morphology and glycoconjugate histochemistry of the palpebral glands of the adult newt,...

JOURNAL. OF MORPHOLOGY 211 165-178 (1992)

Morphology and Glycoconjugate Histochemistry of the Palpebral Glands of the Adult Newt, Notophthalmus viridescens

RANDALL W REYER, WILLISA LIOU, AND CARLIN A. PINKSTAFF Department of Anatomy, Schools of Dentistry and Medicine, West Virginza Uniuersity, Morgantown, West Virginia 26506

ABSTRACT The eyelids of the newt were studied in 10 km serial paraffin and 1-2 pm plastic sections using standard histologid stains and special stains for glycoconjugates. The eyelids contain four different glands. Simple acinar serous and simple acinar mucous glands occur in the skin; unicellular mucous glands occur in the conjunctiva; and convoluted tubular seromucous glands are present in connective tissue beneath the conjunctiva. The first two are identical t o cutaneous glands found elsewhere on the head and body. The simple acinar serous glands are surrounded by myoepithelial cells and release their secretion, which is composed largely of proteins with minimal glycoconjugate content, by a holocrine mechanism. The secretory product of the simple acinar mucous glands is composed of neutral glycoconjugates with a minor content of acidic glycoconjugates; the mucin exhibits strong PAS and PAPD staining and weak staining by AB and PAPS methods. The unicellular conjunctival mucous glands secrete both neutral and acidic glycoconjugates as shown by positive reactions with PAS, PAPD, PAPS, and AB methods. ConvoIuted tubuIar seromucous glands in the ventral eyelid synthesize both proteins and neutral glycoconjugates. The mucous secretions of the conjunctival glands probably provide lubrication and protection for the cornea.

In its adult stage, Notophthalmus uiridescens is aquatic and found in the shallow water of lakes, ponds, and quiet streams. Adult newts may emerge from the water and rest on protruding stones or other objects, but they do not leave the vicinity of the water. Eggs are laid on aquatic plants and hatch into aquatic larvae. These metamor- phose into a terrestrial phase, the red eft, which returns to the water as an adult. The adults have upper and lower eyelids which cover the eye when it is retracted. Both aquatic and aerial vision are employed by adult newts.

Cutaneous glands of amphibians have been studied by numerous investigators since the nineteenth century. References to the early literature were cited by Dawson ('20) and Le Quang Trong ('67a). Much of the early work was on anurans, but there were also a few papers on urodeles. From studies on Nectu- rus maculosus (Dawson, '2O), the axolotl Am- bystoma tigrinum (Le Quang Trong, '67a), and the frog R a m pipiens (Noble and Noble, '44), there is general agreement that there are two kinds of prominent cutaneous glands

located in the dermis: (1) large granular glands producing a serous protein secretion and (2) smaller mucous glands producing a mucous secretion that is stained by the PAS method. Both gland types are simple acinar and open on the surface of the skin by means of a duct of simple squamous to simple cuboidal epithelium that traverses the epidermis. The appearance of these glands varies in sectioned material, depending on the different stages of the secretory cycle, and will be described later. Another type of gland, characteristic of aquatic larval stages, lies within the epidermis and consists of one or two enlarged cells termed Leydig or club cells that secrete a proteinaceous product.

Hoffman and Dent ('77) reported a differ- ence in the secretory response of the granular cutaneous glands to neurotransmitters in anuran and urodelian species. Using skin explants in Holtfreter's salt solution, these authors observed that epinephrine and nor- epinephrine, but not acetylcholine, stimu-

Willisa Liou's current address is Department o f h a t o m y , Chang Gung Medical College, Taiwan, R.O.C.

D 1992 WILEY-LISS, INC.

166 R.W. REYER ET AL.

lated secretion of these glands in Xenopus laevis; this adrenergic response was blocked by the a-blocker ergotamine. By contrast, granular glands of N . viridescens were stimulated by acetylcholine, but not by adrenergic agents. The acetylcholine response was blocked by atropine, an inhibitor of muscar- inic receptors. The cutaneous mucous glands were not stimulated to secrete by either adrenergic or cholinergic neurotransmitters. However, this secretion is under positive hor- monal control by prolactin and thyroxine (Dent et al., '73; Dent, '75).

In his review of vertebrate cutaneous glands, Quay ('72) summarized the character- istics of the two kinds of glands in amphibians and emphasized their distinct identities. He noted that the granular glands are the source of the toxic secretions of some amphibians; he also observed that these glands were surrounded by myoepithelial cells that con- tract to discharge the secretory product from the acini after neural or humoral stimulation. The mucous glands are always stained strongly by the PAS reaction; these glands do not have a distinct investing layer of myoepithelial cells and appear to secrete continuously and spontaneously. Noble ('31) stated that the function of the mucous glands was to provide a lubricant when the animal was in the water and a skin moistener when the animal was on land.

During the course of studies in this laboratory on lens regeneration in newts, many of the eyes, fixed for histological study, included the eyelids, either dorsal alone or both dorsal and ventral. These preparations provided an opportunity to investigate the structure of the glands of the eyelid and the kinds of secretion produced. Four different kinds of glands have been observed in newt eyelids. Two of these glands appear to be uniqueIy found in the eye adnexa, while the other two are similar to cutaneous glands found throughout the newt integument.

MATERIALS AND METHODS

Adult newts (N. uzridescens) were collected locally from ponds or purchased from Lee's Newt Farm, Oak Ridge, TN. They were kept in aquaria at room temperature in tap water that had been dechlorinated with sodium thio- sulfate. They were fed chopped beef liver two or three times a week. Methylene blue (0.214 mg/liter) was added to the culture water as a fungicide. The eyes used for the morphological investigation were from newts used in several studies on regeneration of the lens from the iris. Therefore, the lens had been

removed from the eye and lens regeneration allowed to proceed for a few days to a few weeks before the animals were killed. Follow- ing anesthesia in 0.1% chlorobutanol or chlo- retone (Parke Davis), the animals were decap- itated, the lower jaw removed, and the heads fixed in Bouin's fluid (most specimens) or in Carnoy's fixative. The heads were decalcified in 5% HNO, in 80% ethanol followed by 80% ethanol saturated with MgCO,, or in 5.5% EDTA at pH 7.0. In one group, the eyes and dorsal eyelids were dissected from the orbits in 70% ethanol and processed separately, thereby eliminating the need for decalcifica- tion. The heads or eyes were dehydrated in graded ethanols to 95% ethanol and cleared in a-terpineol. After a toluene rinse, specimens were embedded in Paraplast@ and 10 pm serial sections were cut through the eye in a dorso-ventral meridional plane trans- verse to the pupil, thereby cutting the eyelids in cross-section. These serial sections were mounted on glass slides and stained with Ehrlich's alum hematoxylin and erythrosin (Drury and Wallington, '67), Masson's trichrome (Luna, '68), or Himes-Moriber stains (Himes and Moriber, '56).

Four eyes with eyelids were fixed in ice- cold Karnovsky's fixative (Karnovsky, '65) in 0.1 M sodium cacodylate buffer at pH 7.4; they were then dehydrated through graded ethanols and flat-embedded in butoxyethanol- glycol methacrylate (Polysciences, Inc.). Se- rial sections, 2 pm in thickness, were cut in a dorso-ventral meridional plane with dry glass knives, mounted with water on slides and stained with hematoxylin and erythrosin (Gill et al., '74). Observations also were made on the dorsal eyelids of two other eyes that were originally prepared for another EM study. These specimens were fixed in Karnovsky's fixative in 0.1 M sodium cacodylate buffer at pH 7.3 at room temperature. Specimens were rinsed in three changes of buffer and post- fixed in ice-cold 1% OsO, in the same buffer. After several rinses in buffer, specimens were dehydrated through graded ethanols to pro- pylene oxide and flat-embedded in Epox 812 (Ernest F. Fullam, Inc.). One micrometer thick sections were cut with glass knives into a water-filled boat, transferred to glass slides, dried, and stained with toluidine blue-borax.

Heads to be used for histochemical studies were fixed in 10% formalin containing 1% cetylpyridinium chloride. This fixative was used in an effort to retain acidic glycoconjugates (Williams and Jackson, '56). As before, the eyes and dorsal eyelids were from lentec-

GLYCOCONJUGATE HISTOCHEMISTRY OF EYELID GLANDS 167

tomized animals at different stages of lens regeneration from the iris. The eyes and dorsal eyelids were dissected from the orbits, dehydrated and cleared as before, and embedded in Paraplast Plus@. Serial sections were cut a t 10 pm and mounted as previously described.

The histochemical methods employed were standard techniques for demonstrating glycoconjugates. The periodic acid-Schiff (PAS) method was used to demonstrate glycoconjugates containing vicinal hydroxyl groups that are known to occur in neutral glycoproteins and in some acidic glycoproteins (Mowry, '63). The PAS reaction was also used to test for glycogen; control sections were treated with porcine a-amylase prior to staining with the PAS method (Lillie and Fullmer, '76). Other control sections were pretreated by an amine-aldehyde condensation prior to application of the PAS method (PAS-AAC) in or- der to determine whether or not reactive aldehyde groups from the fixative might have contributed to the overall PAS staining (Pearse, '68). Periodate-reactive acidic glycoconjugates, presumably sialoglycoproteins, were shown using a periodic acid-phenylhydrazine-Schiff (PAPS) method (Reid et al., '84). Alcian blue (-1 a t pH 2.5 was used to demonstrate weakly sulfated andlor non- sulfated acidic glycoconjugates (Spicer et al., '67). Simultaneous demonstration of periodate-reactive and alcianophilic glycoconjugates was achieved using an alcian blue (pH 2.5)-periodic acid-Schiff (AB-PAS) method (Spicer et al., '67). The high iron diamine (HID) method was used to demonstrate sulfated glycoconjugates and a combined high iron diamine-alcian blue (pH 2.5) method (HID-AB) was used for the simultaneous demonstration of sulfated (HID+) and non- sulfated or weakly sulfated glycoconjugates (AB+) (Spicer, '65). The periodic acid-p- diamine (PAPD) method was used to demonstrate neutral glycoconjugates thought to be rich in fucose, galactose, or mannose (Spicer et al., '67).

In one experimental series, serial sections of 10 to 18 eyes were used for each histochemical procedure except for the PAS-a-amylase and the PAS-amine-aldehyde condensation procedures; either five or six eyes were used with the latter two procedures. In another series of 29 eyes, comparisons of PAS staining reactions versus PAPS staining reactions were made, as well as comparisons of PAS staining reactions versus PAPD staining reactions.

All sections illustrated are 10 km paraffin, except where noted.

RESULTS Morphology of eyelid glands

There are three different kinds of glands in the dorsal eyelids of the adult newt: (1) unicellular mucous glands located within the epithelium of the palpebral conjunctiva, (2) simple acinar mucous glands, and (3) simple acinar serous glands. The latter two gland types are located in dermal connective tissue and open by means of ducts through the epithelium on the outer surface of the eyelid (Figs. 1, 2). These three types of glands also occur in the ventral eyelid, as does a fourth gland type, convoluted tubular seromucous glands.

Unicellular mucous glands The conjunctival epithelium varies from

stratified cuboidal or columnar at the fornix to some stratified squamous toward the margin and is two to four cell layers thick. The mucin-secreting cells lie above the basal layer of the conjunctival epithelium and extend to the free epithelial surface in both dorsal and ventral eyelids. The cells have basally located oval to flattened nuclei and the cytoplasm is filled with mucin (Figs. 3, 7). Numerous mucous cells occur in the palpebral conjunctiva and they also are present in the bulbar conjunctiva. The mucin was well preserved in both paraffin-embedded and plastic-embedded specimens.

Simple acinar mucous glands These glands are surrounded by loose con-

nective tissue and lie in the dermis under the epidermis of both dorsal and ventral eyelids. The basal acinar cells are tall pyramidal to columnar cells, while the more apically 10- cated cells are cuboidal to squamous cells. The oval nuclei are basally located. The two cell types can be distinguished easily in paraffin sections stained by Masson's trichrome and Himes-Moriber staining methods. Outer and apically placed cells stain orange-red with Masson's trichrome (Fig. 9) and light yellow with the Himes-Moriber stain (Fig. 2); the more central and basally located cells are strongly stained by the PAS component of the Himes-Moriber stain (Figs. 1,2). In 2 pm methacrylate sections, the centrally placed cells stain red with erythrosin (Fig. 3). In 1 km plastic sections stained with toluidine blue, one readily sees cells with light and dark staining granules interspersed in the acinus. The narrow duct of the acinus opens


Fig. 1. Notophthalmus uiridescens. Dorsal eyelid of adult. Two kinds of simple acinar glands are present: larger serous glands ( S ) with a clear lumen and smaller mucous glands (MI stained by the PAS component. Himes- Moriber stain. BC, bulbar conjunctiva. Ep, epidermis. PC, palpebral conjunctiva. x 100.

Fig. 2. Part of dorsal eyelid showing mucin, stained with PAS, in some cells of the simple acinar mucous glands and a clear or faintly stained lumen in the simple acinar serous glands, one of which has a large, probably polyploid nucleus (arrow). The glands lie between the palpebral epidermis above and the conjunctiva below. Himes-Moriber stain. X 190.

Fig. 3. Dorsal eyelid near margin. Unicellular mucous glands, with darkly stained mucin, in palpebral conjunctiva (arrows) and a simple acinar mucous gland, with a granular mucous secretion, in the connective tissue between the epidermis and the conjunctival epithelium; 2 pm methacrylate section. Hematoxylin and erythrosin. x345.

Fig. 4. Simple acinar mucous gland and duct which is penetrating the palpebral epidermis. Small, non-granular cells form the apical part of the acinus opening into the duct. Secretory cells contain light or dark granules. Clear area in epidermis (arrow) is an artifact. 1 km Epox section. Toluidine blue. ~ 5 2 0 .

onto the surface of the integument (Fig. 4). Myoepithelial cells could not be seen in association with simple acinar mucous glands.

mucous glands and occur in both dorsal and ventral eyelids. The appearance of this gland type depends on the stage of the secretory cycle observed. The various stages are clearly

Simple acinar serous glands seen in 1-2 pm sections of plastic embedded material. In earlv stages of the secretorv CY-

The simple acinar serous glands are larger and more numerous than the simple acinar

cle, the acinar ce"1ls a& filled with nume"ro;s small secretory granules that are strongly


Fig. 5. Two stages in secretory cycle of the simple acinar serous gland. At right is an early stage made up of large, granule-containing cells. At left is a late stage in which a large lumen is filled by a foamy secretion. A single layer of myoepithelial cells surrounds each acinus; these cells are cut both transversely and longitudinally (arrows). 1 Fm Epox section. Toluidine blue. X220.

Fig. 6. An intermediate stage in the secretory cycle of a simple acinar serous gland. The cytoplasm of the large cells is filled by vacuoles and the cell toward the epidermis is breakingdown. Myoepithelial cell sheath (arrows). Small mucous gland is to left of serous gland. 1 km Epox section. Toluidine blue. x 288.

stained by toluidine blue, but virtually unstained by hematoxylin or erythrosin (Fig. 5). The acini increase in size and most of the granules disappear or are no longer stained darkly by toluidine blue. This stage is also characterized by the appearance of vacuoles in the cytoplasm of the acinar cells (Fig. 6). Narrow intercellular spaces separate the acinar cells. Shortly before secretion begins, the acinus is filled by secretory vacuoles that are unstained by toluidine blue. At this stage, one is unable to distinguish individual cells in the acinus (Fig. 5). Myoepithelial cells surround the serous acini (Figs. 5,6).

In sections of paraffin-embedded material, the stages of the secretory cycle can also be discerned. The granular nature of the secretory product a t early stages is sometimes demonstrable by the Himes-Moriber method. Later, acini may contain loosely packed glob- ules or a fibrillar precipitate; or the acini may appear to be empty (Figs. 2, 7, 8). The secretory material is stained a brick-red color of variable intensity with Masson’s trichrome stain. It is stained yellow with the Himes- Moriber stain and light pink with erythrosin. The nuclei are flattened in the basal regions of the acinar cells; large, apparently polyploid

nuclei may be present (Figs. 2, 8, 9). As the onset of secretion nears, the first cells that lose their granules, or whose granules may fuse to give the impression of a homogeneous material, are those located in the apical region of the acinus. These cells appear to break down and, as the secretory material accumulates, a lumen appears in the apical region for the first time (Figs. 7, 8). Myoepi- thelial cells are less obvious in sections of paraffin-embedded specimens, but they can be distinguished with Masson’s trichrome stain, especially at early stages of the secretory cycle (Fig. 9).

Convoluted tubular seromucous glands The convoluted tubular seromucous glands

are found mainly in the ventral eyelids. These glands are composed of simple cuboidal to low columnar epithelial cells surrounding a narrow lumen. The nuclei of these cell are round and basally located in the columnar cells. Secretory granules lie in the cytoplasm apical to the nucleus of the cell. Sections through the eyelid cut the tubule in various planes indicting a convoluted course (Figs. 10 , l l ) . We were unable to determine whether or not the tubules are branched. As the tu-

170 R.W. REYER ET AL

Fig. 7. Two simple acinar serous glands showing the large cells, containing secretory granules, that form the ncinus before the cells break down to release the secretion. A lumen appears to be forming in the gland on the left. Three unicellular mucous glands, stained with PAS, are also visible in the palpehral conjunctiva (arrow). Himes-Moriber stain. x 173.

Fig. 8. Two simple acinar serous glands containing precipitated secretion. Small duct cells a t apex of acinus lie next to channel of duct in epidermis in gland on left. Two large, probably polyploid nuclei are visible (arrows). Himes-Moriher stain. x 210.

Fig. 9. In center of photomicrograph is a simple acinar serous gland in an early stage of the secretory cycle. It is composed of large pale cells with distinct cell boundaries enclosed by a sheath of myoepithelial cells (arrowhead). Smaller, simple acinar mucous gland is in contact with left side of serous gland. Outer cells are stained orange red (clear arrow) and enclose lightly- stained inner cells. Later stage serous gland with probably polyploid nucleus (arrow) at right. Ns, nucleus of serous cell. Nm, nucleus of myoepithelial cell. Masson’s trichrome stain. ~ 2 0 0 .

bule approaches the margin of the ventral eyelid, the cells change to simple cuboidal epithelium and form a duct that opens through the conjunctival epithelium at a short distance from the eyelid margin (Fig. 12). The staining intensity of secretory granules by the PAS component of the Himes-Moriber

method varies, as does the red staining with Masson’s trichrome. That portion of the tubule nearest the duct stains more intensely with the PAS component of the Himes- Moriber stain and does not stain red with Masson’s trichrome stain. That portion of the tubule most distant from the duct stains


Fig. 10. Ventral eyelid. Convoluted, secretoryportion of tubular gland. Region toward duct is at the right. The PAS reaction is darker in the tubule toward the duct than toward the fundus at the left. Himes-Moriber stain. x 110.

Ventral eyelid. Convoluted, secretory portion of tubular gland. Red stain is darker toward the fundus of

gland at the right than toward the duct at the left. Masson’s trichrome stain. x 176.

Fig. 12. Ventral eyelid of adult newt. The long, quite straight duct of a tubular gland opens through the palpebral conjunctiva (arrow). Duct cells are unstained by PAS. Himes-Moriber stain. x 60.

Fig. 11.

strongly with Masson’s trichrome stain, while PAS staining is less intense than in cells near the duct (Figs. 10,111.

Carbohydrate histochemistry of eyelid glands

The application of the Himes-Moriber method, used in this study for the examina- tion of gland morphology, revealed that PAS staining of varying degrees occurred in all glands of the eyelids, except in simple acinar serous glands. The application of other staining methods for demonstrating glycoconjugates yielded additional information about the secretory products that are summarized in Table 1.

The results of staining glands by the PAS method, with and without a-amylase pretreat- ment, were identical; therefore, there was no demonstrable glycogen. The use of aldehyde- containing fixatives did not influence PAS staining of the glands since identical staining reactions were seen in sections stained with the PAS method and in sections exposed to an amine-aldehyde condensation procedure before PAS staining. Dark magenta staining by the PAS method was observed in secretions of the unicellular mucous glands, in the centrally located cells of the simple acinar mucous glands (Fig. 13), and in many of the secretory granules of the cells of the convoluted tubular glands. Cells of simple acinar

172 R W REYER ET AL

TABLE 1. HLstochenml reaetlons forglycoconJugates m the secretcons ofglands m the eyellds of Notophthalmus viridescens'

Glands

S t a n Uniellular mucous Simple acinar mucous Simple acinar serous seromuoous

PAS Magenta Magenta Rare, light magenta Magenta, in part a-amylase Magenta Magenta Rare, light magenta Magenta, in part

PAS-AAC Magenta Magenta Rare, light magenta ND

AB-PAS Dark purple Magenta Rare, light magenta Magenta, in part

Convoluted tubular

PAS

PAPS Lighb pink Light pink 0 0 AB, pH 2.5 Aquamarine Light blue in few cells 0 0

HID Brown granules in rare cells 0 0 0 HID-AB Aquamarine, brown p a n - Light blue in few cells 0 0

ules rare PAPD Intense golden-brown Intense golden-brown 0 Faint golden-brown

'ND, not determind; 0, negative n e d t

serous glands were usually unstained by the PAS method. Occasional small cells a t the base of the duct stained a light magenta and, in rare instances, light magenta cytoplasmic granules were observed, at early stages of the secretory cycle, in more basally located acinar cells (Fig. 13). To varying degrees, glycoconjugates containing vicinal hydroxyl groups are present in all eyelid gland cells.

The PAPS method showed the presence of periodate-reactive acidic glycoconjugates, presumably sialoglycoproteins, as lightly pink stained secretory material in the unicellular mucous glands and in the central acinar cells of the simple acinar mucous glands (Fig. 14); the simple acinar serous glands and the convoluted tubular glands did not stain. Acidic glycoconjugates in unicellular mucous glands and simple acinar mucous glands were also stained by AB at pH 2.5; unicellular mucous glands were strongly stained (Fig. 15), while cells of the simple acinar mucous glands were less intensely stained (Fig. 16). Simple acinar serous glands and convoluted tubular glands were unstained by AB at pH 2.5. There was good agreement of both intensities and loca- tions of staining reactions with the PAPS and AB at pH 2.5 methods. Unicellular mucous glands were stained by both the AB and PAS components of the AB-PAS method; the combination of the magenta of the PAS reaction and the blue of the AB method resulted in dark purple coloration of the mucin. The simple acinar mucous glands were stained magenta by the AB-PAS method. This indi- cates that, apparently, the content of AB- stained acidic glycoconjugate in these glands was not high enough to shift the color to purple. Simple acinar serous glands and convoluted tubular glands were stained only by the PAS component in the AB-PAS method.

A few scattered unicellular mucous glands were stained by the HID method for sulfated glycoconjugates (Fig. 171, but this method failed to stain the secretory products of the cells of any other gland types. When used in conjunction with AB at pH 2.5 (HID-AB), only alcianophilia was observed in most cases, although both AB and HID staining were found occasionally in a few unicellular mucous glands.

The PAPD method stained the secretory products of the unicellular mucous glands and the central cells of the simple acinar mucous glands an intense golden-brown color (Figs. 18, 19); some secretory granules of the convoluted tubular glands were stained a light golden-brown color. The simple acinar serous glands failed to exhibit any golden- brown coloration.

DISCUSSION

In the newt eyelid, there are cutaneous glands in the outer skin and conjunctival glands located in or opening on the palpebral conjunctiva. The serous and mucous cutaneous glands of the eyelids are the same as those found over the rest of the head and correspond to the cutaneous glands which are distributed abundantly over the bodies of different amphibians. The morphological and histochemical evidence supports the conclu- sions of other investigators that there are two kinds of glands in amphibian skin, serous and mucous. The serous glands were originally called granular glands (Dawson, '20; Noble and Noble, '44; Le Quang Trong, '67a; Hoffman and Dent, '77; Neuwirth et al., '79; Mills and Prum, '84).

The morphological features of each gland are quite variable and depend on the stage of


Fig. 13. Dorsal eyelid. Two simple acinar mucous glands stained a dark magenta by Schiffs reagent. One simple acinar serous gland in the center has a clear lumen and large cells with probably polyploid nuclei (arrows). In the latter gland, cytoplasmic granules are stained a light magenta. PAS-hematoxylin. x 200.

Fig. 15. Palpebral conjunctiva of dorsal eyelid. Vacu- oles of unicellular mucous glands and a layer of mucus on the epithelial surface have stained an aquamarine blue. AE-nuclear fast red. x250.

Fig. 16. Margin of dorsal eyelid. Some acinar cells of a simple acinar mucous gland have stained a pale blue (arrow). AB-nuclear fast red. x 250. Fig. 14. Dorsal eyelid. Unicellular mucous glands (ar-

rows) and a simple acinar mucous gland are stained a light pink. PAPS-hematoxylin. ~ 2 5 0 .

the secretory cycle of the gland (Dawson, '20). Dawson described the enlargement of the secretory cells of granular glands and the appearance of cytoplasmic granules which stained red or purple with Mallory's connective tissue stain. He reported the disappear- ance of the plasmalemmas of acinar cells with the resultant filling of the body of the gland with secretory material and described

the appearance of giant nuclei in some of the gland cells during formation of the granular secretion. Dawson also observed enlargement of secretory cells in mucous glands and reported that the nuclei in these enlarged cells were flattened and located basally. He noted that these cells contained a non-granular secretion that was stained blue by Mal- lory's stain or red-violet by thionin.


Fig. 17. Palpebral conjunctiva of dorsal eyelid. Closely- packed granules in the vacuoles of three unicellular mucous glands have stained dark brown. HID. ~400.

Palpebral conjunctiva of dorsal eyelid. Vacu- oles of the unicellular mucous glands have a reticulated, golden brown stain. PAPD. ~400.

Fig. 18.

Le Quang Trong (’67a,b, ’71, ’73, ’74) studied the skin of one urodele, the axolotl (A. tigrinurn), and a number of anurans (Xeno- pus tropicalis, Bufo regularis and other species of the families Bufonidae and Ranidae), using both histological and histochemical procedures. In the granular glands of the axolotl, he observed acinar cells with fine or coarse carminophilic granules and cells filled with granule-containing vacuoles. These granules were not stained by PAS, AB, or standard paraldehyde fuchsin methods. In the anurans, granules in early secretory stages were stained with PAS and AB or PAS alone in some of the species investigated. These staining reactions disappeared in more mature secretory cells. In our study, secretory granules were well preserved in the “granular” simpIe acinar serous glands only in the early stages of the secretory cycle. Some of these secretory granules in the main acinar cells were slightly PAS positive. In later stages, the secretory material had a vacuolated, globular appearance. Le Quang Trong (’67a) also observed cells in the collar regions of some serous acini of the axolotl that appeared to form small lateral, PAS- positive acini. These cells were thought to contribute a glycoconjugate component to the primarily protein secretion of these glands.

Fig. 19. Dorsal eyelid. A simple acinar mucous gland was filled with closely-packed, golden brown granules. Secretions of adjacent, simple acinar serous glands are unstained. PAPD. x 250.

Similar regions were also found in some of the simple acinar serous glands examined in our study, but only minimal staining with the PAS method was observed. In later stages of the secretory cycle, this PAS staining virtually disappeared. The faint PAS reaction sug- gests the possible presence of minute amounts of neutral glycoproteins in these glands, and the negative staining reactions with AB and HID reveal the absence of histochemically demonstrable acidic glycoconjugates in serous gland secretory cells. Because of the insignif- icant glycoconjugate content in the secretory cells of these glands, particularly in the late stages of the secretory cycle, we have chosen to refer to these glands as serous glands.

In all of Le Quang Trong’s studies, the granular glands gave positive reactions for proteins. Based on several histochemical tests, using PAS, AB, colloidal iron, ninhy- drin-Schiff, Biebrich scarlet, mercury orange with and without DTT reduction, DMAB- nitrite and Masson-Hamper1 argentaffine reactions, Navas et al. (’82) concluded that the secretion of the serous skin glands in the urodele, Pleurodeles waltlii, is a basic protein, rich in cysteine, with disulfide bonds and containing tryptophan and tyrosine. No glycoconjugates could be demonstrated in this species. In the different species studied, Le


Quang Trong observed a pattern of secretion for the granular glands similar to that described by Dawson ('20). A lumen was lack- ing in the acinus. Fine granules appeared first. These increased in size and finally be- came vacuolated. Plasma membranes then broke down forming a secretory syncytium. This material was then discharged through an apical canal in a holocrine manner. Our studies confirmed these morphological changes.

Although the chemical composition of the secretions appears to vary depending on the toxins present, the fine structure of the granular, serous glands is quite similar in both urodeles and anurans and correlates well with light microscopic observations. Electron microscopic studies on the urodele, P. waltlii, demonstrated an abundant granular endoplasmic reticulum (GER) and prominent Golgi complex, as well as electron-dense secretory granules in the immature secretory cell. As the cells matured, plasma membranes were lost so that a syncytium was formed, the number of secretory granules increased, and active cytoplasm was confined to the basal part of the cell (Navas et al., '82). Similar observations were made by Neuwirth et al. ('79) on several species of tropical, poison-dart frogs of the family Dendrobatidae and by Mills and Prum ('84) on three species of Rana. In the former, abundant agranular endoplasmic reticulum and free ribosomes were also found, evidence for both protein and steroid secretion. These electron microscopic observations were consistent with the appearance of the serous glands in the newt at the light microscopic level, as reported in this article.

The protein-secreting serous glands of amphibians are the source of a toxin which varies in potency among different species and is similar to tetrodotoxin of the Japanese puffer fish (Brodie, '68). The secretion of this toxin, and its role in protection from preda- tion, may be the main function of the serous glands.

Le Quang Trong ('67a) described two kinds of cells in the acinar mucous glands of the axolotl: (1) those with dense granules that were PAS-positive and AB-negative, and (2) those with both granules and a foamy cytoplasm displaying metachromasia with toluidine blue and staining with PAS and AB. On the basis of these staining reactions, the au- thor believed that the secretory product of these glands was composed of both neutral

and sulfated, acidic glycoconjugates secreted in a merocrine manner.

On the basis of positive staining of newt mucous glands with PAS and PAPD (periodic acid-para-diamine) methods, and negative results with Al3 in the critical electrolyte concentration method, azure A at a basic pH with the pH extinction procedure, and toluidine blue, Hoffman and Dent ('78) concluded that these mucous glands secreted only a neutral glycoconjugate. They also studied the mucous glands by electron microscopy. Elec- tron-dense, membrane-bound secretory granules filled the apical ends of the mucous, secretory cells and appeared to be released into the lumen by exocytosis. The nuclei were basally located and there was abundant GER. Elongated epithelial cells in the neck region connected the secretory cells with the duct which traversed the epidermis and was lined by a single, keratinized stoma cell.

Our histochemical results in the newt are in general agreement with those of Le Quang Trong in the axolotl, except that we found no evidence to suggest the presence of sulfated glycoconjugates in the simple acinar mucous glands of the newts we examined. The secretion appears to be composed mainly of neutral glycoconjugates, agreeing with Hoffman and Dent, but also with a minor component of acidic sialoglycoproteins which they did not observe. The simple acinar mucous glands are probably important in maintaining the normal slimy coat of the adult newt. This layer of mucus is believed to protect fish and aquatic amphibians from invasive micro- organisms.

Histochemical studies on the mucous glands of several anuran species (Le Quang Trong, '67b, '71, '73, '74; Dapson, '70) showed consistent staining with PAS, and variable AB and paraldehyde fuchsin staining. There- fore, the mucous secretion appears to vary between an acidic and a neutral mucous sub- stance in these species. When studied in plastic-embedded material, the mucous glands of anurans differed somewhat in morphology from those of urodeles. The acini contained a larger lumen lined by cuboidal to low columnar secretory cells. A myoepithelial cell sheath was seen in electron micrographs. By means of light and electron microscopy, Els and Henneberg ('90) described two kinds of secretory cells in the ventral skin glands of Rana fuscigula: (1) a principal cell containing secretory granules with an electron-dense core that stained with AB and PAS; and (2) a

176 R.W. REYER E T AL.

solitary cell with secretory granules of a uni- form, medium electron density that stained only with PAS. The authors concluded that the mucous secretion was a mixture of acidic glycoproteins, probably with occasional sul- fation, from the principal cells and neutral glycoproteins from the solitary cells. Similar secretory cells were found in the mucous glands of R. pipiens, R. temporaria, and R. catesbeiana (Mills and Prum, '84). Unlike the situation with urodeles, anuran mucous glands secrete upon adrenergic stimulation (Mills and Prum, '84).

In all of these investigations, there was a clear distinction between the granular, serous glands and the mucous glands. During the development of the skin in R. pipiens tadpoles, these two classes of glands were also distinct since the serous glands were larger and differentiated more rapidly (Bovb- jerg, '63). In anurans, it has been proposed that the mucous secretions function in protection, thermoregulation, osmoregulation, and water absorption. This mucus layer could act as a barrier to larger macromolecules, while allowing passage of smaller molecules such as electrolytes (Els and Henneberg, '90).

There has been disagreement concerning the sheath of smooth muscle fibers or myoepithelial cells surrounding gland acini in the skin of urodeles. Some investigators have described a contractile, muscular layer only in association with serous (granular) glands (Dawson, '20; Quay, '72), while others reported similar muscular sheaths in association with both serous and mucous glands (Le Quang Trong, '67a). With the electron microscope, Hoffman and Dent ('78) observed very thin myoepithelial cells with ellipsoidal nuclei and abundant microfilaments lying between the secretory cells and the basal lamina in the mucous gland of the newt. These cells could not be identified with light microscopy of paraffin sections. Without distinguish- ing between serous and mucous glands, Bani ('69) described the ultrastructure of the myoepithelial cells in the cutaneous glands of Triturus cristatus carnifex. These cells formed a continuous investment between the glandular acinar cells and their basal lamina. In the cytoplasm were many fine filaments, dense bodies, and micropinocytotic vesicles. Other organelles occupied polar caps at the ends of the nuclei. Some modified myoepithelial cells with dispersed mitochondria and agranular ER were also observed. Contrac- tion of the myoepithelial sheath of serous

glands appears to play an active role in the discharge of the lumenal contents, but the myoepithelial layer in mucous glands is probably non-functional. These glands secrete mucin continuously by exocytosis of the secretory granules in the merocrine manner (Hoffman and Dent, '77, '78). In our study, this myoepithelial sheath could be detected in some Masson's-stained, 10 km thick, paraffin sections and was distinct in 1 pm plastic sections. It was seen only around simple acinar serous glands. No observations were made with the electron microscope.

The precise function of the unicellular mucous glands, restricted to the palpebral and bulbar conjunctiva, is less clear than that of the skin glands. In our study, the conjunctival glands were shown to contain large amounts of neutral glycoconjugates, as indicated by positive PAS and PAPD reactions, as well as by reduced PAS staining of sections pretreated with phenylhydrazine in the PAPS method. Strong AB staining and some PAS staining of sections exposed to the PAPS method suggest the presence of sialic acid- containing glycoconjugates (presumably sialoglycoproteins). Occasional gland cells, positive for HID, indicate that a very small component of sulfated glycoconjugates may also be present. The unicellular mucous glands in newt conjunctiva correspond to unicellular mucous glands of the mammalian conjunctiva. In fact, the staining patterns of the newt glands with PAS, AB at pH 2.5 and AB-PAS methods are remarkably similar to those seen in conjunctival unicellular mucous glands of the Japanese monkey (Macaca fuscata), the rat, and man (Yamabayashi, '87; Yamabayashi and Tsukahara, '87; Kess- ing, '68). Srinivasan et al. ('77) also observed that the "goblet cells" of the rat and human conjunctiva stained with PAS, AB at pH 2.0, aldehyde fuchsin, and dialyzed iron methods. These reactions were reduced by neuramini- dase digestion. PAS-staining was reduced even more if this procedure were preceded by saponification. The authors concluded that the goblet cells secreted non-sulfated, acidic mucins containing sialic acid. Kawano et al. ('84, '88) studied goblet cells of the human conjunctiva, using fluorescent, FITC-labelled, sugar specific lectins to identify the sugar moieties of the glycoconjugates. The staining pattern indicated the presence of N-acetyl glucosamine (wheat germ agglutinin-positive), N-acetyl galactosamine (soybean agglutinin-positive, peanut agglutinin-positive),


galactose (Ricinus communis agglutinin-l- positive), and sialic acid (Limulus polyphe- mus agglutinin-positive).

The convoluted tubular seromucous glands in the lower eyelids are rich in neutral glycoprotein-containing secretory granules, as revealed by PAS and PAPD staining. His- tochemical staining methods for acidic glycoconjugates (PAPS, AB, and HID) did not stain secretory products in these glands. The convoluted tubular glands are referred to as seromucous because they appear to contain a much greater concentration of mucin than do the simple acinar serous glands. The secretions from the unicellular mucous glands and the convoluted tubular seromucous glands are discharged onto the surface of the palpebral conjunctiva and from there, spread over the cornea. This mucus probably serves the same protective function for the cornea as the secretion of the simple acinar mucous glands does for the skin. In man, Kessing ('68) proposed that this mucus had a lubricating, cleaning, and protective effect on the conjunctival mucosa.

Compared with mammalian eyelids (Fine and Yanoff, '79), those of the newt are quite simple. Both dorsal and ventral eyelids con- sist of a fold of skin and associated glands on the outside and the conjunctiva with its unicellular mucous glands on the inside. The conjunctival epithelium is stratified cuboidal and continues over the fornix into the bulbar conjunctiva, where it changes to stratified squamous at the beginning of the cornea. The numerous, convoluted tubular seromucous glands in the ventral eyelid are believed to represent both lacrimal and Harderian glands of mammals (Walls, '42). There are no sebaceous glands or sweat glands in newt eyelids.

Our studies not only confirm many of the results of previous investigators, but also clearly demonstrate differences in the secretory products of the glands of the newt eyelids, as shown by histochemical staining methods. We have also shown that the myoepithelial cell sheath is well developed and visible with light microscopy only around the simple acinar serous glands. The changing morphology of the simple acinar serous glands during their secretory cycle is well illustrated and correlated to differences in histochemical staining patterns.

ACKNOWLEDGMENTS

The authors wish to acknowledge the assis- tance of Dr. Nancy G . Dixon with the his-

tochemical procedures. Other technical assis- tance in the laboratory was provided by Dr. Min Ja Song, Ms. Dorothy Heritage, and Ms. Cynthia Phillips. The studies were supported by the WW Medical and Dental Corpora- tions, and by Biomedical Research grant RR05433 and grant EY00196 from the NIH.

Portions of this article are part of a thesis submitted by W.L. to the Faculty of West Virginia University in partial fulfillment of the requirements for the degree of Master of Science.

LITERATURE CITED

Bani, G. (1969) L'ultrastuttura delle cellule mioepiteliali delle ghiandole cutanee di Trzturus cristatus earnifex (Law.). Lo Sperimentale 119t229-253.

Bovbjerg, A.M. (1963) Development of the glands of the dermal plicae in Rana pzpzens. J. Morphol. 113t231- 243.

Brodie, E.D. Jr . (1968) Investigations on the skin toxin of the adult rough-skinned newt, Taricha granulosa. Copeia 1968:307-313.

Dapson, R.W. (1970) Histochemistry of mucus in the skin of the frog, Ranapzpiens. Anat. Rec. 166:615-626.

Dawson, A.B. (1920) The integument of Necturus maculosus. J . Morphol. 34:487-589.

Dent, J.N. (1975) Integumentary effects of prolactin in the lower vertebrates. Am. Zool. 15t923-935.

Dent, J.N., L.A. Eng, and M.S. Forbes (1973) Relations of prolactin and thyroid hormone to molting, skin tex- ture, and cutaneous secretion in the red-spotted newt.

Drury, R.A.B., and E.A. Wallington (1967) Carleton's Histological Technique, 4th Edition. New York: Oxford University Press, pp. 127.

Els, W.J. and R. Henneberg (1990) Histological features and histochemistry of the mucous glands in ventral skin of the frog (Rana fusczgula). Histol. Histopath. 5:343-348.

Fine, B.S., and M. Yanoff (1979) Ocular Histology: A Text and Atlas, 2nd Edition. Hagerstown, Maryland: Harper and Row, pp. 289-317.

Gill, G.W., J.K. Frost, and K.A. Miller (1974) A new formula for a half-oxidized hematoxvlin solution that

J . EXP. ZOO^. 184t369-382.

neither overstains nor requires differentiation. Acta Cytol. 18:300-311.

Himes, M., and L. Moriber (1956) A triple stain for deoxyribonucleic acid, polysaccharides, and proteins. Stain Technol. 3137-70.

Hoffman, C.W., and J.N. Dent (1977) Effects of neurotransmitters upon the discharge of secretory product from the cutaneous glands of the red-spotted newt. J .

Hoffman, C.W., and J.N. Dent (1978) The morphology of the mucous gland and its responses to prolactin in the skin of the red-spotted newt. J. Morphol. 157t79-87.

Karnovsky, M.J. (1965) A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J . Cell Biol. 27:137A-l38A.

Kawano, K., F. Uehara, M. Sameshima, and N. Ohba (1984) Application of lectins for detection of goblet cell carbohydrates of the human conjunctiva. Exp. Eye Res. 38t439-447.

Kawano, K., F. Uehara, and N. Ohba (1988) Lectin- cytochemical study on epithelial mucous glycoprotein of conjunctiva and pterygium. Exp. Eye Res. 47:43-51.

EXP. ZOO^. 202:155-161.


Kessing, S.V. (1968) Mucous gland system of the conjunctiva. A quantitative normal anatomical study. Acta Ophthalmol. 95(Suppl.): 1-133.

Le Quang Trong, N.Y. (1967a) Histogenese et his- tochimie des glandes cutanees de I’axolotl (Ambystoma tigrinum Green). Arch. Zool. Exp. GQn. (Paris) 108:49- 75.

Le Quang Trong, Y. (1967b) Structure et developpement de la peau et des glandes cutanees de Nectophrynoides occidentalis Angel. Arch. Zool. Exp. GQn. (Paris) 108: 589-61 1.

Le Quang Trong, Y. (1971) Etude de la peau et des glandes cutanees de quelques Amphibiens du genre Phrynobatrachus. Bull. I.F.A.N. SQr. A. 33t987-1025.

Le Quang Trong, Y. (1973) Structure et developpement de la peau et des glandes cutanQes de Bufo regularis Reuss. Bull. SOC. 2001. (France) 98t449-485.

Le Quang Trong, Y. (1974) Etude de la peau et des glandes cutanees de Xenopus tropicalis Gray. Bull. I.F.A.N. Ser.A. 36t407-427.

Lillie, R.D., and H.M. Fullmer (1976) Histopathologic Technic and Practical Histochemistry, 4th Edition. New York: McGraw Hill, pp. 629-630.

Luna. L.G. (1968) Manual of Histoloeic Staining Meth- ods’of the Armed Forces Institute of?athology,-3rd Ed. New York: McGraw-Hill, pp. 94-95.

Mills, J.W., and B.E. Prum (1984) Morphology of the exocrine glands of the frog skin. Am. J. Anat. 1 7 1 9 - 106.

Mowry, R.W. (1963) The special value of methods that color both acidic and vicinal hydroxyl groups in the histochemical study of mucins. With revised directions for the colloidal iron stain, the use of alcian blue G8X, and their combinations with the periodic acid-Schiff reaction. Ann. N.Y. Acad. Sci. 106t402-423.

Navas, P., C. Bueno, J. Hidalgo, J . Aijon, and J.L. Lopez- Campos (1982) Secretion and secretory cycle of tegu- mentary serous glands in PZeurodeles waltlii Mich. Basic Appl. Histochem. 26t7-15.

Neuwirth. M.. J.W. Dalv. C.W. Mvers. and L.W. Tice

skin of poison-dart frogs (Dendrobatidae). Tissue Cell llt755-771.

Noble, G.A., and E.R. Noble (1944) On the histology of frog skin glands. Trans. h e r . Microsc. SOC. 63t254- 263.

Noble, G.K. (1931) The Biology of the Amphibia. New York: McGraw-Hill.

Pearse, A.G.E. (1968) Histochemistry. Theoretical and Applied, Vol. 1, 3rd Edition. Boston: Little, Brown and Co., p. 707.

Quay, W.B. (1972) Integument and the environment: Glandular composition, function, and evolution. Am. Zool. 1295-108.

Reid, P.E.. W.L. Dunn. C.W. Ramev. E. Coret, L. True- man, and M.G. Clay (1984) Histbchemical studies of the mechanism of the periodic acid-phenylhydrazine- Schiff (PAPS) procedure. Histochem. J. 16641-649.

Spicer, S.S. (1965) Diamine methods for differentiating mucosubstances histochemically. J . Histochem. Cy- tochem. 13t211-234.

Spicer, S.S., R.G. Horn, andT.J. Leppi (1967) Histochem- istry of connective tissue mucopolysaccharides. In B.M. Wagner and D.E. Smith (eds). The Connective Tissue. Baltimore: Williams and Wilkins Co., pp. 251-303.

Srinivasan, B.D., B.V. Worgul, T. Iwamoto, and G.R. Merriam, Jr . (1977) The conjunctival epithelium. 11. Histochemical and ultrastructural studies on human and rat conjunctiva. Ophthalmic. Res. 9:65-79.

Walls, G.L. (1942) The Vertebrate Eye and its Adaptive Radiation. Bulletin 19, Cranbrook Institute of Science. Bloomfield Hills, Michigan: The Cranbrook Press.

Williams, G., and D.S. Jackson (1956) Two organic fixatives for acid rnucopolysaccharides. Stain Technol. 31: 189-191.

Yamahayashi, S. (1987) Periodic acid-Schiff-Alcian blue: A method for the differential staining of glycoproteins. Histochem. J. 19t565-571.

Yamabayashi, S., and S. Tsukahara (1987) Histochemical studies on the coniunctival goblet cells. I. (Alcian-blue) AE-(periodic acidYSchiff) P’ks staining and PAS-AB staining. Ophthalmic. Res. 19t137-140. (1979) Morphology of the granulir secretory glands in

morphology and glycoconjugate histochemistry of the palpebral glands of the adult newt,...

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