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Lipofuscin and abnormalities in colloid in the equine thyroid gland in relation to age
J. Comp. Path. 1994 Vol. 111, 389 399
L i p o f u s c i n a n d A b n o r m a l i t i e s in C o l l o i d in t h e E q u i n e T h y r o i d G l a n d in R e l a t i o n to Age R. R. Dalefield, D. N. Palmer and R. D. Jolly Department of Veterinary Pathology and Public Health, Massey University, PaImerston North, New Zealand
Summary Lipofuscin accumulation and other histological changes in thyroid tissue, previously reported to be age-related, were studied in 31 horses aged up to 35 years. The number of lipofuscin granules relative to thyrocytes increased from birth to 5 years of age. There was a wide individual variation in the number of lipofuscin granules in thyrocytes in mature horses, but this was not directly related to age. Several abnormalities were identified in thyroid colloid. The prevalence of spherites, lipofuscin granules, nucleated cells and shreds of colloid increased with age, but the prevalence of calcium oxalate crystals, erythrocytes, basophilic zones and solid fragments of colloid did not. In horses younger than 7 years, particularly large lipofnscin granules were found in thyrocytes of a small proportion of follicles which also contained abnormal colloid. Such follicles became more common in older horses without being accompanied by large lipofuscin granules. No correlation was found between granule numbers and frequency of colloid abnormalities. These results cast doubt on the traditional assumption that lipofuscin is indigestible cellular residue, since there was little evidence for excretion of granules. It is postulated that lipofuscin in this tissue may be-a normal stage in lysosomal catabolism.
Introduction Yellow or yellow-brown lipofuscin granules occur in the cytoplasm of a wide range of cells (Brizzee and Ordy, 1981). These granules are irregular in shape and composed of a matrix which contains vacuolar spaces. They are autofluorescent and stain with a variety of histological stains including Schmorl's stain (Elleder, 1981), periodic acid-Schiff and lipid stains (Dolman and Macleod, 1981). Lipofuscin granules accumulate with age in postmitotic cells such as central nervous system neurons (Porta and Hartroft, 1969) and cardiac myocytes (Munnell and Getty, 1968; Ambani et al., 1977) and are reported to increase in number, colour and size in cell types with a low mitotic rate (Dolman and Macleod, 1981). Lipofuscin has been found in human thyrocytes (Heimann, 1966; Lupulescu and Petrovici, 1968; Klinck et al., 1970; Matsubara et al., 1982; Mizukami and Matsubara, 1982; Alexander et al., 1985, Landas et al., 1986; Ohaki et al., 1986), and in thyrocytes of the cat (Ives et al., 1975), dog, monkey, guinea-pig (Gordon et aL, 1984), and horse Correspondence to: R. D. Jolly. 0021-9975/94/080389+ 11 $0[3.00/0
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(Anderson and Capen, 1978). Thyroid lipofuscin granules show both acid phosphatase and beta glucuronidase activity, indicating lysosomal function (Ives et al., 1975). Lipofuscin granules have been assumed to be composed of inert cellular debris, resistant to lysosomal catabolism--inevitable waste products of years of normal cellular metabolism (Dolman and Macleod, 1981). There is some evidence that the degree of cellular activity can affect the quantity of lipofuscin (Dolman and Macleod, 1981 ; Sohal, 1981). Others have argued that lipofuscin is the product of cell damage. The most commonly advanced theory is that the products of free radical-induced peroxidation of membrane lipids crosslink to the amino groups of proteins (Dolman and Macleod, 1981; Lippman, 1983; Nakano et al., 1989), forming polymerized residues which are resistant to lysosomal degradation and accumulate as lipofuscin with time (Brunk and Collins, 1981 ; Zs.-Nagy, 1988). Material with the histological properties of lipofuscin accumulates in the ceroid-lipofuscinoses, a group of fatal inherited neurodegenerative diseases of children and animals. Recently it has been shown that the lipopigment stored in these diseases is composed of discrete chemical species, including a specific protein, subunit c ofmitochondrial ATP synthase protein (Palmer et al., 1986a, b, 1989, 1990). We initiated a study to determine whether protein accumulation may also play a role in the biogenesis oflipofuscin in normal cells. The thyroid gland was chosen because the lysosomal catabolism of a protein, thyroglobulin, is an essential part of thyroid function. Horses were chosen as a suitable species for studying age-related changes in the thyroid gland, because they live longer than other domestic and laboratory animals. As part of the study, the relationship between age and lipofuscin accumulation, follicle size (Doniach, 1978; Studer et al., 1978; Wollman, 1980; Gerber et al., 1987), and the appearance of various unusual structures in thyroid colloid (Doniach, 1978; Reid et al., 1987) were investigated. Materials and Methods
Fixation, Preparation and Staining of Sections Thyroid tissue for histology was obtained from 31 horses, which ranged in age from >300 days of gestation (a premature foal) to 35 years. Horses were killed by shooting or barbiturate overdose for reasons other than systemic or endocrine disease, and the age was obtained from age-brands or from the owner. Samples were fixed with minimal delay in 10% neutral buffered formalin. Serial sections of paraffin waxembedded tissue were stained with haematoxylin and eosin (HE) (Gill et al., 1974) or with Schmorl's stain (Bancroft and Stevens, 1982), or left unstained for fluorescence microscopy. Sections from some horses were also stained with von Kossa's stain (Culling et al., 1985). Counting Procedures The diameters of 30 follicles were measured across a thyroid section cut 5 to 10 mm from the pole of one gland from each horse, after preliminary results (not shown) had determined that 30 follicles were sufficient to give an accurate average diameter and that the site chosen was representative of the rest of the thyroid tissue. Each follicle was given a score from 0 (nil) to 4 (heavy) for the frequency of colloid vacuoles (an
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indicator ofmacropinocytosis) in the peripheral colloid. Abnormalities in colloid were counted and classified in 20 fields, at x 10 magnification, across the widest diameter of an HE-stained section from each horse. Lipofuscin granules and cell nuclei were counted in the thyrocytes of 30 follicles in Schmorl-stained sections of each thyroid, after preliminary results (not shown) had determined that the number of lipofuscin granules per cell was constant throughout both glands and that examination of 30 follicles was sufficient to obtain an accurate estimation of this number. The number of granules per follicle was "regressed" on the number of cells per follicle and the mean number of granules per 50 thyrocytes was calculated from each individual linear regression, after preliminary calculations had shown that the average follicle contained approximately 50 cells in the plane of section.
Statistical Analyses Data were analysed by linear regression. The accuracy of the model was analysed by calculation of the coefficient of determination, R 2 (Cangelosi et al., 1983).
Results
Thywid Colour T h e thyroid tissue from horses in their first decade was dark pink. In older horses it was tan to dark brown, but age was not directly related to depth of colour.
Follicle Size and Activi~ T h e average diameter of follicles (180pm) was not affected by age. T h e diameter of the most peripheral follicles was roughly half that of the more central ones. Except in the p r e m a t u r e foal, the thyroids invariably showed colloid vacuoles in some follicles, but there was no change in overall macropinocytotic activity with age. " C o l d " follicles with squamous thyrocytes and no colloid vacuoles, as described by Studer et al. (1978), were rare or absent in most horses.
Lipofuscin Lipofuscin granules were observed as irregularly shaped bodies in the apical cytoplasm of thyrocytes. T h e y stained dark blue with Schmorl's stain (Fig. la), were yellow in unstained sections and exhibited a yellow-orange fluorescence with fluorescence microscopy (Fig. 1b). These m e t h o d s d e m o n s t r a t e d small granules which were invisible in HE-stained sections, including small granules in the normal follicles of y o u n g horses between 3 and 7 years of age. T h e r e was no relationship between age and affinity for Schmorl's stain. Yellow-brown or brown p i g m e n t e d granules were found in HE-stained sections in the majority of thyrocytes in all horses over 7 years of age. In most horses the size of lipofuscin granules did not appear to vary t h r o u g h o u t the gland. However, in horses y o u n g e r t h a n 7 years, particularly large granules, three to four times the size of other lipofuscin granules in the same section,
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Fig. 1.
Lipofuscin and colloid abnormalities in the equine thyroid gland. (a) Lipofuscin pigment stained with Schmorl's stain in equine thyrocytes. Bar=25 gin. (b) Fluorescence of lipofuscin and of spherites in equine thyroid gland. Fluorescence microscopy. Bar = 25 gin. (c) A large basophilic discrete zone within follicular colloid is marked by "knife chatter", an artefact of sectioning. HE. Bar= 125 gm. (d) Yellow fluorescence of an area of basophilic colloid, as in (c). Thyrocytes also contain fluorescent lipofuscin granules. Fluorescence microscopy. Bar= 100 gm. (e) Spherites within colloid. The pale areas at the periphery of the colloid are colloid vacuoles, indicators of the macropinocytosis process. HE. Bar= 37 gm. (f) The colloid of this follicle contains an oxalate crystal, lipofuscin granules and lipofuscin-iaden macrophages. HE. Bar = 22 gin.
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were found in thyrocytes of a small proportion of follicles which also contained abnormal colloid.
Lipofuscin Accumulation in Relation to Age The majority of follicles contained less than 100 thyrocyte nuclei in cross section and in 24 of 31 horses in which counts were made, the relationship between number of cells and number of lipofuscin granules was linear. In a small minority of larger follicles, containing 100 to 200 cells, the number of lipofuscin granules per cell was less than in the majority of follicles. The mean number of granules per 50 thyrocytes was calculated from each individual linear regression, and plotted against age (Fig. 2). Lipofuscin accumulated rapidly in the first few years of life, but after 5 years the number of lipofuscin granules differed greatly between individuals and was not directly related to age. The depth of colour of lipofuscin granules was consistent within a section but was not related to age. The size of granules appeared to vary between thyroids but was not age-related. Thus, some horses aged 20-30 years had particularly small granules, whereas the largest granules were found in a 5year-old horse, in a minority of follicles which also contained abnormal colloid. Lipofuscin-laden macrophages were occasionally observed in the interstitial connective tissue between follicles in some horses over 20 years of age.
Lipofuscin, Follicle Size and Macropinocytosis No correlation existed between lipofuscin granule numbers and the mean level of macropinocytosis in the gland (R2= 9"45%). No correlation was found between average follicle size and lipofuscin accumulation (R2=2"29%), or between the range of follicle sizes and lipofuscin accumulation (R 2= 0"69%).
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Changes in Colloid In most follicles the colloid stained an even pink colour with HE, but "knife chatter" (Figs l c,d) was a frequent artefact of sectioning. A number of departures from this appearance were seen, some of which increased in incidence with age. These changes were classified and counted in order to determine whether they were age-related or related to the accumulation of lipofuscin in thyrocytes. Some changes affected the appearance of the colloid. These included zones ofbasophilia, usually in a central position in the follicle and sharply demarcated from the normal colloid (Fig. lc). In unstained sections they exhibited yellow fluorescence, in contrast to the rest of the colloid (Fig. l d). In other follicles the colloid was in fragments with sharp edges and angles. In contrast to normal colloid, this colloid also exhibited yellow-green fluorescence in unstained sections. Occasionally shreds of colloid were observed in an otherwise empty follicle. Colloid with this appearance was not fluorescent. Other changes observed were the presence of cells or other structures in the colloid. Erythrocytes were sometimes present. Nucleated cells, presumed to be detached thyrocytes, were an occasional finding. Spherites, rounded basophilic structures which were often multiple in the follicle lumina, were also observed. Some spherites had intensely basophilic centres (Fig. l e) and resembled swollen degenerating cells, suggesting that at least some spherites represented a stage in thyrocyte degeneration. Spherites were fluorescent and the central core was often more fluorescent than the peripheral area (Fig. lb). The majority of spherites did not stain for calcium by yon Kossa's method. Lipofuscin granules in the colloid were rare (Table 1), and did not represent a major proportion of the total lipofuscin observed in the gland. Such granules were located free in the colloid, within detached thyrocytes, within spherites, within basophilic zones, or as large, highly fluorescent clusters within macrophages in the follicle lumina (Fig. 10. Calcium oxalate crystals were sometimes present. Irregularly shaped and colourless in HE-stained sections (Fig. if), they were birefringent and showed a glassy grey to black reaction with von Kossa's stain for calcium. The percentages of follicles showing each type of colloid abnormality in relation to age are shown in Table 1. Few follicles contained abnormal colloid in horses under 15 years of age, but such follicles were common in most horses of 25 years and over. However, there was a striking individual variation, and some aged horses, including the oldest horse in the study, had few follicles containing abnormal colloid. There was a trend for the prevalence ofspherites, lipofuscin in the colloid, nucleated cells in the colloid, and shreds of colloid to increase with age. In contrast, there was no relationship between age and the prevalence of calcium oxalate crystals, erythrocytes, basophilic zones or solid fragments of colloid.
Thyrocyte LipoJ&scin Related to Colloid Abnormalities In an 8-month-old foal and a 1-year-old animal lipofuscin was found only in the thyrocytes of a small number of follicles which contained basophilic or
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fragmented colloid. However, not all follicles containing such colloid contained lipofuscin in their thyrocytes. No lipofuscin granules could be found in normal follicles in unstained, HE-stained or Schmorl-stained serial sections from these two foals. Spherites or basophilic zones were present in 5% of follicles in one 3-yearold horse (but not in a second animal of this age) and in 15% of follicles in a 5-year-old horse. The thyrocytes of these follicles frequently contained particularly large lipofuscin granules. No lipofuscin granules could be seen in the thyrocytes of normal follicles in the HE-stained section from the same two horses, but on examination of unstained sections by light microscopy, small yellow granules were observed. These small granules also stained with Schmorl's stain. In one of two 3-year-old animals similar small granules were found in the follicles, which contained no colloid abnormalities or associated large granules. In a 6-year-old horse, lipofuscin granules were observed in normal follicles in HE-stained sections, but the small number of follicles containing abnormal colloid usually featured lipofuscin granules that were more intensely coloured and three to four times larger. Colloid abnormalities became more common in older horses (Table 1) but were not accompanied by these large lipofuscin granules. No correlation was found between granule numbers and frequency of colloid abnormalities (R 2= 17.41%). Discussion
The number of lipofuscin granules per 50 thyrocytes increased rapidly with age in the younger horses, but there was no age-related increase after 5 years of age, nor was there any increase in size. This is in contrast to the commonly held belief that lipofuscin accumulates with age. The lack of an age-related accumulation of lipofuscin in mature thyroids is unlikely to be restricted to horses. An age-related increase in lipofuscin was found in juvenile cats (Ives et al., 1975), but the study did not include enough older cats for a firm conclusion to be reached about continued accumulation; the oldest cats were aged only 8 years and no increase in lipofuscin deposition was observed between 5 and 8 years. Two studies (Matsubara et al., 1982; Ohaki et al., 1986) purported to show an overall tendency for lipofuscin to continue to increase with age in adult human thyrocytes. In both studies, however, only HEstaining was used, a method found to be misleading in the present study. Greater numbers of granules were visible in the Schmorl-stained thyroids of the older horses than in HE-stained ones. The large individual variation in the amount of lipofuscin in horse thyroids (Fig. 2) accords with studies of human thyroids, in which very little lipofuscin was found in some individuals in all the age-groups studied. The number of lipofuscin granules per cell was independent of either follicle size or the frequency of colloid vacuoles. The latter, indicators of macropinocytosis, were ubiquitous in these equine thyroids, implying that they are a normal feature in horses, as they are in the rat (Lupulescu and Petrovici, 1968). There was no age-related trend in follicle size or range of follicle sizes in
Lipofuscin in Equine Thyroid Glands
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equine thyroids, in contrast to the trend towards increased variability known to occur in man (Doniach, 1978; Wollman, 1980). Large follicles with squamous thyrocytes and greatly slowed colloid turnover develop in the mouse thyroid with age, and may account for 60 to 80% of follicles by 13 months of age (Studer et al., 1978; Gerber et al., 1987). In the two foals aged 8 months and 1 year, lipofuscin was restricted to the thyrocytes of the few follicles that also contained abnormal colloid, suggesting a connection. However, in older horses, lipofuscin was found evenly distributed in normal follicles. Therefore colloid abnormalities were not an essential precursor to lipofuscin accumulation. In contrast to the frequent occurrence of lipofuscin granules in normal thyrocytes, the rare instances of lipofuscin in colloid were accompanied by other abnormalities, namely the presence of spherites, nucleated cells and erythrocytes in the colloid. Spherites were present in the majority (59%) of follicles in which lipofuscin was observed in the colloid. Exfoliated thyrocytes, observed as nucleated cells shortly after exfoliation and possibly as spherites later, may release lipofuscin into the colloid. Calcium oxalate crystals become more numerous with age in h u m a n thyroid tissue (Doniach, 1978; Reid et al., 1987), but this was not so in the horse. The aetiology and pathogenesis of deposition of these crystals are unknown (Doniach, 1978). Lipofuscin granules are generally assumed to be inert residual bodies, resistant to lysosomal catabolism (Dolman and Macleod, 1981; Barden and Brizzee, 1987). If this were true then the quantity of lipofuscin in the equine thyroid should increase progressively with age. However, it does not do so, and therefore lipofuscin in this tissue is either excreted or catabolized. There was little evidence for excretion of lipofuscin. Lipofuscin granules in the colloid were a rare phenomenon; they were usually found in abnormal colloid and were therefore more likely to have been released from exfoliated thyrocytes than excreted from living thyrocytes. Macrophages containing lipofuscin were occasionally observed in the colloid; possibly they migrate to become the lipofuscin-laden macrophages occasionally observed in the interstitial connective tissue, forming a pathway for gradual removal of lipofuscin. However, such macrophages were too rare to account for the lack of an age-related increase in thyrocyte lipofuscin. The results lead to the hypothesis that lipofuscin is not inert waste material but represents a normal though slow stage of lysosomal catabolism in adult horses. This hypothesis can be tested in part by chemical analysis oflipofuscin isolated from equine thyroid tissue. Acknowledgments We acknowledge the help of Mrs E M. Slack and Mrs R Davey in processing samples for histology. This study was funded by the New Zealand Medical Research Council (grant 87/91) and the United States Institute of Communicative Disorders and Stroke, grant number NS 11238 (D.N.R). References Alexander, C. B., Herrera, G: A., Jaffe, K. and Yu, H. (1985). Black thyroid: clinical
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manifestations, ultrastructural findings, and possible mechanisms. Human Pathology, 16, 72-78. Ambani, L. M., Jhung, J. W., Edelstein, L. M. and van Woert, M. H. (1977). Quantitation of melanin in hepatic and cardiac lipofuscin. Experientia, 33, 296-298. Anderson, M. R and Capen, C. C. (1978). The endocrine system. In: Pathology of Laboratory Animals, Vol. "1, K. Benirschke, F. M. Garner and T. C. Jones, Eds, Springer, New York, pp. 423-508. Bancroft, J. D. and Stevens, A. (1982). Theory and Practice of Histological Techniques, 2nd Edit., Churchill Livingstone, Edinburgh, p. 252. Barden, H. and Brizzee, K. R. (1987). The histochemistry of lipofuscin and neuromelanin. Advances in the Biosciences, 64, 339-392. Brizzee, K. R. and Ordy, J. M. (1981). Cellular features, regional accumulation and prospects of modification of age pigments in mammals. In: Age Pigments, R. S. Sohal, Ed., Elsevier/North-Holland Biomedical Press, Amsterdam, pp. l01-154. Brunk, U. T. and Collins, V. R (1981). Lysosomes and age pigments in cultured cells. In: Age Pigments, R. S. Sohal, Ed., Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 243 264. Cangelosi, V. E., Taylor, R H. and Rice, R F. (1983). Correlation and regression analysis: the simple linear case. In: Basic Statistics. A Real WorldAppmach, 3rd Edit., West Publishing Company, St Paul, New York, pp. 314-334. Culling, C. F. A., Allison, R. T. and Barr, W. T. (1985). Cellular Pathology Technique, 4th Edit., Butterworths, London, p. 417. Dolman, C. L. and Macleod, R M. (1981). Lipofuscin and its relation to aging. In: Advances in Cellular Neurobiology, Vol. 2, S. Federoff and L. Hertz, Academic Press, New York, pp. 205-247. Doniach, I. (1978). The thyroid gland. In: Systemic Pathology, Vol. 4, 2nd Edit., Churchill Livingstone, Edinburgh, pp. 1975-2017. Elleder, M. (1981). Chemical characterization of age pigments. In: Age Pigments, R. S. Sohal, Ed., Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 203-241. Gerber, H., Peter, H.J. and Studer, H. (1987). Age related failure of endocytosis may be the pathogenetic mechanism responsible for 'cold' follicle formation in the aging mouse thyroid. Endocrinology, 120, 1758-1764. Gill, G. O., Foost, J. K. and Miller, K. A. (1974). A new formula for half-oxidised haematoxylin that neither overstains nor requires differentiation. Acta Cytologica, 18, 300 311. Gordon, G., Sparano, B. M., Kramer, A. W., Kelly, R. G. and Iatropoulos, M.J. (1984). Thyroid gland pigmentation and minocycline therapy. American Journal of Pathology, 117, 98 109. Heimann, R (1966). Ultrastructure of human thyroid. Acta Endocrinologica, 53 (Suppl. 10), 1 102. Ives, RJ., Haensly, W. E., Maxwell, R A. and McArthur, N. H. (1975). A histochemical and ultrastructural study of lipofuscin accumulation in thyroid follicular cells of aging domestic cats. Mechanisms of Ageing and Development, 4, 399-413. Klinck, G. H., Oertel, J. E. and Winship, T. (1970). Ultrastructure of normal human thyroid. Laboratory Investigation, 22, 2-22. Landas, S. K., Schelper, R. L., Tio, F. O., Turner, J. W., Moore, K. C. and BennettGray, J. (1986). Black thyroid syndrome: exaggeration of a normal process? American Journal of Clinical Pathology, 85, 411 418. Lippman, R. D. (1983). Lipid peroxidation and metabolism in aging: a biological, chemical and medical approach. Review of Biological Research in Aging, 1, 315-342. Lupulescu, A. and Petrovici, A. (1968). Ultrastructure of the Thyroid Gland, William Heinemann Medical Books, London. Matsubara, F., Mizukami, Y. and Tanaka, Y. (1982). Black thyroid. Morphological, biochemical and geriatric studies on the brown granules in the thyroid follicular cells. Acta Pathologica Japonica, 32, 13-22. Mizukami, Y. and Matsubara, F. (1982). Origin of lipofuscin granules in human
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thyroid follicular cell. Archives of Pathology and Laboratory Medicine, 106, 650 651. Munnell, J. F. and Getty, R. (1968). Rate of accumulation of cardiac lipofuscin in the aging canine. Journals of Gerontology, 23, 154 158. Nakano, M., Toshiaki, M., Katoh, H. and Gotoh, S. (1989). Age-related accumulation of lipofuscin in myocardium of Japanese monkey (Macacafuscata). Mechanisms of Ageing and Development, 49, 4t 48. Ohaki, Y., Misugi, K. and Hasegawa, H. (1986). "Black thyroid" associated with minocycline therapy. A report of an autopsy case and review of the literature. Acta PathologicaJaponica, 36, 1367-1375. Palmer, D. N., Fearnley, I. M., Medd, S. M., Walker, J. E., Martinus, R., Bayliss, S. L., Hall, N. A., Lake, B. D., Wolfe, L. and Jolly, R. D. (1990). Lysosomal storage of the DCCD reactive proteolipid subunit of mitochondrial ATP synthase in human and ovine ceroid lipofuscinoses. In: Lipofuscin and CeroidPigments, E. A. Porta, Ed., Plenum Press, New York, pp. 211-223. Palmer, D. N., Husbands, D. R., Winter, P.J., Blunt, J. w. and Jolly, R. D. (1986a). Ceroid-lipofuscinosis in sheep: I Bis(monoacylglycero)phosphate, dolichol, ubiquinone, phospholipids, fatty acids and fluorescence in liver lipopigment lipids. Journal of Biological Chemistry, 261, 1766 1772. Palmer, D. N., Barns, G., Husbands, D. R. and Jolly, R. D. (1986b). Ceroidlipofuscinosis in sheep: II The major component of the lipopigment in liver, kidney, pancreas and brain is low molecular weight protein. Journal of Biological Chemistry, 261, 1773-1777. Palmer, D. N., Martinus, R. D., Cooper, S. M., Midwinter, G. G., Reid, J. C. and Jolly, R. D. (1989). Ovine ceroid lipofuscinosis. The major lipopigment protein and the lipid-binding subunit of mitochondrial ATP synthase have the same NH2terminal sequence. Journal of Biological Chemistry, 264, 5736 5740. Porta, E. A. and Hartroft, W. S. (1969). Lipid pigments in relation to aging and dietary factors (lipofuscins). In: Pigments in Pathology, M. Wolman, Ed., Academic Press, New York, pp. 191 235. Reid, J. D., Choi, C.-H. and Oldroyd, N. O. (1987). Calcium oxalate crystals in the thyroid. Their identification, prevalence, origin, and possible significance. American Journal of Clinical Pathology, 87, 443 454. Sohal, R. S. (1981). Metabolic rate, aging and lipofuscin accumulation. In: Age Pigments, R. S. Sohal, Ed., Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 303 316. Studer, H., Forster, R., Conti, A., Kohler, H., Haeberli, A. and Engler, H. (1978). Transformation of normal follicles into thyrotropin-refractory 'cold' follicles in the aging mouse thyroid gland. Endocrinology, 102, 1576-1586. Wollman, S. H. (1980). Structure of the thyroid gland. In: The Thyroid Gland, M. de Visscher, Ed. In the series Comprehensive Endocrinology, L. Martini, Ed., Raven Press, New York, pp. 1-19. Zs.-Nagy, I. (1988). The theoretical background and cellular autoregulation of biological waste product formation. In: Lipofuscin-1987: State of the Art, I. Zs.-Nagy, Ed., Akademiai Kiado, Budapest and Elsevier Science Publishers, Amsterdam, pp. 23-50.
I Received, March 8th, 1994] Accepted, August 3rd, 1994J
L i p o f u s c i n a n d A b n o r m a l i t i e s in C o l l o i d in t h e E q u i n e T h y r o i d G l a n d in R e l a t i o n to Age R. R. Dalefield, D. N. Palmer and R. D. Jolly Department of Veterinary Pathology and Public Health, Massey University, PaImerston North, New Zealand
Summary Lipofuscin accumulation and other histological changes in thyroid tissue, previously reported to be age-related, were studied in 31 horses aged up to 35 years. The number of lipofuscin granules relative to thyrocytes increased from birth to 5 years of age. There was a wide individual variation in the number of lipofuscin granules in thyrocytes in mature horses, but this was not directly related to age. Several abnormalities were identified in thyroid colloid. The prevalence of spherites, lipofuscin granules, nucleated cells and shreds of colloid increased with age, but the prevalence of calcium oxalate crystals, erythrocytes, basophilic zones and solid fragments of colloid did not. In horses younger than 7 years, particularly large lipofnscin granules were found in thyrocytes of a small proportion of follicles which also contained abnormal colloid. Such follicles became more common in older horses without being accompanied by large lipofuscin granules. No correlation was found between granule numbers and frequency of colloid abnormalities. These results cast doubt on the traditional assumption that lipofuscin is indigestible cellular residue, since there was little evidence for excretion of granules. It is postulated that lipofuscin in this tissue may be-a normal stage in lysosomal catabolism.
Introduction Yellow or yellow-brown lipofuscin granules occur in the cytoplasm of a wide range of cells (Brizzee and Ordy, 1981). These granules are irregular in shape and composed of a matrix which contains vacuolar spaces. They are autofluorescent and stain with a variety of histological stains including Schmorl's stain (Elleder, 1981), periodic acid-Schiff and lipid stains (Dolman and Macleod, 1981). Lipofuscin granules accumulate with age in postmitotic cells such as central nervous system neurons (Porta and Hartroft, 1969) and cardiac myocytes (Munnell and Getty, 1968; Ambani et al., 1977) and are reported to increase in number, colour and size in cell types with a low mitotic rate (Dolman and Macleod, 1981). Lipofuscin has been found in human thyrocytes (Heimann, 1966; Lupulescu and Petrovici, 1968; Klinck et al., 1970; Matsubara et al., 1982; Mizukami and Matsubara, 1982; Alexander et al., 1985, Landas et al., 1986; Ohaki et al., 1986), and in thyrocytes of the cat (Ives et al., 1975), dog, monkey, guinea-pig (Gordon et aL, 1984), and horse Correspondence to: R. D. Jolly. 0021-9975/94/080389+ 11 $0[3.00/0
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(Anderson and Capen, 1978). Thyroid lipofuscin granules show both acid phosphatase and beta glucuronidase activity, indicating lysosomal function (Ives et al., 1975). Lipofuscin granules have been assumed to be composed of inert cellular debris, resistant to lysosomal catabolism--inevitable waste products of years of normal cellular metabolism (Dolman and Macleod, 1981). There is some evidence that the degree of cellular activity can affect the quantity of lipofuscin (Dolman and Macleod, 1981 ; Sohal, 1981). Others have argued that lipofuscin is the product of cell damage. The most commonly advanced theory is that the products of free radical-induced peroxidation of membrane lipids crosslink to the amino groups of proteins (Dolman and Macleod, 1981; Lippman, 1983; Nakano et al., 1989), forming polymerized residues which are resistant to lysosomal degradation and accumulate as lipofuscin with time (Brunk and Collins, 1981 ; Zs.-Nagy, 1988). Material with the histological properties of lipofuscin accumulates in the ceroid-lipofuscinoses, a group of fatal inherited neurodegenerative diseases of children and animals. Recently it has been shown that the lipopigment stored in these diseases is composed of discrete chemical species, including a specific protein, subunit c ofmitochondrial ATP synthase protein (Palmer et al., 1986a, b, 1989, 1990). We initiated a study to determine whether protein accumulation may also play a role in the biogenesis oflipofuscin in normal cells. The thyroid gland was chosen because the lysosomal catabolism of a protein, thyroglobulin, is an essential part of thyroid function. Horses were chosen as a suitable species for studying age-related changes in the thyroid gland, because they live longer than other domestic and laboratory animals. As part of the study, the relationship between age and lipofuscin accumulation, follicle size (Doniach, 1978; Studer et al., 1978; Wollman, 1980; Gerber et al., 1987), and the appearance of various unusual structures in thyroid colloid (Doniach, 1978; Reid et al., 1987) were investigated. Materials and Methods
Fixation, Preparation and Staining of Sections Thyroid tissue for histology was obtained from 31 horses, which ranged in age from >300 days of gestation (a premature foal) to 35 years. Horses were killed by shooting or barbiturate overdose for reasons other than systemic or endocrine disease, and the age was obtained from age-brands or from the owner. Samples were fixed with minimal delay in 10% neutral buffered formalin. Serial sections of paraffin waxembedded tissue were stained with haematoxylin and eosin (HE) (Gill et al., 1974) or with Schmorl's stain (Bancroft and Stevens, 1982), or left unstained for fluorescence microscopy. Sections from some horses were also stained with von Kossa's stain (Culling et al., 1985). Counting Procedures The diameters of 30 follicles were measured across a thyroid section cut 5 to 10 mm from the pole of one gland from each horse, after preliminary results (not shown) had determined that 30 follicles were sufficient to give an accurate average diameter and that the site chosen was representative of the rest of the thyroid tissue. Each follicle was given a score from 0 (nil) to 4 (heavy) for the frequency of colloid vacuoles (an
Lipofuscin in Equine Thyroid Glands
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indicator ofmacropinocytosis) in the peripheral colloid. Abnormalities in colloid were counted and classified in 20 fields, at x 10 magnification, across the widest diameter of an HE-stained section from each horse. Lipofuscin granules and cell nuclei were counted in the thyrocytes of 30 follicles in Schmorl-stained sections of each thyroid, after preliminary results (not shown) had determined that the number of lipofuscin granules per cell was constant throughout both glands and that examination of 30 follicles was sufficient to obtain an accurate estimation of this number. The number of granules per follicle was "regressed" on the number of cells per follicle and the mean number of granules per 50 thyrocytes was calculated from each individual linear regression, after preliminary calculations had shown that the average follicle contained approximately 50 cells in the plane of section.
Statistical Analyses Data were analysed by linear regression. The accuracy of the model was analysed by calculation of the coefficient of determination, R 2 (Cangelosi et al., 1983).
Results
Thywid Colour T h e thyroid tissue from horses in their first decade was dark pink. In older horses it was tan to dark brown, but age was not directly related to depth of colour.
Follicle Size and Activi~ T h e average diameter of follicles (180pm) was not affected by age. T h e diameter of the most peripheral follicles was roughly half that of the more central ones. Except in the p r e m a t u r e foal, the thyroids invariably showed colloid vacuoles in some follicles, but there was no change in overall macropinocytotic activity with age. " C o l d " follicles with squamous thyrocytes and no colloid vacuoles, as described by Studer et al. (1978), were rare or absent in most horses.
Lipofuscin Lipofuscin granules were observed as irregularly shaped bodies in the apical cytoplasm of thyrocytes. T h e y stained dark blue with Schmorl's stain (Fig. la), were yellow in unstained sections and exhibited a yellow-orange fluorescence with fluorescence microscopy (Fig. 1b). These m e t h o d s d e m o n s t r a t e d small granules which were invisible in HE-stained sections, including small granules in the normal follicles of y o u n g horses between 3 and 7 years of age. T h e r e was no relationship between age and affinity for Schmorl's stain. Yellow-brown or brown p i g m e n t e d granules were found in HE-stained sections in the majority of thyrocytes in all horses over 7 years of age. In most horses the size of lipofuscin granules did not appear to vary t h r o u g h o u t the gland. However, in horses y o u n g e r t h a n 7 years, particularly large granules, three to four times the size of other lipofuscin granules in the same section,
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:/
Fig. 1.
Lipofuscin and colloid abnormalities in the equine thyroid gland. (a) Lipofuscin pigment stained with Schmorl's stain in equine thyrocytes. Bar=25 gin. (b) Fluorescence of lipofuscin and of spherites in equine thyroid gland. Fluorescence microscopy. Bar = 25 gin. (c) A large basophilic discrete zone within follicular colloid is marked by "knife chatter", an artefact of sectioning. HE. Bar= 125 gm. (d) Yellow fluorescence of an area of basophilic colloid, as in (c). Thyrocytes also contain fluorescent lipofuscin granules. Fluorescence microscopy. Bar= 100 gm. (e) Spherites within colloid. The pale areas at the periphery of the colloid are colloid vacuoles, indicators of the macropinocytosis process. HE. Bar= 37 gm. (f) The colloid of this follicle contains an oxalate crystal, lipofuscin granules and lipofuscin-iaden macrophages. HE. Bar = 22 gin.
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were found in thyrocytes of a small proportion of follicles which also contained abnormal colloid.
Lipofuscin Accumulation in Relation to Age The majority of follicles contained less than 100 thyrocyte nuclei in cross section and in 24 of 31 horses in which counts were made, the relationship between number of cells and number of lipofuscin granules was linear. In a small minority of larger follicles, containing 100 to 200 cells, the number of lipofuscin granules per cell was less than in the majority of follicles. The mean number of granules per 50 thyrocytes was calculated from each individual linear regression, and plotted against age (Fig. 2). Lipofuscin accumulated rapidly in the first few years of life, but after 5 years the number of lipofuscin granules differed greatly between individuals and was not directly related to age. The depth of colour of lipofuscin granules was consistent within a section but was not related to age. The size of granules appeared to vary between thyroids but was not age-related. Thus, some horses aged 20-30 years had particularly small granules, whereas the largest granules were found in a 5year-old horse, in a minority of follicles which also contained abnormal colloid. Lipofuscin-laden macrophages were occasionally observed in the interstitial connective tissue between follicles in some horses over 20 years of age.
Lipofuscin, Follicle Size and Macropinocytosis No correlation existed between lipofuscin granule numbers and the mean level of macropinocytosis in the gland (R2= 9"45%). No correlation was found between average follicle size and lipofuscin accumulation (R2=2"29%), or between the range of follicle sizes and lipofuscin accumulation (R 2= 0"69%).
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Changes in Colloid In most follicles the colloid stained an even pink colour with HE, but "knife chatter" (Figs l c,d) was a frequent artefact of sectioning. A number of departures from this appearance were seen, some of which increased in incidence with age. These changes were classified and counted in order to determine whether they were age-related or related to the accumulation of lipofuscin in thyrocytes. Some changes affected the appearance of the colloid. These included zones ofbasophilia, usually in a central position in the follicle and sharply demarcated from the normal colloid (Fig. lc). In unstained sections they exhibited yellow fluorescence, in contrast to the rest of the colloid (Fig. l d). In other follicles the colloid was in fragments with sharp edges and angles. In contrast to normal colloid, this colloid also exhibited yellow-green fluorescence in unstained sections. Occasionally shreds of colloid were observed in an otherwise empty follicle. Colloid with this appearance was not fluorescent. Other changes observed were the presence of cells or other structures in the colloid. Erythrocytes were sometimes present. Nucleated cells, presumed to be detached thyrocytes, were an occasional finding. Spherites, rounded basophilic structures which were often multiple in the follicle lumina, were also observed. Some spherites had intensely basophilic centres (Fig. l e) and resembled swollen degenerating cells, suggesting that at least some spherites represented a stage in thyrocyte degeneration. Spherites were fluorescent and the central core was often more fluorescent than the peripheral area (Fig. lb). The majority of spherites did not stain for calcium by yon Kossa's method. Lipofuscin granules in the colloid were rare (Table 1), and did not represent a major proportion of the total lipofuscin observed in the gland. Such granules were located free in the colloid, within detached thyrocytes, within spherites, within basophilic zones, or as large, highly fluorescent clusters within macrophages in the follicle lumina (Fig. 10. Calcium oxalate crystals were sometimes present. Irregularly shaped and colourless in HE-stained sections (Fig. if), they were birefringent and showed a glassy grey to black reaction with von Kossa's stain for calcium. The percentages of follicles showing each type of colloid abnormality in relation to age are shown in Table 1. Few follicles contained abnormal colloid in horses under 15 years of age, but such follicles were common in most horses of 25 years and over. However, there was a striking individual variation, and some aged horses, including the oldest horse in the study, had few follicles containing abnormal colloid. There was a trend for the prevalence ofspherites, lipofuscin in the colloid, nucleated cells in the colloid, and shreds of colloid to increase with age. In contrast, there was no relationship between age and the prevalence of calcium oxalate crystals, erythrocytes, basophilic zones or solid fragments of colloid.
Thyrocyte LipoJ&scin Related to Colloid Abnormalities In an 8-month-old foal and a 1-year-old animal lipofuscin was found only in the thyrocytes of a small number of follicles which contained basophilic or
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fragmented colloid. However, not all follicles containing such colloid contained lipofuscin in their thyrocytes. No lipofuscin granules could be found in normal follicles in unstained, HE-stained or Schmorl-stained serial sections from these two foals. Spherites or basophilic zones were present in 5% of follicles in one 3-yearold horse (but not in a second animal of this age) and in 15% of follicles in a 5-year-old horse. The thyrocytes of these follicles frequently contained particularly large lipofuscin granules. No lipofuscin granules could be seen in the thyrocytes of normal follicles in the HE-stained section from the same two horses, but on examination of unstained sections by light microscopy, small yellow granules were observed. These small granules also stained with Schmorl's stain. In one of two 3-year-old animals similar small granules were found in the follicles, which contained no colloid abnormalities or associated large granules. In a 6-year-old horse, lipofuscin granules were observed in normal follicles in HE-stained sections, but the small number of follicles containing abnormal colloid usually featured lipofuscin granules that were more intensely coloured and three to four times larger. Colloid abnormalities became more common in older horses (Table 1) but were not accompanied by these large lipofuscin granules. No correlation was found between granule numbers and frequency of colloid abnormalities (R 2= 17.41%). Discussion
The number of lipofuscin granules per 50 thyrocytes increased rapidly with age in the younger horses, but there was no age-related increase after 5 years of age, nor was there any increase in size. This is in contrast to the commonly held belief that lipofuscin accumulates with age. The lack of an age-related accumulation of lipofuscin in mature thyroids is unlikely to be restricted to horses. An age-related increase in lipofuscin was found in juvenile cats (Ives et al., 1975), but the study did not include enough older cats for a firm conclusion to be reached about continued accumulation; the oldest cats were aged only 8 years and no increase in lipofuscin deposition was observed between 5 and 8 years. Two studies (Matsubara et al., 1982; Ohaki et al., 1986) purported to show an overall tendency for lipofuscin to continue to increase with age in adult human thyrocytes. In both studies, however, only HEstaining was used, a method found to be misleading in the present study. Greater numbers of granules were visible in the Schmorl-stained thyroids of the older horses than in HE-stained ones. The large individual variation in the amount of lipofuscin in horse thyroids (Fig. 2) accords with studies of human thyroids, in which very little lipofuscin was found in some individuals in all the age-groups studied. The number of lipofuscin granules per cell was independent of either follicle size or the frequency of colloid vacuoles. The latter, indicators of macropinocytosis, were ubiquitous in these equine thyroids, implying that they are a normal feature in horses, as they are in the rat (Lupulescu and Petrovici, 1968). There was no age-related trend in follicle size or range of follicle sizes in
Lipofuscin in Equine Thyroid Glands
397
equine thyroids, in contrast to the trend towards increased variability known to occur in man (Doniach, 1978; Wollman, 1980). Large follicles with squamous thyrocytes and greatly slowed colloid turnover develop in the mouse thyroid with age, and may account for 60 to 80% of follicles by 13 months of age (Studer et al., 1978; Gerber et al., 1987). In the two foals aged 8 months and 1 year, lipofuscin was restricted to the thyrocytes of the few follicles that also contained abnormal colloid, suggesting a connection. However, in older horses, lipofuscin was found evenly distributed in normal follicles. Therefore colloid abnormalities were not an essential precursor to lipofuscin accumulation. In contrast to the frequent occurrence of lipofuscin granules in normal thyrocytes, the rare instances of lipofuscin in colloid were accompanied by other abnormalities, namely the presence of spherites, nucleated cells and erythrocytes in the colloid. Spherites were present in the majority (59%) of follicles in which lipofuscin was observed in the colloid. Exfoliated thyrocytes, observed as nucleated cells shortly after exfoliation and possibly as spherites later, may release lipofuscin into the colloid. Calcium oxalate crystals become more numerous with age in h u m a n thyroid tissue (Doniach, 1978; Reid et al., 1987), but this was not so in the horse. The aetiology and pathogenesis of deposition of these crystals are unknown (Doniach, 1978). Lipofuscin granules are generally assumed to be inert residual bodies, resistant to lysosomal catabolism (Dolman and Macleod, 1981; Barden and Brizzee, 1987). If this were true then the quantity of lipofuscin in the equine thyroid should increase progressively with age. However, it does not do so, and therefore lipofuscin in this tissue is either excreted or catabolized. There was little evidence for excretion of lipofuscin. Lipofuscin granules in the colloid were a rare phenomenon; they were usually found in abnormal colloid and were therefore more likely to have been released from exfoliated thyrocytes than excreted from living thyrocytes. Macrophages containing lipofuscin were occasionally observed in the colloid; possibly they migrate to become the lipofuscin-laden macrophages occasionally observed in the interstitial connective tissue, forming a pathway for gradual removal of lipofuscin. However, such macrophages were too rare to account for the lack of an age-related increase in thyrocyte lipofuscin. The results lead to the hypothesis that lipofuscin is not inert waste material but represents a normal though slow stage of lysosomal catabolism in adult horses. This hypothesis can be tested in part by chemical analysis oflipofuscin isolated from equine thyroid tissue. Acknowledgments We acknowledge the help of Mrs E M. Slack and Mrs R Davey in processing samples for histology. This study was funded by the New Zealand Medical Research Council (grant 87/91) and the United States Institute of Communicative Disorders and Stroke, grant number NS 11238 (D.N.R). References Alexander, C. B., Herrera, G: A., Jaffe, K. and Yu, H. (1985). Black thyroid: clinical
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manifestations, ultrastructural findings, and possible mechanisms. Human Pathology, 16, 72-78. Ambani, L. M., Jhung, J. W., Edelstein, L. M. and van Woert, M. H. (1977). Quantitation of melanin in hepatic and cardiac lipofuscin. Experientia, 33, 296-298. Anderson, M. R and Capen, C. C. (1978). The endocrine system. In: Pathology of Laboratory Animals, Vol. "1, K. Benirschke, F. M. Garner and T. C. Jones, Eds, Springer, New York, pp. 423-508. Bancroft, J. D. and Stevens, A. (1982). Theory and Practice of Histological Techniques, 2nd Edit., Churchill Livingstone, Edinburgh, p. 252. Barden, H. and Brizzee, K. R. (1987). The histochemistry of lipofuscin and neuromelanin. Advances in the Biosciences, 64, 339-392. Brizzee, K. R. and Ordy, J. M. (1981). Cellular features, regional accumulation and prospects of modification of age pigments in mammals. In: Age Pigments, R. S. Sohal, Ed., Elsevier/North-Holland Biomedical Press, Amsterdam, pp. l01-154. Brunk, U. T. and Collins, V. R (1981). Lysosomes and age pigments in cultured cells. In: Age Pigments, R. S. Sohal, Ed., Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 243 264. Cangelosi, V. E., Taylor, R H. and Rice, R F. (1983). Correlation and regression analysis: the simple linear case. In: Basic Statistics. A Real WorldAppmach, 3rd Edit., West Publishing Company, St Paul, New York, pp. 314-334. Culling, C. F. A., Allison, R. T. and Barr, W. T. (1985). Cellular Pathology Technique, 4th Edit., Butterworths, London, p. 417. Dolman, C. L. and Macleod, R M. (1981). Lipofuscin and its relation to aging. In: Advances in Cellular Neurobiology, Vol. 2, S. Federoff and L. Hertz, Academic Press, New York, pp. 205-247. Doniach, I. (1978). The thyroid gland. In: Systemic Pathology, Vol. 4, 2nd Edit., Churchill Livingstone, Edinburgh, pp. 1975-2017. Elleder, M. (1981). Chemical characterization of age pigments. In: Age Pigments, R. S. Sohal, Ed., Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 203-241. Gerber, H., Peter, H.J. and Studer, H. (1987). Age related failure of endocytosis may be the pathogenetic mechanism responsible for 'cold' follicle formation in the aging mouse thyroid. Endocrinology, 120, 1758-1764. Gill, G. O., Foost, J. K. and Miller, K. A. (1974). A new formula for half-oxidised haematoxylin that neither overstains nor requires differentiation. Acta Cytologica, 18, 300 311. Gordon, G., Sparano, B. M., Kramer, A. W., Kelly, R. G. and Iatropoulos, M.J. (1984). Thyroid gland pigmentation and minocycline therapy. American Journal of Pathology, 117, 98 109. Heimann, R (1966). Ultrastructure of human thyroid. Acta Endocrinologica, 53 (Suppl. 10), 1 102. Ives, RJ., Haensly, W. E., Maxwell, R A. and McArthur, N. H. (1975). A histochemical and ultrastructural study of lipofuscin accumulation in thyroid follicular cells of aging domestic cats. Mechanisms of Ageing and Development, 4, 399-413. Klinck, G. H., Oertel, J. E. and Winship, T. (1970). Ultrastructure of normal human thyroid. Laboratory Investigation, 22, 2-22. Landas, S. K., Schelper, R. L., Tio, F. O., Turner, J. W., Moore, K. C. and BennettGray, J. (1986). Black thyroid syndrome: exaggeration of a normal process? American Journal of Clinical Pathology, 85, 411 418. Lippman, R. D. (1983). Lipid peroxidation and metabolism in aging: a biological, chemical and medical approach. Review of Biological Research in Aging, 1, 315-342. Lupulescu, A. and Petrovici, A. (1968). Ultrastructure of the Thyroid Gland, William Heinemann Medical Books, London. Matsubara, F., Mizukami, Y. and Tanaka, Y. (1982). Black thyroid. Morphological, biochemical and geriatric studies on the brown granules in the thyroid follicular cells. Acta Pathologica Japonica, 32, 13-22. Mizukami, Y. and Matsubara, F. (1982). Origin of lipofuscin granules in human
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thyroid follicular cell. Archives of Pathology and Laboratory Medicine, 106, 650 651. Munnell, J. F. and Getty, R. (1968). Rate of accumulation of cardiac lipofuscin in the aging canine. Journals of Gerontology, 23, 154 158. Nakano, M., Toshiaki, M., Katoh, H. and Gotoh, S. (1989). Age-related accumulation of lipofuscin in myocardium of Japanese monkey (Macacafuscata). Mechanisms of Ageing and Development, 49, 4t 48. Ohaki, Y., Misugi, K. and Hasegawa, H. (1986). "Black thyroid" associated with minocycline therapy. A report of an autopsy case and review of the literature. Acta PathologicaJaponica, 36, 1367-1375. Palmer, D. N., Fearnley, I. M., Medd, S. M., Walker, J. E., Martinus, R., Bayliss, S. L., Hall, N. A., Lake, B. D., Wolfe, L. and Jolly, R. D. (1990). Lysosomal storage of the DCCD reactive proteolipid subunit of mitochondrial ATP synthase in human and ovine ceroid lipofuscinoses. In: Lipofuscin and CeroidPigments, E. A. Porta, Ed., Plenum Press, New York, pp. 211-223. Palmer, D. N., Husbands, D. R., Winter, P.J., Blunt, J. w. and Jolly, R. D. (1986a). Ceroid-lipofuscinosis in sheep: I Bis(monoacylglycero)phosphate, dolichol, ubiquinone, phospholipids, fatty acids and fluorescence in liver lipopigment lipids. Journal of Biological Chemistry, 261, 1766 1772. Palmer, D. N., Barns, G., Husbands, D. R. and Jolly, R. D. (1986b). Ceroidlipofuscinosis in sheep: II The major component of the lipopigment in liver, kidney, pancreas and brain is low molecular weight protein. Journal of Biological Chemistry, 261, 1773-1777. Palmer, D. N., Martinus, R. D., Cooper, S. M., Midwinter, G. G., Reid, J. C. and Jolly, R. D. (1989). Ovine ceroid lipofuscinosis. The major lipopigment protein and the lipid-binding subunit of mitochondrial ATP synthase have the same NH2terminal sequence. Journal of Biological Chemistry, 264, 5736 5740. Porta, E. A. and Hartroft, W. S. (1969). Lipid pigments in relation to aging and dietary factors (lipofuscins). In: Pigments in Pathology, M. Wolman, Ed., Academic Press, New York, pp. 191 235. Reid, J. D., Choi, C.-H. and Oldroyd, N. O. (1987). Calcium oxalate crystals in the thyroid. Their identification, prevalence, origin, and possible significance. American Journal of Clinical Pathology, 87, 443 454. Sohal, R. S. (1981). Metabolic rate, aging and lipofuscin accumulation. In: Age Pigments, R. S. Sohal, Ed., Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 303 316. Studer, H., Forster, R., Conti, A., Kohler, H., Haeberli, A. and Engler, H. (1978). Transformation of normal follicles into thyrotropin-refractory 'cold' follicles in the aging mouse thyroid gland. Endocrinology, 102, 1576-1586. Wollman, S. H. (1980). Structure of the thyroid gland. In: The Thyroid Gland, M. de Visscher, Ed. In the series Comprehensive Endocrinology, L. Martini, Ed., Raven Press, New York, pp. 1-19. Zs.-Nagy, I. (1988). The theoretical background and cellular autoregulation of biological waste product formation. In: Lipofuscin-1987: State of the Art, I. Zs.-Nagy, Ed., Akademiai Kiado, Budapest and Elsevier Science Publishers, Amsterdam, pp. 23-50.
I Received, March 8th, 1994] Accepted, August 3rd, 1994J