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*

Abstract

Although the ability of estradiol to enhance pituitary sensitivity to GnRH is established, the underlying mechanism(s) remain undefined. Herein, we find that approximately 9,100 bp of 5' flanking region from the ovine GnRH receptor (oGnRHR) gene is devoid of transcriptional activity in gonadotrope-derived cell lines and is not responsive to either estradiol or GnRH. In stark 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 females were treated with a GnRH antiserum alone or in combination with estradiol. Treatment with antiserum alone reduced pituitary expression of luciferase by 80%. Pituitary expression of luciferase in animals receiving both antiserum and estradiol was approximately 50-fold higher than animals receiving antiserum alone. The estradiol response of the -9,100-bp promoter was equally 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 different 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-specific expression and is robustly responsive to both GnRH and estradiol in transgenic mice. To begin to refine the functional boundaries of the critical cis-acting elements, we next constructed transgenic mice harboring a transgene consisting of 2,700 bp of 5' flanking region from the oGnRHR gene fused to luciferase. As with the -9,100 bp promoter, expression of luciferase in the -2,700 lines was primarily confined to the pituitary gland, brain and testes. Furthermore, the passive immunization-hormonal replacement paradigms described above revealed both GnRH and estradiol responsiveness of the -2,700-bp promoter. Thus, 2,700 bp of proximal promoter from the oGnRHR gene is sufficient for tissue-specific expression as well as GnRH and estradiol responsiveness. Given the inability to recapitulate estradiol regulation of GnRHR gene expression in vitro, transgenic mice may represent one of the few viable avenues for ultimately defining the molecular mechanisms underlying estradiol regulation of GnRHR gene expression.

Does a Nonclassical Signaling Mechanism Underlie an Increase of Estradiol-Mediated Gonadotropin-Releasing Hormone Receptor Binding in Ovine Pituitary Cells?1

“…Thus, the most likely mechanism is that E2 activates GNRHR gene expression. Consistent with this notion, we have established E2 responsiveness of the ovine GNRHR gene promoter in multiple lines of transgenic mice [60]. Furthermore, as has been demonstrated in sheep, this effect of E2 is evident in the absence of GnRH input [9,56,61].…”

“…This, however, is not the case. A canonical ERE is not present in the ovine GNRHR gene [43], and E2 responsiveness is not detectable using standard in vitro analyses of promoter regulation [60]. Thus, it appears that E2 leads to an increase in GNRHR gene expression through a noncanonical pathway.…”

Colocalization of GH, TSH and prolactin, but not ACTH, with ?LH-immunoreactivity: evidence for pluripotential cells in the ovine pituitary

“…It is perhaps surprising, however, that the GnRH receptor has never been visualized immunocytochemically within the mammalian pituitary. This omission appears to be due to the absence of an effective antibody against the GnRH receptor (Duval et al 2000). Clearly, the specific cell types that express GnRH receptors warrant further investigation.…”

Immunoreactive GnRH type I receptors in the mouse and sheep brain

“…We have been unable to visualize GnRHR immunoreactivity in brains that were not perfusion fixed or subjected to antigen retrieval (data not shown). Using an established (Wolfe et al, 2002) goat anti-luciferase polyclonal antibody (1:20; cr2029gap, Cortex Biochemical; San Leandro, CA), we performed a dual-labeling control study on transgenic mice containing the luciferase gene driven by 9100 base pairs of the ovine GnRH receptor promoter (Duval et al, 2000).…”