Expression of a Murine Gonadotropin-Releasing Hormone Receptor-Luciferase Fusion Gene in Transgenic Mice Is Diminished by Immunoneutralization of Gonadotropin-Releasing Hormone

Abstract

A line of transgenic mice harboring a fusion gene consisting of 1900 bp of proximal 5'-flanking region from the murine GnRH receptor gene linked to the complementary DNA encoding luciferase was established to determine whether this promoter can direct tissue-specific expression in vivo and serve as a model for identifying the molecular mechanisms underlying hormonal regulation of this gene. Of 10 tissues screened, luciferase was detected predominantly in pituitary gland, but also in brain and testis. To assess hormonal regulation, luciferase activity was measured in intact males and ovariectomized females treated with an anti-GnRH serum alone, and in combination with testosterone or 17beta-estradiol. No effect of steroid treatment on transgene expression was detected. However, immunoneutralization of GnRH resulted in decreased serum LH concentrations and suppressed pituitary expression of luciferase. Furthermore, the effects of GnRH antiserum could be prevented by the administration of a noncross-reactive GnRH agonist. Thus, 1900 bp of 5'-flanking DNA from the murine GnRH receptor gene are sufficient to target luciferase expression in transgenic mice to established sites of GnRH receptor gene expression. Furthermore, we suggest that GnRH regulation of GnRH receptor gene expression is mediated by regulatory elements residing within 1900 bp of the 5'-flanking region.

Characterization of an Intrinsically Fluorescent Gonadotropin-Releasing Hormone Receptor and Effects of Ligand Binding on Receptor Lateral Diffusion*

“…In contrast to GnRH, antide had no effect on the fraction of mobile receptors. Consistent with this, we have found that the promoter of the GnRHR gene is hormonally regulated in transgenic mice (16). Furthermore, fluorescence photobleaching recovery analysis of the GnRHR-green fluorescent protein fusion reveals fundamental differences in the membrane dynamics of the GnRHR induced by the binding of an agonist vs. that induced by the binding of an antagonist.…”

Membrane rafts and GnRH receptor signaling

“…This syndrome arises as a failure of GnRH neurons to migrate from the olfactory placode to the hypothalamus correctly during embryogenesis resulting in decreased levels of GnRH and subsequent gonadotropins [8–10]. In addition to various naturally occurring mutations in humans, several experimental models also underscore the critical role of GnRH/GnRH receptor (GnRHR) signaling including mutation of the GnRHR gene [11, 12], genetic ablation of gonadotropes [13], immunoneutralization of GnRH [14, 15], and animal models of hypothalamic-pituitary disconnection [16, 17]. Thus, fertility is not only dependent on proper GnRH production and release from the hypothalamus but also its subsequent binding to high affinity GnRHR on pituitary gonadotropes and the coordination of these events is of universal importance in controlling reproduction in mammals.…”

Inside and Outside the Pituitary: Comparative Analysis of Gnrhr Expression Provides Insight into the Mechanisms Underlying the Evolution of Gene Expression

“…The first transgenic studies were performed using the luciferase reporter gene under the control of the mouse (1.9 kb) and ovine (9 or 2.7 kb) 5′ flanking regions of Gnrhr . One major contribution of these models was to quantify promoter activity with high sensitivity in homogenates from several organs and, subsequently, to compare this activity in pituitary and extra‐pituitary sites . Thus, the mouse Gnrhr promoter induced strong luciferase activity (adjusted for protein content) in the pituitary gland as expected and, to a lesser extent, in the whole brain, in which luciferase activity was estimated to be approximately 10% of that in the pituitary .…”

“…Thus, the mouse Gnrhr promoter induced strong luciferase activity (adjusted for protein content) in the pituitary gland as expected and, to a lesser extent, in the whole brain, in which luciferase activity was estimated to be approximately 10% of that in the pituitary . In addition, this promoter led to weak and variable luciferase expression in the testis, corresponding to 1% of the estimated level of expression in the pituitary . In our laboratory, we recently investigated Gnrhr expression in mouse testis but were unable to detect transcripts using conventional real‐time RT‐PCR .…”

“…In addition to the gonads and brain, GnRH‐binding activity and Gnrhr mRNA expression have also been identified in a variety of nongonadotroph cells, including other pituitary lineages, uterus, placenta, prostate, kidney, liver, heart, adrenal gland, spleen or lymphocytes, as well as many tumor cells . In all of these contexts, transgenic models (using luciferase or ALPP ) have not always been relevant for two main reasons . First, the restricted DNA regulatory region driving the transgenic construct may not be sufficient to reproduce all of the features of the endogenous gene expression depending on the cell context.…”