Ultrastructure and immunohistochemical characterization of proteins concerned with the secretory machinery in goat ceruminous glands

Abstract

The expression of soluble N-ethyl-maleimide sensitive fusion attachment protein receptor (SNARE) proteins in apocrine glands has not been fully elucidated. In addition to performing ultrastructural observation of the ceruminous glands in goats, our study focuses on the demonstration of β-defensins, SNARE proteins and Rab3D in these glands with the use of immunohistochemical methods. The secretory cells were equipped with two types of vesicles, Golgi apparatus and abundant rough endoplasmic reticulum (ER). Additionally, in some of them, the characteristic concentric structures composed of rough ER were observed in their circum- and infranuclear parts. The expression of phosphorylated inositol requiring enzyme 1a was also detected. These findings may indicate their ability to produce numerous secretory proteins and the maintenance of homeostasis in the glandular cells. Furthermore, β-defensins were demonstrated as products of the ceruminous glands. The present investigation also revealed the presence of SNARE proteins and Rab3D. It is suggested that these proteins are concerned with the secretory machinery of this gland type.

Figure 1.

Light microscopic structure of the ceruminous glands in the goat. a) The welldeveloped ceruminous glands were detected lying below the epidermis of the external auditory canal, H&E. b) higher magnification of the ceruminous glands, H&E. EC, elastic cartilage; asterisks, ceruminous glands.

Figure 2.

Electron microscopic structure of the ceruminous glands. a) The secretory portion consists of single layer of secretory cells with underlying myoepithelial cells. b) Part of the supranuclear cytoplasm of secretory cells. c) Part of the apical cytoplasm of secretory cells. d) Part of the infranuclear cytoplasm of secretory cells; inset (upper left) shows higher magnification of a concentric structure composed of rough ER (boxed area). Go, Golgi apparatus; L, lumen; Ly, lysosome; M, mitochondria; Mv, microvilli; N, nucleaus; rER, rough endoplasmic reticulum; SV, secretory vesicle; asterisk, a concentric structure composed of rough ER; double arrowhead, a morphological feature suggestive of exocytosis.

Figure 3.

Immunohistochemical staining for the detection of β-defensins in the ceruminous glands. a) β-defensin 1. b) Negative control for β-defensin 1 using absorbed antibodies. c) β-defensin 2. d) Negative control for β-defensin 2 using absorbed antibodies. Arrows, positive-stained granules.

Figure 4.

Immunohistochemical staining for the detection of v-snares in the ceruminous glands. a) VAMP-1. b) negative control for VAMP-1 using absorbed antibodies. c) VAMP-2. d) Negative control for VAMP-2 using absorbed antibodies. e) VAMP-3. f ) Negative control for VAMP-3 using absorbed antibodies. g) VAMP-4. h) Negative control for VAMP-4 using absorbed antibodies. i) VAMP-7. j) Negative control for VAMP-7 using absorbed antibodies. k) VAMP-8. l) Negative control for VAMP-8 using absorbed antibodies. Arrows, positivestained granules; arrowheads, positive reactions in basal cytoplasm.

Figure 5.

Immunohistochemical staining for the detection of t-snares in the ceruminous glands. a) Syntaxin 2. b) Negative control for syntaxin 2 using absorbed antibodies. c) Syntaxin 4. d) Negative control for syntaxin 4 using absorbed antibodies. e) Syntaxin 6. f ) Negative control for syntaxin 6 performed by the incubation with normal mouse IgG. g) SNAP23. h) Negative control for SNAP23 using absorbed antibodies. Arrows, positive-stained granules.

Figure 6.

Immunohistochemical staining for Rab3D detection in the ceruminous glands. a) Rab3D. b) Negative control for Rab3D using absorbed antibodies.

Figure 7.

Immunohistochemical staining for the detection of activated IRE1 in the ceruminous glands. a) pIRE1>; inset (upper left) shows higher magnification of the secretory cells. b) Negative control for pIRE1>>using absorbed antibodies. Arrowheads, positive reactions in basal cytoplasm.