Cytological Differentiation in the Uropygial Gland ROGER C. W A G N E R ' AND ROBERT L. BOORD D e p a r t m e n t of Biological Sciences, University of Delaware, N e w a r k , Delaware 1971 1
ABSTRACT
Ultrastructural changes were studied in the cells undergoing secretory differentiation in zone I of the tubules of the uropygial gland of White Plymouth Rock chickens. A layer of basal cells and four secretory stages are recognized as the cells migrate from the periphery to the lumen of tubules and progressively elaborate a secretion product. Basal cells, containing rough endoplasmic reticulum and free ribosomes, rest on the basement membrane and are the source from which secretory cells arise. Dilated perinuclear cisternae and the proliferation of smooth endoplasmic reticulum in the form of vesicles, invaginated sacs and cuspshaped cisternae indicate the onset of lipogenesis in stage I cells. The perinuclear cisternae are more dilated and the endoplasmic reticulum is composed of saccules and cisternae in stage I1 cells. Stage I11 cells are characterized by concentric lamellae of endoplasmic reticulum surrounding secretory droplets. Dilated cisternae of endoplasmic reticulum and secretory droplets both contain a reticular substance. The perinuclear cisternae of stage 111 cells have returned to normal dimensions. Large mature lucent secretory droplets, lined with electron-dense material, fill the cytoplasm of stage IV cells which degenerate and release their secretory product into the tubule lumen. Spherical membrane-bound compartments containing a mottled substance of moderate electron density occur in basal cells and aIl subsequent secretory stages. These mottled bodies are surrounded by saccules of endoplasmic reticulum in stage I1 cells and are intimately associated with secretory droplets in stage I11 cells, but there is no evidence that they give rise to secretory droplets and their role in secretory differentiation is unknown.
The uropygial gland is a simple branched tubular, lipid-secreting gland situated over the base of the tail of most birds (Lucas and Stettenheim, '72). Its secretory products are highly specialized and consist mainly of esters of fatty acids and triglycerides (Elder, '54; Apandi and Edwards, '64; Haahti et al., '64; Haahti and Fales, '67). Although i t shows gross and cellular morphological similarities to the sebaceous and sebaceous-like glands of mammals and reptiles, its evolutionary relationships are unclear (Quay, '72). Previous authors have recognized differences in the cells lining the walls of the secretory tubules on the basis of light microscopic and histochemical studies (Stern, '05; Hou, '28; Cater and Lawrie, '50; Kanwar, '61; Lucas and Stettenheim, '72), but the morphological correlates of the secretory process at the ultrastructural level have received no attention. The purpose of this study therefore is to describe J . MORPH.,146: 3 9 5 4 1 4 .
the cytological changes that occur during the secretory process in zone I of the tubular walls of the uropygial gland of the domestic chicken. This zone comprises the bulk of the gland and exhibits a preponderance of secretory cells (Lucas and Stettenheim, '72). MATERIALS A N D METHODS
Uropygial glands were removed from normal, male, White Plymouth Rock chickens, eight weeks of age and approximately 1.4 kg body weight. Those processed for light microscopy were sliced in half through the sagittal plane, fixed by immersion in 10% neutral formalin, paraffin embedded, sectioned at 7 pm in frontal and sagittal planes and stained with hematoxylin and eOSin.
Those processed for electron microscopy were cut into 2 mm3 pieces, washed in 1 This study was supported by a University of Delaware Research Foundation Grant.
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0.05 M Tyrodes-cacodylate buffer (pH 7.4) mic reticulum is scant. Rod-shaped mitoand fixed for two hours in 2% glutaralde- chondria are randomly dispersed in the hyde. The tissue was refixed in 1 % osmium cytoplasm. Membrane-bound bodies containing a tetroxide, dehydrated in ethanol and embedded in epon which was polymerized at mottled substance of moderate electron 60°C for 48 hours. Silver sections were density are found in the cytoplasm of basal cut with a diamond knife on a Porter-Blum cells (figs. 3, 5). Basal cells are joined to MT-1 ultramicrotome, counterstained with each other, as well as to more differentisaturated uranyl acetate (10 min.) and ated cell types, by desmosomal junctions Reynolds lead citrate ( 3 min.) and exam- (fig. 4). Tonofilaments radiate from these ined with a Zeiss EM-9 electron micro- junctions and are observed deep in the cytoplasm (fig. 4). scope. RESULTS 2. Secretory cells (Stage I) Light microscopy These cells are located adjacent to basal Cross sections through secretory tubules cells either towards the tubule lumen or reveal layers of cells which exhibit a gra- resting on the basement membrane beside dient of secretory differentiation from the a basal cell (fig. 2 ) . The amount and type periphery toward the lumen (fig. 1). The of endoplasmic reticulum and the degree thick layer of secretory cells indicates that of distention of the perinuclear cisternae the section was taken through zone I of distinguish stage I secretory cells from the gland (Stern, '05; Lucas and Stetten- basal cells. heim, '72). This zone, corresponding to The cytoplasm is packed with smooththe distal ends of secretory tubules, is char- surfaced vesicles of endoplasmic reticulum. acterized by a thin layer of darkly staining Rough endoplasmic reticulum is absent; basal cells and a thicker layer of secretory however, a few ribosomal clusters are prescells. The secretory cells extend from the ent in the peripheral cytoplasm (fig. 6). basal layer to the tubule lumen where they Invaginated vesicles (fig. 7) or cusp-shaped degenerate and are released along with segments (fig. 8) of smooth endoplasmic their secretory products. reticulum frequently appear in clusters. The walls of these structures, which apElectron microscopy pear to form small sacs, are comprised of Thin sections through zone I of secretory a cisterna of smooth endoplasmic reticutubules exhibit a striking progression of lum. The sac is open at one end apparently cellular differentiation indicative of the providing direct access to the cytoplasmic evolution of secretory products within the compartment. The sac contents, however, cytoplasm (fig. 2 ) . This differentiation gra- are devoid of electron-dense materials. The dient parallels the centripetal migration size and form of these structures range of cells from the basal layer to the tubule from small invaginated vesicles to larger lumen. The cells can be arbitrarily classi- lucent sacs. The perinuclear cisternae of stage I cells fied into a basal and four secretory stages on the basis of their ultrastructural changes are markedly dilated. The nuclear envelope as they progressively elaborate a secretion appears beaded in cross section since its swelling is checked only by the isthmuses product. of nuclear pores (fig. 6). The amount of 1. Basal cells cytoplasm relative to the nucleus is greater These cells rest upon the basement mem- than that of basal cells. Mottled bodies observed in basal cells brane which surrounds the tubule wall and appear relatively undifferentiated (figs. are also present in stage I cells and in 2, 3 ) . The nuclei are large and ovoid and greater numbers (fig. 6). At low magnifithe perinuclear cisternae of the nuclear cation, cross sections of these structures envelopes exhibit normal dimensions. The appear similar to mitochondria, but at cytoplasmic volume is small relative to the higher magnification they are distinguishnucleus. The cytoplasm contains rough able from mitochondria by the presence of endoplasmic reticulum and clusters of free a single limiting membrane and the lack ribosomes (figs. 3, 4) but smooth endoplas- of cristae (fig. 8).
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3. Secretory cells (Stage 11) These cells are located centripetal to stage I cells (fig. 2). The configuration of smooth endoplasmic reticulum and changes in the mottled bodies distinguish these cells from stage I secretory cells. The endoplasmic reticulum is comprised primarily of saccules and cisternae rather than vesicles (fig. 9). The perinuclear cisternae remain dilated and breakage of some of the nuclear pore membranes is evident (fig. 9). The mottled bodies are greater in number and have become heterogeneous with respect to size and the amount and electron density of their contents (figs. 9-11). Saccules of endoplasmic reticulum have become oriented in concentric layers around mottled bodies (fig. 10) but as yet no large secretion droplets have formed. 4. Secretory cells (Stage 111)
Stage I11 cells are centripetal to stage I1 cells and their cytoplasm exhibits an advanced stage of endoplasmic and secretory differentiation (fig. 12). Saccules of endoplasmic reticulum form tightly packed concentric lamellae around mottled bodies and larger developing secretion droplets (fig. 13). Secretion droplets contain various amounts of electron-dense material that range from condensed reticular conglomerates to only a thin layer closely associated with the inner wall (figs. 12, 13). Dilated cisternae are found within lamellated whorls of endoplasmic reticulum. Some of these dilated areas contain a reticular material similar to that observed in secretion droplets (fig. 14). Mottled bodies are present in abundance in stage I11 cells and occur frequently in a satellite association with larger secretion droplets (figs. 12, 13). Although they vary in size and electron density, no direct transition or fusion between them and secretion droplets is evident. M a n y of the mottled bodies retain the size and staining characteristics observed in basal cells. The perinuclear cisternae of stage 111 cells have undergone an abrupt decrease in volume and appear similar to the nuclear envelope of basal cells (fig. 12). 5. Secretory cells (Stage IV) These cells comprise the inner-most layers of secretory tubules, which border on
the lumen where holocrine secretion occurs. They constitute the bulk of the cells observed in cross sections of zone I secretory tubules (fig. 1). Large spherical secretion droplets crowd the cytoplasm of stage IV cells (fig. 15). A thin electron-dense coating lines the internal surface of these droplets, which becomes dissociated from this surface in some cases (fig. 16). Lamellae of endoplasmic reticulum are packed tightly adjacent to secretion droplets. The nuclei and perinuclear cisternae appear identical to those of basal cells (fig. 15); however, in some instances enroachment of large droplets results in indentations of the nuclear surface (fig. 16). Mottled bodies, structurally similar to those in basal cells, are scattered throughout the cytoplasm (figs. 15, 16). The entire stage IV cell degenerates, releasing its contents into the tubule lumen where large electron-transparent areas as well as cellular debris are found. This cellular debris consists mainly of closely packed bundles of filaments in a dense matrix (fig. 17). DISCUSSION
The histological characteristics (Stern, '05; Lucas and Stettenheim, '72; Cater and Lawrie, '50), epidermal origin (Gomot, '59), and holocrine secretion of lipoidal substances by the uropygial gland are similar to those of the sebaceous glands of mammals (Ellis and Henrikson, '63) and the lipoidal glands of reptiles (Quay, '72). The evolutionary relationships of these glandular epidermal derivatives are discussed by Quay ('72) who places them along morphogenetic lines of descent in terrestrial vertebrates based on holocrine secretion of keratinizing cells. The uropygial gland arises embryonically as an invagination of the epidermis and its secretory epithelium is similar to that of the epidermis of the skin. Mitotic figures are restricted to the layer of basal cells (Maiti, '68) which give rise to differentiated secretory cells. The generative potential of basal cells is also evident at the ultrastructural level. Rough endoplasmic reticulum and free ribosomes indicate a capacity for protein synthesis necessary for cellular reproduction. The juxtaposition of basal cells with secretory cells also sug-
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gests that basal cells are the stem from which differentiated secretory cells arise. Similar ultrastructural characteristics are found in the basal cells of sebaceous glands (Ellis and Henrikson, '63). Ultrastructural studies of reptilian oil glands are unavailable for comparison. The secretory cells of the uropygial gland have been classified on the basis of their position and morphology. Lucas and Stettentheim ('72) describe basal, intermediate and transitional (including degenerating cells bordering the lumen) layers of cells in zone I of the secretory tubules of the chicken. These layers of cells correspond to the basal cells, stage I and I1 secretory cells and stage I11 and IV secretory cells respectively which are described in this study. Maiti and Ghosh ('69) distinguish flat basal, cuboidal, large lipidladen and small disintegrating cells within the walls of secretory tubules of the pigeon. These correspond with respect to shape, position and morphological features to basal cells, stage I and I1 secretory cells, stage I11 and IV secretory cells, and degenerating stage IV secretory cells respectively. Cross identification of secretory cell types at the light and electron microscope level should be made with caution. Basal cells resting upon the basement membrane correlate well with flat basophilic cells observed with the light microscope. Their intense affinity for hematoxylin can be attributed to ribonucleic acid present in the rough endoplasmic reticulum and ribosomes. Since stage I and I1 secretory cells contain no secretion droplets which would be observable at the light microscope level, their equivalence with intermediate or cuboidal cells is evident. Stage I11 and IV secretory cells both contain secretory droplets which correlate well with lipoid spheres observed in the transitional or large lipidladen cells of light microscopy. More subtle changes occurring during secretory differentiation are detected by electron microscopy. These provide a more detailed cytological basis on which cell types can be distinguished. Proliferation of clear, smooth-surfaced vesicles of endoplasmic reticulum in stage I secretory cells indicates the onset of lipid synthesis (Seikevitz, '63). Development of smooth endoplasmic reticulum is associated
with a surge of lipogenesis in sebaceous cells (Nicolaides, '63), adrenal cortical cells (Brenner, '67), corpus luteum (Bjersing, '67), and testicular interstitial cells (Christianson, '65). Dilation of the perinuclear cisterna may reflect an accumulation of lipid in this compartment since i t is continuous with the endoplasmic reticulum. It is possible that this conspicuous swelling reflects a prominent role of the nuclear envelope in lipogenesis. It is tempting to speculate that invaginated membraneous sacs of stage I cells represent a transition from smooth-surfaced vesicles to the saccular profiles and cisternae of stage I1 and I11 cells. However, the electron transparency of their lumenae suggests that these spaces are occluded from the cytoplasmic compartment and are separate from the endoplasmic compartment as well. It is also unlikely that the inner compartment (the lumen) represents an early secretion droplet since i t is surrounded by the endoplasmic compartment and not the cytoplasm. Secretion droplets probably arise from dilated cisternae of endoplasmic lamellae of stage I11 cells since they contain an electron-dense reticular substance identical to that observed in mature secretion droplets. This origin is similar to that of secretion droplets of sebaceous glands (Palay, '58). The role of mottled bodies during secretory differentiation is unknown. Similar structures have not been reported in sebaceous glands. Their presence in basal cells, prior to endoplasmic reticulum development, implies that their contents are not derived from cellular lipid synthesis (Seikevitz, '63). Furthermore, the formation or reproduction of mottled bodies evidently occurs prior to basal cell division and they are present in all subsequent secretory stages. The concentric arrangement of saccules of endoplasmic reticulum around mottled bodies in stage I1 cells suggests transfer of material from the saccules to the mottled bodies. The observation that a certain proportion of mottled bodies increase in size with a concomitant decrease in electron density supports this suggestion. There is no evidence to indicate that mottled bodies give rise to or contribute to the development of secretorv droulets. Even in stage Ifi cells where both mottled bodies
CHICKEN UROPYGIAL GLAND
and definitive secretory droplets are intimately associated with each other at the hub of whorls of endoplasmic lamellae, the membranes of each type of shucture retain their integrity. It is reasonable to postulate that mottled bodies, as satellites to large secretory droplets, serve an auxiliary role in the packaging of secretory products for subsequent release into the tubule lumen. Palay (’58) postulates that the osmiophilic “husk” lining secretion droplets in sebaceous glands consist of compressed profiles of endoplasmic reticulum. A thin layer of electron-dense material also lines mature secretion droplets of stage IV cells. However, it displays no membranous profiles and may represent segregation of osmiophilic components of the droplet contents. The role of cytoplasmic membrane systems in the synthesis, transport and secretion of proteins is well characterized (Jamieson and Palade, ’67a,b). In contrast, functional correlations between cytoplasmic structures and lipogenesis are poorly understood. The secretory cells of the uropygial gland exhibit conspicuous morphological changes of cytoplasmic membranes which parallel the concomitant evolution of lipid secretions. However, the precise role of these structures in lipogenesis remains to be elucidated. ACKNOWLEDGMENTS
We wish to thank Dr. Harold B. White for suggesting this study and Dr. Paul H. Sammelwitz for generously keeping us supplied with uropygial glands. LITERATURE CITED Apandi, M., and H. M. Edwards, Jr. 1964 Studies on the composition of the secretions of the uropygial gland of some avian species. Poultry Sci., 43: 14451462. Bjersing, L. 1967 On the ultrastructure of granulosa lutein cells in porcine corpus luteum with special reference to endoplasmic reticulum and steroid hormone synthesis. Z. Zellforsch, 82: 187-21 1 . Brenner, R. M. 1967 Fine structure of adrenocortical cells in adult male Rhesus monkeys. Am. J. Anat., 119: 4 2 9 4 5 3 . Cater, D. B . , and N. R. Lawrie 1950 Some his-
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tochemical a n d biochemical observations on the preen gland. J. Physiol., 3: 231-243. Christianson, A. K. 1965 T h e fine structure of testicular interstitial cells i n guinea pigs. J. Cell Biol., 26: 911-935. Elder, W. H. 1954 T h e oil gland of birds. Wilson Bull., 66: &31. Ellis, R. A,, and R. C. Henrikson 1963 T h e ultrastructure of t h e sebaceous glands of m a n . I n . Advances i n Biology of Skin. Vol. IV. T h e Sebaceous Glands. W. Montagna, R . A. Ellis and A. F. Silver, eds. Permagon Press, Oxford. Chap. VI, pp. 94-109. Gomot, L. 1959 Sur l a determination d e la glande Uropygienne des Oiseux. Arch. Anat. Histol. Embryol., 42: 245-281. Haahti, E. 0. A,, a n d H. M. Fales 1967 The uropygiols: identification of the unsaponifiable constituent of a diester wax from chicken preen glands. J. Lipid Res., 8: 131-137. Haahti, E., K. Lagerspetz, T. Nikkari and H. M. Fales 1964 Lipids of the uropygial gland of birds. Comp. Biochem. Physiol., 12: 4 3 5 4 3 7 . Hou, H. C. 1928 Studies on the glandula uropygialis of birds. Chin. Journ. Physiol., 2: 34.5 380. Jamieson, J. D., and G. E. Palade 1967a Intracellular transport of secretory proteins in the pancreatic exocrine cell. I . Role of the peripheral elements of t h e Golgi complex. J . Cell Biol., 34: 577-596. 1967b Intracellular transport of secretory proteins in the pancreatic exocrine cell. 11. Transport of condensing vacuoles and zymogen granules. J. Cell Biol., 34: 597-4315, Kanwar, K . C. 1961 Morphological and histochemical studies on the uropygial gland of pigeon and domestic fowl. Cytologia, 26: 124-136. Lucas, A. M . , and P. R. Stettenheim 1972 Avian Anatomy: Integument. Agricultural Handbook 362. U. S. Dept. Agriculture, Washington, D. C. Maiti, B. 1968 Dynamics of the uropygial gland in the pigeon. Folia Biologica, 16: 4 C ~ 5 4 . Maiti, B., and A. Ghosh 1969 Effect of cortisone on mitotic activity and cell loss in t h e uropygial gland of male pigeons. Acta Anat., 74: 97-103. Nicolaides, W . 1963 H u m a n skin surface lipids - origin, composition and possible function. In: Advances in Biology of Skin. Vol. IV. T h e Sebaceous Glands. W. Montagna, R. A. ElIis and A. F. Silver, eds. Pergamon Press, Oxford. Chap. XI, pp. 167-187. Palay, S. L. 1958 The morphology of secretion. I n : Frontiers in Cytology. S. L. Palay, ed. Yale Univ. Press. Chap. XI, pp. 3 0 5 3 4 2 , Quay, W. B. 1972 Integument and the environment: glandular composition, function, and evolution. Am. Zool., 12: 95-108. Siekevitz, P. 1963 Protoplasm: endoplasmic reticulum and microsomes and their properties. Ann. Rev. Phvsiol.. 25: 1 5 4 0 . Stern, M. 1905 Histologische Beitrage zur Sekretion der Burseldruse. Arch. f. mikros. Anat., 66: 294-311.
PLATE I EXPLANATION O F FIGURE
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Light photomicrograph of a transverse section through zone I of secretory tubules of the uropygial gland. Darkly staining basal cells form a thin layer around the periphery of each tubule. The inner thicker layer consists of lightly staining secretory cells. Hematoxylin and eosin stain. X 300; scale marker, 33.3 pm.
CHICKEN UROPYGIAL G L A N D Roger C . Wagner and Robert L. Boord
PLATE I
40 1
PLATE 2 E X P L A N A T I O N O F FIGURE
2
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Electron micrograph of a section through the wall of a secretory tubule. Progressive cytoplasmic differentiation is evident from the basal cells (B) which rest upon the basement membrane (large arrows) through stages I, I1 and 111 secretory cells. Mottled bodies (small arrows) are observable in basal cells as well as in stage I and 111 secretory cells. x 1,600; scale marker, 6.25 p m .
CHICKEN UROPYGIAL G L A N D Roger C Wagner and Robert L Boord
PLATE 2 .
_
_
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- __. -. -- - -
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PLATE 3 EXPLANATION
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OF FIGURES
3
A basal cell showing the large ovoid nucleus with a normal nuclear envelope. T h e cytoplasm contains rough endoplasmic reticulum, ribosomal clusters, tonofilaments and mottled bodies (arrows). X 12,500; scale marker, 0.80 p m .
4
Higher magnification of a portion of a basal cell showing rough endoplasmic reticulum, ribosomal clusters and desmosomal junctions (arrows) from which tonofilaments radiate into the cytoplasm. X 29,200; scale marker, 0.34 p m .
5
Enlargement of a portion of the cell of figure 3 showing two membranebound bodies with mottled electron-dense contents. X 35,500; scale marker, 0.28 p m .
CHICKEN UROPYGIAL GLAND Roger C. Wagner and Robert L. Boord
PLATE 3
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PLATE 4 EXPLANATION OF FIGURES
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6
A stage I secretory cell exhibiting smooth-surfaced vesicles of endoplasmic reticulum. The perinuclear cisterna is markedly dilated and appears beaded in areas constrained by nuclear pores (small arrows). Mottled bodies (large arrows) are present in the cytoplasm. X 14,400; scale marker, 0.69 pm.
7
A cluster of invaginated vesicles and larger cusp-shaped cisternae of endoplasmic reticulum in the cytoplasm of a type I secretory cell. X 18,000; scale marker, 0.58 pm.
8
Cusps of smooth endoplasmic reticulum exhibiting electron-transparent contents. A mottled body is bounded by a single membrane and exhibits no cristae. x 25,900; scale marker, 0.39 pm.
CHICKEN UROPYCIAL GLAND Roger C . Wagner and Robert L. Board
PLATE 4
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PLATE 5 EXPLANATION OF FIGURES
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9
A stage I1 secretory cell containing cisternae and saccules of smooth endoplasmic reticulum. The perinuclear cisterna is extremely dilated and nuclear pores appear broken in some areas (small arrows). Mottled bodies (large arrows) are still present and some have increased in size. X 12,500; scale marker, 0.80 p m .
10
Three mottled bodies surrounded by layers of saccules of endoplasmic reticulum. Two of the bodies have less electron-dense contents. X 40,600; scale marker, 0.25 fim.
11
Mottled bodies are distinct from mitochondria since they are lighter staining, bounded by a single membrane and are devoid of cristae. X 31,500; scale marker, 0.32 p m .
CHICKEN UROPYGIAL GLAND Roger C. Wagner and Robert L. Boord
PLATE 5
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PLATE 6 E X P L A N A T I O N O F FIGURES
12
A stage 111 secretory cell containing various stages in the development of secretory droplets. The perinuclear cisterna is no longer dilated. Mottled bodies are abundantly present and frequently occur in satellite association with secretory droplets (arrows). X 10,900; scale marker, 0.92 pm.
13 Secretory droplets contain electron-dense contents ranging from condensed reticular conglomerates to a thin layer associated with the inner wall. Concentric lamellae of endoplasmic reticulum encircle secretory droplets and mottled bodies. X 20,300; scale marker, 0.49 pm. 14
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Dilated cisternae are found within whorls of endoplasmic lamellae. Some contain a reticular substance similar to that found in secretory droplets (arrows). x 17,600; scale marker, 0.57pm.
CHICKEN UROPYGIAL GLAND Roger C. Wagner and Robert L. Boord
PLATE 6
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PLATE 7 EXPLANATION O F FIGURES
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15
A stage IV secretory cell showing large luscent secretory droplets which are lined by a thin layer of electron-dense material. Small dense mottled bodies (arrows) are still present. The perinuclear cisterna remains undilated. x 13,700; scale marker, 0.73 p m .
16
Electron-dense material becomes dissociated from the inner wall of secretory droplets in some cases. Note the mottled body in close association with a secretory droplet (arrow). X 11,900; scale marker, 0.84 Fm.
17
The lumen of a secretory tubule exhibiting large electron-transparent areas and cellular debris consisting of filamentous bundles. X 10,500; scale marker, 0.95 p m .
CHICKEN UROPYGIAL GLAND Roger C. Wagner and Robert L. Boord
PLATE 7
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ABSTRACT
Ultrastructural changes were studied in the cells undergoing secretory differentiation in zone I of the tubules of the uropygial gland of White Plymouth Rock chickens. A layer of basal cells and four secretory stages are recognized as the cells migrate from the periphery to the lumen of tubules and progressively elaborate a secretion product. Basal cells, containing rough endoplasmic reticulum and free ribosomes, rest on the basement membrane and are the source from which secretory cells arise. Dilated perinuclear cisternae and the proliferation of smooth endoplasmic reticulum in the form of vesicles, invaginated sacs and cuspshaped cisternae indicate the onset of lipogenesis in stage I cells. The perinuclear cisternae are more dilated and the endoplasmic reticulum is composed of saccules and cisternae in stage I1 cells. Stage I11 cells are characterized by concentric lamellae of endoplasmic reticulum surrounding secretory droplets. Dilated cisternae of endoplasmic reticulum and secretory droplets both contain a reticular substance. The perinuclear cisternae of stage 111 cells have returned to normal dimensions. Large mature lucent secretory droplets, lined with electron-dense material, fill the cytoplasm of stage IV cells which degenerate and release their secretory product into the tubule lumen. Spherical membrane-bound compartments containing a mottled substance of moderate electron density occur in basal cells and aIl subsequent secretory stages. These mottled bodies are surrounded by saccules of endoplasmic reticulum in stage I1 cells and are intimately associated with secretory droplets in stage I11 cells, but there is no evidence that they give rise to secretory droplets and their role in secretory differentiation is unknown.
The uropygial gland is a simple branched tubular, lipid-secreting gland situated over the base of the tail of most birds (Lucas and Stettenheim, '72). Its secretory products are highly specialized and consist mainly of esters of fatty acids and triglycerides (Elder, '54; Apandi and Edwards, '64; Haahti et al., '64; Haahti and Fales, '67). Although i t shows gross and cellular morphological similarities to the sebaceous and sebaceous-like glands of mammals and reptiles, its evolutionary relationships are unclear (Quay, '72). Previous authors have recognized differences in the cells lining the walls of the secretory tubules on the basis of light microscopic and histochemical studies (Stern, '05; Hou, '28; Cater and Lawrie, '50; Kanwar, '61; Lucas and Stettenheim, '72), but the morphological correlates of the secretory process at the ultrastructural level have received no attention. The purpose of this study therefore is to describe J . MORPH.,146: 3 9 5 4 1 4 .
the cytological changes that occur during the secretory process in zone I of the tubular walls of the uropygial gland of the domestic chicken. This zone comprises the bulk of the gland and exhibits a preponderance of secretory cells (Lucas and Stettenheim, '72). MATERIALS A N D METHODS
Uropygial glands were removed from normal, male, White Plymouth Rock chickens, eight weeks of age and approximately 1.4 kg body weight. Those processed for light microscopy were sliced in half through the sagittal plane, fixed by immersion in 10% neutral formalin, paraffin embedded, sectioned at 7 pm in frontal and sagittal planes and stained with hematoxylin and eOSin.
Those processed for electron microscopy were cut into 2 mm3 pieces, washed in 1 This study was supported by a University of Delaware Research Foundation Grant.
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0.05 M Tyrodes-cacodylate buffer (pH 7.4) mic reticulum is scant. Rod-shaped mitoand fixed for two hours in 2% glutaralde- chondria are randomly dispersed in the hyde. The tissue was refixed in 1 % osmium cytoplasm. Membrane-bound bodies containing a tetroxide, dehydrated in ethanol and embedded in epon which was polymerized at mottled substance of moderate electron 60°C for 48 hours. Silver sections were density are found in the cytoplasm of basal cut with a diamond knife on a Porter-Blum cells (figs. 3, 5). Basal cells are joined to MT-1 ultramicrotome, counterstained with each other, as well as to more differentisaturated uranyl acetate (10 min.) and ated cell types, by desmosomal junctions Reynolds lead citrate ( 3 min.) and exam- (fig. 4). Tonofilaments radiate from these ined with a Zeiss EM-9 electron micro- junctions and are observed deep in the cytoplasm (fig. 4). scope. RESULTS 2. Secretory cells (Stage I) Light microscopy These cells are located adjacent to basal Cross sections through secretory tubules cells either towards the tubule lumen or reveal layers of cells which exhibit a gra- resting on the basement membrane beside dient of secretory differentiation from the a basal cell (fig. 2 ) . The amount and type periphery toward the lumen (fig. 1). The of endoplasmic reticulum and the degree thick layer of secretory cells indicates that of distention of the perinuclear cisternae the section was taken through zone I of distinguish stage I secretory cells from the gland (Stern, '05; Lucas and Stetten- basal cells. heim, '72). This zone, corresponding to The cytoplasm is packed with smooththe distal ends of secretory tubules, is char- surfaced vesicles of endoplasmic reticulum. acterized by a thin layer of darkly staining Rough endoplasmic reticulum is absent; basal cells and a thicker layer of secretory however, a few ribosomal clusters are prescells. The secretory cells extend from the ent in the peripheral cytoplasm (fig. 6). basal layer to the tubule lumen where they Invaginated vesicles (fig. 7) or cusp-shaped degenerate and are released along with segments (fig. 8) of smooth endoplasmic their secretory products. reticulum frequently appear in clusters. The walls of these structures, which apElectron microscopy pear to form small sacs, are comprised of Thin sections through zone I of secretory a cisterna of smooth endoplasmic reticutubules exhibit a striking progression of lum. The sac is open at one end apparently cellular differentiation indicative of the providing direct access to the cytoplasmic evolution of secretory products within the compartment. The sac contents, however, cytoplasm (fig. 2 ) . This differentiation gra- are devoid of electron-dense materials. The dient parallels the centripetal migration size and form of these structures range of cells from the basal layer to the tubule from small invaginated vesicles to larger lumen. The cells can be arbitrarily classi- lucent sacs. The perinuclear cisternae of stage I cells fied into a basal and four secretory stages on the basis of their ultrastructural changes are markedly dilated. The nuclear envelope as they progressively elaborate a secretion appears beaded in cross section since its swelling is checked only by the isthmuses product. of nuclear pores (fig. 6). The amount of 1. Basal cells cytoplasm relative to the nucleus is greater These cells rest upon the basement mem- than that of basal cells. Mottled bodies observed in basal cells brane which surrounds the tubule wall and appear relatively undifferentiated (figs. are also present in stage I cells and in 2, 3 ) . The nuclei are large and ovoid and greater numbers (fig. 6). At low magnifithe perinuclear cisternae of the nuclear cation, cross sections of these structures envelopes exhibit normal dimensions. The appear similar to mitochondria, but at cytoplasmic volume is small relative to the higher magnification they are distinguishnucleus. The cytoplasm contains rough able from mitochondria by the presence of endoplasmic reticulum and clusters of free a single limiting membrane and the lack ribosomes (figs. 3, 4) but smooth endoplas- of cristae (fig. 8).
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CHICKEN UROPYGIAL GLAND
3. Secretory cells (Stage 11) These cells are located centripetal to stage I cells (fig. 2). The configuration of smooth endoplasmic reticulum and changes in the mottled bodies distinguish these cells from stage I secretory cells. The endoplasmic reticulum is comprised primarily of saccules and cisternae rather than vesicles (fig. 9). The perinuclear cisternae remain dilated and breakage of some of the nuclear pore membranes is evident (fig. 9). The mottled bodies are greater in number and have become heterogeneous with respect to size and the amount and electron density of their contents (figs. 9-11). Saccules of endoplasmic reticulum have become oriented in concentric layers around mottled bodies (fig. 10) but as yet no large secretion droplets have formed. 4. Secretory cells (Stage 111)
Stage I11 cells are centripetal to stage I1 cells and their cytoplasm exhibits an advanced stage of endoplasmic and secretory differentiation (fig. 12). Saccules of endoplasmic reticulum form tightly packed concentric lamellae around mottled bodies and larger developing secretion droplets (fig. 13). Secretion droplets contain various amounts of electron-dense material that range from condensed reticular conglomerates to only a thin layer closely associated with the inner wall (figs. 12, 13). Dilated cisternae are found within lamellated whorls of endoplasmic reticulum. Some of these dilated areas contain a reticular material similar to that observed in secretion droplets (fig. 14). Mottled bodies are present in abundance in stage I11 cells and occur frequently in a satellite association with larger secretion droplets (figs. 12, 13). Although they vary in size and electron density, no direct transition or fusion between them and secretion droplets is evident. M a n y of the mottled bodies retain the size and staining characteristics observed in basal cells. The perinuclear cisternae of stage 111 cells have undergone an abrupt decrease in volume and appear similar to the nuclear envelope of basal cells (fig. 12). 5. Secretory cells (Stage IV) These cells comprise the inner-most layers of secretory tubules, which border on
the lumen where holocrine secretion occurs. They constitute the bulk of the cells observed in cross sections of zone I secretory tubules (fig. 1). Large spherical secretion droplets crowd the cytoplasm of stage IV cells (fig. 15). A thin electron-dense coating lines the internal surface of these droplets, which becomes dissociated from this surface in some cases (fig. 16). Lamellae of endoplasmic reticulum are packed tightly adjacent to secretion droplets. The nuclei and perinuclear cisternae appear identical to those of basal cells (fig. 15); however, in some instances enroachment of large droplets results in indentations of the nuclear surface (fig. 16). Mottled bodies, structurally similar to those in basal cells, are scattered throughout the cytoplasm (figs. 15, 16). The entire stage IV cell degenerates, releasing its contents into the tubule lumen where large electron-transparent areas as well as cellular debris are found. This cellular debris consists mainly of closely packed bundles of filaments in a dense matrix (fig. 17). DISCUSSION
The histological characteristics (Stern, '05; Lucas and Stettenheim, '72; Cater and Lawrie, '50), epidermal origin (Gomot, '59), and holocrine secretion of lipoidal substances by the uropygial gland are similar to those of the sebaceous glands of mammals (Ellis and Henrikson, '63) and the lipoidal glands of reptiles (Quay, '72). The evolutionary relationships of these glandular epidermal derivatives are discussed by Quay ('72) who places them along morphogenetic lines of descent in terrestrial vertebrates based on holocrine secretion of keratinizing cells. The uropygial gland arises embryonically as an invagination of the epidermis and its secretory epithelium is similar to that of the epidermis of the skin. Mitotic figures are restricted to the layer of basal cells (Maiti, '68) which give rise to differentiated secretory cells. The generative potential of basal cells is also evident at the ultrastructural level. Rough endoplasmic reticulum and free ribosomes indicate a capacity for protein synthesis necessary for cellular reproduction. The juxtaposition of basal cells with secretory cells also sug-
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ROGER C . WAGNER AND ROBERT L. BOORD
gests that basal cells are the stem from which differentiated secretory cells arise. Similar ultrastructural characteristics are found in the basal cells of sebaceous glands (Ellis and Henrikson, '63). Ultrastructural studies of reptilian oil glands are unavailable for comparison. The secretory cells of the uropygial gland have been classified on the basis of their position and morphology. Lucas and Stettentheim ('72) describe basal, intermediate and transitional (including degenerating cells bordering the lumen) layers of cells in zone I of the secretory tubules of the chicken. These layers of cells correspond to the basal cells, stage I and I1 secretory cells and stage I11 and IV secretory cells respectively which are described in this study. Maiti and Ghosh ('69) distinguish flat basal, cuboidal, large lipidladen and small disintegrating cells within the walls of secretory tubules of the pigeon. These correspond with respect to shape, position and morphological features to basal cells, stage I and I1 secretory cells, stage I11 and IV secretory cells, and degenerating stage IV secretory cells respectively. Cross identification of secretory cell types at the light and electron microscope level should be made with caution. Basal cells resting upon the basement membrane correlate well with flat basophilic cells observed with the light microscope. Their intense affinity for hematoxylin can be attributed to ribonucleic acid present in the rough endoplasmic reticulum and ribosomes. Since stage I and I1 secretory cells contain no secretion droplets which would be observable at the light microscope level, their equivalence with intermediate or cuboidal cells is evident. Stage I11 and IV secretory cells both contain secretory droplets which correlate well with lipoid spheres observed in the transitional or large lipidladen cells of light microscopy. More subtle changes occurring during secretory differentiation are detected by electron microscopy. These provide a more detailed cytological basis on which cell types can be distinguished. Proliferation of clear, smooth-surfaced vesicles of endoplasmic reticulum in stage I secretory cells indicates the onset of lipid synthesis (Seikevitz, '63). Development of smooth endoplasmic reticulum is associated
with a surge of lipogenesis in sebaceous cells (Nicolaides, '63), adrenal cortical cells (Brenner, '67), corpus luteum (Bjersing, '67), and testicular interstitial cells (Christianson, '65). Dilation of the perinuclear cisterna may reflect an accumulation of lipid in this compartment since i t is continuous with the endoplasmic reticulum. It is possible that this conspicuous swelling reflects a prominent role of the nuclear envelope in lipogenesis. It is tempting to speculate that invaginated membraneous sacs of stage I cells represent a transition from smooth-surfaced vesicles to the saccular profiles and cisternae of stage I1 and I11 cells. However, the electron transparency of their lumenae suggests that these spaces are occluded from the cytoplasmic compartment and are separate from the endoplasmic compartment as well. It is also unlikely that the inner compartment (the lumen) represents an early secretion droplet since i t is surrounded by the endoplasmic compartment and not the cytoplasm. Secretion droplets probably arise from dilated cisternae of endoplasmic lamellae of stage I11 cells since they contain an electron-dense reticular substance identical to that observed in mature secretion droplets. This origin is similar to that of secretion droplets of sebaceous glands (Palay, '58). The role of mottled bodies during secretory differentiation is unknown. Similar structures have not been reported in sebaceous glands. Their presence in basal cells, prior to endoplasmic reticulum development, implies that their contents are not derived from cellular lipid synthesis (Seikevitz, '63). Furthermore, the formation or reproduction of mottled bodies evidently occurs prior to basal cell division and they are present in all subsequent secretory stages. The concentric arrangement of saccules of endoplasmic reticulum around mottled bodies in stage I1 cells suggests transfer of material from the saccules to the mottled bodies. The observation that a certain proportion of mottled bodies increase in size with a concomitant decrease in electron density supports this suggestion. There is no evidence to indicate that mottled bodies give rise to or contribute to the development of secretorv droulets. Even in stage Ifi cells where both mottled bodies
CHICKEN UROPYGIAL GLAND
and definitive secretory droplets are intimately associated with each other at the hub of whorls of endoplasmic lamellae, the membranes of each type of shucture retain their integrity. It is reasonable to postulate that mottled bodies, as satellites to large secretory droplets, serve an auxiliary role in the packaging of secretory products for subsequent release into the tubule lumen. Palay (’58) postulates that the osmiophilic “husk” lining secretion droplets in sebaceous glands consist of compressed profiles of endoplasmic reticulum. A thin layer of electron-dense material also lines mature secretion droplets of stage IV cells. However, it displays no membranous profiles and may represent segregation of osmiophilic components of the droplet contents. The role of cytoplasmic membrane systems in the synthesis, transport and secretion of proteins is well characterized (Jamieson and Palade, ’67a,b). In contrast, functional correlations between cytoplasmic structures and lipogenesis are poorly understood. The secretory cells of the uropygial gland exhibit conspicuous morphological changes of cytoplasmic membranes which parallel the concomitant evolution of lipid secretions. However, the precise role of these structures in lipogenesis remains to be elucidated. ACKNOWLEDGMENTS
We wish to thank Dr. Harold B. White for suggesting this study and Dr. Paul H. Sammelwitz for generously keeping us supplied with uropygial glands. LITERATURE CITED Apandi, M., and H. M. Edwards, Jr. 1964 Studies on the composition of the secretions of the uropygial gland of some avian species. Poultry Sci., 43: 14451462. Bjersing, L. 1967 On the ultrastructure of granulosa lutein cells in porcine corpus luteum with special reference to endoplasmic reticulum and steroid hormone synthesis. Z. Zellforsch, 82: 187-21 1 . Brenner, R. M. 1967 Fine structure of adrenocortical cells in adult male Rhesus monkeys. Am. J. Anat., 119: 4 2 9 4 5 3 . Cater, D. B . , and N. R. Lawrie 1950 Some his-
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tochemical a n d biochemical observations on the preen gland. J. Physiol., 3: 231-243. Christianson, A. K. 1965 T h e fine structure of testicular interstitial cells i n guinea pigs. J. Cell Biol., 26: 911-935. Elder, W. H. 1954 T h e oil gland of birds. Wilson Bull., 66: &31. Ellis, R. A,, and R. C. Henrikson 1963 T h e ultrastructure of t h e sebaceous glands of m a n . I n . Advances i n Biology of Skin. Vol. IV. T h e Sebaceous Glands. W. Montagna, R . A. Ellis and A. F. Silver, eds. Permagon Press, Oxford. Chap. VI, pp. 94-109. Gomot, L. 1959 Sur l a determination d e la glande Uropygienne des Oiseux. Arch. Anat. Histol. Embryol., 42: 245-281. Haahti, E. 0. A,, a n d H. M. Fales 1967 The uropygiols: identification of the unsaponifiable constituent of a diester wax from chicken preen glands. J. Lipid Res., 8: 131-137. Haahti, E., K. Lagerspetz, T. Nikkari and H. M. Fales 1964 Lipids of the uropygial gland of birds. Comp. Biochem. Physiol., 12: 4 3 5 4 3 7 . Hou, H. C. 1928 Studies on the glandula uropygialis of birds. Chin. Journ. Physiol., 2: 34.5 380. Jamieson, J. D., and G. E. Palade 1967a Intracellular transport of secretory proteins in the pancreatic exocrine cell. I . Role of the peripheral elements of t h e Golgi complex. J . Cell Biol., 34: 577-596. 1967b Intracellular transport of secretory proteins in the pancreatic exocrine cell. 11. Transport of condensing vacuoles and zymogen granules. J. Cell Biol., 34: 597-4315, Kanwar, K . C. 1961 Morphological and histochemical studies on the uropygial gland of pigeon and domestic fowl. Cytologia, 26: 124-136. Lucas, A. M . , and P. R. Stettenheim 1972 Avian Anatomy: Integument. Agricultural Handbook 362. U. S. Dept. Agriculture, Washington, D. C. Maiti, B. 1968 Dynamics of the uropygial gland in the pigeon. Folia Biologica, 16: 4 C ~ 5 4 . Maiti, B., and A. Ghosh 1969 Effect of cortisone on mitotic activity and cell loss in t h e uropygial gland of male pigeons. Acta Anat., 74: 97-103. Nicolaides, W . 1963 H u m a n skin surface lipids - origin, composition and possible function. In: Advances in Biology of Skin. Vol. IV. T h e Sebaceous Glands. W. Montagna, R. A. ElIis and A. F. Silver, eds. Pergamon Press, Oxford. Chap. XI, pp. 167-187. Palay, S. L. 1958 The morphology of secretion. I n : Frontiers in Cytology. S. L. Palay, ed. Yale Univ. Press. Chap. XI, pp. 3 0 5 3 4 2 , Quay, W. B. 1972 Integument and the environment: glandular composition, function, and evolution. Am. Zool., 12: 95-108. Siekevitz, P. 1963 Protoplasm: endoplasmic reticulum and microsomes and their properties. Ann. Rev. Phvsiol.. 25: 1 5 4 0 . Stern, M. 1905 Histologische Beitrage zur Sekretion der Burseldruse. Arch. f. mikros. Anat., 66: 294-311.
PLATE I EXPLANATION O F FIGURE
1
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Light photomicrograph of a transverse section through zone I of secretory tubules of the uropygial gland. Darkly staining basal cells form a thin layer around the periphery of each tubule. The inner thicker layer consists of lightly staining secretory cells. Hematoxylin and eosin stain. X 300; scale marker, 33.3 pm.
CHICKEN UROPYGIAL G L A N D Roger C . Wagner and Robert L. Boord
PLATE I
40 1
PLATE 2 E X P L A N A T I O N O F FIGURE
2
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Electron micrograph of a section through the wall of a secretory tubule. Progressive cytoplasmic differentiation is evident from the basal cells (B) which rest upon the basement membrane (large arrows) through stages I, I1 and 111 secretory cells. Mottled bodies (small arrows) are observable in basal cells as well as in stage I and 111 secretory cells. x 1,600; scale marker, 6.25 p m .
CHICKEN UROPYGIAL G L A N D Roger C Wagner and Robert L Boord
PLATE 2 .
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_
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- __. -. -- - -
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PLATE 3 EXPLANATION
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OF FIGURES
3
A basal cell showing the large ovoid nucleus with a normal nuclear envelope. T h e cytoplasm contains rough endoplasmic reticulum, ribosomal clusters, tonofilaments and mottled bodies (arrows). X 12,500; scale marker, 0.80 p m .
4
Higher magnification of a portion of a basal cell showing rough endoplasmic reticulum, ribosomal clusters and desmosomal junctions (arrows) from which tonofilaments radiate into the cytoplasm. X 29,200; scale marker, 0.34 p m .
5
Enlargement of a portion of the cell of figure 3 showing two membranebound bodies with mottled electron-dense contents. X 35,500; scale marker, 0.28 p m .
CHICKEN UROPYGIAL GLAND Roger C. Wagner and Robert L. Boord
PLATE 3
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PLATE 4 EXPLANATION OF FIGURES
406
6
A stage I secretory cell exhibiting smooth-surfaced vesicles of endoplasmic reticulum. The perinuclear cisterna is markedly dilated and appears beaded in areas constrained by nuclear pores (small arrows). Mottled bodies (large arrows) are present in the cytoplasm. X 14,400; scale marker, 0.69 pm.
7
A cluster of invaginated vesicles and larger cusp-shaped cisternae of endoplasmic reticulum in the cytoplasm of a type I secretory cell. X 18,000; scale marker, 0.58 pm.
8
Cusps of smooth endoplasmic reticulum exhibiting electron-transparent contents. A mottled body is bounded by a single membrane and exhibits no cristae. x 25,900; scale marker, 0.39 pm.
CHICKEN UROPYCIAL GLAND Roger C . Wagner and Robert L. Board
PLATE 4
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PLATE 5 EXPLANATION OF FIGURES
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9
A stage I1 secretory cell containing cisternae and saccules of smooth endoplasmic reticulum. The perinuclear cisterna is extremely dilated and nuclear pores appear broken in some areas (small arrows). Mottled bodies (large arrows) are still present and some have increased in size. X 12,500; scale marker, 0.80 p m .
10
Three mottled bodies surrounded by layers of saccules of endoplasmic reticulum. Two of the bodies have less electron-dense contents. X 40,600; scale marker, 0.25 fim.
11
Mottled bodies are distinct from mitochondria since they are lighter staining, bounded by a single membrane and are devoid of cristae. X 31,500; scale marker, 0.32 p m .
CHICKEN UROPYGIAL GLAND Roger C. Wagner and Robert L. Boord
PLATE 5
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PLATE 6 E X P L A N A T I O N O F FIGURES
12
A stage 111 secretory cell containing various stages in the development of secretory droplets. The perinuclear cisterna is no longer dilated. Mottled bodies are abundantly present and frequently occur in satellite association with secretory droplets (arrows). X 10,900; scale marker, 0.92 pm.
13 Secretory droplets contain electron-dense contents ranging from condensed reticular conglomerates to a thin layer associated with the inner wall. Concentric lamellae of endoplasmic reticulum encircle secretory droplets and mottled bodies. X 20,300; scale marker, 0.49 pm. 14
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Dilated cisternae are found within whorls of endoplasmic lamellae. Some contain a reticular substance similar to that found in secretory droplets (arrows). x 17,600; scale marker, 0.57pm.
CHICKEN UROPYGIAL GLAND Roger C. Wagner and Robert L. Boord
PLATE 6
41 1
PLATE 7 EXPLANATION O F FIGURES
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15
A stage IV secretory cell showing large luscent secretory droplets which are lined by a thin layer of electron-dense material. Small dense mottled bodies (arrows) are still present. The perinuclear cisterna remains undilated. x 13,700; scale marker, 0.73 p m .
16
Electron-dense material becomes dissociated from the inner wall of secretory droplets in some cases. Note the mottled body in close association with a secretory droplet (arrow). X 11,900; scale marker, 0.84 Fm.
17
The lumen of a secretory tubule exhibiting large electron-transparent areas and cellular debris consisting of filamentous bundles. X 10,500; scale marker, 0.95 p m .
CHICKEN UROPYGIAL GLAND Roger C. Wagner and Robert L. Boord
PLATE 7
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