Measurement of vitellogenin gene expression by RT-PCR as a tool to identify endocrine disruption in Japanese medaka (Oryzias latipes)

Abstract

In order to monitor vitellogenin gene expression in the Japanese medaka (Oryzias latipes), a reverse transcription-polymerase chain reaction (RT-PCR) system was developed. To date cDNA for medaka vitellogenin has not been published; therefore, initially a sequence fragment had to be obtained and compared with other known vertebrate vitellogenins. For this, a 1.2 kb cDNA of medaka vitellogenin (M-Vg1.2) was amplified by RT-PCR and cloned into a pCR® II-TOPO bacterial vector. On Northern blot analysis, the antisense cRNA of M-Vg1.2 stained a 5.5 kb gene product found exclusively in female fish, but not in males. Additionally, the 5'-end of medaka vitellogenin cDNA was amplified by 5'-RACE-PCR. The analysed nucleotide sequence of 1.6 kb shared significant similarities with vitellogenins known from other fish species: approximately 72% similarity with mummichog (Fundulus heteroclitus) vitellogenin I and approximately 62% with fathead minnow (Pimephales promelas) vitellogenin. To develop a semiquantitative RT-PCR for the measurement of vitellogenin gene expression, primers specific to a 500 bp sequence of the vitellogenin cDNA (M-Vg0.5) were constructed using the gene product of elongation factor 1α as internal standard. Induction of vitellogenin gene expression was measured in male medaka exposed to 0, 2, 20 and 50 μg l-1 nonylphenol and 0, 2.5, 25 and 100ng l-1 17α -ethinyloestradiol for 7 days. The LOECs for vitellogenin induction in male medaka were 20 μg l-1 and 25ng l-1 for nonylphenol and 17α -ethinyloestradiol, respectively.