Research reportProgesterone receptor expression in the pregnant and parturient rat hypothalamus and brainstem
Introduction
Once pregnancy is established, it is vital that fertility is suppressed and premature activation of the Ferguson reflex is prevented. High levels of progesterone secreted by the corpora lutea maintain pregnancy in the rat by acting at several sites within the brain and periphery. Within the hypothalamus, gonadotrophin-releasing hormone (GnRH) secretion is restrained during pregnancy by the action of progesterone probably acting through an inhibitory GABA-mediated mechanism. Any stimulatory action of oxytocin on uterine contractile activity is blocked by progesterone binding to oxytocin receptors in the myometrium [15]. At parturition magnocellular neurons in the hypothalamic supraoptic and paraventricular nuclei (SON and PVN) are activated and oxytocin is secreted from the neurohypophysis to stimulate uterine contractions [37]. This activation is mediated by neural afferents from the uterus or birth canal that relay in the nucleus tractus solitarius (NTS) in the brainstem [4], from where neurons project directly to SON oxytocin neurons [24]. Parturition is preceded ∼24 h before term by a decrease in circulating progesterone. If this normal decline is prevented by progesterone administration, gestation is prolonged and activation of NTS and SON neurons is inhibited [4]. Fos expression in the NTS and SON in progesterone-treated rats during delayed spontaneous or oxytocin-induced parturition indicates that inhibitory actions of progesterone are progressively withdrawn, first at the uterus, then the NTS and finally the SON [4]. NTS neurons are known to express progesterone receptor (PR) immunoreactivity (-ir) [17] allowing a mechanism by which progesterone could inhibit these neurons through pregnancy.
Several studies have alluded to a genomic action of progesterone on oxytocin neurons. Oxytocin mRNA expression in the magnocellular neurons increases only at term, when progesterone is withdrawn [2], [11], [44], but it is unclear whether PRs in the oxytocin neurons are involved as no PR mRNA or protein has been detected in the non-pregnant rat SON [35], [40]. Nonetheless, identification of PR mRNA in the PVN (e.g. Ref. [44]) raises the possibility that progesterone may mediate its effects via PR in the magnocellular system. Elsewhere in the hypothalamus, PR gene expression is positively regulated by estrogen [17], [36], [40], [43], and as estrogen secretion peaks prior to parturition, stimulation of neuronal PR expression at this time is predicted. Progesterone can also act in the brain via a non-genomic cell membrane-mediated mechanism. In the SON, there is strong evidence for progesterone inhibition of oxytocin neurons in late pregnancy [20] through allosteric modulation of the GABAA receptor by the metabolite allopregnanolone [7]. At present, it is unknown whether NTS neurons are inhibited by progesterone through a similar interaction with GABA mechanisms.
Evidence for the importance of PR-containing neurons that project to the SON in the control of oxytocin release at the end of pregnancy has been revealed by studies using the PR antagonist RU486. Late pregnant rats pretreated with progesterone and given RU486 have enhanced Fos expression within the SON as compared to controls. This suggests that RU486 may cause disinhibition by acting on PR expressing inputs to the SON, rather than acting directly on the SON [4].
The aim of this study was to evaluate irPR expression in the rat hypothalamus and brainstem during late pregnancy and parturition, with particular emphasis on regions with known projections to the SON such as the NTS, PVN, organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), anteroventral periventricular nucleus of the preoptic region (AvPe), posterior periventricular nucleus (Pe), median preoptic nucleus (MnPO) and ventromedial hypothalamus (VMH) [24], [48]. We investigated PR expression in putative noradrenergic neurons (tyrosine hydroxylase (TH)-immunopositive) that project from the NTS to the SON, since these cells are known to be active during parturition [24]. In addition, irPR was examined in the medial preoptic nucleus (MPA) and the arcuate nucleus (ARC) where many neurons normally express PR [5], [8], [13].
Section snippets
Experimental animals
Female Sprague–Dawley rats (body weight 225–250 g, Bantin and Kingman, Hull, UK) were housed five animals per cage at 22 °C on a 12 h light:12 h dark cycle (lights on at 08:00 h) and given standard rat chow and water ad libitum. Age-matched virgin females were mated overnight with sexually experienced males, and those found to have a vaginal plug of coagulated semen (day 0 of pregnancy) were housed singly. All procedures were performed in accordance with requirements of the United Kingdom Home
Results
Immunoreactivity for PR was visualized as a dense black precipitate seen exclusively in cell nuclei. The distribution of irPR within the hypothalamus and brainstem was similar to that previously reported [17], [40]. Removal of endogenous estrogen by ovariectomy completely abolished all irPR staining. Restoration of irPR staining was achieved by estrogen replacement.
Discussion
There is no question of the importance of progesterone in the regulation of the magnocellular system during pregnancy and parturition. This study has revealed that progesterone is likely to exert any PR-mediated actions on neurons that have direct projections to the SON and PVN, rather than on the magnocellular cells themselves. PR were found to be undetectable in the SON and sparsely observed in the PVN during pregnancy and parturition and confirmed reports of low PR expression in virgin rats
Acknowledgements
Many thanks go to Michelle Giles for preliminary work on this project and Dr. Philip Bull for technical assistance. Supported by the BBSRC.
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Present address: Department of Medical Biochemistry, Tenovus Building, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK.