The Coelomocytes of Holothuria leucospilota

Avariety of free cells, known generally as coelomocytes, occurs in the coelomic fluids, haemal systems, and water-vascular systems, and amongst the tissues of holothurians. These cells have been investigated by numerous workers and the results of these investigations are well summarized by Hyman (1955). Although the structure and distribution of each of the various cell-types have received considerable attention, little information is available on the chemical constitution and role of each.

The present paper is concerned with investigations made on the coelomocytes of Holothuria leucospilota Brandt. Attention is focused on aspects of their structure and chemical constitution which might have a bearing on the biosynthesis and deposition of collagen fibres within the Cuvierian tubules.

Specimens of H. leucospilota were obtained, initially, from Heron Island, in the Capricorn Group, Queensland, and subsequently from Caloundra, southern Queensland.

Coelomic fluid surrounds the organs lying in,the body-cavity. The fluid was obtained for study as follows. A specimen was picked up and ejection of its Cuvierian organs prevented by placing a spring-loaded steel paper-clip over its anal end. Grit and sea-water adhering to the animal were removed with a cloth and a longitudinal incision made through the body-wall, in an oral direction from a point midway between oral and anal ends of the animal. The coelomic fluid which ran from this incision was collected in a beaker. If the whole operation was carried out rapidly, the procedure followed minimized the chances of the cloaca! wall rupturing and of the exposure of the sticky Cuvierian tubules. Adult specimens of average size yielded 50 to 80 ml of cloudy coelomic fluid.

Cuvierian tubules and portions of the stem of the respiratory trees were removed from specimens and fixed variously with Bouin’s fluid, Heidenhain’s ‘Susa’, and 5% formalin in sea-water. Sections were stained with Mallory’s aniline blue collagen stain and with Heidenhain’s ‘Azan’.

Coelomocytes present in the coelomic fluid showed a marked tendency to clump together when coelomic fluid was withdrawn from the body of the holothurian and this vitiated attempts to make accurate counts of coelomocyte numbers by the use of a haemocytometer. Centrifugation of the coelomic fluid yielded a greyish mass of coelomocytes. The average volume occupied by coelomocytes from ro ml of coelomic fluid which had been centrifuged at 3,000 r.p.m. for 5 min was 0·08 ml.

Morula-shaped cells (fig. 1) usually comprised about 70% of the total number of coelomocytes found in the coelomic fluid. These cells appeared to correspond with the ‘migratory plasma cells’ of Hamann (1883), the ‘cellules muriformes’ of Herouard (1889) and of Cuenot (1891), and the ‘colourless amoebocytes with spherules’ of Theel (1921) and of Hyman (1955). They also bore a striking resemblance to the ‘ferrocytes’ of the ascidian, Pyura stolonifera (Heller) (Endean, 1955a).

The morula-shaped cells were colourless and spherical at rest. They ranged from 8 to 16 μ, in diameter. Each contained a variable number of refractile globules encased in cytoplasm. In their natural position the globules were mutually compressed, but if the cells were cytolysed with distilled water, the globules that burst forth were spherical. The globules averaged about 1 μ, in diameter, but in the larger cells many were as large as 2 μ.

Observations with the phase-contrast microscope on cytolysing cells revealed that the emitted globules were accompanied by a granulated viscous material which apparently surrounded them in the intact cell. An eccentrically placed nucleus, about 2 · 5 μ, in diameter, was also revealed; this was obscured in life by the globules.

The larger morula-shaped cells (13 to 16μ in diameter) were amoeboid. Blunt pseudopods were put out which extended 2 to 3 μ, from the cells. Into these pseudopods the globules eventually flowed, rolling over one another in the process. The smaller morula-shaped cells did not exhibit such activity and were spherical.

With neutral red (0 · 0001% in sea-water), the smaller non-amoeboid cells stained bright red whilst the larger ones stained brownish red. In both cases when cells so stained were cytolysed with distilled water the globules that emerged were colourless. It would appear that it is the granulated material surrounding them in the intact cell which takes up the stain.

With methylene blue (0 · 0001% in sea-water), the smaller cells stained greenish blue and the larger ones purplish. When cells so stained were cytolysed with distilled water the globules that emerged in both cases were stained a pale blue. It is believed that the granulated fluid normally surrounding the globules is responsible for the metachromasy exhibited. The nuclei of the cells became evident when the obscuring globules were released by cytolysis and these nuclei stained faintly with methylene blue. It was noted that the nucleus was relatively larger in the smaller cells than in the larger ones.

Methyl red indicator was added to a drop of coelomic fluid but it did not penetrate into the interior of any of the coelomocytes.

Morula-shaped cells, identical with those found in the coelomic fluid, occurred in abundance amongst the fibres of the connective tissue in the walls of the respiratory trees and papillae from which the Cuvierian tubules arise. Similar cells occurred also in the central core of the tubules and amongst the fibres present in the tubules. In the tubules, however, the morula-shaped cells were usually large amoeboid ones. (See Endean, 1957.)

The morula-shaped cells appeared to enter the core of each tubule by migrating from the connective tissue of the papilla.

Homogeneous amoebocytes. These cells were from 4 to 5 μ in diameter and consisted essentially of a large nucleus (3 · 5 to 4μ in diameter) containing many chromatin granules and invested by a thin cytoplasmic envelope (fig. 2, A). Usually these cells were spherical but some possessed filamentous pseudopodia (fig. 2, B).

These cells seem to be the primitive cells in holothurian coelomic fluid and they may give rise to other cell types. Transitional stages from homogeneous amoebocyte to morula-shaped cell were commonly found. This transition seemed to occur as follows:

1. Numerous minute vacuoles appeared in the cytoplasm of a homogeneous amoebocyte (fig. 2, C). These vacuoles stained blue with dilute methylene blue but did not stain with dilute neutral red. (In some cases two or three small bodies which stained with neutral red (possibly nucleoli) were observed against the nucleus.)

2. The vacuoles enlarged and there was a concomitant increase in the size of the cell containing them.

3. The vacuoles continued to enlarge and each gradually acquired a peripheral layer of refractile material which stained red with neutral red (fig. 2, D).

4. By the time each vacuole had acquired a refractile layer the cells had attained a diameter of 8 μ and were identical with the smaller morula-shaped cells.

The nucleus becomes smaller during these transition stages and it is possible that nuclear material is utilized in the formation of the globules.

Homogeneous amoebocytes appeared to be formed in the epithelium of the lumen of the respiratory trees, where they occurred in abundance. There was a general tendency for the average size of amoebocytes present in the walls of the respiratory trees to increase from the lumen outwards towards the periphery. This may indicate that there is an outward migration of homogeneous amoebocytes from their sites of origin in the epithelium of the lumen to the coelomic fluid. Should this be the case, it would be expected that there would be a continual influx of homogeneous amoebocytes into the tubules from the epithelium of extensions of the lumen present in the papillae. Apparently there is such an influx because numerous homogeneous amoebocytes (recognized by their nuclear diameter, poverty of cytoplasm, and staining reactions, and by their protrusion of filamentous pseudopodia) occurred amongst the fibres present in the tubules.

Amoebocytes, presumably migrating to the coelomic fluid, were observed in the coelomic lining of the respiratory trees. Also bodies about 4μ, in diameter were observed in the cells of the outer layer of the tubules; these stained with nuclear stains. It was not possible to ascertain whether these bodies were amoebocytes or whether they were the nuclei of the cells of the outer layer. Since some cells possessed more than one of these bodies it is thought probable that some, at least, were homogeneous amoebocytes migrating through these cells to the coelomic fluid.

Approximately 10% of the total number of coelomocytes were large cells (fig. 3), possessing irregular outlines. Long pseudopods radiated from these cells, which contained a small nucleus, granules, vacuoles and a varying number of solid bodies of different sizes. These bodies had irregular shapes and were probably ingested material-in some cases ingested morulashaped cells. It is for this reason that the cells are believed to be phagocytic. Some of the vacuoles stained with dilute methylene blue.

Phagocytes were not observed in the wall of the respiratory trees, in the papillae, or in the tubules.

These cells were spindle-shaped and each ranged from 6 to 10 μ, in length. They comprised about 10 % of the total number of coelomocytes and each possessed a refractive outer cytoplasm which rendered observation of internal structures difficult. Their cytoplasm stained faintly blue with dilute methylene blue and this stain showed the presence in each of a large nucleus. Dilute neutral red revealed the presence of a variable number of small granules in the cytoplasm.

The function of these cells is not known. They were not found in the tubules or in the walls of the respiratory trees or papillae. Similar cells were found in the genus Molpadia by Ohuye (1936).

About 5% of the cells observed in the coelomic fluid were each filled with a rhomboidal crystal. These cells were 9 to I Iμ, long, 7 to 9 μ, wide, and 4 to 5 μ thick. In surface view no cytoplasm was evident, but in side view a thin film could be seen. In a few cells two rhomboidal crystals were united. These cells did not stain with either dilute methylene blue or dilute neutral red. They were not observed in the tubules or walls of the respiratory trees or papillae.

Similar cells have been found in other species of holothurians by Th éel (1921).

Drops of coelomic fluid were placed on slides and a drop of 5% formalin in sea-water added to each. In each case a coagulum was formed which settled on the slides. The drops were then allowed to stand for about 10 min and fluid was then washed from the slides with distilled water. Many cells were lost as a result of this procedure and many were cytolysed, but a proportion remained intact and were suitably fixed.

Coelomocytes treated as above were stained with iron haematoxylin. The nuclei of all cells stained heavily and chromatin granules were evident. The globules of the morula-shaped cells and the vacuoles of their precursors stained blue-black.

With Heidenhain’s Azan the globules in the smaller morula-shaped cells and those released from both large and small cytolysed cells stained blue. However, the globules of many of the larger intact morula-shaped cells stained red. All gradations in intensity of staining from a deep blue to a deep red were observed. Sometimes within the same cell some globules stained blue and others red.

The reasons for such staining are obscure but again evidence is presented that the larger globules consist of two components.

Formalin-fixed morula-shaped cells were stained for polysaccharides by Hotchkiss’s (1948) method. Intact cells gave a strong positive reaction, as did the granulated material from cytolysed cells. The globules themselves, when released, stained only faintly. Intense metachromasy with toluidine blue was exhibited by the globules of the larger formalin-fixed morula-shaped cells, but the globules of smaller cells stained a pale blue or not at all. In the case of the larger cells it was found that the globules, when free, also stained a pale blue and the metachromatic staining observed was given by the granulated material which surrounds the globules in intact cells but which forms a granulated coagulum under the action of formalin when released from these cells.

Since the results of the above tests indicated the presence of highly polymerized polysaccharides it was thought that part, at least, of these might be acid mucopolysaccharides. These substances were sought by using Hale’s (1946) histochemical method, which requires fixation in Carnoy’s fluid. When a drop of this fixative was added to a drop of coelomic fluid a coagulum was formed and all the coelomocytes present were cytolysed. However, clumps of cytolysed cells stained strongly for acid mucopolysaccharides.

Formalin-fixed morula-shaped cells darkened slightly with Sudan black B. It was established that the granules in exudates of the viscous material which formed films around the globules took up the colouring agent and possibly contained a small quantity of neutral lipid.

Formalin-fixed cells were next subjected to the mercuric chloride bromphenol blue technique of Mazia, Brewer, and Alfert (1953) for the histochemical staining of protein. The morula-shaped cells became dark blue, indicating that proteins were present. Free globules from morula-shaped cells stained heavily, indicating the presence of considerable quantities of protein. The granules in exudates from cytolysed cells of the viscous material which forms films around the globules also stained for proteins, but the viscous material itself stained only faintly, if at all.

Because of the similarity between the morula-shaped cells of H. leucospilota and the ferrocytes of Pyura stolonifera it was thought that iron might be present. Inorganic iron was first sought by using the Prussian blue reaction and Humphrey’s (1935) dinitrosoresorcinol test. Iron was not detected. However, when formalin-fixed cells and alcohol-fixed cells were placed in 33% nitric acid at 35° C for 24 h and then stained for iron, positive results were obtained both with ferrocyanide in hydrochloric acid and with dinitrosoresorcinol. Also it was ascertained that the organically bound iron was confined to the globules and little if any appeared to be present in the viscous material surrounding them.

The histochemical tests described above indicate that the globules of the morula-shaped corpuscles each contain an inner core of protein associated with an iron compound. The iron compound is surrounded by a film of polysaccharide, part or all of which is possibly in the form of acid mucopolysaccharide, and in this film granules containing lipid and protein are found.

Since organically bound iron had been detected histochemically in the globules of the morula-shaped coelomocytes, attempts were made to detect it in coelomocytes removed from the coelomic fluid by centrifugation. Care was taken to prevent cytolysis of the coelomocytes from occurring. Samples, each consisting of the coelomocytes from 20 ml of blood, were used.

Four samples were each heated with 10 ml of glass-distilled water to which 2·5 ml of concentrated nitric acid and r ml of concentrated sulphuric acid were added, until all trace of organic material was removed. The samples were then neutralized with NaOH. Two of them were tested for iron by Sandell’s (1944) o-phenanthroline technique. A reddish colour was obtained, indicating the presence of iron. Confirmation of this finding was obtained by acidifying the other two samples with nitric acid and adding potassium ferrocyanide solution. Precipitates of Prussian blue were obtained.

The concentration of iron in the coelomocytes from 20 ml samples of coelomic fluid taken from 7 different specimens was determined by the method of Sandell (1944). The mean value for the iron content of the coelomocytes was found to be 0·12 g per 100 g dry weight of the coelomocytes, with a range of 0·1 to 0·13 g per roe g dry weight.

Haemoglobin has been found in the coelomocytes of several species of holothurians (Howell, 1885; Kollman, 1908; Hogben and van der Lingen, 1928; Kobayashi, 1932). Hyman (1955, p. 147) stated that haemoglobincontaining cells (‘hemocytes’) were ‘found throughout the class’. However, cells answering the description of these hemocytes were not observed in H. leucospilota. Nevertheless, there is the possibility that the iron compound in the morula-shaped coelomocytes of this species is haemoglobin. Examination with a microspectroscope of the coelornic fluid, centrifuged coelomocytes, and suspensions of coelomocytes cytolysed with distilled water, failed to reveal any absorption bands.

The iron compound in the morula-shaped coelomocytes of H. leucospilota shows marked resemblances to the iron compound present in the ferrocytes of the ascidian, Pyura stolonifera. In both cases the compound is present in structurally similar morula-shaped cells at about the same concentrations. In neither case does it appear to have chemical relationship to haem or other similar iron-porphyrin compounds.

In view of this it was decided to investigate other aspects of the chemical constitution of the morula-shaped coelomocytes and to ascertain whether further similarities existed between them and the ferrocytes. In particular, it was decided to investigate the intracellular acidity of the morula-shaped cells and also the reducing properties of the intracellular material.

The coelomic fluid, as determined by the glass electrode, showed a constant pH of 7·7 and was therefore less alkaline than sea-water. When the coelomocytes in 10 ml samples of the coelomic fluid were cytolysed by freezing, the pH of the fluid fell to 7·2-7·4. Possibly the buffering power of the coelomic fluid affected this result. Therefore, the coelomocytes in Io ml samples of coelomic fluid were centrifuged off, and the clear fluid above them was poured off and replaced by equivalent amounts of glass-distilled water. The pH of the resulting mixtures in each case lay between 6·5 and 6·7. It would seem that a small amount of acid is present in the coelomocytes.

Samples of coelomocytes centrifuged off in each case from 10 ml of coelomic fluid were each cytolysed with 5 ml of glass-distilled water. The intracellular material did not reduce dilute potassium permanganate or potassium dichromate solutions.

When 10% formalin or 96% alcohol was added to drops of coelomic fluid placed on slides, the coelomocytes cytolysed and a coagulum in which :filaments were apparent was formed (fig. 4, A). The filaments enmeshed the cytolysed coelomocytes and in many cases appeared to have arisen from them. Adjacent clumps of cytolysed coelomocytes were usually linked by straight filaments which in places were sufficiently thick to form a film.

The above phenomena have a parallel in observations made by Théel (1921) on the coelomic fluid of Mesothuria intestinalis (Ascanius and Rathke). A coagulum similar to that described above was formed in the coelomic fluid of this species when the fluid was placed in a glass tube. Théel (p. 26) believed that there was ‘an abundance of fibrin matter in the coelomic fluid’ and that ‘plasma amoebocytes’ were not present in sufficient numbers to play a major part in the process.

The nature and significance of the coagulum formed by the coelomic fluid of H. leucospilota when treated with alcohol or formalin are not known. However, it was observed that when coelomocytes separated from the coelomic fluid by centrifugation were cytolysed with distilled water, what appeared to be fibrous processes arose from many of the morula-shaped coelomocytes (fig. 4, B). Similar structures did not form in the coelomic fluid from which the coelomocytes were removed. It would seem therefore that the fibrous processes are formed primarily from material present in the coelomocytes.

When coelomocytes, concentrated by centrifugation, were left to stand in a little coelomic fluid, in many cases definite fibres arose from small clumps of cells which had cytolysed and sometimes such fibres arose from single morulashaped cells. All the fibres, however, had a beaded appearance (fig. 4, n). The cells and fibres were placed on slides and fixed with 5% formalin in sea-water. The ‘beads’ stained positively for polysaccharides with Hotchkiss’s (1948) technique and they also exhibited metachromasy with toluidine blue. In the case of the fibres themselves negative results were obtained, but they stained positively though faintly for protein when the histochemical method of Mazia, Brewer, and Alfert (1953) was used. It is believed that the ‘beads’ are globules and that these are linked together by protein fibres.

Hanging drop preparations of coelomocytes were examined at regular intervals. In a few cases short fibres were formed from morula-shaped coelomocytes. The mode of formation was as follows. A projection appeared on the surface of a morula-shaped coelomocyte. In this projection granules showing violent movement were noted and from the projection one or more globules were hurled out, sometimes as far as 16 μ from the cell. The globule or globules were attached by slender fibres to the cell which gave rise to them (fig. 4, c). Other globules accompanied by granules moved out of the cell and passed along the fibres. Sometimes numerous globules arranged themselves like beads along the length of the fibre. The appearance of such fibres is identical with that of the beaded fibres mentioned earlier.

Frequently one or more globules, after moving back and forth along a fibre, would suddenly shoot off in a new direction, each trailing a fibre. Globules which gave rise to such fibres decreased in size and presumably intraglobular material is utilized in the formation of the fibres. Often the globules collided with one another whilst moving back and forth along the fibres. When such a collision occurred the colliding globules appeared to merge temporarily but soon snapped apart from one another (fig. 4, E).

Small meshworks of fibres were sometimes formed but the fibres were never very long and were oriented in all directions. Usually only short fibres were formed from a single morula-shaped cell (fig. 4, F).

Fibre production within the Cuvierian tubules. A satisfactory procedure for observing fibre production in position within the Cuvierian tubules has not yet been devised.

Study of fresh whole mounts of tubules revealed an abundance of morulashaped coelomocytes within the central core of each tubule. Coelomocytes of the same type were also present between the fibres of the tubule connective tissue, but these coelomocytes were in all stages of apparent degeneration. Often the globules had broken free from the cells and frequently such globules were disposed in lines between the fibres.

Sections through the tubules showed that such globules were almost always connected by fibrous processes to the cells from which they were derived and in many cases similar fibrous processes radiated out from the morula-shaped cells themselves and from the emitted globules. However, because of the uncertain action on the morula-shaped cells of the fixatives used (Bouin’s fluid and Susa), it is not possible to equate these fibrous processes with true fibres.

Reflection upon the histology and histochemistry of the Cuvierian tubules and coelomocytes of H. leucospilota will reveal that, of the cells present in the tubules, only the morula-shaped coelomocytes could be involved, directly, in the synthesis of fibrillar material and in the deposition of fibres. The available evidence indicates that the globules of these cells contain materials which are probably precursors of the collagenous connective tissue of the tubules.

It would seem that amoebocytes, formed in the epithelium lining the lumen of the respiratory trees, develop in their cytoplasm vacuoles in which proteinaceous materials are found. The vacuoles enlarge and each gradually acquires a peripheral film of mucopolysaccharide. Subsequently, in some cases at least, these globules pass out of the morula-shaped cells so formed into the interfibrillar substance of the tubules. Here their contained protein and mucopolysaccharide may be utilized in the formation of collagen fibres.

‘Ferrocytes’ that participate in the formation of fibres and are similar in shape to the holothurian morula-shaped cells have been found in the ascidian Pyura stolonifera (Endean, 1955a, 1955b, 1955c). A comparison of the properties possessed by the ferrocytes and holothurian morula-shaped cells is instructive (Table 1).

It can be seen that the morula-shaped coelomocytes of the holothurian and the ferrocytes of the ascidian are similar in many respects. Thus both are structurally similar, both are formed in a comparable fashion, and both can produce fibres. Both contain an acid polysaccharide which appears to be a mucopolysaccharide and both contain unidentified iron compounds which do not appear to have chemical relationship with haem or other familiar ironporphyrin compounds. It is possible that the iron compounds have roles in the polymerization processes which must occur within the globules of both cells. The chemical constitution of these iron compounds will be discussed in a later paper.

The differences exhibited by the morula-shaped cells of the holothurian and the ferrocytes of the ascidian are of importance. In particular, the observations that the intra-globular material of the holothurian morula-shaped cells was proteinaceous whilst the intraglobular material of the ferrocytes appeared to be polysaccharide are of interest because the former cells are involved in the formation of a structural protein (collagen) whilst the latter are concerned with the production of a.structural polysaccharide(tunicin). Other major differences concern the acidity and reducing power of the intracellular material. In the ferrocytes this material is strongly acid and has marked reducing power whilst in the coelomocytes of the holothurian the intracellular material appears to be only faintly acid and does not show similar reducing properties. The manner in which these differences are related to the synthesis of the precursors of tunicin and collagen, if indeed they are, is not known.

In fibrogenic cells of the fowl embryo grown in serum exudate, conspicuous granules ranging from 0·7 to 2μ, in diameter have been found (Jackson and Smith, 1955). These granules are therefore similar in size to the globules contained in the morula-shaped cells of H. leucospilota. Moreover, both granules and globules stain supravitally with neutral red, exhibit metachromasy with toluidine blue, and stain histochemically for polysaccharide and acid polysaccharide. In both cases part at least of the polysaccharide is a mucopolysaccharide. Also both granules and globules contain protein.

It was noted by Jackson and Smith (1955) that the centres of the granules found in the fibroblasts of the embryonic chick were less dense than the periphery and that ‘even after prolonged treatment with periodic acid and Schiff’s reagent only the periphery of the granules stain while the centres remain colourless’ (p. 92). In each globule of the morula-shaped cells of the holothurian studied, a central proteinaceous area is surrounded by a film of acid polysaccharide material. It is intended to ascertain whether the protein and polysaccharide in the granules of the fibroblasts of the developing fowl are similarly distributed and also whether the protein is associated with an iron compound as it is in the morula-shaped cells.

Both granules and globules, when removed from the cells where they are formed, show a tendency to line up and form fibres with a beaded appearance. The chemical nature of the beaded fibres formed by the isolated granules was not stated but each of those formed by the globules of the morula-shaped cells of H. leucospilota consists of globules united by a protein thread.

No normal extrusion of intact globules was observed in the case of the fibroblasts of the developing fowl. By analogy with the mode of formation of fibres from rabbit fibroblasts (Steams, 1940) it might be expected that protoplasmic protruberances would eventually arise from these fibroblasts and that in the vicinity of these protruberances fibres would form.

Globules were observed to be thrown out of the morula-shaped cells of H. leucospilota. These globules were linked by fibres to the cell and it would appear that fibres can arise directly from the intraglobular material. Whether fibres are normally formed in this fashion inside the Cuvierian tubules is not known, but it may be significant that globules are usually released from morulashaped cells occurring amongst the collagenous fibres of the tubules.

The author is indebted to Professor W. Stephenson of the University of Queensland for continued interest and helpful advice and to Dr. M. C. Bleakly of the University of Queensland for reading and discussing the manuscript. The technical assistance given by Mr. J. Jenkins and Mr. C. Illidge is gratefully acknowledged. Enlargements of the author’s negatives and photographs of the author’s drawings were prepared by Mr. E. Hollywood of the Photographic Department, University of Queensland.