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J. Neural Transmission 49, 229--245 (1980)

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9 by Springer-Verlag 1980

Circadian Rhythmicity of the Activity of HydroxyindoleO-Methyl Transferase (HIOMT) in the Formation of Melatonin and 5-Methoxytryptophol in the Pineal, Retina, and Harderian Gland of the Golden Hamster P. P 6 v e t l , ~, M. G. M. B a l e m a n s 3, W. C. L e g e r s t e e 3, and B. Vivien-Roels4, 5 J The Netherlands Institute for Brain Research, Amsterdam, The Netherlands, 2 Department of Anatomy and Embryology, University of Amsterdam, The Netherlands, ~ Zoological Laboratory, Section of Histology and Cell Biology, State University of Utrecht, The Netherlands, 4 Laboratoire de Zoologie et Embryologie Exp6rimentale, Universit6 Louis Pasteur, Strasbourg, France, and Laboratoire de Physiologie Compar6e des R6gulations, CNRS, Strasbourg, France With 5 Figures Received July 15, 1980

Summary The day and night rhythms in the activity of HIOMT in the formation of melatonin and of 5-methoxytryptophol have been determined in the pineal, retina and Harderian gland of the adult male golden hamster. In all hamsters used there was no detectable HIOMT activity in the deep pineal. In the superficial pineal HIOMT activity, involved in the synthesis of melatonin (Mel), was observed to be high at the end of the dark period and at the middle of the light period. Considering the HIOMT activity involved in the production of 5-methoxytryptophol (5-MTL), an increase in 5-MTL synthesis was observed only during the light period. Comparing the peak of Mel-production with that of 5-MTL it appears that during the light period the pineal produces more 5-MTL than Mel. In the Harderian glands, the circadian course of HIOMT activity involved in the synthesis of Mel seems to run parallel to that of the enzyme implicated in 5-MTL synthesis, both being stimulated at the end of the dark period. The activity of HIOMT in 5-MTL production is, however, always approximately 2 times higher than for Mel synthesis.

0300-9564/80/0049/0229/~ 03.40

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P. P6vet, M. G. M. Balemans, W. Co Legerstee~ and B, Vivien-Rcels:

In the retina the synthesis of Mel and 5-MTL is not significantly higher during the dark period than during the light period. However, the production of 5-MTL is larger than that of Mel. It appears that (1) with the exception of the end of the dark period, the extra-pineal synthesis of Mel and 5-MTL is always higher than that in the pineal; (2) the circadian synthesis of 5-methoxyindoles is different in each organ, and (3) in the pineal the circadian activity of HIOMT involved in 5-MTL formation is different from that of the same enzyme involved in the formation of Mel. The results are discussed. Key words: Melatonin, 5-methoxytryptophol, HIOMT, pineal, retina, Harderian gland. Introduction

Two distinct groups of substances isolated from pineal tissue have been proposed to be responsible for the endocrine capabilities of the pineal gland: (1) indoleamines, particularly the 5-methoxyindoles and (2) peptides which are not as yet fully characterized. Within the pineal gland all 5-methoxyindoles are synthesized by the enzyme hydroxyindole-O-methyltransferase (HIOMT). Among 5-methoxyindoles, melatonin was isolated first (Lerner et al., 1959) and has been by far the most extensively studied compound. It is, however, also known that, like melatonin, 5-methoxytryptophol is capable of modifying sexual development and reproduction in birds and mammals (Balemans, 1972, 1973 a, b, 1974; Mclsaac et aI., 1964; Mullen et al., 1979). Initially, H I O M T was thought to be present only in the pineal gland. Subsequently, it was identified in the retina (Cardinali and Rosner, 1971, 1972; Quay, 1965; P~vet et al., 1978), in the Harderian gland (Vlahakes and Wurtman, 1972; Cardinali and Wurtman, 1972; Balemans et al., 1980 b), and in the intestine (Quay and Ma, 1976). Moreover, it has also been demonstrated that the retina in the mole (P~vet et al., 1978) and the Harderian gland in the mole-rat (Balemans et aI., 1980 a) can synthesize 5-methoxyindoles in larger quantities than the pineal. In the Syrian hamster, naturally or artificially shortened daylight induces a pronounced gonadal involution (see for review Reiter, 1978). This effect of short photoperiods is mediated by the pineal gland (see for details Reiter, 1978; Hoffmann, 1979). Melatonin which is considered by many authors as "the" pineal hormone (Wurtman and Ozaki, 1978), of which the synthesis is light/darkdependent, has been shown to inhibit reproductive function in the hamster under certain circumstances (Tamarkin et al., 1976, 1977; Turek et al., 1975). It therefore seemed of importance to study, in the

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hamster, the day and night synthesis of melatonin and of 5-methoxytryptophol in the deep and the superficial pineal and in the retina and the H a r d e r i a n gland. Material and Methods Thirty young adult male golden hamsters (Mesocricetus auratus) (80--90 g), obtained from TNO, Zeist, The Netherlands, were used in this study. They were maintained in light from 6.00 a.m. to 8.00 p.m. and in darkness from 8.00 p.m. to 6.00 a.m. at 25 ~ in constant humidity and received tap water and food ad libiturn. On June 28 and 29, 1979, approximately every two hours (0.30 p.m., 3.00 p.m., 5.30 p.m., 8.30 p.m., 11.00 p.m., 1.30 a.m., 3.30 a.m., 5.30 a.m., 7.30 a.m., and 10.30 a.m.), 3 hamsters were sacrificed. Alter decapitation, the superficial pineal, the part of the brain containing the deep pineal, the retinae and the Harderian glands were quickly removed. The organs were then frozen in liquid nitrogen and conserved at - 8 0 ~ until they were investigated. The time of dissection for all organs (some few minutes) was the same for all animals. During the night the animals were killed in the dark, but the different organs were dissected in light. The detailed method to determine separately the HIOMT activities for the production of Mel and 5-MTL, respectively, has extensively been reported elsewhere (Balemans et al., 1978 a, b). Using this method, the methylation of several 5-hydroxyindoles in the superficial and deep pineal, retina and Harderian gland was analysed by using the 5-hydroxyindoles present in the tissues studied as a substrate. This implies that no extra substrate was added to the incubation medium (20 #1 0.1 M phosphate buffer, pH 8.0 and 10 #1 of (SH)-S-adenosyl methionine containing 1.0 #Ci in H~SOa, pH 2.5). The different organs, slightly disrupted, were incubated at 37 ~ for 60 rain. Then the tissues, together with their corresponding medium, were chromatographed by TLC (Merck DC-Silicagel plates). This bidimensional TLC technique permits the separation of melatonin and 5-methoxytryptophol. As a solvent system for the first direction, chloroform:methanol:acetic acid ( 9 3 : 4 : 3 ) was used. The plates were dried under nitrogen and developed in the second direction in chloroform:methanol:ammonia 25 % ( 6 0 : 3 5 : 5 ) . The spots were then scraped and counted with a liquid scintillation counter (Mark I of Nuclear Chicago). For more details on the techniques and on the controls used we refer to Balernans et al. (1978 b). The HIOMT activities in the synthesis of melatonin and of 5-methoxytryptophol have been determined in the entire pineal and retinae. The very large Harderian glands were cut a~er weighing into numerous small pieces and the HIOMT activities were determined in some of them taken from different parts of both glands. The results were adjusted to be expressed in HIOMT activity for the entire glands. The remaining Harderian gland pieces were placed in 1 ml HC1 0.1 N (with 0.5 % ascorbic acid) and used for serotonin determination. Serotonin was measured by spectrofluorometry

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P. P~vet, M. G. M. Balemans, W. C. Legerstee, and B. Vivien-Rcels:

according to the method of Quay (1963), slightly modified according to Vivien-Rcels (1976). The results were also calculated for the entire glands. Results

Pineal Following the classification proposed by Vollrath (1979), the pineal of the hamster is os type aC. This means that, besides the superficial pineal, a small deep pineal is present. In all hamsters examined, no detectable H I O M T activity was observed in the deep pineal. The results obtained thus concern the superficial pineal only. In the course of a 24-hour period, methylation of N-acetylserotonin to melatonin was observed to be stimulated during the dark as well as during the light period, showing maxima of melatonin production at 3.30a.m. and 0.30p.m. (Fig. 1). The production maximum of melatonin during the dark phase is, however, quite different in shape from that during the light phase. In the dark phase a true peak is observed between 1 a.m. and 5.30 a.m., while in the light phase the decrease of the activity is slow and lasts from 12 a.m. to 8 p.m. This difference might probably be of physiological importance. DPIV "Pineal

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Fig. 1. Circadian rhythm of HIOMT activity in synthesizing melatonin in the pineal of adult male hamsters. Each point represents the mean + S.E.M. Dotted horizontal bar represents the dark phase of the L/D cycle. D P M desintegration per minute


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In the case of the HIOMT activity involved in the production of 5-methoxytryptophol, however, the circadian synthesis occurs quite differently. An increase in the 5-methoxytryptophol synthesis was observed only during the light period showing a high value between 0.30 p.m. and 5.30 p.m. (Fig. 2). Moreover, comparing the peak of melatonin production (Fig. 1) to that of 5-methoxytryptophol (Fig. 2) it appears that during the light period the pineal produces more 5-methoxytryptophol than melatonin, while during the dark period only the production of melatonin was increased. DPIV rPineal

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Fig. 2. Circadian rhythm of HIOMT activity in synthesizing 5-methoxytryptophol in the pineal of adult male hamsters. Each point represents the mean +__S.E.M. Dotted horizontal bar represents the dark phase of the L/D cycle. D P M desintegration per minute

Harderian Gland

The circadian course of HIOMT activity involved in the synthesis of melatonin runs parallel to that of the enzyme implicated in 5-methoxytryptophol synthesis (Fig. 3). The activity of HIOMT in 5-methoxytryptophol production is, however, constantly approximately 2 times higher than that for melatonin production.

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P. P~vet, M. G. M. Balemans, W. C. Legerstee, and B. Vivien-Rcels :

The enzymatic activities are stimulated at the end of the dark period, showing a peak at 5.30 a.m. During the light period a small decrease of the 5-methoxyindole synthesis is observed at 2.30 p.m. (Fig. 3). Interestingly, the serotonin concentration in the gland DPM/2Glands 2500

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Fig. 3. Circadian rhythm of H I O M T activity in synthesizing melatonin ( , ,) or 5-methoxytryptophol ( . . . . . ) in the Harderian glands of adult male hamsters. Each point represents the mean +_ S.E.M. Dotted horizontal bar represents the dark phase of the L/D cycle. DPM desintegration per minute ng/2G~ands 1300

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Fig. 4. Circadian rhythm of serotonin concentration in the Harderian glands of adult male hamsters. Each point represents the mean _+ S.E.M. Dotted horizontal bar represents the dark phase of the L/D cycle


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corresponds exactly, but in an inverse way, with that of the HIOMT activities. A decrease in serotonin concentration corresponds with a peak in enzymatic activities while an increase in serotonin concentration corresponds with a decrease in HIOMT activities. Retina

During the dark period, the synthesis of melatonin and 5methoxytryptophol appears not to be significantly higher than during the light period. However, like in the Harderian gland, the production of 5-methoxytryptophol in the retina is larger than that of melatonin. DPM/2retinae

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Fig. 5. Circadian rhythm of HIOMT activity in synthesizing melatonin ( ) or 5-methoxytryptophol ( ..... ) in the retinae of the adult male hamsters. Each point represents the mean + S.E.M. Dotted horizontal bar represents the dark phase

of the L/D cycle. DPM desintegration per minute

Discussion The results presented in this paper demonstrate that, like the pineal, the retina and the Harderian gland of the hamster can produce melatonin and 5-methoxytryptophol.

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P. P&et, M. G. M. Balemans,W. C. Legerstee, and B. Vivien-Rcels:

No H I O M T activity was observed at all in the deep pineal, a result which can be related to the finding of Moore (1974) (unpublished data, cited in Wiklund, 1974) who failed to detect a circadian rhythm of N-acetyltransferase activity in brain tissue comprising the deep pineal and part of the habenular nuclei. With regard to the relatively large number of pinealocytes found in the deep pineal of the hamster (Sheridan and Reiter, 1970, it seems likely that H I O M T activities would have been found if the deep pineal were identical in function with the superficial one. Our observation thus supports the earlier idea (Wiklund, 1974) that the function of the deep pineal is different from that of the superficial gland. Further research is, however, needed to confirm this assumption. To facilitate the discussion we term the superficial pineal "the pineal", as does the majority of scientists. Our observation that in the hamster pineal melatonin synthesis increases during the dark phase agrees with that of Rollag et al. (1980), Tarnarkin et al. (1979) and Panke et al. (1979). Moreover, using the same lighting conditions as ours, Panke et al. (1979) found a peak in pineal melatonin concentration at about 3.30 a.m., the hour when we observed a peak in melatonin synthesis. This seems to indicate a direct relationship, at least in the hamster, between pineal melatonin synthesis and pineal melatonin concentration. Apparently, the increase of pineal melatonin synthesis observed by us during the light phase is in contradiction with the generally accepted idea that the pineal is able to increase its melatonin synthesis only during the dark phase of the light/dark cycle, while this finding is also not conform to earlier observations in hamster by Rollag et al. (1980), Tamarkin et al. (1979) and Panke et al. (1979) who never observed such a phenomenon. It is difficult to explain this discrepancy between our results and those obtained by other authors. It should, however, be noted that the hamsters used for the present investigation were killed in June. An increase of H I O M T activity in the formation of melatonin during the light phase has already been described in rat sacrificed in June (Balemans et aI., 1979). Unfortunately, Rollag et al. (1980), Tamarkin et al. (1979) and Panke et al. (1979) did not mention the time of the year in which they performed their experiments. Possibly, such a peak in melatonin formation during the light phase could depend on the time of the year. Moreover, it seems that the concept following which the pineal activity is high during the dark phase only, should be used carefully. This opinion has, so far, only been based on results obtained with melatonin production. Our results, however, clearly demonstrate that, considering 5-


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methoxyindoles, the pineal synthesis of 5-methoxytryptophol is also activated during the light phase of the light/dark cycle. From the fact that melatonin has been isolated first and that, consequently, its biological effects were thoroughly studied, it should not be followed that melatonin is physiologically the most important pineal 5-methoxyindo!e. It has, for instance, been known for a long time that 5-methoxytryptophol, like melatonin, is capable of modifying sexual development and reproduction in birds and mammals (see for a review Mullen et al., 1979). Moreover, already in 1972 Balernans demonstrated that the effects of 5-methoxytryptophol could be more specific than those of melatonin, at least in chick, while in 1974 Balemans showed that the combination of 5-methoxytryptophol and melatonin in chick was physiologically most effective. Our present results demonstrate that, with the exception of the pineal at the end of the dark period, the production of 5-methoxytryptophol in the hamster pineal, the retina and the Harderian gland is always larger than that of melatonin. In the pineal and retina of the mole, HIOMT activity for the synthesis of 5-methoxytryptophol is also always higher than that for the formation of melatonin (P~vet and Balemans, unpublished results), and in the pineal and the Harderian gland of the mole-rat the production of 5-methoxytryptophol is also constantly larger than that of melatonin (Balernans et al., 1980 a). It thus appears that 5-methoxytryptophol is of great importance regarding pineal physiology. In the pineal, serotonin can be acetylated to N-acetylserotonin, oxidized to 5-hydroxyindole-3-acetic acid or metabolized to 5hydroxytryptophol. The 5-hydroxyindoles, 5-hydroxytryptophan, 5hydroxytryptamine, 5-hydroxyindole-3-acetic acid, 5-hydroxytryptophol and N-acetylserotonin can all be methylated by HIOMT. This means that at least 5 different methylated products can be formed in the pineal. Although probable, it is not known whether the retina and the Harderian gland are also capable of synthesizing all of these 5-methoxyindoles. In the present investigation, HIOMT activities in the formation of melatonin and in that of 5-methoxytryptophol have been examined because the biological activities of these two 5-methoxyindoles have been extensively studied. This does, however, not mean that we are of the opinion that melatonin and 5-methoxytryptophol are the only 5-methoxyindoles of physiological importance. Some of us (Balemans et al., 1980 b) did, indeed, recently demonstrate that 5-methoxytryptophan may also be of physiological consequence. It is evident that a parallel study of the HIOMT activities involved in the production of all 5-methoxyindoles is needed. To

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date, however, such a study has been performed only in the rat pineal in regard of the methylation of 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxyindole-3-acetic acid and N-acetyl-5-hydroxytryptamine together with 5-hydroxytryptophol (Balernans et al., 1978 a, 1980 b). In the pineal and the Harderian glands a circadian rhythmicity has been observed in H I O M T activity involved in the formation of melatonin and in that of 5-methoxytryptophol. The rhythm observed in the pineal is, however, completely different from that in the Harderian gland. In the pineal, like in the Harderian gland, we observed a peak in H I O M T activity for the formation of melatonin during the dark phase, but the peak was at 3.30 a.m. in the pineal, and at 5.30 a.m. in the Harderian gland. On the other hand, the increase in H I O M T activity determined in the pineal during daytime was not observed in the Harderian gland. As H I O M T activity involved in the formation of 5-methoxytryptophol is concerned, a difference exists between the pineal and the Harderian gland' In the latter the synthesis of 5-methoxytryptophol runs exactly parallel to that of melatonin. In the pineal, on the contrary, there is no parallelism and H I O M T activity for the formation of 5-methoxytryptophol is stimulated only during daytime. In the retina we observed for both H I O M T activities a nonsignificant increase during the dark phase of the cycle. Rhythms in H I O M T activity or in melatonin concentration have been described in the retina of different species (Nagle et al., 1972; Pang et al., 1977; Gern et al., 1978; Joss, 1978). Possibly, our observation that a true rhythm is absent can be ascribed to the methodological procedure used. Although sacrificing hamsters during the dark phase in darkness, we used light for the dissection. In the rat pineal, Balemans (unpublished results) demonstrated--using pineals dissected in light and pineals dissected in darkness--that light does not modify the rhythm but decreases H I O M T activity, especially the amplitude of the peak. On the other hand, Joss (1978), studying the rhythm in H I O M T activity in the pineal and the retina of a lizard and, like us, using light for dissection (personal communication) observed a rhythm in the retina. It would thus be possible that the method used provokes a loss of H I O M T activities. This would, however, mean that, if a rhythm was present in the retina, its amplitude was very low, an observation already made by other authors (Gern et al., 1978). The possibility that seasonal differences might occur can also not be excluded. Comparing the data for the pineal, the two retinae and the two Harderian glands, it appears that the production of 5-methoxy-

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tryptophol by the retinae and the Harderian glands, considered together, is always larger than that of 5-methoxytryptophol by the pineal alone. The same appears for melatonin, with the exception of the end of the night (3.30 a.m.) when melatonin production by the pineal is larger than that by the two other organs combined. When realizing that other organs, such as the intestine, are able to synthesize additional melatonin it becomes apparent that only part of the 5-methoxyindoles is produced by the pineal. This raises the question of the physiological significance of the 5-methoxyindoles in general. It has been hypothesized (Quay, 1974; Bridges et al., 1976; Reiter and Vaughan, 1975; Reiter et al., 1975) that one of the target organs for melatonin and/or 5-methoxyindoles could be the pineal itself. This may mean that the melatonin synthesized in the pineal would influence intracellularly or extracellularly the formation, storage, and/or release of pineal peptidergic effector compounds (see Quay, 1974; Benson, 1977). A similar local action has also been hypothesized for the melatonin synthesized in the retina and the Harderian gland. Wetterberg et al. (1970) suggest, for example, that melatonin may act as a substance inhibiting or facilitating the visual process by modifying the porphyrine content of the Harderian gland. In the retina, Pang and Yew (1979) demonstrated that melatonin plays a role in the pigment aggregation of the retinal pigment epithelium, as was hypothesized by Flight (1976, 1979). Melatonin and 5-methoxytryptophol are known to be present in the general circulation and to play an important role in numerous physiological processes (see AriFns Kappers and P~vet, 1979). The question whether melatonin and 5-methoxytryptophol produced by the retina, the Harderian gland and the intestine are released in the general circulation thus seems of much importance. It has been generally assumed that melatonin, synthesized in the pineal, is released in the general circulation, although this has never been demonstrated. On the contrary, injected melatonin is mainly taken up by the pineal (Wurtrnan et al., 1964). There is, however, some evidence, such as the decrease of blood melatonin concentration after pinealectomy (Pelham et aI., 1972), which permits to accept this assumption. There is likewise no proof that the 5-methoxyindoles synthesized in the retina, the Harderian gland or other organs are released in the general circulation. However, like for the pineal, there is good evidence for such a release, namely the presence of circulating melatonin in pinealectomized rat and trout (Ozaki and Lynch, 1976; Gem et al., 1978), and even the increase of blood melatonin concentration aflcerpinealectomy in sheep (Kennaway et aI., 1977). 17 Journal of Neural Transmission 49/4

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The fact that the pineal, the retina, the Harderian gland and other organs such as the intestine (Holloway et al., 1980) synthesize 5-methoxyindoles which are probably released in the general circulation, prohibits to consider 5-methoxyindoles in general, and melatonin especially, as pineal hormones. They are pineal compounds like they are also retinal, Harderian or intestinal compounds. These considerations oblige us to completely re-evaluate our concept of the role of 5-methoxyindoles, and especially our concept of the mechanism by which the pineal acts on the gonadal axis. As has been mentioned, we observed that in the hamster the rhythm in pineal and Harderian gland H I O M T activities is photoperiod-dependent. This is also the case for the retinal rhythm of H I O M T activity in cyclostomes (Joss, 1978). Eichler and Moore (1975) found that the pineal rhythm in H I O M T activity is light- as well as temperature-dependent, while heat exposure was observed to decrease rat pineal H I O M T activity (Nir et al., 1975). Vivien-Roels et aI. (1979), studying the circannual variations of the circadian rhythm in melatonin concentration in the pineal of a tortoise, found that the rhythm is light-dependent, but that its amplitude is temperature-dependent. Very recently, Vivien-Roels and Arendt (1980) demonstrated, likewise in the tortoise, that in the pineal the circadian production of melatonin seems to be regulated by temperature although the photoperiod appeared to be important for the regulation of the circadian production of serotonin, the precursor of melatonin. It has been suggested that melatonin, one of the methoxyindoles, acts not only directly on the organs by which it is produced, such as the pineal, retina, Harderian gland or intestine (Freire and Cardinali, 1975; Benson and Krasovich, 1977; Holloway et al., 1980), on the hypothalamo-hypophysio-gonadal axis (Quay, 1974; Berndtson and Desjardin, 1974; Turek et aI., 1975), but also directly on the gonads (Peat and Kinson, 1971). Thus, via the 5-methoxyindoles, a system would exist which probably permits to integrate parallelly different informations such as light, temperature, etc. These indolic compounds could be thus of great importance for the adaptation of the endocrine reproductive axis to modifications in the environment. References

Ari~ns Kappers, J., P~vet, P.: The pineal gland of vertebrates including man. Progr. Brain Res. 52 (1979). Balernans, M. G.M.: Age-dependent effects of 5-methoxytryptophol and melatonin on testes and comb growth of the white leghorn (Gallus domesticus, L.). J. Neural Transm. 33, 179--194 (1972).


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Balemans, M. G. M.: The stimulatory effects of several concentrations of 5-methoxytryptophol on testicular growth in the white leghorn (Gallus domesticus, L.). J. Neural Transm. 34, 49--60 (1973 a). Balemans, M.G.M.: The inhibitory effect of 5-methoxytryptophol on ovarian weight, follicular growth and egg production of adult white leghorn hens (Gallus domesticus, L.). J. Neural Transm. 34, 159--169 (1973 b). Balemans, M. G. M.: The pineal gland. A study on the presence and the biological significance of melatonin and 5-methoxytryptophol. Thesis, Utrecht, 1974. Balemans, M. G. M., Bary, F. A. M., Legerstee, W. C.: Seasonal variations in HIOMT activity during the night in the pineal gland of rats of several ages. Melatonin Symposium, September 28--30, Bremen, Federal Republic of Germany (1980 a). Balemans, M. G. M., Legerstee, W.C., Van Benthem, J.: Day and light rhythms in the methylation of N-acetylserotonin/5-hydroxytryptophol in the pineal gland of male rats of different ages. J. Neural Transm. 45, 265--272 (1979). Balemans, M. G. M., Noordegraaf, E. M., Bary, F. A. M., Van Berlo, M. F.: Estimation of the methylating capacity of the pineal gland. With special reference to indole metabolism. Experientia (Basel) 34, 887--888 (1978 a). Balemans, M. G.M., Bary, F. A. M., Legerstee, W.C., Van Benthem, J.: Estimation of the methylating capacity of the pineal gland of the rat with special reference to the methylation of N-acetylserotonin and 5-hydroxytryptophol separately. Experientia (Basel) 34, 1434--1435 (1978 b). Balemans, M. G. M., P~vet, P., Legerstee, W. C., Nero, E.: Melatonin and 5-methoxytryptophol synthesis in the pineal, the retina and the Harderian gland of the mole-rat (Spalax ehrenbergi, Nehring) and in the pineal of the mouse "eyeless". J. Neural Transm. 49, 247--255 (1980 b). Benson, B.: Current status of pineal peptides. Neuroendocrinology 24, 241 to 258 (1977). Benson, B., Krasovich, M.: Circadian rhythm in the number of granulated vesicles in the pinealocytes of mice. Effect of sympathectomy and melatonin treatment. Cell Tiss. Res. 184, 499--506 (1977). Berndtson, W.E., Desjardins, C.: Circulating LH and FSH levels and testicular function in hamsters during light deprivation and subsequent photoperiodic stimulation. Endocrinology 95, 195--199 (1974). Bridges, R., Tamarkin, L., Goldman, B.: Effects of photoperiod and melatonin on reproduction in the Syrian hamster. Ann. Biol. Anim. Biochem. Biophys. 16, 399--408 (1976). Cardinali, D. P., Rosner, J. M.: Retinal localization of the hydroxyindoleO-methyltransferase (HIOMT) in the rat. Endocrinology 89, 301--303 (1971). 17"

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Circadian Rhythmicity of the Activity o~:HIOMT

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