PARENT SESSION

(105) IN VIVO DELIVERY OF ADENOVIRUS EXPRESSING FOLLISTATIN REVEALS A KEY ROLE FOR THE GnRH RECEPTOR ACTIVATING SEQUENCE (GRAS) IN MEDIATING ACTIVIN RESPONSIVENESS OF THE GnRH RECEPTOR GENE PROMOTER IN TRANSGENIC MICE.

Cherrington, Brian¹, Cantlon, Jeremy ¹, Farmerie, Todd¹, Clay, Colin¹, ¹ Colorado State University, Ft. Collins, CO

ABSTRACT- Activity of the murine GnRH receptor (GnRHR) gene promoter in gonadotrope derived T3-1 cells is dependent on autocrine stimulation by activin, an input mediated at an element termed GRAS. Our understanding of activin regulation of the GnRHR gene promoter is, however, based solely on in vitro analyses. To determine if activin responsiveness is evident in vivo, we utilized transgenic mice harboring a fusion gene consisting of 1900 bp of proximal promoter from the murine GnRHR fused to the cDNA encoding luciferase (-1900wt) and a second line containing the same promoter with a loss of function mutation in GRAS (-1900GRAS). In both lines, luciferase expression was confined to the pituitary, brain, and gonads; however, pituitary expression was 10-fold lower in the -1900GRAS animals. To assess activin responsiveness of the transgenes, we used an indirect approach based on in vivo delivery of adenovirus expressing the activin binding protein follistatin (AdCAFS288). In preliminary studies, wild-type mice were ovariectomized and remained untreated or administered increasing doses of AdCAFS288 or adenovirus expressing GFP (AdGFP) as a single intraperitoneal injection. Four days following administration of adenovirus, animals were euthanized and serum was analyzed for FSH concentrations. A viral dose of 3.9x10¹¹pfu of AdCAFS288 effectively suppressed serum FSH whereas FSH was unaffected by AdGFP. In the next series of experiments, -1900wt and -1900GRAS mice were ovariectomized and, 1 week later, administered 3.9x10¹¹pfu of AdCAFS288 or AdGFP. Four days later, tissues were collected and analyzed for luciferase expression and serum concentrations of FSH and LH were determined. Luciferase expression in the -1900wt pituitaries was approximately 50% (P<0.01) of that observed in animals receiving Ad-GFP. In contrast, pituitary expression of luciferase in the -1900GRAS line was unaffected (P>0.05) by AdCAFS288. Consistent with activin neutralization, serum concentrations of FSH but not LH were suppressed in both lines of mice receiving AdCAFS288. Thus, activin responsiveness of the GnRHR gene promoter is effectively recapitulated in transgenic mice. Furthermore, these data establish a crucial role for GRAS in mediating activin responsiveness both in vitro and in vivo.

KEY WORDS: GRAS, GnRH Receptor, Activin, Transgenic Mice