phagocytes in the axial complex of the sea urchin, sphaerechinus granularis (lam.)

Cell Tissue Res. 213, 109-120 (1980) Cell and Tissue Research �9 by Springer-Verlag 1980

Phagocytes in the Axial Complex of the Sea Urchin, Sphaerechinus granularis (Lam.) Fine Structure and X-ray Microanalysis

Sebastian Bachmann, Hannes Pohla, and Alfred Goldschmid

Department of Zoology, University of Salzburg, Salzburg, Austria

Summary. In the axial complex of Sphaerechinus granularis intense phagocytotic activity is encountered. The phagocytes ingest morula cells and other phagocytes; a lysosomal digestion of the phagocytosed cells is suggested. A subdivision into early and late phagocytes is made according to their granular content. Most of the phagocytes possess intranuclear crystalloids that exhibit a filamentous or particulate substructure. Late stages of phagocytes filled with residual bodies and crystalloids leave the axial complex via the lacunar system to be removed through the rectum. X-ray microanalysis reveals a constant presence of iron and sulphur in the crystalloids. The residual bodies contain iron, sulphur, calcium and zinc in varying amounts. This study confirms that one function of the axial complex is excretion.

Key words: Axial complex - Phagocytosis - Intranuclear crystalloids - X-ray microanalysis - Sphaerechinus.

The echinoid axial complex is composed of(a) the axial organ surrounding the axial sinus, (b) the pulsating vessel running along the inside of the axial organ, and (c) the aboral contractile terminal process. Recent investigations (Jangoux and Schaltin 1977; Bachmann and Goldschmid 1978a, b) have elucidated the anatomy, fine structure and innervation of the axial complex. The different regions of this complex, in particular the axial organ, were observed to collect and transform coelomocytes. A proper definition of the function of the echinoid axial complex is still lacking.

Bachmann and Goldschmid (1978a) suggested an excretion of degradated cellular content via the water vascular system; the present study will deal with this

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

Acknowledgements: This study was supported by the Austrian "Fonds zur F6rderung der wissenschaftlichen Forschung" (grants nos. 1811, 3204, 2183). The authors wish to thank Mr. A. Laminger for photographic assistance

0302-766X/80/0213/0109/$02.40

110 S. Bachmann et al.

hypothesis. A n account by Fechter (1973) reported a high concent ra t ion of urea in the axial organ (i.e., the ma in region of the complex) of an echinoid, but the exact site of urea synthesis is still unknown. Most conspicuous among the coelomocytes of the axial complex are the phagocytes, which possess large phagosomes and in t ranuclear crystalloids. A short description of these cells has been given recently by Bachmann and Goldschmid (1978a); however, generally, very few investigations have been focussed on the fine structure and cytochemistry of the coelomocytes (Johnson 1969; Chien et al. 1970, Lecal 1971; Vethamany and F u n g 1972; H6baus 1978). The aim of the present study is to define the precise structure of cytoplasmic inclusions and crystalloids of different phagocytes with regard to the na ture and fate of ingested material. X-ray microanalysis permits the invest igat ion of the elemental composi t ion of small intracellular areas.

Materials and Methods

Adult specimens of both sexes of Sphaerechinus granular& were collected from the Adriatic Sea at the Rovinj Marine Biological Station. They were kept for I to 3 weeks in artificial seawater.

Light Microscopy. Portions of the aboral test with the rectum and axial complex were fixed in Bouin's fluid, dehydrated in ethanol and embedded in Paraplast. 5 gm thick sections were stained using Azan (Heidenhain) and aldehyde-fuchsin.

Scanning Electron Microscopy. Portions of the axial complex were fixed for 2 h in a 3 % glutaraldehyde- paraformaldehyde solution buffered with artificial seawater (pH7.3; 1220 mOsm), dehydrated in ethanol and Frigen, dried in a critical point apparatus and observed in a Cambridge Stereoscan Mark II at 30 kV.

Transmission Electron Microscopy. Tissue was fixed for 3 h in a 2 % OsO 4 solution buffered with seawater (see above), dehydrated in ethanol and embedded in Epon 812. Sections were stained with aqueous uranyl acetate and lead citrate and were examined in a Philips EM 300 electron microscope at 80 kV.

X-ray Microanalysb. Tissue was quickly removed, dissected into small pieces, quenched in liquid nitrogen-cooled propane and freeze-dried in a tissue dryer (Simonsberger et al. 1977) for 50 h. The dried. specimens were embedded in a low viscosity resin (Spurr 1969). Thin sections (300 rim) were mounted on 100-mesh aluminium grids covered with carbon-coated formvar film. Analysis was performed with a Cambridge Stereoscan Mark II in the transmission mode adapted for energy dispersive X-ray microanalysis (Pohla 1980) and equipped with an ORTEC solid state detector. Conditions of analysis were 20 kV and 100 s counting time per point analysis. Relative elemental (atomic) ratios (Fe : S) were calculated as described in Chandler (1976, 1977). The iron concentration was calculated using the continum method (Hall 1979).

Results

A. Fine Structure oJ the Phagocytes

Leukocyte-type cells and granule-fil led phagocytes can be regarded as a single class of phagocytes (Bachmann and Goldschmid 1978a). The different stages of these

Phagocytes in the Axial Complex of Sphaerechinus 111

Fig. 1. Scanning electron micrograph of an early phagocyte in the axial organ. P pseudopodium, x 8,000

Fig. 2. Transmission electron micrograph showing phagocytosis. N nucleus of the phagocytic cell, NI nucleus of the ingested cell. • 7,500

Fig.3. Early phagocyte. M mitochondria, N nucleus, PL primary lysosomes. • 17,700

112 s. Bachmann et al.

cells are characterized by the varying content ofphagosomes or lysosomal granules. Fig. 1 shows a phagocyte moderately filled with ingested material. This cell exhibits long pseudopodia, which are typical for active phagocytes. Transmission electron microscopy reveals a variety of cytoplasmic membrane-bound vacuoles in the phagocytes (Figs. 3, 6). Morphological criteria indicate that the majority of these vacuoles or granules are lysosomes. For different lysosomal stages the terms suggested for the vacuolar system by Dingle (1972) will be employed. According to the amount of ingested granules early and late phagocytes can be differentiated.

Early Phagocytes. The cytoplasm contains conspicuous electron-dense granules (diameter 200-500 nm), the homogeneous content of which is usually separated from the surrounding membrane by a narrow electron-lucid halo (Fig. 3). These granules meet the structural criteria of primary lysosomes.

The rough endoplasmic reticulum and the Golgi apparatus are well developed. The Golgi apparatus is composed of one or more stacks of parallel smooth membranes, frequently with membrane-bound vesicles among the lamellae. Some of these appear to bud from the lameltae and may be related to primary lysosome formation. There is a large quantity ofmicropinocytotic vesicles, some of which are in close contact with the lysosomes. At certain sites, fusion between vesicles and lysosomes appears to occur. The transitional stages between early and late phagocytes are characterized by an increasing number ofphagosomes or secondary lysosomes due to endocytosis of whole cells (Figs. 2, 6).

The phagocytosed cells resemble the early phagocytes. Their cytoplasm contains small electron-lucid blebs and lysosomal bodies; the nucleus exhibits a condensed nucleoplasm (Fig. 2). Another cell type phagocytosed in the axial complex is the morula cell (for description, see Bachmann and Goldschmid 1978 a). Phagocytosis ofmorula cells can be observed mainly at sites surrounding the central cavity of the axial organ (axial sinus). Except for the morula cells, the majority of the early and late phagocytes as well as the phagocytosed cells usually contain an intranuclear crystalloid (Figs. 5, 6).

Late Phagocytes. These phagocytes are scattered throughout the axial complex, but are preferentially concentrated in the periphery of the axial organ near and within the outer lacunae (see below). Their cytoplasm is filled with granules. The large phagocytic granules contain the variously digested residues of phagocytosed cells (Figs. 5, 6). The intranuclear crystalloid of a phagocytosed cell can still be identified even in advanced stages oflysis (Fig. 6). Granules that have not yet terminated their digestive activity (i.e., cytoplasmic remnants are still discernible), may be termed telolysosomes. They contain myelin figures, clusters of electron-dense particles (remnants of nuclear chromatin) and electron-lucid spaces. Numerous residual bodies, the digestive capacity of which appears exhausted, are filled with dense layers and whorls of tight membrane lamellae (average distance between two membranes: 5 nm). In localized areas structurally altered membrane material forms crystalline structures (Figs. 7, 8). Heterolysosomes and residual bodies are limited by a single membrane. The residual bodies are reminiscent of lipofuscin granules in vertebrate liver cells.

Fig.4. Scanning electron micrograph of late phagocytes in the periphery of the axial organ, x 2,800

Fig. 5. Transmission electron micrograph of a late phagocyte displaying few residual bodies (RB) in the cytoplasm. I N intranuclear crystalloid, n nucleolus, x 8,400

Fig. 6. Late phagocyte filled with remnants of phagocytosed ceils. The pycnotic nucleus contains a crystalloid (IN). Note crystalloids (C) in the residual bodies. P early phagocyte, x 7,540


Fig. 7. Residual body in a late phagocyte. Membrane arrays form a crystalline structure (C). x 91,400

Fig. 8. Residual body forming a membranous whorl, x 76,000

Fig. 9. Intranuclear crystalloid of an early phagocyte showing a square array of globular substructure. x 206,100

Fig. 10. Intranuclear crystalloid with a filamentous substructure. Note patches of condensed chromatin in the periphery, x 84,100


Intranuclear Crystalloids. With the exception of a few irregular, rhomboid or hexagonal forms, the crystalloids are generally cubiform in shape. Within the nucleus, these crystalloids are not surrounded by a membrane. The nucleoplasm shows patches of condensed chromatin in the vicinity of the crystalloid. The size of the crystalloids ranges between 0.25 and 1.5 gm in nuclei of intact phagocytes but can increase to 5 gm within the lysosomes after being phagocytosed. Depending on the plane of section, the crystalloids of intact phagocytes exhibit either a lattice-like or particulate substructure (Figs. 9, 10). In the lattice-like pattern filamentous structures are arranged in parallel rows with a periodicity of 9-10nm. In crystalloids with a particulate substructure, the particles measure approximately 7 nm in diameter and reveal a square array.

As mentioned above, the only remnant still discernible of a phagocytosed and digested cell is, if present before, the crystalloid (Fig. 6). In this state, even high magnification reveals little, if any substructure of the crystalloid. The loss of substructure may be due to structural alterations during the constant growth of the crystalloid.

B. Exit-Transport oj the Late Phagocytes

In previous communications (Bachmann and Goldschmid 1978a; 1980), the authors suggested an elimination of waste products via the water vascular system. For soluble products and a few coelomocytes other than late phagocytes this hypothesis might still be tenable, however, concerning the late phagocytes it was recently observed that (i) these cells are predominantly concentrated in the lacunae of the aboral portion of the axial organ, whereas the lacunae of the oral portion appear more or less free of late phagocytes; (ii) near the anus a voluminous lacunar connection between axial organ, dorsoventral mesentery and rectum provides a transport of large amounts of partly disintegrating phagocytes; (iii) after storage at the base of the rectum wall, the meanwhile fully digested cellular content is transported interstitially to the epithelium lining the lumen of the rectum. Here voluminous accumulations are formed which are released into the lumen through rupture of the epithelium. During their storage and transport in the wall of the rectum the crystalloids appear to grow larger and reach their maximum size (up to 10 gm) at the moment of discharge into the lumen of the rectum (Fig. 11).

Thus, the late phagocytes formed in the axial complex are transported via the haemal system to the rectum. Their remnants, the large crystalloids and residual bodies are removed with the faeces.

C. X-ray Microanalysis

The phagocytes contain iron, sulphur, calcium and zinc in a distribution shown in Fig. 12. The intranuclear crystalloids of early and late phagocytes always reveal prominent iron and sulphur peaks (Fig. 12). The iron content of the crystalloids ranges from approximately 5 to 10~, the atomic Fe: S ratio from 8.1 to 9.6: 1. Neither nucleoplasm nor cytoplasm of the phagocytes contain detectable amounts

Fig. 11. Light micrograph of the wall of the rectum. The digested content of late phagocytes is stored at the base (DC). Note the large crystalloids, some of which are still surrounded by a lysosomal membrane (arrows). An accumulation of crystalloids and residual bodies prior to discharge into the lumen of the rectum (L). • 780

g r a n u l e s ( n = 2 6 )

S i 8 8 % m =1,98 r

c. liiiiiiiiiiiiiiiiiiiiill oo%o:1..

Fe 58% m-2 ,25

iiiii!iiiiiiiii!iii!i!iii!iiiiiiiili~ilili~ z~ !!!ili!i!i!i!i!i!i::i!i::iii::i::;::;::;::iii::iii:: ~2%~=1,o, :::::::::::::::::::::::::::::::::::::::::::

c r y s t a l l o i d s ( n = 1 2 )

S ::i::i::i::i 100% m = 1.30 ;.;-;.:,:

C= 0%

Fe 100% m =17,4 i

Zn 14%m=1 ,23

( ~ , , , , ; ~ ~ P : b

2 3 4 5 10 20 30

Fig. 12. X-ray microanalysis. The diagram shows the peak to background (P : b) ratio of S, Ca, Fe and Zn in late phagocyte granules and crystalloids. Next to the dotted bars the percentage of significant peaks; rn median value, n number of measurements. Note that Fe and S are always present in the crystalloids


(i.e., below 0.1%) of iron or any other of the analyzed elements. A weak zinc peak appears in some of the analyzed crystalloids, but this measurement should be considered exceptional. The iron-containing granules necessarily also contain sulphur; this indicates the presence of an ingested crystalloid (Fig. 6). Morula cells do not contain detectable amounts of the elements found in the phagocytes.

Discussion

Evidence exists that the axial complex is directly involved in the process of differentiation and activity of the phagocytes. The present authors introduced the distinction between early and late phagocytes to clarify that they represent different stages of the same cell type. There is most likely a common basic cell type developing either into fibroblasts or into phagocytes, since early phagocytes and fibroblasts in areas of connective tissue of the axial complex show numerous morphological similarities (Bachmann and Goldschmid 1978a). With reference to the above-mentioned resemblance between echinoid phagocytes and vertebrate macrophages (Bachmann and Goldschmid 1978a), the assumption that both cell types carry out similar functions is favored by a number of features. Both cell types can occur as free cells, both are phagocytotic, basophilic and concerned with cellular defense (clotting and ingestion of foreign matter in echinoid phagocytes; Endean 1966; Binyon 1972.

A possible cell-mediated immunologic responsiveness; as proposed for asteroids (Leclerc 1974) and holothuroids (Smith 1977), has yet to be demonstrated in echinoids.

In the present study on Sphaerechinus the terminology of the lysosomal concept (Dingle 1972; Vernon-Roberts 1972; Fawcett 1973) has been applied for the granular bodies and vesicles involved in phagocytotic and digestive processes in analogy to comparable phenomena in vertebrates. However, it should be noted that only cytochemistry at the electron microscopic level can determine the true nature of the granules. Our suggestions seem to be justified in the light of a comparison between the present observations of the phagocytotic process and lysosomal activities described in vertebrate liver and spleen cells (Htibner 1968; Vernon- Roberts 1972; Bucher 1977).

In evertebrates, similar processes are reported from phagocytotic amoebocytes in holothuroids (Baccetti and Rosati 1968), in an ascidian (Fujimoto and Watanabe 1976), and in kidney cells of Mytilus (Pirie and George 1979; George et al. 1980). Moreover, a cytochemical criterion for lysosomal identification, i.e., the ingestion of heavy metal compounds (Dingle 1972), can also be applied to the echinoid phagocytes as they readily ingest and metabolize injected ferritin (Jangoux and Schaltin 1977; unpublished observations of the present authors). Additional tests for specific lysosomal enzymes in the primary and secondary lysosomes are still required. The membrane lamellae detected in the late phagocyte granules are characteristic of residual bodies (Dingle 1972; Weinstock and Albright 1967). The fact that Vevers (1962) detected lipofuscin in the axial organ points to the possibility that lipofuscin is related to some of the phagocyte inclusions. The echinoid phagocytes reveal a well defined ability to release their residual bodies and


crystalloids through rupture of the cell membrane, as seen in the lacunae of the axial complex and in the rectum. In contrast hereto, little is known concerning the extracellular discharge of phagocytic cells in vertebrates apart from elimination of lysosomal residues in bile (Duve and Wattiaux, in Dingle 1972).

Intranuclear crystalloids in echinoid coelomocytes have been described by several authors (for review, see H6baus 1978). According to H6baus, nearly all phagocytes of the coelomic fluid contain crystalloids; the same appears to be true of phagocytes in various organs (Karasaki 1965; Cobb and Sneddon 1977; Bachmann and Goldschmid 1980). The formation and function of the crystalloids are still unknown. Lecal (1971) has termed these structures "centres organisateurs" and assigns to them insufficiently defined regulatory functions. As to the fine structure of the crystalloids, the observations of Karasaki (1965) are highly consistent with our findings. The particulate structure of the crystalloid lattices does not coincide with the structure of ferritin as found in liver cell nuclei (Richter 1961 ; Karasaki 1965). The components detected in the crystalloids are protein and ferric iron (Karasaki 1965; H6baus 1978).

X-ray microanalysis confirmed the presence of iron and revealed that iron is always associated with sulphur, whereas zinc is only occasionally detected. The significance of these findings cannot be fully interpreted at present; sulphur probably belongs to the proteinaceous component of the crystalloid. Ghadially (1979) reports the occurrence of iron and sulphur in rabbit muscle siderosomes. However, it is unlikely that the echinoid crystalloids and vertebrate siderosomes are comparable structures, since echinoids lack iron-containing respiratory pigments (Binyon 1972). Possibly the crystalloids function as collectors in neutralizing heavy metals, as observed in kidney cell nuclei of the rat (Mfiller and Ramin 1963).

In the kidney of the scallop, highly mineralized membrane-limited vacuoles or residual bodies occur that contain high amounts of various metals (George et al. 1978; 1980). These authors report a gradual development of the granules from lysosomal membranous vesicles to residual bodies, which are eventually excreted into the urinary tract. There might exist the necessity for echinoids to neutralize iron when they feed on iron-containing algae (Stevenson and Ufret 1966; H6baus 1978).

The substructure of the echinoid crystalloids is reminiscent of a similar structure in microbodies of vertebrate liver and kidney cells (Hruban and Rechcigl 1969; Lehninger 1975). The microbodies contain urate oxidase, which is an enzyme involved in the synthesis of urea. Since Fechter (1973) reports high concentrations of urea in the echinoid axial organ and intestine, it would be interesting to localize cytochemically the sites of synthesis and storage of urea. The obvious growth of the phagocytosed crystalloids during their migration through the lacunae and intestine wall into the lumen of the rectum might be related to an increasing synthesis of urea, so that the crystalloids are able to synthesize and/or store urea. According to Fechter (1973), in Paracentrotus, at least 1/3 of the total urea of the body is excreted via the intestine.

In conclusion, there is some evidence to link the function of the axial complex with that of the Tiedemann bodies (Bachmann and Goldschmid 1980). Both are organ systems involved in the formation of late phagocytes. Early phagocytes may reach these organs via the haemal system or via epithelial passage (Bachmann and Goldschmid 1978a), morula cells via the water vascular system. Since both axial


c o m p l e x a n d T i e d e m a n n bod ies a re c o n n e c t e d to the l a c u n a r sys tem, b o t h

s t ruc tu res m a y r e m o v e la te p h a g o c y t e s v ia the in tes t ine .

T w o q u e s t i o n s r equ i r e f u r t he r i n v e s t i g a t i o n : (1) a re the i n t r a n u c l e a r

c rys ta l lo ids i n v o l v e d in t he m e t a b o l i s m o f u rea , a n d (2) is the h i g h i r o n c o n t e n t in the c rys ta l lo ids d u e to a necess i ty fo r e l i m i n a t i o n o f i r on?

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Accepted August 25, 1980

phagocytes in the axial complex of the sea urchin, sphaerechinus granularis (lam.)

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