Immunoreactive Luteinizing Hormone-releasing Hormone Research Articles

Immunohistochemical demonstration of LHRH-neurons in young rat hypothalamus: Light and electron microscopy.

Localization and ultrastructural appearance of immunoreactive LHRH neurons were studied in 2-, 8- and 21-day-old rats. The lower half of the diencephalon including the septo-preoptic area was serially cut at 10 microns in the frontal plane, and all sections stained with an anti-LHRH serum were examined light microscopically. Although the antiserum could not demonstrate LHRH-immunoreactive perikarya in adult rats, they were stained in these young rats. The immunoreactive perikarya were scattered in the following regions: nucleus tractus diagonalis, medial septum, medial preoptic area, lateral septum, lateral preoptic area, tuberculum olfactorium, suprachiasmatic area, supraoptic area, pericommisural area, anterior commissure, vascular organ of the lamina terminalis (OVLT), lateral hypothalamic region and lateral tuberal region. The first three regions contained many cells but others showed only sporadical cells. The cells were most frequently observed in 8-day-old rats. Most of the cells appeared to be bipolar showing two immunoreactive processes. Electron microscopy observation was made using the section prepared by a pre-embedding technique. In the immunoreactive perikarya, secretory granules, ribosomal particles, and the particles on the outer surface of endoplasmic reticulum appeared to be immunoreactive, but the Golgi area was immunonegative. The secretory granules appeared along the cytoplasmic membrane but were surprisingly few in number. By contrast, the immunoreactive nerve terminals in the median eminence and in the OVLT contained many granules. These findings suggest that LHRH produced in the perikarya is prompted to be transported and stored in the axonal terminals.

Localization of LH-RH immunoreactivity in the avian retina.

The present study demonstrated numerous LH-RH--positive structures in the paddy-bird (padda orizivora) retina. LH-RH-positive cells were seen in the ganglion cell layer (GL), inner plexiform layer (IPL) and inner nuclear layer (INL). Judging from the morphological features, the former two types of cells may belong to the displaced amacrine cells and the latter to the amacrine cells. LH-RH-positive fibers were found in the nerve fiber layer, GL, IPL, INL and outer plexiform layer.

Decapeptide luteinizing hormone releasing hormone in ovine pineal gland.

Ovine pineal gland extracts were examined throughout the year for content and molecular form of immunoreactive (IMR) LH releasing hormone (LH-RH). The content of IMR LH-RH in pineal glands collected in spring and summer was 160-2230 pg/gland, while the content in pineal glands collected in autumn and winter was lower (31-39 pg/gland). Pineal gland IMR LH-RH, purified by affinity and Sephadex G-25 chromatography, yielded displacement curves parallel to those of hypothalamic IMR LH-RH and synthetic LH-RH in radioimmunoassays employing four antisera which require different regions of the decapeptide for effective binding, suggesting considerable similarities in the structure of the molecule. The majority of pineal gland IMR LH-RH behaved identically to hypothalamic IMR LH-RH and synthetic LH-RH on gel filtration, cation exchange chromatography, high-pressure liquid chromatography and cellulose thin-layer chromatography. However, a small amount of a less positively charged LH-RH species was also present in all pineal gland extracts. Our findings indicate that hypothalamic decapeptide LH-RH occurs in the ovine pineal gland.

The luteinizing hormone releasing hormone (LHRH) neuronal networks of the guinea pig brain. III. The regulation of cyclic gonadotropin secretion

The effects of selected CNS lesions on the ovulatory cycle as well as the distribution of LHRH terminals in the median eminence of the female guinea pig were examined. Radiofrequency lesions were placed in the medial preoptic area (MPOA) or suprachiasmatic nucleus (SCN). Animals were studied for varying lengths of time (1-5 months) following lesion placement. The occurrence of at least two ovulatory cycles was determined by cyclicity in vaginal opening, the presence of viable corpora lutea and measurements of luteal phase elevations in serum progesterone. The distribution of LHRH immunoreactivity was determined by immunocytochemical procedures on Bouin's fixed, paraffin-embedded brain sections. Lesions of the MPOA or SCN resulted in different degrees of loss of LHRH fibers in the zona externa of the ventral and lateral aspects of the infundibular stalk as well as complete loss of terminals throughout the zona interna. Despite depletion of LHRH fibers, all animals with SCN (n = 8) and the majority with MPOA (4/6) lesions showed regular ovulatory cycles. Two females with MPOA lesions presented two different anovulatory syndromes. Anovulation appeared unrelated to the size or site of the lesions or in the degree of depletion of LHRH fibers in the infundibulum. It would appear that these brain areas and their LHRH projections are not essential for cyclic gonadotropic release.

Presence and localization of immunoreactive luteinizing hormone-releasing hormone (LHRH) within the olfactory bulbs of adult male and female rats

A sensitive and specific radioimmunoassay was employed to measure the immunoreactive luteinizing hormone releasing hormone (LHRH) concentration from localized areas within the olfactory bulbs of adult male and female rats. Although no significant sex differences or lateralization were obtained, LHRH concentration within the posterior half of the olfactory bulb was significantly greater than that obtained in the anterior half for both males and females. The presence of LHRH within the olfactory bulbs and localization to areas involved in the chemical stimulation of reproductive processes suggests the possibility that this olfactory bulb LHRH may be involved in mediating pheromonally stimulated endocrinological responses.

Electron microscopic study of immunoreactive LHRH perikarya with special reference to neuronal regulation.

In early postnatal rats, immunoreactive LHRH perikarya in the preoptic area were studied by light and electron microscopy. Synaptic junctions were found between the immunoreactive perikaryon or its process, and the immunonegative nerve fibers. The significance of these synapses is discussed in relation to possible mechanisms by which the activities of LHRH neurons are regulated.

The fetal development of the luteinizing hormone-releasing hormone (LHRH) neuronal systems of the guinea pig brain

We have studied the distribution of LHRH-like immunoreactive material in fetal guinea pig brains beginning at day 25 of gestation. Cells and processes were first detected throughout the peripheral, intracranial and central course of the nervus terminalis at 28 (but not 25) days of gestation. The localization of LHRH in this structure preceded its appearance in the hypothalamus and coincided with the initial detection of immunoreactive LH in the pituitary gland. The possible role of the LHRH neuronal network within the nervus terminals in the development of reproductive function is discussed. Comparisons between the brains of littermates of both sexes were made at each age (days 28 through 60 of gestation) to determine possible differences between the sexes in the development of the LHRH neurosecretory systems. No sexually dimorphic features were evident in these systems throughout the prenatal period except at days 40 and 45. At these ages, differences in the number of LHRH neurons in the arcuate nucleus were found between the sexes in some but not all of the brains examined. These differences in LHRH concentrations may reflect the onset of testicular activity as indicated by an increase in serum testosterone levels. Increased serum testosterone concentrations were observed in the male fetuses beginning at 45 days of gestation. However, cell counts made within this nucleus from days 40 through 60 of gestation indicated no comparable sexual dimorphism in the total neuronal population which appeared to be relatively stable throughout this period of brain growth. The number of immunoreactive LHRH neurons visible throughout the brain increased from days 30 through 45 and fewer LHRH cells were seen on days 50 and 60 of gestation, particularly in the arcuate nucleus. The apparent decrease in visible LHRH neurons was concomitant with an increase in number and more extensive distribution of immunoreactive processes throughout the hypothalamus and in certain extrahypothalamic areas of the brain.

Rat testis immunoreactive LH-RH differs structurally from hypothalamic LH-RH

Abstract Luteinizing hormone releasing hormone immunoreactivity (LH-RH-IR) has been identified in acetic acid extracts of adult rat testes and partially purified by immunoaffinity chromatography. On Sephadex G-100 this material separated into four major peaks of >100K, ∼32K, ∼5K and ≤4K daltons. The ≤4K peak of LH-RH-IR eluted later than synthetic hypothalamic LH-RH decapeptide on Sephadex G-25. Antibody binding studies on the various LH-RH-IR species with antisera specific for different regions of synthetic LH-RH decapeptide indicate that all the testicular LH-RH-IR molecules have C-terminal immunological homology with the hypothalmic decapeptide but differ towards the N-terminus of the decapeptide sequence.

The Effects of Ions, Metabolic Inhibitors, and Colchicine on Luteinizing Hormone-Releasing Hormone Release from Superfused Rat Hypothalami

In the present study, the effects of ions, metabolic inhibitors, and colchicine on the release of immunoreactive LHRH from superfused male rat hypothalami were examined. High extracellular potassium reliably stimulated LHRH release; this K+-evoked release has been attributed to membrane depolarization triggering influx of Na+ and Ca++ and leading to exocytosis of LHRH. As such, stimulated release of LHRH was clearly Ca++ dependent. However, in the absence of Na+ or in the presence of the Na+ channel blocker tetrodotoxin, K+-stimulated release persisted, indicating that Na+ was not obligatory in this release process. Ouabain, a Na+-K+-APase inhibitor, blocked K+-stimulated release of LHRH, although basal release was unaffected. 2,4-Dinitrophenol, which uncouples oxidative phosphorylation, surprisingly resulted in significant elevations in both basal and K+-stimulated LHRH release, whereas metabolic energy inhibitors, cyanide and iodoacetic acid, reduced basal release and completely blocked the K+-stimulate...

Progesterone-induced changes in hypothalamic luteinizing hormone-releasing hormone and catecholamines: differential effects of pentobarbital.

The effects of pentobarbital (Pnt) treatment on the progesterone (P)-induced afternoon increase in the medial basal hypothalamic (MBH) LHRH and serum LH and FSH levels in ovariectomized estradiol benzoate-primed rats were studied. Pnt injection before P blocked the afternoon rise in serum gonadotropins but failed to alter the increase in the MBH LHRH levels. Moreover, when Pnt was injected 150 min after P, the MBH LHRH content continued to rise to levels 25-37% above those seen in control rats. Analyses of LHRH concentrations in discrete hypothalamic nuclei revealed that the Pnt-induced accumulation was confined mainly to the median eminence, with a small increase in the suprachiasmatic nuclei region. P administration increased the MBH norepinephrine activity and concurrently decreased dopamine activity. Pnt was ineffective in suppressing the MBH LHRH response in these rats, but drastically reduced norepinephrine and accelerated dopamine turnovers in the MBH. These studies show 1) no definitive cause and effect relationship of the increments in MBH LHRH either with LH release (or LHRH release) or with changes in hypothalamic catecholamines induced by P treatment, and 2) that the striking rise in the MBH LHRH levels in estradiol benzoate-primed rats may represent formation of new immunoreactive LHRH predominantly in the median eminence region.

Immunohistochemical demonstration of LHRH neurons and their pathways in the rat hypothalamus.

LRHR neurons and fiber tracts have been studied immunohistochemically in rats. After the intraventricular infusion of colchicine, a number of immunoreactive LHRH neurons were consistently evident in the preoptico-septal area and in the diagonal band of Broca. The immunoreactive fiber tracts originating in those areas were classified into two groups: preoptico-terminal and preoptico-infundibular tracts. The former terminated in the OVLT. The latter was further divided into the mediobasal and laterobasal tracts; both were found to terminate in the median eminence. The mediobasal tract ran posteriorly through the ventral portion of the lateral periventricular area. The laterobasal tract ran posteriorly in the medial forebrain bundle and was accompanied by several immunoreactive neurons. The immunoreactive cell bodies were further scattered in the base of the tuberal hypothalamic area. An anterior bilateral Halász cut in the retrochiasmatic region was followed by a great reduction of the immunoreactive materials in the median eminence and by the appearance of the immunoreactive perikarya and in the preoptico-septal area and in the diagonal band of Broca. A horizontal hypothalamic cut, on the other hand, which disconnected the tubero-infundibular tract between the median eminence and the arcuate nuclei, caused neither reduction of the immunoreactive materials in the median eminence nor appearance of the immunoreactive perikarya in the arcuate nuclei. Those results indicate that the bulk of the immunoreactive LHRH is synthesized in the anterior hypothalamic area and transported by the preoptico-terminal and -infundibular tracts but a minor part is produced in the cell bodies located in the basal tuberal hypothalamus and transported to the median eminence.

Light and electron microscopic observation on the appearance of immunoreactive LHRH in perinatal rat hypothalamus.

The appearance and localization of LHRH were studied in the developing hypothalamus of perinatal rats using the unlabelled antibody method. By light microscopy, immunoreactive LHRH was first detected as brown dots on day 18.5 of gestation in the OVLT and on day 19.5 in the median eminence, respectively. When the median eminence was examined by the preembedding immunohistochemistry technique for electron microscopy, the occurrence of immunoreactive LHRH fibers could be demonstrated on day 18.5. These fibers were thin and very occasionally encountered near the surface of the lateral regions of the median eminence. The axoplasm contained a few immunopositive secretory granules and also extragranular immunoreactive products. With development, a gradual increase was noted both in number and size of nerve fibers with a concomitant accumulation of secretory granules within the axoplasm. A possible physiological significance of LHRH is discussed in relation to the onset of hypothalamo-hypophysial system in fetal life.

Plasma Luteinizing Hormone-Releasing Hormone Levels in Normal Women and Patients with Amenorrhea

Plasma levels of immunoreactive luteinizing hormone-releasing hormone (LH-RH) in 89 women with normal menstrual cycles and 109 patients with amenorrhea of various etiologies were determined by specific radioimmunoassay after methanol extraction. The LH-RH levels in patients with hypergonadotropism and Sheehan's syndrome were more than 10 pg/ml, and were significantly higher than basal levels during normal cycles (4.2 +/- 0.3 pg/ml). The mean levels of LH-RH were low in all hypogonadotropic patients with amenorrhea, with the exception of a few patients who showed high LH-RH levels suggestive of a pituitary lesion. The LH-RH levels in patients with polycystic ovary syndrome were comparable with basal levels during normal cycles. It is concluded that, although there is considerable overlap of the plasma LH-RH levels in each group of amenorrheic patients divided according to gonadotropin level, determination of LH-RH levels may be useful for the differentiation of hypothalamic amenorrhea from pituitary amenorrhea.

Distribution of Luteinizing Hormone Releasing Hormone in the Canine Hypothalamus: Effect of Castration and Exogenous Gonadal Steroids

SUMMARY The effect of exogenous gonadal steroids or orchidectomy on the distribution and concentration of luteinizing hormone-releasing hormone (lhrh) in the hypothalamus of the dog was studied. Various hypothalamic areas of the canine brain were extracted with 90% methanol and processed, and lhrh was assayed, using a specific radioimmunoassay. The highest concentration of immunoreactive lhrh was found in the infundibular, premammillary, preoptic, and suprachiasmatic areas in intact male dogs and in anestrous female dogs. Measurable amounts of lhrh were found in the mammillary and lateral hypothalamic regions. The hypothalamus in the male dog had significantly (P < 0.05) higher lhrh concentrations 3 mm lateral to the midsagittal line (the lateral hypothalamic area) as compared with the area in the anestrous female dog. Daily estrogen treatment of female dogs with 0.2 mg/day or 1 mg/day, im, or with testosterone propionate (1 mg/day, im) for 3 weeks increased lhrh concentrations 2.5- to 5-fold in the mammillary, premammillary, and infundibular areas and almost 40-fold in the preoptic, rostral commissural area and in the lateral and dorsal hypothalamic regions. Orchidectomy caused an increase in lhrh concentration in the preoptic and suprachiasmatic area with a concurrent decrease in lhrh in the infundibular area. Therefore, in the dog, lhrh is possibly synthesized or transported (or both) from different regions of the hypothalamus to the median eminence, and the gonadal steroid feedback mechanism is complex, involving multiple sites of action in the hypothalamus and the components of the limbic system.

The effects of ions, metabolic inhibitors, and colchicine on luteinizing hormone-releasing hormone release from superfused rat hypothalami.

In the present study, the effects of ions, metabolic inhibitors, and colchicine on the release of immunoreactive LHRH from superfused male rat hypothalami were examined. High extracellular potassium reliably stimulated LHRH release; this K+-evoked release has been attributed to membrane depolarization triggering influx of Na+ and Ca++ and leading to exocytosis of LHRH. As such, stimulated release of LHRH was clearly Ca++ dependent. However, in the absence of Na+ or in the presence of the Na+ channel blocker tetrodotoxin, K+-stimulated release persisted, indicating that Na+ was not obligatory in this release process. Ouabain, a Na+-K+-APase inhibitor, blocked K+-stimulated release of LHRH, although basal release was unaffected. 2,4-Dinitrophenol, which uncouples oxidative phosphorylation, surprisingly resulted in significant elevations in both basal and K+-stimulated LHRH release, whereas metabolic energy inhibitors, cyanide and iodoacetic acid, reduced basal release and completely blocked the K+-stimulate...

Immunoreactive luteinizing hormone-releasing hormone (LHRH) in ovine pineal glands.

Ovine pineal glands (PGs, n = 168), cerebral cortex(C) and stalk-median eminences (SMEs) were obtained fresh from slaughterhouse sheep and mature ewes during the estrous cycle. Tissues were extracted in 2N acetic acid (aa) followed by either glacial aa or ethanol. Portions of the preparations were further subjected to gel filtration using Sephadex G-25. Neutralized extracts were assayed for LHRH using radioimmunoassay. LHRH immunoreactivity was consistantly detected in the various extracts, ranging from 7-150 pg/PG (0.1-1.7 pg/mg), 0.1-0.3 pg/mg C and 35-72 ng/SME (0.7-1.4 ng/mg). Slopes of binding displacement regression lines for the preparations were not different from that of synthetic LHRH. Chromatographic elution profiles for preparations of SMEs and PGs were similar and the bulk of immunoreactivity eluted in a position coincident with that of synthetic LHRH. Immunoreactivity in cortical extracts ran in advance of the others. PG contents of LHRH immunoreactivity were similar during the estrous cycle.

Effect of Ovariectomy and Adrenalectomy on Luteinizing Hormone-Releasing Hormone in Pituitary Stalk Blood from Female Rats

The spontaneous release of LHRH has been measured in female rats subjected to ovariectomy and adrenalectomy to determine if the release of LHRH is altered in the absence of sex steroids. Release of LHRH was determined by RIA of blood collected from the cut pituitary stalk of rats anesthetized with alphaxalone-alphadolone acetate. The concentration and content of immunoreactive LHRH in pituitary stalk plasma of rats which had been ovariectomized and adrenalectomized (Ovx-Adx) for either 4 or 28 days were comparable to the levels in intact rats at the time of the proestrous surge of LH and significantly increased when compared with levels in intact diestrous rats. The increased levels of LHRH in Ovx-Adx rats were not altered by estradiol benzoate (50 μg) injected sc either 1 h before or at the beginning of the collection of portal blood. The concentration of immunoreactive LH in jugular venous plasma obtained before cutting the pituitary stalk was significantly increased in proestrous rats and in 28-day Ovx...

Comparative aspects of luteinizing hormone-releasing hormone structure and function in vertebrate phylogeny.

Immunoreactive LHRH was detected in hypothalamic and extrahypothalamic brain extracts of the rat, bird (pigeon and chicken), reptile (tortoise and lizard), amphibian (frog and toad), teleost (cichid), and elasmobranch (dogfish) and in the whole brain of the cyclostome (bagfish). The concentration of hypothalamic immunoreactive LHRH was more than 5-fold greater than that of the extrahypothalamic brain. Mammalian and amphibian hypothalamic immunoreactive LHRH yielded displacement curves parallel to those of synthetic LHRH in assays employing four antisera which recognize different regions of the decapeptide, thus suggesting a similarity in the structure of their LHRH. Hypothalamic immunoreactive LHRH from the bird, reptile, teleost, and elasmobranch differed from the mammalian and amphibian peptide in yielding displacement curves nonparallel to those of synthetic LHRH with three different antisera (1076, 743, and 744) which bind between Trp3 and Pro9 of LHRH. The differenece in structure appears to be near Leu7. With antiserum 422 which binds the NH2- and CO2H- termini of LHRH, bird, reptile, and teleost hypothalamic extracts yielded displacement curves parallel to that of synthetic LHRH. Bird, reptile, and teleost hypothalamic extracts showed displacement curves parallel to each other in all assays. In studies on the relative quantitation of LHRH, all four antisera gave similar values of immunoreactive LHRH concentration in mammalian hypothalamic extracts and in amphibian hypothalamic extracts. By contrast, assay of bird, reptile, teleost, and elasmobranch hypothalamic extracts with antiserum 422 gave much higher values than did the other antisera, suggesting that the LHRH peptide is structurally different from mammalian and amphibian LHRH in the region of Leu7 but similar at the NH2- and CO2H-termini. These conclusions are supported by studies on the biological activity of hypothalamic LHRH from the different species using dispersed ovine anterior pituitary cells in culture. The LH release responses to equivalent amounts of immunoreactive LHRH (as measured by antiserum 422 which binds a region of LHRH essential for biological activity) from the various species were similar, indicating that the biologically active region of the molecule has been conserved in evolution. Structural differences in vertebrate hypothalamic immunoreactive LHRH were confirmed by cation exchange and high pressure liquid chromatography. Our findings of differences and similarities in vertebrate LHRH support a contemporary phylogenetic scheme.

Luteinizing Hormone-Releasing Hormone and the Human Menstrual Cycle

Plasma concentrations of LHRH were measured by radioimmunoassay in daily samples obtained from 10 normally ovulating women. The normalcy of each menstrual cycle was determined by measuring luteinizing hormone (LH), follicle-stimulating hormone (FSH), total estrogen, and progesterone concentrations. Six women had consistently measurable immunoreactive LH-releasing hormone (LHRH) in every blood sample. In four, LHRH could not be detected in some of the samples, more frequently during the follicular phase. LHRH levels varied between 10 and 35 pg/ml during the menstrual cycle. At midcycle, coincident with the LH surge, the mean LHRH level (17.6 +/- 4.4 pg/ml) was not significantly different from the mean follicular or luteal phase values. The lowest LHRH level, 11.4 +/- 4.6 pg/ml, was observed on the day of the estrogen surge. A detailed evaluation was made of the specific days of the cycle. Blood samples obtained from an indwelling venous catheter every 20 minutes over a 3 or a 5 hour period during the days of the estrogen surge and the LH surge and on the following day showed no correlation between OHRH, LH, and FSH values.

Luteinizing hormone-releasing hormone in cerebrospinal fluid of women.

The concentration of luteinizing hormone-releasing hormone (LH-RH) in both cerebrospinal fluid (CSF) and plasma in 36 women with various conditions were determined, and the correlation of LH-RH concentrations between plasma and CSF was studied. Paired samples of plasma and CSF were obtained just before operation and were extracted with methanol for LH-RH radioimmunoassay. LH and FSH were also measured by radioimmunoassay. The mean plasma LH-RH levels were 3.79 pg/ml in the follicular phase, 4.15 pg/ml in the luteal phase, 12.73 pg/ml in postmenopausal women and 2.31 pg/ml in pregnant women at term. No immunoreactive LH-RH in the CSF was found in 29 out of 36 subjects, and the remaining 7 subjects showed very low levels (less than 0.95 pg/ml). The LH-RH levels in the CSF of post-menopausal women were not higher their those in the remaining 3 groups, although their plasma levels of LH-RH were significantly (p less than 0.001) elevated. The mean plasma levels of LH and FSH in post-menopausal women were significantly higher than those in normal cyclic women and in pregnant women. The present results suggest that little or no LH-RH is present in the CSF, and it is not clear that there is any correlation between the LH-RH level of plasma and those of CSF.