Production of effective luteinizing hormone antisera by two immunization methods

This paper addresses the production of effective luteinizing hormone antisera by two different immunization methods; the traditional and modified methods. The main difference between these two methods is in the immunization procedure. In the modified method, an additional injection of emulsion with complete Freund's adjuvant and only one booster are applied at the third and 28th day from the first injection, respectively. The results of the study indicated the possibility of producing the antiseria using the modified method in short time and better quality than those produced by the traditional methods. The details of both methods are presented together with the results obtained from the antibodies detection. Comparison between these two methods is also presented along with discussions.     

An electrophysiological dissection of the hypothalamic regions which regulate the pre-ovulatory secretion of luteinizing hormone in the rat

1. Extracellular action potentials were recorded from 299 single units in the preoptic and anterior hypothalamic areas of fifty-two female rats anaesthetized with urethane. The units were categorized by their response to single biphasic pulses (ca. 1 mA; 1 msec duration) applied to the ventromedial/arcuate region of the hypothalamus.2. Experiments with five lactating rats demonstrated that the effective zone of stimulation was confined within the ventromedial/arcuate region. This observation was supported by further evidence obtained during unit recording sessions.3. Antidromic action potentials were recorded from 122 (41%) of the neurones monitored in preoptic and anterior hypothalamic areas. These Type A cells were characterized by their very slow discharge rate (median for spontaneously active units 1.2 spikes/sec) when contrasted with adjacent cells (median 3.9 spikes/sec). Orthodromic action potentials were not observed in thirty-six Type A cells. The antidromically identified neurones had an average conduction rate of 0.32 m/sec. The absolute refractory period of the soma, computed by separation of the IS and SD components of the antidromic action potentials recorded from forty-four neurones, ranged from < 3.0 to > 100 msec. With ten units it was not possible to obtain a soma dendritic (SD) wave by antidromic activation even though the initial segment (IS) wave was seen clearly and the orthodromic potentials consisted of both IS and SD waves.4. Type B cells (32% of population) were excited and/or inhibited by the ventromedial/arcuate stimulation but were not antidromically activated. This post-stimulatory change in discharge rate lasted for up to 400 msec.5. Type C cells (27% of population) showed no change in spontaneous activity after stimulation of the ventromedial arcuate area. These neurones had discharge rates similar to Type B cells.6. The previously reported prooestrous increase in firing rate, recorded from neurones in the ventral part of the preoptic/anterior hypothalamic area was restricted to cell Types B and C.     

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.     

Brainstem projections to the medial preoptic region containing the luteinizing hormone-releasing hormone perikarya in the rat. An immunohistochemical and retrograde transport study.

The afferent projections to the anterior medial preoptic area (MPA) from the brainstem have been studied, in female Wistar rats, by retrograde tracing with horseradish peroxidase (HRP). The HRP was injected by iontophoresis into the preoptic region containing the luteinizing hormone-releasing hormone (LHRH) perikarya. The brain sections including the MPA were reacted with diaminobenzidine (DAB) to reveal the injection site; the LHRH cells were then immunohistochemically identified using DAB with ammonium nickel sulphate. When the injection site incorporated the LHRH cells, the brainstem sections were reacted with the DAB nickel solution to detect lysosomal HRP and then immunohistochemically processed to locate the adrenaline-synthesizing cells using DAB alone. The results confirm the brainstem projections to the MPA from the central grey matter, ventral tegmental area, subcoeruleus area, the dorsal raphe nucleus, the lateral parabrachial nucleus, the raphe pontis nucleus, the raphe obscurus nucleus, the region of the paragigantocellular nucleus and the nucleus of the solitary tract. Given the considerable evidence implicating the ascending adrenergic systems in the regulation of LHRH, we focused our attention on the afferents from the locus coeruleus, area postrema and the adrenaline-synthesizing cell groups (C1-3). The only cells which were retrogradely labelled and immunopositive for phenylethanolamine N-methyltransferase were found in C3.     

Antagonists of luteinizing hormone releasing hormone bind to rat mast cells and induce histamine release

It was reported previously that administration of certain synthetic antagonists of LHRH to rats produced allergy-like symptoms that were attributed to their histamine releasing action. In the present study the interaction of LHRH analogs with rat peritoneal mast cells was investigatedin vitro. Potent antagonists of LHRH showed strongin vitro histamine releasing activity from rat peritoneal mast cells. Membrane preparations of rat pituitary glands showed specific binding of radioiodinated LHRH antagonist as well as LHRH agonist. However, rat peritoneal mast cells and membrane preparations from those cells bound antagonist but not the agonist. Furthermore, the LHRH antagonist did not bind to membranes prepared from tissues such as prostate, liver, kidney, and brain. Competitive displacement curves of the [¹²⁵I]-antagonist with different LHRH analogs showed that the ability of the analogs to compete for binding sites on mast cells was related to their histamine releasing activity. We conclude that histamine release from rat mast cells induced by LHRH analogs is mediated by specific binding of the active peptides to cell membranes. Furthermore, using rat mast cells, the binding assay in conjunction with histamine releasing assay may be utilized to predict thein vivo histamine releasing potential of new LHRH peptides which are of clinical importance.     

Effect of the ratio of follicle-stimulating hormone to luteinizing hormone on rat granulosa cell proliferation and oestradiol-17 beta secretion.

The present study examined the effects of the ratio of follicle-stimulating hormone (FSH) to luteinizing hormone (LH) on granulosa cell proliferation and oestradiol-17 beta secretion. For these studies, ovarian segments from either immature rats or those primed with pregnant mares serum gonadotrophin (PMSG) were incubated for 5 h with [3H]thymidine and FSH (0-100 mIU/ml) with or without equivalent doses of LH. After incubation, granulosa cells were isolated and their mitotic activity estimated by determining the amount of [3H]thymidine incorporated into the DNA. The amount of oestradiol secreted into the media was measured by radioimmunoassay. Compared to granulosa cells from immature ovaries, granulosa cells from PMSG-primed ovaries required significantly less FSH to stimulate incorporation of [3H]thymidine, had a 9-fold higher basal level of oestradiol production and increased oestradiol secretion in response to gonadotrophins. At pharmacological serum levels (10-20 mIU of total gonadotrophin), FSH:LH ratios of less than or equal to 2 increased oestradiol secretion from PMSG-primed ovaries but did not increase the rate of [3H]thymidine incorporation. Conversely, FSH:LH ratios of greater than or equal to 3 stimulated [3H]thymidine incorporation without altering oestradiol secretion. These data demonstrate that granulosa cells of immature follicles not secreting oestradiol are relatively unresponsive to gonadotrophins at any dose tested. Once the capacity for oestradiol secretion develops, then both the dose and ratio of FSH and LH play major roles in determining whether the follicle will grow or secrete oestradiol.     

Nicotine-induced increases in brain luteinizing hormone releasing hormone-like immunoreactivity and in serum luteinizing hormone levels of the male rat

By means of radioimmunoassay procedures luteinizing hormone releasing hormone (LHRH)-, Metenkephalin- and somatostatin-like immunoreactivities have been measured in discrete hypothalamic and preoptic nuclei as well as serum luteinizing hormone (LH) levels. Nicotine (2 mg/kg, i.p.) produced after 5 min a significant and selective increase in LHRH-like immunoreactivity in the median eminence and in the medial preoptic nucleus, associated with increases in serum LH levels but without any changes in Met-enkephalin and somatostatin-like immunoreactivities in the median eminence. The results indicate that the nicotine-induced activation of LHRH-immunoreactive neurons involves an enhanced processing of the precursor peptide to LHRH.     

Descending luteinizing hormone-releasing hormone (LH-RH) nerve fibers to the midbrain of the rat

Using the peroxidase-antiperoxidase complex (PAP) immunohistological method, a luteinizing hormone-releasing hormone (LH-RH)-containing nerve fiber tract was detected in the mesencephalon of intact rats, and in rats bearing a symmetrical cut in the coronal plane at the level of the superior collicle reaching down to the ventral tegmental decussation. This LH-RH fiber tract was found in subependymal position in the mesencephalic central gray. The LH-RH fibers reached the ponto-mesencephalic junction in the intact animals, whereas in the experimental group they were absent behind the cut. These findings indicated that the direction of the LH-RH axons in the mesencephalic central gray is descending.     

Pregnancy outcome following exposure to gonadotrophin-releasing hormone analogue during early pregnancy: comparisons in patients with normal or elevated luteinizing hormone

The outcomes of established pregnancies following the treatmentof infertile women with pituitary down-regulation before andduring treatment with ovulation induction and either intrauterineinsemination or timed intercourse were reviewed. Once startedon gonadotrophin-releasing hormone analogue (GnRHa) treatment,the patients were maintained on GnRHa therapy throughout thefollowing luteal phase to facilitate the start of the next treatmentcycle if no pregnancy was established. This resulted in patientstaking GnRHa until a positive pregnancy test indicated cessationof the treatment. The aim of our study was to determine whetherexposure to GnRHa during early pregnancy constituted a risk.Patients who were diagnosed as having elevated follicular phaseluteinizing hormone (LH) concentrations during their investigationswere analysed as a separate cohort to assess whether this diagnosishad implications with respect to pregnancy outcome. Out of 226recorded clinical pregnancies, 173 were traced and the datacollated: 16 cases resulted in clinical abortions, two wereectopic pregnancies and 155 women had live births at variousages of gestation. There were three pregnancies which were complicatedby congenital abnormalities. Patients with elevated LH concentrationson examination showed a higher rate of total pregnancy lossthan those with normal LH concentrations, despite the fact thatthe LH was suppressed during the cycle in which they conceived.The results suggest that pregnancy outcome is not adverselyaffected by GnRHa administration during the luteal phase ofthe conception cycle, and that the group diagnosed as havingelevated LH concentrations may retain their propensity to higherrates of pregnancy loss even when their LH concentrations aresuppressed during treatment.     

Immunoreactivities for antisera to three putative neuropeptides of the rat melanin-concentrating hormone precursor are coexpressed in neurons of the rat lateral dorsal hypothalamus.

Antisera (AS) raised against rat melanin-concentrating hormone (rMCH) and against two additional peptides sequences derived from the rat MCH precursor (neuropeptide glutamic acid-isoleucineamide (NEI), and neuropeptide glycine-glutamic acid (NGE)) exclusively stained the hypothalamic neurons previously described using AS to salmon MCH, human somatocrinin 1-37 (GRF37) and alpha-MSH. Liquid phase and dot-blot controls for specificity indicated that rMCH-, NEI- and NGE-AS bound epitopes recognized by sMCH-, alpha-MSH- and GRF-37-AS, respectively. The distinct intracellular patterns of immunoreactivity obtained in control animals with rMCH-, NGE- and NEI-AS, as well as the changes observed after intracerebroventricular injection of colchicine matched previous findings using sMCH-, GRF37- and alpha-MSH-AS.     

The optimum dose and mode of administration of luteinizing hormone releasing hormone analogue in in-vitro fertilization: a comparison of three regimens.

Three regimens for administration of the luteinizing hormone releasing hormone analogue (LHRHa), buserelin, were compared in terms of pituitary down-regulation prior to ovarian stimulation for in-vitro fertilization (IVF). Thirty-three patients who had failed to conceive in previous IVF cycles were randomly divided into three groups. Patients in Group I (n = 11) received intranasal (i.n.) buserelin 200 micrograms four hourly for 21 days; patients in Group II (n = 11) received subcutaneous (s.c.) buserelin 500 micrograms twice a day for 7 days, followed by 500 micrograms daily for 14 days; patients in group III (n = 11) received (s.c.) buserelin 500 micrograms three times a day for 7 days. In all three groups administration of buserelin was continued until serum oestradiol levels were less than 100 pmol/l, at which time human menopausal gonadotrophin (HMG) was commenced. All the patients achieved pituitary down-regulation after seven days of treatment with buserelin, except for four patients who developed ovarian cysts and two with polycystic ovarian disease. The total dose of HMG used and the ongoing pregnancy rate were not significantly different between the three groups. A much higher proportion of patients in Group I developed side-effects and found their treatment disruptive to their life-style. Our results suggest that patient variables (e.g. polycystic ovaries, ovarian cysts) rather than the dose and mode of administration of buserelin, are the major determinant of the length of time needed for pituitary down-regulation. The s.c. route is preferable and the smallest dose of buserelin that will produce pituitary down-regulation should be used.     

黄体生成素及其受体在大鼠肝脏的定位

目的观察黄体生成素(LH)及其受体(LHR)在大鼠肝脏的定位分布,为研究LH是否参与肝代谢功能的调节提供形态学依据。方法应用免疫荧光组织化学双标染色技术,在激光共聚焦扫描显微镜下观察染色结果。结果大鼠肝细胞既呈LH阳性又有LHR阳性,LH和LHR免疫反应阳性物质均分布在大鼠肝细胞的细胞质,细胞核为阴性。不同部位的肝细胞LH及其受体免疫反应强度存在差别。结论 LH及其受体存在于大鼠肝脏细胞,可能对肝脏的功能起调节作用。     

Effects of ethanol on hypothalamic luteinizing hormone-releasing hormone (LHRH) in the male rat an immunocytochemical study

Summary These studies were designed to determine if the acute alcohol-induced decreases in luteinizing hormone (LH) seen in previous studies using rats could be due to an inhibitory effect of ethanol (ETOH) on hypothalamic LHRH release. Thus, effects of multiple injections of ETOH on the relative amount of immunoreactive LHRH fibers in the hypothalamus and median eminence (ME) of castrate and intact male rats were determined immunocytochemically. Brains were removed following cardiac perfusion of 10% phosphate-buffered formalin. A block containing the hypothalamus with the ME was isolated from each brain, then postfixed in Bouin's solution. Paraffin sections were rehydrated and stained for LHRH with the peroxidaseantiperoxidase technique using an antiserum to synthetic LHRH conjugated to bovine serum albumen. Differences visualized immunocytochemically between saline-treated intact and castrate rats indicated that the LHRH content of the ME was markedly depleted after castration. Conversely, castrate rats treated with ETOH showed only a slight reduction in immunoreactive LHRH fibers. In ETOH-treated intact animals, the LHRH fiber content of both the hypothalamus and ME appeared to be slightly greater than the saline-treated intact controls. Thus, these data support the hypothesis that ETOH diminishes LHRH release, and hence provides an explanation for the depressed plasma LH levels observed in ETOH-treated intact and castrate rats.This work was supported by Grants NIH-BRSG-3-81 and TAMU-ORR-8-82