- Email: [email protected]
Ultrastructural localization of lectin-binding sites in normal human eccrine and apocrine glands
Journal of Dermatological
Science, 2 (1991)
55-61
55
Elsevier
DESC 00061
Ultrastructural
localization of lectin-binding sites in normal human eccrine and apocrine glands
G. Schaumburg-Lever
‘, G. Metzler
I Department of Dermatology, Eberhard-Karls-Universitiit, (Received
’
and M. Tronnier2
Tiibingen and ’ Department
12 May 1990; accepted 26 September
Key words: Ultrastructure;
Lectin-binding;
of Dermatology, Liibeck, F.R.G.
1990)
Eccrine gland; Apocrine
gland
Abstract
The distribution of carbohydrate residues in eccrine and apocrine glands of normal human skin was studied using a postembedding technique with Lowicryl K4M. Thin sections were incubated with Ulex europaeus agglutinin I (UEA I), wheat germ agglutinin (WGA), peanut agglutinin (PNA), concanavalin A (Con A), soybean agglutinin (SBA), and dolichos biflorus agglutinin (DBA). All lectins except for PNA showed labeling of the plasma membranes of dark cells, clear cells, and apocrine cells. The granules of the eccrine gland were labeled with all lectins except for DBA. The mitochondrial granules of the apocrine gland were not labeled with any lectin, whereas the lysosomal granules showed a positive reaction with all lectins except for PNA. After incubation with PNA, in eccrine glands the granules were the only structure labeled, whereas in apocrine glands the luminal side of the plasma membrane and cytoplasmic vesicles beneath it were the only structures labeled.
Introduction The secretory portion of eccrine glands consists of two types of secretory cells (clear and dark cells) and of myoepithelial cells [ 11. The clear cells are broader at the base than they are at the lumen. By electron microscopy, they show numerous villous folds wherever two clear cells lie side by side. The villous folds of adjacent clear cells interdigitate with one another. In addition, intercellular canaliculi are present between adjoining clear cells [ 11, showing tortuous microvilli protruding into their central opening. The dark Correspondence to: Gundula Schaumburg-Lever, Department of Dermatology, Calwerstrasse, 7, D-7400 Ttibingen, F.R.G.
0923-181 l/91/.$03.50 0 1991 Elsevier Science Publishers
cells contain large electron-dense granules, particularly in their luminal portion [ 11, which are PAS-positive and contain sialomucin. Eccrine glands are merocrine glands. The dark cells excrete by expulsion of their PAS-positive granules into the lumen [ 1, 21 whereas the clear cells excrete precursor sweat into the canaliculi [l-3]. Precursor sweat contains electrolytes and lactate. The secretory portion of the apocrine gland consists of a single layer of secretory cells facing the lumen and of myoepithelial cells [ 11. Apocrine glands show three types of secretion [4] ; merocrine, apocrine, and holocrine. The secretory cells vary in height. After fixation with glutaraldehyde and osmium tetroxide two types of granules can be recognized by electron microscopy: ‘dark’ and
B.V. (Biomedical
Division)
56
‘light’ granules. The ‘dark granules’ are of lysosomal origin. They contain protein, lipids, sialomucin and are PAS-positive by light microscopy [ 11. The ‘light granules’ are derived from mitochondria and possess cristae. In Lowicryl embedded tissue, which is not fixed in osmium tetroxide, the ‘dark granules’ (lysosomal granules) appear as light granules. The ‘light granules’ (mitochondrial granules) appear as dark granules. In order to avoid confusion, the two types of granules will be referred to as lysosomal or mitochondrial granules. Previous investigations by light microscopy involving biotinylated lectins and an avidinbiotin-peroxidase complex [ 5,6] have shown that eccrine glands were positive after incubation with concanavalin A (Con A) [ 5,6], wheat germ agglutinin (WGA) [5, 61, ricinus communis agglutinin (RCA) [6], dolichos biflorus agglutinin (DBA) [ 5, 61, soybean agglutinin (SBA) [5, 61, and Ulex europaeus agglutinin (UEA I) [ 61. Incubation with peanut agglutinin (PNA) showed a negative reaction. However, after incubation with neuraminidase and subsequent incubation with PNA, the cells became positive [ 61 indicating that TABLE
the PNA-binding sites had been masked by neuraminic acid [ 61. Apocrine glands after incubation with FITCconjugated WGA and PNA showed a positive reaction in both the cytoplasm and plasma membrane [7]. Recently the ultrastructural distribution of carbohydrate residues in keratinocytes and of CI-L -fucose in eccrine glands has been studied in our laboratory [ 8,9]. It was shown that lectin-binding sites were evenly distributed within the plasma membrane of keratinocytes after incubation with Con A and WGA. In contrast, PNA-binding sites were present in the plasma membrane between the desmosomes, absent in that portion of the plasma membranes of basal cells facing the basal lamina and almost completely absent within desmosomes. The purpose of the present investigation was to find out: 1) Is there a difference in the distribution of carbohydrate residues between the luminal and the basolateral portion of the plasma membrane of secretory cells? 2) Is there a difference in the lectin-binding pattern between eccrine and apocrine glands?
I
The lectins used, their specificity and binding inhibitors Lectin
Abbreviated
Concanavalia ensiformis Peanut agglutinin Wheat germ agglutinin Ulex europaeus agglutinin Dolichos biflorus agglutinin Soybean agglutinin
Con A PNA WGA UEA DBA SBA
Gal = galactose; GalNAc = N-acetyl-galactosamine; (sialic acid); Man = mannose; Glc = glucose.
name
Major sugar specificity
Binding inhibitor
cc-D-Glc, a-D-Man Gal-p( I-3)-GalNAc (B-( I-4)-D-GlcNAc),
X-D-Methyl-Man lactose NeuNac
NeuNAc
ED-fUCOSe
N-D-fUCOSC
a-D-GalNAc a-D-GalNAc
cc-o-GalNAc a-D-GalNAc
GlcNAc = N-acetyl-glucosamine;
NeuNAc = acetyl-neuraminic
acid
Fig. 1. (a-f) Eccrine gland. (a) Incubation with WGA shows labeling of the plasma membrane (arrow) of a myoepithelial (myo) cell and of the villous folds (asterisks) of adjacent clear cells. x 7,000. (b) Incubation with SBA shows distinct gold particles in the villous folds of two adjacent clear cells. x 7,000. (c) Incubation with Con A, dark cell. There is labeling of the perinuclear space (arrows), the granules (g) and of the cytoplasm in association with glycogen granules (arrowheads). The mitochondria (m) are negative and serve as a built in control. x 20,000. (d) Incubation with UEA I shows distinct labeling of the villous folds of two adjacent clear cells. The mitochondrium (m) is negative. x 20,000. (e) Incubation with PNA shows heavy labeling of the granules of a dark cell. L = lumen of the eccrine gland. x 12,000. (f) Negative control. There is no labeling of the villous folds of two adjacent clear cells. x 20,000.
58
to ing the temperature -35°C and was embedded in Lowicryl at -35°C. The lectins used and their specificity as well as their binding inhibitors are listed in Table I. The immunocytochemical procedure was carried out as previously described [ 81.
Materials and Methods Normal skin of five healthy volunteers was fixed in a mixture of 2% paraformaldehyde and 0.1% glutaraldehyde in PBS (pH 7.2) for 4 h. The specimens were rinsed in PBS, 0.5 M NH&l in PBS [ lo], rinsed again in PBS. The tissue was dehydrated in graded ethanol by gradually lowerTABLE
Results All results are summarized
II
Distribution
in Table II.
of lectin binding sites in eccrine and apocrine glands UEA
WGA
PNA
Con A
SBA
DBA
+ ++ + _ _
++ +++ + _
_ _ _ _
ii +++ ++ _ +
+ ++ _ _ -
++ ++ _ -
++ ++ + _ -
++ + + +++
_ +++
+++ ++ ++ _ + +++
+ + _ _ _ ii
++ ++ _ _ _ _
+++ + ++ _ _ +
+++ _ _ _ ++
++ ++ + +++ _ _ + t+
+ ++ + _ _ _ -
+ +t + _ _ _ -
++ _ _ _ _
_ _ _ _
+ _ _ +
_ _ _ -
_ _ _
Eccrine Glands Clear cells
Plasma membrane:
luminal basolateral desmosomes nucleus, mitochondria nuclear membrane, RER Dark cells
Plasma membrane:
luminal basolateral desmosomes
nucleus, mitochondria nuclear membrane, RER granules Apocrine glands Plasma membrane:
luminal basolateral desmosomes
lysosomal granules mitochondrial granules nucleus, mitochondria nuclear membrane cytoplasmic vesicles beneath the lumen
+++ +++ ;i, + _ _ +
Myoepithelial cells of eccrine and apocrine glands Plasma membrane _ myotilaments _ dense bodies _ nucleus _ nuclear membrane ( + ) occasional,
+ weak, + + moderate,
+ + + strong labeling RER rough endoplasmic
reticulum.
Fig. 2. (a-d) Apocrine gland. (a) Incubation with WGA, apocrine cell undergoing apocrine secretion. L = lumen of the gland. Heavy labeling of the luminal plasma membrane including the villous projections and of the lysomal granules (lyg). x 12,000. (b) Incubation with Con A, apocrine cell with evidence of apocrine secretion. Two types of granules can be seen. The lysomal granules (arrows) are labeled. x 3,000. (c) Incubation with Con A, higher magnification of the two types of granules: the mitochondrial granules (arrows) are negative, whereas the lysosomal granule (lyg) shows good labeling. x 12,000. (d) Incubation with PNA: the luminal plasma membranes as well as cytoplasmic vesicles beneath it (arrows) are labeled. L = lumen ofthe gland. x 20,000.
59
60
Eccrine glands
When incubated with UEA, WGA, Con A, SBA and DBA, gold particles were found in the luminal portion of the plasma membrane as well as in the basolateral portion of both clear and dark cells (Fig. la, b and d). In general the lectin binding sites of the basolateral plasma membrane of clear cells outnumbered the lectin binding sites of the luminal plasma membrane of clear cells, whereas in dark cells the gold particles in the luminal plasma membrane outnumbered those of the basolateral portion of the plasma membrane. The desmosomes showed labeling with UEA (Fig. Id), WGA, and Con A but no labeling with SBA and DBA. The granules of the dark cells (Fig. lc) showed labeling with all lectins except for DBA. After incubation with PNA, the granules in dark cells were the only structures labeled (Fig. le). No PNA binding sites were detected in clear cells. The nuclei and mitochondria were never labeled, no matter which lectin was used (Fig. lc) and thus served as a built in negative control. However, the perinuclear membrane was labeled after incubation with Con A (Fig. lc). Cytoplasmic labeling was seen after labeling with Con A and WGA. The negative controls were always negative (Fig. If) except for an occasional gold particle. Apocrine glands
After incubation with UEA, WGA (Fig. 2a), Con A, SBA, and DBA the luminal and basolateral plasma membranes of the secretory cells were labeled. The number of gold particles associated with the luminal plasma membrane outnumbered those associated with the basolateral plasma membrane except after incubation with SBA and DBA. The nuclei, mitochondria, and mitochondrial granules were never labeled with any lectin (Fig. 2~). Cytoplasmic labeling was seen after incubation with Con A indicating the presence of glycogen. The lysosomal granules showed weak labeling with UEA, SBA, and DBA, moderate and strong labeling with WGA and Con A (Fig. 2b and c) and no labeling with PNA. PNA binding sites were seen in the luminal
plasma membrane as well as in vesicles located directly beneath the plasma membrane. Vesicles located in the center of the cell were not labeled with PNA. The number of gold particles did not seem to depend on the functional state of the cell, i.e. cells with evidence of decapitation (Fig. 2b) did not show heavier or weaker labeling than those not undergoing decapitation secretion. Myoepithelial cells of eccrine and apocrine glands
The plasma membrane was labeled after incubation with WGA and Con A only. The myofilaments were not labeled with any lectin. Discussion The distribution of lectin binding sites in eccrine and apocrine glands was similar to the distribution in keratinocytes of normal epidermis [8]: Con A and WGA binding sites were found within the cytoplasm as well as in the plasma membrane. All lectins except for PNA were associated with the entire plasma membrane and all lectins except for DBA showed a positive reaction in the granules of the dark cells of eccrine glands. The lysosomal granules of apocrine glands were positive after incubation with UEA, WGA, Con A, SBA, and DBA whereas the mitochondrial granules did not show any reaction with any of the lectins. Incubation with PNA revealed an interesting difference between eccrine and apocrine glands: in eccrine glands the granules were positive. Both granules of the apocrine glands, however, were negative, but in apocrine glands the luminal plasma membrane was positive whereas in eccrine glands the luminal membrane was negative. After incubation with Con A gold particles indicating the presence of mannose and glucose residues were found in both glands in the perinuclear space and in other portions of the rough endoplasmic reticulum. The presence of Con A binding sites in these locations is compatible with the assembly of carbohydrate chains. The glycoproteins acquire their core sugars in the endoplasmic reticulum [ 1 l] and then enter the Golgi complex where the carbohydrate units of glyco-
61
the glycoproteins are altered. Subsequently proteins are sorted and transported via separate types of vesicles to the plasma membrane, secretory granules, and lysosomes [ 121. Unfortunately it is difficult to recognize the membranes of a Golgi complex in Lowicryl embedded tissue, which is most likely due to the weak fixative used (0.1% glutaraldehyde and no osmium tetroxide). In eccrine glands the glycoproteins are transported from the Golgi complex to the luminal and basolateral plasma membranes of dark and clear cells. In dark cells more gold particles were found associated with the luminal plasma membrane than with the basolateral plasma membrane, whereas in clear cells heavier labeling was seen in the basolateral plasma membrane. This could possibly be correlated to the functions of the various portions of the plasma membrane: in dark cells secretion occurs by discharging the granules with their glycoproteins through the luminal membrane of the gland [ 31. In clear cells the basolateral membrane is the site of active transport of ions for sweat secretion [3]. It is not known which role the carbohydrate residues play in the production of sweat. The significance of apocrine secretion is unknown. The fact that PNA binding sites in apocrine glands can be detected in the luminal plasma membrane and the vesicles located beneath it most likely can be explained by the fact that terminal galactosyl groups in other locations are masked by sialic acid [ 131. The masked PNAbinding sites most likely split off their terminal sialic acid as they approach the luminal plasma membrane. This is supported by light microscopy. If the tissue is incubated first with neuraminidase and subsequently with PNA, more PNA binding sites can be revealed. It can be concluded: 1) There is a quantitative difference in the distribution of carbohydrate residues between the luminal and basolateral plasma membranes of dark and clear cells: in dark cells the luminal membranes show heavier labeling than the basolateral membranes, whereas in clear cells the basolateral membranes show heavier labeling than the luminal membranes.
2) After incubation with PNA, in eccrine glands the granules of dark cells are the only portion labeled, whereas in apocrine glands both types of granules are negative, but the luminal plasma membranes and cytoplasmic vesicles beneath it are the only structures labeled. Acknowledgement
We thank Birgit Fehrenbacher technical assistance.
for her excellent
References 1 Lever WF, Schaumburg-Lever G: Histopathology of the Skin, 7th edition, Lippencott, Philadelphia, pp 21-26, 1990. 2 Sato K: Biology of eccrine sweat glands, in: Dermatology in General Medicine. Edited by T. Fitzpatrick et al. McGraw-Hill New York, 1987, pp 195-209. 3 Sato K, Kang WH, Saga K, Sato KT: Biology of sweat glands and their disorders. I. Normal sweat gland function. J Am Acad Dermatol 20. 537-563, 1989. 4 Schaumburg-Lever G, Lever WF: Secretion from human apocrine glands. J Invest Dermatol 64: 38-41, 1975. 5 Ookusa Y, Takata K, Nagashima M, Hirano H: Distribution of glycoconjugates in normal human skin using biotinyl lectins and avidin-horseradish peroxidase. Histochem 79: l-7, 1983. 6 Schaumburg-Lever G, Alroy J, Ucci A, Lever WF: Distribution of carbohydrate residues in normal skin. Arch Dermatol Res 276: 216-223, 1984. I Tamaki K, Hino H, Ohara K, Furue M: Lectin-binding sites in Paget’s disease. Br J Dermatol 113: 17-24, 1985. 8 Schaumburg-Lever G: Ultrastructural localization of lectin-binding sites in normal skin. J Invest Dermato194: 465-470, 1990. 9 Metzler G, Schaumburg-Lever G, Liebig K: Ultrastructural localization of keratin and a+fucose in human eccrine sweat glands. Arch Dermatol Res 282: 12-16, 1989. 10 Roth J, Bendayan M, Carlemalm E et al.: Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue. J Histochem Cytochem 29: 663-671, 1981. 11 Stryer L: Protein targeting, 3rd ed, in: Biochemistry. Edited by L. Stryer. WH Freeman and Company, New York, pp 774-776, 1988. 12 Dunphy WG, Rothman JE: Compartmental organization of the Golgi stack. Cell 42: 13-21, 1985. 13 Faraggian T, Jagirdar J, Patil J: The expression of sialic acid-rich coat on the alveolar surface of adult and fetal human lungs. J Histochem Cytochem 34: 107 (abstract), 1986.
Science, 2 (1991)
55-61
55
Elsevier
DESC 00061
Ultrastructural
localization of lectin-binding sites in normal human eccrine and apocrine glands
G. Schaumburg-Lever
‘, G. Metzler
I Department of Dermatology, Eberhard-Karls-Universitiit, (Received
’
and M. Tronnier2
Tiibingen and ’ Department
12 May 1990; accepted 26 September
Key words: Ultrastructure;
Lectin-binding;
of Dermatology, Liibeck, F.R.G.
1990)
Eccrine gland; Apocrine
gland
Abstract
The distribution of carbohydrate residues in eccrine and apocrine glands of normal human skin was studied using a postembedding technique with Lowicryl K4M. Thin sections were incubated with Ulex europaeus agglutinin I (UEA I), wheat germ agglutinin (WGA), peanut agglutinin (PNA), concanavalin A (Con A), soybean agglutinin (SBA), and dolichos biflorus agglutinin (DBA). All lectins except for PNA showed labeling of the plasma membranes of dark cells, clear cells, and apocrine cells. The granules of the eccrine gland were labeled with all lectins except for DBA. The mitochondrial granules of the apocrine gland were not labeled with any lectin, whereas the lysosomal granules showed a positive reaction with all lectins except for PNA. After incubation with PNA, in eccrine glands the granules were the only structure labeled, whereas in apocrine glands the luminal side of the plasma membrane and cytoplasmic vesicles beneath it were the only structures labeled.
Introduction The secretory portion of eccrine glands consists of two types of secretory cells (clear and dark cells) and of myoepithelial cells [ 11. The clear cells are broader at the base than they are at the lumen. By electron microscopy, they show numerous villous folds wherever two clear cells lie side by side. The villous folds of adjacent clear cells interdigitate with one another. In addition, intercellular canaliculi are present between adjoining clear cells [ 11, showing tortuous microvilli protruding into their central opening. The dark Correspondence to: Gundula Schaumburg-Lever, Department of Dermatology, Calwerstrasse, 7, D-7400 Ttibingen, F.R.G.
0923-181 l/91/.$03.50 0 1991 Elsevier Science Publishers
cells contain large electron-dense granules, particularly in their luminal portion [ 11, which are PAS-positive and contain sialomucin. Eccrine glands are merocrine glands. The dark cells excrete by expulsion of their PAS-positive granules into the lumen [ 1, 21 whereas the clear cells excrete precursor sweat into the canaliculi [l-3]. Precursor sweat contains electrolytes and lactate. The secretory portion of the apocrine gland consists of a single layer of secretory cells facing the lumen and of myoepithelial cells [ 11. Apocrine glands show three types of secretion [4] ; merocrine, apocrine, and holocrine. The secretory cells vary in height. After fixation with glutaraldehyde and osmium tetroxide two types of granules can be recognized by electron microscopy: ‘dark’ and
B.V. (Biomedical
Division)
56
‘light’ granules. The ‘dark granules’ are of lysosomal origin. They contain protein, lipids, sialomucin and are PAS-positive by light microscopy [ 11. The ‘light granules’ are derived from mitochondria and possess cristae. In Lowicryl embedded tissue, which is not fixed in osmium tetroxide, the ‘dark granules’ (lysosomal granules) appear as light granules. The ‘light granules’ (mitochondrial granules) appear as dark granules. In order to avoid confusion, the two types of granules will be referred to as lysosomal or mitochondrial granules. Previous investigations by light microscopy involving biotinylated lectins and an avidinbiotin-peroxidase complex [ 5,6] have shown that eccrine glands were positive after incubation with concanavalin A (Con A) [ 5,6], wheat germ agglutinin (WGA) [5, 61, ricinus communis agglutinin (RCA) [6], dolichos biflorus agglutinin (DBA) [ 5, 61, soybean agglutinin (SBA) [5, 61, and Ulex europaeus agglutinin (UEA I) [ 61. Incubation with peanut agglutinin (PNA) showed a negative reaction. However, after incubation with neuraminidase and subsequent incubation with PNA, the cells became positive [ 61 indicating that TABLE
the PNA-binding sites had been masked by neuraminic acid [ 61. Apocrine glands after incubation with FITCconjugated WGA and PNA showed a positive reaction in both the cytoplasm and plasma membrane [7]. Recently the ultrastructural distribution of carbohydrate residues in keratinocytes and of CI-L -fucose in eccrine glands has been studied in our laboratory [ 8,9]. It was shown that lectin-binding sites were evenly distributed within the plasma membrane of keratinocytes after incubation with Con A and WGA. In contrast, PNA-binding sites were present in the plasma membrane between the desmosomes, absent in that portion of the plasma membranes of basal cells facing the basal lamina and almost completely absent within desmosomes. The purpose of the present investigation was to find out: 1) Is there a difference in the distribution of carbohydrate residues between the luminal and the basolateral portion of the plasma membrane of secretory cells? 2) Is there a difference in the lectin-binding pattern between eccrine and apocrine glands?
I
The lectins used, their specificity and binding inhibitors Lectin
Abbreviated
Concanavalia ensiformis Peanut agglutinin Wheat germ agglutinin Ulex europaeus agglutinin Dolichos biflorus agglutinin Soybean agglutinin
Con A PNA WGA UEA DBA SBA
Gal = galactose; GalNAc = N-acetyl-galactosamine; (sialic acid); Man = mannose; Glc = glucose.
name
Major sugar specificity
Binding inhibitor
cc-D-Glc, a-D-Man Gal-p( I-3)-GalNAc (B-( I-4)-D-GlcNAc),
X-D-Methyl-Man lactose NeuNac
NeuNAc
ED-fUCOSe
N-D-fUCOSC
a-D-GalNAc a-D-GalNAc
cc-o-GalNAc a-D-GalNAc
GlcNAc = N-acetyl-glucosamine;
NeuNAc = acetyl-neuraminic
acid
Fig. 1. (a-f) Eccrine gland. (a) Incubation with WGA shows labeling of the plasma membrane (arrow) of a myoepithelial (myo) cell and of the villous folds (asterisks) of adjacent clear cells. x 7,000. (b) Incubation with SBA shows distinct gold particles in the villous folds of two adjacent clear cells. x 7,000. (c) Incubation with Con A, dark cell. There is labeling of the perinuclear space (arrows), the granules (g) and of the cytoplasm in association with glycogen granules (arrowheads). The mitochondria (m) are negative and serve as a built in control. x 20,000. (d) Incubation with UEA I shows distinct labeling of the villous folds of two adjacent clear cells. The mitochondrium (m) is negative. x 20,000. (e) Incubation with PNA shows heavy labeling of the granules of a dark cell. L = lumen of the eccrine gland. x 12,000. (f) Negative control. There is no labeling of the villous folds of two adjacent clear cells. x 20,000.
58
to ing the temperature -35°C and was embedded in Lowicryl at -35°C. The lectins used and their specificity as well as their binding inhibitors are listed in Table I. The immunocytochemical procedure was carried out as previously described [ 81.
Materials and Methods Normal skin of five healthy volunteers was fixed in a mixture of 2% paraformaldehyde and 0.1% glutaraldehyde in PBS (pH 7.2) for 4 h. The specimens were rinsed in PBS, 0.5 M NH&l in PBS [ lo], rinsed again in PBS. The tissue was dehydrated in graded ethanol by gradually lowerTABLE
Results All results are summarized
II
Distribution
in Table II.
of lectin binding sites in eccrine and apocrine glands UEA
WGA
PNA
Con A
SBA
DBA
+ ++ + _ _
++ +++ + _
_ _ _ _
ii +++ ++ _ +
+ ++ _ _ -
++ ++ _ -
++ ++ + _ -
++ + + +++
_ +++
+++ ++ ++ _ + +++
+ + _ _ _ ii
++ ++ _ _ _ _
+++ + ++ _ _ +
+++ _ _ _ ++
++ ++ + +++ _ _ + t+
+ ++ + _ _ _ -
+ +t + _ _ _ -
++ _ _ _ _
_ _ _ _
+ _ _ +
_ _ _ -
_ _ _
Eccrine Glands Clear cells
Plasma membrane:
luminal basolateral desmosomes nucleus, mitochondria nuclear membrane, RER Dark cells
Plasma membrane:
luminal basolateral desmosomes
nucleus, mitochondria nuclear membrane, RER granules Apocrine glands Plasma membrane:
luminal basolateral desmosomes
lysosomal granules mitochondrial granules nucleus, mitochondria nuclear membrane cytoplasmic vesicles beneath the lumen
+++ +++ ;i, + _ _ +
Myoepithelial cells of eccrine and apocrine glands Plasma membrane _ myotilaments _ dense bodies _ nucleus _ nuclear membrane ( + ) occasional,
+ weak, + + moderate,
+ + + strong labeling RER rough endoplasmic
reticulum.
Fig. 2. (a-d) Apocrine gland. (a) Incubation with WGA, apocrine cell undergoing apocrine secretion. L = lumen of the gland. Heavy labeling of the luminal plasma membrane including the villous projections and of the lysomal granules (lyg). x 12,000. (b) Incubation with Con A, apocrine cell with evidence of apocrine secretion. Two types of granules can be seen. The lysomal granules (arrows) are labeled. x 3,000. (c) Incubation with Con A, higher magnification of the two types of granules: the mitochondrial granules (arrows) are negative, whereas the lysosomal granule (lyg) shows good labeling. x 12,000. (d) Incubation with PNA: the luminal plasma membranes as well as cytoplasmic vesicles beneath it (arrows) are labeled. L = lumen ofthe gland. x 20,000.
59
60
Eccrine glands
When incubated with UEA, WGA, Con A, SBA and DBA, gold particles were found in the luminal portion of the plasma membrane as well as in the basolateral portion of both clear and dark cells (Fig. la, b and d). In general the lectin binding sites of the basolateral plasma membrane of clear cells outnumbered the lectin binding sites of the luminal plasma membrane of clear cells, whereas in dark cells the gold particles in the luminal plasma membrane outnumbered those of the basolateral portion of the plasma membrane. The desmosomes showed labeling with UEA (Fig. Id), WGA, and Con A but no labeling with SBA and DBA. The granules of the dark cells (Fig. lc) showed labeling with all lectins except for DBA. After incubation with PNA, the granules in dark cells were the only structures labeled (Fig. le). No PNA binding sites were detected in clear cells. The nuclei and mitochondria were never labeled, no matter which lectin was used (Fig. lc) and thus served as a built in negative control. However, the perinuclear membrane was labeled after incubation with Con A (Fig. lc). Cytoplasmic labeling was seen after labeling with Con A and WGA. The negative controls were always negative (Fig. If) except for an occasional gold particle. Apocrine glands
After incubation with UEA, WGA (Fig. 2a), Con A, SBA, and DBA the luminal and basolateral plasma membranes of the secretory cells were labeled. The number of gold particles associated with the luminal plasma membrane outnumbered those associated with the basolateral plasma membrane except after incubation with SBA and DBA. The nuclei, mitochondria, and mitochondrial granules were never labeled with any lectin (Fig. 2~). Cytoplasmic labeling was seen after incubation with Con A indicating the presence of glycogen. The lysosomal granules showed weak labeling with UEA, SBA, and DBA, moderate and strong labeling with WGA and Con A (Fig. 2b and c) and no labeling with PNA. PNA binding sites were seen in the luminal
plasma membrane as well as in vesicles located directly beneath the plasma membrane. Vesicles located in the center of the cell were not labeled with PNA. The number of gold particles did not seem to depend on the functional state of the cell, i.e. cells with evidence of decapitation (Fig. 2b) did not show heavier or weaker labeling than those not undergoing decapitation secretion. Myoepithelial cells of eccrine and apocrine glands
The plasma membrane was labeled after incubation with WGA and Con A only. The myofilaments were not labeled with any lectin. Discussion The distribution of lectin binding sites in eccrine and apocrine glands was similar to the distribution in keratinocytes of normal epidermis [8]: Con A and WGA binding sites were found within the cytoplasm as well as in the plasma membrane. All lectins except for PNA were associated with the entire plasma membrane and all lectins except for DBA showed a positive reaction in the granules of the dark cells of eccrine glands. The lysosomal granules of apocrine glands were positive after incubation with UEA, WGA, Con A, SBA, and DBA whereas the mitochondrial granules did not show any reaction with any of the lectins. Incubation with PNA revealed an interesting difference between eccrine and apocrine glands: in eccrine glands the granules were positive. Both granules of the apocrine glands, however, were negative, but in apocrine glands the luminal plasma membrane was positive whereas in eccrine glands the luminal membrane was negative. After incubation with Con A gold particles indicating the presence of mannose and glucose residues were found in both glands in the perinuclear space and in other portions of the rough endoplasmic reticulum. The presence of Con A binding sites in these locations is compatible with the assembly of carbohydrate chains. The glycoproteins acquire their core sugars in the endoplasmic reticulum [ 1 l] and then enter the Golgi complex where the carbohydrate units of glyco-
61
the glycoproteins are altered. Subsequently proteins are sorted and transported via separate types of vesicles to the plasma membrane, secretory granules, and lysosomes [ 121. Unfortunately it is difficult to recognize the membranes of a Golgi complex in Lowicryl embedded tissue, which is most likely due to the weak fixative used (0.1% glutaraldehyde and no osmium tetroxide). In eccrine glands the glycoproteins are transported from the Golgi complex to the luminal and basolateral plasma membranes of dark and clear cells. In dark cells more gold particles were found associated with the luminal plasma membrane than with the basolateral plasma membrane, whereas in clear cells heavier labeling was seen in the basolateral plasma membrane. This could possibly be correlated to the functions of the various portions of the plasma membrane: in dark cells secretion occurs by discharging the granules with their glycoproteins through the luminal membrane of the gland [ 31. In clear cells the basolateral membrane is the site of active transport of ions for sweat secretion [3]. It is not known which role the carbohydrate residues play in the production of sweat. The significance of apocrine secretion is unknown. The fact that PNA binding sites in apocrine glands can be detected in the luminal plasma membrane and the vesicles located beneath it most likely can be explained by the fact that terminal galactosyl groups in other locations are masked by sialic acid [ 131. The masked PNAbinding sites most likely split off their terminal sialic acid as they approach the luminal plasma membrane. This is supported by light microscopy. If the tissue is incubated first with neuraminidase and subsequently with PNA, more PNA binding sites can be revealed. It can be concluded: 1) There is a quantitative difference in the distribution of carbohydrate residues between the luminal and basolateral plasma membranes of dark and clear cells: in dark cells the luminal membranes show heavier labeling than the basolateral membranes, whereas in clear cells the basolateral membranes show heavier labeling than the luminal membranes.
2) After incubation with PNA, in eccrine glands the granules of dark cells are the only portion labeled, whereas in apocrine glands both types of granules are negative, but the luminal plasma membranes and cytoplasmic vesicles beneath it are the only structures labeled. Acknowledgement
We thank Birgit Fehrenbacher technical assistance.
for her excellent
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