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Melatonin-induced suppression of gonadotropin titers in male golden hamsters: Effect on gonadal feedback mechanisms
Life Sciences, Vol. 28, pp. 243-250 Printed in the U.S.A.
Pergamon Press
MELATONIN-INDUCED SUPPRESSION OF GONADOTROPIN TITERS IN MALE GOLDEN HAMSTERS: EFFEC T ON GONADAL FEEDBACK MECHANISMS ~ Barbara Tate-Ostroff and Milton H. Stetson 2 Physiology Section School of Life and Health Sciences University of Delaware Newark, Delaware 19711 (Received in final form October 31, 1980) Summar[ Daily subcutaneous injections of melatonin cause testicular regression and a decline in circulating gonadotropin levels in male hamsters maintained on long photoperiods. We examine here if a reduction in gonadotropin levels also occurs in castrates administered melatonin and if m e l a t o n i n - r e g r e s s e d hamsters respond to castration with an increased release of pituitary gonadotropins - a typical "castration response." Groups of intact and castrated male hamsters maintained on a photoperiod of LD 14:10 received subcutaneous injections of 15 ug melatonin/day. Controls received vehicle only. After 7 weeks of injedtions the intact males were castrated. All animals were sacrificed a few days later and serum was assayed fom gonadotr0pin titers. Melatonin injections caused a marked decline in serum gonadotropins in castrates and in intact males also caused testicular regression. In the latter, no "castration response" was observed upon removal of the testes. Thus, daily injections "of melatonin depress serum gonadotropins in castrate and intact males and block the castrationassociated rise in circulating gonadotropins in the latter. Adult male hamsters respond to short photoperiods with a dramatic decrease in testes weight (2), preceeded by a decline in serum gonadotropin levels (3) and accompanied by a decline in serum testosterone levels (4). Exposure to short photoperiods also induces a change in the sensitivity of the hypothalamic-pituitary axis to testosterone feedback. Male hamsters castrated after undergoing photoperiod-induced testicular regression often show an attenuated castration response (5,6) or no castration response
IA portion of this work was presented at the Annual Meeting of the American Society of Zoologists (i). This work was supported by NSF Research Grant PCM 78-06664. 2To whom reprint requests should be sent. 0024-3205/81/030243-08502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
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(7,8,9). Less exogenous testosterone is required to suppress the castration response in these animals than is required in castrates maintained on long photoperiods (5). Male hamsters castrated and then maintained on short photoperiods for prolonged periods also show a decline in serum gonadotropin levels, a phenomenon seemingly independent of testicular feedback (9). The pineal gland appears to play a role in the photoperiodic response of the adult male hamster. Pinealectomized, or superior cervical ganglionectomized hamsters do not undergo testicular regression when exposed to short photoperiods (i0). Moreover, pinealectomized male hamsters exposed to short photoperiods do not demonstrate an increased sensitivity to steroid feedback (4). Melatonin, a methoxyindole found in highest concentrations in the pineal gland of hamsters during the night (ll), may be the pineal product affecting the photoperiodic response. Administration of melatonin to adult male hamsters maintained on long photoperiods induces testicular regression and a decline in serum gonadotropin levels provided the melatonin is administered via injection at the proper time of day (12). The site of action of melatonin remains unknown. There are many possible mechanisms through which melatonin may influence gonadal function including a direct effect on one or more of the components of the hypothalamo-hypophysial-testicular axis. At the hypothalamus and the pituitary it might alter steroid feedback sensitivity similar to that seemingly caused by short photoperiods. It might affect the synthesis and/or release from the hypothalamus of gonadotropin releasing hormone (GnRH). It may interfere with the normal action of GnRH or by some other means decrease gonadotropin release from the anterior pituitary. It might block the action of gonadotropins, decrease the number of available gonadotropin binding sites or interfere with steroid production and/or release at the testes. The experiments we describe here were designed to assess the effect of melatonin on testicular feedback mechanisms in hamsters maintained on long photoperiods. Methods For this study we used adult male hamsters (12 weeks old), that were raised in our laboratory on a photoperiod of LD 14:10 (lights 0600-2000) at 22-25°C. The animals were housed 6 per cage and given food and water ad libitum throughout the experiment. For surgery animals were anesthesized with ether. An initial control group was sacrificed at the beginning of the experiment. Melatonin was administered daily via subcutaneous injections of 15 ug in 0.i ml of ethanol:saline (i:i0). Control animals were given ethanol:saline only. Injections were given between 1915-1945 hours, a time previously reported to be effective in inducing testicular regression (12). Animals were sacrificed via decapitation between 1300-1500 hrs and serum was stored frozen for later radioimmunoassay for LH and FSH using rat hormone kits provided by NIAMDD. Minor modifications of the protocols provided with the kits are described in detail (13,14). Pituitaries were collected at the time of sacrifice, pooled by group and immediately placed in acetone (-20°C). Several weeks later they were homogenized in cold 0.01M phosphobuffered saline
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Melatonin and Gonadal Feedback
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(pH7.6; 1 ml P B S / g l a n d ) , d i l u t e d and a s s a y e d w i t h the serum as desc r i b e d above. T e s t e s w e i g h t s and m e a s u r e m e n t s w e r e taken at the time of sacrifice. S t a t i s t i c a l c o m p a r i s o n s w e r e d o n e using the S t u d e n t ' s "t" test. E x p e r i m e n t I. A d u l t m a l e h a m s t e r s m a i n t a i n e d on LD 14:10 w e r e d i v i d e d into two groups. One r e c e i v e d m e l a t o n i n i n j e c t i o n s and the o t h e r v e h i c l e only. E v e r y 14 days six animals from e a c h g r o u p w e r e l a p a r o t o m i z e d and the l e n g t h and w i d t h of the testis w e r e measured. Testis m e a s u r e m e n t s w e r e used to c o m p u t e an index (length x width) by w h i c h r e g r e s s i o n could be d e s c r i b e d q u a n t i t a t i v e l y . After 7 w e e k s of i n j e c t i o n s 6 a n i m a l s from e a c h g r o u p w e r e sacrificed. All o t h e r animals w e r e c a s t r a t e d and c o n t i n u e d to r e c e i v e injections for one w e e k u n t i l they w e r e sacrificed. E x p e r i m e n t II. A d u l t m a l e s h e l d on LH 14:10 w e r e castrated. On the d a y of s u r g e r y i n j e c t i o n s of e i t h e r m e l a t o n i n or v e h i c l e w e r e begun. E v e r y i0 days b l o o d samples w e r e taken by c a r d i a c puncture b e t w e e n 1 3 0 0 - 1 5 0 0 hrs f r o m 6 m e l a t o n i n - i n j e c t e d a n i m a l s and six v e h i c l e - i n j e c t e d animals. At 60 days all animals w e r e sacrificed. Results A n i m a l s s a c r i f i c e d at the b e g i n n i n g of the e x p e r i m e n t h a d m e a n s e r u m LH levels of 42 f ng/ml and m e a n s e r u m FSH levels of 196~81 ng/ ml. P o o l e d p i t u i t a r y LH c o n t e n t for the g r o u p w a s 36 u g / ~ l a n d and F S H c o n t e n t w a s 79 ug/gland. M e a n testes w e i g h t was 2 9 4 5 - 1 9 0 m g and m e a n testis l e n g t h x w i d t h (TLW) was 23857. E x p e r i m e n t I. "Melatonin i n d u c e d a m a r k e d d e c l i n e in t e s t i c u lar w e i g h t as c o m p a r e d to i n i t i a l c o n t r o l a n i m a l s and to saline c o n t r o l a n i m a l s (p<.001). A f t e r o n l y two w e e k s of i n j e c t i o n s and t h r o u g h o u t the r e s t of the e x p e r i m e n t , the TLW of the m e l a t o n i n g r o u p w a s s i g n i f i c a n t l y lower (fig. i) than that of the v e h i c l e -
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Fig. 1 M e a n ±SEM(n=4-6) testes l e n g t h x w i d t h of intact m a l e h a m s t e r s r e c e i v i n g s u b c u t a n e o u s i n j e c t i o n of saline (open bar) or m e l a t o n i n (hatched bar). Anim a l s w e r e l a p o r a t o m i z e d at 2, 4 and 6 w e e k s of treatment. M e a s u r e m e n t s for the initial c o n t r o l g r o u p (IC) and 8 w e e k g r o u p w e r e o b t a i n e d at sacrifice.
246
Melatonin and Gonadal Feedback
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i n j e c t e d group (p<.001). A f t e r 7 w e e k s of i n j e c t i o n s the m e l a t o n i n - i n j e c t e d animals had a m e a n testes w e i g h t of 226~34 mg while the m e a n testes w e i g h t for s a l i n e - i n j e c t e d animals was signific a n t l y h i g h e r (30261151 mg; p<.05). S e r u m LH and FSH levels in initial c o n t r o l animals and in the s a l i n e - i n j e c t e d and m e l a t o n i n - i n j e c t e d animals of E x p e r i m e n t I are d e p i c t e d in figure 2. A f t e r 7 weeks of treatment, m e l a t o n i n i n j e c t e d animals had s i g n i f i c a n t l y lower LH and FSH levels t h a n did saline i n j e c t e d animals. At w e e k 7 the animals w e r e c a s t r a t e d and s a c r i f i c e d one w e e k later. C a s t r a t i o n did not r e s u l t in an i n c r e a s e in s e r u m LH levels in e i t h e r g r o u p w h i l e s e r u m FSH levels w e r e s i g n i f i c a n t l y e l e v a t e d in the saline c o n t r o l group only. The same trends w e r e seen in p i t u i t a r y g o n a d o t r o p i n levels (fig. 4).
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Fig. 2 Mean~SEM(n=5-6) serum LH and FSH levels in h a m s t e r s r e c e i v i n g saline (open bar) or m e l a t o n i n (solid bar) injections. Initial c o n t r o l (IC) animals w e r e s a c r i f i c e d b e f o r e i n j e c t i o n s began. A f t e r 7 w e e k s of i n j e c t i o n s a group of saline i n j e c t e d and m e l a t o n i n i n j e c t e d a n i m a l s w e r e sacrificed; all o t h e r a n i m a l s w e r e c a s t r a t e d and s a c r i f i c e d one w e e k later. L i m i t of d e t e c t a b i l i t y i0 ng/ml (LH) and 284 ng/ml (FSH).
Vol. 26, No. 3, 1981
Melatonin and Gonadal Feedback
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After 7 weeks of injections pituitary LH (16 ug/gland) and FSH (28 ug/gland) in the animals receiving melatonin were considerably depressed when compared to levels in the saline-injected controls. A week after castration pituitary LH levels remained unchanged while FSH levels in saline controls, but not melatonininjected animals, were markedly elevated (fig. 4). Experiment II: Castrated male hamsters receiving melatonin injections showed a marked decline in serum LH and FSH (fig. 3) when compared to castrates receiving saline injections. The animals were sacrificed after 60 days of injections and pituitary LH and FSH content was measured (fig. 4). Pituitary LH and FSH content in saline controls (92 and 966 ug/gland, respectively) were elevated over those in the melatonin-injected group (64 ug/ gland, LH; 232 ug/gland, FSH). As pituitaries were pooled prior to assay; no statistical comparisons can be made.
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"Fig. 3 Mean~SEM(n=5-6) serum LH and FSH in castrated male hamsters receiving daily injections of saline (solid line) of melatonin (broken line). Blood was taken by cardiac puncture on days 10-50. On day 60 the animals were sacrificed. I n some cases the standard error is obscured by the symbol. The limit of detectability for LH was i0 ng/ml and for FSH it was 284 ng/ml.
248
Melatonln and Gonadal Feedback
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Discussion Melatonin administration to intact male hamsters on long photo. periods results in a rapid decline in testes size with complete regression within 6 weeks (fig. 1). This decline is similar to that induced by short photoperiods but occurs much more rapidly. Total regression is not seen in male hamsters exposed to short photoperiods in our laboratory for 10-12 weeks (6). Similarly, the effect of melatonin administration on serum LH and FSH levels in intact hamsters maintained on long days is very dramatic (fig. 2). Following castration no increase in either serum LH or FSH
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Fig. 4 Pituitary LH and FSH content in intact and and castrated male hamsters on LD 14:10 receiving daily injections of saline (open bar) or melatonin (hatched bar). Initial control animals (IC) were sacrificed before injections began. Left side of graph shows pituitary content in intact animals after 7 weeks of injections. The remaining animals were castrated and sacrificed one week later. On the right are pituitary content in saline-and melatonin-injected castrates sacrificed after 60 days of injections.
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Melatonln and Gonadal Feedback
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occurs in melatonin-injected hamsters. An increase in serum FSH but no increase in serum LH occurs 7 days after castration of saline injected animals. In experiment II after i0 days there is a large castration response for both LH and FSH. Perhaps if we had sampled after i0 days we would have seen an increase in LH also in saline-injected animals. Neither FSH nor LH rises in melatonin injected hamsters. Previous studies have also reported no increase in serum gonadotropin levels following castration of male hamsters that have undergone photoperiod-induced testicular regression (7,8), while others report an attentuated castration response (5,6). It appears from the data presented here that melatonin can induce a change in response to castration similar to that previously reported to be ~nduced by short photoperiods. It is unknown at this time if melatonin and short days induce this change via the same mechanism. It is interesting to note that a change in sensitivity to steroid feedback as measured by suppression of gonadotropin levels in castrates by exogenous testosterone does not occur in pinealectomized male hamsters exposed to short photoperiods (15). It is unclear at this time whether the diminished castration response and the hypersensitivity to testosterone feedback displayed bY photoregressed hamsters are two consequences of the same physiological phenomenon. It does appear, however, that melatonin and the pineal may have a role in both phenonema. The suppression of gonadotropin levels by m e l a t o n i n is also reflected in pituitary gonadotropin content. Pituitary LH and FSH levels are lower in intact hamsters receiving melatonin injections than in saline-injected animals and do not increase following castration (fig. 4). This may reflect an action of melatonin at the level of the hypothalamus, preventing gonadotropin releasing hormone (GnRH) synthesis and/or release, even in the absence of steroid feedback. Or it may reflect an action of m e l a t o n i n at the level of the pituitary, preventing response of the gonadotropes to GnRH. Again this action prevents gonadotropin synthesis and release even in the absence of steroid feedback. In chronic castrates ~fig. 3) m e l a t o n i n administration also results in a gradual decline in serum gonadotropins to undetectable or nearly undetectable levels after 60 days~ In contrast, short photoperiods have been reported to induce a decline in serum gonadotropin levels in castrates (8,9) but the leyels never drop as low as in intact animals. M e l a t o n i n administration does not, therefore, reproduce the effects of short day treatment in chronic castrates. Whether this is a dose-dependent phenomenon remains to be determined. Pituitary gonadotropin content in chronic castrates receiving melatonin injections is remarkably high (fig. 4). Although suppressed when compared to chronic castrates receiving saline injection, the pituitary content in m e l a t o n i n - i n j e c t e d chronic castrates is much greater after 8 weeks of injection than pituitary content of intact animals receiving melatonin injections. It therefore appears that in the complete absence of testicular steroid feedback, melatonin administration over time does not completely suppress gonadotropin synthesis, although continued melatonin administration in the presence of testicular feedback does cause suppression of gonadotropin
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Melatonln and Gonadal Feedback
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synthesis w h i c h apparently cannot be reversed by castration ~ i g . 4}. It is also important to note that castrates which have received melatonin injections for 8 weeks have levels of serum LH and FSH which do not reflect the high pituitary gonadotropin content of these hormones. In contrast, saline injected animals have both extremely high serum gonadotropin levels and pituitary content. Melatonin, therefore, may be more effective in suppressing release of gonadotropins from the pituitary than in suppressing synthesis of gonadotropins in chronic castrates. These data taken together support the hypothesis that exogenous melatonin administration to male hamsters maintained on long photoperiods suppresses gonadotropin synthesis and release through action of melatonin at either the pituitary, hypothalamus or both. References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15.
B. TATE-OSTROFF and M. H. STETSON, Amer. Zoologist 19 969 (1979). S. GASTON and M. MENAKER, Science 158 925-928 (1967). W. E. BERNDTSON and C. DESJARDINS, Endocrinology 95 195-205 (1974). F. W. TUREK, Endocrinology i01 1210-1215 (1977). L. TAMARKIN, J. S. HUTCHISON, B. D. GOLDMAN, Endocrinology 99 1528-1533 (1976). K. S. MATT and M. H. STETSON, Biol. Reprod. 20 739-746 (1979). F. W. TUREK, J. A. ELLIOTT, J. D. ALVIS and M. MENAKER, Biol. Reprod. 13 475-481 (1975). F. W. TUREK, J. A. ELLIOTT, J. D. ALVIS and M. MENAKER, Endocrinology 96 854-860 (1975). B. TATE-OSTROFF an-d M. H. STETSON, Amer. Zoologist 1 8 572 (1978). R. A. HOFFMAN and R. J. REITER, Science 148 1609-1611 (1965). E. S. PANKE, M. D. ROLLAG and R. J. REITER, Endocrinology 104 194-197 (1979). L. TAMARKIN, W. K. WESTROM, A. I. HAMILL and B. D. G O L D M A N , Endocrinology _99 _ 1534-1541 (1976). M. H. STETSON and M. WATSON-WHITMYRE, Biol. Reprod. 16 536-542 (1977). J. D. BAST and G. S. G R E E ~ A L D , Endocrinology 94 1295-1299 (1974). F. W. TUREK, Endocrinology 104 636-640 (1979).
Pergamon Press
MELATONIN-INDUCED SUPPRESSION OF GONADOTROPIN TITERS IN MALE GOLDEN HAMSTERS: EFFEC T ON GONADAL FEEDBACK MECHANISMS ~ Barbara Tate-Ostroff and Milton H. Stetson 2 Physiology Section School of Life and Health Sciences University of Delaware Newark, Delaware 19711 (Received in final form October 31, 1980) Summar[ Daily subcutaneous injections of melatonin cause testicular regression and a decline in circulating gonadotropin levels in male hamsters maintained on long photoperiods. We examine here if a reduction in gonadotropin levels also occurs in castrates administered melatonin and if m e l a t o n i n - r e g r e s s e d hamsters respond to castration with an increased release of pituitary gonadotropins - a typical "castration response." Groups of intact and castrated male hamsters maintained on a photoperiod of LD 14:10 received subcutaneous injections of 15 ug melatonin/day. Controls received vehicle only. After 7 weeks of injedtions the intact males were castrated. All animals were sacrificed a few days later and serum was assayed fom gonadotr0pin titers. Melatonin injections caused a marked decline in serum gonadotropins in castrates and in intact males also caused testicular regression. In the latter, no "castration response" was observed upon removal of the testes. Thus, daily injections "of melatonin depress serum gonadotropins in castrate and intact males and block the castrationassociated rise in circulating gonadotropins in the latter. Adult male hamsters respond to short photoperiods with a dramatic decrease in testes weight (2), preceeded by a decline in serum gonadotropin levels (3) and accompanied by a decline in serum testosterone levels (4). Exposure to short photoperiods also induces a change in the sensitivity of the hypothalamic-pituitary axis to testosterone feedback. Male hamsters castrated after undergoing photoperiod-induced testicular regression often show an attenuated castration response (5,6) or no castration response
IA portion of this work was presented at the Annual Meeting of the American Society of Zoologists (i). This work was supported by NSF Research Grant PCM 78-06664. 2To whom reprint requests should be sent. 0024-3205/81/030243-08502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
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(7,8,9). Less exogenous testosterone is required to suppress the castration response in these animals than is required in castrates maintained on long photoperiods (5). Male hamsters castrated and then maintained on short photoperiods for prolonged periods also show a decline in serum gonadotropin levels, a phenomenon seemingly independent of testicular feedback (9). The pineal gland appears to play a role in the photoperiodic response of the adult male hamster. Pinealectomized, or superior cervical ganglionectomized hamsters do not undergo testicular regression when exposed to short photoperiods (i0). Moreover, pinealectomized male hamsters exposed to short photoperiods do not demonstrate an increased sensitivity to steroid feedback (4). Melatonin, a methoxyindole found in highest concentrations in the pineal gland of hamsters during the night (ll), may be the pineal product affecting the photoperiodic response. Administration of melatonin to adult male hamsters maintained on long photoperiods induces testicular regression and a decline in serum gonadotropin levels provided the melatonin is administered via injection at the proper time of day (12). The site of action of melatonin remains unknown. There are many possible mechanisms through which melatonin may influence gonadal function including a direct effect on one or more of the components of the hypothalamo-hypophysial-testicular axis. At the hypothalamus and the pituitary it might alter steroid feedback sensitivity similar to that seemingly caused by short photoperiods. It might affect the synthesis and/or release from the hypothalamus of gonadotropin releasing hormone (GnRH). It may interfere with the normal action of GnRH or by some other means decrease gonadotropin release from the anterior pituitary. It might block the action of gonadotropins, decrease the number of available gonadotropin binding sites or interfere with steroid production and/or release at the testes. The experiments we describe here were designed to assess the effect of melatonin on testicular feedback mechanisms in hamsters maintained on long photoperiods. Methods For this study we used adult male hamsters (12 weeks old), that were raised in our laboratory on a photoperiod of LD 14:10 (lights 0600-2000) at 22-25°C. The animals were housed 6 per cage and given food and water ad libitum throughout the experiment. For surgery animals were anesthesized with ether. An initial control group was sacrificed at the beginning of the experiment. Melatonin was administered daily via subcutaneous injections of 15 ug in 0.i ml of ethanol:saline (i:i0). Control animals were given ethanol:saline only. Injections were given between 1915-1945 hours, a time previously reported to be effective in inducing testicular regression (12). Animals were sacrificed via decapitation between 1300-1500 hrs and serum was stored frozen for later radioimmunoassay for LH and FSH using rat hormone kits provided by NIAMDD. Minor modifications of the protocols provided with the kits are described in detail (13,14). Pituitaries were collected at the time of sacrifice, pooled by group and immediately placed in acetone (-20°C). Several weeks later they were homogenized in cold 0.01M phosphobuffered saline
Vol. 28, No. 3, 1981
Melatonin and Gonadal Feedback
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(pH7.6; 1 ml P B S / g l a n d ) , d i l u t e d and a s s a y e d w i t h the serum as desc r i b e d above. T e s t e s w e i g h t s and m e a s u r e m e n t s w e r e taken at the time of sacrifice. S t a t i s t i c a l c o m p a r i s o n s w e r e d o n e using the S t u d e n t ' s "t" test. E x p e r i m e n t I. A d u l t m a l e h a m s t e r s m a i n t a i n e d on LD 14:10 w e r e d i v i d e d into two groups. One r e c e i v e d m e l a t o n i n i n j e c t i o n s and the o t h e r v e h i c l e only. E v e r y 14 days six animals from e a c h g r o u p w e r e l a p a r o t o m i z e d and the l e n g t h and w i d t h of the testis w e r e measured. Testis m e a s u r e m e n t s w e r e used to c o m p u t e an index (length x width) by w h i c h r e g r e s s i o n could be d e s c r i b e d q u a n t i t a t i v e l y . After 7 w e e k s of i n j e c t i o n s 6 a n i m a l s from e a c h g r o u p w e r e sacrificed. All o t h e r animals w e r e c a s t r a t e d and c o n t i n u e d to r e c e i v e injections for one w e e k u n t i l they w e r e sacrificed. E x p e r i m e n t II. A d u l t m a l e s h e l d on LH 14:10 w e r e castrated. On the d a y of s u r g e r y i n j e c t i o n s of e i t h e r m e l a t o n i n or v e h i c l e w e r e begun. E v e r y i0 days b l o o d samples w e r e taken by c a r d i a c puncture b e t w e e n 1 3 0 0 - 1 5 0 0 hrs f r o m 6 m e l a t o n i n - i n j e c t e d a n i m a l s and six v e h i c l e - i n j e c t e d animals. At 60 days all animals w e r e sacrificed. Results A n i m a l s s a c r i f i c e d at the b e g i n n i n g of the e x p e r i m e n t h a d m e a n s e r u m LH levels of 42 f ng/ml and m e a n s e r u m FSH levels of 196~81 ng/ ml. P o o l e d p i t u i t a r y LH c o n t e n t for the g r o u p w a s 36 u g / ~ l a n d and F S H c o n t e n t w a s 79 ug/gland. M e a n testes w e i g h t was 2 9 4 5 - 1 9 0 m g and m e a n testis l e n g t h x w i d t h (TLW) was 23857. E x p e r i m e n t I. "Melatonin i n d u c e d a m a r k e d d e c l i n e in t e s t i c u lar w e i g h t as c o m p a r e d to i n i t i a l c o n t r o l a n i m a l s and to saline c o n t r o l a n i m a l s (p<.001). A f t e r o n l y two w e e k s of i n j e c t i o n s and t h r o u g h o u t the r e s t of the e x p e r i m e n t , the TLW of the m e l a t o n i n g r o u p w a s s i g n i f i c a n t l y lower (fig. i) than that of the v e h i c l e -
--
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Fig. 1 M e a n ±SEM(n=4-6) testes l e n g t h x w i d t h of intact m a l e h a m s t e r s r e c e i v i n g s u b c u t a n e o u s i n j e c t i o n of saline (open bar) or m e l a t o n i n (hatched bar). Anim a l s w e r e l a p o r a t o m i z e d at 2, 4 and 6 w e e k s of treatment. M e a s u r e m e n t s for the initial c o n t r o l g r o u p (IC) and 8 w e e k g r o u p w e r e o b t a i n e d at sacrifice.
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i n j e c t e d group (p<.001). A f t e r 7 w e e k s of i n j e c t i o n s the m e l a t o n i n - i n j e c t e d animals had a m e a n testes w e i g h t of 226~34 mg while the m e a n testes w e i g h t for s a l i n e - i n j e c t e d animals was signific a n t l y h i g h e r (30261151 mg; p<.05). S e r u m LH and FSH levels in initial c o n t r o l animals and in the s a l i n e - i n j e c t e d and m e l a t o n i n - i n j e c t e d animals of E x p e r i m e n t I are d e p i c t e d in figure 2. A f t e r 7 weeks of treatment, m e l a t o n i n i n j e c t e d animals had s i g n i f i c a n t l y lower LH and FSH levels t h a n did saline i n j e c t e d animals. At w e e k 7 the animals w e r e c a s t r a t e d and s a c r i f i c e d one w e e k later. C a s t r a t i o n did not r e s u l t in an i n c r e a s e in s e r u m LH levels in e i t h e r g r o u p w h i l e s e r u m FSH levels w e r e s i g n i f i c a n t l y e l e v a t e d in the saline c o n t r o l group only. The same trends w e r e seen in p i t u i t a r y g o n a d o t r o p i n levels (fig. 4).
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Fig. 2 Mean~SEM(n=5-6) serum LH and FSH levels in h a m s t e r s r e c e i v i n g saline (open bar) or m e l a t o n i n (solid bar) injections. Initial c o n t r o l (IC) animals w e r e s a c r i f i c e d b e f o r e i n j e c t i o n s began. A f t e r 7 w e e k s of i n j e c t i o n s a group of saline i n j e c t e d and m e l a t o n i n i n j e c t e d a n i m a l s w e r e sacrificed; all o t h e r a n i m a l s w e r e c a s t r a t e d and s a c r i f i c e d one w e e k later. L i m i t of d e t e c t a b i l i t y i0 ng/ml (LH) and 284 ng/ml (FSH).
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Melatonin and Gonadal Feedback
247
After 7 weeks of injections pituitary LH (16 ug/gland) and FSH (28 ug/gland) in the animals receiving melatonin were considerably depressed when compared to levels in the saline-injected controls. A week after castration pituitary LH levels remained unchanged while FSH levels in saline controls, but not melatonininjected animals, were markedly elevated (fig. 4). Experiment II: Castrated male hamsters receiving melatonin injections showed a marked decline in serum LH and FSH (fig. 3) when compared to castrates receiving saline injections. The animals were sacrificed after 60 days of injections and pituitary LH and FSH content was measured (fig. 4). Pituitary LH and FSH content in saline controls (92 and 966 ug/gland, respectively) were elevated over those in the melatonin-injected group (64 ug/ gland, LH; 232 ug/gland, FSH). As pituitaries were pooled prior to assay; no statistical comparisons can be made.
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"Fig. 3 Mean~SEM(n=5-6) serum LH and FSH in castrated male hamsters receiving daily injections of saline (solid line) of melatonin (broken line). Blood was taken by cardiac puncture on days 10-50. On day 60 the animals were sacrificed. I n some cases the standard error is obscured by the symbol. The limit of detectability for LH was i0 ng/ml and for FSH it was 284 ng/ml.
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Melatonln and Gonadal Feedback
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Discussion Melatonin administration to intact male hamsters on long photo. periods results in a rapid decline in testes size with complete regression within 6 weeks (fig. 1). This decline is similar to that induced by short photoperiods but occurs much more rapidly. Total regression is not seen in male hamsters exposed to short photoperiods in our laboratory for 10-12 weeks (6). Similarly, the effect of melatonin administration on serum LH and FSH levels in intact hamsters maintained on long days is very dramatic (fig. 2). Following castration no increase in either serum LH or FSH
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Fig. 4 Pituitary LH and FSH content in intact and and castrated male hamsters on LD 14:10 receiving daily injections of saline (open bar) or melatonin (hatched bar). Initial control animals (IC) were sacrificed before injections began. Left side of graph shows pituitary content in intact animals after 7 weeks of injections. The remaining animals were castrated and sacrificed one week later. On the right are pituitary content in saline-and melatonin-injected castrates sacrificed after 60 days of injections.
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Melatonln and Gonadal Feedback
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occurs in melatonin-injected hamsters. An increase in serum FSH but no increase in serum LH occurs 7 days after castration of saline injected animals. In experiment II after i0 days there is a large castration response for both LH and FSH. Perhaps if we had sampled after i0 days we would have seen an increase in LH also in saline-injected animals. Neither FSH nor LH rises in melatonin injected hamsters. Previous studies have also reported no increase in serum gonadotropin levels following castration of male hamsters that have undergone photoperiod-induced testicular regression (7,8), while others report an attentuated castration response (5,6). It appears from the data presented here that melatonin can induce a change in response to castration similar to that previously reported to be ~nduced by short photoperiods. It is unknown at this time if melatonin and short days induce this change via the same mechanism. It is interesting to note that a change in sensitivity to steroid feedback as measured by suppression of gonadotropin levels in castrates by exogenous testosterone does not occur in pinealectomized male hamsters exposed to short photoperiods (15). It is unclear at this time whether the diminished castration response and the hypersensitivity to testosterone feedback displayed bY photoregressed hamsters are two consequences of the same physiological phenomenon. It does appear, however, that melatonin and the pineal may have a role in both phenonema. The suppression of gonadotropin levels by m e l a t o n i n is also reflected in pituitary gonadotropin content. Pituitary LH and FSH levels are lower in intact hamsters receiving melatonin injections than in saline-injected animals and do not increase following castration (fig. 4). This may reflect an action of melatonin at the level of the hypothalamus, preventing gonadotropin releasing hormone (GnRH) synthesis and/or release, even in the absence of steroid feedback. Or it may reflect an action of m e l a t o n i n at the level of the pituitary, preventing response of the gonadotropes to GnRH. Again this action prevents gonadotropin synthesis and release even in the absence of steroid feedback. In chronic castrates ~fig. 3) m e l a t o n i n administration also results in a gradual decline in serum gonadotropins to undetectable or nearly undetectable levels after 60 days~ In contrast, short photoperiods have been reported to induce a decline in serum gonadotropin levels in castrates (8,9) but the leyels never drop as low as in intact animals. M e l a t o n i n administration does not, therefore, reproduce the effects of short day treatment in chronic castrates. Whether this is a dose-dependent phenomenon remains to be determined. Pituitary gonadotropin content in chronic castrates receiving melatonin injections is remarkably high (fig. 4). Although suppressed when compared to chronic castrates receiving saline injection, the pituitary content in m e l a t o n i n - i n j e c t e d chronic castrates is much greater after 8 weeks of injection than pituitary content of intact animals receiving melatonin injections. It therefore appears that in the complete absence of testicular steroid feedback, melatonin administration over time does not completely suppress gonadotropin synthesis, although continued melatonin administration in the presence of testicular feedback does cause suppression of gonadotropin
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synthesis w h i c h apparently cannot be reversed by castration ~ i g . 4}. It is also important to note that castrates which have received melatonin injections for 8 weeks have levels of serum LH and FSH which do not reflect the high pituitary gonadotropin content of these hormones. In contrast, saline injected animals have both extremely high serum gonadotropin levels and pituitary content. Melatonin, therefore, may be more effective in suppressing release of gonadotropins from the pituitary than in suppressing synthesis of gonadotropins in chronic castrates. These data taken together support the hypothesis that exogenous melatonin administration to male hamsters maintained on long photoperiods suppresses gonadotropin synthesis and release through action of melatonin at either the pituitary, hypothalamus or both. References i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15.
B. TATE-OSTROFF and M. H. STETSON, Amer. Zoologist 19 969 (1979). S. GASTON and M. MENAKER, Science 158 925-928 (1967). W. E. BERNDTSON and C. DESJARDINS, Endocrinology 95 195-205 (1974). F. W. TUREK, Endocrinology i01 1210-1215 (1977). L. TAMARKIN, J. S. HUTCHISON, B. D. GOLDMAN, Endocrinology 99 1528-1533 (1976). K. S. MATT and M. H. STETSON, Biol. Reprod. 20 739-746 (1979). F. W. TUREK, J. A. ELLIOTT, J. D. ALVIS and M. MENAKER, Biol. Reprod. 13 475-481 (1975). F. W. TUREK, J. A. ELLIOTT, J. D. ALVIS and M. MENAKER, Endocrinology 96 854-860 (1975). B. TATE-OSTROFF an-d M. H. STETSON, Amer. Zoologist 1 8 572 (1978). R. A. HOFFMAN and R. J. REITER, Science 148 1609-1611 (1965). E. S. PANKE, M. D. ROLLAG and R. J. REITER, Endocrinology 104 194-197 (1979). L. TAMARKIN, W. K. WESTROM, A. I. HAMILL and B. D. G O L D M A N , Endocrinology _99 _ 1534-1541 (1976). M. H. STETSON and M. WATSON-WHITMYRE, Biol. Reprod. 16 536-542 (1977). J. D. BAST and G. S. G R E E ~ A L D , Endocrinology 94 1295-1299 (1974). F. W. TUREK, Endocrinology 104 636-640 (1979).