A reverse transcription-polymerase chain reaction bioassay for avian vitellogenin mRNA.
A bioassay based on the measurement of vitellogenin (VTG) mRNA in avian embryo hepatocyte cultures by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was developed. To allow sequence comparison and design of suitable PCR primers, a short region of VTG cDNA was cloned and sequenced for seven species of birds. Cell cultures were prepared from both chicken and herring gull embryos and treated with the estradiol analogue moxestrol 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-treated 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 hepatocyte cultures responded with substantial VTG mRNA induction when treated with 10,000 nM o,p'-DDT. These results suggest that the bioassay will be useful for comparing avian embryo hepatocyte culture concentration-response data in terms of intra- and interspecies sensitivities to pharmacological estrogens or environmental contaminants.
Lorenzen
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Environment Canada, National Wildlife Research Centre, Hull, Quebec, K1A 0H3, Canada.
Casley
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WL
Moon
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TW
D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D001717:Birds / Q000196:embryology / Q000235:genetics*; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D003634:DDT / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D004965:Estrogen Antagonists / Q000494:pharmacology; D004968:Estrogens, Non-Steroidal / Q000494:pharmacology; D004997:Ethinyl Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D005260:Female; D015870:Gene Expression / Q000187:drug effects; D022781:Hepatocytes / Q000737:chemistry; D008099:Liver / Q000196:embryology; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020308:Songbirds / Q000196:embryology / Q000235:genetics; D014819:Vitellogenins / Q000235:genetics*
D004965:Estrogen Antagonists; D004968:Estrogens, Non-Steroidal; D012333:RNA, Messenger; D014819:Vitellogenins; D004997:Ethinyl Estradiol; C011310:moxestrol; D004247:DNA; D003634:DDT; C016340:o,p'-DDT