The presence of activins in those hypothalamic regions containing gonadotropin-releasing hormone (GnRH)-secreting neurons suggests that these peptides may regulate the reproductive function modulating not only pituitary FSH release and biosynthesis, but also hypothalamic GnRH release. The purpose of this study was to evaluate the effects of activin-A, a homodimer of inhibin beta A subunit, on hypothalamic GnRH release in vitro and, because of their well known antithetical effects, to evaluate its interaction with inhibin. In addition, since androgens modulate the release of GnRH from male rat hypothalami, we thought it of interest to study the possible interplay between these steroids and activin on GnRH release. To accomplish this, we employed a hypothalamic organ culture system which enabled us to evaluate GnRH release from individually incubated hemi-hypothalami explanted from male rats. Activin-A stimulated GnRH release in a biphasic manner. The maximal effect was reached at a concentration of 10 ng/ml which increased GnRH output by about 75%. Inhibin abolished the stimulatory effect of a maximally effective concentration of activin-A in a dose-dependent manner, whereas alone it had no effect on GnRH output. As previously shown, testosterone (1 nmol/l) and dihydrotestosterone (DHT, 0.1 nmol/l) suppressed basal GnRH release, but only testosterone was able to inhibit the release of GnRH stimulated by activin-A. Since DHT is a non-aromatizable androgen, we evaluated whether the inhibitory effect of testosterone was due to its in vitro conversion into 17 beta-estradiol. The addition of 4-hydroxyandrostenedione, a steroidal aromatase inhibitor, did not influence the suppressive effect of testosterone on GnRH release stimulated by activin-A. In conclusion, activin-A stimulated hypothalamic GnRH release in vitro and this effect was abolished by inhibin and was blunted by testosterone. These findings suggest that activins may participate in the regulation of the hypothalamic-pituitary-gonadal axis by modulating GnRH release. The ability of testosterone to suppress the release of GnRH stimulated by activin-A indicates that this steroid has a potent negative feedback influence on GnRH release.
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AE Calogero, N Burrello, AM Ossino, P Polosa, and R D'Agata
A. E. Calogero, G. Bagdy, M. L. Moncada, and R. D'Agata
The present study was undertaken to evaluate the serotonin (5-HT) receptor subtype(s) by which 5-HT acts on the pituitary to stimulate ACTH secretion. We tested the effects of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), the 5-HT1C receptor agonist metachloro-phenylpiperazine (m-CPP), which also binds to other 5-HT receptors with lower affinity, and the 5-HT2/1C receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) on basal, corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP)-stimulated ACTH release from primary rat anterior pituitary cell cultures. 5-HT, 8-OH-DPAT and DOI significantly increased basal ACTH release, an effect which was antagonized by 5-HT receptor antagonists. 5-HT and DOI were effective at nanomolar concentrations whereas 8-OH-DPAT was effective at higher concentrations. 5-HT, 8-OH-DPAT (both at 10 nmol/l) and DOI (at higher concentrations) blunted the stimulatory effect of CRH. The suppressive effects of 8-OH-DPAT and DOI on CRH-stimulated ACTH release were antagonized by (−)propranolol, a β-adrenergic receptor antagonist which binds the 5-HT1A receptor with elevated affinity, and ketanserin, a 5-HT2 receptor antagonist respectively. 5-HT, 8-OH-DPAT and DOI showed additive stimulatory effects with AVP but only at the highest concentration tested, whereas m-CPP potentiated AVP-induced ACTH release at concentrations of 1 nmol/l or more. This effect was antagonized by metergoline, a non-selective 5-HT receptor antagonist and mianserin, an antagonist which binds the 5-HT1C receptor with elevated affinity.
Thus (1) 5-HT and selective 5-HT agonists such as 8-OH-DPAT, m-CPP and DOI modulate ACTH release by acting directly at the pituitary level; (2) serotonergic stimulation of basal ACTH release is mediated by both 5-HT2 and 5-HT1A receptors; (3) serotonergic inhibition of CRH-stimulated ACTH release is mediated by 5-HT1A and 5-HT2 receptors; whereas (4) serotonergic potentiation of AVP-stimulated ACTH release is mediated mainly by 5-HT1C receptors.
Journal of Endocrinology (1993) 136, 381–387
A E Calogero, N Burrello, A M Ossino, R F A Weber, and R D'Agata
Brain catecholamines have been implicated in the regulation of gonadotrophin release. It has been recently reported that noradrenaline (NA), applied within the hypothalamic paraventricular nucleus, suppresses the pulsatile release of LH in the rat through a corticotrophin-releasing hormone (CRH)-dependent mechanism. Prolactin (PRL) is also able to suppress hypothalamic GnRH release following activation of the CRH-releasing neurone. Given that PRL stimulates the release of NA from hypothalamic explants and that NA stimulates the release of hypothalamic CRH, we hypothesized that this neurotransmitter may be involved in the intrahypothalamic neuroendocrine circuit mediating the inhibitory effects of PRL on GnRH release. To test this hypothesis, we evaluated the effects of PRL on GnRH release in the presence of α- or β-adrenergic receptor antagonists using a static hypothalamic organ culture system which enabled us to evaluate immunoreactive GnRH (iGnRH) release from individually incubated, longitudinally halved hypothalami. As previously shown, PRL at a concentration of 100 nm inhibited basal iGnRH release by about 35%. Phentolamine, a non-selective α-adrenergic receptor antagonist, prazosin, an α1-receptor antagonist, and yohimbine, an α2-receptor antagonist, overcame the inhibitory effect of PRL on iGnRH release in a concentration-dependent fashion. In contrast, propranolol, a non-selective β-adrenergic receptor antagonist, atenolol, a β1-receptor antagonist, and ICI-118,551, a β2-receptor antagonist, had no effect. None of these compounds had any effect on basal iGnRH release. These findings suggested that an α-adrenergic mechanism is involved in the suppressive effects of PRL on GnRH release. Since the activation of α-adrenergic receptors increases hypothalamic CRH release, we evaluated whether PRL stimulates CRH release via an α-adrenergic mechanism. PRL stimulated basal CRH release by about twofold and this effect was inhibited by phentolamine in a concentration-dependent fashion.
In conclusion, α-, but not β-, adrenergic receptors mediate the inhibitory effects of PRL on GnRH release in vitro. We speculate that, at least under these experimental conditions, PRL inhibits GnRH release through an α-adrenergic mechanism which activates the CRH-secreting neurone.
Journal of Endocrinology (1996) 151, 269–275
A E Calogero, F Raiti, G Nicolosi, N Burrello, R D'Agata, and F Mantero
Endothelins (ETs) are potent vasoconstrictor peptides that also participate in the regulation of endocrine function. Indeed, immunoreactive ET, ET mRNA and ET receptors have been found in the brain and the pituitary gland and ETs stimulate arginine vasopressin, LH, FSH, TSH and gonadotrophin-releasing hormone and inhibit prolactin release in vitro. The present study was undertaken to evaluate the effects of ET-1 and ET-3, two members of this family, on corticotrophin-releasing hormone (CRH) release by explanted male rat hypothalami in vitro and on ACTH release by primary pituitary cell culture. ET-3 decreased basal CRH release in a concentration-related manner. The lowest effective concentration tested was 3 nmol/l but a more pronounced inhibitory effect was obtained at a concentration of 10 nmol/l. On the other hand, ET-1 did not have any detectable effect on basal CRH release. Neither ET-1 nor ET-3 had any effect on the release of CRH stimulated by potassium chloride. ET-1 increased basal ACTH release, whereas ET-3 did not have any effect. Both ET-1 and ET-3 suppressed the release of ACTH stimulated by 1 nmol CRH/l.
These data suggest that both ET-1 and ET-3 are able to modulate the hypothalamic-pituitary-adrenal axis function in vitro. However, they act at different levels and seem to have opposite effects. Indeed, while ET-1 stimulated pituitary ACTH release, ET-3, the peptide produced mainly in the brain, inhibited hypothalamic CRH release in vitro.
Journal of Endocrinology (1994) 140, 419–424
AE Calogero, A Barreca, N Burrello, I Palermo, G Giordano, R D'Agata, and E Vicari
Corticotrophin-releasing hormone (CRH), a neuropeptide which modulates gonadal function during stress, is expressed by several cell types of the rat ovary and is able to suppress oestrogen release from rat granulosa cells. The mechanism of this effect is, however, not known. Since insulin-like growth factor (IGF)-I is produced by rat granulosa cells and exerts a synergistic role with FSH on granulosa cell steroidogenesis, we hypothesised that CRH may suppress oestrogen release from granulosa cells by inhibiting IGF-I release and/or stimulating the release of its binding protein (IGFBP-3). To test this hypothesis, granulosa cells were obtained from immature female Sprague-Dawley rats primed with diethylstilboestrol, and hormone concentrations were measured in the conditioned medium by radioimmunoassay. CRH suppressed oestrogen and IGF-I release stimulated by FSH used at a concentration of 1 IU/l, whereas it did not have any statistically significant effect on oestrogen and IGF-I release in basal conditions or in response to 5 IU/l FSH. The suppressive effects of CRH on oestrogen and IGF-I release were antagonised by a selective CRH receptor antagonist. CRH had no effects on IGFBP-3 release. CRH did not have any effect on oestrogen release stimulated by increasing concentrations of IGF-I and its suppressive effect on FSH-stimulated oestrogen release was overcome by the addition of low doses of exogenous IGF-I. In conclusion, CRH suppressed the release of oestrogen and IGF-I, but not of IGFBP-3. Thus, the inhibitory effects of CRH on oestrogen release could be mediated, partly, by a suppression of the autocrine/paracrine action of IGF-I.
AE Calogero, MA Palumbo, AM Bosboom, N Burrello, E Ferrara, G Palumbo, F Petraglia, and R D'Agata
The central nervous system (CNS) is able to synthesize and/or metabolize steroid hormones. These neuroactive steroids are capable of modulating several brain functions and, among these, they seem to regulate the hypothalamic-pituitary-gonadal (HPG) axis. Indeed, recent observations have shown that 5 alpha-pregnane-3 alpha-ol-20-one (allopregnanolone), one of the most abundant naturally occurring neuroactive steroids, suppresses ovulation and sexual behaviour when administered within the CNS. The present study was undertaken to evaluate the effects of allopregnanolone and its inactive stereoisomer, 5 alpha-pregnane-3 beta-ol-20-one, upon the release of gonadotropin-releasing hormone (GnRH) from individually-incubated hemihypothalami. Allopregnanolone suppressed GnRH release in a concentration-dependent manner with maximal activity in the nanomolar range, a range at which this neurosteroid is capable of playing a biological action. The specificity of allopregnanolone suppression of GnRH release was provided by the lack of effect of its known inactive stereoisomer. To evaluate the involvement of gamma-aminobutyric acidA (GABAA) receptor, we examined the effects of two neurosteroids with GABA-antagonistic properties, pregnanolone sulfate (PREG-S) and dehydroepiandrosterone sulfate (DHEAS), and of bicuculline, a selective antagonist of the GABA binding site on the GABAA receptor, on allopregnanolone (10 nM)-suppressed GnRH release. Both PREG-S and bicuculline overcame the inhibitory effects of allopregnanolone on GnRH release, whereas DHEAS did not. To substantiate the involvement of the GABAA receptor further, we tested the effects of muscimol, a selective agonist for this receptor, which suppressed GnRH release. In conclusion, allopregnanolone suppressed hypothalamic GnRH release in vitro and this effect appeared to be mediated by an interaction with the GABAA receptor. We speculate that the inhibitory effect of allopregnanolone on the HPG axis may also be caused by its ability to suppress hypothalamic GnRH release.