Ultrastructure of the Apocrine-Sebaceous Anal Scent Gland of the Woodchuck, Marrnota rnonax: Evidence for Apocrine and Merocrine Secretion by a Single Cell Type J A N E T D. SMITH AND GAIL W. HEARN Department of Anatomy, The Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129, and Department of Biology, Beaver College, Glenside, Pennsylvania 19038
ABSTRACT
Light and electron microscope studies of t h e woodchuck anal scent gland revealed t h a t i t is composed of apocrine and sebaceous components emptying into a common duct. In t h e apocrine acini, t h e single secretory cell type showed evidence of both merocrine and apocrine secretion. Merocrine secretion resulted in t h e release of t h e contents of apical secretory granules while apocrine secretion released apical caps of cytoplasm by a process involving the following: (1)formation of a n apical cap, usually containing no organelles or secretory granules; (2) appearance of a single row of flattened vesicles forming a n incomplete barrier between t h e apical cap and t h e remaining cell cytoplasm; and (3) fusion between vesicles and plasmalemma, causing progressive constriction of t h e neck of t h e apical cap and eventual cap release. Since both merocrine and apocrine secretory processes have been reported in three other types of apocrine glands, i t is likely t h a t t h e occurrence of both processes in a single cell is a general characteristic of apocrine cells. Several features apparently unique to these particular apocrine cells were observed, including secretory granules of a single morphological type and a population of small dense-cored basal vesicles of unknown function. Therefore, i t would appear t h a t , just as with merocrine cells, apocrine cells from different types of glands also have distinctive morphologies which probably reflect real differences in their functions and products.
Since Schiefferdecker's report ('171, histologists have separated glandular cells into three categories based on their mode of secretion: merocrine cells, which release their product from secretory granules, usually without loss of cytoplasm; apocrine cells which pinch off a portion of apical cytoplasm (the apical cap) as part of t h e secretory product; and holocrine cells which accumulate secretory product within t h e cytoplasm and release i t by disintegration of t h e entire cell. However, in recent years, numerous workers have questioned t h e existence of t r u e apocrine, i.e., "decapitation'' secretion. For example, electron microscope studies on t h e supposedly apocrine cells of t h e human axillary apocrine gland failed to demonstrate conclusively either apocrine secretion or t h e expected cell debris in ANAT. REC. (1979) 193: 269-292.
t h e glandular lumen, and revealed instead a merocrine mechanism of secretion (Montes e t al., '60; Montagna, '62; Biempica and Montes, '65). Although secretory cells with apical caps were frequently observed, the caps were considered to be a n artifact of fixation (Munger, '64, '65, '71). Other investigators, however, have continued to argue for t h e additional existence of a t r u e apocrine secretory process in t h e human axillary apocrine gland (Charles, '59; Kurosumi et al., '59; Hibbs, '62; Hashimoto e t al., '66; Schaumberg-Lever and Lever, '75). And, in studying t h e ultrastructure of the closely related human ceruminous gland, Kawabata ('64) and later Main and Lim ('76) found eviReceived June 27, '78. Accepted Sept. 5, '78.
269
2 70
JANET D. SMITH AND GAIL W. HEARN
dence t h a t t h e single type of apocrine cell observed could carry out both apocrine and merocrine secretion. One goal of the present study was to determine t h e mode or modes of secretion in a different type of apocrine cell, namely those of a mammalian scent gland. A second goal was to provide a basic description of a typical mammalian scent gland a t the electron microscope level. Light microscope studies have indicated t h a t many such glands are composed of a n apocrine and a sebaceous component (Mykytowycz, '70). Examples include t h e metatarsal glands of the black-tailed deer, Odocoileus hemionus (Quay and Muller-Schwarze, '701, t h e lateral gland of the short-tailed shrew, Blarina breuicauda (Pearson, '461, t h e a n a l gland of Richardson's ground squirrel Citellus richardsonii (Sleggs, '261, all of which produce a n alarm odor (Muller-Schwarze, '711; and t h e inguinal glands of t h e rabbit, Oryctolagus cuniculus, which produce a n odor indicating sexual and social status (Mykytowycz, '66). Despite the recent interest in mammalian chemical communication (Muller-Schwarze and Mozell, '77) we know of only one previous electron microscope study of a mammalian scent gland, Kneeland's ('66) report on t h e lemur antibrachial organ. Furthermore t h e antibrachial organ would appear to be a somewhat atypical scent gland since i t lacks a sebaceous component. We therefore undertook to provide a n ultrastructural description of a typical apocrine-sebaceous mammalian scent gland, t h e anal gland of t h e woodchuck, Marmots monax. Field studies on a variety of ground squirrels including t h e woodchuck indicate t h a t these glands produce a n alarm odor (Hamilton, '34). MATERIALS AND METHODS
For routine electron microscopy t h e lateral and/or medial anal glands from each of 1 3 woodchucks were fixed either by perfusion of anesthetized animals with glutaraldehyde (1.25% followed by 2.5%) in 0.1 M sodium cacodylate, pH 7.2, or by immersion fixation of the dissected glands for two to eight hours in 2.5% cacodylate-buffered glutaraldehyde. The glands were minced, postfixed with 1%OsO, and then with uranyl acetate, dehydrated and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate, carboncoated, and examined in a Philips 200 or JEOL 100-C transmission electron microscope at a n accelerating voltage of 80 kv.
For localization of acid phosphatase activity, samples were fixed for a minimum of two hours in 2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.2, rinsed thoroughly (18-72 hours) in several changes of cacodylate buffer containing 5% sucrose and then incubated in a 37°C waterbath in a reaction mixture containing t h e following: 25 mg cytidine 5'-mOnOphosphate (CMP) (sodium salt, Sigma Chemical Co.); 12 ml deionized H,O; 10 mlO.l M Tris maleate buffer, pH 5.2; 3 ml 1%aqueous lead nitrate; and 5% sucrose. Prior to incubation t h e reaction mixture was filtered through Whatman no. 50 filter paper, incubated for one hour in a 37°C waterbath, and refiltered. After incubation t h e samples were fixed for 90 minutes in 1%OsO, in 0.1 M cacodylate buffer, and then dehydrated and embedded in Epon. Further processing was as described above, except that some sections were observed without staining. The thick pasty secretory product collected from the common duct of t h e anal glands of four woodchucks was pooled and processed using glutaraldehyde fixation and OsO, postfixation followed by routine dehydration and embedding. One-micron sections of all samples were stained with toluidine blue and observed by light microscopy. RESULTS
Gross anatomy The wall of t h e woodchuck anal canal contains three separate anal glands, two disposed laterally and one on t h e ventromedial surface of t h e canal. As shown in figure 1,each gland is a n anatomically complex structure containing both apocrine and sebaceous elements. Ducts from several apocrine and sebaceous units empty into t h e distal end of a single common duct which in t u r n communicates with t h e anal canal near t h e anocutaneous junction. A common sheath of striated skeletal muscle surrounds t h e three glands. When aroused, t h e woodchuck can evert its anal glands, apparently as a result of voluntary contraction of these striated muscles. In t h e everted state, t h e glands present as papillae whose tips represent t h e base of the common duct. Light microscopy Histologically, t h e various regions of t h e gland were well-defined. The apocrine units were simple coiled tubular or tubuloalveolar
WOODCHUCK APOCRINE-SEBACEOUS ANAL SCENT GLAND
glands, whose secretory portion consisted of a single layer of cuboidal to columnar cells resting on a n incomplete layer of myoepithelial cells (fig. 2). Each apocrine unit had a wide and distinct lumen, usually filled with a substance which stained deeply with toluidine blue. In 1 p sections i t was often possible to distinguish dark-staining secretory granules at t h e apical or lumenal border of t h e cells. A smaller number of secretory cells had prominent apical caps, t h a t is, clear rounded projections of t h e apical cytoplasm which appeared to be pinching off from the rest of the cell in a process of t r u e apocrine secretion. In randomly cut sections apical caps were sometimes observed apparently free in t h e lumen. The apocrine duct was lined by cuboidal cells similar in appearance to secretory cells but lacking large accumulations of secretory granules, and lacking apical caps. Near t h e secretory acini the duct walls did contain myoepithelial cells. From reconstructions of serial sections, i t was found t h a t t h e duct, although initially quite wide in t h e region of t h e secretory cells, narrowed considerably as it approached t h e common duct. The apocrine duct could be clearly distinguished from t h e common duct since the latter was lined by a minimally keratinized stratified squamous epithelium. Also opening into the base of t h e common duct were several sebaceous acini each consisting of a solid mass of cells without a lumen (fig. 3). The ducts of the sebaceous elements, if present, were extremely short. In t h e anal gland these sebaceous units were not associated with hair follicles. The acini showed a classical histology, with small, apparently undifferentiated cells located at their periphery and large lipid-laden mature cells located centrally. The latter appeared to undergo holocrine secretion accompanied by cellular degeneration. We observed no major differences in histology or ultrastructure between medial and lateral glands or between glands fixed by perfusion or immersion. Similarly, there was no obvious correlation between gland structure or size and t h e sex of the animal. Currently we are investigating t h e effects of age and seasonal variation. Ultrastructure of apocrine components The apocrine portions of t h e anal gland were composed of a t least three morphologically distinct cell types, t h e secretory or apocrine cell, the myoepithelial cell, and t h e duct cell.
271
By electron microscopy, the secretory cells showed a definite ultrastructural polarization (fig. 41, with a large, euchromatic nucleus located basally along with most of t h e rough endoplasmic reticulum. A. well-developed Golgi zone occupied a supranuclear position, and large electron-opaque, membranebounded secretory granules were found underlying t h e apical plasma membrane. The secretory granules all appeared to be of a single type and contained a homogeneous finely granular material similar in electron density to t h e material filling t h e glandular lumen. Mitochondria and smooth endoplasmic reticulum were found throughout t h e cell but tended to be more heavily concentrated in the apical cytoplasm and, to a lesser extent, at t h e base of t h e cells. Free ribosomes, most of them apparently organized into polysomes, were scattered throughout t h e cytoplasm. Several specializations of t h e plasma membrane were observed on secretory cells including apical microvilli, desmosomes, lateral interdigitations, basal infoldings, a n d apical t i g h t junctions. Within this general ultrastructural framework, two entirely different secretory processes were observed, one apocrine and t h e other merocrine in nature. Evidence for a n apocrine-like secretion was observed in those secretory cells which had developed a n apical cap. At t h e electron microscope level (fig. 51, the apical cap was a region of cytoplasm containing microfilaments and occasional free ribosomes, but devoid of secretory granules, mitochondria and other organelles. The plasmalemma in t h e region of the apical cap was generally smooth, with very few microvilli. The caps were often partially segregated from t h e rest of t h e cell by a n incomplete row of small, flat, electron-lucent vesicles (fig. 5, inset). At t h e level of this incomplete demarcation, t h e apical cap developed a constriction, and images were observed which could be interpreted as successive stages in the fusion of t h e demarcation vesicles, and the progressive constriction of t h e neck of t h e apical cap, i.e., as stages in a process of true apocrine secretion. It was observed t h a t secretory granules, although normally not found within a n apical cap, were often located in moderately large numbers immediately beneath t h e layer of demarcation vesicles, suggesting t h a t some mechanism acts specifically to exclude them and most other cytoplasmic elements from the apical cap. Hence, t h e contents of t h e secre-
272
JANET D. SMITH AND GAIL W. HEARN
tory granules were not released as a result of this putative apocrine secretion. Apical caps were observed free in t h e lumen of t h e gland, but without serial sections i t is difficult to exclude the possibility t h a t they might be continuous with a secretory cell in some other plane of section. The contents of t h e apical secretory granules were released by a process of merocrine secretion (fig. 6). In cytochemical studies these vacuoles were shown to be consistently negative for acid phosphatase activity using CMP as substrate, whereas several Golgi-associated cisternae and small vesicles in the same cell contained reaction product (fig. 7). I t would appear t h a t t h e acid phosphatase-negative apical granules do not represent lysosomes, nor do they normally fuse with lysosomes as might be expected if they represented phagosomes. Therefore, t h e apical granules probably contain nonlysosomal secretory product elaborated by the apocrine cells themselves. This product is released via merocrine secretion, i.e., exocytosis. The apical granules appear to develop from large, less electron-opaque condensing vacuoles in the Golgi region. There was no evidence for t h e disruption or dissolution of granules in t h e apical cytoplasm, or for t h e existence of more than one mature granule type. Mitochondria were always readily distinguishable from secretory granules. One feature which, to our knowledge, has not been previously reported in apocrine glands, was the presence of small electronopaque, membrane-bounded granules at t h e basal end of many secretory cells (figs. 4, 8 ) . These tended to occur mainly where the secretory cell was in direct contact with t h e basal lamina and where basal infoldings of the plasmalemma were absent. These basal granules characteristically showed a dense central region separated from t h e membrane by a clear, electron-lucent zone approximately 1217 nm wide. The granules were usually rounded and from 70-250 nm in diameter. However, rod-shaped, crescent-shaped a n d irregular granules were also observed. There was considerable variation from animal to animal in t h e frequency of basal granules, but in several samples they were found in almost every secretory cell and tangential sections of apocrine units revealed large accumulations in the basal cytoplasm. Exactly what these granules represent is not evident. They are clearly distinct from t h e apical granules,
which are larger and have no electron-lucent peripheral zone. The cells of t h e apocrine duct were similar to the secretory cells except t h a t they tended to be cuboidal rather t h a n columnar, their apical microvilli were few in number and they contained very few, if any, secretory granules. The myoepithelial cells were very similar to those described by numerous a u t h o r s i n eccrine sweat glands and axillary apocrine sweat glands (Ellis, '67). The cells formed a n incomplete layer between t h e secretory cells and the basal lamina, and also sent cytoplasmic projections up between secretory cells for considerable distances, although they were never seen to reach the lumen. Desmosomes and interdigitations of t h e plasma membrane were found between neighboring myoepithelial cells and between myoepithelial and secretory cells. Myoepithelial cells contained relatively few cytoplasmic organelles. These were usually localized just beneath the plasma membrane or near t h e nucleus. The cells did however contain abundant filaments scattered throughout t h e cytoplasm. These were associated with t h e numerous dense bodies characteristic of myoepithelial and smooth muscle cells. Also present in abundance were pinocytotic vesicles which were especially numerous on the cell surface adjacent to t h e basal lamina. In addition to t h e secretory and myoepithelial cells, t h e secretory portion of the apocrine gland sometimes contained cells with euchromatic nuclei and a n abundance of free ribosomes but lacking secretory granules, extensive rough endoplasmic reticulum or a well-developed Golgi. It is not clear whether these cells, which were few in number, represent a cell type distinct from secretory and myoepithelial cells, a n undifferentiated cell, or a resting stage of a secretory cell. Within t h e lumen of t h e secretory portion of t h e apocrine units, we occasionally encountered degenerating polymorphonuclear leukocytes and lymphocytes, and both cell types were also observed between secretory cells in the wall of t h e gland, presumably in t h e process of crossing t h e epithelium to reach the lumen. In the lumen, these nucleated cells, with their normal complement of cytoplasmic organelles, were readily distinguishable from apical caps. At t h e basal ends of the secretory and myoepithelial cells, beyond the basal lamina, t h e apocrine units were surrounded by a loose
WOODCHUCK APOCRINE-SEBACEOUS ANAL SCENT GLAND
connective tissue rich in lymphatic capillaries, blood capillaries and wandering cells including mast cells, lymphocytes and neutrophils. The blood capillaries were of t h e fenestrated type. On several occasions unmyelinated nerves containing predominantly electron-lucent synaptic vesicles were observed near myoepithelial cells.
Ultrastructure of sebaceous components The sebaceous acini of the anal gland were located superficial to t h e modified apocrine glands, and often appeared to empty directly into t h e base of the common duct, without any true sebaceous duct (fig. 1).The peripherallylocated immature cells were usually somewhat flattened, with elongated nuclei (fig. 9). They contained moderate amounts of rough and smooth endoplasmic reticulum, numerous free ribosomes, and a well developed Golgi region, but few if any lipid-containing cytoplasmic droplets. In t h e more highly differentiated centrally located cells, t h e nuclei were rounded with prominent nucleoli. Both t h e number and size of t h e individual cytoplasmic lipid droplets were greatly increased. These droplets were often associated with bundles of tonofilaments which were abundant in t h e cytoplasm. Smaller lipid droplets were scattered throughout t h e cytoplasm, while larger ones were often closely associated with t h e nucleus, sometimes deforming i t considerably. Although several different types of cytoplasmic inclusions were observed, including membranous whorls, accumulations of granular material, and large lysosomes, we did not find any regular grid-like arrays of smooth endoplasmic reticulum such as reported in t h e sebaceous glands of other rodents and certain nonhuman primates (Palay, '58; Bell, '74a). Neighboring sebaceous cells, immature as well as mature, showed extensive interdigitation of t h e plasma membrane at shared surfaces and desmosomes were also common. The latter were generally associated with prominent intracytoplasmic tonofilaments. Lymphocytes were occasionally observed between t h e cells of t h e sebaceous acini.
Ultrastructure of secretory product The secretory product in t h e common duct of t h e anal gland is a pasty yellowish-white material which can be collected using roughsurfaced glass rods. Electron microscope examination of pooled secretory material from four woodchucks (fig. 10) revealed the pres-
273
ence of squames of epithelial cells shed from t h e wall of t h e common duct, as well a s numerous rod-shaped and rounded bacteria. Bacteria had not been observed within t h e individual apocrine or sebaceous units, nor in t h e apocrine ducts. They thus appeared to be limited to t h e lumen of t h e common duct. From samples of unfixed secretory material plated on agar and cultured in broth, Proteus mirabilis and Proteus vulgaris were consistently isolated. Membrane-bounded apical caps could not be positively identified in t h e secretory material, and i t seems likely t h a t they break down into unrecognizable components by the time t h e secretory product reaches the common d u c t where i t can be conveniently collected. DISCUSSION
The data presented here support t h e hypothesis t h a t both merocrine and apocrine secretory processes occur in t h e apocrine cells of t h e woodchuck anal scent gland. The release of material from t h e apical, membranebounded secretory granules occurred via a merocrine pathway (fig. 61, while apical caps appeared to pinch off from the remainder of t h e cell cytoplasm as a result of classical apocrine secretion (fig. 5 ) . Apocrine secretion involved: (1)formation of a smooth apical cap usually devoid of microvilli and containing few if any organelles; ( 2 ) appearance of a single row of flattened vesicles oriented to form a n incomplete line of demarcation between t h e apical cap and t h e remainder of cell; and (3) gradual fusion of t h e demarcation vesicles with one another and with the plasma membrane, bringing about a constriction of the neck of t h e apical cap at t h e level of t h e vesicles, and eventual release of t h e cap into t h e glandular lumen. It should be noted t h a t this proposed sequence differs somewhat from t h a t suggested by Schaumberg-Lever and Lever ('75), who reported t h a t , in the human axillary apocrine gland, constriction and separation of t h e apical cap occurred some distance above (i.e., adlumenal to) t h e demarcation vesicles. That apical caps a r e involved in true apocrine secretion and a r e not merely fixation artifacts is suggested by t h e following: (1)apical caps have now been observed in apocrine cells from a variety of glands by workers using different fixatives and fixation procedures (Montes e t al., '60; Hashimoto et al., '66; Kneeland, '66; Schaumberg-Lever and Lever,
274
JANET D. SMITH AND GAIL W. HEARN
'75; Main and Lim, '76); (2) apical caps, with t h e i r organelle-free cytoplasm segregated from the remainder of t h e cell by flattened demarcation vesicles (fig. 5 , inset), have a distinctive ultrastructure suggestive of regional intracellular specialization rather than fixation artifact; (3) constriction of t h e neck of the apical cap usually occurred at t h e level of the demarcation vesicles, suggesting t h a t t h e vesicles were actively involved in t h e process of apocrine secretion. Therefore our data suggest that in addition to merocrine secretion, true apocrine secretion also occurs in t h e apocrine cells of t h e woodchuck anal scent gland. Since both modes of secretion have also been reported in t h e human axillary apocrine gland (Charles, '59; Hashimoto et al., '66; Schaumberg-Lever and Lever, '75), in t h e apocrine cells of t h e human ceruminous gland (Kawabata, '64; Main and Lim, '761, and in the apocrine cells of the lemur antibrachial organ (Kneeland, '66), it is possible t h a t t h e occurrence of both apocrine and merocrine secretion processes is characteristic of apocrine cells in general. The function of t h e observed apocrine secretion remains a matter of speculation. Since secretory granules are excluded from apical caps, true apocrine secretion does not result in the release of t h e granule contents. Nor does the apical cap usually contain any other type of secretory granules, organelles or inclusions which might comprise a well-defined secretory product. The contents of t h e apical membranebounded secretory granules were apparently released into t h e glandular lumen only as a result of merocrine secretion (fig. 6). In contrast with studies on human apocrine glands which reported two or three types of secretory granules in the cytoplasm (Charles, '59; Kurosumi et al., '59; Bell, '74b; Main and Lim, '761, we observed only a single morphological type. Furthermore, t h e electron-opaque matrix of the granules which we observed was quite homogeneous and contained none of t h e globules, grains, locules, and pale areas often illustrated in t h e granules of t h e human axillary apocrine gland (Charles, '59; Kurosumi et al., '59; Bell, '74b). These differences in granule morphology may to some extent reflect real differences in t h e nature of t h e secretory product in different apocrine glands. We, like Main and Lim ('761, found no evidence for t h e transformation of mitochondria into granules, a process which had been suggested earlier by
several investigators (Charles, '59; Kurosumi et al., '61; Yasada et al., '62; Munger, '65). Finally, we did not observe t h e intracytoplasmic rupture or dissolution of secretory granules reported in t h e apocrine cells of human axillary apocrine glands (Montes e t al., '60; Biempica and Montes, '65; Bell, '74b). What we did observe was a classical picture of merocrine secretion characterized by t h e formation of large, relatively electron-lucent vacuoles (condensing vacuoles) in t h e immedia t e vicinity of t h e Golgi; the apparent maturation of t h e condensing vacuoles into somewhat smaller, more electron-opaque granules located in t h e apical cytoplasm; and the release of t h e granular contents via exocytosis. That these homogeneous apical granules in apocrine cells actually represent secretory vacuoles and not lysosomes or phagolysosomes is indicated not only by t h e ultrastructural evidence for the classical pathway of merocrine secretion, but also by the data on acid phosphatase localization. Our results showed t h a t even in cells where t h e Golgi saccules and small lysosomal vesicles contained acid phosphatase reaction product (fig. 71, the apical granules were always negative for this lysosoma1 marker. This agrees with t h e light microscope observations of Biempica and Montes ('651, who reported t h a t in the human axillary apocrine sweat gland t h e lysosomes a n d mature secretory granules were clearly distinguishable. However, in t h e electron microscope they observed a gradation of reaction product, heaviest in young secretory granules and light or absent in mature granules. We saw no staining of secretory granules at any stage of their development in our material. This difference may reflect a variation in the intensity of t h e cytochemical reaction, a real difference in t h e nature of t h e secretory product in t h e different apocrine glands, or a difference in substrate specificity, since Biempica and Montes used P-glycerophosphate rather t h a n cytidine 5'-monophosphate as a substrate. Actual fusion of t h e membrane of the secretory granules and t h e plasmalemma was only infrequently observed, suggesting either t h a t i t occurs very rapidly or t h a t i t occurs relatively rarely. It may be t h a t t h e secretory granules are stored for varying periods in t h e apical cytoplasm and t h a t their contents are then released singly or in a concerted fashion in response to some stimulus. Certainly, we
WOODCHUCK APOCRINE-SEBACEOUS ANAL SCENT GLAND
did observe images which suggested t h e concerted release of secretory product (fig. 6). What the nature of the stimulus triggering this postulated release might be is open to debate. Synaptic regions of unmyelinated nerves were observed near t h e basement membrane of apocrine acini, suggesting t h a t nerve impulses may act on t h e apocrine cell either directly, or indirectly via t h e contraction of myoepithelial cells. Alternatively, the presence of fenestrated capillaries surrounding apocrine acini may indicate a sensitivity to hormonal influence. In t h e basal cytoplasm we observed small round or elongated vesicles, each containing a central dense matrix or core separated from t h e vesicle membrane by a clear zone of regular width. The relative abundance of these vesicles varied greatly from animal to animal. In some specimens numerous examples were present in almost every cell, whereas in others they were barely detectable. The morphology of these granules does not appear to correspond directly to anything previously described in apocrine cells. Kurosumi et al. ('59) described numerous vesicles in t h e basal cytoplasm but these lacked dense cores. Bell ('74b) described dense-cored basal vesicles which she interpreted as immature secretory granules, but t h e cores were irregular and eccentrically placed. In appearance t h e vesicles we observed resemble either certain types of lysosomes (Swift and Hruban, '64; Daems et al., '691, or certain of t h e endocrine granules in entero-endocrine cells (Forssmann et al., '69). We cannot rule out either possibility since, in t h e specimens used for acid phosphatase localization, basal granules happened to be extremely rare. Biempica and Montes ('651, in their light microscope study of acid phosphatase localization in t h e axillary apocrine sweat gland did comment t h a t reactivity was occasionally observed in t h e basal portion of t h e cell, and Montagna and Parks ('48)made similar observations in t h e anal sac of t h e dog. What t h e function of a basally located lysosome might be is not immediately evident. On t h e other hand, a n endocrine function is also possible, and there a r e several precedents for cells which appear to have both exocrine and endocrine functions (Nabeyama, '75). The fact t h a t t h e basal granules were most numerous at points where t h e apocrine cells abutted directly on t h e basal lamina without intervening myoepithelial cells might indicate a release of product from t h e apocrine cell into t h e
275
underlying fenestrated capillaries. However, no morphological evidence for this putative release process was observed. Further experiments will be necessary to clarify t h e nature of these basal granules and to explain the large difference in their numbers from animal to animal. Large numbers of bacteria were identified by electron microscopy in secretory material collected from t h e common duct, but they were not observed in the ducts or secretory acini of t h e apocrine elements themselves. Several of t h e same bacteria (notable Proteus vulgaris and Proteus rnirabilis) were consistently isolated from a number of different animals. I t is possible that, as previously suggested (Shelley, '56; Gorman et al., '741, these bacteria may be important in producing the characteristic odors of the gland by metabolizing certain of the secretory products. In summary, our data suggest, first, t h a t in t h e anal scent gland of t h e woodchuck, t h e apocrine cell is capable of both apocrine and merocrine secretion. Merocrine secretion results in the release of t h e contents of t h e apical vacuoles, while t h e functional significance of apocrine secretion is less clear. Second, our findings indicate t h a t just as merocrine cells from different glands have distinctive morphologies, so too do apocrine cells. Thus t h e apocrine cells of t h e woodchuck anal scent gland differ from those of t h e human axillary apocrine sweat gland and t h e human ceruminous gland in t h e morphology of their secretory vacuoles and the presence of a population of dense-cored basal vesicles. These morphological findings probably reflect differences in t h e nature of the secretory product released by these functionally distinct types of apocrine cells. It should be clear t h a t morphological as well as physiological differences a r e to be expected between apocrine cells from different glands. Finally, a number of problems remain to be investigated, including the nature of t h e dense-cored basal granules seen i n t h e apocrine cells. Also of fundamental interest is t h e relative importance of sebaceous and apocrine cells in producing t h e odorous material used by t h e animal in chemical communication. ACKNOWLEDGMENTS
We thank Dorothy Foster for technical assistance and Lynda Harrison for secretarial assistance.
276
JANET D. SMITH AND GAIL W. HEARN LITERATURE CITED
Bell, M. 1974a A comparative study of the ultrastructure of the sebaceous glands of man and other primates. J. Invest. Dermatol., 62: 132-143. 1974b The ultrastructure of human axillary apocrine glands after epinephrine injection. J. Invest. Dermatol., 63: 147-159. Biempica, L., and L. F. Montes 1965 Secretory epithelium of the large axillary sweat glands. Am. J. Anat., 217: 47-72. Charles, A. 1959 An electron microscope study of the human axillary apocrine gland. J. Anat., 93: 226-232. Daems, W. T., E. Wisse and P . Brederoo 1969 Electron microscopy of the vacuolar apparatus. In: Lysosomes in Biology and Pathology, Vol. 1. J. T. Dingle and H. B. Fell, eds. Amsterdam, North-Holland Publishing Co., pp. 64-112. Ellis, R. A. 1967 Eccrine, sebaceous and apocrine glands. In: Ultrastructure of Normal and Abnormal Skin. A. S. Zelickson, ed. Philadelphia, Lea and Febiger, pp. 132-162. Forssmann, W. G., L. Orci, R. Pictet, A. E. Renold and C. Rouiller 1969 The endocrine cells in the epithelium of the gastrointestinal mucosa of the rat. An electron microscope study. J. Cell Biol., 40: 692-715. Gorman, M. L., D. B. Nedwell and R. M. Smith 1974 An analysis of the contents of the anal scent pockets of Herpestes aurpunctatus (Carnivora: Viverridae). J . Zool. (London), 272: 389-399. Hamilton, W. J., Jr. 1934 The life history of the rufescent woodchuck, Marmota monax rufescens Howell. Ann. Carnegie Mus., 23: 85-178. Hashimoto, K., B. L. Gross and W. F. Lever 1966 Electron microscopic study of apocrine secretion. J. Invest. Dermatol., 46: 378-390. Hibbs. R . G. 1962 Electron microscopy of h u m a n apocrine sweat glands. J. Invest. Dermatol., 38: 77-84. Kawabata, I 1964 Electron microscope studies on the human ceruminous gland. Arch. Histol. Jap., 25: 165-187. Kneeland, J . E. 1966 Fine structure of the sweat glands of the antebrachial organ of Lemur catta. Z. Zellforsch., 73: 521-533. Kurosumi, K., T. Kitamura. and T. Iijima 1959 Electron microscopic studies on human axillary apocrine sweat glands. Arch. Histol. Jap., 26: 523-566. Kurosumi, K.. M. Yamagishi and M. Sekine 1961 Mitochondrial deformation and apocrine secretory mechanism in the rabbit submandibular organ as revealed by electron microscopy. Z. Zellforsch., 55: 297-312. Main, T., and D. Lim 1976 The human external auditory canal secretory system - an ultrastructural study. Laryngoscope, 86- 1164-1176. Montagna, W. 1962 The Structure and Function of Skin. Second ed. New York, Academic Press. Montagna, W., and H. F. Parks 1948 A histochemical study of the anal sac of the dog. Anat. Rec., 200: 297-315.
Montes, L. F., B. L. Baker and A. C. Curtis 1960 The cytology of the large axillary sweat glands in man. J. Invest. Dermatol., 35: 273-291. Muller-Schwarze, D. 1971 Pheromones in the blacktailed deer (Odocoileus hemionus colurnbranusi. Anim. Behav., 29: 141-152. Muller-Schwarze, D., and M. M. Mozell, eds. 1977 Chemical Signals in Vertebrates. New York, Plenum Press. Munger, B. L. 1964 The ultrastructure and histochemistry of human apocrine gland cells. J. Cell Biol., 23: 64 (Abstract). 1965 The cytology of apocrine sweat glands. I. Cat and Monkey. Z. Zellforsch., 67: 373-383. 1971 The histology and cytology of the sweat glands. In: The Skin. E. B. Helwig and F. K. Mostofi, eds. Baltimore, Williams and Wilkins, pp. 47-64. Mykytowycz, R. 1966 Observations on odoriferous and other glands in the Australian wild rabbit, Oryctolagus cuniculus (L.),and the hare, Lepus europaeus P. 11. The inguinal glands. CSIRO Wildl. Res., 22: 49-64. 1970 The roll of skin glands in mammalian communication. In: Communication by Chemical Signals. J. W. Johnston, D. G. Moulton and A. Turk, eds. New York, Appleton-Century-Crofts, pp. 327-360. Nabeyama, A. 1975 Presence of cells combining features of two different cell types in the colonic crypts and pyloric glands of the mouse. Am. J. Anat., 242: 471-484. Palay, S . L. 1958 Morphology of secretion. In: Frontiers of Cytology. S. L. Palay, ed. New Haven, Yale University Press, pp. 305-342. Pearson, 0. P. 1946 Scent glands of the short-tailed shrew. Anat. Rec., 94: 615-625. Quay, W. B., and D. Muller-Schwarze 1970 Functional histology of integumentary glandular regions in black-tailed deer (Odocoileus hemionus colurnbranusi. J . Mammal., 51: 675-694. Schaumberg-Lever, G., and W. F. Lever 1975 Secretion from human apocrine glands: an electron microscopic study. J. Invest. Dermatol., 64: 38-41. Schiefferdecker, P. 1917 Die Hautdrusen des Menschen u n d des S a u g e t i e r e s , i h r e biologische und rassenanatomische Bedeutung sowie die Muscularis sexualis. Zentralbl. Biol., 37: 534-562. Shelley, W. B. 1956 The role of apocrine sweat in the production ofaxillaryodor. J. SOC.Cosmet. Chem., 7: 171-175. Sleggs, G. F. 1926 The adult anatomy and histology of the anal glands of the Richardson ground squirrel, Citellus richardsonii, Sabine. Anat. Rec., 32: 1-43. Swift, H., and Z. Hruban 1964 Focal degradation as a biological process. Fed. Proc., 23: 1026-1037. Yasada, K., R. A. Ellis and W. Montagna 1962 The fine structural relationship between mitochondria and light granules in the human apocrine sweat glands. Okajima Folia Anat. Jap., 38: 455-483.
PLATES
PLATE 1 EXPLANATION OF FIGURE
1 Diagrammatic representation of one of the three anal glands of the woodchuck, showing its communication with the anal canal. Each gland consists of multiple sebaceous and apocrine components which empty into the base of a single common duct. The apocrine secretory acini lie deeper in the surrounding loose connective tissue, and communicate with the common duct via the ducts of the apocrine gland. In contrast, the sebaceous acini characteristically lie closer to the common duct and appear to empty directly into its basal end.
278
WOODCHUCK APOCRINE SEBACEOUS A N A L S C E N T (;LAND Janet D Smith and (:ail W Hparn
f'l.A'lE 1
279
PLATE 2
2
Light microscopic view of a n apocrine secretory acinus and an apocrine duct lnsteriskl Secretory acini consisted of a single layer of cuboidal t o columnar secretory or apocrine cells resting on a n incomplete layer of myoepithelial cells The apocrine cells often contained accumulations of d a r k ~ s t a i n i n gsecretory granules in their apical cytoplasm (arrowhead, lower r i g h t ) . and many showed evidence of apical cap formation (arrow1 In contrast. t h e cells of t h e apocrine duct showed little e v dence of either secretory granules or apical caps However, like t h e wall of the acinus. t h e wall of t h e duct did contain myoepithelial cells. These a r e particularly evident where t h e duct has heen sectioned somewhat tangentially (arrowhead. upper left). One-micron Epon section stained with toluidine blue. X ,150.
:i Light microscopic view of several of the sebaceous acini of t h e anal gland Note a t
t h e periphery of each cluster of cells t h e small undifferentiated cells. and more centrally t h e larger differentiated cells with their accumulation of multiple cytoplasmic lipid droplets. The sebaceous acini. like t h e apocrine, were surrounded by a rich plexus of lymphatic and blood capillaries In t h e anal gland. sebaceous cells were not ociated with hair follicles. One-micron Epon section stained with toluidine hlue X 350
280
WOODCHUCK APOCRINE-SEUACEOUS ANAL S C E N T G L A N D Janet D. Smith and tiall W Hearn
PLATE 2
PLATE 3
4
282
The secretory cells of a n apocrine acinus contain a basal nucleus. a well-developed supranuclear Golgi apparatus (arrow) often with numerous condensing vacuoles, and electron-opaque secretory granules in t h e apical cytoplasm. These cells, with their apical microvilli and their secretory granules immediatelv beneath t h e apical plasma membrane. have not formed apical caps. The cells rest on an incomplete layer of myoepithelial cells. Note also t h e collection of small dense granules in t h e basal cytoplasm of a neighboring secretory cell (arrowhead). The n a t u r e and function of these granules is unclear 6.000
WOODCHUCK APOCKINE SEBACEOUb A N A L SC'ENT GLAND J a n e t D Smith and Gail W Hearn
PI ATE :I
283
PLATE 4 EXPLANATIOK OF F I G U R E S
284
5
Two neighboring apocrine secretory cells with apical caps which protrude into t h e lumen. Microvilli are typically lacking on t h a t portion of t h e plasma memhrane which covers t h e apical cap. The caps usually contain no secretory granules or other cytoplasmic organelles, although these may be present in great numbers in the c y t o ~ plasm immediately below t h e cap. At higher magnification ( i n s e t ) it is often possible to detect a n incomplete row of flattened vesicles located a t t h e level of t h e constriction between t h e apical cap and t h e remainder of the cytoplasm (arrowheadsl. These vesicles appear to fuse with one another, leading to t h e eventual separation of t h e apical cap from t h e cell. X 11,800, inset X 15,600.
6
At several points [arrowheads),t h e membranes of t h e secretory granules of apocrine secretory cells have fused with each other a n d with t h e plasma membrane to release their contents into t h e lumen in a process of t r u e merncrine secretion. The arrow indicates t h e level of the apical tight junctions between secretory cells. This sample was incubated for acid phosphatase activity and shows no localization of enzyme within t h e secretory granules. X 15.300
WOODCHUCK APOCRINE SEBACEOUS ANAI, SCENT G L A N D .Janet D
S m i t h and Gail W
PI.A'I'b: 4
Hearn
285
PL.ATE 5
286
7
Apocrine secretory cells incubated for acid phosphatase activity show dark reaction product over small vesicles and some cisternae in t h e Golg, region (arrowheads].hut t h e apical granules are always negative for this lysosomal marker enzyme. This indicates t h a t t h e apical granules probably a r e neither lysosomes nor phagw Iysosomes. and t h a t they would therefore appear to represent secretory vacuoles containing a nonlysosomal product synthesized by the apocrine cell. X 5,500
8
Two cytoplasmic processes from apocrine secretory cells a r e interposed between a myoepithelial cell and the basal lamina surrounding t h e apocrine acinus. The apocrine cell cytoplasm contains numerous small granules with dense cores which are separated from t h e limiting membrane of t h e granule by a clear zone of regular width. Such granules were abundant in t h e basal cytoplasm of some samples. especially in locations such a s here, where t h e apocrine cells or cell processes abutted directly on t h e basal lamina. Also common in t h e basal cytoplasm were numerous clear vesicles probably representing pinocytotic vesicles. and multivesicular bodies. See also figure 4 for a coinparison between basal granules and apical secretory granules. X 15.000.
WOODCHUCK APOCRINE SEBACEOUS ANAL SCENT G L A N D J a n e t D Smith and Gail W Hearn
PIATE 5
287
9
288
Piirtiotih of twu sehacciius a c i n i art' w p a r a t r d t i v c~illagrnou.;connective t i s h u t > c i w taining B netwtirk uf fianestrated hlui~dc;rpillaries In each aciiius t h e smaller p e ~ riphei a1 cells with few lipid droplets rtxpresrnt iindiffcrentiated stern cells. while f sizr and niimher are t h e larger central c e l l s containing lipid droplets i ~ increasing of iiii'frrrntiating. The seh;iceiius cells contain prominent bundles of tmofilarnents iarrowheadl in t h e cytoplasni. a n d a r e jiiined to o n e another via dark Iy btiiininp desniosorncs X 4.600.
WOODCHUCK APOCHINE SERA('EOUS A N A L SC'EkT G L A N D J a n e t 1) S m i t h a n d Gail W Hear"
1'I.A'lt f i
289
PLATE 7 E X P L A N A T I O N OF FIGITRE
10 Thin sections through fixed secretorv material collected from t h e common duct of
t h e anal gland showed squames of epithelial cells (arrow) probably shed from t h e wall of t h e common duct itself, a s well as numerous round a n d rod-shaped bacteria x
290
um.
WOODCHIJCK APOCKINE SEBACEOUS ANAI. SCENT G L A N D J a n e t Ll S m i t h and Gail W Hearn
['[.ATE T
29 1
ABSTRACT
Light and electron microscope studies of t h e woodchuck anal scent gland revealed t h a t i t is composed of apocrine and sebaceous components emptying into a common duct. In t h e apocrine acini, t h e single secretory cell type showed evidence of both merocrine and apocrine secretion. Merocrine secretion resulted in t h e release of t h e contents of apical secretory granules while apocrine secretion released apical caps of cytoplasm by a process involving the following: (1)formation of a n apical cap, usually containing no organelles or secretory granules; (2) appearance of a single row of flattened vesicles forming a n incomplete barrier between t h e apical cap and t h e remaining cell cytoplasm; and (3) fusion between vesicles and plasmalemma, causing progressive constriction of t h e neck of t h e apical cap and eventual cap release. Since both merocrine and apocrine secretory processes have been reported in three other types of apocrine glands, i t is likely t h a t t h e occurrence of both processes in a single cell is a general characteristic of apocrine cells. Several features apparently unique to these particular apocrine cells were observed, including secretory granules of a single morphological type and a population of small dense-cored basal vesicles of unknown function. Therefore, i t would appear t h a t , just as with merocrine cells, apocrine cells from different types of glands also have distinctive morphologies which probably reflect real differences in their functions and products.
Since Schiefferdecker's report ('171, histologists have separated glandular cells into three categories based on their mode of secretion: merocrine cells, which release their product from secretory granules, usually without loss of cytoplasm; apocrine cells which pinch off a portion of apical cytoplasm (the apical cap) as part of t h e secretory product; and holocrine cells which accumulate secretory product within t h e cytoplasm and release i t by disintegration of t h e entire cell. However, in recent years, numerous workers have questioned t h e existence of t r u e apocrine, i.e., "decapitation'' secretion. For example, electron microscope studies on t h e supposedly apocrine cells of t h e human axillary apocrine gland failed to demonstrate conclusively either apocrine secretion or t h e expected cell debris in ANAT. REC. (1979) 193: 269-292.
t h e glandular lumen, and revealed instead a merocrine mechanism of secretion (Montes e t al., '60; Montagna, '62; Biempica and Montes, '65). Although secretory cells with apical caps were frequently observed, the caps were considered to be a n artifact of fixation (Munger, '64, '65, '71). Other investigators, however, have continued to argue for t h e additional existence of a t r u e apocrine secretory process in t h e human axillary apocrine gland (Charles, '59; Kurosumi et al., '59; Hibbs, '62; Hashimoto e t al., '66; Schaumberg-Lever and Lever, '75). And, in studying t h e ultrastructure of the closely related human ceruminous gland, Kawabata ('64) and later Main and Lim ('76) found eviReceived June 27, '78. Accepted Sept. 5, '78.
269
2 70
JANET D. SMITH AND GAIL W. HEARN
dence t h a t t h e single type of apocrine cell observed could carry out both apocrine and merocrine secretion. One goal of the present study was to determine t h e mode or modes of secretion in a different type of apocrine cell, namely those of a mammalian scent gland. A second goal was to provide a basic description of a typical mammalian scent gland a t the electron microscope level. Light microscope studies have indicated t h a t many such glands are composed of a n apocrine and a sebaceous component (Mykytowycz, '70). Examples include t h e metatarsal glands of the black-tailed deer, Odocoileus hemionus (Quay and Muller-Schwarze, '701, t h e lateral gland of the short-tailed shrew, Blarina breuicauda (Pearson, '461, t h e a n a l gland of Richardson's ground squirrel Citellus richardsonii (Sleggs, '261, all of which produce a n alarm odor (Muller-Schwarze, '711; and t h e inguinal glands of t h e rabbit, Oryctolagus cuniculus, which produce a n odor indicating sexual and social status (Mykytowycz, '66). Despite the recent interest in mammalian chemical communication (Muller-Schwarze and Mozell, '77) we know of only one previous electron microscope study of a mammalian scent gland, Kneeland's ('66) report on t h e lemur antibrachial organ. Furthermore t h e antibrachial organ would appear to be a somewhat atypical scent gland since i t lacks a sebaceous component. We therefore undertook to provide a n ultrastructural description of a typical apocrine-sebaceous mammalian scent gland, t h e anal gland of t h e woodchuck, Marmots monax. Field studies on a variety of ground squirrels including t h e woodchuck indicate t h a t these glands produce a n alarm odor (Hamilton, '34). MATERIALS AND METHODS
For routine electron microscopy t h e lateral and/or medial anal glands from each of 1 3 woodchucks were fixed either by perfusion of anesthetized animals with glutaraldehyde (1.25% followed by 2.5%) in 0.1 M sodium cacodylate, pH 7.2, or by immersion fixation of the dissected glands for two to eight hours in 2.5% cacodylate-buffered glutaraldehyde. The glands were minced, postfixed with 1%OsO, and then with uranyl acetate, dehydrated and embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate, carboncoated, and examined in a Philips 200 or JEOL 100-C transmission electron microscope at a n accelerating voltage of 80 kv.
For localization of acid phosphatase activity, samples were fixed for a minimum of two hours in 2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.2, rinsed thoroughly (18-72 hours) in several changes of cacodylate buffer containing 5% sucrose and then incubated in a 37°C waterbath in a reaction mixture containing t h e following: 25 mg cytidine 5'-mOnOphosphate (CMP) (sodium salt, Sigma Chemical Co.); 12 ml deionized H,O; 10 mlO.l M Tris maleate buffer, pH 5.2; 3 ml 1%aqueous lead nitrate; and 5% sucrose. Prior to incubation t h e reaction mixture was filtered through Whatman no. 50 filter paper, incubated for one hour in a 37°C waterbath, and refiltered. After incubation t h e samples were fixed for 90 minutes in 1%OsO, in 0.1 M cacodylate buffer, and then dehydrated and embedded in Epon. Further processing was as described above, except that some sections were observed without staining. The thick pasty secretory product collected from the common duct of t h e anal glands of four woodchucks was pooled and processed using glutaraldehyde fixation and OsO, postfixation followed by routine dehydration and embedding. One-micron sections of all samples were stained with toluidine blue and observed by light microscopy. RESULTS
Gross anatomy The wall of t h e woodchuck anal canal contains three separate anal glands, two disposed laterally and one on t h e ventromedial surface of t h e canal. As shown in figure 1,each gland is a n anatomically complex structure containing both apocrine and sebaceous elements. Ducts from several apocrine and sebaceous units empty into t h e distal end of a single common duct which in t u r n communicates with t h e anal canal near t h e anocutaneous junction. A common sheath of striated skeletal muscle surrounds t h e three glands. When aroused, t h e woodchuck can evert its anal glands, apparently as a result of voluntary contraction of these striated muscles. In t h e everted state, t h e glands present as papillae whose tips represent t h e base of the common duct. Light microscopy Histologically, t h e various regions of t h e gland were well-defined. The apocrine units were simple coiled tubular or tubuloalveolar
WOODCHUCK APOCRINE-SEBACEOUS ANAL SCENT GLAND
glands, whose secretory portion consisted of a single layer of cuboidal to columnar cells resting on a n incomplete layer of myoepithelial cells (fig. 2). Each apocrine unit had a wide and distinct lumen, usually filled with a substance which stained deeply with toluidine blue. In 1 p sections i t was often possible to distinguish dark-staining secretory granules at t h e apical or lumenal border of t h e cells. A smaller number of secretory cells had prominent apical caps, t h a t is, clear rounded projections of t h e apical cytoplasm which appeared to be pinching off from the rest of the cell in a process of t r u e apocrine secretion. In randomly cut sections apical caps were sometimes observed apparently free in t h e lumen. The apocrine duct was lined by cuboidal cells similar in appearance to secretory cells but lacking large accumulations of secretory granules, and lacking apical caps. Near t h e secretory acini the duct walls did contain myoepithelial cells. From reconstructions of serial sections, i t was found t h a t t h e duct, although initially quite wide in t h e region of t h e secretory cells, narrowed considerably as it approached t h e common duct. The apocrine duct could be clearly distinguished from t h e common duct since the latter was lined by a minimally keratinized stratified squamous epithelium. Also opening into the base of t h e common duct were several sebaceous acini each consisting of a solid mass of cells without a lumen (fig. 3). The ducts of the sebaceous elements, if present, were extremely short. In t h e anal gland these sebaceous units were not associated with hair follicles. The acini showed a classical histology, with small, apparently undifferentiated cells located at their periphery and large lipid-laden mature cells located centrally. The latter appeared to undergo holocrine secretion accompanied by cellular degeneration. We observed no major differences in histology or ultrastructure between medial and lateral glands or between glands fixed by perfusion or immersion. Similarly, there was no obvious correlation between gland structure or size and t h e sex of the animal. Currently we are investigating t h e effects of age and seasonal variation. Ultrastructure of apocrine components The apocrine portions of t h e anal gland were composed of a t least three morphologically distinct cell types, t h e secretory or apocrine cell, the myoepithelial cell, and t h e duct cell.
271
By electron microscopy, the secretory cells showed a definite ultrastructural polarization (fig. 41, with a large, euchromatic nucleus located basally along with most of t h e rough endoplasmic reticulum. A. well-developed Golgi zone occupied a supranuclear position, and large electron-opaque, membranebounded secretory granules were found underlying t h e apical plasma membrane. The secretory granules all appeared to be of a single type and contained a homogeneous finely granular material similar in electron density to t h e material filling t h e glandular lumen. Mitochondria and smooth endoplasmic reticulum were found throughout t h e cell but tended to be more heavily concentrated in the apical cytoplasm and, to a lesser extent, at t h e base of t h e cells. Free ribosomes, most of them apparently organized into polysomes, were scattered throughout t h e cytoplasm. Several specializations of t h e plasma membrane were observed on secretory cells including apical microvilli, desmosomes, lateral interdigitations, basal infoldings, a n d apical t i g h t junctions. Within this general ultrastructural framework, two entirely different secretory processes were observed, one apocrine and t h e other merocrine in nature. Evidence for a n apocrine-like secretion was observed in those secretory cells which had developed a n apical cap. At t h e electron microscope level (fig. 51, the apical cap was a region of cytoplasm containing microfilaments and occasional free ribosomes, but devoid of secretory granules, mitochondria and other organelles. The plasmalemma in t h e region of the apical cap was generally smooth, with very few microvilli. The caps were often partially segregated from t h e rest of t h e cell by a n incomplete row of small, flat, electron-lucent vesicles (fig. 5, inset). At t h e level of this incomplete demarcation, t h e apical cap developed a constriction, and images were observed which could be interpreted as successive stages in the fusion of t h e demarcation vesicles, and the progressive constriction of t h e neck of t h e apical cap, i.e., as stages in a process of true apocrine secretion. It was observed t h a t secretory granules, although normally not found within a n apical cap, were often located in moderately large numbers immediately beneath t h e layer of demarcation vesicles, suggesting t h a t some mechanism acts specifically to exclude them and most other cytoplasmic elements from the apical cap. Hence, t h e contents of t h e secre-
272
JANET D. SMITH AND GAIL W. HEARN
tory granules were not released as a result of this putative apocrine secretion. Apical caps were observed free in t h e lumen of t h e gland, but without serial sections i t is difficult to exclude the possibility t h a t they might be continuous with a secretory cell in some other plane of section. The contents of t h e apical secretory granules were released by a process of merocrine secretion (fig. 6). In cytochemical studies these vacuoles were shown to be consistently negative for acid phosphatase activity using CMP as substrate, whereas several Golgi-associated cisternae and small vesicles in the same cell contained reaction product (fig. 7). I t would appear t h a t t h e acid phosphatase-negative apical granules do not represent lysosomes, nor do they normally fuse with lysosomes as might be expected if they represented phagosomes. Therefore, t h e apical granules probably contain nonlysosomal secretory product elaborated by the apocrine cells themselves. This product is released via merocrine secretion, i.e., exocytosis. The apical granules appear to develop from large, less electron-opaque condensing vacuoles in the Golgi region. There was no evidence for t h e disruption or dissolution of granules in t h e apical cytoplasm, or for t h e existence of more than one mature granule type. Mitochondria were always readily distinguishable from secretory granules. One feature which, to our knowledge, has not been previously reported in apocrine glands, was the presence of small electronopaque, membrane-bounded granules at t h e basal end of many secretory cells (figs. 4, 8 ) . These tended to occur mainly where the secretory cell was in direct contact with t h e basal lamina and where basal infoldings of the plasmalemma were absent. These basal granules characteristically showed a dense central region separated from t h e membrane by a clear, electron-lucent zone approximately 1217 nm wide. The granules were usually rounded and from 70-250 nm in diameter. However, rod-shaped, crescent-shaped a n d irregular granules were also observed. There was considerable variation from animal to animal in t h e frequency of basal granules, but in several samples they were found in almost every secretory cell and tangential sections of apocrine units revealed large accumulations in the basal cytoplasm. Exactly what these granules represent is not evident. They are clearly distinct from t h e apical granules,
which are larger and have no electron-lucent peripheral zone. The cells of t h e apocrine duct were similar to the secretory cells except t h a t they tended to be cuboidal rather t h a n columnar, their apical microvilli were few in number and they contained very few, if any, secretory granules. The myoepithelial cells were very similar to those described by numerous a u t h o r s i n eccrine sweat glands and axillary apocrine sweat glands (Ellis, '67). The cells formed a n incomplete layer between t h e secretory cells and the basal lamina, and also sent cytoplasmic projections up between secretory cells for considerable distances, although they were never seen to reach the lumen. Desmosomes and interdigitations of t h e plasma membrane were found between neighboring myoepithelial cells and between myoepithelial and secretory cells. Myoepithelial cells contained relatively few cytoplasmic organelles. These were usually localized just beneath the plasma membrane or near t h e nucleus. The cells did however contain abundant filaments scattered throughout t h e cytoplasm. These were associated with t h e numerous dense bodies characteristic of myoepithelial and smooth muscle cells. Also present in abundance were pinocytotic vesicles which were especially numerous on the cell surface adjacent to t h e basal lamina. In addition to t h e secretory and myoepithelial cells, t h e secretory portion of the apocrine gland sometimes contained cells with euchromatic nuclei and a n abundance of free ribosomes but lacking secretory granules, extensive rough endoplasmic reticulum or a well-developed Golgi. It is not clear whether these cells, which were few in number, represent a cell type distinct from secretory and myoepithelial cells, a n undifferentiated cell, or a resting stage of a secretory cell. Within t h e lumen of t h e secretory portion of t h e apocrine units, we occasionally encountered degenerating polymorphonuclear leukocytes and lymphocytes, and both cell types were also observed between secretory cells in the wall of t h e gland, presumably in t h e process of crossing t h e epithelium to reach the lumen. In the lumen, these nucleated cells, with their normal complement of cytoplasmic organelles, were readily distinguishable from apical caps. At t h e basal ends of the secretory and myoepithelial cells, beyond the basal lamina, t h e apocrine units were surrounded by a loose
WOODCHUCK APOCRINE-SEBACEOUS ANAL SCENT GLAND
connective tissue rich in lymphatic capillaries, blood capillaries and wandering cells including mast cells, lymphocytes and neutrophils. The blood capillaries were of t h e fenestrated type. On several occasions unmyelinated nerves containing predominantly electron-lucent synaptic vesicles were observed near myoepithelial cells.
Ultrastructure of sebaceous components The sebaceous acini of the anal gland were located superficial to t h e modified apocrine glands, and often appeared to empty directly into t h e base of the common duct, without any true sebaceous duct (fig. 1).The peripherallylocated immature cells were usually somewhat flattened, with elongated nuclei (fig. 9). They contained moderate amounts of rough and smooth endoplasmic reticulum, numerous free ribosomes, and a well developed Golgi region, but few if any lipid-containing cytoplasmic droplets. In t h e more highly differentiated centrally located cells, t h e nuclei were rounded with prominent nucleoli. Both t h e number and size of t h e individual cytoplasmic lipid droplets were greatly increased. These droplets were often associated with bundles of tonofilaments which were abundant in t h e cytoplasm. Smaller lipid droplets were scattered throughout t h e cytoplasm, while larger ones were often closely associated with t h e nucleus, sometimes deforming i t considerably. Although several different types of cytoplasmic inclusions were observed, including membranous whorls, accumulations of granular material, and large lysosomes, we did not find any regular grid-like arrays of smooth endoplasmic reticulum such as reported in t h e sebaceous glands of other rodents and certain nonhuman primates (Palay, '58; Bell, '74a). Neighboring sebaceous cells, immature as well as mature, showed extensive interdigitation of t h e plasma membrane at shared surfaces and desmosomes were also common. The latter were generally associated with prominent intracytoplasmic tonofilaments. Lymphocytes were occasionally observed between t h e cells of t h e sebaceous acini.
Ultrastructure of secretory product The secretory product in t h e common duct of t h e anal gland is a pasty yellowish-white material which can be collected using roughsurfaced glass rods. Electron microscope examination of pooled secretory material from four woodchucks (fig. 10) revealed the pres-
273
ence of squames of epithelial cells shed from t h e wall of t h e common duct, as well a s numerous rod-shaped and rounded bacteria. Bacteria had not been observed within t h e individual apocrine or sebaceous units, nor in t h e apocrine ducts. They thus appeared to be limited to t h e lumen of t h e common duct. From samples of unfixed secretory material plated on agar and cultured in broth, Proteus mirabilis and Proteus vulgaris were consistently isolated. Membrane-bounded apical caps could not be positively identified in t h e secretory material, and i t seems likely t h a t they break down into unrecognizable components by the time t h e secretory product reaches the common d u c t where i t can be conveniently collected. DISCUSSION
The data presented here support t h e hypothesis t h a t both merocrine and apocrine secretory processes occur in t h e apocrine cells of t h e woodchuck anal scent gland. The release of material from t h e apical, membranebounded secretory granules occurred via a merocrine pathway (fig. 61, while apical caps appeared to pinch off from the remainder of t h e cell cytoplasm as a result of classical apocrine secretion (fig. 5 ) . Apocrine secretion involved: (1)formation of a smooth apical cap usually devoid of microvilli and containing few if any organelles; ( 2 ) appearance of a single row of flattened vesicles oriented to form a n incomplete line of demarcation between t h e apical cap and t h e remainder of cell; and (3) gradual fusion of t h e demarcation vesicles with one another and with the plasma membrane, bringing about a constriction of the neck of t h e apical cap at t h e level of t h e vesicles, and eventual release of t h e cap into t h e glandular lumen. It should be noted t h a t this proposed sequence differs somewhat from t h a t suggested by Schaumberg-Lever and Lever ('75), who reported t h a t , in the human axillary apocrine gland, constriction and separation of t h e apical cap occurred some distance above (i.e., adlumenal to) t h e demarcation vesicles. That apical caps a r e involved in true apocrine secretion and a r e not merely fixation artifacts is suggested by t h e following: (1)apical caps have now been observed in apocrine cells from a variety of glands by workers using different fixatives and fixation procedures (Montes e t al., '60; Hashimoto et al., '66; Kneeland, '66; Schaumberg-Lever and Lever,
274
JANET D. SMITH AND GAIL W. HEARN
'75; Main and Lim, '76); (2) apical caps, with t h e i r organelle-free cytoplasm segregated from the remainder of t h e cell by flattened demarcation vesicles (fig. 5 , inset), have a distinctive ultrastructure suggestive of regional intracellular specialization rather than fixation artifact; (3) constriction of t h e neck of the apical cap usually occurred at t h e level of the demarcation vesicles, suggesting t h a t t h e vesicles were actively involved in t h e process of apocrine secretion. Therefore our data suggest that in addition to merocrine secretion, true apocrine secretion also occurs in t h e apocrine cells of t h e woodchuck anal scent gland. Since both modes of secretion have also been reported in t h e human axillary apocrine gland (Charles, '59; Hashimoto et al., '66; Schaumberg-Lever and Lever, '75), in t h e apocrine cells of t h e human ceruminous gland (Kawabata, '64; Main and Lim, '761, and in the apocrine cells of the lemur antibrachial organ (Kneeland, '66), it is possible t h a t t h e occurrence of both apocrine and merocrine secretion processes is characteristic of apocrine cells in general. The function of t h e observed apocrine secretion remains a matter of speculation. Since secretory granules are excluded from apical caps, true apocrine secretion does not result in the release of t h e granule contents. Nor does the apical cap usually contain any other type of secretory granules, organelles or inclusions which might comprise a well-defined secretory product. The contents of t h e apical membranebounded secretory granules were apparently released into t h e glandular lumen only as a result of merocrine secretion (fig. 6). In contrast with studies on human apocrine glands which reported two or three types of secretory granules in the cytoplasm (Charles, '59; Kurosumi et al., '59; Bell, '74b; Main and Lim, '761, we observed only a single morphological type. Furthermore, t h e electron-opaque matrix of the granules which we observed was quite homogeneous and contained none of t h e globules, grains, locules, and pale areas often illustrated in t h e granules of t h e human axillary apocrine gland (Charles, '59; Kurosumi et al., '59; Bell, '74b). These differences in granule morphology may to some extent reflect real differences in t h e nature of t h e secretory product in different apocrine glands. We, like Main and Lim ('761, found no evidence for t h e transformation of mitochondria into granules, a process which had been suggested earlier by
several investigators (Charles, '59; Kurosumi et al., '61; Yasada et al., '62; Munger, '65). Finally, we did not observe t h e intracytoplasmic rupture or dissolution of secretory granules reported in t h e apocrine cells of human axillary apocrine glands (Montes e t al., '60; Biempica and Montes, '65; Bell, '74b). What we did observe was a classical picture of merocrine secretion characterized by t h e formation of large, relatively electron-lucent vacuoles (condensing vacuoles) in t h e immedia t e vicinity of t h e Golgi; the apparent maturation of t h e condensing vacuoles into somewhat smaller, more electron-opaque granules located in t h e apical cytoplasm; and the release of t h e granular contents via exocytosis. That these homogeneous apical granules in apocrine cells actually represent secretory vacuoles and not lysosomes or phagolysosomes is indicated not only by t h e ultrastructural evidence for the classical pathway of merocrine secretion, but also by the data on acid phosphatase localization. Our results showed t h a t even in cells where t h e Golgi saccules and small lysosomal vesicles contained acid phosphatase reaction product (fig. 71, the apical granules were always negative for this lysosoma1 marker. This agrees with t h e light microscope observations of Biempica and Montes ('651, who reported t h a t in the human axillary apocrine sweat gland t h e lysosomes a n d mature secretory granules were clearly distinguishable. However, in t h e electron microscope they observed a gradation of reaction product, heaviest in young secretory granules and light or absent in mature granules. We saw no staining of secretory granules at any stage of their development in our material. This difference may reflect a variation in the intensity of t h e cytochemical reaction, a real difference in t h e nature of t h e secretory product in t h e different apocrine glands, or a difference in substrate specificity, since Biempica and Montes used P-glycerophosphate rather t h a n cytidine 5'-monophosphate as a substrate. Actual fusion of t h e membrane of the secretory granules and t h e plasmalemma was only infrequently observed, suggesting either t h a t i t occurs very rapidly or t h a t i t occurs relatively rarely. It may be t h a t t h e secretory granules are stored for varying periods in t h e apical cytoplasm and t h a t their contents are then released singly or in a concerted fashion in response to some stimulus. Certainly, we
WOODCHUCK APOCRINE-SEBACEOUS ANAL SCENT GLAND
did observe images which suggested t h e concerted release of secretory product (fig. 6). What the nature of the stimulus triggering this postulated release might be is open to debate. Synaptic regions of unmyelinated nerves were observed near t h e basement membrane of apocrine acini, suggesting t h a t nerve impulses may act on t h e apocrine cell either directly, or indirectly via t h e contraction of myoepithelial cells. Alternatively, the presence of fenestrated capillaries surrounding apocrine acini may indicate a sensitivity to hormonal influence. In t h e basal cytoplasm we observed small round or elongated vesicles, each containing a central dense matrix or core separated from t h e vesicle membrane by a clear zone of regular width. The relative abundance of these vesicles varied greatly from animal to animal. In some specimens numerous examples were present in almost every cell, whereas in others they were barely detectable. The morphology of these granules does not appear to correspond directly to anything previously described in apocrine cells. Kurosumi et al. ('59) described numerous vesicles in t h e basal cytoplasm but these lacked dense cores. Bell ('74b) described dense-cored basal vesicles which she interpreted as immature secretory granules, but t h e cores were irregular and eccentrically placed. In appearance t h e vesicles we observed resemble either certain types of lysosomes (Swift and Hruban, '64; Daems et al., '691, or certain of t h e endocrine granules in entero-endocrine cells (Forssmann et al., '69). We cannot rule out either possibility since, in t h e specimens used for acid phosphatase localization, basal granules happened to be extremely rare. Biempica and Montes ('651, in their light microscope study of acid phosphatase localization in t h e axillary apocrine sweat gland did comment t h a t reactivity was occasionally observed in t h e basal portion of t h e cell, and Montagna and Parks ('48)made similar observations in t h e anal sac of t h e dog. What t h e function of a basally located lysosome might be is not immediately evident. On t h e other hand, a n endocrine function is also possible, and there a r e several precedents for cells which appear to have both exocrine and endocrine functions (Nabeyama, '75). The fact t h a t t h e basal granules were most numerous at points where t h e apocrine cells abutted directly on t h e basal lamina without intervening myoepithelial cells might indicate a release of product from t h e apocrine cell into t h e
275
underlying fenestrated capillaries. However, no morphological evidence for this putative release process was observed. Further experiments will be necessary to clarify t h e nature of these basal granules and to explain the large difference in their numbers from animal to animal. Large numbers of bacteria were identified by electron microscopy in secretory material collected from t h e common duct, but they were not observed in the ducts or secretory acini of t h e apocrine elements themselves. Several of t h e same bacteria (notable Proteus vulgaris and Proteus rnirabilis) were consistently isolated from a number of different animals. I t is possible that, as previously suggested (Shelley, '56; Gorman et al., '741, these bacteria may be important in producing the characteristic odors of the gland by metabolizing certain of the secretory products. In summary, our data suggest, first, t h a t in t h e anal scent gland of t h e woodchuck, t h e apocrine cell is capable of both apocrine and merocrine secretion. Merocrine secretion results in the release of t h e contents of t h e apical vacuoles, while t h e functional significance of apocrine secretion is less clear. Second, our findings indicate t h a t just as merocrine cells from different glands have distinctive morphologies, so too do apocrine cells. Thus t h e apocrine cells of t h e woodchuck anal scent gland differ from those of t h e human axillary apocrine sweat gland and t h e human ceruminous gland in t h e morphology of their secretory vacuoles and the presence of a population of dense-cored basal vesicles. These morphological findings probably reflect differences in t h e nature of the secretory product released by these functionally distinct types of apocrine cells. It should be clear t h a t morphological as well as physiological differences a r e to be expected between apocrine cells from different glands. Finally, a number of problems remain to be investigated, including the nature of t h e dense-cored basal granules seen i n t h e apocrine cells. Also of fundamental interest is t h e relative importance of sebaceous and apocrine cells in producing t h e odorous material used by t h e animal in chemical communication. ACKNOWLEDGMENTS
We thank Dorothy Foster for technical assistance and Lynda Harrison for secretarial assistance.
276
JANET D. SMITH AND GAIL W. HEARN LITERATURE CITED
Bell, M. 1974a A comparative study of the ultrastructure of the sebaceous glands of man and other primates. J. Invest. Dermatol., 62: 132-143. 1974b The ultrastructure of human axillary apocrine glands after epinephrine injection. J. Invest. Dermatol., 63: 147-159. Biempica, L., and L. F. Montes 1965 Secretory epithelium of the large axillary sweat glands. Am. J. Anat., 217: 47-72. Charles, A. 1959 An electron microscope study of the human axillary apocrine gland. J. Anat., 93: 226-232. Daems, W. T., E. Wisse and P . Brederoo 1969 Electron microscopy of the vacuolar apparatus. In: Lysosomes in Biology and Pathology, Vol. 1. J. T. Dingle and H. B. Fell, eds. Amsterdam, North-Holland Publishing Co., pp. 64-112. Ellis, R. A. 1967 Eccrine, sebaceous and apocrine glands. In: Ultrastructure of Normal and Abnormal Skin. A. S. Zelickson, ed. Philadelphia, Lea and Febiger, pp. 132-162. Forssmann, W. G., L. Orci, R. Pictet, A. E. Renold and C. Rouiller 1969 The endocrine cells in the epithelium of the gastrointestinal mucosa of the rat. An electron microscope study. J. Cell Biol., 40: 692-715. Gorman, M. L., D. B. Nedwell and R. M. Smith 1974 An analysis of the contents of the anal scent pockets of Herpestes aurpunctatus (Carnivora: Viverridae). J . Zool. (London), 272: 389-399. Hamilton, W. J., Jr. 1934 The life history of the rufescent woodchuck, Marmota monax rufescens Howell. Ann. Carnegie Mus., 23: 85-178. Hashimoto, K., B. L. Gross and W. F. Lever 1966 Electron microscopic study of apocrine secretion. J. Invest. Dermatol., 46: 378-390. Hibbs. R . G. 1962 Electron microscopy of h u m a n apocrine sweat glands. J. Invest. Dermatol., 38: 77-84. Kawabata, I 1964 Electron microscope studies on the human ceruminous gland. Arch. Histol. Jap., 25: 165-187. Kneeland, J . E. 1966 Fine structure of the sweat glands of the antebrachial organ of Lemur catta. Z. Zellforsch., 73: 521-533. Kurosumi, K., T. Kitamura. and T. Iijima 1959 Electron microscopic studies on human axillary apocrine sweat glands. Arch. Histol. Jap., 26: 523-566. Kurosumi, K.. M. Yamagishi and M. Sekine 1961 Mitochondrial deformation and apocrine secretory mechanism in the rabbit submandibular organ as revealed by electron microscopy. Z. Zellforsch., 55: 297-312. Main, T., and D. Lim 1976 The human external auditory canal secretory system - an ultrastructural study. Laryngoscope, 86- 1164-1176. Montagna, W. 1962 The Structure and Function of Skin. Second ed. New York, Academic Press. Montagna, W., and H. F. Parks 1948 A histochemical study of the anal sac of the dog. Anat. Rec., 200: 297-315.
Montes, L. F., B. L. Baker and A. C. Curtis 1960 The cytology of the large axillary sweat glands in man. J. Invest. Dermatol., 35: 273-291. Muller-Schwarze, D. 1971 Pheromones in the blacktailed deer (Odocoileus hemionus colurnbranusi. Anim. Behav., 29: 141-152. Muller-Schwarze, D., and M. M. Mozell, eds. 1977 Chemical Signals in Vertebrates. New York, Plenum Press. Munger, B. L. 1964 The ultrastructure and histochemistry of human apocrine gland cells. J. Cell Biol., 23: 64 (Abstract). 1965 The cytology of apocrine sweat glands. I. Cat and Monkey. Z. Zellforsch., 67: 373-383. 1971 The histology and cytology of the sweat glands. In: The Skin. E. B. Helwig and F. K. Mostofi, eds. Baltimore, Williams and Wilkins, pp. 47-64. Mykytowycz, R. 1966 Observations on odoriferous and other glands in the Australian wild rabbit, Oryctolagus cuniculus (L.),and the hare, Lepus europaeus P. 11. The inguinal glands. CSIRO Wildl. Res., 22: 49-64. 1970 The roll of skin glands in mammalian communication. In: Communication by Chemical Signals. J. W. Johnston, D. G. Moulton and A. Turk, eds. New York, Appleton-Century-Crofts, pp. 327-360. Nabeyama, A. 1975 Presence of cells combining features of two different cell types in the colonic crypts and pyloric glands of the mouse. Am. J. Anat., 242: 471-484. Palay, S . L. 1958 Morphology of secretion. In: Frontiers of Cytology. S. L. Palay, ed. New Haven, Yale University Press, pp. 305-342. Pearson, 0. P. 1946 Scent glands of the short-tailed shrew. Anat. Rec., 94: 615-625. Quay, W. B., and D. Muller-Schwarze 1970 Functional histology of integumentary glandular regions in black-tailed deer (Odocoileus hemionus colurnbranusi. J . Mammal., 51: 675-694. Schaumberg-Lever, G., and W. F. Lever 1975 Secretion from human apocrine glands: an electron microscopic study. J. Invest. Dermatol., 64: 38-41. Schiefferdecker, P. 1917 Die Hautdrusen des Menschen u n d des S a u g e t i e r e s , i h r e biologische und rassenanatomische Bedeutung sowie die Muscularis sexualis. Zentralbl. Biol., 37: 534-562. Shelley, W. B. 1956 The role of apocrine sweat in the production ofaxillaryodor. J. SOC.Cosmet. Chem., 7: 171-175. Sleggs, G. F. 1926 The adult anatomy and histology of the anal glands of the Richardson ground squirrel, Citellus richardsonii, Sabine. Anat. Rec., 32: 1-43. Swift, H., and Z. Hruban 1964 Focal degradation as a biological process. Fed. Proc., 23: 1026-1037. Yasada, K., R. A. Ellis and W. Montagna 1962 The fine structural relationship between mitochondria and light granules in the human apocrine sweat glands. Okajima Folia Anat. Jap., 38: 455-483.
PLATES
PLATE 1 EXPLANATION OF FIGURE
1 Diagrammatic representation of one of the three anal glands of the woodchuck, showing its communication with the anal canal. Each gland consists of multiple sebaceous and apocrine components which empty into the base of a single common duct. The apocrine secretory acini lie deeper in the surrounding loose connective tissue, and communicate with the common duct via the ducts of the apocrine gland. In contrast, the sebaceous acini characteristically lie closer to the common duct and appear to empty directly into its basal end.
278
WOODCHUCK APOCRINE SEBACEOUS A N A L S C E N T (;LAND Janet D Smith and (:ail W Hparn
f'l.A'lE 1
279
PLATE 2
2
Light microscopic view of a n apocrine secretory acinus and an apocrine duct lnsteriskl Secretory acini consisted of a single layer of cuboidal t o columnar secretory or apocrine cells resting on a n incomplete layer of myoepithelial cells The apocrine cells often contained accumulations of d a r k ~ s t a i n i n gsecretory granules in their apical cytoplasm (arrowhead, lower r i g h t ) . and many showed evidence of apical cap formation (arrow1 In contrast. t h e cells of t h e apocrine duct showed little e v dence of either secretory granules or apical caps However, like t h e wall of the acinus. t h e wall of t h e duct did contain myoepithelial cells. These a r e particularly evident where t h e duct has heen sectioned somewhat tangentially (arrowhead. upper left). One-micron Epon section stained with toluidine blue. X ,150.
:i Light microscopic view of several of the sebaceous acini of t h e anal gland Note a t
t h e periphery of each cluster of cells t h e small undifferentiated cells. and more centrally t h e larger differentiated cells with their accumulation of multiple cytoplasmic lipid droplets. The sebaceous acini. like t h e apocrine, were surrounded by a rich plexus of lymphatic and blood capillaries In t h e anal gland. sebaceous cells were not ociated with hair follicles. One-micron Epon section stained with toluidine hlue X 350
280
WOODCHUCK APOCRINE-SEUACEOUS ANAL S C E N T G L A N D Janet D. Smith and tiall W Hearn
PLATE 2
PLATE 3
4
282
The secretory cells of a n apocrine acinus contain a basal nucleus. a well-developed supranuclear Golgi apparatus (arrow) often with numerous condensing vacuoles, and electron-opaque secretory granules in t h e apical cytoplasm. These cells, with their apical microvilli and their secretory granules immediatelv beneath t h e apical plasma membrane. have not formed apical caps. The cells rest on an incomplete layer of myoepithelial cells. Note also t h e collection of small dense granules in t h e basal cytoplasm of a neighboring secretory cell (arrowhead). The n a t u r e and function of these granules is unclear 6.000
WOODCHUCK APOCKINE SEBACEOUb A N A L SC'ENT GLAND J a n e t D Smith and Gail W Hearn
PI ATE :I
283
PLATE 4 EXPLANATIOK OF F I G U R E S
284
5
Two neighboring apocrine secretory cells with apical caps which protrude into t h e lumen. Microvilli are typically lacking on t h a t portion of t h e plasma memhrane which covers t h e apical cap. The caps usually contain no secretory granules or other cytoplasmic organelles, although these may be present in great numbers in the c y t o ~ plasm immediately below t h e cap. At higher magnification ( i n s e t ) it is often possible to detect a n incomplete row of flattened vesicles located a t t h e level of t h e constriction between t h e apical cap and t h e remainder of the cytoplasm (arrowheadsl. These vesicles appear to fuse with one another, leading to t h e eventual separation of t h e apical cap from t h e cell. X 11,800, inset X 15,600.
6
At several points [arrowheads),t h e membranes of t h e secretory granules of apocrine secretory cells have fused with each other a n d with t h e plasma membrane to release their contents into t h e lumen in a process of t r u e merncrine secretion. The arrow indicates t h e level of the apical tight junctions between secretory cells. This sample was incubated for acid phosphatase activity and shows no localization of enzyme within t h e secretory granules. X 15.300
WOODCHUCK APOCRINE SEBACEOUS ANAI, SCENT G L A N D .Janet D
S m i t h and Gail W
PI.A'I'b: 4
Hearn
285
PL.ATE 5
286
7
Apocrine secretory cells incubated for acid phosphatase activity show dark reaction product over small vesicles and some cisternae in t h e Golg, region (arrowheads].hut t h e apical granules are always negative for this lysosomal marker enzyme. This indicates t h a t t h e apical granules probably a r e neither lysosomes nor phagw Iysosomes. and t h a t they would therefore appear to represent secretory vacuoles containing a nonlysosomal product synthesized by the apocrine cell. X 5,500
8
Two cytoplasmic processes from apocrine secretory cells a r e interposed between a myoepithelial cell and the basal lamina surrounding t h e apocrine acinus. The apocrine cell cytoplasm contains numerous small granules with dense cores which are separated from t h e limiting membrane of t h e granule by a clear zone of regular width. Such granules were abundant in t h e basal cytoplasm of some samples. especially in locations such a s here, where t h e apocrine cells or cell processes abutted directly on t h e basal lamina. Also common in t h e basal cytoplasm were numerous clear vesicles probably representing pinocytotic vesicles. and multivesicular bodies. See also figure 4 for a coinparison between basal granules and apical secretory granules. X 15.000.
WOODCHUCK APOCRINE SEBACEOUS ANAL SCENT G L A N D J a n e t D Smith and Gail W Hearn
PIATE 5
287
9
288
Piirtiotih of twu sehacciius a c i n i art' w p a r a t r d t i v c~illagrnou.;connective t i s h u t > c i w taining B netwtirk uf fianestrated hlui~dc;rpillaries In each aciiius t h e smaller p e ~ riphei a1 cells with few lipid droplets rtxpresrnt iindiffcrentiated stern cells. while f sizr and niimher are t h e larger central c e l l s containing lipid droplets i ~ increasing of iiii'frrrntiating. The seh;iceiius cells contain prominent bundles of tmofilarnents iarrowheadl in t h e cytoplasni. a n d a r e jiiined to o n e another via dark Iy btiiininp desniosorncs X 4.600.
WOODCHUCK APOCHINE SERA('EOUS A N A L SC'EkT G L A N D J a n e t 1) S m i t h a n d Gail W Hear"
1'I.A'lt f i
289
PLATE 7 E X P L A N A T I O N OF FIGITRE
10 Thin sections through fixed secretorv material collected from t h e common duct of
t h e anal gland showed squames of epithelial cells (arrow) probably shed from t h e wall of t h e common duct itself, a s well as numerous round a n d rod-shaped bacteria x
290
um.
WOODCHIJCK APOCKINE SEBACEOUS ANAI. SCENT G L A N D J a n e t Ll S m i t h and Gail W Hearn
['[.ATE T
29 1
