Ovothiol ensures the correct developmental programme of the sea urchin Paracentrotus lividus embryo

Abstract

Ovothiols are π-methyl-5-thiohistidines produced in great amounts in sea urchin eggs, where they can act as protective agents against the oxidative burst at fertilization and environmental stressors during development. Here we examined the biological relevance of ovothiol during the embryogenesis of the sea urchin Paracentrotus lividus by assessing the localization of the key biosynthetic enzyme OvoA, both at transcript and protein level, and perturbing its protein translation by morpholino antisense oligonucleotide-mediated knockdown experiments. In addition, we explored the possible involvement of ovothiol in the inflammatory response by assessing ovoA gene expression and protein localization following exposure to bacterial lipopolysaccharide. The results of the present study suggest that ovothiol may be a key regulator of cell proliferation in early developing embryos. Moreover, the localization of OvoA in key larval cells and tissues, in control and inflammatory conditions, suggests that ovothiol may ensure larval skeleton formation and mediate inflammatory processes triggered by bacterial infection. This work significantly contributes to the understanding of the biological function of ovothiols in marine organisms, and may provide new inspiration for the identification of the biological activities of ovothiols in humans, considering the pharmacological potential of these molecules.

Keywords: cell proliferation; embryonic development; marine organisms; ovothiol; oxidative stress; sea urchin.

Figure 1.

OvoA mRNA localization in P. lividus embryos. For FISH detection, ovoA antisense/DAPI merge (on left), ovoA antisense probe signal (middle) and images in bright field (BF, on right) are shown for some representative P. lividus developmental stages. (A1–A3) 32-cell stage; (B1–B3) gastrula; (C1–C3) prism stage; (D1–D3) pluteus larva. For CISH detection, bright field images are shown for the same developmental stages (A4–D4). White arrows indicate the cells with stronger signal (for details refer to the text). FISH pictures were taken at confocal microscope (Zeiss LSM 700) at 20× magnification and maximum intensity Z-projections were obtained through ImageJ software. CISH pictures were taken at optical microscope (Zeiss Axio Imager M1) at 20× magnification.

Figure 2.

OvoA protein expression during P. lividus development. Top: representative western blot (WB) experiment showing the immunopositive bands OvoA (75 kDa) and actin (42 kDa) for all the examined developmental stages, from left to right: unfertilized eggs (UE), zygote (Zi), 64-blastomere stage (64bl), early blastula (EB), swimming blastula (SB), mesenchyme blastula (MB), late gastrula (LG), prism (Pr), pluteus larva (Pl). Bottom: bar-chart showing the OvoA/actin optical densitometry (OD) ratio resulting from WB analyses performed on embryos derived from three different females. Data are presented as means ± s.d. (n = 3 biological replicates). *p < 0.05 represents significance compared to UE; #p < 0.05 and ##p < 0.01 represent significance compared to Zi; §p < 0.05 represents significance compared to 64bl.

Figure 3.

OvoA spatial expression in P. lividus early developmental stages. For immunohistochemistry experiments (IHC), OvoA immunofluorescence (in red, left side), nuclei (labelled in blue with DAPI, middle) and images in bright field are shown for: (A1–A3) unfertilized eggs; (B1–B3) fertilized eggs; (C1–C3) 2-cell stage; (D1–D3) 4-cell stage; (E1–E3) 32-cell stage. In OvoA protein panel, white arrows indicate OvoA signal (for details refer to the text). epn = egg pronucleus, spn = sperm pronucleus, S-n = S-phase (interphase) nucleus, M-n = M-phase (mitosis) nucleus. Pictures were taken at confocal microscope (Zeiss LSM 700) at 20× magnification.

Figure 4.

OvoA immunolocalization in P. lividus late developmental stages. (a) For immunohistochemistry experiments (IHC), OvoA immunofluorescence (in red, left side), nuclei (labelled in blue with DAPI, middle) and images in bright field are shown for: (A1–A3) gastrula stage; (B1–B3) early echinopluteus; (C1–C3) plutei larvae. (b) For pluteus stage two zoom insights are reported, showing (i) OvoA signal in intestine, and (ii) stomach and primary mesenchyme cells (PMCs). White arrows indicate OvoA signal (for details refer to the text). Pictures were taken at confocal microscope (Zeiss LSM 700) at 20× magnification.

Figure 5.

Phenotypic and molecular characterization of P. lividus pluteus larvae exposed to LPS treatments. (a) Phenotype observation of larvae exposed to various combinations of LPS concentrations and time points, and untreated larvae: (A1–B2) untreated larvae; (C1–C4) larvae after 1 h of exposure to 10 µg ml⁻¹ of LPS; (D1–E2) larvae treated with 100 µg ml⁻¹ of LPS for 1 h or 4 h (arrows indicate pigment cells inside the larva). (b) OvoA (in green) and Msp130 (in purple) immunostaining of untreated larvae (A–D′), and after treatment with 10 (E–H′) and 100 µg ml⁻¹ (I–L′) of LPS for 1 h. g = gut; m = mouth; white asterisks = cells possibly skeletogenic; yellow arrow = skeletogenic OvoA-positive cells; white arrows = non-skeletogenic OvoA-positive cells. Brightness/contrast was adjusted only on the double immunostainings with Msp130, on the zoomed images.

Figure 6.

Apoptotic nuclei detection in OvoA knockdown sea urchin embryos. (a) TUNEL signal, nuclei staining (Hoechst) and bright field images are shown for both ctrl-MASO (A1–A3) and ovoA-MASO embryos (B1–B3). (b) TUNEL/Hoechst merged signal from two additional representative ovoA-MASO embryos. White arrows indicate the TUNEL-positive nuclei.

Figure 7.

Proliferating nuclei staining in OvoA knockdown sea urchin embryos. (a) EdU signal, nuclei staining (Hoechst) and bright field images are shown for embryos injected with ctrl-MASO (A1–A3), ovoA-MASO alone (B1–B3) and co-injected with ovoA synthetic mRNA (C1–C3). (b) EdU/Hoechst merged signal from two additional representative ovoA-MASO embryos. White arrows indicate the EdU-positive nuclei. Pictures were taken with a confocal microscope (Zeiss LSM 700) at 20× magnification.