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However, 1 mg melatonin (MEL) administered in the morning completely prevents the ... This result is specific of MEL since morning injections of 1 mg 5-MT.

j. Neural Transmission 55, 85-93 (1982)

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N~wa/~msm~sion (g) by Springer-Verlag 1982

Morning Injections of Large Doses of Melatonin, but not of 5-Methoxytryptamine, Prevent in the Hamster the Antigonadotropic Effect of 5-Methoxytryptamine Administered Late in the Afternoon P. P~vet 1'2 and C. Haldar-Misra 1. The Netherlands Institute for Brain Research, Amsterdam, The Netherlands. 2 Department of Anatomy and Embryology, University of Amsterdam, The Netherlands With 3 Figures Received March 16, 1982

Summary Late-afternoon-administered 5-methoxytryptamine (5-MT) induces gonadal atrophy in male hamsters kept under long photoperiods (14 hL/ 10 hD). Morningqnjection of 1mg 5-MT does not show antigonadal effects. However, 1mg melatonin (MEL) administered in the morning completely prevents the antigonadotropic effect of 5-MT administered in the late afternoon. This result is specific of MEL since morning injections of 1mg 5-MT do not present this counterantigonadotropic property. When interpreted according to Reiter's concept of down-regulation of MEL receptors, these results suggest that the physiological effects of 5-MT are indirectly due to MEL. Key words: melatonine, 5-methoxytryptamine, pineal, hamster, photoperiod.

Introduction It is now evident that the pineal gland is principally involved in long term adaptation o f functions such as reproduction to environmental conditions (Pdvet, 1976; Pd~et et aL, 1981b; Hoffmann, 1979, * IBRO/UNESCO fellow.

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1981; Reiter, 1973, 1980). The mechanism by which the pineal acts on the gonadal axis is, however, not yet known. Although its classification as a true pineal hormone is still controversial (Pdraet et aL, 1981b, P&aet and Haldar-Misra, 1982 a), melatonin (MEL) appears to be implicated in this p h e n o m e n o n of adaptation. MEL, when properly administered, indeed duplicates in the hamster most of the effects of the photoperiod on the reproductive axis (see review in Reiter, 1980; Hoffmann, 1981). Recently, however, we demonstrated that 5-methoxytryptamine (5-MT), like MEL a product ofserotonin metabolism, is also able to duplicate the effect of the photoperiod on the hamster reproductive function, More especially it was shown that 5-MT acts in the same way as does MEL (P&et et aL, 1981 a, b; P&et and Haldar-Misra, 1982 a, b). These results raise the question of the relationship between both 5-methoxyindoles. According to Reiter (1980), the interaction of MEL with its own receptors (down regulation) could determine how MEL acts, depending on its mode of administration, as either an anti- or a counterantigonadotropic agent (details in Reiter, 1980). Considering this hypothesis, the apparent similarities between the physiological effects of MEL and those of 5-MT suggest the possible existence of a mechanism of regulation of 5-MT receptors by 5-MT or a poasible action of 5-MT on the MEL receptors (or vice versa). In the present investigation, we tested this receptor hypothesis for 5-MT. Use was made of an experimental model developed by Reiter's group (Chen et aL, 1980) in which large doses of MEL injected in the morning prevent, possibly by desensitization of the melatonin receptors, the testicular atrophy induced by late afternoon injected MEL.

Materials and Methods Experiments have been performed at two consecutive times in MarchApril, 1981, and in May-June, 1981. As the results obtained in both experiments were similar, the results of the last experiment are reported in the present paper only. Fifty-two young adult male golden hamsters (Mesocricetusauratus) (6080 g at the onset of the study) obtained from TNO, Zeist, The Netherlands, were used. They were maintained in light from 4 a.m. to 6 p.m. (14 hL/10 hD) at 20 ~ in constant humidity and received tap water and food ad libitum. After two weeks of acclimatization to our laboratory conditions, the animals were divided into 6 groups: Group I (12 hamsters)-control group. Four of the animals were injected daily (6 days on 7) with vehicles between 9 and 9.30 a.m.; four others be-

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tween 3.30 and 4 p.m., and the last four between 9 and 9.30 p.m. and between 3.30 and 4 p.m. Group II (8 hamsters) received 25/Jg 5-MT daily (6 days on 7) between 3.30 and 4 p.m. Group Ill (8 hamsters) received 1 mg 5-MT daily (6 days on 7) between 9 and 9.30a.m. Group I V (8 hamsters) received lmg MEL daily (6 days on 7) between 9 and 9.30 a.m. Group V(8 hamsters) received daily (6 days on 7) 1 mg of 5-MY between 9 and 9.30a.m. and 25/ag of 5-MT between 3.30 and 4p.m.. Group VI (8 hamsters) received daily (6 days on 7) lmg of MEL between 9 and 9.30a.m. and 25/~g of 5-MT between 3.30 and 4p.m. After 8 weeks of treatment, the animals were sacrificed by decapitation and the testes and accessory sex organs (seminal vesicles, coagulating gland and secretory content) were dissected and weighed. The results were statistically analysed using Student's T-test and are given as means 4- S.E.M. (see legends). 5-MT and MEL, obtained from Sigma Chemical Co. (St. Louis, Missouri, USA), was dissolved in some drops of ethanol 100% and then mixed with saline 0.9%. 5-MT and MEL were injected subcutaneously in the back of the animals in a volume of 0.1ml. Control groups received the vehicle only (drops of ethanol in saline 0.9%).

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Fig. 1. Absolute (cross hatched bars) and relative (dotted bars) testicular weight of hamsters kept under long photoperiod and submitted to various treatments. Vertical lines at top of bars signify standard errors..00, **, AA, OO: p ~ 0.001 Controlcontrol animals; 2512g 5-MTanimals receiving daily 25#g 5-MT in the late afternoon; img 5-MTanimals receiving daily 1mg 5-MT in the morning; 1 rngMEL animals receiving daily ling MEL in the morning; ling 5-MTq-25/tg 5-MT animals receiving daily img 5-MT in the morning and 25/2g 5-MT in the afternoon; 1mg MEL +25lag 5-MT animals receiving daily 1mg MEL in the morning and 25 ktg 5-MT in the afternoon

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Results In male adult hamsters daily injections of 25/ag of 5-MT given late in the afternoon caused a highly significant reduction in both the testicular (Fig. 1) and accessory organ weights (Fig.2). When injected in the morning at a dose of 1 mg, 5-MT did not induce any significant decrease in the testis or accessory organ weights (Figs. 1, 2). The same was observed when 1 mg of MEL was injected. However, l mg MEL injected in the morning completely prevented the antigonadotropic effect of 5-MT administered late in the afternoon, a result which is specific for MEL since a large dose of 5-MT did not have the same effect (Figs. 1, 2). Comparing the body weight of the hamsters of the different groups at the end of the experiment, it appeared that none of these treatments had any effect on somatic development (Fig. 3). It should be noted that, at the end of the eight weeks, injection of ling 5-MT/ day provoked a necrosis of the skin at the site of the injections. Autopsy of the animals after the experiments demonstrated that the necrosis was restricted to the skin.

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Morning Injections of Large Doses of Melatonin

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Discussion

Recently it has been established that 5-MT must be administered late in the light phase of the light/dark cycle to exert an antigonadotropic effect in male hamsters kept in tong photoperiod (Pdvet et al., 1981a, b). When injected in the beginning of the light phase, 5-MT does not present this effect (Pivet et al., 1981a). As melatonin is concerned, a similar observation had already been made by many teams (Tamarkin et al., 1876; Reiter et al., 1976). Different hypotheses have been proposed to explain these properties of melatonin. Among those, the hypothesis of Reiter (1980) is presently most elaborated. According to this author, the effects would be explained by a MEL down-regulation of its own receptors. After MEL has acted on its receptors it renders them temporarily insensitive to additional melatonin, i.e., the receptors become transiently refractory. A-s endogenously produced MEL is normally secreted in a late stage of the dark phase, early in the light phase the receptors would be in a desensitized state and thus unable to respond to exogenously injected melatonin. Late in the afternoon the sensibility of the receptors would be re-established. An injection of MEL at this time is capable of acting and a reproductive atrophy ensues. According to such a hypothesis, the injection of 1mg of MEL in the morning would prevent the gonadal inhibitory effect of MEL

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administered in the afternoon because such a large dose would probably prolong the insensitivity of the receptors. Clearly, we cannot use a similar receptor hypothesis to explain our results obtained with 5-MT because lmg 5-MT injected in the morning has no effect on the capacity of 5-MT administered late in the afternoon to induce gonadal atrophy. A large dose of 5-MT has no effect but, on the contrary, large doses of MEL completely prevent the normally inhibitory afternoon injection of 5-MT. This result is most important because it demonstrates that 5-MT and MEL are probably physiologically closely related compounds. Using the concept of Reiter on MEL receptor regulation it would be possible to explain our results in two different ways: (1) 5-MT would not have its own receptors but interact with melatonin receptors. Morning injections of large doses of MEL would render the MEL receptors insensitive explaining why 5-MT injected in the afternoon, which normally would act on these receptors, has no effect. Such an action for 5-MT on MEL receptors is very probable, the more so since Wilson and Harr5 (1981) observed that 5-MT antagonizes MEL on melatonin binding sites in the skin of adult Xenopus laevis with a potency relative to melatonin of 1.02. In the mammalian brain in which the MEL receptors are probably similar to those in the amphibian skin (Messenger and Warner, 1977), such properties of 5-MT on MEL receptors have also been observed showing, however, less potency (Cardinali and Vacas, 1981)o According to such a hypothesis lmg 5-MT in the morning should, however, also have acted on MEL receptors and thus have prevented the inhibitory effect of 5-MT administered in the afternoon. As this was not the case this explanation cannot be used. (2) In the pineal gland or in other organs, such as the retina or the Harderian gland, 5-MT could possibly be acetylated by [email protected] and thus be transformed into MEL. Although such a metabolic pathway has never been demonstrated, its existence cannot be excluded (Balemans et al., 1980). The physiological properties of 5-MT would thus be indirectly due to MEL explaining the present effect of i mg MEL on 5-MT administered in the afternoon. The negative result obtained with a large dose of 5-MT would be due to the fact that, N-acetyltransferase being activated only during the dark period of the light/dark cycle (Klein, 1979), 5-MT injected in the morning could not be transformed into MEL. This second explanation of the findings seems more plausible. In earlier publications (Pdvet et al., 1981 a, b) we explained that the similarities of the effect of 5-MT and MEL could be due to a transfor-

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mation of 5-MT into MEL, a MEL-induced increase of 5-MT, or an independent effect of 5-MT and of MEL. The present results seem to indicate that the first proposition is accurate. They would again stress the importance of MEL. However, this interpretation .is valid only if we use Reiter's hypothesis on down-regulation of melatonin receptors. Are we justified in doing so?

This theory of Reiter explains some apparent paradoxical activities of MEL and perhaps of 5-MT in terms of their effects on reproduction. It is therefore of heuristic value but needs more information on the structure and physiology of the melatonin receptors, an area of pinealogy which until now has been relatively neglected (see review in Cardinali and Vacas, 1981). Moreover, some experimental results obtained by different independent teams cannot be explained by such a concept. Three daily injections of melatonin, for example, are effective in pinealectomized and normal hamsters irrespective of the fact whether they are performed during daytime or at night (Tamarkin et aL, 1977 a; Goldman et al., 1979). In hamsters, maintained in long photoperiod, injections of MEL at midnight-the receptors following Reiter's hypothesis then being very sensitivehave no effect on the gonadal axis (Tamarkin et al., 1977 b), while MEL injected late in the dark period-the receptors following Reiter's hypothesis being then desensitized by endogenously produced MEL-induces gonadal atrophy (Tamarkin et al., 1977b). Considering 5-MT, the observations that in male hamsters (1) 5-MT, continuously available from subcutaneous deposits, prevents the antigonadotropic effects of daily afternoon injections of 5-MT (Pivet and Haldar-Misra, 1982 c); that (2) 5-MT injected in the morning as well as continuously available 5-MT prevents short photoperiodically induced gonadal atrophy (Piz,et and Haldar-Misra, 1982 a, b), can also not be explained by the MEL receptor theory of Reiter. In conclusion, when interpreted in using Reiter's concept of down-regulation of MEL receptors, the present results would suggest that the physiological effects of 5-MT are indirectly due to the effect of MEL. However, as many results in the literature are in contradiction with this concept, such a conclusion should be drawn with precautions. The present results anyway clearly demonstrate that the physiological effects of MEL and 5-MT are closely related.

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Acknowledgements The authors wish to thank Prof. J. Arifins Kappers for his stimulating interest in this study and for critically reading the manuscript, Miss E. De Graaf for technical help, Miss J. Sels and M. Hesseling for secretarial aid.

References Balemans, M. G. M., Bary, F. A. M,, Legerstee, W. C., Van Bentbem, J.: Seasonal variation in HIOMT activity during the night in the pineal gland of 21-day old male Wistar rats. J. Neural Transm. 49, 107-116 (1980). Cardinali, D. P., Vacas, M. I.: Molecular endocrinology of melatonin: Receptor sites in brain and peripheral organs. In: Melatonin--Current Status and Perspectives (Birau, N., Scbloot, W,, eds.), pp. 237-246. Oxford: Pergamon Press. 1981. Chen, H.J., Brainard, G.C., Reiter, R.J.: Melatonin given in the morning prevents the suppressive action on the reproductive system of melatonin given in late afternoon. Neuroendocrinology 31, 129-132 (1980). Goldman, B., Hall, V., Hollister, C., Roychoudhury, P., Tamarkin, L., Westro, W.: Effect ofmelatonin on the reproductive system in intact and pinealectomized male hamsters maintained under various photoperiods. Endocrinology 104, 82-88 (1979). Hoffmann, K.: Photoperiod, pineal, melatonin and reproduction in hamsters. In: The Pineal Gland of Vertebrates Including Man (Prog. Brain. Res., Vol. 52) (Arians Kappers, J., Pgvet, P., eds.), pp. 397-417. Amsterdam: Elsevier/North-Holland Biomed. Press. 1979. Hoffmann, K.: Photoperiodic function of the mammalian pineal organ. In: The Pineal Organ: Photobiology-Biochronometry-Endocrinology (Oksche, A., Pdvet, P., eds.), pp. 123-133. Amsterdam: Elsevier/NorthHolland Biomed. Press. 1981. Klein, D. C.: Circadian rhythms in the pineal gland. In: Endocrine Rhythms (Krieger, D.T., ed.), pp. 203-220. New York: Raven Press. 1979. Messenger, E.A., Warner, E. A.: The action ofmelatonin on single amphibian pigment cells in tissue culture. Br. J. Pharmacol. 61, 607-614 (1977). Pdvet, P.: Correlations between pineal gland and sexual cycle. Thesis, University of Amsterdam, 1976. Pdvet, P., Haldar-Misra, C.: Effect of 5-methoxytryptamine on the testicular atrophy induced by experimental or natural short photoperiods in the golden hamster. J. Neural Transm. 55, 69-84 (1982 a). Pgvet, P., Haldar-Misra, C.: Daily 5-methoxytryptamine injections inhibit short-day induced testicular atrophy in golden hamsters. J. Neural Transm. 55, 95-99 (1982 b). Pgvet, P., Haldar-Misra, C.: (in prep., 1982 c). Pdvet, P., Haldar-Misra, C., Oca~ T.: Effect of 5-methoxytryptophan and 5-methoxytryptamine on the reproductive system of the male golden hamster. J. Neural Transm. 51, 303-311 (1981a).

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P&et, P., Haldar-Misra, C., Ocal, T.: The independency of an intact pineal gland of the inhibition by 5-methoxytryptamine of the reproductive organ in the male hamster. J. Neural Transm. 52, 95-106 (1981b). Reiter, R.J.: Pineal control of a seasonal reproductive rhythm in male golden hamsters exposed to natural daylight and temperature. Endocrinology 92, 423-430 (1973). Reiter, R.J.: The pineal and its hormones in the control of reproduction in mammals. Endocr. Rev. /, 109--131 (1980). Reiter, R.J., Blask, D. E., Johnson, L. Y., Rudeen, P. K., Vaughan, M. K., Waring, P.J.: Melatonin inhibition of reproduction in the male hamster: its dependency on time of day of administration and on an intact and sympathetically innervated pineal gland. Neuroendocrinology 22, 107116 (1976). Tamarkin, L., Westrom, W. K., Hamill, A. I., Goldman, B. D.: Effect ofmelatonin on the reproductive systems of male and female hamsters: a diurnal rhythm in sensitivity to melatonin. Endocrinology 99, 1534-1541 (1976). Tamarkin, L., Hollister, C. W., Lefebvre, N.G., Goldman, B.D.: Melatonin induction of gonadal quiescence in pinealectomized Syrian hamsters. Science 198, 953-955 (1977a). Tamarkin, L., Lefebvre, N. G., Hollister, C. W., Goldman, B. D.: Effect ofmelatonin administered during the night on reproductive function in the Syrian hamster. Endocrinology 101, 631-634 (1977 b). WiIson,J. iv., Harry, F. M.: Low affinity binding sites for melatonin in the skin of adult Xenopus laevis. XIth Conf. Europ. Soc. Comp. Endocrinol.,Jerusalem, 10-14 August 1981. Authors' address: Dr. P. P&et, The Netherlands Institute for Brain Research, IJdijk 28, 1095 KJ Amsterdam, The Netherlands.

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