Chicken Luteinizing Hormone-releasing Hormone Research Articles

Colocalization of arginine-vasotocin and chicken luteinizing hormone-releasing hormone-I (cLHRH-I) in the preoptic–hypothalamic region of the chicken

To characterize a possible relationship between chicken luteinizing hormone-releasing hormone-I (cLHRH-I) and arginine-vasotocin (AVT) we performed immunocytochemical double-stainings throughout the preoptic–hypothalamic region of the chicken brain. This study clearly reveals a partial colocalization between both neuropeptides. Single-labeled neurons, containing either cLHRH-I or AVT are found intermingled with double stained cells, immunoreactive (ir) for both peptides. A significant number of double-labeled perikarya is found in the preoptic area, more specifically in the ventral and external portion of the supraoptic nucleus (SOv and SOe) and in the medial preoptic nucleus (MPOv). At the level of the anterior hypothalamus, double-labeled cells are predominantly observed near the third ventricle in the nucleus paraventricularis magnocellularis (PVN) and the nucleus periventricularis hypothalami (PHN). Next to this colocalization, a number of cLHRH-I-ir cell bodies are found in close apposition to AVT-ir fiber profiles in the very same areas. Taken together, these data are the first to provide morphological evidence indicating that the AVT system might be involved in the regulation of cLHRH-I release and thus of reproductive functions in birds.

LH responses to chicken luteinizing hormone-releasing hormone I and II in laying, incubating, and out of lay turkey hens

An experiment was conducted to assess the relative in vivo and in vitro activities of chicken LH-RH-I and -II in laying, incubating and out-of-lay turkey hens. The highest plasma concentrations of LH were measured in laying turkey hens, whereas hypophyseal concentrations were highest in incubating hens (I) and lowest in the laying hens at the end of the laying period (EL). Hypophyseal and plasma concentrations of LH decreased with aging in laying hens (L) and the greater decrease occurred in the hypophyses. An in vitro hypophyseal acute challenge with 2-min pulses of cLHRH I or II (10 −7 M) using a perifusion technique resulted in an increase in the release of LH in out-of-lay (OL) and incubating (I) hens, but not in laying (L) hens. Although both peptides elicited comparable responses in I hens, cLHRH II was more effective in OL hens. This difference was attributable to a greater amplitude of the response, whose duration was unchanged. Hypophyseal desensitization to a subsequent stimulation was observed in OL hens when the interval between stimulations was 30 min, but this did not occur at 60- or 120-min intervals. In vivo, the injection of cLHRH I or II, at doses of 10 −8 and 10 −10M/kg B.W. stimulated increases in the plasma concentrations of LH, which were initiated within 1 min of injection in OL and I hens but from 5 to 20 min postinjection in L hens. The responses were dose-related and greater immediate responses were measured with cLHRH I than with cLHRH II. Also, after the injection of cLHRH II at the 10 −8 M/kg B.W. dose, the shape of the LH response consisted of an initial increase, followed by a more sustained phase during which LH concentrations were either stable (I hens) or continued to increase (L and OL hens) from 20 to 60 min after injection. In contrast, the injection of cLHRH I at doses of 10 −8 or 10 −10 M/kg or cLHRH II at a dose of 10 −10 M/kg in I and OL hens, produced a peak of LH concentrations in plasma within 5 min and thereafter declined gradually. The difference in the in vivo responses to LHRH I and II could not be attributed to a greater potency of cLHRH II, but to a more prolonged action. In summary, the responses to both forms of chicken LH-RH varies markedly with the stage of the reproductive cycle (L, I, and OL) and differs between the in vivo and in vitro situations. Although cLHRH II may be more active than cLHRH I, controversy still surrounds its precise physiological role.

Appearance of an arginine vasotocin receptor of large molecular size in the uterus (shell gland) of the hen at oviposition.

The molecular mass of the arginine vasotocin receptor in membrane fractions of the uterus (shell gland) of hens was estimated by autoradiography of [125I]-labelled arginine vasotocin binding protein after SDS-PAGE. A band of approximately 67 kDa was found in all of the samples taken from laying hens before and after oviposition and from nonlaying hens. In addition to this band, another band of approximately 95 kDa was found in samples from laying hens taken immediately before and after oviposition. Both bands were reduced by the presence of unlabelled arginine vasotocin or mesotocin, but the reduction was greater by arginine vasotocin than by mesotocin. These two bands were not reduced by chicken luteinizing hormone-releasing hormone-I (Gln8-GnRH), chicken luteinizing hormone-releasing hormone-II (His5,Trp7,Tyr8-GnRH) and chicken angiotensin-II (Val5-angiotensin-II). The appearance of the arginine vasotocin receptor of the larger molecular size may be related to oviposition in hens.

Absence of an effect of naloxone, an opioid antagonist, on luteinizing hormone release in vivo and luteinizing hormone-releasing hormone I release in vitro in intact, castrated, and food restricted cockerels

The possibility that the tonic secretion of luteinizing hormone (LH) and chicken luteinizing hormone-releasing hormone I (LHRH-I) is regulated by an inhibitory action of endogenous opioid peptides was investigated in cockerels using the opiate receptor antagonist, naloxone. Baseline concentrations of plasma LH in the experimental cockerels were increased by surgical castration or reduced by limiting food intake. Baseline and K +-induced releases of LHRH-I from perifused mediobasal-preoptic hypothalami from castrated cockerels were higher than those from hypothalami from intact cockerels. Similarly, baseline and K +-induced releases of LHRH-I from perifused mediobasal hypothalami from fully fed cockerels were higher than those from the hypothalami from fasting cockerels. Intravenous injections of 0.1, 1, or 10 mg naloxone/kg body weight failed to increase the concentration of plasma LH in castrated, intact, fully fed, or fasted cockerels. Perifusion of mediobasal-preoptic hypothalami from castrated or intact cockerels with 200 μ M naloxone or mediobasal hypothalami from fully fed or fasted cockerels with 10 μ M naloxone failed to stimulate the release of LHRH-I. These observations suggest in the cockerel that endogenous opioid peptides may not play an obligatory role in the inhibitory control of the tonic secretion of luteinizing hormone.

Changes in pituitary responsiveness during the ovulatory cycle of the Japanese quail, in vitro.

Anterior pituitary glands from ovulating Japanese quail (Coturnix coturnix) were used to investigate variation in sensitivity to chicken luteinizing hormone-releasing hormone (cLHRH I; Gln8-LHRH). Grouping the pituitaries by ovulatory stage provided preliminary evidence of changes in sensitivity to LHRH during the ovulatory cycle. Pituitaries taken from quail before the preovulatory LH surge were responsive to cLHRH I, while pituitaries from the other times of the cycle showed minimal response to cLHRH I. Female pituitary glands release less LH than those of males. These data indicate a change in sensitivity to LHRH in the female quail that may be due to changes in gonadal steroids or the pool of releaseable LH from the pituitary.

Steroids modulate the release of luteinizing hormone from quail pituitary cells

An enzymatically dispersed pituitary preparation from male Japanese quail ( Coturnix coturnix) was used to study the effects of gonadal and adrenal steroids on gonadotropin release. Cells were preincubated for 18 hr with or without steroids and then challenged with chicken luteinizing hormone-releasing hormone (cLH-RH I; Gln 8-LH-RH). Preincubation with testosterone (T; 10 n M) significantly suppressed ( P<0.05) luteinizing hormone release in response to cLH-RH I (10 ng/ml). Preincubation with 5α-dihydrotestosterone (5α-DHT) (10 n M) caused even further suppression of LH-RH-stimulated LH release while the same concentration of 5β-dihydrotestosterone and estradiol-17β had no effect. In addition, preincubation with corticosterone (10 n M) significantly ( P<0.01) suppressed the amount of LH released in response to cLH-RH I. Pituitary cells from immature males, when stimulated with cLH-RH I, released LH in a dose-related manner. Neither T nor 5α-DHT (10 n M) altered the effect of LH-RH. These data suggest that T and 5α-DHT play a role in mediating LH release in the avian pituitary while 5β-reduced androgens have no effect. These appears to be no androgen effect in the immature quail. In addition, corticosterone seems to be a factor in controlling gonadotropin secretion in the quail.

Effects of two kinds of chicken luteinizing hormone-releasing hormone (LH-RH), mammalian LH-RH and its analogs on the release of LH and FSH in Japanese quail and chicken

A newly isolated and characterized chicken luteinizing hormone-releasing hormone-II (chicken LH-RH-II, Miyamoto et al., 1984) had luteinizing hormone (LH) and follicle-stimulating hormone (FSH) releasing activity in vitro and in vivo in Japanese quail; the activity was almost equal to chicken LH-RH-I and mammalian LH-RH. These three LH-RHs induced the release of LH several times higher than that of FSH in vitro and also in vivo. No significant difference between chicken LH-RH-I and LH-RH-II was observed in LH releasing activity in vitro using chicken pituitary gland in the same incubating condition as in quail. Another experiment indicated that no synergism existed between chicken LH-RH-I and -II and that there was neither LH nor FSH releasing activity in [ d-Phe 2, Pro 3, d-Phe 6]-LH-RH or in mesotocin. However, the same potency as in the chicken LH-RH-II was observed in [ d-Ala 6, des-Gly 10]-LH-RH ethylamide, a superactive analog in mammals. The results indicate that an avian adenohypophysis differs from a mammalian adenohypophysis in its responsiveness to LH-RH suggesting that an avian LH-RH receptor may have a lower specificity in ”recognition” of LH-RH molecules than a mammalian LH-RH receptor has.

Effects of chicken (Gln 8)- and mammalian (Arg 8)-luteinizing hormone-releasing hormones on the release of gonadotrophins in vitro and in vivo from the adenohypophysis of Japanese quail

Both chicken luteinizing hormone-releasing hormone (Gln 8-LH-RH) and mammalian luteinizing hormone-releasing hormone (Arg 8-LH-RH) increased the release of FSH and LH from adenohypophysial halves of adult male Japanese quail when the halves were incubated in a medium containing LH-RH. Elevation of the FSH release was less marked than that of the LH release irrespective of the LH-RH species. A pulse stimulation (10 min) of adenohypophysial halves with chicken as well as mammalian LH-RH in vitro resulted in a burst of both FSH and LH release. The increment of the FSH release was smaller than that of the LH release. A single intravenous injection of each LH-RH to adult male quail induced rapid increases of plasma FSH and LH levels within 5 min. The plasma levels of FSH and LH returned to their initial levels at varying times after the injection depending on the dose of LH-RH. Again, the increment of the FSH level was smaller than that of the LH level, showing a similar profile to the in vitro pulse stimulation experiment. No significant difference in ability to stimulate FSH and LH secretions from the quail adenohypophysis was detected between chicken and mammalian LH-RHs under either in vivo or in vitro conditions, whereas chicken LH-RH has been reported to be far less potent than mammalian LH-RH when tested on the rat adenohypophysis.

Discrete Hypothalamic Distribution of Luteinizing Hormone-Releasing Hormone (LHRH) Content and of LHRH-Degrading Activity in Laying and Nonlaying Hens1

Hypothalamic enzymatic luteinizing hormone-releasing hormone (LHRH)-degrading activity (LHRH-DA) may play a physiologic role in the neuroendocrine control of LHRH in mammals. The present study analyzes the existence and possible physiologic role of LHRH-DA in birds. The LHRH content in discrete hypothalamic samples of laying and nonlaying hens was correlated to their LHRH-DA. Degrading activity was assessed by high-performance liquid chromatography (HPLC) of chicken LHRH and of its degradation fragments. Luteinizing hormone-releasing hormone content was estimated by radioimmunoassay. Luteinizing hormone-releasing hormone content of discrete medial preoptic, infundibulum, and arcuate samples, as well as serum LH and progesterone levels, were higher (P less than 0.05) in laying than in nonlaying hens. The LHRH content of these hypothalamic areas was also higher (P less than 0.05) than those of immediately adjacent areas, in both animal groups. Luteinizing hormone-releasing hormone-degrading activity, which generates LHRH1-5 as the main degradation fragment, was higher (P less than 0.01) in the infundibulum of laying than in nonlaying hens. It was also higher (P less than 0.01) in samples from the infundibulum and medial preoptic area than in immediately adjacent lateral samples. Finally, LHRH-DA, showing a similar HPLC profile of degradation fragments, was also present in areas of low or undetectable LHRH content.(ABSTRACT TRUNCATED AT 250 WORDS)

Trp 7,Leu 8]LH-RH in reptilian brain

Luteinizing hormone-releasing hormone (LH-RH) immunoreactive peptides in acetic acid extracts of lizard ( Cordylis nigra) brain were studied by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. Four different LH-RH immunoreactive peptides were detected. The major form co-eluted with salmon brain LH-RH, [Trp 7,Leu 8]LH-RH, in a cation exchange and three reverse phase HPLC systems which were specifically designed to separate a range of LH-RH analogues. The interaction of this major LH-RH immunoreactive peptide with a number of antisera directed against different regions of mammalian, chicken and salmon LH-RH was similar to the relative interaction of [Trp 7,Leu 8]LH-RH with these antisera. These data strongly indicate that the major form of lizard brain LH-RH is identical to salmon brain LH-RH ([Trp 7,Leu 8]LH-RH). The three additional molecular forms of immunoreactive LH-RH in lizard brain appear to differ from mammalian LH-RH in the middle to C-terminal region of the molecule.

Isolation and structural characterization of chicken hypothalamic luteinizing hormone releasing hormone.

Studies on partially purified chicken hypothalamic luteinizing hormone releasing hormone (LHRH) utilizing chromatography, radioimmunoassay with region-specific antisera, enzymic inactivation, and chemical modification established that the peptide is structurally different from mammalian hypothalamic LHRH. These studies demonstrated that arginine in position 8 is substituted by a neutral amino acid. On the basis of conformational criteria and evolutionary probability of amino acid interchange for arginine, the most likely substitution was glutamine. We therefore synthesized [Gln8]-LHRH and established that it had identical chromatographic, immunologic, and biological properties to the natural chicken peptide. In concurrent studies, purification of 17 micrograms of an LHRH from 249,000 chicken hypothalami was achieved using acetic acid extraction, immuno-affinity chromatography, and cation exchange and reverse phase high performance liquid chromatography. Amino acid composition and sequence analyses confirmed the structure of this form of chicken LHRH as pGlu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH2.

A comparison of the luteinizing hormone-releasing activities of synthetic chicken luteinizing hormone-releasing hormone (LH-RH), synthetic porcine LH-RH, and buserelin, and LH-RH analogue, in the domestic fowl

The luteinizing hormone-releasing activities of synthetic chicken luteinizing hormone-releasing hormone (chLH-RH), synthetic porcine LH-RH (pLH-RH), and an analogue of LH-RH (buserelin, d-Ser-(Bu t) 6-des-Gly 10-LH-RH ethylamide) were compared in the domestic fowl. In adult cockerels, intravenous injections of 0.5 or 1 μg chLH-RH/kg released the same amount of LH as the same doses of pLH-RH; subcutaneous injections of 0.5 or 1 μg buserelin/kg were about twice as effective as the same doses of pLH-RH. In laying hens, injections of 1, 10, 20, and 50 μg buserelin induced more sustained releases of LH than the corresponding doses of pLH-RH. Daily injections of 1 or 10 μg buserelin/bird or of 10 μg pLH-RH/bird for 12 days synchronized the timing of most ovipositions showing that the injections of releasing hormone could induce preovulatory surges of LH. In contrast with mammals, daily injections of buserelin in laying hens did not reduce pituitary responsiveness to the analogue. It is concluded that the structural difference between mammalian and chicken LH-RH does not affect their LH-releasing activities in the domestic fowl. Although the LH-releasing activity of buserelin in the hen is greater than that of pLH-RH, the difference in activity is not as great as that observed in most mammals. This view is strengthened by the finding that chronic treatment with buserelin, which exerts an antagonistic effect on ovulation in mammals, does not do so in the domestic hen.

Biological properties of chicken luteinizing hormone-releasing hormone: gonadotropin release from rat and chicken cultured anterior pituitary cells and radioligand analysis.

We have examined the biological properties of chicken LHRH, which has been isolated and characterized in our laboratory, using primary monolayer cultures of rat and chicken anterior pituitary cells. The radioligand receptor analysis using rat pituitary LHRH receptor was also performed. Biological activities of mammalian LHRH and a more potent analog, [D-Leu6]des-Gly10-LHRH N-ethylamide ([D-Leu6] LHRH analog), were compared with those of chicken LHRH. The ED50 values for [D-Leu6]LHRH analog, mammalian LHRH, and chicken LHRH were, respectively, 0.0166, 0.455, and 18.2 nM for LH secretion and 0.0089, 0.263, and 8.28 nM for FSH secretion from rat pituitary cells. Relative potencies of chicken LHRH were 2.5% that of mammalian LHRH for LH and 3.2% that for FSH. On the other hand, the ED50 for chicken LHRH for LH release from chicken anterior pituitary cells was 1.03 nM, indicating that the biological potency of chicken LHRH acting on chicken pituitary cells was higher than that of chicken LHRH acting on mammalian pituitary cells. The ID50 of chicken LHRH to inhibit the binding of 125I-labeled [D-Leu6]LHRH analog to rat anterior pituitary membrane was 708.5 nM, and was 200 times that of mammalian LHRH (3.39 nM). These results suggest that the substitution of arginine residue of mammalian LHRH for glutamine residue of chicken LHRH causes a decrease in the binding affinity of the hormone for the mammalian LHRH receptor and that the mammalian LHRH receptor has a binding site for the cationic center of the arginine residue of LHRH. On the other hand, chicken pituitary LHRH receptor seems to have more broad specificity than mammalian LHRH receptor.

Synthesis and biological activity of [D-Trp 6] chicken luteinizing hormone-releasing hormone

An agonist of chicken hypothalamic luteinizing hormone-releasing hormone (cLH-RH). [D-Trp 6] cLH-RH, was synthesized and tested for luteinizing hormone (LH)-releasing activity using dispersed chicken anterior pituitary cells, as well as for binding to rat anterior pituitary membrane receptors. cLH-RH and mammalian LH-RH (mLH-RH) gave identical dose-response curves in stimulating chicken LH release ( ED 50=1.6 and 1.8×10 −9 M respectively) and similar estimates of potency. The [D-Trp 6] analogs of cLH-RH and mLH-RH stimulated LH release at lower doses ( ED 50=7.0 and ∼7.0×10 −11 M respectively) and were approximately 20-fold more potent. In contrast to the activity in the chicken bioassay, cLH-RH bound to rat anterior pituitary membrane receptors with a much lower affinity than did mLH-RH and had a relative potency of 2%. [D-Trp 6] cLH-RH was approximately 100-fold more potent than cLH-RH in the rat receptor assay while [D-Trp 6] mLH-RH was 28-fold more active than mLH-RH. These data demonstrate that substitution of Gly 6 of LH-RH with D-Trp enhances the LH release from chicken pituitary cells to a similar extent to that observed in mammals, and indicate that the approaches used to produce active LH-RH analogs in mammals are likely to be applicable to birds.

Structure of chicken hypothalamic luteinizing hormone-releasing hormone. II. Isolation and characterization.

Avian luteinizing hormone-releasing hormone (LH-RH) has been isolated from 249,000 chicken hypothalami and shown to differ structurally from mammalian hypothalamic LH-RH. Purification was achieved by acetic acid extraction, anti-LH-RH affinity chromatography, and cation exchange and reverse phase high performance liquid chromatography. The isolated peptide eluted as a single peak on reverse phase high performance liquid chromatography. Acid hydrolysis of the peptide yielded integral molar ratios of amino acids and a composition identical with that of mammalian decapeptide LH-RH, except for the presence of an additional glutamic acid residue and the absence of arginine. The isoelectric point of chicken LH-RH (7.3) is consistent with the glutamic acid representing a glutamine residue. We therefore synthesized [Gln8]LH-RH and established that it has chromatographic properties identical with natural chicken LH-RH. These studies indicate that the structure of chicken hypothalamic LH-RH is: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Gln-Pro-Gly-NH2.

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, Glx 2, Gly 2, 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 [Gln 8]-LH-RH.

PREVENTION OF POSITIVE FEEDBACK IN THE HEN (GALLUS DOMESTICUS) BY ANTIBODIES TO LUTEINIZING HORMONE RELEASING HORMONE

MRC Unit of Reproductive Biology, 2 Forrest Road, Edinburgh, EH3 9ER and * Agricultural Research Council's Poultry Research Centre, King's Buildings, West Mains Road, Edinburgh, EH9 3JS (Received 22 August 1977) The decapeptide luteinizing hormone releasing hormone (LH-RH) stimulates the release of luteinizing hormone (LH) in birds as well as in mammals (Van Tienhoven & Schally, 1972) and a substance immunochemically similar to LH-RH is present in the chicken hypothalamus (Jeffcoate, Sharp, Fraser, Holland & Gunn, 1974). Avian LH-RH has still to be isolated and sequenced, however, and there is some doubt about whether the decapeptide is the naturally occurring LH-RH in the bird (Jackson, 1971). In the hen, release of LH is induced by the positive feedback action of progesterone (Wilson & Sharp, 1975) which is presumably associated with the release of chicken LH-RH. To gain further information about the identity of chicken LH-RH and to investigate the site

Ovarian ascorbic acid depleting factors in the chicken hypothalamus.

A substance active in the ovarian ascorbic acid (OAA) depletion assay was found in acid extracts of chicken hypothalamus but not in extracts of cerebral cortex. The depletions evoked by these extracts were not proportional to the logarithm of the dose, and the extracts were equally active in intact and hypophysectomized assay rats. This effect was not due to the presence of LH in the extracts for the active substance had no effect in the 32P assay, and it passed through a dialysis membrane with an exclusion limit of under 12,000 molecular weight. The activity of this small molecular weight fraction was destroyed by incubation with thioglycollate. Purification of this fraction on a column of Sephadex G-25 produced a single peak with OAA depleting activity. This fraction also contained vasopressor activity, and the elution volume of the fraction was the same as that of synthetic arginine vasotocin (AVT). Because of the many similarities of AVT and the active compound in the extracts, it was concluded that AVT was the primary active substance of the extracts. The possible relationships of AVT to chicken luteinizing hormone-releasing factor are discussed. (Endocrinology85: 113, 1969)