Neuroestrogen-Dependent Transcriptional Activity in the Brains of ERE-Luciferase Reporter Mice following Short- and Long-Term Ovariectomy

Abstract

Previous work has demonstrated that estrogen receptors are transcriptionally active in the absence of ovarian estrogens. The current work aims to determine whether brain-derived estrogens influence estrogen receptor-dependent transcription after short- or long-term loss of ovarian function. Experiments were conducted using estrogen response element (ERE)-Luciferase reporter mice, which express the gene for luciferase driven by consensus ERE, allowing for the quantification of ERE-dependent transcription. Brain regions examined were hippocampus, cortex, and hypothalamus. In Experiment 1, short-term (10 d) ovariectomy had no impact on ERE-dependent transcription across brain regions compared with sham surgery. In Experiment 2, chronic intracerebroventricular administration of the aromatase inhibitor letrozole significantly decreased transcriptional activity in 10-d-old ovariectomized mice across brain regions, indicating that the sustained transcription in short-term ovariectomized mice is mediated at least in part via actions of neuroestrogens. Additionally, intracerebroventricular administration of estrogen receptor antagonist ICI-182,780 blocked transcription in 10-d-old ovariectomized mice across brain regions, providing evidence that sustained transcription in ovariectomized mice is estrogen receptor dependent. In Experiment 3, long-term (70 d) ovariectomy significantly decreased ERE-dependent transcription across brain regions, though some residual activity remained. In Experiment 4, chronic intracerebroventricular letrozole administration had no impact on transcription in 70 d ovariectomized mice across brain regions, indicating that the residual ERE-dependent transcription in long-term ovariectomized mice is not mediated by neuroestrogens. Overall, the results indicate that ERE-dependent transcription in the brain continues after ovariectomy and that the actions of neuroestrogens contribute to the maintenance of ERE-dependent transcription in the brain following short-term, but not long-term, loss of ovarian function.

Keywords: cortex; estradiol; estrogen; hippocampus; hypothalamus; neuroestrogen.

Graphical abstract

Figure 1.

A–D, Experimental timelines for Experiment 1 (A), Experiment 2 (B), Experiment 3 (C), and Experiment 4 (D). Sham, Sham surgery; OVX, ovariectomy.

Figure 2.

Impact of short-term ovariectomy on ERE-dependent transcription. ERE-Luciferase reporter mice were ovariectomized (OVX) or underwent sham surgery (Intact). Approximately ten days later, mice were killed and luciferase activity was measured in the brain (cortex, hypothalamus, and hippocampus) and uterus. A, Short-term ovariectomy had no impact on luciferase activity as measured by relative light units per microgram of protein (RLU/μg) across brain regions. There was no effect of brain region or interaction between treatment and brain region. B, Short-term ovariectomy resulted in a near significant (p = .057) decrease in luciferase activity in the uterus. Data are presented as means ± SEM normalized to percent Intact.

Figure 3.

Impact of aromatase inhibition and estrogen receptor antagonism on ERE-dependent transcription following short-term ovariectomy. ERE-Luciferase reporter mice were ovariectomized (OVX) or underwent sham surgery (Intact). Beginning ∼7 days after surgeries, OVX mice received chronic i.c.v. delivery of vehicle (OVX + aCSF), the aromatase inhibitor letrozole (OVX + Letrozole) or the estrogen receptor antagonist ICI 182,780 (OVX + ICI). Intact mice received vehicle (Intact + aCSF). Three days later, mice were killed and luciferase activity was measured in the cortex, hypothalamus, and hippocampus. There was a main effect of treatment (p < .05) on luciferase activity as measured by relative light units per microgram of protein (RLU/μg) across brain regions. Post hoc testing revealed that luciferase activity was not impacted by short-term ovariectomy (OVX + aCSF vs. Intact + aCSF), but was decreased in ovariectomized mice by aromatase inhibition (OVX + aCSF vs. OVX + Letrozole, p < .05) and by estrogen receptor antagonism (OVX + aCSF vs. OVX + ICI, p < .05). There was no effect of brain region or interaction between treatment and brain region. Data are presented means ± SEM normalized to percent Intact + aCSF.

Figure 4.

Impact of long-term ovariectomy on ERE-dependent transcription. ERE-Luciferase reporter mice were ovariectomized (OVX) or underwent sham surgery (Intact). Approximately 70 days later, mice were killed and luciferase activity was measured in the cortex, hypothalamus, and hippocampus. Long-term ovariectomy significantly decreased luciferase activity as measured by relative light units per microgram of protein (RLU/μg) across brain regions (p < .05). There was no effect of brain region or interaction between treatment and brain region. Data are presented as means ± SEM normalized to percent Intact.

Figure 5.

Impact of aromatase inhibition on ERE-dependent transcription following long-term ovariectomy. ERE-Luciferase reporter mice were ovariectomized (OVX) or underwent sham surgery (Intact). Beginning ∼70 days after surgeries, OVX mice received chronic i.c.v. delivery of vehicle (OVX + aCSF), or the aromatase inhibitor letrozole (OVX + Letrozole). Intact mice received vehicle (Intact + aCSF). Three days later, mice were killed and luciferase activity was measured in the cortex, hypothalamus, and hippocampus. There was a main effect of treatment (p < .05) on luciferase activity as measured by relative light units per microgram of protein (RLU/μg) across brain regions. Post hoc testing revealed that luciferase activity was significantly decreased following long-term ovariectomy (OVX + aCSF vs. Intact + aCSF, p < .05), but was not impacted by aromatase inhibition in ovariectomized mice (OVX + aCSF vs. OVX + Letrozole). There was no effect of brain region or interaction between treatment and brain region. Data are presented as means ± SEM normalized to percent Intact + aCSF.