Responsiveness of the ovine gonadotropin-releasing hormone receptor gene to estradiol and gonadotropin-releasing hormone is not detectable in vitro but is revealed in transgenic mice

Although the ability of estradiol to enhance pituitary sensitivity to GnRH is established, the underlying mechanism(s) remain undefined. Herein, we fi nd that approximately 9,100 bp of 5' flanking region from the ovine GnRH re ceptor (oGnRHR) gene is devoid of transcriptional activity in gonadotrope-d erived cell lines and is not responsive to either estradiol or GnRH. In sta rk contrast, this same 9,100 bp promoter fragment directed tissue-specific expression of luciferase in multiple lines of transgenic mice. To test for hormonal regulation of the 9,100-bp promoter, ovariectomized transgenic fem ales were treated with a GnRH antiserum alone or in combination with estrad iol. Treatment with antiserum alone reduced pituitary expression of lucifer ase by 80%. Pituitary expression of luciferase in animals receiving both an tiserum and estradiol was approximately 50-fold higher than animals receivi ng antiserum alone. The estradiol response of the -9,100-bp promoter was eq ually demonstrable in males. In addition, a GnRH analog (D-Ala-6-GnRH) that does not cross-react with the GnRH antiserum restored pituitary expression of luciferase in males passively immunized against GnRH to levels not diff erent from castrate controls. Finally, treatment with both estradiol and D- Ala-6-GnRH increased pituitary expression of luciferase to a level greater than the sum of the individual treatments suggesting synergistic activation of the transgene by these two hormones. Thus, despite the complete absence of transcriptional activity and hormonal responsiveness in vitro, 9,100 bp of proximal promoter from the oGnRHR gene is capable of directing tissue-s pecific expression and is robustly responsive to both GnRH and estradiol in transgenic mice. To begin to refine the functional boundaries of the criti cal cis-acting elements, we next constructed transgenic mice harboring a tr ansgene consisting of 2,700 bp of 5' flanking region from the oGnRHR gene f used to luciferase. As with the -9,100 bp promoter, expression of luciferas e in the -2,700 lines was primarily confined to the pituitary gland, brain and testes. Furthermore, the passive immunization-hormonal replacement para digms described above revealed both GnRH and estradiol responsiveness of th e -2,700-bp promoter. Thus, 2,700 bp of proximal promoter from the oGnRHR g ene is sufficient for tissue-specific expression as well as GnRH and estrad iol responsiveness. Given the inability to recapitulate estradiol regulatio n of GnRHR gene expression in vitro, transgenic mice may represent one of t he few viable avenues for ultimately defining the molecular mechanisms unde rlying estradiol regulation of GnRHR gene expression.