Image_2_Short-term effects of estradiol and bisphenol A on gene expression associated with early head mineralization in the seabass Dicentrarchus labrax.tif

IntroductionNatural and synthetic estrogens are pollutants found in aquatic ecosystems at low concentrations reaching ng.L-1 to μg.L-1. At these concentrations, they are able to interfere with the fish endocrine system. When waterborne exposure occurs at early life stages, when blood estrogens concentrations are low, this may have significant consequences for estrogen-sensitive functions such as skeletal development.MethodsTo better understand how (xeno)estrogens may affect early head mineralization, 12 days post-hatch larvae of the European seabass Dicentrarchus labrax were experimentally exposed for 4 days to the natural estrogen estradiol E2 and to the xenoestrogen bisphenol A (BPA), both used at either regulatory concentration of water quality or a 100 times higher concentration. Head mineralization level was assessed using Alizarin red staining, together with the relative quantification of mRNA expression levels of several genes playing key roles in skeletogenesis and estrogen signaling pathways.ResultsWe showed that (xeno)estrogen exposure at early larval stage increases the expression of skeleton-associated genes: matrix proteins encoding genes (col1a2, col2a1a, col2a1b, bgp1a, bgp1b, sparc), proteolytic enzyme encoding genes (ctsk) and transcription and signaling factors (sox9a, sox9b, ihha, runx2, rankl). Although transcriptional overexpression of these genes was significant in larvae exposed to 40 ng.L-1 E2 and to 1.6 and 160 μg.L-1 BPA, increased mineralization was detected only in E2-exposed larvae, suggesting a difference in head skeleton development and remodeling in BPA-treated larvae.DiscussionOur results suggest that these phenotypic differences could be due to the implication of other estrogenic signaling pathways involving both nuclear and membrane-bound estrogen receptors (ERs and GPERs), but also estrogen-related receptors (ERRs). This study brings new insights into the regulatory mechanisms of skeletogenesis by E2 and BPA and into the effects of waterborne exposure to (xeno)estrogens on the early skeletal development of teleost fishes.</p

Image_1_Short-term effects of estradiol and bisphenol A on gene expression associated with early head mineralization in the seabass Dicentrarchus labrax.tiff

IntroductionNatural and synthetic estrogens are pollutants found in aquatic ecosystems at low concentrations reaching ng.L-1 to μg.L-1. At these concentrations, they are able to interfere with the fish endocrine system. When waterborne exposure occurs at early life stages, when blood estrogens concentrations are low, this may have significant consequences for estrogen-sensitive functions such as skeletal development.MethodsTo better understand how (xeno)estrogens may affect early head mineralization, 12 days post-hatch larvae of the European seabass Dicentrarchus labrax were experimentally exposed for 4 days to the natural estrogen estradiol E2 and to the xenoestrogen bisphenol A (BPA), both used at either regulatory concentration of water quality or a 100 times higher concentration. Head mineralization level was assessed using Alizarin red staining, together with the relative quantification of mRNA expression levels of several genes playing key roles in skeletogenesis and estrogen signaling pathways.ResultsWe showed that (xeno)estrogen exposure at early larval stage increases the expression of skeleton-associated genes: matrix proteins encoding genes (col1a2, col2a1a, col2a1b, bgp1a, bgp1b, sparc), proteolytic enzyme encoding genes (ctsk) and transcription and signaling factors (sox9a, sox9b, ihha, runx2, rankl). Although transcriptional overexpression of these genes was significant in larvae exposed to 40 ng.L-1 E2 and to 1.6 and 160 μg.L-1 BPA, increased mineralization was detected only in E2-exposed larvae, suggesting a difference in head skeleton development and remodeling in BPA-treated larvae.DiscussionOur results suggest that these phenotypic differences could be due to the implication of other estrogenic signaling pathways involving both nuclear and membrane-bound estrogen receptors (ERs and GPERs), but also estrogen-related receptors (ERRs). This study brings new insights into the regulatory mechanisms of skeletogenesis by E2 and BPA and into the effects of waterborne exposure to (xeno)estrogens on the early skeletal development of teleost fishes.</p

Morphology of control and UV-treated embryos and larvae.

<p>a) Control embryo at 74 h pf; b) control larva at hatching; c) UV treated embryos at 74 hpf showing microcephaly, short and large body; d) UV-treated larvae at hatching showing microphtalmy, short body and curved tail. Scale bars represent 500 µm.</p

Percent survival relative to controls of treated groups exposed to different cumulative UV-doses in the range 7.2–720 mJ.cm⁻².

<p>Error bars represent standard deviations of means (STD).</p

Microsatellite marker loci transmission in three putative androgenetic progenies (A1, B1 and C2) produced with a UV-dose of 60 mJ.cm⁻².

<p>Genotypes of progenies showing only discriminant paternal alleles are presented in bold characters. For putative androgenetic progenies, homozygous or haploid alleles are only written once since genotyping cannot distinguish between the presence of one or two copies of the same allele. N represents the number of analyzed individuals in each progeny.</p><p>Microsatellite marker loci transmission in three putative androgenetic progenies (A1, B1 and C2) produced with a UV-dose of 60 mJ.cm⁻².</p

Representative examples of flow-cytometry histograms obtained from nuclear suspensions (5–10,000 counts) of Propidium Iodide (PI) stained larval samples.

<p>a) Control diploid (2n) larva (CV: 5%); b) haploid (n) larva produced with a UV-dose of 60 mJ.cm⁻² (CV: 10%). DNA values on the X-axis are reported in arbitrary units expressed as fluorescent channel numbers (PE-A). G2 represent mitotic peaks.</p

Percent survival relative to controls of hatched larvae issued from the different UV-irradiation treatments (7.2–72 mJ.cm⁻².min⁻¹) lasting 0.5–12 min.

<p>Error bars represent standard deviations of means (STD).</p

Absorbance spectrum of non-irradiated (control) egg extracts at pH8 (dotted line) and pH3 (solid line).

<p>Wavelengths cover almost the entire UV spectrum (UVC: 200–280 nm, UVB: 280–315 nm, and UVA: 315–400 nm). Absorption is shown as arbitrary units.</p