Embryonic ethanol exposure alters expression of sox2 and other early transcripts in zebrafish, producing gastrulation defects.

Ethanol exposure during prenatal development causes fetal alcohol spectrum disorder (FASD), the most frequent preventable birth defect and neurodevelopmental disability syndrome. The molecular targets of ethanol toxicity during development are poorly understood. Developmental stages surrounding gastrulation are very sensitive to ethanol exposure. To understand the effects of ethanol on early transcripts during embryogenesis, we treated zebrafish embryos with ethanol during pre-gastrulation period and examined the transcripts by Affymetrix GeneChip microarray before gastrulation. We identified 521 significantly dysregulated genes, including 61 transcription factors in ethanol-exposed embryos. Sox2, the key regulator of pluripotency and early development was significantly reduced. Functional annotation analysis showed enrichment in transcription regulation, embryonic axes patterning, and signaling pathways, including Wnt, Notch and retinoic acid. We identified all potential genomic targets of 25 dysregulated transcription factors and compared their interactions with the ethanol-dysregulated genes. This analysis predicted that Sox2 targeted a large number of ethanol-dysregulated genes. A gene regulatory network analysis showed that many of the dysregulated genes are targeted by multiple transcription factors. Injection of sox2 mRNA partially rescued ethanol-induced gene expression, epiboly and gastrulation defects. Additional studies of this ethanol dysregulated network may identify therapeutic targets that coordinately regulate early development.

Artificial oocyte activation improves cycles with prospects of ICSI fertilization failure: a sibling oocyte control study.

RESEARCH QUESTION: Does artificial oocyte activation improve clinical outcomes for patients at risk of intracytoplasmic sperm injection (ICSI) fertilization failure? DESIGN: In this study, sibling oocytes from patients with previous ICSI failure or severe teratozoospermia were divided equally into two groups, half for artificial oocyte activation (AOA) with ionomycin after conventional ICSI and the other half for conventional ICSI only (non-AOA). The fertilization rates, cleavage rates, transferable embryo rates and blastulation rates of the two groups were compared first; the clinical pregnancy and live birth rates were also compared to assess the efficiency and safety of AOA. RESULT: The outcomes of the AOA group were significantly better than those of the conventional ICSI group in terms of the fertilization (50.38% versus 33.86%, respectively, P < 0.001), cleavage (59.16% versus 39.04%, respectively, P < 0.001) and transferable embryo rates (43.51% versus 26.69%, respectively, P < 0.001). The blastulation (43.53% versus 36.11%, respectively), implantation (26.83% versus 15.79%, respectively), clinical pregnancy (38.46% versus 25%, respectively) and live birth rates (38.46% versus 16.67%, respectively) were not significantly different. CONCLUSION: This study showed that AOA improved some aspects of cycles at risk of ICSI failure by increasing the fertilization and transferable embryo rates. But blastulation, pregnancy and implantation rates were not improved. The study is limited by its small size and absence of data on cumulative outcomes.

Cryopreservation of embryos and larvae of the edible sea urchin loxechinus albus (Molina, 1782).

The natural population of the edible red sea urchin, Loxechinus albus, is decreasing due to overfishing. The embryos and larvae of the species are highly useful for monitoring marine pollution, which makes it necessary to conserve gametes, embryos and larvae to facilitate their use in diverse areas of aquaculture and environmental quality monitoring. This need can be met by cryopreserving individuals representing the different developmental stages to provide an ongoing supply of genetic material of the species. The present study establishes a reproducible protocol for cryopreserving red sea urchin blastula and larvae. Toxicity tests were conducted in the first stage of this study using two permeable cryoprotectors, dimethyl sulfoxide (Me2SO) and propylene glycol (PG), at three concentrations (5%, 10% and 15%). The tests were repeated in the second stage, but mixing the cryoprotectors with 0.04 M of trehalose (TRE), a non-permeable cryoprotector. Cryopreservation tests were conducted in the third stage employing different freezing rates: 2 °C/min, 3 °C/min, 3.5 °C/min, 4 °C/min and 4.5 °C/min, using the cryoprotectors that yielded the highest post-incubation survival rates. The highest post-freezing survival rates for blastula (76 ± 7%) and larvae (79 ± 7%) were obtained with Me2SO at 10% + 0.04 M of trehalose, with freezing rates of 3 °C/min and 4.5 °C/min, respectively.

Differential sensitivity of developmental stages of the South American toad to a fungicide based on fludioxonil and metalaxyl-M.

Agricultural fungicide application in Argentina has increased twice since 2008, with Maxim® XL (2.5% fludioxonil +1% metalaxyl-M) as one of the most used fungicide formulation. The toxicity of this pesticide on Rhinella arenarum was assessed by means of continuous (from embryo and larval development) and 24-h pulse exposure standardized bioassays. Lethality was concentration- and exposure time-dependent. Maxim® XL caused a progressive lethal effect along the bioassays with higher toxicity on embryos than larvae, obtaining 50% lethal concentrations at 96, 336, and 504 h of 10.85, 2.89, and 1.71 mg/L for embryos, and 43.94, 11.79, and 5.76 mg/L for larvae respectively. Lethal 504-h no observed effect concentration values for embryos and larvae were 1 and 2.5 mg/L respectively. A stage-dependent toxicity of Maxim® XL was also demonstrated within the embryo development, with early stages more sensitive than the later ones, and blastula as the most sensitive developmental stage. The risk quotients obtained for chronic risk assessment determined a potential threat for the survival and continuity of R. arenarum populations under these conditions. The results indicate that the levels of the fungicide reaching amphibian habitats could be risky for the early development of this amphibian species. This study also emphasizes the necessity to evaluate the chronic effects of fungicides in pesticide risk assessment.

Apoptosis-related genes induced in response to ketamine during early life stages of zebrafish.

Increasing evidence supports that ketamine, a widely used anaesthetic, potentiates apoptosis during development through the mitochondrial pathway of apoptosis. Defects in the apoptotic machinery can cause or contribute to the developmental abnormalities previously described in ketamine-exposed zebrafish. The involvement of the apoptotic machinery in ketamine-induced teratogenicity was addressed by assessing the apoptotic signals at 8 and 24 hpf following 20min exposure to ketamine at three stages of early zebrafish embryo development (256 cell, 50% epiboly and 1-4 somites stages). Exposure at the 256-cell stage to ketamine induced an up-regulation of casp8 and pcna at 8 hpf while changes in pcna at the mRNA level were observed at 24 hpf. After the 50% epiboly stage exposure, the mRNA levels of casp9 were increased at 8 and 24 hpf while aifm1 was affected at 24 hpf. Both tp53 and pcna expressions were increased at 8 hpf. After exposure during the 1-4 somites stage, no meaningful changes on transcript levels were observed. The distribution of apoptotic cells and the caspase-like enzymatic activities of caspase-3 and -9 were not affected by ketamine exposure. It is proposed that ketamine exposure at the 256-cell stage induced a cooperative mechanism between proliferation and cellular death while following exposure at the 50% epiboly, a p53-dependent and -independent caspase activation may occur. Finally, at the 1-4 somites stage, the defence mechanisms are already fully in place to protect against ketamine-insult. Thus, ketamine teratogenicity seems to be dependent on the functional mechanisms present in each developmental stage.

Differential gene expression patterns during embryonic development of sea urchin exposed to triclosan.

Triclosan (TCS; 2,4,4'-trichloro-2'-hydroxydiphenyl ether) is a broad-spectrum antibacterial agent used in common industrial, personal care and household products which are eventually rinsed down the drain and discharged with wastewater effluent. It is therefore commonly found in the aquatic environment, leading to the continual exposure of aquatic organisms to TCS and the accumulation of the antimicrobial and its harmful degradation products in their bodies. Toxic effects of TCS on reproductive and developmental progression of some aquatic organisms have been suggested but the underlying molecular mechanisms have not been defined. We investigated the expression patterns of genes involved in the early development of TCS-treated sea urchin Strongylocentrotus nudus using cDNA microarrays. We observed that the predominant consequence of TCS treatment in this model system was the widespread repression of TCS-modulated genes. In particular, empty spiracles homeobox 1 (EMX-1), bone morphogenic protein, and chromosomal binding protein genes showed a significant decrease in expression in response to TCS. These results suggest that TCS can induce abnormal development of sea urchin embryos through the concomitant suppression of a number of genes that are necessary for embryonic differentiation in the blastula stage. Our data provide new insight into the crucial role of genes associated with embryonic development in response to TCS. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 426-433, 2017.

Embryotoxicity of nitrophenols to the early life stages of zebrafish (Danio rerio).

The nitrophenols (NPs) are water-soluble compounds. These compounds pose a significant health threat since they are priority environmental pollutants. In this study, 2-Nitrophenol (2NP) and 2,4-dinitrophenol (DNP) were examined for embryo and early life stage toxicity in zebrafish (Danio rerio). Acute toxicity and teratogenicity of 2NP and DNP were tested for 4 days using zebrafish embryos. The typical lesions observed were no somite formation, incomplete eye and head development, tail curvature, weak pigmentation (≤48 hours postfertilization (hpf)), kyphosis, scoliosis, yolk sac deformity, and nonpigmentation (72 hpf). Also, embryo and larval mortality increased and hatching success decreased. The severity of abnormalities and mortalities were concentration- and compound-dependent. Of the compounds tested, 2,4-DNP was found to be highly toxic to the fish embryos following exposure. The median lethal concentrations and median effective concentrations for 2NP are 18.7 mg/L and 7.9 mg/L, respectively; the corresponding values for DNP are 9.65 mg/L and 3.05 mg/L for 48 h. The chorda deformity was the most sensitive endpoint measured. It is suggested that the embryotoxicity may be mediated by an oxidative phosphorylation uncoupling mechanism. This article is the first to describe the teratogenicity and embryotoxicity of two NPs to the early life stages of zebrafish.

[ENGAGEMENT OF SEROTONIN-MODULATING ANTICONSOLIDATION PROTEIN IN REGULATION OF EMBRYOGENESIS OF LYMNEAE STAGNALIS AND LEWIS SARCOMA IN HYBRID MICE Fl C57B2/6 X DBA].

The article concerns study of the effects of a novel serotonin-modulating anticonsolidation protein (SMAP) being in a linear relationship with serotonin level, on embryogenesis of Lymneae stagnalis and Lewis sarcoma in hybrid mice Fl C57B2/6 X DBA. Inhibition of embryogenesis of Lymneae stagnalis on the stage of four blastomers and late blastula, lack of changes on the stage of trochofora and acceleration of metamorphosis under the effects of SMAP in a dose-dependent manner was observed. Short-term retardation (during the first 10 days) of development of Lewis sarcoma in mice and survival of 25% of transferring animals under high doses of SMAP was revealed. Cytostatic activity for high doses of SMAP and their effects on the duration of single phases of the cell cycle is proposed.

Dose-dependent nuclear ß-catenin response segregates endomesoderm along the sea star primary axis.

In many invertebrates, the nuclearization of ß-catenin at one pole of the embryo initiates endomesoderm specification. An intriguing possibility is that a gradient of nuclear ß-catenin (nß-catenin), similar to that operating in vertebrate neural tube patterning, functions to distinguish cell fates in invertebrates. To test this hypothesis, we determined the function of nß-catenin during the early development of the sea star, which undergoes a basal deuterostomal mode of embryogenesis. We show that low levels of nß-catenin activity initiate bra, which is expressed in the future posterior endoderm-fated territory; intermediate levels are required for expression of foxa and gata4/5/6, which are later restricted to the endoderm; and activation of ets1 and erg in the mesoderm-fated territory requires the highest nß-catenin activity. Transcription factors acting downstream of high nß-catenin segregate the endoderm/mesoderm boundary, which is further reinforced by Delta/Notch signaling. Significantly, therefore, in sea stars, endomesoderm segregation arises through transcriptional responses to levels of nß-catenin activity. Here, we describe the first empirical evidence of a dose-dependent response to a dynamic spatiotemporal nß-catenin activity that patterns cell fates along the primary axis in an invertebrate.

The increase in maternal expression of axin1 and axin2 contribute to the zebrafish mutant ichabod ventralized phenotype.

ß-Catenin is a central effector of the Wnt pathway and one of the players in Ca(+)-dependent cell-cell adhesion. While many wnts are present and expressed in vertebrates, only one ß-catenin exists in the majority of the organisms. One intriguing exception is zebrafish that carries two genes for ß-catenin. The maternal recessive mutation ichabod presents very low levels of ß-catenin2 that in turn affects dorsal axis formation, suggesting that ß-catenin1 is incapable to compensate for ß-catenin2 loss and raising the question of whether these two ß-catenins may have differential roles during early axis specification. Here we identify a specific antibody that can discriminate selectively for ß-catenin1. By confocal co-immunofluorescent analysis and low concentration gain-of-function experiments, we show that ß-catenin1 and 2 behave in similar modes in dorsal axis induction and cellular localization. Surprisingly, we also found that in the ich embryo the mRNAs of the components of ß-catenin regulatory pathway, including ß-catenin1, are more abundant than in the Wt embryo. Increased levels of ß-catenin1 are found at the membrane level but not in the nuclei till high stage. Finally, we present evidence that ß-catenin1 cannot revert the ich phenotype because it may be under the control of a GSK3ß-independent mechanism that required Axin's RGS domain function.

FoxA4 favours notochord formation by inhibiting contiguous mesodermal fates and restricts anterior neural development in Xenopus embryos.

In vertebrates, the embryonic dorsal midline is a crucial signalling centre that patterns the surrounding tissues during development. Members of the FoxA subfamily of transcription factors are expressed in the structures that compose this centre. Foxa2 is essential for dorsal midline development in mammals, since knock-out mouse embryos lack a definitive node, notochord and floor plate. The related gene foxA4 is only present in amphibians. Expression begins in the blastula -chordin and -noggin expressing centre (BCNE) and is later restricted to the dorsal midline derivatives of the Spemann's organiser. It was suggested that the early functions of mammalian foxa2 are carried out by foxA4 in frogs, but functional experiments were needed to test this hypothesis. Here, we show that some important dorsal midline functions of mammalian foxa2 are exerted by foxA4 in Xenopus. We provide new evidence that the latter prevents the respecification of dorsal midline precursors towards contiguous fates, inhibiting prechordal and paraxial mesoderm development in favour of the notochord. In addition, we show that foxA4 is required for the correct regionalisation and maintenance of the central nervous system. FoxA4 participates in constraining the prospective rostral forebrain territory during neural specification and is necessary for the correct segregation of the most anterior ectodermal derivatives, such as the cement gland and the pituitary anlagen. Moreover, the early expression of foxA4 in the BCNE (which contains precursors of the whole forebrain and most of the midbrain and hindbrain) is directly required to restrict anterior neural development.

3-(5-)-Amino-o-diarylisoxazoles: regioselective synthesis and antitubulin activity.

A regioselective synthesis of both 5-amino- and 3-aminodiarylisoxazoles substituted with polyalkoxyaryl pharmacophores has been validated. Starting materials for the synthetic scheme were easily available from plant extracts. The targeted molecules were further tested in the phenotypic sea urchin embryo assay to identify compounds with antimitotic microtubule destabilizing activity. Structure-activity relationship studies suggested that the structural features essential for potent antiproliferative activity include: 1) 5-aminoisoxazole bridge linking biaryl substituents (rings A and B); 2) unsubstituted 5-amino group; 3) 3,4,5-methoxy substituted benzene and 4-methoxy benzene pharmacophores as rings A and B, respectively. The most potent compounds also showed strong in vitro cytotoxicity in NCI60 anticancer drug screen against a panel of 60 human cancer cell lines, including multi-drug resistant cells.

Lethal and sublethal toxicity of the industrial chemical epichlorohydrin on Rhinella arenarum (Anura, Bufonidae) embryos and larvae.

Lethal and sublethal toxicity of the major chemical used in epoxide compounds, epichlorohydrin (ECH) was evaluated on the early life cycle of the common South American toad, Rhinella arenarum (Anura, Bufonidae). The stages evaluated were (according to Del Conte and Sirlin): early blastula (S.3-S.4), gastrula (S.10-S.12), rotation (S.15), tail bud (S.17), muscular response (S.18), gill circulation (S.20), open mouth (S.21), opercular folds (S.23) and complete operculum (S.25). The LC50 and EC50 values for lethal and sublethal effects were calculated. The early blastula was the most sensitive stage to ECH both for continuously and pulse-exposures (LC50-24h=50.9 mg L(-1)), while S.20 was the most resistant (LC50-24h=104.9 mg L(-1)). Among sublethal effects, early blastula was also the most sensitive stage (LOEC-48 h=20 mg L(-1)) and it has a Teratogenic Index of 2.5, which indicates the teratogenic potential of the substance. The main abnormalities were persistent yolk plugs, cell dissociation, tumors, hydropsy, oral malformations, axial/tail flexures, delayed development and reduced body size. ECH also caused neurotoxicity including scarce response to stimuli, reduction in the food intake, general weakness, spasms and shortening, erratic or circular swimming. Industrial contamination is considered an important factor on the decline of amphibian populations. Considering the available information about ECH's toxicity and its potential hazard to the environment, this work shows the first results of its developmental toxicity on a native amphibian species, Rhinella arenarum.

Oocyte-specific H2A variant H2af1o is required for cell synchrony before midblastula transition in early zebrafish embryos.

Oocyte-specific histone variants have been expected to play significant roles in early embryonic development, but the exact evidence and the biological function have remained unclear. Here, we present evidence that H2af1o, an oocyte-specific H2A variant, is required for cell synchrony before midblastula transition in early zebrafish embryos. The H2A variant is oocyte specific, peaks in mature eggs, and is supplied to early embryos. We constructed a series of deletion plasmids of the zebrafish h2af1o tagged with EGFP and determined the main key function regions including nuclear localization signal of N-terminal 25 amino acids and nucleosome binding region of 110-122 amino acid sequence in the C-terminus by microinjecting them into one-cell-stage zebrafish embryos. In comparison with ubiquitous H2A.X, the H2af1o was revealed to confer a more open structure than canonical H2A in the nucleosomes. Furthermore, we conducted the h2af1o-specific morpholino knockdown analysis in early embryos of zebrafish and revealed its biological function for maintaining cell synchrony division because the H2af1o deficiency disturbed cell synchrony in early cleavages before midblastula transition. Therefore, our current findings provided the first case to understand the biological function of maternal oocyte-specific histone variants in vertebrates.

ERK signaling pathway regulates embryonic survival and eye development in goldfish, Carassius auratus.

The extracellular signal-regulated kinase (ERK) is one of the three major types of mitogen-activated protein kinases. Previous studies showed that ERKs mediate various signaling pathways for cell proliferation, differentiation, survival and transformation in mammals. In the present study, we use goldfish as a model system and demonstrate that ERK kinases play important roles in promoting embryonic survival and regulate development of eye and trunk in vertebrates. ERKs are highly expressed in multiple tissues including lens epithelial cells, lens fiber cells, retina, brain, muscle and heart of adult goldfish. Injection of the dominant negative ERK mutant (DNM-ERK) into the fertilized eggs of goldfish significantly inhibited ERK activity at blastula stage, and completely blocked ERK activity at gastrula and later stages. As a result, the blastula cells were induced into apoptosis, and majority of the injected embryos were lethal at embryonic stages. At the molecular level, inhibition of ERK activity by DNM-ERKs suppressed phosphorylation of Bad at Ser-112 to promote apoptosis. Similar results were observed when MEK activity was inhibited by U0126 treatment. The survived embryos display significant abnormality in the phenotypes of both eye and trunk. Associated with the abnormality in the eye development, phosphorylation in Pax-6 and expression of HSF4 were significantly decreased and expression of the ß-crystallin gene was also downregulated. These results provide novel information regarding the roles of ERKs in regulating vertebrate development.

Petroacetylene, a new polyacetylene from the marine sponge Petrosiasolida that inhibits blastulation of starfish embryos.

A new C30 linear polyacetylene compound designated petroacetylene (1) has been isolated from the marine sponge Petrosia solida Hoshino 1981, collected off the coast of Amami-Oshima, Kagoshima Prefecture, Japan. The structure was elucidated on the basis of spectroscopic data and chemical means. Petroacetylene (1) inhibited blastulation of starfish embryos at a concentration of 3.1 µg mL(- 1) or greater.

Early developmental gene regulation in Strongylocentrotus purpuratus embryos in response to elevated CO2 seawater conditions.

Ocean acidification, or the increased uptake of CO(2) by the ocean due to elevated atmospheric CO(2) concentrations, may variably impact marine early life history stages, as they may be especially susceptible to changes in ocean chemistry. Investigating the regulatory mechanisms of early development in an environmental context, or ecological development, will contribute to increased understanding of potential organismal responses to such rapid, large-scale environmental changes. We examined transcript-level responses to elevated seawater CO(2) during gastrulation and the initiation of spiculogenesis, two crucial developmental processes in the purple sea urchin, Strongylocentrotus purpuratus. Embryos were reared at the current, accepted oceanic CO(2) concentration of 380 microatmospheres (µatm), and at the elevated levels of 1000 and 1350 µatm, simulating predictions for oceans and upwelling regions, respectively. The seven genes of interest comprised a subset of pathways in the primary mesenchyme cell gene regulatory network (PMC GRN) shown to be necessary for the regulation and execution of gastrulation and spiculogenesis. Of the seven genes, qPCR analysis indicated that elevated CO(2) concentrations only had a significant but subtle effect on two genes, one important for early embryo patterning, Wnt8, and the other an integral component in spiculogenesis and biomineralization, SM30b. Protein levels of another spicule matrix component, SM50, demonstrated significant variable responses to elevated CO(2). These data link the regulation of crucial early developmental processes with the environment that these embryos would be developing within, situating the study of organismal responses to ocean acidification in a developmental context.

Splice blocking of zygotic sox31 leads to developmental arrest shortly after Mid-Blastula Transition and induces apoptosis in zebrafish.

Here we report that splice blocking morpholinos (Sb MO) against zebrafish sox31 elicit developmental arrest, likely through creating a series of dominant negative splicing variants. Embryos injected with the Sb MO develop normally before the Mid-Blastula Transition (MBT); however, they do not initiate epiboly. Microarray analysis of mRNAs collected at the dome stage revealed that the Sb MO impairs activation of a large set of zygotic genes and reduces degradation of maternal mRNA during MBT. Furthermore, an apoptotic response occurs in Sb morphants at about 6hpf. SoxB1 family genes including sox31 thus play an essential role for early embryos traversing the transitional stage.

Comparative toxicity of antifouling compounds on the development of sea urchin.

In the present study, embryotoxicity experiments using the sea urchin Lytechinus variegatus were carried out to better clarify the ecotoxicological effects of tributyltin (TBT) and triphenyltin (TPT) (the recently banned antifouling agents), and Irgarol and Diuron (two of the new commonly used booster biocides). Organisms were individually examined to evaluate the intensity and type of effects on embryo-larval development, this procedure has not been commonly used, however it showed to be a potentially suitable approach for toxicity assessment. NOEC and LOEC were similar for compounds of same chemical class, and IC10 values were very close and showed overlapping of confidence intervals between TBT and TPT, and between Diuron and Irgarol. In addition, IC10 were similar to NOEC values. Regardless of this, the observed effects were different. Embryo development was interrupted at the gastrula and blastula stages at 1.25 and 2.5 µg l(-1) of TBT, respectively, whereas pluteus stage was reached with the corresponding concentrations of TPT. Furthermore, embryos reached the prism and morula stages at 5 µg l(-1) of TPT and TBT, respectively. The effects induced by Irgarol were also more pronounced than those caused by Diuron. Pluteus stage was always reached at any tested Diuron concentration, while embryogenesis was interrupted at blastula/gastrula stages at the highest concentrations of Irgarol. Therefore, this study proposes a complementary approach for interpreting embryo-larval responses that may be employed together with the traditional way of analysis. Consequently, this application leads to a more powerful ecotoxicological assessment tool focused on embryotoxicity.

Advances in the cryopreservation of sea-urchin embryos: Potential application in marine water quality assessment.

Among the most widely used biological techniques in marine pollution assessment, the sea-urchin embryo-larval bioassay is in an advanced developmental stage. Cryopreservation might help to overcome the problem of the spawning seasonality and therefore strengthen the use of those embryo-larval bioassays. This work investigates different factors influencing cryopreservation of sea-urchin embryos, including the cooling rates and holding temperatures, the seeding, or the impact of plunging into liquid nitrogen. The blastula stage yielded better results than the fertilised egg, and the most effective cryoprotectants combination was dimethyl sulfoxide 1.5M plus trehalose 0.04M. The optimised protocol developed begins with an initial hold at 4°C for 2min, followed by cooling at -1°Cmin(-1) to -12°C. At this point a seeding step was incorporated with a 2min hold, followed by a second cooling at -1°Cmin(-1) to -80°C. After a final hold of 2min the cryovials are transferred into liquid nitrogen for storage. Although the cryopreservation processes might cause a delay in the development of sea-urchin embryos, high larval growth (71-98% of controls) was obtained when cryopreserved blastulae were further incubated for 72-96h in artificial seawater. We conclude that embryo-larval bioassays with cryopreserved sea-urchin blastulae are suitable for use in marine pollution monitoring programs and may be considered as an acceptable solution to the reproductive seasonality of sea-urchin species.