Analysis of multiple positive feedback paradigms demonstrates a complete absence of LH surges and GnRH activation in mice lacking kisspeptin signaling

Abstract

Kisspeptin stimulates gonadotropin-releasing hormone (GnRH) neurons via the kisspeptin receptor, Kiss1r. In rodents, estrogen-responsive kisspeptin neurons in the rostral hypothalamus have been postulated to mediate estrogen-induced positive feedback induction of the preovulatory luteinizing hormone (LH) surge. However, conflicting evidence exists regarding the ability of mice lacking Kiss1r to display LH surges in response to exogenous hormones. Whether the discrepancy reflects different mouse strains used and/or utilization of different surge-induction paradigms is unknown. Here, we tested multiple hormonal paradigms in one Kiss1r knockout (KO) model to see which paradigms, if any, could generate circadian-timed LH surges. Kiss1r KO and wild-type (WT) females were ovariectomized, given sex steroids in various modes, and assessed several days later for LH levels in the morning or evening (when surges occur). Serum LH levels were very low in all morning animals, regardless of genotype or hormonal paradigm. In each paradigm, virtually all WT females displayed clear LH surges in the evening, whereas none of the KO females demonstrated LH surges. The lack of LH surges in KO mice reflects a lack of GnRH secretion rather than diminished pituitary responsiveness from a lifetime lack of GnRH exposure because KO mice responded to GnRH priming with robust LH secretion. Moreover, high cfos-GnRH coexpression was detected in WT females in the evening, whereas low cfos-GnRH coexpression was present in KO females at all time points. Our findings conclusively demonstrate that WT females consistently display LH surges under multiple hormonal paradigms, whereas Kiss1r KO mice do not, indicating that kisspeptin-Kiss1r signaling is mandatory for GnRH/LH surge induction.

Keywords: GPR54; GnRH; Kiss1; Kiss1r; LH surge; estradiol; kisspeptin; positive feedback.

FIG. 1

Mean serum LH levels in adult female WT and Kiss1r KO mice exposed to a positive feedback regimen of constant elevated E₂. Two days after E₂ implantation, blood was collected from mice at either a morning (approx. 1000 h) or an evening (lights off; 1800 h) time point. *Significantly different from all other groups (P < 0.01). All data are expressed as the mean ± SEM for each group.

FIG. 2

Mean serum LH levels in female WT and Kiss1r KO mice exposed to a positive feedback regimen of low E₂ followed by an injection of EB to induce rising E₂. The day after EB injection, blood was collected from mice at either a morning (1000 h) or an evening (lights off; 1800 h) time point. *Significantly different from all other groups (P < 0.01). All data are expressed as the mean ± SEM for each group.

FIG. 3

Mean serum LH levels in female WT and Kiss1r KO mice exposed to a positive feedback paradigm of low E₂ followed by morning injections of EB and then, 1 day later, P. The day of the P injection, blood was collected from mice at either a morning (approx. 1100 h) or an evening (lights off; 1800 h) time point. *Significantly different from all other groups (P < 0.01). All data are expressed as the mean ± SEM for each group.

FIG. 4

Mean serum LH levels in female WT and Kiss1r KO mice exposed to a positive feedback regimen of constant elevated E₂ after several days of GnRH priming. A) LH levels in mice 15 min after the final GnRH priming injection. GnRH stimulated robust LH secretion in both genotypes, indicating a responsive pituitary. *Significantly different from non-GnRH treated group (P < 0.01). B) Two days after the last GnRH priming treatment, mice were OVX and given E₂, and blood was collected 2 days later at a morning (1000 h) or an evening (lights off; 1800 h) time point. **Significantly different from all other groups. All data are expressed as the mean ± SEM for each group.

FIG. 5

Representative photomicrographs of cfos mRNA coexpression in GnRH neurons in WT and Kiss1r KO female mice exposed to an LH surge paradigm of constant elevated E₂ and sacrificed in the evening. Yellow arrows denote examples of GnRH cells (red fluorescence) with significant cfos (silver grains) coexpression. Blue arrows designate example GnRH neurons that did not have cfos induction.

FIG. 6

Mean percent of GnRH neurons coexpressing cfos in female mice exposed to an LH surge paradigm of constant elevated E₂. Two days after E₂ implantation, brains were collected from mice at either a morning (1000 h) or an evening (1800 h) time point. Brains were assayed for cfos induction in GnRH cells using double-label ISH. Mean levels of cfos-GnRH coexpression in mice from different time-points were quantified in three brain regions within the GnRH neural system as well as the entire GnRH population. *Significantly different from all other groups (P < 0.01). All data are expressed as the mean ± SEM for each group.