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A Histological and Histochemical Study of the Glands of the External Auditory Meatus of the Ox
Brit. vet.]. (1965),
121, 223
A HISTOLOGICAL AND HISTOCHEMICAL STUDY OF THE GLANDS OF THE EXTERNAL AUDITORY MEATUS OF THE OX By S. D. A.
FERNANDO
D epartment of Veterinary Science, University of Ceylon, ColOJ;nho
-=============
SUMMAR Y
In the external auditory meatus of the ox, two types of secretory cells were encountered in the apocrine glands. These were (a) cells with a clear cytoplasm, and (b) cells with vacuolated cytoplasm. Cells with clear cytoplasm secreted by budding off apical cytoplasm, while cells with vacuolated cytoplasm secreted by rupture of their luminal border. The apocrine glands secret ed phospholipids, protein-bound lipids, acid mucopolysaccharides, glycogen and diastase resistant P.A.S. positive carbohydrates. Alkaline and acid phosphatases were also found in the secretions. The sebaceous gla nds secreted large amounts of neutral lipids. INTROD UCT IO N
The dim ensions of the glandular appendages of the skin of the ox were r eported by T akeda (195 I) in the course of a comparative study of the sizes of these glands in various species of animals. Nay & Dowling (1957) and Nay (1959) reported on the size and shape of the sweat glands of cattle. Findlay & Yang ( I 950) described their histology, while Yang (1952) investigat ed the histochemistry of these glands. Most of the histochemical investigations of Yang yielded negative results. This study was undertaken to gain information on the gla ndular appendages of the external a uditory canal and to add to the present knowledge of the skin of the ox. MATERIALS AND METHODS
Skin tissues were taken at slaughter from the skin lining the external auditory canal of five Ayrshire cattle. These tissues were stretched and pinned out on corks prior to immersion in the appropriate fixatives. Tissue blocks for lipid histochemistry were fixed in formol calcium cadmium and frozen sections 10 microns thick were made. These sections were coloured by Sudan Black in 70 per cent alcohol to demonstrate lipids. Baker's acid h aematein method (Pearse, 1960) and Menschik's nile blue method (Menschik, 1953) were used to demonstrate phospholipids. Cholesterol and its esters were studied by Schultze's method (Pearse, 1960) . Berenbaum's st ain ( 1958) was employed for the study of protein-bound lipids. Tissue blocks for enzyme study were prepared by the m ethod d escribed by
224
BRITISH VETERINARY JOURNAL,
121,5
Fernando (1963a). Alkaline phosphatase was demonstrated according to the coupling azo dye technique (Pearse, 1960) using sodium alpha naphthyl phosphate and 5-chloro-ortho toluidine in o· I M "tris" buffer. Acid phosphatases were demonstrated by the standard coupling azo dye method of Pearse (1960), using sodium alpha naphthyl phosphate and ortho diansidine in 0·1 M veronal acetate buffer at pH 5. Nucleic acids were investigated by the use of the Feulgen and the Gallocyaninchromalum methods (Pearse, 1960). Mallory's phosphotungstic acid haematoxylin was employed to demonstrate the myoepitheiial cells and Weigert's iron haematoxylin was used for routine histological study.
.
RESULTS
Histological Observations The skin which lines the external auditory canal of the ox carries three main appendages: I. Hairs 2. Sebaceous glands 3. Apocrine glands The hairs vary greatly in their development and stiffness. There is a preponderant growth of stiff hairs in an outward direction from the regions bounding the anti tragic notch, and the borders of the ear. The rest of the lining skin bears finer hairs which diminish progressively in size and stiffness as the skin passes from the cartilagenous to the bony meatus. The larger coarser hairs have their papillae embedded much deeper in the corium than the smaller lanugo hairs covering the other parts of the ear canal. The sebaceous glands are composed of two to five acini. These glands are round and compact and occur in the upper third of the dermis. Their average measurements are 240-280 microns in breadth and 150- 350 microns in depth. Actively secreting acini open via their short ducts into a larger common duct which opens directly to the exterior or into a hair follicle below the level of the dermo-epidermal junction. The apocrine glands of the ox are the predominant glandular elements of the external auditory canal (Fig. I). They are simple, long, tubular glands occurring in the deeper layers of the dermis and subcutaneous tissue These glands are heavily coiled and are as large or even larger than the sebaceous glands. In the proximal part of the cartilagenous auditory canal they are much larger than the sebaceous glands. These glands open via their long ducts into a hair follicle just below the opening of a sebaceous duct, or they open directly on to the skin surface. The glands are composed of an inner secretory layer surrounded by a closely enveloping myoepithelial layer resting on a hyaline basement membrane. Two types of secretory cells are observed. One type of cell is cuboidal or columnar in shape and has a large, round basal nucleus and a clear cytoplasm (Fig. I and 2). The other type of cell has a cytoplasm which contains many large vacuoles (Fig. 3). Both types of cells are met with in the same ear.
PLATE I
c
b
a Fig. I . Skin of the extern a l aud itory meatus of t he ox , show ing th e distri b uti on of the se baceous a nd apocrin e g la nd s. (a ) Vacuo lated cells, a nd (b ) ta ll co lum nar ce lls of th e a pocrine g lands; (c) se baceous g la nds. (Weigert H a nd E x 140.)
a
a
b _ _ _......
F ig . 2. The apocrine g la nds of the extern a l a ud itory mea tus of the ox. (a ) Tall columna r cell s w ith a la rge round ed nucle us. (b ) T he nu clei of a few m yoepith e lia l cells outside the secretory cells. (Weigert H a nd Ex 585. )
Fig. 3. T he vacuola ted ce lls of th e apocrine g la nds in the ear of the ox . (a ) Cells in the ph ase of active secret ion . (H & Ex 585. )
F ern a ndo , Brit. vet.]. ,
121,
5
PLATE II
F ig. 4 . Sudano phi lic material in the secretory cells of t he a pocrine g lands . (Sudan Black B x 585. )
a
Fig. 5. Protein-bound lipids in the apocrine gla nds of the ox. (a ) Pro tein-bound li pids in the secre tory cells. (Acetone Sudan Black B x 585.) F ernando , Brit. vet. ]' ,
121,
5
GLANDS OF THE EAR OF THE OX
225
The myoepithelial layer is composed of elongated cells with a strongly basophilic nucleus. These cells are disposed along the length of the gland and lie on the thin basement membrane. Methods of secretion. The tall columnar cells with clear cytoplasm have apical blebs protruding into the lumen of the glands. These blebs are budded off into the lumen to form one homogeneous mass of secretion. The vacuolated cells, however, secrete by another mechanism. The luminal border of these cells ruptures and allows the secretions to escape into the lumen of the gland. The sebaceous glands secrete by the holocrine method. Their secretions are poured into the hair follicles and there mix with secretions of the apocrine glands to form cerumen or ear wax found coating the external auditory canal.
Histochemical 0 bservations Sudan Black B stain revealed the presence of lipids in the mature sebaceous glands. These lipids were all neutral in character as shown by Cain's nile blue stain. Immature acini and the peripheral cells of mature acini contained droplets of phospholipids. Cholesterol or its esters could not be demonstrated in the sebaceous and apocrine glands of the ear. The apocrine ceruminous glands revealed the presence of large amounts of lipids in the cytoplasm as well as in the secretions contained in the lumen of the glands (Fig. 4). These lipids were mainly phospholipids and protein-bound lipids (Fig. 5). Periodic acid-Schiff positive substances. Those substances that stained positively with periodic acid-Schiff (P.A.S.) but which were removed by digestion with saliva for 30 minutes at 37°C were assumed to be glycogen. The sebaceous glands contained glycogen in discrete granules in the less mature peripheral cells. The mature acini contained no glycogen. Glycogen was also present in the cytoplasm ' of the secretory cells of the apocrine ceruminous glands. Glycogen in the secretions contained within the lumen were mixed with other P.A.S. positive substances. P.A.S. positive saliva-resistant substances. Substances belonging to this class were absent in the sebaceous glands. The apocrine glands, however, showed the presence of large quantities of these materials in the cytoplasm of the secretory cells and within the contents of the lumina of the glands. The P.A.S. positive substances did not belong to the class of unsaturated lipids, as pyridine extraction for 18 hours at 37°C did not prevent a positive P.A.S. reaction, and, furthermore, the P.A.S. reaction was not blocked by bromination. Acid mucopolysaccharides. No acid mucopolysaccharides were contained in the sebaceous glands. The secretory cells of the apocrine gland contained these substances confined to the luminal margins of the cells. The secretions within the lumina of these glands also contained acid mucopolysaccharides. D.N.A. and R.N.A. Deoxyribonucleic acids were found in the nuclei of the
BRITISH VETERINARY JOURNAL, 121,5
sebaceous and apocrine cells. Their nucleoli, however, revealed the presence of ribonucleic acids only. Ribonucleic acids were found in the cytoplasmic network between the lipid droplets of immature sebaceous cells. In the lipid-laden mature sebaceous cells R.N.A. was seen in traces. The cytoplasm of the ap
Histological observations show that the ~ebaceous glands of the ox are similar to those of the external auditory canal of other species of mammals: man (Montagna, Noback & Zak, 1948), cat (Montagna, 1949) and pig (Fernando, 19 63b). Investigations of series of apocrine glands showed secretions in the lumen having varying degrees of homogeneity. These observations lend support to the work of Findlay & Yang (1950) who said that the final homogeneous mass of secretion found in the lumen of the gland was a result of the transformation of a coarse primary secretion. Histochemical findings differ considerably from those of Yang (1952). He was able to show the presence oflipids, cholesterol and glycogen in the sebaceous glands of the ox. Cholesterol esters were not observed in the present study, while phospholipids were seen in the immature sebaceous cells. Yang said that no lipids, phospholipids, glycogen, mucoproteins, D.N.A. and acid phosphatases were present in the apocrine glands. The present work has shown all the above m entioned substances to be present along with protein bound lipids, acid mucopolysaccharides, R.N.A. and alkaline phosphatases. ACKNOWLEDGEMENTS
My thanks are due to Prof. J. McCunn, Messrs M. G. A. D. Scott and R. F. S. Creed of the Royal Veterinary College, London, for their advice and facilities provided and Mr. H. Burgess for the microphotographs. REFERENCES
M. C. (1954), Nature, Lond., 174, 190. S. D. A. (1963a). C£DIlon vet. J., 11, 39. FERNANDO, S. D. A. (1963b). C£DIlon vet. J., II, 112. FINDLAY, J. D. & YANG, S. H. (1950). J. agric. Sci., 40, 126. BERENBAUM, FERNANDO,
GLANDS OF THE EAR OF THE OX MENSCHIK, Z. ( 1953). Stain T echnol., 28, 13. MONTAGNA, W. (1949). ). Morph., 85, 423. MONTAGNA, W., No BACK, C. R. & ZAK, F. G. (1948). Amer. J. Anat., 83, 409. NAY, T. (1959). Aust.J. agric. Res., 10, 12I. NAY, T. & DOWLING, D . F. (1957) . Aust. J . agric. Res., 8, 385. PEARSE, A. G. E. ( 1960) . 'Histochemistry Theoretical & Applied'. 2nd edn. Churchill. TAKEDA, S. ( 1951 ). Okajima's Folia anat.japon., 23, 357. YAN G, S. H. (1952). ]. agric. Sci., 42, 155.
(Receivedjor publication 21 December, 1964)
D
227
London:
121, 223
A HISTOLOGICAL AND HISTOCHEMICAL STUDY OF THE GLANDS OF THE EXTERNAL AUDITORY MEATUS OF THE OX By S. D. A.
FERNANDO
D epartment of Veterinary Science, University of Ceylon, ColOJ;nho
-=============
SUMMAR Y
In the external auditory meatus of the ox, two types of secretory cells were encountered in the apocrine glands. These were (a) cells with a clear cytoplasm, and (b) cells with vacuolated cytoplasm. Cells with clear cytoplasm secreted by budding off apical cytoplasm, while cells with vacuolated cytoplasm secreted by rupture of their luminal border. The apocrine glands secret ed phospholipids, protein-bound lipids, acid mucopolysaccharides, glycogen and diastase resistant P.A.S. positive carbohydrates. Alkaline and acid phosphatases were also found in the secretions. The sebaceous gla nds secreted large amounts of neutral lipids. INTROD UCT IO N
The dim ensions of the glandular appendages of the skin of the ox were r eported by T akeda (195 I) in the course of a comparative study of the sizes of these glands in various species of animals. Nay & Dowling (1957) and Nay (1959) reported on the size and shape of the sweat glands of cattle. Findlay & Yang ( I 950) described their histology, while Yang (1952) investigat ed the histochemistry of these glands. Most of the histochemical investigations of Yang yielded negative results. This study was undertaken to gain information on the gla ndular appendages of the external a uditory canal and to add to the present knowledge of the skin of the ox. MATERIALS AND METHODS
Skin tissues were taken at slaughter from the skin lining the external auditory canal of five Ayrshire cattle. These tissues were stretched and pinned out on corks prior to immersion in the appropriate fixatives. Tissue blocks for lipid histochemistry were fixed in formol calcium cadmium and frozen sections 10 microns thick were made. These sections were coloured by Sudan Black in 70 per cent alcohol to demonstrate lipids. Baker's acid h aematein method (Pearse, 1960) and Menschik's nile blue method (Menschik, 1953) were used to demonstrate phospholipids. Cholesterol and its esters were studied by Schultze's method (Pearse, 1960) . Berenbaum's st ain ( 1958) was employed for the study of protein-bound lipids. Tissue blocks for enzyme study were prepared by the m ethod d escribed by
224
BRITISH VETERINARY JOURNAL,
121,5
Fernando (1963a). Alkaline phosphatase was demonstrated according to the coupling azo dye technique (Pearse, 1960) using sodium alpha naphthyl phosphate and 5-chloro-ortho toluidine in o· I M "tris" buffer. Acid phosphatases were demonstrated by the standard coupling azo dye method of Pearse (1960), using sodium alpha naphthyl phosphate and ortho diansidine in 0·1 M veronal acetate buffer at pH 5. Nucleic acids were investigated by the use of the Feulgen and the Gallocyaninchromalum methods (Pearse, 1960). Mallory's phosphotungstic acid haematoxylin was employed to demonstrate the myoepitheiial cells and Weigert's iron haematoxylin was used for routine histological study.
.
RESULTS
Histological Observations The skin which lines the external auditory canal of the ox carries three main appendages: I. Hairs 2. Sebaceous glands 3. Apocrine glands The hairs vary greatly in their development and stiffness. There is a preponderant growth of stiff hairs in an outward direction from the regions bounding the anti tragic notch, and the borders of the ear. The rest of the lining skin bears finer hairs which diminish progressively in size and stiffness as the skin passes from the cartilagenous to the bony meatus. The larger coarser hairs have their papillae embedded much deeper in the corium than the smaller lanugo hairs covering the other parts of the ear canal. The sebaceous glands are composed of two to five acini. These glands are round and compact and occur in the upper third of the dermis. Their average measurements are 240-280 microns in breadth and 150- 350 microns in depth. Actively secreting acini open via their short ducts into a larger common duct which opens directly to the exterior or into a hair follicle below the level of the dermo-epidermal junction. The apocrine glands of the ox are the predominant glandular elements of the external auditory canal (Fig. I). They are simple, long, tubular glands occurring in the deeper layers of the dermis and subcutaneous tissue These glands are heavily coiled and are as large or even larger than the sebaceous glands. In the proximal part of the cartilagenous auditory canal they are much larger than the sebaceous glands. These glands open via their long ducts into a hair follicle just below the opening of a sebaceous duct, or they open directly on to the skin surface. The glands are composed of an inner secretory layer surrounded by a closely enveloping myoepithelial layer resting on a hyaline basement membrane. Two types of secretory cells are observed. One type of cell is cuboidal or columnar in shape and has a large, round basal nucleus and a clear cytoplasm (Fig. I and 2). The other type of cell has a cytoplasm which contains many large vacuoles (Fig. 3). Both types of cells are met with in the same ear.
PLATE I
c
b
a Fig. I . Skin of the extern a l aud itory meatus of t he ox , show ing th e distri b uti on of the se baceous a nd apocrin e g la nd s. (a ) Vacuo lated cells, a nd (b ) ta ll co lum nar ce lls of th e a pocrine g lands; (c) se baceous g la nds. (Weigert H a nd E x 140.)
a
a
b _ _ _......
F ig . 2. The apocrine g la nds of the extern a l a ud itory mea tus of the ox. (a ) Tall columna r cell s w ith a la rge round ed nucle us. (b ) T he nu clei of a few m yoepith e lia l cells outside the secretory cells. (Weigert H a nd Ex 585. )
Fig. 3. T he vacuola ted ce lls of th e apocrine g la nds in the ear of the ox . (a ) Cells in the ph ase of active secret ion . (H & Ex 585. )
F ern a ndo , Brit. vet.]. ,
121,
5
PLATE II
F ig. 4 . Sudano phi lic material in the secretory cells of t he a pocrine g lands . (Sudan Black B x 585. )
a
Fig. 5. Protein-bound lipids in the apocrine gla nds of the ox. (a ) Pro tein-bound li pids in the secre tory cells. (Acetone Sudan Black B x 585.) F ernando , Brit. vet. ]' ,
121,
5
GLANDS OF THE EAR OF THE OX
225
The myoepithelial layer is composed of elongated cells with a strongly basophilic nucleus. These cells are disposed along the length of the gland and lie on the thin basement membrane. Methods of secretion. The tall columnar cells with clear cytoplasm have apical blebs protruding into the lumen of the glands. These blebs are budded off into the lumen to form one homogeneous mass of secretion. The vacuolated cells, however, secrete by another mechanism. The luminal border of these cells ruptures and allows the secretions to escape into the lumen of the gland. The sebaceous glands secrete by the holocrine method. Their secretions are poured into the hair follicles and there mix with secretions of the apocrine glands to form cerumen or ear wax found coating the external auditory canal.
Histochemical 0 bservations Sudan Black B stain revealed the presence of lipids in the mature sebaceous glands. These lipids were all neutral in character as shown by Cain's nile blue stain. Immature acini and the peripheral cells of mature acini contained droplets of phospholipids. Cholesterol or its esters could not be demonstrated in the sebaceous and apocrine glands of the ear. The apocrine ceruminous glands revealed the presence of large amounts of lipids in the cytoplasm as well as in the secretions contained in the lumen of the glands (Fig. 4). These lipids were mainly phospholipids and protein-bound lipids (Fig. 5). Periodic acid-Schiff positive substances. Those substances that stained positively with periodic acid-Schiff (P.A.S.) but which were removed by digestion with saliva for 30 minutes at 37°C were assumed to be glycogen. The sebaceous glands contained glycogen in discrete granules in the less mature peripheral cells. The mature acini contained no glycogen. Glycogen was also present in the cytoplasm ' of the secretory cells of the apocrine ceruminous glands. Glycogen in the secretions contained within the lumen were mixed with other P.A.S. positive substances. P.A.S. positive saliva-resistant substances. Substances belonging to this class were absent in the sebaceous glands. The apocrine glands, however, showed the presence of large quantities of these materials in the cytoplasm of the secretory cells and within the contents of the lumina of the glands. The P.A.S. positive substances did not belong to the class of unsaturated lipids, as pyridine extraction for 18 hours at 37°C did not prevent a positive P.A.S. reaction, and, furthermore, the P.A.S. reaction was not blocked by bromination. Acid mucopolysaccharides. No acid mucopolysaccharides were contained in the sebaceous glands. The secretory cells of the apocrine gland contained these substances confined to the luminal margins of the cells. The secretions within the lumina of these glands also contained acid mucopolysaccharides. D.N.A. and R.N.A. Deoxyribonucleic acids were found in the nuclei of the
BRITISH VETERINARY JOURNAL, 121,5
sebaceous and apocrine cells. Their nucleoli, however, revealed the presence of ribonucleic acids only. Ribonucleic acids were found in the cytoplasmic network between the lipid droplets of immature sebaceous cells. In the lipid-laden mature sebaceous cells R.N.A. was seen in traces. The cytoplasm of the ap
Histological observations show that the ~ebaceous glands of the ox are similar to those of the external auditory canal of other species of mammals: man (Montagna, Noback & Zak, 1948), cat (Montagna, 1949) and pig (Fernando, 19 63b). Investigations of series of apocrine glands showed secretions in the lumen having varying degrees of homogeneity. These observations lend support to the work of Findlay & Yang (1950) who said that the final homogeneous mass of secretion found in the lumen of the gland was a result of the transformation of a coarse primary secretion. Histochemical findings differ considerably from those of Yang (1952). He was able to show the presence oflipids, cholesterol and glycogen in the sebaceous glands of the ox. Cholesterol esters were not observed in the present study, while phospholipids were seen in the immature sebaceous cells. Yang said that no lipids, phospholipids, glycogen, mucoproteins, D.N.A. and acid phosphatases were present in the apocrine glands. The present work has shown all the above m entioned substances to be present along with protein bound lipids, acid mucopolysaccharides, R.N.A. and alkaline phosphatases. ACKNOWLEDGEMENTS
My thanks are due to Prof. J. McCunn, Messrs M. G. A. D. Scott and R. F. S. Creed of the Royal Veterinary College, London, for their advice and facilities provided and Mr. H. Burgess for the microphotographs. REFERENCES
M. C. (1954), Nature, Lond., 174, 190. S. D. A. (1963a). C£DIlon vet. J., 11, 39. FERNANDO, S. D. A. (1963b). C£DIlon vet. J., II, 112. FINDLAY, J. D. & YANG, S. H. (1950). J. agric. Sci., 40, 126. BERENBAUM, FERNANDO,
GLANDS OF THE EAR OF THE OX MENSCHIK, Z. ( 1953). Stain T echnol., 28, 13. MONTAGNA, W. (1949). ). Morph., 85, 423. MONTAGNA, W., No BACK, C. R. & ZAK, F. G. (1948). Amer. J. Anat., 83, 409. NAY, T. (1959). Aust.J. agric. Res., 10, 12I. NAY, T. & DOWLING, D . F. (1957) . Aust. J . agric. Res., 8, 385. PEARSE, A. G. E. ( 1960) . 'Histochemistry Theoretical & Applied'. 2nd edn. Churchill. TAKEDA, S. ( 1951 ). Okajima's Folia anat.japon., 23, 357. YAN G, S. H. (1952). ]. agric. Sci., 42, 155.
(Receivedjor publication 21 December, 1964)
D
227
London: