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Harderian glands in mice: Fluorescence, peroxidase activity and fine structure
0040-8166/82/00160135$02.00
TISSUE & CELL 198214 (1) 135-148 0 1982Longman Group Ltd
JUDY M. STRUM and CHARLES
R. SHEAR
HARDERIAN GLANDS IN MICE: FLUORESCENCE, PEROXIDASE ACTIVITY AND FINE STRUCTURE Key words:
Electron
microscopy,
porphyrins,
lipid, secretion,
pigment.
ABSTRACT. The Harderian glands of albino mice are composed of tubule-alveoli which contain two secretory cell types. The most common cell (type A) displayed a natural red fluorescence due to the presence of porphyrins. Lipid droplets in this cell and along its apical border were often intensely fluorescent. The less common cell (type B) did not fluoresce. The type B cell contained unusual lipid droplets surrounded by concentric layers of membranes, and sometimes displayed cylindrical organelles believed to be associated with the formation of pigment. A dense red-brown pigment was observed in the lumens of a few tubule-alveoli and it did not fluoresce, but areas where pigment formation was taking place fluoresced brightly. Myoepithelial cells, containing thick and thin filaments, were found underlying both secretory cell types. Fenestrated capillaries and adrenergic and cholinergic nerve endings were abundant in the adjacent connective tissue. Endogenous peroxidase activity was identified in both secretory cell types and was found localized only within tubules and vesicles of the smooth endoplasmic reticulum.
Introduction THE Harderian gland is located in the orbit, just behind the eyeball, in animals possessing a third eyelid (Davis, 1929). In mice the gland is composed almost exclusively of secretory tubules and alveoli and has therefore been classified as tubulo-alveolar. Histochemical studies showed that the gland contains large amounts of lipoprotein and a porphyrin pigment bound to lipids (Cohn, 1955). The Harderian gland produces an oily secretion that is deposited upon the concave surface of the third eyelid (a fold of tissue immediately covering the eye). This lipid secretion undoubtedly lubricates the eye and we believe it also contains compounds and enzymes having antibacterial properties. During a study examining the effects of light on the mouse Harderian gland, we Department of Anatomy, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, Maryland 21201. Received 14 September 1981. Revised 30 October 1981.
noticed a number of interesting characteristics of the normal gland that had not heretofore been reported, and they form the basis of this paper. Materials and Methods Morphology
Fifteen adult male and female mice from strains GR, BALB/c, CD-l and chimeric BALB/cx C57BL were examined in this study. They were maintained in clear plastic cages in a standard animal facility under cyclical light conditions (12 hr light/l2 hr dark cycle). The mice were killed by cervical dislocation and the Harderian glands were fixed either (1) by immediately injecting fixative into the orbit or (2) by perfusing fixative through the ventricle. The gland was then removed and pieces of it were fixed at room temperature overnight in the same fixative: 2 % paraformaldehyde-2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3 (Karnovsky, 1965). The pieces of tissue were then transferred to 0.2 M 135
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sodium cacodylate buffer, pH 7.3 in the cold for about 48 hr. Post-fixation was for 1 hr at 4°C in 1% 0~04 in 0.1 M sodium cacodylate buffer, pH 7.3. The tissues were dehydrated in ethanol, stained en bloc with 2% uranyl acetate in 50% ethanol and embedded in Epon 812 (Luft, 1961). Semithin sections were cut, stained with 1 y0 toluidine blue 0 in 1% sodium borate and examined by light microscopy. Thin sections were then cut from selected blocks of tissue, stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963) and examined in a Philips EM201 electron microscope. Fluorescence
The Harderian glands from three female CD-1 mice were used for cutting frozen sections which were then examined and photographed in a fluorescence microscope. One gland from each animal was fixed in 2% paraformaldehyde-25 % glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3 overnight at room temperature and then transferred to 0.1 M phosphate buffer containing 7% sucrose, pH 7.3, and stored in a refrigerator. The other three glands were removed and immediately frozen by plunging them into 2-methylbutane (isopentane) cooled by liquid nitrogen. They were then stored in liquid nitrogen until sectioned. Frozen sections 10 pm thick were cut from blocks of fixed and unfixed frozen glands using a SLEE cryostat (Slee International Inc., New York). The sections were mounted, examined and photographed in ultraviolet light in an Olympus fluorescence microscope using a BG 12 excitation filter and a 530 nm barrier filter. Cytochemistry To
detect the activity of endogenous peroxidase in Harderian glands, the glands were fixed in 1.4 % paraformaldehyde and 1.7 % glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3 for only 30min at room temperature (Karnovsky, 1965). The pieces of tissue were then placed into 0.2 M sodium cacodylate buffer, pH 7.4 and stored overnight in the refrigerator. The next day the pieces of tissue were incubated for 2+ hr in the following medium: 0.5 M Tris-HCl buffer, pH 7.6; 0,05 % 3,3’-diaminobenzidine tetrahydrochloride (Sigma Chem. Co., St Louis, Missouri) and 0.0025 % H202. Following incubation the pieces of tissue were
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rinsed three times in buffer and postosmicated at 4°C in 1% 0~04 in 0.1 M sCollidine buffer, pH 7.4. After dehydration through increasing concentrations of ethanol the tissues were embedded in Epon 812. One pm thick sections were cut to evaluate the peroxidase staining and thin sections were then cut from selected areas and examined (either unstained or stained lightly with lead) in the electron microscope. Controls for this technique included Harderian gland tissue incubated in complete media containing 1O-2 M sodium azide, and tissue incubated in media lacking either Ha02 or 3,3’-diaminobenzidine tetrahydrochloride (DAB). Freeze fracture
One female BALB/c mouse was etherized and perfused via the ventricle with 2% paraformaldehyde-2.5 % glutaraldehyde fixative prepared in 0.1 M sodium cacodylate buffer, pH 7.3. The Harderian glands were carefully dissected out and placed into the same fixative overnight. Small pieces of the gland were then rinsed well with 0.1 M sodium cacodylate buffer, pH 7.3 and were gradually impregnated with 20% glycerol in 0.1 M cacodylate buffer, pH 7,3. The samples were frozen in partially solidified Freon 22 and freeze-fractured at - 110°C in a Balzers 301 apparatus (Balzers AG, Balzers, Liechtenstein). They were then shadowed with platinum-carbon. The replicas were cleaned in Clorox, rinsed in distilled water, mounted on uncoated 200 mesh grids and examined in the electron microscope. Observations Fluorescence and light microscopy
A red fluorescence was clearly seen within the Harderian glands of mice when frozen sections were examined in a fluorescence microscope under ultraviolet light. Tissue which was fixed before the frozen sections were cut showed the best morphological preservation. An intense red fluorescence was observed only in some tubulo-alveoli and it quenched or faded rapidly. The fluorescence was often brightest along the apical luminal borders of the cells but also was associated with lipid droplets in the cells (Figs. I A, 1B). The dense red-brown pigment in the lumens of some tubulo-alveoli
HARDERIAN
GLANDS
IN
MICE
rarely displayed fluorescence (Figs. I A, 1B). However, less dense areas where the pigment was being formed often displayed a red fluorescence (Figs. 1A, 1B). By light microscopy the Harderian gland was seen to be composed of many secretory tubules and alveoli (tubulo-alveoli). The cells comprising both of these structures were filled with lipid droplets. Close observation of semithin plastic sections revealed two secretory cell types (Fig. 2). The most common (type A cell) occupied approximately 90% of the secretory units, and had large lipid droplets, while the less common (type B cell) had darkly stained lipid droplets in its cytoplasm. The broad base of the type B cell was often seen at the outer edge of tubulo-alveoli, but both cell types extended to the lumens. Myoepithelial cells were also present beneath portions of both secretory cell types but they were difficult to identify by light microscopy. Ducts were rarely observed in sections of the gland, although a few small ones (Fig. 3) lined by cuboidal epithelial cells (Fig. 4) were identified. We believe the tubules serve as initial ‘ducts’ to carry the secretory material from the more distal (alveolar) portions of the gland. Near the medial border of the gland several large excretory ducts were also observed. They were lined by a stratified epithelium and emptied onto the concave surface of the third eyelid. In control female mice, some tubulo-alveolar lumens (roughly 15% but great variation was observed among different glands) contained a dense red-brown pigment, but pigment was only rarely observed in the glands of male mice. Ektron
microscopy
Differences between the two secretory cell types was best seen by electron microscopy. The common type A secretory cell was filled with large homogeneous lipid droplets (Fig. 5). The cytoplasm was dense and occupied by mitochondria, polysomes, smooth endoplasmic reticulum, Golgi regions and only a few scanty profiles of rough endoplasmic reticulum. The less common type B cell had unusual lipid droplets that were surrounded by concentric layers of membranes (Fig. 5). Its cytoplasm was less dense than that of the type A cell but it had more rough endoplasmic reticulum as well as an equal
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amount of smooth endoplasmic reticulum. Both of these secretory cells rested upon a basal lamina and extended to a secretory lumen (Fig. 6). A few microvilli projected from these cells into the lumen, where lipid droplets were released (Fig. 7). Prominent gap junctions were identified on/y between the two different secretory cell types (A and B) (Fig. 8). In the connective tissue adjacent to the secretory tubulo-alveoli, fenestrated capillaries were observed (Fig. 9). Nerve endings, containing both clear and dense-cored vesicles (Fig. IO), were also seen close to the secretory cells, myoepithelial cells, and capillaries. Many type B cells contained unusual cylindrical structures smaller in width than mitochondria. When these structures were sectioned longitudinally, two dense lines lying parallel to one another and approximately 0.18 pm apart were observed (Fig. I I ). When cut in cross-section (Fig. 12) the dense structures were found to be curved or circular. The dense structures were contained within a double-layered membrane which sometimes had ribosomes attached to its outer surfaces, suggesting that the endoplasmic reticulum enveloped and synthesized the cylinders. In fact, the densities themselves appeared to consist of two fused, or immediately apposed membranes of endoplasmic reticulum (Fig. 14). Ribosomes were also observed in the cytoplasm between the two parallel densities, and tubules were seen in the ‘cores’ of the cylinders. The cylindrical shape of these structures was confirmed by freeze-fracture preparations of type B cells (Fig. 13). The pigment within the lumens of certain tubulo-alveoli had an unusual appearance by electron microscopy (Fig. 15). A number of dense structures of various shapes and lengths were observed. Some of these densities were nearly identical to the cylindrical structures just described (insert, Fig. 15). Myoepithelial cells (Fig. 16) were present underlying portions of the two types of secretory cells. The myoepithelial cells had hemidesmosomes, caveolae, some smooth endoplasmic reticulum, ribosomes and cytoplasmic densities. They also contained two populations of filaments (Fig. 16). The thick filaments measured 12-17 nm in diameter and the thin filaments 6-7 nm.
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Endogenous peroxidase activity was identified by ultrastructural cytochemistry in both A and B secretory cell types (Fig. 17). The dense reaction product indicative of the enzyme was localized only within certain profiles of smooth endoplasmic reticulum, which appeared either tubular or vesicular depending upon the plane of section. In contrast, endogenous peroxidase activity in the lacrimal and thyroid gland from the same mouse (incubated under identical conditions) was found in ‘expected’ locations, such as in the nuclear envelope, rough endoplasmic reticulum, certain Golgi saccules, etc. Discussion The natural red fluorescence observed in Harderian glands examined in ultraviolet
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light is due to the presence of porphyrins (Cohn, 1955). In mice only protoporphyrin is present (Bittner and Watson, 1946). The very dense red-brown pigment observed in some secretory lumens is said to be porphyrin (Grafflin, 1942; Bittner and Watson, 1946), but it did not fluoresce. However, the less dense pigment just peripheral to it, and discrete yellowish tan pigment in some lumens fluoresced brightly. For years the Harderian gland was thought to be a storage organ for pigment (porphyrin) formed elsewhere in the body. However, Cohn (1955) presented evidence suggesting that the gland might synthesize the pigment, and more recently it was reported that homogenates of the gland synthesize porphyrins (Kennedy, 1970). We observed that lipid droplets in the A cells
Fig. 1A. Two tubule-alveoli within the Harderian gland display a natural red fluorescence in ultraviolet light due to the presence of porphyrin. Lipid droplets, apical cytoplasm and forming pigment fluoresce, but dense red-brown pigment does not. Compare with Fig. 2. x 125. Fig. 1B. Identical section viewed by phase microscopy structures. Dense pigment (P), forming pigment (arrow).
to more clearly demonstrate x 125.
Fig. 2. Light micrograph of tubule-alveolus in Harderian gland of albino mouse. Type A secretory cells filled with lipid droplets occupy most of the structure, but a type B secretory cell (arrow) is also present. x 205. Fig. 3. Light micrograph of a small duct (arrow) among secretory tubule-alveoli. lining epithelium varies in thickness from one to two cell layers. x 155. Fig. 4. Electron micrograph of simple cuboidal duct in the Harderian gland of a mouse. x 6500.
epithelial
The
cells (EP) lining a small
Fig. 5. Electron micrograph showing basal portions of the two types of secretory cells in the Harderian gland of the mouse. Cell type A (A) is most common and contains large lipid droplets (L) in a dense cytoplasm filled with many organelles. The less common type B cell (B) has a less dense cytoplasm and contains lipid droplets surrounded by concentric layers of membranes (arrow). x 22,000. Fig. 6. Electron micrograph of apical cytoplasm of a type B secretory cell with its typical ‘membranous lipid droplets’ (arrows). Both secretory cells abut on lumens (L) and contain a few apical microvilli. x 14,000. Fig. 7. Electron micrograph showing lipid droplet and its surrounding membranes being secreted from a type B cell into the lumen (L). Occasionally cytoplasmic fragments accompany the release of lipid from type A cells, so merocrine and apocrine mechanisms of secretion appear to occur in the mouse Harderian gland. x 29,000. Fig. 8. Electron micrograph showing a long gap junction (arrows) existing between a type A cell and a type B cell indicating that cell-to-cell communication occurs between them. x 67,500. Fig. 9. Electron micrograph of a fenestrated capillary (CAP) in Harderian gland of mouse. Processes of two myoepithelial cells (MY) are also evident underlying portions of two type A cells which are rich in smooth endoplasmic reticulum. x 23,000.
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displayed a red fluorescence, but those in the B secretory cells did not. Thus, on the basis of fluorescence we conclude that porphyrins are present in the lipid droplets of the A secretory cells, often around the borders of lumens and in areas where pigment is being formed. The dense red-brown pigment may also contain porphyrin, but it presumably is in a different form and does not fluoresce. It was in 1963 that Woodhouse and Rhodin discovered two kinds of secretory cells (which they designated type A and type B) in the Harderian gland of the albino mouse. Based upon morphological differences in these cells they proposed a possible mode of secretion by each cell type (Woodhouse and Rhodin, 1963). In their scheme the type A cells appeared either light or dark depending upon their ‘stage in the secretory cycle’. We observed a few ‘light’ A cells in our sections, but based upon their ultrastructural appearance we believe they represent ‘older’ cells about to be replaced. By electron microscopy the type B secre-
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tory cells had a less dense cytoplasm than the type A cells. However, they were most easily identified by their unusual lipid droplets which were surrounded by concentric layers of membranes. These membranes are probably rich in phospholipids, since these compounds are abundant in the gland (Cohn, 1955). The B cells also synthesized unusual ‘cylinders’ which appeared to contribute to the formation of pigment in the lumens of the glands. Other lipids are apparently also associated with pigment formation. In contrast to the type A cells, the type B cells did not display a red fluorescence. Brownscheidle and Niewenhuis (1978) and Watanabe (1980) suggested that type B cells selectively secrete porphyrins, but our fluorescence evidence indicates that this is not true. The porphyrins appear to be secreted only by type A cells. Once released, the porphyrins become oxidized (Cohn, 1955) and combine with other products secreted by the cells. In some lumens where pigment precursors or small pigment accum-
Fig. 10. Electron micrograph of nerve endings clear vesicles are most abundant, but dense-cored Fig. 11. Longitudinal cell. x 53,000.
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section of unusual
(arrows) near secretory cell. Small vesicles are also present. x 27,000.
parallel densities (arrows)
present in type B
Fig. 12. Cross-sections of densities show them to be curved structures contained within (or surrounded by) a membranous sac (arrows). This image combined with that shown in Fig. 11 indicated these structures were cylindrical. x 71,000. Fig. 13. A fortuitous freeze-fracture through a type B cell reveals cylindrical structure (CYL) enveloped in membranous ‘fold’, and lipid droplet surrounded by concentric layers of membrane (arrow). x 45,000. Fig. 14. Electron micrograph revealing how dense walls of cylinders form by fusion of membranes (arrow). Compare this image with that seen in Fig. 13. x 65,000. Fig. 15. Electron micrograph of pigment in the lumen of the Harderian gland. Note cylindrical structures. Insert shows a cylindrical density (identical to those observed in type B cells) being extruded into area where pigment is forming. x 24,300; insert, x 36,000. Fig. 16. A portion of a myoepithelial cell (MY) and a type B cell (B). Note the thick and thin filaments within the myoepithelial cell. The central filaments are sectioned obliquely. x 97,000. Fig. 17. Electron micrograph of type A cell in Harderian gland incubated for presence of endogenous peroxidase activity. The dense reaction product is found associated only with the smooth endoplasmic reticlum of the cell and is confined to tubules (T) and vesicles (V). Insert shows vesicles (V) of smooth endoplasmic reticulum containing peroxidase activity in type B cell. x 26,000; insert x 37,000.
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ulations are present, chemical reactions take place which ultimately transform the porphyrins and surrounding material into the dense red-brown pigment that is no longer fluorescent. The fine structure of myoepithelial cells underlying the secretory cells in mouse Harderian gland has been described by Chiquoine (1958), Woodhouse and Rhodin (1963) and Watanabe (1980). However, to date only thin filaments have been reported in these cells. In this study we observed both thick and thin filaments. Based upon the size and appearance of similar filaments in smooth muscle cells (Somlyo et al., 1973) we believe the thick (12-17 nm) filaments represent mostly myosin and the thin (6-7 nm) filaments primarily actin. Gap junctions were identified by transmission electron microscopy only between the type A and B cells. We did not observe such junctions between type A cells. However, a more thorough freeze-fracture study of the gland would be required to establish this with certainty. Since gap junctions permit communication from cell to cell (Ham and Cormack, 1979) it is likely that the synthesis of products within the type B cell is regulated via ‘signals’ from the type A cell and vice versa. In addition to its exocrine function the Harderian gland might also have an endocrine function. A number of investigators have noticed that the gland is altered by hormones. For example, Boas and Scow (1954) reported that Harderian glands in adult rats atrophied following either thyroidectomy or hypophysectomy. In a more recent study, Wetterberg and Schapiro (1970) found that neonatal injections of thyroxine into rats caused porphyrins to appear in the Harderian gland sooner than normal, whereas neonatal hydrocortisone treatment retarded the appearance of porphyrins. The abundance of smooth endoplasmic reticulum in the secretory epithelial cells is also characteristic of steroid secreting endocrine cells (although cells involved in cholesterol and lipid synthesis may also have large amounts of smooth endoplasmic reticulum). The most direct indication that these secretory cells might play a role in steroid synthesis comes from a report that the Harderian gland in rats selectively accumulates the precursor of a particular pheromone steroid (Brooksbank
STRUM
et al., 1973). Fenestrated
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capillaries in the Harderian gland also coincide with a possible endocrine function, since fenestrated capillaries are characteristically associated with endocrine glands. Thus the Harderian gland appears to be regulated by endocrine hormones and may synthesize certain steroids (endocrine function) as well as synthesizing and secreting lipid and porphyrins into a lumen (exocrine function). Unmyelinated nerve endings were abundant in the connective tissue close to the secretory cells, myoepithelial cells and capillaries. Two types of nerve endings were observed in association with all of the above structures: small clear cholinergic vesicles and dense-cored adrenergic vesicles. Both types of nerve endings lacked Schwann cell investments and occasionally displayed focal peripheral densities along the cytoplasmic surfaces of their membranes. However, no direct synaptic contacts were observed and a basal lamina intervened between the endings and the cells. Tashiro et al. (1940) many years ago reported that acetylcholine injected into rats induced secretion from the Harderian gland and others have demonstrated dual innervation (i.e. cholinergic and adrenergic) (Huhtala et al., 1977; Watanabe, 1980). Most investigators have suggested that acetylcholine stimulates the myoepithelial cells to contact and thus promote the release of material by the secretory cells they embrace, but the role of norepinephrine in regulating secretion by the gland is not understood. Endogenous peroxidase was present within the secretory cells of the mouse Harderian gland, but its distribution was unusual. The enzyme was found only within profiles of smooth endoplasmic reticulum, which is reminiscent of its appearance in microperoxisomes of other cells (Novikoff et al., 1973). However, the peroxidatic activity of catalase in microperoxisomes is demonstrated at an alkaline pH of 9 or 10 (Novikoff and Goldfischer, 1969) whereas we incubated the Harderian gland at pH 7.6. Moreover, in our study lacrimal gland and thyroid gland from the same mouse (incubated at the same time under identical conditions) had endogenous peroxidase activity localized within the endoplasmic reticulum, nuclear envelope, and certain Golgi saccules, etc., which is the ‘typical’ distribution of peroxidases in many
HARDERIAN
GLANDS
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types (Herzog and Miller, 1970; Strum and Karnovsky, 197Oa, b; Essner, 1971). To our knowledge the only report of peroxidase staining (at a neutral pH) which resembles what we see here, is in peroxisomes of acatalasemic mice (Feinstein, 1970; Goldfischer and Essner, 1970). The peroxisomes in the liver and kidney of these mice showed peroxidase activity, but there are no reports of peroxidase staining patterns in cells of other tissues known to synthesize endogenous peroxidase such as the thyroid, salivary and lacrimal glands. For this reason we are unable to compare our present findings of different peroxidase staining patterns in different glands of the same mouse. In acatalasemic mice it has been shown that the catalase in peroxisomes is partially dissociated even at a neutral pH into subunits that have enhanced peroxidase activity (Goldfischer and Essner, 1970; Feinstein et al., 1971). For this reason it is not necessary to cell
use alkaline treatment (as is usually required) to dissociate the subunits so they will display peroxidase activity. In cells of the Harderian gland the staining of the smooth endoplasmic reticulum at pH 7.6 might also indicate an unstable or partially dissociated enzyme, but further studies are necessary to establish whether or not this is true. Acknowledgements
The authors wish to thank MS Marietta McAtee for excellent technical assistance, Dr Paul Reier for freeze-fracturing the Harderian gland, and Mrs Eloise DeLong for typing the manuscript. This research was supported by PHS Grant Number ROl CA20764 awarded by the National Cancer Institute, DHHS, and by PHS Grant Number ROl AM20131 awarded by the National Institute of Arthritis, Metabolism and Digestive Diseases, DHHS.
References BITTNER, J. J. and WATSON, C. J. 1946. The possible association between porphyrins and cancer in mice. Cancer Res., 6, 337-343. BOAS, N. F. and SCOW, R. 0. 1954. Apparent exopthalmos in the rat following cortisone treatment or thyroidectomy. Endocrinology, 55, 148-155. BROOKSBANK,B. W. L., WILSON, D. A. A. and CLOUGH, G. 1973. The in-t&o uptake of sH-androsta-4,16dien-3-one in tissues of the adult male rat. J. Endowin., 57, i-ii. BROWNSCHEIDLE,C. M. and NIEWENHUIS, R. J. 1978. Ultrastructure of the Harderian gland in male albino rats. Anat. Rec., 190, 73.5-754. CHIQUOINE, A. D. 1958. The identification and electron microscopy of myoepithelial cells in the Harderian gland. Anat. Rec., 132, 569-583. COHN, S. A. 1955. Histochemical observations on the Harderian gland of the albino mouse. J. Histochem. Cytochem., 3,342-353. DAVIS, F. A. 1929. The anatomy and histology of the eye and orbit of the rabbit. Trans. Am. Opthalmol. Sot., 27, 401-441. ESSNER, E. 1971. Localization of endogenous peroxidase in rat exorbital lacrimal gland. J. Histochem. Cytochem., 19,216-225. FEINSTEIN,R., SAVOL, R. and HOWARD, J. B. 1971. Conversion of catalatic to peroxidatic activity in livers of normal and acatalasemic mice. Enzymologia, 41, 345-358. FEINSTEIN,R. N. 1970. Acatalasemia in the mouse and other species. Biochem. Generics, 4, 135-155. GOLDFISCHER, S. and ESSNER, E. 1970. Peroxidase activity in peroxisomes (microbodies) of acatalasemic mice. J. Histochem. Cytochem., 18, 482-489. GRAFFLIN, A. L. 1942. Histological observations upon the porphyrinexcreting Harderian gland of the albino. rat. Am. J. Anat., 71, 43-64. HAM, A. W. and CORMACK, D. H. 1979. Histology, 8th edn, pp. 200-201. J. B. Lippincott Co., Philadelphia. HERZOG, V. and MILLER, F. 1970. Die Lokalisation endogener Peroxydase in der Glandula parotis der Ratte. Z. Zellforsch. mikrosk. Anat., 107, 403-420.
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HUHTALA, A., HUIKURI, K. T., PALKAMA, A. and TERVO, T. 1977. Innervation adrenergic and cholinergic nerve fibers. Anat. Rec., 188, 263-272. KARNOVSKY, M. J. 1965. A formaldehyde-glutaraldehyde microscopy. J. Cell Biol., 27, 137A. KENNEDY, G. Y. 1970. Harderoporphyrin:
a new porphyrin
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of the rat Harderian
fixative of high osmolarity from the Harderian
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for use in electron
glands of the rat. Camp.
Biochem. Physiol., 36, 21-36. LUFT, J. H. 1961. Improvements of epoxy resin embedding methods. J. biophys. biochem. Cytol., 9,409-414. NOVIKOFF, A. B. and GOLDFISCHER,S. 1969. Visualization of peroxisomes (microbodies) and mitochondria with diaminobenzidine. J. Histochem. Cytochem., 17, 675-680. NOVIKOFF, A. B., NOVIKOFF, P. M., DAVIS, C. and QUINTANA, N. 1973. Studies on microperoxisomes. V. Are microperoxisomes ubiquitous in mammalian cells? J. Histochem. Cytochem., 21, 737-755. REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol., 17, 208-212. SOMLYO, A. P., DEVINE, C. E., SOMLYO, A. and RICE, R. V. 1973. Filament organization in vertebrate smooth muscle. Phil. Trans. R. Sot. Land. (B), 265, 223-229. STRUM,J. M. and KARNOVSKY, M. J. 1970a. Cytochemical localization of endogenous peroxidase in thyroid follicular cells. J. CeN Biol., 44, 655-666. STRUM, J. M. and KARNOVSKY, M. J. 1970b. Ultrastructural localization of peroxidase in submaxillary acinar cells. J. Ultrastruct. Res., 31, 323-336. TASHIRO, S., SMITH, C. C., BADGER, E. and KEZUR, E. 1940. Chromodacryorrhoea, a new criterion for biological assay of acetylcholine. Proc. Sot. exp. Biol. Med., 44, 658-661. WATANABE, M. 1980. An autoradiographic, biochemical and morphological study of the Harderian gland of the mouse. J. Morph., 163, 349-365. WATSON, M. L. 1958. Staining of tissue sections for electron microscopy with heavy metals. J. biophys.
biochem. Cytol., 4, 475478. WETTERBERG,L. and SCHAPIRO, S. 1970. Harderian gland: development and influence of early hormonal treatment on porphyrin content. Science, 168,996-998. WOODHOUSE,M. A. and RHODIN, J. A. G. 1963. The ultrastructure of the Harderian gland of the mouse with particular reference to the formation of its secretory product., J. Ultrustruct. Ices., 9,76-98.
TISSUE & CELL 198214 (1) 135-148 0 1982Longman Group Ltd
JUDY M. STRUM and CHARLES
R. SHEAR
HARDERIAN GLANDS IN MICE: FLUORESCENCE, PEROXIDASE ACTIVITY AND FINE STRUCTURE Key words:
Electron
microscopy,
porphyrins,
lipid, secretion,
pigment.
ABSTRACT. The Harderian glands of albino mice are composed of tubule-alveoli which contain two secretory cell types. The most common cell (type A) displayed a natural red fluorescence due to the presence of porphyrins. Lipid droplets in this cell and along its apical border were often intensely fluorescent. The less common cell (type B) did not fluoresce. The type B cell contained unusual lipid droplets surrounded by concentric layers of membranes, and sometimes displayed cylindrical organelles believed to be associated with the formation of pigment. A dense red-brown pigment was observed in the lumens of a few tubule-alveoli and it did not fluoresce, but areas where pigment formation was taking place fluoresced brightly. Myoepithelial cells, containing thick and thin filaments, were found underlying both secretory cell types. Fenestrated capillaries and adrenergic and cholinergic nerve endings were abundant in the adjacent connective tissue. Endogenous peroxidase activity was identified in both secretory cell types and was found localized only within tubules and vesicles of the smooth endoplasmic reticulum.
Introduction THE Harderian gland is located in the orbit, just behind the eyeball, in animals possessing a third eyelid (Davis, 1929). In mice the gland is composed almost exclusively of secretory tubules and alveoli and has therefore been classified as tubulo-alveolar. Histochemical studies showed that the gland contains large amounts of lipoprotein and a porphyrin pigment bound to lipids (Cohn, 1955). The Harderian gland produces an oily secretion that is deposited upon the concave surface of the third eyelid (a fold of tissue immediately covering the eye). This lipid secretion undoubtedly lubricates the eye and we believe it also contains compounds and enzymes having antibacterial properties. During a study examining the effects of light on the mouse Harderian gland, we Department of Anatomy, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, Maryland 21201. Received 14 September 1981. Revised 30 October 1981.
noticed a number of interesting characteristics of the normal gland that had not heretofore been reported, and they form the basis of this paper. Materials and Methods Morphology
Fifteen adult male and female mice from strains GR, BALB/c, CD-l and chimeric BALB/cx C57BL were examined in this study. They were maintained in clear plastic cages in a standard animal facility under cyclical light conditions (12 hr light/l2 hr dark cycle). The mice were killed by cervical dislocation and the Harderian glands were fixed either (1) by immediately injecting fixative into the orbit or (2) by perfusing fixative through the ventricle. The gland was then removed and pieces of it were fixed at room temperature overnight in the same fixative: 2 % paraformaldehyde-2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3 (Karnovsky, 1965). The pieces of tissue were then transferred to 0.2 M 135
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sodium cacodylate buffer, pH 7.3 in the cold for about 48 hr. Post-fixation was for 1 hr at 4°C in 1% 0~04 in 0.1 M sodium cacodylate buffer, pH 7.3. The tissues were dehydrated in ethanol, stained en bloc with 2% uranyl acetate in 50% ethanol and embedded in Epon 812 (Luft, 1961). Semithin sections were cut, stained with 1 y0 toluidine blue 0 in 1% sodium borate and examined by light microscopy. Thin sections were then cut from selected blocks of tissue, stained with uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963) and examined in a Philips EM201 electron microscope. Fluorescence
The Harderian glands from three female CD-1 mice were used for cutting frozen sections which were then examined and photographed in a fluorescence microscope. One gland from each animal was fixed in 2% paraformaldehyde-25 % glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3 overnight at room temperature and then transferred to 0.1 M phosphate buffer containing 7% sucrose, pH 7.3, and stored in a refrigerator. The other three glands were removed and immediately frozen by plunging them into 2-methylbutane (isopentane) cooled by liquid nitrogen. They were then stored in liquid nitrogen until sectioned. Frozen sections 10 pm thick were cut from blocks of fixed and unfixed frozen glands using a SLEE cryostat (Slee International Inc., New York). The sections were mounted, examined and photographed in ultraviolet light in an Olympus fluorescence microscope using a BG 12 excitation filter and a 530 nm barrier filter. Cytochemistry To
detect the activity of endogenous peroxidase in Harderian glands, the glands were fixed in 1.4 % paraformaldehyde and 1.7 % glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3 for only 30min at room temperature (Karnovsky, 1965). The pieces of tissue were then placed into 0.2 M sodium cacodylate buffer, pH 7.4 and stored overnight in the refrigerator. The next day the pieces of tissue were incubated for 2+ hr in the following medium: 0.5 M Tris-HCl buffer, pH 7.6; 0,05 % 3,3’-diaminobenzidine tetrahydrochloride (Sigma Chem. Co., St Louis, Missouri) and 0.0025 % H202. Following incubation the pieces of tissue were
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rinsed three times in buffer and postosmicated at 4°C in 1% 0~04 in 0.1 M sCollidine buffer, pH 7.4. After dehydration through increasing concentrations of ethanol the tissues were embedded in Epon 812. One pm thick sections were cut to evaluate the peroxidase staining and thin sections were then cut from selected areas and examined (either unstained or stained lightly with lead) in the electron microscope. Controls for this technique included Harderian gland tissue incubated in complete media containing 1O-2 M sodium azide, and tissue incubated in media lacking either Ha02 or 3,3’-diaminobenzidine tetrahydrochloride (DAB). Freeze fracture
One female BALB/c mouse was etherized and perfused via the ventricle with 2% paraformaldehyde-2.5 % glutaraldehyde fixative prepared in 0.1 M sodium cacodylate buffer, pH 7.3. The Harderian glands were carefully dissected out and placed into the same fixative overnight. Small pieces of the gland were then rinsed well with 0.1 M sodium cacodylate buffer, pH 7.3 and were gradually impregnated with 20% glycerol in 0.1 M cacodylate buffer, pH 7,3. The samples were frozen in partially solidified Freon 22 and freeze-fractured at - 110°C in a Balzers 301 apparatus (Balzers AG, Balzers, Liechtenstein). They were then shadowed with platinum-carbon. The replicas were cleaned in Clorox, rinsed in distilled water, mounted on uncoated 200 mesh grids and examined in the electron microscope. Observations Fluorescence and light microscopy
A red fluorescence was clearly seen within the Harderian glands of mice when frozen sections were examined in a fluorescence microscope under ultraviolet light. Tissue which was fixed before the frozen sections were cut showed the best morphological preservation. An intense red fluorescence was observed only in some tubulo-alveoli and it quenched or faded rapidly. The fluorescence was often brightest along the apical luminal borders of the cells but also was associated with lipid droplets in the cells (Figs. I A, 1B). The dense red-brown pigment in the lumens of some tubulo-alveoli
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rarely displayed fluorescence (Figs. I A, 1B). However, less dense areas where the pigment was being formed often displayed a red fluorescence (Figs. 1A, 1B). By light microscopy the Harderian gland was seen to be composed of many secretory tubules and alveoli (tubulo-alveoli). The cells comprising both of these structures were filled with lipid droplets. Close observation of semithin plastic sections revealed two secretory cell types (Fig. 2). The most common (type A cell) occupied approximately 90% of the secretory units, and had large lipid droplets, while the less common (type B cell) had darkly stained lipid droplets in its cytoplasm. The broad base of the type B cell was often seen at the outer edge of tubulo-alveoli, but both cell types extended to the lumens. Myoepithelial cells were also present beneath portions of both secretory cell types but they were difficult to identify by light microscopy. Ducts were rarely observed in sections of the gland, although a few small ones (Fig. 3) lined by cuboidal epithelial cells (Fig. 4) were identified. We believe the tubules serve as initial ‘ducts’ to carry the secretory material from the more distal (alveolar) portions of the gland. Near the medial border of the gland several large excretory ducts were also observed. They were lined by a stratified epithelium and emptied onto the concave surface of the third eyelid. In control female mice, some tubulo-alveolar lumens (roughly 15% but great variation was observed among different glands) contained a dense red-brown pigment, but pigment was only rarely observed in the glands of male mice. Ektron
microscopy
Differences between the two secretory cell types was best seen by electron microscopy. The common type A secretory cell was filled with large homogeneous lipid droplets (Fig. 5). The cytoplasm was dense and occupied by mitochondria, polysomes, smooth endoplasmic reticulum, Golgi regions and only a few scanty profiles of rough endoplasmic reticulum. The less common type B cell had unusual lipid droplets that were surrounded by concentric layers of membranes (Fig. 5). Its cytoplasm was less dense than that of the type A cell but it had more rough endoplasmic reticulum as well as an equal
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amount of smooth endoplasmic reticulum. Both of these secretory cells rested upon a basal lamina and extended to a secretory lumen (Fig. 6). A few microvilli projected from these cells into the lumen, where lipid droplets were released (Fig. 7). Prominent gap junctions were identified on/y between the two different secretory cell types (A and B) (Fig. 8). In the connective tissue adjacent to the secretory tubulo-alveoli, fenestrated capillaries were observed (Fig. 9). Nerve endings, containing both clear and dense-cored vesicles (Fig. IO), were also seen close to the secretory cells, myoepithelial cells, and capillaries. Many type B cells contained unusual cylindrical structures smaller in width than mitochondria. When these structures were sectioned longitudinally, two dense lines lying parallel to one another and approximately 0.18 pm apart were observed (Fig. I I ). When cut in cross-section (Fig. 12) the dense structures were found to be curved or circular. The dense structures were contained within a double-layered membrane which sometimes had ribosomes attached to its outer surfaces, suggesting that the endoplasmic reticulum enveloped and synthesized the cylinders. In fact, the densities themselves appeared to consist of two fused, or immediately apposed membranes of endoplasmic reticulum (Fig. 14). Ribosomes were also observed in the cytoplasm between the two parallel densities, and tubules were seen in the ‘cores’ of the cylinders. The cylindrical shape of these structures was confirmed by freeze-fracture preparations of type B cells (Fig. 13). The pigment within the lumens of certain tubulo-alveoli had an unusual appearance by electron microscopy (Fig. 15). A number of dense structures of various shapes and lengths were observed. Some of these densities were nearly identical to the cylindrical structures just described (insert, Fig. 15). Myoepithelial cells (Fig. 16) were present underlying portions of the two types of secretory cells. The myoepithelial cells had hemidesmosomes, caveolae, some smooth endoplasmic reticulum, ribosomes and cytoplasmic densities. They also contained two populations of filaments (Fig. 16). The thick filaments measured 12-17 nm in diameter and the thin filaments 6-7 nm.
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Endogenous peroxidase activity was identified by ultrastructural cytochemistry in both A and B secretory cell types (Fig. 17). The dense reaction product indicative of the enzyme was localized only within certain profiles of smooth endoplasmic reticulum, which appeared either tubular or vesicular depending upon the plane of section. In contrast, endogenous peroxidase activity in the lacrimal and thyroid gland from the same mouse (incubated under identical conditions) was found in ‘expected’ locations, such as in the nuclear envelope, rough endoplasmic reticulum, certain Golgi saccules, etc. Discussion The natural red fluorescence observed in Harderian glands examined in ultraviolet
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light is due to the presence of porphyrins (Cohn, 1955). In mice only protoporphyrin is present (Bittner and Watson, 1946). The very dense red-brown pigment observed in some secretory lumens is said to be porphyrin (Grafflin, 1942; Bittner and Watson, 1946), but it did not fluoresce. However, the less dense pigment just peripheral to it, and discrete yellowish tan pigment in some lumens fluoresced brightly. For years the Harderian gland was thought to be a storage organ for pigment (porphyrin) formed elsewhere in the body. However, Cohn (1955) presented evidence suggesting that the gland might synthesize the pigment, and more recently it was reported that homogenates of the gland synthesize porphyrins (Kennedy, 1970). We observed that lipid droplets in the A cells
Fig. 1A. Two tubule-alveoli within the Harderian gland display a natural red fluorescence in ultraviolet light due to the presence of porphyrin. Lipid droplets, apical cytoplasm and forming pigment fluoresce, but dense red-brown pigment does not. Compare with Fig. 2. x 125. Fig. 1B. Identical section viewed by phase microscopy structures. Dense pigment (P), forming pigment (arrow).
to more clearly demonstrate x 125.
Fig. 2. Light micrograph of tubule-alveolus in Harderian gland of albino mouse. Type A secretory cells filled with lipid droplets occupy most of the structure, but a type B secretory cell (arrow) is also present. x 205. Fig. 3. Light micrograph of a small duct (arrow) among secretory tubule-alveoli. lining epithelium varies in thickness from one to two cell layers. x 155. Fig. 4. Electron micrograph of simple cuboidal duct in the Harderian gland of a mouse. x 6500.
epithelial
The
cells (EP) lining a small
Fig. 5. Electron micrograph showing basal portions of the two types of secretory cells in the Harderian gland of the mouse. Cell type A (A) is most common and contains large lipid droplets (L) in a dense cytoplasm filled with many organelles. The less common type B cell (B) has a less dense cytoplasm and contains lipid droplets surrounded by concentric layers of membranes (arrow). x 22,000. Fig. 6. Electron micrograph of apical cytoplasm of a type B secretory cell with its typical ‘membranous lipid droplets’ (arrows). Both secretory cells abut on lumens (L) and contain a few apical microvilli. x 14,000. Fig. 7. Electron micrograph showing lipid droplet and its surrounding membranes being secreted from a type B cell into the lumen (L). Occasionally cytoplasmic fragments accompany the release of lipid from type A cells, so merocrine and apocrine mechanisms of secretion appear to occur in the mouse Harderian gland. x 29,000. Fig. 8. Electron micrograph showing a long gap junction (arrows) existing between a type A cell and a type B cell indicating that cell-to-cell communication occurs between them. x 67,500. Fig. 9. Electron micrograph of a fenestrated capillary (CAP) in Harderian gland of mouse. Processes of two myoepithelial cells (MY) are also evident underlying portions of two type A cells which are rich in smooth endoplasmic reticulum. x 23,000.
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displayed a red fluorescence, but those in the B secretory cells did not. Thus, on the basis of fluorescence we conclude that porphyrins are present in the lipid droplets of the A secretory cells, often around the borders of lumens and in areas where pigment is being formed. The dense red-brown pigment may also contain porphyrin, but it presumably is in a different form and does not fluoresce. It was in 1963 that Woodhouse and Rhodin discovered two kinds of secretory cells (which they designated type A and type B) in the Harderian gland of the albino mouse. Based upon morphological differences in these cells they proposed a possible mode of secretion by each cell type (Woodhouse and Rhodin, 1963). In their scheme the type A cells appeared either light or dark depending upon their ‘stage in the secretory cycle’. We observed a few ‘light’ A cells in our sections, but based upon their ultrastructural appearance we believe they represent ‘older’ cells about to be replaced. By electron microscopy the type B secre-
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tory cells had a less dense cytoplasm than the type A cells. However, they were most easily identified by their unusual lipid droplets which were surrounded by concentric layers of membranes. These membranes are probably rich in phospholipids, since these compounds are abundant in the gland (Cohn, 1955). The B cells also synthesized unusual ‘cylinders’ which appeared to contribute to the formation of pigment in the lumens of the glands. Other lipids are apparently also associated with pigment formation. In contrast to the type A cells, the type B cells did not display a red fluorescence. Brownscheidle and Niewenhuis (1978) and Watanabe (1980) suggested that type B cells selectively secrete porphyrins, but our fluorescence evidence indicates that this is not true. The porphyrins appear to be secreted only by type A cells. Once released, the porphyrins become oxidized (Cohn, 1955) and combine with other products secreted by the cells. In some lumens where pigment precursors or small pigment accum-
Fig. 10. Electron micrograph of nerve endings clear vesicles are most abundant, but dense-cored Fig. 11. Longitudinal cell. x 53,000.
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section of unusual
(arrows) near secretory cell. Small vesicles are also present. x 27,000.
parallel densities (arrows)
present in type B
Fig. 12. Cross-sections of densities show them to be curved structures contained within (or surrounded by) a membranous sac (arrows). This image combined with that shown in Fig. 11 indicated these structures were cylindrical. x 71,000. Fig. 13. A fortuitous freeze-fracture through a type B cell reveals cylindrical structure (CYL) enveloped in membranous ‘fold’, and lipid droplet surrounded by concentric layers of membrane (arrow). x 45,000. Fig. 14. Electron micrograph revealing how dense walls of cylinders form by fusion of membranes (arrow). Compare this image with that seen in Fig. 13. x 65,000. Fig. 15. Electron micrograph of pigment in the lumen of the Harderian gland. Note cylindrical structures. Insert shows a cylindrical density (identical to those observed in type B cells) being extruded into area where pigment is forming. x 24,300; insert, x 36,000. Fig. 16. A portion of a myoepithelial cell (MY) and a type B cell (B). Note the thick and thin filaments within the myoepithelial cell. The central filaments are sectioned obliquely. x 97,000. Fig. 17. Electron micrograph of type A cell in Harderian gland incubated for presence of endogenous peroxidase activity. The dense reaction product is found associated only with the smooth endoplasmic reticlum of the cell and is confined to tubules (T) and vesicles (V). Insert shows vesicles (V) of smooth endoplasmic reticulum containing peroxidase activity in type B cell. x 26,000; insert x 37,000.
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ulations are present, chemical reactions take place which ultimately transform the porphyrins and surrounding material into the dense red-brown pigment that is no longer fluorescent. The fine structure of myoepithelial cells underlying the secretory cells in mouse Harderian gland has been described by Chiquoine (1958), Woodhouse and Rhodin (1963) and Watanabe (1980). However, to date only thin filaments have been reported in these cells. In this study we observed both thick and thin filaments. Based upon the size and appearance of similar filaments in smooth muscle cells (Somlyo et al., 1973) we believe the thick (12-17 nm) filaments represent mostly myosin and the thin (6-7 nm) filaments primarily actin. Gap junctions were identified by transmission electron microscopy only between the type A and B cells. We did not observe such junctions between type A cells. However, a more thorough freeze-fracture study of the gland would be required to establish this with certainty. Since gap junctions permit communication from cell to cell (Ham and Cormack, 1979) it is likely that the synthesis of products within the type B cell is regulated via ‘signals’ from the type A cell and vice versa. In addition to its exocrine function the Harderian gland might also have an endocrine function. A number of investigators have noticed that the gland is altered by hormones. For example, Boas and Scow (1954) reported that Harderian glands in adult rats atrophied following either thyroidectomy or hypophysectomy. In a more recent study, Wetterberg and Schapiro (1970) found that neonatal injections of thyroxine into rats caused porphyrins to appear in the Harderian gland sooner than normal, whereas neonatal hydrocortisone treatment retarded the appearance of porphyrins. The abundance of smooth endoplasmic reticulum in the secretory epithelial cells is also characteristic of steroid secreting endocrine cells (although cells involved in cholesterol and lipid synthesis may also have large amounts of smooth endoplasmic reticulum). The most direct indication that these secretory cells might play a role in steroid synthesis comes from a report that the Harderian gland in rats selectively accumulates the precursor of a particular pheromone steroid (Brooksbank
STRUM
et al., 1973). Fenestrated
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capillaries in the Harderian gland also coincide with a possible endocrine function, since fenestrated capillaries are characteristically associated with endocrine glands. Thus the Harderian gland appears to be regulated by endocrine hormones and may synthesize certain steroids (endocrine function) as well as synthesizing and secreting lipid and porphyrins into a lumen (exocrine function). Unmyelinated nerve endings were abundant in the connective tissue close to the secretory cells, myoepithelial cells and capillaries. Two types of nerve endings were observed in association with all of the above structures: small clear cholinergic vesicles and dense-cored adrenergic vesicles. Both types of nerve endings lacked Schwann cell investments and occasionally displayed focal peripheral densities along the cytoplasmic surfaces of their membranes. However, no direct synaptic contacts were observed and a basal lamina intervened between the endings and the cells. Tashiro et al. (1940) many years ago reported that acetylcholine injected into rats induced secretion from the Harderian gland and others have demonstrated dual innervation (i.e. cholinergic and adrenergic) (Huhtala et al., 1977; Watanabe, 1980). Most investigators have suggested that acetylcholine stimulates the myoepithelial cells to contact and thus promote the release of material by the secretory cells they embrace, but the role of norepinephrine in regulating secretion by the gland is not understood. Endogenous peroxidase was present within the secretory cells of the mouse Harderian gland, but its distribution was unusual. The enzyme was found only within profiles of smooth endoplasmic reticulum, which is reminiscent of its appearance in microperoxisomes of other cells (Novikoff et al., 1973). However, the peroxidatic activity of catalase in microperoxisomes is demonstrated at an alkaline pH of 9 or 10 (Novikoff and Goldfischer, 1969) whereas we incubated the Harderian gland at pH 7.6. Moreover, in our study lacrimal gland and thyroid gland from the same mouse (incubated at the same time under identical conditions) had endogenous peroxidase activity localized within the endoplasmic reticulum, nuclear envelope, and certain Golgi saccules, etc., which is the ‘typical’ distribution of peroxidases in many
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types (Herzog and Miller, 1970; Strum and Karnovsky, 197Oa, b; Essner, 1971). To our knowledge the only report of peroxidase staining (at a neutral pH) which resembles what we see here, is in peroxisomes of acatalasemic mice (Feinstein, 1970; Goldfischer and Essner, 1970). The peroxisomes in the liver and kidney of these mice showed peroxidase activity, but there are no reports of peroxidase staining patterns in cells of other tissues known to synthesize endogenous peroxidase such as the thyroid, salivary and lacrimal glands. For this reason we are unable to compare our present findings of different peroxidase staining patterns in different glands of the same mouse. In acatalasemic mice it has been shown that the catalase in peroxisomes is partially dissociated even at a neutral pH into subunits that have enhanced peroxidase activity (Goldfischer and Essner, 1970; Feinstein et al., 1971). For this reason it is not necessary to cell
use alkaline treatment (as is usually required) to dissociate the subunits so they will display peroxidase activity. In cells of the Harderian gland the staining of the smooth endoplasmic reticulum at pH 7.6 might also indicate an unstable or partially dissociated enzyme, but further studies are necessary to establish whether or not this is true. Acknowledgements
The authors wish to thank MS Marietta McAtee for excellent technical assistance, Dr Paul Reier for freeze-fracturing the Harderian gland, and Mrs Eloise DeLong for typing the manuscript. This research was supported by PHS Grant Number ROl CA20764 awarded by the National Cancer Institute, DHHS, and by PHS Grant Number ROl AM20131 awarded by the National Institute of Arthritis, Metabolism and Digestive Diseases, DHHS.
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HUHTALA, A., HUIKURI, K. T., PALKAMA, A. and TERVO, T. 1977. Innervation adrenergic and cholinergic nerve fibers. Anat. Rec., 188, 263-272. KARNOVSKY, M. J. 1965. A formaldehyde-glutaraldehyde microscopy. J. Cell Biol., 27, 137A. KENNEDY, G. Y. 1970. Harderoporphyrin:
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of the rat Harderian
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Biochem. Physiol., 36, 21-36. LUFT, J. H. 1961. Improvements of epoxy resin embedding methods. J. biophys. biochem. Cytol., 9,409-414. NOVIKOFF, A. B. and GOLDFISCHER,S. 1969. Visualization of peroxisomes (microbodies) and mitochondria with diaminobenzidine. J. Histochem. Cytochem., 17, 675-680. NOVIKOFF, A. B., NOVIKOFF, P. M., DAVIS, C. and QUINTANA, N. 1973. Studies on microperoxisomes. V. Are microperoxisomes ubiquitous in mammalian cells? J. Histochem. Cytochem., 21, 737-755. REYNOLDS, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol., 17, 208-212. SOMLYO, A. P., DEVINE, C. E., SOMLYO, A. and RICE, R. V. 1973. Filament organization in vertebrate smooth muscle. Phil. Trans. R. Sot. Land. (B), 265, 223-229. STRUM,J. M. and KARNOVSKY, M. J. 1970a. Cytochemical localization of endogenous peroxidase in thyroid follicular cells. J. CeN Biol., 44, 655-666. STRUM, J. M. and KARNOVSKY, M. J. 1970b. Ultrastructural localization of peroxidase in submaxillary acinar cells. J. Ultrastruct. Res., 31, 323-336. TASHIRO, S., SMITH, C. C., BADGER, E. and KEZUR, E. 1940. Chromodacryorrhoea, a new criterion for biological assay of acetylcholine. Proc. Sot. exp. Biol. Med., 44, 658-661. WATANABE, M. 1980. An autoradiographic, biochemical and morphological study of the Harderian gland of the mouse. J. Morph., 163, 349-365. WATSON, M. L. 1958. Staining of tissue sections for electron microscopy with heavy metals. J. biophys.
biochem. Cytol., 4, 475478. WETTERBERG,L. and SCHAPIRO, S. 1970. Harderian gland: development and influence of early hormonal treatment on porphyrin content. Science, 168,996-998. WOODHOUSE,M. A. and RHODIN, J. A. G. 1963. The ultrastructure of the Harderian gland of the mouse with particular reference to the formation of its secretory product., J. Ultrustruct. Ices., 9,76-98.