Cell and Tissue Research

Cell Tissue Res (1981) 217:105-115

,~, Springer-Verlag 1981

Immunohistochemical Evidence for the Presence of Melatonin in the Pineal Gland, the Retina and the Harderian Gland* B. Vivien-Roels *.1, P. P6vet z'3, M.P. D u b o i s 4, J. A r e n d t 5, and G . M . B r o w n 6 1 Laboratoire de Zoologie et d'Embryologie exp+rimentale, ULP, and Laboratoire de Physiologic Compar~e des R6gulations, CNRS, Strasbourg, France; 2 The Netherlands Institute for Brain Research, Amsterdam, The Netherlands; 3 Department of Anatomy and Embryology, University of Amsterdam, Amsterdam, The Netherlands; 4 Laboratoire de Neuroendocrinologie sexuelle, INRA, Nouzilly, France; 5 Department of Clinical Biochemistry, University of Surrey, Guildford, England; 6 Department of Neurosciences, Me Master University, Hamilton, Canada

Summary. The presence o f melatonin is demonstrated in the pineal gland, the retina and the Harderian gland in some m a m m a l i a n and n o n - m a m m a l i a n vertebrates, using a specific fluorescence labelled a n t i b o d y technique. F o u r different potent antibodies against melatonin have been used and compared. In the pineal gland o f hamsters, mice, rats a n d snakes, specific fluorescence, mostly restricted to the cytoplasm of the cells, is detected in pinealocytes. Fluorescence is also detected in the pineal organ o f fishes, tortoises a n d lizards, but it has not been possible, f r o m cryostat sections o f fresh tissue, to assert which kind of cell is reacting (photoreceptor cells or interstitial ependymal cells). In the retina, fluorescence is almost exclusively restricted to the outer nuclear layer. In the Harderian gland o f m a m m a l s and reptiles, fluorescence is localized in the secretory cells o f the alveoli and mostly restricted to the cytoplasm surrounding the nucleus. These results are discussed in relation to the concept o f melatonin synthesis at extrapineal sites independent o f pineal production. Key words: Melatonin Immunohistochemistry

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Pineal gland -

Retina -

Harderian

gland -

Since its discovery by Lerner (1958), melatonin has been considered as the principal h o r m o n a l agent o f the pineal gland (e.g., Reiter et al. 1975; W u r t m a n and Ozaki 1978). However, the original view o f m e l a t o n i n as a substance unique to pineal m u s t Send offprint requests to: Dr. B. Vivien-Roels, Laboratoire de Zoologie et d'Embryologie exp6rimentale

12, rue de l'Universit~, 67000 Strasbourg - France * Parts of this work have been presented in the Xth Conference of Comparative Endocrinologists, Sorrento, May 20-25, 1979 (Vivien-Roels and Dubois 1980) and the VIth International Congress of Endocrinology, Melbourne, February 10-16, 1980 (Vivien-Roels et al. 1980) ** The author wishes to thank Professor Lutz Vollrath who has accepted her in his laboratory for a short period, Doctor George M. Bubenik for his suggestions and critical remarks, Dr. L.J. Grota for producing the melatonin diazobenzoic acid-BSA and Dr. Castro for preparing one of the melatonin derivates

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now be revised, since melatonin has been found to be present and synthesized in numerous other tissues such as the retina (Quay 1965; Cardinali and Rosner 1971, 1972; Bubenik et al. 1974, 1976a, 1978; Pang et al. 1976, 1977; Prvet et al. 1978; Gern and Ralph 1979; Wainwright 1979), the Harderian gland (Wetterberg et al. 1970; Bubenik et al. 1976b; Pang et al. 1976; Prvet et al. 1980; Balemans et al. in prep.) and the intestine (Raikhlin et al. 1975; Quay and Ma 1976; Bubenik et al. 1977; Holloway et al. 1980). The recent development of immunohistochemical methods now permits the visualization of melatonin containing cells and until now, data concerning the localization of melatonin are quite exclusively restricted to the rat (Bubenik et al. 1974, 1976a, b; Freund et al. 1977). It is known that the pineal organ of non-mammalian vertebrates contains, like the retina, photoreceptor cells and that phylogenetically the pinealocytes of the mammalian pineal gland derive from photoreceptor cells. As it has been proved that the synthesis of melatonin is light dependent and that the pineal gland, the retina and the Harderian gland are able to synthesize melatonin, we have tried, using four different potent antibodies against melatonin, to identify the cells containing melatonin in these organs. Materials and Methods Experiments were performed on freshwater and marine fishes, reptiles and mammals. Five to twelve animals of each of the following species were used: freshwater fishes: Salmofario L. (trout); marine fishes: Ciliata mustella L. (five beard rockling), Taurulus bubalis E. (seascorpion); reptiles: Testudo hermanni G. (tortoise), Lacerta agilis L. (lizard), Natrix tesselata L. (snake); mammals: Rattus norvegicus (Wistar rat), Mus musculus (mouse), Mesocricetus auratus (hamster). Hamsters, mice and rats were maintained in light from 6.00 h to 20.00 h and in darkness from 20.00 h to 6.00 h at 25 ~ C in constant humidity and received tap water and food ad libitum. The other animals were kept under natural conditions of photoperiod and temperature. They were killed between December and February during the light period (between 9.00 h and 12.00 h). Tortoises were killed in January at 11.00 h, 16.00 h and 23.00 h. Following decapitation, the pineal glands, eyes and Harderian glands were quickly removed, frozen in liquid nitrogen, stored in a cryostat at - 20 ~C and sectioned at 10 Ixm. The sections used for immunohistochemical localization of melatonin were fixed 2 s in alcohol or in an acetone-alcohol mixture. Antisera Four different antisera raised against melatonin were used for this study: (a) Antiserum S 20 produced by immunizing sheep with melatonin coupled to bovine serum albumin through an N-p-carboxybenzyl bridge (de Silva and Snieckus 1978); (b) antiserum S 12 produced by immunizing sheep with melatonin coupled to bovine serum albumin via a diazobenzoic acid bridge (Wurzburger et al. 1976); (c) antiserum GS 531 produced by immunizing sheep with melatonin coupled to thyroglobulin; characteristics of the antiserum are reported in Table 1; (d) antiserum 19605: the detailed procedure concerning the preparation of this antiserum is reported in another paper (Ravault et al., in prep.). The immunogen used is melatonin bound to a protein (human serum albumin HSA - in the first period of immunisation, bovine thyroglobulin in the second) by the way of a lateral arm bearing a terminal amine (this melatonin derivate was prepared by Dr. Castro, Nancy, France). The linkage between the - NH2 end of the derivate and the free - NH z of the conjugated protein used glutaraldehyde. Reaction products were kept as aliquots at - 2 0 ~ C. First Period of Immunization (in Rabbits). Each aliquot was emulsified with the same volume of complete Freund's adjuvant and intradermally injected in multiple sites, weekly during one month.

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Table 1. Cross reaction: Antibody antimelatonin GS 531

Compound

~o cross-reaction pg melatonin x 100 pg analogue at 50 ~ displacement

6-hydroxymelatonin 5-methoxytryptophol 5-methoxytryptophan N-acetylserotonin 5-methoxytryptamine 5-methoxyindoleacetic acid N-acetyltryptamine 5-hydroxyindoleacetic acid 5-hydroxytryptamine 5-hydroxytryptophan N-acetyltryptophan tryptophan

0.43 1.04 2.22 0.05 0.04 0.08 0.015 < 0.011 < 0.011 < 0.011 < 0.011 < 0.011

Three weeks after the last injection, rabbits were bled at the marginal vein of the ear and their serum tested for the presence of antibodies: a) a precipitation ring test using melatonin conjugated with bovine thyroglobulin (instead of liSA) showed a ring of precipitation at the interface between serum and immunogen; b) passive immunohemolysis using red blood cells coated with melatonin derivate by the way of bisdiazoted benzidine according to Rangel (1968) gave curves of hemolysis with a titer (dilutions of antiserum corresponding to 50 ~ hemolysis) of 1/2,000 for rabbit 19605.

Second Period of Immunization. After a resting period of several months, new series of immunization were made according to the same protocol, but using thyroglobulin (instead of HSA) to be conjugated with melatonin derivate, and killed Bordetella pertussis suspension (instead of Mycosachesium) in the complete adjuvant. Moreover, the rabbit underwent an intravenous booster injection of the immunogen without adjuvant one month after the end ofintradermal immunization. They were bled six days later. The serum from this last bleeding was used for immunocytochemistry. The histochemical demonstration of melatonin was carried out according to the indirect method of Coons (1957). In the first step, sections were incubated in the antiserum diluted 1/20 to 1/100 in phosphate buffer saline (PBS) (containing 30/00 human serum albumin or bovine serum albumin, in order to avoid a possible cross reaction) overnight at 4 ~C. In a second step, preceded by washing the PBS, anti-rabbit or anti-sheep globulin labelled with fluorescein isothiocyanate (Institut Pasteur or Wellcome Laboratories) was employed in a dilution of 1/50. Controls were carried out using antiserum saturated with an excess of melatonin and by using the second antibody alone. Sections were examined with a Leitz SM LUX fluorescence microscope.

Results

Comparison o f the Antisera I n all t i s s u e s , s i m i l a r r e s u l t s w e r e o b t a i n e d w i t h all f o u r a n t i s e r a . B e c a u s e o f s p a c e limitation, only representative findings are shown.

Pineal Organ. I n t h e t r o u t , as well as i n t h e s e a s c o r p i o n t h e p i n e a l p s e u d o s t r a t i f i e d epithelium, with numerous infoldings delimiting a narrow central lumen, contains t w o c a t e g o r i e s o f cells: p h o t o r e c e p t o r cells a n d e p e n d y m a l cells ( O m u r a a n d O g u r i

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1969). In the tortoise, the pineal organ appears as a follicular structure. The pineal parenchyma contains two categories of ceils: "photoreceptor cells" and interstitial ependymal cells (Vivien-Roels 1976). In the snake, the pineal organ is a compact glandular structure containing only one category of cells: the pinealocytes (Vivien 1964; Petit 1976). In the rat, mouse and hamster, the pineal parenchyma contains pinealocytes and glial cells (ref. in: P6vet 1977). Using the four antisera in fishes (Fig. 1 a), as well as in tortoises (Fig. 1 c and d) or lizards, some pineal cells show a distinct specific fluorescence. Fig. l d demonstrates that the fluorescence is restricted to the cytoplasm of cells. In the snake (Fig. 1 b), the pineal of which consists of pinealocytes only, fluorescence appears quite obvious in most of the pinealocytes, with only some differences in the intensity in individual cells. In all of the mammals used in this study, some pineal cells show a specific fluorescence and reacting cells appear regularly distributed through the pineal parenchyma (Fig. 2a and b). Some differences exist in the number of reacting cells from one species to another; in the hamster killed during the day-time only a small number of pineal cells are fluorescent while in the rat or the mouse, numerous cells are reacting and show a strong fluorescence. In all the control investigations, saturation of the antisera with an excess of melatonin induced almost complete disappearance of the fluorescence. In one experiment, tortoises were killed at different times of the 24 h-cycle, but we were unable to detect differences either in the fluorescence intensity, or in the number of reacting cells. This results confirms our previous data (Vivien-Roels et al. 1979) demonstrating, by means of radioimmunoassay, the lack of circadian variations in the pineal melatonin content during hibernation. Retina. Using the four antisera in sagittal or parasagittal sections of the retina of

fishes, reptiles and mammals, specific fluorescence was almost exclusively confined to the outer nuclear layer (Fig. 3 a-d). No differences were observed in the intensity of fluorescence or in the number of reacting cells throughout the outer nuclear layer. A weak fluorescence, likely due to diffusion, may appear from time to time in the inner nuclear layer. In the tortoise retina, as in the pineal organ, we were unable to find differences either in the fluorescence intensity or in the number of reacting cells in animals killed at different times of the 24 h-cycle in winter. Harderian Gland. In the reptiles and the mammals used in this study, the Harderian

gland was a large lobulated gland, situated in the orbital cavity behind the eyes. In fishes, the Harderian gland is lacking. Using each of the antisera, specific fluorescence was observed in the secretory cells of the alveoli of reptiles and mammals, and was mostly restricted to the cytoplasm surrounding the nucleus. Fig. 2c and d show the results obtained in mouse znd hamster. Differences in the number of reacting cells appear from one alveolus to another; in some alveoli, most of the cells are fluorescent (Fig. 2c), while in the other alveoli no reacting cells were observed (Fig. 2d). In most of the alveoli, a red fluorescence, due to the presence of porphyrins is observable, which is unaffected by saturation of the antiserum with melatonin, while the specific yellow fluorescence of the melatonin containing cells disappears after saturation.

Fig. 1. a Immunohistochemical demonstration of melatonin in the pineal organ of a trout. L central lumen. Antiserum 19605 1/100 ( • 560). b Immunohistochemical demonstration of melatonin in the pineal gland of a snake. Note the strong fluorescence of most of the pinealocytes. Antiserum 19605 1/100 ( • 875). e Immunohistochemical demonstration o f melatonin in the pineal organ o f a tortoise. Antiserum $20 1/10 ( x 560). d Higher magnification of melatonin-immunoreactive cells in the tortoise pineal organ. Fluorescence is restricted to the cytoplasm of the cells, nuclei are non-fluorescent (arrows). Antiserum 19605 1/100 ( • 1,400)

Fig. 2. a Immunohistochemical demonstration ofmelatonin in the hamster pineal gland. Note the small n u m b e r of reacting cells regularly distributed through the pineal parenchyma. Antiserum 19605 1/100 ( • 560). b Immunoreactive cells in the mouse pineal gland; most of the pinealocytes are fluorescent. Antiserum 19605 1/100 ( x 560). e Melatonin in the Harderian gland of the mouse. Note the fluorescence restricted to the cytoplasm surrounding the nucleus in the secretory cells of the alveoli. Antiserum GS 531 1/10 ( • 560). d Melatonin in the Harderian gland of the hamster. Note the lack of fluorescence in some alveoli (arrow). Antiserum $20 1/10 ( • 560)

Fig. 3. a Sagittal section o f tortoise retina, stained by Gabe's ferric trihematoxylin, on Outer nuclear layer; in inner nuclear layer; gc ganglion cell layer ( • 750). b Sagittal section of tortoise retina. Melatonin is confined to the outer nuclear layer (on). Antiserum 19605 1/100 ( • 750). c Parasagittal section of rockling retina. Note the intense fluorescence of the outer nuclear layer (on). Antiserum 19605 1/100 ( • 560). d Immunohistochemical demonstration of melatonin in the hamster retina. Note the strong fluorescence o f the outer nuclear layer (on). Antiserum S 12 1/10 (• 680)

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Discussion

Our immunohistochemical results clearly demonstrate (i) the localization of melatonin in the perikaryon of some pineal cells in mammals as well as in fishes and reptiles and (ii) the existence of extrapineal tissues containing melatonin. The presence ofmelatonin in the retina, which corroborates the previous results of Bubenik et al. (1974), is of special interest since in lower vertebrates the pineal organ, like the retina, contains photoreceptor cells and in mammals, P6vet and Collin (1976) and P+vet et al. (1977) have shown that the pinealocytes phylogenetically evolved from photoreceptor cells. Melatonin is almost completely localized in the outer nuclear layer of the retina; these results suggest that in the pineal organ of lower vertebrates, melatonin would be synthesized by photoreceptor cells rather than by ependymal cells. Nevertheless, this remains to be demonstrated. As in fishes, tortoises and lizards, not all of the cells showed a specific fluorescence and since there are two categories of cells, we have tried to identify which category of cell is reactive, by using serial sections, the one coloured by classical methods and the other used for immunofluorescence, or in some cases by counter colouring the sections used for immunofluorescence by Evans blue. However, it appeared that none of these methods was successful and for the moment, in cryostat sections of fresh tissue, it is not possible to ascertain which kind of cell is reacting (photoreceptor cells or interstitial ependymal cells) in the pineal epithelium of fishes, tortoises and lizards. In the pineal gland of the snake, rat and mouse, our results corroborate the observations of Bubenik et al. (1974) who noted a specific fluorescence in almost all the parenchymal cells of the rat pineal gland. In the hamster, similar results to those of Bubenik et al. (1974) in the rat, were found, except that only a few cells were reacting in hamsters killed during the day. Except in the snake where only pinealocytes are present in the pineal, due to the technique used, we have not been able to determine whether the immunocytochemically stained cells are glial cells or represent pinealocytes. However, in comparison with the small number of glial cells immunocytochemicatly or ultrastructurally determined, it appears that the cells stained in our study are not glial in nature but evidently pinealocytes. Only electron microscopical immunocytochemistry, however, will permit to give a definitive answer. In all of the mammals used in this study and in the snake, we were unable to demonstrate the cortical localization of melatonin immunoreactive cells, as described by Freund et al. (1977). While it is not possible to exclude a problem of differences of cell sensitivity to the antiserum, this discrepancy, in our opinion, could be due to interspecies variations (Freund et al. used Sprague-Dawley rats, while Bubenik et al. and ourselves used Wistar rats). Specificity of the antisera is unlikely to be a problem as we obtained the same distribution of fluorescent cells with the four antisera tested in this study. The presence of melatonin in the Harderian gland of mammals and reptiles as detected with our immunohistochemical method, also confirms the previous results of Bubenik et al. (1976a, b) in the rat and Pang et al. (1976) in the rat and chicken. Our results are also in agreement with the biochemical data such as: (i) The presence of hydroxyindole-O-methyltransferase (HIOMT), the enzyme required to convert N-acetyl serotonin to melatonin in retinal tissue and in the

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Harderian gland, demonstrated by many authors (Quay 1965; Wetterberg et al. 1970; Vlahakes and Wurtman 1972; Cardinali and Wurtman 1972; Cremer-Bartels et al. 1975; Cremer-Bartels 1979; Joss 1978; Wainwright 1979). (ii) The capacity of HIOMT to synthesize melatonin, demonstrated in the eye of the mole (P6vet et al. 1978), in the retina and the Harderian gland of the hamster (P6vet et al. 1980) and the mole-rat (Balemans et al., in preparation). (iii) The ability of the retina to synthesize melatonin from serotonin in vitro recently demonstrated in the trout by Gem and Ralph (1979). These data strongly support the idea of melatonin synthesis by the retina and the Harderian gland. The functional significance of retinal and Harderian gland melatonin is unknown. Some data confirm that, as in the pineal, extrapineal melatonin concentrations also show circadian fluctuations. Gem et al. (1978) have shown that in the trout, retinal melatonin concentrations are high during photophase and low during scotophase, while Bubenik et al. (1978) in the rat found higher amounts of melatonin during the dark phase in the retina as well as in the Harderian gland; in the tortoise killed during hibernation we found no differences in the fluorescence reaction of the retina between day and night. Recently, Pang and Yew (1979) have reported a possible role of melatonin in the pigment aggregation of the retinal pigment epithelium, and in the Harderian gland Wetterberg et al. (1970) suggested that melatonin may act as a substance inhibiting or facilitating the visualization process by a modification of the porphyrin content. But for the moment and despite the numerous reports on this subject the mechanisms of action ofmelatonin are not yet known; it seems very likely that melatonin is synthesized in extrapineal tissues; however, its physiological significance remains to be elucidated. References Brown GM, Grota LJ (1980) Use of immunologic techniques in the examination of neurotransmitters and neuromodulators. In: Hanin I, Koslow S (eds) Physicochemical methodologies in Psychiatric Research. Raven Press, New York, in press Bubenik GA, Brown GM, Uhlir I, Grota IA (1974) Immunohistological localization of Nacetylindolalkylamines in pineal gland, retina and cerebellum. Brain Res 81:233-242 Bubenik GA, Brown GM, Grota LJ (1976a) Immunohistochemical localization of melatonin in the rat Harderian gland. J Histochem Cytochem 24:1173-1178 Bubenik GA, Brown GM, Grota IA (1976b) Differential localization of N-acetylated indolealkylamines in CNS and the Harderian gland using immunohistology. Brain Res 118:417-427 Bubenik GA, Brown GM, Grota IA (1977) Immunohistological localization of melatonin in the rat digestive system. Experientia 33:662-663 Bubenik GA, Purtill RA, Brown GM, Grota LJ (1978) Melatonin in the retina and the Harderian gland. Ontogeny, diurnal variations and melatonin treatment. Exp Eye Res 27:323-333 Cardinali DP, Rosner JM (1971) Retinal localization of the hydroxyindole-O-methyltransferase (HIOMT) in the rat. Endocrinology 89:301 Cardinali DP, Rosner JM (1972) Ocular distribution of hydroxyindole-O-methyltransferase (HIOMT) in the duck (Anasplatyrhinchos). Gen Comp Endocrinol 18:407-409 Cardinali DP, Wurtman RJ (1972) Hydroxyindole-O-methyltransferase in rat pineal, retina and Harderian gland. Endocrinology 91:247-252 Coons AH (1957) The application of fluorescent antibodies to the study of naturally occurring antigens. Ann N Y Acad Sci 69:658-662 Cremer-Bartels G (1979) The effect of pteridines on multiple forms of hydroxyindole-Omethyltransferase (HIOMT) in the retina and the pineal gland. In: Ariens Kappers J, P6vet P (eds) The pineal gland of vertebrates including man. Progr Brain Res 52:231-239

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Cremer-Bartels G, Hollwich F, Kotulla W (1975) Melatoninbiosynthese in der S/iugetierretina in Abh/ingigkeit v o n d e r Adaptation an Belichtung. Klin Monatsbl Augenheilkd 167(1): 88-93 Freund D, Arendt J, Vollrath L (1977) Tentative immunohistochemical demonstration of melatonin in the rat pineal gland. Cell Tissue Res 181:239-244 Gem W, Ralph C (1979) Melatonin synthesis by the retina. Science 204:183-185 Gem W, Owens D, Ralph C (1978) The synthesis of melatonin by the trout retina. J Exp Zoology 206:263-270 Holloway WR, Grota IA, Brown GM (1980) Determination of immunoreactive melatonin in the colon of the rat by immunocytochemistry. Histochem Cytochem 28:255-262 Joss JMP (1978) A rhythm in hydroxyindole-O-methyltransferase (HIOMT) activity in the Scincid lizard, Lampropholas guichenoti. Gen Comp Endocrinol 36:521-525 Lerner AB, Case JD, Takahashi Y, Lee TH, Mori W (1958) Isolation of melatonin, the pineal gland factor that lightens melanocytes. J Am Chem Soc 80:2587 Omura Y, Oguri M (1969) Histological studies on the pineal organ of 15 species of teleosts. Bull Jpn Soc Scient Fisheries 35:991-1000 Pang SF, Yew DT (1979) Pigment aggregation by melatonin in the retinal pigment epithelium and choroid of guinea-pigs Cavia porcellus. Experientia 35:231-233 Pang SF, Brown GM, Grota IA, Rodman RL (1976) Radioimmunoassay ofmelatonin in pineal glands, Harderian glands, retina and sera of rats and chickens. Fed Proc 35:691 Pang SF, Brown GM, Grota LJ, Chambers JW, Rodman RL (1977) Determination of N-acetylserotonin and melatonin activities in the pineal gland, retina, Harderian gland, brain and serum of rats and chickens. Neuroendocrinology 23:1-13 Petit A (1976) Contribution ~il'~tude de r6piphyse des reptiles: le complexe 6piphysaire des Lacertiliens et des Ophidiens. Etude embryologique, structurale, ultrastructurale; analyse qualitative et quantitative de la s~rotonine dans des conditions nonnales et exprrimentales. Th~se Doct d'Etat, Strasbourg Prvet P (1977) On the presence of different populations of pinealocytes in the mammalian pineal gland. J Neural Transm 40:289-304 P+vet P, Collin JP (1976) Les pinralocytes de mammifrre: diversit+, homologie, origine. Etude chez la taupe adulte (Talpa europaea L.). J Ultrastruct Res 57:22-31 Prvet P, Kappers JA, Voute AM (1977) Morphologic evidence for differentiation of pinealocytes from photoreceptor cells in the adult noctule bat (Nyctalus noctula, Schreber). Cell Tissue Res 182: 99-I 11 Prvet P, Balemans MGM, Bary PAM, Noordegraaf EM (1978) The pineal gland of the mole (Talpa europaea L.). Activity of hydroxyindole-O-methyltransferase (HIOMT) in the formation of melatonin 5-methoxytryptophol in the eyes and the pineal gland. Ann Biol Anim Bioch Biophys 18:259-265 P6vet P, Balemans MGM, Legerstee WC, Vivien-Roels B (1980) Circadian rhythmicity of the activity of hydroxyindole-O-methyltransferase (HIOMT) in the formation of melatonin and 5-methoxytryptophol in the pineal retina, and Harderian gland of the golden hamster. J Neurol Transm 49:229-245 Quay WB (1965) Retinal and pineal hydroxyindole-O-methyltransferase activity in vertebrates. Life Science 4:983 Quay WB, MaYH (1976) Demonstration of gastrointestinal hydroxyindole-O-methyltransferase. IRCS Med Sci 4:563 Raikhlin NT, Kvetnoy IM, Tolkachev VN (1975) Melatonin may be synthesized in enterochromaffin cells. Nature 255:344-345 Rangel H (1968) Studies on passive haemolysis mediated by antiserum globulin antibodies. Immunology 14:197-211 Reiter R J, Vaughan MK, Vaughan GM, Sorrentino SJ, Donofrio RJ (1975) The pineal gland as an organ of internal secretion. In: Altschule MD (ed). Cambridge, MA MIT Press Silva OS de, Snieckus W (1978) Indole-N-alkylation of tryptamines via dianion and phthalimido intermediates. New potential indolealkylamine haptens. Canad J Chem 56:1621-1627 Vivien JH (1964) Organisation et structure de l'organe pineal d'un Ophidien, Tropidonotus natrix. J Microsc (Paris) 3:57 Vivien-Roels B (1976) L'rpiphyse des chrloniens: 6tude embryologique, structurale, ultrastructurale, analyse qualitative et quantitative de la srrotonine darts des conditions normales et exp6rimentales. Th+se de Doctorat d'Etat no 990, Strasbourg

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Vivien-Roels B, Dubois MP (1980) Immunohistochemical indentification of melatonin in the pineal gland and the retina of lower vertebrates. Xth Conference of European comparative endocrinologists, Sorrento (Italy), May 21-25, 1979. Gen Comp Endocrinol 40(3):368 Vivien-Roels B, Arendt J, Bradtke J (1979) Circadian and circannual fluctuations of pineal indoleamines (serotonin and melatonin) in Testudo hermanni Gmelin (Reptilia, Chelonia) I. Under natural conditions of photoperiod and temperature. Gen Comp Endocrinol 37:197-210 Vivien-Roels B, P6vet P, Arendt J, Brown GM, Dubois MP (1980) Immunohistochemical evidence of the presence of melatonin in the pineal gland, the retina and the Harderian gland of mammalian and non mammalian vertebrates. Vlth international congress of endocrinology, Melbourne (Australia), February 10-16, 1980 Vlahakes GJ, Wurtman ILl (1972) A Mg++dependent hydroxy-O-methyltransferase in the rat Harderian gland. Biochem Biophys Acta 261:184-198 Wainwright SD (1979) Development of hydroxyindole-O-methyltransferase activity in the retina of the chick embryo and young chick. J Neurochem 32:1099-1103 Wetterberg L, Geller E, Yuwiller A (1970) Harderian gland: an extraretinal photoreceptor influencing the pineal gland in neonatal rats. Science 167:884-885 Wurtman RJ, Ozaki Y (1978) Physiological control of melatonin synthesis and secretion: mechanisms generating rhythms in melatonin, methoxytryptophol and arginine-vasotocin levels and effects on the pineal of endogenous catecholamines, the estrous cycle and environmental lightening. In: Nir I, Reiter ILl, Wurtman RJ (eds) The pineal gland. Springer-Verlag, Wien, p 59-71 Wurzburger ILl, Kawashima K, Muller RL, Spectar S (1976) Determination of rat pineal gland melatonin content by radioimmunoassay. Life Sci 18:867

Accepted January 30, 1981