Monohydroxylated polycyclic aromatic hydrocarbons influence spicule formation in the early development of sea urchins (Hemicentrotus pulcherrimus).

We previously demonstrated that monohydroxylated polycyclic aromatic hydrocarbons (OHPAHs), which are metabolites of polycyclic aromatic hydrocarbons (PAHs), act on calcified tissue and suppress osteoblastic and osteoclastic activity in the scales of teleost fish. The compounds may possibly influence other calcified tissues. Thus, the present study noted the calcified spicules in sea urchins and examined the effect of both PAHs and OHPAHs on spicule formation during the embryogenesis of sea urchins. After fertilization, benz[a]anthracene (BaA) and 4-hydroxybenz[a]anthracene (4-OHBaA) were added to seawater at concentrations of 10(-8) and 10(-7) M and kept at 18 °C. The influence of the compound was given at the time of the pluteus larva. At this stage, the length of the spicule was significantly suppressed by 4-OHBaA (10(-8) and 10(-7) M). BaA (10(-7) M) decreased the length of the spicule significantly, while the length did not change with BaA (10(-8) M). The expression of mRNAs (spicule matrix protein and transcription factors) in the 4-OHBaA (10(-7) M)-treated embryos was more strongly inhibited than were those in the BaA (10(-7) M)-treated embryos. This is the first study to demonstrate that OHPAHs suppress spicule formation in sea urchins.

Development of the GABA-ergic signaling system and its role in larval swimming in sea urchin.

The present study aimed to elucidate the development and γ-amino butyric acid (GABA)-ergic regulation of larval swimming in the sea urchin Hemicentrotus pulcherrimus by cloning glutamate decarboxylase (Hp-gad), GABAA receptor (Hp-gabrA) and GABAA receptor-associated protein (Hp-gabarap), and by performing immunohistochemistry. The regulation of larval swimming was increasingly dependent on the GABAergic system, which was active from the 2 days post-fertilization (d.p.f.) pluteus stage onwards. GABA-immunoreactive cells were detected as a subpopulation of secondary mesenchyme cells during gastrulation and eventually constituted the ciliary band and a subpopulation of blastocoelar cells during the pluteus stage. Hp-gad transcription was detected by RT-PCR during the period when Hp-Gad-positive cells were seen as a subpopulation of blastocoelar cells and on the apical side of the ciliary band from the 2 d.p.f. pluteus stage. Consistent with these observations, inhibition of GAD with 3-mercaptopropioninc acid inhibited GABA immunoreactivity and larval swimming dose dependently. Hp-gabrA amplimers were detected weakly in unfertilized eggs and 4 d.p.f. plutei but strongly from fertilized eggs to 2 d.p.f. plutei, and Hp-GabrA, together with GABA, was localized at the ciliary band in association with dopamine receptor D1 from the two-arm pluteus stage. Hp-gabarap transcription and protein expression were detected from the swimming blastula stage. Inhibition of the GABAA receptor by bicuculline inhibited larval swimming dose dependently. Inhibition of larval swimming by either 3-mercaptopropionic acid or bicuculline was more severe in older larvae (17 and 34 d.p.f. plutei) than in younger ones (1 d.p.f. prism larvae).

Effects of monocrotophos pesticide on serotonin metabolism during early development in the sea urchin, Hemicentrotus pulcherrimus.

Organophosphate pesticides can interfere with the serotonergic nervous system and potentially lead to malformations and behavioral abnormalities during early development in sea urchin. To investigate the mechanism by which monocrotophos (MCP) pesticide disrupts the serotonergic nervous system, we evaluated its effects on serotonin metabolism. Fertilized embryos of sea urchin were incubated with 40% MCP pesticide at nominal concentrations of 0.01, 0.10 and 1.00mg/L, and the effects on tryptophan hydroxylase of Hemicentrotus pulcherrimus (HpTPH), serotonin reuptake transporter (SERT), monoamine oxidase (MAO), and serotonin levels were investigated. The results indicated that MCP pesticide disturbed the baseline pattern of HpTPH and SERT mRNA expression and MAO activity during early development in H. pulcherrimus. When serotonin should be quickly metabolized at 36-hpf stage, HpTPH and SERT expression was decreased and MAO activity was induced by MCP pesticide, leading to the impairment of serotonergic synaptic activity. But when serotonin should be metabolized at low levels during the other six stages, MCP pesticide induced HpTPH and SERT expression, resulting in the improvement of serotonergic synaptic activity. We concluded that this metabolic disturbance is one of the major mechanisms by which MCP pesticides affect the serotonergic nervous system and potentially contribute to various developmental abnormalities.

Distinct embryotoxic effects of lithium appeared in a new assessment model of the sea urchin: the whole embryo assay and the blastomere culture assay.

Early embryogenesis is one of the most sensitive and critical stages in animal development. Here we propose a new assessment model on the effect of pollutant to multicellular organism development. That is a comparison between the whole embryo assay and the blastomere culture assay. We examined the LiCl effect on the sea urchin early development in both of whole embryos and the culture of isolated blastomeres. The mesoderm and endoderm region were capable to differentiate into skeletogenic cells when they were isolated at 60-cell stage and cultured in vitro. The embryo developed to exogastrula by the vegetalizing effect of the same LiCl condition where ectodermal region changed their fate to endoderm, while the isolated blastomeres from the presumptive ectoderm region differentiated into skeletogenic cells in the culture with LiCl. The effect of LiCl to the sea urchin embryo and to the dissociated blastomere is a unique example where same cells response distinctly to the same agent depend on the condition around them. Present results show the importance of examining the process in cellular and tissue levels for the exact understanding on the morphological effect of chemicals and metals.

Larval arm resorption proceeds concomitantly with programmed cell death during metamorphosis of the sea urchin Hemicentrotus pulcherrimus.

Sea urchins are excellent models to elucidate metamorphic phenomena of echinoderms. However, little attention has been paid to the way that their organ resorption is accomplished by programmed cell death (PCD) and related cellular processes. We have used cytohistochemistry and transmission electron microscopy to study arm resorption in competent larvae of metamorphosing sea urchins, Hemicentrotus pulcherrimus, induced to metamorphose by L-glutamine treatment. The results show that: (1) columnar epithelial cells, which are constituents of the ciliary band, undergo PCD in an overlapping fashion with apoptosis and autophagic cell death; (2) squamous epithelial cells, which are distributed between the two arrays of the ciliary band, display a type of PCD distinct from that of columnar epithelial cells, i.e., a cytoplasmic type of non-lysosomal vacuolated cell death; (3) epithelial integrity is preserved even when PCD occurs in constituent cells of the epithelium; (4) secondary mesenchyme cells, probably blastocoelar cells, contribute to the elimination of dying epithelial cells; (5) nerve cells have a delayed initiation of PCD. Taken together, our data indicate that arm resorption in sea urchins proceeds concomitantly with various types of PCD followed by heterophagic elimination, but that epithelial organization is preserved during metamorphosis.