Seasonal Changes in Secretory Granules and Crystalloid Inclusions of Bat Thyroid Parafollicular Cells
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J. Cell Sci. 6, 821-841 (1970) 821 Printed in Great Britain SEASONAL CHANGES IN SECRETORY GRANULES AND CRYSTALLOID INCLUSIONS OF BAT THYROID 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 calcitonin (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 thyroids 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.