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 Mice1
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-d...
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Veröffentlicht in: | Endocrinology (Philadelphia) 2000-03, Vol.141 (3), p.1001-1010 |
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Zusammenfassung: | 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
expressio |
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ISSN: | 0013-7227 1945-7170 |
DOI: | 10.1210/endo.141.3.7391 |