histochemical and electron microscopic observations on the salt secreting lacrymal glands of marine...

Histochemical and Electron Microscopic Observations on the Salt Secreting Lacrymal Glands of Marine Turtles

JOHN H. ABEL, JR. AND RICHARD A. ELLIS Department of Biology, Brown University, Providence, Rhode Island, and M t . Desert Islaiid Biological Laboratory, Salisbury Cove, Maine

ABSTRACT The lobular, compound, branched, tubular, salt-secreting lacrymal glands of two marine turtles, Cheloiiia mydas and Caretta caretta are similar in structure and in histochemical reactivity. Blood from the centrolobular arteries flows through a rich capillary bed counter to the flow of tubule secretion. The capillary endo- thelium is reactive for adrnosine triphosphatase ( ATPase). Nerves containing cholinesterase pervade the connective tissue. At the blind ends of the secretory tubules small basophilic peripheral cells contain an abundance of glycogen, monoamine oxidase (MAO) and phosphorylase but little succinic dehydrogenase (SDH) or cytochrome oxidase (CTO). Non-mitochondria1 ATPase is concentrated at the luminal interface of these cells. The larger principal cells, lining the major portion of the secretory tubules, are rich in SDH and CTO but contain relatively little glycogen, MA0 or phosphorylase. Broad intercellular channels reactive for mucopolysaccharide are formed by intermeshing, pleomorphic microvilli that fringe the extensive lateral surfaces of the principal cells. The cytoplasm of these cells contains profiles of smooth- surfaced endoplasmic reticulum ( SSER), abundant mitochondria, and prominent Golgi membranes. Profiles of SSER and small membrane bound vesicles fill the apical cytoplasm but mitochondria are lacking. The luminal secretory border of the cell is extremely limited in area.

Two types of epithelial cells line the duct system: basal cells that react strongly for non-specific esterase and MAO; and goblet cells containing mucopolysaccharide, acid phosphatase, cholinesterase, and ATPase.

The principal cells, close to the arterial blood supply, contain the highest con- centrations of oxidative enzymes and have special modifications of the cell surface consistent with their role in salt concentration and secretion.

Osmoregulation in marine reptiles is aided by head glands that have an extra- renal function in the elimination of salt (Schmidt-Nielsen and Fange, '58). In the loggerhead turtle (Caretta caretta) and the Atlantic green turtle (Chelonia mydas) a salt solution hypertonic to blood is se- creted by modified lacrymal glands that lie against the posterior wall of the orbit (Peters, 1892; Schmidt-Nielsen and Fange '58). Each gland consists of approximately 100 lobules that are filled with myriad, closely packed secretory tubules.

Histologically, the salt glands of the sea turtles resemble in their organization other glands that are modified for osmoregulation in different marine vertebrates. The nasal glands of marine birds (Marples, '32; F h g e et al., '58a) and the rectal glands of marine elasmobranchs (Fange and Fugelli, '63; Bulger, '63) are both compound, branched tubular structures.

AM. J. ANAT., 118: 337-358.

Many of the morphological similarities among these salt-secreting glands are lost, however, when they are examined at the cellular level. For example, the principal secretory cells in the salt glands of marine turtles (Ellis and Abel, '64) have specializations of the cell surface that are entirely different in form from the secretory cells in the nasal glands of marine birds (Doyle, '60; Komnick, '63c) or in the rectal glands of elasmobranchs (Bulger, '63; Doyle, '62a, b ) . These are, however, merely vari- ations in form, since in each instance the same effect is achieved; that is, the area of the absorptive surfaces of the cell is greatly expanded.

In this study the unusual features of the turtle salt gland as well as the char- acteristics that it shares with the salt-secreting glands of other marine vertebrates are explored by histochemical and electron microscopic techniques. The sites of

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338 JOHN H. ABEL, JR. AND RICHARD A . ELLIS

specific enzymatic activity are identified within the tissues of the gland and the fine structure of the principal secretory cells is delineated. This data is correlated, wherever possible, with biochemical and cytological observations on other salt glands and with the general problem of salt secretion.

MATERIALS AND METHODS

Ten vigorous young Atlantic green turtles (Chelonia m y d a s ) raised in aquaria at the marine laboratory of Duke Univer- sity and two loggerhead turtles (Caret ta cmet ta ) captured fresh locally were used for these studies. After the animals were drugged with chloral hydrate or with nem- butal, the salt glands were removed intact from the posterior wall of the orbit and weighed. Single lobules were dissected from the salt gland and rapidly frozen in dry ice, sectioned at 7-60 u in an Interna- tional cryostat and used to demonstrate monoamine oxidase (Glenner et al., '57), phosphorylase (Takeuchi and Kuriaki, '55; Ellis and Montagna, '58), cytochronie oxidase (Burstone, '59), and succinic dehydrogenase (Nachlas et al., ' 5 7 ) activity. Other segments of the glands were appro- priately fixed in 10% neutral formalin, formal calcium or chloral formalin sectioned on the cryostat and tested for alkaline phosphatase (Gomori, '52; Pearse, '61), acid phosphatase (Gomori, '50; Burstone, 'SS), a naphthyl esterase (Nachlas and Seligman, '49), non-mitochondria1 ATPase (Wachstein and Meisel, '57), and cholinesterase (Koelle and Friedenwald, '49). Sudan black B staining was used to demonstrate lipids and phospholipids ( Gomori, '52); alcian blue was used to demonstrate acid mucopolysaccharides (Pearse, '61 ).

Still other portions of the glands were fixed in Zenkers, Bouins, or Hellys' fluids for histological study. The following techniques were applied to 2-6 u paraffin sections: toluidine blue at pH 2.5, 3 .8 , 4.5, 5.5 and 7 (Montagna et al., '51), toluidine blue for permanent metachromasia (Hess and Hollander, '47), toluidine blue following sulfation (Mowry, ' 58 ) , toluidine blue following hyaluronidase digestion (Gersh and Catchpole, '48), PAS (McManus, '48) with saliva, diastase and/or hyaluronidase digestions as controls, hematoxylin and

eosin, and Mallory's triple connective tissue stain (Holde and Isler, '58; Baker, '58). Mitochondria were demonstrated by the Altman technique following fixation in Regaud's fluid. The glands of two turtles were perfused by the method of Williams ('48) to demonstrate blood vessels.

Tissue for electron microscopy was fixed in phosphate-buffered 2% osmium tetrox- ide (Millonig, '61) and dehydrated with acetone; the fixed tissue was embedded in Epon (Luft, '61), sectioned and stained either with lead hydroxide (Karnovsky , '61), or lead citrate (Reynolds, '63). The sections were examined in an RCA-EMU- 3F electron microscope equipped with a double condenser and a 40 u platinum objective aperture. Electron micrographs were taken at initial magnifications 4,100 to 12,000 and enlarged photographically as desired. One micron thick sections of epon embedded tissues were mounted on glass slides and stained with toluidine blue.

RESULTS AND OBSERVATIONS

Gross morphology The two species of marine turtles that

were examined have salt glands which are essentially similar in all histological de- tails. These paired lacrymal glands located within the orbits, posterior and dorsal to the eye, constitute between 0.05 to 0.1% of the total body weight. In lateral view they are roughly pyrimidal in form, with the bulges of individual secretory lobules clearly visible at the surface. Dense sheaths of interlobular connective tissue separate the lobules. Internally each of the 100 or more secretory lobules consists of myriad tightly-packed branched tubules that radiate from a central excretory canal (fig. 1). The central canals coalesce into one large tortuously infolded duct which opens externally to the posterior canthus of the eye. Histologically, therefore, the modified lacrymal glands of marine turtles may be classified as compound, branched tubular glands.

Broad sheaths of intralobular connective tissue surround the central canals while only a thin sleeve of peritubular con-

* Green turtles were secured through the courtesy of Dr. Klaus Fisher; Duke University.

T H E SALT GLANDS OF M A R I N E TURTLES 339

nective tissue encloses each tubule. Two types of nerve fibers course through the connective tissue of the glands: one gives a strong positive reaction for cholinesterase; the other is reactive for monoamine oxidase. The large fibers that react for cholinesterase in the inter- and intralobular connective tissue send branches that form a rich plexus around each tubule (fig. 2 ) . Nerves reactive for monoamine oxidase form a diffuse perilobular net- work.

Many large arteries revealed by staining and by vascular perfusions lie embedded in the interlobular connective tissue. Smaller arteries branch from the larger ones, pene- trate the lobules through broad sheaths of intralobular connective tissue and send 8- 16 small arterioles along the central canals. Radiating from these arterioles towards the periphery of the lobule are numerous extensions that branch and rebranch to form a rich capillary bed paralleling the branched secretory tubules (fig. 1 ). Many circumtubular and intertubular connectives further augment this system so that at any one point each tubule is associated with 4-6 capillaries. At the periphery of the lobules the capillaries enter venous sinuses that fuse together and drain into larger interlobular veins. Some venous elements are present within the intralobular connective tissue and appear to drain the vessels supplying the central canal.

The secretory tubules The blind terminal segments of secre-

tory tubules lie just beneath the interlobular connective tissue and run centripetally towards the center of each lobule. In the loggerhead turtle the tubules average 1 mm in length and generally bifurcate two or three times within this distance. Near the blind end of each tubule the central lumen is extremely small; centripetally it increases slightly in size and reaches its largest diameter when the tubule joins with the central canal. The h e bore of the lumen indicates that the single layer of truncated epithelial cells have a very limited secretory surface.

Three distinct cell types may be identified among the cells lining the tubules. At the blind ends of the tubule are small terminal cells with scant cytoplasm and a

high affinity for basic dyes. These com- prise approximately a tenth of the cells lining the secretory tubules in the imma- ture green turtles. Most of the remainder of each tubule is paved with principal secretory cells. These are small near the periphery of the lobule but they gradually increase in size as they approach the central canals. Broad intercellular channels are apparent along the lateral surfaces of adjacent secretory cells, except at their apex where they are joined by junctional complexes (fig. 15). Cross-sections of the secretory cells reveal large folds projecting from the main cell body that interlock with similar processes of adjacent cells. The cell boundaries thus formed are extremely irregular and fit together like the pieces of a jigsaw puzzle (fig. 4 ) . The broad bases of the secretory cells rest upon a well-developed basement membrane that separates them from the adjacent peritubular connective tissue. No myoepithelial elements have been detected with either the light or electron microscope.

The principal and peripheral cells contain a large central nucleus with a single nucleolus, bits of heterochromatin and a homogeneous karyoplasm. There are no obvious differences in nuclear structures between the principal and peripheral cells.

The third cell type of the tubules is mor- phologically unrelated to the secretory cells. This small oval cell lies against the basement membrane of the tubule, its apical surface never extends more than one- quarter the distance to the lumen, (fig. 17) and the bases of adjoining secretory cells surround it on all sides. The highest concentration of these cells is found in portions of tubules nearest the central canal while none occurs at the blind ends.

Tubule c ytochemistry There are striking cytochemical differ-

ences between the peripheral cells and the principal secretory cells. The peripheral cells are sudanophobic; they have few mitochondria and little succinic dehydrogenase and cytochrome oxidase activity (figs. 5, 6) . The centripetal principal cells, however, are sudanophilic (fig. 7), have large numbers of mitochondria and exhibit a large amount of oxidative enzyme activity (figs. 5, 6) . Conversely, the pe-

340 JOHN H. ABEL, JR. AND RICHARD A. ELLIS

ripheral cells contain more glycogen and react more intensely for phosphorylase and monoamine oxidase than do the principal cells. Occasionally small basophilic clumps are evident in the peripheral cells.

Different phosphatases are extremely abundant within these glands but are sepa- rately and discretely localized to specific areas. Acid phosphatase activity is strong- est nearest the central canal and appears as a sharply localized band near the cell membrane at the basal surface of the principal secretory cells (fig. 8). Alkaline phosphatase is restricted to the connective tissue (fig. 9) and blood vessel endothe- lium. Non-mitochondria1 ATPase is sharply localized as a dense line on or near the plasma membrane at the apical cell surfaces of the peripheral cells (fig. 10). ATPase activity is also evident within the connective tissue, the endothelial cells lining the vascular system and as a thin perinuclear ring in each of the tubule cells.

The broad intercegllular channels between adjacent tubule cells contain abundant mucopolysaccharides. They stain a bright blue green with alcian blue and an intense pink after the PAS reaction even when the sections are treated with diastase, saliva or hyaluronidase (fig. 11). A red y metachromasia is evident following sulfation with toluidine blue at pH 5 or after the technique of Hess and Hollander (fig. 4 ) . The lateral borders of the peripheral cells are not as strongly metachromatic as the borders of the principal cells. The basement membrane is PAS positive but does not stain metachromatically (fig. 11). Using these same techniques it is also possible to make distinctions between the ground substance of the connective tissue and the intercellular mucopolysaccharides since the sugary constituents associated with the connective tissue at best exhibit only a purple (8) metachromasia and are faintly PAS positive. A negative image of the intercellular channels is apparent when the tissues are stained with acid dyes. The intercellular channels are not readily demonstrated by hematoxylin and eosin staining.

The small “basal” cells are differentiated cytochemically from the remainder of their tubule cells by the positive reaction of their cytoplasm for a naphthyl esterase

(fig. 3 ) . The secretory cells have no esterase activity. In addition the “basal” cells contain few mitochondria, are low in oxidative enzymes and have an acidophilic cytoplasm.

Cytology of principal secretory cells Electron micrographs show that the

channels between opposed secretory cells are packed with long pleomorphic microvilli that extend from and fringe the cell margins (fig. 13). In some regions the microvilli are truly digitiform while in other areas they appear as flat folds. The microvilli of adjacent secretory cells intermesh loosely with one another and are linked by occasional desmosomes. Irregu- larly arranged clear spaces appear between them forming tortuous intercellular channels that average 1.5 in width (fig. 14). At the base of each cell the microvilli are sparse and may be flattened against the basement membrane (fig. 16). No fibrous elements or other cytoplasmic organelles extend outward into the microvilli.

Scant irreguIar short microvilli extend outward from the apical cell surface into the lumen of the tubule (fig. 15). No open or direct connection was observed between the intercellular channels and the lumen of the secretory tubules. With- out exception the lateral and luminal surfaces of the cell are separated by a con- tinuous system of prominent terminal bars and intermittent desmosomes that resemble the junctional complexeS found in other epithelia (fig. 15).

A large number of small irregularly shaped profiles of endoplasmic reticulum, predominantly of the smooth surfaced form are distributed evenly throughout the central and basal cytoplasm (fig. 13). Smooth surfaced vesicles are sometimes evident near the lateral margins of the cells immediately subjacent to the micro- villous borders (fig. 14), and they are al- ways present in large numbers in the cell apex (fig. 15). In addition vesicles are often associated with the stacked cisternae of the Golgi apparatus that usually assumes a supranuclear position.

Abundant mitochondria are distributed evenly throughout the basal cytoplasm. None of these organelles are located in the villous processes fringing the cells or

.

THE SALT GLANDS OF MARINE TURTLES 341

in the apical cytoplasm bordering the tubular lumen. The mitochondria take a va- riety of forms but most are seen as long or short rodlets. Numerous tightly-packed cristae are oriented more or less parallel to the short axis of the mitochondria, and a variable number of large dense granules are suspended within the matrix. The in- tramitochondrial granules are nearly spher- ical in outline and although some are larger they average 200-300 A in diameter.

Glycogen granules are very abundant within the basal cytoplasm of the tubule cells. Clumps of glycogen are frequently sequestered within alternated, looped mitochondria.

The small basal cells, that lie flat against the basement membrane of the tubule, are covered by the microvilli of overlying secretory cells. Unlike the secretory cells. the basal cells have a nearly smooth plasma membrane. There are only a few mitochondria, but numerous smooth-surfaced vesicles and some glycogen granules are present in the cytoplasm. The nucleus is prominent, comprising nearly one-half of the cell volume. Small fila- ments, tentatively identified as tonofila- ments, may be clumped in twisted masses or scattered diffusely through the cytoplasm; they are a distinguishing charac- teristic of the basal cells (fig. 16).

The central canals Two distinct cell types form the strati-

fied mucous epithelium that lines the central canals. Immediately subjacent to the surrounding capsule of intralobular connective tissue are several layers of small cells with scant cytoplasm and large nuclei. Along the epithelial surface lining the lumen of the canal itself is a single row of large closely packed, goblet cells, with basal nuclei (figs. 7, 8). The goblet cells appear to be in various stages of mucus synthesis and release since there are many transitional, intermediate-sized ones lining the canal. Those that have dis- charged their contents into the lumen show only a vacuolated membrane-bound sac projecting outward into the lumen.

Central canal cytochemistry Central canal cells may be distinguished

readily from the tubule cells since their

cytoplasm contains extremely high con- centrations of a naphthyl esterase (fig. 12) and a few mitochondria (figs. 5-7). The large mucus cells differ from the small underlying epithelial cells in giving intense reactions for cholinesterase (fig. 2), acid phosphatase (fig. 8) and ATPase in their apical caps. The mucous secretion of the goblet cells is highly PAS positive and stains metachromatically with toluidine blue above pH 3. These staining reactions are unaffected by previous digestion of the tissue section with saliva, diastase or hyaluronidase.

DISCUSSION

The salt-secreting lacrymal glands of marine turtles are comprised of a heteroge- neous population of cells. Along the length of the branched secretory tubules, from the center to the periphery of each lobule, the cells vary progressively in size, shape and chemical reactivity. At the blind ends of the secretory tubules the terminal cells are small in size; they contain glycogen, monoamine oxidase and phosphorylase in high concentration but only low levels of succinic dehydrogenase and cytochrome oxidase. The apical surfaces of these cells are reactive for adenosine triphosphatase. Centripetally there is a gradual transition between the terminal cells and the principal secretory cells, and the principal secretory cells themselves also change progressively in size and increase in histochemical reactivity toward the center of the lobule. The principal cells contain lipids, succinic dehydrogenase and cytochrome oxidase in high concentration but little glycogen, monoamine oxidase or phosphorylase. The intercellular channels between the principal cells become progressively more conspicuous toward the center of the lobule; and the mitochondria within the cells become more numerous. Small basal cells that are found among the principal secretory cells in the turtle salt gland have not been reported in the salt glands of birds (Komnick, '63a, b, c ) and seem to be unique in the turtle. Where the secretory tubules join the central canal, the strikingly different cells of the central canal are admixed with the principal cells. The central canal cells contain high con- centrations of non-specific esterase and

342 JOHN H . ABEL, JR. A N D RICHARD A. ELLIS

monoamine oxidase but they have low levels of succinic dehydrogenase and cytochrome oxidase. Small peripheral and large principal secretory cells have also been reported in the salt gland of the duck (Scothorne, '58, '59; Ellis et al., '63) in the herring gull (Fiinge et al., '58) and in the rectal gland of the dogfish (Bulger, ' 6 3 ) . There is, therefore, overwhelming evidence that the epithelium of the salt- secreting glands in birds, reptiles and fishes cannot be considered as a homogeneous cell population.

The salt-secreting nasal glands of marine birds, the rectal glands of elasmobranchs and the lacrymal glands of marine turtles have many common cytochemical and cytological features. All are lobulated, branched, tubular glands. In all these species the secretory segments of the glands are composed of two different cell types, small terminal cells that probably have a generative function (Ellis et al., '63) and larger principal cells that are active in salt secretion (Doyle, '60; Kom- nick, '63c). The principal cells have abundant mitochondria, high levels of succinic dehydrogenase, and cytochrome oxidase; they stain deeply for phospholipid, and have a surface coat of mucopolysaccharide. Acid phosphatase is usually present at the base of the principal cells. Both the basal and lateral absorptive surfaces of the principal cells are extensively folded to increase their surface area while the apical secretory surface of the principal cells is very small. The principal cells usually contain little glycogen, phosphorylase or monoamine oxidase. All of these factors are probably relevant to the salt-secreting function of the glands but three seem par- ticularly sienificant : ( 1 ) the absorptive surface of the principal cells is vast rela- tive to the restricted secretory surface, (2 ) a mucopolysaccharide is associated with the absorptive surfaces of the principal cells, and (3) the principal cells are packed with mitochondria rich in the oxidative enzymes that aid in the synthesis of ATP.

The surface area of the principal cells that is exposed to the extracellular fluid is extensive; in constrast, the cell surface that borders the lumen is extremely narrow (fig. 16). By a conservative approxi- mation, the area of the absorptive surface

of these cells exceeds the area of the secretory surface by a factor greater than 1000 : 1 . The intercellular channels that run from the perimeter of the tubule to the junctional complexes bordering the lumen allow extracellular fluid to bathe both the lateral and basal surfaces. These surfaces are expanded by the irregular outlines of the principal cells and by the fringe of microvilli that border the intercellular channels. The pleomorphism of the microvilli suggests that this surface of the cell is active in pinocytosis and there is evidence of pinocytotic vesicles in the adjacent cytoplasm. In the rectal gland of elasmobranchs the salt-secreting cells also have highly folded basal surfaces. By a different modification of the cell surface the principal cells in the salt glands of marine birds achieve the same goal and Fawcett ('62) cites this cell type as an ultimate example of a cell with an extensive basal absorptive surface. The significance of these observations in salt secretion is not entirely clear, but two possibilities present themselves : ( 1 ) the expanded surface area provides for rapid exchange of metabolites, permitting the cells to sustain high levels of activity, ( 2 ) the expanded surface area may permit the more efficient trapping of ions.

Cytochemical techniques reveal that the broad intercellular channels between the lateral margins of the principal cells in the turtle salt gland are intensely PAS positive and exhibit a variable degree of metachromasia, indicating that a nega- tively charged carbohydrate, probably a mucopolysaccharide or glycoprotein, re- sides between the secretory cells. Muco- polysaccharides have been reported between the secretory cells in the rectal gland of the dogfish (Bulger, '63), and within the principal cells of the nasal glands in ducks (Ellis et al., '63). Many cells have a surface coat of mucopolysaccharide, the "glycocalyx" and Bennett ('63) has pro- posed that the anionic charge of the glycocalyx could produce an effective means of attracting, trapping and concentrating cations. In fact some mucopolysaccharides do have the capacity for binding cations and can function as ion exchange resins (Farber, '60). The abundant mucopolysaccharides associated with the absorptive

THE SALT GLANDS OF MARINE TURTLES 343

surfaces of salt secreting cells in the lacry- ma1 gland of turtles, in the nasal glands of birds and in the rectal glands of elasmobranchs supports the hypothesis that the glycocalyx is linked with electrolyte trans- port.

Mitochondria are packed within the cytoplasm of the secretory cells in the turtle salt glands. Abundant mitochondria are also conspicuous within the secretory cells of the avian salt gland (Doyle, '60; Faw- cett, '62; Komnick, '63c) and elasmobranchs rectal gland (Bulger, '63). They are concentrated at the absorptive surfaces of these cells. Strong reactions for both cytochrome oxidase and succinic dehydrogenase occur in the principal cells; suggesting a high level of oxidative phos- phorylation. The large numbers of mitochondria, their production of high energy compounds, and their close association with the plasma membranes of the absorptive surfaces suggest a primary role in salt secretion.

The peculiar vascular architecture of the salt gland of the turtle probably con- tributes to its capacity to concentrate and secrete electrolytes. Arteries in the central lobular connective tissue ramify into a capillary plexus paralleling the secretory tubules, and the capillaries eventually empty into venous sinuses at the periphery of each lobule. In the salt-glands of marine birds (Fange et al., '58) the blood flow is counter to the pathway of salt secretion, and the arterial blood circulates first through the mid-portion of each lobule. Schmidt-Nielsen ('61 ) has shown that such a countercurrent flow cannot function in ion concentration like the countercurrent multiplier system found in the mammalian kidney. The circulatory pattern in the turtle salt gland, however, does expose the most metabolically active secretory cells (i.e., those near the center of the lobule) to fresh arterial blood. A similar pattern is repeated in the non-homologous salt-secreting nasal glands of marine birds. This system may be adaptive for optimal salt secretion.

From the histochemical evidence pre- sented, the lacrymal salt-secreting glands of marine turtles appear to have a double innervation. The nerves positive for cholinesterase are probably cholinergic fibers

of the parasympathetic nervous system, and the nerves reactive for monoamine oxidase are probably adrenergic fibers of the sympathetic nervous system. The cholinergic fibers ramify through the peritubular connective tissue but the adrenergic fibers seem to be restricted primarily to the perilobular connective tissue. This suggests that the cholinergic secretory fibers may affect the interlobular arteries. Metacholine evokes secretion in the turtle salt gland ( Schmidt-Nielsen and F h g e , '58), and in the avian salt gland epineph- rine blocks secretion ( F h g e et al., '58b). Indications of the effect of acetylcholine on the secretory cells may also be drawn from the literature. Borut and Schmidt- Nielsen ('63) showed that metacholine causes an increase in the rate of respiration in avian salt gland tissue slices, but NaCl in increased concentration inhibits respiration. Salt secretion then is medi- ated at least partially by the nervous system and is not a direct effect of an osmotic load on the secretory epithelium.

LITERATURE CITED Baker, J. R. 1958 Principles of Biological Tech-

nique. Methuen and Co., Ltd., London. Bennett, H. S. 1963 Morphological aspects of ex-

tracellular polysaccharides. J. Histochem. Cyto- chem., 11: 14-23.

Borut, A., and K. Schmidt-Nielsen 1963 Res- piration of avian salt-secreting gland in tissue slice experiments. Am. J. Physiol., 204: 573- 581.

Bulger, R. E. 1963 Fine structure of the rectal (salt-secreting) gland of spiny dogfish, Squalus acanthias. Anat. Rec., 147: 95-127.

Burstone, M. S. 1958 Histochemical compari- son of naphthol AS phosphates for the demonstration of phosphatases. J. Natl. Cancer Inst.,

_- 1959 New histochemical techniques for the demonstration of tissue oxidase (cytochrome oxidase). J. Histochem. Cytochem., 7: 112-122,

Doyle, W. L. 1960 The principal cells of the salt-gland of marine birds. Exptl. Cell Res., 21: 386-393.

-- 1962a Secretory cells of the rectal salt- gland of an elasmobranch, Urolophus. Anat. Rec., 142: 228 (abstr.).

196213 Tubule cells of the rectal salt- gland of Urolophus. Am. J. Anat., 111: 223- 238.

Ellis, R. A., and J. H. Abel 1964 Intercellular channels in the salt secreting glands of marine turtles. Science, 144: 1340-1342.

Ellis, R. A., C. C. Goertemiller, Jr., R. A. DeLellis and Y. H. Kablotsky 1963 The effect of a salt water regimen on the development of the

20: 601-616.

344 JOHN H. ABEL, JR. AND RICHARD A. ELLIS

salt glands of domestic ducklings. Dev. Biol., 8: 286.

Ellis, R. A., and W. Montagna 1958 Histology and cytochemistry of human skin XV. Sites of phosphorylase and amylo-1 , 6-glucosidase activity. J. Histochem. Cytochem., 6: 201-207.

Fange, R., and K. Fugelli 1963 The rectal salt gland of elasmobranchs and osmoregulation in Chimaeroid Fishes. Sarcia, 10: 27-34.

Fange, R., K. Schmidt-Nielsen and H. Osaki 1958a The salt gland of the herring gull. Biol. Bull., 115: 161-171.

Fange, R., K. Schmidt-Nielsen and M. Robinson 195813 The control of secretion from the avian salt gland. Am. J. Physiol., 195: 321-326.

Farber, S. J. 1950 Mucopolysaccharides and sodium metabolism. Circulation, 22: 941-953.

Fawcett, D. W. 1962 Physiologically significant specializations of the cell surface. Circulation, 26: 1105-1126.

Gersh, I., and H. R. Catchpole 1948 The organization of ground substance and basement membrane and its significance in tissue injury, disease and growth. Am. J. Anat., 85: 457- 523.

Glenner, G. G., H. G. Burtner and G. W. Brown, Jr. 1957 The histochemical demonstration of monoamine oxidase activity by tetrazolium salts. J. Histochem. Cytochem., 5: 591-600.

Gomori, G. 1950 A n improved histochemical technique for acid phosphatase. Stain Tech., 25: 81-86. - 1952 Microscopic Histochemistry: Prin-

ciples and Practice. Univ. of Chicago Press, Chicago, Ill.

Hess, M., and F. Hollander 1947 Permanent metachromatic staining of mucus in tissue sections and smears. J. Lab. Clin. Med., 32: 905.

Holde, P., and H. Isler 1958 The effect of phosphomolybdic acid on the stainability of connective tissue by various dyes. J. Histochem. and Cytochem., 6: 265-270.

Karnovsky, M. J. 1961 Simple methods for “staining with lead” at high pH in electron microscopy. J. B. B. C., 11: 729.

Koelle, G. B., and J. S. Friedenwald 1949 A histochemical method for localizing cholinesterase activity. Proc. SOC. Exp. Biol. Med., 70: 617-622.

Komnick, H. 1963a Elecktronenmickroskopische Untersuchungen zur funktionellen Morphologie des Ionentransportes in der Salzdruse von Lams argentatus (I . Bau und Feinstruktur der Salz- druse). Protoplasma, 56: 274-314.

1963b Elecktronenmickroskopische Un- tersuchungen zur funktionellen Morphologie des Ionentransportes in der Salzdriise von Larus argentatus (11. Funktionelle Morphologie Blut- gefasse). Protoplasma, 56: 385-419. - 1963c Electronenmickroskopische Un- tersuchungen zur funktionellen Morphologie des Ionentransportes in der Salzdriise von Larzcs

argentatus (111. Funktionelle Morphologie der Tubulusepithelzellen). Protoplasma, 56: 605- 636.

Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. €3. B. C., 9: 409-414.

Marples, B. J. 1932 The structure and development of the nasal glands o f birds. Proc. Zool. SOC. (London), 4: 820-844.

McManus, J. F. A. 1948 The periodic acid routine applied to the kidney. Am. J. Pathol.,

Millonig, G. 1961 A modified procedure for lead staining of thin sections. J. C. B., 11: 736-740.

Montagna, W., H. B. Chase and H. P. Melaragno 1951 Histology and cytochemistry of human skin. I. Metachromasia in the mons pubis. J. Natl. Cancer Inst., 12: 591-597.

Mowry, R. 1958 Observations on the use of sulfuric acid in ether. J. Histochem. Cytochem., 6: 82.

Nachlas, M. M., and S. M. Seligman 1949 The histochemical demonstration of esterase. J. Natl. Cancer Inst., 9: 415-425.

Nachlas, M. M., K. C. Tsou, E. DeSouza, C. S. Cheng and S. M. Seligman 1957 Cytochem- ical demonstration of succinic dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole. J. Histochem. Cytochem., 5: 420- 436.

Pearse, A. G. E. 1961 Histochemistry, Theo- retical and Applied. Little, Brown and Co., Boston.

Peters, A. 1892 Beitrog zur kenntriss der harder’ schen druse. Arch. Mikr. Anat., 36: 192.

Reynolds, E. S. 1963 The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. C. B., 17: 208-212.

Schmidt-Nielsen, K. 1961 Concentrating mech- anism of the kidney from a comparative view- point. Amer. Heart J., 62: 579-586.

Schmidt-Nielsen, K., and R. Fange 1958 Salt glands in marine reptiles. Nature, 182: 783- 784.

Scothorne, R. J. 1958 A histochemical study of the nasal (supraorbital) gland of the duck. Nature, 181: 732.

1959 The nasal glands of birds: A histological and histochemical study of the in- active gland in the domestic duck. J. Anat., 93: 246-256.

Takeuchi, T., and H. Kuriaki 1955 Histo- chemical method for glucosan phosphorylase and transglycolase. J. Histochem. Cytochem., 3: 153-160.

Wachstein, M., and E. Meisel 1957 Histochem- istry of hepatic phosphatases at a physiological pH. Am. J. Clin. Pathol., 27: 13.

Williams, T. W. 1948 The visualization of vertebrate capillary beds by intravascular pre- cipitation of lead chromate. Anat. Rec., 100:

24: 643-653.

115-126.

PLATES

PLATE 1

EXPLANATION O F FIGURES

1 This photomicrograph illustrates the basic histological organization and the vascular system of a lobule in the lacrymal gland of the green turtle. The branch- ing secretory tubules are small at the periphery of the lobule and increase steadily in size centripetally, where they join the central canal (arrows). At the left a lnrge artery ( A j penetrates the lobule and branches to form arterioles that extend along the central canal. From the arterioles capillaries radiate between the secretory tubules to the periphery of the lobule where they empty into venous sinusoids. Lead chromate perfusion, counterstained with hematoxylin and eosin, ( X 70 ) .

2 Cholinesterase activity is restricted to large interlobular nerve fibers (arrows), smaller intralobular fibers that form a peritubular net-work and the goblet cells ( G ) lining the central canals. Acetylthiocholine iodide substrate, 60 p formalin fixed cryostat section, incubation one hour, ( x 60).

Strong alpha-naphthol esterasc activity may be demonstrated in the small basal cells near the perimeter of each tubule (arrows). In this preparation the channels between the principal cells were also reactive for this enzyme, but this was not a consistent finding. Ten microns cryostat section, a naphthol acetate substrate, 5 minutes incubation at room temperature, ( x 450).

4 The broad intercellular channels between the secretory cells stain metachromatically with toluidine blue. The irregular surfaces of the cells intermesh with one another to form a jigsaw puzzle pattern. Hess and Hollander technique for permanent metachromasia, ( x 430).

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5 The principal secretory cells have cytochrome oxidase within cytoplasmic granules; the intercellular channels and the nuclei are unstained. The peripheral cells and the cells lining the central canal react weakly. Paraphenylene Diamine base, In- cubation 15 minutes, ( x 100).

6 The pattern of succinic dehydrogenase activity in the gland parallels that of cytochrome oxidase, the reaction is weak in the cells at the periphery of the lobule, intense in the principal secretory cells and weak in the cells lining the central canals. Tetranitro blue tetrazolium, incubated 15 minutes, ( x 110).

This micrograph shows only a small portion of a lobule in the area of its central canal. The cytoplasm of the principal secretory cells is intensely sudanophilic while the nuclei and intercellular channels are unstained. The cells lining the central canal as well as the surrounding connective tissue contain little stain- able lipid. A large artery (arrow) bisects the field. Ten microns cryostat section, Sudan black B, ( x 190). Acid phosphatase activity is restricted to a narrow band along the base of the principal secretory cells ( B urrows) and to the goblets of the mucous cells lining the central canal (G arrows). Twelve microns cryostat section, Naphthol AS-B1 tion, Sudan black B ( x 190).

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EXPLANATION OF FIGURES

9 Alkaline phosphatase activity is restricted to the blood vessels and connective tissue in the turtle salt gland. Twenty microns cryosat section, sodium a naphthyl phosphate substrate, 20 minute incubation, ( X 100).

Non-mitochondria1 ATPase activity is demonstrated within the blood vessels and the connective tissue. A thin dense line of ATPase is also present along the lumen at the periphery of each tubule (arrows). None of the enzyme is detect- able along the borders or within the principal secretory cells. Twelve microns cryostat section, Wachstein and Meisel method, substrate disodium salt ATP, 30 minutes incubation, ( X 200).

Intense PAS positive, diastase resistant material is found within the well developed basement membrane surrounding each tubule, within the broad intercellular channels at the lateral surfaces of the secretory cells, and around each of the blood vessels. The ground substance of the connective tissue and cytoplasm of the secretory cells is also moderately reactive. Five microns paraffin section, 30 minutes, digestion with diastase at 37"C, PAS stain, ( X 400).

12 Alpha-napthylesterase activity is restricted primarily to the cytoplasm of cells lining the central canal. The tubule cells do not give any more reaction than in the control sections incubated without substrate. Twelve micron cryostat section, a naphthyl acetate substrate, 5 minutes incubation at room temperature, ( X 300).

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EXPLANATION OF FIGURE

13 A secretory cell sectioned at the level of the nucleus, and the cytoplasm of the several adjacent cells are shown in this electron micrograph. Mitochondria, glycogen, and small smooth-surfaced vesicles of endoplasmic reticulum fill the cytoplasm around the nucleus. Tall pleomorphic microvilli pack the intercellular spaces forming broad tortuous intercellular channels. The microvilli of adjacent cells in- terdigitate and are connected by occasional desmosomes (arrows), ( x 10,800).

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PLATE 5

E X P L A N A T I O N O F F I G U R E S

14 Portions of the intercellular channels surrounding three cells are shown in this electron micrograph; the intercellular spaces lying between the projecting microvilli of adjacent cells are irregular and variable in width. Cytoplasmic organelles are excluded from the microvilli but mitochondria and small smooth-surfaced vesicles (arrows) are evident near their bases, ( X 24.120).

In this electron micrograph seven cells are sectioned at their apices. Junctional complexes separate the lateral and luminal surfaces of the cells, and the interdigitating microvilli on the adjoining lateral surfaces end abruptly at these points. The apical cytoplasm is packed with numerous vesicles of nearly uniform size but there are no mitochondria and little glycogen in this zone. The small tubular lumen, contains a few villous projections from the apical surface, ( x 26,300).

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16 In this electron micrograph the basal portions of several salt-secreting cells are extended along their apposed basement membranes. The long villous processes on the sides of the principal cells intermesh laterally and they are flattened against the basement membrane. A small basal cell (arrows), wedged among the principal cells, contains few mitochondria and little glycogen but numerous profiles, of SSER and some filamentous cytoplasmic elements. The surface of the basal cell is nearly smooth and bears no villous process, ( X 14,700).

17 Portions of three secretory tubules with adjoining connective tissue and capillaries are included in this photomicrograph. The large principal cells, outlined by the negative images of broad intercellular channels extend from the small central lumen to the basement membrane. Small basal cells are interposed between bases of the secretory cells (arrows). One micron section, stained with toluidine blue, epon embedded, ( X 640).

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histochemical and electron microscopic observations on the salt secreting lacrymal glands of marine...

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