Developmental and activin -mediated regulation of gonadotrope gene expression.

摘要

Activin is an important hormonal regulator of both follicle-stimulating hormone (FSH) and gonadotropin-releasing hormone (GnRH) receptor expression. However, the molecular mechanisms of this regulation were unknown. The work described in this dissertation provides insight into this regulation, and identifies basic helix-loop-helix (bHLH) transcription factors as potentially important developmental regulators of gonadotrope gene expression.;Activin regulation of an ovine FSHbeta-subunit reporter gene requires three regulatory elements within the -985 to +759 region of the gene, at -972/961, -166 and -133. In electromobility shift assays (EMSA), the -166 and -133 regions bind specific LbetaT2 nuclear proteins, and the -972/-961 region binds Smad4 protein. Unexpectedly, the homeodomain proteins Pbx1 and Prep1 were found to bind to the -133 site, as well as the homologous region of the mouse FSHbeta-subunit gene in vivo. The -133 site is also required for full GnRH responsiveness of the reporter gene, and overexpression of a dominant negative form of Prep1 reduces activin and GnRH responsiveness of the reporter gene.;Activin regulation of the GnRH receptor gene similarly requires several regulatory elements: a Smad-binding element (SBE) that overlaps the GnRH receptor-activating sequence (GRAS element), two downstream elements homologous to Smad half-sites, and adjacent E-Boxes (consensus binding sites for bHLH proteins). The GRAS SBE is required for full activin response, is sufficient to confer activin responsiveness, and in EMSA binds Smad4 protein. Both E-Boxes are also required for full activin response, and specific alphaT3-1 nuclear proteins bind to the 3' Ebox site in EMSA.;Interestingly, the 3' E-Box is required for basal expression of the GnRH receptor gene as well. Mutation of this E-Box causes a significant decrease in GnRH receptor reporter expression. The bHLH protein Neurod1 binds to the 3' EBox in EMSA, and activates reporter gene expression in transfections, as do the related proteins Ascl 1 and Neurod4. The in vivo role of Neurod1 was investigated in Neurod1 knockout mice. While GnRH receptor gene expression is detected in Neurod1 null embryos, expression of the LHbeta-subunit gene is not, suggesting a more general role for Neurod1, and perhaps other bHLH family members, in gonadotrope gene expression and development.