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(P734) Cloning and Sequencing of Brain Aromatase in Medaka and its Response to Developmental Exposure to Estradiol.
Kuhl, Adam*,1, Brouwer, Marius1, 1 University of Southern Mississippi, Ocean Springs, MS
ABSTRACT- Aromatization of androgen by aromatase is the main source of estrogen in both males and females. This estrogen is required for the development of primary and secondary sex characteristics. Transcription of aromatase can be regulated by both endogenous and environmental estrogens. There are two isoforms of aromatase sequenced in several teleost species, one mainly expressed in the brain and the other in the ovary. In medaka, only the ovarian form has been sequenced. This project focused on cloning and sequencing of brain aromatase in medaka and measuring its expression in fish exposed to estradiol for two weeks after hatch. Cloning was performed by RT-PCR using RNA extracted from the brain of adult medaka. Using degenerate primers designed from tilapia brain aromatase, a 930 bp partial cDNA product was cloned and sequenced. 3' and 5' RACE kits from Invitrogen were used to obtain the entire 1897 bp cDNA sequence. Estradiol treatments were conducted in a flow through system from hatch to day 14. Fish were exposed to treatments of 0, 0.01, 0.05, 0.1, 0.2, 0.5, and 2.0 ppb estradiol. On day 0, 3, 5, 9, and 14, fish were sampled for RNA extraction. Using the known sequence of the aromatases, gene specific primers were designed and expression of brain and ovarian aromatase was determined in all treatments through relative quantitative RT-PCR. Results show that the amino acid sequence of medaka aromatase shows 68% homology to medaka ovarian aromatase and 84% and 67% homology to tilapia and goldfish brain aromatase, respectively. Preliminary data suggest that ovarian aromatase is not expressed in developing medaka and brain aromatase expression increases directly with estradiol exposure. Adults from the estradiol exposure showed a dose dependent female to male sex reversal. These results suggest that brain aromatase catalyzed conversion of testosterone to estradiol causes sex reversal of medaka.
Key words: Aromatase, Medaka, Sex-reversal, Endocrine Disruption
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