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

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 pCRR H-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 1 alpha as internal standard. Induction of vitellogenin gene expression was measured in male medaka exposed to 0, 2, 20 and 50 micrograms l-1 nonylphenol and 0, 2.5, 25 and 100 ng l-1 17 alpha-ethinyloestradiol for 7 days. The LOECs for vitellogenin induction in male medaka were 20 micrograms l-1 and 25 ng l-1 for nonylphenol and 17 alpha-ethinyloestradiol, respectively.