Induction of gene expression in sheepshead minnows (Cyprinodon variegatus) treated with 17beta-estradiol, diethylstilbestrol, or ethinylestradiol: the use of mRNA fingerprints as an indicator of gene regulation.
The recent interest in hormonally active environmental contaminants has sparked a drive to find sensitive methods to measure their effects on wildlife. A molecular-based assay has been developed to measure the induction of gene expression in sheepshead minnows (Cyprinodon variegatus) exposed in vivo to the natural and pharmaceutical estrogens 17beta-estradiol, ethinylestradiol, and diethylstilbestrol. This method used differential display reverse transcriptase polymerase chain reaction assays to compare the expression of individual mRNAs from control and estrogen-exposed fish. Forty-eight differentially expressed cDNAs were isolated by this method, including cDNAs for vitelline envelope proteins and vitellogenin. The mRNA expression patterns for fish injected with a pharmacological dose of estradiol (5 mg/kg) were identical to those obtained in fish receiving constant aqueous exposure to 212 ng estradiol/liter. Further, the cDNA "fingerprint" pattern observed in the estradiol-treated fish also matched that obtained in fish receiving continuous-flow aqueous exposures to 192 ng ethinyl estradiol/liter and a nominal concentration of 200 ng diethylstilbestrol/liter. The results demonstrate a characteristic expression pattern for genes upregulated by exposure to a variety of natural and anthropogenic estrogens and suggest this approach may be valuable to examine the potential effects of environmental contaminants on other endocrine-mediated pathways of reproduction, growth, and development.
Denslow
N D
ND
Department of Biochemistry, University of Florida, Gainesville, Florida 32610, USA.
Bowman
C J
CJ
Ferguson
R J
RJ
Lee
H S
HS
Hemmer
M J
MJ
Folmar
L C
LC
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D002347:Carps; D003530:Cyprinidae / Q000235:genetics*; D016172:DNA Fingerprinting; D018076:DNA, Complementary / Q000737:chemistry; D004054:Diethylstilbestrol / Q000494:pharmacology*; D004527:Egg Proteins / Q000737:chemistry / Q000235:genetics; D004958:Estradiol / Q000494:pharmacology*; D004997:Ethinyl Estradiol / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D008099:Liver / Q000737:chemistry; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis*; D011956:Receptors, Cell Surface; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014819:Vitellogenins / Q000097:blood / Q000235:genetics; D015027:Zebrafish; D000072216:Zona Pellucida Glycoproteins
D018076:DNA, Complementary; D004527:Egg Proteins; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D014819:Vitellogenins; D000072216:Zona Pellucida Glycoproteins; D004997:Ethinyl Estradiol; D004958:Estradiol; D004054:Diethylstilbestrol