J. Cell Sci. 6, 821-841 (1970) 821 Printed in Great Britain

SEASONAL CHANGES IN SECRETORY GRANULES AND CRYSTALLOID INCLUSIONS OF BAT PARAFOLLICULAR CELLS

E. A.NUNEZ Department of Radiology, Cornell University Medical School, New York, Neiv York 10021, U.S.A. R. P. GOULD Department of Anatomy, Middlesex Hospital Medical School, London, W.\, England AND S. J. HOLT Conrtauld Institute of Biochemistry, Middlesex Hospital Medical School, London, W. 1, England

SUMMARY Parafollicular cells of active Autumn bats sometimes contain many large dense secretory granules (diameter 1—2 /*m) enclosed within ribosome-studded membranes. At the same time, parafollicular cells containing small granules were often characterized by the presence of slender crystalloid rods, up to several microns in length. Parafollicular cells with large granules become depleted of them following exposure of non- hibernating bats to cold (4 °C) for 12-48 h. This treatment causes no change in the cells con- taining small granules, although partial degranulation in such cells was seen in early hibernating (November) bats. On the basis of these and previous studies it is suggested that the two types of granules in bat parafollicular cells have different fates during the animal's annual cycle of physiological activity.

INTRODUCTION Interest has recently been renewed in the parafollicular cells of the thyroid gland because of increasing evidence that they are responsible for the production of calci- tonin (Foster, Maclntyre & Pearse, 1964; Pearse, 1966/1; Bauer & Teitelbaum, 1966; Bussolati & Pearse, 1967; Carvalheira & Pearse, 1967; Matsuzawa & Kurosumi, 1967; Young, Care & Duncan, 1968; Capen & Young, 1967; Ericson, 1968), the hormone which lowers plasma calcium (Foster et al. 1964; Hirsch, Gauthier & Munson, 1963). Since the level of blood calcium is known to undergo seasonal alterations in hibernators (Riedesel & Folk, 1954; Biorck, Johansson & Veige, 1956; Riedesel, 1957; Azzali, 1968), we have been examining the fine structure of the parafollicular cell in active and hibernating bats in an effort to determine possible relationships between parafollicular cells and hibernation. These studies have revealed that the ultrastructure of the parafollicular cells of bats caught in Spring, in early Summer, and during part of the cycle of hibernation (Nunez et al. 1967; Nunez, Gould & Holt, 1969) 822 E. A. Nunez, R. P. Gould and S. J. Holt is similar to that described for the rat (Ekholm & Ericson, 1968), cow (Capen & Young, 1967), pig (Young et al. 1968), dog (Tashiro, 1964), opossum (Azzali, 1966) and the tree shrew (Pearse, 1968). These cells contain numerous small dense granules which occupy a large part of the cell. They have an average diameter of about 0-2 /im and possess a smooth limiting membrane. Bats caught in late Summer, however, have some parafollicular cells which contain large dense granules (1-5 /un) bounded by ribosome- studded membranes (Nunez, Gould & Holt, 1968), and since these large granules may be functionally important during the onset of hibernation we have now extended our observations to bats caught during the Autumn. In addition, we have placed active, non-hibernating bats (caught in late October) into a cold environment for 12 to 48 h so that any changes which occur in the initial stages of hibernation could be studied. The results of this study, taken together with our earlier observations (Nunez et al. 1967, 1968, 1969), suggest that the two types of secretory granules present in bat thyroid parafollicular cells have different fates during the annual cycle of physiological activity.

MATERIAL AND METHODS The thyroid glands of adult bats of the species Myotis Iticifugus and Eptesicus serotimis were used for this study. Active, non-hibernating bats were caught in their natural habitats in mid- September and in late October. Some of the active October animals were kept in the re- frigerator (4 °C) for 12, 24 and 48 h. Several early hibernating bats (Eptesicus) from a group collected in the previous mid-November were also studied. All animals were anaesthetized with ether or by an injection of chloral hydrate. The whole thyroid glands were removed and fixed for 2 h in 6-25 % glutaraldehyde in 0-067 M cacodylate buffer, pH 7-3, or for 6 h in Karnovsky's cacodylate-buffered formalin/glutaraldehyde mixture (Karnovsky, 1965). After washing in cacodylate buffer, the tissues were post-fixed in phosphate- buffered 1 % osmium tetroxide, pH 7-4, for 1-2 h. Tissue blocks were dehydrated and embedded in Epon. Ultrathin sections were stained with a 4 % aqueous uranyl acetate solution (Watson, 1958) and/or lead citrate (Reynolds, 1963) and examined in a Philips EM 200 electron microscope.

OBSERVATIONS Some of the parafollicular cells in the active, non-hibernating bats captured during Autumn contain the large dense granules enclosed in ribosome-dotted membranes, previously described in detail in August bats (Nunez et al. 1968). These cells are rare and are usually found together with the more numerous parafollicular cells character- ized by the presence of many small dense granules (Figs. 2, 3, 9). We estimate that less than 3% of bat parafollicular cells contain large granules. In September, the parafollicular cells with large granules possessed them in groups of 2-4, which were usually situated near the basement membrane (Fig. 2). The largest granules were in most cases round and measured about 1-5 /tm in diameter. The other type of parafollicular cell with large numbers of small dense granules occasionally showed oval to round structures approximately 0-4 /im in size and of moderate density (Fig. 2). At this time, the plasma membranes of adjacent parafollicular cells show complex interdigitations (Fig. 2). Parafollicular cell large granules 823 Structural differences were noted in parafollicular cells of bats caught in late October. Firstly, more large granules were seen in these cells (Fig. 8). Secondly, conspicuous structures of medium density were found in some of the parafollicular cells containing the small granules. These structures were variable in size, and com- monly appeared as slender rods, several microns in length with rounded or tapering ends (Fig. 3). They possess a limiting unit membrane and their content appears to have a crystalline sub-structure (Fig. 4). They were usually found in groups of 2 or 3 in those regions of the cell packed with the small secretory granules (Fig. 3). Occasional lysosome-like bodies and small multivesicular bodies were also seen at this time (Fig. 3). The thyroid glands of active bats caught in late October and placed in the cold for 12 h also had parafollicular cells which contained numerous large round granules. After this treatment, the granules were found to lie enclosed within larger, irregular vesicles, the membranes of which were unevenly studded with ribosomes (Fig. 5). These large granules varied in size, the largest being about i-o /tm in diameter (Fig. 5). A few small dense granules were also seen throughout the cytoplasmic matrix of these cells, which were always separated from the luminal colloid by the cytoplasm of the lining follicular cells. Further marked changes occurred following exposure of the bats to cold for 24-48 h. In particular, almost all the dense content had disappeared from the irregular cytoplasmic vesicles (Figs. 6, 7). Further, the degranulated cells possessed variable numbers of multivesicular bodies (Fig. 6) and the granules in either type of parafollicular cell remained unchanged after exposing the animals to cold. Thorough examination of the thyroid glands of the early hibernating bats (Nov- ember) again failed to reveal any large granules in the parafollicular cells (Nunez et al. 1967), but, as before, there were various degrees of degranulation in the cells which normally contain small granules (Fig. 9). The material persisting in these discharged granules was arranged as 4 or 5 concentric lamellae (Fig. 9).

DISCUSSION Our present findings together with those of our earlier studies (Nunez et al. 1967, 1969), suggest that the large dense granules may represent a second class of para- follicular cell secretory granule. This view is based on the observations, summarized in Fig. 1, that parafollicular cells containing large granules are found only during the Autumn, while those containing small granules alone are found throughout the year. Moreover, the large granules degranulate rapidly with the onset of hibernation, while the smaller degranulate slowly during the early months of hibernation. Histochemical studies have shown that the thyroid parafollicular cells appear to have two functions. First, they are thought to be involved in the production of (Pearse, 19666; Bussolati & Pearse, 1967) and, second, in some species, parafollicular cells can incorporate and metabolize the amino-acid precursors of bio- genic amines (Falck et al. 1964; Ritzen, Hammarstrom & Ullberg, 1965; Erspamer 1966; Larson, Owman & Sundler, 1966; Pearse, 1966a). More recently, it has been shown that parafollicular cells of active bat are able to incorporate 5-hydroxy- tryptophan (Gershon & Nunez, 1970). 53 CEL 6 824 E- A. Nunez, R. P. Gould and S. jf. Holt Interest has now become focussed on the possible subcellular location of calcitonin and biogenic amines. The results of recent biochemical studies (Bauer & Teitelbaum, 1966), and ultrastructural studies (Pearse, 19666; Matsuzawa & Kurosumi, 1967; Capen & Young, 1967; Young et al. 1968; Ericson, 1968) strongly suggest that the small granules are the intracellular storage sites of calcitonin. Moreover, the observa- tions of Matsuzawa & Kurosumi (1967) and Ericson (1968), that the number of these granules decreases greatly following experimental elevation of blood calcium, offers convincing evidence in favour of our postulate (Nunez et al. 1969) that the changes in form and number of the small granules in parafollicular cells during hibernation

• Small granules with solid dense cores Vacuoles with large granules o Degranulated small granules Empty vacuoles o Resynthesized small granules

(Nunez eta/. 1969) (Nunez eta/. 1967)

May June Aug. /Sept. Nov. v Dec. Jan. Feb. Mar. Apr.

-Active bats •* ' • Hibernating bats-

Sept. Oct' .12 24 48h • Active bats ^Cold (4 °C) bats-* Present study Fig. 1. Diagram summarizing the seasonal appearance of small and large secretory granules of bat thyroid parafollicular cells. reflect the release of calcitonin in response to the transient high blood-calcium level which develops in hibernating bats (Azzali, 1968). However, this rise in bat blood calcium is followed by a hypocalcaemic state during extended hibernation (Riedesel & Folk, 1954; Riedesel, 1957). It is well known in non-hibernating animals that recovery from experimentally induced hypocalcaemia requires parathyroid hormone (Tashjian, 1965; Johnston & Deiss, 1966; Anast, Arnaud, Rasmussen & Tenenhouse, 1967; Tashjian, 1966). It is therefore of interest that bat parathyroids in late hibernation appear to be in a non- secretory state (E. A. Nunez, to be published), and this, in conjunction with the postulated secretion of calcitonin, could account for the reported low levels of blood calcium in bats in deep hibernation. As for the parafollicular cells containing large granules, two alternative functions may be ascribed to them. Firstly, their granules may be a special form of the smaller kind and so also store calcitonin. The rapid depletion of these large granules in the first 48 h of experimentally induced hibernation is therefore consistent with the steep transitory rise in blood calcium which is known to occur in the initial phases of hiber- nation (Azzali, 1968), while the slow depletion of the small granules may be the Parafollicular cell large granules 825 expression of an important control system which prevents an otherwise harmful degree of hypercalcaemia which might develop during prolonged hibernation. Secondly, the large granules may be storage sites for some quite different cell product, such as the biogenic amines, e.g. serotonin, which are known to be incorporated by the parafollicular cells of some species (Gershon'& Ross, 1966), and to be present in others (Falck et al. 1964). This possibility, to which we have referred before (Nunez et al. 1967), has now been investigated and it has been shown that serotonin is, in fact, present in some parafollicular cells in active bat thyroids (Gershon & Nunez, 1970). This finding is of interest, for it is known that these substances alter the capacity of the thyroid gland to incorporate radioactive iodine (Zizine, 1959; Carlier & Keil, i960; Maraud, Stoll & Sparfel, 1962; D'Addabbo, Giorgino & Fanfani, 1963) and therefore, presumably, to change the output of thyroxine. Thus the depression of thyroid activity which accompanies hibernation in some species (Lachiver, 1952; Vidovic & Popovic, 1954; Petrovic & Kayser, 1958) may also occur in the bat and be partly controlled by the local action of serotonin released from the large granules. The rod-like crystalline inclusions found in Autumn in the bat parafollicular cells containing large numbers of small granules closely resemble the crystalline bodies found in a variety of mammalian cells (Fedorko & Hirsch, 1965; Hadek & Swift, i960) and, in particular, those of the interstitial cells of the human testis (Fawcett & Burgos, i960). Although the nature of such crystalloids is not known, in some cells they appear to consist of protein and to possess enzyme activity (Miller, De Harven & Palade, 1966). It seems unlikely, as suggested by Tandler & Shipkey (1964) for tumour cells, that in the bat thyroid they represent secretory material which cannot be liberated from the cell, since they are found only before the onset of secretion by the para- follicular cells.

The authors wish to express their gratitude to Dr Roy C. Swan, for the use of a Philips EM 200 electron microscope in the Department of Anatomy, Cornell University Medical School, New York, and to the University of London and the Wellcome Foundation for financial support in obtaining the European bats used in this study. A Philips EM 200 electron microscope was also provided in the Courtauld Institute of Biochemistry through the generosity of the Fleming Memorial Fund for Medical Research. This investigation was supported by Public Health Service Research Grant No. AM 12957-01 from the National Institute of Arthritis and Metabolic Diseases and by a Public Health Service Research Career Development Award (1-K4-GM-29,958-01) to Dr E. Nunez from the National Institute of General Medical Sciences.

REFERENCES ANAST, C, ARNAUD, C. D., RASMUSSEN, H. & TENENHOUSE, A. (1967). Thyrocalcitonin and response to parathyroid hormone. J. din. Invest. 46, 57-64. AZZALI, G. (1966). Sullo sviluppo delle cellule parafollicolari della tiroide di Opossum (Di- delphys azarae) e di Xenarthra (Dasypus novemcinctus). Ateneo parmense 37, 569-614. AZZALI, G. (1968). Ultrastructure of parafollicular cells. In Calcitonin: Proceedings of Symposium on Thyrocalcitonin and C cells (ed. S. Taylor), pp. 152-166. London: Heinemann Medical Books. BAUER, W. C. & TEITELBAUM, S. L. (1966). Thyrocalcitonin activity of particulate fractions of the thyroid gland. Lab. Invest. 15, 323-329. 53-2 826 E. A. Nunez, R. P. Gould and S. J. Holt BIORCK, G., JOHANSSON, B. & VEIGE, S. (1956). Some laboratory data on hedgehogs, hibernat- ing and nonhibernating. Ada physiol. scand. 37, 281-294. BUSSOLATI, G. & PEARSE, A. G. E. (1967). Immunofluorescent localization of calcitonin in the 'C cells of pig and dog thyroid. J. Endocr. 37, 205-210. CAPEN, C. C. & YOUNG, D. M. (1967). The ultrastructure of the parathyroid glands and thyroid parafollicular cells of cows with parturient paresis and hypocalcemia. Lab. Invest. 17, 7I7-737- CARLIER, J. & KEIL, C. (i960). Action de la 5-hydroxytryptamine et du 5-hydroxytryptophane sur la thyroide du lapin. J. Physiol. Path. gen. 52, 42-44. CARVALHEIRA, A. F. & PEARSE, A. G. E. (1967). The cytology and cytochemistry of the ' C' cells in the thyroid gland of the pig. Jl R. microsc. Soc. 86, 203-213. D'ADDABBO, A., GIORGINO, R. & FANFANI, G. (1963). Studio di una nuova azione della 5-idros- sitryptamina: inibizione della captazione de mI nel ratto. Folia endocr. (Rome) 16, 577-590. EKHOLM, R. &ERICSON, L. E. (1968). The ultrastructure of the parafollicular cells of the thyroid gland in the rat. J. Ultrastruct. Res. 23, 378-402. ERICSON, L. E. (1968). Degranulation of the parafollicular cells of the rat thyroid by vitamin D2-induced hypercalcemia. J. Ultrastruct. Res. 24, 145-149. ERSPAMER, V. (1966). In Handbook of Experimental. Pharmacology, vol. 19 (ed. V. Erspamer), p. 245. Heidelberg: Springer. FALCK, B., LARSON, B., MECKLENBURG, C. VON, ROSENGREN, E. & SVENAEUS, K. (1964). On the presence of a second specific cell system in mammalian thyroid gland. Ada physiol. scand. 62, 491-492. FAWCETT, D. W. & BURGOS, M. H. (i960). Studies on the fine structure of the mammalian testis. II. The human interstitial tissue. Am. J. Anat. 107, 245-270. FEDORKO, M. E. & HIRSCH, J. G. (1965). Crystalloid structures in granules of guinea pig basophils and human mast cells. J. Cell Biol. 26, 973-976. 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. G. E. (1964). Calcitonin production and the mito- chondrion-rich cells of the dog thyroid. Nature, Lond. 203, 1029-1030. GERSHON, M. D. & NUNEZ, E. A. (1970). Histochemical and radioautographic studies of serotonin and parafollicular cells in the thyroid gland of the pre-hibernating bat. Endocrino- logy (in the Press). GERSHON, M. D. & Ross, L. (1966). Location of sites of 5-hydroxytryptamine storage and metabolism by radioautography. J. Physiol., Lond. 186, 477-492. HADEK, R. & SWIFT, H. (i960). A crystalloid inclusion in the rabbit blastocyst. J. biophys. biochem. Cytol. 8, 836-842. HIRSCH, P. F., GAUTHIER, G. F. & MUNSON, P. L. (1963). Thyroid hypocalcemic principle and recurrent latyngeal nerve injury as factors affecting the response to parathyroidectomy in rats. 73, 244-252. JOHNSTON, C. C. Jr. & DEISS, W. P. Jr. (1966). An inhibitory effect of thyrocalcitonin on calcium release in vivo and on metabolism in vitro. Endocrinology 78, 1139-1143. KARNOVSKY, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27, 137A-138A. LACHIVER, F. (1952). Cycle saisonnier de l'iodemie d'un hibernant, le lerot (Eliomys quercinus L.). C. r. Se'anc. Soc. Biol. 146, 245-248. LARSON, B., OWMAN, C. & SUNDLER, F. (1966). Monoaminergic mechanisms in parafollicular cells of the mouse thyroid gland. Endocrinology 78, 1109-1114. MARAUD, R., STOLL, R. & SPARFEL, A. (1962). Action de la se>otonine sur la thyroide de l'embryon de poulet. C. r. Se'anc. Soc. Biol. 156, 1375-1377. MATSUZAWA, T. & KUROSUMI, K. (1967). Morphological changes in the parafollicular cells of the rat thyroid gland after administration of calcium shown by electron microscopy. Nature, Lond. 213, 927-928. MILLER, F., DE HARVEN, E. & PALADE, G. E. (1966). The structure of eosinophil leukocyte granules in rodents and in man. J. Cell Biol. 31, 349-362. NUNEZ, E. A., GOULD, R. P., HAMILTON, D. W., HAYWARD, J. S. & HOLT, S. J. (1967). Seasonal changes in the fine structure of the basal granular cells of the bat thyroid. J. Cell Sci. 2, 401-410. Parafollicular cell large granules 827

NUNEZ, E. A., GOULD, R. P. & HOLT, S. J. (1968). Observations on the dense granules in the bat thyroid parafollicular cells. In Colcitonin: Proceedings of Symposium on Thyrocalcitonin and C cells (ed. S. Taylor), pp. 204-214. London: Heinemann Medical Books. NUNEZ, E. A., GOULD, R. P. & HOLT, S. J. (1969). A study of granule formation in the bat parafollicular cell. J. Cell Sri. 5, 12-29. PEARSE, A. G. E. (1966a). 5-Hydroxytryptophan uptake by dog thyroid 'C cells and its possible significance in polypeptide hormone production. Nature, Lond. 211, 598-600. PEARSE, A. G. E. (19666). The cytochemistry of the thyroid ' C cells and their relationship to calcitonin. Proc. R. Soc. B 164, 478-487. PEARSE, A. G. E. (1968). Common cyrochemical and ultrastructural characteristics of cells producing polypeptide hormones (the APUD series) and their relevance to thyroid and ultimobranchial C cells and calcitonin. Proc. R. Soc. B 170, 71-80. PETROVIC, A. & KAYSER, C. (1958). Variations saisonnieres du seuil reactionnel de la thyro'ide a la thyreostimuline chez le hamster (Cricetus cricetus). J. Physiol. Path. gen. 50, 446—450. REYNOLDS, F. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy, jf. Cell Biol. 17, 208-212. RIEDESEL, M. L. (1957). Serum magnesium levels in mammalian hibernation. Trans. Kans. Acad. Sri. 60, 99-141. RIEDESEL, M. L. & FOLK, C. F. Jr. (1954). Serum magnesium changes in hibernation. ^4??;. J. Physiol. 179, 665. RITZEN, M., HAMMARSTROM, L. & ULLBERG, S. (1965). Autoradiographic distribution of 5-hydroxytryptamine and 5-hydroxytryptophan in the mouse. Biochem. Pharmacol. 14, 313-321. TANDLER, B. & SHIPKEY, F. H. (1964). Ultrastructure of Warthin's tumor. II. Crystalloids. y. Ultrastruct. Res. n, 306-314. TASHIRO, K. (1964). Electron microscopic studies of the parafollicular cells in the thyroid gland of the dog. Okajimas Folio anat.jap. 39, 191-210. TASHJIAN, A. H. Jr. (1965). of plasma calcium: effects of actinomycin D, para- thyroidectomy and thyrocalcitonin. Endocrinology 77, 373-381. TASHJIAN, A. H. Jr. (1966). Effects of parathyroidectomy and cautery of the thyroid gland on the plasma calcium level of rats with autotransplanted parathyroid glands. Endocrinology 78, 1144-1153. 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. y. biophys. biochem. Cytol. 4, 475-478. YOUNG, B. A., CARE, A. D. & DUNCAN, T. (1968). Some observations on the light cells of the thyroid gland of the pig in relation to thyrocalcitonin production, y. Anat. 102, 275-288. ZIZINE, L. (1959). Action de la serotonine sur l'activite thyroidienne de rat. C. r. Se'anc. Soc. Biol. 153, 1156-1158. (Received 20 October 1969) 828 E. A. Nunez, R. P. Gould and S. J. Holt

ABBREVIATIONS ON PLATES b blood vessel m mitochondrion bm basement membrane mvb multivesicular body c colloid n nucleus cr crystalloid pcx parafoUicular cell containing small f follicular cell secretory granules gc Golgi complex pc2 parafoUicular cell containing large secre- I lamellae tory granules Ig large dense secretory granule sg small dense secretory granules ly lysosome V cytoplasmic vacuole

Fig. 2. Cluster of thyroid parafoUicular cells from a bat caught in mid-September. Most parafoUicular cells (pCx) contain small dense secretory granules (sg) which occupy large areas of the cytoplasm. Occasionally, structures of medium density (cr) are found among the secretory granules. The plasma membranes of adjacent parafoUicular cells exhibit considerable interdigitation (arrows). A cell (pc») with several large dense granules (Ig) is situated near the blood vessel (b). Also seen are Golgi complexes (gc) and nuclei (n). (Myotis.) x 20000. Parafollicular cell large granules 829

m 830 E. A. Nunez, R. P. Gould and S. J. Holt

Fig. 3. Parts of the cytoplasm of two October parafoUicular cells with small granules are present in this micrograph. Several medium to long moderately dense structures (cr) are found among the secretory granules. The slender forms of these cytoplasmic inclusions often reach several microns in length. Also present are the adjacent plasma membranes (arrow), lysosome-like bodies (ly), small multivesicular bodies (mvb), part of a nucleus (n) and mitochondria (m). (Myotis.) x 21000. Parafollicular cell large granules 831 832 E. A. Nunez, R. P. Gould and S. J. Holt

Fig. 4. At higher magnification the unit membrane (arrows) and the parallel linear densities of the crystalloids are more evident, x 100 000. Parafollicular cell large granules 834 E. A. Nunez, R. P. Gould and S. J. Holt

Fig. 5. Electron micrograph of part of the thyroid gland of an active, non-hibernating bat caught in late October and placed into the cold for 12 h. Parafollicular cells with small (pCi) and large granules (pc») are present. The cells contain many cytoplasmic vacuoles which enclose dense round bodies (Ig) of various sizes. Small dense granules (arrows) are found throughout the cytoplasm of these cells. Colloid (c) and follicular cells (/) are also seen. (Eptesicus.) x 18000. Parafollicular cell large granules «35

#••'•/•. per 836 E. A. Nunez, R. P. Gould and S. J. Holt

Fig. 6. This micrograph shows a large granule-containing parafollicular cell in the thyroid of an active bat kept in the cold for 24 h. The cytoplasmic vacuoles (v) are now almost all depleted of their dense content, but one with dense material is still present (arrow). Small dense granules (sg) and multivesicular bodies (mvb) are scattered throughout the cytoplasmic matrix. Colloid (c) and follicular cells (/) are also shown. (Eptesicus.) x 22000. Parafollicular cell large granules

•*• 838 E. A. Nunez, R. P. Gould and S. J. Holt

Fig. 7. A fieldshowin g parafollicular cells of types pc1 and pc, from a non-hibernating bat kept in the cold for 48 h. Depletion of large granules from the cell pc2 is now complete, leaving many empty cytoplasmic vacuoles (v). Follicular cells (/), basement membrane (bm) and a blood vessel (b) are also present in this field. (Myotis.) x 25000. Fig. 8. Parafollicular cells with small pc± and large pc, granules from an active bat caught in late October. The large granules are enclosed in membranes unevenly studded with ribosomes (arrow). Also seen in the micrograph are the basement membrane (bni) and a nucleus (n). (Myotis.) x 25000. ParafoUicular cell large granules 839

54 C E I. 6 840 E. A. Nunez, R. P. Gould and S. J. Holt

Fig. 9. A micrograph showing part of a small-granule type parafoUicular cell from an early hibernating bat (mid-November). Most of the granules now have a hollow core surrounded by a cylindrical array of 4-6 concentric lamellae (/). (Eptesiats.) x 40000. Parafollicular cell large granules 841