In Vitro Cell.Dev.Biol.--Animal 31:528-535, July/August 1995 © 1995Society for In VitroBiology 1071-2690/95 $05.00 + 0,00 FORMATION OF SPICULES BY SCLEROCYTES FROM THE FRESHWATER SPONGE EPHYDATIA MUELLERI IN SHORT-TERM CULTURES IN VITRO GEORG IMSIECKE, RENATE STEFFEN, MARCIO CUSTODIO, RADOVAN BOROJEVIC, ANDWERNER E. G. Mi]LLERa lnstitut far Physiologische Chemie, Universitat, Duesbergweg6, D-55099 Mainz, Germany (G. 1., R. S., W. E. G. M.) and Departamento de BioquCmica,lnstituto de Qufmica, Universidade Federal do Rio de Janeiro, Caixa Postal 68021, 21944-970 Rio de Janeiro, Brazil (M. C., R. B.) (Received 29 August 1994; accepted 28 December 1994) SUMMARY Cells from the freshwater sponge Ephydatia muelleri were isolated by dissociating hatching gemmules, During the first 24 h the cells reaggregated, but the aggregates progressively disintegrated again to single cells, among which the spicule- forming sclerocytes were recognized. Such cultures were used to study spicule (megascleres) formation in vitro. The isolated sclerocytes formed the organic central axial filament onto which they deposited inorganic silicon. The size of the spicules (200 to 350/am in length) as well as the rate of spicule formation (1 to 10/am/h) under in vitro conditions were similar to the values measured in vivo. Immediately after completion of spicule formation, or even before, the sclerocyte could start formation of a new spicule; 5% of the cells were in the process of forming two spicules simultaneously. Cultivation of sclerocytes in the absence of silicon resulted in the formation of the axial filament only. We succeeded in maintaining the sclerocytes in a proliferating and spicule-forming state for up to 3 mo. These results demonstrate that the establishment of short-term cell cultures from E. muelleri is possible; however, future studies must be undertaken to identify the growth factors required for a permanent culture of sponge cells. Key words: sponges; Ephydatia muelleri; spicules; sclerocytes; cell culture. INTRODUCTION The phylum Porifera (sponges) is considered to be the lowest mul- ticellular Metazoa. Sponges comprise several thousand species, among which approximately 120 species live in freshwater (Penney and Racek, 1968). Marine forms have been known since the Pro- terozoic period, >1 billion yr ago (Orlov, 1971). The oldest fossil freshwater sponge, Spongilla gutenbergiana, was described from the Middle Eocene (Miiller et al., 1982), whereas spicules from fresh- water sponges were already known from the Cretaceous of Patagonia (Ott and Volkheimer, 1972). The body of the sponges is permeated by water, entering through inhalant pores, flowing through the frequently very complex aquifer- ous system of channels and choanocyte chambers, and leaving through oscule(s). The sponge's internal environment is separated from the external water by exopinacocytes, and from the water flowing in the aquiferous system by endopinacocytes and choanocytes. Sponges are classified by the chemical nature of their skeleton. The largest group, Demospongiae, which are found in all aquatic envi- ronments, are characterized by siliceous (Si02) spicules, supple- mented with collagen and, often, spongin, which also belongs to the collagen family. The spicules have a one-(monactine) to four-(tetrac- tine) rayed organization (Bergquist, 1978; Simpson, 1984). Little is known about the course and the control of spicule devel- opment, in either marine and freshwater sponges. Besides electron microscopic studies, light microscopic investigations using sandwich 1To whom correspondence should be addressed. cultures have been performed to study the spicule formation. Using the freshwater sponge Ephydatiafluviatilis, Weissenfels and Land- schoff (1977) and Weissenfels (1989) demonstrated that the forma- tion of spicules starts in sclerocytes within a specific vesicle. After the production of an axial organic filament, silicon is deposited around it, and the whole process of forming a spicule (190/am in length and 6 to 8/am in diameter) is completed after 40 h, at 21 ° C. Several aspects of spicule formation still remain unclear: a) It is not known if a single sclerocyte or several sclerocytes together with "'helper cells" contribute to spicule formation (Simpson, 1984). b) The final fate of the sclerocyte after the formation of the spicule is unknown; Minchin (1909) assumed that sclerocytes degenerate after completion of the spicules. The alternative view would be moving away from the recently formed spicules, and initiating a new cycle of spicule formation, c) Moreover, since the contribution of Evans (1899), it remains unclear if sclerocytes represent a specific cell type, or may originate from more than one type of precursor cells. To ap- proach an understanding of spicule formation under more defined conditions, primary cultures of cells from the freshwater sponge E. muelleri have been established. We used the gemmules--asexual reproduction bodies--as starting material for the establishment of primary cultures of sclerocytes. MATERIALSAND METHODS Animals Gemmules of the freshwater sponge E. muelleri (Lieberkiihn, 1855; Pori- fera: Demospongiae: Spongillidae) were collected in the river Wied near 528