harderian gland ultrastructure of the black sea bottlenose dolphin (tursiops truncatus ponticus)

JOURNAL OF MORPHOLOGY 220:207-221 (1994)

Harderian Gland Ultrastructure of the Black Sea Bottlenose Dolphin (Tursiops truncatus ponticus)

NATALJE D. BODYAK AND LILY V . STEPANOVA Severtsov Institute of Evolutionary Morphology and Ecology ofAnimals, Russian Academy of Science, 11 7071 Moscow, Russia

ABSTRACT Examination of the Harderian gland structure of the Black Sea bottlenose dolphin, Tursiops truncatus ponticus, at macroscopic, microscopic, and electron microscopic levels shows significant sexual dimorphism. The epithelial cells of male and female glands are different cell types, capable of producing chemically different products. Secretory cells in both sexes contain secretion granules that produce a secretion consisting mainly of proteins and carbohydrates, but thought to be sex-specific in composition. The female glands also contain lipid secretion granules. It is suggested that in the bottlenose dolphin the Harderian gland functions to produce sexually distinct pheromones and may have other physiological activities, e.g., participating in local immunological or endocrine-related reactions. o 1994 Why-Liss, Inc.

The Harderian gland. discovered in 1694 by a Swiss scienti2 and'named in his honor, is the largest orbital gland. It has been observed in all vertebrate classes. Among mammals the Harderian gland has been described in marsupials and most species of eutheri- ans. Interest toward this gland has increased in the last decade because of the concept that it is a multfunctional organ that performs the roles of lubrication of the eyeball, produc- tion of chemical signals, and possible participation in photoreception somehow associated with the function of the pineal gland.

Although the gland has been investigated for 300 years, it has been studied intensively in mammals only in some species of rodents and lagomorphs. The criteria for distinguish- ing the Harderian gland among other similar structures within the orbit are based upon histological data from these species. These criteria indicate that the Harderian gland is tubuloalveolar, and that in contrast to the lacrimal gland its secretory tubes have a relatively wide lumen. The Harderian glandular cells are known to produce an abundance of lipids secreted by exocytosis, whereas the secretion of the lacrimal glands is rich in proteins and carbohydrates and contains no lipids (Sakai, '81, '89). Some information in the literature, however, indicates that the Harderian gland of the mammals secretes not only lipids but also glycosaminoglycans and proteins.

Among 26 species of mammals studied by histochemical methods, Harderian gland secretion in all but five contained lipids (Paule and Hayes, '58). Identification of mammalian orbital glands is made difficult in part because of the lack of information about their phylogenetic interrelations. In some animals the Harderian glands may include lobules consisting of secretory cells that synthesize nonlipid products. For instance, the lobules of the Harderian gland in the greater marsupial glider (Sakai and van Lennep, '84) and in hedgehog (see review by Sakai, '81) contain lacrimallike tubules. Moreover, the Hard- erian gland of some rodents-the mongolian gerbil (Johnston et al., '831, the midday gerbil (Sakai and Yohro, '811, the woodmouse (Johnston et al., '87), and the pseudorat (Johnston et al., '85)-are characterized by the presence of mucus-producing cells in the ducts, although the gland in these animals secretes lipids. Electron microscopic investigation of the Harderian gland of the nine- banded armadillo revealed the possibility that two kinds of secretory cells might be present, one type synthesizing lipids, the other type

Address reprint requests to Natalie Bodyak, Severtsov Institute Evolutionary Morphology & Ecology of Animals RAN, 33 Lenin- sky Prospect, 117071 Moscow, Russia.

Q 1994 WILEY-LlSS, INC

208 N.D. BODYAK AND L.V. STEPANOVA

glycosaminoglycans and proteins (Weaker, '81). Another interesting phenomenon is the process of so-called harderinisation in the rat, in which secretory cells similar t o those of the Harderian gland were shown to occur in the acini and ducts of the lacrimal gland (Sashima et al., '89).

The above-mentioned instances of overlap- ping cell types in the Harderian and lacrimal glands provide grounds for the hypothesis that they are phylogenetically related. The possibility that the specialized functions of the two kinds of glands can be combined in a single kind cannot be ruled out. The facts mentioned above provoke doubts on the valid- ity of the distinctive identification criteria proposed by Sakai ('81). Study of the ultrastructure of secretory cells from different species of mammals belonging to a variety of taxonomic groups, especially of mammals lacking lacrimal glands, would be helpful for determining interrelationships of the orbital glands. Cetaceans are a very interesting group for this kind of investigation, as whales and dolphins are reported to have well-developed Harderian glands but as a rule no lacrimal glands (Putter, '03).

MATERIALS AND METHODS

The Harderian gland was examined in one male and two female Black Sea bottlenose dolphins, Tursiops truncatusponticus, which had lived in captivity in a marine mammal center. All three were in good physical condi- tion at the times of their deaths by accidents in the summers of 1984, 1989, and 1991. Pieces of tissue from both wide and narrow portions of the gland were fixed in 2.5% glu- taraldehyde in 0.2 M S-collidine buffer (pH 7.3) immediately after death, and then post- fixed in 1% osmium tetroxide in the same buffer. Tissues were dehydrated in a graded ethanol series, cleared in propylene oxide, and embedded in Epon 812-Araldite. Sec- tions were obtained with glass knives on an LKB-I11 ultramicrotome. Semithin sections were mounted on glass slides and stained by the method of Humpry and Pittman ('74) and examined by light microscopy. This method makes possible the recognition of protein products (blue staining), carbohydrates, (crimson staining) and lipids (from light blue to green). Only silver-grey ultrathin sections were used in subsequent trans- mission electron microscopic (TEM) studies. To provide the proper control of conditions, the ultrathin sections from all three animals were stained simultaneously with uranyl ac-

etate and lead citrate in an Ultrastainer LKB and then examined with a JEM-100C electron microscope. Frozen sections stained with Sudan I11 or Sudan black B were examined for lipid detection.

RESULTS

The Harderian gland of Tursiops truncatus ponticus forms a ribbon that completely encircles the ocular globe. It is more developed in the rostra1 portion of the orbit. The gland is closely associated with the ocular musculature. In both sexes, it consists of many lobules, separated from each other by thin layers of connective tissue. Lobules are closely packed with ramifying secretory tubes. The width of the alveolar lumen varies from narrow to wide. The shape of the lumen may be round, oval, or ramifying, depending on the plane of the section (Fig. 1A). The interstitium commonly contains blood vessels and elements of lymphoid tissue: lymphocytes and plasma cells. Plasma cells often are aggregated either around blood vessels or apart from them. Small capillaries have fenestrated walls (Fig. 1B).

Secretory tubules in all parts of the gland of both sexes are lined by columnar epithelial cells usually resting on myoepithelial cells but sometimes in direct contact with the basal lamina. The basally located, multilo- bate nuclei possess prominent nucleoli. A considerable amount of heterochromatin is distributed peripherally or in dispersed clumps. Centrioles, observed in some secretory cells, provide evidence of mitotic capabil- ity. Apical surfaces of the cells bear slender microvilli. The basal lamina may be smooth or infolded. The basal borders may have microvillus-like processes, and basolateral borders of adjoining cells have interdigitating and thin, microvillus-like processes. Junc- tional complexes include desmosomes and tight junctions. The Golgi complex and the rough endoplasmic reticulum (RER) are prominently developed organelles of the secretory cells in both sexes. These are the main characteristics shared by male and female glands.

Female Harderian gland Epithelium of the female gland has only

one type of cells. Profiles of RER in the secretory cells are aligned in parallel, and make many anastomoses (Fig. 2 ) . Some cisternae are engorged with secretory products and irregular in shape, particularly in the

HARDERIAN GLAND ULTRASTRUCTURE OF TURSIOPS 209

Fig. 1. Tursiops truncatus ponticus. A. Light micrograph illustrating the female gland lobule packed with ramifying secretory tubules. Stained by method of Hum- pry and Pittman ('74). Scale bar = 10 wm. B. Transmis- sion electron micrograph (TEM) of typical capillary in

connective tissue septa of female Harderian gland. Endo- thelium shows fenestrae (arrows). Erythrocyte (El nearly fills lumen. N, nucleus of secretory cell. Scale bar = 0.5 wm.


Fig. 2. Turswps truncatus ponticus. TEM of tubule cells of female Harderian gland showing basal nuclei and illustrating elongated cisternae of rough endoplasmic

apical part of the cells (Fig. 3). Proteins synthesized in the rough endoplasmic reticulum accumulate in the secretory granules, which in thin sections are seen to be membrane- bound and moderately electron-dense. In semithin sections the granules stain blue. The fine, granular content of these secretory granules at the EM level has the same electron density as that of the RER cisternae (Fig. 4). The secretory granules may fuse and form aggregations in the apical region of the cell (Figs. 3,5). Numerous small vesicles are present throughout the cytoplasm and often surround the secretory granules (Fig. 6). It is possible that they participate in transporting the secretory product to the granules or in transferring it from the granules to the cell surface, but no exocytosis of these vesicles was observed.

The Golgi complex is located in the supra- nuclear region. Its stacked cisternae form an arc (Fig. 7A). Small Golgi vesicles of different

reticulum (RER), juxtanuclear Golgi complex (Gc) and abundant secretory (Sg) and lipid granules (L) with dark rims. Lm, lumen. Scale bar = 2 pm.

electron density are present on both concave and convex surfaces. In addition, heavily electron-dense granules located in the region of the Golgi complex are formed by separation from its widened cisternae (Fig. 7B). In semithin sections stained with fuchsin these granules were bright crimson, thus indicating their carbohydrate nature.

A characteristic feature of the female gland’s secretory cells is the presence of su- danophilic lipid granules surrounded by narrow electron-dense rims (Figs. 5, 6). They occur throughout the cytoplasm but are often concentrated in the apical region. Some- times such granules are observed in the lumen of tubules. Mitochondria of the secretory cells are relatively large and often elongated and ramifying in section. Smooth endoplasmic reticulum is sparse, consisting of short tubules distributed throughout the cytoplasm. The secretory cells may export products of synthesis by exocytosis or by apocrine

HARDERIAN GLAND ULTRASTRUCTURE OF TURSZOPS 21 1

Fig. 3. Tursiops truncatus ponticus. TEM of the apical ends of secretory cells in the female Harderian gland. Note that cisternae of RER are dilated, and that cell surface has abundant microvilli (Mv). Moderately elec-

secretion. Sometimes secretory cells appear dead, suggesting that the whole secretory contents have been released into the lumen.

Male Harderian gland In the male there are two morphologically

distinct types of secretory cells, in contrast to the single type in the female gland. Cell type I is characterized by a diversity of secretory granules, whereas cell-type I1 contains a single type of granule (Fig. 8). In cell type I two kinds of granules are most abundant: electron-lucent (light) granules with a net- like content, and moderately electron-dense (gray) granules having a finely granular content. Sometimes the granules of the gray type contain electron-dense, round inclusions. The gray and light granules are distributed throughout the cytoplasm but often are more abundant in the apical portion of the cell, where light granules may fuse to form aggregations (Fig. 9A). The gray granules do not aggregate apically and are always discrete. In stained semithin sections, the light-type granules remain unstained, whereas those of

tron-dense granules (Mg) fuse, whereas heavily electron- dense granules (Hg) do not fuse. L, lipid granules; Lm, lumen. Scale bar = 1 bm.

the gray-type stain purple. In addition to the light and gray granules, cell type I contains dark heavily electron-dense granules that may contain moderately electron-dense inclusions (Fig. 8), and complex lysosome-like granules (Fig. 9B) that could not be identified at the light microscopic level. Cisternae of RER with moderately electron-dense contents are chiefly aligned baso-apically along the periphery in cell type I (Figs. 8,9C). The Golgi complex consists of a stack of thin, separated cisternae accompanied by Golgi vesicles (Fig. 9C).

Type I1 cells generally are associated in groups distributed along the tubules; thus sometimes type I cells are not included in a section. The single type of granule in the type I1 cell contains two regions differing in electron-density. There is a sharp distinction between lighter and more heavily electron- dense regions (Fig. 10A). In semithin sections these granules stain lilac. Profiles of the RER cisternae in cell type I1 are relatively short and not as numerous as in cell type I, but ribosomes are abundant. Cisternae of the


Fig. 4. Tursiops truncatus ponticus. TEM of portion of female glandular cell at the level of Golgi saccules. Note moderately electron dense (gray) granules (GI, con- tainingfine-grained contents similar to those in the RER; two such granules are fused. Cisternae of Golgi complex

(Gc) are accompanied by abundant Golgi vesicles (V). Basolateral plasmalemma (Pm) shows interdigitation of adjacent cells. Lb, lamellar body; M, mitochondrion. Scale bar = 0.3 pm.

Golgi complex are relatively short and dis- tended, compared with those in cell type I. Their content is electron-lucent or displays a thin network. Golgi vesicles are less abundant than in type I cells (Fig. 10B,C).

DISCUSSION

Differences between glandular epithelial cells of the Harderian gland in male and female Black Sea bottlenose dolphins have been described in this report (see Table 1). The female gland has only one type of secretory cells, whereas the male gland possesses two distinct types. Secretory cells of the two sexes differ with respect to the form and distribution of their endoplasmic reticulum, Golgi complex, and secretory granules. Do these differences indicate the existence of distinct genetically determined cell types in males and females?

Specialization of the glandular cells is associated with their ability to synthesize and secrete a variety of specific products. Morpho- logically distinct cell types differ in composi-

tion, abundance, distribution, and details of structure of their organelles (Alberts et al., '83). Cells with well-developed smooth endoplasmic reticulum produce predominately lipids rather than peptides, whereas those with well-developed RER produce predominately peptides. Active synthesis of carbohydrates is correlated with the presence of a well-developed Golgi complex. The forms of endoplasmic reticulum and Golgi complex are highly variable among eukaryotic cells (Fawcett, '81; Terasaki et al., '86), including secretory cells. Lee and Chen ('88) have shown that at the time of cell division in secretory cells the endoplasmic reticulum separates into small fragments, whereas the Golgi complex disap- pears. The morphologies of these organelles in daughter cells are identical to those in the parental cell. In a series of experiments on transplantation of the endoplasmic reticulum and Golgi complex into heterologous cells, it was found that after transplantation these organelles undergo rapid reconstitution according to a pattern specific for the


Fig. 5. Tursiops truncatus ponticus. TEM of apical ends of the female glandular cells. A. Some gray granules are coalesced into clumped masses (M). Lipid granules (L) have an electron-lucent interior but an electron-dense rim. Lm, lumen; Ly, lysosome; Pm, plasmalemma; RER,

rough endoplasmic reticulum. B. Dark granules (D) remain separated. Lamellar bodies (Lb) are associated with fused mass of moderately electron dense (gray) granules. L, lipid granules; RER, rough endoplasmic reticulum. Scale bars = 0.5 wm.

donor cells (Paiement, '86; Dominguez and Paiement, '89; Paiement et al., '88, '89, '90). During this process, elements of donor cells are used as construction materials. The authors conclude that intrinsic membrane fac-

tors are important for the three-dimensional organization of these organelles and deter- mine the pattern of their reconstitution.

The above-mentioned results confirm the hypothesis that the shape of the biosynthetic

2 14 N.D. BODYAK AND L.V. STEPANOVA

Fig. 6. Tursiops truncatus ponticus. Enlarged view showing organelles and inclusions in female glandular cells. Note that gray granules (G) are closely associated

apparatus is genetically predetermined for every cell type (Gluecksohn-Waelsch, '79; So- kolov and Stepanova, '89). Apart from variations in the biosynthetic apparatus of secretory cells, we have found that there is high variability in structure of the secretory granules; yet one or another morphologically distinct type or type-set of granules is inherent for a particular cell type (Sokolov and Stepa- nova, '89). In cells of the adenohypophysis, for example, each of the seven cell types responsible for the secretion of one of the seven hormonally active peptides possesses its own characteristic granules. The cell-type specific granules also have been described for unicellular intestinal glands (Zufarov et al., '83). It is reasonable to assume that the morphology of secretory granules (their size, shape, electron density, tinctorial features) is determined mainly by the chemical and structural properties of the substances they contain (Sat0 and Spicer, '80; Larkin et al., '87).

A previous study on the morphology of anal glands in the beaver has revealed pro- nounced sexual dimorphism in the structure of the epithelial cells. Secretory granules in

with small transport vesicles and that lipid granules (L) have an electron-dense rim. Ly, lysosome with lamellar body; M, mitochondria. Scale bar = 0.2 pm.

these cells vary in correlation with biochemi- cally revealed intersexual variations in the chemical composition of secreted material (Sokolov et al., '92). Thus we agree with the generalization that the functional specialization of a secretory cell is expressed morphologically by a characteristic portrait of its biosynthetical apparatus and its specific secretory granules. In the present study, our observations on the RER, Golgi complex, and secretory granules of the Harderian gland cells in both sexes of the bottlenose dolphin indicate that these cells are active producers of peptides and carbohydrates. This conclusion cor- responds completely to the data of Uskova and colleagues ('751, obtained with the help of chemical analysis. According to their study, the secretion of the orbital gland of Tursiops truncatus is notable for its rather high content of proteins (6.56-7.5 mg/ml) and carbohydrates (3-4 mg/ml) whereas their weight ratio appears to be 2:l. Since male and female secretory cells differ in the structure of their biosynthetic apparatus and secretory granules, it may be that male and female


Fig. 7. Tursiops truncatusponticus. TEM of the Golgi apparatus in female glandular cell. A. Golgi complex consists of stacked Golgi saccules and Golgi vesicles (arrows). Both gray (G) and dark (D) granules are seen in the vicinity of the Golgi zone. Scale bar = 0.3 pm. B.

Note the formation of dark granules (D) by separation from cisternae of the Golgi complex (arrow). M, mitochondria; Pm, lateral plasmalemma showing interdigitation; RER, rough endoplasmic reticulum. Scale bar = 0.5 pm.

glands produce different peptides and carbohydrates.

Synthesis of carbohydrates in the secretory cells of the Harderian gland of Tursiops truncatus has been confirmed in a recent study (Tarpley and Ridgway, '91) revealing the presence of both acid and neutral gly-

cosaminoglycans in these cells, detected by histochemical methods. Their study prob- ably refers to the glands of males, although no clear distinction of the sex of the animals under study was made. Comparing their electron microscopic and histochemical data, the authors concluded that electron-dense dark


Fig. 8. Tursiops truncatusponticus. TEM of the male Harderian gland. Note two types of cells: cell type I (GI) shows two kinds of granules, gray (G) and dark (D); cell type I1 (CII) has only one kind of granule. Golgi complex

(Gc) is well developed. Lm, lumen, RER, rough endoplasmic reticulum. Scale bar = 2 Fm. Insert Enlarged view showing dark granules, one with gray inclusion. Scale bar = 0.4 bm.

granules (found in cells belonging to the male cell type I1 according to our classification) contained only neutral glycosaminoglycans, whereas the electron-lucent light granules

(found in male cell type I) contained both acid and neutral glycosaminoglycans. Lipid analysis, based on material from three animals, proved that lipids constituted < 1% of

HARDERIAN GLAND ULTRASTRUCTURE OF TURSIOPS

Fig. 9. Tursiops truncatus ponticus. TEM of secretory cells of type I in the male Harderian gland. A. Apical end of the cell. Electron-lucent (light) granules (E) are aggregated. Moderately electron-dense (gray) granules (G) do not aggregate, Lm, lumen. Scale bar = 0.4 pm. B. Lysosome-like granules in apical end of secretory cell.

the total mass of the secretions. The lipid fraction consisted of phospholipids and neutral lipids, including cholesterol esters, free fatty acids, and triglycerides. The latter was the predominant component. Origin of the lipids was not discussed.

217

Scale bar = 0.4 pm. C. Golgi complex (Gc) shows thin saccules and associated Golgi vesicles. Elongate cisternae of RER contain moderately electron-dense substance. Gray granules (G) have fine-grained contents. N, nucleus. Scale bar = 0.7 pm.

Our data support the hypothesis that lipids are produced principally as a secretion of the female gland, since smooth endoplasmic reticulum was found only in cells of this sex. Some part of the phospholipid fraction, however, might originate from membrane struc-


Fig. 10. Turscops trurzcatus jwnticus. TEM of secretory cells of type I1 in the male Harderian gland. A. Apical end of cell, showing secretory process (Sp) of one cell protruding into the lumen of the tubule. Arrows indicate a “demarcation membrane.” Granules (GI have both

gray and dark regions. Ds, desmosome; R, ribosomes. Scale bar = 0.2 pm. B, C. Golgi complexes (Gc) in two cells of type 11. M, mitochondria; N, nuclei; RER, rough endoplasmic reticulum. Scale bar = 0.4 pm.


TABLE 1. Ultrastructural differences between secretory cells of the Harderian gland of the Black Sea bottlenose dolphin

Female Male

Cell type number One Two

Traits Type I Type I1

Rough endoplasmic Cisternae are elongated, reticulum their contents moderately

electron-dense

Smooth endoplasmie Short tubules sparsely dis- reticulum persed throughout the

cytoplasm Golgi complex Elongate, arc-shape, stacked

cisternae with moderately electron-dense contents

Granules Three types: 1. Lipid, with dark rim 2. Moderately electron-

dense (gray) contents; fuse

3. Heavily electron-dense; do not fuse

Cisternae are elongated, situated along cell periphery, their contents moderately electron-dense electron-dense

Cisternae short, situated in basal portion of the cell, their contents moderately

Not observed Not observed

Relatively short dilated cisternae, contents electron- lucent

Four types: 1. Electron-lucent (light);

2. Moderately electron- fuse

dense (gray), contents fine-grained, some with dark inclusion; do not fuse

3. Electron-dense (dark), some with gray inclusions; do not fuse

4. Lysosome-like granules may fuse

Cisternae dilated, not stacked, contents electron- lucent

One type: 1. Granules stained partly

gray and partly black with distinct border between gray and black parts; do not fuse

tures, including those of desquamated cells as well as those from cytoplasmic fragments extruded during the process of apocrine secretion.

Cell composition appears to be the same in all parts of the gland; hence we see no basis for the suggestion by Waller and Harrison ('78) that the narrow and wide parts of the orbital gland of the dolphin are histologically different. These authors distinguished the conjunctival and the Harderian glands.

Our data indicate that lipids, proteins, and glycosaminoglycans are synthesized by the bottlenose dolphin Harderian gland. Accord- ing to Sakai's ('81) criteria, Harderian glands secrete lipids, whereas proteins and carbohydrates are secreted by lacrimal glands. Since lipid secretion appears to be largely restricted to the female in the dolphin, the female gland would be classified as Harderian and the male gland as lacrimal by these criteria. Adoption of these criteria offers considerable difficulty. For instance, the gland of the dog that consists of mixed tubules and synthesizes muco- polysaccharides and lipids (Martin et al., '88) is sometimes identified as Harderian (see review by Kennedy, '70) and sometimes as su- perficial lacrimal or nictitans (see review by Sakai, '81). Among Australian marsupials the Harderian gland was found in two related

species of marsupial mice, whereas a third species was considered to have nictitans glands solely for the reason that they gave a positive reaction for the presence of muco- polysaccharides (Sakai and van Lennep, '84).

We believe that synthesis of nonlipid substances by the Harderian gland has been undeservedly ignored by the authors of the studies cited above. It is accepted as generally true that Harderian glands of nonmamma- lian tetrapods secrete glycosoaminoglycans but not lipids. For example, the Harderian glands of the frog, Rana esculenta (DiMatteo et al., '89)' and the snake, Bothrops jararaca (Lopes et al., '741, contain no lipids and be- long to the mixed sero-mucoid type. In the lizards Crotaphytus collaris and Phrynosoma cornutum, the Harderian glands secrete mucus, and the secretory cells of Podarcis sicula possess both mucous and serous granules. Moreover, the gland of the latter species can be divided into three parts differing in their tinctorial properties. The intermediate part of this gland resembles the lacrimal gland, which is also present in the eye orbit (Baccari et al., '90). The Harderian glands of birds also are of the mucous type but also contain some amount of lipids as well (Wight et al., '71). Secretory cells of the turkey, Meleagris meleagris gallopavo, are known to be rich in


nonsecretory lipid granules located in the basal part of the cells (Maxwell et al., '86). From our results and those of some previous workers, it appears that there is no universal cell type characteristic for the Harderian gland.

Functions of the Harderian glands of bottlenose dolphin and other mammals have not been clarified. In cetaceans the gland is sup- posed to produce a secretion protecting the eye from seawater (Waller and Harrison, '78; Tarpley and Ridgway, '91), mechanical par- ticles, and microorganisms (Tarpley and Ridg- way, '91), or it may reduce water resistance in the orbital zone (Uskova et al., '75). Since the Harderian gland epithelium lies in direct contact with lymphoid tissue, the source of the secretory immunoglobulins (Scicchitano et al., '86), participation of the Harderian gland in local immunological reactions should be regarded as plausible. Another intriguing possibility is the participation of the Hard- erian gland in chemical communication. The Harderian gland is thought to produce pheromones that play an important role in the coordination of social behavior in some rodents (Thiessen et al., '76; Payne, '77, '79).

It is traditionally believed that dolphins lack any chemical senses, as the brain of cetaceans is anosmic owing to the total ab- sence of olfactory bulbs. However, several recent investigations have contradicted this belief. According to Kuznetsov ('861, bottlenose dolphins are sensitive to many odor- ants. Another corroboration comes from some observations on the behavior of dolphins in captivity (Lilly, '62). In addition, professional trainers comment that the gland in bottlenose dolphin constantly secretes its products into the environment. Rate of excretion can be regulated, as the secretory tubules that serve also as reservoirs are closely associated with muscular fibers (Tarpley and Ridgway, '91). These observations, along with the fact that the Harderian gland of Tursiops truncatus ponticus demonstrates significant sexual differences, provide support for the idea that the gland participates in communicative processes and may serve as a source of sexual pheromones.

The high degree of development of the vascular network and the large numbers of fenestrations of the capillaries in the Hard- erian gland's interstitium are noteworthy. Many secretory cells lie directly on the basal plasmalemma, so that proximity to capillaries is close. Fenestrated capillaries were found

to lie around tubular and acinar glands in the anal gland of the woodchuck (Smith and Hearn, '79) and in the stroma of the tubular plantar gland of the laboratory mouse (Quick et al., '84). Many mammalian species have skin glands in some locations showing an abundance of highly fenestrated capillaries, whereas glands in other locations lack this feature (unpubl. observations).

It is possible that the presence of fenestrated vessels, i.e., vessels with increased per- meability, is an indication of the gland's endocrine activity. The suggestion that mammalian skin glands can combine both exocrine and endocrine functions has been previously expressed (Montagna and Yun, '62; Sokolov et al., '89). The same may also apply to the bottlenose dolphin Harderian gland.

ACKNOWLEDGMENTS

We appreciate the helpful comments of Vladimir S. Lebedev and Valentin D. Mil- gram during revisions of this manuscript.

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