A reverse transcription-polymerase chain reaction bioassay for avian vitellogenin mRNA

A bioassay based on the measurement of vitellogenin (VTG) mRNA in avian emb ryo hepatocyte cultures by semiquantitative reverse transcription-polymeras e chain reaction (RT-PCR)was developed. To allow sequence comparison and de sign of suitable PCR primers, a short region of VTG cDNA was cloned and seq uenced for seven species of birds. Cell cultures were prepared from both ch icken and herring gull embryos and treated with the estradiol analogue moxe strol or the organochlorine insecticide o,p ' -DDT. Using primers based on an area of the VTG gene that was identical for herring gull and chicken, in vitro VTG mRNA induction was observed for both moxestrol- and o,p ' -DDT-t reated cultures. Herring gull embryo hepatocyte cultures responded with VTG mRNA induction at moxestrol concentrations of 1 nM compared with 10 nM in chicken embryo hepatocyte cultures. Both herring gull and chicken embryo he patocyte cultures responded with substantial VTG mRNA induction when treate d with 10,000 nM o,p ' -DDT. These results suggest that the bioassay will b e useful for comparing avian embryo hepatocyte culture concentration-respon se data in terms of intra-and interspecies sensitivities to pharmacological estrogens or environmental Contaminants. (C) 2001 Academic Press.