ultrastructural studies on the coronet cells of the saccus vasculosus of the freshwater stickleback,...

Z. Zellforsch. 147, 551--565 (1974) © by Springer-Verlag 1974

Uhrastructural Studies on the Coronet Cells of the Saccus vasculosus of the Freshwater Stickleback,

Gasterosteus aculeatus form leiurus*

Michael Ben jamin **

Department of Zoology, University College of Wales, Aberystwyth

Received December 17, 1973

Summary. EM studies on the coronet cells of the freshwater stickleback showed that the cells were characterised by abundant SER, perinuclear fibrils, microtubulcs, free ribosomes and mitochondria. Acid phosphatase-positive dense bodies were present, but there was no alkaline phosphatase activity nor were there any glycogen granules in the coronet cells. On the surface of the coronet cell and its globules was a layer of Ruthenium red-stainable material which almost completely disappeared when the fish were kept in 70 % seawater for 1 week. Quantitative E ~ of fish kept in freshwater or 70 % seawater for 1 week showed that the SEI~ tubules of seawater-maintained fish had a wider lumen than those of freshwater-maintained fish. The volume occupied by the Golgi apparatus also increased but there was no change in the volume of the cytoplasm occupied by mitochondria. Both the morphology of the coronet cells and their response to 70% seawater support the hypothesis that the saccus vasculosus is an osmo(iono)regulatory structure.

Key words: Saccus vasculosus - - Freshwater stickleback - - Coronet cell - - Seawater adaptation - - Electron microscopy, morphometry.

Introduction The saccus vasculosus is an ependymal s t ructure tha t protrudes from the

caudal in fundibular wall of the diencephalon immedia te ly behind the p i tu i t a ry gland in some teleosts and elasmobranchs (cf. Dorn 1955). Structural ly, the most highly specialised cells in the saceus vasculosus are the coronet cells or crown cells. Current hypotheses on the funct ion of the saceus vasculosus are therefore largely concerned with these cells.

Since the earlier EM invest igat ions of Ba rgmann and Knoop (1955) and Kurota - ki (1961), detailed u l t ras t ruc tura l studies on the coronet cells have been confined to the ray, Dasyatis aka]ei (Watanabe, 1966) and the ra inbow trout , Salmo gairdneri irideus (Jansen and Flight, 1969), al though Lanzing and Lennep (1970) have surveyed a large n u m b e r of teleosts belonging to different orders. There has been a great debate as to whether the saecus vasculosus is a sensory or secretory structure. Several authors have proposed a sensory funct ion, e.g., D a m m e r m a n n (1910), Kuro tak i (196 l) and Harraeh (1970) and others have proposed a secretory function, e.g., B a r g m a n n (1954), Kamer and Verhagen (1954), Ba rgmann and Knoop (1955), Stahl and Scite (1960), Kamer et al. (1960), Jansen and K a me r (1961), Sundaraj and Prasad (1963), Kamer (1965), K h a n n a and Singh (1967) and Marquet et al. (1972).

* This work formed part of a thesis submitted for the degree of Doctor of Philosophy in 1973 and for which the author was in receipt of an S.R.C. studentship. ** Present adress: Department of Cellular Biology and Histology, St. Mary's Hospital Medical School, Paddington, London W2.

552 M. Benjamin

Recently, J a n s e n (1969) and Jansen and West (1971) proposed tha t the saccus vasculosus t ranspor ts cations, while Emanuelsson and Mecklenburg (1972) and Mecklenburg (1973) have suggested an osmoregulatory funct ion for the saccus vasculosus. I n m a n y of the a t t empts to discover the funct ion of the saccus vasculosus, the exper imental an imal has been the ra inbow trout , Salmo gairdneri irideus (Jansen and Kamer , 1961; Jansen , 1969; J ansen and Flight, 1969; Jansen and West, 1971; Emanue lsson and Mecklenburg, 1972; Mecklenburg, 1973). I t is ob- viously impor t an t to s tudy as m a n y fish as possible in order to avoid a ny generali- sat ion tha t one species is the same as another.

The present s tudy a t t empts to establish basic u l t ras t ruc tura l da ta on the coronet cells of Gasterosteus aculeatus form leiurus--a hitherto uninves t iga ted spec ies - -and to invest igate the effect of a highly saline env i ronment on the coronet cells. Because most changes in the funct ional state of highly specialised cells are accompanied by quan t i t a t ive ra ther t h a n qual i ta t ive al terat ions in ul tra- s t ructure (Weibe], 1972), the morphometr ic methods of Weibel (1969) were used to assess the effect of altering the env i ronmenta l salinity.

Materials and Methods Collection o] Fish. To avoid any possibility of variation in saccus vasculosus structure

because of seasonal differences or sexual immaturity, all the fish used in this study were adults collected during the months of June to August. The sticklebacks were collected by hand netting in the river Rheidol, Cardiganshire, about 1 mile from its mouth at Aberystwyth harbour. They were transported to the laboratory in rivel~vater in large polythene containers, and kept until needed in glass tanks that contained 20-30 litres of well-aerated tapwater.

Electron Microscopy Material was fixed at 5°C in 3 % glutaraldehyde in either s-co]lidine or cacodylate buffer at pH 7.4, rinsed overnight in buffer and post-fixed in 1.33% buffered osmium tetroxide. The material was then dehydrated in graded alcohols, treated with propylene oxide and embedded in TAAB resin. Ultrathin sections were cut on an LKB ultrotome, double stained in lead citrate (Reynolds, 1963) and uranyl acetate and examined on an AEI EM6 B electron microscope. In order to localize acid mucopolysaccharides ultrastructural]y, Ruthe- nium red (TAAB laboratories) was added to both the glutaraldehyde and osmium tetroxide, according to the method of Anderson (1968). Acid phosphatase was located by the method of Holt and Hicks (1962) using Gomori's incubation medium. However it was not necessary to cut frozen sections of the saccus vasculosus as the latter is very small and consists essentially of a single layer of cells. The whole saccus vasculosus was placed in the incubation medium. To demonstrate glycogen granules ultrastrueturally, sections were placed in 2% periodic acid for 30 min, rinsed in double distilled water and stained in lead citrate for 30 min (Perry, 1967). The cadmium incubation method of Mizutani and Barrnett (1965) was used to demonstrate alkaline phosphatase activity.

Experimental Studies. To investigate the effect of salinity changes on coronet cell ultrastructure, 10 adult sticklebacks were acclimatized to laboratory conditions for 1 week before the experiment and then 5 fish were transferred to 70 % seawater, the other fish were transferred to another tank containing freshwater. Material was prepared for electron microscopy as before, and 20 random electron micrographs of the coronet cells (4 from each animal) from animals of both groups were taken at X20000. The final magnification of the printed micrographs was x 40000. The relative area of the micrographs occupied by the various organelles shown in Table 1 was determined by point counting volumetry (Weibel, 1969). Membrane profile concentrations of the smooth endoplasmic reticulum (SEI~) were estimated by the method of Weibel (1969). Significant differences between the 2 groups were assessed by Student's T test.

In addition, the saccus vasculosi of a further 10 sticklebacks that had been kept in either freshwater or 70 % seawater as above, were stained with Ruthenium red and examined without further staining.

Saccus vasculosus of the Freshwater Stickleback 553

Table 1. Organelles in the coronet cell of G. aculeatus, form leiurus selected for analysis

SER - including membranes and cavities Golgi apparatus - flattcne4 lamellae and vesicles Multivesicular and Dense bodies - considered jointly Mitochondria Free ribosomes

R e s u l t s

Morphological Studies

The numerous coronet cells were the most conspicuous feature of the saecus vasculosus of the freshwater stickleback. They were separated from one another at their apical poles by supportive cells (Fig. 1) and were in direct contact with the cerebrospinal fluid tha t filled the lumen of the saceus. At their basal surfaces, the coronet cells were separated f rom the sinuses by a basement membrane, and by the cytoplasm of the mesenchymal cells tha t bordered the sinuses.

The nucleus of the coronet cell was an oval structure in the basal region of the cell. I t contained little heterochromatin, but a conspicuous nucleolus tha t was si tuated peripherally (Fig 2). Bundles of fine fibrils were present t ha t often en- compassed as much as half the circumference of the nucleus (Fig. 2). There were numerous mitochondria throughout the cel l--part icularly at either pole (Figs. 3, 4). Typically they contained 1 or 2 small, dense granules in their matr ix (Fig. 4). Small tubules of SEI% and groups of free ribosomes were profusely scattered throughout the cell (Fig. 5). Interspersed between the tubules of SEt~ were numerous fibrils and microtubules tha t were mainly directed towards the head region of the coronet cell. The Golgi apparatus was poorly developed and consisted of a few vesicles and flat tened eisternae, al though multivesicular and dense bodies were common in the apical port ion of the cell (Fig. 6). Both dense and multivesicular bodies varied great ly in size, but were usually oval or round, and rarely of irregular shape. Some coronet ceils contained isolation bodies, i.e., concentric whorls of paired, smooth-surfaced membranes tha t surrounded a number of organelles e.g., mitochondria, ribosomes. I n both the apical and basal regions of the coronet cell there were small R E R tubules, while aeanthosomes (coated vesicles) were sometimes found in the apical regions.

The coronet cell bulged out into a rounded head region at the level of the free surface of the supportive cell. This head region bore on its surface occasional microvilli and free cilia and numerous stalked globules (Fig. 3). A transverse section of the stalk or globule showed a 9d-0 arrangement of microtubules similar to tha t in non-motile cilia (Fig. 7). Also present in the globules were numerous vesicles, whose membrane on its cytoplasmic side had a fuzzy appearance. This was due to numerous bristle-like structures tha t were evenly spaced and projected perpendicularly into the cytoplasm (Fig. 7). These bristles were approximate ly 10-20 nm in length, with a centre to centre spacing of 20-25 nm. They were best observed in thin sections exactly perpendicular to the cell membrane. There was great variat ion in the arrangement of the vesicles. Sometimes these were great ly enlarged and occasionally they were f lat tened into parallel arrays of cisternae.

37 Z Zellforsch., Bd. 147

2

Fig. 1. Portions of two coronet cells (C) separated by a wedge-shaped supportive cell (S). G globules, L lumen of saccus v~sculosus. × 11200

Fig. 2. A bundle of perinuclear fibrils (arrows) in a coronet cell. N U C nucleolus. ×45000

3

S~ccus vasculosus of the Freshwater Stickleback 555

4

Fig. 3. The head region of a coronet cell bearing numerous stalked globules (G) and containing a conspicuous group of mitochondria (M). × 20000

Fig. 4. A group of mitochondria (M) in the basal region of a coronet cell. I M G intra-mitochondrial granule, N nucleus. × 45000

37*

6

7 Fig. 5. SEI~ and ribosomes (R) in the main body of a coronet cell. X 45000

Fig. 6. Golgi vesicles (V) and cisternae (C) in a coronet cell. MVB multivesicular body con- raining small vesicles (SV) and dense material (D). X 60000

Fig. 7. The globules of the coronet cell. Note the bristle-like structures (arrows) on the membranes of the globules (G) and the vesicles (V). M 9 ~-0 arrangement of microtubules. X 45 000


Table 2. Percentage of the coronet cell volume (excluding the nucleus and globules) occupied by various organclles (means 4- s.e.)

Organelle Animals Animals maintained maintained in in 70% seawater freshwater

SER membrane space 32.6 -[= 1.85 22.0 ± 1.43

Golgi apparatus 6.7 i 1.09 3.6 4- 0.65

Multivesicular and Dense bodies 2.9 4-0.77 2.3 4-0.33 Mitochondria 9.2 4-0.91 7.2 4-0.73 Free ribosomes 7.3 4-0.56 3.9 ±0.48

Where there were significant differences between 2 means (P > 5 % ), these means were under- lined.

Although dense material did occur in the vesicles, the latter were usually empty. Basal bodies were common at the point of a t tachment of the stalk to the rounded head of the coronet cell, but only rarely did they have striated rootlets. Around the basal bodies were characteristic perieentriolar bodies or satellites. Globules, in what were assumed to be various stages of formation were seen on the coronet cell surfaces. Numerous free globules, together with occasional lymphocytes and phagocytes were present in the saccus lumen. "Da rk" coronet cells were sometimes found in the saccus epithelium. These differed from the normal coronet cells in having a darker ground substance and a more conspicuous dilation of the SEI{. The globules of the dark cells also had a correspondingly denser matr ix than those of the light ceils.

The Go]gi apparatus contained small and varied amounts of acid phosphatase- positive material and the dense and mu]tivesicular bodies contained large amounts (Fig. 8). Occasionally, acid phosphatase act ivi ty was noticed at the free surface of the coronet ee]l directly beneath the plasma membrane, while some of the globules (both those a t tached to the cell and those lying free in the lumen) showed intense acid phosphatase act ivi ty in a region tha t corresponded to the fuzz coat (Fig. 9). No par t of the coronet cells contained any conspicuous glycogen deposits or showed any alkaline phosphatase activity. The main morphological features of the coronet cell of the freshwater stickleback are summarised in Fig. 10.

Experimental Studies The results of the morphometr ic analysis of the coronet cells of animals kept

in freshwater or 70 % seawater for 1 week are summarized in Table 2. Animals maintained in the hypertonic medium showed a marked increase in the amount of cytoplasm of the coronet cell occupied by SER membrane space al though the membrane profile concentrat ion remained the same (seawater, 2.2~ 0.16; freshwater, 2.2~:0.13). Similarly, there was no significant change in the cytoplasmic volume occupied by multivesieular and dense bodies or by mitochondria, a l though there was a large increase in the volume of the Golgi apparatus, and in the number of free ribosomes.

558 M. Benjamin

8

9 Fig. 8. Acid phosphatase-positive dense bodies (arrows). × 30000

Fig. 9. Acid phosph~tase-10ositive material (arrows) in a globule lying free in the saecus v~sculosus lumen. × 45 000

The layer of R u t h e n i u m red-s ta inable ma te r i a l a round the head and globules of the coronet cells of control an imals kep t in f reshwater for 1 week a lmos t com- p le te ly d i sappea red when fish were kep t in 70 % seawater for 1 Week (Figs. 11, 12). I n s eawa te r - adap t ed animals , R u t h e n i u m red-s ta inable ma te r i a l r emained in the in terce l lu lar spaces and was occasional ly present in t race l lu lar ly . This perhaps


Fig. 10. A drawing of a typical coronet cell. A acanthosome. C cilium. DB dense bodies. D desmosome. F fibrils. GL globule. GO Golgi apparatus. I intra-mitoehondrial granules. I S isolation bodies. M mierovillus. M I C mierotubules. M I T mitochondria. M V B multivesi-

cular body. N nucleus. P perinuclear fibrils. R ribosomes. SC supportive cell

indicates t ha t the difference in the dis t r ibut ion of the stain in the 2 groups of

sticklebacks was not caused by a leaching out of s tain from the saccus vaseulosus

of the seawater -adapted animals.

Discussion

According to Bock (1928) the st ickleback lacks a saccus vasculosus. No ex- planat ion can be offered for this s t a t ement as all the freshwater st icklebacks used

560 M. Benjamin

11

12 Fig. 11. Ruthenium red-stainable material in the surface of the coronet cell and globules

(arrows) from a stickleback kept ia freshwater for 1 week. × 45000

Fig. 12. Absence of Ruthenium red-stainable material from the surface of the coronet cell and globules. Note the presence of Ruthenium red deposits on some vesicles within the globules

(arrows). X 30000

in this investigation always had a typical saccus vaseulosus. Furthermore, the migra to ry stickleback, G. aculeatus form trachurus, also has a saeeus vaseulosus

(Dorn, 1955, lit., Mullein, 1959; Benjamin, 1973).

Saccus vaseulosus of the Freshwater StieMeback 561

Bundles or whorls of fibrils around the nucleus (i.e., perinuclear fibrils) were characteristic of the coronet cells in the freshwater stickleback. Most authors have noticed a small number of fibrils in these cells in other animals, though more conspicuous groups have been recorded in the ray (Watanabe, 1966) and in Salmo trutta, Anguilla australis and Cnidoglanis macrocephalus (Lanzing and Lennep, 1970). There are also perinuclear fibrils in peritoneal maerophages (PetrJs et al., 1962), rat ependymal cells (Brightman and Palay, 1963), epithelioid cells (Sutton and Weiss, 1966), lymphoblasts and myeloblasts (Anderson, 1966), and leukaemic cells (Ross and Harnden, 1969). Among the roles that have been postulated for perinue]ear fibrils are a transport role, by the provision of a surface for the move- ment of substances or organel]es, and a "skeletal" role in mMntaining the con- figuration of the cell against such distortions that a transport of substances within the cell may produce (Sutton and Weiss, 1966).

Sundaraj et al. (1966) have proposed that the function of the saeeus vaseulosus in Notopterus notopterus is to regulate the amount of glucose in the eerebrospinM fluid. They suggest that the coronet cells of this fish convert glycogen to glucose. In view of the complete absence of glycogen from the coronet cells of the stickleback, this hypothesis cannot be considered applicable to M] teleost species.

The dense bodies in the coronet cells of the freshwater stickleback were acid phosphatase-positive. Although dense bodies are a common feature of coronet cells (Watanabe, 1966; Jansen and Flight, 1969; Marquet et al., 1972) only Zim- merman and Altner (1970) and Lanzing and Lennep (1970) have similarly demonstrated acid phosphatase activity. According to Jansen (1969) there is no acid phosphatase in the saeeus vaseulosus of Salmo gairdneri. As the dense bodies showed no signs of fusion with autophagic or phagocytic vacuoles, it is difficult to speculate on their function.

The polarisation of mitoehondria in the coronet cells of the freshwater stickleback has also been found in eels and bullheads (Kurotaki, 1961) and in Notopterus (Sundaraj and Prasad, 1963). However, in Salmo gairdneri (Jansen and Flight, 1969) there is a clear zone at the base of the coronet cell. ']2here were conspicuous intramitoehondrial granules in the coronet cells of the freshwater stickleback. Although Jansen and Flight (1969) noticed them in Salmo gairdneri, only a few species of fish contain these granules in their mitoehondria (Lanzing and Lennep, 1970). When Roullier (1960) looked for a pat tern in the distribution of these granules in various tissues, he found that they were espeeiMly prominent in tissues concerned with active transport. Peaehey (1965) considers them to be cation binding sites tha t accumulate divalent cations.

I t has recently been suggested that the saeeus vasculosus is an osmo(iono)- regularory organ (Jansen, 1969; Jansen and West, 1971; Emanuelsson and Meck- lenburg, 1972). Considering that aquatic environments can range from freshwater to hypersaline media, teleosts maintain their osmotic pressure remarkably con- stant. I t is thought tha t the saceus vasculosus regulates the osmotic or the ionic conditions in the eerebrospinM fluid. When the saceus vasculosus is viewed in this light, a functional interpretation can be suggested for some of the specialised cytological features in the coronet ceils. The intramitochondrial granules and their role as possible cation binding sites in several tissues have already been mentioned.

562 M. Benjamin

In spite of their basically similar molecular architecture, cell membranes are highly specialised physiologically, particularly as regards the selective absorption of non-electrolytes and electrolytes by energy-dependent transport processes. Various investigators have suggested that at least some of these properties may be determined by additional coats of macromolecules on one or both surfaces of the bimolecular leaflet. Gupta and Berridge (1966) noticed a regular array of subunits on the cytoplasmic surface of the plasma membrane in the rectal papillae of Calliphora erythrocephala. The rectal papillae are believed to be engaged in active transport. Anderson and Harvey (1966) found that extensive areas of the plasma membrane in goblet cells actively transport massive amounts of potassium ions. Kallio et al. (1971) showed tha t the membranes at the ruffled borders of mammalian osteoclasts have a similar coating. Osteoclasts are ion transporting cells, as they play a major role in the resorption of bone. In view of all these findings it is noteworthy that the globules of the coronet cells have a membrane with a similar morphological specialisation.

In addition to the spine-like projections on the cytoplasmic side of the globule membranes, there was a layer of Ruthenium red-stainable material on the outside of the globules. According to Luft (1964) an opaque layer of Ruthenium red material at the surfaces of cells shows that acid mucopolysaccharides are present. Bennett (1963) coined the term "glycocalyx" to describe the glycoprotein and mucopolysaccharide coats tha t exist at the surface of many different types of cells. The glycocalyx is thought to be a dynamic structure with various physiological roles. Using colloidal iron solutions, Lanzing and Lennep (1970) demonstrated an acid mucopolysaecharide coat lining the luminal surface of the globules of Perca- lates colonorum. Tani and Ametani (1971) have pointed out that the most notable properties of mucopolysaccharides are their high viscosity, hydration ability, ion binding characteristics, ability to regulate diffusion, and their interaction with macromoleeules, According to Katchalsky (1964) surface coats can be regarded as polyelectroly~es. They can render large quantities of ions inactive by immobi- lising them in the vicinity of their charged groups. Over a wide range of ionisation, such molecules oppose any changes in osmotic pressure, and therefore act as an osmotic buffer.

The disappearance of this l%utheninm red layer from animals kept in seawater suggests an alteration in the physiological state of the cell. I t is well known tha t the cell membrane of non-growing cells is constantly being replaced at a remarkable rate (Warren and Glick, 1968) and thus the complete disappearance of a glycocalyx within one week would seem feasible. Torpier and Montagnier (1970) showed tha t B H K 21/13 cells transformed by various oneogenic viruses have a greater affinity for Ruthenium red at their cell surface than normal cells. Paintrand and Rosenfeld (1972) noticed an increased thickness in the Ruthenium red layer in leueemie leukocytes. In the intermediate segment of the nephron in Xenopus laevis there is a highly developed intracellular channel system filled with acid mucopolysaeeharides. Jonas and g6hlich have found tha t the intracellular channels are strongly underdeveloped and contain little acid mucopolysaecharide in animals kept in saltwater for 2 months, but tha t the contents return when the animals are kept in freshwater. These are essentially similar to the changes shown in the glycocalyx on coronet cells of the freshwater stickleback.


Several authors have suggested tha t the saccus vasculosos secretes acid mucopolysaeeharides (e.g., Kamer et al., 1960; Jansen and Kamcr , 1961; K h a n n a and Singh, 1967; Emanuelsson and Mecklenburg, 1972). Emanuelsson and Mecklen- burg (1972) proposed tha t this secretion accounts for the mechanism of osmo- regulation. According to these authors the coronet cells of marine fish and of freshwater fish kept in seawater actively secreted acid mucopolysaccharides into the cerebrospinal fluid. If these findings apply to the stickleback, it would seem tha t the coronet cells of this fish lose their glycocalyx when they actively secrete acid mucopolysaccharides.

The u l t ras t ruc tura l changes within the coronet cells when sticklebacks were kept in 70 % seawater for one week support the idea tha t the saecus vaseulosus is osmo(iono)regulatory. The dilated cavities of SER and the large Golgi appara tus suggest tha t the coronet cells are more active in seawater t han freshwater. Meck- lenburg (1973) kept ra inbow t rout in seawater and also found tha t this t r ea tmen t enlarged the Golgi apparatus. As in the present invest igat ion, he also found tha t there was a striking increase in the number of free ribosomes in seawater-maintained fish.

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Dr. Michael Benjamin Department of Cellular Biology and Histology St. Mary's Hospital Medical School Paddington, London W 2 England

ultrastructural studies on the coronet cells of the saccus vasculosus of the freshwater stickleback,...

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