Fish Physiology and Biochemistry vol. 7 nos 1-4 pp 3-10 (1989) Kugler Publications, Amsterdam/Berkeley
Roots of fish endocrinology, a perspective Aubrey Gorbman Department of Zoology, University of Washington, Seattle, WA 98195 U.S.A. Keywords: brain, pituitary, neurosecretory cells, thyroid, gonad, pancreas, adrenal cortex
The organizers of this symposium have assigned to me the task of defining the roots of what we may call fish endocrinology. Botanically, this has been a challenging task because, in large part, what is a root in this field is a fruit or offshoot of the findings and techniques developed in mammalian endocrinology. In theory it should be possible for each field of endocrinology to trace back a continuous path to its origins and to illustrate its conceptual development. With few exceptions the progress in each of these fields has been gradual and stepwise. Tracing such slow development from the early purely morphological studies to the current status of a given field is both laborious and, as a rule, of small interest. Accordingly, this survey has been made more selective and focused by taking up relatively few of the key events in the history of piscine endocrinology. If there appear to be gaps in the picture created by this approach, it is hoped that they are justified by the greater interest and clarity of the fewer details. There have been remarkably few developments in fish endocrinology that have been seminal for mammalian endocrinology. Two in particular can be mentioned at the outset: the discovery by Marine and Lenhart (1910) of the relationship of iodine to thyroid gland pathology in trout, and the recognition of endocrine secretory nerve cells in the central nervous systems of fishes by Carl Speidel and by Ernst Scharrer (1919 and 1928). In common with all other endocrinology, fish endocrinology began as a morphological science and
gradually entered an experimental phase. There will be no attempt here to review the beginnings of endocrine morphological study, although in many ways it was the microstructure of endocrine organs that provided the roots for the subsequent functional approaches. This is particularly true of the observed seasonal cycles of cytological detail that led eventually to further studies utilizing surgery, hormonal measurements, and other manipulative approaches for analysis of endocrine mechanisms.
Brain and pituitary The use of hypophysectomized test animals has always provided the ultimate criteria in studies of pituitary gland function. Successful and complete surgical hypophysectomy is difficult in most vertebrates and particularly difficult in small aquatic organisms like fishes. Undoubtedly, this is one of the main barriers that has held up the isolation and purification of piscine pituitary hormones, since these procedures require appropriate bioassay at some points. This may be why we are at this late date finally seeing the purification of some of the teleost pituitary hormones, about fifty years after the first mammalian adenohypophysial hormones became available in purified form. Yet, in one area, the relationship of the brain to the pituitary, it was the fish that provided the lead for the mammal. The now famous conclusion of Ernst Scharrer (1928) that the droplet-filled
4
Fig. 1. Diagrams showing locations of perikarya, LC and SC, in the caudal spinal cord of the loach. Their endings (NE) are in the structure he named the urohypophysis (U), a neurohaemal region. A represents a normal fish and B the degranulated urohypophysis of a fish injected with hypertonic salt solution after transection of the cord at a point shown by the arrow. Enami (1955, 1959).
perikarya and axons of certain diencephalic neurons of a small cyprinid fish, the 'Elritze', represented secretory properties stimulated a new field of research in neurosecretion. Scharrer's proposition was followed by nerve tract cutting and blocking experiments by Bargmann (1949, 1954) that showed that the secretory granules in cells described by Scharrer moved in the tracts toward the pituitary gland. Eventually Bargmann and others identified these neural structures with the production of neurohypophysial hormones. It is difficult today to imagine the disbelief, and even antagonism that was at first directed toward the concept of neurosecretion while it was based only on morphological studies in a fish. Even as late as 1941 Scharrer was obliged to preface his meeting presentations and published papers defensively with justifications for the idea that nerve cells may secrete. For example (E. Scharrer, 1941): 'The proposition that the nervous system fulfills a part of its functions through a glandular apparatus of its own seems at first sight to raise more difficulties than, for instance, the suggestion that the germ cells might produce sex hormones. It appears, however, from a number of findings made in recent years, that gland-like cells occur within the nervous tissues of invertebrates and vertebrates from worms up
Fig. 2. Ventral and side views of trout reared in iodine-poor water, showing gross thyroid tumors which could be reduced by iodine-treatment. Marine and Lenhart (1910).
to and including man.' Ernst and Berta Sharrer, through their persistance and the persuasiveness of the evidence they produced, eventually recruited interest and experimental work that put neurosecretion on a firm footing. However, theirs was not the first presentation of this idea. Speidel (1919) had already described enlarged granulated cells in the caudal spinal cord of skates and referred to them as 'gland cells of internal secretion'. He soon extended these observations (1922) to 30 species of fish that included elasmobranchs, ganoids and teleosts. It should be remembered that Speidel's several morphological papers on neurosecretory cells only implied an endocrine functional property, but they must be considered at the root of the concept of neurosecretion. Furthermore, they formed the basis for the eventual recognition of the caudal neurosecretory system of fishes as an endocrine unit. Unfortunately Speidel's work stimulated no further work on the 'urohypophysis' until, in the 1950's, Enami (1955, 1959) published several strikingly illustrated papers on the subject (Fig. 1). The urohypophysis, or urophysis, as it is now more commonly called, became a full-fledged endocrine organ when the laboratories of H.A. Bern and K. Lederis undertook its study. Along with the cor-
5 puscles of Stannius, it has the distinction of being a vertebrate organ that is limited in its occurrence to the fishes.
Thyroid gland The first descriptions of fish thyroid glands date from 1844. In Simon's study of that date is the report that the salmon has no thyroid gland! The diffuseness of the distribution of the thyroid follicles in most bony fishes (Gudernatsch 1911) has made surgical thyroidectomy impossible as a rule. Accordingly, experimental analysis of thyroid function has had to follow routes other than this traditional one, particularly by use of antithyroid drugs. One fortunate tool that was available early to thyroid researchers was the analysis of iodine metabolism, since this element is peculiar to the thyroid hormone. It was this fact that enabled Marine and Lenhart (1910) to show that endemic goiter of trout, like that of humans, is due to deficient regional iodine supply (Fig. 2). They were able to reduce visible thyroid tumors of hatchery salmonids by supplementing their environmental iodine levels. Observation of thyroid tumors in fish head kidneys led Baker (1955) and Chavin (1956) to the discovery of another characteristic feature of fish thyroid follicles, a 'normal' tendency to wander from their pharyngeal region of origin to other anatomical sites, predominantly to the nephric region. Although the molecular structure of thyroxine was described by Harington in 1926, and it was synthesized in 1927, it was not available in pure form for experiments with fish until the 1930's. Grobstein and Bellamy (1939), for example, tested the effect of thyroid on fin growth in swordtails by feeding them cattle thyroid tissue. Most commonly, even into the 1950's, a preparation called 'thyroid powder' was fed to fish orally or injected as a suspension. Thyroid powder was prepared by grinding acetone-extracted and dried thyroid glands of pigs or cattle (for example: Hopper, 1952, observed the effect of thyroid powder on growth of Lebistes). Some blame for confusion and non-reproducibility of results in this era must be laid to the irregularity
Fig. 3. Measurements of thyroid gland volume (dashed line) and epithelial cell height (solid line) in the herring in two phases of the life cycle: I, the time of metamorphosis (at 40 mm length) and II, the time of egg-laying (70 to 80 mm length). Buchmann (1940). Note similarity of the phenomena at metamorphosis to the measurements of plasma T3 and T4 in smoltifying salmon (see Dickhoff and Sullivan (1987).
of iodine and hormone content of these thyroid preparations, depending on a number of variables in their manufacture (see Lynn and Wachowski 1951). One of the remarkable aspects of thyroid study in the fishes is the difficulty of showing that the thyroid hormone has an important function in the adult, a function basic enough to explain why the hormone and the gland that synthesizes it, have evolved and persisted through all vertebrate forms. There is no consistently demonstrable action on calorigenesis or respiratory metabolism (see Etkin et al. 1940) as in higher vertebrates. However, there is a well established action on guanine metabolism in the skin that results in a 'silvering' of certain fish species. There are seasonal changes in plasma thyroid histology (see Buchmann 1940, Fig. 3) or in plasma hormone levels that may correlate with such phenomena as catadromous migration or developmental progress from the parr to the smolt stage in salmonids. Study of the possible influence of thyroid hormone on developmental phenomena in fishes was sparked by the discovery by Gudernatsch (1912) that the metamorphosis of tadpoles into frogs is stimulated by feeding thyroid tissue. Unfortunately, it has been difficult or impossible to precipitate more than a few minor features of 'smoltification' in salmonids by thyroid hormone treatments other than integumentary silvering. Re-
6
Fig. 5. Photographs of the female bitterling (Rhodeus amarus) depositing eggs in the open shells of a freshwater mussel through their long ovipositors, with the male in attendance for their fertilization. Bretschneider and de Wit (1947).
Fig. 4. Illustration from Harms (1929). Mudskippers (Periophthalmus) are shown on the walls of the terrarium after feeding thyroid hormone. Harms reported that the height climbed and the length of the stay out of water correlated with the total dose of thyroid hormone.
cently Inui and Miwa (1985) reported that thyroid hormone stimulates metamorphic changes in young flatfish including migration of the eye. This has been the clearest instance of thyroid-evoked fish development to my knowledge. One of the more remarkable claims in this connection was by Harms (1929) who treated Periophthalmus and certain blennies with thyroid hormone and found that they emerged from the water and became terrestrial, or at least more amphibious. Harms also proposed the height that thyroid-treated Periophthalmusclimbed on the aquarium wall as an assay for thyroid hormone (Fig. 4). He called the phenomenon 'Landtierwerdung' and believed, thus, that the thyroid hormone might have been the basis for the emergence of vertebrates from the aquatic to the land environment. After almost 60 years this claim remains to be substantiated, so we may consider it one of the roots of fish endocrinology that failed to prosper.
Gonad The field of fish gonadal endocrinology also contains some puzzling early features that in their day cast as much shadow as light. Gonadectomy to show the effect of hormonal absence appears to have been only rarely practiced (see Pickford and Atz 1957). As a result, many of the experiments testing the effects of administration of presumed hormonal extracts lacked the proper controls. One of the first reports of this kind of test is that of Wunder (1931) who injected 'extractum testiculi' and Progynon B into the bitterling Rhodeus amarus, and stimulated development of integumentary nuptial coloration, or 'Hochzeitskleid'. He observed also a rapid stimulation of the growth in length of the ovipositor, a structure used by the female bitterling to deposit eggs for their protection within the shells of clams (Fig. 5). In the following years, and particularly after the publication of Fleischmann and Kann's papers in 1932 and 1934 which proposed the ovipositor of the intact female bitterling as a bioassay for female hormones (Fig. 6), this became widely used. Unfortunately, it soon became a confused issue as to what the test measures. Kleiner et al. (1935) and others proposed it as an assay for male hormone. A later view by Van Koersveld (1949) was that the bitterling tests are
7
Fig. 6. Diagram of a female bitterling showing a scale of length of the ovipositor for use as a bioassay for pregnancy or for androgens. Kleiner et al. (1935, 1936).
'totally unsuitable qualitatively as well as quantitatively'. Fish sexual endocrinology developed in a more orderly manner after the amphibians (mostly Xenopus) took over the burden as a poikilotherm pregnancy test. In the light of later understanding of steroid hormonal metabolism and interconversion (especially aromatization of androgens), and characterization of specificity of some of the steroid receptors, some of the confusion generated by the bitterling ovipositor may become understandable. However, as far as this reviewer knows, nobody has returned to this formerly popular research area to clarify it definitively. A measure of the frustration of reviewers in evaluating the early status of fish gonadal endocrinology is epitomized in the statement by Pickford and Atz (1957) 'a systematic attack on the problem of the identity of the sex hormones throughout the vertebrates is one of the fundamental needs of comparative endocrinology'. Ultimately this requirement by Pickford and Atz has been largely achieved, utilizing techniques and advances in understanding provided by mammalian endocrinology.
from Pickford and Atz, an uncertainty as to the nature of the steroids themselves, and in some instances, at least, the identity of the tissue that secretes them. This contrasts with the situation today in which dependable chromatographic separations and immunoassay can be used to identify and quantify with confidence the corticosteroids of fishes, even when the levels are low. In the absence of such techniques, and with use of morphology and crude biochemical indicators (e.g., lipid or ascorbic acid content), it was for about 50 years a matter of contention among endocrinologists (until about 1930) whether the anterior (the present fish interrenal tissue) or posterior (the present corpuscles of Stannius) interrenals of bony fishes are the functional steroidogenic equivalents of the higher vertebrate adrenal cortex. Stannius, for example, favored his corpuscles at first, but was an early convert (1854) to the other camp. S. Vincent, a prominent worker in the field in the late 19th century, similarly changed his mind in 1927 (Vincent and Curtis 1927). Chester Jones (1957) has reviewed this phase of the beginnings of fish adrenal physiology and anatomy. To the comparative endocrinologist, the evolution of the hormonal system with which he or she works is of great interest. Thus the status of these systems in fishes, and in the most primitive fishes in particular, is of paramount importance. One evolutionary puzzle that has no apparent solution is why the corpuscles of Stannius are found only in bony fishes and have no equivalent in higher vertebrates, or in cartilaginous fish species, or in cyclostomes. Just as puzzling has been the fact that a corticoadrenal equivalent tissue has yet to be firmly identified in cyclostomes. There have been a number of presumptive interrenal tissue structures described in lampreys but none has been established beyond question as corticosteroidogenic. In the other group of cyclostomes, the hagfishes, even a presumptive interrenal tissue has yet to be described.
Cortico-adrenal equivalents Pancreatic hormones, insulin The early history of the corticosteroid secreting tissues of fishes was subject to some of the same difficulties that were indicated by the above citation
Morphological studies of the endocrine pancreas of fishes after the pioneering work of Langerhans
8 the first to find that extracts of the fish pancreas relieve symptoms of diabetes in man. Isolation of mammalian insulin was accomplished in 1922, and fish insulins were isolated soon afterward, between 1924 and 1926 (Jensen et al. 1929). Among the first insulins to be sequenced were those of the cod (Reid et al. 1968) and of hagfish (Peterson et al. 1975). The fish pancreas also figured in the early phases of isolation of glucagon. In the year before Staub et al. (1953) isolated mammalian glucagon Audy and Kerly (1952) reported glucagon-like activity in angler fish pancreatic extracts.
Conclusion
Fig. 7. A frequently reproduced figure of the dissection of an angler fish, Lophius piscatorius showing the large single principal pancreatic lobe (Brockmann body) at the arrow. From Buddenbrock (1950), after Bargmann.
(1869) established several interesting facts. Though the fish pancreas contains islets of endocrine cells, there are some species in which the endocrine tissue is concentrated in an organ known variously as the 'principal islet' or Brockmann body (Fig. 7). Furthermore, in the cyclostomes there is no exocrine pancreas and the endocrine cells are clustered in a swelling of the anterior intestine known as the Langerhans organ or follicles. Fish played a prominant role in studies of pancreatic physiology, in part because of the above anatomical facts. The famous pancreatectomy experiments of von Mering and Minkowski (1890) in dogs established the relationship between the pancreas and carbohydrate metabolism. However, initial attempts to extract the pancreatic hormone, insulin, were thwarted because proteolytic enzymes in ground pancreas digested the hormone. One obvious and successful solution to this problem was in the use of Brockmann bodies, such as those of the angler fish, Lophius (McLeod 1922; McCormick and Noble 1924). Diamare and Kuljabko (1904) reported that extracts of angler fish principal islets are active in altering carbohydrate metabolism in the fish. Rennie and Fraser (1906) were apparently
In this abbreviated overview of the beginnings of piscine endocrine research no attempt has been made to be encyclopedic. Instead, some of the insightful, as well as some of the fumbling exploratory earlier efforts in this field have been outlined. Some of the more recent roots and rootlets have been omitted because they are more obvious and, to take up one or two of them would require consideration of many more recent rapid developments. For example, it was knowledge of the developmental relationships of fish and amphibian ultimobranchial bodies that made possible the fuller understanding of thyroidal calcitonin in mammals, and led to the use of salmon calcitonin in the clinic. At the interface between academic fish endocrinology and fish aquaculture there has been rapid recent progress in purification and sequencing of fish pituitary and pancreatic hormones, as well as better understanding of the role of endocrines in fish development, growth and reproduction. But these developments clearly have been based on the general fund of related information that already existed. There has been a recognizable field of fish endocrinology since the early nineteenth century. Explanation of why a biologist enters this field is no doubt the same as for any comparative physiologist: an interest in exploring the nature of a particular mechanism in this vertebrate group for comparison and contrast with other vertebrates. The further value of work in fish endocrinology for some of us is in the strategic value of data obtained from
9 fishes in explaining the evolution of these mechanisms in vertebrates in general. Others are motivated by the usefulness of endocrine data in promoting fish as an agricultural resource. However, all fish endocrinologists should be encouraged by the realization that what they discover today may be at the root of important developments in this field in the future.
References cited Audy, G. and Kerly, M. 1952. The content of glycogenolytic factor in pancreas from different species. Biochem .J. 52: 77-78. Baker, K.F., Berg, O., Nigrelli, F., Gorbman, A. and Gordon, M. 1955. Functional thyroid tumors in kidneys of platyfish. Cancer Res. 15: 118-123. Baker-Cohen, K.F. 1959. Renal and other heterotopic thyroid tissue in fishes. In Comparative Endocrinology pp. 283-301. Edited by A. Gorbman. Wiley, New York. Bargmann, W. 1949. Uber die neurosekretorische Verknupfung von Hypothalamus and Neurohypophyse. Z. Zellforsch. Mikros. Anat. 34: 610-634. Bargmann, W. 1954. Das Zwischenhirn-Hypophysensystem. Springer Verlag, Berlin. Bretschneider, L.H. and Duyvene de Wit, J.J. 1947. Sexual Endocrinology of Non-mammalian Vertebrates. Elsevier, New York, Amsterdam. Buchmann, H.H. 1940. Hypophyse und Thyroidea im Individualizyklus des Herings. Zool. Jahrb., Abt. Anat. 66: 191-262. von Buddenbrock, W. 1950. Vergleichende Physiologie. Band IV. Hormone. Verlag Birkhauser, Basel. Chavin, W. 1956. Thyroid distribution and function in the goldfish. Carassiusauratus L. J. Exp. Zool. 133: 259-279. Chester Jones, 1. 1957. The Adrenal Cortex. Cambridge University Press, Cambridge. Dahlgren, U. 1914. On the electric motor nerve centers in the skates (Rajidae). Science 40: 862-863. Diamare, V. 1906. Weitere Beobachtungen fiber den Experimentaldiabetes nach Pankreas Extirpation bei Selachiern. II Mitt., Zbl. Physiol. 20: 617-620. Diamare, V. 1908. Vergleichende anatomisch-physiologische Studien ber den Pankreas Diabetes. III. Mitt. Zbl. Physiol. 21: 863-869. Diamare, V. and Kuljabko, A. 1904. Zur Frage nach der physiologischen Bedeutung der Langerhansschen Inseln im Pankreas. Zbl. Physiol. 18: 432-435. Dickhoff, W.W. and Sullivan, C.V. 1987. Involvement of the thyroid gland in smoltification, with special reference to metabolic and developmental processes. Am. Fish. Soc. Symp. 1: 197-210. Enami, M. 1955. Caudal neurosecretory system in the eel (Anguillajaponica). Gunma J. Med. Sci. 4: 23-36. Enami, M. 1959. The morphology and functional significance of
the caudal neurosecretory system of fishes. In Comparative Endocrinology pp. 697-724. Edited by A. Gorbman. John Wiley and Sons, New York. Etkin, W., Root, R.W. and Mofshin, B.P. 1940. The effect of thyroid feeding on oxygen consumption of the goldfish. Physiol. Zool. 13: 415-429. Fleischmann, W. and Kann, S. 192. Uber eine Funktion des weiblichen Sexualhormons bei Fischen (Wachstum der Legerohre des Bitterlings). Pflug. Arch. Ges. Physiol. 230: 662-667. Fleischmann, W. and Kann, S. 1934. Uber die Wachstum der Legerohre des Bitterlings unter dem EinfluB des weiblichen Sexualhormons. Pflu. Arch. Ges. Physiol. 234: 130-136. Grobstein, C. and Bellamy, A.W. 1939. Some effects of feeding thyroid to immature fishes (Platypoecilus). Proc. Soc. Exp. Biol. Med. 41: 363-365. Gudernatsch, J.F. 1911. The thyroid gland of the teleosts. J. Morph. 21: 709-782. Gudernatsch, J.R. 1912. Feeding experiments on tadpoles. 11.A further contribution to the knowledge of organs with internal secretion. Arch. Ent. Mech. Org. 35: 457-483. Harms, J.W. 1929. Die Realization von Genen und die consekutive Adaptation. I. Phasen in der Differenzierung der Anlagenkomplexe und die Frage der Landtierwerdung. Zeitsch. wiss. Zool. 133: 211-397. Hopper, A.F. 1952. Growth and maturation response of Lebistes reticulatus to treatment with thyroid powder. J. Exp. Zool. 119: 205-217. Inui, Y. and Miwa, S. 1985. Thyroid hormone induces metamorphosis of flounder larvae. Gen. Comp. Endocrinol. 60: 450-454. Jensen, H., Wintersteiner, O. and Geiling, E.M.K. 1929. Studies on crystalline insulin; isolation of crystalline insulin from fish islets (cod and pollock) and from pigs' pancreas; activity of crystalline insulin and further remarks on its preparation. J. Pharmacol. Exp. Therap. 36: 115-128. Kleiner, I.S., Weisman, A.I. and Barowsky, H. 1935. An investigation of the new biologic test for hormones in pregnancy urine. J. Am. Med. Assoc. 104: 1318-1319. Kleiner, .S., Weisman, A.I., Mishkind, D.I. and Coates, C.W. 1936. The female bitterling as a biologic test animal for male hormone. Zoologica 21: 244-250. Landgrebe, F.W. 1941. The role of the pituitary and thyroid in the development of teleosts. J. Exp. Biol. 18: 162-169. Langerhans, P. 1869. Beitrage zur mikroskopischen Anatomie der Bauchspeicheldruse. Diss., Berlin. Lynn, W.G. and Wachowski, H.E. 1951. The thyroid gland and its functions in cold-blooded vertebrates. Quart. Rev. Biol. 26: 123-168. Marine, D.J. and Lenhart, C.H. 1910. On the occurrence of goitre (active thyroid hyperplasia) in fish. Johns Hopkins Hosp. Bull., 21: 95-98. Marine, D.J. and Lenhart, C.H. 1910. Observations and experiments on the so-called thyroid carcinoma of brook trout (Salvelinusfontinalis) and its relation to ordinary goitre. J. Exp. Med. 12: 311-337. Marine, D.J. 1911. Further observations and experiments on the
10 so-called thyroid carcinoma of the brook trout (Salvelinus fontinalis)and its relation to endemic goitre. J. Exp. Med. 13: 455-475. von Mering, I. and Minkowski, 0. 1890. Diabetes mellitus nach Pankreasextirpation. Arch. exp. Pathol. und Pharmakol. 26: 371-387. MacLeod, J.J.R. 1922. The source of insulin. A study of the effect produced on blood sugar by extracts of the pancreas and principal islets of fishes. J. Metabol. Res. 2: 149-172. McCormick, N.A. and Noble, E.C. 1924. The yield of insulin from fish. Contr. Can. Biol. Fish. N.S. 2: 115-128. Miwa, S. and Inui, Y. 1985. Effects of thyroxine and thiourea on the parr-smolt transformation of amago salmon (Oncorhynchus rhodurus).Bull. Nat. Inst. Aquaculture, Japan 4: 41-52. Peterson, J.D., Steiner, D.F., Emdin, S.O. and Falkmer, S. 1975. The amino acid sequence of the insulin from a primitive vertebrate, the Atlantic hagfish, (Myxine glutinosa). J. Biol. Chem. 250: 5183-5191. Pickford, G.E. and Atz, J.W. 1957. The Physiology of the Pituitary Gland of Fishes. New York Zoological Society, New York. Reid, K.P. Grant and Youngson, A. 1968. The sequence of amino acids in insulin isolated from islet tissue of the cod (Gadus callarius). Biochem. Jour. 10: 289-296.
Rennie, J.and Fraser, T. 1906. The islets of Langerhans in relation to diabetes. Biochem. J. 2: 7-19. Robertson, O.H. 1949. Production of the silvery smolt stage in rainbow trout by intramuscular injection of mammalian thyroid extract and thyrotrophic hormone. J. Exp. Zool. 110: 337-355. Scharrer, E. 1928. Die Lichtempfindlichkeit blinder Elritzen (Untersuchungen fiber das Zwischenhirn der Fische.) Zeitschr. vergl. Physiol. 7: 1-38. Scharrer, E. 1941. Neurosecretion. I. The nucleus preopticus of Fundulus heteroclitus L.J. Comp. Neurol. 74: 81-92. Simon, J. 1844. On the comparative anatomy of the thyroid gland. Phil. Trans. Roy. Soc. Ser. B. 134: 295-303. Speidel, C.C. 1919. Gland cells of internal secretion in the spinal cord of the skates. Publ. Carnegie Inst. Washington, no. 281. Speidel, C.C. 1922. Further comparative studies in other fishes of cells that are homologous to the large irregular cells in the spinal cord of the skates. J. Comp. Neurol. 34: 303-317. Stannius, H. 1854. Zootomie der Fische und Amphibien. Berlin. Staub, A., Sinn, L.G. and Behrens, O.K. 1953. Purification and crystallization of hyperglycemicglycogenolytic factor (HgF). Science 117: 628-629. Vincent, S. and Curtis, F.R. 1927. A note on the teleostean adrenal bodies. J. Anat. 62: 110-120.
Roots of fish endocrinology, a perspective Aubrey Gorbman Department of Zoology, University of Washington, Seattle, WA 98195 U.S.A. Keywords: brain, pituitary, neurosecretory cells, thyroid, gonad, pancreas, adrenal cortex
The organizers of this symposium have assigned to me the task of defining the roots of what we may call fish endocrinology. Botanically, this has been a challenging task because, in large part, what is a root in this field is a fruit or offshoot of the findings and techniques developed in mammalian endocrinology. In theory it should be possible for each field of endocrinology to trace back a continuous path to its origins and to illustrate its conceptual development. With few exceptions the progress in each of these fields has been gradual and stepwise. Tracing such slow development from the early purely morphological studies to the current status of a given field is both laborious and, as a rule, of small interest. Accordingly, this survey has been made more selective and focused by taking up relatively few of the key events in the history of piscine endocrinology. If there appear to be gaps in the picture created by this approach, it is hoped that they are justified by the greater interest and clarity of the fewer details. There have been remarkably few developments in fish endocrinology that have been seminal for mammalian endocrinology. Two in particular can be mentioned at the outset: the discovery by Marine and Lenhart (1910) of the relationship of iodine to thyroid gland pathology in trout, and the recognition of endocrine secretory nerve cells in the central nervous systems of fishes by Carl Speidel and by Ernst Scharrer (1919 and 1928). In common with all other endocrinology, fish endocrinology began as a morphological science and
gradually entered an experimental phase. There will be no attempt here to review the beginnings of endocrine morphological study, although in many ways it was the microstructure of endocrine organs that provided the roots for the subsequent functional approaches. This is particularly true of the observed seasonal cycles of cytological detail that led eventually to further studies utilizing surgery, hormonal measurements, and other manipulative approaches for analysis of endocrine mechanisms.
Brain and pituitary The use of hypophysectomized test animals has always provided the ultimate criteria in studies of pituitary gland function. Successful and complete surgical hypophysectomy is difficult in most vertebrates and particularly difficult in small aquatic organisms like fishes. Undoubtedly, this is one of the main barriers that has held up the isolation and purification of piscine pituitary hormones, since these procedures require appropriate bioassay at some points. This may be why we are at this late date finally seeing the purification of some of the teleost pituitary hormones, about fifty years after the first mammalian adenohypophysial hormones became available in purified form. Yet, in one area, the relationship of the brain to the pituitary, it was the fish that provided the lead for the mammal. The now famous conclusion of Ernst Scharrer (1928) that the droplet-filled
4
Fig. 1. Diagrams showing locations of perikarya, LC and SC, in the caudal spinal cord of the loach. Their endings (NE) are in the structure he named the urohypophysis (U), a neurohaemal region. A represents a normal fish and B the degranulated urohypophysis of a fish injected with hypertonic salt solution after transection of the cord at a point shown by the arrow. Enami (1955, 1959).
perikarya and axons of certain diencephalic neurons of a small cyprinid fish, the 'Elritze', represented secretory properties stimulated a new field of research in neurosecretion. Scharrer's proposition was followed by nerve tract cutting and blocking experiments by Bargmann (1949, 1954) that showed that the secretory granules in cells described by Scharrer moved in the tracts toward the pituitary gland. Eventually Bargmann and others identified these neural structures with the production of neurohypophysial hormones. It is difficult today to imagine the disbelief, and even antagonism that was at first directed toward the concept of neurosecretion while it was based only on morphological studies in a fish. Even as late as 1941 Scharrer was obliged to preface his meeting presentations and published papers defensively with justifications for the idea that nerve cells may secrete. For example (E. Scharrer, 1941): 'The proposition that the nervous system fulfills a part of its functions through a glandular apparatus of its own seems at first sight to raise more difficulties than, for instance, the suggestion that the germ cells might produce sex hormones. It appears, however, from a number of findings made in recent years, that gland-like cells occur within the nervous tissues of invertebrates and vertebrates from worms up
Fig. 2. Ventral and side views of trout reared in iodine-poor water, showing gross thyroid tumors which could be reduced by iodine-treatment. Marine and Lenhart (1910).
to and including man.' Ernst and Berta Sharrer, through their persistance and the persuasiveness of the evidence they produced, eventually recruited interest and experimental work that put neurosecretion on a firm footing. However, theirs was not the first presentation of this idea. Speidel (1919) had already described enlarged granulated cells in the caudal spinal cord of skates and referred to them as 'gland cells of internal secretion'. He soon extended these observations (1922) to 30 species of fish that included elasmobranchs, ganoids and teleosts. It should be remembered that Speidel's several morphological papers on neurosecretory cells only implied an endocrine functional property, but they must be considered at the root of the concept of neurosecretion. Furthermore, they formed the basis for the eventual recognition of the caudal neurosecretory system of fishes as an endocrine unit. Unfortunately Speidel's work stimulated no further work on the 'urohypophysis' until, in the 1950's, Enami (1955, 1959) published several strikingly illustrated papers on the subject (Fig. 1). The urohypophysis, or urophysis, as it is now more commonly called, became a full-fledged endocrine organ when the laboratories of H.A. Bern and K. Lederis undertook its study. Along with the cor-
5 puscles of Stannius, it has the distinction of being a vertebrate organ that is limited in its occurrence to the fishes.
Thyroid gland The first descriptions of fish thyroid glands date from 1844. In Simon's study of that date is the report that the salmon has no thyroid gland! The diffuseness of the distribution of the thyroid follicles in most bony fishes (Gudernatsch 1911) has made surgical thyroidectomy impossible as a rule. Accordingly, experimental analysis of thyroid function has had to follow routes other than this traditional one, particularly by use of antithyroid drugs. One fortunate tool that was available early to thyroid researchers was the analysis of iodine metabolism, since this element is peculiar to the thyroid hormone. It was this fact that enabled Marine and Lenhart (1910) to show that endemic goiter of trout, like that of humans, is due to deficient regional iodine supply (Fig. 2). They were able to reduce visible thyroid tumors of hatchery salmonids by supplementing their environmental iodine levels. Observation of thyroid tumors in fish head kidneys led Baker (1955) and Chavin (1956) to the discovery of another characteristic feature of fish thyroid follicles, a 'normal' tendency to wander from their pharyngeal region of origin to other anatomical sites, predominantly to the nephric region. Although the molecular structure of thyroxine was described by Harington in 1926, and it was synthesized in 1927, it was not available in pure form for experiments with fish until the 1930's. Grobstein and Bellamy (1939), for example, tested the effect of thyroid on fin growth in swordtails by feeding them cattle thyroid tissue. Most commonly, even into the 1950's, a preparation called 'thyroid powder' was fed to fish orally or injected as a suspension. Thyroid powder was prepared by grinding acetone-extracted and dried thyroid glands of pigs or cattle (for example: Hopper, 1952, observed the effect of thyroid powder on growth of Lebistes). Some blame for confusion and non-reproducibility of results in this era must be laid to the irregularity
Fig. 3. Measurements of thyroid gland volume (dashed line) and epithelial cell height (solid line) in the herring in two phases of the life cycle: I, the time of metamorphosis (at 40 mm length) and II, the time of egg-laying (70 to 80 mm length). Buchmann (1940). Note similarity of the phenomena at metamorphosis to the measurements of plasma T3 and T4 in smoltifying salmon (see Dickhoff and Sullivan (1987).
of iodine and hormone content of these thyroid preparations, depending on a number of variables in their manufacture (see Lynn and Wachowski 1951). One of the remarkable aspects of thyroid study in the fishes is the difficulty of showing that the thyroid hormone has an important function in the adult, a function basic enough to explain why the hormone and the gland that synthesizes it, have evolved and persisted through all vertebrate forms. There is no consistently demonstrable action on calorigenesis or respiratory metabolism (see Etkin et al. 1940) as in higher vertebrates. However, there is a well established action on guanine metabolism in the skin that results in a 'silvering' of certain fish species. There are seasonal changes in plasma thyroid histology (see Buchmann 1940, Fig. 3) or in plasma hormone levels that may correlate with such phenomena as catadromous migration or developmental progress from the parr to the smolt stage in salmonids. Study of the possible influence of thyroid hormone on developmental phenomena in fishes was sparked by the discovery by Gudernatsch (1912) that the metamorphosis of tadpoles into frogs is stimulated by feeding thyroid tissue. Unfortunately, it has been difficult or impossible to precipitate more than a few minor features of 'smoltification' in salmonids by thyroid hormone treatments other than integumentary silvering. Re-
6
Fig. 5. Photographs of the female bitterling (Rhodeus amarus) depositing eggs in the open shells of a freshwater mussel through their long ovipositors, with the male in attendance for their fertilization. Bretschneider and de Wit (1947).
Fig. 4. Illustration from Harms (1929). Mudskippers (Periophthalmus) are shown on the walls of the terrarium after feeding thyroid hormone. Harms reported that the height climbed and the length of the stay out of water correlated with the total dose of thyroid hormone.
cently Inui and Miwa (1985) reported that thyroid hormone stimulates metamorphic changes in young flatfish including migration of the eye. This has been the clearest instance of thyroid-evoked fish development to my knowledge. One of the more remarkable claims in this connection was by Harms (1929) who treated Periophthalmus and certain blennies with thyroid hormone and found that they emerged from the water and became terrestrial, or at least more amphibious. Harms also proposed the height that thyroid-treated Periophthalmusclimbed on the aquarium wall as an assay for thyroid hormone (Fig. 4). He called the phenomenon 'Landtierwerdung' and believed, thus, that the thyroid hormone might have been the basis for the emergence of vertebrates from the aquatic to the land environment. After almost 60 years this claim remains to be substantiated, so we may consider it one of the roots of fish endocrinology that failed to prosper.
Gonad The field of fish gonadal endocrinology also contains some puzzling early features that in their day cast as much shadow as light. Gonadectomy to show the effect of hormonal absence appears to have been only rarely practiced (see Pickford and Atz 1957). As a result, many of the experiments testing the effects of administration of presumed hormonal extracts lacked the proper controls. One of the first reports of this kind of test is that of Wunder (1931) who injected 'extractum testiculi' and Progynon B into the bitterling Rhodeus amarus, and stimulated development of integumentary nuptial coloration, or 'Hochzeitskleid'. He observed also a rapid stimulation of the growth in length of the ovipositor, a structure used by the female bitterling to deposit eggs for their protection within the shells of clams (Fig. 5). In the following years, and particularly after the publication of Fleischmann and Kann's papers in 1932 and 1934 which proposed the ovipositor of the intact female bitterling as a bioassay for female hormones (Fig. 6), this became widely used. Unfortunately, it soon became a confused issue as to what the test measures. Kleiner et al. (1935) and others proposed it as an assay for male hormone. A later view by Van Koersveld (1949) was that the bitterling tests are
7
Fig. 6. Diagram of a female bitterling showing a scale of length of the ovipositor for use as a bioassay for pregnancy or for androgens. Kleiner et al. (1935, 1936).
'totally unsuitable qualitatively as well as quantitatively'. Fish sexual endocrinology developed in a more orderly manner after the amphibians (mostly Xenopus) took over the burden as a poikilotherm pregnancy test. In the light of later understanding of steroid hormonal metabolism and interconversion (especially aromatization of androgens), and characterization of specificity of some of the steroid receptors, some of the confusion generated by the bitterling ovipositor may become understandable. However, as far as this reviewer knows, nobody has returned to this formerly popular research area to clarify it definitively. A measure of the frustration of reviewers in evaluating the early status of fish gonadal endocrinology is epitomized in the statement by Pickford and Atz (1957) 'a systematic attack on the problem of the identity of the sex hormones throughout the vertebrates is one of the fundamental needs of comparative endocrinology'. Ultimately this requirement by Pickford and Atz has been largely achieved, utilizing techniques and advances in understanding provided by mammalian endocrinology.
from Pickford and Atz, an uncertainty as to the nature of the steroids themselves, and in some instances, at least, the identity of the tissue that secretes them. This contrasts with the situation today in which dependable chromatographic separations and immunoassay can be used to identify and quantify with confidence the corticosteroids of fishes, even when the levels are low. In the absence of such techniques, and with use of morphology and crude biochemical indicators (e.g., lipid or ascorbic acid content), it was for about 50 years a matter of contention among endocrinologists (until about 1930) whether the anterior (the present fish interrenal tissue) or posterior (the present corpuscles of Stannius) interrenals of bony fishes are the functional steroidogenic equivalents of the higher vertebrate adrenal cortex. Stannius, for example, favored his corpuscles at first, but was an early convert (1854) to the other camp. S. Vincent, a prominent worker in the field in the late 19th century, similarly changed his mind in 1927 (Vincent and Curtis 1927). Chester Jones (1957) has reviewed this phase of the beginnings of fish adrenal physiology and anatomy. To the comparative endocrinologist, the evolution of the hormonal system with which he or she works is of great interest. Thus the status of these systems in fishes, and in the most primitive fishes in particular, is of paramount importance. One evolutionary puzzle that has no apparent solution is why the corpuscles of Stannius are found only in bony fishes and have no equivalent in higher vertebrates, or in cartilaginous fish species, or in cyclostomes. Just as puzzling has been the fact that a corticoadrenal equivalent tissue has yet to be firmly identified in cyclostomes. There have been a number of presumptive interrenal tissue structures described in lampreys but none has been established beyond question as corticosteroidogenic. In the other group of cyclostomes, the hagfishes, even a presumptive interrenal tissue has yet to be described.
Cortico-adrenal equivalents Pancreatic hormones, insulin The early history of the corticosteroid secreting tissues of fishes was subject to some of the same difficulties that were indicated by the above citation
Morphological studies of the endocrine pancreas of fishes after the pioneering work of Langerhans
8 the first to find that extracts of the fish pancreas relieve symptoms of diabetes in man. Isolation of mammalian insulin was accomplished in 1922, and fish insulins were isolated soon afterward, between 1924 and 1926 (Jensen et al. 1929). Among the first insulins to be sequenced were those of the cod (Reid et al. 1968) and of hagfish (Peterson et al. 1975). The fish pancreas also figured in the early phases of isolation of glucagon. In the year before Staub et al. (1953) isolated mammalian glucagon Audy and Kerly (1952) reported glucagon-like activity in angler fish pancreatic extracts.
Conclusion
Fig. 7. A frequently reproduced figure of the dissection of an angler fish, Lophius piscatorius showing the large single principal pancreatic lobe (Brockmann body) at the arrow. From Buddenbrock (1950), after Bargmann.
(1869) established several interesting facts. Though the fish pancreas contains islets of endocrine cells, there are some species in which the endocrine tissue is concentrated in an organ known variously as the 'principal islet' or Brockmann body (Fig. 7). Furthermore, in the cyclostomes there is no exocrine pancreas and the endocrine cells are clustered in a swelling of the anterior intestine known as the Langerhans organ or follicles. Fish played a prominant role in studies of pancreatic physiology, in part because of the above anatomical facts. The famous pancreatectomy experiments of von Mering and Minkowski (1890) in dogs established the relationship between the pancreas and carbohydrate metabolism. However, initial attempts to extract the pancreatic hormone, insulin, were thwarted because proteolytic enzymes in ground pancreas digested the hormone. One obvious and successful solution to this problem was in the use of Brockmann bodies, such as those of the angler fish, Lophius (McLeod 1922; McCormick and Noble 1924). Diamare and Kuljabko (1904) reported that extracts of angler fish principal islets are active in altering carbohydrate metabolism in the fish. Rennie and Fraser (1906) were apparently
In this abbreviated overview of the beginnings of piscine endocrine research no attempt has been made to be encyclopedic. Instead, some of the insightful, as well as some of the fumbling exploratory earlier efforts in this field have been outlined. Some of the more recent roots and rootlets have been omitted because they are more obvious and, to take up one or two of them would require consideration of many more recent rapid developments. For example, it was knowledge of the developmental relationships of fish and amphibian ultimobranchial bodies that made possible the fuller understanding of thyroidal calcitonin in mammals, and led to the use of salmon calcitonin in the clinic. At the interface between academic fish endocrinology and fish aquaculture there has been rapid recent progress in purification and sequencing of fish pituitary and pancreatic hormones, as well as better understanding of the role of endocrines in fish development, growth and reproduction. But these developments clearly have been based on the general fund of related information that already existed. There has been a recognizable field of fish endocrinology since the early nineteenth century. Explanation of why a biologist enters this field is no doubt the same as for any comparative physiologist: an interest in exploring the nature of a particular mechanism in this vertebrate group for comparison and contrast with other vertebrates. The further value of work in fish endocrinology for some of us is in the strategic value of data obtained from
9 fishes in explaining the evolution of these mechanisms in vertebrates in general. Others are motivated by the usefulness of endocrine data in promoting fish as an agricultural resource. However, all fish endocrinologists should be encouraged by the realization that what they discover today may be at the root of important developments in this field in the future.
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the caudal neurosecretory system of fishes. In Comparative Endocrinology pp. 697-724. Edited by A. Gorbman. John Wiley and Sons, New York. Etkin, W., Root, R.W. and Mofshin, B.P. 1940. The effect of thyroid feeding on oxygen consumption of the goldfish. Physiol. Zool. 13: 415-429. Fleischmann, W. and Kann, S. 192. Uber eine Funktion des weiblichen Sexualhormons bei Fischen (Wachstum der Legerohre des Bitterlings). Pflug. Arch. Ges. Physiol. 230: 662-667. Fleischmann, W. and Kann, S. 1934. Uber die Wachstum der Legerohre des Bitterlings unter dem EinfluB des weiblichen Sexualhormons. Pflu. Arch. Ges. Physiol. 234: 130-136. Grobstein, C. and Bellamy, A.W. 1939. Some effects of feeding thyroid to immature fishes (Platypoecilus). Proc. Soc. Exp. Biol. Med. 41: 363-365. Gudernatsch, J.F. 1911. The thyroid gland of the teleosts. J. Morph. 21: 709-782. Gudernatsch, J.R. 1912. Feeding experiments on tadpoles. 11.A further contribution to the knowledge of organs with internal secretion. Arch. Ent. Mech. Org. 35: 457-483. Harms, J.W. 1929. Die Realization von Genen und die consekutive Adaptation. I. Phasen in der Differenzierung der Anlagenkomplexe und die Frage der Landtierwerdung. Zeitsch. wiss. Zool. 133: 211-397. Hopper, A.F. 1952. Growth and maturation response of Lebistes reticulatus to treatment with thyroid powder. J. Exp. Zool. 119: 205-217. Inui, Y. and Miwa, S. 1985. Thyroid hormone induces metamorphosis of flounder larvae. Gen. Comp. Endocrinol. 60: 450-454. Jensen, H., Wintersteiner, O. and Geiling, E.M.K. 1929. Studies on crystalline insulin; isolation of crystalline insulin from fish islets (cod and pollock) and from pigs' pancreas; activity of crystalline insulin and further remarks on its preparation. J. Pharmacol. Exp. Therap. 36: 115-128. Kleiner, I.S., Weisman, A.I. and Barowsky, H. 1935. An investigation of the new biologic test for hormones in pregnancy urine. J. Am. Med. Assoc. 104: 1318-1319. Kleiner, .S., Weisman, A.I., Mishkind, D.I. and Coates, C.W. 1936. The female bitterling as a biologic test animal for male hormone. Zoologica 21: 244-250. Landgrebe, F.W. 1941. The role of the pituitary and thyroid in the development of teleosts. J. Exp. Biol. 18: 162-169. Langerhans, P. 1869. Beitrage zur mikroskopischen Anatomie der Bauchspeicheldruse. Diss., Berlin. Lynn, W.G. and Wachowski, H.E. 1951. The thyroid gland and its functions in cold-blooded vertebrates. Quart. Rev. Biol. 26: 123-168. Marine, D.J. and Lenhart, C.H. 1910. On the occurrence of goitre (active thyroid hyperplasia) in fish. Johns Hopkins Hosp. Bull., 21: 95-98. Marine, D.J. and Lenhart, C.H. 1910. Observations and experiments on the so-called thyroid carcinoma of brook trout (Salvelinusfontinalis) and its relation to ordinary goitre. J. Exp. Med. 12: 311-337. Marine, D.J. 1911. Further observations and experiments on the
10 so-called thyroid carcinoma of the brook trout (Salvelinus fontinalis)and its relation to endemic goitre. J. Exp. Med. 13: 455-475. von Mering, I. and Minkowski, 0. 1890. Diabetes mellitus nach Pankreasextirpation. Arch. exp. Pathol. und Pharmakol. 26: 371-387. MacLeod, J.J.R. 1922. The source of insulin. A study of the effect produced on blood sugar by extracts of the pancreas and principal islets of fishes. J. Metabol. Res. 2: 149-172. McCormick, N.A. and Noble, E.C. 1924. The yield of insulin from fish. Contr. Can. Biol. Fish. N.S. 2: 115-128. Miwa, S. and Inui, Y. 1985. Effects of thyroxine and thiourea on the parr-smolt transformation of amago salmon (Oncorhynchus rhodurus).Bull. Nat. Inst. Aquaculture, Japan 4: 41-52. Peterson, J.D., Steiner, D.F., Emdin, S.O. and Falkmer, S. 1975. The amino acid sequence of the insulin from a primitive vertebrate, the Atlantic hagfish, (Myxine glutinosa). J. Biol. Chem. 250: 5183-5191. Pickford, G.E. and Atz, J.W. 1957. The Physiology of the Pituitary Gland of Fishes. New York Zoological Society, New York. Reid, K.P. Grant and Youngson, A. 1968. The sequence of amino acids in insulin isolated from islet tissue of the cod (Gadus callarius). Biochem. Jour. 10: 289-296.
Rennie, J.and Fraser, T. 1906. The islets of Langerhans in relation to diabetes. Biochem. J. 2: 7-19. Robertson, O.H. 1949. Production of the silvery smolt stage in rainbow trout by intramuscular injection of mammalian thyroid extract and thyrotrophic hormone. J. Exp. Zool. 110: 337-355. Scharrer, E. 1928. Die Lichtempfindlichkeit blinder Elritzen (Untersuchungen fiber das Zwischenhirn der Fische.) Zeitschr. vergl. Physiol. 7: 1-38. Scharrer, E. 1941. Neurosecretion. I. The nucleus preopticus of Fundulus heteroclitus L.J. Comp. Neurol. 74: 81-92. Simon, J. 1844. On the comparative anatomy of the thyroid gland. Phil. Trans. Roy. Soc. Ser. B. 134: 295-303. Speidel, C.C. 1919. Gland cells of internal secretion in the spinal cord of the skates. Publ. Carnegie Inst. Washington, no. 281. Speidel, C.C. 1922. Further comparative studies in other fishes of cells that are homologous to the large irregular cells in the spinal cord of the skates. J. Comp. Neurol. 34: 303-317. Stannius, H. 1854. Zootomie der Fische und Amphibien. Berlin. Staub, A., Sinn, L.G. and Behrens, O.K. 1953. Purification and crystallization of hyperglycemicglycogenolytic factor (HgF). Science 117: 628-629. Vincent, S. and Curtis, F.R. 1927. A note on the teleostean adrenal bodies. J. Anat. 62: 110-120.
