Gynogeneticteleost, Poeciiia Formosa, and That of Other Poeciliid Fishes1

[CANCER RESEARCH 37, 3751-3755, October 1977] A Comparative Study of the Pretumorous Thyroid Gland of the GynogeneticTeleost, PoeciIia formosa, and That of Other Poeciliid Fishes1

A. D. Woodhead and Paula M. Scully Biology Department,BrookhavenNationalLaboratory,Upton,New York11973

SUMMARY sexually reproducing) teleost fish, Poeci!ia formosa, the Amazon molly, as a research tool for oncogenic studies. A laboratory clone of the gynogenetic fish, Poeci!ia for Offspring of a particular clone of fish are genetically identi mosa (the Amazon molly), showed a high incidence of cal with each other and with any member of that clone; invasive thyroid hyperplasia when injected with isogenic tissue transplants within clones are accepted and survive fish cells with damaged DNA. The sensitivity of the response indefinitely. Using this fish system, Hart and Setlow ob indicated that the thyroid gland of this species might differ tamed evidence suggesting that pynimidine dimers in DNA, from that typically found in fish. Comparisons were made as a result of the exposure of fish cells to UV radiation in between the thyroid of P. formosa of several ages and that vitro, induced hyperplasia and hypertrophy of the thyroid of five closely related species that had been kept under gland of isogenic recipient fish. The thyroid tissue was the same laboratory conditions. In the five related species, invasive into both soft and hard tissue, and in some expeni the thyroid gland was composed of a few, uniform, colloid ments such thyroid lesions developed in 100%of the recipi filled follicles lined with cuboidal epithelial cells, scattered ent fish. When UV exposure was followed by enzymic pho around the ventral aorta. The thyroid gland of P. formosa toreactivation â€”(aDNA repair system specific for pymimidine was more extensive and, in addition to normal follicles, dimers)â€”there were far fewer lesions in the injected fish.2 contained areas of microfollicles and afollicular groups of This work suggested that P. formosa might have consid epithelial cells. The follicular cells were hypentrophied and, enable potential research use in cancer studies. Such exper in some areas of the gland, there was hyperplasia of the iments, however, can be readily interpreted and evaluated connective tissue. There was an increase in the abundance only against a background knowledge of the biology of the of these atypical elements with advancing age. The ap fish. Although there is a considerable body of information pearance of the thyroid gland of P. formosa resembled on the origin and the evolutionary sequence of unisexuality that seen in the stages of development of thyroid tumors in this genus (7, 8, 13), there have been few other biological in some highlyinbredstrainsoffish. studies. Hart et a!. (5) have provided the basis parameters Young Amazon mollies appear to have a high probability for handling and breeding the Amazon molly. of developing invasive thyroid hyperplasia under the influ The present study describes the microanatomy of the ence of cells with damaged DNA, yet in young fish the thyroid gland. We undertook this survey because the high response is quite precise and specific. P. formosa seems sensitivity of P. formosa to the development of thyroid to provide good advantages as a test animal for oncogenic growths following the injection of cells with damaged DNA studies. suggested that the gland might be atypical. The clones of P. formosa used in the experiments of Hart and Setlow (6) and in the present work were originally collected from a fresh INTRODUCTION water pool near Brownsville, Texas, by Dr. C. P. Haskins in 1946, and they have been kept in laboratory aquaria since A major problem in cancer research studies is to minimize that time. We also examined the thyroid of the bisexual genetic variation within a strain of test animals. Long-term species, Poeci!ia latipinna and Poecilia sphenops, the pre genetic stability of a strain allows reliable repetition of ex sumed parental species of the Amazon molly (7). Glands peniments and reduces the range of response to carcino from other closely related Poeciliidae (live-bearers) were genic agents. To achieve these ends, pedigreed inbred included , namely Poeci!ia (Lebistes) reticu!ata, Gambusia strains of rodents have been maintained oven many years; yucatana, and P!atypoeci!ius maculatus. nevertheless, separated colonies may quickly differ as a result of gene drift, mutation, on contamination of the popu lation. MATERIALS AND METHODS Hart and Setlow (6) used clones of the gynogenetic (non Surveys of the thyroid gland were made in 3 groups each I Supported by Contract YOL-CP-50202 from the National cancer Institute of 5 Amazon mollies, ages 3 to 4 months, 1 year, and 18 to and bythe UnitedStatesEnergyResearchandDevelopmentAdministration.

By acceptance of this article, the publisher and/or recipient acknowledges 2 A. B. Setlow, A. W. Hart, and A. D. Woodhead, Evidence That Pyrimidine the U. S. Government's right to retain a nonexclusive, royalty-free license Dimars in DNA Can Give Rise to Tumors, submitted for publication to in and to any copyright covering this paper. Proceedings of the National Academy of Science of the United States of Received October 28, 1976; accepted July 11, 1977. America.

OCTOBER 1977 3751

24 months. For comparison, the thyroids were examined regular and rounded, with squamous or cuboidal epithelial from 5 malesand 5 females,agesabout 10to 12months, of cells 5 to 7 @.&mhigh.Homogenous colloid almost filled the the bisexuals P. latipinna, P. sphenops, and G. yucatana, follicles, but there were absorption vacuoles at its edges, which had been maintained in tanks in our laboratory for at suggesting that there was some secretory activity in prog least 3 months before the thyroids were examined. Thyroid ness. hyperplasia readily occurs in artificially raised fish in me The Thyroid Gland of Young P. formosa . The thyroid sponse to iodine deficiency in the water or the diet or to the gland of young Amazon mollies (ages 3 to 4 months) was presence of antithyroid compounds in the food (9, 10, 11, already slightly larger, both in its anterior-posterior extent 12). Our aquarium environment was iodine rich; iodine was and in the number of follicles present, than the gland of added in making up the dilute seawater in amounts suffi adults of the closely related species described above. It was cient to prevent the development of goiter. Nevertheless, to composed principally of regular, colloid-filled follicles lying ensure that the thyroids would be comparable, we adopted adjacent to the ventral aorta and at the bases of the gill the measure of keeping these species together in the same arches. Towards the region of the 2nd and 3rd branchial environment before examination. arches, however, the gland contained several small islets of The guppies, P. reticu!atus, were taken from an untreated microfollicles dispersed among the connective tissue; the population that had been kept by Dr. A. Comfort for aging epithelial cells of these microfollicles were hypertrophied. studies (2). Serial sections through the heads of P. macu!a The areas of microfollicles appeared well vasculanized by tus, the platyfish, were kindly made available by Dr. S. comparison with other regions of the thyroid. Holtzmann of the Medical Department of Brookhaven Na The Thyroid Gland of Adult P. formosa . By the age of 12 tional Laboratory, Upton, New York. months, the gland of the Amazon molly had an appearance For histological study, the heads of the fish were fixed in very different from that of the adults of the related species. buffered fonmalin, decalcified, embedded, and serially sec The thyroid was much larger, and the mass of the gland was tioned at 8 @M;every5th section was mounted and stained composed of numerous microfollicles, widely dispersed with hematoxylin and eosin. In the Poeciliidae, as in most singly, or in small groups of 2 to 3, among the connective teleost fish, the thyroid gland is diffuse and consists of tissue surrounding the aorta. Many afollicular groups of numerous discrete follicles lying in the connective tissue epithelial cells were present. There had been a further in around the ventral aorta. Comparisons of the thyroid gland crease in the anterior-posterior extent of the gland, which were made in a number of ways. The extent of the gland extended backwards over the bulbus arteniosusand termi along its anterior-posterior axis was assessed by counting nated behind the ventricle. A few follicles were found the number of sections over which it extended. The extent around the junction of the ventricle with the sinus venosus. of the gland in the dorsoventral plane and in the lateral There was a slight increase in the spread of the gland in the plane was also measured. We used the total number of dorsoventral plane. The dispersion of the gland laterally follicles as an indication of the size of the gland, counting was greatly increased, and there was abundant thyroid tis them in each section along its length. Measurements were sue in the gill bases,often penetrating the gill filaments. No made of the diameter of 60 follicles and of the height of 50 thyroid follicles were found in extrapharyngeal sites. epithelial cell heights in thyroid tissue from the anterior end The structure of the thyroid was not uniform. The anterior of the gland, behind the aortic bifurcation. The staining part of the gland was composed of large numbers of micro reaction of the colloid and the general appearance of the follicles, separated by lange areas of loose connective tis gland were recorded. sue, which would seemto allow further growth and spread of follicles. Many single epithelial cells and small afollicular RESULTS groups of 3 to 4 cells occurred in this area of the gland. The epithelial cells of the afolliculan groups and those of the Themewas a striking difference between the appearance microfollicles were hypertrophied, with a rounded, balloon of the thyroid of the gynogenetic P. formosa and that of the like appearance (Fig. 2). Their cytoplasm had beocme closely related bisexual species, the thyroid of the adult foamy and thin, and the nuclei were basal in position. These Amazon molly showing folliculam hypemplasia, hypertrophy cells measured about 10 @m.Insome of the microfollicles, of the epithelial cells, and some hypemplasiaof the connec the walls of the epithelial cells were indistinct and colloid, tive tissue towards the posterior region of the gland (Fig. 1). and cell cytoplasm had merged. Toward the bulbus artenio The Thyroid Gland In the Bisexual Species of Poecili sus some normal thyroid follicles were found that were idae. The appearance of the thyroid gland was similar in all larger, were regular in appearance, and contained colloid. of the bisexual species studied, and there were no differ They closely adhered to the blood vessel. The overall ap ences seen between the glands of male and female fish. The pearance of the gland suggested that there was little secre thyroid consisted of relatively few large follicles, distributed tory activity. in the stroma along the ventral aorta. The gland began just Counts of the total number of follicles in the gland of the in front of the aortic bifurcation and extended posteriorly several species confirmed these observations (Chart 1). The along the vessel, terminating immediately behind the bul difference shown tends to be an underestimate, because bus arteniosus. The amount of thyroid tissue reached its counts were made only of normal follicles and microfollicles maximum development in the region of the 2nd to 4th in the gland of P. formosa; the groups of afolliculam cells branchial arches and then narrowed, follicles becoming were not counted. Measurementsof follicle diameter also closely grouped around the aorta. There were no follicles in illustrate the difference in the thyroids (Chart 2), and again the gills or in extrapharyngeal sites. Individual follicles were the difference is undenemphasized, since no measurements

3752 CANCERRESEARCHVOL. 37

0 50 were found in other parts of the body (15). IN@J@E@l We examined the thyroids of 5 adult Amazon mollies 0 OF FOWCLES from the same original clone that had been continuously @ 4000 maintained by Dr. C. P. Haskins in his laboratory and I' that he kindly made available to us. His fish were smaller @2000 than ours, but the thyroid again showed similar mompholog â€˜4$ P. formosa P spheno@,s P lotipinna G yuCOtOnO ical features (hyperplasia of the connective tissue), al though these features were not as pronounced as in our Chart 1. Distribution diagrams of the thyroid tissue in 1-year-old â€˜P.for largerfish. mosa and in 3 closelyrelatedPoeciliidae.

DISCUSSION

The histological appearance of the thyroid gland of 1- year-old P. formosa was quite different from that of closely related species. It was characterized by extensive follicular hypemplasia, epithelial cell hypentrophy, and proliferation of U) Li thyroid gland connective tissue. The extent of these fea -J U tunes progressed with age. The thyroid gland of closely -J 0 related Poeciliidae, kept in our laboratory in the same U- aquarium system, did not exhibit any thyroid hypemplasia, U- 0 showing that the condition was not a goitrogenic response to low levels of iodine in the environment, unless P. formosa @ 40 has atypically high iodine requirements. :D z 20 Some species of xiphophonid fish (swondtails) have been found to be very prone to the development of thyroid tumors (1). In Xiphophorus montezumae and Xiphophorus pyg maeus, thyroid tumors develop spontaneously in older fish. The progressive stages in their development in X. montezu mae have been followed in detail by Berg et a!. (1). Very young fish have a small typical thyroid, but with increasing I 23456 789IOlII2I3I4@5I6I7I8l92O size of the fish, there is a rapid increase in the number of FOLLICLE DIAMETER thyroid follicles, accompanied by hypertrophy of the follicu Chart2.ComparisonofthedistributionoffolliclediameterinP.formosa Ianepithelial cells until, finally, a thyroid tumor develops in and other species. old individuals. Highly inbred strains of swomdtails were more susceptible than were less intensely inbred strains, were made of the afollicular groups. and the authors concluded that genes influencing the The Thyroid Gland of P. formosa, 18 to 24 Months. With growth of thyroid cell elements accumulated in members of some exceptions, the thyroid gland of fish of 18 months of theinbredstrain. age was similar in appearance to that described for 1-year The changes seen in the thyroid gland of the Amazon old fish, although generally the gland showed an increase in molly resembled those found during the development of extent. However, in a few fish, the gland had enlarged much tumors in X. montezumae. Hyperplasia of the connective more, and the extent of the areas of microfollicles and tissue, which was prominent in the posterior region of the afollicular groups of cells had increased. Toward the heart, thyroid of the molly, did not occur in swordtails, although it the gland retained a normal appearance, but a few of the is a common feature of the tumorous thymoidal tissue of follicles now appeared to have become inactive; flattened hatchery-reared trout (3). The morphology of the thyroid of epithelial cells enclosed dense homogenous colloid with no old P. formosa also clearly resembled the thyroid tumors vacuolation. There were also occasional restricted regions that are found in some old guppies, P. reticulatus, in which at the edges of the gland where muscle tissue was being themeis proliferation of large numbers of microfollicles and invaded by the follicles. hypertrophy of the folliculan epithelial cells (14, 15). These In the majority of fish aged 24 months, the thyroid gland similarities suggest to us that the thyroid gland of our clone had greatly enlarged and occupied much of the phamyngeal of P. formosa may be unusually sensitive to agents and area. In general, the gland could be divided into 3 regions; stimuli that provoke neoplasia, and this conclusion is around the heart, there were many large inactive follicles, strengthened by the finding that the incidence of sponta filled with colloid. Peripherally, and towards the anterior of neous invasive thyroid hyperplasia approaches 100% in very the gland, there was a predominance of microfollicles, old fish (15). Such thyroid growths may be linked to the while the outermost areas contained many small afollicular gynogenetic mode of reproduction and the accumulation of groups of cells distributed among loose connective tissue. genes regulating thyroid growth, as suggested by Berg et The thyroid tissue was invasive into both muscle and bone. a!. (1). This study was made in order to provide a back There was considerable variability in the appearance of the ground knowledge of the Amazon molly against which the gland both within and between individuals. No metastases findings from oncogenic studies could be evaluated. This

OCTOBER 1977 3753

The Pretumorous Thyroid Gland of P. formosa Ia I1L U@'â€¢a â€¢@ .

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Fig. 1. a, low-power photomicrograph of the thyroid gland of P. sphenops. Arrows, thyroid follicles. b, low-power photomicrograph of the thyroid gland of P. formosa.top arrows, afollicular groups;bottom arrows, thyroid follicles. Fig. 2. High-power photomicrograph of the thyroid gland of P. formosa to show the hypertrophied epithalial calls. Double arrows, hypertrophied apithelial cells; single arrow, afollicular group.

OCTOBER 1977 3755

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1977 American Association for Cancer Research. A Comparative Study of the Pretumorous Thyroid Gland of the Gynogenetic Teleost, Poecilia formosa, and That of Other Poeciliid Fishes

A. D. Woodhead and Paula M. Scully

Cancer Res 1977;37:3751-3755.

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