Compound deficiencies in multiple fibroblast growth factor signalling components differentially impact the murine gonadotrophin-releasing hormone system

Abstract

Gonadotrophin-releasing hormone (GnRH) neurones control the onset and maintenance of fertility. Aberrant development of the GnRH system underlies infertility in Kallmann syndrome [KS; idiopathic hypogonadotropic hypogonadism (IHH) and anosmia]. Some KS patients harbour mutations in the fibroblast growth factor receptor 1 (Fgfr1) and Fgf8 genes. The biological significance of these two genes in GnRH neuronal development was corroborated by the observation that GnRH neurones were severely reduced in newborn transgenic mice deficient in either gene. In the present study, we hypothesised that the compound deficiency of Fgf8 and its cognate receptors, Fgfr1 and Fgfr3, may lead to more deleterious effects on the GnRH system, thereby resulting in a more severe reproductive phenotype in patients harbouring these mutations. This hypothesis was tested by counting the number of GnRH neurones in adult transgenic mice with digenic heterozygous mutations in Fgfr1/Fgf8, Fgfr3/Fgf8 or Fgfr1/Fgfr3. Monogenic heterozygous mutations in Fgfr1, Fgf8 or Fgfr3 caused a 30-50% decrease in the total number of GnRH neurones. Interestingly, mice with digenic mutations in Fgfr1/Fgf8 showed a greater decrease in GnRH neurones compared to mice with a heterozygous defect in the Fgfr1 or Fgf8 alone. This compounding effect was not detected in mice with digenic heterozygous mutations in Fgfr3/Fgf8 or Fgfr1/Fgfr3. These results support the hypothesis that IHH/KS patients with digenic mutations in Fgfr1/Fgf8 may have a further reduction in the GnRH neuronal population compared to patients harbouring monogenic haploid mutations in Fgfr1 or Fgf8. Because only Fgfr1/Fgf8 compound deficiency leads to greater GnRH system defect, this also suggests that these fibroblast growth factor signalling components interact in a highly specific fashion to support GnRH neuronal development.

Fig. 1

The number of gonadotrophin-releasing hormone (GnRH) neurones in the preoptic/hypothalamus region of mice with monogenic heterozygous defects in fibroblast growth factor signalling components. (a) Fgfr1 ^+/− mice (n = 7) have significantly fewer GnRH neurones than wild-type (WT) mice (n = 8; P = 0.046). (B) Fgf8 ^+/− mice (n = 4) have significantly fewer GnRH neurones than wild-type mice (n = 4; P < 0.02). (c) Fgfr3 ^+/− mice (n = 5) have significantly fewer GnRH neurones than wild-type mice (n = 6; P = 0.01). Asterisks represent significant differences. Each bar represents the mean ± SEM.

Fig. 2

Gonadotrophin-releasing hormone (GnRH) system in Fgfr1 ^+/−, Fgf8 ^+/− or Fgfr1 ^+/− / Fgf8 ^+/− mice. Representative photomicrographs showing GnRH-immunoreactive (IR) neurones at the level of the organum vasculosum lamina terminalis (OVLT) of (a) wild-type (WT) (n = 7), (b) Fgfr1 ^+/− (n = 5), (c) Fgf8 ^+/− (n = 7) and (d) Fgfr1 ^+/− / Fgf8 ^+/− (n = 5) mice. Note that the number of GnRH-IR neurones is reduced in Fgfr1 ^+/−, Fgf8 ^+/− and Fgfr1 ^+/− / Fgf8 ^+/− mice compared to wild-type mice. Moreover, the reduction in the number of GnRH-IR neurones in Fgfr1 ^+/− / Fgf8 ^+/ mice is visually more prominent compared to Fgfr1 ^+/− or Fgf8 ^+/− mice. Scale bar = 100 μm. (e, f) Total number and distribution of GnRH neurones in mice deficient in Fgfr1 and/or Fgf8.(e) anova followed by post-hoc test showed the total number of GnRH-IR neurones in the preoptic/hypothalamus was significantly lower in Fgfr1 ^+/− (n = 5), Fgf8 ^+/− (n = 7) or Fgfr1 ^+/− / Fgf8 ^+/− (n = 5) mice than in wild-type (n = 7) mice (P < 0.0001 by anova). Moreover, the loss of GnRH neurones was greater in Fgfr1 ^+/− / Fgf8 ^+/− mice than in Fgfr1 ^+/− and Fgf8 ^+/− mice. (f) The rostral–caudal distribution of GnRH neurones in wild-type, Fgfr1 ^+/−, Fgf8 ^+/− and Fgfr1 ^+/− / Fgf8 ^+/− mice showed that mice of all genotypes retained the basic pattern of having more GnRH neurones near the OVLT. Each data point represents the mean ± SEM.

Fig. 3

The number of gonadotrophin-releasing hormone (GnRH) neurones in the preoptic/hypothalamus region of mice with digenic or monogenic heterozygous deficiencies in various fibroblast growth factor signalling components. (a) anova followed by post-hoc test showed GnRH neuronal number was significantly lower in Fgfr3 ^+/− (n = 6), Fgf8 ^+/− (n = 5) or Fgfr3 ^+/− / Fgf8 ^+/− (n = 7) mice compared to wild-type mice (WT) (n = 7; P < 0.0001 by anova). (b) GnRH neuronal number was significantly lower in Fgfr1 ^+/− (n = 6), Fgfr3 ^+/− (n = 5) or Fgfr1 ^+/− / Fgfr3 ^+/− (n = 5) mice compared to wild-type mice (n = 5; P < 0.0001 by anova). Different letters represent significant differences. Each bar represents the mean ± SEM.

Fig. 4

Hypothetical model illustrating the temporal sequence in which fibroblast growth factor (FGF) signalling components may affect gonadotrophin-releasing hormone (GnRH) neuronal development. In this model, we hypothesise that Fgf8-dependent activation of fibroblast growth factor receptor (FGFR) 1 is required for GnRH neuronal fate specification [i.e. approximately embryonic day (E) 10.5]. After fate specification, the same population of GnRH neurones becomes dependent on Fgfr3 for maintenance (11). Thus, deficiencies in Fgfr1, Fgf8 or Fgfr3 alone could compromise the GnRH system by affecting genesis (Fgfr1 and Fgf8) or maintenance (Fgfr3). Fgfr3 mutation in addition to Fgfr1 or Fgf8 did not further worsen the GnRH system defect because the target population was already absent as a result of earlier fate specification failure. Conversely, because Fgfr1 and Fgf8 both act on the fate specification stage, their effects can be additive. At present, it is unclear which FGF ligand(s) is required for the activation of Fgfr3 at the post-fate specification stage. Cumulatively, the data obtained in the present study suggest that the proper development of the mouse GnRH system requires the intricate orchestration of multiple FGF/FGFR signals in a time-specific manner.