Luteinizing Hormone-releasing Hormone Gene Expression Research Articles

Manganese Induces IGF-1 and Cyclooxygenase-2 Gene Expressions in the Basal Hypothalamus during Prepubertal Female Development

Precocious puberty is a significant child health problem, especially in girls, because 95% of cases are idiopathic. Our earlier studies demonstrated that low-dose levels of manganese (Mn) caused precocious puberty via stimulating the secretion of luteinizing hormone-releasing hormone (LHRH). Because glial-neuronal communications are important for the activation of LHRH secretion at puberty, we investigated the effects of prepubertal Mn exposure on specific glial-derived puberty-related genes known to affect neuronal LHRH release. Animals were supplemented with MnCl(2) (10 mg/kg) or saline by gastric gavage from day 12 until day 22 or day 29, then decapitated, and brains removed. The site of LHRH release is the medial basal hypothalamus (MBH), and tissues from this area were analyzed by real-time PCR for transforming growth factor α (TGFα), insulin-like growth factor-1 (IGF-1), and cyclooxygenase-2 (COX-2) messenger RNA levels. Protein levels for IGF-1 receptor (IGF-1R) were measured by Western blot analysis. LHRH gene expression was measured in the preoptic area/anteroventral periventricular (POA/AVPV) region. In the MBH, at 22 days, IGF-1 gene expression was increased (p < 0.05) with a concomitant increase (p < 0.05) in IGF-1R protein expression. Mn also increased (p < 0.01) COX-2 gene expression. At 29 days, the upregulation of IGF-1 (p < 0.05) and COX-2 (p < 0.05) continued in the MBH. At this time, we observed increased (p < 0.05) LHRH gene expression in the POA/AVPV. Additionally, Mn stimulated prostaglandin E(2) and LHRH release from 29-day-old median eminences incubated in vitro. These results demonstrate that Mn, through the upregulation of IGF-1 and COX-2, may promote maturational events and glial-neuronal communications facilitating the increased neurosecretory activity, including that of LHRH, resulting in precocious pubertal development.

Luteinizing Hormone-Releasing Hormone I (LHRH-I) and Its Metabolite in Peripheral Tissues

Luteinizing hormone-releasing hormone (LHRH) was first isolated in the mammalian hypothalamus and shown to be the primary regulator of the reproductive system through its initiation of pituitary gonadotropin release. Since its discovery, this form of LHRH (LHRH-I) has been shown to be one of many structural variants with a variety of roles in both the brain and peripheral tissues. Enormous interest has been focused on LHRH-I and LHRH-II and their cognate receptors as targets for designing therapies to treat cancers of the reproductive system. LHRH-I is processed by a zinc metalloendopeptidase EC 3.4.24.15 (EP24.15) that cleaves the hormone at the fifth and sixth bond of the decapeptide (Tyr(5)-Gly(6)) to form LHRH-(1-5). We have previously reported that the autoregulation of LHRH gene expression can also be mediated by its processed peptide, LHRH-(1-5). Furthermore, LHRH-(1-5) has also been shown to be involved in cell proliferation. This review will focus on the possible roles of LHRH and its processed peptide, LHRH-(1-5), in non-hypothalamic tissues.

Regulation of type II luteinizing hormone-releasing hormone (LHRH-II) gene expression by the processed peptide of LHRH-I, LHRH-(1-5) in endometrial cells.

Luteinizing hormone-releasing hormone (LHRH) was first isolated in the mammalian hypothalamus and shown to be the primary regulator of the reproductive system through its initiation of pituitary gonadotropin release. Since its discovery, this form of LHRH (LHRH-I) has been shown to be one of many structural variants with a variety of roles in both the brain and peripheral tissues. Enormous interest has been focused on LHRH-I, LHRH-II, and their cognate receptors as targets for designing therapies to treat cancers of the reproductive system. LHRH-I is processed by a zinc metalloendopeptidase EC 3.4.24.15 (EP24.15) that cleaves the hormone at the Tyr(5)-Gly(6) bond. We have previously reported that the autoregulation of LHRH gene expression can also be mediated by its processed peptide, LHRH-(1-5). Given its importance in the brain, we have investigated the role of the specific processed peptide of LHRH-I, LHRH-(1-5), within Ishikawa cells, a human endometrial cell line. Using real-time polymerase chain reaction, we observed that LHRH-(1-5) upregulates LHRH-II mRNA expression in Ishikawa cells but does not exert any influence on LHRH-I mRNA levels. This is in contrast to the effects of LHRH-I, which affects the expression of LHRH-I mRNA. Our findings support a potential role for LHRH-(1-5) as a processed metabolite in the endometrium. Further investigations are needed to determine the role of this processed metabolite and to identify specific pathways involved in LHRH-(1-5) signaling.

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.

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.

Glutamatergic signaling through the N-methyl-D-aspartate receptor directly activates medial subpopulations of luteinizing hormone-releasing hormone (LHRH) neurons, but does not appear to mediate the effects of estradiol on LHRH gene expression.

Although estradiol (E2) triggers phasic increases in LH-releasing hormone (LHRH) synthesis and release, the neurocircuitry responsible for these changes is unclear. We used an ovariectomized, E2-treated animal model to investigate the possibility that glutamate, through N-methyl-D-aspartate (NMDA) receptors (NMDAR), communicates E2 signals to LHRH neurons. A neuroanatomical analysis of the region containing LHRH neurons revealed that approximately 80% of LHRH neurons in medial, but less than 40% in lateral, nuclei of the preoptic area contained NMDAR1 mRNA. Consistent with this distribution pattern, NMDA doubled LHRH mRNA levels in medial neurons, but increased them by less than 30% in cells of the lateral nuclei. Steroids did not alter NMDAR1 mRNA levels in LHRH neurons or change the percentage of LHRH neurons expressing the gene. Furthermore, in contrast to the regionalized effects of NMDA, E2 treatment increased LHRH mRNA levels to the same extent in medial and lateral neurons, and MK801 failed to block E2-induced changes in LHRH gene expression. These results demonstrate that glutamatergic signaling via NMDA receptors is direct and preferentially targets LHRH neurons in medial nuclei of the preoptic area. However, it is unlikely that NMDAR activation mediates E2-dependent increases in LHRH mRNA levels before the LH surge.

Midline Nasal Tissue Influences Nestin Expression in Nasal-Placode-Derived Luteinizing Hormone-Releasing Hormone Neurons during Development

Neurons differentiating into the luteinizing hormone-releasing hormone (LHRH) neuroendocrine phenotype are derived from the nasal placode. Cells within the vomeronasal organ anlage that turn on LHRH gene and peptide expression subsequently migrate into the forebrain where they influence reproductive function. The molecular and cellular cues regulating differentiation and migration of these cells are unknown. Discovery of developmental markers can indicate proteins directing or associated with differentiation. Analysis of such markers after manipulation of external cues can elucidate important extracellular differentiation signals. Embryonic LHRH neurons were examined in vivo for Mash-1 and nestin, two factors that delineate precursor populations in PNS and forebrain CNS cells. Nestin, but not Mash-1, was detected in early expressing LHRH cells in the vomeronasal organ anlage. These results were duplicated in LHRH neurons maintained in vitro in nasal explants. Such LHRH cells expressed nestin mRNA but not Mash-1 mRNA and were also negative for three other olfactory epithelial developmental transcription factors, Math4A, Math4C/neurogenin1, and NeuroD mRNA. Experimental manipulation of nasal explants revealed dual expression of nestin protein and LHRH in cells proximal to the vomeronasal organ anlage that was dependent upon midline cartilaginous/mesenchymal tissues. Prolonged nestin expression in LHRH cells after midline removal is consistent with nasal midline tissues modulating differentiation of LHRH neurons from the nasal placode.

Effects of Nitric Oxide Synthase Blockade on the Acute Response of the Reproductive Axis to Ethanol in Pubertal Male Rats

The effects of ethanol (EtOH) and nitric oxide (NO) are well known in the adult male rat reproductive axis. In the present study, we investigate the effects of EtOH, NO, and their interaction on key genes and reproductive hormone levels in mid-(45-day) and late pubertal(55-day) male rats. Using three different NO synthase blockers—N-nitro-L-arginine methyl ester (L-NAME), N;-nitro-L-arginine (L-NA), and 7-nitroi-ndazole—we show that it is possible to block, in part, some of the disruptive effects of EtOH. L-NAME totally prevented the EtOH-induced fall in serum testosterone in both 45- and 55-day-old rats (p < 0.05 and p < 0.001, respectively). On the other hand, the D-NAME, an inactive isomer of L-NAME, did not protect testosterone from suppression caused by EtOH. Similarly, L-NA and 7-nitroindazole prevented the suppression of testosterone caused by EtOH in 55-day-old animals (p < 0.001 L-NA and p <0.05 for 7-nitroindazole), but not in the 45-day-old rats. Serum luteinizing hormone (LH) was significantly reduced by EtOH in all the studies in both age groups. L.-NAME (but not D-NAME) and L-NA prevented this inhibition in 55-day-old animals (p < 0.001 for L-NAME and p < 0.01 for L-NA). However, only L-NA was able to prevent the effects of EtOH on LH in the 45-day-old rats. 7-Nitroindazole was unable to prevent the decrease in LH in either age group. Despite changes in the other reproductive hormones, there were no consistent changes in hypothalamic concentrations of either LH releasing hormone (LHRH) or its precursor. pro-LHRH. No treatment caused any change in steady-state levels of β-LH mRNA. There were no consistent changes in pro-LHRH mRNA; but, interestingly, in 45-day-old rats, L-NA given with or without EtOH lead to a significant fall in LHRH gene expression. Our findings indicate that the acute suppressive effects of EtOH on the hypothalamic-pituitary-gonadal axis of the purbertal male rat can be at least partially prevented by NO synthase blockade.

Effects of Nitric Oxide Synthase Blockade on the Acute Response of the Reproductive Axis to Ethanol in Pubertal Male Rats

The effects of ethanol (EtOH) and nitric oxide (NO) are well known in the adult male rat reproductive axis. In the present study, we investigate the effects of EtOH, NO, and their interaction on key genes and reproductive hormone levels in mid- (45-day) and late pubertal (55-day) male rats. Using three different NO synthase blockers--N'omega-nitro-L-arginine methyl ester (L-NAME), N'omega-nitro-L-arginine (L-NA), and 7-nitroindazole--we show that it is possible to block, in part, some of the disruptive effects of EtOH. L-NAME totally prevented the EtOH-induced fall in serum testosterone in both 45- and 55-day-old rats (p < 0.05 and p < 0.001, respectively). On the other hand, the D-NAME, an inactive isomer of L-NAME, did not protect testosterone from suppression caused by EtOH. Similarly, L-NA and 7-nitroindazole prevented the suppression of testosterone caused by EtOH in 55-day-old animals (p < 0.001 L-NA and p < 0.05 for 7-nitroindazole), but not in the 45-day-old rats. Serum luteinizing hormone (LH) was significantly reduced by EtOH in all the studies in both age groups. L-NAME (but not D-NAME) and L-NA prevented this inhibition in 55-day-old animals (p < 0.001 for L-NAME and p < 0.01 for L-NA). However, only L-NA was able to prevent the effects of EtOH on LH in the 45-day-old rats. 7-Nitroindazole was unable to prevent the decrease in LH in either age group. Despite changes in the other reproductive hormones, there were no consistent changes in hypothalamic concentrations of either LH releasing hormone (LHRH) or its precursor, pro-LHRH. No treatment caused any change in steady-state levels of beta-LH mRNA. There were no consistent changes in pro-LHRH mRNA; but, interestingly, in 45-day-old rats, L-NA given with or without EtOH lead to a significant fall in LHRH gene expression. Our findings indicate that the acute suppressive effects of EtOH on the hypothalamic-pituitary-gonadal axis of the pubertal male rat can be at least partially prevented by NO synthase blockade.

Luteinizing hormone-releasing hormone gene expression differs in young and middle-aged females on the day of a steroid-induced LH surge

LHRH mRNA levels were examined in young and middle-aged female rats at 4 times (10:00 h, 14:00 h, 18:00 h and 20:00 h) on the day of a steroid-induced LH surge by in situ hybridization with a digoxigenin-labeled riboprobe. Young, but not middle-aged females, exhibited dynamic temporal changes in the number of LHRH mRNA positive neurons detected in the organum vasculosum of the lamina terminalis–preoptic area (OVLT–POA) continuum. Specifically, fewer LHRH mRNA positive neurons were detected at 18:00 h compared with the number detected at 14:00 h and 20:00 h ( P<0.01) in the OVLT–POA of young females. All LHRH mRNA positive neurons present in 4 anatomically matched sections through the rostral POA of young and middle-aged animals were digitized for detailed computer-assisted analysis of the hybridization reaction product. The mean hybridization area ( P<0.00025) and integrated optical density per cell ( P<0.006) were reduced in middle-aged compared to young females consistent with a relative age-related decline in LHRH mRNA levels. Moreover, an age-related reduction in cellular and/or regional hybridization area was noted at each of the time points examined ( P<0.05– P<0.001). These data confirm earlier reports of dynamic changes in LHRH mRNA levels on the day of an LH surge. Furthermore, they support a role for age-related alterations in LHRH gene expression in the disruption of regular estrous cyclicity in middle-aged females.

Transforming growth factor-beta and astrocytic conditioned medium influence luteinizing hormone-releasing hormone gene expression in the hypothalamic cell line GT1.

On the basis of our previous observations indicating that a principle [possibly transforming growth factor-beta1 (TGFbeta1)] secreted by type 1 astrocytes may increase the release of LHRH in the GT1 cell line, it was deemed of interest to analyze whether TGFbeta1 might influence LHRH gene expression in addition to LHRH release in GT1-1 neurons. The effects of TGFbeta1 on the levels of LHRH messenger RNA (mRNA) present in GT1-1 cells have been compared to those found after either coculture of these cells with type 1 astrocytes or exposure of GT1-1 cells to the conditioned medium in which type 1 astrocytes were grown for 24 h. The data obtained indicate that 1) TGFbeta1 increases LHRH mRNA levels 1 and 6 h after the beginning of treatment; longer exposures (24 h) bring about a decrease in LHRH gene expression; 2) a significant stimulatory effect of TGFbeta1 (1 and 6 h of exposure) is also evident on LHRH release; 3) the exposure to the conditioned medium of type 1 astrocytes is able to increase LHRH gene expression in GT1-1 cells at 1 h; LHRH mRNA levels show a small decrease after 6 h of exposure, which becomes more evident at 24 h; and 4) the coculture of GT1-1 cells with type 1 astrocytes is not able to modify LHRH mRNA levels at any time considered. The present data support the concept that glial cells are able to control, possibly through the release of TGFbeta, the gene expression of LHRH in hypothalamic neurons.

Transforming growth factor-beta and astrocytic conditioned medium influence luteinizing hormone-releasing hormone gene expression in the hypothalamic cell line GT1

Transforming growth factor-beta and astrocytic conditioned medium influence luteinizing hormone-releasing hormone gene expression in the hypothalamic cell line GT1

Amplitude and frequency modulation of pulsatile luteinizing hormone-releasing hormone release.

1. A variety of neuroendocrine approaches has been used to characterize cellular mechanisms governing luteinizing hormone-releasing hormone (LHRH) pulse generation. We review recent in vivo microdialysis, in vitro superfusion, and in situ hybridization experiments in which we tested the hypothesis that the amplitude and frequency of LHRH pulses are subject to independent regulation via distinct and identifiable cellular pathways. 2. Augmentation of LHRH pulse amplitude is proposed as a central feature of preovulatory LHRH surges. Three mechanisms are described which may contribute to this increase in LHRH pulse amplitude: (a) increased LHRH gene expression, (b) augmentation of facilitatory neurotransmission, and (c) increased responsiveness of LHRH neurons to afferent synaptic signals. Neuropeptide Y (NPY) is examined as a prototypical afferent transmitter regulating the generation of LHRH surges through the latter two mechanisms. 3. Retardation of LHRH pulse generator frequency is postulated to mediate negative feedback actions of gonadal hormones. Evidence supporting this hypothesis is reviewed, including results of in vivo monitoring experiments in which LHRH pulse frequency, but not amplitude, is shown to be increased following castration. A role for noradrenergic neurons as intervening targets of gonadal hormone negative feedback actions is discussed. 4. Future directions for study of the LHRH pulse generator are suggested.

Gene expression in a subpopulation of luteinizing hormone-releasing hormone (LHRH) neurons prior to the preovulatory gonadotropin surge

Gene expression in luteinizing hormone-releasing hormone (LHRH) neurons was analyzed during the periovulatory period to (1) characterize temporal patterns of LHRH gene expression and their relationship(s) to gonadotropin surges, and (2) determine if any such changes are uniform or dissimilar at different rostrocaudal levels of the basal forebrain. The number of neurons expressing mRNA for the decapeptide, and the relative degree of expression per cell were analyzed using in situ hybridization and quantitative image analysis. Rats were killed at 1800 hr on metestrus (Met), 0800 hr, 1200 hr, 1800 hr, and 2200 hr on proestrus (Pro), or 0200 hr, 0800 hr, and 1800 hr on estrus (E; n = 5-6 rats/group). All sections were processed for LHRH mRNA in a single in situ hybridization assay. Sections were atlas matched and divided into four rostrocaudal groups for analysis: vertical limb of the diagonal band of Broca (DBB), rostral preoptic area/organum vasculosum of the lamina terminalis (rPOA/OVLT), medial preoptic area (mPOA), and suprachiasmatic/anterior hypothalamic area (SCN/AHA). Plasma LH and FSH levels from all animals were analyzed by RIA. The labeling intensity per cell was similar among all time points at all four rostrocaudal levels. The number of cells expressing LHRH mRNA, however, varied as a function of time of death during the estrous cycle, and this temporal pattern varied among the four anatomical regions. At the level of the mPOA, the number of cells was highest at 1200 hr on Pro, and then declined and remained low throughout the morning of E. At the level of the rPOA/OVLT, the greatest number of LHRH neurons was noted later in Pro, at 1800 hr, dropping rapidly to lowest numbers at 2200 hr. No significant changes in LHRH cell number occurred at the DBB or SCN/AHA levels. At all anatomical levels, the secondary surge of FSH was unaccompanied by any change in the number of neurons expressing LHRH mRNA. These data demonstrate that (1) the number of detectable LHRH mRNA-expressing cells fluctuates during the periovulatory period and (2) peak numbers of LHRH-expressing cells are attained in the mPOA before the onset of the LH surge and before peak LHRH cell numbers are seen at more rostral levels. A model is proposed in which gene expression in this subpopulation of LHRH neurons may be activated by preovulatory estrogen secretion and acutely reduced following the proestrous surge of progesterone.

Developmental expression of the genes encoding transforming growth factor alpha and its receptor in the hypothalamus of female rhesus macaques.

Studies in female rats have shown that transforming growth factor alpha (TGF alpha) stimulates release of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual maturation, and that expression of the TGF alpha gene in the hypothalamus increases during both the initiation of normal puberty and after hypothalamic lesions that induce sexual precocity. Since blockade of epidermal growth factor receptors (EGFR), which mediate TGF alpha actions, delayed the normal timing of puberty, it was postulated that TGF alpha/EGFR contributes to the neuroendocrine process that underlies the initiation of normal female puberty. The present study was undertaken to examine the hypothesis that hypothalamic expression of the TGF alpha gene and its receptor changes in relation to the stage of sexual development in nonhuman primates, and to determine whether these changes are accompanied by corresponding alterations in LHRH gene expression. DNA fragments complementary to the coding regions of the rhesus monkey TGF alpha, EGFR and LHRH genes were cloned by reverse transcription-polymerase chain reaction (RT-PCR), sequenced and used to prepare monkey-specific antisense RNA probes. A quantitative RT-PCR was developed in which the cloned sequences were utilized to prepare RNA standards for the quantitation of tissue mRNA levels. Both TGF alpha and EGFR mRNA levels in the medial basal hypothalamus and preoptic area of female monkeys were elevated during neonatal life (1 week to 6 months of age), when FSH secretion is also high, decreased during juvenile development (8-18 months of age), when secretion of both FSH and LH is low, and markedly increased during the expected time of puberty (30-36 months of age). No such changes were observed in either the cerebellum or the cerebral cortex, two brain regions irrelevant to neuroendocrine reproductive control. In contrast to the pronounced alterations in hypothalamic TGF alpha/EGFR gene expression observed during sexual development, LHRH mRNA levels did not vary significantly during this time. Hybridization histochemistry revealed the presence of both TGF alpha and EGFR mRNAs in cells scattered throughout the hypothalamus, but more predominantly in the median eminence, suprachiasmatic nuclei, optic chiasm and cells along the wall of the third ventricle. These results demonstrate that increases in TGF alpha and EGFR gene expression, specific to the neuroendocrine brain, occur during developmental phases in which gonadotropin output is also elevated--most noticeably at the time of puberty.(ABSTRACT TRUNCATED AT 400 WORDS)

Luteinizing hormone-releasing hormone gene expression in the bovine brain: anatomical localization and regulation by ovarian state.

Experiments were conducted to identify neurons in the bovine brain that express the LHRH gene and to determine whether LHRH mRNA levels are influenced by the ovaries. Two groups of postpubertal heifers were utilized: heifers killed during the mid-luteal phase of the estrous cycle (LUTEAL, n = 5) and heifers killed 14-16 wk following ovariectomy (OVX, n = 5). In situ hybridization was performed through use of a 32P-end-labeled deoxyoligonucleotide (59 mer) complementary to the human LHRH mRNA sequence. LHRH-expressing neurons were identified in the diagonal band of Broca, the preoptic area, and the anterior hypothalamus in a manner consistent with immunocytochemical localization. Reduced silver grains, proportional to LHRH mRNA content, were quantified (in pixels, 45x objective) with an image analysis system. Expected serum hormone concentration differences between endocrine states were confirmed by radioimmunoassay for progesterone (LUTEAL > OVX, p < 0.01) and for LH (OVX > LUTEAL, p < 0.01). Compared to the OVX group, LUTEAL heifers had 34% fewer LHRH-expressing neurons (p < 0.05); on the average, these neurons possessed 28% fewer pixels/cell (p < 0.01), indicating fewer copies of LHRH mRNA per cell. When the numbers of pixels in all labeled cells were totalled, LUTEAL animals had 57% fewer pixels (p < 0.05) than did the OVX females--probably reflecting a decrease in LHRH synthetic capacity in the LUTEAL animals. Therefore, during the mid-luteal phase of the bovine estrous cycle, ovarian steroid (i.e., luteal progesterone) suppression of LHRH release (as reflected by serum LH) is coincident with decreased LHRH mRNA in the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential Regulation of Luteinizing Hormone‐Releasing Hormone and Galanin Messenger Ribonucleic Acid Levels by Alpha1Adrenergic Agents in the Ovariectomized Rat

α(1) -Adrenergic control of luteinizing hormone-releasing hormone (LHRH) and galanin mRNA levels was examined in ovariectomized rats. Each rat was ovariectomized and a permanent bilateral cannula was implanted 1 mm dorsal to the preoptic area. Eleven to 14 days later, each rat received one of three treatments: prazosin (α(1) antagonist, n = 8), methoxamine (α(1) agonist, n = 5) or control (no drug, n = 7). Each drug was suspended in a polyacrylamide gel matrix and delivered to the preoptic area via the bilateral cannula. After 24 h of continuous exposure to the adrenergic agents (or control), rats were anesthetized, decapitated and brains were stored in liquid nitrogen until sectioned (7 μm) on a cryostat. In situ hybridization was performed using a [(32) P]-end-labelled 59mer complementary to LHRH mRNA. Reduced silver grains, proportional to LHRH mRNA content, were quantified for number of LHRH cells per section, number of grains per labelled cell and total number of grains in labelled cells. Compared to the controls, prazosin caused a 32% decrease (P<0.05) in the number of cells expressing the LHRH gene. The LHRH cells from untreated animals had a median of 53 grains/cell with a smooth distribution above and below the median. Treatment with prazosin reduced the median number of grains/cell to 36 (P<0.05). When the number of grains in labelled cells were totalled, prazosin decreased (P<0.01) the number of grains by 47%. Surprisingly, methoxamine caused no quantitative changes in any of the parameters examined. This might be explained if LHRH transcription in control animals was proceeding at near-maximal rates supported, in part, by an endogenous α(1) ligand. Alternatively, continuous exposure to this agonist may have resulted in desensitization to its stimulatory effects. When anatomically matched brain tissue sections from these animals were examined for galanin mRNA, no differences among experimental groups were detected. In conclusion, administration of an α(1) -adrenergic antagonist into the preoptic area suppressed levels of LHRH mRNA but not galanin mRNA. Therefore, the data suggest that an endogenous α(1) ligand, such as norepinephrine (or epinephrine), is required to maintain a high level of LHRH gene expression in the ovariectomized rat.

Glucocorticoid receptors in LHRH neurons.

Luteinizing hormone-releasing hormone (LHRH)-producing neurons constitute the final pathway for regulation of reproductive endocrine function by the central nervous system. Chronically elevated levels of glucocorticoids exert an inhibitory effect on reproductive function. Although this is thought to be mediated in part via modulation of classical transmitters and peptides which regulate LHRH synthesis and release, it is also possible that glucocorticoids may regulate LHRH neurons directly. We localized glucocorticoid receptors (GR) in LHRH neurons in the rat central nervous system using immunocytochemistry. In males and randomly cycling females 10-24% of LHRH neurons in the medial septum-diagonal bands of Broca, and preoptic regions colocalized nuclear GR. Ovariectomy increased the percentage of GR/LHRH neurons at the level of the organum vasculosum of the lamina terminalis to 34%, half of which showed both nuclear and cytoplasmic GR. Treatment with estradiol reversed this effect. We suggest that the actions of glucocorticoids on reproductive endocrine function are mediated partly through direct modulation of LHRH gene expression and/or release by activated GR. Moreover, GR in LHRH neurons may provide a mechanism by which the gonadal steroid progesterone can affect LHRH neurons directly, despite a lack of progesterone receptors in these neurons.

Effects of gamma-aminobutyric acid receptor agonists and antagonist on LHRH-synthesizing neurons as detected by immunocytochemistry and in situ hybridization.

Gamma-aminobutyric acid (GABA) is thought to play an important role in the regulation of luteinizing hormone-releasing hormone (LHRH) release but its role in the regulation of LHRH gene expression and LHRH synthesis is not known. We hypothesized that since GABA appears to have primarily inhibitory effects on LHRH cells (at the level of the cell body), GABA may act to decrease LHRH gene expression and peptide synthesis. This hypothesis was tested by examining the effect of GABA receptor activation and GABA receptor blockade on LHRH mRNA and peptide levels employing in situ hybridization and immunocytochemistry. Cells in the preoptic area (POA) of ovariectomized (ovx) rats were selectively exposed in vivo to specific GABA-ergic receptor agonists or an antagonist for up to 24 h. THIP, a specific GABAA receptor agonist, did not have a significant effect on either the intensity of LHRH immunoreactivity, or the number of LHRH-ir cells, observed as compared to controls. Baclofen, a GABAB receptor agonist appeared to decrease the number of cells with the greatest intensity of LHRH immunoreactivity, compared to controls. In situ hybridization, with either a tritiated RNA probe or a 32P-labelled oligonucleotide, complementary to LHRH mRNA, revealed that THIP either had no effect on the labelling intensity (32P-labelled oligonucleotide) or (contrary to our hypothesis) a slight excitatory effect on the level of LHRH mRNA detected per cell (tritiated RNA probe). Bicuculline (a specific GABAA receptor antagonist) decreased both the number of labelled cells observed per section through the POA, and the intensity of labelling observed in sections hybridized with the 32P-labelled oligonucleotide. These results suggest that in the POA GABAA receptors do not exert an inhibitory effect on LHRH gene expression, but rather could affect LH perhaps by electrically inhibiting LHRH neurons. In contrast, baclofen appeared to exert an inhibitory effect on LHRH gene expression, since the number of grains per labelled cell in the POA of baclofen-treated rats was lower than the grains per labelled cell of control rats. Also, similar to the results obtained with immunocytochemistry, in situ hybridization following baclofen treatment suggested that activation of GABAB receptors is able to reduce the number of neurons with the highest levels of LHRH mRNA. Thus, in the POA, GABA acting through GABAB receptors could be effective through changes in mRNA or peptide synthesis.

Activation of intracellular pathways with forskolin and phorbol ester increases LHRH mRNA level in the rat hypothalamus superfused in vitro

Two intracellular signal transduction mechanisms such as cAMP-protein kinase a and phosphatidylinositol (PI) turnover-protein kinase c are known to be dually involved in the regulation of luteinizing hormone-releasing hormone (LHRH) release. However, it is not yet evident that the activation of two intracellular pathways affects the LHRH gene expression. The present study aims, therefore, to determine whether the activation of two intracellular pathways affects changes in LHRH mRNA. To this end, we took advantage of an in vitro superfusion system, where rat hypothalamic tissues were superfused with media containing forskolin (FKN) and/or phorbol-12-myristate-13-acetate (PMA). Superfusates were collected at 10-min intervals and LHRH release was determined by radioimmunoassay. After a 2-h superfusion period, the post-superfusion hypothalami were recovered and poly (A) RNA fractions were isolated. Alterations in LHRH mRNA in response to FKN and/or PMA were determined by an RNA-blot hybridization assay using a 32P-end-labeled LHRH oligonucleotide (29-mer) probe. In vitro perfusion of hypothalamic fragments with PMA and/or FKN stimulated LHRH release as well as LHRH mRNA. The combined infusion of FKN and PMA did not produce an additive effect on the LHRH mRNA levels, but it was effective in synergistically increasing LHRH secretion in vitro. These data clearly demonstrate that the biosynthetic machinery of HRH is influenced by activation of two intracellular pathways, both cAMP-protein kinase a and phosphatidyl-inositol turnover-protein kinase c, indicating the transsynaptic regulation of hypothalamic LHRH gene expression.