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Ultrastructure of the corpuscles of stannius in the garpike (Lepisosteus platyrhynchus)
GtNERAl
AND
COMPARATIVE
Ultrastructure TAPAN
KUMAR
DcJpartmrrlt
ENDOCRINOLOGY
46, 29-41
(1982)
of the Corpuscles of Stannius (Lepisosteus platyrhynchus)
BHATTACHARYYA,
of Zooloy~. University ‘.Departnwnt oj’2oo1og.v.
DAVID
GORDON
BUTLER.
of Toronto. _25 Harbortl Strrrt, Unir9ersit.v Scarborough College. Outark) MIC IA4. Catlada Accepted
February
in the Garpike AND JOHN H. YOUSON *
Toronto, Ontario M5.S IAI, OJ Twotzto. West Hill.
ad
27, 1981
The ultrastructural organization of the white corpuscles in the kidneys of the garpike, Lapisostc~us platwhpnchus. was studied following intravascular perfusion fixation. The white corpuscles are located within the kidneys and near the great dorsal vessels of the garpike and they possess all the ultrastructural features of corpuscles of Stannius (CS) which are identified in teleosts and in the other holostean. the bowfin. In contrast to the bowfin, however, the garpike CS reveals a strikingly different anatomic distribution and the presence of two cell types, that can be differentiated on the basis of cell size and shape, various ultrastructural characteristics. and relative abundance. This evidence suggests a possible divergence of corpuscular structure, and, possibly. function among holostean fishes.
The corpuscles of Stannius (CS) which occur uniquely in two groups of bony fishes, i.e., Holostei and Teleostei, are putative endocrine organs associated with the kidneys. Numerous experiments on teleosts have indicated that CS are involved in electrolyte metabolism, but to date, the mechanics of their action is not thoroughly understood. Surgical extirpation of the CS in different species results in elevation of serum calcium and potassium, and lowering of blood sodium, magnesium, chloride, and phosphate levels (Fontaine, 1964; Chan er al., 1967; Butler, 1969. 1972; Pang, 1971; Kenyon rt al., 1980). Hypercalcemia resulting from stanniectomy can be prevented by homotransplantation of the corpuscles or injection of corpuscular extracts (Jones et al.. 1967: Fenwick and Forster. 1972: Pang et al., 1973). The current consensus among comparative endocrinologists is that the CS produce an active principle, which probably maintains calcium homeostasis by inhibiting excess calcium influx across the gills through inhibition of the function of branchial Caz+-activated ATPase (Pang ct crl., 1974; Fenwick, 1976: So and Fenwick, 1979). Removal of Stannius corpuscles led
to enhanced gill calcium influx and decreased outflux in the European eel (Milet et al., 1979). A second hypothesis about the role of the CS was based on earlier experiments which indicated that the CS secrete a pressor substance since stanniectomy results in decreased blood pressure and corpuscular extracts elevated the blood pressure of rats and eels (Jones et ul., 1966: Sokabe et al.. 1970). Although the chemical nature of CS secretions has not been clearly ascertained, cytophysiological analysis has indicated corpuscular endocrine functions. and morphological studies in teleosts have revealed a variation in their histological organization in different families (Krishnamurthy and Bern. 1969). Ultrastructural studies have established that corpuscular cells are equipped with the machinery for synthesis and secretion of proteinaceous products and that the corpuscles in different teleost species may possess one or two types of cells (Ogawa. 1967; Tamasulo et al.. 1970: Wendelaar Bonga et nl., 1977. 1980; Bhattacharyya and Butler, 1978; Aida ct cf!., 1980a). Changes in the fine structure of the corpuscular cells also reflect their response 29 00166480/82/010029-13$01.00/O Copyright @ 1982 by Academic Press. Inc. All nghfs of reproduction in any form reserved
30
BHATTACHARYYA,
to environmental fluctuations in salinity or pituitary hormone stimulation (Tomasulo ef al.. 1970: Carpenter and Heyl, 1974; Cohen et al.. 1975; Olivereau and Olivereau, 1978; Aida et al., 1980b). As a contrast to the information available in Teleostei, there are no physiological data about the role of the CS in Holostei, which are freshwater survivors of ancient oceandwelling forms (Weichert, 1965). Earlier incomplete ontogenic studies on the holostean and teleostean CS revealed that they are numerous in Holostei, but a reduction in number is accompanied by an increase in size and a more posterior position in Teleostei (Garrett, 1942; De Smet. 1962). A preliminary study on the identity and distribution of the CS in the garpike, Lepisosteus sp. (Bhattacharyya et al., 1981). revealed that there is a reduced number of CS compared to the other holostean. the bowfin (Anzia calva) (Youson et al., 1976). The CS are present in both species as white corpuscles which can be distinguished from yellow corpuscles (adrenocortical homolog) by the absence of 3/3-hydroxysteroid dehydrogenase activity in the former (Bhattacharyya et al.. 1981). Ultrastructurally. the CS in A. calrla contain predominantly one type of cell with secretory granules and are similar to that described in most teleost species (Youson and Butler, 1976). In view of the difference in number and distribution of the CS between Amia and Lepisosteus sp. (Bhattacharyya et al., 1981), it seemed worthwhile to make a detailed examination of the ultrastructure of the CS in the garpike. This report is intended to serve as a baseline description of the tine structural morphology of the CS in this species for future cytophysiological investigations and presents some morphological evidence of cellular dichotomy in the garpike CS. MATERIALS AND METHODS Florida garpike (Lrpisosrrus plafyrhynchus) obtained from commercial fish dealers were transported to the Iaboratory where they were kept for at least 21
BUTLER,
AND
YOUSON
days in experimental aquaria with recycled dechlorinated tap water at lo- 15”. They were fed minnows or goldfish two times per week. A total of 16 specimens was examined and the animals ranged in body weight from 35 to 45 gm. Prior to experiments the animals were anesthetized in a dilute solution of tricaine methanesulfonate and the viscera were exposed through a midventral incision from the cloaca to the branchial region. For light microscopy, a piece of tissue which extended from the pericardial cavity to the cloaca containing the kidneys, accompanying blood vessels (dorsal aorta, posterior cardinal veins, and renal veins), and the vertebral column was removed and fixed in Bouin’s fluid for a few weeks for decalcification. Following storage in 70% ethanol, the samples were dehydrated. embedded in paraffin. and serial sections stained with hematoxylin and eosin or with a combination of periodic acid-Schiff, acid hemalum, and aqueous orange Cl (Lillie. 1965). For electron microscopy. the kidneys and adjoining white corpuscles (CS) in anesthetized animals were routinely fixed by intravascular perfusion. In a single, nonperfused animal white corpuscles were removed and fixed by immersion. Animals were perfused through the dorsal aorta or the heart and the perfusate was pumped from a 25-m] hypodermic plastic syringe fitted with lntramedic polyethylene tubing (Adams) and a hypodermic needle. The syringe was operated from an infusion pump (Model 975, Harvard Apparatus Co.. Inc.) with aflowrate of 0.81-1.1 mllmin. Fixation by aldehyde fixative usually followed a prerinsing of blood vessels with Tyrode solution or a fluid made of normal saline, procaine-HCl, and polyvinylpyrrolidone (Forssmann et al., 1977). The fixatives used were 2.5% glutaraldehyde in phosphate buffer (Millonig, 1962). and 2.5% glutaraldehyde or 2% formaldehyde-2.5% glutaraldehyde in 0.1 M cacodylate buffer (Flickinger. 1967) or 0.5% picric acid in formaldehyde-glutaraldehyde solution (Ito and Karnovsky. 1968). Pieces of fixed kidney were sliced in small fragments under a binocular microscope with the tissue being submerged
in fixative.
The
tissue
fragments
were
further immersed in the fixative for 2 hr at 4” followed by washing in cold buffer and postfixation in 1% buffered OsO, for 2 hr at 4”. Following dehydration in graded alcohols, the pieces were infiltrated with Araldite or Epon- Araldite mixtures after passing through propylene oxide. Plastic blocks were sectioned with glass knives using a Porter-Blum MT 2 microtome set to give silver-gray or pale-gold sections. These were mounted on coated or uncoated 200 mesh grids and stained with uranyl acetate and lead citrate solutions. Thick sections from plastic blocks were stained with 1% methylene blue or toluidine blue in 1% borax solutions or methylene blue-basic fuchsin solution (Bennett t~f crl.. 1976) for study
and orientation.
The grids
ULTRASTRUCTURE
OF
STANNIUS
were examined in a Philips 201 electron microscope operated at 60 kV. White corpuscles from a total of five animals were considered for measurements of nuclear and cell size, and number and diameter of secretory granules. From each animal six representative C, and C1 cell types were selected for these measurements which were made from electron micrographs with an ultrastructure size calculator (Polaron). The diameter of at least 100 mature secretory granules was measured from the two cell types which were differentiated by various cytomorphological criteria.
RESULTS
The five to seven white corpuscles (corpuscles of Stannius) in L. platyrllynchus occur as round to oval structures around the midportion of each kidney and are associated with postcardinal and renal veins and, occasionally, the adrenocortical tissue, i.e., yellow corpuscles (Fig. la, Ic). They are recognized as well-vascularized whitish masses projecting slightly from the surface of the kidney and are confined to the middle region of the kidneys where little or no nephric tissue is apparent but where hemopoietic tissue is abundant. Their position within these cords of hemopoietic tissue is variable. On a few occasions they are juxtaposed with yellow corpuscles near the posterior cardinal veins but more commonly they are found isolated deeper within the hemopoietic tissue (Fig. lb). They range in diameter from 50 to 400 pm and in length from 300 pm to 2.0 mm. The white corpuscles are limited from the yellow corpuscles and the surrounding hemopoietic tissue by a capsule of collagen fibrils and fibroblasts. The glandular parenchyma of each white corpuscle consists of two-layered solid cellular cords or islets which are convoluted and which anastomose with each other (Fig. 2). Following immersion fixation the capillaries appear collapsed, but perfused organs reveal extensive capillaries irrigating all regions of the parenchyma. The cells are basophilic with hematoxylin and eosin and demonstrate a variable staining
CORPUSCLES
IN
GARPIKE
31
intensity with periodic acid-Schiff. Granules in the cytoplasm are periodic acidSchiff positive (Fig. lc) and are dispersed around the nucleus or more concentrated in two poles. A few nongranular cells with smaller nuclei also may be present. Ultrastrrrcturc
The lobules of granulated epithelial cells are bounded by a thin basal lamina and neighboring lobules of corpuscular cells are separated by an interstitium containing small and round or tortuous capillaries. unmyelinated nerve fibers. and connective tissue cells (Fig. 3). The endothelial cells in the capillary wall are fenestrated and rest upon a continuous basal lamina. The interlobular space also contains a few inconspicuous collagen bundles and a granular precipitate. Two types of epithelial cells make up the parenchymal population in each corpuscle and these are provisionally classified as C, and C, cells. A rough approximation showed that C, cells comprise about only 5- 10% of the total population in all animals. The C, cell is characterized by abundant cytoplasmic granules (Fig. 3) and is oval to round in shape (diameter, 7- 10 pm). It possesses a round central nucleus (average diameter, 4 pm) with one usually eccentric nucleolus and irregular patches of heterochromatin. The presence of a number of long parallel or slender wavy stacks of rough endoplasmic reticulum (RER) and isolated slender cisternae near the cell surface are identifying features of this cell type (Fig. 4). The cisternae of the RER are usually disposed as a branching interconnected network and may be continuous with perinuclear cisternae of the nuclear envelope. Free ribosomes are scattered throughout the cytoplasm. The Golgi apparatus consists of one or more sets of compacted saccules (Fig. 4) and small prosecretory granules are observed near their concave or lateral margins. The saccules may also possess electron-dense material apparently in
32
BHATTACHARYYA,
BUTLER,
AND
YOUSON
ULTRASTRUCTURE
OF STANNIUS
the process of condensation. Numerous smooth or coated vesicles are dispersed near the region of the Golgi complex and the cell surface. The mitochondria are round, oval, elongated, or irregularly branched with dense matrices, and lamellar cristae often oriented in parallel fashion; the mitochondria occur in a cytoplasmic area unoccupied by secretion granules. Other cellular constituents are randomly dispersed microtubules, bundles of filaments, lysosome-like dense bodies, autophagic vacuoles, and multivesicular bodies. The cell surface possesses coated pits often concentrated near the capillary zone. Secretory granules are noted in close proximity to the plasma membrane although no clear evidence of their involvement in exocytosis was observed. In some cells clear vesicles are present in the cytoplasmic area close to the pits of the plasma membrane. The presence of numerous (200-250/tell) secretory granules is a conspicuous feature of this cell type (Fig. 5). The granules are either distributed uniformly throughout the cytoplasm or they may show a heavy concentration toward the vascular pole. Their matrices are homogeneously amorphous and electron-opaque and are confined within a close-fitting membrane. The granules are mostly of round to oval shape and their diameter varies from 0.20 to 0.35 pm (average size about 0.25 pm). Granule appearance and distribution varies from cell to cell and may reflect functional activity. In a presumed storage phase, the cell is
CORPUSCLES
IN GARPIKE
33
loaded mostly with granules and a few protiles of the mitochondria and RER (Fig. 5). Other cells shows a reduced granulation, very prominent Golgi complex, numerous coated vesicles, multivesicular bodies, autophagic vacuoles, and polymorphic mitochondria. This latter form suggests a secretory or synthetic stage of this cell type (Fig. 4). Some animals had a few C, cells with a nucleus of irregular profile. slightly dilated cisternae of the nuclear envelope, and dense patches of heterochromatin (Fig. 91. The mitochondria of the cells possess electron-lucent matrices and cisternae of RER are distended and difficult to recognize, and are restricted to perinuclear positions. The secretory granules are clumped toward the cell periphery and their limiting membranes are often very dilated forming an electron-lucent halo around the granule. Other granules appear as small remnant of the dense granular mass and are frequently less dense in appearance. Some C, cells possessed numerous lysosome-like bodies and small multivesicular bodies. Interestingly, C, cells situated within a cluster of such transformed C, cells did not manifest this kind of alteration. The C, cell is oval (diameter, 6-8.5 pm), polygonal, or frequently triangular in section. It appears compressed between C, cells and it sends out elongated cellular processes between this more abundant cell type (Figs. 6, 7). Nuclei are oval (diameter about 5 pm) or irregular in profile and the nucleolus is usually inconspicuous. Com-
FIG. la. Transverse section of the middle of the kidney ofL. plaryrh~nchus demonstrating renal (R) and hemopoietic (H) tissues and the position of a white (WC) and yellow (YC) corpuscle with respect to the posterior cardinal vein (PCV). x40. FIG. lb. A light micrograph of a transverse section through the kidney of a garpike. A large white corpuscle (WC) maintains a close relationship to the posterior cardinal vein (PCV) and is completely surrounded by hemopoietic tissue (H). ~36. FIG. lc. An encapsulated white corpuscle (WC) is clearly separated from the cords of vacuolated (V) cells of the yellow corpuscle and hemopoietic tissue (H). The cells of the white corpuscle stain as dark (D) and light (L) cells with periodic acid-Schiff. x400. FIG. 2. A toluidine blue-stained plastic section through a perfused white corpuscle (WC). Cellular cords loaded with granules are profusely supplied with capillaries (CL H. hemopoietic tissue. x420.
34
BHATTACHARYYA,
BUTLER,
AND
YOUSON
FIG. 3. A survey electron micrograph of several lobules of epithelial cells from a corpuscle of Stannius. The predominant cell type (C,) is oval or round. possesses a spherical nucleus, and is filled with secretory granules (SC). A less abundant second cell type (C,) is compressed between C, cells. The C2 cells have irregular nuclear outline and fewer secretory granules. Connective tissue cells (CT) and a capillary (C) are present in the interlobular space. x 3800.
pared to C, cells, the cytoplasmic matrix is of low electron density and there is a paucity of cell organelles. In particular, slender, elongate RER cisternae are rarely observed but instead cisternae are short, dilated, and may possess a flocculent content of moderate electron density. The Golgi complex is very prominent, often ring-like in profile and may possess a number of associated enlarged vacuoles (Fig. 8). Dumbbellshaped mitochondria with dense matrices and a few parallel lamellar cristae, and free
ribosomes are commonly noted. The cell also contains many smooth and coated vesicles, small vacuoles, lysosome-like dense bodies, and a few small lipid droplets, the latter being of rare occurrence in C, cells. Secretory granules of C, cells are more widely scattered and fewer in number (501 cell) than those of C, cells (Figs. 6, 8). While the majority of granules are round or oval. others are rod-shaped or are irregular in profile and may show a variable electron
FIG. 4. Enlarged view of C, cells. The identifying characteristics of this cell are slender parallel arrays of RER (er) and a Golgi complex (G) with flattened sac&es. A number of coated vesicles (arrowhead) are present near the Golgi complex. The C2 cell shows an elongated nucleus and short dilated cisternae of RER (arrow). x 12,100. FIG. 5. A C, cell possesses mostly round, electron-dense secretory vesicles (SC), a few isolated profiles of RER (arrowhead). and mitochondria (M). x 11,700. 35
FIG. 6. A C2 cell appears squeezed between C, cells and is sparsely populated with secretory granules which are scattered and frequently rod-shaped (arrowhead). A prominent Golgi complex (G) and numerous mitochondria (M). with lamellar cristae, are also observed in the C, cells. C, cells possess many round secretory granules (SG) and elongated parallel profiles of RER (er). x7900. FIG. 7. A C, cell is depleted of secretory granules and displays RER organized in the form of short isolated cistemae (arrowhead). An adjacent C, cell is filled with granules. M, Mitochondria: I, interdigitation of the plasma membrane. x 11,900. 36
FIG. 8. A C2 cell with profiles of hypertrophied saccules of the Golgi complex (G). The cell contains a few secretory granules which are dispersed, and some of which appear in rod-shaped or commashaped profiles (arrowhead). Neighboring C, cells have clusters of larger secretory granules. x 11.900. FIG. 9. A group of transformed C, cells show nuclei of irregular outline with dilated nuclear envelopes. The mitochondria (M) appear pale due to loss of matrix density and cisternae of the RER are dilated (arrowhead). A Cz cell appears of normal configuration. Compare with Fig. 3. x5200. 37
38
BHATTACHARYYA,
density. The size of the round granules varies from 0.10 to 0.25 pm, the average size being 0.15 pm. Their dense matrices may incompletely fill up the membranous vesicle or may be eccentric in location. A few large granules have granular or net-like matrices with a partly electron-dense content, which is often separated from the limiting membrane by a large electron-lucent space. A number of C, cells possess a totally granule-depleted cytoplasm and a large number of RER sacs and free ribosomes (Fig. 7). Others are characterized by numerous mitochondria, intercommunicating short cisternae of RER, numerous polyribosomes, a conspicuous Golgi apparatus with dilated vacuoles, numerous multivesicular bodies, smooth and coated vesicles. parallel bundles of microtubules, and a few small secretory granules. This ultrastructural picture may indicate actively synthetic or secretory stage of the C, cell.
BUTLER,
AND
YOUSON
species may development pear in Amia ric duct and
also be related to the ontogenic of this organ, since the CS apas outgrowths of the pronephopisthonephric tubules, but in Lepisostelrs spp., originate only from the pronephric duct (De Smet, 1962). A physiological explanation is lacking to account for this extremely large number in the bowfin and its gradual evolutionary concentration in other bony fishes. Based on ultrastructural criteria, such as the arrangement and profiles of the Golgi apparatus and RER. especially the size, shape, and abundance of secretory granules, two cell types seem to be a characteristic of this organ in the garpike. The existence of two cell types has been documented in the CS of the stickleback. Gasterosteus awleatus. the European eel, Atzguilfa arlguiffa, the killifish, Flrtlciulrrs hetc~rocfitlrs. the goldfish, Carassius auratus (Wendelaar Bonga and Greven, 1975; Wendelaar Bonga et al., 1980), the trout, Salmo gairdrzpri (Krishnamurthy and Bern, DISCUSSION 1969; Meats et al., 1978). and the coho salThe present study shows that the white mon, Otzcorhynchus kisrrtc.h (Aida et ~1.. corpuscles within the kidneys of the garpike 1980a). They are present as principal (type are the corpuscles of Stannius (CS) which C,) and a rare (type C,) cell types (Meats et have been recognized in another holostean al.. 1978). However, other teleost species, (Youson and Butler, 1976; Youson et ~1.. i.e.. the Atlantic salmon, Safmo salar (Car1976) and in teleosts (Oguri, 1966; To- penter and Heyl, 1974), the guppy, Lchistcs masulo er al., 1970; Cohen cjt al., 1975). rrticulatrrs (Tomasulo et (IL., 1970), the cod, However, certain morphological features of Gadus tnorrhrra, the plaice, Pkut-ot~~~ctcs the CS in the garpike are strikingly different platessa (Wendelaar Bonga and Greven, from the CS of its closest holostean rela- 1975), and the toadfish, Opsarllrs talr tive, the bowfin. In the latter species, the (Bhattacharyya and Butler, 1978), are corpuscles are widely scattered throughout characterized by the presence of a single the kidney, which is believed to be the more cell type with large secretory granules. The ancestral organization that ultimately cul- presence of two cell types (C, and C,) in the minated into more compactness and poste- garpike is noteworthy, since the other rior position in Teleostei (Garrett, 1942; De holostean. the bowfin (Youson and Butler. Smet, 1962: Youson et al., 1976). In con- 1976) possesses only a single cell type. trast to the bowfin, the garpike manifests a The C, cells of the garpike are uldistinctive reduction in number and re- trastructurally comparable to the principal gional concentration of the CS indicating an cell type noted in most teleosts and indicate intermediate phylogenetic trend among the possible endocrine function of this bony fishes. However, this difference in organ. Generally, the cytological profile of gross anatomic distribution in these two the CS reveals that the corpuscular cells are
ULTRASTRUCTURE
OF
STANNIUS
packed with mitochondria, Golgi saccules, RER, and numerous secretory granules. The latter structures arise from the Golgi apparatus in other fishes (Youson and Butler, 1976; Wendelaar Bonga et al., 1977; Bhattacharyya and Butler, 1978) and signify a mechanism in the CS which is consistent with the secretion of protein or polypeptide hormones (Fawcett ct al., 1969). The morphological evidence for endocrine secretion is also indicated by a profuse vascular investment, there being an extensive capillary network supplying all regions of the glandular tissue. Ultrastructural studies have not clearly documented the mode of discharge of secretory granules from the CS under normal or stimulated conditions. However, the preferential location of the secretory granules toward the capillary pole. and the presence of coated pits on the cell surface suggestsan exocytotic mode of discharge of granules as is generally observed in protein-secreting endocrine glands (Wendelaar Bonga et al.. 1977: Bhattacharyya and Butler, 1978). However, it has been proposed that corpuscular cells may disintegrate during stress periods in hyperosmotic medium resulting in release of intact protein granules into capillaries (Meats ct NI., 1978). The presence of secretory granules in extracellular spaces in the bowfin was believed to be related to mechanical injury (Youson and Butler, 1976). In the present material there was no evidence of the apocrine or holocrine secretion by the glandular cells that was often described in earlier literature (Lopez, 1969). Therefore, exocytosis may be the principal mode of liberation of secretory vesicles after their origin and packaging from the Golgi region. The secretory activity of the C, cell is apparently related to environmental calcium concentration, the cells being more active in animals in seawater than in fresh water (Wendelaar Bonga ct crl., 1976; Meats cr al.. 1978; Olivereau and Olivereau, 1978: Wendelaar Bonga. 1980: Aidart ~1.. 1980a).
CORPUSCLES
IN GARPIKE
39
The presence of some transformed C, cells in the garpike is difficult to explain but may indicate a possible functional hypoactivity of this cell type in fresh water leading to cellular degeneration. This is also supported by the observation that most of the C, cells seemed to be in an inactive or a storage phase of the secretory cycle because they contained a heavy accumulation of secretory granules together with a paucity of cellular organelles. On the other hand, these transformed cells of the garpike CS also resemble degenerating parenchymal cells of the salmon CS (Carpenter and Heyl. 1974) during the spawning migration from seawater to fresh water. Furthermore, during a study of the CS under freshwater and seawater conditions, cellular degeneration of the mullet (Mlrgi/ cc>ph~lrrs) CS was observed in fresh water but not in seawater (Johnson, 1972). The C, cells in the garpike have a general morphological similarity and a relative scarcity which is characteristic of the second cell type found in other teleosts (Krishnamurthy and Bern, 1969; Wendelaar Bonga and Greven. 1975: Meats et ~1.. 1978; Aida PI a/.. 1980a). In stickleback corpuscles, activity of the C, type cell is a function of the concentration of sodium and potassium ions in the media (Wendelaar Bonga et al., 1976) while the C2 cells of the trout CS are active in media of low ionic and osmotic strength and relatively inactive when the salinity increases (Meats et al., 1978). On the other hand, type 2 cells in the coho salmon are more active in the seawater smolt and are inactive in fresh water (Aida et al., 1980a). As a contrast with C, cells of the garpike, the C, celis display a greater degree of cytologic variability, they did not appear to be in an inactive storage phase loaded with secretory granules, and presented no evidence of cellular degeneration. There is no documented evidence of corpuscular participation in electrolyte metabolism among holostean fishes, and it is unclear whether the two types of cells in
40
BHATTACHARYYA,
BUTLER,
this species release different endocrine factors, or they represent alterations of the same cell type. The functional significance of the garpike CS and its cell types presently remains speculative. ACKNOWLEDGMENTS This investigation was supported by Grant A2359 to D. G. Butler and Grant A5945 to J. H. Youson from the Natural Sciences and Engineering Council of Canada.
REFERENCES Aida, K.. Nishioka, R. S.. and Bern, H. A. (1980a). Changes in the corpuscles of Stannius of coho salmon (Uncorhynchus kisutch) during smoltification and seawater adaptation. Gen. Camp. Endocrinol. 41, 296-304. Aida, K., Nishioka, R.’ S., and Bern. H. A. (1980b). Degranulation of the Stannius corpuscles of coho salmon (Oncorhynchus kisutch) in response to ionic changes in vitro. Gen. Camp. Endocrinol. 41,305-313. Bennett, H. S., Wyrick, A. D.. Lee, S. W., and McNeil, J. H.. Jr. (1976). Science and art in preparing tissues embedded in plastic for light microscopy with special reference to glycol methacrylate, glass knives and simple stains. Stair1 Tech. 51,71-97. Bhattacharyya, T. K., and Butler. D. G. (1978). Fine structure of the corpuscles of Stannius in the toadlish. J. Morphol. 155, 271-286. Bhattacharyya, T. K., Butler, D. G., and Youson, J. H. (1981). Distribution and structure of the adrenocortical homologue in the garpike. Amer. J. Anat. 160, 231-246. Butler, D. G. (1969). Corpuscles of Stannius and renal physiology in the eel (Anguilla rostrata). J. Fish. Res. Board
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Butler, D. G. (1972). Failure to observe changes in selected metabolites following removal of the Stannius corpuscles from the freshwater North American eel, Anguilla rastrata. J. Fish. Res. Board. Canad. 29, 1362- 1364. Carpenter, S. J.. and Heyl, H. L. (1974). Fine structure of the corpuscles of Stannius of Atlantic salmon during the freshwater spawning journey. Gen. Camp.
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Chan, D. K. 0.. Jones, I. C., Henderson, I. W., and Rankin, J. C. (1967). Studies on the experimental alteration of water and electrolyte composition of the eel (Anguilla anguilla L.). J. Endocrinol. 37, 297-317. Cohen, R. S.. Pang, P. K. T.. and Clark, N. B. (1975).
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Ultrastructure of the Stannius corpuscles of the killitish, Fundulus heferoclirus. and its relationship to calcium regulation. Gen. Camp. Endocrinol. 27, 413-423. De Smet, D. (1%2). Considerations on the Stannius corpuscles and interrenal tissues of bony fishes. especially based on researches into Amia. Acta Z&d. Stdckholm 43, 201-219. Fawcett. D. W.. Lona. J. A.. and Jones. A. L. (1969). The ultrastructure of endocrine glands. Recent Prog. Harm. Res. 25, 315-380. Fenwick. J. C. (1976). Effect of stanniectomy on calcium-activated adenosine-triphosphatase activity in the gills of freshwater adapted North American eels, Anguilla rostratu Le Sueur. Geti. Camp.
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Fenwick, J. C.. and Forster, M. E. (1972). Effects of stanniectomy and hypophysectomy on total plasma cortisol level in the eel (Anguilla anguilla L.). Gen. Camp. Endocrinol. 19, 184-191. Flickinger. C. J. (1967). The prenatal development of Sertoli cells in the mouse. Z. Zellforsch. 125, 480-496. Fontaine, M. (1964). Corpuscles de Stannius et regulation ionique (Ca, K et Na) du milieu interieur de I’Anguille (Anguilla anguillu L.). C. R. Sac. Biol. 259, 875-878. Forssman. W. G., Ito, S.. Weihe. E., Aoki, A., Dym, M., and Fawcett, D. W. (1977). An improved perfusion fixation method for the testis. Anat. Rec. 188, 307-314. Garrett, F. S. (1942). The development and phylogeny of the corpuscles of Stannius in ganoid and teleostean fishes. J. Morphol. 70, 41-67. Ito, S., and Karnovsky. M. J. (1968). Formaldehyde-glutaraldehyde fixatives containing trinitro compounds. J. Cell Biol. 39, l68a (Abstract) Johnson, D. W. (1972). Variations in the interrenal and corpuscles of Stannius of Mugil cephalus from the Colorado river and its estuary. Gen. Camp. Endocrinol.
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Jones, I. C., Henderson, I. W.. Chan. D. K. 0.. Rankin, J. C.. Mosley. W.. Brown, J. J., Lever. A. F.. Robertson, I. S., and Tree, M. (1966). Pressor activity in extracts from the corpuscles of Stannius from the European eel (Anguilla anguilla L.). J. Endocrinol. 24, 393-408. Jones, I. C., Henderson. I. W.. Chan, D. K. 0.. and Rankin. J. C. (1967). Steroids and pressor substances in bony fish with special reference to adrenal cortex and the corpuscles of Stannius of the eel (Anguilla anguilla L.). In “Proc. Int. Congr. Steroid Horm.. Milan, 1966,” Int. Congr. Ser. Excerpta Med. 132, 136- 145. Kenyon. C. J., Jones, I. C.. and Dixon. R. N. B. (1980). Acute responses of the freshwater eel
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OF
STANNIUS
anguilla) to extracts of the corpuscles of Stannius opposing the effects of Stanniosomatiectomy. Grn. Camp. Endocrinol. 41, 531-538. Krishnamurthy, Y. G., and Bern, H. A. (1969). Correlative histologic study of the corpuscles of Stannius and juxtaglomerular cells of teleost fishes. (Anguilla
Gen.
Cornp.
Et~doc~rinol.
13, 313-335.
Lillie. R. D. (1965). “Histopathologic Technic and Practical Histochemistry.” McGraw-Hill, New York. Lopez, E. (1969). Etude histophysiologique des corpuscles de Stannius de Sa/nr<>s&r L. au tours de divers &apes de son cycle vital. Gen. Camp. Endoc~rinol.
12, 339-349.
Meats. M., Ingleton, P. M.. Jones, 1. C.. Garland, H. 0.. and Kenyon. C. J. (1978). Fine structure of the corpuscles of Stannius of the trout, Salvo guirdtwri: Structural changes in response to increased environmental salinity and calcium ions. Gets. Cornp.
Endocrinol.
36, 45 I-561.
Milet, C., Peignoux-Deville, J., and Martelly, E. (1979). Gill calcium fluxes in the eel, Anguilla angui//cr (L): Effects of Stannius corpuscles and ultimobranchial body. Camp. Biochem. Physiol. 63. 63-70. Millonig, G. (1962). Further observations on a phosphate buffer for osmium solutions in fixation. If? “Proc. Fifth International Congress for Electron Microscopy” (S. S. Breeze. Jr., ed.). Vol. 2. p, 8, Academic Press. New York. Ogawa. M. (1967). Fine structure of the corpuscles of Stannius and the interrenal tissue in the goldfish. Z. Zdljbrsd~.
81, 174-
189.
Oguri. M. (1966). Electron microscopic observations on the corpuscles of Stannius of goldfish. B/r//. Japun
SW.
SC;. Fislt.
32, 902-908.
Olivereau. M., and Olivereau. J. (1978). Prolactin, hypercalcemia and corpuscles of Stannius in seawater eels. Cell Tisslrr Rrs. 186, 81-96. Pang, P. K. T. (1971). The relationship between the corpuscles of Stannius and serum electrolyte regulation in the kilhtish. Furd~r/us h~~/~roc/ifcts. J. Exp.
Zod.
178,
l-8.
Pang, P. K. T.. Pang. R. K., and Sawyer, W. H. (1973). Effects of environmental calcium and replacement therapy in the killifish. Furzd~l~s heteroclifus. after the surgical removal of the corpuscles of Stannius. Entlocrindogy 93, 705-710. Pang, P. K. T.. Pang, R. K., and Sawyer. W. H. (1974). Environmental calcium and the sensitivity of the killifish (Fu&u/rrs /re/eror/i/us) in bioassay for the hypocalcemic response to Stannius cor-
CORPUSCLES
41
IN GARPIKE
puscles for killifish Endortinolog.v
and cod (Gadus nwrrhucr).
94, 548 - 555.
So.. Y. P., and Fenwick, J. C. (1979). In view and itz vifro effects of Stannius corpuscles extract on the branchiai uptake of 45Ca in stanniectomized North American eels (An&//a rostrafa ). Grn. Camp. Endocrid. 34, 143- 149. Sokabe. H.. Nishimura, H., Ogawa, M., and Oguri. M. (1970). Determination of renin in the corpuscles of Stannius of the teleost. Cert. Cwnp. Endocried.
14, 510-5
16.
Tomasulo. J. A., Belt, W. D.. and Hayes, E. R. (1970). The tine structure of the Stannius body of the guppy and its response to deionized water. Amer. J. Anaf.
129, 307-328.
Weichert, C. K. (1965). “Anatomy of the Chordates.” McGraw-Hill Book Company, Toronto. Wendelaar Bonga. S. E. (1980). Effect of synthetic salmon calcitonin and low ambient calcium on plasma calcium, ultimobranchial cells. Stannius bodies. and prolactin cells in the teleost. Gasfcrosfrus
awleurlts.
Gerr.
Camp.
Encloc~ritwl.
40,
99- 108. Wendelaar Bonga, S. E., and Greven. J. A. (1975). A second cell type in Stannius bodies of two euryhaline teleost species. Cull Tis~tte Res. 159, 287- 290. Wendelaar Bonga, S. E., Greven. J. A. A.. and Veenhuis. M. (1976). The relationship between the ionic composition of the environment and the secretory activity of the endocrine cell types of Stannius corpuscles in the teleost Gusferosfros ac~ulrrrfus.
Cell
Tissue
Rcs.
175, 297-312.
Wendelaar Bonga, S. E., Greven, J. A. A., and Veenhuis, M. (1977). Vascularization. innervation and ultrastructure of the endocrine cell types of Stannius corpuscles in the teleost, Gnstc,rr>sfrrrs uculearus.
J. Morpiwl.
153, 225-244.
Wendelaar Bonga, S. E., van der Meij. J. C. A., and Pang, P. K. T. (1980). Evidence for two secretory cell types in the Stannius bodies of the teleosts Funddrts hc~feroclifus and Carassirrs tmmfus. Ccl1 Tissue
Rrs.
212. 295-306.
Youson, J. H.. and Butler, D. G. (1976). The fine structure of the adrenocortical homologue and the corpuscles of Stannius of Amiu c,a/vcr L. Acta Zoo/. Sfodholrn 57, 217-238. Youson, J. H.. Butler, D. G., and Chan, A. T. C. (1976). Identification and distribution of the adrenocortical homolog. chromaffin tissue and the corpuscles of Stannius in Amia cu/,fa L. Grrl. Corwp.
Em/wrino/.
29, 198-21
1.
AND
COMPARATIVE
Ultrastructure TAPAN
KUMAR
DcJpartmrrlt
ENDOCRINOLOGY
46, 29-41
(1982)
of the Corpuscles of Stannius (Lepisosteus platyrhynchus)
BHATTACHARYYA,
of Zooloy~. University ‘.Departnwnt oj’2oo1og.v.
DAVID
GORDON
BUTLER.
of Toronto. _25 Harbortl Strrrt, Unir9ersit.v Scarborough College. Outark) MIC IA4. Catlada Accepted
February
in the Garpike AND JOHN H. YOUSON *
Toronto, Ontario M5.S IAI, OJ Twotzto. West Hill.
ad
27, 1981
The ultrastructural organization of the white corpuscles in the kidneys of the garpike, Lapisostc~us platwhpnchus. was studied following intravascular perfusion fixation. The white corpuscles are located within the kidneys and near the great dorsal vessels of the garpike and they possess all the ultrastructural features of corpuscles of Stannius (CS) which are identified in teleosts and in the other holostean. the bowfin. In contrast to the bowfin, however, the garpike CS reveals a strikingly different anatomic distribution and the presence of two cell types, that can be differentiated on the basis of cell size and shape, various ultrastructural characteristics. and relative abundance. This evidence suggests a possible divergence of corpuscular structure, and, possibly. function among holostean fishes.
The corpuscles of Stannius (CS) which occur uniquely in two groups of bony fishes, i.e., Holostei and Teleostei, are putative endocrine organs associated with the kidneys. Numerous experiments on teleosts have indicated that CS are involved in electrolyte metabolism, but to date, the mechanics of their action is not thoroughly understood. Surgical extirpation of the CS in different species results in elevation of serum calcium and potassium, and lowering of blood sodium, magnesium, chloride, and phosphate levels (Fontaine, 1964; Chan er al., 1967; Butler, 1969. 1972; Pang, 1971; Kenyon rt al., 1980). Hypercalcemia resulting from stanniectomy can be prevented by homotransplantation of the corpuscles or injection of corpuscular extracts (Jones et al.. 1967: Fenwick and Forster. 1972: Pang et al., 1973). The current consensus among comparative endocrinologists is that the CS produce an active principle, which probably maintains calcium homeostasis by inhibiting excess calcium influx across the gills through inhibition of the function of branchial Caz+-activated ATPase (Pang ct crl., 1974; Fenwick, 1976: So and Fenwick, 1979). Removal of Stannius corpuscles led
to enhanced gill calcium influx and decreased outflux in the European eel (Milet et al., 1979). A second hypothesis about the role of the CS was based on earlier experiments which indicated that the CS secrete a pressor substance since stanniectomy results in decreased blood pressure and corpuscular extracts elevated the blood pressure of rats and eels (Jones et ul., 1966: Sokabe et al.. 1970). Although the chemical nature of CS secretions has not been clearly ascertained, cytophysiological analysis has indicated corpuscular endocrine functions. and morphological studies in teleosts have revealed a variation in their histological organization in different families (Krishnamurthy and Bern. 1969). Ultrastructural studies have established that corpuscular cells are equipped with the machinery for synthesis and secretion of proteinaceous products and that the corpuscles in different teleost species may possess one or two types of cells (Ogawa. 1967; Tamasulo et al.. 1970: Wendelaar Bonga et nl., 1977. 1980; Bhattacharyya and Butler, 1978; Aida ct cf!., 1980a). Changes in the fine structure of the corpuscular cells also reflect their response 29 00166480/82/010029-13$01.00/O Copyright @ 1982 by Academic Press. Inc. All nghfs of reproduction in any form reserved
30
BHATTACHARYYA,
to environmental fluctuations in salinity or pituitary hormone stimulation (Tomasulo ef al.. 1970: Carpenter and Heyl, 1974; Cohen et al.. 1975; Olivereau and Olivereau, 1978; Aida et al., 1980b). As a contrast to the information available in Teleostei, there are no physiological data about the role of the CS in Holostei, which are freshwater survivors of ancient oceandwelling forms (Weichert, 1965). Earlier incomplete ontogenic studies on the holostean and teleostean CS revealed that they are numerous in Holostei, but a reduction in number is accompanied by an increase in size and a more posterior position in Teleostei (Garrett, 1942; De Smet. 1962). A preliminary study on the identity and distribution of the CS in the garpike, Lepisosteus sp. (Bhattacharyya et al., 1981). revealed that there is a reduced number of CS compared to the other holostean. the bowfin (Anzia calva) (Youson et al., 1976). The CS are present in both species as white corpuscles which can be distinguished from yellow corpuscles (adrenocortical homolog) by the absence of 3/3-hydroxysteroid dehydrogenase activity in the former (Bhattacharyya et al.. 1981). Ultrastructurally. the CS in A. calrla contain predominantly one type of cell with secretory granules and are similar to that described in most teleost species (Youson and Butler, 1976). In view of the difference in number and distribution of the CS between Amia and Lepisosteus sp. (Bhattacharyya et al., 1981), it seemed worthwhile to make a detailed examination of the ultrastructure of the CS in the garpike. This report is intended to serve as a baseline description of the tine structural morphology of the CS in this species for future cytophysiological investigations and presents some morphological evidence of cellular dichotomy in the garpike CS. MATERIALS AND METHODS Florida garpike (Lrpisosrrus plafyrhynchus) obtained from commercial fish dealers were transported to the Iaboratory where they were kept for at least 21
BUTLER,
AND
YOUSON
days in experimental aquaria with recycled dechlorinated tap water at lo- 15”. They were fed minnows or goldfish two times per week. A total of 16 specimens was examined and the animals ranged in body weight from 35 to 45 gm. Prior to experiments the animals were anesthetized in a dilute solution of tricaine methanesulfonate and the viscera were exposed through a midventral incision from the cloaca to the branchial region. For light microscopy, a piece of tissue which extended from the pericardial cavity to the cloaca containing the kidneys, accompanying blood vessels (dorsal aorta, posterior cardinal veins, and renal veins), and the vertebral column was removed and fixed in Bouin’s fluid for a few weeks for decalcification. Following storage in 70% ethanol, the samples were dehydrated. embedded in paraffin. and serial sections stained with hematoxylin and eosin or with a combination of periodic acid-Schiff, acid hemalum, and aqueous orange Cl (Lillie. 1965). For electron microscopy. the kidneys and adjoining white corpuscles (CS) in anesthetized animals were routinely fixed by intravascular perfusion. In a single, nonperfused animal white corpuscles were removed and fixed by immersion. Animals were perfused through the dorsal aorta or the heart and the perfusate was pumped from a 25-m] hypodermic plastic syringe fitted with lntramedic polyethylene tubing (Adams) and a hypodermic needle. The syringe was operated from an infusion pump (Model 975, Harvard Apparatus Co.. Inc.) with aflowrate of 0.81-1.1 mllmin. Fixation by aldehyde fixative usually followed a prerinsing of blood vessels with Tyrode solution or a fluid made of normal saline, procaine-HCl, and polyvinylpyrrolidone (Forssmann et al., 1977). The fixatives used were 2.5% glutaraldehyde in phosphate buffer (Millonig, 1962). and 2.5% glutaraldehyde or 2% formaldehyde-2.5% glutaraldehyde in 0.1 M cacodylate buffer (Flickinger. 1967) or 0.5% picric acid in formaldehyde-glutaraldehyde solution (Ito and Karnovsky. 1968). Pieces of fixed kidney were sliced in small fragments under a binocular microscope with the tissue being submerged
in fixative.
The
tissue
fragments
were
further immersed in the fixative for 2 hr at 4” followed by washing in cold buffer and postfixation in 1% buffered OsO, for 2 hr at 4”. Following dehydration in graded alcohols, the pieces were infiltrated with Araldite or Epon- Araldite mixtures after passing through propylene oxide. Plastic blocks were sectioned with glass knives using a Porter-Blum MT 2 microtome set to give silver-gray or pale-gold sections. These were mounted on coated or uncoated 200 mesh grids and stained with uranyl acetate and lead citrate solutions. Thick sections from plastic blocks were stained with 1% methylene blue or toluidine blue in 1% borax solutions or methylene blue-basic fuchsin solution (Bennett t~f crl.. 1976) for study
and orientation.
The grids
ULTRASTRUCTURE
OF
STANNIUS
were examined in a Philips 201 electron microscope operated at 60 kV. White corpuscles from a total of five animals were considered for measurements of nuclear and cell size, and number and diameter of secretory granules. From each animal six representative C, and C1 cell types were selected for these measurements which were made from electron micrographs with an ultrastructure size calculator (Polaron). The diameter of at least 100 mature secretory granules was measured from the two cell types which were differentiated by various cytomorphological criteria.
RESULTS
The five to seven white corpuscles (corpuscles of Stannius) in L. platyrllynchus occur as round to oval structures around the midportion of each kidney and are associated with postcardinal and renal veins and, occasionally, the adrenocortical tissue, i.e., yellow corpuscles (Fig. la, Ic). They are recognized as well-vascularized whitish masses projecting slightly from the surface of the kidney and are confined to the middle region of the kidneys where little or no nephric tissue is apparent but where hemopoietic tissue is abundant. Their position within these cords of hemopoietic tissue is variable. On a few occasions they are juxtaposed with yellow corpuscles near the posterior cardinal veins but more commonly they are found isolated deeper within the hemopoietic tissue (Fig. lb). They range in diameter from 50 to 400 pm and in length from 300 pm to 2.0 mm. The white corpuscles are limited from the yellow corpuscles and the surrounding hemopoietic tissue by a capsule of collagen fibrils and fibroblasts. The glandular parenchyma of each white corpuscle consists of two-layered solid cellular cords or islets which are convoluted and which anastomose with each other (Fig. 2). Following immersion fixation the capillaries appear collapsed, but perfused organs reveal extensive capillaries irrigating all regions of the parenchyma. The cells are basophilic with hematoxylin and eosin and demonstrate a variable staining
CORPUSCLES
IN
GARPIKE
31
intensity with periodic acid-Schiff. Granules in the cytoplasm are periodic acidSchiff positive (Fig. lc) and are dispersed around the nucleus or more concentrated in two poles. A few nongranular cells with smaller nuclei also may be present. Ultrastrrrcturc
The lobules of granulated epithelial cells are bounded by a thin basal lamina and neighboring lobules of corpuscular cells are separated by an interstitium containing small and round or tortuous capillaries. unmyelinated nerve fibers. and connective tissue cells (Fig. 3). The endothelial cells in the capillary wall are fenestrated and rest upon a continuous basal lamina. The interlobular space also contains a few inconspicuous collagen bundles and a granular precipitate. Two types of epithelial cells make up the parenchymal population in each corpuscle and these are provisionally classified as C, and C, cells. A rough approximation showed that C, cells comprise about only 5- 10% of the total population in all animals. The C, cell is characterized by abundant cytoplasmic granules (Fig. 3) and is oval to round in shape (diameter, 7- 10 pm). It possesses a round central nucleus (average diameter, 4 pm) with one usually eccentric nucleolus and irregular patches of heterochromatin. The presence of a number of long parallel or slender wavy stacks of rough endoplasmic reticulum (RER) and isolated slender cisternae near the cell surface are identifying features of this cell type (Fig. 4). The cisternae of the RER are usually disposed as a branching interconnected network and may be continuous with perinuclear cisternae of the nuclear envelope. Free ribosomes are scattered throughout the cytoplasm. The Golgi apparatus consists of one or more sets of compacted saccules (Fig. 4) and small prosecretory granules are observed near their concave or lateral margins. The saccules may also possess electron-dense material apparently in
32
BHATTACHARYYA,
BUTLER,
AND
YOUSON
ULTRASTRUCTURE
OF STANNIUS
the process of condensation. Numerous smooth or coated vesicles are dispersed near the region of the Golgi complex and the cell surface. The mitochondria are round, oval, elongated, or irregularly branched with dense matrices, and lamellar cristae often oriented in parallel fashion; the mitochondria occur in a cytoplasmic area unoccupied by secretion granules. Other cellular constituents are randomly dispersed microtubules, bundles of filaments, lysosome-like dense bodies, autophagic vacuoles, and multivesicular bodies. The cell surface possesses coated pits often concentrated near the capillary zone. Secretory granules are noted in close proximity to the plasma membrane although no clear evidence of their involvement in exocytosis was observed. In some cells clear vesicles are present in the cytoplasmic area close to the pits of the plasma membrane. The presence of numerous (200-250/tell) secretory granules is a conspicuous feature of this cell type (Fig. 5). The granules are either distributed uniformly throughout the cytoplasm or they may show a heavy concentration toward the vascular pole. Their matrices are homogeneously amorphous and electron-opaque and are confined within a close-fitting membrane. The granules are mostly of round to oval shape and their diameter varies from 0.20 to 0.35 pm (average size about 0.25 pm). Granule appearance and distribution varies from cell to cell and may reflect functional activity. In a presumed storage phase, the cell is
CORPUSCLES
IN GARPIKE
33
loaded mostly with granules and a few protiles of the mitochondria and RER (Fig. 5). Other cells shows a reduced granulation, very prominent Golgi complex, numerous coated vesicles, multivesicular bodies, autophagic vacuoles, and polymorphic mitochondria. This latter form suggests a secretory or synthetic stage of this cell type (Fig. 4). Some animals had a few C, cells with a nucleus of irregular profile. slightly dilated cisternae of the nuclear envelope, and dense patches of heterochromatin (Fig. 91. The mitochondria of the cells possess electron-lucent matrices and cisternae of RER are distended and difficult to recognize, and are restricted to perinuclear positions. The secretory granules are clumped toward the cell periphery and their limiting membranes are often very dilated forming an electron-lucent halo around the granule. Other granules appear as small remnant of the dense granular mass and are frequently less dense in appearance. Some C, cells possessed numerous lysosome-like bodies and small multivesicular bodies. Interestingly, C, cells situated within a cluster of such transformed C, cells did not manifest this kind of alteration. The C, cell is oval (diameter, 6-8.5 pm), polygonal, or frequently triangular in section. It appears compressed between C, cells and it sends out elongated cellular processes between this more abundant cell type (Figs. 6, 7). Nuclei are oval (diameter about 5 pm) or irregular in profile and the nucleolus is usually inconspicuous. Com-
FIG. la. Transverse section of the middle of the kidney ofL. plaryrh~nchus demonstrating renal (R) and hemopoietic (H) tissues and the position of a white (WC) and yellow (YC) corpuscle with respect to the posterior cardinal vein (PCV). x40. FIG. lb. A light micrograph of a transverse section through the kidney of a garpike. A large white corpuscle (WC) maintains a close relationship to the posterior cardinal vein (PCV) and is completely surrounded by hemopoietic tissue (H). ~36. FIG. lc. An encapsulated white corpuscle (WC) is clearly separated from the cords of vacuolated (V) cells of the yellow corpuscle and hemopoietic tissue (H). The cells of the white corpuscle stain as dark (D) and light (L) cells with periodic acid-Schiff. x400. FIG. 2. A toluidine blue-stained plastic section through a perfused white corpuscle (WC). Cellular cords loaded with granules are profusely supplied with capillaries (CL H. hemopoietic tissue. x420.
34
BHATTACHARYYA,
BUTLER,
AND
YOUSON
FIG. 3. A survey electron micrograph of several lobules of epithelial cells from a corpuscle of Stannius. The predominant cell type (C,) is oval or round. possesses a spherical nucleus, and is filled with secretory granules (SC). A less abundant second cell type (C,) is compressed between C, cells. The C2 cells have irregular nuclear outline and fewer secretory granules. Connective tissue cells (CT) and a capillary (C) are present in the interlobular space. x 3800.
pared to C, cells, the cytoplasmic matrix is of low electron density and there is a paucity of cell organelles. In particular, slender, elongate RER cisternae are rarely observed but instead cisternae are short, dilated, and may possess a flocculent content of moderate electron density. The Golgi complex is very prominent, often ring-like in profile and may possess a number of associated enlarged vacuoles (Fig. 8). Dumbbellshaped mitochondria with dense matrices and a few parallel lamellar cristae, and free
ribosomes are commonly noted. The cell also contains many smooth and coated vesicles, small vacuoles, lysosome-like dense bodies, and a few small lipid droplets, the latter being of rare occurrence in C, cells. Secretory granules of C, cells are more widely scattered and fewer in number (501 cell) than those of C, cells (Figs. 6, 8). While the majority of granules are round or oval. others are rod-shaped or are irregular in profile and may show a variable electron
FIG. 4. Enlarged view of C, cells. The identifying characteristics of this cell are slender parallel arrays of RER (er) and a Golgi complex (G) with flattened sac&es. A number of coated vesicles (arrowhead) are present near the Golgi complex. The C2 cell shows an elongated nucleus and short dilated cisternae of RER (arrow). x 12,100. FIG. 5. A C, cell possesses mostly round, electron-dense secretory vesicles (SC), a few isolated profiles of RER (arrowhead). and mitochondria (M). x 11,700. 35
FIG. 6. A C2 cell appears squeezed between C, cells and is sparsely populated with secretory granules which are scattered and frequently rod-shaped (arrowhead). A prominent Golgi complex (G) and numerous mitochondria (M). with lamellar cristae, are also observed in the C, cells. C, cells possess many round secretory granules (SG) and elongated parallel profiles of RER (er). x7900. FIG. 7. A C, cell is depleted of secretory granules and displays RER organized in the form of short isolated cistemae (arrowhead). An adjacent C, cell is filled with granules. M, Mitochondria: I, interdigitation of the plasma membrane. x 11,900. 36
FIG. 8. A C2 cell with profiles of hypertrophied saccules of the Golgi complex (G). The cell contains a few secretory granules which are dispersed, and some of which appear in rod-shaped or commashaped profiles (arrowhead). Neighboring C, cells have clusters of larger secretory granules. x 11.900. FIG. 9. A group of transformed C, cells show nuclei of irregular outline with dilated nuclear envelopes. The mitochondria (M) appear pale due to loss of matrix density and cisternae of the RER are dilated (arrowhead). A Cz cell appears of normal configuration. Compare with Fig. 3. x5200. 37
38
BHATTACHARYYA,
density. The size of the round granules varies from 0.10 to 0.25 pm, the average size being 0.15 pm. Their dense matrices may incompletely fill up the membranous vesicle or may be eccentric in location. A few large granules have granular or net-like matrices with a partly electron-dense content, which is often separated from the limiting membrane by a large electron-lucent space. A number of C, cells possess a totally granule-depleted cytoplasm and a large number of RER sacs and free ribosomes (Fig. 7). Others are characterized by numerous mitochondria, intercommunicating short cisternae of RER, numerous polyribosomes, a conspicuous Golgi apparatus with dilated vacuoles, numerous multivesicular bodies, smooth and coated vesicles. parallel bundles of microtubules, and a few small secretory granules. This ultrastructural picture may indicate actively synthetic or secretory stage of the C, cell.
BUTLER,
AND
YOUSON
species may development pear in Amia ric duct and
also be related to the ontogenic of this organ, since the CS apas outgrowths of the pronephopisthonephric tubules, but in Lepisostelrs spp., originate only from the pronephric duct (De Smet, 1962). A physiological explanation is lacking to account for this extremely large number in the bowfin and its gradual evolutionary concentration in other bony fishes. Based on ultrastructural criteria, such as the arrangement and profiles of the Golgi apparatus and RER. especially the size, shape, and abundance of secretory granules, two cell types seem to be a characteristic of this organ in the garpike. The existence of two cell types has been documented in the CS of the stickleback. Gasterosteus awleatus. the European eel, Atzguilfa arlguiffa, the killifish, Flrtlciulrrs hetc~rocfitlrs. the goldfish, Carassius auratus (Wendelaar Bonga and Greven, 1975; Wendelaar Bonga et al., 1980), the trout, Salmo gairdrzpri (Krishnamurthy and Bern, DISCUSSION 1969; Meats et al., 1978). and the coho salThe present study shows that the white mon, Otzcorhynchus kisrrtc.h (Aida et ~1.. corpuscles within the kidneys of the garpike 1980a). They are present as principal (type are the corpuscles of Stannius (CS) which C,) and a rare (type C,) cell types (Meats et have been recognized in another holostean al.. 1978). However, other teleost species, (Youson and Butler, 1976; Youson et ~1.. i.e.. the Atlantic salmon, Safmo salar (Car1976) and in teleosts (Oguri, 1966; To- penter and Heyl, 1974), the guppy, Lchistcs masulo er al., 1970; Cohen cjt al., 1975). rrticulatrrs (Tomasulo et (IL., 1970), the cod, However, certain morphological features of Gadus tnorrhrra, the plaice, Pkut-ot~~~ctcs the CS in the garpike are strikingly different platessa (Wendelaar Bonga and Greven, from the CS of its closest holostean rela- 1975), and the toadfish, Opsarllrs talr tive, the bowfin. In the latter species, the (Bhattacharyya and Butler, 1978), are corpuscles are widely scattered throughout characterized by the presence of a single the kidney, which is believed to be the more cell type with large secretory granules. The ancestral organization that ultimately cul- presence of two cell types (C, and C,) in the minated into more compactness and poste- garpike is noteworthy, since the other rior position in Teleostei (Garrett, 1942; De holostean. the bowfin (Youson and Butler. Smet, 1962: Youson et al., 1976). In con- 1976) possesses only a single cell type. trast to the bowfin, the garpike manifests a The C, cells of the garpike are uldistinctive reduction in number and re- trastructurally comparable to the principal gional concentration of the CS indicating an cell type noted in most teleosts and indicate intermediate phylogenetic trend among the possible endocrine function of this bony fishes. However, this difference in organ. Generally, the cytological profile of gross anatomic distribution in these two the CS reveals that the corpuscular cells are
ULTRASTRUCTURE
OF
STANNIUS
packed with mitochondria, Golgi saccules, RER, and numerous secretory granules. The latter structures arise from the Golgi apparatus in other fishes (Youson and Butler, 1976; Wendelaar Bonga et al., 1977; Bhattacharyya and Butler, 1978) and signify a mechanism in the CS which is consistent with the secretion of protein or polypeptide hormones (Fawcett ct al., 1969). The morphological evidence for endocrine secretion is also indicated by a profuse vascular investment, there being an extensive capillary network supplying all regions of the glandular tissue. Ultrastructural studies have not clearly documented the mode of discharge of secretory granules from the CS under normal or stimulated conditions. However, the preferential location of the secretory granules toward the capillary pole. and the presence of coated pits on the cell surface suggestsan exocytotic mode of discharge of granules as is generally observed in protein-secreting endocrine glands (Wendelaar Bonga et al.. 1977: Bhattacharyya and Butler, 1978). However, it has been proposed that corpuscular cells may disintegrate during stress periods in hyperosmotic medium resulting in release of intact protein granules into capillaries (Meats ct NI., 1978). The presence of secretory granules in extracellular spaces in the bowfin was believed to be related to mechanical injury (Youson and Butler, 1976). In the present material there was no evidence of the apocrine or holocrine secretion by the glandular cells that was often described in earlier literature (Lopez, 1969). Therefore, exocytosis may be the principal mode of liberation of secretory vesicles after their origin and packaging from the Golgi region. The secretory activity of the C, cell is apparently related to environmental calcium concentration, the cells being more active in animals in seawater than in fresh water (Wendelaar Bonga ct crl., 1976; Meats cr al.. 1978; Olivereau and Olivereau, 1978: Wendelaar Bonga. 1980: Aidart ~1.. 1980a).
CORPUSCLES
IN GARPIKE
39
The presence of some transformed C, cells in the garpike is difficult to explain but may indicate a possible functional hypoactivity of this cell type in fresh water leading to cellular degeneration. This is also supported by the observation that most of the C, cells seemed to be in an inactive or a storage phase of the secretory cycle because they contained a heavy accumulation of secretory granules together with a paucity of cellular organelles. On the other hand, these transformed cells of the garpike CS also resemble degenerating parenchymal cells of the salmon CS (Carpenter and Heyl. 1974) during the spawning migration from seawater to fresh water. Furthermore, during a study of the CS under freshwater and seawater conditions, cellular degeneration of the mullet (Mlrgi/ cc>ph~lrrs) CS was observed in fresh water but not in seawater (Johnson, 1972). The C, cells in the garpike have a general morphological similarity and a relative scarcity which is characteristic of the second cell type found in other teleosts (Krishnamurthy and Bern, 1969; Wendelaar Bonga and Greven. 1975: Meats et ~1.. 1978; Aida PI a/.. 1980a). In stickleback corpuscles, activity of the C, type cell is a function of the concentration of sodium and potassium ions in the media (Wendelaar Bonga et al., 1976) while the C2 cells of the trout CS are active in media of low ionic and osmotic strength and relatively inactive when the salinity increases (Meats et al., 1978). On the other hand, type 2 cells in the coho salmon are more active in the seawater smolt and are inactive in fresh water (Aida et al., 1980a). As a contrast with C, cells of the garpike, the C, celis display a greater degree of cytologic variability, they did not appear to be in an inactive storage phase loaded with secretory granules, and presented no evidence of cellular degeneration. There is no documented evidence of corpuscular participation in electrolyte metabolism among holostean fishes, and it is unclear whether the two types of cells in
40
BHATTACHARYYA,
BUTLER,
this species release different endocrine factors, or they represent alterations of the same cell type. The functional significance of the garpike CS and its cell types presently remains speculative. ACKNOWLEDGMENTS This investigation was supported by Grant A2359 to D. G. Butler and Grant A5945 to J. H. Youson from the Natural Sciences and Engineering Council of Canada.
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