The role of cAMP response element-binding protein in estrogen negative feedback control of gonadotropin-releasing hormone neurons

Abstract

The mechanisms through which estradiol (E2) regulates gonadotropin-releasing hormone (GnRH) neurons to control fertility are unclear. Previous studies have demonstrated that E2 rapidly phosphorylates cAMP response element-binding protein (CREB) in GnRH neurons in vivo. In the present study, we used GnRH neuron-specific CREB-deleted mutant mice [GnRH-CREB knock-outs (KOs)] with and without global cAMP response element modulator (CREM) deletion (global-CREM KOs) to investigate the role of CREB in estrogen negative feedback on GnRH neurons. Evaluation of GnRH-CREB KO mice with and without global CREM deletion revealed normal puberty onset. Although estrus cycle length in adults was the same in controls and knock-out mice, cycles in mutant mice consisted of significantly longer periods of diestrus and less estrus. In GnRH-CREB KO mice, basal levels of luteinizing hormone (LH) and the postovariectomy increment in LH were normal, but the ability of E2 to rapidly suppress LH was significantly blunted. In contrast, basal and postovariectomy LH levels were abnormal in GnRH-CREB KO/global-CREM KO mice. Fecundity studies showed that GnRH-CREB KO with and without global CREM deletion were normal up to ∼9 months of age, at which time they became prematurely reproductively senescent. Morphological analysis of GnRH neurons revealed a significant reduction (p < 0.01) in GnRH somatic spine density of GnRH-CREB KO mice compared to control females. These observations implicate CREB within the GnRH neuron as an important target for E2's negative feedback actions. They also indicate that the rapid modulation of CREB by E2 is of physiological significance in the CNS.

Figure 1.

A, CREB expression in CREB mutant mouse line. The photomicrographs show nuclear CREB immunoreactivity (black nuclei, red arrowhead) in GnRH neurons (brown cytoplasm, yellow arrowhead) in CREB^loxP/loxP mouse and lack of CREB expression in GnRH neurons (brown cytoplasm, yellow arrowhead) in GnRH-CREB KO mouse. Scale bars, 10 μm. B, Histogram shows the percentage of GnRH neurons expressing CREB in control (C57BL/6, CREB^loxP/loxP, CREM^+/+), GnRH-CREB KO, global-CREM KO, and GnRH-CREB KO/global-CREM KO mice. The histogram shows the means (+SEM); n = 7–14. ***p < 0.001 (ANOVA with Tukey's post hoc test).

Figure 2.

A, B, Puberty onset in CREB mutant mice. Vaginal opening (A) and first estrus (B) in transgenic and control mice. The mean (+SEM) days of vaginal opening and first estrus are given for each of the six genotypes (n = 8–14). *p < 0.05 (ANOVA with Tukey's post hoc test).

Figure 3.

Abnormal estrus cyclicity in CREB knock-out mice. A–D, The number of cycles (A), cycle length (B), and percentage of time spent in estrus (C) and diestrus (D) in CREB mutant mice. E, Representative estrus cycle profiles (for individual mice over a 21 d period) demonstrating the alterations in estrus cyclicity in CREB KO mice compared with controls. Both control and mutant mice show clearly distinguishable estrus cycle phases and proestrus–metestrus intermediate phases at similar frequency. The histograms show the means (+SEM) for each of the six genotypes; n = 8–16. *p < 0.05; **p < 0.01; ***p < 0.001 (ANOVA with Tukey's post hoc test). E, Estrus; P, proestrus; D, diestrus; M, metestrus.

Figure 4.

Fecundity of CREB mutant female mice. A, B, The histograms show the mean (+SEM) number of litters (A) and number of pups/litter (B) born to mutant and control female mice per 6 months. C, The delivery intervals in young (Y; first 6 months of testing) and old (O; after 6 months of age) mice are given for each of the six genotypes (n = 4–6). **p < 0.01; ***p < 0.001 (ANOVA with Tukey's post hoc test).

Figure 5.

Effects of GnRH-specific deletion of CREB on estrogen negative feedback mechanisms. A, Chart summarizing the serum LH levels in mice before OVX (intact), 1 week after OVX, and 3 h after treatment with E2. B, C, Intact serum LH levels (B) and the increment from basal LH level (C) after OVX. D, LH levels decrease after treatment with E2 normalized to post-OVX LH levels. The histograms show the means (+SEM); n = 9–16. *p < 0.05; **p < 0.01; ***p < 0.001 (ANOVA with Tukey's post hoc test).

Figure 6.

Somatic spine density of GnRH neurons in CREB knock-out mice. A, B, Confocal stacks (400 nm optical thickness) of GnRH-immunolabeled neurons from CREB^loxP/loxP (A) and GnRH-CREB KO (B) mice. Insets, Typical somatic spines are shown in confocal stacks from corresponding white boxes in GnRH neurons in A and B. Scale bar, 5 μm. C–E, Spine densities for the somata (C), soma circumferences (D), and numbers of spines on GnRH neurons (E) of control (C57BL/6, CREB^loxP/loxP) and GnRH-CREB KO mice. The histograms show the means (+SEM); n = 4–6. **p < 0.01 (ANOVA with Tukey's post hoc test).