Isolation and characterization of chicken hypothalamic luteinizing hormone-releasing hormone

A peptide having gonadotropin-releasing activity was isolated in a yield of 2.5 μg from an extract of 2,000 chicken hypothalami. The biopotency was monitored using rat anterior pituitary cell culture system. The peptide differs from mammalian Luteinizing Hormone-Releasing Hormone (LH-RH) in its behavior during chromatographic separation (ionexchange and high performance liquid chromatography) and in its reaction towards anti-LH-RH antiserum directed against the C-terminal region of the LH-RH molecule. The peptide (chicken LH-RH) stimulates secretion of both LH and FSH from rat anterior pituitary cells. The biological potency of this peptide was about 4 % of that of the authentic decapeptide estimated in the rat anterior pituitary system. The amino acid composition is (Ser, Pro, Glx₂, Gly₂, Leu, Tyr, His, Trp), which differs from mammalian LH-RH only in that one Arg residue is replaced by a Glx residue. Based on the behavior on CM cellulose chromatography and the reaction towards anti-LH-RH antiserum, one possible structural candidate for this peptide (chicken LH-RH) is [Gln⁸]-LH-RH.     

Purification and characterization of luteinizing hormone-releasing hormone from codfish brain

We have purified luteinizing hormone-releasing hormone (LH-RH) from codfish brain and have demonstrated its identity with salmon LH-RH (sLH-RH). An antiserum raised against sLH-RH was used in a specific radioimmunoassay (RIA) to monitor purification and to manufacture an immunoaffinity chromatography column for the initial purification step. The cross-reactivity of the sLH-RH RIA with mammalian LH-RH was 0.1%. Acid extracts of codfish brains were sequentially purified by immunoaffinity chromatography, gel-filtration chromatography, and three steps of reverse-phase HPLC. The purified material and synthetic sLH-RH coeluted on reverse-phase HPLC and exhibited similar biological activity in a dispersed pituitary cell bioassay. Furthermore, the amino acid composition of the purified material was identical to salmon LH-RH. These results suggest that there is structural conservation of LH-RH between these species of teleost fish.     

Catecholamine stimulation of androgen production by rat Leydig cells. Interactions with luteinizing hormone and luteinizing hormone-releasing hormone

The mechanism(s) of the development of response to catecholamines (CA) by Leydig cells in culture was investigated with the use of primary culture of purified Leydig cells of adult rats. The interactions of a CA agonist, isoproterenol (ISOP), with luteinizing hormone (LH) and a luteinizing hormone-releasing hormone agonist analog (LHRHa) on production of androgen by the Leydig cells were also studied. Cells incubated with ISOP for 3 h increased release of cyclic adenosine 3',5'-monophosphate (cAMP) to similar extents at 0, 3, and 24 h of culture. The beta-agonist did not increase androgen release at 0 h but had a concentration-dependent effect at 3, 24, and 48 h of culture, with maximal effects at 24 h. LH stimulated high increases in production of cAMP and androgen by the cells at 0-24 h of culture. Leydig cell beta-receptors decreased with culture time. Low concentrations but not high levels of LH had additive effects with ISOP on androgen release. ISOP showed a complex interaction with LHRHa on androgen release. Chronic exposure of Leydig cells to LHRHa reduced basal androgen release as well as release of androgen stimulated by ISOP, forskolin, and LH. These studies suggest that the development of response to CA by rat Leydig cells is a postreceptor, postcAMP event and showed that CA can interact with LH or LHRH to regulate Leydig cell function.     

Hydrophobic, aza-glycine analogues of luteinizing hormone-releasing hormone

The effect of combination of the hydrophilic aza-Gly substitution (NHNHCO) at position 10 with hydrophobic, unnatural D-amino acids in position 6 on the potency of luteinizing hormone-releasing hormone (LH-RH) analogues has been investigated. Previously the aza-Gly residue was shown to provide protection from enzymatic cleavage and lead to potency increases in a less hydrophobic series. The compounds were prepared by coupling of the corresponding nonapeptide acids with semicarbazide hydrochloride by the N,N'-dicyclohexylcarbodiimide/1-hydroxybenzotriazole procedure. The required nonapeptide acids were prepared by the solid phase method on chloromethyl-polystyrene resin using HF/anisole deprotection. The products were purified by preparative reversed-phase high-performance liquid chromatography. The analogues were tested in a rat estrous cyclicity suppression assay designed to show the paradoxical antifertility effects of these compounds. The potencies of [6-(3-benzimidazol-2-yl)-D-alanine), 10-aza-glycine] LH-RH and [6-(3-(5,6-dimethylbenzimidazol-2-yl)-D-alanine), 10-aza-glycine] LH-RH are 40 and 190 times that of LH-RH respectively. The most active compound in this series is [6-(3-(2-naphthyl)-D-alanine), 10-aza-glycine] LH-RH with a potency 230 times that of LH-RH. This compound is 2.3 times as potent as the standard ([D-Trp6, Pro9-NHEt] LH-RH) and appears to be the most potent LH-RH agonist reported.     

Receptors for luteinizing hormone-releasing hormone.

Antisera to luteinizing hormone-releasing hormone (LH-RH) confer on Araldite sections of occasional rat pituitaries moderate immunocytochemical staining to the large secretory granules of gonadotrophs. Treatment of the sections with LH-RH before anti-LH-RH yields strong staining in all animals, irrespective of presence or absence of staining without pretreatment. This enhancement of staining is specific for LH-RH and is a high affinity, saturable reaction. Staining with or without LH-RH pretreatment is absent when anti-LH-RH absorbed with insolubilized LH-RH is used. Staining is inhibited by carboxyterminally-deficient LH-RH, unaffected by aminoterminally deficient LH-RH.     

Simultaneous measurement of luteinizing hormone-releasing hormone and luteinizing hormone during estradiol-induced luteinizing hormone surges in the ovariectomized ewe

Sequential bleeding and push-pull perfusion of the hypothalamus were used to characterize luteinizing hormone (LH) and LH-releasing hormone (LHRH) release in ovariectomized (OVX) ewes after injection of corn oil or estradiol benzoate (EB). Push-pull cannulae were surgically implanted into the stalk median eminences of 24 OVX ewes. Seven to 14 days later each of 20 animals was given an i.m. injection of 50 micrograms EB. Blood samples and push-pull perfusate were collected at 10-min intervals for 6-12 h beginning 12-15 h after EB injection. Four OVX ewes were given i.m. injections of corn oil 7 days after implantation of push-pull cannulae. Blood samples and push-pull perfusate were collected at 10-min intervals for 4 h between 18 and 22 h after injection of corn oil. Luteinizing hormone remained below 2 ng/ml throughout most of the sampling periods in 9 of 20 EB-treated ewes. In 5 of these 9 LHRH also was undetectable, whereas in 4 LHRH was detectable (1.84 +/- 0.29 pg/10 min), but did not increase with time. Preovulatory-like surges of LH occurred in 11 EB-treated ewes, but LHRH was undetectable in 5. In 4 of 6 ewes showing LH surges and detectable LHRH, sampling occurred during the onset of the LH surge.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum luteinizing hormone and ovulatory response to luteinizing hormone-releasing hormone in the estrous and anestrous domestic cat.

A heterologous double antibody radioimmunoassay was developed to measure changes in serum luteinizing hormone (LH) concentrations in estrous and anestrous queens (female domestic cats), following a single injection of varying doses (0--25 microgram) of luteinizing hormone-releasing hormone (LH-RH). No increase in serum LH was detected in any of the estrous or anestrous queens following a single saline injection. Treatment with LH-RH resulted in a sharp increase in serum LH concentration in both estrous and anestrous queens. Ovulations as observed by the presence of corpora lutea at laparoscopy occurred in none of four, one of four, two of four and four of four estrous queens receiving 0, 5, 10 or 25 microgram of LH-RH, respectively. Mean serum LH concentration of the ovulating queens was maintained at a higher level and did not return to basal level at the same time as that of nonovulating queens. The data show that: LH-RH can cause release of LH in both estrous and anestrous queens and induce ovulation in the estrous cat; the magnitude of LH response is influenced by the stage of the reproductive cycle; and the duration during which LH is maintained above basal level may play a significant role in ovulation induction in this coitus-induced ovulatory species.     

Hypothalamic deafferentation and luteinizing hormone-releasing hormone effects on secretion of luteinizing hormone in prepubertal pigs

The control of luteinizing hormone (LH) secretion was investigated in ovariectomized, prepubertal Yorkshire pigs by comparing the effects of anterior (AHD), complete (CHD), and posterior (PHD) hypothalamic deafferentation to sham-operated controls (SOC). Gilts (n = 16) were assigned randomly to treatments, fitted with an indwelling jugular catheter, and ovariectomized 2 days before deafferentation or sham-operation (Day 0). Blood for radioimmunoassay (RIA) of LH was collected sequentially at 20-min intervals for a period of 2 h before and 24, 48, 72, and 96 h after hypothalamic deafferentation or SOC. Episodic LH release after AHD or CHD was abolished (p less than 0.01), but not after PHD or SOC. Concentrations of serum LH in AHD and CHD dropped (p less than 0.01) at 24 and 48 h after surgery. Levels of LH before and after surgery in PHD and SOC were similar (p greater than 0.05). Infusion of 25 micrograms LH-releasing hormone (LHRH) i.v. at 72 and 96 h after hypothalamic deafferentation and SOC increased (p less than 0.01) serum LH to peak levels within 15 min. after infusion; LH returned to basal levels 60-80 min later. By 96 h after surgery, LH response to LH-releasing hormone (LHRH) was less in AHD and CHD as compared with the response at 72 h postinjection. Concentrations of LH in PHD and SOC were similar (p greater than 0.05) at 72 and 96 h, respectively. The results from this study clearly indicate that neural stimuli originating or traversing the neural areas rostral to the median eminence are required for secretion of LH in the pig.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulsatile infusion of luteinizing hormone-releasing hormone: Effects on luteinizing hormone secretion and ovarian function in prepuberal gilts

Ten intact and hypophysial stalk-transected (HST), prepuberal Yorkshire gilts, 112–160 days old, were subjected to a pulsatile infusion regimen of luteinizing hormone-releasing hormone (LHRH) to investigate secretion profiles of luteinizing hormone (LH) and ovarian function. A catheter was implanted in a common carotid artery and connected to an infusion pump and recycling timer, whereas an indwelling external jugular catheter allowed collection of sequential blood samples for radioimmunoassay of LH and progesterone. In a dose response study, intracarotid injection of 5 μg LHRH induced peak LH release (5.9 ± 0.65 ng/ml; mean ± SE) within 20 min, which was greater (P < 0.001) than during the preinjection period (0.7 ± 0.65 ng/ml). After HST, 5 μg LHRH elicited LH release in only one of three prepuberal gilts. Four intact animals were infused with 5 μg LHRH (in 0.1% gel phosphate buffer saline, PBS) in 0.5-ml pulses (0.1 ml/min) at 1.5-h intervals continuously during 12 days. Daily blood samples were obtained at 20-min intervals 1 h before and 5, 10, 20, 40, 60 and 80 min after one LHRH infusion. Plasma LH release occurred in response to pulsatile LHRH infusion during the 12-day period; circulating LH during 60 min before onset of LHRH infusion was 0.7 ± 0.16 ng/ml compared with 1.3 ± 0.16 ng/ml during 60 min after onset of infusion (P < 0.001). Only one of four intact gilts ovulated, however, in response to LHRH infusion. This animal was 159 days old, and successive estrous cycles did not recur after LHRH infusion was discontinued. Puberal estrus occurred at 252 ± 7 days in these gilts and was confirmed by plasma progesterone levels. These results indicate that intracarotid infusion of 5 μg LHRH elicits LH release in the intact prepuberal gilt, but this dosage is insufficient to cause a consistent response after HST.     

Ultrastructural immunocytochemical localization of luteinizing hormone-releasing hormone (LH-RH) in the median eminence of the guinea pig

Summary LH-RH was localized at the ultrastructural level in axons and nerve terminals of the median eminence of the male guinea pig. LH-RH positive neuronal profiles were most concentrated in the medial-dorsal aspect of the infundibular stalk and in the post-infundibular median eminence at the level immediately following separation of the stalk from the base of the brain. LH-RH containing axon profiles were most abundant in the palisade zone; nerve terminals in contact with the hypophysial portal vasculature were relatively rare. The hormone was present within granules that measured 900–1,200 Å in axons of the palisade zone and 400–800 Å in nerve terminals abutting on the portal plexus. The differently sized granules represent heterogeneous populations.Supported in part by U.S. Public Health Service grant HD-09636 from the National Institutes of Health and RR-00167 to the Wisconsin Regional Primate Research Center from the National Institutes of Health. Primate Center Publication No. 15-031The authors wish to thank Dr. Sandy Sorrentino, Jr. for the gift of antiserum to LH-RH and Dr. Ludwig Sternberger for the peroxidase.antiperoxidase complex     

Thermal stress reduces serum luteinizing hormone and bioassayable hypothalamic content of luteinizing hormone-releasing hormone in hens

Studies were conducted to evaluate the effects of acute (24 h) thermal stress on anterior pituitary function in hens. Circulating levels of luteinizing hormone (LH) were measured and the ability of the pituitary to respond to luteinizing hormone-releasing hormone (LHRH) challenge was determined. Moreover, bioassayable hypothalamic LHRH content was assessed by using dispersed anterior pituitary cells. In two separate experiments, circulating levels of LH were reduced in hens exposed to acute thermal stress (35 degrees C). Injection of LHRH did not result in significant differences in release of LH between normothermic and hyperthermic hens. However, the hypothalamic content of bioassayable hypothalamic releasing activity from hyperthermic hens were significantly reduced compared with normothermic hens. Taken together, these data suggest that the reproductive decline in the acutely heat-stressed hen is mediated by reduced LH releasing ability of the hypothalamus.     

Binding of luteinizing hormone-releasing hormone analogues to dispersed rat pituitary cells

The binding of luteinizing hormone-releasing hormone (LHRH) to dispersed rat pituitary cells was studied by using ¹²⁵I-labeled analogues of the neurohormone: a superactive agonist [D Ser (Bu^t)⁶]LHRH(1–9) ethylamide and an antagonist DpGlu¹, DPhe², DTrp^3,6-LHRH. Although these cells were exposed to proteolytic enzymes, their ability to respond to LHRH stimulation by gonadotropin release, is preserved. The time course of binding of the two analogues at different temperatures has demonstrated that highest specific binding is evident at 4°C and that equilibrium is reached after 90 min of incubation at this temperature. Incubation of pituitary cells with the labeled analogues together with increasing concentrations of LHRH or unlabeled analogues exhibited parallel competition curves, suggesting binding to the same receptor sites but with different affinities. Biologically inactive analogues of LHRH or unrelated peptides such as TRH did not compete for binding sites. K_a values for the agonist, LHRH and the antagonist were 2.1 × 10⁹M^?1, 0.92 × 10⁸M^?1 and 0.76 × 10⁹M^?1, respectively, and the binding capacity was 116 fmoles/10⁶ pituitary cells.     

Pituitary responsiveness to luteinizing hormone-releasing hormone in prepubertal and postpubertal male ferrets

The pituitary response to three different doses of exogenously administered LHRH was examined in prepubertal (9-wk-old) and postpubertal (32-wk-old) male ferrets. The doses of 5, 10, and 15 ng LHRH/kg body weight tested in this study produced dose-related increases in circulating LH concentrations in both pre- and postpubertal groups. In addition, a significant effect of age on LH response was observed, with the prepubertal animals demonstrating significantly greater serum LH values in response to the two higher doses than the postpubertal males. Prepubertal ferrets also exhibited a significant increase in endogenous LH pulse amplitude in sampling periods following exogenous administration of LHRH compared to baseline pulse amplitudes in periods prior to the LHRH infusions. These results suggest that the low frequency of endogenous LH pulses previously observed in prepubertal ferrets is not due to unresponsiveness of the pituitary gland to LHRH. Thus, suppression of the hypothalamo-hypophyseal axis observed in the prepubertal ferret is probably mediated at the level of the hypothalamus.