Cell Tiss. Res. 154, 399--408 (1974) �9 by Springer-Verlag 1974

Cytologic Observations on the Secretory Cells of the Chin (Submandibular) Gland

and Brown Inguinal Gland in the Rabbit

E v e r e t t H e a t h

Department of Veterinary Anatomy and Physiology, University of Ibadan, Ibadan, Nigeria

Received June 3, 1974

Summary. Smooth endoplasmic reticulum is more plentiful in the secretory cells of both chin and brown inguinal glands than rough endoplasmic reticulum. The smooth reticulum has a relatively dense content in chin glands while in inguinal glands it often appears empty and swollen, resulting in a characteristic cytoplasmic meshwork. Stacks of Golgi cisternae, scattered in the supranuelear region of all cells, are most numerous in male chin glands, while lysosomes are more frequent in female chin glands. Rounded secretory granules in inguinal glands and irregularly shaped secretory lakes in chin glands occur primarily in the supranuclear region. Deformed mitochondria are seen only in close association with the secretory lakes in the chin glands of bucks with normal libido and of a doe treated with depotestosterone. Secretory lakes are numerous and widespread in the chin glands of a buck with poor libido and in a buck castrated 2 weeks previously. At 1 month following castration the male chin gland closely resembles those of does. No cytologic effect of castration or depotestosterone treatment was seen in the inguinal glands. Both chin and brown inguinal secretory cells apparently release their secretions by exocytosis of juxtaluminal secretory vesicles rather than by an "apocrine" mechanism.

Key words: Rabbit - - Skin glands - - Endoplasmic reticulum - - Mitochondria.

In t roduct ion

Both the chin and brown inguinal g lands of r abb i t s are specialized sudor iparous glands. Bo th are b ranched t ubu l a r glands wi th cuboidal to low co lumnar epi the l ium and basa l ly a r ranged myoepi the l ia l cells. Bo th enlarge a t p u b e r t y and bo th are larger in ma tu re males t h a n females (Lyne et al., 1964; Mykytowycz , 1966; Wales and Ebl ing, 1971). The older designat ion, brown, is used here for h lguinal glands, ins tead of apocrine, since the l a t t e r is i nappropr i a t e and the former t e rm clear ly dis t inguishes this g land from the accompany ing white sebaceous inguinal gland.

Func t iona l differences between these two glands are suggested by thei r different locat ions in the body and b y the i r a p p a r e n t l y different pheromona l roles in irLtra- specific communica t ion (Mykytowycz , 1970; Myky towycz and Dudzinski , 1972). Whi le the chin g land is said to secrete a t e r r i to r ia l marker , depos i ted on objects b y ch in-mark ing (Mykytowycz , 1968), the brown inguinal g land is bel ieved to secrete e i ther a sexual a t t r a c t a n t (Strauss and Ebling, 1970) or an individual ident i f ier (Goodrich and Mykytowycz , 1972).

Endocr inologic exper iments have shown t h a t while tes tos te rone s t imula tes and cs t radiol inhibi ts the growth of bo th glands, the chin g land is far more sensit ive to es t radiol inhib i t ion t han is the brown inguinal g land (Wales and Ebl ing, 1971).

Send o//print requests to: Everett Heath, Department of Veterinary Anatomy and Physiology, University of Ibadan, Ibadan, Nigeria.

400 E. Heath

Figs. 1--5

Secretory Cells in Rabbit Skin Glands 401

To date there has been only one report concerned with the electron micro- scopic (EM) appearance of the chin gland (Kurosumi et al., 1961), and only one EM report on the brown inguinal gland (Kfihnel and Wrobel, 1969). The present s tudy of both glands extends these observations.

Materials and Methods Chin and inguinal glands were collected from 20 rabbits (Oryctolagus cuniculus) and pre-

pared for electron microscopy. Six collections were made from intact bucks with normal libido; one collection from a mature buck with poor libido (Heath, 1972); and two collections from bucks respectively castrated 2 weeks and 1 month prior to collection. Of the does from which glands were collected, five were estrual; three were pseudopregnant; and one had been injected 6 days previously with 25 mg depotestosterone subcutaneously (Wales and Ebling,

1971). Specimens were first immersed in 2 % glutaraldehyde in phosphate or cacodylate buffer

at pH 7.2 for 4 to 6 hours, and secondarily fixed for 1 hour in 1% osmium tetroxide in phos- phate buffer at pH 7.2. A few specimens were incubated by Gomori's method for the demon- stration of acid phosphatase (Barka and Anderson, 1965) prior to osmium tetroxide post- fixation.

The fixed specimens were dehydrated in ethanol, passed through propylene oxide and embedded in Epon-Araldite. As part of the dehydration sequence all specimens except those incubated for acid phosphatase were placed for 10 minutes in 70% alcohol containing 0.3 % uranyl acetate. Thick sections (2 ~) were stained with 1% methylene blue in 1% aqueous boric acid for light microscopy. Thin sections of 300 to 700 A thickness were routinely stained with 2% aqueous uranyl acetate followed by Reynold's (1963) lead citrate solution and then examined in an RCA EMU-3F electron microscope. Two or three epoxy-embedded samples of each gland were examined by both light and electron microscopy.

Results

Light Microscopy

Chin Gland. Chin glandular tubules are loosely arranged and highly irregular in shape. The secretory epithelium is usual ly cuboidal with an even luminal surface (Fig. 1). Occasional vacuoles with a homogeneous content are present wi thin the cytoplasm. Vacuoles are par t icular ly large in the chin glands of the buck with poor libido and the buck castrated 2 weeks prior to coitus. The nuclei of secretory cells are usual ly round and central ly located. Mitotic figures were not seen.

Figs. 1--4. Photomicrographs of thick Epon-Araldite sections stained with methylene blue Fig. 1. Section of chin gland from mature buck, illustrating cuboidal epithelium. C capillary;

v cytoplasmic vacuoles with homogenous content, x 1000 Fig. 2. Section from same gland as in Fig. 1, illustrating variation in height and appearance of secretory epithelium in chin gland of intact mature buck. X tangential section; Y cross

section of tubules with pyramid-shaped low columnar cells. X 1000 Fig. 3. Brown inguinal gland from mature doe. C capillary; cv cytoplasmic vacuoles with

heterogenous content; X tangential section with suggestion of apical blebs, x 1000 Fig. 4. Brown inguinal gland from mature buck. Darker staining of some cells with methylene blue probably artifactual. C capillary, v cytoplasmic vacuoles with homogeneous content.

X 1000 Fig. 5. Apical part of columnar cells in brown inguinal gland of mature buck. G stacks of Golgi cisternae; M mitochondria; ma macula adhaerens; rg round granules; sr meshwork of

smooth endoplasmic reticulum, x 13000

402 E. Heath

Figs. 6--8

Secretory Cells in Rabbit Skin Glands 403

Variations in the height and appearance of the secretory epithelium are often evident in the chin glands of intact, mature bucks (Fig. 2). In some tubules the epithelium is nearly squamous, in others low columnar. Tangential sections of the latter give a false appearance of apical cytoplasmic blebs (see tubule X, Fig. 2).

The chin glandular epithelium in does is more consistently cuboidal than in bucks. No differences between the chin glands of estrual, pregnant and pseudo- pregnant does were observed.

B r o w n Ingu ina l Gland. Inguinal glandular tubules are t ightly arranged and most frequently ovoid to circular in profile with occasional branching. The secretory epithelium is more frequently low columnar than cuboidal in both male and female glands. The luminal surface of the secretory cells is usually smooth with only a rare suggestion of apical cytoplasmic blebs (Fig. 3). The latter were occasion- ally observed in cells with large cytoplasmic vacuoles containing a homogeneous material.

The cytoplasm in both male and female glands appears granular. Vacuoles with heterogeneous content are more frequent in the secretory cells of does (Fig. 3). In both sexes some cells stain more darkly with methylene blue than other cells (Fig. 4) ; this is probably an artifact. The round nuclei are close to the base of the cells. No mitotic figures were seen.

As in the female chin glands, no differences were observed between the inguinal glands of estrual, pregnant and pseudopregnant does.

Electron Microscopy

Chin Gland. Secretory epithelial cells of chin glands are consistently joined by juxtaluminal junctional complexes. These usually consist of a macula adhaerens

and a more juxtaluminal zonula adhaerens (Fig. 9). Below these complexes inter- cellular spaces continue around interlocking protuberances and depressions of adjacent cells. Basally myocpithelial cells also contribute folds to intermesh with those of the secretory cells. No junctional complexes were seen between myoepi- thelial and secretory cells. A distinct basal lamina was evident outside the myo- epithelial cells. Nuclei of the secretory cells arc unremarkable. Single nucleoh are usually centrally located in relation to small amounts of chromatin which is in part peripherally arranged along the nuclear envelope.

A few scattered cisternae of rough endoplasmic reticulum are located peri- nuclearly in all secretory cells (Fig. 8). Ribosomal clusters are distributed through- out the cytoplasm. Stacks of Golgi cisternae are scattered in the supranuclear region and are most numerous in the low columnar cells of normal male glands. Only in the chin glands of the buck with poor libido and of the buck castrated 2 weeks prior to collection were Golgi stacks clearly arranged into a fully developed circular complex.

Figs. 6 and 7. Juxtaluminal cytoplasm of columnar cells in brown inguinal gland of mature buck. M mitochondria; ma macula adhaerens; mv microvilli; p pinocytotic invagination;

r ribosomes; v secretory vesicle; za zonula adhaerens. • 40000 Fig. 8. Supranuclear region of columnar cell in chin gland of mature buck. L secretory lakes; M mitochondria; dM characteristically deformed mitochondrion; N nucleus; rr rough endo-

plasmic reticulum; sr smooth endoplasmic reticulum, x 40000

27 Cell Tiss. Res. 154

404 E, Heath

Figs. 9--12

Secretory Cells in Rabbit Skin Glands 405

The most striking characteristics of the cytoplasm of chin gland secretory cells are: profuse profiles of smooth endoplasmic reticulum ; scattered secretory vacuoles or lakes of moderately dense homogeneous material; and, in the chin glands of intact bucks and the depotestosterone treated doe only, deformed mitochondria of euboidal and low columnar cells. Lakes of moderately dense homogeneous mem- brane-bounded material were most frequent in the supranuclear region (Figs. 8, 9). Particularly numerous and large lakes are evident in the glands of the buck with poor libido and of the buck castrated 2 weeks prior to collection.

When present, characteristically deformed mitoehondria always are located in close association with secretory lakes (Fig. 8). This mitochondrial deformation consists of partial elongation, and flattening as described by Kurosumi et al. (1961). The flattened portion is filled by numerous longitudinally arranged cristae. I t should be emphasized that such elongated and flattened mitochondria were not seen in the chin glands of the castrated bucks (Fig. 10) nor in the female glands except for those of the doe treated with depotestosterone. Variations in the num- bers and arrangement of mitochondrial cristae were observed, however, in the chin glands of all animals (Fig. 8).

The ubiquitous smooth endoplasmic reticulum is particularly evident in the supranuclear region where some of its membranes are continuous with the mem- branes surrounding lake material. Continuity was also seen between the smooth reticulum and Golgi cisternae. The latter and their related vacuoles, however, often appear empty while elements of the smooth endoplasmic reticulum contain a material of similar density to that within the lakes.

Various other cytoplasmic inclusions, occasionally observed, are lysosomes and pigment granules. Glycogen deposits were not seen. Tests for acid phosphatase activity revealed a higher incidence of lysosomes in female glands, where they occur in both the supranuclear (Fig. 11) and the juxtaluminal regions (Fig. 12).

Particular attention was paid to the juxtaluminal portions of cuboidal to low columnar secretory cells. Except for a narrow region alongside junctional complexes which contain primarily microfibrillar material, the juxtaluminal region is occupied by ribosomal clusters, smooth endoplasmic reticulum and vesicles of highly variable size, shape and density of content (Fig. 9). Many of these vesicles are closely aligned with folds in the plasma membrane. Microvilli are present along the entire luminal cell surface. Frequent pinocytotic invaginations occur between microvilli.

B r o w n I n g u i n a l Gland. Secretory epithelial cells of the brown inguinal glands are also consistently joined by juxtaluminal junctional complexes consisting of a m a c u l a adhaerens and a more juxtaluminal zonu la adhaerens (Figs. 5, 6). Below

Fig. 9. Juxtaluminal cytoplasm of cuboidal cell in chin gland of mature buck. ma macula adhaerens; mv mierovilli; p pinocytotic invagination; r ribosomes; v secretory vesicles; • 40000 Fig. 10. Supranuclear region of a columnar cell in chin gland of buck castrated one month

previously. L secretory lakes; M mitochondria; r ribosomal clusters. • 40000 Fig. 11. Lysosome in juxtaluminal cytoplasm of chin gland of doe. Ly lysosome; mv micro-

villi. Incubated by the acid phosphatase technique. • 80000 Fig. 12. Lysosomes in supranuclear region of the chin gland of doe. Labels and treatment as

for specimen in Fig. 11

27*

406 E.I-Ieath

these complexes the closely aligned plasma membranes of adjacent cells show only occasional interlocking laminar folds. The same interlocking arrangement exists between secretory and myoepithelial cells, but there are no basal junctional complexes. A distinct basal lamina is always present. The nuclei of brown inguinal secretory cells resemble those of the chin gland.

Ribosomal clusters and cisternae of rough endoplasmic reticulum are wide- spread with some concentration in the basal region of most cells. Small stacks of Golgi cisternae are scattered in the supranuclear region. Mitochondria of variable shape are distributed throughout the cytoplasm. These have relatively few cristae and a notably dense matrix (Fig. 5). Relatively few lysosomes were demonstrated by the acid phosphatase technique in these cells.

The most striking characteristics of the cytoplasm of brown inguinal secretory cells are the numerous round granules and the unique meshwork of smooth endo- plasmic reticulum. In both male and female glands these were both consistent in location and appearance. The round granules have a moderately dense and rela- tively homogeneous content and are membrane-bounded. They occur in largest number in the central regions of the secretory cells and never in the juxtaluminal region. Occasional round granules appear to be interconnected. No consistent relationship of round granules to other cytoplasmic organelles was observed.

The unusual meshwork appearance of the smooth endoplasmic reticulum, caused by the apparent swelling of intracisternal spaces, results in a marked dimunition of extracisternal space. The cytoplasmic lines of the meshwork thus consist of the closely aligned membranes of adjacent cisternae. The effect at lower magnification is particularly dramatic where the cisternae appear to be empty (Fig. 5). This complex smooth endoplasmic reticulum is frequently continuous with the rough endoplasmic reticulum. Occasional continuity between smooth endoplasmic reticulum and Golgi cisternae was also seen.

In the juxtaluminal region of brown inguinal secretory cells there were numer- ous vesicles of variable size, shape and density of content (Figs. 6, 7). Palisading of these vesicles beneath the plasma membrane is frequent. Ribosomes and ribo- somal clusters are also present here, as well as mitochondria. The smooth endo- plasmic reticulum also extends into the juxtaluminal region. The luminal cell surfaces are covered with microvilli and there are occasional pinocytotic invagin- ations of the plasma membrane.

Discussion

I t must first be emphasized that there is no convicing evidence that either the chin gland or the brown inguinal gland secretes in an apocrine manner. Kiihnel and Wrobel (1969) assumed the existence of apocrine secretion in the brown inguinal gland despite Montagna's (1950) earlier work to the contrary. The cyto- plasmic protrusions or apical blebs observed in light and electron microscopy are most likely artifacts resulting from displacement of the juxtaluminal portion of the cell, that is, that portion near or above the junctional complexes, into the lumen, concomitant with dissolution of luminal content during dehydration. The Kurosumi et al. (1961) micrograph of an apical bleb being "constricted at the base" in a chin gland more likely represents the cytoplasmic protrusion of two separate cells in tangential section.

Secretory Cells in Rabbit Skin Glands 407

I t seems most likely that both the chin gland and brown inguinal gland release their secretions by means of exocytosis. The predominance of moderately dense vesicles in the ]uxtaluminal region of secretory cells in both glands suggests that such vesicles are involved in the release of secretory material. Palisading of secre- tory vesicles beneath the plasma membrane {Fig. 6) supports this suggestion (Lacy et al., 1968). The relative complexity of the cell-lumen interface in these cells, however, makes it difficult to distinguish processes of pinocytosis and exoeytosis. Both processes are probably present and together must represent a mechanism by which membrane constituents can be recycled (Orci et al., 1973).

Goodrich and Mykytowycz (1972) have demonstrated lipids and proteins in the secretions of both glands, but the protein concentration is higher in males than females. Further, carbohydrates bound to proteins, though present in the secretions of both glands, are a major component only in the male chin gland secretion.

Both glands of both sexes have the machinery for protein synthesis, i.e., some scattered cisternae of rough endoplasmic reticulum and numerous free ribosomes. However, there is no structural evidence for increased protein synthesis in male glands. Montagna (1950) did not report any sexual difference in cytoplasmic basophilia for brown inguinal glands.

Preliminary comparison of pooled glandular extracts with standard lipids, (including cholesterol, palmitate, methyl palmitate, tripalmitate and cholesterol palmitate) by thin-layer chromatography suggests the presence of hydrocarbons, non-glyceryl esters, free fa t ty acids and cholesterol in both chin and brown inguinal glands (Goodrich and Mykytowycz, 1972). Triglycerides, diglycerides and mono- glycerides were other major components of the extracts from chin glands. These observations have been confirmed for chin glands (Wolf and Heath, unpublished). Both palmitate, an abundant tissue fat ty acid, and cholesterol are lipids arising by extramitochondrial synthesis (Harper, 1973). The smooth endoplasmic reti- culum which is plentiful in both chin and brown inguinal glands may be involved in this synthesis. Free ribosomes, ribosomal clusters and the scattered Golgi stacks, however, cannot be overlooked in a consideration of total lipid synthesis.

Strauss and Ebling (1970) have reported on the basis of unpublished data collected by Wales and Ebling that brown inguinal gland secretion has a steroidal pattern on mass spectrometry, whereas chin gland secretion does not. I t is difficult to speculate why this should be so.

I t should be noted that the chin glands of the buck with poor libido and the buck castrated 2 weeks prior to collection both had well developed Golgi complexes as well as more numerous cytoplasmic lakes of secretory material. In contrast the chin gland of the buck castrated 1 month prior to collection more closely resembled the female glands in this respect (Fig. 8). Further, in none of the abnormal three males was mitochondrial elongation and flattening evident in chin gland cells. These observations suggest that the stimulatory effect of testosterone (Wales and Ebling, 1971) may be expressed in temporally different ways at the intracellular level.

The mitochondrial changes in the chin gland in particular should be the sub- ject of further experimentation. In the present study, the most dramatic change in the depotestosterone treated doe was the appearance of the characteristic mitochondrial deformation referred to. Its consistent occurrence in association

408 E. Heath

wi th lakes of secre tory ma te r i a l in the most act ive chin glands suggests t h a t the deformed mi tochondr ia m a y funct ion in energizing a more rap id synthesis . This hypothesis , however, l ike the more specula t ive suggestion of in t r ami tochondr i a l synthesis of secre tory ma te r i a l (Kurosumi et al., 1961) stil l awai t s conf i rmat ion.

The observat ion t h a t lysosomes are more numerous in the chin glands of ma tu re does t han in the brown inguinal g lands or in bo th glands of bucks is in keeping wi th the reduced secre tory role of the chin g land in does. Chin-marking is a lmost exclus ively a male behaviora l p a t t e r n (Mykytowycz, 1968; Hea th , 1972). The act of ch in-marking u n d o u b t e d l y assists in expressing secret ion out of the g landula r ducts . The general cytologic s imi la r i ty of male and female chin gland, however, suggests t h a t in t race l lu lar synthesis is also occurring in the chin glands of ma tu re does. An increased number of lysosomes here m a y be associa ted with a removal of undischarged secre tory mate r ia l and re la ted membranes .

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