Leptin receptor null mice with reexpression of LepR in GnRHR expressing cells display elevated FSH levels but remain in a prepubertal state

Abstract

Leptin signals energy sufficiency to the reproductive hypothalamic-pituitary-gonadal (HPG) axis. Studies using genetic models have demonstrated that hypothalamic neurons are major players mediating these effects. Leptin receptor (LepR) is also expressed in the pituitary gland and in the gonads, but the physiological effects of leptin in these sites are still unclear. Female mice with selective deletion of LepR in a subset of gonadotropes show normal pubertal development but impaired fertility. Conditional deletion approaches, however, often result in redundancy or developmental adaptations, which may compromise the assessment of leptin's action in gonadotropes for pubertal maturation. To circumvent these issues, we adopted a complementary genetic approach and assessed if selective reexpression of LepR only in gonadotropes is sufficient to enable puberty and improve fertility of LepR null female mice. We initially assessed the colocalization of gonadotropin-releasing hormone receptor (GnRHR) and LepR in the HPG axis using GnRHR-IRES-Cre (GRIC) and LepR-Cre reporter (tdTomato or enhanced green fluorescent protein) mice. We found that GRIC and leptin-induced phosphorylation of STAT3 are expressed in distinct hypothalamic neurons. Whereas LepR-Cre was observed in theca cells, GRIC expression was rarely found in the ovarian parenchyma. In contrast, a subpopulation of gonadotropes expressed the LepR-Cre reporter gene (tdTomato). We then crossed the GRIC mice with the LepR null reactivable (LepR(loxTB)) mice. These mice showed an increase in FSH levels, but they remained in a prepubertal state. Together with previous findings, our data indicate that leptin-selective action in gonadotropes serves a role in adult reproductive physiology but is not sufficient to allow pubertal maturation in mice.

Keywords: HPG axis; gonadotropes; obesity; puberty; sexual maturation.

Fig. 1.

LepR and GnRHR (GRIC-reporter gene) are expressed in different hypothalamic cell populations. Bright-field and fluorescent images show the distribution of leptin-induced STAT3 phosphorylation (pSTAT3-ir) and GnRHR (GRIC-tdTomato) in the arcuate nucleus (Arc; A–C), in the ventromedial nucleus of the hypothalamus (VMH; D–E), and in the dorsomedial nucleus of the hypothalamus (DMH; G–I). 3V, Third ventricle. Scale bar: 200 μm.

Fig. 2.

Leptin receptor (LepR) is expressed in a subpopulation of cells expressing GnRHR in the pituitary gland. A–C: fluorescent images showing that subsets of LepR-expressing cells (LepR-Cre tdTomato; A) display LH-β immunoreactivity (LH-β-ir; B) in males (arrows indicate dual-labeled cells). C: merged. D: bar graphs showing the percentage of colocalization between LepR-tdTom and LH-β-ir in the pituitary gland of male and female mice (n = 3 males and n = 5 females). E and F: fluorescent images showing the distribution of LepR-reporter gene (tdTom; E) (arrows mark these cells) and GRIC-reporter gene (eGFP; F) (arrow indicates a cell in the parenchyma) in the mouse ovary. CL, corpus luteum; Gr, granulosa layer. Scale bar: 100 μm in A–C and 200 μm in E and F.

Fig. 3.

Reexpression of LepR selectively in GnRHR cells causes no change in body weight of LepR^loxTB mice. A: agarose gel demonstrating Cre-induced DNA recombination (higher band) of LepR^loxTB in the pituitary (but not in the hypothalamus and tail) of GRIC LepR^loxTB mice (n = 2). B: graph showing the progression of body weight of wild-type, LepR^loxTB, and GRIC LepR^loxTB female mice (n = 3–8 for each time point). *P < 0.05 and ****P < 0.0001 per one-way ANOVA, Tukey's post hoc multiple-comparison test.

Fig. 4.

Reexpression of LepR selectively in GnRHR cells causes no improvement of the reproductive phenotype of LepR^loxTB mice. A–C: bright-field images showing hematoxylin and eosin stained sections of the ovary of controls in diestrus (A), LepR^loxTB (B), and GRIC LepR^loxTB (C) mice. Note the presence of corpus luteum (CL) only in the ovary of the control mouse. D: digital image showing the uterine morphology of controls in diestrus, LepR^loxTB, and GRIC LepR^loxTB mice. E: bar graphs comparing uterus weight of controls in diestrus (n = 11), LepR^loxTB (n = 11), and GRIC LepR^loxTB (n = 5) adult mice. No difference between uterus weight of LepR^loxTB and GRIC LepR^loxTB mice was observed. ****P < 0.001 compared with wild-type control mice per one-way ANOVA, Tukey's post hoc multiple comparison test. Scale bar: 1 mm.

Fig. 5.

Increased FSH levels following endogenous reexpression of LepR in GnRHR cells. A and B: expression of GnRH (A) and GnRHR (B) in the hypothalamus of diestrus control (n = 7), LepR^loxTB (n = 6), and GRIC LepR^loxTB (n = 4) mice. C–F: expression of GnRHR (C), LH-β (D), FSH-β (E), and CGA mRNAs (F) in the pituitary gland of diestrus wild-type (n = 7), LepR^loxTB (n = 6), and GRIC LepR^loxTB (n = 4) mice. G: expression of LH-β, FSH-β, and CGA mRNAs in the pituitary gland of LepR^loxTB (n = 7) and db/db (n = 5) mice. All data were normalized to the housekeeping gene RPS29. H: serum levels of FSH in diestrus wild-type littermates (n = 14), LepR^loxTB (n = 12), and GRIC LepR^loxTB (n = 6) mice. Increase in FSH levels was observed in GRIC LepR^loxTB female mice compared with littermate controls in diestrus and LepR^loxTB female mice. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with wild-type control mice per one-way ANOVA, Tukey's post hoc multiple-comparison test.