Unilateral ovariectomy-induced luteinizing hormone-releasing hormone content changes in the two halves of the mediobasal hypothalamus

The luteinizing hormone-releasing hormone (LHRH) content of the two halves of the mediobasal hypothalamus (MBH) has been studied two weeks after unilateral or bilateral ovariectomy (OVX) in the rat. In control animals, the LHRH content of the right-side MBH was higher than that of the left side. Right-side OVX induced a significant increase in LHRH content on the right-side MBH while left-side OVX was followed by elevated LHRH content on the left-side MBH. In contrast, bilateral OVX induced a significant decrease on the right-side MBH. The observation that unilateral OVX induced increased LHRH content in the ipsilateral half of the MBH to OVX, supports the view that a neural pathway might connect the ovary and the MBH.     

Immunocytochemical localization of luteinizing hormone-releasing hormone in neurons in the medial basal hypothalamus of the female rat

Summary Luteinizing hormone-releasing hormone (LHRH)-neurons were localized with a LHRH antiserum (WP-1) in the medial basal hypothalamus (MBH) of the female rat using the sagittal hypothalamic slice preparation in combination with the peroxidase-antiperoxidase immunocytochemical technique of Sternberger (1979). Numerous darklystained perikarya were visualized in the cell-poor zone, lateral arcuate nucleus and the median eminence. The processes of these neurons contributed to the intensely-stained fiber bundle above the tubero-infundibular sulcus. The fibers in this tract run in a rostrocaudal plane in the lateral external zone of the median eminence. Also, numerous fibers course into the internal zone of the median eminence, perpendicular to the rostrocaudal plane. Several LHRH-immunoreactive perikarya also were identified in the periventricular area caudal to the optic chiasm (retrochiasmatic area). The LHRH neurons were small (10 m) bipolar neurons with fibers (dendrites) exiting from either pole which showed very little branching. It appears that the sagittal slice in combination with the appropriate fixation procedure and LHRH antiserum enabled us to demonstrate the presence of LHRH-immunoreactive perikarya in the MBH of the female rat.Supported by Health Research Services Foundation Grant W-22 and NIH Grant NS16419     

P2X receptors are expressed on neurons containing luteinizing hormone-releasing hormone in the mouse hypothalamus

Expression of P2X₁, P2X₂, P2X₃, P2X₄, P2X₅ and P2X₆ receptors, members of a family of ATP-gated cation channels, on neurons containing luteinizing hormone-releasing hormone (LHRH) in the mouse hypothalamus was studied with double-labeling fluorescence immunohistochemistry. This study demonstrated that different combinations of P2X receptor subunits were expressed on the perikarya and axon terminals of LHRH-producing neurons. It was shown for the first time that P2X₂, P2X₄, P2X₅ and P2X₆ receptor subunits were expressed on the perikarya of LHRH-producing neurons and P2X₂ and P2X₆ on their axon terminals. These results suggest that activation of P2X receptors by ATP via different homomeric or heteromeric P2X receptors at both presynaptic and postsynaptic sites could be involved in the regulation of LHRH secretion at the forebrain level.     

LHRH类似物在乳猪体内的药代动力学研究

目的 :研究黄体激素释放激素类似物 (luteinizinghormone releasinghormoneanalogue ,LHRH -A)在健康乳猪体内的药代动力学规律。方法 :采用氯胺T法 ,用12 5I标记LHRH A ,放化纯度 96.2 %。按 1mg·kg 1体重于乳猪颈背部两侧分别肌注 ,药后 2 1个不同时点分别取血 0 .2 0～ 0 .2 5ml做血药浓度测定 ,用大型药理学计算软件DAS统计其药代动力学参数。结果 :LHRH A在体内的分布、消除以二室模型方式进行。Tmax :1 .1 667± 0 .40 82h ,Cmax :98.82 0 0± 69.5 5 86μg·L 1,T1 /2 β :5 5 8.3 71 4± 3 76.93 2 8h ,AUC :5 5 65 .1 2 41± 3 5 60 .1 962 μg·L 1·h 1,Vd/F :0 .0 0 2 2± 0 .0 0 1 6L。结论 :LHRH A在乳猪体内的分布、消除方式为二室模型 ,且消除较慢     

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 micrograms 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 microns 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.     

Access of luteinizing hormone-releasing hormone neurons to the vasculature in the rat

Luteinizing hormone-releasing hormone is secreted into the hypophysial portal vasculature through which it controls the release of the gonadotropins. The peptide also acts as a neurotransmitter in various loci within the central nervous system. It is not known whether these roles are performed by separate populations of luteinizing hormone-releasing hormone neurons. Some recent tracing experiments suggest that this is the case (Silverman et al., J. Neurosci. 7, 2312, 1987; Jennes and Stumpf, Neuroscience 18, 403, 1986). One aspect of this question was addressed in the current study by intraperitoneal introduction of Fluoro-Gold (a retrograde tracer) into male and female rats under various age and hormonal conditions. Brain sections from the anterior olfactory nuclei to the median eminence were treated for the immunocytochemical demonstration of luteinizing hormone-releasing hormone. In all cases, regardless of the age, sex or hormonal condition of the animal, the Fluoro-Gold tracer was found in more than 90% of the luteinizing hormone-releasing hormone neurons. We conclude that virtually all luteinizing hormone-releasing hormone neurons in the rat secrete outside the blood-brain barrier, including those which project to sites within the central nervous system.     

Characterization of high affinity monoclonal antibodies against the luteinizing hormone-releasing hormone

Four hybridoma clones (BKL1, BKL2, BKL5 and BKL6), secreting monoclonal antibodies against the decapeptide luteinizing hormone releasing hormone (LHRH), were obtained by the fusion of Sp 2/0-Ag14 myeloma cells with spleen cells of a Balb/k mouse immunized with a conjugate of thyroglobulin-LHRH. All four monoclonal antibodies belong to the IgG1 subclass. The antibodies cross-reacted in RIA from 9.3 to 21% with 4-10 LHRH and less than 1% with 7-10 LHRH, 1-3 LHRH and LHRH-OH; 4-6 LHRH showed no cross-reaction. A RIA based on the BKL2 antibody and able to detect 4 pg LHRH per tube was developed. An extract of rat hypothalamus was submitted to Sephadex G50 separation and a single peak of LHRH immunoreactivity corresponding to synthetic LHRH, was detected with the antibody BKL2. When this material was further analyzed by HPLC, we found that the major peak of immunoreactivity co-migrated with synthetic LHRH. The data shows that the antibodies should be useful tools for biochemical and physiological studies on LHRH.     

Estrogen modulates norepinephrine-induced accumulation of adenosine cyclic monophosphate in a subpopulation of immortalized luteinizing hormone-releasing hormone secreting neurons from the mouse hypothalamus

A subpopulation of luteinizing hormone-releasing hormone (LHRH)-producing cells that express the intermediate filament protein vimentin and the neuronal marker neurofilament 145, but not neurofilament 200 nor glial fibrillary acidic protein, has been isolated from GT1-7 cultures. These cells express the mRNA encoding estrogen receptor alpha (ERalpha) and respond to physiological concentrations of 17beta-estradiol (E2) by reducing the accumulation of cyclic adenosine monophosphate induced by norepinephrine, but not that induced by direct activation of adenylate cyclase. These results indicate that the activity of LHRH-producing neurons may be directly modulated by estrogen. In addition, they are suggestive of an estrogen-dependent desensitization of the beta-adrenoceptor in these cells.     

Antagonistic analogs of luteinizing hormone-releasing hormone are mast cell secretagogues

The histamine-releasing activity of luteinizing hormone-releasing hormone (LHRH) antagonistic analogs has been documented. Antagonists of LHRH elicited in vitro histamine release from mast cells obtained from previously unexposed rats. Intradermal injection of the antagonists caused increased local skin permeability. Anaphylactoid reactions followed subcutaneous injection of the antagonists and in some cases these edematous reactions were accompanied by increased serum histamine levels. These studies show that these small peptides can cause mast cell degranulation and suggest that the neuropeptide, LHRH, may have modulating effects on the immune system.     

Treatment of uterine leiomyomas with luteinizing hormone-releasing hormone antagonist Cetrorelix

The efficacy of the luteinizing hormone-releasing hormone antagonist Cetrorelix (SB-75) in the medical management of uterine leiomyomas (fibromas) was evaluated. Cetrorelix was administered to 18 pre- menopausal women with myomas with a mean age of 33.3 years, who had been candidates for hysterectomy. The initial dose of Cetrorelix was 5 mg twice daily s.c. for the first 2 days and thereafter 0.8 mg was given twice daily s.c. for at least 3 months. The mean duration of the treatment was 4.4 months. Before the therapy with Cetrorelix, the mean uterine volume, measured by ultrasonography, was 395.4 +/- 69.2 ml (range 89-1166). Sixteen patients showed a progressive reduction in uterine volume from 410.4 +/- 77.1 to a mean of 230.8 +/- 52.6 ml at 3 months. All patients became amenorrhoeic and had hot flushes. After treatment with Cetrorelix, a surgical myomectomy was performed in 12 women. One of the patients subjected to myomectomy after therapy with Cetrorelix became pregnant. These patients have been followed for up to 25 months and only in one case has the uterine volume increased after therapy. Three patients had good responses to therapy with Cetrorelix and it was decided to follow them only by observation. One patient became pregnant 2 months later. In the other patient, the uterine volume remained unchanged for the duration of the follow-up of 2 years and the third patient showed an increase after 21 months. In three patients, it was necessary to perform total hysterectomy. In 14 patients, serum concentrations of luteinizing hormone, follicle stimulating hormone and oestradiol decreased after the administration of the first dose of Cetrorelix and continued at subnormal values throughout therapy. In 15 patients who were not subjected to total hysterectomy, menstrual function returned at 1 month after cessation of treatment. Overall results support the use of Cetrorelix for the management of uterine leiomyomas.      

Chicken II luteinizing hormone-releasing hormone inhibits the M-current of bullfrog sympathetic neurons

The M-current of dissociated bullfrog sympathetic neurons, measured by the whole-cell patch clamp technique, is powerfully inhibited by chicken II LH-RH (luteinizing hormone-releasing hormone), with 50% inhibition near 1 nM. Chicken II LH-RH is approximately 100 times more potent than salmon LH-RH, and at least 1000 times more potent than other known naturally occurring LH-RH analogs (chicken I LH-RH, mammalian LH-RH, and lamprey LH-RH). This high potency makes chicken II LH-RH a candidate for the endogenous transmitter mediating the late, slow excitatory postsynaptic potential (EPSP) in bullfrog sympathetic ganglia.     

Effect of follicle stimulating hormone treatment on the pituitary response to luteinizing hormone-releasing hormone in post-menopausal women

To study the role of exogenous follicle stimulating hormone(FSH) in the attenuation of luteinizing hormone (LH) responseto luteinizing hormone-releasing hormone (LHRH) during ovulationinduction in women, 10 healthy post-menopausal women were treatedwith FSH (225 IU/day) for 5 days and normal saline (2 ml/day)for another 5 days. The two regimens were given consecutivelyin a 10 day experiment. The regimen for the first 5 days wasrandomly chosen and was given to the women in an alternate way.The response of LH to an i.v. injection of 10 µg LHRHwas investigated twice on day 1 (i.e. before the onset of treatmentand 12 h later) and once on days 2, 5 and 10 of the experiment(0900 h). Basal FSH and LH values before the onset of treatmenton day 1 were similar in the five women who started with thesaline and the five who started with the FSH regimen. BasalFSH values increased significantly during treatment with FSH,while LH and oestradiol values remained unchanged throught thewhole experiment. LH increment 30 min post –LHRH did notchange significantly either during the first 24 h or duringthe whole experiment regardless of the starting regimen. Theseresults demonstrate that in post-menopausal women the responseof LH to LHRH is not affected by exogenous administration ofFSH. It is suggested that exogenous FSH does not show activitieson gonadotrophin secretion similar to those ascribed to a gonadotrophinsecretion similar to those ascribed to a gonadotrophin surgeattenuating factor.     

Immunohistochemical localization of luteinizing hormone-releasing hormone (LHRH) in the hypothalamus of adult female hamsters treated neonatally with monosodium glutamate or hypertonic saline

The immunohistochemical localization of luteinizing hormone-releasing hormone (LHRH) was studied in paraffin and vibratome-sectioned tissue from adult female hamsters that were treated neonatally with monosodium glutamate (MSG) or hypertonic saline. There appeared to be a reduction in LHRH-positive fibers in the median eminence of animals with an MSG-induced lesion of the arcuate nucleus in paraffin-embedded tissue. However, when unembedded tissue was cut on a vibratome, the distribution of LHRH-positive fibers and perikarya was similar in both groups of animals. Fibers were seen coursing through the periventricular area and lateral hypo-thalamus to the median eminence. In addition, LHRH-positive fibers were seen in the organum vasculosum of the lamina terminalis, subfornical organ, septal and preoptic areas, fasciculus retroflexus, habenular complex, and several regions in the basal forebrain. Animals that were pretreated with colchicine had LHRH-positive perikarya in the medial habenular nucleus, diagonal band of Broca, and the medial olfactory tract.     

Sleep deprivation increases somatostatin and growth hormone-releasing hormone messenger RNA in the rat hypothalamus

We studied the effect of sleep deprivation (SD) on the amount of somatostatin (SRIF) and growth hormone-releasing hormone (GHRH) mRNA in rat hypothalamic nuclei. According to earlier studies SRIF possibly facilitates REM sleep and GHRH slow-wave sleep. Adult male rats were sleep deprived by the gentle handling method either for 6 h during the first half of the light phase or for 12 h during the dark phase. Undisturbed rats sacrificed at the same time as the SD rats served as controls. After oligonucleotide in situ hybridization the amount of SRIF and GHRH mRNA was measured in brain sections by image analysis and cell count. SD increased the amount of SRIF mRNA in the arcuate nucleus (ARC). In the periventricular nucleus (PE) there was no effect. The amount of GHRH mRNA increased in the paraventricular nucleus (PA) in the 6 h SD group but no effect was detected in ARC. In the periventromedial hypothalamic area (pVMH) the amount of GHRH mRNA was higher in the control rats sacrificed in the morning (09.00 hours) than in the afternoon (15.00 hours), and SD had no effect. We conclude that SRIF cells in ARC and GHRH cells in PA are modulated by sleep loss, which is in accordance with the possible sleep regulatory function of these neuropeptides.     

Acetylcholinesterase and luteinizing hormone-releasing hormone distinguish separate populations of terminal nerve neurons

The terminal nerve is composed of a morphologically heterogeneous population of unipolar, bipolar and multipolar neurons located in the nasal and intracranial cavities of vertebrates. The question has arisen as to whether these neurons are neurochemically heterogeneous and therefore possibly functionally different as well. Among the substances localized in the terminal nerve are acetylcholinesterase and luteinizing hormone-releasing hormone-like immunoreactive material. We have developed a double-label procedure, combining immunocytochemistry and enzyme histochemistry to determine whether these two substances are localized within different populations of terminal nerve neurons. Compatibility of the two procedures was accomplished by modifications of the fixative and primary antibody solutions. In the immunocytochemical step, the avidin-biotin-peroxidase complex coupled to a new chromogen, Chromo-red, produced a bright red reaction product in neurons containing luteinizing hormone-releasing hormone-like material. This reaction product was easily differentiated from the black silver-intensified acetylcholinesterase label. In both neonatal and adult preparations, a large population of terminal neurons contained the acetylcholinesterase label only, whereas a smaller population contained both acetylcholinesterase and luteinizing hormone-releasing hormone-like material. The acetylcholinesterase-containing population of neurons was concentrated peripherally and included multipolar neurons. In contrast neurons with the two substances co-localized were unipolar or bipolar and were concentrated centrally. The simultaneous visualization of acetylcholinesterase and luteinizing hormone-releasing hormone-like material in the same tissue section enable the differentiation of two separate neurochemically defined populations of terminal neurons. The distribution of these two neuronal types was the same in neonatal and adult animals. These data provide support for a functional diversity of terminal neurons.     

Aging changes in synaptology of luteinizing hormone-releasing hormone neurons in male rat preoptic area

This study was undertaken to examine some aspects of the anatomical substrate for reproductive senescence. Immunocytochemically identified luteinizing hormone-releasing hormone neurons and their processes in the male rat brain preoptic area were compared in young adult (2-4 months), middle-aged (12-14 months) and old (20-23 months) animals. At the light microscopic level there were no age-dependent differences in total numbers or sizes of LHRH neurons nor in their distribution in the brain. Examination of these neurons at the electron microscopic level did reveal significant differences in certain organelles and in the degree and kind of synaptic input. Random sections of middle-aged luteinizing hormone-releasing hormone neurons more frequently passed through the nucleolus and the incidence of nematosomes was higher than in luteinizing hormone-releasing hormone neurons from the young and old animals. Quantitative measures of synaptic input to luteinizing hormone-releasing hormone soma and dendrites as well as to unidentified neurons in the same thin section were made. These are reported as percent of membrane that showed synaptic structure. Dendrites of both luteinizing hormone-releasing hormone and nonidentified neurons were more densely innervated than perikarya. The density of synaptic input to luteinizing hormone-releasing hormone neurons was significantly greater than that to nonidentified neurons in young and middle-aged animals, but was equal to that of nonidentified neurons by old age. Age-related changes were noted in synaptic organization with the most significant change being an increased input to luteinizing hormone-releasing hormone perikarya. Indeed, synaptic input to luteinizing hormone-releasing hormone perikaryal membrane was increased three-fold by middle age and ten-fold by old age. Density of synaptic input to luteinizing hormone-releasing hormone dendritic membrane did not change with age. There were no aging changes in percentage of membrane with synaptic structure in nonidentified elements. Synapses were also classified on the basis of their synaptic vesicle content. There were proportionately more synaptic boutons containing round clear than pleomorphic vesicles in the young sample. The proportion of synapses with pleomorphic vesicles increased with age onto both luteinizing hormone-releasing hormone perikarya and their dendrites. The proportion of boutons containing some electron dense-core vesicles along with clear vesicles decreased with age onto both luteinizing hormone-releasing hormone and nonidentified neurons and their processes.     

Local degradation of luteinizing hormone-releasing hormone (LH-RH) in the rat central nervous system

Peptidases inactivating luteinizing hormone-releasing hormone (LH-RH) were found to be present in two subcellular fractions prepared from the following areas of the central nervous system of male and female rats: thalamus, hypothalamus, cortex and cerebellum. LH-RH immunoreactivity was also detected in these fractions after peptidase activity had been removed by heat denaturation. These findings support the concept that LH-RH may have a role in modulating neuronal activity within the central nervous system (CNS).