Cytological and functional properties of the thyroid of a chimaeroid fish, Hydrolagus colliei

GESJERAL

AND

COMPARATIVE

Cytological

13, 285302

EXDOCRINOLOGY

and

Functional

of a Chimaeroid YASUMITSU Department

of Zoology,

NAIL41 University

(1969)

Properties

of the

Fish, Hydrolagus AND

AUBREY

of Washington,

Received

April

Thyroid

colliei GQRBMAN

Seattle,

Washington

$8105

28, 1969

Cytological features of the ratfish (holocephalian) thyroid gland are described from the ultrastructural level. A series of vacuolar structures were found in thyroid cells which could be readily interpreted to indicate successive (a) resorption of colloid fro,m the follicular lumen, (b) fusion of the resulting vacuolar organelles with lysosomes, (c) formation of protein crystalloids and loss of the vacuolar limiting membrane. Treatment with either thiourea or mammalian TSH appea.red to stimulate this process. Although radioautography (at the E. M. level) was done only with two specimens, results indicated that iodine organification occurs in the eolloid, and that resorption vacuoles contain already protein bound iodine, which is further metabolized in the intracellular organelles.

The Holocephalian (chimaeroid) fishes based not only on descriptions of normal remain the least studied vertebrates from thyroid cellular structure, but also on histothe point of view of endocrine organ mor- chemical localization of acid phosphatase~ phology. This group apparently evolved on incorporation and radioauto&apby of from a primitive selachian line (Romer, radioiodine, and on responses to thyrotro~~o 1950) but its pituitary gland differs in im- hormone and goitrogenic drug injections. portant respects from that of the modern The experimental procedures were perelasmobranchs. For example, there is no formed in an effort to derive the ~~n~tio~a~ ventral lobe, but there is an extracranial significance of some of the ~~tra~el~~la~ adenohypophysial lobe named the Rachenstructures that were found. dachhypophyse (Fujita, 1963; Altner: MATERIALS APU’D METHOD8 1964). Because of the chimaeroid’s evolutionary position among the most primitive A tota! of twenty-nine ‘katfish,” Hyckolagus of living vertebrates, it has been considered colliei was used in this study. They were cc& important to learn more about the mor- lected by otter trawl near Friday Harbor, Washphology of its thyroid gland, as well as ington and placed in a large concrete basin wnth flow of sea. water at the Friday about the regulation of function of this or- a continuous Laboratories of the University of Washgan. We report here the first of such studies, Harbor ington. A few thyroid specimens were fixed as devoted. principally to features revealed by freshly as possib!e on board the collecting boat. electr’on microscopy. but this did not appear to he more advantageous The observations presented here are than fixation a.fter holding at the laboratory. Animals used in this study varied from XI t,a 1400 g in body weight and were not. selected as to ses. The dates of capture va.ried from August, 1967 to March? 1968. Distribution of the animals studied in the different phases of this study is summarized in Table 1. TSH Injection. The thyrotropic preparation used was a partly purified bovine TSH (supplied

‘This study was aided in part by research Grant NB04881 from the National Institutes of Health. We express our appreciation also for valuable counsel and use of some of their facilities and supplies to Professor R. A. Cloney, Zoology Department, Professor Russell Ross, Pathology Department, and Professor Thomas H. Shepard, Pediatrics Department. 285

286

NAKAI

PROTOCOL

Treatment

Number of specimens

None TSH injection Thiourea injection Radioiodine injection

20 3 4 2

AND

GORBMAN

TABLE 1 OF STUDY OF RATFISH Length

range

(cm) 17-58 38-55 35-50 38-45

by the N.I.H.), dissolved in saline solution. It was injected intraperitoneally in a dose of 10 I.U. per day for 1 or 3 days and the specimen was killed on the fourth day. Goitrogen Injection. Thiourea dissolved in stiine solution was injected intraperitoneally in a dose of 10 mg per day for 2 or 3 days. Radioautography. One animal was killed 4 hr after intraperitoneal injection of 232 /& of radioiodine”‘, and another after 72 hr. Sections were cut after fixation by the procedures described below. The radioautographic procedure is one modified by R. Ross (University of Washington) from one described by Gramboulan (1965). In this ;procedure thin sections are affixed to subbed glass ‘slides. and covered with molten photographic ‘emulsion. After exposure and development of the : photographic emulsion, the section and autograph are transferred to a grid for electron microscopic examination. Acid Phosphatase. The technique of Miller and Palade (1964) was used to show the distribution of acid phosphatase in frozen sections of thyroid tissue. In this case fixation was in 2.5% glutaraldehyde buffered at pH 7.4 with sodium cacodylate. Sections 20-50 p thick were incubated in a modified Gomori solution (Miller and Palade, 1964) for 20-40 min, postfixed in 2% osmium tetroxide and embedded in Epon. Electron Microscopy. Some of the thyroid glands of normal ratfishes were placed in a cold fixative consisting of equal parts of 4% osmium tetroxide and Millonig’s phosphate buffer (Millonig, 1962) at pH 7.4 for 2 hr; others were fixed in 2.5% glutaraldehyde solution buffered with Millonig’s phosphate buffer at pH 7.4 for 2 hr and then postfixed with 2% osmium tetroxide solution at pH 7.4 with Millonig’s phosphate for two hours at 4”. Tissue blocks were dehydrated in graded concentrations of alcohol, followed by propylene oxide and embedded in Epon 812 (Luft, 1961). Sections for electron microscopy were cut on a Porter-Blum microtome with glass knives, mounted on copper grids and stained for 3 min ‘in saturated uranyl acetate and for 3 min in lead

THYROID

Date

range

August, 1967-March, September, 1967 September, 1967 March, 1968

1968

hydroxide (Millonig, 1961). Sections for light microscopy were cut at a thickness of 1.0 p and stained with methylene blue and azure II (Richardson et al., 1960). The preparations were examined and photographed in a RCA EMU-3G electron microscope operated at 50 kV. RESULTS

The ratfish thyroid gland is a solid encapsulated organ near the anterior extremity of the lower jaw, under the small tongue. It is composed of follicles that are extremely varied in size and shape. Smaller follicles tend to a spherical shape, but the larger ones may be tubular and produced into various configurations. Generally the follicular epithelium consists of .cuboidal or columnar cells, and follicular epithelium falls mostly between 15 and 50 ,U in height (Fig. 1). The thyroid follicular epithelium is typically simple, but areas where the epithelium is several cells thick can be found in Figs. 1 and 2. The free luminal surfaces of the follicular cells have an irregular outline characterized by a few microvilli and a central flagellum. The microvilli vary widely in number and length, but generally there are fewer than in higher vertebrate thyroids. The oval or round nucleus is consistently basal in position. The fine particles are most densely accumulated at the nuclear membrane, and the nucleolus, composed of aggregatks of the particles, is found near the center of the nucleus (Figs. 3, 13). The follicular epithelial cells are closely adjacent, separated by a lateral plasma membrane and a narrow intercellular space (Figs. 6, 8, 16). The intercellular space is about 200 ii in width and is continuous with the pericapillary space (Fig. 6). At the base of t,he cell the plasma mem-

CHIMAEROlD

THYROID

TJLTRASTRUCTURE

2a

lqlc. 1. Thyroid follicles of a normal ratfish. The follicular epithelium consists of cuboidal or co:~m~~~ cells and is typically simple, but in some areas there are several layers of nuclei and the epithelium is pseltdostratified. Generally, the oval or round nuclei are basal in position. X11 follicles are enclosed by expi&r& and connective tissue. Y 1,300. pIG. 2. Thyroid follicles of ratfish injected intraperitoneally with beef TSH, 10 T.V. per day for 3 days alId killed ox> the fourth day. Kute the large and small vacuoles in the follicular lumen at the upper right,, arrd ‘;he loss of stainable colioid from other follicles. Dark nuclei of decreased size and many large lucent basal . ,. vacuoles are in the foihcular cells at upper left and mrddle of this figure. Epltheha! height, is on the average decreased. particularly in follicles lacking stainable colloid. X 1,300.

288

NAKAI

AND

GORBMAN

?IG. 3. Thyroid follicular epithelial cells of an adult rat&h. A few microvilli project into the foll iicle The nucleus (N) lies in the lower sector. Several Golgi apparati (G) are visible in the sul palun nen (FL). In the apical portions are numerous small vesicles (V) and many inclusion bodies (1[B), XlUC clear region. a bundle of fine parallel filaments. BM, basement membrane; rER, rough surfaced er Idoeat :h containing X5,400. da smic reticulum. body; M, m 1PIG. 4. Small vesicles (V) in the apices of follicle cells. FL, follicle lumen; IB, inclusion chc mdria; G, Golgi apparatus. X 14,000.

CHIMAEROID

THYROID

brane is irregularly folded. Occasionally the basal folding together with basement membrane when most highly developed and branched, projects deeply into the cell (Figs. 5, 6). All follicles are enclosed by capillaries (Figs. 1, 2) I and between each capillary and the plasma membrane of the epithelial cells there is a pericapillary space that is filled with the endothelial and epithelial basement membranes, and connective tissue cells and fibrils (Fig. 5’). Desmosomes occur also between the endothelial processes, and no pores are discernible. In the cytoplasm of the endothelium many pinocytotic vesicles are observed (Fig. 4). The Golgi apparatus, which consists of smooth membranes, vacuoles, and vesicles, is most frequently encountered in the supranuciear region of the cell (Figs. 4, 9)) and less frequently in the region of the nucleus. Golgi structures are never observed in the infranuclear region. There is much variation in the shape, size, and electron density of t’he Golgi vacuoles and vesicles. A few vesicles and vacuoles in the Golgi region resemble the small less-dense granules that are found in the apical cytoplasm (Fig. 4). Mitochondria are scattered throughout the cytoplasm at random; in the basal region of the cytoplasm they are found among the elements of the rough-surfaced endoplasmic reticulum. Most of the mitochondria are oval or elongated in shape and they usually have crests. Although rough-surfaced endoplasmic reoiculum is found throughout the cytoplasm, it is usually dilated and most abundant in the middle and basal region of the thyroid cells; its contained spaces are occupied by a homogeneous less-dense substance (Figs. 3, 6). In the apical region of the cell, roughsurfaced endoplasmic reticulum is relatively rare and attached ribosomes are scarce. In the apical cytoplasm three kinds of vesicles and granules can be seen. The first type appears as small, round and less-dense vesicles (0.1-0.3 ,U in diameter). These are generally most numerous in the apical part of the cell and they are ordinarily clustered around the Golgi apparatus (V in Fig. 4). It is difficult to distinguish the smallest

ULTRASTRUCTURE

less-dense vesicles from Golgi vesicles. The contents of such vesicles that are foun beneath the apical plasma membrane, have the same density and texture as the dloid in the follicular lumen (Fig. 4). It is suggested that the small less-dense vesicles found in the apical cytoplasm are derived from the Golgi apparatus The second type of vesicle is large, lessdense and most often spherical in outline (0.6-2.0 p in diameter) (D in Fig. 7)? but some of these are relatively irregular in outline and are slightly more electron-dense than the follicular colloid. However, the content of most of these droplets is homogeneous and similar to the colloid of the follicular lumen in electron density. ‘Fhey are limited by a single membrane. In the middle portion of the thyroid cells some of these droplets contain a few filaments (I& in Fig. 8). Blending with the second vesicle type and seemingly derived from it by a continuous process, is a series of granules, round or oval in shape and distinguished by their content’s which are extraordinarily variable in structure and density. Although most of them comain an apparently homogeneous dense substance, others display a fingerprint like structure, or bundles of wavy fine filaments, and sometimes a, heterogeneous content. As a class we will refer to them here as ‘Linclusion bodies” (Figs.. 8, 9, 10). A third type, referred to here as small. dense granules (0.2-1.0 k in diameter) (Figs. 7, 15) frequently may be observed near the Golgi apparatus, and more rarely just below the apical plasma membrane appearing from the point of view of size as if several had fused with each other. ever, no actual observation of such fusion to the apical plasma membrane or of seeretion into the follicular lumen has yet, been seen. Frequently around the large less-dense droplets several of the small dense granules are observable and in some cases they are in actual contact with each other by fusing of both limiting membranes (Fig. 7). Figure 16 illustrates a small dense granule just incorporated within a large droplet, and the

290

NAKAI

AND

GORBMAN

FIG. 5. Section through the basal regions of several ratfish thyroid follicular epithelial cells, and part of a capillary wall. Note the folded course of the basal plasma membrane (arrows) of the thyroid cells. EiVD, endothelium; PS, pericapillary space; B, blood cell. X6,003. FIG. 6. Note that the folded basal plasma membrane (arrows) of the thyroid epithelial cells, together with epithelial basement membrane, are irregularly projected into the thyroid cells. END, endothelium; M, mitochondria; PM, lateral plasma membrane; rER, rough-surfaced endoplasmic reticulum. X16,OCO.

CHIMAEROID

THYROID

ULTRASTRUCTURE

29i

Fra. 7. Note the apparent fusion betw-een large less-dense droplet (D) and a small dense gramde (DC&). DG, small dense granule; IG, intermediate form of dense granule. X46,006. FIG. 8. Cross section through the supra-nuclear region of ratfish thyroid follicular cells. n’ote that two less-dense droplets (DJ contain a few filaments and in another one (Dz) small vesicular formation 5 seen. Rough-surfaced endoplasmic reticulum (rER) in some places is void of dense particles. IB, inclusion body, PM, lateral plasma membrane of thyroid epithelial cell. X19,000.

292

NAKAI

AND

GORBMAN

through the Golgi level of thyroid follicular epithelial cell. Note FIG. 9. Cross section bodies (IB). Arrows indicate microtubule. G, Golgi apparatus; M, mitochondria; DG, x 19,000. FIG: 10. Note that the crystalloids (Cr) consisting of numerous fine filaments are dense granule is incorporated within a less-dense droplet (D). IB, inclusion body; x 18,000. FIG. 11. The longitudinal and oblique sections of the crystalloids (Cr) consisting arrangements. X18,000.

an array of inclusion small dense granule. exposed. G, Golgi of fine

The small apparatus. filamentous

CHIMAEROID

THYROID

inside limiting membrane has disappeared. -4t this moment, or stage, the rest of the large droplet is still occupied by homogeneous less-dense substance and has none of the fine filamentous structure. Generally, these fused and compounded figures are observed in the apical part of the thyroid cell, but most of the separate small dense granules appear to be situated in the deeper portion of the cells. As the changes following incorporation of small dense into the large less-dense droplets apparently progress, a small number of characteristic fine filaments becomes observable, as well as intravesicular, small granular structures, and a few dense masses (Figs. 8, 16, 18) ; in presumably more advanced stages the dense intravesicular structures appear to be more numerous. Such complex inclusion bodies are found much more frequently in the adult ratfish thyroid than in younger ones. The proportion of intravesicular fibers to granular elements increases until most of the inclusion body is occupied by parallel fibers within the membrane (Fig. 9). The originally spherical inclusion body then gradually elongates as the bundle of fibers straightens out (Figs. 3, 4, 8, 17, 18). Organelles consisting of groups of straight parallel fibers or filaments we have referred to here as crystalloids. The investing membrane around the crystalloid disappears from one end (Figs. 10, 11) finally leaving the filamentous bundle free in the cytoplasm. In the test’s for the acid phosphatase reaction, the reaction product was found localized intracellularly only in the small dense granules and in the inclusion bodies. Within the inclusion bodies phosphatase reaction product, was seen only on the dense granular portion of the organelle, presumably derived from the fusion with small dense granules ; no reaction product ever was observed on the filamentous portion of the inclusion body (Figs. 12a, 12b). In some large less-dense droplets, scattered deposits of phosphatase reaction product were found (Fig. 12~). Although, in such instances no small dense granular material was noted, it was quite possible that’ this material

ULTRASTRUCTURE

FIGS.

action not in formatix~ thyroid FIG. ucts in

293

12a,b. Acid phosphatase reaction. The reproducts are seen in the dense substance, but the filsmentous struet,ure (F) or vesIcuiaz (V) of the inclusion body in the normai cell. I ig. 12a, > 36,000; Fig. 12b, X28,000. 12~. Dense acid phosphataae reaction proda large less-dense droplet. X28,000.

294

FIGS.

day for

NAKAI

AND

GORBMAN

13-18. Thyroid follicular cells of a ratfish injected 1 or 3 days and killed on second or fourth day.

intraperitoneally

with

a dose of TSH

10 I.U.

per

FIG. 13. Thyroid follicular epithelial cells after TSH 30 I.U. administration. Note the rough-surfaced endoplasmic reticulum (rER) in the basal cytoplasm of the cell is extremely enlarged compared to controls. BM, basement membrane; END, endothelium of capillary; B, blood cell; FL, follicle lumen. X5,600.

might have been visible in another plane of section. As summarized in Table 2, thiourea treatment produced no changes in mean cell height, or in follicular dimensions in the ratfish thyroid. However, bovine TSH affect,ed both of these parameters. But despite the fact that thiourea did not affect these dimensions, it evoked cytological changes which at the electron microscopic level were similar to those that followed

TSH-treatment. Both of these agents caused an increase in irregularity of shape of the apical border of the epithelial cells. This irregularity was due to an increased number of microvilli and sheet-like projections, which by folding over entrapped bits of colloid (Figs. 13, 14, 15; compare with Figs. 3, 4). TSH treatment provoked a remarkable increase in the volume of rough endoplasmic reticular structures in the thyroid cells.

CHIMAEROID

THYROID

UbTRASTRUCTCP.E

FIG. 14. Large and small rough-surfaced endoplasmic reticuium (rER) plasma membrane. FL, follicle lumen; IB, inclusion hody; NI, mitochondria. x 116,000. FIG. 15. Large less-dense droplet (D) in the wide cytoplasmic projection. dense granule. TRH 30 I.U. administration. X24,000.

295

are found beneath the apical TSH 30 1.11. administraiion. FL,

follicle

lumen;

DG,

small

296

NAKAI

AND

GORBMAN

FIG. 16. Note the small dense gramde (DG) within the large less-dense droplet (D) . Arr JW shows absence of limiting membrane of the dense granule (DG). IB, inclusion body. PM, lateral plasma memxane. TSH 10 I.U. administration. X26,000. FIG. 17. Cross section through the supra-nuclear region of the follicular epithelial cells. A large number of inclusion bodies (IB) are found compared to controls. rER, rough-surfaced endoplasmic reticulum; PM, plasma membrane of thyroid epithelial cells. PM, lateral plasma membrane. TSH 10 I.U. administration. X 16,000.

CHIMAEROID

THYROID

TABLE 2 EFFECT OF THYROTROPIN OR GOITROGEN ox DIMENSIONS OF THYROID FOLLICLES IN RATFISH (measurements averaged for 20 follicles per specimen) Mean cell height Treatment Kane Thiourea, 30 mg TSH, 10 units TSH, 30 units

(4 34.3 33.4 42.6 34.0

F 1.1

& 2.0 + 2.1 i 1.6

Mean ratio: Colloid diametera Cell height 2.3 2.2 3.7 3.7

f. + + +

0.2 0.1 0.5 0.3

a Colloid diameter was calculated by subtracting twice the cell height from the mean diameter of each follicle (mean of longest and shortest diameters).

Particularly in the basal region (Figs. 2, 13) the cisternae of the rough ER were so dilated that at low magnifications this region appeared without structure. The swelling of rough ER was not limited to the basal parts of cells. It was apparent even in the apical region (Figs. 13, 14) where, in control specimens the ER is relatively inThiourea produced a much conspicuous. less developed increase in rough ER than did TSH treatment. A third system of cell structures clearly responsive to TSH treatment were the inclusion bodies. These were strikingly increased in relative number (Fig. 317). TSH treatment also apparently increased the relative number of small dense granules in the Golgi area, although this was not as easily evaluated the changes already mentioned. A certain proportion of nuclei always was found in a condensed hyperchromatic form. TSH increased the proportion of such condensed nuclei (Fig. 2). In radioautographs silver grains were sparsely distribated over the thyroid follicular lumen and no radioautographic grains were seen over any of the intracelluiar granules in the thyroid epithelial cells at 4 $I after lzsI injection. Three days folSowing intraperitoneal injection of lz51, a large number of grains were diffusely distributed over the thyroid follicular lumen.

297

ULTRASTRUCTURE

However, even after this longer time only a few silver grains were seen randomly distributed over the cytoplasm of thyroid epithelial cells. Some grains were localized near the microvilli and few were localized over the less-dense droplets located beneath the apical cytoplasmic membrane (Figs. 19, 20). Although very few grains were seen in the follicular cells of the thyroid gla no localization was ever seen over any the kinds of granules in the basal portion of the cell. From these results, it appears that the site of iodination of thyroglobulin in the thyroid gland of the ratfish is the follicular lumen. It is possible that lessdense droplet labeled with radioiodine might be derived from the follicular colloid. DISCUSSION

Numerous electron microscopic studies have appeared in which an attempt. is made to elucidate the origin of droplets and vesicles in the apical cytoplasm of the thyroid gland (Ekholm and Sjastrand, 1957; Wissig, 1960, 1963; Fujita, 1963, 1966a). The principal problem is whether the large less-dense droplet in the apical cytoplasm of t,hyroid cells represents resorbed colloid from the follicular lumen, or whether it is a secretory droplet derived from t,he Golgi apparat.us. Autoradiographic investigations in mammals have made iiv appear most probable that the large lessdense droplet is not a pre-secretary substance, but rather the result of an absorption process (Kayes et al., 1962; Stein and Gross, 1964; Sheldon et al.? 1965). Furthermore, it is likely that the small less-dense vesicle is a secretory substance derived from the Golgi region (Nadler et al., 19627 1964; Van Heyningen and Nadler, 1965) in higher vertebrates. In ratfish thyroid follicular cells, the large less-dense droplets and small less-dense vesicles found by us appear to be equivalent to similar organelles found by the authors cited above in a variety of other vertebrates (Wissig, 1963; Fujita et al., 1966a, 1966b). The similarit” are in location, forni and density. Althou

FIG. 18. Three inclusion bodies (IB), two of which contain several kinds of small inclusion body contains small dense granules and much fine filamentous structure membrane is incomplete. TSH 10 I.U. administration. X 18,000.

vesicles. and the

The largest surrounding

LYb

NAKAl

FIG _ 19. Electron microscopic radioautograph intrar leritoneally with 232 PC of lzsI and killed 3 of IT over the follicular oolloid and some in the FIG . 20. Sam& animal as in Fig. 19. Note the rinernk crane. X23,000,

ANlJ

WJHJ5MAN

shows a portion of a thyroid follicle of a ratfish injec :ted days after injection. Some of radioautographiclocalizal tion microvillous cytoplasmic processes. X20,000. grain over less-dense droplet beneath the apical plat gma

CHIMAEROID

THYROID

there are various kinds of granules and vesicles in the thyroid follicular cells, they have been placed in three classes according to size, form and electron density in most higher vertebrates (Wissig, 1963) as well as in eels (Fujita et al., 1966b), and lampreys (Fujita et al., 1966a). Thus, the follicular cells of the thyroid gland of the ratfish, in which we have recognized three kinds of granules and vesicles, are similar in this respect. In the thyroid follicular cells of a teleost, Xeriola, Fujita and Machino (1965) reported that only less dense droplets as well as lysosome-like bodies containing small vesicles and membranous structures were found. In Seriola dense granules were not observed except, perhaps, in the form of inclusion bodies in lysosomes. The three vesicular organelle types in ratfish thyroid cells frequently display intermediate or transitional forms. However, these intermediates are in some instances formed by fusions, apparently, since at the ultrastructural level certain primary or typical features still can be distinguished. As Lo electron microscopic observations of inclusion bodies and crystals in the thyroid follieular cells: several reports have been published in rats and chicks (Yoshimura and Irie, 1961), in a teleost fish, Seriola (Fujita and Machino, 1965)) in tadpoles and toads (Coleman et al., 1968) and in senile rats (Youson and Van Heyningen, 1968). Yoshimura and Irie (1961) described crystalloid structures in normal and stimulated rat and chicken thyroid cells, and they considered that the crystalloid might be derived from the colloid droplet, because of its affinity for periodic acid Schiff stain and because they increased in number and size after TSH-stimulation. In Seriola thyroid, Pujita and Machino (1965) observed that the inclusion bodies contain aggregates of numerous fine filaments and crystals consisting of groups of thick needle-like fib& ; they suggested that the structures are older or altered colloid droplets. Recently Yousen and Van IIeyningen (1968) reported that crystalline thyroid inclusions were present only in. the thyroid follicular cells of old rats and suggested that they might be derived from the dense granules, because of

ULTRASTRUCTURE

299

morphological similarit,ies between crystals and dense granules. In all the thyroid follicular cells of ratfishes we studied inclusion bodies of this kind were found, but they were relatively fewer in young than in adult ratfishes. The ratfish inclusion bodies presented some variation in structure and content (filaments, vesicles, dense masses) ? and it seems to us that, they form a complete series. This series begins development with apparent ingestion of a eolloid droplet into the cytoplasm. Progressing stages in this series can be judged in part by position in the cell and the demonstration of acid phosphatase reaction product. Infrequently, we observed small dense granules and large less-dense droplets in close contact with each other, and in some instances they were fused and mutually incorporated. Examples of fusion of these two types of granules and. droplets were found most frequently in the ratfish thyroid glands stimulated with TSH or thiourea. As suggest’ed previously (Seljelid, 1965; Wetzel et al., 1965), t’he rapidity of lysosomal incorporation int,o the thyroid colloid droplet, which appears to occur in several species, may be too rapid in ratfish thyroid cells to provide more than the relatively rare figures that we have illustrated here in fixed tissues (Fig. 16). Generally, the colloid droplets containing no filaments (presumably newly formed ones) were found beneath the apical plasma membrane and inclusion bodies, especially t’he figures representing apparemly recent fusion were also near the apical surface. In organelles in which dense granules were just fusing with large less-dense droplets no Elaments were observed. In contrast, small dense granules are numerous in the Golgi region and in the apical cytoplasm of the follicular cells. This distribution suggests that the small dense granules (lysosomes) are synthesized in the Golgi apparatus aad that they directly move to the region beneath the apical plasma membrane. 41 would appear that the colloid droplets in this region is fused wifh the lgsosome-type small-dense granule thus introducing enzymes and acid phosphatase reactivity into the droplet. The large less-dense droplet thus formed, in which a small amount of

300

NAKAI

AND

acid phosphatase reaction product is diffusely distributed, has neither filamentous nor vesicular structure. Presumably it is subject to action of the hydrolytic enzymes just transferred from the dense granule. Most of the inclusion bodies in the thyroid follicular cell contain a discrete segregated dense mass which consists of numerous fine particles and, in addition, filamentous or vesicular structures. By use of electron microscopic cytochemical techniques we found that the acid phosphatase reaction product is localized only on the dense mass, but no reaction product is found on the filamentous and vesicular structures in the inclusion bodies. Possibly, the vesicular structures are formed temporarily during the hydrolytic process in the colloid droplet and with time they disappear from the inclusion bodies. From this study the inclusion body may be considered an altered colloid droplet whose properties and structure are derived from a hydrolytic process and other reactions to fusion with the lysosome small dense bodies. Cytochemical studies characterizing hydrolysis of thyroid colloid have been made by Wollman and Burstone (1964)) Novikoff (1963) and Seljelid (1965, 1967). Wollman and Burstone (1964) observed the acid phosphatase reaction product in intracellular granules and colloid droplets of the rat, and suggested t,hat the intracellular colloid droplets might be the site of thyroxine release. Recently Wetzel et al. (1965) and Seljelid (1965, 1967) using electron microscopic techniques reported that the colloid droplets of the rat which appear initially in the apical cytoplasm, contain no phosphatase reaction product. They suggested that as colloid droplets lacking reaction product move from the apical cytoplasm toward the base of the rat thyroid cell, they acquire acid phosphatase. Wetzel et al. (1965) and Seljelid (1965, 1967) have illustrated instances of dense granule-colloid droplet fusion and they have made the point that this phenomenon is rarely observed in the follicle cells of rats; thus, the relative infrequency of observed granular fusion in this study seems to be approximately equivalent. Yoshimura and Irie (1961) have de-

GORBMAN

scribed crystalloids in the normal and stimulated thyroid cells of rats and chicken, and recently Yousen and Van Heyningen (1968) have emphasized the relationship between dense granules and the crystals in the senile rat thyroids. The crystalloids found in all the follicular cells of ratfish thyroid glands studied by us consist of bundles of numerous long wavy filaments surrounded by an incomplete limiting membrane; generally they are located in the deeper regions of the follicular cells. Since all intermediate phases can be found, it can be supposed that they are final products of the inclusion bodies after completion of the enzymatic processes which enable the colloid droplets to release thyroid hormone into the cytoplasm. The crystalloid thus may be considered to be a digestive or other reaction product resulting from interaction with the lysosomes, but their chemical properties and their fate are unknown. The origin of the large less-dense cytoplasmic droplet from the colloid has been disputed by many investigators, and several inconclusive earlier radioautographic studies have been made using rat thyroid (Nadler et al., 1962; Kayes et al., 1962; and Ibrahim and Budd, 1965). Stein and Gross (1964) were able to show by means of appropriate radioiodine experiments that the intracellular colloid droplets contain radioactivity that originated from the luminal colloid. Sheldon et al. (1965) similarly, in suppressed and thyrotropin-stimulated mouse thyroid glands, provided evidence that the large cytoplasmic droplets are derived from the luminal colloid. By use of 3H-leucine radioautography Nadler et al. (1962) had reached a similar conclusion. The radioautographic evidence that we have from the ratfish thyroid gland indicates that the first binding of radioactive iodine in organic form takes place in the colloid, not in the intracellular large less-dense droplets. It may be concluded that in the ratfish thyroid gland the site of iodination of thyroglobulin is in the follicular lumen and that the large less-dense droplets are derived from rather than contribute to the follicular colloid. In comparison with the 125I radioautography of the higher vertebrate thy-

CHIMAEROID

THYROID

raids the radioautographic grains in the ratfish thyroid gland are relatively scarce. This may indicate a relatively slower turnover of the thyroglobulin. In these experiments there was a thyroid cytological response to both beef TSH and to the goitrogen, thiourea. The pattern of the response was simiIar, so it may be assumed that the thiourea treatment evoked secretion of endogenous TSH. If this assumption is correct, it suggests that these halocephalian fishes have the same type of thyroxine-feedback mechanism regulating TSH release as do higher vertebrates. The cytological response that we observed is a complex one, involving increased resorptive activity at the cell apex, greatly increased formation of inclusion body org,anelles (Pigs. 13, 17) and a striking increase in volume of rough endoplasmic reticulum (Fig. 13). It would appear, therefore, that TSH activation of ratfish thyroid cells involves stimulation of a basic and extensive series of enzyme controlled mechanisms, probably involving new protein synthesis. REFERENCES ALTTNER, II. (1964). Vergleichende Untersuchungen iiber Cytologie and Vascularization des Saccus Vasculosus der Chondrichthyes. Z. Zelljorsch. 64:

570692.

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