An approach to quantitate maternal transcripts localized in sea urchin egg cortex using RT-qPCR with accurate normalization.

The sea urchin egg cortex is a peripheral region of eggs comprising a cell membrane and adjacent cytoplasm, which contains actin and tubulin cytoskeleton, cortical granules and some proteins required for early development. Method for isolation of cortices from sea urchin eggs and early embryos was developed in 1970s. Since then, this method has been reliable tool to study protein localization and cytoskeletal organization in cortex of unfertilized eggs and embryos during first cleavages. This study was aimed to estimate the reliability of RT-qPCR to analyze levels of maternal transcripts that are localized in egg cortex. Firstly, we selected seven potential reference genes, 28S, Cycb, Ebr1, GAPDH, Hmg1, Smtnl1 and Ubb, the transcripts of which are maternally deposited in sea urchin eggs. The candidate reference genes were ranked by five different algorithms (BestKeeper, CV, ΔCt, geNorm and NormFinder) based on calculated level of stability in both eggs as well as isolated cortices. Our results showed that gene ranking differs in total RNA and mRNA samples, though Ubb is most suitable reference gene in both cases. To validate feasibility of comparative analysis of eggs and isolated egg cortices, we selected Daglb-2 as a gene of interest, which transcripts are potentially localized in cortex according to transcriptome analysis, and observed increased level of Daglb-2 in egg cortices by RT-qPCR. This suggests that proposed RNA isolation method with subsequent quantitative RT-qPCR analysis can be used to determine cortical association of transcripts in sea urchin eggs.

Isolation, characterization, and ecotoxicological application of marine mammal skin fibroblast cultures.

Marine mammal cell cultures are a multifunctional instrument for acquiring knowledge about life in the world's oceans in physiological, biochemical, genetic, and ecotoxicological aspects. We succeeded in isolation, cultivation, and characterization of skin fibroblast cultures from five marine mammal species. The cells of the spotted seal (Phoca largha), the sea lion (Eumetopias jubatus), and the walrus (Odobenus rosmarus) are unpretentious to the isolation procedure. The sea otter (Enhydra lutris) fibroblasts should be isolated by trypsin disaggregation, while only mechanical disaggregation was suitable for the beluga whale (Delphinapterus leucas) cells. The cell growth parameters have been determined allowing us to find the optimal seeding density for continuous and effective cultivation. The effects of nonpathogenic algal extracts on proliferation, viability, and functional activity of marine mammal cells in vitro have been presented and discussed for the first time.

Maternal control of early patterning in sea urchin embryos.

Sea urchin development has been studied extensively for more than a century and considered regulative since the first experimental evidence. Further investigations have repeatedly supported this standpoint by revealing the presence of inductive mechanisms that alter cell fate decisions at early cleavage stages and flexibility of development in response to environmental conditions. Some features indicate that sea urchin development is not completely regulative, but actually includes determinative events. In 16-cell embryos, mesomeres and macromeres represent multipotency, while the cell fate of most vegetal micromeres is restricted. It is known that the mature sea urchin eggs are polarized by the asymmetrical distribution of some maternal mRNAs and proteins. Spatially-distributed maternal factors are necessary for the orientation of the primary animal-vegetal axis, which is established by both maternal and zygotic mechanisms later in development. The secondary dorsal-ventral axis is conditionally specified later in development. Dorsal-ventral polarity is very liable during the early cleavages, though more recent data argue that its direction may be oriented by maternal asymmetry. In this review, we focus on the role of maternal factors in initial embryonic patterning during the first cleavages of sea urchin embryos before activation of the embryonic genome.

The contribution of apoptosis and necrosis in freezing injury of sea urchin embryonic cells.

Sea urchins have recently been reported to be a promising tool for investigations of oxidative stress, UV light perturbations and senescence. However, few available data describe the pathway of cell death that occurs in sea urchin embryonic cells after cryopreservation. Our study is focused on the morphological and functional alterations that occur in cells of these animals during the induction of different cell death pathways in response to cold injury. To estimate the effect of cryopreservation on sea urchin cell cultures and identify the involved cell death pathways, we analyzed cell viability (via trypan blue exclusion test, MTT assay and DAPI staining), caspase activity (via flow cytometry and spectrophotometry), the level of apoptosis (via annexin V-FITC staining), and cell ultrastructure alterations (via transmission electron microscopy). Using general caspase detection, we found that the level of caspase activity was low in unfrozen control cells, whereas the number of apoptotic cells with activated caspases rose after freezing-thawing depending on cryoprotectants used, also as the number of dead cells and cells in a late apoptosis. The data using annexin V-binding assay revealed a very high apoptosis level in all tested samples, even in unfrozen cells (about 66%). Thus, annexin V assay appears to be unsuitable for sea urchin embryonic cells. Typical necrotic cells with damaged mitochondria were not detected after freezing in sea urchin cell cultures. Our results assume that physical cell disruption but not freezing-induced apoptosis or necrosis is the predominant reason of cell death in sea urchin cultures after freezing-thawing with any cryoprotectant combination.

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%).

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.

Expression pattern of vascular endothelial growth factor 2 during sea urchin development.

The VEGF family in the sea urchin is comprised of three members designated Vegf1 through Vegf3. In this study, we found a high level of similarity between the PDGF/VEGF domain of the predicted gene Sp-Vegf2 in the sea urchin Strongylocentrotus purpuratus and the same domain of a gene that we found in a closely related sea urchin, Strongylocentrotus intermedius. The sequence of the Si-Vegf2 cDNA was determined, and the expression of the Si-Vegf2 mRNA throughout early sea urchin development was studied by RT-PCR and in situ hybridization. Also we analyzed phylogenetic relationships of Si-Vegf2 and other members of the PDGF and VEGF families. We have found that the Si-Vegf2 present during the time span from the egg to the 4-arm pluteus stage. This mRNA is uniformly distributed in eggs, cleaving embryos and early blastulae. At the gastrula stage, the Si-Vegf2 transcripts are localized in the ventrolateral clusters of primary mesenchyme cells, and later, at the prism stage, they are detected in the forming apex. At the early pluteus stage, Si-Vegf2 mRNAs are found in two groups of mesenchyme cells in the scheitel region on the apical pole. We have determined that Si-Vegf2 is a mesenchyme-expressed factor but its developmental function is unknown.