Vanadium Modulates Proteolytic Activities and MMP-14-Like Levels during Paracentrotus lividus Embryogenesis.

The increasing industrial use of vanadium (V), as well as its recent medical use in various pathologies has intensified its environmental release, making it an emerging pollutant. The sea urchin embryo has long been used to study the effects induced by metals, including V. In this study we used an integrated approach that correlates the biological effects on embryo development with proteolytic activities of gelatinases that could better reflect any metal-induced imbalances. V-exposure caused morphological/morphometric aberrations, mainly concerning the correct distribution of embryonic cells, the development of the skeleton, and the embryo volume. Moreover, V induced a concentration change in all the gelatinases expressed during embryo development and a reduction in their total proteolytic activity. The presence of three MMP-like gelatinases (MMP-2, -9, and -14) was also demonstrated and their levels depended on V-concentration. In particular, the MMP-14-like protein modified its expression level during embryo development in a time- and dose-dependent manner. This enzyme also showed a specific localization on filopodia, suggesting that primary mesenchyme cells (PMCs) could be responsible for its synthesis. In conclusion, these results indicate that an integrated study among morphology/morphometry, proteolytic activity, and MMP-14 expression constitutes an important response profile to V-action.

Vanadium Toxicity Monitored by Fertilization Outcomes and Metal Related Proteolytic Activities in Paracentrotus lividus Embryos.

Metal pharmaceutical residues often represent emerging toxic pollutants of the aquatic environment, as wastewater treatment plants do not sufficiently remove these compounds. Recently, vanadium (V) derivatives have been considered as potential therapeutic factors in several diseases, however, only limited information is available about their impact on aquatic environments. This study used sea urchin embryos (Paracentrotus lividus) to test V toxicity, as it is known they are sensitive to V doses from environmentally relevant to very cytotoxic levels (50 nM; 100 nM; 500 nM; 1 µM; 50 µM; 100 µM; 500 µM; and 1 mM). We used two approaches: The fertilization test (FT) and a protease detection assay after 36 h of exposure. V affected the fertilization percentage and increased morphological abnormalities of both egg and fertilization envelope, in a dose-dependent manner. Moreover, a total of nine gelatinases (with apparent molecular masses ranging from 309 to 22 kDa) were detected, and their proteolytic activity depended on the V concentration. Biochemical characterization shows that some of them could be aspartate proteases, whereas substrate specificity and the Ca2+/Zn2+ requirement suggest that others are similar to mammalian matrix metalloproteinases (MMPs).

A single-cell RNA-seq analysis of Brachyury-expressing cell clusters suggests a morphogenesis-associated signal center of oral ectoderm in sea urchin embryos.

Brachyury is a T-box family transcription factor and plays pivotal roles in morphogenesis. In sea urchin embryos, Brachyury is expressed in the invaginating endoderm, and in the oral ectoderm of the invaginating mouth opening. The oral ectoderm is hypothesized to serve as a signaling center for oral (ventral)-aboral (dorsal) axis formation and to function as a ventral organizer. Our previous results of a single-cell RNA-seq (scRNA-seq) atlas of early Strongylocentrotus purpuratus embryos categorized the constituent cells into 22 clusters, in which the endoderm consists of three clusters and the oral ectoderm four clusters (Foster et al., 2020). Here we examined which clusters of cells expressed Brachyury in relation to the morphogenesis and the identity of the ventral organizer. Our results showed that cells of all three endoderm clusters expressed Brachyury in blastulae. Based on expression profiles of genes involved in the gene regulatory networks (GRNs) of sea urchin embryos, the three clusters are distinguishable, two likely derived from the Veg2 tier and one from the Veg1 tier. On the other hand, of the four oral-ectoderm clusters, cells of two clusters expressed Brachyury at the gastrula stage and genes that are responsible for the ventral organizer at the late blastula stage, but the other two clusters did not. At a single-cell level, most cells of the two oral-ectoderm clusters expressed organizer-related genes, nearly a half of which coincidently expressed Brachyury. This suggests that the ventral organizer contains Brachyury-positive cells which invaginate to form the stomodeum. This scRNA-seq study therefore highlights significant roles of Brachyury-expressing cells in body-plan formation of early sea urchin embryos, though cellular and molecular mechanisms for how Brachyury functions in these processes remain to be elucidated in future studies.

Bioactive Potential of Two Marine Picocyanobacteria Belonging to Cyanobium and Synechococcus Genera.

Coccoid cyanobacteria produce a great variety of secondary metabolites, which may have useful properties, such as antibacterial, antiviral, anticoagulant or anticancer activities. These cyanobacterial metabolites have high ecological significance, and they could be considered responsible for the widespread occurrence of these microorganisms. Considering the great benefit derived from the identification of competent cyanobacteria for the extraction of bioactive compounds, two strains of picocyanobacteria (coccoid cyanobacteria < 3 µm) (Cyanobium sp. ITAC108 and Synechococcus sp. ITAC107) isolated from the Mediterranean sponge Petrosia ficiformis were analyzed. The biological effects of organic and aqueous extracts from these picocyanobacteria toward the nauplii of Artemia salina, sea urchin embryos and human cancer lines (HeLa cells) were evaluated. Methanolic and aqueous extracts from the two strains strongly inhibited larval development; on the contrary, in ethyl acetate and hexane extracts, the percentage of anomalous embryos was low. Moreover, all the extracts of the two strains inhibited HeLa cell proliferation, but methanol extracts exerted the highest activity. Gas chromatography-mass spectrometry analysis evidenced for the first time the presence of ß-N-methylamino-l-alanine and microcystin in these picocyanobacteria. The strong cytotoxic activity observed for aqueous and methanolic extracts of these two cyanobacteria laid the foundation for the production of bioactive compounds of pharmacological interest.

Low-dose action of tryptanthrin and its derivatives against developing embryos of the sea urchin Strongylocentrotus intermedius.

Nine tryptanthrin derivatives, including tryptanthrin itself, were synthesized using different methods, including oxidation of the corresponding isatins to obtain 1-4, the reaction of tryptanthrin 1 with hydrazine and its derivatives to obtain 5-7, and aldol condensation of 1 with acetone and methylethylketone to obtain 8 and 9. The action of 1-9 in doses corresponding to the IC50 against developing embryos of the sea urchin Strongylocentrotus intermedius and in the sperm test allowed us to estimate to potency of all the compounds and to determine which were cytotoxic. In addition, these studies showed that compounds 3, 4, 8, and 9 had a stimulatory effect at lower doses. In particular, the tryptanthrin derivatives stimulated the larval stages of development in surviving embryos at concentrations lower than the IC50.

Release of Sticky Glycoproteins from Chlamydomonas Flagella During Microsphere Translocation on the Surface Membrane.

Chlamydomonas flagella display surface motility such that small polystyrene beads (microspheres) attached to the flagellar membrane move bidirectionally along the flagellum. This surface motility enables cells to glide on a solid substrate to which they are attached by the flagellar surface. Previous studies suggested that microsphere movement and gliding motility result from the movement of transmembrane glycoprotein(s) within the plane of the plasma membrane, driven by intraflagellar transport (IFT), which utilizes cytoplasmic dynein and kinesin-2. However, it is not well understood how a cell can continuously glide in one direction further than a single flagellar length. Here we show that, during microsphere translocation on the flagella of a non-motile mutant, pf18, some flagellar glycoproteins, including FMG-1B and FAP113, detach from the membrane and attach to the microspheres. We propose that such relocation of surface glycoproteins underlies the ability to glide over a long distance. Surface motility is likely common to cilia/flagella of various organisms, as a similar microsphere movement is observed in the apical ciliary tuft in sea urchin embryos.

Extremely stable high molecular mass soluble multiprotein complex from eggs of sea urchin Strongylocentrotus intermedius with phosphatase activity.

It was proposed that most biological processes are performed by different protein complexes. In contrast to individual proteins and enzymes, their complexes usually have other biological functions, and their formation may be important system process for the expansion of diversity and biological functions of different molecules. Identification and characterization of embryonic components including proteins and their multiprotein complexes seem to be very important for an understanding of embryo function. We have isolated and analyzed for the first time a very stable multiprotein complex (SPC; approximately 1100 kDa) from the soluble fraction of extracts of the sea urchin embryos. By fast protein liquid chromatography (FPLC) gel filtration the SPC was well separated from other extract proteins. Stable multiprotein complex is stable in different drastic conditions but dissociates moderately in the presence of 8M urea + 1.0M NaCl. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis data, this complex contains many major, moderate and minor proteins with molecular masses from 10 to 95 kDa. The SPC was destroyed by 8M urea or SDS, and its components were separated using thin layer chromatography, ion-exchange chromatography, gel filtration, and reverse phase chromatography. Using matrix-assisted laser desorption/ionization mass spectrometry of partially dissociated SPC, it was shown that the complex contains not only proteins (10-95 kDa) but also few dozens of peptides with molecular masses from 2 to 9.5 kDa. Short peptides form very strong complexes, which at the treatment of SPC with urea or SDS can be partially break down into smaller complexes having different peptide compositions. Reverse phase chromatography of these complexes after all type of abovementioned chromatographies led to detection from 6 to 11 distinct peaks corresponding to new complexes containing up to a few dozens of peptides. The SPCs possess alkaline phosphatase activity. Progress in the study of embryos protein complexes can help to understand their biological functions.

Gadolinium perturbs expression of skeletogenic genes, calcium uptake and larval development in phylogenetically distant sea urchin species.

Chelates of Gadolinium (Gd), a lanthanide metal, are employed as contrast agents for magnetic resonance imaging and are released into the aquatic environment where they are an emerging contaminant. We studied the effects of environmentally relevant Gd concentrations on the development of two phylogenetically and geographically distant sea urchin species: the Mediterranean Paracentrotus lividus and the Australian Heliocidaris tuberculata. We found a general delay of embryo development at 24h post-fertilization, and a strong inhibition of skeleton growth at 48h. Total Gd and Ca content in the larvae showed a time- and concentration-dependent increase in Gd, in parallel with a reduction in Ca. To investigate the impact of Gd on the expression of genes involved in the regulation of skeletogenesis, we performed comparative RT-PCR analysis and found a misregulation of several genes involved in the skeletogenic and left-right axis specification gene regulatory networks. Species-specific differences in the biomineralization response were evident, likely due to differences in the skeletal framework of the larvae and the amount of biomineral produced. Our results highlight the hazard of Gd for marine organisms.

Autophagy as a defense strategy against stress: focus on Paracentrotus lividus sea urchin embryos exposed to cadmium.

Autophagy is used by organisms as a defense strategy to face environmental stress. This mechanism has been described as one of the most important intracellular pathways responsible for the degradation and recycling of proteins and organelles. It can act as a cell survival mechanism if the cellular damage is not too extensive or as a cell death mechanism if the damage/stress is irreversible; in the latter case, it can operate as an independent pathway or together with the apoptotic one. In this review, we discuss the autophagic process activated in several aquatic organisms exposed to different types of environmental stressors, focusing on the sea urchin embryo, a suitable system recently included into the guidelines for the use and interpretation of assays to monitor autophagy. After cadmium (Cd) exposure, a heavy metal recognized as an environmental toxicant, the sea urchin embryo is able to adopt different defense mechanisms, in a hierarchical way. Among these, autophagy is one of the main responses activated to preserve the developmental program. Finally, we discuss the interplay between autophagy and apoptosis in the sea urchin embryo, a temporal and functional choice that depends on the intensity of stress conditions.

Genome-wide assessment of differential effector gene use in embryogenesis.

Six different populations of cells were isolated by fluorescence-activated cell sorting from disaggregated late blastula- and gastrula-stage sea urchin embryos according to the regulatory states expressed in these cells, as reported by recombineered bacterial artificial chromosomes producing fluorochromes. Transcriptomes recovered from these embryonic cell populations revealed striking, early differential expression of large cohorts of effector genes. The six cell populations were presumptive pigment cells, presumptive neurogenic cells, presumptive skeletogenic cells, cells from the stomodeal region of the oral ectoderm, ciliated band cells and cells from the endoderm/ectoderm boundary that will give rise both to hindgut and to border ectoderm. Transcriptome analysis revealed that each of these domains specifically expressed several hundred effector genes at significant levels. Annotation indicated the qualitative individuality of the functional nature of each cell population, even though they were isolated from embryos only 1-2 days old. In no case was more than a tiny fraction of the transcripts enriched in one population also enriched in any other of the six populations studied. As was particularly clear in the cases of the presumptive pigment, neurogenic and skeletogenic cells, all three of which represent precociously differentiating cell types of this embryo, most specifically expressed genes of given cell types are not significantly expressed at all in the other cell types. Thus, at the effector gene level, a dramatic, cell type-specific pattern of differential gene regulation is established well before any significant embryonic morphogenesis has occurred.

Toxicity mechanisms of ionic silver and polymer-coated silver nanoparticles with interactions of functionalized carbon nanotubes on early development stages of sea urchin.

Exposures of aquatic organisms to multiple contaminants are likely to take place in estuarine and coastal areas and combined effects on early life stages have to be examined. Among emerging contaminants, ionic silver (Ag(+)) and silver nanoparticles (AgNps) have demonstrated contrasting effects on marine invertebrates, but their interactions with functionalized carbon nanotubes (f-SWCNTs) have not yet been investigated in details. In order to observe the impacts and understand the toxicity mechanism of Ag(+) and polymer-coated AgNps, and their combined effects with f-SWCNTs, successive development stages of embryos of sea urchin, Strongylocentrotus droebachiensis, were exposed to Ag(+), AgNps and f-SWCNTs, separately and in mixtures using moderate environmental concentrations. We also assessed long-term effects of treatments under recovery conditions. Morphological endpoints such as archenteron elongation, primary and secondary mesenchyme cells fate, pigment cells migration, spiculogenic cells and gut development indicated different effects of silver and nanosilver forms during successive development stages. Whereas Ag(+) induced vegetalization and extrusion of mesenchyme cells on early embryos; f-SWCNTs+Ag(+) strongly interfered with gut regionalization in late larvae. Sensitive blastocoelar cells got vacuolized and shapeless with AgNps, but not with mixtures with f-SWCNTs. Increased concentrations of Ag(+) and f-SWCNTs+Ag(+) led to the most disruptive effects during development, but f-SWCNTs+Ag(+) caused the highest mortality rate during the recovery period, which indicated far-reaching effects driven by f-SWCNTs and their ability to keep silver more available during exposure period.

Effect of bisphenol A on P-glycoprotein-mediated efflux and ultrastructure of the sea urchin embryo.

Usage of bisphenol A (BPA) in production of polycarbonate plastics has resulted in global distribution of BPA in the environment. These high concentrations cause numerous negative effects to the aquatic biota, among which the most known is the induction of endocrine disruption. The focus of this research was to determine the effects of two experimentally determined concentrations of BPA (100nM and 4µM) on cellular detoxification mechanisms during the embryonic development (2-cell, pluteus) of the rocky sea urchin (Paracentrotus lividus), primarily the potential involvement of multidrug efflux transport in the BPA intercellular efflux. The results of transport assay, measurements of the intracellular BPA and gene expression surveys, for the first time indicate the importance of P-glycoprotein (P-gp/ABCB1) in defense against BPA. Cytotoxic effects of BPA, validated by the immunohistochemistry (IHC) and the transmission electron microscopy (TEM), induced the aberrant karyokinesis, and consequently, the impairment of embryo development through the first cell division and retardation.

Modelization of the regulation of protein synthesis following fertilization in sea urchin shows requirement of two processes: a destabilization of eIF4E:4E-BP complex and a great stimulation of the 4E-BP-degradation mechanism, both rapamycin-sensitive.

Fertilization of sea urchin eggs involves an increase in protein synthesis associated with a decrease in the amount of the translation initiation inhibitor 4E-BP. A highly simple reaction model for the regulation of protein synthesis was built and was used to simulate the physiological changes in the total 4E-BP amount observed during time after fertilization. Our study evidenced that two changes occurring at fertilization are necessary to fit with experimental data. The first change was an 8-fold increase in the dissociation parameter (koff1) of the eIF4E:4E-BP complex. The second was an important 32.5-fold activation of the degradation mechanism of the protein 4E-BP. Additionally, the changes in both processes should occur in 5 min time interval post-fertilization. To validate the model, we checked that the kinetic of the predicted 4.2-fold increase of eIF4E:eIF4G complex concentration at fertilization matched the increase of protein synthesis experimentally observed after fertilization (6.6-fold, SD = 2.3, n = 8). The minimal model was also used to simulate changes observed after fertilization in the presence of rapamycin, a FRAP/mTOR inhibitor. The model showed that the eIF4E:4E-BP complex destabilization was impacted and surprisingly, that the mechanism of 4E-BP degradation was also strongly affected, therefore suggesting that both processes are controlled by the protein kinase FRAP/mTOR.

Synthesis and antineoplastic properties of (1H-1,2,3-triazol-1-yl)furazans.

A method of 3-amino-4-[5-aryl(heteroaryl)-1H-1,2,3-triazol-1-yl)]furazan synthesis was optimized. Condensation of these compounds with 2,5-dimethoxytetrahydrofuran resulted in a series of previously unknown 4-[5-aryl(heteroaryl)-1H-1,2,3-triazol-1-yl)]-3-(pyrrol-1-yl)furazans. All target compounds were evaluated for both antimitotic microtubule destabilizing effect in a phenotypic sea urchin embryo assay and cytotoxicity in a panel of 60 human cancer cell lines. Pyrrolyl derivatives of triazolylfurazans were determined as antiproliferative compounds. The most potent microtubule targeting compounds 7a and 7e are of interest for further trials as antineoplastic agents.

Cell-to-substratum adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus.

Some plant lectins, Concanavalin agglutinin (Con A), succinyl Con A and wheat germ agglutinin (WGA) increased the adhesion of dissociated embryonic cells of the sea urchin,Pseudocentrotus depressus, to the substratum (plastic and glass surface) in vitro. Other plant lectins,Ulex europeus agglutinin (UEA) andDolichos biflorus agglutinin (DBA) had no effect on the cell-to-substratum interaction. A specific monocarbohydrate inhibitor of lectins, α-methyl-D-mannoside, inhibited the Con A-induced cell-to-substratum adhesion of dissociated embryonic cells. This observation suggests that the Con A-induced cell-to-substratum adhesion may be attributed to the Con A-carbohydrate interaction. In Millipore-filtered sea water (MPFSW) containing Con A (0.1 mg/ml), dissociated embryonic cells adhered to the substratum for more than 6 h at 18°C, while in MPFSW as control, almost all the dissociated cells were released from the substratum after 1 h. A scanning electron microscopic study showed that dissociated embryonic cells adhered to the substratum were surrounded by an extracellular fibrous material, when the cells were cultured in MPFSW containing Con A. The induction of the extracellular fibrous material by Con A was inhibited by α-methyl-D-mannoside. The appearance of this material may be related to the cell-to-substratum adhesion of dissociated cells. Sequential extractions of Con A-treated dissociated cells with Triton X 100 and urea solubilized most of the cellular components, leaving the fibrous material on the surface. Biochemical conponents of the isolated fibrous material included sea urchin fibronectin, Con A and minor components (88 and 140 kilodalton proteins). Fibronectin preformed in the cells was excreted after the dissociation, while the 88 and 140 kilodalton proteins were synthesized and released to the extracellular space.

Distribution and redistribution of pigment granules in the development of sea urchin embryos.

Pigment granules (PGs) are embeded in the cortex of embryos of the Japanese sea urchins,Hemicentrotus pulcherrimus and Anthocidaris crassispina. PGs in the cortex actively retreated from the vegetal pole area at the 4-cell stage and then a notable PG-distribution gradient formed along the egg axis (the polar redistribution of PGs). The polar redistribution of PGs in the cortex occurred at the same time after fertilization even in solutions of microtubule disrupting reagents such as Colcemid, vinblastine sulfate or griseofulvin. Consequently, the polar redistribution of PGs was not associated with the microtubules. However, the polar redistribution of PGs was interrupted in seawater containing cytochalasin B (CB), dithiothreitol (DTT) or tetracaine, and the distribution pattern of PGs in the cortex was definitely disturbed. Moreover, CB, DTT and tetracaine altered the division pattern of vegetal blastomeres at the 4th cleavage which is normally unequal so that all the blastomeres divided equally. Microtubule disrupting reagents did not have such an effect on the cleavage pattern. Thus the cortical movement along the egg axis reflected by the polar redistribution of PGs seems to correlate with the micromere formation.