Changes in prodynorphin gene expression and neuronal morphology in the hypothalamus of postmenopausal women

Abstract

Human menopause is characterised by ovarian failure, gonadotrophin hypersecretion and hypertrophy of neurones expressing neurokinin B (NKB), kisspeptin (KiSS)-1 and oestrogen receptor (ER) alpha gene transcripts within the hypothalamic infundibular (arcuate) nucleus. In the arcuate nucleus of experimental animals, dynorphin, an opioid peptide, is colocalised with NKB, kisspeptin, ER alpha and progesterone receptors. Moreover, ovariectomy decreases the expression of prodynorphin gene transcripts in the arcuate nucleus of the ewe. Therefore, we hypothesised that the hypertrophied neurones in the infundibular nucleus of postmenopausal women would express prodynorphin mRNA and that menopause would be accompanied by changes in prodynorphin gene transcripts. In the present study, in situ hybridisation was performed on hypothalamic sections from premenopausal and postmenopausal women using a radiolabelled cDNA probe targeted to prodynorphin mRNA. Autoradiography and computer-assisted microscopy were used to map and count labelled neurones, measure neurone size and compare prodynorphin gene expression between premenopausal and postmenopausal groups. Neurones expressing dynorphin mRNA in the infundibular nucleus of the postmenopausal women were larger and exhibited hypertrophied morphological features. Moreover, there were fewer neurones labelled with the prodynorphin probe in the infundibular nucleus of the postmenopausal group compared to the premenopausal group. The number of dynorphin mRNA-expressing neurones was also reduced in the medial preoptic/anterior hypothalamic area of postmenopausal women without changes in cell size. No differences in cell number or size of dynorphin mRNA-expressing neurones were observed in any other hypothalamic region. Previous studies using animal models provide strong evidence that the changes in prodynorphin neuronal size and gene expression in postmenopausal women are secondary to the ovarian failure of menopause. Given the inhibitory effect of dynorphin on the reproductive axis, decreased dynorphin gene expression could play a role in the elevation in luteinising hormone secretion that occurs in postmenopausal women.

Figure 1

Computer-microscope maps showing the distribution of neurones expressing prodynorphin mRNA in representative sagittal sections of a premenopausal (A) and postmenopausal woman (B). Each symbol represents one labelled neurone. There were decreased numbers of neurones expressing prodynorphin mRNA in the infundibular nucleus and the medial preoptic/anterior hypothalamic area of postmenopausal women. Abbreviations: ac, anterior commissure; AHA, anterior hypothalamic area, fx, fornix; INF, infundibular nucleus; MB, mammillary body; oc, optic chiasm; POA, preoptic area; PH, posterior hypothalamus. Scale bar = 4 mm.

Figure 2

Alterations in prodynorphin gene expression and neuronal morphology in the infundibular nucleus (A, B, C) or the preoptic/anterior hypothalamic area (D, E, F) of premenopausal (Pre) and postmenopausal (Post) women. The graphs show the number of prodynorphin mRNA-expressing neurones/section (top), profile area of prodynorphin mRNA-expressing neurones (middle) and the number of autoradiographic grains per labelled neurone (bottom). There were decreased numbers of prodynorphin mRNA-expressing neurones detected in the infundibular nucleus of postmenopausal women accompanied by a marked increase in cell size. A similar decline in cell number was seen in the preoptic/anterior hypothalamic area of postmenopausal women without differences in the size of labelled neurones. Values are expressed as mean ± SEM. * p < 0.05, **p ≤ 0.002.

Figure 3

Photomicrographs of prodynorphin mRNA-expressing neurones in the infundibular nucleus (A, B) and medial preoptic nucleus (C, D) in premenopausal (A, C) and postmenopausal (B, D) women. The grains mark the location of prodynorphin mRNA and the sections are counterstained with toluidine blue. Prodynorphin neurones in the infundibular nucleus (but not the preoptic area) of the postmenopausal group had larger somata and displayed hypertrophied morphological features. Scale bar = 10 μm (applies to A–D).