The synthesis and secretion of progesterone in the corpus luteum are regulated by both endocrine and paracrine/ autocrine factors which affect the steroidogenic cells. Evidence suggests that these cells communicate via cell-cell junctional proteins, the connexins. Previously we have shown that connexin-43 is expressed in both human and baboon (Papio hamadryus anubis) corpora lutea, with differential expression throughout luteal development, but is not detectable in corpora albicantia. We have examined the effect of human chorionic gonadotropin (hCG), oxytocin, clomiphene citrate and the anti-progesterone onapristone on expression of connexin-43 protein in the early luteal phase 1-5 days after the mid-cycle luteinizing hormone (LH) surge (LH+ 1-5 days), the mid-luteal phase 6-10 days after the LH surge (LH+ 6-10 days), and the late luteal phase 11-15 days after the LH surge (LH+ 11-15 days) in corpora lutea obtained from normal adult cycling females. Connexin-43 was localized by immunohistochemistry in cultured cells from all the three stages. Western blot analysis of the treated cells indicated the presence of two bands at 43 and 45 kDa. The band at 45 kDa was found to be phosphorylated connexin-43, indicating the presence of functional gap junctions. hCG (10 IU/ml) stimulated the expression of connexin-43 throughout luteal development; however, maximum expression occurred in the early luteal phase with a significantly greater expression of the non-phosphorylated protein. In contrast, in the mid-luteal phase, the expression of the phosphorylated protein was predominant. Oxytocin (200 mU/ml) also stimulated connexin-43 expression throughout luteal development with similar effects on the phosphorylated and non-phosphorylated protein in the early and mid-luteal phase; however, compared with hCG, oxytocin had a greater effect on mid-luteal phase connexin-43 expression. In the presence of both hCG and oxytocin, the expression of connexin-43 was significantly higher than the control only in the late luteal phase. Both clomiphene citrate and onapristone suppressed connexin-43 expression, and concomitant addition of hCG did not counteract their effect. In the context of our previous studies, it is concluded that, together with LH/hCG and the steroid hormones, oxytocin is involved in cell-cell contact-dependent communication in the corpus luteum.

The cloning of the mineralocorticoid receptor (MR) 30 years ago was the start of a new era of research into the regulatory processes of MR signalling at target genes in the distal nephron, and subsequently in many other tissues. Nuclear receptor (NR) signalling is modified by interactions with coregulatory proteins that serve to enhance or inhibit the gene transcriptional responses. Over 400 coregulatory proteins have been described for the NR super family, many with functional roles in signalling, cellular function, physiology and pathophysiology. Relatively few coregulators have however been described for the MR although recent studies have demonstrated both ligand and/or tissue selectivity for MR-coregulator interactions. A full understanding of the cell, ligand and promoter-specific requirements for MR-coregulator signalling is an essential first step towards the design of small molecular inhibitors of these protein-protein interactions. Tissue-selective steroidal or non-steroidal modulators of the MR are also a desired therapeutic goal. Selectivity, as for other steroid hormone receptors, will probably depend on differential expression and recruitment of coregulatory proteins.

The mineralocorticoid receptor (MR) differs from the other steroid receptors in that it responds to two physiological ligands, aldosterone and cortisol. In epithelial tissues, aldosterone selectivity is determined by the activity of 11β-hydroxysteroid dehydrogenase type 2, while in other tissues, including the heart and regions of the central nervous system, cortisol is the primary ligand for the MR where it may act as an antagonist. Clinical trials have demonstrated the potential of MR antagonists in the treatment of cardiovascular disease, though their use has been limited by concurrent hyperkalaemia. In order to better target the MR, an understanding of the structural determinants of tissue- and ligand-specific MR activation is needed. Interactions of the MR have been identified, which exhibit ligand discrimination and/or specificity. These interactions include those of the ligand-binding domain with ligand, with the N-terminal domain and with putative co-regulatory molecules. Agonist and antagonist binding have been characterised using chimeras between the human MR and the glucocorticoid receptor or the zebra fish MR together with molecular modelling. The interaction between the N-terminus and the C-terminus is aldosterone dependent but is unexpectedly antagonised by cortisol and deoxycorticosterone in the human MR. Nuclear receptor-mediated transactivation is critically dependent on, and modulated by, co-regulatory molecules. Proteins that interact with the MR in the presence of either aldosterone or cortisol, but not both, have been identified. The successful identification of ligand-specific interactions of the MR may provide the basis for the development of novel MR ligands with tissue specificity.

Primary aldosteronism, or Conn syndrome, is the most common endocrine cause of hypertension. It is associated with a higher risk of cardiovascular, metabolic and renal diseases, as well as a lower quality of life than for hypertension due to other causes. The multi-systemic effects of primary aldosteronism can be attributed to aldosterone-mediated activation of the mineralocorticoid receptor in a range of tissues. In this review, we explore the signalling pathways of the mineralocorticoid receptor, with a shift from the traditional focus on the regulation of renal sodium–potassium exchange to a broader understanding of its role in the modulation of tissue inflammation, fibrosis and remodelling. The appreciation of primary aldosteronism as a multi-system disease with tissue-specific pathophysiology may lead to more vigilant testing and earlier institution of targeted interventions.

It has been documented that stress or glucocorticoids have conflicting effects on memory under different conditions. However, it is not fully understood why stress can either impair or enhance memory. Here, we have examined the performance of six age groups of Wistar rats in a water maze spatial task to evaluate the effects of stress under different conditions. We found that the impairment or enhancement effect of an 'elevated platform' (EP) stress on memory was dependent on previous stress experience and on age. EP stress impaired memory retrieval in water maze naive animals, but enhanced rather than impaired memory retrieval in young water maze stress-experienced animals. Furthermore, exogenously applied corticosterone or foot shock stress before water maze training prevented the impairment of memory retrieval that should be induced by treatment with corticosterone or foot shock before the 'probe trial'. Again, memory retrieval was enhanced in young animals under these conditions, and this enhancement can be prevented by the glucocorticoid receptor antagonist RU 38486. Thus, glucocorticoid receptor activation not only induced impairment of memory but also increased the capacity of young animals to overcome a later stress. The present findings suggest that the effect of stress on memory can be switched from impairment to enhancement dependent on both stress experience and age.

Abstract

Developmental changes in pituitary glucocorticoid receptor (GR) mRNA were examined during gestation and early neonatal life using in situ hybridization. Pituitaries were harvested from sheep fetuses at days 60–80, 100–120, 130–135, 140–142 and term, and from lambs of days 0–7 and 30–60, and adults. GR mRNA was present in the pars distalis by day 60, levels increased through gestation, and there was a redistribution of GR mRNA, resulting in a relatively greater abundance at the base of the pars distalis. At term, there was a significant (P<0·05 compared with the day 140–142 fetuses) elevation of GR mRNA, which was maintained in the newborn lamb, reaching highest levels at days 30–60 of neonatal life. GR mRNA was undetectable in the pars intermedia until day 120, but subsequently increased to high levels at term. Interestingly, the expression of GR mRNA in the pars intermedia dropped precipitously in the newborn (P<0·05 compared with term), though levels recovered in the older lambs and adults. The regional and cellular distribution of GR mRNA correlated closely with the presence of immuno-reactive GR (irGR) in the pituitary; the majority of irGR was present in the nuclei. Intrafetal infusion of cortisol (12 h; 5 μg/min) in late gestation (day 135) had no effect on GR mRNA expression in either the pars distalis or pars intermedia. These results indicated that, in the fetal pituitary, (1) the GR gene is expressed in both the pars distalis and pars intermedia, (2) levels of GR mRNA in the fetal pituitary correlated with the distribution of nuclear irGR, (3) GR mRNA is present at higher levels in the inferior aspect of the pars distalis, its abundance increases immediately prior to parturition and is maintained in the newborn, and (4) cortisol infusion for 12 h does not affect GR mRNA in either region of the pituitary, suggesting that, in the short term, glucocorticoids do not directly regulate GR synthesis.

Journal of Endocrinology (1995) 144, 483–490

ABSTRACT

The responses of the fetal sheep pituitary to corticotrophin-releasing hormone (CRH) change during gestation with maximum output of ACTH around days 120–130, and decreased ACTH output near term. However, there is no information available concerning the extent to which these responses may be modulated by alterations in the number of CRH receptors. Therefore we measured specific CRH-binding sites, and changes in binding characteristics in membrane preparations from fetal sheep anterior pituitaries collected at days 65–70, 85–88, 100–110, 125–130 and at term (approximately 145 days). Binding assays were carried out using ¹²⁵I-labelled Tyr-ovine CRH (¹²⁵I-Tyr-oCRH), incubated with crude membrane fractions for 90 min at 22 °C. Binding was time- and temperature-dependent, linear with protein concentration, saturable and specific for oCRH. Scatchard analysis of binding data for individual tissues revealed a single class of CRH-binding sites with high affinity (K _d ≃1 nmol/l) that did not change significantly with gestational age. However, the number of CRH-binding sites increased progressively from days 65–70 to a maximum at days 125–130, then decreased at term. These results demonstrate the presence of specific CRH-binding sites in the fetal sheep anterior pituitary. Furthermore, the change in CRH receptor number with advancing pregnancy follows a similar time-course to the changes reported previously in responsiveness of the fetal sheep anterior pituitary to exogenous CRH stimulation in vivo. These results suggest that alterations in CRH receptor number may contribute to changes in responsiveness of the fetal sheep anterior pituitary to CRH during gestation.

Journal of Endocrinology (1991) 130, 223–229

Abstract

Oxytocin has been identified in both non-human primate and human corpora lutea of the menstrual cycle by RIA, immunocytochemistry and HPLC. Evidence for the transcription of the oxytocin gene in this tissue using PCR is available. Oxytocin receptors have been characterized by biochemical procedures. However, there is some debate as to whether the oxytocin identified in these tissues is biologically active and has a role in luteal function. In this study we have demonstrated that oxytocin isolated by gel chromatography of tissue extracts from the baboon and the human corpus luteum is biologically active as determined in a rat uterine bioassay. Since both oxytocin and its receptors are present in these tissues, it is suggested that oxytocin in the human and non-human primate corpora lutea has a functional role.

Journal of Endocrinology (1995) 147, 525–532

Abstract

The level of 11 β-hydroxysteroid dehydrogenase (11β-HSD) mRNA in the fetal sheep liver increases dramatically between day 130 and term (term=day 145), but the causal factors remain unknown. The present study was designed to determine the effects of exogenous glucocorticoid on the fetal hepatic 11 β-HSD gene expression. Dexamethasone (dex; 2 μg/min over 15 min every 2 h) or saline was infused into chronically-catheterized fetal sheep at day 130 of gestation for 4 days. At the end of infusion, the lower right lobe of the liver was collected, total cellular RNA extracted and subjected to Northern blot analysis. It was found that the level of the hepatic 11 β-HSD mRNA in dex-treated fetuses was about four times higher than that in the saline-treated controls. To examine whether changes occur in the response of hepatic 11 β-HSD gene expression to glucocorticoids in adulthood, we also treated non-pregnant ewes with dex (10 mg/day) for 4 days. By contrast, this treatment regime in adult sheep produced a small but significant decrease in hepatic 11 β-HSD mRNA levels.

We also determined whether age-specific changes in the hepatic level of 11 β-HSD mRNA following dex treatment were reflected in the level of 11 β-HSD enzyme activity. Hepatic 11 β-HSD activity was determined by a standard in vitro conversion assay using cortisol and cortisone as physiological substrates. In both fetal and adult livers, 11-oxoreductase activity (cortisone→cortisol) was predominant. Following dex treatment, there was a significant increase in the fetal hepatic level of both 11β-dehydrogenase (cortisol→cortisone) and 11-oxoreductase activities. Furthermore, the C-11 activation index, an indicator of glucocorticoid net gain, was also increased in the fetal liver by dex. In marked contrast, dex treatment in the adult did not alter the C-11 activation index though it produced a significant decrease in the hepatic level of both 11 β-dehydrogenase and reductase activities. In summary, these results indicate that (1) exogenous glucocorticoid exerts opposite effects on hepatic 11 β-HSD gene expression in fetal and adult sheep; (2) dex-induced age-specific changes in the level of 11β-HSD mRNA are carried though to the level of 11β-HSD protein; and (3) since 11 β-HSD reductase activity is predominant in both fetal and adult sheep livers, the liver may be a potential extra-adrenal source of cortisol. Furthermore, we speculate that (1) the dramatic increase in the fetal hepatic 11 β-HSD mRNA level at term may be due to the elevated fetal plasma concentration of glucocorticoid; and (2) glucocorticoid-induced increases in the fetal hepatic 11β-HSD gene expression and the resultant increase in the C-11 activation index during the last days of fetal life may play a crucial role in fetal organ maturation and in the endocrine mechanisms leading to parturition.

Journal of Endocrinology (1994) 143, 121–126

Abstract

Parturition and fetal organ maturation in sheep are associated with increased activity of the fetal hypothalamic-pituitary-adrenal (HPA) axis during late pregnancy. However, the factors responsible for HPA activation remain unclear. In the fetal pituitary, levels of pro-opiomelanocortin (POMC) mRNA increase, but the numbers of binding sites for corticotrophin-releasing hormone (CRH), and ACTH responsiveness to exogenous CRH decline during the last 20 days of pregnancy. We have examined regulation of CRH binding, pituitary ACTH responsiveness, and levels of POMC mRNA in cultures of adenohypophysial cells from term fetal sheep. After a 4-day stabilization period, output of immunoreactive (ir) ACTH was increased over 48 h in a dose-dependent fashion by both CRH and arginine vasopressin (AVP) but decreased by cortisol. Subsequent output of ir-ACTH to a 3-h challenge with 100 nm CRH was attenuated after pretreatments with CRH, AVP or cortisol; the effect of CRH being greater than that of cortisol or AVP. At the end of 48 h of treatment with CRH, AVP or cortisol, there was a 40–50% reduction in the number of CRH-binding sites, but the levels of POMC mRNA decreased significantly only after cortisol treatment and were not altered significantly by CRH or AVP.

We conclude that under the conditions of these experiments, CRH and AVP increase ir-ACTH output without increasing the level of steady-state POMC mRNA, but may contribute to loss of pituitary responsiveness to CRH by down-regulation of CRH receptor number. Cortisol exerts negative feedback on POMC mRNA and decreases the number of CRH receptors. Thus, any one or all of CRH, AVP and cortisol could be responsible for the decline in CRH binding in the fetal sheep pituitary during late pregnancy. Although CRH and AVP may affect secretion of ir-ACTH, the present results do not support a role for these neuropeptides in affecting the level of POMC mRNA in the fetal sheep pituitary.

Journal of Endocrinology (1994) 143, 199–208