269 Activin-A stimulates hypothalamic gonadotropin-releasing hormone release by the explanted male rat hypothalamus: interaction with inhibin and androgens A E Calogero, N Burrello, A M Ossino, P Polosa and R D’Agata Division of Andrology, Department of Internal Medicine, University of Catania, 95123 Catania, Italy (Requests for offprints should be addressed to A E Calogero, Istituto di Medicina Interna e Specialita` Internistiche, Ospedale Garibaldi, Piazza S.M. di Gesu`, 95123 Catania, Italy) Abstract The presence of activins in those hypothalamic regions had no effect on GnRH output. As previously shown, containing gonadotropin-releasing hormone (GnRH)- testosterone (1 nmol/l) and dihydrotestosterone (DHT, secreting neurons suggests that these peptides may regulate 0·1 nmol/l) suppressed basal GnRH release, but only the reproductive function modulating not only pituitary testosterone was able to inhibit the release of GnRH FSH release and biosynthesis, but also hypothalamic stimulated by activin-A. Since DHT is a non-aromatizable GnRH release. The purpose of this study was to evaluate androgen, we evaluated whether the inhibitory effect of the effects of activin-A, a homodimer of inhibin âA testosterone was due to its in vitro conversion into 17â- subunit, on hypothalamic GnRH release in vitro and, estradiol. The addition of 4-hydroxyandrostenedione, a because of their well known antithetical effects, to evalu- steroidal aromatase inhibitor, did not influence the sup- ate its interaction with inhibin. In addition, since andro- pressive effect of testosterone on GnRH release stimulated gens modulate the release of GnRH from male rat by activin-A. hypothalami, we thought it of interest to study the possible In conclusion, activin-A stimulated hypothalamic interplay between these steroids and activin on GnRH GnRH release in vitro and this effect was abolished by release. To accomplish this, we employed a hypothalamic inhibin and was blunted by testosterone. These findings organ culture system which enabled us to evaluate GnRH suggest that activins may participate in the regulation of release from individually incubated hemi-hypothalami the hypothalamic–pituitary–gonadal axis by modulating explanted from male rats. Activin-A stimulated GnRH GnRH release. The ability of testosterone to suppress the release in a biphasic manner. The maximal effect was release of GnRH stimulated by activin-A indicates that reached at a concentration of 10 ng/ml which increased this steroid has a potent negative feedback influence on GnRH output by about 75%. Inhibin abolished the GnRH release. stimulatory effect of a maximally effective concentration of Journal of Endocrinology (1998) 156, 269–274 activin-A in a dose-dependent manner, whereas alone it Introduction 1985, 1986, Rivier et al. 1985, Vale et al. 1986). Although these glycoproteins were first isolated from the gonads, Activins and inhibins are dimer glycoproteins sharing a they were since found to be widely distributed (Meunier common â-subunit. Inhibin is a heterodimer of an et al. 1988) and it was hypothesized that they may also act inhibin-specific á-subunit and one of the two closely on the hypothalamus indirectly to regulate FSH secretion related â-subunits (âA or âB), thus forming two different (Franchimont et al. 1989). The presence of âA and âB inhibins: inhibin-A and inhibin-B. Activins originate from subunit-containing neurons in the septal and preoptic the dimerization of two â-subunits. Hence, they exist in regions of the hypothalamus, where gonadotropin- three different molecular forms: activin-A, a homodimer releasing hormone (GnRH)-containing neurons are of the âA subunit, activin-B, a homodimer of the âB mainly located, suggests that activins may participate in the subunit, and activin-AB, a heterodimer (see Ying 1988 for regulation of GnRH release (Vale et al. 1988). More review). recently, the activin receptor ActRII has been found Activins and inhibins were initially identified for their expressed in several hypothalamic areas involved in capability to stimulate and inhibit, respectively, the release neuroendocrine regulation, such as the suprachiasmatic, of pituitary follicle-stimulating hormone (FSH) (Ling et al. supraoptic, paraventricular, and arcuate nuclei (Cameron Journal of Endocrinology (1998) 156, 269–274 ? 1998 Journal of Endocrinology Ltd Printed in Great Britain 0022–0795/98/0156–0269 $08.00/0 Downloaded from Bioscientifica.com at 09/29/2021 04:43:37PM via free access 270 A E CALOGERO and others · Activin-A and hypothalamic GnRH et al. 1994), adding further circumstantial evidence for a design consisted of 2 passages of the hemi-hypothalami possible physiological role of activins at the hypothalamic into different wells (one hemi-hypothalamus/0·5 ml) level. These observations prompted us to examine the every 6 h (total length of the experiment 12 h). This effects of activin-A on GnRH release by employing a interval of incubation was selected on the basis of prelimi- hypothalamic organ culture system which enabled us to nary studies which showed that activin-A had no effect study GnRH release from individually incubated hemi- after 45 min and 2 h of incubation (data not shown), but hypothalami explanted from male rats. Given that activins was fully effective after 6 h. Tissue transfer from one well and inhibins exert antithetical effects (Ying 1988), we to the other was carried out with a 3#3 mm nylon mesh evaluated their interaction on GnRH release. Moreover, grid (nylon monofilament 44 µ; Small Parts, Miami, FL, based on the observation that testosterone and its 5á- USA). In the second well, a number of hemi-hypothalami reduced derivative dihydrotestosterone (DHT) suppress for each experiment were exposed to a depolarizing GnRH release (Calogero et al. 1993), we also examined concentration (60 mmol/l) of potassium chloride (KCl) to whether these androgens may modulate the effects of test tissue viability by identifying the GnRH response. activin-A on GnRH release in vitro. Three different protocols were used in this study. Protocol 1 was designed to evaluate the effects of activin-A, inhibin, testosterone and DHT upon un- Materials and Methods stimulated GnRH release. For this purpose, each hemi- hypothalamus was incubated in M199 without test Materials substance in the first well (the concentration of GnRH was Human recombinant activin-A was generously donated by used as basal GnRH release for that given tissue) and Genentech Inc. (South San Francisco, CA, USA). Bovine exposed to graded concentrations of activin-A or inhibin inhibin was purchased from Peninsula Laboratories Europe and to 1 nmol/l testosterone or 0·1 nmol/l DHT in the Ltd (St Helens, Merseyside, UK). Testosterone and DHT second well (treated GnRH release). The effects of were purchased from Sigma Chimica (Milan, Italy). the zero concentration of activin-A, inhibin, testosterone, 4-Hydroxyandrostenedione (4OH-A, CGP-31349) was a and DHT were evaluated by incubating the hemi- gift from Ciba-Geigy S.p.A. (Origgio, VA, Italy). 125I- hypothalami with M199 alone also in the second well. Labeled GnRH was purchased from New England Protocol 2 was designed to evaluate the effects of Nuclear (Boston, MA, USA). Steroids were solubilized in inhibin, testosterone, and DHT on activin-A (10 ng/ml)- absolute ethanol and the highest concentration of ethanol stimulated GnRH release. For this purpose, the hemi- used in the tissue culture was 0·01%. At this concentration, hypothalami were incubated in M199 without test ethanol did not have any detectable effect on GnRH substance in the first well (basal GnRH release) and ex- release (data not shown). posed to graded concentrations of inhibin plus activin-A, testosterone (1 nmol/l) plus activin-A or DHT (0·1 nmol/l) plus activin-A in the second well (treated GnRH release). Removal and incubation of hypothalami The effect of activin-A alone was determined by incu- Experiments were performed using intact male Sprague- bating the hemi-hypothalami allocated to this treatment Dawley rats weighing 200–225 g (8–9 weeks old) (Charles in M199 containing 10 ng/ml activin-A in the second River, Calco, CO, Italy). Animals were housed for 4–5 well. days in a temperature-controlled room (22 )C) with lights Protocol 3 was designed to evaluate the effect of on from 0700 to 1900 h. Rat chow and tap water were 4OH-A on testosterone-suppressed, activin-A-stimulated available ad libitum. The rats were killed by decapitation GnRH release. For this purpose, the hemi-hypothalami and hypothalami explanted and halved longitudinally as were incubated in M199 alone in the first well (basal previously reported (Calogero et al. 1993). The hemi- GnRH release) and incubated with activin-A (10 ng/ml) hypothalami (one tissue per well, 48-multiwell plates; alone, activin-A (10 ng/ml) plus testosterone (1 nmol/l) or Costar, Cambridge, MA, USA) were placed in a water- activin-A (10 ng/ml) plus testosterone (1 nmol/l) plus jacketed incubator at 37 )C under an atmosphere of 95% 4-OH-A (1 µmol/l) in the second well (treated GnRH release). air and 5% CO2. Preincubation and incubation were carried out using medium-199 with modified Earle’s salt (Gibco, Paisley, Strathclyde, UK) containing 0·1% bovine GnRH radioimmunoassay serum albumin (fraction V; Sigma) and 20 µmol/l baci- tracin (zinc salt, Aldrich, Milwaukee, WI, USA) (M199). The concentration of GnRH in the incubation medium was measured by RIA as previously reported (Calogero et al. 1993). Standard solutions and medium samples Experimental protocols were measured in duplicate. GnRH antiserum, used at a The hemi-hypothalami were preincubated for 30 min in final dilution of 1:15 000, bound 40·8&1·9% (n=14) M199 before the experiments were run. The experimental 125I-GnRH (~3200 c.p.m.). Nonspecific binding was Journal of Endocrinology (1998) 156, 269–274 Downloaded from Bioscientifica.com at 09/29/2021 04:43:37PM via free access Activin-A and hypothalamic GnRH · A E CALOGERO and others 271 Figure 2 Effects of increasing concentrations of inhibin (INH) on hypothalamic GnRH release stimulated by activin-A (ACT).