Luteinizing Hormone-releasing Hormone Content Research Articles

Regulation of luteinizing hormone-releasing hormone (LHRH) secretion by melatonin in the ewe. II. Changes in N-methyl-D,L-aspartic acid-induced LHRH release during the stimulation of luteinizing hormone secretion by melatonin.

Injection of N-methyl-D,L-aspartic acid (NMDA), a glutamatergic agonist, cause an acute release of LH. The amount of LH released is modulated by photoperiod in sheep; it is larger in animals in which reproductive activity has been inhibited by long days than in those photostimulated by short days. The purpose of the present experiment was to determine whether this effect of photoperiod on LH response to NMDA is mediated by melatonin and whether it reflects a change in the amount of LHRH released in response to NMDA. Twenty-one ovariectomized ewes, bearing an s.c. implant of estradiol and kept in long days (16L:8D), received s.c. implants of melatonin on Day 0. The effect of a single injection of NMDA (5 mg/kg i.v.) on LHRH and simultaneous LH secretion was studied at three different periods of the stimulation of LH secretion by the melatonin treatment: first, during inhibition of LH secretion by long days (on Day -1, n = 6); secondly, during the onset of the LH increase (on Day 39, n = 9); and thirdly, during maximum LH secretion (on Day 74, n = 6). The stimulation of LHRH secretion by administration of NMDA was characterized by a large and long increase in LHRH concentration on Day -1 and Day 39. There was no significant difference between these two periods in terms of duration and amplitude of the LHRH release. On Day 74, the stimulation was much shorter than on Day -1 and Day 39 and was followed by a phase of decreased pulsatility of LHRH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) 27 and PACAP 38 on the Release of Luteinizing Hormone (LH) from Cultured Bovine Anterior Pituitary Cells.

The effect of pituitary adenylate cyclase-activating polypeptide (PACAP) on the release of luteinizing hormone (LH) from cultured bovine anterior pituitary cells was determined and compared with that of luteinizing hormone-releasing hormone (LHRH). PACAP 27 significantly increased the release of bovine LH at a dose as low as 10-14 M. PACAP 38 also significantly increased the release of LH and the minimal effective dose was 10-12 M. Maximal LH release induced by PACAP 27 (10-7 M) and PACAP 38 (10-7 M) were 40.5% and 71.6%, respectively of control levels. LHRH significantly increased the release of LH at a dose as low as 10-14 M. The increase in LH reached a maximum level which was 3-fold above the basal level at the highest concentration of LHRH tested (10-7 M, P<0.001). LH release induced by PACAP 27, PACAP 38, and LHRH was in a dose-dependent manner. The results suggest that PACAP 27, PACAP 38, and LHRH stimulate the release of LH in the cattle by acting directly at anterior pituitary cells.

Induction of Feline Acquired Immune Deficiency Syndrome by Feline Leukemia Virus: Immuno- and Neuroendocrine Dysfunctions

Young cats, when chronically infected with feline leukemia virus (FeLV), developed feline acquired immune deficiency syndrome (FAIDS). The syndrome was associated with a sequence of dysfunctions in the hypothalamic-pituitary-gonadal (HPG) and the immune system, manifested in the reduction of luteinizing hormone-releasing hormone (LHRH), follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone in blood plasma. The average FSH and LH (in plasma or lymphocyte), testosterone, and LHRH concentrations in the 20 FeLV-infected cats were measured by radioimmunoassay. The results were compared with those of the 12 control cats that were not FeLV-infected. Four weeks after infection, the plasma LHRH concentration in the infected cats showed a 43% reduction. Five to six weeks after infection, the content of FSH and LH in lymphocyte was reduced by 50% and 28%, respectively, whereas, the plasma FSH and LH was reduced by 52% and 42%, respectively. A significant reduction in testosterone content was detected at Week 11 of infection. The onset of the immuno- and neuroendocrine dysfunctions in FAIDs cats followed this sequence: hypothalamus, lymphocyte, pituitary, adrenal gland, and gonads. Indirect immunofluorescence assay showed the presence of FeLV cytoplasmic antigens in the fibers of the hypothalamic preoptic region and the Leydig cells. The possible causal relationship between the dysfunction of the lymphocyte and HPG systems and the presence of FeLV was discussed.

Evidence for a protein-like factor from "rete testis" fluid that suppresses luteinizing hormone-releasing hormone (LHRH) pulses (LHRH statin): a new hormonal activity?

Signals that modulate LH-releasing hormone (LHRH) pulse frequency are fundamental mechanisms for regulating important reproductive processes. Gonadal steroids are presently considered to account for the entire gonadal feedback mechanism that modulates LHRH secretion. However, we have previously suggested that a testicular protein(s) present in charcoal-treated rete testis fluid (ctRTF) can suppress LH pulsatility in the ram. The present experiments were aimed at determining whether the disappearance of LH pulses induced by ctRTF administration implicate a hypothalamic or a pituitary site of action. Thus, we have examined the effects of ctRTF peripheral administration on 1) the LH response to LHRH, 2) LHRH portal blood levels, and 3) LHRH content in hypothalamic tissue. Finally, the effects of ctRTF administered into the third ventricle on plasma LH levels were assessed. The present results show that a testicular protein(s) is able to suppress LHRH pulse frequency without affecting amplitude and without any effect on the LH response to LHRH (LHRH Statin). The observation that an active dose administered by the intracerebroventricular route is 0.0005 the active dose needed by the peripheral route reinforces this evidence. These data lead to the new concept that the testicular signals that govern LHRH pulse frequency may be not only steroids, but also proteins.

Evidence for a protein-like factor from "rete testis" fluid that suppresses luteinizing hormone-releasing hormone (LHRH) pulses (LHRH statin): a new hormonal activity?

Signals that modulate LH-releasing hormone (LHRH) pulse frequency are fundamental mechanisms for regulating important reproductive processes. Gonadal steroids are presently considered to account for the entire gonadal feedback mechanism that modulates LHRH secretion. However, we have previously suggested that a testicular protein(s) present in charcoal-treated rete testis fluid (ctRTF) can suppress LH pulsatility in the ram. The present experiments were aimed at determining whether the disappearance of LH pulses induced by ctRTF administration implicate a hypothalamic or a pituitary site of action. Thus, we have examined the effects of ctRTF peripheral administration on 1) the LH response to LHRH, 2) LHRH portal blood levels, and 3) LHRH content in hypothalamic tissue. Finally, the effects of ctRTF administered into the third ventricle on plasma LH levels were assessed. The present results show that a testicular protein(s) is able to suppress LHRH pulse frequency without affecting amplitude and without any effect on the LH response to LHRH (LHRH Statin). The observation that an active dose administered by the intracerebroventricular route is 0.0005 the active dose needed by the peripheral route reinforces this evidence. These data lead to the new concept that the testicular signals that govern LHRH pulse frequency may be not only steroids, but also proteins.

Luteinizing hormone-releasing hormone (LHRH) in rat prostate: characterization of LHRH peptide, messenger ribonucleic acid expression, and molecular processing of LHRH in intact and castrated male rats.

Continuous administration of LHRH agonist suppresses the pituitary-gonadal axis, achieving chemical castration. Thus, LHRH agonist has been used as an alternative (to surgical castration) for the treatment of steroid-dependent prostate cancer. However, recent reports have demonstrated that LHRH agonist had a direct inhibiting effect on prostate cancer cell proliferation and that cancerous prostate tissue contained a LHRH-like peptide. In this paper we are reporting for the first time that the normal rat ventral prostate contained immunoactive and bioactive LHRH as well as its precursor molecule, pro-LHRH. Our investigation showed that the LHRH concentration in prostate increased 2 weeks after castration from 1.68 +/- 0.09 to 3 +/- 0.2 pg/mg tissue (P < 0.001). At the same time, the concentration of pro-LHRH decreased from 149 +/- 6.5 to 68 +/- 6.8 pg/mg tissue (P < 0.001). Furthermore, intact rat prostate expressed LHRH mRNA, which increased 13-fold 2 weeks after castration. In summary, the prostate of intact Sprague-Dawley rats has the capacity to produce the LHRH precursor and process it to the mature decapeptide, and this production/processing increases significantly after castration.

Modulation of N-type calcium channels in bullfrog sympathetic neurons by luteinizing hormone-releasing hormone: kinetics and voltage dependence

Inhibition of Ca channel current by luteinizing hormone-releasing hormone (LHRH) was studied in freshly dissociated bullfrog sympathetic ganglion neurons using whole-cell recording. LHRH inhibited up to 80% of the high-threshold Ca channel current with a half-maximally effective concentration of about 20 nM. LHRH inhibited omega-conotoxin-sensitive but not nimodipine-sensitive current and also did not inhibit Bay K 8644-enhanced currents, suggesting that LHRH inhibits N-type but not L-type channels. Inhibition was faster at higher concentrations of LHRH, reaching a limiting time constant of 2 sec at 0.3-3 mM LHRH. The rate of recovery from block (tau approximately 19 sec) was independent of LHRH concentration. Inhibition of N-type current by LHRH was highly sensitive to the gating state of the channel. Though strongly effective if applied when channels were mostly in the resting state, LHRH had little effect if applied rapidly during a long depolarization that opened the channels. Inhibition could be relieved if channels were activated by short, large test depolarizations or by long, smaller depolarizations. The state-dependent properties of LHRH block could be simulated by a model that assumes that inhibition by LHRH results from activated G-proteins binding to N-type channels and that (1) G-protein binding stabilizes closed gating states and (2) activation of G-protein-bound channels destabilizes the binding of the G-protein to the channel.

Beta-endorphin regulation of LHRH release at the median eminence level: immunocytochemical and physiological evidence in hens.

We studied the effect that beta-endorphin (beta END) might have at the median eminence (ME) on luteinizing hormone (LH)-releasing hormone (LHRH) during the ovulatory cycle of domestic hens. Thus, we assessed (a) the immunocytochemical distribution of beta END and LHRH in the hen ME, (b) the temporal changes in ME and preoptic area (POA) LHRH and beta END content, in both a spontaneous and a premature C2 ovulatory model. The premature C2 ovulation occurs 6-7 h after the administration of progesterone (P4) injected 14 h before the spontaneous second (C2) ovulation of a sequence and therefore 7-8 h earlier than expected, (c) the ME in vitro release of beta END in both models, and (d) the effect of beta END and naloxone on in vitro ME-LHRH release in the two models. In the hen, beta END cell bodies are located in the periarcuate area with axons projecting to both the ME and the POA. LHRH perikarya are located in the medial POA and anterior hypothalamus and project to the ME and infundibulum through the ventrolateral hypothalamus. In the spontaneous C2 ovulatory model, both beta END and LHRH content in the ME remained unchanged during the 14 h preceding the C2 ovulation. However, POA-LHRH content was increasing at the time of the LH surge (4 h before the expected C2 ovulation) and remained elevated until the C2 ovulation occurred. In contrast, POA-beta END content was lowest at the time of the LH surge and remained low until the C2 ovulation occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropeptide Y Regulation of LHRH Release in the Median Eminence: Immunocytochemical and Physiological Evidence in Hens

It has been suggested that hypothalamic median eminence (ME) might be a control site for luteinizing hormone-releasing hormone (LHRH) release. Thus, stimulatory and/or inhibitory inputs acting at this site might be involved in regulating LHRH release from the ME and, therefore, luteinizing hormone (LH) release from the anterior pituitary. Since a role for neuropeptide Y (NPY) on LH release has been suggested, we have hypothesized that NPY might act in the ME to control preovulatory LHRH release in hens. To examine this possibility we have determined: (a) the immunocytochemical distribution of LHRH and NPY in the ME of the hen, (b) the basal and NPY-stimulated release of LHRH in vitro from the ME of hens undergoing a natural or a premature preovulatory surge of LH, and (c) the tissue content of LHRH and NPY in microdissected MEs, at various times before and during a natural or a premature preovulatory surge of LH. A potential role for NPY on LHRH release in the ME is suggested for the following reasons. (a) There are opportunities for synaptic interactions between NPY and LHRH-containing axons at this site. LHRH-containing cell bodies localized in the anterior hypothalamus/medial preoptic area project to the ME. NPY-containing perikarya, concentrated in the ventromedial aspect of the arcuate nucleus, might contact LHRH processes going to the ME and/or might themselves send axons to the ME, (b) Addition of NPY to the incubation media increases LHRH release from microdissected ME tissue of hens killed at the time of the natural preovulatory surge of LH, but not in hens killed 7 h before the occurrence of this surge. However, the stimulatory effect of NPY on LHRH release can be induced at this latter time when a premature LH surge is elicited. While the natural preovulatory surge of LH occurs 4 h before the second ovulation in a sequence (C2 ovulation), administration of progesterone (P4) 10-14 h before the expected natural C2 ovulation advances the natural LH surge by 7-8 h. Thus, NPY might act as a physiological stimulus of LHRH release at the ME during the preovulatory surge of LH. This is suggested since in vitro basal LHRH release from denervated ME tissue does not change before and during the natural or the premature LH surge. Therefore, preovulatory release of LHRH in vivo might be under a continuous drive from stimulatory inputs to the LHRH neuron and NPY might be one of these stimulating factors.(ABSTRACT TRUNCATED AT 400 WORDS)

Enzyme-linked immunosorbent assay (ELISA) for luteinizing hormone releasing hormone (LH-RH) using a heterobifunctional cross-linking agent, N-[beta-(4-diazophenyl)ethyl]maleimide.

Luteinizing hormone-releasing hormone (LH-RH) was first labeled with an enzyme, beta-D-galactosidase (beta-Gal; EC 3.2.1.23), using N-[beta-(4-diazophenyl)ethyl]maleimide (DPEM) as a heterobifunctional cross-linking agent. An antigen was similarly prepared by coupling LH-RH to mercaptosuccinylated bovine serum albumin with DPEM and was used for the immunization of rabbits for antibodies against LH-RH. A new, simple enzyme-linked immunosorbent assay (ELISA) for LH-RH was developed by using the principle of direct competition between LH-RH and beta-Gal-labeled LH-RH for anti-LH-RH IgG which had been adsorbed to the plastic surface of microtiter plates. LH-RH concentrations lower than 50 pg/assay well were measurable reproducibly by the ELISA, the sensitivity of which was found to be about 6250 times greater than the corresponding high performance liquid chromatography (HPLC) procedure. The specificity of this ELISA seems to be primarily toward the C-terminal region of LH-RH, showing a cross-reaction with the LH-RH6-10 fragment to the same extent as with LH-RH, but no cross-reaction with the LH-RH1-3 and LH-RH4-6 fragments. Using this assay, LH-RH levels were easily measured in the blood and urine of rats following the administration of LH-RH in a single dose of 0.5 mg/kg i.v. The present, newly developed ELISA is a nonradioactive, inexpensive and rapid method, and might be useful for elucidating experimental hypothalamic-pituitary-gonad interactions.

Presence of high concentrations of LHRH in human follicular fluid

Health workers took plasma and human follicular fluid samples from 7 women with uterine fibroids at the First Affiliated Hospital in Heilongijiam China to determine whether luteinizing hormone releasing hormone (LHRH) is in human follicular fluid (HFF) and if it is in HFF what is the relationship between its presence in HFF and in plasma. Laboratory personnel used the LHRH specific radioimmunoassay kit by Immuno Technology Service Production B.V. to detect LHRH concentration in HFf and in plasma. Not only was LHRH present in HFf but its mean concentration was significantly greater than it was in plasma (369.3 pg/ml vs. 29.1 mg/ml; p<.05). LHRH levels in HFF ranged from 99 to 600 pg/ml and in plasma from 13 to 72 pg/ml. Since the LHRH in HFF and in the plasma did not cross-react with the LHRH-like protein in the kit the researchers determined that the LHRH in HFF was not an LHRH-like protein. They proposed 2 possible reasons for the LHRH in HFF to be higher than that in plasma. Certain cells in the human ovary may synthesize and secrete LHRH or the LHRH derives from the plasma where it concentrates in the HFF. They believed the 1st proposed reason to be the most likely reason. Nevertheless they suggested that researchers conduct more detailed tests to determine the origin of LHRH in HFF.

Central peptidergic mechanisms controlling reproductive hormone secretion: novel methodology reveals a role for the natriuretic peptides.

A variety of neural factors can influence reproductive hormone secretion by neuromodulatory actions within the hypothalamus or neuroendocrine actions within the anterior pituitary gland. Passive immunoneutralization and antagonist administration protocols have suggested physiological roles for a number of these factors; however, both experimental approaches have severe technical limitations. We have developed novel methodology utilizing cytotoxin cell targeting with neuropeptides linked to the toxic A chain of the plant cytotoxin ricin. With this methodology we can target and destroy in vivo or in vitro cells bearing receptors for that peptide. Ricin A chain conjugated to atrial natriuretic peptide (ANP), a neuropeptide known to pharmacologically inhibit luteinizing hormone-releasing hormone (LHRH) release, was injected into the cerebroventricular system of intact, cycling rats and ovariectomized rats. Cytotoxin conjugate treatment significantly lengthened the estrous cycle. In ovariectomized rats the luteinizing hormone surge induced by steroid priming was completely inhibited. LHRH content of the median eminences of these rats was not significantly altered. These data suggest that ANP binding to clearance receptors in the hypothalamus displaces the C-type natriuretic peptide (CNP) from the shared clearance receptor, making more CNP available to inhibit LHRH release. In the absence of cells bearing the clearance receptor all available CNP binds to the ANPR-B receptor and exerts its effect via an inhibitory interneuron, since LHRH fibers are spared by this treatment.

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)

Dopaminergic regulation of luteinizing hormone-releasing hormone release at the median eminence level: immunocytochemical and physiological evidence in hens.

Theoretically, the most effective inhibitory control of hypophysiotropic luteinizing hormone-releasing hormone (LHRH) release might occur through a presynaptic inhibition of LHRH neuronal terminals at the median eminence (ME) level. Since: (a) we have recently reported the existence of synaptic contacts between dopamine- and LHRH-containing processes in the ewe ME, and (b) nutritional deprivation induces an ovulatory failure in both birds and mammals, we have assessed the possibility that the anovulatory state induced by feed withdrawal (FW) in laying hens, might be caused by a dopaminergic inhibition of LHRH release at the ME level. Laying hens at the start (35 weeks old) and end (75 weeks old) of their commercial egg-laying life were killed at 0, 1, 2 and 4 days after FW. Serum luteinizing hormone (LH) and progesterone (P4), in vitro release of LHRH by isolated ME, and LHRH content in ME and preoptic area (POA) were determined by RIA. ME content of dopamine (DA) and its main metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were assessed by LCED. The distribution of LHRH and tyrosine hydroxylase (TH)-containing processes at the ME level of the hen was determined immunocytochemically. In the hen, LHRH-containing cell bodies are localized in the anterior hypothalamus and medial POA. LHRH-containing axons project toward the ME and infundibulum through the ventral-lateral hypothalamus. TH-containing perikarya are concentrated in the arcuate nucleus and in the adjacent part of the periventricular nucleus, dorsal to the arcuate. TH-containing axons converge toward the ME and descend into the infundibulum. Dense concentrations of TH- and LHRH-containing processes are located in the lateral and mediobasal portions of the external layer of the ME, providing opportunities for synaptic interactions between them. Ovulatory failure and regression of the ovary and reproductive tract occurred 2-3 days after FW at the end, but not at the beginning of the hen's commercial egg-laying life. After FW, hens at the end of their productive life had higher (p less than 0.01) tuberoinfundibular DA turnover, produced less LHRH, and had lower serum LH and P4 than hens undergoing FW at the beginning of their productive life. In addition, in vitro release of HRH from denervated ME tissue of hens undergoing FW at the end of their commercial egg-laying life was higher and was reversed in a dose-dependent fashion by DA, but not by serotonin. Thus, the ovulatory failure associated with FW in laying hens might be caused by a presynaptic inhibition of in vivo LHRH release at the level of ME hypothalamic neuronal terminals.

Direct antitumor effects of LH-RH analogs.

Luteinizing hormone releasing hormone (LH-RH) is a hypothalamic decapeptide secreted in a pulsatile way which leads to an increase in pituitary LH-RH receptor numbers and gonadotropin secretion [1]. Exposure of the pituitary gland to supraphysiological concentrations of LH-RH or continuously to long-acting LH-RH agonists causes downregulation of LH-RH receptors and loss of responsiveness (see review [2]). The presence of LH-RH or LH-RH-like peptides has not only been detected in the hypothalamus, but also in human biological fluids, the central nervous system, and a variety of normal and tumor tissues (see review [3]). This, together with the presence of LH-RH receptors in a wide range of normal and tumor tissues [3], has resulted in the suggestion that LH-RH may have a paracrine or autocrine role in the local regulation of cell biological processes. In the present report we summarize the literature with special emphasis on direct antitumor actions of LH-RH analogs on classical hormone-regulated cancers, i.e., of the breast, the prostate, and the ovaries.

Content and distribution pattern of luteinizing hormone-releasing hormone (LHRH) in the hypothalamus of neonatally estrogenized female rats

The rostro-caudal distribution of luteinizing hormone-releasing hormone (LHRH) in the hypothalamus of neonatally estrogenized (10 μg EB for 1–5 days), sterile female rats was compared with that of cycling rats at 50, 100 and 240 days of age. Serial slices of 400 μm in thickness in total were cut from a hypothalamic block, and the LHRH content of each slice was measured by radioimmunoassay. Two peaks of LHRH in the rostro-caudal direction were apparent in all groups. The rostral peak corresponded to the LHRH-producing cells in the septal-preoptic region and storage of LHRH in the organum vasculosum lamina terminalis (OVLT), while the caudal peak corresponded to that of LHRH in the median eminence. Thus, the total LHRH content of the regions of the hypothalamus was calculated separately either anterior or posterior to the suprachiasmatic nucleus, which were designated as the rostral hypothalamus (RH) and mediobasal hypothalamus (MBH), respectively. The total content of LHRH in the 50-day-old sterile rats was significantly higher than that in the estrous females in both RH and MBH. This was also true for the MBH of 240-day-old sterile females in comparison to that of estrous females but not so in 100-day-old females. The present results suggest that sterility in neonatally estrogenized rats is not due to a reduction in LHRH content of the hypothalamus but, rather, is due to changes in LHRH-releasing systems. Furthermore, in the control females, ovariectomy performed at 22 days of age induced a striking decrease in LHRH content in the MBH at 50 days of age, while it remained unchanged after ovariectomy in the neonatally estrogenized sterile rats. These findings suggest that the hypothalamic mechanism involved in the release of LHRH after ovariectomy was damaged by neonatal treatment with estrogen.

Porcine Lymphocytes Secrete a Bioactive and Immunoreactive LH-like Factor in Response to LHRH and Concanavalin A

Lymphocyte conditioned medium (CM) was prepared by incubating 0 (cell-free control) or 4 x 10(6) lymphocytes/ml in serum-supplemented RPMI containing 0, 10(-9), 10(-7), and 10(-5) M luteinizing hormone releasing hormone (LHRH), 10(-5) M LHRH antagonist (LHRHA), or 10(-7) M LHRH + 10(-5) M LHRHA. Treatments were applied with and without 10 micrograms/ml concanavalin A (con A), and media were analyzed for LH. Aliquots of the CM from cultures incubated for 48 h were later applied to porcine granulosa cell cultures (suspended to 1.25 x 10(5) cells in 450 mul). Thereafter, 50 mul of CM were added to granulosa cell cultures. Media were collected after 12, 24, and 48 h and progesterone determined. Immunoreactive LH increased with time of incubation in lymphocyte CM but not cell-free CM. LH content of lymphocyte CM increased as LHRH concentration increased. LHRHA significantly reduced the amount of LH measured. The presence of con A in the medium resulted in maximal concentrations of LH, irrespective of dose of LHRH or LHRHA. Cell-free CM containing LHRH, LHRHA, and/or con A did not affect progesterone production by granulosa cells at any of the time periods. Lymphocyte CM containing LHRH caused a dose-dependent increase in progesterone production at 48 h. This stimulation was blocked by lymphocyte CM containing LHRHA. Lymphocyte CM containing con A also stimulated progesterone production at all of the LHRH concentrations studied. This response was not inhibited by lymphocyte CM containing the LHRHA.(ABSTRACT TRUNCATED AT 250 WORDS)

A potential code of luteinizing hormone-releasing hormone-induced calcium ion responses in the regulation of luteinizing hormone secretion among individual gonadotropes

Luteinizing hormone-releasing hormone (LHRH) induces two Ca2+ responses in single gonadotropes: a Ca2+ spike/plateau or oscillation. Similar receptor-mediated Ca2+ signals have been reported in many cell types but their functional significance is obscure. Accordingly, we have determined the concentration-response properties of LHRH-induced luteinizing hormone (LH) release at the single cell level. We demonstrate a critical single cell LHRH threshold for LH release. Each gonadotrope had a particular LHRH threshold value and a range of different single cell thresholds was distributed in the gonadotrope population. The physiological significance of the threshold was demonstrated by a striking reduction (delta ED50 = 153 nM) of the LHRH threshold immediately before the preovulatory surge of LH release. The metestrous phenotype of secretion resembled a quantal process in contrast with the graded process of the proestrous phenotype. That is, the quantity of hormone secreted per metestrous gonadotrope was independent of LHRH concentration and more all-or-none than graded. The LHRH threshold and the quantal secretion process of metestrous gonadotropes was further studied by measuring cytosolic Ca2+ using fura-2 and digital imaging microscopy. We provide evidence suggesting that the Ca2+ spike/plateau and oscillation are the respective responses to subthreshold and suprathreshold concentrations of LHRH. It is proposed therefore that the Ca2+ oscillation and spike/plateau response form a binary intracellular signaling code that functions as an on-off switch. It is further proposed that this potential code unraveled here for the regulation of hormone secretion may also regulate other gonadotrope functions. Thus, while the Ca2+ spike/plateau response is strongly associated with LH release, it may be associated with reduced levels of LH-beta mRNA, and reduced numbers of LHRH receptors. Conversely, while the Ca2+ oscillation appears to be unrelated to LH release, it may be associated with increased levels of LH-beta mRNA, and increased numbers of LHRH receptors. This model may explain in molecular terms the long-standing observation that an invariant, albeit pulsatile, pattern of LHRH release is sufficient to support the preovulatory surge of LH release.

Hypothalamic luteinizing hormone-releasing hormone content and serum luteinizing hormone levels in male rats during wallerian degeneration of sympathetic nerve terminals after superior cervical ganglionectomy

The main hypothesis of this study was that sympathetic neurons located at the superior cervical ganglia (SCG) control luteinizing hormone (LH) releasing mechanisms by acting at a hypothalamic site. To test this, medial basal hypothalamus (MBH) luteinizing hormone-releasing hormone (LHRH) content and serum LH levels were measured in male rats subjected to superior cervical ganglionectomy (SCGx) or sham-operation 14 or 38 h earlier, at the time of degeneration of nerve endings post-SCGx. Significantly augmented MBH LHRH levels and decreased circulating LH were found in SCGx rats. In animals subjected to SCGx 14 h earlier and receiving a single injection of the alpha 1-adrenergic blocker prazosin, the beta-adrenergic blocker propranolol or a mixture of both drugs 45 min before sacrifice, only the injection of prazosin prevented the decrease of plasma LH levels. Neither treatment prevented the increase in MBH LHRH content. When prazosin was given every hour starting from the 10th to the 13th h after surgery, it was effective to prevent both the increase of MBH LHRH content and the decrease of serum LH found during sympathetic nerve degeneration. Similar repetitive injections of propranolol resulted in the greatest depression of serum LH, and in the greatest increase of MBH LHRH observed. Serum LH response to LHRH injection was similar in SCGx and sham-operated rats. The data indicate that SCG neurons exert, through inhibitory alpha 1-, and weaker, stimulatory beta-adrenoceptors, a significant influence on LHRH release at a supra-hypophysial site.

Naltrexone Does Not Reverse the Inhibitory Effect of Chronic Restraint on Gonadotropin Secretion in the Intact Male Rat

There is considerable evidence suggesting that endogenous opioids may play an important role in acute stress-induced decreases in luteinizing hormone (LH) release. Studies were undertaken to analyze the role of endogenous opioids in chronic stress-induced decrease in circulating LH and follicle-stimulating hormone (FSH). Chronic restraint (6 h daily over 4 days) evoked a decrease in circulating LH and FSH. Naltrexone treatment, (2 mg/kg three times daily) during the 4 days of restraint, caused an increase in plasma concentrations of LH and FSH, and antagonized the LH suppressory effect of morphine (10 mg/kg) administration. Despite this, naltrexone treatment was ineffective in preventing the inhibitory effect of chronic restraint stress on circulating LH and FSH. Chronic restraint also induced a decrease in hypothalamic LH-releasing hormone (LHRH) content in saline-treated rats. On the contrary, in naltrexone-treated rats, chronic restraint evoked an increase in hypothalamic LHRH content. Thus endogenous opioids and chronic stress seem to act by different mechanisms on the hypothalamic LHRH neuron. In unstressed orchidectomized rats, naltrexone administration did not modify circulating LH, but increased plasma concentrations of LH in acutely restrained rats. These data suggest that endogenous opioids may mediate gonadotropin secretion during acute stress, but not during chronic stress.