Autofluorescence mediated red spherulocyte sorting provides insights into the source of spinochromes in sea urchins.

Red spherule cells (RSCs) are considered one of the prime immune cells of sea urchins, but their detailed biological role during immune responses is not well elucidated. Lack of pure populations accounts for one of the major challenges of studying these cells. In this study, we have demonstrated that live RSCs exhibit strong, multi-colour autofluorescence distinct from other coelomocytes, and with the help of fluorescence-activated cell sorting (FACS), a pure population of live RSCs was successfully separated from other coelomocytes in the green sea urchin, Strongylocentrotus droebachiensis. This newly developed RSCs isolation method has allowed profiling of the naphthoquinone content in these cells. With the use of ultra high-performance liquid chromatography, UV absorption spectra, and high-resolution tandem mass spectrometry, it was possible to identify sulphated derivatives of spinochrome C, D, E and spinochrome dimers, which suggests that the RSCs may play an important biological role in the biogenesis of naphthoquinone compounds and regulating their bioactivity.

[Effect of exogenous factors on the induction of spicule formation in sea urchin embryonic cell cultures].

The effect of exogenous factors on the realization of the spicule formation program in two sea urchin species, Strongylocentrotus intermedius and S. nudus, has been studied in primary embryonic cell cultures derived from the blastula and gastrula stages. It has been shown that the process of spicule formation depends on the type of substrate and the composition of the medium. An original finding is that calf or horse serum necessary for spicule formation in vitro can be replaced by a complex of factors including insulin, transferrin, and lectins. Methods allowing control over the growth and differentiation of marine invertebrate embryonic cells in vitro open prospects for their application to practical problems such as the establishment of cell cultures producing certain mineral structures.

Isolation of oogonia from ovaries of the sea urchin Strongylocentrotus nudus.

The presence of oogonia in the ovaries of adult females is typical in species with a broadcast spawning reproductive strategy, including invertebrates and lower vertebrates. In sea urchins, difficulties in the study of oogonia arise from the small number of these cells and the lack of specific markers for their identification. Therefore, more reliable methods are needed for identifying and manipulating oogonial cells in quantities sufficient for experimentation. Homologs of the DEAD-box RNA helicase vasa expressed in germline cells have been proposed for use as markers to detect germline cells in diverse species. We have developed a method for the isolation of sea urchin oogonia by using immunocytochemistry with vasa antibodies, together with reverse transcription and the polymerase chain reaction to detect the expression of Sp-vasa and Sp-nanos2 homologs and a morphological approach to identify germline cells in sea urchin ovaries and cell fractions isolated from the ovarian germinal epithelium. This method has allowed us to obtain 15%-18% of small oogonia with 70%-75% purity from the total amount of isolated germ cells. Our findings represent the first methodological basis for obtaining cell populations containing sea urchin oogonia; this method might be useful as a tool for further investigations of the early stages of sea urchin oogenesis.

Derivation of muscles of the Aristotle's lantern from coelomic epithelia.

Transmission electron microscopy was employed to study structural changes in the lantern muscles occurring during the transition from young to adult in the sea urchin Strongylocentrotus nudus. A comparative examination of four major lantern muscles (compass depressors, compass elevators, protractors and retractors) suggests that myogenesis involves four consecutive stages. At the initial stage, the muscles show the organization of a mesentery delimited by pseudostratified coelomic epithelia, which are composed of peritoneal cells spanning the whole height of each epithelium, and myoepithelial cells, which are clustered together to fill the interstices between the basal processes of the peritoneal cells. During the next stage, the clusters of myoepithelial cells partly "sink" into the underlying connective tissue. At the third stage of muscularization, the myoepithelial cells increase in size and further invade the underlying connective tissue so that the myoepithelium splits into an apical peritoneal layer and a deeper mass of myoepithelial cells immersed in the connective tissue. However, these two layers are connected by a continuous basal lamina. This is thus the first description of an intermediate developmental stage between pseudostratified myoepithelim and genuine echinoderm muscles. For such a myoepithelium, we propose the term "immersed myoepithelium". At the most advanced stage of myogenesis, the myocytes detach completely from the epithelium to form subepithelial muscle bundles. Myogenesis in the sea urchin takes a long time during which continuous myogenic differentiation occurs in the coelomic epithelium and the newly formed myocytes and associated neurons penetrate into the underlying connective tissue.

Nutritive phagocyte incubation chambers provide a structural and nutritive microenvironment for germ cells of Strongylocentrotus droebachiensis, the green sea urchin.

Here we characterize the germinal epithelia of both sexes of Strongylocentrotus droebachiensis, the green sea urchin, throughout its annual gametogenic cycle, using light and electron microscopy and cytochemistry. In both sexes, germinal epithelia include two interacting cellular populations: nutritive phagocytes (NPs) and germ cells. After spring spawning, NPs accumulate nutrients; amitotic oogonia and often mitotic spermatogonia occur in clusters beneath NPs; and subsequent gametogenic stages are residual or absent. During the summer, NP nutrients are mobilized for use in vitellogenesis by residual primary oocytes or to support limited spermatogenesis. In addition, some residual primary oocytes may degenerate and be phagocytized by NPs. Significant nutrient mobilization from NPs and substantial gonial cell mitoses (indicative of new gametogenesis) occur in the fall. In both sexes, all of these changes are facilitated by NPs that form basal incubation chambers near the gonadal wall and within which germ cells are surrounded by nutrients released from the NPs. In females, germ cells at several stages of gametogenesis may be housed in separate chambers in the same NP. Primary oocytes also carry out jelly coat formation, meiosis, and cortical granule translocation within NP incubation chambers. In males, many NPs cooperate to provide large continuous chambers that contain spermatogenic cells at diverse stages. In both sexes these chambers persist throughout the year and isolate gametogenesis from the gonadal lumen. NPs become slender and shorten as their nutrients are depleted. Ova or spermatozoa are stored in the gonadal lumen. Post-spawning, NPs phagocytize differentiated germ cells while simultaneously enclosing intact gonial and residual gametogenic cells in basal chambers near the gonadal wall. In light of our observations, we suggest investigating proteins that may be important in the structural, phagocytic, and nutritive functions of NPs and for which corresponding genes have already been identified in the genome of S. purpuratus, the closely related purple sea urchin.