Increase In Luteinizing Hormone-releasing Hormone Release Research Articles

Effect of Manganese on Luteinizing Hormone-Releasing Hormone Secretion in Adult Male Rats

Recently studies have demonstrated that low doses of (Mn(+2)) in the form of manganese chloride can stimulate specific puberty-related hormones and advance signs of pubertal development in immature female and male rats. In the present study, we used an in vitro system to evaluate the ability of 0, 50, 250, and 500 microM doses of Mn(+2) to stimulate luteinizing hormone-releasing hormone (LHRH) secretion and to assess the hypothalamic mechanism of this action in adult male Sprague-Dawley rats. We demonstrated that Mn(+2) at 500 microM, but not the lower doses, increased LHRH release, nitric oxide (NO) synthase (NOS) activity, and the content of cyclic cGMP in the medial basal hypothalamus. Inhibition of NOS with a competitive inhibitor (Nomega-nitro-L-arginine methyl ester hydrochloride) prevented the Mn-induced increase in LHRH release. Additionally, methylene blue and KT5823, specific inhibitors of guanylyl cyclase and protein kinase G (PKG), respectively, also blocked the stimulatory effect of Mn(+2) on LHRH release. These in vitro studies demonstrated that the hypothalamic mechanism of Mn(+2) action in adult males is by activation of the NOS/NO system, resulting in increases in cGMP and PKG and thus the secretion of LHRH from the nerve terminals. These results indicate Mn(+2) can cause LHRH release in adult males, and this action is discussed in relation to age, gender, as well as mechanistic and functional differences between adult and immature animals.

An increase in in vivo release of LHRH and precocious puberty by posterior hypothalamic lesions in female rhesus monkeys (Macaca mulatta)

We have previously shown that a decrease in gamma-aminobutyric acid (GABA) tone and a subsequent increase in glutamatergic tone occur in association with the pubertal increase in luteinizing hormone releasing hormone (LHRH) release in primates. To further determine the causal relationship between developmental changes in GABA and glutamate levels and the pubertal increase in LHRH release, we examined monkeys with precocious puberty induced by lesions in the posterior hypothalamus (PH). Six prepubertal female rhesus monkeys (17.4 +/- 0.1 mo of age) received lesions in the PH, three prepubertal females (17.5 +/- 0.1 mo) received sham lesions, and two females received no treatments. LHRH, GABA, and glutamate levels in the stalk-median eminence before and after lesions were assessed over two 6-h periods (0600-1200 and 1800-2400) using push-pull perfusion. Monkeys with PH lesions exhibited external signs of precocious puberty, including significantly earlier menarche in PH lesion animals (18.8 +/- 0.2 mo) than in sham/controls (25.5 +/- 0.9 mo, P<0.001). Moreover, PH lesion animals had elevated LHRH levels and higher evening glutamate levels after lesions, whereas LHRH changes did not occur in sham/controls until later. Changes in GABA release were not discernible, since evening GABA levels already deceased at 18-20 mo of age in both groups and morning levels remained at the prepubertal levels. The age of first ovulation in both groups did not differ. Collectively, PH lesions may not be a good tool to investigate the mechanism of puberty, and, taking into account the recent findings on the role of kisspeptins, the mechanism of the puberty onset in primates is more complex than we initially anticipated.

Stimulation of luteinizing hormone-releasing hormone (LHRH) gene expression in GT1-7 cells by its metabolite, LHRH-(1-5).

Given the central role of the decapeptide LHRH in reproduction and reproductive behavior, it is important to focus on delineating the possible effects of this gene and its products in the regulation of hormone-dependent reproductive processes. In the female, ovulation is preceded by a marked increase in LHRH release; the increase in LHRH release culminates in a preovulatory LH surge, which coincides with a period of sexual receptivity. In contrast to the belief that the proteolytic metabolism of LHRH serves only as a degradative process that removes excess LHRH and attenuates signal transduction through the LHRH receptor, we hypothesized that a metabolite of the decapeptide, LHRH-(1-5), can directly regulate LHRH neuronal function. This study demonstrates the ability of LHRH-(1-5) peptide to regulate LHRH gene expression in the LHRH neuronal cell line, the GT(1-7) cell. The results show that LHRH-(1-5) stimulated LHRH gene expression at the posttranscriptional level. In contrast to the LHRH suppression of its own gene expression, the coadministration of LHRH with the metalloendopeptidase, EC 3.4.24.15, an endopeptidase known to cleave LHRH to form LHRH(1-5), shows a reversal of effect, a stimulation of LHRH gene expression. Finally, the effect of LHRH-(1-5) on LHRH gene expression appears to be mediated by the calcium/calmodulin-dependent protein kinase. The present study supports the hypothesis that the physiological metabolite of LHRH, LHRH-(1-5), is functionally capable of regulating the reproductive neuroendocrine system.

The inhibitory effect of anandamide on luteinizing hormone-releasing hormone secretion is reversed by estrogen.

Because Delta-9-tetrahydrocannabinol (THC) inhibited luteinizing hormone-releasing hormone (LHRH) in male rats, we hypothesized that the endocannabinoid, anandamide (AEA), would act similarly. AEA microinjected intracerebroventricularly (i.c.v.) decreased plasma luteinizing hormone (LH) at 30 min in comparison to values in controls (P < 0.001). The cannabinoid receptor 1 (CB1-r)-specific antagonist, [N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-chlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide] (AM251), produced a significant elevation in plasma LH (P < 0.01). AEA (10(-9) M) decreased LHRH release from medial basal hypothalami incubated in vitro. These results support the concept that endogenous AEA inhibits LHRH followed by decreased LH release in male rats. In ovariectomized (OVX) female rats, AEA i.c.v. also inhibited LH release, but in this case AM251 had an even greater inhibitory effect than AEA. In vitro, AEA had no effect on LHRH in OVX rats. It seems that endogenous AEA inhibits LHRH followed by decreased LH release in OVX rats but that AM251 has an inhibitory action in this case. In striking contrast, in OVX, estrogen-primed (OVX-E) rats, AEA i.c.v. instead of decreasing LH, increased its release. This effect was completely blocked by previous injection of AM251. When medial basal hypothalami of OVX-E rats were incubated, AEA increased LHRH release. The synthesized AEA was higher in OVX-E rats than in OVX and males, indicating that estrogen modifies endocannabinoid levels and effects. The results are interpreted to mean that sex steroids have profound effects to modify the response to AEA. It inhibits LHRH and consequently diminishes LH release in males and OVX females, but stimulates LHRH followed by increased LH release in OVX-E-primed rats.

Oestradiol-dependent and -independent modulation of tyrosine hydroxylase mRNA levels in subpopulations of A1 and A2 neurones with oestrogen receptor (ER)alpha and ER beta gene expression.

Oestradiol (E2) induces luteinizing hormone-releasing hormone (LHRH) hypersecretion, thereby triggering LH surge release in ovariectomized (OVX) rats. Neural signals responsible for the surge are marked by a morning increase in LHRH gene expression and an afternoon increase in LHRH release. Evidence suggests that subpopulations of noradrenergic neurones may be responsible for one or both of these signals. To further investigate this issue, we examined effects of E2 on the activity of A1 and A2 noradrenergic neurones, as reflected in changes in tyrosine hydroxylase (TH) mRNA expression, on the day of LH surge release. We then used dual-label in situ hybridization to determine whether E2-induced changes occurred primarily in A1 and A2 subdivisions wherein most noradrenergic neurones expressed oestrogen receptor (ER)alpha and/or ER beta mRNA. We found that in all subdivisions, levels of TH mRNA were higher in E2- than oil-treated rats at 12.00 h. These differences resulted from a decline in TH mRNA expression in oil-treated rats, as well as a rise in levels in E2-treated rats between 10.00 h and 12.00 h. During the afternoon, TH mRNA expression in most A1 and A2 subdivisions peaked at 14.00 h when LH surge release began. However, in all but the middle and caudal A2 subdivisons, levels were similar in E2-treated and control rats at this time. This was attributable to a widespread increase in TH mRNA expression between 12.00 h and 14.00 h in OVX rats. There was no evidence that E2 induced changes in TH mRNA expression preferentially in regions wherein most neurones contained ER alpha or ER beta mRNA. Our findings suggest that E2 activation of middle and caudal A2 neurones, in conjunction with the widespread E2-independent activation of noradrenergic neurones in other subdivisions, may play a role in the induction of LH surge release.

N-Methyl D,L-Aspartate Induces the Release of Luteinizing Hormone-Releasing Hormone in the Prepubertal and Pubertal Female Rhesus Monkey as Measured by in Vivo Push-Pull Perfusion in the Stalk-Median Eminence

The role of the excitatory amino acid glutamate, N-methyl d-aspartate (NMDA) receptor agonist, in stimulating in vivo luteinizing hormone-releasing hormone (LHRH) release in the stalk-median eminence of conscious prepubertal and pubertal female rhesus monkeys was evaluated using push-pull perfusion. In Exp 1, the effects of iv bolus injection of N-methyl d,l-aspartate (NMA) on LHRH release were examined. Injection of NMA induced an increase in LHRH release in all maturational stages of monkeys. Although the LHRH response to NMA tended to be larger in the older groups, only the duration of the LHRH response in the midpubertal group was significantly longer than that in the prepubertal group. In Exp 2, the effects of direct infusion of NMA (0.1, 1, and 100 μm) into the stalk-median eminence on LHRH release were similarly examined. NMA infusion stimulated LHRH release in pubertal monkeys, whereas it did not induce any consistent changes in LHRH release in prepubertal monkeys except for the highest dose. These data suggest that: 1) the systemic injection of NMA is more effective than direct infusion of NMA; and 2) the prepubertal LHRH neurosecretory system is capable of responding to NMDA, although the responsiveness may undergo developmental changes. Therefore, stimulation of NMDA receptors may contribute to the pubertal changes in the LHRH neurosecretory activity.

N-methyl D,L-aspartate induces the release of luteinizing hormone-releasing hormone in the prepubertal and pubertal female rhesus monkey as measured by in vivo push-pull perfusion in the stalk-median eminence.

The role of the excitatory amino acid glutamate, N-methyl D-aspartate (NMDA) receptor agonist, in stimulating in vivo luteinizing hormone-releasing hormone (LHRH) release in the stalk-median eminence of conscious prepubertal and pubertal female rhesus monkeys was evaluated using push-pull perfusion. In Exp 1, the effects of i.v. bolus injection of N-methyl D,L-aspartate (NMA) on LHRH release were examined. Injection of NMA induced an increase in LHRH release in all maturational stages of monkeys. Although the LHRH response to NMA tended to be larger in the older groups, only the duration of the LHRH response in the midpubertal group was significantly longer than that in the prepubertal group. In Exp 2, the effects of direct infusion of NMA (0.1, 1, and 100 microM) into the stalk-median eminence on LHRH release were similarly examined. NMA infusion stimulated LHRH release in pubertal monkeys, whereas it did not induce any consistent changes in LHRH release in prepubertal monkeys except for the highest dose. These data suggest that: 1) the systemic injection of NMA is more effective than direct infusion of NMA; and 2) the prepubertal LHRH neurosecretory system is capable of responding to NMDA, although the responsiveness may undergo developmental changes. Therefore, stimulation of NMDA receptors may contribute to the pubertal changes in the LHRH neurosecretory activity.

An increase in glutamate release follows a decrease in gamma aminobutyric acid and the pubertal increase in luteinizing hormone releasing hormone release in the female rhesus monkeys.

Previously we have shown that release of gamma-aminobutyric acid (GABA) in the stalk-median eminence (S-ME) is high in prepubertal monkeys and that a decrease in GABA release triggers the onset of puberty. However, it is still unclear how disinhibition of the luteinizing hormone releasing hormone (LHRH) neuronal system from GABA input is followed (or accompanied) by an increase in stimulatory signals, such as glutamatergic input to LHRH neurons. To clarify the temporal relationship between the reduction of the GABAergic inhibitory signal and the enhancement of the glutamatergic stimulatory signal in the control of LHRH release at the onset of puberty, we conducted two experiments using a push-pull perfusion method. In the first experiment, we measured developmental changes in release of LHRH, GABA, and glutamate in the S-ME. LHRH levels were very low in prepubertal monkeys, increased to higher levels in early pubertal monkeys, with the highest LHRH levels occurring in mid-pubertal monkeys. As we previously observed, GABA levels were high in prepubertal monkeys and then decreased in early- and mid-pubertal monkeys. In contrast, glutamate levels were very low in prepubertal monkeys, increased dramatically in early pubertal monkeys, and then slightly decreased in mid-pubertal monkeys, although mid-pubertal levels remained much higher than prepubertal levels. In the second experiment, we measured GABA, glutamate and LHRH in the same samples obtained from prepubertal monkeys which were infused with an antisense oligodeoxynucleotide (AS) for glutamic acid decarboxylase (GAD) 67 mRNA into the S-ME. GAD67 is a catalytic enzyme for GABA synthesis from glutamate, and AS GAD67 mRNA interferes with GAD67 synthesis. Infusion of the AS GAD67 induced a decrease in GABA release, which subsequently resulted in an increase in LHRH release. Surprisingly, glutamate release also increased several hours after the decrease in GABA release, and the increased LHRH release continued. These data are interpreted to mean that a decrease in GABA synthesis by interference with GAD67 synthesis and the reduction of GABA release in the S-ME trigger an increase in LHRH release, but that a subsequent increase in glutamate release in the S-ME further contributes to the pubertal increase in LHRH release at the onset of puberty. The data further support our hypothesis that GAD plays an important role in the mechanism of the onset of puberty.

Glucagon-like peptide-1 stimulates luteinizing hormone-releasing hormone secretion in a rodent hypothalamic neuronal cell line.

To examine the influence of the putative satiety factor (GLP-1) on the hypothalamo-pituitary-gonadal axis, we used GT1-7 cells as a model of neuronal luteinizing hormone- releasing hormone (LHRH) release. GLP-1 caused a concentration-dependent increase in LHRH release from GT1-7 cells. Specific, saturable GLP-1 binding sites were demonstrated on these cells. The binding of [125I]GLP-1 was time-dependent and consistent with a single binding site (Kd = 0.07+/-0.016 nM; binding capacity = 160+/-11 fmol/mg protein). The specific GLP-1 receptor agonists, exendin-3 and exendin-4, also showed high affinity (Ki = 0.3+/-0.05 and 0.32+/-0.06 nM, respectively) as did the antagonist exendin-(9-39) (Ki = 0.98+/-0.24 nM). At concentrations that increased LHRH release, GLP-1 (0.5-10 nM) also caused an increase in intracellular cAMP in GT1-7 cells (10 nM GLP-1: 7.66+/-0.4 vs. control: 0.23+/-0.02 nmol/mg protein; P < 0.001). Intracerebroventricular injection of GLP-1 at a single concentration (10 microg) produced a prompt increase in the plasma luteinizing hormone concentration in male rats (GLP-1: 1.09+/-0.11 vs. saline: 0.69+/-0.06 ng/ml; P < 0.005). GLP-1 levels in the hypothalami of 48-h-fasted male rats showed a decrease, indicating a possible association of the satiety factor with the low luteinizing hormone levels in animals with a negative energy balance.

Nitric oxide synthase content of hypothalamic explants: increase by norepinephrine and inactivated by NO and cGMP.

Release of luteinizing hormone (LH)-releasing hormone (LHRH), the hypothalamic peptide that controls release of LH from the adenohypophysis, is controlled by NO. There is a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers in the lateral median eminence, intermingled with terminals of the LHRH neurons. To study relations between NOS and LHRH in this brain region, we measured NOS activity in incubated medial basal hypothalamus (MBH). NOS converts [14C]arginine to equimolar quantities of [14C]citrulline plus NO, which rapidly decomposes. The [14C]citrulline serves as an index of the NO produced. NOS basal activity was suppressed by incubation of the tissue with an inhibitor of NOS, nitroarginine methyl ester (NAME) (10(-5) M). Furthermore, incubation of MBH explants for 30 min with norepinephrine (NE) increased NOS activity and the increase was prevented by prazosine (10(-5) M), an alpha 1-adrenergic receptor blocker; however, direct addition of NE to the tissue homogenate or to a preparation of MBH synaptosomes did not alter enzyme activity, which suggested that NE increased the content of NOS during incubation with the tissue. After purification of NOS, the increase in enzyme content induced by NE was still measurable. This indicates that within 30 min NE increased the synthesis of NOS in vitro. Incubation of MBH or the MBH homogenate with various concentrations of sodium nitroprusside (NP), a releaser of NO, reduced NOS activity at high concentrations (> or = 0.9 mM), which were associated with either a reduction of stimulation or a plateau of LHRH release. Finally, incubation of either MBH or the homogenate with cGMP, a major mediatior of NO action, at concentrations that increased LHRH release also reduced NOS activity. These results indicate that NO at high concentrations can inactivate NOS and that cGMP can also inhibit the enzyme directly. Therefore, the increased NOS activity induced by activation of alpha 1 receptors by NE is inhibited by NO itself and a principal product of its activity, cGMP, providing negative feedback on NOS. In central nervous system (CNS) infections with high concentrations of inducible NOS produced by glial elements, the high concentrations of NO and cGMP produced may suppress LHRH release, resulting in decreased gonadotropin and gonadal steroid release.

Role of glutamic acid decarboxylase in the prepubertal inhibition of the luteinizing hormone releasing hormone release in female rhesus monkeys

To investigate further the role of GABA in the onset of puberty, this study examines whether glutamic acid decarboxylase (GAD), the catalytic enzyme for GABA synthesis, is involved in the suppression of luteinizing hormone releasing hormone (LHRH) before puberty in rhesus monkeys. First, both GAD67 and GAD65 mRNAs were detectable by reverse transcription-PCR analysis in the preoptic area, medio-basal hypothalamus, posterior hypothalamic area, and hippocampus of the monkey brain. Second, effects of antisense oligodeoxynucleotides (D-oligos) for GAD67 and GAD65 mRNAs on LHRH release were examined in conscious female rhesus monkeys at the prepubertal stage using a push-pull perfusion method. The GAD67 or GAD65 antisense D-oligos or scrambled D-oligos were infused directly into the stalk-median eminence. Both the GAD67 and the GAD65 antisense D-oligos induced a large and prompt increase in LHRH release, whereas the scrambled D-oligos did not induce any significant effect. The results suggest that the removal of GABA inhibition by interfering with GAD synthesis is effective in increasing LHRH release in prepubertal monkeys. Third, the specificity of the antisense D-oligos on GAD levels was examined by incubating basal hypothalami with D-oligos in vitro and subsequent Western blot analysis. The antisense D-oligos consistently decreased the proteins GAD67 and GAD65 compared with respective control D-oligos. We conclude that the decrease of tonic GABAergic inhibition and maturational changes in GAD synthesis may be critical factors for the onset of puberty in nonhuman primates.

Steroid modulation of neuropeptide Y-induced luteinizing hormone releasing hormone release from median eminence fragments from male rats.

Neuropeptide Y (NPY) has been shown to stimulate hypothalamic release of luteinizing hormone-releasing hormone (LHRH) both in vitro and in vivo. In female rats, NPY facilitation of LHRH release is greatly augmented in advance of preovulatory LHRH surges, likely via the actions of ovarian steroids. However, the role of NPY in regulating LHRH release in male rats and the effects of testicular hormones on LHRH responses to NPY in males are not well understood. The objective of the present studies was to determine whether NPY stimulates LHRH release in vitro from hypothalamic tissue of male rats, and whether these effects could be modulated by testosterone (T). Mediobasal hypothalamic (MBH) or median eminence (ME) fragments from either sham-operated or castrated male rats (7 days) were placed in superfusion chambers and superfused with M199 for a 30-min baseline, 30-min challenge with NPY (10(-7)M), and a final 30-min challenge with 56 mM KCl. One-milliliter fractions were collected every 10 min and average LHRH release values over the 30-min periods were compared among groups. NPY (10(-7)M) produced a significant increase in LHRH release from the MBH and ME from intact animals. In contrast, the same dose of NPY did not stimulate LHRH release from tissues from castrated animals; only with a higher dose of NPY (10(-6)M) were the effects of NPY on LHRH release significant. Potassium challenge (56 mM KCl) significantly stimulated LHRH release from the ME of both intact and castrate male rats suggesting that all tissues were able to respond to a stimulus, and that castration did not alter the responsiveness of the LHRH neuron to a nonspecific secretagogue. To determine the extent to which T regulates the sensitivity of LHRH responses to NPY, male rats were castrated and implanted with T capsules that maintained either low (1.24 +/- 0.06 ng/ml) or high (2.17 +/- 0.31 ng/ml) physiological plasma levels of T. Treatment with the higher dose of T restored the ability of NPY to stimulate LHRH release from the ME tissues. These results demonstrate that NPY stimulates LHRH release from the hypothalamus in vitro, and that gonadal steroids, in this case T and/or its metabolites, modulate the responsiveness of the LHRH neuron to NPY. Based on these data from intact and castrate-derived tissues, it appears that steroids are necessary to maintain LHRH responsiveness to NPY receptor stimulation.

Control of luteinizing hormone-releasing hormone pulse generation in nonhuman primates.

1. The pulsatile release of luteinizing hormone-releasing hormone (LHRH) is critical for reproductive function. However, the exact mechanism of LHRH pulse generation is unclear. The purpose of this article is to review the current knowledge on LHRH pulse generation and to discuss a series of studies in our laboratory. 2. Using push-pull perfusion in the stalk-median eminence of the rhesus monkey several important facts have been revealed. There is evidence indicating that LHRH neurons themselves have endogenous pulse-generating mechanisms but that the pulsatility of LHRH release is also modulated by input from neuropeptide Y (NPY) and norepinephrine (NE) neurons. The release of NPY and NE is pulsatile, with their pulses preceding or occurring simultaneously with LHRH pulses, and the neuroligands NPY and NE and their agonists stimulate LHRH pulses, while the antagonists of the ligands suppress LHRH pulses. 3. The pulsatile release of LHRH increases during the estrogen-induced LH surge as well as the progesterone-induced LH surge. These increases are partly due to the stimulatory effects of estrogen and progesterone on NPY neurons. 4. An increase in pulsatile LHRH release occurs at the onset of puberty. This pubertal increase in LHRH release appears to be due to the removal of tonic inhibition from gamma aminobutyric acid (GABA) neurons and a subsequent increase in the inputs of NPY and NE neurons to LHRH neurons. 5. There are indications that additional neuromodulators are involved in the control of the LHRH pulse generation and that glia may play a role in coordinating pulses of the release of LHRH and neuromodulators. 6. It is concluded that the mechanism generating LHRH pulses appears to comprise highly complex cellular elements in the hypothalamus. The study of neuronal and nonneuronal elements of LHRH pulse generation may serve as a model to study the oscillatory behavior of neurosecretion.

Gamma-Aminobutyric acid is an inhibitory neurotransmitter restricting the release of luteinizing hormone-releasing hormone before the onset of puberty.

To test the hypothesis that the pubertal increase in luteinizing hormone-releasing hormone (LHRH) release is withheld by a dominant inhibitory neuronal system, the role of gamma-aminobutyric acid (GABA), a known inhibitory neurotransmitter, in the control of LHRH release was examined in conscious female monkeys at the prepubertal and pubertal stages using a push-pull perfusion method. GABA, bicuculline (a GABAA receptor blocker), and 2-hydroxysaclofen (a GABAB receptor blocker) were directly infused into the stalk-median eminence while perfusates were collected for LHRH determination. Bicuculline, but not saclofen, induced a large and prompt increase in LHRH release in prepubertal monkeys, whereas it stimulated LHRH release slightly in pubertal monkeys. In contrast, GABA suppressed LHRH release in pubertal, but not prepubertal, monkeys. These differential effects of GABA and the GABA antagonist on LHRH release in the two developmental stages were due to an age factor rather than to the steroid hormonal background. Moreover, GABA release in the stalk-median eminence of prepubertal monkeys was much higher than that in pubertal monkeys. Thus, the results suggest that in the prepubertal period there is a powerful GABA inhibition of the LHRH neurosecretory system: infusions of GABAA, but not GABAB, antagonists stimulate LHRH release by removal of the endogenous GABA inhibition, whereas exogenous GABA is ineffective because of high endogenous GABA levels. The decrease of this tonic inhibition may be a key factor for the onset of puberty in non-human primates.

Role of nitric oxide in the control of luteinizing hormone-releasing hormone release in vivo and in vitro.

Nitric oxide (NO) synthase, the enzyme which converts arginine into citrulline plus NO, a highly active free radical, has been found in many neurons in the brain, including neurons in the hypothalamus. Our previous experiments showed that norepinephrine-induced prostaglandin E2 release from hypothalamic explants incubated in vitro is mediated by NO. Since the release of luteinizing hormone-releasing hormone (LHRH) is also driven by norepinephrine and prostaglandin E2, we hypothesized that NO might also control pulsatile release of LHRH in vivo, resulting in turn in pulsatile release of luteinizing hormone (LH). To ascertain the role of NO in control of pulsatile LH release in vivo, an inhibitor of NO synthase, NG-monomethyl-L-arginine (NMMA), was microinjected into the third cerebral ventricle (1 mg/5 microliters) of conscious castrate male rats at time 0 and 60 min later; blood samples were taken every 10 min during this period. NMMA blocked pulsatile LH release within 20 min, and plasma LH concentration declined further without pulses after the injection at 60 min. Pulsatile release of LH was not altered in diluent-injected controls. NMMA did not alter pulsatile release of follicle-stimulating hormone, which suggests that its release does not require NO. Incubation of medial basal hypothalami with norepinephrine (10 microM) induced an increase in LHRH release that was inhibited by NMMA (300 microM). NMMA alone did not alter basal LHRH release, whereas it was augmented by sodium nitroprusside (100 microM), which releases NO spontaneously. This augmentation was prevented by hemoglobin (2 micrograms/ml), which binds the NO released by nitroprusside. Our previous experiments showed that norepinephrine-induced release of prostaglandin E2 is mediated by NO. Nitric oxidergic neurons were visualized in the median eminence adjacent to the LHRH terminals. The combined in vivo and in vitro results indicate that the pulsatile release of LHRH induced by norepinephrine is brought about by alpha 1-adrenergic activation of NO synthase. NO then induces prostaglandin E2 release that activates exocytosis of LHRH secretory granules into the portal vessels to induce pulsatile LH release.

Role of 5-hydroxytryptamine receptors on luteinizing-hormone-releasing hormone release in the ovariectomized, estradiol-treated rat

The present experiments examined the effect of ketanserin [5-hydroxytryptamine-2 (5-HT 2) antagonist] and 8-hydroxy-2-(di- n-propylamino) tetralin (8-OH-DPAT) (5-HT 1 agonist) on the in vitro release of luteinizing-hormone-releasing hormone (LHRH) from the medial basal hypothalamuspreoptic area-suprachiasmatic nucleus region (MBH-POA-SCN) of ovariectomized (OVX), estradiol-(E 2) treated rats using in vitro superfusion techniques. Regularly cycling female Holtzman rats (250–300 g) were maintained on a photoperiod of 0500–1900 h light at 22 ± 2° C. Rats were ovariectomized (25–30 days) and received Silastic E 2 implants (150 μg E 2/ml sesame oil) SC 48 h prior to the in vitro superfusion. Following a control period of Krebs-Ringer Phosphate (KRP) superfusion, ketanserin (5HT 2 receptor antagonist, 1 × 10 −6 M) significantly increased LHRH release ( p < 0.05). Subsequent superfusion of 5-HT (1 × 10 −8 M) significantly decreased ( p < 0.05) the effect of ketanserin on LHRH release. The 5-HT 2 antagonist Lilly 53857 (1 × 10 −6 M or 1 × 10 −5 M) did not increase LHRH release above control levels. Neither 5-HT nor quipazine had a significant effect on LHRH release at 1 × 10 −6 M. Superfusion of 8-OH-DPAT (5-HT 1 receptor agonist 1 × 10 −5 M) significantly ( p < 0.01) increased LHRH release but subsequent superfusion of 8-OH-DPAT + pindolol (mixed 5-HT 1a,1b and a β-adrenergic receptor antagonist, 1 × 10 −6 M) or pindolol alone had no effect on LHRH release. These results suggest that the 5-HT 1 receptor plays a role in LHRH release and this effect may be related to the opposing effects of postsynaptic and autoreceptors. However, the failure of Lilly 53857 to reproduce the stimulatory effect of ketanserin on LHRH release suggests that 5-HT 2 receptors in the MBH-POASCN may not modify LH release during the estrous cycle.

Seasonal changes in thyroid-gonadal interactions in the edible dormouse, Glis glis.

This study was conducted to determine changes in thyroid-gonadal interaction in the edible dormouse during the phase of the annual cycle that corresponds to the end of the breeding season (from June to September). We evaluated intra-hypothalamic luteinizing hormone-releasing hormone (LHRH) content, and plasma concentrations of luteinizing hormone (LH), testosterone, thyroid-stimulating hormone (TSH) and thyroxine (T4) in three groups of dormice: (1) controls; (2) dormice receiving sufficient T4 supplementation to maintain June levels in control animals until September, thus counteracting the seasonal reduction of T4 that normally begins in July; and (3) thyroidectomized dormice. The effect of thyroidectomy was only detectable in June, when plasma T4 concentration in the control group was maximal, and consisted of a significant decrease in plasma testosterone levels. This provides strong support for the hypothesis that thyroid function positively influences gondal function during the breeding season. The T4 supplementation resulted in a decrease in hypothalamic LHRH concentration, suggesting that an increased LHRH release led to the observed stimulated hypophyseal secretion of LH in June and September and the increased circulating testosterone levels in September. There was no detectable effect in July and August. These results show that thyroid axis activation of the hypothalamic-pituitary-gondal system is only possible during certain phases of the annual cycle, particularly evidenced here during the breeding season. They also reinforce our conclusions drawn from the thyroidectomy results. Conversely, the summer testicular regression which normally occurs after the breeding season is no longer controlled by plasma T4 levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Mating Behavior on Luteinizing Hormone-Releasing Hormone Release in Female Rabbits as Monitored with Push-Pull Cannulae

To examine the effect of mating behavior on luteinizing hormone-releasing hormone (LHRH) release, intact New Zealand female rabbits were implanted with push-pull cannulae (PPC) aimed at the tuberal region of the hypothalamus and perfused with modified Krebs-Ringer phosphate medium at 11-13 microliters/min. In the mating experiments, does (n = 10) were initially perfused for a control period of 60-170 min followed by a mating period (100-160 min) which included the introduction of the male rabbit for an average time period of 30 min. Two groups of LHRH release patterns were observed: positive and negative responders. In the positive LHRH responders (n = 5), a clear rapid increase in LHRH release following mounting by the male occurred with a significant increase in the mean LHRH release (1.83 +/- 0.33 to 3.27 +/- 0.80 pg/10 min, p less than 0.040), in the mean LHRH amplitude (1.97 +/- 0.46 to 4.33 +/- 1.29 pg, p less than 0.022) and in the amplitude of the largest LHRH pulse (2.13 +/- 0.43 to 7.58 +/- 3.65 pg, p less than 0.022). In the negative LHRH responders (n = 5), no changes in LHRH release were detected although all rabbits ovulated, with some becoming pregnant. It appears from histological analysis that the difference between these two patterns of responses following mating are due to different cannula placements. In the positive responders, the tip of the PPC was localized in the tuberal region whereas in the negative responders, the placements were more dorsal and, in some cases, anterior.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-Like Growth Factor I: A Possible Metabolic Signal Involved in the Regulation of Female Puberty

In both rats and primates, including humans, serum levels of insulin-like growth factor I (IGF-I) increase during the onset of puberty, suggesting a role for IGF-I in this process. We examined the ability of IGF-I to affect the release of hypothalamic luteinizing hormone releasing hormone (LHRH) in prepubertal female rats. Our results indicate that IGF-I acts on the median eminence, which contains the highest density of type 1 IGF receptors in the brain, to elicit a dose-related increase in LHRH release. In this regard, a minimal effective dose of 10 ng/ml (p less than 0.05) and a maximal effective dose of 100 ng/ml (p less than 0.01) was observed. IGF-II and insulin were one order of magnitude less effective. The results demonstrate that IGF-I has the capability to act directly upon the median eminence to effect release of LHRH, thereby suggesting a role for IGF-I in facilitating peripubertal changes in LHRH release. Thus, IGF-I may represent one of the 'metabolic signals' thought to be involved in the initiation of puberty.

Involvement of transforming growth factor alpha in the release of luteinizing hormone-releasing hormone from the developing female hypothalamus.

Little is known about the presence of trophic factors in the hypothalamus and the role they may play in regulating the functional development of hypothalamic neurons. We have investigated the ability of epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) to affect the release of luteinizing hormone-releasing hormone (LHRH), the neuropeptide that controls reproductive development. We have also determined whether the genes encoding EGF and TGF-alpha are expressed in the prepubertal female hypothalamus. Northern blot analysis of poly(A)+ RNA utilizing a single-stranded EGF cDNA probe failed to reveal the presence of EGF mRNA in either the hypothalamus or the cerebral cortex at any age studied (fetal day 18 to postnatal day 36). In contrast, both a complementary RNA probe and a double-stranded TGF-alpha cDNA recognized in these regions a 4.5-kilobase (kb) mRNA species identical to TGF-alpha mRNA. The abundance of TGF-alpha mRNA was 3-4 times greater in the hypothalamus than in the cerebral cortex. Both EGF and TGF-alpha (2-100 ng/ml) elicited a dose-related increase in LHRH release from the median eminence of juvenile rats in vitro. They also enhanced prostaglandin E2 (PGE2) release. The transforming growth factors TGF-beta 1 and -beta 2 were ineffective. Only a high dose of basic fibroblast growth factor was able to increase LHRH and PGE2 release. Blockade of the EGF receptor transduction mechanism with RG 50864, a selective inhibitor of EGF receptor tyrosine kinase activity, prevented the effect of both EGF and TGF-alpha on LHRH and PGE2 release but failed to inhibit the stimulatory effect of PGE2 on LHRH release. Inhibition of prostaglandin synthesis abolished the effect of TGF-alpha on LHRH, indicating that PGE2 mediates TGF-alpha-induced LHRH release. The results indicate that the effect of EGF and TGF-alpha on LHRH release is mediated by the EGF/TGF-alpha receptor and suggest that TGF-alpha rather than EGF may be the physiological ligand for this interaction. Since in the central nervous system most EGF/TGF-alpha receptors are located on glial cells, the results also raise the possibility that--at the median eminence--TGF-alpha action may involve a glial-neuronal interaction, a mechanism by which the trophic factor first stimulates PGE2 release from glial cells, and then PGE2 elicits LHRH from the neuronal terminals.