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The pars distalis nerves and metamorphosis in Rana temporaria
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
The Pars Distalis
36, 497-501 (1978)
Nerves and Metamorphosis
in Rana temporaria
STIG ARONSSON Deportment
of Zoology,
Utziversity of Giiteborg,
Fuck, S-400 33 Giiteborg,
Sweden
Accepted October 17, 1978 The length of prometamorphosis in the tadpoles of Roan temporurio is influenced by different treatments: (1) thyroidectomy, (2) propylthiouracil, (3) l-thyroxine, (4) low temperature. In normally developing tadpoles the fluorescent pars distaiis fibers persist until climax. Even when prometamorphosis is artificially prolonged, irrespective of the reason, the fibers still persist until climax. When the prometamorphic period is artificially shortened. or when a prolongation of prometamorphosis is interrupted by thyroxin-induced climax, the fibers disappear. Thus the disappearance of the aminergic nerves is a metamorphic event associated with climax.
It has been shown with the aid of the Falck-Hillarp fluorescence technique that there are aminergic fibers in the pars distalis of Rarza tevnporaria tadpoles from early premetamorphosis, and that these fibers disappear at the metamorphic climax (Aronsson, 1976). Adult frogs lack such fibers (Enemar and Falck, 1965; Prasada Rao and Hartwig, 1974; Aronsson, 1976). At Gosner’s stage 41 (Gosner, 1960) fluorescent pars distalis fibers are seen in the microscope, but already at the following stage, i.e., the onset of climax, they have all disappeared. Thus the disappearance of the nerves is at least temporally related to the thyroxin-controlled, revolutionary events that constitute climax. The aim of the present work is to study the occurrence of the pars distalis fibers in tadpoles with experimentally prolonged or shortened prometamorphosis. MATERIALS
AND METHODS
Ram tetnpororin eggs were collected from ponds in Gothenburg and surrounding areas and were placed in tap water. The tadpoles were staged according to Gosner (1960). The experiments were performed in the following way. Group I. Twenty-four tadpoles were thyroidectomized (TX) and eight tadpoles were sham operated as controls. After 6 weeks the sham operated controls had passed climax. Twelve of the TX tadpoles (now at
stage 36) were placed in tanks with 200,ppb thyroxin in the water (I-thyroxinnatrium, Nyegaard and Co, Oslo) (TxT) while 12 tadpoles were kept in tap water (TxW). When the TxT tadpoles showed external signs of climax after 6 days, all the TxT and TxW tadpoles were processed according to the F&k-Hillarp technique (Falck and Owman, 1965). See below. Group II. Twenty-four tadpoles at stage 36 were placed in water with 200 mg of the goitrogen propyithiouracil (pu) (Tiotil, Pharmacia Uppsala) per liter (PU). Eight tadpoles were kept in tap water as controls. Six weeks after the controls had passed climax, eight of the PU tadpoles (now at stages 39-40) were kept in pu-water (PU), eight tadpoles were s,imuitaneously treated with pu and thyroxin (PUT) and eight tadpoles were placed in tap water (PUW) thus enabling their thyroid glands to resume activity. When the PUT tadpoles showed external signs of climax ,ali PU, PUT, and PUW tadpoles were processed eccordkng to the Falck-Hillarp technique. Group III. Sixteen tadpoles at stage 36 were treated with 200 ppb thyroxin by immersion (NT) while eight tadpoles were kept in tap water and developed in ‘a normal way. After 3 days eight of the NT tadpoles were freeze-dried. After six days, when the &T tadpoles showed external signs of climax, the normal tadpoles were at stage 41, and all the tadpoles were processed according to Falck-Hillarp. Group IV. The metamorphosis of 20 tadpoles was retarded by keeping them at a low temperature. They were collected as eggs on April 5, 1976, kept at +I0 until October 1976, (stages 37-39), kept at +4’+between October 1976 and April 1977 (stage 39),’ and were then removed into +lO”. They had developed to stages 39-41 by June 1977 and were still at thosemstages in September 1977. Ten tadpoles were, then transferred to water at + 18”, where they passed into climax in ‘10days
497 ~~6-6480~78/0364-049Y$01.~~0 Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form resewed.
STIG ARONSSON
498
while the tadpoles at + 10” were still at stages 39-4 1. All tadpoles were processed according to Falck-Hillarp. The animals were killed by decapitation and the hypophysis with the adjacent part of the brain was dissected out and immediately frozen in liquid propane cooled by liquid nitrogen. The sphenoid cartilage was left to protect the hypophysis. The specimens were freeze-dried and processed for fluorescence microscopy according to the histochemical method of Falck and Hillarp (Falck and Owman, 1965). The paraformaldehyde treatment was performed at +80” for 1 hr with paraformaldehyde equilibrated in air with 55-60% relative humidity. Specimens not treated with paraformaldehyde were used as controls for the specificity of the fluorescence. The tissue was serially sectioned at 7pm in the sagittal and transverse planes and mounted in Entellan (Merck) for fluorescence microscopy. For fluorescence microscopy, a Leitz Orthoplan with incident light and a high-pressure mercury lamp. CS2OOW-4, was used with excitation filters BG 3, BG 38, barrier filters K 470, and dichroic mirror TK 455. The concepts of prometamorphosis and climax are used according to Etkin (1968). The onset of prometamorphosis corresponds to stage 36 and the onset of climax to stage 42.
RESULTS
When tadpoles at prometamorphic stages are treated with thyroxin by immersion (see Materials and Methods) the induced climax is somewhat abnormal in comparison with the spontaneous, normal climax. Thus the order of the external tissue changes that imply climax is different. Often the tip of the tail begins to regress and the jaws begin to form before the emergence of the forelimbs, and the, differentiation of the hind feet corresponds to stages 37-39. The external signs of climax appear after 5-6 days. Group Z
When the TxT tadpoles showed external signs of climax after thyroxin treatment for 6 days, the hypophysial monoaminergic fiber pattern was of the normal appearance of climax stages, i.e., an accumulation ,of fluorescent material around the capillaries of eminentia mediana and a fluorescent fiber network in the pars intermedia but no
fluorescent fibers in the pars distalis. The TxW tadpoles did not develop beyond stage 37; they showed a normal prometamorphic hypophysis so far as the monoaminergic fibers are concerned, i.e., an accumulation of fluorescent material around the capillaries of the eminentia mediana, a fluorescent-fiber network in the pars intermedia and fluorescent fibers in the pars distalis. Group ZZ
The PU tadpoles developed to stages 39-41 but did not pass these stages. The pattern of fluorescence was of ordinary prometamorphic appearance, i.e., fibers in pars distalis. The PUT tadpoles showed climax signs after thyroxin treatment for 6 days. As in normal climax tadpoles, fluorescent material accumulated around the capillaries of the eminentia mediana and in the pars intermedia there was a fluorescent network. In the pars distalis, however, a phenomenon heretofore unknown appeared: many fluorescent spots were distributed over the gland. No fibers could be detected among these strongly fluorescent spots. Some of these spots are granular and intracellular while others seem to be amorphous and intercellular. Their fluorescence,.however, is not specific; it appears without paraformaldehyde treatment and persists after treatment with water vapor. The PUW tadpoles were at stage 41 when they were freeze-dried. They showed the same type of fluorescent spots in pars distalis as the PUT tadpoles at climax, but in addition, fluorescent fibers were observed among the spots. Group ZZZ
The NT tadpoles that were thyroxin treated for 3 days and then processed according to Falck-Hillarp showed the normal prometamorphic fluorescence appearance, i.e., fibers in the pars distalis. Their external morphological status corre-
PARS
DISTALiS
NERVES
AND
METAMORPHOSIS
499
sponded to stage 41 and no climax signs could be seen.However, thoseNT tadpoles that were thyroxin treated for 6 days showed external climax signs and no fluorescentfibers in the pars distalis. The tadpoles that were kept at low temperatures for about 16 months until they were freeze-dried, (stages 39-4I) showed ~uorescent fibers in the pars distaffs like normal prometamorphic tadpoles. In the tadpoles that were brou~t into climax at -t-18”,no such fibers appeared. Although no accurate morphometric studies have been carried out, it may be noted that the partes distalis of thyroidectomized and pu-treatedtadpoles in sagittal and transversesectionsare about 1.5times as large as.normal ones (length scale).This results in altered relations between the hypophys~a1 parts. Becauseof the enlarged pars distalis, the pars intermedia and pars nervosa adopt a more rostro-dorsal position than in normal tadpolesof corresponding stages..However, the pattern and distribut~on of the fluorescent fibers do not change.
FIG. 1. Transverse section of the ~rni~e*~~a mediana (ME) and the pars distalis (PD) at lata pro-. metamorphosis. c, Capillaries in the emirienda mediana; s, sinusoid capiliaries in the pars dj~~~~i~. The arrows indicate the fluorescent fibers in the pars distalis. (x250).
pars intermedia. At the onset of climax, however, when the known thyr~~i~induced emergenceof the forelimbs occurs and the tail begins to regress,the pars distalis nerves suddenly disappear. As cornpared with the pars iuterme~ia with its densefluorescentnetwork, the pars di$alis has few delicate varicose fibers. No &erations in distribution or number of the fib&s are observed during the period between DISCUSSION stage 2.5and th” onset of climhx. (Figs. 1 and 2) (Aronsson, 1976).This ex~erime~~a~ It has long been known that thyroxin is study shows that the occ~~e~ce’a responsible for metamorphic changes in amphibians (Cudernatsch, 1912). Since then much work has been done in the vast field of amphibian metamorphosis, with referenceto hormonal mechanisms{for review see Etkin, 1968; Dodd and Dodd, 1976).It is supposedthat the tissuesgradually become more sensitive to thyroid hormones during devel,opmentand it is ciear that at climax some tissues differentiate, e.g., the hind limbs, while others, e.g., the tail, undergo regressions. In normally developingRana ternporaria tadpoles the monoaminergic pars distalis FIG. 2. Transverse section Of tha ~m~~e~~ia nerves appear ait early premetamorphosis, mediana and the pars distalis at, the first stage of at the same stage (25) as the ~rninerg~c climax. There are no fluorescent 4%&s in the pars nerves in the eminentia mediana and the distalis. Abbreviations as in Pig. 1. (x120).
500
STIG ARONSSON
tribution of the pars distalis fibers are similar to those of normal preclimax stages. In the tadpoles of group II abundant fluorescent sports occurred. Their fluorescence is not of aminergic origin. In the tadpoles of group I such spots did not occur. That means that chemical but not surgical thyroidectomy followed by thyroxin treatment gives the spots. If these unspecific fluorescent structures were accumulated hormone products, they should occur in group I as well as in group II. It may be supposed that they are pharmacological phenomena. In the studies described above it has been shown that a prolongation of the -prometamorphic period, irrespective of the methods used to bring this about, leads to a preservation of the fluorescent pars distalis fibers until climax. When the period is shortened the fibers still disappear at climax, and when a prolongation of prometamorphosis is interrupted by thyroxin-induced climax, the fibers also disappear. The disappearance of the nerves is thus associated with climax and it may be supposed that the same mechanisms which control the effects of thyroxin on other tissues are involved in the disappearance of the pars distalis fibers. Moreover, the nerves themselves, which are of hypothalamic origin (Aronsson, in preparation) may be a part of the thyroid controlling system. According to the theory of Dodd and Dodd (1976), during late prometamorphosis and early climax there is an “abnormal” secretion of TSH (and TRH) because the metamorphosing tissues consume an increasing amount of thyroxin. The portal system of the eminentia mediana is not fully developed until climax (Etkin, 1965; Aronsson, 1976) and perhaps the neurovascular link between the hypothalamus and the pars distalis during prometamorphosis is not sufficient to account for this abnormal secretion so accessory, TSH-stimulating nerves are required. During adult life such a state never occurs and
the nerves are unnecessary, which may explain their disappearance at climax. An effective inhibitory nervous influence presupposes a complete innervation as in the pars intermedia (Enemar and Falck, 1965) so a stimulatory role for the few pars distalis nerves is more probable. Anyhow, it may be supposed that the nerves play some part in the control of secretion and/or release of pars distalis hormones. The larval tissues which differentiate or are transformed during metamorphosis are adapted to adult functions while those ones that regress have only larval functions. Since the pars distalis fibers, or at least their biogenic amines, disappear at spontaneous climax (Aronsson, 1976) and at exogenous thyroxin-induced climax (this work), the fibers must be regarded as exclusively larval structures. The nerves disappear at the transition from “fish-life” to “tetrapod-life.” Teleosts (Bern et al. 1971) and the lungfish, Lepidosiren (Zambrano and Iturriza, 1973), have an aminergic pars distalis innervation while there are no reports of such an innervation in tetrapods The function, if any, of the pars distalis nerves of Rana temporaria must be restricted to preclimax stages and maybe to water life. However, the possibility that the nerves are evolutionary vestiges must not be excluded. ACKNOWLEDGMENTS This investigation was supported by Grant B 2180-032 from the Swedish Natural Science Research Council. I am indebted to Professor Anders Enemar for his encouraging interest in this study, to Miss Barbro Liifnertz for skillful technical assistance, and to Miss Eva Myrin for typewriting and corrections.
REFERENCES Aronsson, S. (1976). The ontogenesis of monoaminergic nerve fibers in the hypophysis of Rana temporaria with special reference to the pars distalis. Cell Tiss. Res. 171, 437-448. Bern, H. A., Zambrano, D., and Nishioka, R. S.
PARS
DISTALIS
NERVES
(197 1). Comparison of the innervation of the pituitary of two euryhaline teleost fishes, Gillichrhys mirubilis and Tilapia mossambica, with special reference to the origin and nature of type “B” fibres. Mem. Sot. Endocrinol. No. 19, 817-822. Dodd, M. H. I., and Dodd, J. M. (1976). The biology of metamorphosis. In “Physiology of the Amphibia” (B. Lofts, ed.), Vol. III, pp. 467-599. Academic Press, New York. Enemar, A., and Falck, B. (1965). On the presence of adrenergic nerves in the pars intermedia of the frog, Rana temporaria. Gen. Comp. Endocrinol. 5, 577-583. Etkin, W. (1965). The phenomena of amphibian metamorphosis: IV, The development of the median eminence. J. Murphol. 116, 371-378. Etkin, W. (1968). Hormonal control of amphibian metamorphosis. In “Metamorphosis” (W. Etkin and L. I. Gilbert, eds.), pp. 313-348. NorthHolland, Amsterdam. Falck, B., and Owman, Ch. (1965). A detailed meth-
AND
METAMORPHOSIS
581
odological description of the fluorescence method for the cellular demonstration of biogenic monoamines. Acta Univ. Lund. Sect. 2. No. 7. C.W.K. Gleerup, Lund. Gosner, K. L. (1960). A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16, 183-190. Gudernatsch, J. F. (1912). Feeding experiments on tadpoles: I, The influence of specific organs given as food on growth and differentiation. A contribution to the knowledge of organs with internal secretion. Wilhelm Roux Arch. EntwickIungsmech. Organismen 35,457-483. Prasada Rao, P. D., and Hartwig, H+ G. (1974). Monoaminergic tracts of the diencephalon and innervation of the pars intermedia in Rana temporaria. Cell Tiss. Res. 151, 1-26. Zambrano, D., and Iturriza, F. C. (1973). Hypothalamic-hypophysial relationships in the South American lungfish Lepidosiren paradoxa. Gen. Comp. Endocrinol. 20, 256-273.
AND
COMPARATIVE
ENDOCRINOLOGY
The Pars Distalis
36, 497-501 (1978)
Nerves and Metamorphosis
in Rana temporaria
STIG ARONSSON Deportment
of Zoology,
Utziversity of Giiteborg,
Fuck, S-400 33 Giiteborg,
Sweden
Accepted October 17, 1978 The length of prometamorphosis in the tadpoles of Roan temporurio is influenced by different treatments: (1) thyroidectomy, (2) propylthiouracil, (3) l-thyroxine, (4) low temperature. In normally developing tadpoles the fluorescent pars distaiis fibers persist until climax. Even when prometamorphosis is artificially prolonged, irrespective of the reason, the fibers still persist until climax. When the prometamorphic period is artificially shortened. or when a prolongation of prometamorphosis is interrupted by thyroxin-induced climax, the fibers disappear. Thus the disappearance of the aminergic nerves is a metamorphic event associated with climax.
It has been shown with the aid of the Falck-Hillarp fluorescence technique that there are aminergic fibers in the pars distalis of Rarza tevnporaria tadpoles from early premetamorphosis, and that these fibers disappear at the metamorphic climax (Aronsson, 1976). Adult frogs lack such fibers (Enemar and Falck, 1965; Prasada Rao and Hartwig, 1974; Aronsson, 1976). At Gosner’s stage 41 (Gosner, 1960) fluorescent pars distalis fibers are seen in the microscope, but already at the following stage, i.e., the onset of climax, they have all disappeared. Thus the disappearance of the nerves is at least temporally related to the thyroxin-controlled, revolutionary events that constitute climax. The aim of the present work is to study the occurrence of the pars distalis fibers in tadpoles with experimentally prolonged or shortened prometamorphosis. MATERIALS
AND METHODS
Ram tetnpororin eggs were collected from ponds in Gothenburg and surrounding areas and were placed in tap water. The tadpoles were staged according to Gosner (1960). The experiments were performed in the following way. Group I. Twenty-four tadpoles were thyroidectomized (TX) and eight tadpoles were sham operated as controls. After 6 weeks the sham operated controls had passed climax. Twelve of the TX tadpoles (now at
stage 36) were placed in tanks with 200,ppb thyroxin in the water (I-thyroxinnatrium, Nyegaard and Co, Oslo) (TxT) while 12 tadpoles were kept in tap water (TxW). When the TxT tadpoles showed external signs of climax after 6 days, all the TxT and TxW tadpoles were processed according to the F&k-Hillarp technique (Falck and Owman, 1965). See below. Group II. Twenty-four tadpoles at stage 36 were placed in water with 200 mg of the goitrogen propyithiouracil (pu) (Tiotil, Pharmacia Uppsala) per liter (PU). Eight tadpoles were kept in tap water as controls. Six weeks after the controls had passed climax, eight of the PU tadpoles (now at stages 39-40) were kept in pu-water (PU), eight tadpoles were s,imuitaneously treated with pu and thyroxin (PUT) and eight tadpoles were placed in tap water (PUW) thus enabling their thyroid glands to resume activity. When the PUT tadpoles showed external signs of climax ,ali PU, PUT, and PUW tadpoles were processed eccordkng to the Falck-Hillarp technique. Group III. Sixteen tadpoles at stage 36 were treated with 200 ppb thyroxin by immersion (NT) while eight tadpoles were kept in tap water and developed in ‘a normal way. After 3 days eight of the NT tadpoles were freeze-dried. After six days, when the &T tadpoles showed external signs of climax, the normal tadpoles were at stage 41, and all the tadpoles were processed according to Falck-Hillarp. Group IV. The metamorphosis of 20 tadpoles was retarded by keeping them at a low temperature. They were collected as eggs on April 5, 1976, kept at +I0 until October 1976, (stages 37-39), kept at +4’+between October 1976 and April 1977 (stage 39),’ and were then removed into +lO”. They had developed to stages 39-41 by June 1977 and were still at thosemstages in September 1977. Ten tadpoles were, then transferred to water at + 18”, where they passed into climax in ‘10days
497 ~~6-6480~78/0364-049Y$01.~~0 Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form resewed.
STIG ARONSSON
498
while the tadpoles at + 10” were still at stages 39-4 1. All tadpoles were processed according to Falck-Hillarp. The animals were killed by decapitation and the hypophysis with the adjacent part of the brain was dissected out and immediately frozen in liquid propane cooled by liquid nitrogen. The sphenoid cartilage was left to protect the hypophysis. The specimens were freeze-dried and processed for fluorescence microscopy according to the histochemical method of Falck and Hillarp (Falck and Owman, 1965). The paraformaldehyde treatment was performed at +80” for 1 hr with paraformaldehyde equilibrated in air with 55-60% relative humidity. Specimens not treated with paraformaldehyde were used as controls for the specificity of the fluorescence. The tissue was serially sectioned at 7pm in the sagittal and transverse planes and mounted in Entellan (Merck) for fluorescence microscopy. For fluorescence microscopy, a Leitz Orthoplan with incident light and a high-pressure mercury lamp. CS2OOW-4, was used with excitation filters BG 3, BG 38, barrier filters K 470, and dichroic mirror TK 455. The concepts of prometamorphosis and climax are used according to Etkin (1968). The onset of prometamorphosis corresponds to stage 36 and the onset of climax to stage 42.
RESULTS
When tadpoles at prometamorphic stages are treated with thyroxin by immersion (see Materials and Methods) the induced climax is somewhat abnormal in comparison with the spontaneous, normal climax. Thus the order of the external tissue changes that imply climax is different. Often the tip of the tail begins to regress and the jaws begin to form before the emergence of the forelimbs, and the, differentiation of the hind feet corresponds to stages 37-39. The external signs of climax appear after 5-6 days. Group Z
When the TxT tadpoles showed external signs of climax after thyroxin treatment for 6 days, the hypophysial monoaminergic fiber pattern was of the normal appearance of climax stages, i.e., an accumulation ,of fluorescent material around the capillaries of eminentia mediana and a fluorescent fiber network in the pars intermedia but no
fluorescent fibers in the pars distalis. The TxW tadpoles did not develop beyond stage 37; they showed a normal prometamorphic hypophysis so far as the monoaminergic fibers are concerned, i.e., an accumulation of fluorescent material around the capillaries of the eminentia mediana, a fluorescent-fiber network in the pars intermedia and fluorescent fibers in the pars distalis. Group ZZ
The PU tadpoles developed to stages 39-41 but did not pass these stages. The pattern of fluorescence was of ordinary prometamorphic appearance, i.e., fibers in pars distalis. The PUT tadpoles showed climax signs after thyroxin treatment for 6 days. As in normal climax tadpoles, fluorescent material accumulated around the capillaries of the eminentia mediana and in the pars intermedia there was a fluorescent network. In the pars distalis, however, a phenomenon heretofore unknown appeared: many fluorescent spots were distributed over the gland. No fibers could be detected among these strongly fluorescent spots. Some of these spots are granular and intracellular while others seem to be amorphous and intercellular. Their fluorescence,.however, is not specific; it appears without paraformaldehyde treatment and persists after treatment with water vapor. The PUW tadpoles were at stage 41 when they were freeze-dried. They showed the same type of fluorescent spots in pars distalis as the PUT tadpoles at climax, but in addition, fluorescent fibers were observed among the spots. Group ZZZ
The NT tadpoles that were thyroxin treated for 3 days and then processed according to Falck-Hillarp showed the normal prometamorphic fluorescence appearance, i.e., fibers in the pars distalis. Their external morphological status corre-
PARS
DISTALiS
NERVES
AND
METAMORPHOSIS
499
sponded to stage 41 and no climax signs could be seen.However, thoseNT tadpoles that were thyroxin treated for 6 days showed external climax signs and no fluorescentfibers in the pars distalis. The tadpoles that were kept at low temperatures for about 16 months until they were freeze-dried, (stages 39-4I) showed ~uorescent fibers in the pars distaffs like normal prometamorphic tadpoles. In the tadpoles that were brou~t into climax at -t-18”,no such fibers appeared. Although no accurate morphometric studies have been carried out, it may be noted that the partes distalis of thyroidectomized and pu-treatedtadpoles in sagittal and transversesectionsare about 1.5times as large as.normal ones (length scale).This results in altered relations between the hypophys~a1 parts. Becauseof the enlarged pars distalis, the pars intermedia and pars nervosa adopt a more rostro-dorsal position than in normal tadpolesof corresponding stages..However, the pattern and distribut~on of the fluorescent fibers do not change.
FIG. 1. Transverse section of the ~rni~e*~~a mediana (ME) and the pars distalis (PD) at lata pro-. metamorphosis. c, Capillaries in the emirienda mediana; s, sinusoid capiliaries in the pars dj~~~~i~. The arrows indicate the fluorescent fibers in the pars distalis. (x250).
pars intermedia. At the onset of climax, however, when the known thyr~~i~induced emergenceof the forelimbs occurs and the tail begins to regress,the pars distalis nerves suddenly disappear. As cornpared with the pars iuterme~ia with its densefluorescentnetwork, the pars di$alis has few delicate varicose fibers. No &erations in distribution or number of the fib&s are observed during the period between DISCUSSION stage 2.5and th” onset of climhx. (Figs. 1 and 2) (Aronsson, 1976).This ex~erime~~a~ It has long been known that thyroxin is study shows that the occ~~e~ce’a responsible for metamorphic changes in amphibians (Cudernatsch, 1912). Since then much work has been done in the vast field of amphibian metamorphosis, with referenceto hormonal mechanisms{for review see Etkin, 1968; Dodd and Dodd, 1976).It is supposedthat the tissuesgradually become more sensitive to thyroid hormones during devel,opmentand it is ciear that at climax some tissues differentiate, e.g., the hind limbs, while others, e.g., the tail, undergo regressions. In normally developingRana ternporaria tadpoles the monoaminergic pars distalis FIG. 2. Transverse section Of tha ~m~~e~~ia nerves appear ait early premetamorphosis, mediana and the pars distalis at, the first stage of at the same stage (25) as the ~rninerg~c climax. There are no fluorescent 4%&s in the pars nerves in the eminentia mediana and the distalis. Abbreviations as in Pig. 1. (x120).
500
STIG ARONSSON
tribution of the pars distalis fibers are similar to those of normal preclimax stages. In the tadpoles of group II abundant fluorescent sports occurred. Their fluorescence is not of aminergic origin. In the tadpoles of group I such spots did not occur. That means that chemical but not surgical thyroidectomy followed by thyroxin treatment gives the spots. If these unspecific fluorescent structures were accumulated hormone products, they should occur in group I as well as in group II. It may be supposed that they are pharmacological phenomena. In the studies described above it has been shown that a prolongation of the -prometamorphic period, irrespective of the methods used to bring this about, leads to a preservation of the fluorescent pars distalis fibers until climax. When the period is shortened the fibers still disappear at climax, and when a prolongation of prometamorphosis is interrupted by thyroxin-induced climax, the fibers also disappear. The disappearance of the nerves is thus associated with climax and it may be supposed that the same mechanisms which control the effects of thyroxin on other tissues are involved in the disappearance of the pars distalis fibers. Moreover, the nerves themselves, which are of hypothalamic origin (Aronsson, in preparation) may be a part of the thyroid controlling system. According to the theory of Dodd and Dodd (1976), during late prometamorphosis and early climax there is an “abnormal” secretion of TSH (and TRH) because the metamorphosing tissues consume an increasing amount of thyroxin. The portal system of the eminentia mediana is not fully developed until climax (Etkin, 1965; Aronsson, 1976) and perhaps the neurovascular link between the hypothalamus and the pars distalis during prometamorphosis is not sufficient to account for this abnormal secretion so accessory, TSH-stimulating nerves are required. During adult life such a state never occurs and
the nerves are unnecessary, which may explain their disappearance at climax. An effective inhibitory nervous influence presupposes a complete innervation as in the pars intermedia (Enemar and Falck, 1965) so a stimulatory role for the few pars distalis nerves is more probable. Anyhow, it may be supposed that the nerves play some part in the control of secretion and/or release of pars distalis hormones. The larval tissues which differentiate or are transformed during metamorphosis are adapted to adult functions while those ones that regress have only larval functions. Since the pars distalis fibers, or at least their biogenic amines, disappear at spontaneous climax (Aronsson, 1976) and at exogenous thyroxin-induced climax (this work), the fibers must be regarded as exclusively larval structures. The nerves disappear at the transition from “fish-life” to “tetrapod-life.” Teleosts (Bern et al. 1971) and the lungfish, Lepidosiren (Zambrano and Iturriza, 1973), have an aminergic pars distalis innervation while there are no reports of such an innervation in tetrapods The function, if any, of the pars distalis nerves of Rana temporaria must be restricted to preclimax stages and maybe to water life. However, the possibility that the nerves are evolutionary vestiges must not be excluded. ACKNOWLEDGMENTS This investigation was supported by Grant B 2180-032 from the Swedish Natural Science Research Council. I am indebted to Professor Anders Enemar for his encouraging interest in this study, to Miss Barbro Liifnertz for skillful technical assistance, and to Miss Eva Myrin for typewriting and corrections.
REFERENCES Aronsson, S. (1976). The ontogenesis of monoaminergic nerve fibers in the hypophysis of Rana temporaria with special reference to the pars distalis. Cell Tiss. Res. 171, 437-448. Bern, H. A., Zambrano, D., and Nishioka, R. S.
PARS
DISTALIS
NERVES
(197 1). Comparison of the innervation of the pituitary of two euryhaline teleost fishes, Gillichrhys mirubilis and Tilapia mossambica, with special reference to the origin and nature of type “B” fibres. Mem. Sot. Endocrinol. No. 19, 817-822. Dodd, M. H. I., and Dodd, J. M. (1976). The biology of metamorphosis. In “Physiology of the Amphibia” (B. Lofts, ed.), Vol. III, pp. 467-599. Academic Press, New York. Enemar, A., and Falck, B. (1965). On the presence of adrenergic nerves in the pars intermedia of the frog, Rana temporaria. Gen. Comp. Endocrinol. 5, 577-583. Etkin, W. (1965). The phenomena of amphibian metamorphosis: IV, The development of the median eminence. J. Murphol. 116, 371-378. Etkin, W. (1968). Hormonal control of amphibian metamorphosis. In “Metamorphosis” (W. Etkin and L. I. Gilbert, eds.), pp. 313-348. NorthHolland, Amsterdam. Falck, B., and Owman, Ch. (1965). A detailed meth-
AND
METAMORPHOSIS
581
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