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Pineal Regulation of Body Blanching in Amp hibian Larvaexs
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Pineal Regulation of Body Blanching in Amphibian Larvae JOSEPH T. B A G N A R A Department of Zoology, University of Arizona, Tucson, Ariz. (U.S.A.)
Among the many problems being investigated during the current renaissance of interest in the pineal organ, is the role of the pineal in pigmentary phenomena. This is due largely to the discovery that pineal elements are photoreceptive and to the fact that mammalian pineals contain a potent melanophore contracting agent. Integration of these two facts makes possible new interpretations of earlier observations which seemed unrelated. As early as 1911, Von Frisch observed that the pineal area of the teleost Phoxinus was able to regulate melanophore response by virtue of its sensitivity to light. About this time Babak (1910) and Laurens (1915) showed that larvae of several amphibian species became pale when placed in the dark, Fuchs (1914) attempted to explain the latter by advancing a theory based upon Von Frisch’s work on Phoxinus. Fuchs felt that larvae produce, as a result of their ordinary life processes, certain substances which cause melanophores to contract. Thus, when larvae are placed in darkness and are, therefore, free from external stimulation, their melanophores contract under the influence of these endogenous metabolic substances. When light is provided, however, the pineal is stimulated which results in an inhibition of the endogenously induced melanophore contraction. Laurens (1916) was opposed to the view of Fuchs and in an intensive study involving localized illumination of the pineal area of the head of larval Ambystoma opacum and A . maculatum concluded that the epiphysis has no influence on the reactions of larvae to light and to darkness. His arguments were so convincing that McCord and Allen (1917) who discovered that extracts of beef pineals cause blanching in Rana pipiens tadpoles, concluded that any pineal induced pigmentary changes must occur independent of environmental conditions. Subsequent to this early work, only sporadic references concerning the pineal and pigmentation appeared. Scharrer (1928) confirmed the observations of Von Frisch, and Young (1935) observed that pallor which occurs in lampreys on transference from light to darkness is abolished after removal of the pineal complex. Beall et al. (1937) extended the observations of McCord and Allen by demonstrating that the melanophore contracting substance present in beef and sheep pineals is an unsaturated nitrogenous base and Bors and Ralston (1951) observed that pineal extracts of both pig and man induce melanophore contraction in larval and adult Xenopus. Recently, Lerner et al. (1958) have isolated a potent, direct acting, melanophore contracting agent from beef pineal glands. They References p.-503/504
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have identified this compound as melatonin (N-acetyl-5-methoxytryptamine). Presumably, melatonin is the active melanophore contracting agent found in the pineal of other mammalian species. Simmonet et al. (1952) have also demonstrated the presence of a melanophore contracting agent in the mammalian pineal. Moreover, their experiments imply that the pineal may actually play a role in melanophore regulation, for they observed that melanophores of hypophysectomized adult Rana esculenta expand after pinealectomy. Not long ago, while studying the tail-darkening reaction ofXenopuslarvae (Bagnara, 1957, 1960a), an event which occurs when the larvae are placed in the dark, we had reason to compare this response with the body-blanching reaction which occurs at the same time. Tail-darkening is mediated by the direct action of light on fin melanophores. Apparently, a photochemical system is involved, because full expansion of tail melanophores requires over 30 min exposure to darkness while re-contraction occurs about 5 min after light is restored. Temporal factors involved in the paling of the body of these larvae are in marked contrast with tail darkening. Onset of body lightening becomes obvious in 10-15 min and by 20 min melanophore contraction is quite strong. Upon restoration of illumination these melanophores re-expanded very slowly, requiring about an hour for complete expansion. Temporal events in body lightening and subsequent recovery did not seem to be consistent with mediation by a photochemical system. Instead they implied that a hormonal mechanism was involved with relatively rapid onset of melanophore contraction corresponding to release of a stored hormone and slow recovery concordant with gradual loss of this principle from the circulation. With the implication of a hormonal mechanism in the body-blanching reaction, suspicion arose that possibly the melanophore contracting principle of the pineal is the active agent. This suspicion grew even stronger in view of the fact that the paling reaction occurs when eyeless larvae are placed in darkness (Laurens, 1915, 1917; Bagnara, unpublished). It seemed possible, therefore, that the pineal is the photoreceptor necessary for the paling reaction. Following this line of reasoning an hypothesis was established which explained the mechanism of the bodyblanching reaction completely in terms of two aspects of pineal physiology, photoreception, and endocrine functions. Briefly, the hypothesis states that when amphibian larvae are placed in darkness the pineal is affected by the absence of light causing it to release and produce a melanophore contracting agent, the action of which, causes the blanching reaction. The first publication of this hypothesis (Bagnara, 1960b) included data showing that pinealectomized Xencrpus larvae are not capable of performing the blanching reaction when placed in the dark. Confirmation of this point was derived from the study of Eakin (1961) which pointed out that Hyla regilla larvae, deprived of frontal organ and eyes, show a weaker blanching reaction when placed in the dark than do controls without eyes. Since, initial publication of the hypothesis, additional supporting data have accumulated, much of which is presented in a recent paper (Bagnara, 1963). Despite the availability of additional support, this hypothesis remains:a generality awaiting information which will clarify many specific points. The identity of the melanophore contracting agent is still unknown. It is attractive to suppose that melatonin is the active substance, however, this compound
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has never been found in the pineals of lower vertebrates. Moreover, if a hormone is indeed invclved, the specific site of release of this substance is not known. Another question concerns the relationship of other chromatophore-stimulating systems to the blanching reaction. For instance, how does body blanching occur in the presence of the hypophysis with its melanophore expanding factor? These are only a few of the problems which must be understood before the hypothesis advanced above can be accepted. It is the aim of this paper to completely define the body-blanching reaction of larval amphibians, to present all the data which led to the formulation of the pineal hypothesis, and to clarify some of the unsolved problems pertaining to this hypothesis. I. THE B L A N C H I N G REACTION
Although it is generally known that amphibian larvae become light when they are placed in the dark (Babak, 1910; Laurens, 1916, 1917; Bagnara, 1960a) specific temporal factors in the blanching reaction are known for Xenopus only (Bagnara, 1963). These were obtained by determining the M.I. (melanophore index) of tadpoles which were kept in the dark for varying lengths of time. Temporal events of a typical blanching reaction are shown in Fig. 1. In this experiment tadpoles were kept in the dark for Light
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Fig. 1. Melanophore responses, expressed in melanophore index (M. I.), with time, of Xenopus larvae (stages 4 8 4 9 ) during development of, and recovery from, the body-blanchingreaction induced by 28-min exposure to darkness (shaded area). (From Bagnara, 1963).
28 min before being returned to lighted conditions. Onset of melanophore contraction occurred very quickly and maximum contraction was attained in less than 30 min. When the tadpoles were returned to the light, recovery of the expanded condition proceeded slowly, requiring over an hour. Rapid onset of the blanching reaction implies that only relatively short exposures to darkness are necessary to induce melanophore contraction. This was shown to be true from experiments in which larvae were placed in the dark for brief periods. References p . 503j504
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Fig. 2 demonstrates the melanophore responses of tadpoles placed in the dark for 45 sec. With this short exposure, only a slight reaction is evident, however, the pattern of contraction is similar to that shown for the 28-min exposure. Temporal factors involved in the blanching reaction do not favor an explanation based upon the presence of a photochemical system in individual melanophores. First Dor knes s
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Fig. 2. Melanophore responses, expressed in melanophore index (M.I.), with time, of Xenopus larvae (stages 4849) during development of, and recovery from, the body-blanching reaction induced by 45-sec exposure to darkness (shaded area indicated by arrow). (From Bagnara, 1963).
of all, synthesis of an effective amount of a photosensitive melanophore contracting agent would probably require quite a few minutes. For example, synthesis of enough of the photosensitive material for mediation of the tail-darkening reaction requires about 30 min in the dark (Bagnara, 1957). The body-blanching reaction, however, begins in only a few minutes after larvae are placed in the dark, and, moreover, an exposure to darkness of only 45 sec is sufficient to initiate some response. Even more important is that, upon return to normal illumination, recovery from blanching requires a long time. If recovery of the expanded state involves light inactivation of the photochemical stimulation, only a few minutes should suffice for melanophore re-expansion. It seems more likely that some sort of hormonal substance mediates body blanching. Implicit in this suggestion is that some melanophore contracting hormone is stored and its release leads to rapid melanophore contraction when larvae are placed in the dark even for only short periods. Persistence of blanching would depend upon continued production and release of the active agent. It seems reasonable to consider that the long recovery period corresponds to inactivation or catabolism of the active melanophore contracting agent. The blanching reaction is not a pexliarity of Xenopus larvae. However, it is more easily observed in this species than in many others because melanophores are large, relatively few in number, symmetrically shaped and unobscured by overlying free melanin. The latter presents a real problem in observing accurately melanophores
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Fig 3 . Melanophores in fin at base of tail of Hylu urenicolor larvae. A = under normal illumination; note melanophore expansion. B = in darkness for 1 h; note prominent melanophore contraction, x loo.
Fig. 4. Xenopus larvae, stages 37-38. A = under noranalillumination. B = in darkness for 1 h. Note young melanophores of head, expanded in A, but contracted in B, x 50. (From Bagnara, 1963). References p . 503j504
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in many species such as those of Rana, Hyla, and Bufo. Nevertheless, the blanching reaction has been clearly seen in larvae of many amphibian species. Fig, 3 illustrates melanophore contraction, induced by darkness for 1 h, in skin at the base of the tail of larval Hyla arenicolor. Contraction is evident in both epidermal and dermal melanophores. With the latter it is so profound that several overlapping melanophores which normally appear as one large cell separate out as discrete individual melanophores. The ontogeny of the blanching reaction in Xenopus is interesting, because the onset of this reaction coincides with the first appearance of clearly differentiated melanophores (Bagnara, 1961a, 1963). At stage 37/38 (stages of Nieuwkoop and Faber, 1956) when a few melanophores are beginning to form on the dorsal surface
Fig. 5. Xenopus larvae, stage 42. A = normal larva under normal illumination. B = normal larva in darkness for 1 h; note melanophore contraction. C = ‘pinealectomized’ larva in darkness for 1 h ; note lack of melanophore contraction, x 15.
Fig. 6. Xenopus larvae (stage 54).A = pinealectomized larva in darkcess for 1 h ; note lack of melanophore contraction. B = normal larva in darkness for 1 h; note melanophore contraction. C = normal larva treated with 0.1 pg melatonin; note melanophore contraction, x 5. (From Bagnara, 1963).
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of the head and trunk, these young chromatophores contract when larvae are placed in the dark (Fig. 4). It is interesting that those melanophores located over the forebrain contract more rapidly than those over the hindbrain. The blanching reaction is particularly prominent in early larval stages. In Fig. 5 the blanching reaction of a larva at stage 42 is illustrated. Melanophores of this larva are quite punctate. A weaker, but very distinct reaction is seen in later larval stages. In these stages, melanophore contraction is not restricted to the integument; prominent melanophore contraction can be seen on nerves, blood vessels, and internal organs (Fig. 6). As metamorphosis approaches, body blanching becomes correspondingly weak. 11. P H O T O R E C E P T I O N A N D T H E B L A N C H I N G R E A C T I O N
Implicit in the hypothesis that the pineal is responsible for blanching is the presence of photoreceptors in some part of the pineal complex of amphibians. Recently,
Fig. 7. Electron micrograph of a photoreceptorin the frontal organ of a Xenopus embryo (stage 35/36). L = lamellar array in outer segment; M = mitochondria; ER = endoplasmic reticulum; C = centriolar connecting piece. References p . S03jSOS
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photoreceptive structures have been found in amphibian pineal elements which resemble those found in retinas of lateral eyes. While most of these observations have been made on adult amphibians (Eakin and Westfall, 1961; Kelly, 1962; Oksche and Von Harnack, 1962), Eakin has demonstrated photoreceptor elements in the frontal organ of larval HyZa regilla. Such structures are also present in larvae of Xenopus. In this species, just as with other amphibians, the photoreceptor cell is composed of a highly specialized lamellar outer segment which is joined by means of a centriolar connecting piece to a cytoplasmic inner segment characterized by a dense mitochondria1 array and endoplasmic reticulum. Of particular importance in Xenopus is the fact that photoreceptor elements are well differentiated by stage 35/36 (Fig. 7). This in marked contrast with the relatively undifferentiated macroscopic state of the epiphysis, which, at this stage, still appears to be rudimentary, not yet having separated into its component frontal and pineal organs. It would appear that this precocious differentiation of the epiphyseal photoreceptor structures is directly related to the fact that such young embryos can perform the blanching reaction. The specific location of pineal photoreceptors which might be most important in regulating the blanching reaction is uncertain. Judging from Eakin’s observations (1961) that eyeless HyZa regilla larvae, deprived of frontal organs, show a weaker blanching reaction than eyeless controls, one would surmize that this component of the pineal complex contains photoreceptors which are active in this response. However, the fact that some blanching occurred in these experiments, together with the observations that only partial suppressionof blanching occurs in Xenopus larvae which have been deprived of frontal organ, suggest that perhaps photoreceptor elements of the pineal region itself are very important. It is obvious that pineal photoreceptors are quite sensitive. This is assumed from our observation (Bagnara, 1963) that total darkness is necessary for complete development of this blanching reaction. Xenopus larvae illuminated only by a photographic ‘safelight’fail to blanch. A salient feature of the photoreceptive aspect of the blanching reaction is that the response occurs as a result of lack of illumination. Undoubtedly, this is one reason that Laurens (1916) discounted any influence of light on the pineal organ. He attempted to obtain an active melanophore response by illuminating the pineal directly, thinking that this structure should react as a result of a positive photostimulus. Recently, the concept that the pineal is affected by a lack of light stimulation has gained support from the experiments of Dodt and Heerd (1962). They have recorded stimulation of pineal nerve fibers by darkness in adult Rana temporaria. 111. H O R M O N A L M E C H A N I S M I N T H E B L A N C H I N G R E A C T I O N
Evidence that the pineal releases an hormonal agent which effects the blanching reaction comes from several sources. The first direct experiments (Bagnara, 1960b, 1963) involved cautery of the diencephalic roof. Older XenDpus larvae, ‘pinealectomized’ in this manner, consistently failed to blanch when placed in the dark. The results of typical experiments of this nature are depicted in Fig. 6. Similar results were obtained by Brick (1962) for Ambystoma opacum and by Kelly (1962, 1963) for A .
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opacum and Taricha torosa. Kelly interpreted his results cautiously when he noted that 5-10 days after pinealectomy larvae returned to normal melanophore behavior. He suggested that operative trauma might be a great factor in loss of the blanching reaction. This possibility was taken into account in the original experiments on Xenopus larvae. At that time, larvae were observed to have recovered fully from postoperative effects before they were tested. Some tests were not performed until 3 days after operation. As a further check against the possible existence of indirect effects stemming from subtle prolonged trauma, tests were carried out on very young larvae of Xenopus which had been deprived of the epiphysis in embryonic stages, In order to eliminate the epiphysis completely, in the face of the profound regenerative capacity of the embryonic pineal, the whole top of the prosencephalon was removed. Such larvae developed quite well except that the cephalic region was reduced in size. These tadpoles were never able to carry out the blanching reaction (Fig. 5C). Pineal transplants have also suggested the presence of a melanophore contracting agent. Thurmond (personal communication) has observed localized melanophore contraction in the vicinity of pineal grafts on the head of larval Hyla regilla. A similar response was obtained by Kelly (1963) with transplants of pineal in the fin of larval Taricha torosa. With the latter, the response was variable and transitory. Localized melanophore contraction in the vicinity of the pineal has been observed also during the normal blanching reaction. As the blanching begins, the first melanophores to contract are those lying on the brain in the immediate vicinity of the pineal (Fig. 8). During recovery from the blanching reaction, no great disparity in contraction or expansion is seen between peripheral melanophores and those near the pineal. This is
Fig. 8. Xenopus larvae (stage 48). A = during onset of blanching reaction and B = during recovery from blanching reaction. Melanophore index about 3 for both larvae, but melanophores near pineal (arrow) are more contracted during onset than during recovery, x 17. (From Bagnara, 1963). References p. 503lS04
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Fig. 9. For legend see p. 499.
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taken as an indication that during onset of blanching, melanophores near the pineal are exposed more quickly to ‘pineal hormone’ than are those more distantly located. In the course of recovery, all melanophores are in more or less that same state because the hormonal agent is uniformly distributed in the circulation. In formulating the original hypothesis that dark induced blanching results from the direct action of a ‘pineal hormone’ on melanophores, many alternative possibilities were examined. One of these concerned the chance that the pineal could operate indirectly through the hypophysis by inhibiting release of chromatotropic hormone. This alternative, however, was discounted immediately for two reasons. First of all, hypophyseal pigmentary effects take a relatively long time to occur, far longer than it takes the blanching reaction to take place. Secondly, in the blanching reaction of larvae containing guanophores in the dorsal integument, melanophore contraction is not accompanied by guanophore expansion. It is known that hypophyseal chromatotropic hormone (intermedin, MSH, etc.) induces not only melanophore expansion, but also guanophore contraction (Bagnara, 1958,1961b). Hence, if we were to consider that the blanching reaction results from a reduction of circulating chromatotropic hormone, guanophores should expand concomitantly. Nothwithstanding the above reasoning, Brick (1962) suggested recently in an abstract, that the pineal exerts its pigmentary effects indirectly through the hypophysis. His argument is based, apparently, on the facts that pinealectomized larvae of Ambystoma opacum normally display melanophores which are no more expanded than those of unoperated controls and that larvae, both pinealectomized and hypophysectomized, display melanophores which are no more expanded than hypophysectomized controls. Brick’s argument holds only for an hypothesis which states that the pineal exerts pigmentary effects during normal conditions of illumination. I know of no such hypothesis, nor can I think of any evidence which would support it. Moreover, Brick does not even suggest it. Because of Brick’s publication and because Kelly (1962, 1963) emphasizes Brick’s conclusion, we have found it necessary to undertake some experiments which show clearly that control of the blanching reaction does not operate through the hypophysis. These experiments utilized hypophysectomized tadpoles of both Rana pipiens and Hyla arenicolor. As a result of the lack of hypophyseal chromatotropic hormone in such larvae, melanophores are contracted and guanophores are expanded. When these hypophysectomizedtadpoles were placed in the dark for 1 h, melanophores which were already contracted, contracted even more (Fig. 9A,B). In order to obtain a clearer picture of this reaction, an experiment was performed in which a group of hypophysectomized tadpoles was immersed in water containing 1 ,ug of
Fig. 9. Melanophores of hypophysectomized Hyla arenicolor larvae. A, B, C, D = dorsal surface between eyes; E, F = near base of tail. A = normal illumination, note rnelanophores somewhat contracted. B = in darkness for 1 h; note rnelanophores more contracted than in A. C and E = immersed in 1 p g MSH per ml of water for 1 h; note rnelanophore expansion. D and F = irnqersed in 1 p g MSH per ml of water and kept in dark for 1 h; note prominent melanophore contraction. C, E and D, F = each taken from same animal, x 100. References p. 503l504
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MSH (Armour D216-155c) per ml of water. This treatment caused prominent melanophore expansion. Some of these MSH-treated hypophysectomized tadpoles were placed in the dark for 1 h while others, left under normal illumination, served as controls. As is shown in Fig. 9 C, D, E and F, those tadpoles placed in the dark blanched while their controls retained expanded melanophores. Clearly then, it seems that the mechanism of the blanching reaction does not involve alterations in the elaboration of chromatotropic hormone from the hypophysis, for the reaction occurs perfectly well in the absence of the hypophysis. These experiments seem also to clarify a point which previously has been only implied. That is, that in the blanching reaction of normal larvae, the ‘pineal hormone’ over-rides the melanophore expanding stimulation of endogenous chromatotropic hormone. IV. N A T U R E OF T H E B L A N C H I N G H O R MO N E
All of the experiments described so far allude to mediation of the blanching reaction by a ‘pineal hormone’. The identity of the specific hormone active in this response is unknown, however, we have suggested (Bagnara, 1960b, 1963) that it is melatonin. This assumption has been made on tenuous grounds and is shrouded with indirect evidence. At the moment, however, no other hormone appears as a likely candidate except melatonin, the physiology of which fits all the prerequisites for the blanching reaction. Of direct importance is the fact that melatonin is a powerful melanophore contracting agent, probably the most potent compound known. Lerner and Case (1959) point out that it is 105 times as effective as noradrenalin and we have shown that the minimal effective dose of melatonin required for contraction of Xenopus melanophores is O.OOO1 pg per ml of water in which the tadpoles swim (Bagnara, 1963). On the basis of several points including the occurrence of body blanching in the face of hypophyseal chromatophore stimulation, the rapid onset of the blanching reaction, the relatively short period required for inactivation of the ‘pineal hormone’ during recovery from the blanching reaction, and the occurrence of the tail-darkening reaction concomitant with blanching, it seems that the natural melanophore contracting agent is one that is active at very low concentration. Melatonin certainly fulfils this requirement. It should be noted also that the character of pallor induced by melatonin is identical to that which occurs during normal blanching (Fig. 6). Deep melanophores on blood vessels, nerves, and various organs as well as those in the integument contract markedly. Moreover, the response of Xenopus melanophores with time to a dose of melatonin of 0.001 pg per ml of water (Burgers and Van Oordt, 1962) bears striking resemblance to the naturally occurring blanching reaction. Of considerable significance is the localization of melatonin in the pineal. Thus far, its occurrence has been demonstrated only in mammals (Lerner and Case, 1959; Prop and Ariens Kappers, 1961), however, it does not seem too unreasonable to suggest that this indole is also present in the amphibian pineal. We have tried to isolate melatonin from extracts of amphibian pineals using chromatographic separation, but thus far, our results are uncertain (Shaskan, Bagnara, and Obika, un-
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published). The difficulty in demonstrating the presence of melatonin in the amphibian pineal is probably due to the fact that only very small amounts of this substance are present. This is certainly implied from the observation that only very small quantities of melatonin are necessary to cause melanophore contraction, and from the observations of Lerner and Case (1959) and Prop and Ariens Kappers (1961), that the mammalian pineal contains very minute amounts of melatonin. An additional indirect indication that melatonin is the natural blanching hormone is derived from some recent experiments (Quay and Bagnara, unpublished) in which a vast array of indoles have been tested for their melanophore contracting capacities. Among about 50 indoles tested, only melatonin had a readily discernible melanophore contracting effect. Because many of these compounds were structurally very similar to melatonin, it is concluded that the blanching reaction is mediated by a highly specific compound. Apparently, this specificity resides only in melatonin. As suggested in the original hypothesis and as was implied in the proof that blanching does not result indirectly through the hypophysis, the ‘pineal hormone’ should act directly on melanophores. Lerner and Case (1959) have shown that his is the case for isolated frog skin and Novales (1963) reports the same for melanophores in hanging drop cultures of salamander neural crest. From unpublished experiments from our laboratory, Obika observed that melatonin causes contraction of melanophores present in neural crest-epidermis explants of axolotl and, moreover, Shaskan showed that melanophores in isolated tail of Xenopus, expanded by MSH, can be induced to contract by addition of melatonin. DISCUSSION A N D SUMMARY
In this paper I have attempted to present and evaluate evidence concerning the role
of the pineal in regulating pigmentary phenomena. This evidence, much of it circumstantial and indirect, supports the hypothesis advanced 3 years ago (Bagnara, 1960b), that the pineal operates normally in regulating the enigmatic body-blanching reaction of amphibian larvae. The now well-documented photoreceptive properties of the pineal complex are integrated with the known melanophore contracting principle of the pineal. Previously, these two properties stood isolated and little understood. Similarly, an explanation for the body-blanching reaction was wanting. Although final proof of the hypothesis is still to be obtained, both recent and older findings all seem concordant with the original formulation. Thus, a logical explanation now exists for 3 previously unexplained problems. It is obvious that one of the most important pieces of information still needed is the positive identification of melatonin in the amphibian pineal. At the moment we can only infer its presence from knowledge that it exists in mammalian pineals. Even when it is found, eventually, in the amphibian pineal, this will not be enough. Its presence must be identified in specific pineal structures and moreover, its release from these or related structures must be correlated with temporal events in the blanching reaction. Unpublished experiments of Imai, working in our laboratory, attempted to approach the latter problem by utilizing various histological techniques in studying References p . 5031504
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the pineal of Xenopus larvae fixed before or during the blanching reaction. No stainable differences between tadpoles of these two groups were found. There exists considerable information concerning secretory activity of the pineal (for literature see Kelly, 1962), however, there is as yet no way of knowing whether this can be correlated with the amphibian-blanching reaction. Prop and Ariens Kappers (1961) have attempted histochemically to localize indoles, such as melatonin, in the pineal of the rat. Thus far they have been unsuccessful and they suggest, quite logically, that this may be due to the fact that these compounds are present in such small amounts. In looking back at some of the older literature, one wonders why an hypothesis such as the one championed here was not advanced earlier. One reason, perhaps, concerns the fact that the previous investigators thought in terms of an active light stimulus, rather than reactivity based upon the absence of light. Probably another reason is that some of the early workers dealing with pigmentary changes were not overly discriminatory about how long their animals were kept in darkness. It is well-known that amphibian larvae kept in darkness for a long time do not show blanching, but rather become quite dark. Laurens (1915) was aware of these differences and, in fact, referred toithe blanching reaction as a primary response and to the darkening reaction as a secondary response. Assuming now, that the primary response is of pineal mediation, how is the secondary response controlled? One interpretation can be made by comparing the secondary response with the known
Fig. 10. Xenopus larvae (stage 53), eyes removed for 3 days. A = normal illumination; note melanophores quite expanded. B = in darkness for 1 h; note melanophores are contracted but not as prominently as in Fig. 6B, x 4.
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darkening effect displayed by tadpoles which have been blinded for about a week or more. Possibly, in both groups the lack of light stimulation of lateral eyes leads to continual release of hypophyseal chromatotropic hormone with consequent potent melanophore expansion. This would be similar to observations of Burgers et al. (1963) who found that Xenopus tadpoles are darkened on dark backgrounds because of persistent MSH release. With such potent melanophore expansion the blanching reaction could not manifest itself, and hence these tadpoles would appear dark. This is the indication provided by some experiments currently in progress in our laboratory wherein it appears that blinded tadpoles observed on successive days became darker in color and are progresssively less able to perform the blanching reaction (Fig. 10). In reviewing the current status of pineal effects of pigmentation our original ideas are unchanged. In effect, all of the information presented in this review has supported and amplified the original hypothesis concerning the regulation of the amphibian-blanching reaction. In the absence of light, through the action of photoreceptors present in either or in both the frontal organ and pineal organ, the pineal is stimulated to release a direct acting melanophore contracting principle, probably melatonin which quickly causes blanching of larvae. Upon resumption of illumination, release of the melanophore contracting agent ceases, and, gradually, normal metabolic processes reduce the effective quantity of circulating hormone to a level insufficient for melanophore stimulation. As a result, melanophores re-expand because of unchanged hypophyseal influences, and the larva takes on a normal pigmentary state. The blanching reaction is not restricted to Xenopus, it seems to be a general response among amphibian larvae.
ACKNOWLEDGEMENTS
The author is indebted to Dr. M. Obika, Dr. K. Imai, E. G . Shaskan, and William J. Smith for their help on various aspects of this study. Most of this work was supported by NSF grants GI4347 and 024030.
REFERENCES BABAK,E., (1910); Zur chromatischen Hautfunktion der Amphibien. Pfliigers Arch. ges. Physiol., 131, 87-118. BAGNARA, J . T., (1957); Hypophysectomy and the tail-darkening reaction in Xenopus. Proc. Soc. exp. Biol. ( N . Y.),94, 572-575. BAGNARA, J. T., (1958); Hypophyseal control of guanophores in anuran larvae. J. exp. Zool., 137, 264-265. BAGNARA, J. T., (1 960a); Tail melanophores of Xenopus in normal development and regeneration. Biol. Bull., 118, 1-8. J. T., (1960b); Pineal regulation of the body lightening reaction in amphibian larvae. BAGNARA, Science, 132, 1481-1483. BAGNARA, J. T., (1961a); Onset of pineal and hypophyseal regulation of melanophores in Xenopus. Amer. Zool.. 1, 339-340. BAGNARA, J. T., (1961b); Stimulation of guanophores and melanophores by MSH peptides. Amer. Zool., 1, 435.
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BAGNARA, J. T., (1963); The pineal and the body lightening reaction of larval amphibians. Gen. comp. Endocr., 3, 86-100. BEALL,D. SHAPIRO, H. A., AND ZWARENSTEIN, H., (1937); The melanophore contracting principle of the pineal. Chem. Jnd. (London), 56, 190. BORS,O., AND RALSTON, W. C., (1951); A simple assay of mammalian pineal extracts. Proc. SOC. exp. Biol. ( N . Y.),77, 807-808. BRICK,I., (I 962); Relationship of the pineal to the pituitary-melanophore effector system in Amblystoma opacum. Anat. Rec., 142, 239. BURGERS, A. C. J., IMAI, K., ANDVAN OORDT,G. J., (1963); The amount of melanophore-stimulating hormone in single pituitary glands of Xenopus Iaevis kept under various conditions. Gen. comp. Endocr., 3, 53-57. BURGERS, A. C. J., AND VANOORDT,G. J., (1962); Regulation of pigment migration in the amphibian melanophore. Gen. comp. Endocr., Suppl. Vol. 1,99-109. DODT,E., AND HEERD,E., (1962); Mode of action of pineal nerve fibers in frogs. J. Neurophysiol., 25, 405420. EAKIN,R. M., (1961); Photoreceptors in the amphibian frontal organ. Proc. nut. Acad. Sci. (Wash.), 47, 1084-1088. EAKIN,R. M., AND WESTFALL, J. A., (1961); The development of photoreceptors in the stirnorgan of the treefrog, Hyla regilla. Embryologia, 6, 84-98. FUCHS, R. F., (1 914); Der Farbenwechsel und die chromatische Hautfunktion der Tiere. Winterstein’s Handbuch der vergleichenden Physiologie, Vol. 3, 1. Halfte. 2. Teil. Jena, Fischer (S. 1189-1657). KELLY,D. E., (1962); Pineal organs: Photoreception, secretion, and development. Amer. Scientist, 50, 597-625. KELLY,D. E., (1963); Pineal oigan of the newt. A development study. Z. Zellforsch., 58, 693-713. LAURENS, H., (1915); The reactions of the melanophores of Amblystoma larvae. J. exp. Zool., 18, 577-638. LAURENS, H., (1916); The reactions of the melanophores of Amblystoma. The supposed influence of the pineal organ. J. exp. Zool., 20, 237-261. LAURENS, H., (1917); The reactions of the melanophores of Amblystoma tigrinum larvae to light and darkness. J. exp. Zool., 23, 195-205. LERNER,A. B., AND CASE,J. D., TAKAHASHI, Y., LEE,T. H., AND MORI,W., (1958); Isolation of melatonin, the pineal gland factor that lightens melanocytes. J. Amer. Chem. Soc., 80, 2587. LERNER, A. B., AND CASE,J. D., (1959); Pigment cell regulatory factors. J. invest. Derm., 32,211-221. MCCORD,C. P., AND ALLEN,F. P., (1917); Evidences associating pineal gland function with alterations in pigmentation. J. exp. Zool., 23, 207-224. P. D., AND FABER, J., (1956); Normal Table of Xenopus laevis (Daudin). Amsterdam, NIEUWKOOP, North-Holland Publishing Company. NOVALES, R. R., (1963); Responses of cultured melanophores t o the synthetic hormones a-MSH, melatonin, and epinephrine. Ann. N . Y. Acad. Sci., 100, 1035-1047. M., (1962) ;Elektronmikroskopische Untersuchungen am Stirnorgan OKSCHE,A., AND VONHARNACK, (Frontalorgan, Epiphysenendblase) von Rana temporaria und Rana esculenta. Naturwissenschafen, 18,429430. PROP,N., AND ARIENSKAPPERS, J., (1961); Demonstration of some compounds present in the pineal organ of the albino rat by histochemical methods and paper chromatography. Acta anat., 45, 90-109. SCHARRER, E., (1928); Die Lichtempfindlichkeit bei Elritzen (Untersuchungen iiber das Zwischenhim der Fischen). Z . vergl. Physiol., 7 , 1-38. H., THIEBLOT,L., AND S ~ G A LV.,, (1952) ; Interrelation epiphyso-hypophysaire et effet SIMMONET, expanso-melanophorique. Ann. Endocr., 13, 340-344. VONFRISCH, K., (1911); Beitrage zur Physiologie der Pigmentzellen in der Fischhaut. PJliigers Arch. ges. Physiol., 138, 319-387. YOUNG,J. Z., (1935); The photoreceptors of lampreys. 11. The functions of the pineal complex. J. exp. Biol., 12, 254-270.
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DISCUSSION KELLY: It is tempting to postulate the production of melatonin by the amphibian pineal organ since mammalian pineals are a common source of melatonin, and since amphibian melanophores respond to mammalian melatonin by contracting similarly to the way they do in vivo in the dark. However, we need precise, direct information that ( I ) melatonin is indeed involved in the in vivo amphibian melanophore response, and (2) that if it is involved, it actually comes from the pineal organ proper. Obtaining such localized information is indeed difficult as I am aware from my own attempts to produce discrete epiphysectomies in amphibian larvae. We do have presently subsidiary electron microscopical information indicating a photoreceptor function in amphibian pineal organs, and it is reasonable to suspect that such a function may be related by some path with melanophore behavior. Similar evidence t o confirm the production and release of melatonin within the pineal organs themselves is still scarce in amphibian systems, and it is such information that we badly‘need in order to verify the reality of Dr. Bagnara’chypothesis.
BAGNARA: Thank you very much Dr. Kelly for your comment. Certainly, one must first of all demonstrate the presence of melatonin in the amphibian before we can say that the hypothesis mentioned is right. It will not be easy to demonstrate melatonin or at least the enzyme that manufactures melatonin in the amphibian pineal organ. Once we have found melatonin in the amphibian we should also demonstrate that it is present in the pineal organ. Moreover, we will have to demonstrate that the substance is released during the blanching reaction and that the content of melatonin in the blood and in the pineal is concordant with the temporal balance in the blanching reaction. Although these problems will not be easily solved I think they are of the utmost importance. TAYLOR: In view of the fact that it has been found that melatonin inhibits thyroid function I should like to ask Dr. Bagnara whether some effect of melatonin on the metamorphosis of amphibians has been demonstrated. BAGNARA: The only thing I could say about the possible role of the pineal organ in metamorphosis is the following. Amphibian larvae raised in the dark metamorphose much earlier than larvae raised under normal conditions of illumination. This is just the opposite from what one would expect. Because metamorphosis of amphibians is under the influence of thyroxine and if in darkness melatonin is rapidly produced one would expect that the production of thyroxine should be inhibited. Therefore, metamorphosis would be postponed. I may state again that at this stage of research it is too early to say something definite about this problem. OKSCHE:I wonder whether the perivascular elementary granules in the epiphysis of Rana esculenta could indicate the presence of melatonin or related substances.
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Eakin et af. (1963) were not able to detect melatonin in the epiphysis of larval Hyfa regilla. On the other hand, the electron micrographs of these authors do not show any granules of this type. May I ask Dr. Kelly whether he has seen perivascular granules in adult Rana pipiens?
KELLY:Our micrographs do show some granules and vacuoles present in somewhat different parts of the cells than the ones described by you. Most of them have also a different appearance. Therefore, I think that our pictures do not confirm nor deny the observation mentioned by you.
Pineal Regulation of Body Blanching in Amphibian Larvae JOSEPH T. B A G N A R A Department of Zoology, University of Arizona, Tucson, Ariz. (U.S.A.)
Among the many problems being investigated during the current renaissance of interest in the pineal organ, is the role of the pineal in pigmentary phenomena. This is due largely to the discovery that pineal elements are photoreceptive and to the fact that mammalian pineals contain a potent melanophore contracting agent. Integration of these two facts makes possible new interpretations of earlier observations which seemed unrelated. As early as 1911, Von Frisch observed that the pineal area of the teleost Phoxinus was able to regulate melanophore response by virtue of its sensitivity to light. About this time Babak (1910) and Laurens (1915) showed that larvae of several amphibian species became pale when placed in the dark, Fuchs (1914) attempted to explain the latter by advancing a theory based upon Von Frisch’s work on Phoxinus. Fuchs felt that larvae produce, as a result of their ordinary life processes, certain substances which cause melanophores to contract. Thus, when larvae are placed in darkness and are, therefore, free from external stimulation, their melanophores contract under the influence of these endogenous metabolic substances. When light is provided, however, the pineal is stimulated which results in an inhibition of the endogenously induced melanophore contraction. Laurens (1916) was opposed to the view of Fuchs and in an intensive study involving localized illumination of the pineal area of the head of larval Ambystoma opacum and A . maculatum concluded that the epiphysis has no influence on the reactions of larvae to light and to darkness. His arguments were so convincing that McCord and Allen (1917) who discovered that extracts of beef pineals cause blanching in Rana pipiens tadpoles, concluded that any pineal induced pigmentary changes must occur independent of environmental conditions. Subsequent to this early work, only sporadic references concerning the pineal and pigmentation appeared. Scharrer (1928) confirmed the observations of Von Frisch, and Young (1935) observed that pallor which occurs in lampreys on transference from light to darkness is abolished after removal of the pineal complex. Beall et al. (1937) extended the observations of McCord and Allen by demonstrating that the melanophore contracting substance present in beef and sheep pineals is an unsaturated nitrogenous base and Bors and Ralston (1951) observed that pineal extracts of both pig and man induce melanophore contraction in larval and adult Xenopus. Recently, Lerner et al. (1958) have isolated a potent, direct acting, melanophore contracting agent from beef pineal glands. They References p.-503/504
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have identified this compound as melatonin (N-acetyl-5-methoxytryptamine). Presumably, melatonin is the active melanophore contracting agent found in the pineal of other mammalian species. Simmonet et al. (1952) have also demonstrated the presence of a melanophore contracting agent in the mammalian pineal. Moreover, their experiments imply that the pineal may actually play a role in melanophore regulation, for they observed that melanophores of hypophysectomized adult Rana esculenta expand after pinealectomy. Not long ago, while studying the tail-darkening reaction ofXenopuslarvae (Bagnara, 1957, 1960a), an event which occurs when the larvae are placed in the dark, we had reason to compare this response with the body-blanching reaction which occurs at the same time. Tail-darkening is mediated by the direct action of light on fin melanophores. Apparently, a photochemical system is involved, because full expansion of tail melanophores requires over 30 min exposure to darkness while re-contraction occurs about 5 min after light is restored. Temporal factors involved in the paling of the body of these larvae are in marked contrast with tail darkening. Onset of body lightening becomes obvious in 10-15 min and by 20 min melanophore contraction is quite strong. Upon restoration of illumination these melanophores re-expanded very slowly, requiring about an hour for complete expansion. Temporal events in body lightening and subsequent recovery did not seem to be consistent with mediation by a photochemical system. Instead they implied that a hormonal mechanism was involved with relatively rapid onset of melanophore contraction corresponding to release of a stored hormone and slow recovery concordant with gradual loss of this principle from the circulation. With the implication of a hormonal mechanism in the body-blanching reaction, suspicion arose that possibly the melanophore contracting principle of the pineal is the active agent. This suspicion grew even stronger in view of the fact that the paling reaction occurs when eyeless larvae are placed in darkness (Laurens, 1915, 1917; Bagnara, unpublished). It seemed possible, therefore, that the pineal is the photoreceptor necessary for the paling reaction. Following this line of reasoning an hypothesis was established which explained the mechanism of the bodyblanching reaction completely in terms of two aspects of pineal physiology, photoreception, and endocrine functions. Briefly, the hypothesis states that when amphibian larvae are placed in darkness the pineal is affected by the absence of light causing it to release and produce a melanophore contracting agent, the action of which, causes the blanching reaction. The first publication of this hypothesis (Bagnara, 1960b) included data showing that pinealectomized Xencrpus larvae are not capable of performing the blanching reaction when placed in the dark. Confirmation of this point was derived from the study of Eakin (1961) which pointed out that Hyla regilla larvae, deprived of frontal organ and eyes, show a weaker blanching reaction when placed in the dark than do controls without eyes. Since, initial publication of the hypothesis, additional supporting data have accumulated, much of which is presented in a recent paper (Bagnara, 1963). Despite the availability of additional support, this hypothesis remains:a generality awaiting information which will clarify many specific points. The identity of the melanophore contracting agent is still unknown. It is attractive to suppose that melatonin is the active substance, however, this compound
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has never been found in the pineals of lower vertebrates. Moreover, if a hormone is indeed invclved, the specific site of release of this substance is not known. Another question concerns the relationship of other chromatophore-stimulating systems to the blanching reaction. For instance, how does body blanching occur in the presence of the hypophysis with its melanophore expanding factor? These are only a few of the problems which must be understood before the hypothesis advanced above can be accepted. It is the aim of this paper to completely define the body-blanching reaction of larval amphibians, to present all the data which led to the formulation of the pineal hypothesis, and to clarify some of the unsolved problems pertaining to this hypothesis. I. THE B L A N C H I N G REACTION
Although it is generally known that amphibian larvae become light when they are placed in the dark (Babak, 1910; Laurens, 1916, 1917; Bagnara, 1960a) specific temporal factors in the blanching reaction are known for Xenopus only (Bagnara, 1963). These were obtained by determining the M.I. (melanophore index) of tadpoles which were kept in the dark for varying lengths of time. Temporal events of a typical blanching reaction are shown in Fig. 1. In this experiment tadpoles were kept in the dark for Light
i.5
I
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A
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o
t
7
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- Deep melanophores
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42 49 56 63
Fig. 1. Melanophore responses, expressed in melanophore index (M. I.), with time, of Xenopus larvae (stages 4 8 4 9 ) during development of, and recovery from, the body-blanchingreaction induced by 28-min exposure to darkness (shaded area). (From Bagnara, 1963).
28 min before being returned to lighted conditions. Onset of melanophore contraction occurred very quickly and maximum contraction was attained in less than 30 min. When the tadpoles were returned to the light, recovery of the expanded condition proceeded slowly, requiring over an hour. Rapid onset of the blanching reaction implies that only relatively short exposures to darkness are necessary to induce melanophore contraction. This was shown to be true from experiments in which larvae were placed in the dark for brief periods. References p . 503j504
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Fig. 2 demonstrates the melanophore responses of tadpoles placed in the dark for 45 sec. With this short exposure, only a slight reaction is evident, however, the pattern of contraction is similar to that shown for the 28-min exposure. Temporal factors involved in the blanching reaction do not favor an explanation based upon the presence of a photochemical system in individual melanophores. First Dor knes s
Light
M'121
o Deep melonophores
't
A Superficial melanophores
( 1 5 1 1 1 1 1 1 30 1 351 0
10
15
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Time ( m i d
Fig. 2. Melanophore responses, expressed in melanophore index (M.I.), with time, of Xenopus larvae (stages 4849) during development of, and recovery from, the body-blanching reaction induced by 45-sec exposure to darkness (shaded area indicated by arrow). (From Bagnara, 1963).
of all, synthesis of an effective amount of a photosensitive melanophore contracting agent would probably require quite a few minutes. For example, synthesis of enough of the photosensitive material for mediation of the tail-darkening reaction requires about 30 min in the dark (Bagnara, 1957). The body-blanching reaction, however, begins in only a few minutes after larvae are placed in the dark, and, moreover, an exposure to darkness of only 45 sec is sufficient to initiate some response. Even more important is that, upon return to normal illumination, recovery from blanching requires a long time. If recovery of the expanded state involves light inactivation of the photochemical stimulation, only a few minutes should suffice for melanophore re-expansion. It seems more likely that some sort of hormonal substance mediates body blanching. Implicit in this suggestion is that some melanophore contracting hormone is stored and its release leads to rapid melanophore contraction when larvae are placed in the dark even for only short periods. Persistence of blanching would depend upon continued production and release of the active agent. It seems reasonable to consider that the long recovery period corresponds to inactivation or catabolism of the active melanophore contracting agent. The blanching reaction is not a pexliarity of Xenopus larvae. However, it is more easily observed in this species than in many others because melanophores are large, relatively few in number, symmetrically shaped and unobscured by overlying free melanin. The latter presents a real problem in observing accurately melanophores
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Fig 3 . Melanophores in fin at base of tail of Hylu urenicolor larvae. A = under normal illumination; note melanophore expansion. B = in darkness for 1 h; note prominent melanophore contraction, x loo.
Fig. 4. Xenopus larvae, stages 37-38. A = under noranalillumination. B = in darkness for 1 h. Note young melanophores of head, expanded in A, but contracted in B, x 50. (From Bagnara, 1963). References p . 503j504
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in many species such as those of Rana, Hyla, and Bufo. Nevertheless, the blanching reaction has been clearly seen in larvae of many amphibian species. Fig, 3 illustrates melanophore contraction, induced by darkness for 1 h, in skin at the base of the tail of larval Hyla arenicolor. Contraction is evident in both epidermal and dermal melanophores. With the latter it is so profound that several overlapping melanophores which normally appear as one large cell separate out as discrete individual melanophores. The ontogeny of the blanching reaction in Xenopus is interesting, because the onset of this reaction coincides with the first appearance of clearly differentiated melanophores (Bagnara, 1961a, 1963). At stage 37/38 (stages of Nieuwkoop and Faber, 1956) when a few melanophores are beginning to form on the dorsal surface
Fig. 5. Xenopus larvae, stage 42. A = normal larva under normal illumination. B = normal larva in darkness for 1 h; note melanophore contraction. C = ‘pinealectomized’ larva in darkness for 1 h ; note lack of melanophore contraction, x 15.
Fig. 6. Xenopus larvae (stage 54).A = pinealectomized larva in darkcess for 1 h ; note lack of melanophore contraction. B = normal larva in darkness for 1 h; note melanophore contraction. C = normal larva treated with 0.1 pg melatonin; note melanophore contraction, x 5. (From Bagnara, 1963).
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of the head and trunk, these young chromatophores contract when larvae are placed in the dark (Fig. 4). It is interesting that those melanophores located over the forebrain contract more rapidly than those over the hindbrain. The blanching reaction is particularly prominent in early larval stages. In Fig. 5 the blanching reaction of a larva at stage 42 is illustrated. Melanophores of this larva are quite punctate. A weaker, but very distinct reaction is seen in later larval stages. In these stages, melanophore contraction is not restricted to the integument; prominent melanophore contraction can be seen on nerves, blood vessels, and internal organs (Fig. 6). As metamorphosis approaches, body blanching becomes correspondingly weak. 11. P H O T O R E C E P T I O N A N D T H E B L A N C H I N G R E A C T I O N
Implicit in the hypothesis that the pineal is responsible for blanching is the presence of photoreceptors in some part of the pineal complex of amphibians. Recently,
Fig. 7. Electron micrograph of a photoreceptorin the frontal organ of a Xenopus embryo (stage 35/36). L = lamellar array in outer segment; M = mitochondria; ER = endoplasmic reticulum; C = centriolar connecting piece. References p . S03jSOS
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photoreceptive structures have been found in amphibian pineal elements which resemble those found in retinas of lateral eyes. While most of these observations have been made on adult amphibians (Eakin and Westfall, 1961; Kelly, 1962; Oksche and Von Harnack, 1962), Eakin has demonstrated photoreceptor elements in the frontal organ of larval HyZa regilla. Such structures are also present in larvae of Xenopus. In this species, just as with other amphibians, the photoreceptor cell is composed of a highly specialized lamellar outer segment which is joined by means of a centriolar connecting piece to a cytoplasmic inner segment characterized by a dense mitochondria1 array and endoplasmic reticulum. Of particular importance in Xenopus is the fact that photoreceptor elements are well differentiated by stage 35/36 (Fig. 7). This in marked contrast with the relatively undifferentiated macroscopic state of the epiphysis, which, at this stage, still appears to be rudimentary, not yet having separated into its component frontal and pineal organs. It would appear that this precocious differentiation of the epiphyseal photoreceptor structures is directly related to the fact that such young embryos can perform the blanching reaction. The specific location of pineal photoreceptors which might be most important in regulating the blanching reaction is uncertain. Judging from Eakin’s observations (1961) that eyeless HyZa regilla larvae, deprived of frontal organs, show a weaker blanching reaction than eyeless controls, one would surmize that this component of the pineal complex contains photoreceptors which are active in this response. However, the fact that some blanching occurred in these experiments, together with the observations that only partial suppressionof blanching occurs in Xenopus larvae which have been deprived of frontal organ, suggest that perhaps photoreceptor elements of the pineal region itself are very important. It is obvious that pineal photoreceptors are quite sensitive. This is assumed from our observation (Bagnara, 1963) that total darkness is necessary for complete development of this blanching reaction. Xenopus larvae illuminated only by a photographic ‘safelight’fail to blanch. A salient feature of the photoreceptive aspect of the blanching reaction is that the response occurs as a result of lack of illumination. Undoubtedly, this is one reason that Laurens (1916) discounted any influence of light on the pineal organ. He attempted to obtain an active melanophore response by illuminating the pineal directly, thinking that this structure should react as a result of a positive photostimulus. Recently, the concept that the pineal is affected by a lack of light stimulation has gained support from the experiments of Dodt and Heerd (1962). They have recorded stimulation of pineal nerve fibers by darkness in adult Rana temporaria. 111. H O R M O N A L M E C H A N I S M I N T H E B L A N C H I N G R E A C T I O N
Evidence that the pineal releases an hormonal agent which effects the blanching reaction comes from several sources. The first direct experiments (Bagnara, 1960b, 1963) involved cautery of the diencephalic roof. Older XenDpus larvae, ‘pinealectomized’ in this manner, consistently failed to blanch when placed in the dark. The results of typical experiments of this nature are depicted in Fig. 6. Similar results were obtained by Brick (1962) for Ambystoma opacum and by Kelly (1962, 1963) for A .
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opacum and Taricha torosa. Kelly interpreted his results cautiously when he noted that 5-10 days after pinealectomy larvae returned to normal melanophore behavior. He suggested that operative trauma might be a great factor in loss of the blanching reaction. This possibility was taken into account in the original experiments on Xenopus larvae. At that time, larvae were observed to have recovered fully from postoperative effects before they were tested. Some tests were not performed until 3 days after operation. As a further check against the possible existence of indirect effects stemming from subtle prolonged trauma, tests were carried out on very young larvae of Xenopus which had been deprived of the epiphysis in embryonic stages, In order to eliminate the epiphysis completely, in the face of the profound regenerative capacity of the embryonic pineal, the whole top of the prosencephalon was removed. Such larvae developed quite well except that the cephalic region was reduced in size. These tadpoles were never able to carry out the blanching reaction (Fig. 5C). Pineal transplants have also suggested the presence of a melanophore contracting agent. Thurmond (personal communication) has observed localized melanophore contraction in the vicinity of pineal grafts on the head of larval Hyla regilla. A similar response was obtained by Kelly (1963) with transplants of pineal in the fin of larval Taricha torosa. With the latter, the response was variable and transitory. Localized melanophore contraction in the vicinity of the pineal has been observed also during the normal blanching reaction. As the blanching begins, the first melanophores to contract are those lying on the brain in the immediate vicinity of the pineal (Fig. 8). During recovery from the blanching reaction, no great disparity in contraction or expansion is seen between peripheral melanophores and those near the pineal. This is
Fig. 8. Xenopus larvae (stage 48). A = during onset of blanching reaction and B = during recovery from blanching reaction. Melanophore index about 3 for both larvae, but melanophores near pineal (arrow) are more contracted during onset than during recovery, x 17. (From Bagnara, 1963). References p. 503lS04
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Fig. 9. For legend see p. 499.
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taken as an indication that during onset of blanching, melanophores near the pineal are exposed more quickly to ‘pineal hormone’ than are those more distantly located. In the course of recovery, all melanophores are in more or less that same state because the hormonal agent is uniformly distributed in the circulation. In formulating the original hypothesis that dark induced blanching results from the direct action of a ‘pineal hormone’ on melanophores, many alternative possibilities were examined. One of these concerned the chance that the pineal could operate indirectly through the hypophysis by inhibiting release of chromatotropic hormone. This alternative, however, was discounted immediately for two reasons. First of all, hypophyseal pigmentary effects take a relatively long time to occur, far longer than it takes the blanching reaction to take place. Secondly, in the blanching reaction of larvae containing guanophores in the dorsal integument, melanophore contraction is not accompanied by guanophore expansion. It is known that hypophyseal chromatotropic hormone (intermedin, MSH, etc.) induces not only melanophore expansion, but also guanophore contraction (Bagnara, 1958,1961b). Hence, if we were to consider that the blanching reaction results from a reduction of circulating chromatotropic hormone, guanophores should expand concomitantly. Nothwithstanding the above reasoning, Brick (1962) suggested recently in an abstract, that the pineal exerts its pigmentary effects indirectly through the hypophysis. His argument is based, apparently, on the facts that pinealectomized larvae of Ambystoma opacum normally display melanophores which are no more expanded than those of unoperated controls and that larvae, both pinealectomized and hypophysectomized, display melanophores which are no more expanded than hypophysectomized controls. Brick’s argument holds only for an hypothesis which states that the pineal exerts pigmentary effects during normal conditions of illumination. I know of no such hypothesis, nor can I think of any evidence which would support it. Moreover, Brick does not even suggest it. Because of Brick’s publication and because Kelly (1962, 1963) emphasizes Brick’s conclusion, we have found it necessary to undertake some experiments which show clearly that control of the blanching reaction does not operate through the hypophysis. These experiments utilized hypophysectomized tadpoles of both Rana pipiens and Hyla arenicolor. As a result of the lack of hypophyseal chromatotropic hormone in such larvae, melanophores are contracted and guanophores are expanded. When these hypophysectomizedtadpoles were placed in the dark for 1 h, melanophores which were already contracted, contracted even more (Fig. 9A,B). In order to obtain a clearer picture of this reaction, an experiment was performed in which a group of hypophysectomized tadpoles was immersed in water containing 1 ,ug of
Fig. 9. Melanophores of hypophysectomized Hyla arenicolor larvae. A, B, C, D = dorsal surface between eyes; E, F = near base of tail. A = normal illumination, note rnelanophores somewhat contracted. B = in darkness for 1 h; note rnelanophores more contracted than in A. C and E = immersed in 1 p g MSH per ml of water for 1 h; note rnelanophore expansion. D and F = irnqersed in 1 p g MSH per ml of water and kept in dark for 1 h; note prominent melanophore contraction. C, E and D, F = each taken from same animal, x 100. References p. 503l504
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MSH (Armour D216-155c) per ml of water. This treatment caused prominent melanophore expansion. Some of these MSH-treated hypophysectomized tadpoles were placed in the dark for 1 h while others, left under normal illumination, served as controls. As is shown in Fig. 9 C, D, E and F, those tadpoles placed in the dark blanched while their controls retained expanded melanophores. Clearly then, it seems that the mechanism of the blanching reaction does not involve alterations in the elaboration of chromatotropic hormone from the hypophysis, for the reaction occurs perfectly well in the absence of the hypophysis. These experiments seem also to clarify a point which previously has been only implied. That is, that in the blanching reaction of normal larvae, the ‘pineal hormone’ over-rides the melanophore expanding stimulation of endogenous chromatotropic hormone. IV. N A T U R E OF T H E B L A N C H I N G H O R MO N E
All of the experiments described so far allude to mediation of the blanching reaction by a ‘pineal hormone’. The identity of the specific hormone active in this response is unknown, however, we have suggested (Bagnara, 1960b, 1963) that it is melatonin. This assumption has been made on tenuous grounds and is shrouded with indirect evidence. At the moment, however, no other hormone appears as a likely candidate except melatonin, the physiology of which fits all the prerequisites for the blanching reaction. Of direct importance is the fact that melatonin is a powerful melanophore contracting agent, probably the most potent compound known. Lerner and Case (1959) point out that it is 105 times as effective as noradrenalin and we have shown that the minimal effective dose of melatonin required for contraction of Xenopus melanophores is O.OOO1 pg per ml of water in which the tadpoles swim (Bagnara, 1963). On the basis of several points including the occurrence of body blanching in the face of hypophyseal chromatophore stimulation, the rapid onset of the blanching reaction, the relatively short period required for inactivation of the ‘pineal hormone’ during recovery from the blanching reaction, and the occurrence of the tail-darkening reaction concomitant with blanching, it seems that the natural melanophore contracting agent is one that is active at very low concentration. Melatonin certainly fulfils this requirement. It should be noted also that the character of pallor induced by melatonin is identical to that which occurs during normal blanching (Fig. 6). Deep melanophores on blood vessels, nerves, and various organs as well as those in the integument contract markedly. Moreover, the response of Xenopus melanophores with time to a dose of melatonin of 0.001 pg per ml of water (Burgers and Van Oordt, 1962) bears striking resemblance to the naturally occurring blanching reaction. Of considerable significance is the localization of melatonin in the pineal. Thus far, its occurrence has been demonstrated only in mammals (Lerner and Case, 1959; Prop and Ariens Kappers, 1961), however, it does not seem too unreasonable to suggest that this indole is also present in the amphibian pineal. We have tried to isolate melatonin from extracts of amphibian pineals using chromatographic separation, but thus far, our results are uncertain (Shaskan, Bagnara, and Obika, un-
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published). The difficulty in demonstrating the presence of melatonin in the amphibian pineal is probably due to the fact that only very small amounts of this substance are present. This is certainly implied from the observation that only very small quantities of melatonin are necessary to cause melanophore contraction, and from the observations of Lerner and Case (1959) and Prop and Ariens Kappers (1961), that the mammalian pineal contains very minute amounts of melatonin. An additional indirect indication that melatonin is the natural blanching hormone is derived from some recent experiments (Quay and Bagnara, unpublished) in which a vast array of indoles have been tested for their melanophore contracting capacities. Among about 50 indoles tested, only melatonin had a readily discernible melanophore contracting effect. Because many of these compounds were structurally very similar to melatonin, it is concluded that the blanching reaction is mediated by a highly specific compound. Apparently, this specificity resides only in melatonin. As suggested in the original hypothesis and as was implied in the proof that blanching does not result indirectly through the hypophysis, the ‘pineal hormone’ should act directly on melanophores. Lerner and Case (1959) have shown that his is the case for isolated frog skin and Novales (1963) reports the same for melanophores in hanging drop cultures of salamander neural crest. From unpublished experiments from our laboratory, Obika observed that melatonin causes contraction of melanophores present in neural crest-epidermis explants of axolotl and, moreover, Shaskan showed that melanophores in isolated tail of Xenopus, expanded by MSH, can be induced to contract by addition of melatonin. DISCUSSION A N D SUMMARY
In this paper I have attempted to present and evaluate evidence concerning the role
of the pineal in regulating pigmentary phenomena. This evidence, much of it circumstantial and indirect, supports the hypothesis advanced 3 years ago (Bagnara, 1960b), that the pineal operates normally in regulating the enigmatic body-blanching reaction of amphibian larvae. The now well-documented photoreceptive properties of the pineal complex are integrated with the known melanophore contracting principle of the pineal. Previously, these two properties stood isolated and little understood. Similarly, an explanation for the body-blanching reaction was wanting. Although final proof of the hypothesis is still to be obtained, both recent and older findings all seem concordant with the original formulation. Thus, a logical explanation now exists for 3 previously unexplained problems. It is obvious that one of the most important pieces of information still needed is the positive identification of melatonin in the amphibian pineal. At the moment we can only infer its presence from knowledge that it exists in mammalian pineals. Even when it is found, eventually, in the amphibian pineal, this will not be enough. Its presence must be identified in specific pineal structures and moreover, its release from these or related structures must be correlated with temporal events in the blanching reaction. Unpublished experiments of Imai, working in our laboratory, attempted to approach the latter problem by utilizing various histological techniques in studying References p . 5031504
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the pineal of Xenopus larvae fixed before or during the blanching reaction. No stainable differences between tadpoles of these two groups were found. There exists considerable information concerning secretory activity of the pineal (for literature see Kelly, 1962), however, there is as yet no way of knowing whether this can be correlated with the amphibian-blanching reaction. Prop and Ariens Kappers (1961) have attempted histochemically to localize indoles, such as melatonin, in the pineal of the rat. Thus far they have been unsuccessful and they suggest, quite logically, that this may be due to the fact that these compounds are present in such small amounts. In looking back at some of the older literature, one wonders why an hypothesis such as the one championed here was not advanced earlier. One reason, perhaps, concerns the fact that the previous investigators thought in terms of an active light stimulus, rather than reactivity based upon the absence of light. Probably another reason is that some of the early workers dealing with pigmentary changes were not overly discriminatory about how long their animals were kept in darkness. It is well-known that amphibian larvae kept in darkness for a long time do not show blanching, but rather become quite dark. Laurens (1915) was aware of these differences and, in fact, referred toithe blanching reaction as a primary response and to the darkening reaction as a secondary response. Assuming now, that the primary response is of pineal mediation, how is the secondary response controlled? One interpretation can be made by comparing the secondary response with the known
Fig. 10. Xenopus larvae (stage 53), eyes removed for 3 days. A = normal illumination; note melanophores quite expanded. B = in darkness for 1 h; note melanophores are contracted but not as prominently as in Fig. 6B, x 4.
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darkening effect displayed by tadpoles which have been blinded for about a week or more. Possibly, in both groups the lack of light stimulation of lateral eyes leads to continual release of hypophyseal chromatotropic hormone with consequent potent melanophore expansion. This would be similar to observations of Burgers et al. (1963) who found that Xenopus tadpoles are darkened on dark backgrounds because of persistent MSH release. With such potent melanophore expansion the blanching reaction could not manifest itself, and hence these tadpoles would appear dark. This is the indication provided by some experiments currently in progress in our laboratory wherein it appears that blinded tadpoles observed on successive days became darker in color and are progresssively less able to perform the blanching reaction (Fig. 10). In reviewing the current status of pineal effects of pigmentation our original ideas are unchanged. In effect, all of the information presented in this review has supported and amplified the original hypothesis concerning the regulation of the amphibian-blanching reaction. In the absence of light, through the action of photoreceptors present in either or in both the frontal organ and pineal organ, the pineal is stimulated to release a direct acting melanophore contracting principle, probably melatonin which quickly causes blanching of larvae. Upon resumption of illumination, release of the melanophore contracting agent ceases, and, gradually, normal metabolic processes reduce the effective quantity of circulating hormone to a level insufficient for melanophore stimulation. As a result, melanophores re-expand because of unchanged hypophyseal influences, and the larva takes on a normal pigmentary state. The blanching reaction is not restricted to Xenopus, it seems to be a general response among amphibian larvae.
ACKNOWLEDGEMENTS
The author is indebted to Dr. M. Obika, Dr. K. Imai, E. G . Shaskan, and William J. Smith for their help on various aspects of this study. Most of this work was supported by NSF grants GI4347 and 024030.
REFERENCES BABAK,E., (1910); Zur chromatischen Hautfunktion der Amphibien. Pfliigers Arch. ges. Physiol., 131, 87-118. BAGNARA, J . T., (1957); Hypophysectomy and the tail-darkening reaction in Xenopus. Proc. Soc. exp. Biol. ( N . Y.),94, 572-575. BAGNARA, J. T., (1958); Hypophyseal control of guanophores in anuran larvae. J. exp. Zool., 137, 264-265. BAGNARA, J. T., (1 960a); Tail melanophores of Xenopus in normal development and regeneration. Biol. Bull., 118, 1-8. J. T., (1960b); Pineal regulation of the body lightening reaction in amphibian larvae. BAGNARA, Science, 132, 1481-1483. BAGNARA, J. T., (1961a); Onset of pineal and hypophyseal regulation of melanophores in Xenopus. Amer. Zool.. 1, 339-340. BAGNARA, J. T., (1961b); Stimulation of guanophores and melanophores by MSH peptides. Amer. Zool., 1, 435.
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BAGNARA, J. T., (1963); The pineal and the body lightening reaction of larval amphibians. Gen. comp. Endocr., 3, 86-100. BEALL,D. SHAPIRO, H. A., AND ZWARENSTEIN, H., (1937); The melanophore contracting principle of the pineal. Chem. Jnd. (London), 56, 190. BORS,O., AND RALSTON, W. C., (1951); A simple assay of mammalian pineal extracts. Proc. SOC. exp. Biol. ( N . Y.),77, 807-808. BRICK,I., (I 962); Relationship of the pineal to the pituitary-melanophore effector system in Amblystoma opacum. Anat. Rec., 142, 239. BURGERS, A. C. J., IMAI, K., ANDVAN OORDT,G. J., (1963); The amount of melanophore-stimulating hormone in single pituitary glands of Xenopus Iaevis kept under various conditions. Gen. comp. Endocr., 3, 53-57. BURGERS, A. C. J., AND VANOORDT,G. J., (1962); Regulation of pigment migration in the amphibian melanophore. Gen. comp. Endocr., Suppl. Vol. 1,99-109. DODT,E., AND HEERD,E., (1962); Mode of action of pineal nerve fibers in frogs. J. Neurophysiol., 25, 405420. EAKIN,R. M., (1961); Photoreceptors in the amphibian frontal organ. Proc. nut. Acad. Sci. (Wash.), 47, 1084-1088. EAKIN,R. M., AND WESTFALL, J. A., (1961); The development of photoreceptors in the stirnorgan of the treefrog, Hyla regilla. Embryologia, 6, 84-98. FUCHS, R. F., (1 914); Der Farbenwechsel und die chromatische Hautfunktion der Tiere. Winterstein’s Handbuch der vergleichenden Physiologie, Vol. 3, 1. Halfte. 2. Teil. Jena, Fischer (S. 1189-1657). KELLY,D. E., (1962); Pineal organs: Photoreception, secretion, and development. Amer. Scientist, 50, 597-625. KELLY,D. E., (1963); Pineal oigan of the newt. A development study. Z. Zellforsch., 58, 693-713. LAURENS, H., (1915); The reactions of the melanophores of Amblystoma larvae. J. exp. Zool., 18, 577-638. LAURENS, H., (1916); The reactions of the melanophores of Amblystoma. The supposed influence of the pineal organ. J. exp. Zool., 20, 237-261. LAURENS, H., (1917); The reactions of the melanophores of Amblystoma tigrinum larvae to light and darkness. J. exp. Zool., 23, 195-205. LERNER,A. B., AND CASE,J. D., TAKAHASHI, Y., LEE,T. H., AND MORI,W., (1958); Isolation of melatonin, the pineal gland factor that lightens melanocytes. J. Amer. Chem. Soc., 80, 2587. LERNER, A. B., AND CASE,J. D., (1959); Pigment cell regulatory factors. J. invest. Derm., 32,211-221. MCCORD,C. P., AND ALLEN,F. P., (1917); Evidences associating pineal gland function with alterations in pigmentation. J. exp. Zool., 23, 207-224. P. D., AND FABER, J., (1956); Normal Table of Xenopus laevis (Daudin). Amsterdam, NIEUWKOOP, North-Holland Publishing Company. NOVALES, R. R., (1963); Responses of cultured melanophores t o the synthetic hormones a-MSH, melatonin, and epinephrine. Ann. N . Y. Acad. Sci., 100, 1035-1047. M., (1962) ;Elektronmikroskopische Untersuchungen am Stirnorgan OKSCHE,A., AND VONHARNACK, (Frontalorgan, Epiphysenendblase) von Rana temporaria und Rana esculenta. Naturwissenschafen, 18,429430. PROP,N., AND ARIENSKAPPERS, J., (1961); Demonstration of some compounds present in the pineal organ of the albino rat by histochemical methods and paper chromatography. Acta anat., 45, 90-109. SCHARRER, E., (1928); Die Lichtempfindlichkeit bei Elritzen (Untersuchungen iiber das Zwischenhim der Fischen). Z . vergl. Physiol., 7 , 1-38. H., THIEBLOT,L., AND S ~ G A LV.,, (1952) ; Interrelation epiphyso-hypophysaire et effet SIMMONET, expanso-melanophorique. Ann. Endocr., 13, 340-344. VONFRISCH, K., (1911); Beitrage zur Physiologie der Pigmentzellen in der Fischhaut. PJliigers Arch. ges. Physiol., 138, 319-387. YOUNG,J. Z., (1935); The photoreceptors of lampreys. 11. The functions of the pineal complex. J. exp. Biol., 12, 254-270.
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DISCUSSION KELLY: It is tempting to postulate the production of melatonin by the amphibian pineal organ since mammalian pineals are a common source of melatonin, and since amphibian melanophores respond to mammalian melatonin by contracting similarly to the way they do in vivo in the dark. However, we need precise, direct information that ( I ) melatonin is indeed involved in the in vivo amphibian melanophore response, and (2) that if it is involved, it actually comes from the pineal organ proper. Obtaining such localized information is indeed difficult as I am aware from my own attempts to produce discrete epiphysectomies in amphibian larvae. We do have presently subsidiary electron microscopical information indicating a photoreceptor function in amphibian pineal organs, and it is reasonable to suspect that such a function may be related by some path with melanophore behavior. Similar evidence t o confirm the production and release of melatonin within the pineal organs themselves is still scarce in amphibian systems, and it is such information that we badly‘need in order to verify the reality of Dr. Bagnara’chypothesis.
BAGNARA: Thank you very much Dr. Kelly for your comment. Certainly, one must first of all demonstrate the presence of melatonin in the amphibian before we can say that the hypothesis mentioned is right. It will not be easy to demonstrate melatonin or at least the enzyme that manufactures melatonin in the amphibian pineal organ. Once we have found melatonin in the amphibian we should also demonstrate that it is present in the pineal organ. Moreover, we will have to demonstrate that the substance is released during the blanching reaction and that the content of melatonin in the blood and in the pineal is concordant with the temporal balance in the blanching reaction. Although these problems will not be easily solved I think they are of the utmost importance. TAYLOR: In view of the fact that it has been found that melatonin inhibits thyroid function I should like to ask Dr. Bagnara whether some effect of melatonin on the metamorphosis of amphibians has been demonstrated. BAGNARA: The only thing I could say about the possible role of the pineal organ in metamorphosis is the following. Amphibian larvae raised in the dark metamorphose much earlier than larvae raised under normal conditions of illumination. This is just the opposite from what one would expect. Because metamorphosis of amphibians is under the influence of thyroxine and if in darkness melatonin is rapidly produced one would expect that the production of thyroxine should be inhibited. Therefore, metamorphosis would be postponed. I may state again that at this stage of research it is too early to say something definite about this problem. OKSCHE:I wonder whether the perivascular elementary granules in the epiphysis of Rana esculenta could indicate the presence of melatonin or related substances.
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Eakin et af. (1963) were not able to detect melatonin in the epiphysis of larval Hyfa regilla. On the other hand, the electron micrographs of these authors do not show any granules of this type. May I ask Dr. Kelly whether he has seen perivascular granules in adult Rana pipiens?
KELLY:Our micrographs do show some granules and vacuoles present in somewhat different parts of the cells than the ones described by you. Most of them have also a different appearance. Therefore, I think that our pictures do not confirm nor deny the observation mentioned by you.