Transformation of the endostyle of the anadromous sea lamprey, Petromyzon marinus L., during metamorphosis

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

30,

243-257 (1976)

Transformation of the Endostyle of the Anadromous Sea Lamprey, Petromyzon marinus L., during Metamorphosis I.

Light

Microscopy

and Autoradiography

GLENDA M. WRIGHT ANDJOHN

withl*V

H. YOUSON

Department of Zoology, Scarborough College, University of Toronto, West Hill, Ontario, Canada MIC lA4 Accepted April 19, 1976 Routine light microscopy and autoradiogtaphy with izsI were used to examine the transformation in structure and iodine-binding capacity of the endostyle throughout (stages 1 to 4) metamorphosis in the anadromous sea lamprey, Petromyzon marinus L. The endostyle began to transform prior to the first external signs of metamorphosis (prometamorphosis) and was already replaced by thyroid follicles by the end of stage 2 of metamorphosis. Many of these follicles appeared to have formed as a result of proliferation of the epithelia (type II and V cells) at the angle of the original lumina of the endostyle. However, it is possible that all cell types of the ammocoete endostyle, with the exception of the type I cells, may be involved in the formation of at least some follicles. The colloid of the newly formed follicles was not stainable with periodic acid-Schiff, suggesting that the thyroglobulin which is synthesized or released by metamorphosing lamprey may have different chemical properties than thyroglobulin of the adult. Large numbers of pigmented granules within some of the remaining epithelial cells may reflect their involvement in the storage of thyroid hormone or in its release during metamorphosis. rZ51 was bound within the transforming endostyle and its replacement throughout metamorphosis, but the localization of the radioiodine varied during the reconstruction. During prometamorphosis and early stage 1 the distribution of iodine resembled that of ammocoetes. In subsequent stages, at first not all follicles demonstrated the ability to bind iodine (end of stage 2), but there was a progressive increase in binding to the end of the metamorphic period, It is suggested that the binding of iodine in the newly formed follicles reflects the involvement of cells of the ammocoete endostyle. There also appears to be a variable differentiation of cells within the developing follicles.

The larval lamprey (ammocoete) does not possess a typical vertebrate thyroid gland but instead has an endostyle (subpharyngeal gland) which has long been considered to function in a similar manner to the thyroid gland (Miiller, 1873), mainly because of its ability to metabolize and store iodine and to synthesize thyroid hormones (Gorbman and Creaser, 1942; Olivereau, 1955; Leloup, 1955; Barrington and Franchi, 1956; Clements-Merlini, 1960a; Fujita 1 This work is a portion of a thesis to be submitted to the University of Toronto in partial fulfillment for the degree of Doctor of Philosophy.

and Honma, 1969). Early microscopists were able to establish that during metamorphosis of the larvae the endostyle undergoes a morphological transformation to produce a thyroid gland in the adult (Schneider, 1879). Marine (1913) described in a series of metamorphosing stages of the landlocked sea lamprey, Petromyzon marinus, five morphologically distinct endostylar epithelial cell types (I to V) and the formation of the follicles of the adult mainly from epithelial types IV and some type II cells of the original ammocoete endostyle. These observations were supported by subsequent studies on other species of lamprey 243

Copyright All rights

@ 1976 by Academic Press, inc. of reproduction in any form reserved.

244

WRIGHT

AND

(Kraentzel, 1933; Leach, 1939), but Sterba (1953) felt that type III cells made a major contribution to the formation of future follicles of the adult thyroid in Petromyzon planer-i. Therefore, there is no definite agreement as to which cells of the endostyle actually contribute to the development of the adult thyroid gland. In addition, there is no evidence as to when the transformation of the endostyle is initiated in metamorphosis and, although the cells appear to bind iodine at some time during metamorphosis (Clements-Merlini, 1960b), it is not known whether this ability is prevalent throughout the entire phase of metamorphosis. A number of these earlier studies on the transformation of the endostyle have been limited by small numbers of animals available for examination and by the absence of a clearly defined method of staging the animals at the various phases of metamorphosis. However, metamorphosis in the landlocked sea lamprey, P. marinus, has recently been divided into stages 1 to 4 based on the external morphological development (Manion and Stauffer, 1970; Beamish and Potter, 1972). In addition, our research group has been able to capture large numbers of the anadromous form of this species during metamorphosis. The timing of the period of metamorphosis in the anadromous sea lamprey is well synchronized with the landlocked species (Beamish and Potter, 1975) and staging is comparable (unpublished data). Therefore, we have taken the opportunity to reexamine the transformation of the endostyle during metamorphosis in the lamprey. The present report describes the point of initiation of transformation in the endostyle and the morphological events of the transformation of the endostyle to adult thyroid follicles. With the use of radioiodine and autoradiography, attempts are made to establish whether the transforming endostyle retains the ability to metabolize and bind iodine throughout the entire period of

YOUSON

metamorphosis and whether the radioiodine can be used as a useful marker in establishing which cells of the endostyle contribute to the formation of the thyroid follicles of the adult. MATERIALS

AND METHODS

Metamorphosing and larval (ammocoete) stages of anadromous sea lamprey, P. mnrinus L., were obtained in August, 1974 and July, 1975 by means of an electrical-shocking device (Tibbles, 1961) from Dennis stream near St. Stephen, New Brunswick. The lengths and weights of the animals were determined and the ammocoetes were classified as to their age (Beamish and Potter, 1975) and the metamorphosing animals as to their stage of external development (Manion and Stauffer, 1970). A third group of animals collected in early July, 1975 at the onset of metamorphosis (Beamish and Potter, 1975) were considered as representing animals during prometamorphosis (unpublished data). Transforming animals collected in later July, 1975 were all in the stages 1 and 2 of metamorphosis and some were permitted to reach more advanced stages before they were used. A few of the animals were used immediately after their collection and transport to the Huntsman Marine Laboratory at St. Andrews, New Brunswick. The majority were transported to the laboratory at Scarborough College, University of Toronto, and were maintained at 20 2 1” in glass tanks containing aerated, dechlorinated tap water and a few inches of river siit. A total of 48 ammocoetes, ranging in size from 9.2 to 14.1 cm (at least 4 or 5 yr old) were used for a preliminary autoradiographic study in order to determine the appropriate dosage and time interval necessary for the appearance of enough labeled iodine in the endostyle to identify the known iodine-binding cells. IZsI (NalZSI carrier free, New England Nuclear) in isotonic saline was injected intraperitoneally into ammocoetes in dosages ranging from 0.5 to 10.0 $3/g body weight. The animals were sacrificed at intervals from 1 to 6 hr and at 24 hr intervals up to 72 hr. Immersion of ammocoetes of similar size in dechlorinated tap water containing 0.5 &i 1Z51/ml of water for various time intervals between 24 and % hr was also included in the preliminary investigation. As a result of these investigations, the most appropriate dosage and time interval was then used in a study of the metamorphosing sea lamprey. Twenty-two animals, ranging in size from 11.1 to 14.1 cm, and representing the 4 stages of metamorphosis, a prometamorphic stage, and an immature adult (macrophthalmia) stage, each received an intraperitoneal injection of 0.5 &i/g body weight of 12*16 hr before their sacrifice. All animals were anaesthetized in a solution of tricaine methanesulfonate

(MS-222) according to Thorson (1959) and a portion of the pharynx containing the endostyle or transforming endostyle was fixed in Bouin’s fluid for at least 24 hr, dehydrated in a graded series of ethanol, cleared in Terpineol, and embedded in Tissue-Prep (Fisher Scientific). The tissues were then serially sectioned at a thickness of 7 pm, mounted on acid-cleaned glass slides (Baserga and Malamud, 1969), hydrated, oxidized with periodic acid according to Sawicki and Rowinski (1%9), rinsed in distilled water, air dried, and dip-coated using Kodak NT-B2 Nuclear Track Emulsion according to the method of Kopriwa and Leblond (1%2). The sections were exposed to the emulsion for a period of 1 week, developed in Kodak D19, rinsed in distilled water, and fixed in Kodak fixer. The developed slides were post-stained with Schilf’s reagent (prepared according to Lillie, 1951), counterstained with Mayer’s acid haemalum (modified by Lillie, 1942), and mounted in Permount. Sections from the endostyle of ammocoetes which had received similar injections of rz51 were prepared for autoradiography in the same manner. A total of 7 uninjected ammocoetes and 42 uninjected prometamorphic and metamorphosing lamprey were utilized for routine light microscopy. Serial sections of a thickness of 10&m were made from the pharyngeal region of these animals and were prepared for observation in the light microscope as described above. Several of these slides were also dip-coated with emulsion, exposed, and developed to serve as controls for the autoradiographic procedure.

RESULTS Ammocoete

Endostyle

The general organization and structure of the endostyle in ammocoetes of anadromous sea lamprey resembled previous descriptions of this species (Marine, 1913; Clements-Merlini, l%Oa) and other species (Leach, 1939; Sterba, 1953; Barrington and Franchi, 1956) of lamprey. The endostyle of the ammocoete extended beneath the pharynx from the first to fifth gill arches, and was open to the pharynx by a slit-like duct situated approximately at the level of the third gill arch. Anterior to the duct the consisted of two straight endostyle epithelial-lined tubes (Fig. 1) and posteriorly the tubes were divided into two straight lateral chambers and a medial chamber that coiled posteriorly to the duct. The two straight lateral chambers extended

posteriorly beyond the coil to the end of the gland. The lumina of the tubes of the endostyle were lined with five epithelial cell types, as previously described (Marine, 1913), although the distribution of these cell types varied throughout the various regions. Wedge-shaped, type I cells formed four large fan-shaped glandular tracts (two dorsal and two ventral) in each tube (Fig. 2). The apical processes of these cells terminated at the glandular opening extending into the endostylar lumen. These cell types did not demonstrate the capacity to bind radioiodine (Fig. 2). Tall, ciliated columnar, type II cells were situated adjacent to the openings of the glandular tracts (Fig. 2) and could be further subdivided into 3 types: IIa, IIb, and IIc (Barrington and Franchi, 1956). Type IIa cells were located below the ventral glandular tract opening, type IIb were between the ventral and dorsal tract opening and had some ability to bind iodine (Fig. 2), and type IIc were just dorsal to the opening of the dorsal glandular tract. Type IIc cells demonstrated the presence of brown granules within their apical cytoplasm and had the ability to bind more iodine than the other type II cells (Fig. 2). Broad ciliated columnar type III cells contained brown granules in their apical cytoplasm and they continued from the type IIc cells over the dorsal glandular tract where they were low columnar and perhaps immature (Barrington and Franchi, 1956). They gradually became tall columnar more dorsally and eventually joined the type IV cells which were situated at the top of the triangular-shaped epithelial ridge. Type III cells were the major cell type involved in the binding of radioiodine (Fig. 2). Type IV cells were low columnar cells with few cilia, and were more closely packed than type III cells. This cell type did not bind iodine and was further characterized by the presence of apical cytoplasmic protrusions into the lumen of the endostyle. The majority of type V cells were low cuboidal and nonciliated when lining the outer wall of the

246

WRIGHT

AND

YOUSON

TRANSFORMATION

OF

endostylar lumen (Fig. 2) but were of columnar shape at the point where they joined the type IIa cells at the angle of the endostylar lumen. These cells were originally classified as type IV by Marine (1913). Leach (1939) classified them as type II, while Clements-Merlini (1960a) referred to them as transitional type II cells. Transforming

Endostyle

The transformation of the endostyle of the ammocoete to the thyroid gland of the adult in anadromous sea lamprey was similar to that of the landlocked species described by Marine (1913) and that of other species of lamprey (Kraentzel, 1933; Leach, 1939; Sterba, 1953). The first signs of transformation in the endostyle appeared in prometamorphic animals when the external features of metamorphosis (Manion and Stauffer, 1970) were not yet evident. This period was marked by a shrinkage of the type I epithelial cells composing the glandular tracts and an overall shrinkage of the endostyle. Concomitant with early changes in the endostyle of prometamorphic animals were changes in the entire pharyngeal region. The area between the ventral portion of the endostyle and the integument was thickened and cartilage was now located beneath the transforming endostyle (Fig. 3). In addition, numerous

1LAMPREY

ENDOSTYLE

247

small blood sinuses were present on either side of the transforming endostyle. Transformation of the endostyle was a progressive event, for animals within each stage of external metamorphosis demonstrated various degrees of change in the endostyle. However, the majority of animals in any one stage showed generally more advanced changes in the endostyle than animals in previous stages of metamorphosis (Figs. 3 and 4). Throughout the length of the endostyle the epithelial cell types II, III, IV, and V became reduced in numbers as the gland decreased in size (Fig. 5). Mitotic figures were seen among the type II ceils and the type V cells joining the type II cells at the angle of the endostylar lumen (Fig. 5). The opening of the ventral glandular tracts was closed by prometamorphosis and this was followed by a closing of the dorsal tract in stage 1 animals. In both prometamorphic and stage 1 animals bound iodine was seen localized throughout the entire gland within the apical cytoplasm and in a region just above the cell apex of cell types IIc, III and V (Fig. 6). The concentration of iodine followed a similar pattern to that found in the ammocoete (Fig. 2). In addition, there was a greater accumulation of brown granules within epithelial cell types IIc and III of the transforming endostyle compared to similar

FIG. 1. A transverse section through the ventral branchial region showing the anterior portion of the ammocoete endostyle (E) consisting of two epithelial lined tubes which are situated beneath the pharynx (P). Epithelial type 1 cells make up the prominent glandular tracts (GT) which are seen within the triangular-shaped epithelial ridge that extends into the endostylar lumen (L) of each tube. Cartilage (C) is located on either side of the gland. x44 FIG. 2. A light microscopic autoradiograph showing the iodine-binding epithelia of the endostyle from an ammocoete sacrificed 6 hr after injection of iZ51. Cell types IIbOb), IIc(Zc), III(3) and V(5) incorporate iZsI. Note the absence of label over the dorsal (Id) and ventral (Iv) cell type of the glandular tract epithelia and type IIa(2a) cells. Endostylar lumen (L), openings to glandular tracts (arrows). x273. FIG. 3. A transverse section through the ventral branchial region of a prometamorphic lamprey showing the anterior portion of the transforming endostyle (E). Note the collapsed appearance of the entire endostyle, the reduction in size of its glandular tracts (CT) and the altered position of the cartilage (C) as compared to Fig. I. x44. FIG. 4. A transverse section through the ventral branchial region of a stage 1 metamorphic lamprey showing the anterior portion of the transforming endostyle (E). Note the extensive reduction in size of the endostyle and its glandular tracts (arrows) and the thickening of the tissue beneath the endostyle. Numerous blood sinuses (S) have formed adjacent to the endostyle and cartilage (C) is now positioned directly beneath it. x56.

248

WRIGHT

AND

YOUSON

TRANSFORMATION

OF LAMPREY

cells of the endostyle of ammocoetes. Some granules larger than the cell nucleus were concentrated in the basal cytoplasm of the type III cells (Figs. 7 and 8). The duct connecting the endostyle with the pharynx was closed by the end of stage 1 of the metamorphic period. Within the coiled region of the transforming endostyle type III cells demonstrated iodine concentrated within their apical cytoplasm and above their cell apex (Fig. 8) and there were also specific regions within the lumen of the endostyle which were heavily labeled (Fig. 7). During stage 1 of metamorphosis there were some cells among the iodine-binding group throughout the entire region that were more active in the accumulation of iodine. The entire cytoplasm of some type V cells were seen to be heavily labeled with radioiodine (Fig. 6). Animals in stage 2 of metamorphosis possessed a greatly reduced endostylar region and this coincided with the development of the tongue muscle (Fig. 9). The developing tongue muscle was first seen as a thickened tissue region above the anterior region of the gland eventually growing over the entire gland. Early in this stage all the endostylar cell types were still recognized but identification became more difficult as the remaining epithelia became more compressed and convoluted and the endostyle lumen disappeared (Fig. 10).

ENDOSTYLE

249

Type I cells were absent by the stage 2 metamorphic period and blood cells were seen in their place. Anteriorly, bound iodine was seen only in small localized regions which seemed to be lumina formed by the epithelia at the angle of the original lumen of the endostyle (Fig. 10). Mitotic figures could still be observed among some of the epithelial cells, especially among type II and V cells at the corner of the original lumen. The coiled region was a mass of disorganized cells, many of which contained brown granules throughout their cytoplasm. Some small follicles, Containing bound iodine within their lumina, were seen among this disorganized cell mass (Fig. 11). Posteriorly, two small follicles present were believed to be remnants of the two original lumina of the endostyle. Some epithelial cell types III could be identified by the presence of brown granules and were believed to comprise some of the surrounding follicular epithelia. These posterior follicles demonstrated little ability to bind iodine and their lumina were frequently filled with cells (Fig. 12). As the transformation of the endostyle progressed during the stage 2 metamorphic period, only a faint outline of the endostylar structure was recognizable. Anteriorly, two masses of cells were seen corresponding to the two anterior tubes of the original ammocoete endostyle. Each cell mass con-

FIG. 5. A transverse section through a portion of the anterior region of the transforming endostyle of a stage 1 metamorphic lamprey showing the presence of a mitotic figure (arrow) among the epithelia at the angle of the endostylar lumen (L). All epithelial cell types have become reduced in number and only a few dorsal (Id) and ventral (Iv) type 1 cells remain. x234. Inset: higher magnification of the mitotic figure (arrow) found at the angle of the endostylar lumen. x687. FIG. 6. A light microscopic autoradiograph of the anterior portion of the transforming endostyle of a stage 1 metamorphic lamprey showing the distribution of bound 125I among the remaining epithelial cell types. Similar to the ammocoete in Fig. 2, type III (3), type II (2) and type V (5) cells all bind izSI. The entire cytoplasm of some type V cells can be seen to be labeled with radioiodine (arrows). Type IV (4) cells and type I (1) cells demonstrate no bound iodine. Blood vessel (BV), endostylar lumen (L). x273. FIG. 7. A light microscopic autoradiograph of the coiled region of the transforming endostyle of a stage 1 metamorphic lamprey showing Iz51 concentration in the epithelial cells and in the lumen (L). Granules (arrows) are present within type III (3) cells. Type II (2) cells, type I (l), type V (5). x273. FIG. 8. A light microscopic autoradiograph of the coiled region of a stage 1 metamorphic lamprey showing type III (3) epithelial cells lining part of the endostylar lumen (L). Large granules (G) are present in the basal portion of these cells and ‘2SI-labeling can be seen at the periphery of the lumen and over the apical cytoplasm of the cells (arrows). x 1120.

250

WRIGHT

AND

YOUSON

TRANSFORMATION

OF LAMPREY

mined one or two follicles and iodine was seen accumulated within their lumina (Fig. 13). However, not all the follicles formed during this period in the transformation of the endostyle demonstrated the presence of bound iodine. The cell masses seen in the anterior region were not present in the midregion, formerly the duct and coil of the ammocoete endostyle. Here there were numerous follicles of various sizes (Fig. 14). The position of some follicles corresponded to areas where the former endostylar lumina had been present. Clumps of cells observed within some follicular lumina demonstrated a positive periodicacid Schiff (PAS) reaction and possessed brown granules within their cytoplasm. Iodine accumulation was also seen within some of these clumps of cells. Follicles possessed cells with brown granules within their cytoplasm and therefore the follicles appeared to be comprised of type IIc and type III epithelial cells. The lumina of the follicles did not contain any colloid which could be demonstrated with PAS, but there was considerably more iodine accumulation in the follicles of the later stage 2 period of metamorphosis than in the earlier stage. In animals of stage 3 of metamorphosis no endostylar structure remained but instead the area of the endostyle had been replaced by a region containing numerous follicles. Anteriorly, the follicles demon-

ENDOSTYLE

2.51

strated iodine accumulation within the center and at the periphery of their lumina (Fig. 15). Cells containing brown granules were located in cell nests between follicles and within the follicular epithelia. Posteriorly, some follicles were very large and contained a heterogeneous mass of dense PAS-positive material, pycnotic nuclei, and brown granules within their lumina. This was reminiscent of the midregion of stage 2 animals and some of this material within the lumina showed the presence of labeled iodine. In general, stage 3 metamorphic animals showed more cellular organization and the presence of more bound iodine within the entire region compared to animals of earlier stages. Thyroid follicles within the animals of stage 4 of metamorphosis and the macrophthalmia stage were much larger and more numerous than in previous stages of metamorphosis. There was less cellular material (cell nests) between follicles and the connective tissue surrounding the follicles was more compact (Fig. 16), yet mitotic figures were still present among some of the follicular epithelial cells. Labeled iodine was observed in the periphery of the follicular lumina and over intensely PAS-positive material within some lumina. The macrophthalmia stage demonstrated iodine accumulation in almost all follicles within the new thyroid region, although the majority of the lumina contained no colloid stainable with PAS.

FIG. 9. The anterior portion of the transforming endostyle of an early stage 2 metamorphic lamprey. The muscle of the tongue (TM) has begun to differentiate and is seen above the reduced endostyle (E). x 124. FIG. 10. A higher magnification of a region similar to E in Fig. 9. This light microscope autoradiograph of an early stage 2 metamorphic lamprey demonstrates that ‘*‘I is less concentrated than in previous stages but is localized within small follicles (arrows) as the epithelial cells become compressed and convoluted and the endostylar lumen is lost. x375. FIG. 11. A light microscopic autoradiograph of the coiled region of the transforming endostyle of an early stage 2 metamorphic lamprey showing several small follicles (arrows) containing bound iZsI in their lumina. Between follicles are disorganized aggregations of cells many of which contain numerous large granules (G). x375. FIG. 12. A light microscopic autoradiograph of the posterior region of the transforming endostyle of an early stage 2 metamorphic lamprey showing two follicles with their lumina Wed with cells (F). Labeling with i2rI is limited. x273.

252

WRIGHT

AND

YOUSON

TRANSFORMATION

OF LAMPREY

DISCUSSION The transformation of the endostyle during metamorphosis in anadromous P. marinus begins prior to the first external signs of metamorphosis as described by Manion and Stauffer (1970) in landlocked P. marinus. During this earliest phase of metamorphosis, the prometamorphic period, there is a simultaneous initiation of transformation in other internal organs (unpublished data). Therefore, it does not seem likely from a morphological standpoint, that the early initiation of transformation in the structure of the endostyle during metamorphosis has any significance to the metamorphic event. It is of interest however, that the process of transformation of the endostyle to thyroid follicles is nearly completed by the end of stage 2 of metamorphosis, while the kidney and alimentary canal, for example, are relatively immature (unpublished data). This may be indicative of the importance of a functioning thyroid to the organism in the latter stages of metamorphosis. The present investigation demonstrates that the transforming endostyle binds radioiodine throughout metamorphosis. However, the distribution of this iodine varies throughout the various stages. In the prometamorphic period and stage 1, the endostyle is shrunken but the epithelial cell types IIc, III, and V are the major iodinebinding cells, as in the endostyle of am-

ENDOSTYLE

253

mocoetes (Barrington and Franchi, 1956; Clement+Merlini, l%Oa). However, these cells appear to have a reduced capacity to bind iodine, a feature which may be related to the diminishing numbers of epithelial cells in the shrinking endostyle. On the other hand, the cells may have a limited ability to bind radioiodine due to the reduction in thyroglobulin synthesis and/or the synthesis of the enzymes responsible for iodine uptake and iodination. An earlier study of the transforming endostyle of one lamprey during early metamorphosis also suggested that iodine uptake was reduced compared to the endostyle of the ammocoete (Leloup and Fontaine, l%O). The reduced capacity for iodine binding also exists in animals of early stage 2 in regions where the cells of the endostyle are compressed and disorganized. However, in these animals the binding of iodine was prevalent in isolated regions where cells had formed small follicles at the angles of the original lumen of the endostyle. A possible explanation for these regional differences in iodine-binding capacity may be that iodination of thyroglobulin-like protein in the endostyle of ammocoetes takes place almost entirely in the region of the apical plasma membrane and in the lumen of the endostyle (Fujita, 1972). Therefore, the loss of the free surfaces of the compressed epithelial cells of the endostyle during metamorphosis may limit these cells from

FIG. 13. A light microscopic autoradiograph of the anterior portion of the transforming endostyle of a late stage 2 metamorphic lamprey. Only a faint outline of the original endostyle structure remains. Bound 9 is seen within the lumina of follicles (arrows) at the angle of the original lumen. x328. FIG. 14. A light microscopic autoradiograph of the midregion of the transforming endostyle of a late stage 2 metamorphic lamprey showing the difference in rz51 incorporation among follicles. Two follicles do not contain label and have cell clumps (C) within their lumina, while the other follicles show intense rz51 accumulation within their luminal centers and periphery (arrows). x351. FIG. 15. A light microscopic autoradiograph of the anterior region of the new thyroid of a stage 3 metamorphic lamprey. Follicles contain bound rz51 within their follicular cells and their lumina (arrows). The tongue muscle (TM) is located above the thyroid. x273. FIG. 16. A light microscopic autoradiograph of the anterior region of the thyroid of a newly metamorphosed lamprey (macrophthalmia). Follicles are larger than in previous stages and are surrounded by a compact connective tissue (CT). lzsI is seen localized around the periphery of all the follicles (arrows). The content of the follicular lumen does not stain with PAS. x351.

254

WRIGHT

AND

YOUSON

iodinating newly synthesized thyroglobustages of metamorphosis. It is of interest lin, while the follicular cells in the newly that the majority of mitotic figures seen in formed follicles possess a free surface and prometamorphic and stage 1 period of have the potential for the iodination of metamorphosis were within the epithelial thyroglobulin. type II and columnar type V cells (Marine, Near the end of stage 2 the disappearance 1913, type IV) at the angle of the original of the endostyle and the accumulation of endostylar lumina, for many thyroid folliradioiodine in numerous follicles reflects a cles seem to be formed at this location. The condition similar to that of the mature origin of the thyroid follicles of lamprey in adult. It is possible that these follicles con- this manner was first suggested by tain type II, type III, and some type V cells Kraentzel (1933). Therefore, the active cell of the ammocoete which retained their abildivision among these and other cell types ity to bind iodine throughout the metamormay be indicative of their involvement in phic event. Not all the follicles at this stage follicle formation. It is worth noting that have this capacity and they may originate Olivereau (1956) observed abundant mitotic from epithelial cells which in the am- figures within the type V and type II epithemocoete had a limited ability to bind iodine. lial cells of the endostyle of ammocoetes However, by the end of metamorphosis al- after administration of thyrotrophic hormost all follicles demonstrated the ability to mone (TSH). In addition, Percy et al. bind iodine and therefore there must be a (1975) have observed “TSH cells” within variable differentiation of these cells within the pituitary of transforming lamprey which the follicles. appeared synthetically active during The increase in the binding of iodine metamorphosis. Although there has been within the transforming endostyle during no direct evidence of the production of metamorphosis of lamprey is in contradicTSH in the lamprey, it could be possible tion to previous observations of Clemthat TSH may play some part in the transents-Merlini (1960b) that cells during formation of the endostyle during metathe initial stage of metamorphosis, though morphosis. disoriented, concentrated more radioiodine In the transforming endostyle the remainthan during follicular formation. A possible ing epithelial cell types IIc and especially reason for this discrepancy could lie in the type III demonstrate larger concentrations fact that a very high dosage of 1311 was of brown pigment granules within their cygiven (20 &i/animal) by Clement+Merlini toplasm compared to these cells in the en(1960b) as compared to an average dosage dostyle of the ammocoete (Marine, 1913; of lzsI Of approximately 1.5 &i/animal in Kraentzel, 1933; Leach, 1939; Sterba, the present investigation. Our preliminary 1953). These granules in the cells of the experimentation with ammocoetes revealed ammocoete have been referred to as reprethat the intraperitoneal injection of lzsI in senting “thyroidal granules,” suggesting dosages exceeding 1.0 @i/g body weight that they have a function of hormone stortended to produce autoradiographs with age (Barrington and Franchi, 1956). In eleccombined labeling over iodine-binding and tron microscopic studies of the ammocoete noniodine-binding cells. endostyle the granules have been considThere has been no previous report of any ered as representing “residual bodies” cell proliferation during endostylar trans- (Egeburg, 1965) or phagosomes (Fujita and formation in lamprey, however in this in- Honma, 1968). Therefore, the granules are vestigation mitotic figures were seen within possibly a form of lysosome which may be the transforming endostyle during the involved in either thyroidal biosynthesis or prometamorphic period and throughout all proteolysis preceding hormone release

TRANSFORMATION

OF

(Barrington and Sage, 1972). In addition, many of these granules incorporate lz51 suggesting they might contain iodinated thyroglobulin (Fujita and Honma, 1%9). This function, if attributed to the granules within the cells of the transforming endostyle, would suggest that there may be increased hormone synthesis during the early stages of metamorphosis. Sterba (1953) noted that these granules persisted during metamorphosis and eventually disappeared as follicular colloid was formed. Although the thyroid follicles of the adult lamprey occasionally possess a heterogeneous material within their lumina (Fujita and Honma, 1966), it is not present to the extent demonstrated in the newly formed follicles during metamorphosis. The clumps of cells present in the lumina of the developing follicles appear to bind iodine and therefore indicate the presence of iodinated thyroglobulin-like protein. However, the radioactivity in these clumps could also have resulted from the diffusion of iodinated material released from the surrounding follicular epithelial cells or the release of stored hormone from the granules present within the cells of the clumps. The follicles of the metamorphosing lamprey contain little colloid which is stainable with PAS, although iodinated protein was present within the lumen of most follicles. In contrast, the adult lamprey has PASpositive colloid in addition to the presence of bound iodine in almost all follicular lumina (Olivereau, 1952). It may be that the thyroglobulin synthesized and secreted by the metamorphosing lamprey may have some difference in its chemical properties from that of the adult. Suzuki and Kondo (1973) have found sedimentation values of 3-5 S and 19 S in thyroglobulin of ammocoetes of Lampetra reissneri. The proportion of 19 to 3-5 S thyroglobulin increases during development of the ammocoetes, but the 3-5 S was still the major thyroglobulin in the larger ammocoetes prior to metamorphosis. In contrast, the

LAMPREY

ENDOSTYLE

255

adult lamprey mainly produces the 19 S thyroglobulin. Therefore, it is likely that during metamorphosis the production of the 3-5 S thyroglobulin by the transforming endostyle decreases and is eventually replaced by 19 S thyroglobulin synthesis. Whether these differences would affect the staining properties of the colloid is not known, for the appearance of a stainable colloid within the follicles of the adult thyroid has been suggested by Leach (1939) to occur simultaneously with the onset of feeding. It would be of interest to establish whether the change in composition of the thyroglobulin molecule occurs during metamorphosis and, if so, when the change occurs. This could prove to be a valuable asset to our knowledge of the metamorphic event of lamprey, in light of our information on the importance of thyroid hormones to amphibian metamorphosis (Etkin, 1968). It could not be definitely established in this study which cells of the ammocoete endostyle contribute to the adult follicles. The early disorganization of the endostyle at transformation makes it difficult to follow the cell types through to follicle formation. Due to the numerous granules within the cells lining the newly formed follicles we support Sterba (1953) and ClementsMerlini (1960b) in suggesting that type III cells of the ammocoete are most certainly involved. However, we do not preclude the possibility that all epithelial cell types, with the exception of type I cells, may take part in the follicle formation (Hoheisel, 1970). Further characterization of this event may be forthcoming after an examination of the transforming endostyle in the electron microscope. ACKNOWLEDGMENTS This investigation was supported by grant No. A5945 from the National Research Council of Canada and a grant from the Canadian National Sportsmen’s Show to J. H. Y. The authors express appreciation to Messrs. E.C. Ooi and S. Zaks who helped in the collection of animals in this study.

256

WRIGHT

AND

REFERENCES Barrington, E. J. W., and Franchi, L. L. (1956). Some cytological characteristics of thyroidal function in the endostyle of the ammocoete larva. Quart. J. Microscop. Sri. 91, 393-409. Barrington, E. J. W., and Sage, M. (1972). The endostyle and thyroid gland. In “The Biology of Lampreys” (M. W. Hardisty and I. C. Potter, eds.), Vol. 2, pp. 105-134. Academic Press, New York. Baserga, R., and Malamud, D. (1969). “Modern Methods in Experimental Pathology. Autoradiography. Techniques and Application.” Harper and Row, New York. Beamish, F. W. H.. and Potter, I. C. (1972). Timing of changes in the blood, morphology, and behavior of Petromyzon marinus during metamorphosis. J. Fish. Res. Bd. Canad. 29, 1277-1282. Beamish, F. W. H., and Potter, I. C. (1975). The biology of the anadromous sea lamprey (Petromyzon marinus L.) in New Brunswick. J. Zool. 177, 57-72. Clement+Merlini, M. (1960a). The secretory cycle of iodoproteins in ammocoetes. I. A radioautographic time study of the subpharyngeal gland. J. Morphol.

106, 337-356.

Clements-Merlini, M. (1960b). The secretory cycle of iodoproteins in ammocoetes. II. A radioautographic study of the transforming larval thyroid gland. J. Morphol. 106, 357-364. Egeburg, J. (1965). Iodine-concentrating cells in the endostyle of ammocoetes. Z. Zellforsch. Mikrosk. Anat. 68, 102-115. Etkin, W. A. (1968). Hormonal control of amphibian metamorphosis. In “Metamorphosis” (W. A. Etkin and L. I. Gilbert, eds.), pp. 313-348. Appleton-Century-Crofts, New York. Fujita, H. (1972). Morphological aspects on the site of iodination of thyroglobulin in the thyroid gland. Arch. Histol. Japan 34, 109-141. Fujita, H., and Honma, Y. (1966). Electron microscopical studies on the thyroid of a cyclostome, Lampetra japonica during its upstream migration. Z. Zellforsch.

Mikrosk.

Anat.

73, 559-575.

Fujita, H., and Honma, Y. (1968). Some observations on the fine structure of the endostyle of larval lampreys, ammocoetes of Lampetra japonica. Gen. Comp. Endocrinol. 11, 111-131. Fujita, H., and Honma, Y. (1969). Iodine metabolism of the endostyle of larval lampreys, ammocoetes of Lampetru juponica. Electron microscopic autoradiography of iz51 Z. Zellforsch. Mikrosk. Anat.

98, 525-537.

Gorbman, A., and Creaser, C. W. (1942). Accumulation of radioactive iodine by the endostyle of larval lampreys and the problem of homology of the thyroid. J. Exp. Zool. 89, 391-401.

YOUSON

Hoheisel, G. (1970). Untersuchungen zur funktionellen Morphologie des Endostyls und der Thyreoidea vom Bachneunauge (Lampetra planeri Bloch). II. Untersuchungen an der Thyreoidea. Gegenbaurs Morph. Jahrb. 114, 337-358. Kopriwa, B., and Leblond. C. P. (1962). Improvements in the coating technique of radioautography. J. Histochem. Cytochem. 10, 269-284. Kraentzel, F. (1933). Contribution a I’etude de la Lamproie Jluviatilis

jluviatile

Morphol.

65, 549-605.

Lampetra

(Petromyzon)

L. 1. La transformation de l’endostyle en glande thyroide. Arch. Biol.. Paris 44, 469517. Leach, W. J. (1939). The endostyle and thyroid gland of the brook lamprey, Zchthyomyzon fossor. J. Leloup, J. (1955). Metabolism de I’iode et fonctionnement endostylaire chez l’ammocoete de Lampetra planeri. J. Physiol. (London) 47, 671-677. Leloup, J., and Fontaine, M. (1960). Iodine metabolism in the lower vertebrates. Ann. N.Y. Acad. Sci. 86, 316-353. Lillie, R. D. (1942). An improved acid hematoxylin formula. Stain Technol. 17, 89. Lillie, R. D. (1951). Simplification of the manufacture of Schiff reagent for use in histochemical procedures. Stain Technol. 26, 163-165. Manion, P. J., and Stauffer, T. M. (1970). Metamorphosis of the landlocked sea lamprey, Petromyzon marinus. J. Fish. Res. Bd. Canad. 27, 1735-1746. Marine, D. (1913). The metamorphosis of the endostyle (thyroid gland) of the ammocoete branchialis (larval land-locked Petromyzon marinus (Jordan) or Petromyzon dorsatus (Wilder). J. Exp. Med. 17, 379-395.

Miller, W. (1873). Uber die Hypobranchialrinne der Tunicaten und deren Vorhandensein bei Amphioxus und den Cyclostomen. Jena. Z. Med. Naturwiss.

7, 327-332.

Olivereau, M. (1952). Etude histologique et autoradiographique de la glande thyroide de la lamproie (Petromyzon marinus marinus L.) Arch. Anat. Microsc. Morphol. Exp. 41, I-10. Olivereau, M. (1955). Presence d’iode lit organiquement dans certaines cellules endostylaires et dans la thyroide. Bull. Ass. Anat. 92, 1113-1132. Olivereau, M. (1956). Endostyle de I’ammocoete (Lampetra planeri Bloch) et hormone thyreotrope. C. R. Ass. Anat. 43, 636-657. Percy, R., Leatherland, J. F., and Beamish, F. W. H. (1975). Structure and ultrastructure of the pituitary gland in the sea lamprey, Petromyzon marinus, at different stages in its life cycle. Cell Tissue Res. 157, 141-164. Sawicki, W., and Rowinski, J. (1969). Periodic acidSchiff reaction combined with quantitative autoradiography of H3-thymidine and S35-sulfate

TRANSFORMATION

OF LAMPREY

labeled epithelial cells of colon. Histochemistry 19, 288-294. Schneider, A. (1879). Anatomic und Entwicklungsgeschichte von Petromzon und Ammocoetes. Beit. verge. Annr. Enrwick. Wilber. G. Reimer, Berlin. Sterba, G. (1953). Die Physiologie und Histogenese der Schilddruse und des Thymus beim Bacheunauge (Lnmpetra planeri Bloch) als Grundlagen phylogenetischer Studien uber die Evolution der innersekretorischen Kiemendarmderivate. Wiss. Z. Friedrich-Schiller Univ. Jena. 2, 239-298.

ENDOSTYLE

257

Suzuki, S., and Kondo, Y. (1973). Thyroidal morphogenesis and biosynthesis of thyroglobulin before and after metamorphosis in the lamprey, Lampetra reissneri. Gen. Comp. Endocrinol. 21, 45 l-460. Thorson, T. B. (1959). Tricaine methanesulfonate (MS-222) as an anaesthetic for sea lamprey, Petromyzon marinus. Copeia 2, 163-165. Tibbles, J. J. (1961). Preparations for lamprey control in Lake Huron. Fish. Res. Bd. Can., Progr. Rep. Biol. Station Technol. Unit, London, Ontario. 2, 32-35.