Melanocortins mimic the effects of leptin to restore reproductive function in lean hypogonadotropic ewes

Abstract

Background/aims: Leptin restores gonadotropic function in lean hypogonadotropic animals by an unknown mechanism. We aimed to test the hypothesis that restoration of gonadotropic function is a result of an upregulation of central acetylated melanocortin production.

Methods and results: Lean ovariectomised (OVX) ewes received intracerebroventricular (i.c.v.) infusions of leptin (or vehicle) for 3 days, which upregulated proopiomelanocortin (POMC) mRNA and restored pulsatile luteinizing hormone (LH) secretion. A melanocortin agonist (MTII), but not naloxone treatment, reinstated pulsatile LH secretion in lean OVX ewes. We treated (i.c.v.) lean OVX ewes with leptin (or vehicle) and measured peptide levels and post-translational modification in the arcuate nucleus (ARC). Levels of beta-endorphin (beta-END) were lower in lean animals, with no effect of leptin treatment. Desacetyl-alpha-MSH was the predominant form of alpha-melanocyte-stimulating hormone (alpha-MSH) in the ARC and levels were similar in all groups. In another group of lean and normal-weight OVX ewes, we measured the different forms of alpha-MSH in ARC, hypothalamus (ARC-removed) and the preoptic area (POA). Acetylated alpha-MSH levels were lower in lean animals in the terminal beds of the hypothalamus and POA but not the ARC.

Conclusions: Leptin corrects the hypogonadotropic state in the lean condition by upregulation of POMC gene expression, and may increase transport and acetylation of melanocortins to target cells in the brain. Melanocortin treatment restores LH secretion in lean animals.

Fig. 1

The effect of third ventricular infusion of aCSF (a) or leptin (10 μg/h; b) for 72 h on plasma LH levels in lean hypogonadotropic OVX ewes. 10-min blood samples were collected for 6 h prior to the infusion and for the final 6 h of the infusion period. c Analysis of mean LH concentration (ng/ml), LH pulse amplitude (ng/ml), LH interpulse interval (min) and LH pre-pulse nadir (ng/ml). Arrowheads indicate an LH pulse. * p < 0.05 and ** p < 0.01 compared to pre-infusion values.

Fig. 2

Effect of third ventricular infusion of aCSF or leptin (50 μg/h) for 72 h on NPY, POMC and AgRP gene expression in the ARC of the hypothalamus of lean hypogonadotropic OVX ewes, as determined by in situhybridization. a–f POMC gene expression in lean control (aCSF) and lean leptin-treated hypogonadotropic ewes. g–l NPY gene expression in lean control (aCSF) and lean leptin-treated hypogonadotropic ewes. m–r AgRP gene expression in lean control (aCSF) and lean leptin-treated hypogonadotropic ewes. Darkfield and brightfield photomicrographs are taken at 10× and 40× magnification, respectively. Data are means ± SEM. * p < 0.05 compared to control.

Fig. 3

The effect of LV infusion of aCSF (a) or melanocortin agonist MTII (10 μg/h; b) for 3 h on plasma LH levels in lean hypogonadotropic OVX ewes. c Analysis of mean LH concentration, LH pulse amplitude, LH interpulse interval (min) and LH pre-pulse nadir. Arrowheads indicate an LH pulse. * p < 0.05 and ** p < 0.01 compared to pre-treatment values.

Fig. 4

The effect of LV infusion of aCSF or naloxone for 3 h with (a, b) or without (c, d) sex steroids, estrogen (E) and progesterone (P), on plasma LH levels in lean hypogonadotropic OVX ewes. e Analysis of mean LH concentration, LH pulse amplitude, LH interpulse interval (min), and LH pre-pulse nadir. Arrowheads indicate an LH pulse.

Fig. 5

Mean peptide levels of NPY (a), des-α-MSH (b), and β-END (c) in the ARC of normal, lean, and lean leptin-treated (50 μg/h) OVX ewes. All data are presented as means ± SEM. d HPLC/EIA profile of MSH standards, des-α-MSH, act-α-MSH and di-act-α-MSH as well as representative HPLC/EIA profiles in the ARC and cerebellum. e Analysis of mean LH concentration (ng/ml), LH pulse amplitude (ng/ml), LH interpulse interval (min) and LH pre-pulse nadir (ng/ml) presented as means ± SEM. * p < 0.05 and ** p < 0.01 compared to ewes of normal body weight; ‸ p < 0.05 and ‸‸ p < 0.01 compared to pre-treatment −4 to 0 h LH values; ^† p < 0.05 compared to lean −4 to 0 h LH interpulse interval.

Fig. 6

Mean (±SEM) peptide levels of des-α-MSH and act-α-MSH in the ARC (a), hypothalamus (HYP; ARC-removed; b) and POA (c) of normal weight (solid bar) and lean hypogonadotropic (open bar) ewes and HPLC/EIA representative profiles for each nuclei (the continuous line represents normal weight ewes and the dotted line represents lean hypogonadotropic ewes). * p < 0.05 and ** p < 0.01 compared to normal-weight controls.