Characterization of estrogen receptor isotypes and estrogen receptor regulated genes in largemouth bass

The discovery of multiple estrogen receptor (ER) isotypes has complicated our understanding of the molecular events involved in transcriptional regulation of estrogen-responsive genes. The comprehensive goal of the research presented in this dissertation was to determine the impact of 17beta-estradiol (E 2) and nonylphenol (NP) on the regulation of the ER isotypes and the ER regulated gene, vitellogenin (Vtg) in largemouth bass (LMB). The overall hypothesis was that the ER isotypes are differentially expressed in E 2 target tissues, and that their transactivation potential varied when stimulated by E2. Furthermore, similar expression and activity profiles produced by E2 and NP would indicate that NP acts, at least in part, as an ER agonist.;To test this hypothesis, the full length cDNAs for LMB ER isotypes were cloned from endocrine sensitive tissues, and three isotypes were found corresponding to alpha, beta and gamma. Based on these sequences, real-time PCR assays were designed to measure changes in mRNA levels of the three isotypes and Vtg, in females during the normal reproductive season. In liver, only ER alpha paralleled plasma E2 levels and Vtg mRNA expression. In the gonad, ER beta and ER gamma expression predominated, but were highest early in the fall, before plasma E2 levels increased. Injection of males with E2 or NP, corroborated the normal in vivo expression studies, indicating that ER alpha, and to a lesser extent, ER gamma are induced by these agents. These studies also indicated that NP acts as an ER agonist. Further verification of NP agonist activity was obtained by macroarray analysis of NP and E 2 treated fish. Many known E2-reponsive genes such as the Vtgs and choriogenins were induced by both E2 and NP in LMB, however, the induction of signal peptidase only by NP implies that alternate modes of action that are independent of ERs may exist.;Finally, an in vitro transient transfection assay was developed to determine differences in the activation potential of the ER isotypes. The activity of ER alpha was greater than ER beta when stimulated with E 2 and NP. In addition, ER alpha activity was suppressed by ER beta when the receptors were activated. Overall, these results suggest that the ER isotypes are functionally distinct, but work in a combinatorial manner to regulate E2-responsive genes.