The Contribution of the Circadian Gene Bmal1 to Female Fertility and the Generation of the Preovulatory Luteinizing Hormone Surge - PubMed (original) (raw)

The Contribution of the Circadian Gene Bmal1 to Female Fertility and the Generation of the Preovulatory Luteinizing Hormone Surge

Karen J Tonsfeldt et al. J Endocr Soc. 2019.

Abstract

In rodents, the preovulatory LH surge is temporally gated, but the timing cue is unknown. Estrogen primes neurons in the anteroventral periventricular nucleus (AVPV) to secrete kisspeptin, which potently activates GnRH neurons to release GnRH, eliciting a surge of LH to induce ovulation. Deletion of the circadian clock gene Bmal1 results in infertility. Previous studies have found that Bmal1 knockout (KO) females do not display an LH surge at any time of day. We sought to determine whether neuroendocrine disruption contributes to the absence of the LH surge. Because Kiss1 expression in the AVPV is critical for regulating ovulation, we hypothesized that this population is disrupted in Bmal1 KO females. However, we found an appropriate rise in AVPV Kiss1 and Fos mRNA at the time of lights out in ovariectomized estrogen-treated animals, despite the absence of a measureable increase in LH. Furthermore, Bmal1 KO females have significantly increased LH response to kiss-10 administration, although the LH response to GnRH was unchanged. We then created Kiss1- and GnRH-specific Bmal1 KO mice to examine whether Bmal1 expression is necessary within either kisspeptin or GnRH neurons. We detected no significant differences in any measured reproductive parameter. Our results indicate that disruption of the hypothalamic regulation of fertility in the Bmal1 KO females is not dependent on endogenous clocks within either the GnRH or kisspeptin neurons.

Keywords: Bmal1; GnRH; LH; circadian rhythms; kisspeptin.

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Figures

Figure 1.

Figure 1.

Bmal1 KO females do not display an LH surge at lights out, despite increased AVPV Kiss1 and Fos mRNA. (A) LH values at ZT4 and ZT12 (lights off) in an induced surge model (n = 5 to 10; two-way ANOVA, effect of genotype; F1,24 = 4.99, P = 0.035); Sidak post hoc multiple comparisons test). (B) Kiss1 mRNA fold change relative to WT ZT12 from AVPV micropunches (n = 4 to 9; two-way ANOVA, effect of time; F1,22 = 14.44, P = 0.0010). (C) Fos mRNA fold change relative to WT ZT4 from AVPV micropunches (n = 4 to 9; two-way ANOVA, effect of time; F1,22 = 9.147, P = 0.0062). Different letters indicate significantly different groups (P < 0.05).

Figure 2.

Figure 2.

Bmal1 KO females have normal LH response to GnRH, but heightened LH response to kisspeptin. (A) LH values before and after exposure to 1 µg/kg GnRH (n = 6; two-way ANOVA, effect of time; F1,10 = 88.88, P < 0.0001; Sidak multiple comparisons test). (B) LH values before and after exposure to 30 nmol of kiss-10 (n = 6; two-way ANOVA significant for time and genotype interaction; F1,10 = 8.4, P = 0.0159; Sidak multiple comparisons test). Different letters indicate significantly different groups (P < 0.05).

Figure 3.

Figure 3.

Bmal1 KO females have prolonged and heightened LH response to kisspeptin, which is not due to loss of Bmal1 in the liver. (A) LH values following 30 nmol of kiss-10 administration in WT and Bmal1 KO females (n = 3 to 4; two-way ANOVA, significant for time and genotype interaction; F6,30 = 3.35, P = 0.012). (B) Area under the curve (AUC) of LH response during 45 min (n = 3 to 4; P = 0.022; unpaired t test). (C) LH values 10 min after kiss-10 administration (0.01 mg/kg to 3 mg/kg) in WT vs Bmal1 KO females fit with a four-parameter logistical curve (n = 4; comparison of fits, P = 0.159). (D) LH values before and 10 min after 2 mg/kg kiss-10 (challenge 1), and then from a second challenge 1 h later (challenge 2) (n = 6, two-way ANOVA, significant effect of time and genotype; F3,40 = 8.168, P = 0.0001; Sidak multiple comparisons test). (E) LH values following 30 nmol of kiss-10 in Bmal1fl/fl and Albumin-Bmal1−/− females (n = 3; two-way ANOVA significant for time; F6,24 = 14.26, P < 0.0001). (F) AUC of LH response during 45 min (n = 3; P = 0.13; unpaired t test).

Figure 4.

Figure 4.

qPCR reveals no significant differences among reproductively relevant hypothalamic and pituitary genes. (A) Arcuate micropunches show no differences between WT and Bmal1 KO expression of Dynorphin or Kiss1R (t test, n = 4 to 5; Kiss1R, P = 0.05). (B) AVPV micropunches show no significant differences between WT and Bmal1 KO expression of V1a or tyrosine hydroxylase (TH; t test, n= 4 to 5). (C) POA micropunches show no differences between WT and Bmal1 KO expression of Kiss1R or GnRH (t test, n = 5). (D) Whole pituitary shows no significant differences among FSHβ, LHβ, GnRH-R, and Kiss1R (t test, n = 3 to 4; Kiss1R, P > 0.05).

Figure 5.

Figure 5.

Validation of conditional KO in GnRH-Bmal1−/− and Kiss-Bmal1−/− mice. (Ai) GnRH (magenta), (Aii) BMAL1 (green), and (Aiii) merged image with DAPI (blue) from a Bmal1fl/fl mouse. GnRH neurons are identified by arrowheads in the isolated and merged images. (Bi) GnRH (magenta), (Bii) BMAL1 (green), and (Biii) merged image of with DAPI (blue) from a GnRH-Bmal1−/− mouse. (C) Quantification of the percentage of GnRH and Bmal1 colocalization in Bmal1fl/fl and GnRH-Bmal1−/− mice (t test, n = 3 to 4; P < 0.003). (D) Representative PCR product from genomic DNA of micropunches showing Bmal1 recombination in the AVPV and ARC of the Kiss-Bmal1−/− mouse, but not in regions lacking Kiss1 expression (~0.57-kb band). Recombination in a Bmal1 KO tail sample is shown as a positive control. ***P < 0.001. Cere., cerebellum; NTC, no template control; Thal., thalamus.

Figure 6.

Figure 6.

Bmal1 KO in GnRH or Kiss1 neurons does not affect pubertal onset or estrous cycling. (A) Age (d) and (B) weight (g) of vaginal opening in Bmal1fl/fl, GnRH-Bmal1−/−, and Kiss-Bmal1−/− female mice (n = 3 to 6; one-way ANOVA not significant). (C) Percentage of time spent in each stage during 25 consecutive days of vaginal cytology (n = 7 to 13, two-way ANOVA significant for effect of stage; F2,51 = 52.15, P < 0.0001). (D) Average cycle length across 25 consecutive days of vaginal cytology (n = 7 to 13; one-way ANOVA not significant). (E) Number of Graafian follicles per section observed in ovaries in light/dark (LD) and constant darkness (DD) conditions (n = 2 to 15, two-way ANOVA significant for effect of lighting; F1,24 = 24.42, P < 0.0001; Sidak post hoc multiple comparisons test). (F) Number of corpora lutea observed per section in LD and DD conditions (n = 2 to 15; two-way ANOVA not significant). **P < 0.01, ***P < 0.001.

Figure 7.

Figure 7.

GnRH-Bmal1−/− and Kiss-Bmal1−/− have normal fecundity. (A) Time to first litter as measured by days after pairing (n = 3 to 5; one-way ANOVA not significant). (B) Average number of litters produced in 100 d of pairing, including 25 d of observation following removal of male (n = 3 to 5; one-way ANOVA not significant). (C) Number of pups per litter during recorded period (n = 3 to 5, one-way ANOVA not significant).

Figure 8.

Figure 8.

GnRH-Bmal1−/− and Kiss-Bmal1−/− produce an LH surge at lights off and have normal GnRH and kiss-10 LH responsiveness. (A) LH levels at lights off in an induced surge paradigm (for nonsurging example, please see Fig. 1A) (n = 5 to 9, one-way ANOVA not significant). (B) LH levels before and 10 min after 1 μg/kg GnRH administration (n = 3 to 9; two-way ANOVA significant for time; F1,30 = 56.89, P < 0.0001; Sidak multiple comparisons test). (C) LH levels before and 10 min after 30 nmol of kiss-10 administration (n = 3 to 8; two-way ANOVA significant for time; F1,28 = 43.61, P < 0.0001; Sidak multiple comparisons test). Different letters indicate significantly different groups (P < 0.05).

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