.7
129.5 ± 7.87
794 ± 61
*P < .01 when compared with respective control.
TABLE 6. The effects of morphine on mean ± SEM serum LH and FSH levels in rats treated with a chronic series (15 days) or morphine or saline injections Group
N
LH ng/ml
FSH ng/ml
Morphine Saline
10
N.D.f 18.2 ± 5.2
756.4 ± 50.7 808.8 ± 34.4
f N.D. = not detectable. Lower limit detectable with this assay is approximately 4.5 ng/ml.
372
CICERO, MEYER, BELL AND KOCH
morphine, on either the pituitary or hypothalamus, was provided by the findings that morphine significantly depleted serum LH levels. Consequently, it seems likely that the narcotics in some way block the release of LH which leads to a reduction in serum testosterone levels and a subsequent reduction in the weights of the secondary sex organs. Whether the narcotics inhibit LH secretion by an action in the hypothalamus (e.g., suppression of the LH-releasing factor) or in the pituitary is unknown, at present, but is currently under investigation. The observation that serum levels of only LH, and not FSH, were reduced by chronic morphine administration is somewhat puzzling, particularly if only one hypothalamic releasing factor exists for these two gonadotropins. These findings either suggest that LH-RF and FSH-RF are indeed different hormones or that the narcotics directly interfere with the secretion of LH, but not FSH, by the pituitary. On the basis of currently available information it is not possible to choose between these two alternative explanations. The effects of narcotics on serum testosterone levels and the weights of the secondary sex organs, appears to be dependent upon the age of the animal. That is, in the present studies, serum testosterone levels were reduced to a greater degree in older animals than they were in younger ones, whereas the weights of the secondary sex organs were less affected in 60 and 90-dayold animals than in the 45-day-old animals. In conclusion, the data presented in this paper indicate that the narcotics lower testosterone levels in the male rat, and subsequently the wet tissue weights of the
Endo • 1976 Vol 98 • No 2
secondary sex organs, by interfering in some way with normal hypothalamic-pituitary function. References 1. Barraclough, C , and C. Sawyer, Endocrinology 57: 329, 1955. 2. Sawyer, C , In Nalbandov, A. (ed.), Advances in Neuroendocrinology, University of Illinois Press, Urbana, 1963, p. 444. 3. Wikler, A., In Baker, A. (ed.), Clinical Neurology, vol. 2, Harper and Row, New York, 1971, p. 1. 4. George, R., In Clouet, D. (ed.), Narcotic Drugs: Biochemical Pharmacology, Plenum Press, New York, 1971, p. 283. 5. Cicero, T. J., E. R. Meyer, R. Bell, and W. G. Wiest, Res Commun Chem Pathol Pharmacol 7: 17, 1974. 6. Cicero, T. J., E. R. Meyer, W. G. Wiest, J. W. Olney, and R. D. Bell, / Pharmacol Exp Ther 192: 542, 1975. 7. Cicero, T. J., R. D. Bell, W. G. Wiest, J. H. Allison, K. Polakoski, and E. Robins, N Engl J Med 292: 882, 1975. 8. Azizi, F., A. G. Vagenakis, C. Longcope, S. H. Ingbar, and L. E. Braverman, Steroids 22: 467, 1973. 9. Mendelson, J. H., J. E. Mendelson, and V. D. Patch, Fed Proc 33: 232, 1974 (abstract). 10. Mendelson, J. H., J. E. Mendelson, and V. D. Patch, J Phannacol Exp Ther 192: 211, 1975. 11. Martin, W. R., D. R. Jasinski, C. A. Haertzen, D. C. Kay, B. E. Jones, P. A. Mansky, and R. W. Carpenter, Arch Gen Psychiat 28: 286, 1973. 12. Cushman, P., AmJ Med 55: 452, 1973. 13. Cushman, P., N Y State J Med 72: 1261, 1972. 14. Gibson, R., and J. TingstadJ Pharm Sci 59: 426, 1970. 15. Cicero, T. J., and E. R. Meyer, J Pharmacol Exp Ther 184: 404, 1973. 16. Shaw, J. H., and R. O. Greep, Endocrinology 44: 520, 1949. 17. Midgley, A., Endocrinology 79: 10, 1966. 18. Midgley, A.JClinEndocrinolMetab27: 295,1967. 19. Moore, C. R., In Allen, E., C. H. Danforth and E. A. Doisy (eds.), Sex and Internal Secretions, Williams and Wilkins, Baltimore, 1939, p. 389.
