Japanese medaka (Oryzias latipes): developmental model for the study of alcohol teratology

Abstract

BACKGROUND: Animal models are necessary to investigate the mechanism of alcohol-induced birth defects. We have used Japanese medaka (Oryzias latipes) as a non-mammalian model to elucidate the molecular mechanism(s) of ethanol teratogenesis. METHODS: Medaka eggs, within 1 hr post-fertilization (hpf) were exposed to waterborne ethanol (0-1000 mM) in hatching solution for 48 hr. Embryo development was observed daily until 10 days post-fertilization (dpf). The concentration of embryonic ethanol was determined enzymatically. Cartilage and bones were stained by Alcian blue and calcein, respectively and skeletal and cardiovascular defects were assessed microscopically. Genetic gender of the embryos was determined by PCR. Levels of two isoenzymes of alcohol dehydrogenase (Adh) mRNAs were determined by semi-quantitative and real-time RT-PCR. RESULTS: The concentration of ethanol required to cause 50% mortality (LC 50 ) in 10 dpf embryos was 568 mM, however, the embryo absorbed only 15-20% of the waterborne ethanol at all ethanol concentrations. The length of the lower jaw and calcification in tail fin cartilaginous structures were reduced by ethanol exposure. Active blood circulation was exhibited at 501 hpf in embryos treated with 0-100 mM ethanol; active circulation was delayed and blood clots developed in embryos treated with 200-400 mM ethanol. The deleterious effects of ethanol were not gender-specific. Moreover, ethanol treatment was unable to alter the constitutive expression of either Adh5 or Adh8 mRNA in the medaka embryo. CONCLUSIONS: Preliminary results suggested that embryogenesis in medaka was significantly affected by ethanol exposure. Phenotypic features normally associated with ethanol exposure were similar to that observed in mammalian models of fetal alcohol syndrome. The results further indicated that medaka embryogenesis might be used as an alternative non-mammalian model for investigating specific alterations in gene expression as a means to understand the molecular mechanism(s) of ethanol-induced birth defects.

Disruption of circulation by ethanol promotes fetal alcohol spectrum disorder (FASD) in medaka (Oryzias latipes) embryogenesis

“…Further analysis of Adh and Aldh mRNAs also indicates that embryos at early stages of development are more sensitive to ethanol than the embryos at late stages ( Figure 5 and Figure 6). Our previous studies showed that exposure to ethanol disrupted the circulation status of the embryos in a dose-dependent manner (Wang et al, 2006). Other morphological parameters like craniofacial, cardiovascular and skeletal deformities by ethanol were also found to be dose-specific in medaka (Wang et al, 2006).…”

“…Our previous studies showed that exposure to ethanol disrupted the circulation status of the embryos in a dose-dependent manner (Wang et al, 2006). Other morphological parameters like craniofacial, cardiovascular and skeletal deformities by ethanol were also found to be dose-specific in medaka (Wang et al, 2006). Recently, we have demonstrated that medaka embryos exposed to ethanol for 48 hpf have reduced cellular macromolecular constituents (Protein, RNA and DNA) which might have a direct effect on the embryonic growth during morphogenesis (Wu et al, 2008).…”

“…Methods of animal maintenance, egg collection, RNA, and protein preparation and purification, semi-quantitative relative (rRT-PCR) and quantitative real-time PCR (qRT-PCR) techniques were previously described (Dasmahapatra et al, 2005;Wang et al, 2006Wang et al, , 2007bWu et al, 2008). The major modification made in the present experimental protocol was in the maintenance of medaka embryos during ethanol treatment.…”

“…The embryos were sacrificed at 6 (~144 hpf) day post fertilization (dpf) after determining the circulation status of the embryos. RNA was extracted from the intact embryo (embryos with yolk) by Trizol reagent (Wang et al, 2006). Total protein was extracted from embryos (after the yolk was removed) by 3.5% perchloric acid precipitation (Wu et al, 2008).…”

“…We are developing Japanese medaka embryogenesis as an alternative/ complementary model to the zebrafish to study the molecular mechanism of ethanol toxicity (Wang et al, 2006(Wang et al, , 2007a(Wang et al, , 2007bWu et al, 2008). We have demonstrated that medaka embryos exposed to ethanol for 48 hour post fertilization (hpf) have developed several phenotypic features in cardiovascular, craniofacial and skelatal systems which are comparable to the FASD features observed in human (Wang et al, 2006). Moreover, the effects at the cellular level have shown that ethanol is able to reduce total protein, RNA and DNA contents of the embryos in a dose-and time-dependent manner (Wu et al, 2008).…”