LHRH Release Research Articles

Effects of Tumor-Induced Hyperprolactinemia on LH Secretion following Stimulation of the Medial Preoptic Area, Pituitary Responsiveness and the Estrogen-Induced LH Surge

In the present study we utilized the 7315a PRL- and ACTH-secreting tumor to induce a hyperprolactinemic (HP) state sufficient to profoundly suppress the postcastration LH rise in female rats. Tumor-induced prolactin levels which ranged 2,000-3,000 ng/ml substantially reduced the LH rise in both ovariectomized (OVX) and OVX + estradiol-17 beta (E2)-treated rats. Bilateral electrochemical stimulation (ECS, 100 microA DC for 60 s) of the ventral diagonal band of Broca-medial preoptic area (DBB-MPOA) resulted in comparable LH responses in control and HP rats in the presence of absence of estradiol. Transient decreases in PRL release occurred following ECS of the DBB-MPOA. Pituitary responsiveness was assessed with two LHRH challenges spaced 60 min apart at doses of 25 and 50 ng LHRH/100 g body weight. The mean maximal LH increments (delta LH) to some of these LHRH challenges were decreased in HP rats. Finally, the LH surge induced in the afternoon in OVX + E2-treated rats was diminished 71% by the presence of the PRL-secreting tumor.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Electrical Stimulation of the Medial Basal Hypothalamus on thein VivoRelease of Luteinizing Hormone-Releasing Hormone in the Prepubertal and Peripubertal Female Monkey*

In this study the hypothesis that the LHRH neurosecretory system of the prepubertal female monkey has the capacity to function in a manner comparable to that of monkeys in more mature stages of development was tested. Using push-pull perfusion in the stalk-median eminence, effects of electrical stimulation of the medial basal hypothalamus on in vivo LHRH release were determined in conscious prepubertal, early pubertal, and midpubertal monkeys. After a 180-min period of baseline sample collection, electrical stimulation was applied six times at 90-min intervals via a monopolar electrode, the tip of which was 1-2 mm rostro-dorsal to the perfusion site. Control experiments were performed in the same manner, but without electrical stimulation. During control perfusions, the mean LHRH level remained stable. Mean (+/- SEM) LHRH release for the entire perfusion period in control experiments was 0.5 +/- 0.2, 2.4 +/- 0.4, and 2.2 +/- 0.7 pg/ml.10 min for the prepubertal (n = 6), early pubertal (n = 4), and midpubertal (n = 6) groups, respectively. Mean LHRH release in the prepubertal group was significantly lower than that in either of the older groups (P less than 0.05). In contrast, in all three age groups, repeated electrical stimulation of the medial basal hypothalamus resulted in 1) a short latency increase in LHRH release occurring within 20 min after each stimulation, and/or 2) a gradual increase in mean LHRH release over several hours. In the prepubertal group (n = 4), mean LHRH levels were 0.8 +/- 0.5 pg/ml.10 min during the 90 min before the first electrical stimulation and increased to 6.1 +/- 2.9 pg/ml.10 min during the 90 min after the sixth stimulation. This degree of responsiveness was similar to that of the older age groups. Mean LHRH levels before stimulation were 1.3 +/- 0.6 and 2.4 +/- 1.1 pg/ml.10 min in the early pubertal (n = 5) and midpubertal (n = 5) groups, respectively, and increased to 7.8 +/- 3.5 and 6.0 +/- 1.8 pg/ml.10 min, respectively, after the sixth stimulation. These increases in LHRH concentration with electrical stimulation were significant for all three age groups (P less than 0.03-0.001), while there were no significant differences between age groups. The temporal patterns of these responses suggest that electrical stimulation elicits LHRH release with a similar magnitude in all three age groups by 1) depolarizing LHRH neurons directly, and/or 2) stimulating multineuronal systems that synapse with LHRH neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Alterations in Opioid Parameters in the Hypothalamus of Rats with Estradiol-Induced Polycystic Ovarian Disease*

The distribution and density of selectively labeled mu-, delta-, and kappa-opioid binding sites were examined by in vitro radioautography in the hypothalamus of normal, estradiol valerate (EV)-injected, and estradiol (E2)-implanted female rats. Hypothalamic beta-endorphin concentration was also examined by RIA in these three groups of animals. Quantitative analysis of film radioautographs demonstrated a selective increase in mu-opioid binding in the medial preoptic area of EV-treated, but not of E2-implanted rats. However, both these estrogenized groups exhibited a reduction in the density of delta-opioid binding in the suprachiasmatic nucleus. Statistically significant changes between either estrogenized groups were not observed for kappa-opioid binding. Results on the hypothalamic concentration of beta-endorphin indicated a marked reduction in EV-injected animals with respect to controls. In contrast, the E2-implanted animals exhibited beta-endorphin concentrations similar to controls. The present results confirm the increase in opioid receptor binding previously reported in the hypothalamus of EV-treated rats and further demonstrate that this increase is confined to the medial preoptic area and exclusively concerns mu-opioid receptors. The concomitant reduction in beta-endorphin levels observed in the same group of animals suggests that the observed increase in mu-opioid binding could reflect a chronic up-regulation of the receptor in response to compromised beta-endorphin input. Given the restriction of this effect to the site of origin of LHRH neurons and the demonstrated inhibitory role of opioids on LHRH release, it is tempting to postulate that such up-regulation could lead to the suppression of the plasma LH pattern that characterizes polycystic ovarian disease in the EV-treated rat.

Cocaine Effects on Luteinizing Hormone-Releasing Hormone-Stimulated Anterior Pituitary Hormones in Female Rhesus Monkey*

The effects of acute cocaine administration on synthetic LHRH-stimulated anterior pituitary hormones (LH, FSH, and PRL) were studied in 6 female rhesus monkeys during the follicular phase of the menstrual cycle (days 4-7). Integrated plasma samples were collected every 10 min for 40 min before iv administration of cocaine (0.4 or 0.8 mg/kg) or an equal volume of vehicle control solution. Synthetic LHRH (100 micrograms, iv) was administered 10 min after cocaine or placebo-cocaine administration, and 10 plasma samples were collected for an additional 100 min. LHRH stimulated a significant increase in LH within 10 min after placebo-cocaine administration (P less than 0.05) and after each dose of cocaine (P less than 0.0001). Cocaine (0.4 mg/kg) significantly enhanced LHRH stimulation of LH compared to placebo or 0.8 mg/kg cocaine administration (P less than 0.01). FSH increased significantly within 20-30 min after LHRH alone (P less than 0.008) and after 0.4 mg/kg cocaine (P less than 0.0001). LHRH-stimulated FSH levels also were significantly higher after 0.4 mg/kg cocaine than after placebo or 0.8 mg/kg cocaine (P less than 0.01). These data indicate that cocaine does not suppress LHRH stimulation of pituitary gonadotropins, and low doses of cocaine significantly enhance LH and FSH release. Consequently, cocaine does not compromise anterior-pituitary function at the level of the gonadotroph and may stimulate hypothalamic release of endogenous LHRH. PRL levels were unchanged by LHRH and placebo-cocaine administration. After LHRH and cocaine administration, PRL levels decreased significantly (P less than 0.05-0.01) and remained suppressed throughout the 110-min postcocaine sampling period. These data indicate that cocaine's significant suppression of PRL is not blocked by LHRH. These findings are consistent with dopaminergic inhibitory control of PRL and suggest that cocaine's inhibition of dopamine reuptake down-regulates pituitary lactotroph activity in rhesus monkey.

Galanin Stimulates Luteinizing Hormone-Releasing Hormone Secretion from Arcuate Nucleus-Median Eminence Fragmentsin Vitro: Involvement of an α-Adrenergic Mechanism

Galanin (GAL), a 29-amino acid peptide originally isolated from porcine intestine, has been shown to be widely distributed not only in the gut, but also in the central nervous system. Several studies have shown that GAL participates in the hypothalamic regulation of PRL, GH, and LH secretion. In this study, we evaluate the effects of rat GAL (rGAL) on LHRH and prostaglandin (PG) E2 release from arcuate nucleus-median eminence fragments in vitro. Fragments were incubated for 30-min periods in Krebs-Ringer bicarbonate buffer containing the different test substances. The addition to the medium of rGAL in concentrations ranging from 5-1000 nM increased the release of both LHRH and PGE2 in a concentration-dependent manner. The ED50 values were approximately 55 and 80 nM for LHRH and PGE2, respectively. rGAL-induced LHRH and PGE2 release were related, as suggested by the finding that the addition to the medium of indomethacin (10 microM), a PG synthesis blocker, completely blocked rGAL-induced LHRH release. In addition, an active catecholaminergic system appears to be necessary for obtaining the stimulatory effect of rGAL. The addition to the medium of the alpha-adrenergic blocker phentolamine or prazosin impaired the ability of rGAL to release both LHRH and PGE2. rGAL-induced stimulation of LHRH and PGE2 release was blocked by phentolamine at doses of 1-10 microM, while prazosin was able to block it at doses as low as 0.1 microM. In summary, rGAL stimulates LHRH and PGE2 release from arcuate nucleus-median eminence fragments in vitro in a dose-dependent fashion. Such an effect is blocked by both indomethacin and alpha-adrenergic blockers, indicating that rGAL-induced stimulation of LHRH secretion is exerted through alpha-adrenergic receptors and requires PGE2 as an intracellular mediator.

Role of Neuropeptide Y in Reproductive Functiona

NPY acts both at the hypothalamus and the anterior pituitary gland to modulate reproductive hormone secretion. Within the hypothalamus, NPY stimulates LHRH secretion in the presence of physiological levels of estrogen and suppresses pulsatile LHRH release following ovariectomy. Intracerebroventricular injection of NPY antiserum blocks or delays the LH surge in steroid-primed ovariectomized rats, thereby adding support for a physiological role of NPY in the neuroendocrine events preceding ovulation. Blockade of alpha 2 adrenergic receptors decreases NPY-stimulated LH release in steroid-primed rats implying a potential noradrenergic mediation of NPY activity. Physiological levels of progesterone do not augment, and may actually suppress NPY-induced LHRH secretion in vitro from median eminences obtained from estrogen-primed ovariectomized rats. The physiological role of progesterone, if any, in modulating NPY effects on LHRH release remains to be determined. Little, if anything, is known about the NPY receptor in the median eminence or the intracellular mechanisms which transduce the NPY signal into activation of LHRH release in estrogen-treated ovariectomized rats although translocation of intracellular calcium is required. Equally puzzling is the mechanism of desensitization of the LHRH-releasing mechanisms of the median eminence of ovariectomized rats or the specific site of NPY suppression of pulsatile LHRH secretion. NPY is released into the hypothalamo-hypophysial portal circulation and this appears correlated with LHRH secretion before the LH surge. NPY affects LH and FSH release from anterior pituitary cells in vitro and enhances LHRH-induced LH secretion. Taken together, the studies described above suggest an important physiological role for NPY as a modulator of neuroendocrine activity which culminates in the preovulatory surge of LH.

Endogenous Opioid Peptides Mediate the Interleukin-1 Induced Inhibition of the Release of Luteinizing Hormone (LH)-Releasing Hormone and LH*

We have reported recently that central administration of both the alpha- and beta-subtypes of the cytokine interleukin-1 (IL-1) inhibited the estrogen-progesterone-induced LH surge in ovariectomized (ovx) rats. This inhibition was probably due to a central effect, since IL-1 alpha and IL-1 beta also suppressed the in vitro LHRH output from the hypothalami of steroid-primed ovx rats. Whether IL-1 inhibits LHRH release by a direct action or via some other neuronal system is not known. Since IL-1 reportedly stimulates the release of POMC peptides, which are known to be inhibitory to the LHRH-LH axis, we have tested the hypothesis that the inhibitory influence of IL-1 may be mediated via activation of hypothalamic opioid peptides. Ovx rats, preimplanted with cannulae in the third ventricle of the brain, were injected with 30 micrograms estradiol benzoate, followed by 2 mg progesterone 48 h later. Three hours after P injection, IL-1 alpha, IL-1 beta, or saline (SAL) was injected intracerebroventricularly (30 ng/3 microliters) at 1300 h, followed immediately by iv infusion of SAL or the opiate antagonist naloxone hydrochloride (NAL; 2 mg/0.6 ml.h) for 2 h. Plasma LH levels were measured in blood samples withdrawn hourly until 1800 h. Both IL-1 alpha and IL-1 beta blocked the afternoon LH surge. NAL infusion into control SAL-injected rats did not alter the LH surge; however, it reversed the IL-1 alpha- and IL-1 beta-induced suppression of the LH surge. To determine whether this reversal of IL-1 suppression of the LH surge was due to NAL action at the hypothalamic level, the preoptic area-medial basal hypothalamus of similarly primed ovx rats was obtained at 1300 h and incubated in vitro in the presence of 10 nM IL-1 alpha or IL-1 beta with or without 100 micrograms/ml NAL. Both subtypes of IL-1 suppressed LHRH output significantly. NAL alone did not affect LHRH release, but it completely reversed the inhibitory effects of the cytokine on LHRH release. These results suggest that IL-1 alpha and IL-1 beta inhibit LHRH-LH release by stimulating the activity of hypothalamic endogenous opioid peptide systems.

Neuropeptide Y Regulation of LHRH Release in Ewe Median Eminence

Neuropeptide Y Regulation of LHRH Release in Ewe Median Eminence

Hypothalamic Prolactin Stimulates the Release of Luteinizing Hormone-Releasing Hormone from Male Rat Hypothalamus*

Previous works from our laboratory and others have shown that there is a PRL-like immunoreactive protein with immunological, chromatographic, and biological characteristics identical to those of pituitary PRL, and this is widely distributed in the rat central nervous system. Since pituitary PRL is important in controlling hypothalamic LHRH release, we have hypothesized that hypothalamic PRL-like immunoreactive protein might serve a similar role, that of an endogenous neuromodulator influencing hypothalamic LHRH release. To this end, we have examined the effect of PRL antiserum and normal rabbit serum on the release of immunoreactive LHRH from rat hypothalamic fragments cultured in vitro. In the first experiment, LHRH release from hypothalami of intact rats, bathed in PRL antiserum (1:200 in Krebs-Ringer bicarbonate buffer), was significantly lower than that from hypothalami bathed in normal rabbit serum (1:200 in Krebs-Ringer bicarbonate buffer) for 90 min of incubation. It was, however, possible that the PRL, immunoneutralized in the first experiment, was material that represented contamination from pituitary PRL. Therefore, we repeated the experiment using hypothalami from animals that had been hypophysectomized 2 weeks before death. Again, PRL antibody significantly inhibited the release of LHRH compared with that by hypothalami incubated in normal rabbit serum. Since testosterone is important to LHRH synthesis, a third experiment was carried out using hypothalami from hypophysectomized male rats that had been implanted sc with testosterone-containing capsules 72 h before death. By 72 h serum testosterone levels had normalized. PRL antibody added to medium containing hypothalamic explants from these animals substantially inhibited in vitro LHRH release, a pattern essentially similar to that seen in intact and hypophysectomized animals without testosterone replacement. From these studies we have concluded that hypothalamic PRL is an important neuromodulator that promotes the release of LHRH from the hypothalamus. Testosterone, at least under the experimental conditions employed, appears not to be essential in this hypothalamic PRL-LHRH interaction.

The Effect of in Vitro Ethanol Exposure on Lhrh Release from Perifused Rat Hypothalami

A variety of indirect data suggest that the luteinizing hormone (LH) lowering effects of ethanol (ETOH) are mediated at a hypothalamic level decreasing the synthesis and/or release of LH-releasing hormone (LHRH). Little direct data support this concept, however. The current study was, therefore, designed utilizing a perifusion system with frequent sampling for LHRH with and without ethanol added to determine if ethanol had a direct effect on basal or stimulated LHRH release. A variety of secretagogues, including dopamine, norepinephrine, naloxone, prostaglandin E2, and a high dose of potassium were utilized. Ethanol at a dose of 300 mg% did not alter either basal or secretagogue-stimulated LHRH release from the hypothalami of ethanol-naive male rats. Thus, ethanol did not appear to have a direct effect on LHRH in this system. Alterations in LHRH release by ethanol may occur at a suprahypothalamic level, involving neurotransmitter-LHRH interactions. Alternatively, the well-described lowering effect of ethanol on LH may be secondary to a direct pituitary locus of action, or involve a metabolic breakdown product of ethanol rather than ethanol itself.

Negative feedback regulation of pulsatile LH secretion during treatment with an LHRH antagonist in rams.

Suppression of LH and testosterone secretion in sexually active rams by the short-term administration of an LHRH antagonist results in a compensatory increase in the release of LHRH from the hypothalamus. This is inferred from the observed increase in the frequency of LH pulses in peripheral blood during the period of recovery when the pituitary regains its responsiveness to LHRH. To investigate the nature of the inhibitory feedback signal which triggers this compensatory response, a single intravenous injection of 1 mg of an LHRH antagonist (28 micrograms/kg; N-Ac-D-pCl-Phe 1, D-pCl-Phe 2, D-Trp 3, D-hArg (Et 2) 6, D-Ala 10, LHRH) was given to groups of intact, testosterone-implanted castrated and castrated rams housed under stimulatory short days. Pulsatile LH secretion was monitored in blood samples collected every 10 min for 34 h. The treatment caused an immediate blockade of LH pulses in all three groups of rams followed by a progressive recovery of LH secretion from 12-30 h. Compared to the pretreatment period, intact rams showed a significant increase in frequency of LH pulses during the recovery period. Castrated rams did not show this increase, with or without supplementary testosterone. Since the circulating testosterone concentration decreased after the blockage of LH secretion in the intact rams, but not in the castrated or testosterone-implanted castrated rams, we conclude that it is the reduction in the steroid negative feedback signal which leads to a compensatory increase in the activity of the LH pulse generator.

Interleukin-1 Inhibits the Ovarian Steroid-Induced Luteinizing Hormone Surge and Release of Hypothalamic Luteinizing Hormone-Releasing Hormone in Rats*

Interleukin-1 (IL-1), a polypeptide cytokine secreted by activated macrophages, has been postulated as a chemical messenger between the immune and endocrine systems. IL-1-immunopositive neurons and fibers have been visualized in the human and rat hypothalamus, and IL-1 receptors are present in the rat brain. We have examined the effects of human recombinant IL-1 (alpha- and beta-subtypes) on LH release in vivo and hypothalamic LHRH release in vitro. Ovariectomized rats were primed with estradiol benzoate, and progesterone was injected 48 h later to elicit a LH surge in the afternoon. IL-1 alpha and IL-1 beta were injected either intracerebroventricularly (icv) via a preimplanted cannula in the third ventricle of the brain or iv. Systemic injection of IL-1 alpha or IL-1 beta (58.8 pmol at 1300 and 1500 h) failed to influence the afternoon LH surge seen in saline-injected control rats. However, IL-1 beta (1.76 pmol) administered icv at 1300 and 1500 h or a single icv injection at 1300 h blocked the progesterone-induced LH surge. Similar icv injections of IL-1 alpha also significantly suppressed the afternoon LH surge compared to that in saline-injected control rats. However, IL-1 alpha was relatively less effective than the beta-subtype, since the LH surge was detected in some rats. To ascertain whether suppression of the LH surge was due to inhibition of LHRH release, the medial basal hypothalamus-preoptic area of estradiol benzoate-progesterone-treated ovariectomized rats was incubated with and without IL-1. Both IL-1 alpha and IL-1 beta, at concentrations of 0.1 nM and higher, significantly suppressed LHRH release in vitro from the medial basal hypothalamus-preoptic area. In contrast, IL-1 (10 nM) was completely ineffective in suppressing LHRH release from the microdissected median eminence. These results demonstrated an overall inhibitory effect of icv IL-1 on the LHRH-LH axis and suggest that suppression of the steroid-induced LH surge by IL-1 may primarily be due to inhibition of LHRH release at hypothalamic sites located within the blood-brain barrier.

An Opioid-Neuropeptide-Y Transmission Line to Luteinizing Hormone (LH)-Releasing Hormone Neurons: A Role in the Induction of LH Surge*

We tested the hypothesis that a decrease in hypothalamic inhibitory opioid tone produced by naloxone (NAL) will activate neuropeptide-Y (NPY) neurosecretion in 17 beta-estradiol (E2)-primed ovariectomized (ovx) rats. NPY neurosecretion was assessed in two ways. First, we studied the effects of iv saline (controls) or NAL infusion (2 mg/h) between 1100-1400 h on NPY concentrations in seven microdissected sites in the medial basal hypothalamus (MBH) and preoptic area in association with the increase in the rate of LH secretion, and then we examined the effects of NAL on the in vitro release of NPY and LHRH from the MBH of E2-primed ovx rats. We observed that in control rats, NPY concentrations in selected hypothalmic sites (median eminence, medial preoptic area, and arcuate nucleus) increased either just before LH rise at 1400 h or in association with the moderate LH surge in the afternoon. NAL infusion advanced the onset and amplified the magnitude of LH surge in the afternoon. In association with this augmentation of LH response, NAL infusion significantly increased NPY concentrations selectively in the median eminence, medial preoptic area, and arcuate nucleus. During NAL infusion at 1300 h and at the end of infusion at 1400 h, NPY concentrations in these sites increased compared to preinfusion levels at 1100 h and corresponding control levels at 1300 h. During the post-NAL infusion period until 1800 h, NPY levels remained elevated in these sites, but were not significantly different from those in control rats, which also displayed increments at this time. Further, NAL increased the in vitro efflux of both NPY and LHRH from the MBH; the increased release of two neuropeptides was dose related between 0.01-0.5 mg/ml, with the maximal increase occurring at 1.0 mg/ml NAL. Cumulatively, these studies show that 1) in association with the spontaneous LH surge in E2-primed rats, NPY concentration increased only in sites confined to the preoptic-tuberal pathway, which previously has been shown to mediate the induction of the LH surge; and 2) a decrease in the inhibitory opioid tone imposed by NAL readily augmented the hypothalamic NPY neurosecretion concomitant with an increase in duration and magnitude of the LH surge. These findings are in accord with the thesis that a decrease in the inhibitory opioid tone by the neural clock, postulated to occur before the LH surge, initiates a chain of neurosecretory events that may include a site-specific activation of NPYergic neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Oestrogen positive feedback stimulates the synthesis of LHRH mRNA in neurones of the rostral diencephalon of the rat

Our aim was to determine whether oestrogen administered to ovariectomized rats in a manner which stimulates the release of LHRH also stimulates the synthesis of LHRH mRNA. Adult female Wistar rats were ovariectomized under halothane anaesthesia between 09.00 and 11.00 h of dioestrus, immediately injected with either oil or 10 micrograms oestradiol benzoate (OB) and then killed by cervical dislocation between 16.00 and 17.00 h of the next day (presumptive pro-oestrus). In-situ hybridization carried out with a 30 mer, 32P-labelled, oligonucleotide probe complementary to LHRH mRNA showed that the concentrations of LHRH mRNA in perikarya in the medial preoptic area, diagonal band and medial septum were significantly greater in OB-compared with oil-treated rats. Plasma LH concentrations were significantly increased in three out of four of the OB-treated rats. These results show for the first time that as well as stimulating LHRH and LH release, oestrogen positive feedback also stimulates the synthesis of LHRH mRNA.

Glutamate Receptors of the Non-N-Methyl-D-Aspartic Acid Type Mediate the Increase in Luteinizing Hormone-Releasing Hormone Release by Excitatory Amino Acidsin Vitro

The present study was designed to analyze in detail the effects of L-glutamate (L-Glu) and its agonists on the release of LHRH from arcuate nucleus-median eminence (AN-ME) fragments in vitro. Fragments from adult male rats were incubated in Krebs-Ringer bicarbonate buffer in the presence of different concentrations of L-Glu, kainate (KA), N-methyl-D-aspartic acid (NMDA), and quisqualate (QA). L-Glu at 10-20 mM evoked a significant increase in basal LHRH release, while D-glutamate at similar concentrations was ineffective. Partial depolarization with 14 mM K+ significantly augmented the release of LHRH induced by L-Glu. L-Glu-induced LHRH release was blunted in a dose-related manner by the specific KA/QA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione. Exposure to KA or QA significantly increased LHRH release at concentrations (1 mM) much lower than those required for L-Glu. In the presence of 14 mM K+ the potencies of KA and QA (0.075 and 0.1 mM, respectively) were significantly enhanced. 6,7-Dinitroquinoxaline-2,3-dione blocked KA-induced LHRH release, while AP-7, a competitive NMDA receptor antagonist, was inactive in preventing L-Glu- and KA-induced LHRH release. LHRH secretion from AN-ME fragments was unaffected by NMDA at concentrations up to 10 mM in the different media tested. A significant stimulatory effect of NMDA at 20 mM was observed when fragments were incubated in Mg2+-free medium. These results show the stereoselectivity of L-Glu to enhance LHRH release from AN-ME fragments in vitro. Moreover, in view of the respective potencies of excitatory amino acid agonists (KA = QA greater than L-Glu greater than NMDA) and the selective antagonism of excitatory amino acid effects, they provide evidence that non-NMDA receptors primarily mediate the excitatory actions of L-Glu on LHRH release from nerve terminals in the AN-ME.

Infusion of Progestins into the Hypothalamus of Female New Zealand White Rabbits: Effect onin VivoLuteinizing Hormone-Releasing Hormone Release as Determined with Push-Pull Perfusion*

Previously, we have reported that intermittent infusion of progesterone (P4; 10 ng/ml) into the hypothalamus of conscious unrestrained female New Zealand White rabbits stimulates LHRH release in vivo. To further investigate this phenomenon, in the present studies, the effect of pulsatile (six pulses; 10 min on, 30 min off) and continuous infusion of P4 (Exp I) and 4-pregnen-20 alpha-ol-3-one or 20 alpha-hydroxyprogesterone (20 alpha-OH-P; Exp II) on LHRH release were studied in vivo using push-pull cannulae. Furthermore, the effect of pulsatile infusion (six pulses; 10 min on, 30 min off) of low doses of the following three progestins [5 beta-pregnan-3 beta-ol-20-one (pregnanolone), 5 alpha-pregnan-3 alpha-ol-20-one (3 alpha-5 alpha-P), and 20 alpha-OH-P] into the hypothalamus of does using push-pull cannulae were examined in Exp III. In Exp I, continuous infusion of P4 at 10 ng/ml was unable to stimulate mean LHRH release. However, pulses of P4 at 0.01 ng/ml (n = 4) were found to significantly increase the amplitude of the largest LHRH pulse (control period, 1.18 +/- 0.41; versus treatment period, 3.15 +/- 0.75 pg; P less than 0.035) as well as the frequency of LHRH pulses (control period, 0.72 +/- 0.26; treatment period, 1.37 +/- 0.12 pulses/h; P less than 0.035). On the other hand, there was no effect of pulses of P4 at 0.001 ng/ml (n = 4) on the activity of the LHRH neural apparatus. In Exp II, pulses of 20 alpha-OH-P at 10 ng/ml (n = 4) were found to significantly increase the mean LHRH release (control period, 1.24 +/- 0.10; treatment period, 2.07 +/- 0.52 pg/10 min) as well as the amplitude of the largest LHRH pulse (control period, 0.99 +/- 0.36; treatment period, 8.15 +/- 3.75 pg). Interestingly, continuous infusion of 20 alpha-OH-P (10 ng/ml) also significantly increased the mean amplitude (control period, 0.58 +/- 0.34; treatment period, 1.90 +/- 0.29 pg) as well as the amplitude of the largest LHRH pulse (control period, 0.64 +/- 0.37; treatment period, 4.30 +/- 1.97 pg).(ABSTRACT TRUNCATED AT 400 WORDS)

Leukotriene C4-induced release of LHRH into the hypophyseal portal blood and of LH into the peripheral blood

Intracerebroventricular (i.c.v.) administration of leukotriene (LT) C4 at doses of 2, 0.5 and 0.2 μg/rat significantly stimulated (3–12 fold) the release of LH into the peripheral blood of male rats. Injection of anti-LHRH serum had no effect on LTC4- stimulated LH release, but did block PGE2- stimulated LH release. I.c.v.- infused LTC4 also stimulated the release of LHRH into the hypophyseal portal blood. This is the first report of an in vivo action of LTC4 on the release of a hypothalamic releasing factor (LHRH) and a pituitary hormone (LH). These observations, plus in vitro results, clearly show that LTC4 stimulates LH release by acting on both the hypothalamus, causing LHRH release, and on the pituitary. Then the action of LTC4 on LH release in vivo is quite different from the indirect action of PGE2.

Postovariectomy Decline in the in vitro Release of LH-Releasing Hormone Is Not Manifested in Aging Female Rats

Aging of the female reproductive system is associated with a transition from a regular cyclic to an irregular cyclic and finally to an acyclic mode of function. We addressed the question: is the transition associated with a decline in the secretory response of the LHRH neuron to an external stimulus and with a change in steroid regulation of the secretory function of the LHRH neuron? Three groups of virgin female rats ranging from 2 to 7 months of age were used: intact, ovariectomized (8 days) and ovariectomized treated with estradiol-17 beta for 49-50 h; explants of the median eminence area (MEA) were incubated in vitro and LHRH release in response to the combined actions of copper and prostaglandin E2 was used as a criterion for secretory function. Based on vaginal cytology, young (2-3 months) females exhibited regular estrous cycles, the transition to irregular cycles manifested at 5 months of age (2/3 of animals) and to acyclicity at 7 months (all animals). In intact animals, there was a doubling in the magnitude of basal as well as stimulated LHRH release which paralleled the increase in MEA content of the peptide. By comparison to intact, stimulated LHRH release was markedly (greater than or equal to 50%; p less than 0.001) attenuated in 2- and 3-month-old ovariectomized rats but not in 5- and 7-month-old rats; thus, stimulated LHRH release at 5-7 months was approximately 2.5-fold greater than at 2-3 months of age. At each age group, estradiol treatment did not alter the magnitude of stimulated LHRH release. Ovariectomy with or without estradiol did not alter basal release nor the MEA content of LHRH.(ABSTRACT TRUNCATED AT 250 WORDS)

Central administration of corticotrophin-releasing factor in the sheep: effects on secretion of gonadotrophins, prolactin and cortisol

Stress interferes with the normal secretion of LH and FSH from the anterior pituitary gland and therefore exerts a deleterious effect on reproductive function. Evidence suggests that the stress-induced disruption of gonadal function is due to a central action of corticotrophin-releasing factor (CRF) to inhibit the release of LHRH into the hypophysial-portal circulation. The following studies were undertaken to investigate further the role of CRF in regulating gonadotrophin release in the sheep and to determine whether central administration of this peptide can alter the secretion of other hormones (e.g. prolactin and cortisol) known to be released under conditions of stress. In contrast to other species, injection of CRF into the third ventricle of the sheep brain caused a dose-related stimulation of LH secretion. The pulse frequency and mean levels of LH were increased significantly following central administration of CRF. In contrast to this effect, central administration of CRF did not alter the plasma concentration of FSH but caused a marked and dose-related stimulation of prolactin and cortisol secretion. The stimulatory effect of CRF on prolactin secretion was reversed by i.v. administration of the opioid antagonist naloxone, suggesting that endogenous opioid peptides mediate the central effect of CRF on the release of prolactin, but not cortisol. In conclusion, these data demonstrate that administration of CRF causes a dose-related stimulation of LH and prolactin release from the anterior pituitary gland and cortisol from the adrenal gland. In the case of prolactin, endogenous opioid peptides are likely to mediate this response.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of Nerve Growth Factor in Female Sexual Development*

The ovary is innervated by noradrenergic and peptidergic fibers. Treatment of neonatal rats with antibodies to nerve growth factor (NGF Ab) resulted in failure of the sympathetic (noradrenergic and neuropeptide-Y) nerves to develop. Partial loss of sensory innervation, represented by calcitonin gene-related peptide fibers, was also observed. Follicular growth was stunted, and production of androgens and estradiol was reduced. The timing of first ovulation was delayed, estrous cyclicity was disrupted, and fertility was compromised. Plasma LH levels were elevated, and LH pulsatility was enhanced, suggesting primary ovarian failure. A normal appearance of tyrosine hydroxylase-, LHRH-, and neuropeptide-Y-immunoreactive neurons in the hypothalamus, as determined by immunocytochemistry, suggested that neonatal immunosympathectomy did not directly affect hypothalamic reproductive function. In vitro release of LHRH from median eminence nerve terminals in response to prostaglandin E2 was, however, reduced in NGF Ab-treated rats. Normalization of the response by prior in vivo exposure of the animals to physiological estradiol levels, suggested that the diminished LHRH output was due at least in part to estrogen deficiency. Although ovarian dysfunction induced by immunosympathectomy may be related to alterations in vascular tone, the striking loss of perifollicular noradrenergic innervation caused by NGF Ab suggests that the absence of the nonvascular norepinephrine stimulus to follicular steroidogenesis is a primary factor responsible for the alterations observed. The results indicate that development of the sympathetic innervation of the ovary is NGF dependent and that NGF, by supporting the differentiation and survival of the innervating neurons, contributes to the acquisition of mature ovarian function.