Corticosterone Blocks Ovarian Cyclicity and the LH Surge via Decreased Kisspeptin Neuron Activation in Female Mice

Abstract

Stress elicits activation of the hypothalamic-pituitary-adrenal axis, which leads to enhanced circulating glucocorticoids, as well as impaired gonadotropin secretion and ovarian cyclicity. Here, we tested the hypothesis that elevated, stress-levels of glucocorticoids disrupt ovarian cyclicity by interfering with the preovulatory sequence of endocrine events necessary for the LH surge. Ovarian cyclicity was monitored in female mice implanted with a cholesterol or corticosterone (Cort) pellet. Cort, but not cholesterol, arrested cyclicity in diestrus. Subsequent studies focused on the mechanism whereby Cort stalled the preovulatory sequence by assessing responsiveness to the positive feedback estradiol signal. Ovariectomized mice were treated with an LH surge-inducing estradiol implant, as well as Cort or cholesterol, and assessed several days later for LH levels on the evening of the anticipated surge. All cholesterol females showed a clear LH surge. At the time of the anticipated surge, LH levels were undetectable in Cort-treated females. In situ hybridization analyses the anteroventral periventricular nucleus revealed that Cort robustly suppressed the percentage of Kiss1 cells coexpressing cfos, as well as reduced the number of Kiss1 cells and amount of Kiss1 mRNA per cell, compared with expression in control brains. In addition, Cort blunted pituitary expression of the genes encoding the GnRH receptor and LHβ, indicating inhibition of gonadotropes during the blockage of the LH surge. Collectively, our findings support the hypothesis that physiological stress-levels of Cort disrupts ovarian cyclicity, in part, through disruption of positive feedback mechanisms at both the hypothalamic and pituitary levels which are necessary for generation of the preovulatory LH surge.

Figure 1.

Cort disrupts estrous cyclicity in the female mouse. A, Mean serum Cort measured on the final day of implantation in Veh- and Cort-treated female mice. B, Representative profiles depicting estrous cyclicity in female mice, as measured by vaginal cytology, before (d 1–22, baseline) and during the period of implantation (d 23–38, implant) with a cholesterol (Veh, top panel) or Cort pellet (2 lower panels). P, proestrus; E, estrus; M, metestrus; D, diestrus. C, Average estrous cycle length during the baseline and implant periods in female mice treated with Veh or Cort. D, Average time spent in each stage of the cycle during the baseline and implant periods in Veh and Cort mice; n = 6 animals/group. Values were analyzed by one- or two-way ANOVA with group (Veh vs Cort) and time (baseline period vs implant period) as factors. *, P < .001, Veh vs Cort.

Figure 2.

Cort blocks the estradiol-induced LH surge. Mean (±SEM) serum Cort (ng/mL, A) and LH (ng/mL, B) in adult female mice ovariectomized and exposed to a positive feedback regimen of constant elevated estradiol along with either a cholesterol (Veh) or Cort implant. Two days after surgery, blood was collected in the morning (am; 10 am) or evening (pm; 6 pm) of the expected LH surge in female mice. C, Percentage of female mice, treated with either Veh or Cort, that responded to the estradiol stimulus with an LH surge; n = 6 animals/group. Values were analyzed by two-way ANOVA with group (Veh vs Cort) and time (am vs pm) as factors or χ² analysis (LH surge incidence). *, P < .001, Veh vs Cort.

Figure 3.

Cort inhibits Kiss1 expression at the time of the LH surge. Representative dark-field photomicrographs showing Kiss1 mRNA (silver grain clusters) expressed in the AVPV/PeN at the time of the expected LH surge (6 pm) in brains collected from female mice exposed to a surge-inducing estradiol stimulus, as well as a pellet containing cholesterol (Veh) (A) or Cort (B). C, Mean (±SEM) number of Kiss1 mRNA-containing cells in the AVPV/PeN of Veh- and Cort-treated females. D, Mean (±SEM) level of Kiss1 mRNA per cell in the AVPV/PeN of Veh- and Cort-treated females. E, Mean (±SEM) total Kiss1 mRNA in the AVPV/PeN of Veh- and Cort-treated females. n = 6 animals per group. Values were analyzed by one-way ANOVA. *, P < .01, Veh vs Cort; 3V, third ventricle.

Figure 4.

Cort inhibits neuronal activation of AVPV/PeN Kiss1 cells. Representative low- and high-power photomicrographs showing Kiss1 mRNA-containing cells (red fluorescence) and cfos mRNA (silver grain clusters; representative of neuronal activation) in the AVPV/PeN at the time of the expected LH surge (6 pm) in brains collected from female mice exposed to a surge-inducing estradiol stimulus, as well as a pellet containing cholesterol (Veh) (A) or Cort (B). Dashed box indicates area of higher magnification. Yellow arrowheads denote examples of Kiss1 cells with significant cfos coexpression, as determined by an automated image processing system. Blue arrowheads designate examples of Kiss1 neurons that did not have notable cfos induction. C, Mean (±SEM) number of Kiss1 mRNA-containing cells in the AVPV/PeN of Veh- and Cort-treated females. D, Mean (±SEM) percentage of Kiss1 mRNA-containing cells that coexpress cfos mRNA in the AVPV/PeN of Veh- and Cort-treated females; n = 6 per group. Values were analyzed by one-way ANOVA. *, P < .01, Veh vs Cort; 3V, third ventricle.

Figure 5.

Cort does not reduce the level of ERα mRNA expression in Kiss1 cells. Representative photomicrographs of brains collected at 6 pm from female mice exposed to a surge-inducing estradiol stimulus, as well as a pellet containing cholesterol (Veh) (A) or Cort (B). Low- and high-power photomicrographs show Kiss1 mRNA containing cells (red fluorescence) and ERα mRNA (silver grain clusters) in the AVPV/PeN. Dashed box indicates area of higher magnification presented. Yellow arrowheads denote examples of Kiss1 cells with significant ERα coexpression. Blue arrowheads designate examples of Kiss1 neurons that did not have ERα induction. C, Mean (±SEM) number of Kiss1 mRNA-containing cells in the AVPV/PeN of Veh- and Cort-treated females. D, Mean (±SEM) percentage of Kiss1 mRNA-containing cells that coexpress ERα mRNA in the AVPV/PeN of Veh- and Cort-treated females; n = 6 per group. Values were analyzed by one-way ANOVA. **, P < .01; Veh vs Cort. 3V, third ventricle.

Figure 6.

Cort inhibits pituitary gene expression at the time of the LH surge. Quantitative RT-PCR analysis of Lhβ (A) and Gnrhr (B) mRNA was performed on individual mouse pituitary glands collected at the time of the expected LH surge (6 pm) from female mice exposed to a surge-inducing estradiol stimulus, as well as a pellet containing cholesterol (Veh) or Cort n = 6 per group. Ct values for Lhβ and Gnrhr were compared with Gapdh using the 2^−ΔΔCt method and analyzed by one-way ANOVA. Data are represented as mean fold change ± SEM. *, P < .05, Veh vs Cort.