Nelly A. Odintsova

Expression of Pigment Cell-Specific Genes in the Ontogenesis of the Sea Urchin Strongylocentrotus intermedius

One of the polyketide compounds, the naphthoquinone pigment echinochrome, is synthesized in sea urchin pigment cells. We analyzed polyketide synthase (pks) and sulfotransferase (sult) gene expression in embryos and larvae of the sea urchin Strongylocentrotus intermedius from various stages of development and in specific tissues of the adults. We observed the highest level of expression of the pks and sult genes at the gastrula stage. In unfertilized eggs, only trace amounts of the pks and sult transcripts were detected, whereas no transcripts of these genes were observed in spermatozoids. The addition of shikimic acid, a precursor of naphthoquinone pigments, to zygotes and embryos increased the expression of the pks and sult genes. Our findings, including the development of specific conditions to promote pigment cell differentiation of embryonic sea urchin cells in culture, represent a definitive study on the molecular signaling pathways that are involved in the biosynthesis of pigments during sea urchin development.

Gal4-Gene-Dependent Alterations of Embryo Development and Cell Growth in Primary Culture of Sea Urchins

Primary cell cultures from sea urchins have a low proliferative level that prevents the establishment of long-term cultures. To increase expression levels of the genes regulating cell growth in sea urchins, and thus enhance cell growth, we used the transcriptional activator gene Gal4 found earlier in yeast. Sea urchin embryos were treated with plasmid DNA containing the Gal4 gene. Expression of the transgene was confirmed by reverse transcriptase polymerase chain reaction. When the fully functional gene was used, embryos effectively formed teratoma-like structures after 50 to 55 hours of cultivation. In contrast, the Gal4 gene, devoid of acidic activating regions, possessed little activity as a teratogen. The Gal4-treated cells in blastula-derived culture showed higher DNA synthesis and higher proliferative activity than control cells. We suggest that formation of the teratoma-like structures in embryos, activation of DNA synthesis, and significant increase of cell number in embryo-derived cell cultures could be attributed to Gal4 gene action.

Cryopreservation of the cells and larvae of marine organisms

This paper reviews original and literature data on the cryoresistance of the cells of marine organisms. The technology for the cryopreservation of these objects includes the selection of freezing conditions and the use, in addition to traditional cryoprotectants, of combinations of exogenous lipids, antioxidants, and disaccharide trehalose as a membrane stabilizing agent. We propose an approach for the preservation of marine invertebrate cells. The approach is based on the use of biologically active substances obtained from the tissues of marine organisms. Our results demonstrated the synergistic activity of these components of cryoprotective mixtures, and, at the same time, the specificity of antioxidant effects. An analysis of the factors that determine the choice of cryoprotectors was performed for various cell types. The development of the cryopreservation methods of marine organisms provides an opportunity for their wide application in both developmental biology and in marine biotechnology and serves as an important prerequisite for the cryobank creation.

The fatty acid profile changes in marine invertebrate larval cells during cryopreservation.

The development of cryopreservation methods for embryonic cells and larvae of sea animals offers a great potential for marine biotechnology. Larval cells of bivalves and sea urchins were frozen to -196 degrees C using traditional cryoprotectants (Me(2)SO and trehalose) and the cryoprotective mixture developed by us. In addition to Me(2)SO and trehalose, this mixture contained an exogenous lipid extract from mussel tissues and antioxidants. A positive effect of antioxidants (alpha-tocopherol acetate, ascorbic acid or echinochrome, the quinoid pigment of sea urchins) on cell viability became significant only in the presence of exogenous lipids. Antioxidants added to cryoprotective mixtures did not reveal visible cryoprotective activity when used separately. To better understand the mechanism of the protective effect of exogenous lipids on cell membranes of sea animals, a comparative analysis of the fatty acid (FA) composition of total lipids in larval cells before and after freezing was carried out using a gas-liquid chromatography. The results indicate that freezing-thawing has direct effects on the FA composition of major lipid classes in marine invertebrate cells, and these effects can vary depending on the provenance of the cells. We have found that (I) both cell viability and the FA profile of cell lipids after cryopreservation depend on the cryoprotectants used; (II) an amount of saturated, monoenic and polyenic FAs changes significantly after cryopreservation. We assume that the addition of the exogenous lipid extract in form of liposomes could promote a renewal of disturbance areas and prevent from membrane damages during freezing-thawing.

Pigment cell differentiation in sea urchin blastula-derived primary cell cultures

The quinone pigments of sea urchins, specifically echinochrome and spinochromes, are known for their effective antioxidant, antibacterial, antifungal, and antitumor activities. We developed in vitro technology for inducing pigment differentiation in cell culture. The intensification of the pigment differentiation was accompanied by a simultaneous decrease in cell proliferation. The number of pigment cells was two-fold higher in the cells cultivated in the coelomic fluids of injured sea urchins than in those intact. The possible roles of the specific components of the coelomic fluids in the pigment differentiation process and the quantitative measurement of the production of naphthoquinone pigments during cultivation were examined by MALDI and electrospray ionization mass spectrometry. Echinochrome A and spinochrome E were produced by the cultivated cells of the sand dollar Scaphechinus mirabilis in all tested media, while only spinochromes were found in the cultivated cells of another sea urchin, Strongylocentrotus intermedius. The expression of genes associated with the induction of pigment differentiation was increased in cells cultivated in the presence of shikimic acid, a precursor of naphthoquinone pigments. Our results should contribute to the development of new techniques in marine biotechnology, including the generation of cell cultures producing complex bioactive compounds with therapeutic potential.

Vascular endothelial growth factors: A comparison between invertebrates and vertebrates

This review aims to summarize recent data concerning the structure and role of the members of the vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGFR) families in the context of early development, organogenesis and regeneration, with a particular emphasis on the role of these factors in the development of invertebrates. Homologs of VEGF and/or VEGFR have been found in all Eumetazoa, in both Radiata and Bilateria, where they are expressed in the descendants of different germ layers and play a pivotal role in the development of animals with and without a vascular system. VEGF is a well-known angiogenesis regulator, but this factor also control cell migration during neurogenesis and the development of branching organs (the trachea) in invertebrate and vertebrate species. A possible explanation for the origin of Vegf/Vegfr in the animal kingdom and a pathway of Vegf/Vegfr evolution are discussed.

Freezing tolerance of sea urchin embryonic cells: Differentiation commitment and cytoskeletal disturbances in culture

This study focuses on the freezing tolerance of sea urchin embryonic cells. To significantly reduce the loss of physiological activity of these cells that occurs after cryopreservation and to study the effects of ultra-low temperatures on sea urchin embryonic cells, we tested the ability of the cells to differentiate into spiculogenic or pigment directions in culture, including an evaluation of the expression of some genes involved in pigment differentiation. A morphological analysis of cytoskeletal disturbances after freezing in a combination of penetrating (dimethyl sulfoxide and ethylene glycol) and non-penetrating (trehalose and polyvinylpyrrolidone) cryoprotectants revealed that the distribution pattern of filamentous actin and tubulin was similar to that in the control cultures. In contrast, very rare spreading cells and a small number of cells with filamentous actin and tubulin were detected after freezing in the presence of only non-penetrating cryoprotectants. The largest number of pigment cells was found in cultures frozen with trehalose or trehalose and dimethyl sulfoxide. The ability to induce the spicule formation was lost in the cells frozen only with non-penetrating cryoprotectants, while it was maximal in cultures frozen in a cryoprotective mixture containing both non-penetrating and penetrating cryoprotectants (particularly, when ethylene glycol was present). Using different markers for cell state assessment, an effective cryopreservation protocol for sea urchin cells was developed: three-step freezing with a low cooling rate (1-2°C/min) and a combination of non-penetrating and penetrating cryoprotectants made it possible to obtain a high level of cell viability (up to 65-80%).

Development of the Muscle System and Contractile Activity in the Mussel Mytilus trossulus (Mollusca, Bivalvia)

The development of contractile apparatus was subjected to comparative analysis during ontogenesis of the mussel Mytilus trossulus. Indirect immunofluorescence with the polyclonal antibody against mussel twitchin, a protein of thick filaments, and fluorescent phalloidin as a marker of filamentous cell actin were used to monitor changes in the developing muscle system at different larval stages. The first definitive muscle structures were found at the late trochophore stage (36 h after fertilization) and starting from the midveliger stage (96h), striated muscles, which are never present in adult mussels, were distinctly seen. The striated muscle periodicity was 1.25 μm in both mussel larvae and adult scallop. The contractile activities of veliger and adult muscles were measured using an electronic signal-processing video workstation. This work is the first complex study of morphological, biochemical, and physiological characteristics of the muscle system in the larvae and adult molluscs.

Influence of the activator of transcription GAL4 on growth and development of embryos and embryonic cells in primary cultures of sand dollar

In order to solve many tasks of biotechnology, constant lines of the cells of marine invertebrates with a high growth potential are required, which are absent at present. We used the universal activator of transcription gal4 to change the degree of expression of genes of growth factors in embryonic sea urchin cells and, thereby, increase their proliferative activity. The fertilized sea urchin eggs and dissociated embryonic cells at the blastula stage were treated with plasmids containing both the functional gene gal4 and the gene devoid of the regions encoding the activator domain. The transfection of embryonic sea urchin eggs with the functional gene led to cell dedifferentiation and formation of tumor-like structures in the embryos or increased number of embryonic cells in culture. In the cells obtained from the transfected embryos, the pigments were found within two months of cultivation, whose absorption spectrum coincided with that of echinochrome.

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.