J. Cell Set. 2, 401-410 (1967) 401 Printed in Great Britain

SEASONAL CHANGES IN THE FINE STRUCTURE OF THE BASAL GRANULAR CELLS OF THE BAT THYROID E.A.NUNEZ Department of Radiology, Cornell University Medical School, New York City R. P. GOULD Department of Anatomy, The Middlesex Hospital Medical School, London D. W. HAMILTON Department of Anatomy, Harvard Medical School, Boston J. S. HAYWARD Department of Zoology, University of Alberta, Edmonton, Canada AND S. J. HOLT The Courtauld Institute of Biochemistry, The Middlesex Hospital Medical School, London

SUMMARY The fine structure of the thyroid gland of non-hibernating, hibernating, and intermittently aroused hibernating bats was examined. It was found that in addition to the ordinary, follicular cell, another widespread thyroid cell type is present in all bats examined. This cell is situated in the basal region of the thyroid follicle and is characterized by a cytoplasm full of secretory- like granules. In the basal cells of bats captured in April and June the granules consist of an extremely dense core and are of a uniform size averaging from 0-1-0-5 /* i*1 diameter. In bats caught in August the solid dense granules vary greatly in size and large granules of diameters from 2 to 5 H are common. These large granules are often found concentrated in groups in the most basal region of the follicular epithelium. Hibernating bats are characterized by partly or totally degranulated basal thyroid cells. The cytoplasmic granules in the partly degranulated cell vary greatly in appearance, ranging from solid dense granules to empty vesicles. In totally degranulated basal cells, empty vesicles fill the cytoplasmic matrix. The granular endoplasmic reticulum of the basal thyroid cell also shows seasonal changes, while the Golgi complex remains a well-developed organelle throughout the year. These observations suggest that the thyroid basal granular cell is involved in secretory activities; its possible functional role is discussed.

INTRODUCTION Animals that undergo hibernation are characterized by seasonal alterations in many physiological functions (Hoffman, 1965). One of the more striking changes occurs in the rate of oxygen consumption, which is elevated during the summer and greatly decreased during hibernation (Erikson, 1956; Popovic, 1955). Since cellular respiration is considered to be under thyroidal regulation (Tata, Ernster & Lindberg, 1962) there have been many investigations of the effect of seasonal changes on the thyroid gland of animals that undergo hibernation (Hoffman, 1965). During hibernation the thyroid gland involutes (Hoffman & Zarrow, 1958) and its capacity to concentrate circulating 402 E. A. Nunez, R. P. Gould, D. W. Hamilton, J. S. Hayward and S. J. Holt radioiodine is abolished (Vidovic & Popovic, 1954). It is therefore surprising that, although numerous electron-microscope studies of the thyroid have been made which have greatly contributed to our knowledge of this gland (Monroe, 1953; Ekholm & Sjostrand, 1957; Wissig, i960; Herman, i960; Irie, i960; Fujita, 1963; Muramoto, 1964; Fujita & Machino, 1965), none has dealt in any detail with the thyroid of hibernating animals. The present investigation is an electron-microscope study of the thyroid gland of bats collected in spring, summer and during hibernation, and after experimental arousal of hibernating animals. The fine structure, occurrence, and seasonal changes of the basal granular thyroid cells are reported and the significance of the findings is discussed.

MATERIAL AND METHODS Adult normal bats of both sexes and of the species Myotis lucifugus (U.S.A.) and Nyctala noctula (England) were used in this study. Non-hibernating bats were cap- tured in their natural habitats in April (Nyctala), June (Myotis) and August (Nyctala). Hibernating bats (Nyctala) were collected in late November from a tree. The animals were killed by ether inhalation and the thyroid glands removed and placed into either Karnovsky's fixative (Karnovsky, 1965) for 6 h, or 6-25 % glutaraldehyde in o-o67M cacodylate buffer, pH 7-3, for 2 h. Following fixation, the tissues were washed in ice-cold 0-25 M sucrose buffered at pH 7-4 with 0-067M cacodylate. Thyroids were also removed from hibernating bats used in a study of the arousal process. The hibernating bats were kept in this state for 8 weeks at 4°-io °C, except that every third to fifth day they were aroused by removal from the cold room and fed mealworms (Tenebrio) to ensure their survival over the experimental period. During the feeding period and immediately after returning to the cold room, the bats exhibited considerable activity for an hour before their return to hibernating body temperature. At the end of the 8th week, the bats were killed, the thyroid glands removed, placed in Karnovsky's fixative for 6 h and then washed in the sucrose/cacodylate buffer. All tissues were post-fixed in phosphate-buffered 1 % osmium tetroxide, pH 7-4, for 1 h and then dehydrated through a graded series of ethanol solutions. The tissues were embedded in either Epon or Araldite. Thin sections were cut with glass or diamond knives on either a Porter-Blum MT-2 or Huxley ultramicrotome, mounted on copper grids and stained with 4% uranyl acetate (Watson, 1958) and/or lead citrate (Reynolds, 1963). The sections were studied in a Philips EM 200 or an AEI EM 6 electron microscope. For light microscopy, 1-2 fi sections were obtained from the embedded material and stained with a 1 % solution of toluidine blue.

RESULTS Two varieties of follicle predominate in the bat thyroid gland. One is composed entirely of follicular cells (Fig. 1), while the other contains, in addition to the ordinary follicular cell, a widespread basal cell characterized by a cytoplasm filled with dense Seasonal changes in bat thyroid 403 granules (Fig. 3). The thyroid follicular cell of the active bat is cuboidal (Figs. 1, 3) and rests upon the follicular basement membrane (Fig. 1). It invariably possesses apical microvilli, which are short, irregular in shape, and unevenly spaced along the surface of the cells (Figs. 1, 2). Very rarely, cilia are seen projecting into the colloid (Fig. 2). The filaments of the cilia appear to arise from the apical region of the cyto- plasmic matrix. Terminal bars are prominent (Figs. 1, 3), and the apposed lateral plasma membranes of adjoining cells are approximately straight (Fig. 2). Nuclei are oval, centrally located, and have regular outlines. Oval to elongate mitochondria are found mainly in the basal half of the cell. They are usually in close contact with the granular endoplasmic reticulum and occasionally appear to be completely enfolded by its cisternae (Figs. 1-3). The baSally located endoplasmic reticulum consists of ribosome-dotted cisternae, characteristically widely dilated and containing a material of low electron density. The Golgi complex is relatively inconspicuous and usually situated in the apical half of the cell (Fig. 2). It consists of several dilated saccules and occasional vesicles. Beneath the apical surface are numerous small vesicles of light- to-medium density (Figs. 1, 2). Dense, lysosome-like bodies are present in the supra- nuclear half of the cell (Figs. 1-3). Colloid droplets are small and not abundant; when present they are found in the apical half of the cell (not shown in micrographs). The basal cell in the non-hibernating bat is an irregularly shaped granular epi- thelial cell, usually larger than the follicular cell. Such cells are usually found grouped together as irregular sheets and masses in the basal portion of the follicular epi- thelium (Fig. 3). They are separated from the luminal colloid by the cytoplasm of the follicular cells, and those most basally situated rest on the follicular basement mem- brane (Figs. 3-8). The salient feature of the basal epithelial cell are the numerous secretory-like granules that fill extensive areas of the cytoplasmic matrix (Figs. 3-8). The granules are characterized by an extremely dense core, sometimes surrounded by an outer light rim, and are individually bounded by a smooth membrane (Fig. 5, inset). They exhibit seasonal changes in size in the non-hibernating bat. In April and June the granules are fairly uniform in size, averaging from o-i to 0-5 ji in diameter (Figs. 3, 4). In the August animals the granules vary greatly in size (Fig. 5) and large, dense granules of 2-5 /i diameter are common (Fig. 6). Often, a group of very large, dense granules fills the entire cytoplasmic matrix of those cells situated in the most basal region of the follicle (Fig. 8). In basal cells of both spring and summer animals there is a well-developed Golgi complex, showing three to five groups of slightly dilated saccules that are often arranged in a roughly circular or horseshoe-like shape (Figs. 4, 5 and 7). In the centre of the Golgi complex are found numerous vesicles, vacuoles, and discrete granules. Some of the vacuoles contain dense material and granules similar in appearance to the granules distributed throughout the cell body (Figs. 4, 7). Long cilium-like structures (possibly centrioles) and multivesicular bodies are associated with the Golgi zone (Fig. 7). Large spherical vacuoles, which often contain flocculent material or small eccentrically placed dense granules are found distributed throughout the cytoplasmic matrix (Figs. 3, 4). The granular endoplasmic reticulum and clusters of free ribosomes are much more abundant in the basal cells of the summer animals (Figs. 5, 6) than in those of the spring animals (Figs. 3, 4). In 26 Cell Sci. 2 404 E. A. Nunez, R. P. Gould, D. W. Hamilton, J. S. Hayward and S. J. Holt the latter the granular endoplasmic reticulum consists mostly of numerous individual rod-like profiles. On the other hand, the prominent granular endoplasmic reticulum in the basal cell of the August animals consists of long tubular channels which are mostly arranged in parallel stacks or whorls of concentric lamellae (Figs. 5, 6). The whorls occasionally enclose a centrally located mass of cytoplasm that contains several large dense granules. The mitochondria are round to elongate and display no parti- cular orientation or concentration in the cell (Figs. 3, 5). Irregularly shaped nuclei) often with deeply indented nuclear membranes, are eccentrically located in the cell (Fig. 3). The lateral plasma membranes show a great deal of contortion and compli- cated interdigitations (Figs. 3, 4). During hibernation the fine structure of the*basal granular cells shows further changes. They are either partly or totally degranulated, which results in their having a lighter cytoplasmic texture than that of the basal granular cell of non-hibernating bats (Figs. 9, 10). The granules of the partly degranulated basal cell vary strikingly in appearance, ranging from solid dense granules to empty vesicles (Fig. 9). Between these two extremes there are various kinds of intermediate forms which range from solid, dense granules with various degrees of electron density to vesicles containing small rod- and membrane-like structures. These might represent steps in the trans- formation of the solid granules into empty vesicles. The solid dense granules are of a uniform size and average from o-i to 0-5 /i in diameter. Large, dense granules similar to those which characterize the basal granular cells of the August bat are not found in the basal cells of hibernating bats. In totally degranulated basal cells the cytoplasmic matrix is filled with vesicles whose diameter appears to be larger than that of the dense granules present in the partly degranulated basal cell (Fig. 10). The vesicles are irregular in shape and contain a fine-granular homogenous material. Vesicles with broken membranes as well as residual fragments of membranes are often found, which may indicate that the vesicles disintegrate. The nuclei of degranulated basal cells are irregular in shape and are centrally located (Fig. 9). The Golgi complex is well-developed, consisting of flattened sacs, vacuoles, and numerous smaller vesicles (Fig. 9). The profiles of the granular endo- plasmic reticulum, which are in the form of short slender rods, are not conspicuous in partly degranulated basal cells (Fig. 9), and are practically absent in totally de- granulated cells (Fig. 10). Clusters of free ribosomes are not conspicuous in degranu- lated basal cells. The mitochondria are similar in shape to those present in the cells of the active bat. The lateral plasma membranes do not show the numerous inter- digitations which characterize the basal granular cells of the spring and summer animals. Intermittent arousal of hibernating bats results in basal granular cells characterized by a cytoplasmic texture that is lighter than that of the basal granular cell of the non- hibernating bat (Figs. 11, 12). The cytoplasmic matrix of these cells, filled with solid dense granules, is devoid of empty vesicles similar to those found in degranulated basal cells. The Golgi complex is not well developed and rough surfaced endoplasmic reticulum is rarely found. Seasonal changes in bat thyroid 405

DISCUSSION The present study demonstrates that in the bat thyroid gland there are two distinct kinds of cell, the follicular and the basal granular cells. The fine structure of the follicular cell of the non-hibernating bat reveals no major differences from the follicular cells of the rat (Wissig, i960) and other mammals (Irie, i960). However, during the hibernating period noticeable changes in the ultrastructure of the follicular cell occur and these changes will be reported in a subsequent communication. Since a second widespread type of cell does not characterize the thyroid gland of other mammals (Wissig, i960; Irie, i960), the presence of large numbers of basal granular cells in the bat thyroid raises the question of the possible role of these cells. Their ultrastructure suggests a secretory function. They have an abundance of dense, secretory-like granules and a well-developed Golgi complex, structures which, in other cells, have been shown to be closely involved in secretory activities (Bencosme & Pease, 1959; Roth & Luse, 1964). The large size of many of the dense granules and the concentration of groups of these in the most basal region of the follicle in the August animal, compared with those in April and June, might be an expression of the final stages in the preparation for such secretory activity. Also, the appearance of degranulated basal thyroid cells in the November bats suggests that the basal cells have secreted their product during the period of hibernation. Although the present evidence cannot permit a conclusion as to the function of the basal granular cell, three possibilities can be envisaged which will be described later on. Concurrent with the changes in the morphology of the dense granules are changes in the granular endo- plasmic reticulum. In non-hibernating bats the granular endoplasmic reticulum is a conspicuous cytoplasmic organeUe, consisting of numerous short, rod-like profiles in the cells of bats captured in April and June, and of long, tubular channels arranged in parallel stacks or whorls in the cells of the August animals. On the other hand, the granular endoplasmic reticulum is inconspicuous in the basal cells of hibernating bats, consisting of short, slender profiles. These seasonal changes in the organelle, con- sidered to be the site of protein synthesis (Nadler, Young, Leblond & Mitmaker, 1964), suggest alterations in protein production, with the highest level occurring during late summer and the lowest level occurring during hibernation. Further studies are to be made of the alteration in the balance of protein synthesis that appears to occur in the basal granular cell during different times of the year. As stated earlier three possibilities can be envisaged as to the functional role of these cells. The first is that they are parathyroid. This possibility can be excluded on the grounds that the parathyroid was found embedded as a distinct entity in the thyroid gland of the bat and its cells are quite distinct in ultrastructure from the basal granular cells. The second possibility is that these cells may be part of the widespread paraganglionic chromafftn system. They do have some resemblance to chromaffin tissue, particularly in the granules, many of which are of the same order of size as adrenalin-noradrenalin granules (Coupland, 1965). Histochemical tests for adrenalin, noradrenalin and other catecholamines will have to be applied before the problem can be resolved, but it might be noted that there is some light-microscopic evidence

26-2 406 E. A. Nunez, R. P. Gould, D. W. Hamilton, J. S. Hayward and S. J. Holt that in the dog thyroid some cells show a high tryptophan content (Pearse, 1966). It is possible that the granules of these basal cells represent 5-hydroxytryptamine. If these granules do contain 5-hydroxytryptophan, then there are two interesting possi- bilities for identification of basal granular cells. The first is that they may be invading mast cells, the second is that they may be argentafEn or enterochromaffin cells ana- logous to those found in the digestive system. The first possibility can almost be eliminated on morphological grounds by comparing the fine structure of known mast cells (Fernando & Movat, 1962; Fujita, 1965) with that of basal granular cells. On morphological grounds the possibility is much more attractive that basal granular cells may be analogous to enterochromaffin cells. The thyroid is a gut derivative and might reasonably be expected to contain similar cell types. The few published micrographs of gut enterochromaffin cells (Greep, 1966; Helander, 1962) show that they contain numerous dense granules. The third possibility is that the basal granular cell represents the endocrine system responsible for the production of thyrocalcitonin. The first evidence that such a hormone existed was presented by Copp, Cameron, Cheney, Davidson & Henze (1962). These authors considered the hormone, which acts by lowering the plasma calcium level, to be of parathyroid origin, and called it calcitonin. However, recent studies have shown that calcitonin is of thyroid origin and it has been renamed thyrocalci- tonin (Foster, Baghdiantz, Kumar, Slack, Soliman & Maclntyre, 1964; Dale, Roth & Garcia, 1965; Hirsch, Gauthier & Munson, 1963; Hirsch, 1964). Several reports have suggested that the thyroid light or parafollicular cell is the source of thyrocalci- tonin (Foster, Maclntyre & Pearse, 1964; Pearse, 1966; Bauer & Teitelbaum, 1966). Although thyroid light cells are rarely found in the epithelium of thyroid follicles, constituting approximately 1 % of all epithelial cells in the thyroid gland of the normal rat (Stux, Thompson, Isler & Leblond, 1961), the possibility exists that the basal granular cell of the bat thyroid might be homologous with the thyroid light cell. This view is based firstly on the resemblance of degranulated basal cells of the hibernating bat to the published micrographs of the light cell of the normal rat (Young & Leblond) 1963). Secondly, the striking similarity of the basal granular cells of intermittently aroused hibernating bats to the light granular cells described in thiouracil-induced rat thyroid tumours suggests that they are derived from the same cell type (Nunez, Money & Becker, 1967). Furthermore, the fact that in the present findings there is an apparent increase in the number of basal granular cells from April to August, that very large dense granules are present at the end of this period, and that the appearance of the cytoplasm of the degranulated cells of the hibernating bat indicates granular discharge at a time when a state of hypocalcaemia exists (Riedesel & Folle, 1954; Riedesel, 1957), are together consistent with the suggestion that the basal granular cells might be the source of thyrocalcitonin in the bat. This possibility is being in- vestigated by studies on the physiological activity of cell fractions containing the dense granules, and by experiments on the effect of raised blood calcium levels on the integrity of the basal granular cell. Finally, the presence of such a widespread granular cell system in the bat thyroid compared to the absence of a similar cellular system in the thyroid glands Seasonal changes in bat thyroid 407 of other mammals suggests that this cell might have a special role in the regu- lation of the hibernation process. Manipulative experiments are planned to test this possibility.

The authors are indebted to Dr David V. Becker, Director, Radioisotope Department, Cornell University Medical School, New York City, for his continued support. They also thank the Fleming Memorial Fund for Medical Research and the Science Research Council for grants for the purchase of electron microscopes and ancillary equipment. The expert technical assis- tance of Mrs Man Roman is gratefully acknowledged. This study was conducted in the Department of Cytochemical Research, Courtauld Institute of Biochemistry, The Middlesex Hospital Medical School, London, while E. A.N. was a recipient of a Public Health Fellowship from the National Institute of Arthritis and Metabolic Diseases, U.S.A. Some of the material for this study was collected while R.P.G. was holder of a Sir Henry Wellcome Travelling Fellowship and a Research Fellow in the Department of Anatomy, Harvard Medical School.

REFERENCES BAUER, W. C. & TEITELBAUM, S. L. (1966). Thyrocalcitonin activity of particulate fractions of the thyroid gland. Lab. Invest. 15, 323-329. BENCOSME, S. A. & PEASE, D. C. (1958). Electron microscopy of the pancreatic islets. Endocrin- ology 63, 1-13. COPP, D. H., CAMERON, E. G., CHENEY, B. A., DAVIDSON, A. G. E. & HENZE, K. G. (1962). Evidence for calcitonin—a new hormone from the parathyroid that lowers serum calcium. Endocrinology 70, 638—649. COUPLAND, R. E. (1965). The Natural History of the Chromaffin Cell. London: Longmans, Green. DALE, D. C, ROTH, S. I. & GARCIA, G. E. (1965). Effect of calcium on parathyroid secretion. Endocrinology 77, 725-729. EKHOLM, R. & SJOSTRAND, S. (i957). The ultrastructural organization of the mouse thyroid gland. J. Ultrastruct. Res. 1, 179-199. ERIKSON, H. (1956). Observations on the metabolism of arctic ground squirrels (CiteUusparryi) at different environmental temperatures. Acta physiol. scand. 36, 66. FERNANDO, N. V. P. & MOVAT, H. Z. (1962). The fine structure of the mast cell. Expl molec. Path. 2, 450-463. FOSTER, G. V., BAGHDIANTZ, A., KUMAR, M. A., SLACK, E., SOLIMAN, H. A. & MACINTYRE, I. (1964). Thyroid origin of calcitonin. Nature, Lond. 202, 1303-1305. FOSTER, G. V., MACINTYRE, I. & PEARSE, A. C. E. (1964). Calcitonin production and the mitochondrion-rich cells of the dog thyroid. Nature, Lond. 203, 1029-1030. FUJITA, H. (1963). Electron microscope studies on the thyroid glands of the domestic fowl, with special reference to the mode of secretion and the occurence of a central flagellum in the follicular cell. Z. Zellforsch. mikrosk. Anat. 60, 615-632. FUJITA, H. & MACHINO, M. (1965). Electron microscope studies on the thyroid gland of a teleost, Seriola quinqueradiata. Anat. Rec. 152, 81-97. FUJITA, T. (1965). Uber die Granula der Gewebmastzellen der Ratte. Z. Zellforsch. mikrosk. Anat. 66, 66-82. GREEP, R. O. (1966). Alimentary tract. In Histology, p. 516. New York: McGraw-Hill. HELANDER, H. F. (1962). Ultrastructure of fundus glands of mouse gastric mucosa. J. Ultra- struct. Res. (Suppl.), 4, 65-123. HERMAN, L. (i960). An electron microscope study of the salamander thyroid during hormonal stimulation. .7- biophys. biochem. Cytol. 7, 143-150. HIRSCH, R. F. (1964). Thyrocalcitonin: Hypocalcaemic hypophosphatemic principle of the thyroid gland. Science, N.Y. 146, 412-413. HIRSCH, R. F., GAUTHIER, G. F. & MUNSON, P. L. (1963). Thyroid hypocalcaemic principle and recurrent laryngeal nerve injury as factors affecting the response to parathyroidectomy in rats. Endocrinology 13, 244-252. 408 E. A. Nunez, R. P. Gould, D. W. Hamilton, J. S. Hayward and S. J. Holt HOFFMAN, R. A. (1965). Terrestrial animals in cold: hibernators. Adaption to the environment. In Handbook of Physiology, Section 4, 379-403. Am. Physiol. Soc. HOFFMAN, R. A. & ZARROW, M. X. (1958). A comparison of seasonal changes and the effect of cold on the thyroid gland of the male rat and ground squirrel (Citellus tridecimlineatus). Acta endocr., Copenh. 27, 77-84. IRIE, M. (i960). Electron microscope observations on the various mammalian thyroid glands. Archvm histol. jap. 19, 39. KARNOVSKY, M. J. (1965). A formaldehyde/glutaraldehyde fixative of high osmolality for use in electron microscopy, j. Cell Biol. 27, 137A-138A. MONROE, B. G. (1953). Electron microscopy of the thyroid. Anat. Rec. 116, 345-361. MUROMOTO, K. (1964). Comparative electron microscope studies of the thyroid gland of lower vertebrates. Okajimas Folio anat. jap. 39, 321-343. NADLER, N. J., YOUNG, B. A., LEBLOND, C. P. & MITMAKER, B. (1964). Elaboration of thyro- globulin in the thyroid follicle. Endocrinology 74, 333-354. NUNEZ, E. A., MONEY, W. & BECKER, D. V. (1967). The fine structure of thiouracil-induced transplantable thyroid tumours in non-inbred, intact rats. Endocrinology (in the Press). PEARSE, A. G. E. (1966). The cytochemistry of the thyroid C cells and their relationship to calcitonin. Proc. R. Soc. B 164, 478-487. POPOVIC, V. (1955). R61e de la grande thyroide dans le sommeil hibernal. Arh. biol. Nauka 7, 25-37- REYNOLDS, F. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in elec- tron microscopy. J. Cell Biol. 17, 208-213. RIEDESEL, M. L. (1957). Serum magnesium levels in mammalian hibernation. Trans. Kansas Acad. Sci. 60, 99-141. RIEDESEL, M. L. & FOLLE, C. F. Jr. (1954). Serum magnesium changes in hibernation. Am. J. Physiol. 179, 665. ROTH, L. M. & LUSE, S. A. (1964). Fine structure of the neurohypophysis of the opossum (Didelphus virgimana). J. Cell Biol. 20, 459-472. STUX, M., THOMPSON, B., ISLER, H. & LEBLOND, C. P. (1961). The ' Light Cells' of the thyroid gland in the rat. Endocrinology 68, 292-307. TATA, J. R., ERNSTER, L. & LmDBERG, O. (1962). Control of basic metabolic rate by thyroid hormones and cellular function. Nature, Lond. 193, 1058-1060. VIDOVIC, V. L. & POPOVIC, V. (1954). Studies on the adrenal and thyroid glands of the ground squirrel during hibernation. J. Endocr. 11, 125-133. WATSON, M. L. (1958). Staining of tissue sections for electron microscopy with heavy metals. J. biophys. biochem. Cytol. 4, 475-478. WlSSiG, S. L. (i960). The anatomy of secretion in the follicular cells of the thyroid gland. 1. The fine structure of the gland in the normal rat. J. biophys. biochem. Cytol. 7, 419-431. YOUNG, B. A. & LEBLOND, C. P. (1963). The light cell as compared to the follicular cell of the thyroid gland of the rat. Endocrinology 73, 669—686.

{Received 3 December 1966—Revised 4 April 1967) Seasonal changes in bat thyroid 409

ABBREVIATIONS out autophagic vacuole gc granular cell av apical vesicles Ic light cell btn basement membrane m mitochondrion bv blood vessel mv microvilli c colloid mvb multivesicular body cd colloid droplet n nucleus til cilium np nuclear pore cs cell surface P cell process d desmosome r ribosomes db dense body s intercellular space dc dense cell V vesicle er endoplasmic reticulum vac vacuole fc follicular cell to whorl 8 Golgi complex

Figs. 1-10 are electron micrographs of the thyroid glands of non-hibernating and hibernating bats (Nyctala). 4io E. A. Nunez, R. P. Gould, D. W. Hamilton, J. S. Hayward and S. J. Holt

Fig. i. Some follicles of the April bat consist entirely of ordinary follicular cells. Portions of three such follicles in contact with colloid (c) are seen in the micrograph. The upper follicle contains a follicular cell with a pale appearance (Ic). x ioooo. Fig. 2. At a higher magnification the apical regions of the follicular cells have small microvilli (mv), numerous apical vesicles (av), a small Golgi complex (g) and dense bodies (db). When found in this region, mitochondria (m) are associated with dis- tended cisternae of the endoplasmic reticulum (er). Cilia (ct/) are occasionally seen projecting into the colloid (c). The lateral membranes of adjoining cells are approxi- mately straight (arrows), x 30000. Journal of Cell Science, Vol. 2, No. 3

E. A. NUNEZ, R. P. GOULD, D. W. HAMILTON, (Facing p. 410) J. S. HAYWARD AND S. J. HOLT Fig. 3. In addition to lining follicular cells (/c), the second type of follicle found in the April bat thyroid contains numerous basal granular cells (gc). The nuclei (n) of these are round to irregular in shape and often show prominent indentations. Numerous light vacuoles (vac) are present, many of which contain small dark granules. The lateral membranes of these cells show complex interdigitations (arrows). The field also shows a blood vessel (bv), colloid (c) and desmosomes (d). x 7500. Journal of Cell Science, Vol. 2, No. 3

mm,

»•.

E. A. NUNEZ, R. P. GOULD, D. W. HAMILTON, J. S. HAYWARD AND S. J. HOLT Fig. 4. Micrograph showing portions of three basal granular cells (April bat). Well- developed Golgi complexes are evident (g). The granular endoplasmic reticulum consists of short, slender rod-like profiles (er) dotted with ribosomes. Free ribosomes are also scattered throughout the cytoplasm. Arrows point to the lateral membranes which show complex interdigitations. x 23000. Journal of Cell Science, Vol. 2, No. 3

E. A. NUNEZ, R. P GOULD, E. W. HAMILTON, J. S. HAYWARD AND S. J. HOLT Fig. 5. The basal granular cells of August bats contain dense granules that vary greatly in size. Prominent endoplasmic reticulum (er) is present at this time and may form whorls or parallel stacks. A well-developed Golgi complex (j>) can be seen in three of the granular cells. Colloid (c) is present at the upper right of the picture, x 12000. The inset shows a group of granules at higher magnification and reveals the light rim surrounding many of them, x 30000. Journal of Cell Science, Vol. 2, No. 3

5<

E. A. NUNEZ, R. P. GOULD, D. W. HAMILTON, J. S. HAYWARD AND S. J. HOLT Fig. 6. A group of very large dense granules in a basal granular cell (August bat). Stacks of endoplasmic reticulum, heavily dotted with ribosomes, are typically asso- ciated with the larger granules, x 26000. Journal of Cell Science, Vol. 2, No. 3

E. A. NUNEZ, R. P. GOULD, D. W. HAMILTON, J. S. HAYWARD AND S. J. HOLT Fig. 7. The Golgi complex of the basal granular cell is well developed in all non- hibernating bats examined. In sections it is seen as three to five groups of flattened saccules with slightly dilated ends (g). Numerous small vesicles (D), clear vacuoles and granules with contents of variable density (arrows) are also present in this zone. The field also shows a multivesicular body (vwb) and a cilium-like structure («/). x 40000. Fig. 8. Groups of very large dense granules, as shown here, are occasionally seen in the most basal regions of the thyroid follicles of August bats, and appear to fill the entire cytoplasm of the cell. Colloid (c), follicular cells (/c), the perifollicular space (s), a granular cell (gc) and an adjacent blood vessel (bv) are also present in this field. Journal of Cell Science, Vol. 2, No. 3

E. A. NUNEZ, R. P. GOULD, D. W. HAMILTON, J. S. HAYWARD AND S. J. HOLT Fig. 9. Electron micrograph showing several basal degranulated cells of the thyroid gland of a hibernating bat (November). The cytoplasm appears lighter than that of the basal cells of non-hibernating animals. Many of the granules contain dark, rod-like structures (single arrow) and vesicles (double arrow), x 20000. Journal of Cell Science, Vol. 2, No. 3

E. A. NUNEZ, R. P. GOULD, D. W. HAMILTON, J. S. HAYWARD AND S. J. HOLT Fig. 10. Electron micrograph showing totally degranulated basal cell. The vesicles (u) that fillth e cytoplasmic matrix are irregular in shape and often have broken membranes (v,). The region between membrane fragments is very light (arrows), x 20000. Figs, n, 12. Electron micrographs of basal granular cells of intermittently aroused hibernating bats. These micrographs show that the basal granular cells appear very light compared with the adjacent follicular cells (Jc), and contain dense, secretory-like granules. Fig. 11, x 7500; Fig. 12, x 17000. Journal of Cell Science, Vol. 2, No. 3

E. A. NUNEZ, R. P. GOULD, D. W. HAMILTON, J. S. HAYWARD AND S. J. HOLT