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AMER. ZOOL., 13:899-905 (1973). The Evolution of Thyroidal Function in Fishes MARTIN SAGE University of Texas, Marine Science Institute, Port Aransas, Texas 78373 SYNOPSIS. Although the thyroid gland evolved from the gut, there is no evidence that thyroxine functions as part of the gastrointestinal endocrine system nor does it have any major function analogous to the control of glucose by the pancreatic islets. The control of the thyroid evolved from the pituitary control of the gonad suggesting that an early role of thyroxine was in reproduction. This idea is supported by the presence of cycles of thyroid activity associated with reproduction in both elasmobranchs and teleosts. In teleosts thyroxine is necessary for gonadal maturation. The numerous other effects of thyroxine in teleosts may have evolved from this maturational effect or have been added to it during the course of teleost evolution. The role of the thyroid gland in fishes changed and been added to in the teleosts. has been reviewed many times (Goldsmith, It may also be meaningful to ask why some 1949; Fleischmann, 1951; Lynn and Wa- hormones such as thyroxine and prolactin chowski, 1951; Gorbman, 1955, 1959; Berg have acquired such a variety of functions, et al., 1959; Hoar, 1959; Leloup and Fon- whereas many other hormones appear to taine, 1960; Roche, 1960; Dodd and Matty, have retained a single or relatively few func1964; Bern and Nandi, 1964 Matty, 1966; tions throughout the whole of vertebrate Barrington, 1968; Gorbman, 1969; Barring- evolution. This approach may at least proton and Sage, 1972). The thyroid has been vide the framework on which to organize implicated in almost every aspect of teleost the data already available, while more hopephysiology including growth, differentia- fully it may provide some impetus and dition, metamorphosis, maturation, reproduc- rection for future research. tion, the integument, respiration, various The presence of a thyroid gland and its aspects of metabolism, behavior, the central ability to produce the thyroid hormones nervous system, seasonal adaptation, tem- thyroxine and triiodothyronine are striking perature tolerance, osmoregulation, and characteristics of vertebrate organization. several others (see reviews, especially Dodd The thyroid gland is composed of hollow and Matty, 1964). The resulting confusion follicles of cells surrounding a liquid colloid. has probably discouraged people from work- This colloid forms a reserve of potential ing in this field with the result that there hormone. The thyroid gland is the only verhas been very little published since the sub- tebrate endocrine organ with such an extraject was last reviewed and no recent work cellular store. In most agnathans and telehas led to any new insight into the role of osts, thyroid follicles are scattered around thyroid hormones in teleosts. There is no the ventral aorta while in other vertebrates justification at this time for yet another the follicles are aggregated into one or two review, whereas there is a great need for an discrete thyroid glands. Such an evolution attempt to discern some order in the already of scattered elements into a compact gland available data. Accordingly, I have chosen is a common feature of endocrine evolution. to examine what we know of the evolution- Similiar changes have occurred in the evoluary history of the thyroid gland, its function tion of the adrenal medulla and cortex and and its control in the hope that from such to a lesser extent the pancreatic islets. The an approach we may suggest reasonable possible advantages of such a trend are not hypotheses for the original functions of the often discussed. It is, however, known that thyroid and how these functions may have part of the control of the human adrenal is exerted by modifying the flow of blood through the gland (Dobbie et al., 1968). Supported by N.S.F. Grant GB 22995. 899 900 MARTIN SAGE Such a method of control is only possible in a compact structure with an independent blood supply. The thyroid is unique amongst endocrine organs in that we can clearly trace its evolution from another structure present in the protochordates. A clue to this origin is seen in the lamprey where at metamorphosis the thyroid differentiates from parts of the larval endostyle, a complex structure in the floor of the pharynx (Barrington and Sage, 1972^. Strikingly similar endostyles are found in the cephalochordate amphioxus and in the tunicates (Barrington, 1959). We also know that in spite of anatomical differences these endostyles have the biosynthetic properties of thyroid glands (Barrington, 1968; Salvatore, 1969) as well as other activities associated with the alimentary nature of the endostyle (Barrington, 1965). In the protochordates the endostyle is a longitudinal groove in the mid-ventral line of the pharynx lined with alternating longitudinal bands of glandular and ciliated cells. It produces a mucous secretion that is swept dorsally by the cilia and is used to trap food particles from the pharyngeal current of water. It may have a similar though less important role in feeding in the larval lamprey although the bulk of the mucus used in feeding is produced elsewhere (Newth, 1930). There is considerable confusion as to which of the endostyle components contribute to the adult thyroid gland. Type 3 cells, which are the main iodine-binding cells, together with type 4 and 5 cells have been implicated while there is general agreement that the prominent glandular tracts of the endostyle disappear. The different accounts presented by various authors may be due to the fact that not all of the work has been carried out using the same species. Some of the thyroid follicles derive their lumen directly from the endostyle chambers and, although the ducts to the pharynx are lost, there are still ciliated cells in the lamprey follicles. Ciliated cells are also seen in the thyroid glands of elasmobranchs and more occasionally in higher vertebrates especially in embryos. Iodine binding is intracellular in both the hagfish thyroid (Tong et al., 1962; Water- man and Gorbman, 1963) and the larval lamprey endostyle (Barrington and Franchi, 1956), although in mammals and other vertebrates it occurs extracellularly at the cell membrane. Treatment of most vertebrates with thyroxine results in an accumulation of extracellular colloid, but in the endostyle the accumulation is intracellular (Barrington and Sage, 1963). Thus, although the homolog of the lumen of the thyroid follicle is the extracellular endostyle chamber, the homolog of the thyroid colloid is intracellular in the iodine binding cells of the endostyle. It is clear that the thyroid gland evolved from the pharyngeal endostyle. We should, therefore, consider the possibility that thyroid hormones may be a part of the gastrointestinal endocrine system. In the past thyroxine has been claimed to promote the absorption of various substances from the gut in mammals. But these effects have generally not been substantiated leaving at the present time only a possible enhancement of the uptake of carotene (Pitt-Rivers and Tata, 1959). In the lower vertebrates there seems to be no evidence of any gastrointestinal role. It seems certain that pancreatic endocrine tissue, like the thyroid gland, also arose originally from the gut. It now controls the level of glucose in the blood although it may have once been a component of the gastrointestinal system where it probably enhanced uptake from the gut. At first sight the effect of thyroxine on cholesterol levels in the blood of mammals might also seem to indicate that thyroidal function evolved in a similar manner to that of the pancreas. Cholesterol is a major component of cell membranes and unlike other membrane components it is freely exchangeable (Graham and Green, 1967), thus maintenance of some minimal level of blood cholesterol may be a necessity. However, although the state of thyroid physiology does alter cholesterol levels in man, the presence of a latent period following the administration of hormone suggests that thyroxine is not used in any short-term control mechanism. In mammals this latency may be of a few hours and in lower, poikilothermous, vertebrates it may THYROID FUNCTION IN FISH be much longer. We must conclude that there is no evidence that thyroxine has a prime role in gastrointestinal physiology nor in the regulation of any blood component. The only feature of thyroid physiology that the gland's evolution from the gut may possibly help us to understand is the oral effectiveness of thyroxine, an unusual feature for a hormone but one which may be a consequence of its original secretion by the endostyle into the gut and the small size of the thyroxine molecule. Another approach to the problem of the original role of thyroxine is to consider the evolution of the control of the thyroid gland. The glycoprotein TSH is the major influence over the thyroid gland in most vertebrates. However we have no evidence for its presence in the agnatha. Early demonstrations of an effect of thiourea on the cytology of the endostyle were interpreted as evidence of a pituitary control of the endostyle, but the effects of thiourea treatment have since been shown to occur even in hypophysectomized larval lampreys (Barrington and Sage, 1966) while hypophysectomy of adult lampreys does not inhibit thyroid activity (Larsen and Rosenkilde, 1971; Pickering, 1972). The only glycoprotein hormone presumed to be present in the lamprey is a gonadotropin (Dodd et al., I960). We may suppose that TSH evolved from such a glycoprotein by gene duplication and later divergence of the two genes. Such duplications may be quite frequent even though they do not always lead to a new hormone. At the present time in the rat there are two insulin molecules differing in a single amino acid and this is presumably the result of such a gene duplication (Steiner et al., 1969). Similar interpretations can be applied to the peccaries which have more than one vasopresin in the neurohypophysis (Heller, 1963). Not only is there no evidence for a separate TSH molecule in the agnatha, there is no evidence of any pituitary control of either the thyroid or the thyroidal activity in the endostyle. It is thus unlikely that the agnathan gonadotropin molecule has acquired any control over the thyroid in this group. 901 In elasmobranchs we can confirm the earlier observations of Dodd and his coworkers (unpublished data summarized by Dodd et al., 1963) that TSH activity is present and is localized in the ventral lobe (Jackson and Sage, 1973). This is also the site of the gonadotropin activity (Dodd et al., 1960) and it remains to be demonstrated whether or not there are two separate hormones or a single one with the properties of both a TSH and a gonadotropin. In teleosts, as in higher vertebrates, the presence of at least two glycoprotein hormones, TSH and a gonadotropin, is well established (Sage and Bern, 1971). Although TSH and gonadotropin have evolved as distinct hormones in the teleosts, the controls over these hormones remain closely linked together (Sage and Bern, 1971). Thus, thyroxine exerts a direct negative feedback on both TSH and gonadotropin cells. Similarly, sex steroids influence both of these pituitary cells (Sage and Bromage, 1970). Thyroxine has effects on the nervous system of teleosts and, as in higher vertebrates, is involved in a feedback control of TSH release acting at the hypothalamic level (Peter, 1971). The direct activity of nerve fibers from the brain differs in being inhibitory on TSH cells and stimulatory on the gonadotropin cells (Sage and Bern, 1971). The in vivo response of both cells to androgen differs from the response to estrogens, but these differences are not seen in organ-cultured pituitaries (Sage and Bromage, 1970) indicating that within the brain there are common pathways to the control of both cell types. Peter (1970) has shown that the hypothalamic centers controlling TSH and gonadotropic cells are close together in the nucleus lateralis tubes. The view that the control of TSH evolved from the control of the original gonadotropin is further strengthened by the structural similarity of mammalian thyrotropin releasing hormone, TRH (pyro Glu, His, Pro NH2), to part of the gonadotropin releasing hormone, LHRH (pyro Glu, His, Trp, Ser, Tyr, Gly, Leu, Arg, Pro, Gly NH,). This brief survey of the evolution of control of the thyroid indicates a very close relationship between the control of the thyroid and the control of the gonad and, not 902 MARTIN SAGE surprisingly therefore, there are many accounts of thyroid activity paralleling reproductive activity (see reviews, especially Gorbman, 1969). So close are these parallels that it is difficult to imagine how the thyroid could have any effect that is not closely related to reproduction. A third and final approach to the original role of thyroxine and how it evolved into other functions is to consider the known effects of the hormone in the various groups of fishes and to look for common factors. This may seem to be the obvious approach to have started with until you realize that we have no idea what role, if any, is played by thyroxine in the agnatha. Apart from a direct effect of thyroxine on iodine binding in the endostyle (Barrington and Sage, 1966) I do not know of any effect that has been demonstrated for thyroxine in the agnatha. We should consider the possibility that thyroxine production may be a consequence of the alimentary functions of the endostyle and that thyroxine may not have acquired any endocrine functions in the agnatha. If this is so, we need to identify the selective pressures that have maintained the production of thyroxine in the myxinoids which do not have an endostyle. In the elasmobranchs there is very little work on the role of the thyroid gland. However, the gland does show cyclic changes associated with seasonal migration (Woodhead, 1966). We have recently shown that in Dasyatis sabina the cyclic activity in the thyroid is clearly related to reproductive development and to the reproductive cycle and not to seasonal environmental changes since immature animals do not show the cycle of activity seen in adults (Sage and Jackson, unpublished). Differences have also been reported in the size of the thyroid gland in male and female dogfish (Woodhead, 1966). There is, thus, circumstantial evidence that thyroid function in elasmobranchs is associated with reproduction. Amongst the many roles attributed to thyroxine in teleosts, we find a parallel to the above association of thyroid activity with elasmobranch reproduction. The thyroid shows cyclic activity associated with the reproductive cycle of teleosts (see reviews) and this can be isolated from seasonal factors (Bromage and Sage, 1968). Evidence from the evolution of the control of the thyroid, from the control of reproduction and from the role of thyroxine in elasmobranchs suggests that some function associated with reproduction was the original role of the thyroid hormones after the thyroid evolved from the endostyle. The origin of such a role will not be considered here, but the effect of thyroxine appears to be exerted on gonadal maturation (Ball, 1960). The exact nature of the effect, whether it occurs in both sexes and whether it is direct or indirect is not known and requires further work. The possibility that the relationship between the thyroid and reproduction is due to effects of gonadal hormones on the thyroid rather than thyroxine affecting reproduction also needs to be examined. Sex steroids can stimulate thyroid activity in teleosts (Matty et al., 1958; Singh, 1969; Sage and Bromage, 1970). Ovarian tissue also binds iodine (Leloup and Fontaine, 1960), and the enlargement of the ovaries prior to reproduction might decrease the availability of iodine to the thyroid which in turn might be followed by compensatory changes in the thyroid. It would thus seem entirely plausible that although the control system of the thyroid evolved from that controlling the gonad, the relationship allowed for changes in the thyroid that would compensate for the effects of reproduction on the thyroid. The alternative idea that thyroxine influences reproductive function is supported by evidence that thyroidectomy or treatment of fish with antithyroidal compounds inhibits gonadal development (for references, see Ball, 1960; Dodd and Matty, 1964) and degenerative changes in the ovary of captive sturgeons can be reversed by thyroid treatment (see review by Ball, 1960). Thyroxine has also been reported to produce precocious sexual development (Grobstein and Bellamy, 1939). Thus, there may be a reciprocal effect, the reproductive hormones influencing thyroid activity and the thyroid exerting an influence over the reproductive system. These two interacting activities appear to be inextricably interwoven. THYROID FUNCTION IN FISH If we now examine the various known effects of thyroxine in teleosts we can see that some of them are closely related to each other. The involvement of thyroxine in gonadal maturation may have led to other morphogenic effects in growth, development, metamorphosis and on the integument, although the effect on growth may be largely synergistic with growth hormone. In many teleosts reproduction is seasonal. A cycle of thyroid activity associated with reproduction would therefore be an ideal coordinating mechanism for seasonal adaptation to environmental changes such as changes in sensitivity to temperature. This may be one of the most important roles of thyroxine in teleosts (Hoar, 1959). The effect of thyroxine in stimulating oxygen consumption is well known in mammals and it has often been looked for in lower vertebrates with a consensus of reviewers considering that there is no such effect in fish. The reported positive results are, to quote Gorbman and Bern (1962, p. 163) "well known for their exceptional nature." In spite of this, Ruhland (1969, 1971) has recently demonstrated positive effects of thyroxine on teleost respiration and a reduction in oxygen consumption following treatment with thiourea of radiothyroidectomy. From personal experience I know that to obtain consistent results in such experiments it is necessary to take exceptional care in standardizing the experimental conditions (e.g. time of day, lighting, handling of fish). This is characteristic of experiments on fish behavior and may indicate that the respiratory changes are secondary to the well-known effects of thyroxine on fish behavior (Hoar et al., 1955; Sage, 1968; Woodhead, 1970). Increased swimming activity may stimulate the thyroid (Higgs and Eales, 1971). Conversely respiratory changes following altered behavior may mask the demonstration of a direct respiratory response to thyroxine. The effect of thyroxine on fish behavior is doubtless due to the effect of thyroxine on the central nervous system which may have evolved from the feedback mechanism controlling TSH release by the action of thyroxine on the hypothalamus. 903 We have so far related many of the known effects of thyroxine in fish to two primary effects: an effect of thyroxine on maturation and an effect on the nervous system. These are not separate since one of the major effects of thyroxine that may have persisted throughout vertebrate evolution is the effect of thyroxine on the maturation of the nervous system. This is well known in man. Whether the effect of thyroxine on cholesterol metabolism is related to this is unknown. In conclusion, in spite of the evolution of the thyroid from the gut, there is no evidence that thyroxine was originally a gastrointestinal hormone, although we cannot exclude it from being so in the endostyle. Since the control of the thyroid evolved from the control of the gonad, an early role of the thyroid associated with reproduction would thus seem most likely. This association has been borne out by recent observations in elasmobranchs. In teleosts, thyroxine is necessary for the maturation of the gonads and, perhaps, also other structures including the nervous system. An effect on the nervous system which evolved as part of the control of TSH release may have further evolved to allow effects on behavior and thus perhaps on respiration. The maturational effects of thyroxine may have been extended to other morphogenic actions on growth, development, metamorphosis, and the integument, while the cycle of thyroid activity associated with an annual gonadal cycle would allow the use of thyroxine as a control of seasonal adaptation which is a major role of thyroxine in teleost fishes. Finally, we may ask why thyroxine should have acquired so many functions. The answer may be that thyroxine became available for new roles after the endostyle was no longer needed for feeding. It is analogous to the rapid evolutionary radiations that follow paedomorphosis (de Beer, 1951) when genetic material is released for new roles. Prolactin is another hormone with a diversity of functions. It presumably evolved from growth hormone by gene duplication and like thyroxine has acquired a host of activities during the adaptive radiation of the teleosts. 904 MARTIN SAGE REFERENCES Ball, J. N. 1960. Reproduction in female bony fishes. Symp. Zool. 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