Simultaneous determination of androgenic and estrogenic endpoints in the threespine stickleback (Gasterosteus aculeatus) using quantitative RT-PCR.

A method to evaluate the expression of three hormone responsive genes, vitellogenin (estrogens), spiggin (androgens), and an androgen receptor (ARbeta) using real-time PCR in threespine stickleback is presented. Primers were designed from previously characterised spiggin and ARbeta sequences, while a homology cloning strategy was used to isolate a partial gene sequence for stickleback vitellogenin (Vtg). Spiggin mRNA was significantly higher in kidneys of field-caught males compared to females by greater than five orders of magnitude while ARbeta levels were only 1.4-fold higher in males. Female fish had four order of magnitude higher liver Vtg expression than wild-captured males. To determine the sensitivity of these genes to induction by hormones, male and female sticklebacks were exposed to 1, 10 and 100 ng/L of methyltestosterone (MT) or estradiol (E2) in a flow-through exposure system for 7 days. Spiggin induction in females, and Vtg induction in males were both detectable at 10 ng/L of MT and E2, respectively. MT exposure did not induce ARbeta expression in the kidneys of female stickleback. In vitro gonadal steroid hormones production was measured in testes and ovaries of exposed stickleback to compare gene expression endpoints to an endpoint of hormonal reproductive alteration. Reduction in testosterone production in ovaries at all three MT exposure concentrations, and ovarian estradiol synthesis at the 100 ng/L exposure were the only effects observed in the in vitro steroidogenesis for either hormone exposure. Application of these methods to assess both androgenic, estrogenic, and anti-steroidogenic properties of environmental contaminants in a single fish species will be a valuable tool for identifying compounds causing reproductive dysfunction in fishes.