Seasonal Variation of Neuropeptide Y Actions on Growth Hormone and Gonadotropin-ll Secretion in the Goldfish: Effects of Sex Steroids

The effects of neuropeptide Y (NPY) on growth hormone (GH) and gonadotropin-II (GtH-II) release in different reproductive stages were studied using perfused pituitary fragments of female goldfish. The GH and GtH-II release responses to 5-min pulses of NPY were relatively small in sexually regressed fish (July), intermediate in recrudescent fish (December), and maximal in sexually mature (= prespawning) fish (May). To test if sex steroids can modulate NPY action, the effects of in vivo implantation of 17β-estradiol (E₂) and testosterone (T) (both at 100 μg/g dosage) on NPY-induced GH and GtH-II secretion were examined. In sexually regressed goldfish, implantation of T significantly enhanced NPY-induced GH and GtH-ll release from perfused pituitary fragments; implantation of E₂ potentiated the NPY-induced GtH-II, but not GH release. However, steroid implantation did not affect responses to NPY when this experiment was repeated using pituitaries from sexually mature fish. To test the hypothesis that steroids may act directly at the level of the pituitary to potentiate NPY action, pituitary fragments taken from sexually regressed goldfish were incubated with 100 nM T for 24 h, and the GH and GtH-ll responses to 5-min challenges of NPY assessed in the presence of T. Both GH and GtH-ll responses to NPY were not affected by treatment with T in vitro, suggesting that T does not act directly at the level of the pituitary. Since we have found that gonadotropin-releasing hormone (GnRH) in part mediates the effects of NPY on GH and GtH-ll release, the possibility that steroids may potentiate the actions of NPY on GnRH release were also examined. In sexually regressed fish, NPY did not alter GnRH release either from pituitary fragments or preoptic anterior hypothalamic slices. When fish were pretreated with E₂ and T by in vivo implantation, NPY significantly stimulated the release of GnRH. Taken together, these results demonstrate that: 1) there is a seasonal variation of NPY action on GH and GtH-ll release in the female goldfish; 2) sex steroids, especially T, potentiate the effects of NPY on GH and GtH-ll release in sexually regressed fish, when the endogenous steroid levels are low; and 3) the seasonality of NPY actions and the potentiation by steroids may be mediated, at least in part, by enhanced stimulation of GnRH release.     

Agonist-specific and sexual stage-dependent inhibition of gonadotropin-releasing hormone-stimulated gonadotropin and growth hormone release by ryanodine: relationship to sexual stage-dependent caffeine-sensitive hormone release

Differential utilization of intracellular Ca2+ stores with specific functional characteristics could be a potential mechanism for coupling various stimuli to specific cellular responses. In the goldfish pituitary, both gonadotropes and somatotropes possess multiple intracellular Ca2+ stores that are differentially coupled to agonist-evoked exocytosis. We investigated the role of ryanodine receptor/Ca2+-release channels (RyR) in basal and gonadotropin-releasing hormone (GnRH)-evoked hormone secretion from cultured gonadotropes and somatotropes using radioimmunoassay for gonadotropin (GTH-II) and growth hormone (GH). As is the case in vivo, the basal and evoked secretion of both hormones varied with seasonal reproductive status. GnRH-stimulated hormone release was three-fold higher in cells from sexually mature animals compared to those in a sexually regressed state. Nanomolar doses of ryanodine evoked significant GTH-II and GH secretion, suggesting that ryanodine-sensitive Ca2+ stores can couple to exocytosis in both cell types. In gonadotropes, 10 microM ryanodine abolished cGnRH-II-evoked GTH-II release in both sexually mature and sexually regressed fish, while sGnRH signalling was mediated by ryanodine-sensitive Ca2+ stores in cells from sexually regressed fish only. Ryanodine-sensitive Ca2+ stores in somatotropes were only involved in cGnRH-II-stimulated GH release during gonadal regression. In contrast, sGnRH-stimulated, but not cGnRH-II-stimulated, GH release was significantly reduced by 1 microM xestospongin C. Although hormone release stimulated by mobilizing caffeine-sensitive Ca2+ pools was also markedly seasonal, it was largely independent of ryanodine-sensitive Ca2+ stores. Ryanodine-sensitive Ca2+ stores in both cell types are not active downstream of ionomycin, BayK 8644, protein kinase C or cyclic adenosine monophosphate signalling pathways, suggesting difference from a classical Ca2+-induced Ca2+ release system. Ours study is the first to suggest that RyR2 may be involved in the seasonal plasticity of pituitary function, which may be related to cyclic changes observed in reproduction and growth.     

Sex steroid regulation of brain glutamic acid decarboxylase (GAD) mRNA is season-dependent and sexually dimorphic in the goldfish Carassius auratus

GABA, the major inhibitory neurotransmitter of the vertebrate brain, has been shown to play an important role in vertebrate reproduction by regulating LH release and sexual behavior. We have studied the expression of the GABA-synthesizing enzyme, glutamic acid decarboxylase (GAD), in goldfish throughout the reproductive cycle in May (mature), November (early gonadal recrudescence) and February (late gonadal recrudescence) and in response to implanted sex steroids. Levels of GAD67 and GAD65 mRNA levels in the hypothalamus of both males and females were highest in the early stages of gonadal recrudescence. In the telencephalon, a different seasonal pattern of GAD expression was evident. The telencephalic expression GAD67, GAD65 and a novel isoform, GAD3, were highest in sexually mature fish in May. Five-day intraperitoneal implantation of gonad-intact fish with testosterone (T), estradiol (E2) or progesterone (P4) did not affect GAD expression in November and February. This is in contrast to results in May when sex differences in steroid responsiveness were evident. Progesterone decreased hypothalamic GAD67 and GAD65 in females and was without effect in males. All other treatments did not alter GAD67, GAD65 or GAD3 expression in the hypothalamus. Both T and P4 decreased GAD67 and GAD65 levels in the telencephalon of male goldfish but had no effect in females. Serum sex steroid levels in control and implanted mature males and females in May were similar so it is unlikely that sex differences in the GAD responses were a result of differences in serum sex steroid levels. These contrasting effects of sex steroids on males and females suggest important sex differences in the regulation of the GADs in sexually mature goldfish.     

The Role of Oxytocin Release in the Mediobasal Hypothalamus of the Sheep in Relation to Female Sexual Receptivity

In vivo microdialysis and retrodialysis were used to investigate the role of oxytocin (OXY) release in the mediobasal hypothalamus (MBH) of the ewe in the control of sexual receptivity. Initial experiments showed that OXY release was significantly increased in ovariectomized animals treated with progesterone and oestradiol when they were sexually receptive towards males and received intromissions. No such increases were seen during tests where the ewes were receptive but the males were prevented from achieving intromission. By contrast, OXY release was significantly reduced in tests where the ewes were not receptive to the male. In a second experiment artificial vaginocervical stimulation (VCS) was found to significantly increase OXY release when the animals were treated with oestradiol and this effect was potentiated by progesterone priming. OXY release in the MBH was not significantly altered by VCS in the presence of progesterone priming alone. Plasma OXY concentrations were significantly increased by VCS following all three hormone treatments but no one treatment was significantly more effective than another. Noradrenaline release in the MBH was only significantly increased following VCS when progesterone priming was given before oestradiol treatment. No effects of VCS on release of GABA, glutamate or dopamine were seen but their basal concentrations were significantly increased by the combined steroid treatment compared to oestradiol alone. In a third experiment it was found that OXY (10 μM) infused bilaterally into the MBH of receptive ewes, by retrodialysis, significantly decreased sexual receptivity and increased the release of noradrenaline and GABA. Finally, in a fourth experiment is was shown that multiple intromissions significantly reduced sexual receptivity. These results show that OXY release in the MBH is increased by natural or artificial VCS and that this is modulated by sex steroids. The inhibitory action of OXY infusions into the MBH on sexual receptivity suggests that a functional role of this peptide within this brain region might be to mediate decreases in sexual receptivity which we observed following multiple intromissions. It is probable that the behavioural effects of this peptide are primarily mediated directly rather than through a modulation of noradrenaline and GABA release.     

Ovarian Steroid Regulation of Glutamic Acid Decarboxylase Gene Expression in Individual Hypothalamic Nuclei

The possibility that steroids exert their effects on luteinizing hormone (LH) and prolactin release and female sexual behaviour via altering γ-aminobutyric acid (GABA) synthesis within the hypothalamus was investigated. Ovariectomized rats were treated with either oil, 5 μg oestradiol benzoate (OB) or 5 μg OB plus 0.5 mg progesterone (P) 48 h later; autopsy was carried out 54 h after the OB injection. At this time, both steroid treatments stimulated prolactin release and lordotic activity. OB alone exerted a negative feedback effect on LH release while OB plus P stimulated an LH surge. The brains of the rats in these three treatment groups were microdissected and glutamic acid decarboxylase messenger nbonucleic acid (GAD mRNA) was estimated in specific hypothalamic areas obtained from individual brains; these areas included the preoptic area (POA), ventromedial nucleus (VMN), anterior medial portion of the zona incerta (Zl) and the arcuatemedian eminence area (ARC/ME). GAD mRNA concentrations were assessed by the slot-blotting technique and expressed as a ratio of GAD mRNA: β-actin mRNA. Both steroid treatments significantly reduced GAD mRNA in the ARC/ME and Zl and OB plus P also reduced the concentration in the POA, while OB alone had no effect in this area. Neither treatment affected GAD mRNA in the VMN. These results indicate that when steroids stimulate LH and prolactin release and female sexual behaviour, they reduce GABA activity probably by reducing GABA synthesis in the POA, Zl and ARC/ME. This suggests that GABA normally has an inhibitory influence on these parameters. GABA synthesis does not however, appear to be involved in the negative feedback effects of steroids on LH release as measured 54 h after OB treatment.     

PACAP Stimulation of Gonadotropin-II Secretion in Goldfish Pituitary Cells: Mechanisms of Action and Interaction with Gonadotropin Releasing Hormone Signalling

Pituitary adenylate cyclase-activating polypeptide (PACAP) has recently been shown to be a hypophysiotropic factor in the goldfish. In this study, we examined the mechanisms of PACAP action on goldfish maturational gonadotropin (GTH-II) release using primary cultures of pituitary cells. The GTH-II response to mammalian PACAP1-38 (mPACAP) was inhibited by a PACAP receptor antagonist suggesting a receptor-mediated action. Addition of either an adenylate cyclase inhibitor or a protein kinase A (PKA) inhibitor reduced the mPACAP-induced GTH-II release. In addition, when GTH-II release was already stimulated by either forskolin or 8-bromo-cAMP (8Br-cAMP), mPACAP did not further increase GTH-II secretion. These results strongly implicated the involvement of an adenylate cyclase/cAMP/PKA pathway in PACAP-stimulated GTH-II release. Although mPACAP induced a rise in intracellular Ca2+ level in identified gonadotropes, results with voltage-sensitive Ca2+ channel inhibitors indicated that the GTH-II responses to mPACAP, forskolin and 8Br-cAMP did not depend upon Ca2+ entry through these channels. Two protein kinase C (PKC) inhibitors did not affect mPACAP-elicited GTH-II release, and mPACAP further increased GTH-II secretion in the presence of PKC activators. These results indicate that PKC-dependent elements are not essential for the stimulatory action of mPACAP in gonadotropes. Interestingly, while GTH-II responses to a stimulatory concentration of mPACAP were additive to responses elicited by maximal effective concentrations of two endogenous gonadotropin releasing hormones (GnRHs), a subthreshold concentration of mPACAP potentiated GnRH and PKC activator stimulation of GTH-II secretion. Similarly, submaximal concentrations of forskolin potentiated the GTH-II response to the PKC activator, tetradecanoyl phorbol acetate. These data suggest that PACAP and its cAMP-dependent signalling mechanisms provide an alternate stimulatory input to goldfish gonadotropes and may influence the effectiveness of the major neuroendocrine control exerted by the PKC-dependent GnRH signalling pathway.     

Role of sex and sex steroids in mediating pituitary-adrenal responses to acute buspirone treatment in sheep

Systematic characterisation of sex differences in the serotonergic modulation of the hypothalamic-pituitary-adrenal (HPA) axis may assist with our understanding of why stress-related disorders are disproportionately represented in women. In this study, we examined the acute effects of buspirone, a serotonergic 1A receptor subtype agonist, on the endocrine endpoints of adrenocorticotrophin (ACTH) and cortisol secretion in gonadectomised male and female sheep. Each sheep was treated with an acute i.v. injection containing vehicle or buspirone (0.03, 0.1 and 0.3 mg/kg) in the presence and absence of sex steroid replacement (SSR). In males, SSR treatment consisted of testosterone (2 x 200 mg s.c. pellets) and, in females, the mid-luteal phase of the oestrus cycle was simulated by treatment with oestradiol (1 cm s.c. implant) and an intravaginal controlled internal drug release device containing 0.3 g progesterone. ACTH, cortisol, testosterone and progesterone were measured in jugular blood. Basal ACTH levels were higher in males, whereas basal cortisol levels were higher in females, regardless of sex steroid status. The magnitude of the increase in ACTH and cortisol secretion following buspirone treatment was dose-dependent. There were no differences in the ACTH responses of males and females to buspirone treatment, either in the presence or absence of sex steroid replacement. However, although the cortisol response to buspirone was greater in females, there was no discernable effect of sex steroid status in addition to this sex difference on either basal or buspirone-stimulated cortisol release. We conclude that the larger basal and buspirone-stimulated cortisol response measured in females may reflect a sex difference, either in the sensitivity of the adrenal gland to ACTH or in the catecholaminergic innervation of the adrenal gland. The lack of effect of sex and sex steroids in the ACTH secretory response to buspirone may indicate that the sex differences in serotonergic modulation of the HPA axis, as reported previously by our group, were mediated via serotonergic receptor subtypes other than the 1A receptor.     

DERMAL NEUROVASCULAR DYSFUNCTION IN TYPE 2 DIABETES

The present article summarizes recent observations obtained in our laboratory which clearly indicate that sex steroids exert relevant effects on the peripheral nervous system. In particular, the following important points have emerged: (1) Steroids exert stimulatory actions on the synthesis of the proteins proper of the peripheral myelin (e.g., glycoprotein Po and peripheral myelin protein 22) in vivo and on the Schwann cells in culture; (2) in many cases the actions of hormonal steroids are not due to their native molecular forms but rather to their metabolites (e.g., dihydroprogesterone and tetrahydroprogesterone in the case of progesterone; dihydrotestosterone and 5 alpha-androstane-3 alpha,17 beta -diol in the case of testosterone); (3) the mechanism of action of the various steroidal molecules may involve both classical (progesterone and androgen receptors) and nonclassical steroid receptors (GABA, receptor); and finally, (4) the stimulatory action of steroid hormones on the proteins of the peripheral myelin might have clinical significance in cases in which the rebuilding of myelin is needed (e.g., aging, peripheral injury, demyelinating diseases, and diabetic neuropathy).     

Activational and Organisational Effects of Gonadal Steroids on Sex-Specific Acetylcholine Release in the Dorsal Hippocampus

Acetylcholine (ACh) release in the dorsal hippocampus increases during stress, exploration or learning, exhibiting sex-specific 24-h release profile. We review the role of gonadal steroids on the ACh release in the dorsal hippocampus. In our studies, we found that male rats showed higher extracellular ACh levels than females, but gonadectomy decreased ACh levels in both sexes of rats and subsequently eliminated the sex difference. To examine the sex difference under comparable gonadal steroid levels, we implanted steroid capsules after gonadectomy. Oestradiol supplementation maintained circulating oestradiol to the levels in proestrous female rats, whereas testosterone capsules maintained circulating testosterone to the levels similar to intact male rats. Under comparable gonadal steroids levels, ACh levels were sex-specific. Testosterone replacement in orchidectomised rats clearly restored ACh levels, which were greater than ovariectomised testosterone-primed rats. Similarly, oestradiol replacement in ovariectomised rats successfully restored ACh levels, which were higher than orchidectomised oestradiol-primed rats. These results suggest sex-specific activational effects of gonadal steroids on ACh release. To further examine the organisational effect, female pups were neonatally treated with oil, testosterone, oestradiol, or dihydrotestosterone. These rats were bilaterally ovariectomised and a testosterone capsule was implanted at postnatal week 8. Neonatal treatment of either testosterone or oestradiol clearly increased ACh levels, whereas neonatal dihydrotestosterone treatment failed to change levels. These results suggest that: (i) gonadal steroids maintain the sex-specific ACh release in the dorsal hippocampus and (ii) neonatal activation of oestrogen receptors is sufficient to mediate masculinisation of the septo-hippocampal cholinergic system.     

The Effect of Gonadal Steroids on Vasoactive Intestinal Peptide Concentration and Release from Mediobasal Hypothalamus and the Anterior Pituitary Gland

The effects of chronic administration of sex steroids on the content of vasoactive intestinal peptide (VIP) in the mediobasal hypothalamus and anterior pituitary were studied in adult rats. Gonadectomy had no effect on VIP concentration in the mediobasal hypothalamus or anterior pituitary gland. Estradiol benzoate (1 μg/100 g body wt/day) administered for 10 days decreased mediobasal hypothalamus VIP concentration of ovariectomized rats whereas it produced no change in mediobasal hypothalamus VIP content of orchidectomized rats. Testosterone propionate (100 μg/100 g body wt/day) administration decreased mediobasal hypothalamus VIP content in both sexes. Estradiol administration caused an increase whereas testosterone treatment resulted in a decrease in anterior pituitary VIP levels in both sexes. The effect of chronic administration of the sex steroids on VIP release from the mediobasal hypothalamus and anterior pituitary was also investigated. Estradiol increased evoked VIP release from the mediobasal hypothalamus and decreased mediobasal hypothalamus VIP content whereas testosterone decreased both mediobasal hypothalamus release and content. Chronic treatment with estradiol enhanced anterior pituitary VIP release and content while testosterone decreased both parameters studied. The data indicate that anterior pituitary VIP content is under the control of gonadal hormones and that the increased anterior pituitary VIP found after estradiol administration may be due to an augmented release from the mediobasal hypothalamus and probably an increase in anterior pituitary VIP synthesis.     

Intracellular calcium involvement in pituitary adenylate cyclase-activating polypeptide stimulation of growth hormone and gonadotrophin secretion in goldfish pituitary cells

The involvement of intracellular Ca(2+) stores and their regulatory mechanisms in mediating pituitary adenylate cyclase-activating polypeptide (PACAP) stimulation of growth hormone (GH) and maturational gonadotrophin (GTH-II) secretion from goldfish pituitary cells was investigated using a cell column perifusion system. Pretreatment with caffeine abolished the GH and GTH-II responses to PACAP. Dantrolene attenuated PACAP-elicited GTH-II release but did not affect the GH response, whereas ryanodine and 8-bromo-cADP ribose did not alter PACAP-induced GH and GTH-II release. Two endoplasmic/sarcoplasmic reticulum Ca(2+) ATPase (SERCA) inhibitors, thapsigargin and cyclopiazonic acid, augmented PACAP-induced GTH-II release; similarly, thapsigargin elevated GH responses to PACAP. Treatment with carbonyl cyanide m-chlorophenylhydrazone, a mitochondrial uncoupler, reduced PACAP-stimulated GH release; however, inhibition of the mitochondrial Ca(2+) uniport by Ru360 did not affect GH and GTH-II responses. The phosphatidyl inositol (PI)-specific phospholipase C (PLC) inhibitor ET-18-OCH(3) inhibited, whereas the phosphatidyl-choline (PC)-specific PLC inhibitor D609 enhanced, PACAP-stimulated GH and GTH-II responses. On the other hand, the IP(3) receptor blocker xestospongin D had no effect on PACAP-induced GTH-II response and potentiated the GH response. These results suggest that, despite some differences between GH and GTH-II cells, PACAP actions in both cell types generally rely on a caffeine-sensitive, but a largely ryanodine receptor-independent, mechanism. PC-PLC and some SERCA negatively modulate PACAP actions but mitochondrial Ca(2+) stores per se are not important. A novel PI-PLC mechanism, which does not involve the traditional IP(3)/Ca(2+) pathway, is also suggested.     

Neuroactive amino acids influence gonadotrophin output by a suprapituitary mechanism in either rodents or primates

Systemic administration of excitatory amino acids (Glu or NMA) rapidly enhances LH release (rodents or primates) and GABA blocks this action (rodents). In the present study rodent and primate pituitaries were incubated with NMA, Glu and/or GABA, with or without added LH-RH. Only LH-RH affected LH release. Therefore the probable site of NMA, Glu or GABA action on LH release in both rodents and primates is suprapituitary.     

Estrogen configures sexual dimorphism in the preoptic area of C57BL/6J and ddN strains of mice

Immunohistochemistry using a calbindin D28k antibody revealed a marked sex difference in neuronal distribution in the central portion of the medial preoptic area in C57BL/6J and ddN strains of mice when the animals were sacrificed on D65 (D1 = the day of birth). Male mice had a distinct ellipsoidal cell aggregate, whereas females lacked such a structure. This sex difference was not observed in Nissl‐stained sections. Co‐localization of calbindin D28k and the neuron‐specific nuclear protein NeuN confrmed that the cells in the aggregate were neurons. The aggregates were larger in males than in females in both strains. When observed on D65, males orchidectomized on D1 had smaller aggregates. However, daily injections of 2 μg estradiol benzoate through D1–D5 as well as a single injection of 100 μg testosterone propionate on D1 enlarged the aggregates in females, but a single injection of 100 μg dihydrotestosterone on D1 had no effect on the female phenotype. Similar endocrine manipulations had no effects in adult animals of both sexes. Thus, the calbindin‐immunoreactive cell aggregates in the preoptic area of C57BL/6J and ddN mice are homologous to the sexually dimorphic nucleus of the rat preoptic area in terms of the morphology and sex steroid‐dependent organization. J. Comp. Neurol. 518:3618–3629, 2010. © 2010 Wiley‐Liss, Inc.     

Ammonia stimulates the release of taurine from cultured astrocytes

The effect of ammonia on the release of the neuroactive amino acids taurine (TAU), γ-aminobutyric acid (GABA) and -aspartate ( -ASP), an analog of -glutamate ( -GLU), from cultured rat cortical astrocytes was studied. NH₄Cl (1 and 5 mM) induced the release of TAU. TAU release was reduced when Na⁺ was removed, and was almost completely abolished when Cl⁻ was omitted. In contrast, TAU basal release was enhanced upon removal of Na⁺ or Cl⁻. Ammonia inhibited the release of GABA and -ASP. Ammonia-induced release of astroglial TAU may modify the neuronal excitability accompanying hyperammonemic conditions.     

Analysis of the Inhibitory Effect of Oestradiol on Functional GABA/5-HT Relationship in the Rat Suprachiasmatic Area

The purpose of this study was to test the capacity of oestradiol to modulate the stimulating effect of a-aminobutyric acid (GABA) on serotonin (5-HT) metabolism, previously described in the Suprachiasmatic area of the male rat. After an in vivo stimulation of GABA transmission by systemic administration of a GABA-transaminase inhibitor (amino-oxyacetic acid) or a GABA_B agonist (RS-baclofen), the 5-HT metabolism was studied in the Suprachiasmatic area of ovariectomized, and ovariectomized oestradiol-treated rats. Amino-oxyacetic acid or RS-baclofen treatment increased the endogenous content of 5-HT in the Suprachiasmatic area of males and ovariectomized rats. These two treatments were without effect in ovariectomized oestradiol-treated rats. GABA transmission stimulation induced by amino-oxyacetic acid treatment failed to affect the release and synthesis of 5-HT in ovariectomized oestradiol-treated rats while it increased these two parameters of 5-HT metabolism in the Suprachiasmatic area of male and ovariectomized rats. To investigate the main target of oestradiol effect, comparative studies of the serotoninergic and GABAergic metabolism in the Suprachiasmatic area were performed in the three experimental groups. Under our experimental conditions the endogenous 5-HT metabolism was similar between ovariectomized and ovariectomized oestradiol-treated rats. Nevertheless, 5-HT metabolism was higher in the two female groups than in the male group. Neither GABA metabolism nor GABAergic response to GABA-related drug treatment differed between ovariectomized, and ovariectomized oestradiol-treated rats. However, the turnover of GABA was higher when compared to the two female groups. It is concluded that the lack of 5-HT responsiveness to GABA transmission stimulation in ovariectomized oestradiol-treated rats was not related to an effect of oestradiol on 5-HT metabolism or to an effect of the steroid on GABA turnover. Furthermore, our results suggest a sex difference in the activity of serotoninergic and GABAergic systems in the Suprachiasmatic area.     

Steroid profiles in quail brain and serum: Sex and regional differences and effects of castration with steroid replacement

Both systemic and local production contribute to the concentration of steroids measured in the brain. This idea was originally based on rodent studies and was later extended to other species, including humans and birds. In quail, a widely used model in behavioural neuroendocrinology, it was demonstrated that all enzymes needed to produce sex steroids from cholesterol are expressed and active in the brain, although the actual concentrations of steroids produced were never investigated. We carried out a steroid profiling in multiple brain regions and serum of sexually mature male and female quail by gas chromatography coupled with mass spectrometry. The concentrations of some steroids (eg, corticosterone, progesterone and testosterone) were in equilibrium between the brain and periphery, whereas other steroids (eg, pregnenolone (PREG), 5α/β‐dihydroprogesterone and oestrogens) were more concentrated in the brain. In the brain regions investigated, PREG sulphate, progesterone and oestrogen concentrations were higher in the hypothalamus‐preoptic area. Progesterone and its metabolites were more concentrated in the female than the male brain, whereas testosterone, its metabolites and dehydroepiandrosterone were more concentrated in males, suggesting that sex steroids present in quail brain mainly depend on their specific steroidogenic pathways in the ovaries and testes. However, the results of castration experiments suggested that sex steroids could also be produced in the brain independently of the peripheral source. Treatment with testosterone or oestradiol restored the concentrations of most androgens or oestrogens, respectively, although penetration of oestradiol in the brain appeared to be more limited. These studies illustrate the complex interaction between local brain synthesis and the supply from the periphery for the steroids present in the brain that are either directly active or represent the substrate of centrally located enzymes.     

Inhibition of luteinizing hormone secretion in the ewe by progesterone: associated changes in the release of gamma-aminobutyric Acid and noradrenaline in the preoptic area as measured by intracranial microdialysis

Progesterone inhibits the pulsatile release of luteinizing hormone (LH) in sheep by an action in the brain to suppress the release of LH-releasing hormone (LHRH). In addition, progesterone blocks the preovulatory surge of LH in this species. The neural basis of this inhibitory action is unknown, but as LHRH cells do not appear to contain progestin receptors other neural systems must mediate the action of this ovarian steroid on LH release. This study focuses on a possible role for the inhibitory amino-acid GABA and the monoamines (noradrenaline, adrenaline, dopamine and serotonin). The technique of microdialysis was used to monitor changes in these substances in the vicinity of the LHRH cell bodies (in the preoptic area) both before and following the administration of progesterone. Levels of this steroid, similar to those measured during the mid-luteal phase of the oestrous cycle, inhibited LH release and this was associated with significant alterations in the release of GABA and noradrenaline (but not adrenaline, dopamine or serotonin). Specifically, progesterone augmented GABA while noradrenaline release was depressed. Whether steroid actions on these neurotransmitters were mediated by opioids was also investigated. This possibility arises because of the reported involvement of opioids in progesterone negative feedback in the ewe. The long-acting opioid antagonist, naltrexone, was administered and GABA and noradrenaline release monitored for a further period both in the presence and absence of progesterone. Naltrexone significantly depressed GABA release in steroid-treated (but not untreated) ewes suggesting that the actions of progesterone on GABA are mediated by the endogenous opioid peptides. However, noradrenaline release was unaltered. In an earlier study we demonstrated that GABA release fell prior to the LH surge while noradrenaline release increased. These data, in conjunction with those from the present study, suggest that the mechanism by which progesterone is able to inhibit the preovulatory surge of LH in the ewe is by enhancing GABA and depressing noradrenaline release in the vicinity of the LHRH cell bodies. As opioid tone is also reported to fall prior to the surge, the interaction between opiate and GABAergic systems in the regulation of gonadotrophin secretion warrants further investigation.     

Acute Action of Oestrogen on Medial Preoptic Gamma-Aminobutyric Acid Neurons: Correlation with Oestrogen Negative Feedback on Luteinizing Hormone Secretion

The acute effects of oestrogen on the medial preoptic area (MPOA) γ-aminobutyric acid (GABA) system were examined by delivering an intravenous bolus of 17β-oestradiol (5 μg/100 g body wt) to conscious ovariectomized rats implanted with microdialysis probes. Fifteen-min blood samples were taken to determine the time-course of negative feedback effects of oestrogen on luteinizing hormone (LH) secretion. Two h after administration of 17β-oestradiol, GABA release from the MPOA was significantly elevated compared with vehicle-treated controls (P<0.05). The rise in GABA levels continued until the end of the experiment, 4 h after 17β- oestradiol, at which time it was over 50% higher than controls (P<0.01). The pulsatile pattern of LH secretion was significantly depressed 2 and 3 h after administration of 17β-oestradiol compared with controls (P<0.05). To determine the effects of the 17β-oestradiol treatment on pituitary responsiveness to LH-releasing hormone (LHRH), a further group of rats were given exogenous LHRH (50ng/100g body wt, intravenously) before and 3 h after vehicle or 17β-oestradiol treatment and blood samples taken to determine the effect on LH secretion. The maximal LH response to LHRH in 17β-oestradiol-treated rats was approximately 50% that of control-treated values. This study demonstrates the acute and potent action of 17β-oestradiol on GABA release in the MPOA and lends support to a genomic site of action for oestrogen in modulating neural elements regulating GABA release from the MPOA. These results, showing a parallel decrease in LH secretion with increased GABA levels in the MPOA, suggest a role for GABA elements within the MPOA as a site of oestrogen negative feedback on LH secretion.