Metamorphosis Although ontogenetic metamorphosis occurs in many groups of animals, it is best known in insect and amphibians.

Metamorphosis can be defined as a series of abrupt ppostembryonic changes involving structural, physilogical, biochemical, and behavioral transformation. Recent summaries of amphibian metamorphosis are by Gilbert and Frieden (1981) and Fox (1984) and M. Dodd and J. Dodd (1976). Three major kinds of changes occur during amphibian metamorphosis (1) regression of structures and function that are significant only to be larvae; (2) transformation of larval structures into a formm suitablee for adult use; and (3) development af structures and function de novo that are essensial to the adult. Three metamorphic stages defined by Etkin(1932) are referencd commonly by experimental biologists. (1) premetammorphosis, characterized by considerable growth and development of larval structures but not metaporphic changes; among amphiboians this phase is unique to anurans; (2) prometamorphosis, a period of continued growth, especially of limbs, and initiation of minor metamorphic changes; and (3) climax, the period of radical changes that culminate in the loss of most larval characters; in anurans the beginning of this periode. All of the events of larval growth and metaporphosis are controlled ultimately by hormones. Throughout the stages of metamorphosis there is a finely tuned integration of the endocrine glands, the products of which influence the morphological and physiological changes (Table 7-1). Gundernatsch (1912) discovered that metamorphosis in tadpoles of Rana temporaria was precipitated by feeding the tadpoles on thyroid glands of horses. This observation marked the beginning of the science of experimental endocrinology and the intitation of studies of the endocrine control of amphibian metamorphosis. Nearly all of the experimental work on amphibian metamorphosis has dealt with only three species of anurans: Xenopus laevis, Rana catesbeina, and R.pipiens. Far less extensive studies have been with the salamanders Ambystoma gracile and A. tigrinum, and the anurans Bufo bufo and R.temporaria. Consequently, realistic comparisons among taxa are not possible. The material presented here is only a brief synthesis of the extensive experimental work on the hormonal control of amphibian metamorphosis. Much more comprehensive coveerage is presented by M.Dodd and J.Dodd (1976) and A. White and Nicoll ( 1981 ). Thyroid Because of the obvious action of its products on metamorphosis in experimental animals (Table 72),the thyroid commonly is considered to be the keystone of amphibian metamorphosis. The primary product of the tiroid are two hormones-tetraodonthyronine (T4, thyroxin) and triiodonthyronine (T3). The paired thyroid glands are composed of aggregates of spherical, cystlike follicles. Each follicles is lined with a secretory epithelium that surrounds the follicular cavity. The tiroids increases in size during larval development, both by the proliferation of follicles and by an increase in the volume of the entire follicle. Both T3 and T4 are made and stored within the thyroid gland. Synthesis of both hormones occures by the iodination of thyrosine that are present on a specific protein, thyroglobulin. When the thyroid glands is stimulates to produce thyroid hormones, T3 and T4 are relased form the large thyroglobulin molecules and more into the bloodstream.

Amphibians, like other organisms,acquire iodine solely from dietary source. As metamorphosis proceeds, certain histological changes occurs within the thyroid glands. The secretory epithelial cell become progressively more columnar, reaching a peak at metamorphosis, only to regress in adults. Furthermore, during metamorphosis the thyroid gland in anurans undergoes an increase in the amount of rough endoplasmic reticulum and golgi apparatus-chnges presumably associated with the synthesis and secretion of thyroid hormones. Theree is in an apparent surge in the production of all thyroid hormones at metamorphic climax (stage 41-44) in Rana catesbeiana (A. White and Nicoll, 1981). However, in Xenopus laevis , the level of T4 rises gradually from late premetamorphic staages to a peak at midclimax, T3 is not detectable until late prometamorphosis, reaching a maximum earlier than the peak of T4 (Leloup and Buscaglia, 1977). Likewise, in Ambystoma gracile levels of T4 are grater in metamorphosing animals than in larvae or transformaed individuals( Eagleson and McKeown, 1987). These concluing are all based on measurements by radiommunoassay of the relative amounts of plasma protein-boumd iodin, T3 and T4 in the blood. Several reasons could account for the observed themporal differences in circulatory hormone levels among these species. For example, undoubtedly there s a lag time between the actual secretion and subsequent binding of thyroid hormones, the extent of which may vary intespecyfically. Moreover, in many cases circulating plasma levels of hormones do not accurately reflect intracellular concentrations. Cell apparently retain T3 better than T4 as evidenced by the fact that in target cell, T3 binding to cytoplasmic reseptor protein is about 250 times greather than binding bt T4 (Kistler et al., 1977). Consequently, it has been proposed that the actual role of intracellular receptor molecules for thyroid hormones in amphibians larvall tissues may be to ensure adequate retention of T3 by the cell during metamorphosis. Unfortunately, attemps to explains the observed differences in potencies between T3 and T4 in amphibians by measured differences in reseptors binding have not led to consistent result. Apparently the sensitivity of tissues to thyroid hormones change during development. Cells that once were less responsive to T4 my gradually (or abruptly) acquire the abylity to responsive to T4 my gardually (or abruptly) acquired the ability ro respond (i.e., bind) ot this hormone. Such changes unquestionable play an important role in the ability of a tissue to complete the process of metamorphosis. Why tissue respond to the various thyroid hormones in different ways and at different times is not clear. Perhaps insight into the control of metamorphosis may be obtained by better understanding how cells selectively bind T3 and T4 to intracellular receptor molecules.

Interrenals
Like the thyroid, these small glands, consisting of cords of cell surrounded by connective tissue and lying under the dorsal aorta, also undergo ultrastructural changes during development. For example, in Xenopus laevis the interrenal tissue apparently is inactive during premetamorphosis but shows an increase in activity through prometamorphosis, reaching a peak at early metamorphic climax and then regressing (Rapola, 1963). Three interrenal steroids have been identified by radioimmunoassay in serum of tadpoles of Rana catesbeiana (Krug et al,1978). Aldosterone is first detectable in premetamorphosis and remains at a low level until completion of metamorphosis, when it occurs at the slightly higher level characteristic of adults. Corticosterone is present at a low level during late

premetamorphosis and rises rapidly in early prometamorphosis to reach a peak at about Stage 40: the level declines somewhat to midclimax and then increases rapidly to the concentration occuring in adults. Cortisol is present in small amounts in early premetamorphic stages and increases slowly until midprometamorphosis, from which point there is a rapid increase to midclimax and then a decline to the low concentrations characteristics of adults. Not surprisingly, the result of radioimmunoassay of serum of Rana indicate earlier production of steroids than was suspected originally from histological examination of interrenals in Xenopus. It is likely that the sensitve radioimmunoassay procedure more accurately reflects the synthetic capabilities of the interrenal glands. The activity of the interrenal cells can be stimulated by adrenocorticotropic hormone (M. Dodd and J.Dodd, 1976), thereby suggesting that the activity of these glands is under the control of the pars distalis of the pituitary. The exact influence of interrenal steroids on metamorphosis is unknown. The rise in corticosterone levels during prometamorphosis is asscociated with regression of the intestine, which is induced by thyroid hormones. The profile of cortisol levels in serum coincides with the pattern of growth of the hindlimbs in Rana catesbeiana, and the peak level of cortisol is associated with the beginning of rapid resorption of the tail. Because interrenal steroids presumably act synergistically with thyroid hormones to promote tail regression, it has been suggested that cortisol and corticosterone facilitate thyroid-induced metamorphosis. Adenohyphophysis The anterior lobe or pars distalis of the pituitary is the source of many hormones. The secretion that are active during larval development and metamorphosis are hormones that stimulate other endocrine glands or promote growth. Prolactin and possibly a growth hormone seem to direct growth and development by acting on perpheral endocrine organs and by controlling the acctivity of the thyroid gland. There is disagreement as to whether both prolactin and a growth hormone are present in amphibians. It has been suggested that a single hormones with prolactin- and gowth hormones-like properties acts as the somatropic agent in larval amphibians. Generally, this hormone is reffered to as amphibians prolactin. Althought the evidence for a somathropic role of proactin among different species is not consistent, indirect but compelling evidence exists that an endogeneous prolactin-like hormone is responsible for suppressing metamorphosis in Ambystoma, Bufo, and Rana. Prolactin apparently is a potent anabolic and somathrpic agnet in spesific tissues (e. G., caudal tissue in tadpoles and gills in salamander larvae), but it may do little to drive general metabolism in a direction most suitable for overall growth. Perhaps, as suggested by frye et al (1972), there is a selective advantage provided by the employment of a hormone that promotes growth in certain tissues but does not mobilize important energy stores, such as fat bodies, for the promoiton of the body growth, because growth is followed by a considerable expenditure of energy for structural transformation. Prolactin may favor growth in larval amphibians by stimulation of intestinal absorption of amino acids and glucose. Prolactin antagonized the actions of thyroids hormones in certain larval organs. For example, prolactin reduces tail regression and antagonizes thyroid promotion of hindlimb growth, gill regression, water and sodium loss in certain larval tissues, and nitrogen excertion (A. White and Nicoll, 1981). However, prolactin does not inhibit several other responses to thyroid hormons, including pancreatic dehydration, or retinal dehydrogenase activity. Furthermore, prolactin also may

exert a direct inhibitory action on the thyroid gland. Because , metamorphosis proceeds at the expense of growth, it might be expected that antimetamorphic and growth-promoting actions of hormones are intimately related. There is no evidence for the separation of these actions. Prolactin antagonizes the action of thyroid hormones at the level of the target organ. This organ specificity of prolactin antagonisem may be an important factor in determining the correct sequence and rate of tissue regression or differentiation during metaporphosing. The pituitary gland of amphibians, like all vertebrates, is directy controlled by t ehypotalamus in the brain. Dopamine, secreted by the hypotalamus, is a regulator of prolactin secretion . dopamine lowers circulating levelsof prolactn and therefore accelerates metamorphosis in larval amphibians. There is evidence for an inhibitory effect of a dopamine agonist on prolactin secretion in the viviparous Nectophrynoides occidentalis (Zuber-Vogeli, 1968). It has been suggested the aminergic fibers regulate (possibly by tonic inhibition) the release of prolactin during prometamorphosis, the surge of prolactin secretion in late climax may be caused by realise from the inhibitory influence of these fibers, which regress t the onset of metamorphic cimax. Newts (Notophthalamus and Triturus) undergo a second metamorphosis when the terrestrial efts return to water. However, in contrast to the clea prolactin-thyroid antagonism that exists in the metamorphosis of larva to subadults, there is no consistent evidence for a prolactine-thyroid synergism in the control of the second metamorphosis in newts (M. Dodd and J. Dodd, 1976).

Hormonal Integration
A model for the control of anuran development and activation of the metamorphic climax proposed by Etkin (1968) has been modified by M. Dodd and J. Dodd (1976) and A. White and Nicoll (1981). This model can beb cutlined, as follows (fig. 7-1). 1. During premetamorphosis, the median eminence of the hypothalamus is undeveloped, and the brain exerts little or no control over adenohypophysial function. Consequently, secretion of prolactin is high and secretion of thyroid-stimulating hormone (and thus thyroid hormone levels) is low. Therefore, prolactin can promote larval growth without interference from thyroid hormones. Negative feedback of thyroid hormone on secretion of thyroidstimulating hormone is operative. 2. During early prometamorphosis, the rate of secretion of thyroid hormone is high, but this is not reflected in increased plasma proteinbound iodine or by radioimmunoassay-detectable plasma T3 or T4 levels, probably because of the rapid clearance of the thyroid hormones. The increased secretion of thyroid hormones presumably results from rising levels of thyroid-stimulating hormone; this increase probably reflects gradual development of hypothalamus influence on the adenohypophisys. The rate of secretion of thyroid hormones continues to increase, so that late in prometamorphosis the capasity of tissues to bind and utilize thyroid hormones is saturated. Consequently, the continually increasing output of thyroid hormones results in surge in plasma levels of these hormones. 3. The rising level of thyroid hormones also promotes development of the median eminence and the establishment of portal vascular conection between the hypothalamus and the adenohyphophysis. As this process progresses, more thyrotropin-releasing hormone is able

to reach the pituitary to stimulating increased secretion of thyroid hormones. The increase in thyroid hormones promotes further development of the median eminence. Thus, a positive feedback loop is established. 4. While hypothalamic control of pituitary function is developing, secretion of prolactin comes inhibitory control, and the circulating levels of prolactin decrease progressively. Thus, prolactin antagonism of thyroid hormone action on peripheral tissues is reduces, alloowing development to proceed more rapidly. 5. Late in prometamorphosis, the median eminence and its vascular connections with the hyphophysis have developed substailly. The saturation of tissues with thyroid hormones results in rapid and complete transformation (climax). Blood levels of prolactin are greatly reduced duuring this period, reflecting maximum hyphotaamic inhibition. Thus, at this stage of development the prolactin-mediated inhibition of thyroid hormone action is minimized. 6. During metamorphic climax, the positive feedback interaction of the hypothalamohyphophysial-thyroid axis presumably are lost. It is not clear how this comes about. Possibly the aminergic fibers that innervate the adenohyphophysis in larvae are involved in both the positive and negative feedback loops. These fibers disappear during metamorphic climax. Thus rising levels of thyroid hormones during late prometamorphosis may act on the hypothalamus and cause these fibers to increase secretion of thyroid-stimulating hormone, thereby accounting for the positive feedback. Rising levels of thyroid hormones also may cause the ecentual degeneration of these fibers. Thus neural stimulating hormone is lost, and the inhibitory action off thyroid hormone directly on the pituitary can operate unopposed.

OTHER BIOCHEMICAL CHANGES

The morphological and physiological changes that take place during metaporphosis are accompanied by, if not driven by, nonhormonal biochemical changes. Electrophoretic and radioimmunoassay investigations indicate that marked changes take place in the blood in late prometamorphosis and during metamorphic climax (Broyles, 1981), and numerous biochemical changes also occur during organ differentiation and maturation(Smith-Gill and carver, 1981;Atkinson, 1981). The changes in hormone levs in the blood have been discusses in the preceding section.

Serum Proteins
One important function of serum is to maintain osmotc equilibrium between blood anf tissues fluids. An increase in the total protein concrentation at metamorphosis serves to increase the osmotic preasure of the blood and hence its water-retainng capacyti, an important adaptive switch considering the translation from the aquatic environment of larvae to the terrestrial envronment of postmethamorpic amphibians. Albumin, with its lower molecular weight, exerts two to three times the osmotic pressure per unit weight in comparison with the globulins. Not surprisingly, albumin increses proportionately more thn globulins durn gmetamorphosis. At normal blood pH, albumin has great ion-

binding capasity. An increase in the ion-binding capacity per unit volume of blood at metamorphosis fulfills the greater transport needs associated with metabolism and erection in the terrestrial environment (Freiden, 1961)/ The contrentation of serum proteins more than doubles during metamorphosis in various Rana (Just et al., 1977), and at least 20% of the increase in R. Catesbeana is due to an increase in serum albumin, which rises more than 10-fold (Feldhof, 1971). At least part of the increase in serum albumin during metamorphosis is due to an increase rate of synthesis in late prometamorphosis in R. Cateabeana (Ledford and Frieden, 1973); serum albumin peaks at early cllimax and declines at late climax to just about the same level as in midprometamorphosis. Albumin levels remain high in froglets, indicating a decreased rate of albumin degredation. In species of ambystomatid salamanders (Nussbaum, 1974), there are increases in the albumin/globulin ratio and total protein concentration. However, in the aquatic Cryptobranchus alleganiensis there are no changes in serum proteins (Nickerson nad Mays, 1973). The hypothesis that the increase in albumin attendant with metamorphosis is designed by natural selection to meet the osmotic needs of the organism in the terrestrial environment (Frieden et al., 1957) was proposed as an adaptive aspect of amphibian metamorphosis. Subsequent investigations on Bufo arenarum from humids and arid habitats (Bertini and Cei, 1960) and varous ambystomatid salamanders (Nussbaum, 1974) demonstrated that there is both interspecific (Ambystoma macrodactylum and Rhyacotriton olympicus) and intraspecific (Dicamptodon ensatus and B. Arenarum) evidence taht higher concentrations of albumin are characteristic of transformed individuals of populations adapted to more arid environments. The absence of metamorphic changes in serum protein concentration in cryptobranchus is understandable. However, the aquatic Xenopus laevis exhibits a marked change in albumin/globulin ratio and total protein concentration betweeen larvae and adults. In comparison with Rana, this change is slow and steady and continues after morpholoogical metamorphoosis the differences between Rana and Xenopus are reasonable in that a rapid change in serum proteins in Rana is necessary for the froglet to survive in the terrestrial environment, whereas Xenopsus remains aqutic. Then, why should concentrations of serum proteins change in Xenopus and not in Cryptobranchus? The latter lives in permanent rivers, whereas Xenopus lives in ponds that may dry up during droughts; the frog aestivate in the soil during droughts and thus are confronted with the osmoregulatory demands faced by terrestrial frog such as Rana. Iron Transport, metabolism, and storage Cerulopasmin is the principal copper protein of serum and is important in donating copper to cells for the shynthesis and assembly of chytocrome oxidase; also it is a molecular link between iron and copper metabolism. The ferroxidase activity of

ceruplasmin is important in the mobilization of iron from ferrritin, an intracellular ironstorage protein. Levels of ceruloplasmin increase gradually through premetamorphosis and rapidly during prometamorphosis to peak at the beginning of metaporphic climax in Rana grylio (Frieden, 1968). There is a significant increase in ferritin reducing activity in the liver in early morphic climax (Osaki et al, 1974), which undoubtedly plays a role in the mobilization of iron from the liver. The marked increase in ceruloplasmin is probably important in mobilization of iron from both the liver and larval red blood cells and the transfer of iron to serum transferrin. Transferrin is required as a donor of iron to the immature, differentiating cells that synthesize hemoglobin. Red Blood Cells and Hemoglobins Morphological differences between larval and adult red blood cells have been noted in numerus species of anrans (Broyles,1981). During metamorphois, larger larval red blood cells are repaced by smaller adult red blood cells; the midpoint of this transition is at midclimax. The mature, differentiated red blood cell retains its nucleus in both larval and adult blood cells, but the nuclei