fine structure of the axial complex of sphaerechinus granularis (lam.) (echinodermata: echinoidea)

Cell Tiss. Res. 193, 107-123 (1978) Cell and Tissue Research �9 by Springer-Verlag 1978

Fine Structure of the Axial Complex of Sphaerechinus granularis (Lam.) (Echinodermata: Echinoidea)

Sebastian Bachmann and Alfred Goldschmid

Department of Zoology, University of Salzburg, Austria (Head: Prof. Dr. H. Adam)

Summary. Three regions of the axial complex in Sphaerechinus granularis can be distinguished: 1) The axial organ which protrudes from one side of the axial sinus; the sinus septum which separates the sinus from the body cavity and encloses the stone canal; the pulsating vessel which runs along the inside of the axial organ. 2) The blindly-ending terminal sinus in which the pulsating vessel broadens out to the contractile terminal process. 3) The ampulla of the stone canal which connects the axocoel and water vascular system and which opens out through the madreporite.

A single-layered, monociliated coelomic epithelium surrounds all regions of the axial complex. This epithelium contains smooth muscle cells at the contractile areas. Canaliculi, surrounded by basal lamina, are formed through infolding of epithelia; they end blindly in the fluid- and connective tissue -matrix of the inner structures.

The lacunae of the dorso-ventral mesentery connect the periesophageal and the perianal haemal ring with the axial organ. The axial organ contains many coelomocytes rich in pigment and granules. These coelomocytes are separated into compartments by elastic fibres. Phagocytosis of whole cells and transformational stages of coelomocytes suggest storage and degradation functions. An excretory function via the water vascular system is also suggested.

Key words: Axial complex - Sphaerechinus - Heamal system - Epithelial muscle cells - Coelomocytes - Function.

The centrally-positioned axial complex of the sea urchin and its connections to the haemal and water vascular systems has given rise to many speculations concerning its function.

Send offprint requests to: Dr. A. Goldschmid, Universit[it Salzburg, Zoologisches Institut, Akademiestr. 26, A-5020 Salzburg, Austria

Acknowledgements. This study was supported by the Austrian "Fonds zur Frrderung der wissenschaftlichen Forschung" (grant no. 1811, 3204 and 3494). We thank Mr. P. Chang for his help with the translation of the manuscript, and Mr. A. Laminger and Mr. G. Sulzer for photographic work

108 s. Bachmann and A. Goldschmid

Tiedemann (1816) described the axial organ as a heart-formed contractile channel which is connected to the ring lacunae. The wall of the organ was thought to contain circularly arranged muscle fibres. A general survey of its anatomy and histology can be found in later publications (Hoffmann, 1871; K6hler, 1883; Hamann, 1887; Perrier, 1887; Prouho, 1887; Leipoldt, 1893). The descriptions given in handbooks by Hyman (1955) and Cuenot (1948) are mostly based on these early investigations. Since there were still differences regarding microanatomy and histology of the axial complex of Echinoids and especially the interpretation of its function it became the object of more recent research (Boolootian and Campbell, 1964; Millott, 1967; Vevers, 1967; Rieder, 1975). There exists only one electron microscopic study on the axial complex of Psammechinus miliaris (Jangoux and Schalting, 1977).

The present work deals with the axial complex of Sphaerechinus granularis and provides a detailed fine structural analysis of its different regions. A light microscopic investigation expands the earlier work. The fine structural results obtained form the basis for a new discussion and interpretation of the organ's function and are hoped to instigate further physiological and ultrahistochemical research.

Materials and Methods

Adult specimens of both sexes of Sphaerechinusgranularis were obtained from the Adriatic from a depth of 4 meters. They were kept for not longer than two weeks in artificial sea water aquaria.

Light microscopy: Small portions of the arboral test with the axial complex were fixed in Bouin's medium. After conventional embedding in paraplast, 7 I~m serial sections of vertically- and horizontally- orientated specimens were made. The sections were stained using Azan (Heidenhain), Masson trichrome (Goldner), aldehydefuchsin, and connective tissue stain (Gomori).

Electron microscopy: Portions of the axial complex were fixed in paraformaldehyde-glutaraldehyde (Karnovsky, 1965) at pH 7.3 for 2 h, rinsed three times for 20 min in filtered sea water, and postfixed with 1 ~ osmium tetroxide buffered in filtered sea water for 1 h. After dehydration, the specimens were embedded in Epon 812 (Luft, 1961). Ultrathin sections were stained with uranyl acetate and lead citrate (Reynolds, 1963), and examined in a Zeiss EM 9 and Philips EM 300 at 80 kV. Semi-thin sections were stained with azur II.

Results

Light Microscopy

The outer surface of the axial complex is a single layer of monociliated cells (epithelium of the somatocoel). The inner cavities are lined with coelomic epithelium which originates from the axocoel and the hydrocoel. Certain portions of the coelomic epithelium contain numerous muscle cells with basal muscle fibres. Terminal process, pulsating vessel and axial organ contain a fluid matrix in which differently oriented connective tissue fibres, canaliculi and free cells (leucocyte-type and morula cells) are found. These cells contain crystalline and granular inclusions respectively and stain light brown to black with azan.

Axial Complex of Sphaerechinus granularis 109

Fig. 1. Semischematic longitudinal representation of the axial complex of Sphaerechinus. Axial organ (AO), ampulla of stone canal (AP), axial sinus (AS), chambered portion (CP), dorso-ventral mesentery (DM), infoldings of canaliculi (IC), madreporic canals (MC), madreporite (MP), opening between axial sinus and water vascular system (OA), peripheral lacunae (PL), pulsating vessel (PV), ramifications of the pulsating vessel (RP), stone canal (SC), septum of the axial sinus (SS), terminal process (TP), terminal sinus (TS)

110 S. Bachmann and A. Goldschmid

The axial organ shows, in cross sections, the following structures (Fig. 1): A chambered portion, subdivided by connective tissue fibres, surrounds the major part of the eccentric axial sinus. A septum surrounding the stone canal separates the axial sinus from the body cavity. The branched pulsating vessel is fused with the inner side of the axial organ and runs along its oral-aboral axis in the axial sinus. The chambered portion contains numerous polygonal compartments whose connective tissue walls do not always appear to be continuous (Figs. 2, 3). In these compartments, one finds one to three morula and leucocyte-type cells which appear to be able to wander freely between compartments. Most of the granular accumulation is located at the periphery of the chambered portion. The numerous haemal lacunae and canaliculi of the axial complex are partially filled with free coelomocytes, coagulated liquid, and granules (Figs. 2, 3). The lacunae form a branched network covering the surface of the chambered portion and connect directly to the rest of the haemal system. The epithelium of the somatocoel forms the outer wall of the lacunae, whose lumen is partially lined with basal lamina to which single cells adhere. Clearly defined boundaries to the chambered portion are lacking and the lacunae seem to connect freely with the inside of the axial organ (Fig. 3). The canaliculi show a continuous endothelial layer covered exteriorly with basal lamina (Figs. 3, 4, 5). These canaliculi originate from infoldings ofsomatocoel and axocoel.

The pulsating vessel, surrounded by the epithelium of the axial sinus, is subdivided into several lumina, whose widths depend on the degree of contraction at fixation (Fig. 4). The epithelium of the pulsating vessel possesses numerous myoepithelial cells. One can occasionally observe a dense layer of elastic fibres beneath the epithelium. The lumina of the pulsating vessel do not have an epithelial lining and contain, besides free coelomocytes, dense connective tissue coils and canaliculi. These canaliculi, which run mainly along the long axis of the pulsating vessel, also seem to be infoldings of the coelomic epithelium, as are the canaliculi of the chambered portion. In contrast to the latter, they contain muscle cells (Fig. 4).

At the aboral end of the axial organ the chambered portion terminates while the pulsating vessel runs into the terminal process. At this point, the axial sinus is separated from the stone canal by a thin septum, which ends further aboral, at which point a communication exists to the stone canal and the ampulla situated below, and opening out via, the madreporite (Fig. 1). The lumina of the madreporic canals contain free cells and cell debris of the same type which are observed in the chambered portion (Fig. 7). The epithelium of the madreporic canals is heavily ciliated.

The terminal process, situated in a closed cavity (terminal sinus), is fused along the length of its oral side with the wall of the sinus. The structures of the pulsating vessel and the terminal process are almost identical (Fig. 6). They communicate with each other by way of their lumina and epithelial canaliculi. The latter are more numerous in the terminal process (Fig. 5), and again contain muscle cells with longitudinally running fibres. The epithelium of the terminal process is particularly rich in muscle fibres which can extend inwardly and which are anchored to the dense elastic and collagenous fibres. The dorso-ventral mesentery extending from the esophagus accompanies the axial organ and widens at its aboral end, where it fuses with the rectum, the septum of the terminal sinus and the aboral ring lacuna. This

Fig. 2. Chambered portion of the axial organ bordering the axial sinus (AS); a number of compartments (C) are filled with cells, some of which contain large amounts of granules (arrows). Ramifications of the pulsating vessel (RP). Semi-thin section, x 560

Fig. 3. Peripheral region o f the chambered portion (CP), showing epithelial canaliculi (EC) densely filled with morula cells. Infolding ofa canaliculus (arrow); lumen of a lacuna (L) which commianicates with the chambered portion. • 450

Fig. 4. Two ramifications of the pulsating vessel (RP), the lower one in a contracted state. Epithelial canaliculi (EC), muscle fibres (arrows). Note free cells in the lumen of the axial sinus (FC). Semi-thin section, x 560

Fig. 5. Portion of a longitudinal section of the terminal process showing a large number of canaliculi (EC) and coils of connective tissue (CT). Septum of the terminal sinus (TS). x 110


Fig. 6. Peripheral portion of the terminal process, showing canaliculi (EC) and free cells (FC) in the fluid matrix. Coelomic epithelium (CE); muscle fibres (arrow). Semi-thin section, x 560

Fig. 7. Cross-section of the madreporic canals (MP) partly filled with cellular and granular accumulation (arrow). Cilia (C). x 560

mesentery has, like the axial organ, epithelial canaliculi and haemal lacunae. The lacunae are covered with myoepithelial cells.

In addition to the above-mentioned connections, there are the following connecting pathways in the axial complex: both the axial sinus with the lumen of the pulsating vessel and the terminal sinus with the lumen of the terminal process communicate via the epithelial canaliculi. The chambered portion makes open contact at the sinus side with the lumen of the branches of the pulsating vessel. On the somatocoel side, there is contact with the haemal system (periesophagial ring, intestine) via the outer lacunae.

The lumen and the canaliculi of the terminal process fuse with the canaliculi and the lacunar spaces of the dorso-ventral mesentery. This provides a possible pathway for exchange of material between the whole axial complex, the intestine, and the aboral haemal system, the latter of which connects to the gonads.

Electron Microscopy

Electron micrographs at low magnification show that the axial complex of Sphaerechinus is not a true tissue, but rather, a loose assembly of canaliculi, connective tissue fibres and different single cells. These elements lie freely in a


matrix which resembles and connects with the haemolymph in the haemal lacunae. Only the epithelia of the axial complex represent true tissues.

a) Coelomic Epithelia of the Axial Complex. The monociliated epithelial cells contain a medium to strongly granulated cytoplasm with many vesicles (50- 100 nm) and large spherical vacuoles (1-2 Ixm). Some cells have large electron-dense lysosome-like bodies. Cytoplasmic extensions with lengths of several micra form interdigitations with neighbouring cells and with the basal lamina. The extensions may surround whole cells. Such cells appear to be in various stages of lysis. The surfaces of cells facing the lumen bear numerous microvilli and often form protrusions. Most of the cells possess one long cilium with the 9 + 2 arrangement of the axial filaments. It arises from a deep invagination which is regularly surrounded by a collar of 9 - 12 radial lamellae connected by a mucus-like substance. The latter forms a mucus film on the inner ridges of the lamellae. At the base of the invagination and on the apices of the lamellae one can see transitional stages of vesicles indicating pinocytotic activity. The cilium has a diameter of about 200 nm. Its peripheral filaments end in a centriole, whereas the central filaments seem to terminate in a thickening of the shaft. A second centriole is normally at right angles to the ciliary rootlet (Fig. 8) which shows a characteristic cross-striation. The rootlet extends over a remarkably long distance into the cytoplasm and lies close to a Golgi apparatus with flat curved cisternae and to elongated mitochondria of the crista type. The nucleus is spherical or irregularly shaped; the chromatin is clustered centrally and marginally in the nucleoplasm. The nucleus possesses one to two nucleoli and is surrounded by a partially voluminous cisterna. Endoplasmic reticulum is sparsely developed.

The epithelia of the contractile parts contain numerous smooth muscle cells (Fig. 9). The perikarya of the muscle cells differ from the epithelial cells by reason of the fact that they have a less intensive cytoplasmic granulation. The muscle cells form irregularly shaped cell processes interdigitating with neighbouring cells. Muscle fibres are always situated near the basal lamina. Single or bundled muscle fibres, surrounded by basal lamina, can reach far into connective tissue zones. The sarcoplasm of contracted muscle cells is folded and forms longitudinal ridges with the basal lamina. These ridges do not contain myofilaments but single mitochondria. Between basal lamina and sarcoplasm there is always an electron- lucent cleft with a width of 30-50 nm (Fig. 9).

The single muscle fibre consists of filaments with two different diameters (20- 40nm and 8-10 nm). Cross striations and Z-lines are not discernible. However longitudinal sections of the filament bundles show irregular transversely or obliquely arranged dense zones. In some fibres the filaments show a V-shaped feathery arrangement.

Naked axons lying side by side are observed at the base of contractile epithelia (Fig. 16). They contain small clear and larger membrane-bound vesicles (for details concerning the innervation of the axial complex, see Bachmann and Goldschmid, in preparation).

There is usually a zonula occludens near the cell surface followed proximally by a zonula adhaerens and a macula adhaerens which is sometimes expanded to a

Fig. 8. Electron micrograph of the apical portion of monociliated epithelial cells. Centrioles (CS), ciliary rootlet (CR), microfilaments (M), mitochondria (MI). The cells are connected by a macula adhaerens (MA) and septate junctions (S J). • 46,000

Fig. 9. Contracted myoepithelial cells lining the axial sinus. The sarcoplasm of the basal muscle fibres (MF) forms interdigitations with the basal lamina (BL) which is connected with elastic fibres (EF). Nucleus (N), mitochondria (M/), lamellae (LA) belonging to the base of a cilium (C). x 10,000


small spherical dilatation. Septate junctions can be observed basally to the connections just mentioned (Fig. 8).

b) Basal Lamina and Connective Tissue. The basal lamina consists of finely granulated material (Fig. 9). Its thickness measures 0.2-1 nm and appears to be correlated with the number of elastic fibres embedded. The elastic fibres (Figs. 9, 11) consist of thin (8-10 nm) and relatively short filaments. They occur within, or close to, the basal lamina underlying contractile epithelia and are also situated in the chambered portion of the axial organ. There they form the "walls" of the compartments (Fig. 2) observed in the light microscope. Most of the connective tissue consists of collagenous fibres. Their filaments measure 80-150nm in diameter and show a characteristic cross-striation with a periodicity of 60 nm (Fig. 11). Bundles of collagenous filaments are found in all contractile parts of the axial complex.

c) Free Cells in the Axial Complex. There are four types of free cells in the liquid matrix of the inner structures and the cavities of the axial complex.

Type L Morula cells: they contain large, unit membrane-bound spherules which leave little space for the cytoplasm (Fig. 12). They are filled with homogenous material or are heterogenously granulated. In some cells, the spherules are partially to completely empty, and localized breakdown of the cell membrane can be observed. The nucleus often appears to be pycnotic or even missing.

Type II. Phagocytes: their structure is comparable to the macrophages or histiocytes of vertebrates. They are provided with large phagocytotic pseudopodia and elongated narrow processes. Their cytoplasm appears electron-lucent and is filled with large vacuoles which are partly membrane-bound. The vacuoles have a spherical or irregular polygonal form due to the tightness of their packing in the cell. They contain lamellate bodies and clusters of electron-dense granula (Fig. 13). In the sparse ground cytoplasm there are singly lying lysosomes and mitochondria. The nucleus is lobed and surrounded by a narrow cisterna. Crystalline bodies of more or less cubical shape are often found in the dense nucleoplasm (Fig. 13). At high magnification these crystals show a regular pattern of parallel striation. Phagocytes are concentrated mainly in the periphery of the axial organ. The lamellate bodies and the electron-dense granules can be found in vacuoles or lying free in the matrix of all inner structures of the axial complex (Fig. 15).

Type IlL Leucocyte-type cells: They are round or possess a few short and stout pseudopodia which can encompass whole cells, some of which seem to undergo a partial breakdown of the cell membrane (Fig. 14). The cytoplasm is filled with small vesicles and electron-dense lysosomes measuring 200-500 nm in diameter. A Golgi apparatus with one to two dictyosomes is found close to the nucleus. Numerous mitochondria are also present. The nucleus normally shows a deep indentation and may also contain a crystalline body as observed in the phagocytes.

Fig. 10. Cross-sections of a canaliculus in the terminal process. The epithelium is surrounded by basal lamina (BL) and contains muscle fibres (MF). Nucleus (N), cilia (C) in the lumen. • 4500

Fig. 11. Connective tissue fibres in the septum of the terminal sinus. Collagenous (CF) and elastic fibres (EF) can be distinguished. • 38,600

Fig. 13. Phagocyte, showing large granular (GR) and tametlate (LA) phagosomes. Nucleus (N) containing a crystalline body. x 18,850. Inset: striated fine structure of a nuclear crystal from a different cell. x 44,700

Fig. 14. Leucocyte-type cells in the axial organ. The cytoplasm contains mitochondria (M/), lysosomes (LY), and a Golgi apparatus (GO). The outer cell membrane is partly interrupted (arrows). Nucleus. (N). Y ~ 1 ~


Fig. 16. Transverse section of the septum of the axial sinus. A dense granule-containing connective tissue layer (CL) lies between the epithelia of the somatocoel (SO) and the axocoel (AX). The somatocoelic epithelium shows cross-sectioned muscle fibres (CM); the muscle fibres of the axocoelic epithelium are longitudinally cut (LM). Note the accumulation of naked axons in the nerve plexus (NP). x 4000

Type IV. Fibrocytes: They are structurally similar to the leucocyte-type cells. They differ from the latter in that they either contain, or are in close contact with, membrane-bound collagenous fibres (Fig. 15).

d) The Septa of the Axial and the Terminal Sinus. The epithelia of both septa are particularly numerous in muscle cells of the type described above. A well developed connective tissue layer is found between the epithelia (Fig. 16). It consists of collagenous and elastic fibres and contains free cells of all four types. There are also free vacuoles, granules and small vesicles. The only difference between the two septa is in the arrangement of the muscle fibres of inner and outer epithelia, which both display a parallel arrangement in the septum of the terminal sinus. In the septum of the axial sinus, the muscle fibres of the axocoelic (inner) epithelium run circularly whereas those of the somatocoelic (outer) epithelium run longitudinally to the oral- aboral axis of the organ.


Discussion

Observations of the axial complex in vivo show alternating and periodic contractions of the pulsating vessel and the terminal process as well as of the septa of the axial and the terminal sinuses (Boolootian and Campbell, 1964; Millott, 1967, Strenger, 1973; Rieder, 1975; Jangoux and Schaltin, 1977). Several authors (on asteroids: Teuscher, 1876; Pietschmann, 1906; Bargmann and v. Hehn, 1968; on echinoids: Hoffmann, 1871; Boolootian and Campbell, 1964) conclude from these contractions that the axial complex functions as a heart. A closed circulatory system in echinoderms is lacking and the light microscope findings rather suggest that the pulsations serve to transport free cells and cell debris between the different regions and between coelomic cavities of the organ. A motor function in the haemal system is carried out by contractile lacunae as they are found on the digestive tract (Strenger, 1973). Burton (1964) claims a so-called ebb- and flow-movement of haemolymph for these lacunae. A similar mechanism for the pulsating vessel and for the terminal process has also been proposed in a recent investigation of the axial complex ofPsammechinus (Jangoux and Schaltin, 1977). Transport of material and cells in the larger cavities and in the canaliculi furthermore is carried out by the ciliated epithelial lining. Contractions of the mesenteric lacunae, for example, are evidently involved in the transport of haemolymph to and from the axial organ (Rieder, 1975).

The myoepithelial cells are responsible for the contractions. Holland (1970) finds only a few muscle cells in the crinoid axial organ, whereas in asteroids (Bargmann and v. Hehn, 1968; Leclerc, 1974) and in Psammechinus (Jangoux and Schaltin, 1977) a large number of muscle cells in contractile parts of the axial complex have been found. The muscle fibres do not display typical Z-lines (Bargmann and v. Hehn, 1968) nor the so called J-granules (Kawaguti, 1964).

The large number of monociliated epithelial cells with an invaginated pit surrounded by a corona of lamellae resemble choanocytes, or collar cells, as described for various coelomic cavities of Echinoderms (Norrevang and Wingstrand, 1970; Florey and Cahill, 1977). Pinocytotic vesicles in the collar region as well as microvilli and protrusions of the cell surface suggest highly active transport functions. Phagocytosis is carried out by coelomocytes which invade the epithelia. Whole, phagocytozed cells and their lysis-like degradation can be observed. The occurrence of septate junctions between muscle cells and between muscle and epithelial cells indicate possible intercellular exchange of material (Gilula et al., 1970).

Our results indicate that a glandular function of the epithelial cells suggested by Millott and Vevers (1964) and Holland (1970) cannot be attributed to the epithelial cells of Sphaerechinus, for neither typical secretory granules nor a well developed endoplasmic reticulum could be observed. However, according to Jangoux and Schaltin (1977) the somatocoelic epithelium of the axial organ secretes acid mucopolysaccharides, which aggregate in granular form at the cell apices.

The epithelial canaliculi frequently possess a longitudinal layer of muscle fibres, which could support contractions in the pulsating parts of the axial complex. The canaliculi generally connect the inner structures of the organ with the coelomic cavities since they appear to remain open to the latter. A passage of free cells via the


epithelium and the basal lamina, especially in places where the basal lamina is covered only with thin cell processes has been observed 0angoux and Schaltin, 1977, "diap6d6se). In contrast to Millott's (1967) findings, an open communication of the peripheral canaliculi with the haemal lacunae does not exist.

Bargmann and v. Hehn (1968) refer to a structural analogy between the axial organ of Asterias and the renal glomerulus of vertebrates. The structure of the canaliculi of Asterias, where muscle cells and "epicytes" with thin processes cover the outside of the basal lamina lining the lumen, does not correspond to the structure of the echinoid canaliculi. In Echinoids, the lumen of the canaliculi, on the contrary, is always lined by epithelial cells and the basal lamina covers these cells from the outside. An analogy to the glomerulus is, therefore, not applicable.

The inner structures of the axial complex do not represent a true tissue (Holland, 1970; Jangoux and Schaltin, 1977). The chambered portion, the pulsating vessel and the terminal process rather consist of free cells, connective tissue fibres, and a considerable amount of cell debris embedded in a fluid matrix, covered by coelomic epithelia and penetrated by canaliculi. The "compartments" in the chambered portion of the axial organ consist mainly of numerous loosely aggregated elastic fibres which are located around groups of free cells. Beneath the contractile epithelia, elastic fibres usually interwoven with the basal lamina, may function antagonistically to the contractions of the muscle fibres, whereas bundles of collagenous fibres in deeper regions may have a supporting function.

The free cells of the axial complex and of its cavities are coelomocytes identical to those found in the somatocoel. Schinke (1950) showed that these cells are derived from the test and from the coelomic epithelium. Ferritin injected into the somatocoel (Jangoux and Schaltin, 1977) is rapidly ingested through endocytosis of the coelomocytes and can soon be found in the axial organ. These coelomocytes are the leucocyte-type cells (leucocytes: Chien et al., 1970; phagocytes: Stang-Voss, 1971; hyaline cells: Jangoux and Schaltin, 1977). The leucocyte-type cells probably appear in different stages; the phagocytes with large lamellate bodies and crystalline inclusions as well as the more compact fibrocytes can thus be suggested to derive from only one basic cell type (Fontaine and Lambert, 1977).

The leucocyte-type cells reach the axial organ from the somatocoel via the infoldings of the canaliculi and via passage through the epithelium. Active movement is based on their different kinds ofpseudopodia (Millott, 1967). Jangoux and Schaltin (1977) suggest that the cells invading the axial organ are being "transformed and neutralized" in the chambered portion. According to our findings, coelomocytes can directly penetrate the somatocoelic epithelium as indicated by transitional stages. The aggregation of "used cells" densely packed with phagosomes and their obvious transformation suggests that the axial organ is a centre for storage and degradation of cells and waste material. A degradation is indicated by the occurrence of necrotic cells and cell debris.

The morula cells have been variously described (echinochrome containing cells and uncolored spherule cells: Chien et al., 1970; white and red eleocytes: Stang- Voss, 1971). According to Jangoux and Schaltin (1977), the morula cells with electron-dense granules (white eleocytes of Stang-Voss, 1971) are supposed to contain proteins, whereas the morula cells with granular content (the red eleocytes of Stang-Voss) should contain pigment, mucous material and iron. Iron containing


crystalloids in phagocytes have been known for some time and have recently been described by H6baus (1978). Phagocytosis ofmorula cells by leucocyte-type cells as well as different breakdown stages are often observed.

Millott (1966, 1969) describes intense clotting of coelomocytes in the lacunae and canaliculi caused by injury or by injection of foreign cells. He concludes from these observations that the axial organ is involved in defense mechanisms. Leclerc (1974) claims an immune function on the grounds of an antibody reaction observed in the asteroids axial organ. Furthermore he describes formation of germinal cells in the aboral region of the asteroid axial organ. Although there are similar connections between axial complex, aboral haemal system and gonads in Echinoids, we did not find any indications to support the above hypothesis that germinal cells are produced in the axial complex and migrate to the gonads.

The axial organ seems to be only of secondary importance for the organism since extirpation experiments did not show significant decrease in survival rate (Verchowskaja, 1931; Schinke, 1950). The regeneration of the axial organ was studied by Millott and Farmanfarmaian (1967). In contrast to the findings of Millott (1969), the axial complex does not seem to function as a site for cell production since we did not observe any mitotic figures.

According to our results, the axial organ plays a prominent role in the storage of waste material transported in different kinds of free cells. A direct communication to the haemal system and to the water vascular system is evident, based on the work of Boolootian and Campbell (1964). These authors injected dye into the axial sinus and found it spread rapidly throughout the water vascular system. We, therefore, suggest a preparatory function in the excretion of waste products and an exit-transport via the axial sinus, the water vascular system, and the tube feet. In contrast to Jangoux and Schaltin (1977), we observed free cells and cell debris also in the axial sinus. Contractions of the sinus septum as well as ciliary movement of the epithelia can transport these elements to the opening communication between the axial sinus and the water vascular system. Fechter (1965) did not observe a considerable exchange of liquid or material through the madreporite. However, we frequently found coelomocytes in transitional stages fill- ing the madreporic canals. This suggests a passage of granules, or whole cells, to the outside via the madreporite.

A transport mechanism through the epithelium of the tube feet has been indicated by Coleman (1969); epidermal cells of the tube feet should be involved in ion-exchange serving an excretory function (see also Pequignat, 1966). Cobb (1977) observed a passage of necrotic cells via interstitial phagocytes in the gills of Echinus esculentus. The latter observation can contribute to our hypothesis of excretion. In addition, the Tiedemann's bodies have been described as "cleaning organs" of the water vascular system (Bargmann and Behrens, 1964, Asteroids); in Echinoids too they should be further investigated in connection with the hypothesis of excretion on a cellular basis.

Histochemical investigations (Vevers, 1967; Jangoux and Schaltin, 1977) have not greatly contributed to the understanding of the function of the axial complex. In addition to our findings, a comprehensive chemical analysis of the fluid from the outer haemal lacunae, axial sinus, terminal sinus and water vascular system will help to elucidate the function of the axial complex.


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Accepted July 14, 1978

fine structure of the axial complex of sphaerechinus granularis (lam.) (echinodermata: echinoidea)

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