Interaction of endogenous chicken gonadotrophin-releasing hormone-I and -II on chicken pituitary cells

1988 ◽  
Vol 117 (1) ◽  
pp. 43-49 ◽  
Author(s):  
J. A. King ◽  
J. S. Davidson ◽  
R. P. Millar
Keyword(s):  
Gnrh Receptor ◽  
Receptor Type ◽  
Single Type ◽  
Receptor Subtypes ◽  
Pituitary Cells ◽  
Gnrh Antagonists ◽  
Single Class ◽  
Releasing Hormone ◽  
Lh Release ◽  
Gonadotrophin Releasing Hormone

ABSTRACT The presence of two endogenous forms of gonadotrophin-releasing hormone (GnRH) in the chicken hypothalamus (chicken GnRH-I ([Gln8]GnRH) and chicken GnRH-II ([His5,Trp7,Tyr8]GnRH)), and the stimulation of gonadotrophins by both forms, suggests the possible existence of GnRH receptor subtypes and gonadotroph subtypes in the chicken pituitary. This question was investigated by assessing the effects of various combinations of the two known forms of chicken hypothalamic GnRH and antagonist analogues of GnRH on LH release from dispersed chicken anterior pituitary cells in both static and perifused systems. The relative inhibition of chicken GnRH-I-stimulated and chicken GnRH-II-stimulated LH release by 12 GnRH antagonists did not differ significantly, suggesting a single GnRH receptor type. Chicken GnRH-II was approximately sixfold more potent than chicken GnRH-I in releasing LH. Release of LH in response to maximal doses of chicken GnRH-I and chicken GnRH-II and to a mixture of both was similar and the two peptides were not additive in their effects, consistent with the presence of a single type of LH gonadotroph and a GnRH receptor which binds both forms of GnRH. Each form of GnRH desensitized cells to subsequent stimulation with the other form, providing additional evidence for a single type of LH gonadotroph. These findings suggest that chicken GnRH-I and -II stimulate gonadotrophin release through a single GnRH receptor type on a single class of LH gonadotroph in the chicken pituitary. J. Endocr. (1988) 117,43–49

Gonadotrophin-releasing hormone (GnRH) overcomes GnRH antagonist-induced suppression of LH secretion in primates

1986 ◽  
Vol 110 (1) ◽  
pp. 145-150 ◽  
Author(s):  
G. R. Marshall ◽  
F. Bint Akhtar ◽  
G. F. Weinbauer ◽  
E. Nieschlag
Keyword(s):  
Gnrh Antagonist ◽  
Receptor Occupancy ◽  
Gnrh Receptor ◽  
Dependent Manner ◽  
Response Curves ◽  
Gnrh Antagonists ◽  
Releasing Hormone ◽  
Gonadotrophin Secretion ◽  
Gonadotrophin Releasing Hormone ◽  
Lh Secretion

ABSTRACT If the suppressive effects of gonadotrophin-releasing hormone (GnRH) antagonists on gonadotrophin secretion are mediated through GnRH-receptor occupancy alone, it should be possible to restore serum gonadotrophin levels by displacing the antagonist with exogenous GnRH. To test this hypothesis, eight adult crab-eating macaques (Macaca fascicularis), weight 4·7–7·6 kg, were subjected to the following treatment regimens. A GnRH-stimulation test was performed before and 4, 12 and 24 h after a single s.c. injection of the GnRH antagonist (N-Ac-d-p-Cl-Phe1,2,d-Trp3,d-Arg6,d-Ala10)-GnRH (ORG 30276). The stimulation tests were performed with 0·5, 5·0 or 50 μg GnRH given as a single i.v. bolus. Blood was taken before and 15, 30 and 60 min after each bolus for analysis of bioactive LH and testosterone. The GnRH-challenging doses were given as follows: 0·5 μg GnRH was injected at 0 and 4 h, followed by 5·0 μg after 12 h and 50 μg after 24 h. One week later, 5·0 μg GnRH were given at 0 and 4 h, followed by 50 μg after 12 h and 0·5 μg after 24 h. Finally, after another week, the GnRH challenges began with 50 μg at 0 and 4 h, followed by 0·5 μg at 12 h and 5·0 μg at 24 h. This design permitted comparison of the LH and testosterone responses with respect to the dose of GnRH and the time after administration of GnRH antagonist. The areas under the response curves were measured and statistical evaluation was carried out by means of non-parametric two-way analysis of variance followed by the multiple comparisons of Wilcoxon and Wilcox. Four hours after the antagonist was injected, the LH and testosterone responses to all three doses of GnRH were suppressed. At the lowest dose of GnRH (0·5 μg) the responses remained reduced even after 24 h, whereas the higher doses of GnRH elicited an LH and testosterone response at 12 and 24 h which was not significantly different from that at 0 h. These data demonstrate that the suppression of LH secretion by a GnRH antagonist in vivo can be overcome by exogenously administered GnRH in a dose- and time-dependent manner, thus strongly supporting the contention that GnRH antagonists prevent gonadotrophin secretion by GnRH-receptor occupancy. J. Endocr. (1986) 110, 145–150

Effects of crude and highly purified bovine inhibin (Mr 32 000 form) on gonadotrophin production by ovine pituitary cells in vitro: inhibin enhances gonadotrophin-releasing hormone-induced release of LH

1990 ◽  
Vol 127 (1) ◽  
pp. 149-159 ◽  
Author(s):  
S. Muttukrishna ◽  
P. G. Knight
Keyword(s):  
Primary Cultures ◽  
Suppressive Effect ◽  
Pituitary Cells ◽  
Dependent Manner ◽  
Α Subunit ◽  
Releasing Hormone ◽  
Lh Release ◽  
Gonadotrophin Releasing Hormone

ABSTRACT Primary cultures of ovine pituitary cells (from adult ewes) were used to investigate the actions of steroid-free bovine follicular fluid (bFF) and highly-purified Mr 32 000 bovine inhibin on basal and gonadotrophin-releasing hormone (GnRH)-induced release of FSH and LH. Residual cellular contents of each hormone were also determined allowing total gonadotrophin content/well to be calculated. As in rats, both crude and highly purified inhibin preparations promoted a dose (P < 0·001)- and time (P < 0·001)-dependent suppression of basal and GnRH-induced release of FSH as well as an inhibition of FSH synthesis, reflected by a fall in total FSH content/well. However, while neither inhibin preparation affected basal release of LH or total LH content/well, GnRH-induced LH release was significantly (P< 0·001) increased by the presence of either bFF (+ 75%) or highly-purified inhibin (+ 64%) in a dose- and time-dependent manner. This unexpected action of bFF on GnRH-induced LH release was abolished in the presence of 5 μl specific anti-inhibin serum, confirming that the response was indeed mediated by inhibin. Furthermore, neither oestradiol-17β (1 pmol/l–10 nmol/l) nor monomeric α-subunit of bovine inhibin (2·5–40 ng/ml) significantly affected basal or GnRH-induced release of LH. These in-vitro findings for the ewe lend support to a number of recent in-vivo observations and indicate that, in addition to its well-documented suppressive effect on the synthesis and secretion of FSH, inhibin may actually facilitate LH release in this species, in marked contrast to its action in the rat. Journal of Endocrinology (1990) 127, 149–159

Influence of the characteristics of pulses of gonadotrophin releasing hormone on the dynamics of luteinizing hormone release from perifused sheep pituitary cells

1983 ◽  
Vol 98 (3) ◽  
pp. 411-421 ◽  
Author(s):  
R. P. McIntosh ◽  
J. E. A. McIntosh
Keyword(s):  
Luteinizing Hormone ◽  
Anterior Pituitary ◽  
Pulse Width ◽  
Pulse Amplitude ◽  
Threshold Value ◽  
Pituitary Cells ◽  
Hormone Release ◽  
Releasing Hormone ◽  
Lh Release ◽  
Gonadotrophin Releasing Hormone

The effects were studied of varying the frequency, width and amplitude of pulses of gonadotrophin releasing hormone (GnRH) on the release of LH from anterior pituitary cells. Dispersed sheep cells supported in Sephadex were perifused with medium for 10 h and stimulated with different constant pulse patterns of GnRH. The timing of release of LH was measured by radioimmunoassay of the effluent fractions. Pulses of GnRH ranging in duration from 2 min every 8 min to 16 min every 128 min, and in concentration from 1·7 pmol/l to 250 nmol/l were applied to the cells, as well as continuous stimulation. Comparisons of differences between LH release patterns among samples of the same preparation of cells were used to demonstrate the effects of different GnRH stimulatory regimes. It was concluded that (1) the frequency of GnRH stimulation was important to the nature of LH release (periods shorter than about 16 min between pulses reduced LH output and caused faster desensitization of response), (2) the pulse width of GnRH input was important (the rising edge of the pulse produced greater LH output per unit of GnRH input than did continued application of GnRH within a pulse and wider pulses combined with shorter periods reduced LH output) and (3) over a threshold value of 5–10 nmol GnRH/1 pulse amplitude had little further influence on LH output or rate of desensitization in dispersed cells. These findings reinforce the hypothesis that the rising edge of the GnRH pulse is the major stimulant to LH release.

Effects of 31 kDa bovine inhibin on FSH and LH in rat pituitary cells in vitro: antagonism of gonadotrophin-releasing hormone agonists

1988 ◽  
Vol 119 (2) ◽  
pp. 233-241 ◽  
Author(s):  
P. G. Farnworth ◽  
D. M. Robertson ◽  
D. M. de Kretser ◽  
H. G. Burger
Keyword(s):  
Pituitary Cells ◽  
Sprague Dawley ◽  
Releasing Hormone ◽  
Pretreatment Period ◽  
Lh Release ◽  
Gonadotrophin Releasing Hormone ◽  
Constitutive Synthesis ◽  
Rat Pituitary Cells ◽  
Median Effective Concentration

ABSTRACT The effects of 31 kDa bovine inhibin on the release of FSH and LH stimulated by gonadotrophin-releasing hormone (GnRH) or its agonist analogue buserelin have been studied using 5-day-old cultures of pituitary cells prepared from adult male Sprague–Dawley rats. Exposure of cultures to increasing concentrations of inhibin for 3 days before and during a 4-h stimulation with GnRH resulted in the progressive suppression of both basal and stimulated gonadotrophin release. At the highest inhibin concentrations FSH release was abolished (inhibin median inhibitory concentration (IC50) = 0·15 U/ml) whereas LH release was suppressed by 75% (IC50 = 0·93 U/ml). To correct for the reduced size of the FSH pool resulting from inhibin pretreatment, the amount of FSH or LH released by an agonist was expressed as a proportion of the total hormone available for release in each case. Following this adjustment, concentrations of inhibin producing maximal effects increased the GnRH median effective concentration for FSH release 4·1-fold and that for LH release 2·2-fold, with inhibin IC50 values of 0·45 and 0·32 U/ml respectively. Inhibin also suppressed the maximum proportion of both FSH and LH that excess GnRH released in 4 h by 36%, with IC50 values of 0·53 and 0·76 U/ml respectively. These effects were not changed by reduction of the inhibin pretreatment period from 3 days to 1 day or by exclusion of inhibin during the stimulation period. After a 3-day pretreatment, inhibin inhibited gonadotrophin release by buserelin less effectively than that by GnRH, but the pattern of antagonism was the same. The results show that purified bovine inhibin antagonizes the release of both FSH and LH stimulated by either GnRH or buserelin in vitro by reducing the apparent potency of GnRH agonists and by decreasing the proportion of total available gonadotrophin that can be released by an excess of GnRH agonist. Higher concentrations of inhibin are required for these common actions against stimulated release of FSH and LH than for the inhibition of FSH tonic synthesis/basal release, indicating one or more secondary sites of inhibin action in addition to its primary selective action to suppress the constitutive synthesis of FSH. J. Endocr. (1988) 119, 233–241

Effect of chronic exposure to a gonadotrophin-releasing hormone agonist on in vitro responsiveness of ovine pituitary cells to inhibin, activin and oestradiol

1994 ◽  
Vol 140 (3) ◽  
pp. 483-493 ◽  
Author(s):  
S Muttukrishna ◽  
P G Knight
Keyword(s):  
Gnrh Agonist ◽  
Inhibitory Effect ◽  
Pituitary Cells ◽  
Cell Content ◽  
Releasing Hormone ◽  
Basal Release ◽  
Lh Release ◽  
Gonadotrophin Releasing Hormone

Abstract To investigate the extent to which the direct actions of inhibin, activin and oestradiol on pituitary output of FSH and LH are dependent on the presence of functional gonadotrophin-releasing hormone (GnRH) receptors, we have compared the effects of these agents on cultured ovine pituitary cells derived from control and GnRH agonist-suppressed ewes. Chronic treatment with GnRH agonist reduced plasma LH and FSH levels (P<0·01) and abolished GnRH-induced release of LH and FSH both in vivo and in vitro. As expected, basal LH release and LH cell content in vitro were drastically reduced in GnRH agonist-suppressed cells (P<0·001). However, basal FSH release and FSH cell content were approximately twofold higher than in control cells (P<0·001). Irrespective of whether the cells had been desensitized to GnRH, inhibin and oestradiol were both found to suppress basal FSH release and FSH cell content in a dose-dependent fashion (P<0·001). Although inhibin had no effect on basal release of LH from control cells, it markedly enhanced GnRH-induced release (P<0·001). In contrast, inhibin increased (P<0·001) basal LH release from GnRH agonist-suppressed cells (which were unresponsive to the GnRH challenge). Inhibin had no overall effect on total LH content/well for either control or GnRH agonist-suppressed cells. Treatment with oestradiol, on the other hand, reduced total LH content/well, an effect which was more pronounced with GnRH agonist-suppressed cells (−44%; P<0·001) than with control cells (−14%, P<0·01). Whereas in control cells activin had no significant effect on any aspect of FSH production examined, in GnRH agonist-treated cells activin enhanced basal FSH release, residual cell content and total FSH content/well (P<0·001). Altering GnRH receptor status also modified the LH response to activin. With control cells activin increased basal release (P<0·001), decreased GnRH-induced release (P<0·001) and increased total LH content/well (P<0·001). With GnRH agonist-treated cells, however, activin had a uniform inhibitory effect on each aspect of LH production examined (P<0·001 in each case). It was concluded that desensitization of ovine gonadotrophs to GnRH by chronic agonist treatment results in a paradoxical enhancement of FSH output in vitro but has little effect on the responsiveness of the cells (in terms of gonadotrophin release and content) to either inhibin or oestradiol. In contrast, GnRH agonist treatment leads to qualitative changes in cellular reponsiveness to activin. Journal of Endocrinology (1994) 140, 483–493

Effects of modifiers of cytoskeletal structures on the dynamics of release of LH from sheep anterior pituitary cells stimulated with gonadotrophin-releasing hormone, K+ or phorbol ester

1987 ◽  
Vol 112 (2) ◽  
pp. 289-298 ◽  
Author(s):  
R. P. McIntosh ◽  
J. E. A. McIntosh ◽  
L. Starling
Keyword(s):  
Initial Response ◽  
Cytochalasin D ◽  
Receptor Interaction ◽  
Release Mechanism ◽  
Pituitary Cells ◽  
Short Term ◽  
Releasing Hormone ◽  
Cell Components ◽  
Lh Release ◽  
Gonadotrophin Releasing Hormone

ABSTRACT This study investigated the importance of reorganization of cell components by cytoskeletal structures to the short-term dynamic changes in LH release from dispersed sheep pituitary cells in perifusion, when stimulated with different dynamic patterns of gonadotrophin-releasing hormone (GnRH). The changes in rate of LH release investigated were the initial response to GnRH, desensitization, change of dose–response during desensitization, and recovery of sensitivity between pulses of stimulation. Cytochalasin D and colchicine were used to modify microfilament and microtubule action respectively. To determine whether receptor movement after binding of agonist was involved in the altered responses, K+ and phorbol 12-myristate 13-acetate (PMA) were used as stimulants because they cause LH release independently of agonist-receptor interaction. After 3 and 48 h culture on dextran beads and 2–3 h incubation in the presence and absence of 2–48 μmol cytochalasin D/l, or 8 or 250 μmol colchicine/l, aliquots of collagenase-dispersed sheep pituitary cells were stimulated at 37 °C in tubes or in a multicolumn perifusion system with 850 pmol GnRH/l, 109 mmol K+/l or 10 nmol PMA/l. Fractions of supernatant or effluent were collected at intervals and LH concentrations measured by radioimmunoassay. Control samples were treated in the same way but without stimulation. Maximal, reversible enhancement of LH release over the first 20 min following stimulation with all secretogogues was observed after incubation of cells in 6 μmol cytochalasin/l. Desensitization behaviour, the supramaximal response, and the ability of cells to recover sensitivity to repeated pulses of GnRH were not altered by this modifier of microfilament polymerization at 6 or 24 μmol/ml. Colchicine at 8 μmol/l caused no changes in LH release. At 250 μmol/l, colchicine reduced the initial response of cells to GnRH stimulation but its action at this relatively high level may not be specific; there was no other major change in desensitization patterns, nor recovery of sensitivity to pulsed GnRH stimulation. Each treatment affected cellular responses similarly before and after culture. From studying the details of the dynamics of the short-term responses of gonadotrophs, we conclude that transport of cell components involving microfilaments and microtubules is unlikely to be a major limitation on the rate of LH release during desensitization, the supramaximal response, or the recovery of sensitivity between pulses of GnRH. This suggests that biochemical reactions rather than physical translocation may be rate-limiting in these processes. In addition, although inhibition of microfilament action does appear to enhance the earliest observed response to stimulation of the LH-release mechanism, this occurs after protein kinase C activation and is probably not related to impairment of processes such as polymerization and sequestration of agonist-bound GnRH receptors because the effects are also observed with K+ and PMA, stimulants acting independently of agonist-receptor interaction. J. Endocr. (1987) 112, 289–298

Pituitary gonadotrophin-releasing hormone receptor up-regulation in vitro: dependence on calcium and microtubule function

1985 ◽  
Vol 107 (1) ◽  
pp. 49-56 ◽  
Author(s):  
L. S. Young ◽  
S. I. Naik ◽  
R. N. Clayton
Keyword(s):  
Luteinizing Hormone ◽  
Gnrh Receptor ◽  
Ionophore A23187 ◽  
Pituitary Cells ◽  
Hormone Release ◽  
Releasing Hormone ◽  
Luteinizing Hormone Release ◽  
Gnrh Receptors ◽  
Gonadotrophin Releasing Hormone

ABSTRACT Exogenous cyclic adenosine nucleotides increase gonadotrophin-releasing hormone (GnRH) receptors in intact cultured rat pituitary cells in a similar manner to that observed with GnRH itself. In this study the calcium and microtubule dependency of GnRH receptor up-regulation was examined in vitro. Treatment of pituitary cells in Ca2+ and serum-containing media with either GnRH (1 nmol/l), K+ (58 mmol/l) or dibutyryl cyclic AMP (dbcAMP; 1 mmol/l) for 7–10 h routinely resulted in a 50–100% increase in GnRH receptors. Incubation of pituitary cells with the calcium channel blocker verapamil, for 7 h, or the calcium chelator EGTA, for 10 h, had no effect on basal receptor levels but prevented the increase in GnRH receptors stimulated by either GnRH, K+ or dbcAMP. Luteinizing hormone release measured with the same stimulators over a 3-h period was prevented by both verapamil and EGTA. Calcium ionophore (A23187) increased GnRH receptors by 40–60% at low concentrations (10 and 100 nmol/l) while higher concentrations (10 and 100 μmol/l) reduced receptor levels. Luteinizing hormone release was not increased by receptor-stimulating concentrations of A23187, but was by higher concentrations (10 μmol/l). None of these pretreatments, for up to 10 h, impaired the subsequent LH response of the cells to increasing doses of GnRH. Vinblastine (1 μmol/l did not affect basal receptor levels but markedly reduced the increase in GnRH receptors stimulated by GnRH, K+ and dbcAMP. This concentration of vinblastine had no effect on LH release. These results indicate that receptor stimulation by GnRH, K+ and dbcAMP is a calcium-dependent process requiring the integrity of the microtubule system and there is a different calcium requirement for the processes of GnRH receptor up-regulation and LH secretion. J. Endocr. (1985) 107, 49–56

Melatonin inhibits luteinizing hormone releasing hormone (LHRH) induction of LH release from fetal rat pituitary cells

1995 ◽  
Vol 184 (2) ◽  
pp. 109-112 ◽  
Author(s):  
Atsuhiko Hattori ◽  
Damon C. Herbert ◽  
Mary K. Vaughan ◽  
Ken Yaga ◽  
Russel J. Reiter
Keyword(s):  
Luteinizing Hormone ◽  
Pituitary Cells ◽  
Luteinizing Hormone Releasing Hormone ◽  
Releasing Hormone ◽  
Fetal Rat ◽  
Rat Pituitary ◽  
Lh Release ◽  
Rat Pituitary Cells

Varying the patterns and concentrations of gonadotrophin-releasing hormone stimulation does not alter the ratio of LH and FSH released from perifused sheep pituitary cells

1986 ◽  
Vol 109 (2) ◽  
pp. 155-161 ◽  
Author(s):  
J. E. A. McIntosh ◽  
R. P. McIntosh
Keyword(s):  
Dose Response ◽  
Dose Interval ◽  
Follicular Phase ◽  
Pituitary Cells ◽  
Ovulatory Cycle ◽  
Short Term ◽  
Releasing Hormone ◽  
Dissociated Cells ◽  
Gonadotrophin Releasing Hormone

ABSTRACT Our aim was to determine whether release of LH and FSH can be controlled differentially by the characteristics of applied signals of stimulatory gonadotrophin-releasing hormone (GnRH) alone, free of the effects of steroid feedback or other influences from the whole animal. The outputs of both gonadotrophins were significantly correlated (r≈0·90; P<0·0005) when samples of freshly dispersed sheep pituitary cells were perifused in columns for 7 h with medium containing a range of concentrations of GnRH in various patterns of pulses. Hormone released in response to the second, third and fourth pulses from every column was analysed in detail. Dose–response relationships for both LH and FSH were very similar when cells were stimulated with 5–8500 pmol GnRH/1 in 5-min pulses every hour. When GnRH was delivered in pulses at a maximally stimulating level, the outputs of both hormones increased similarly with increasing inter-pulse intervals. Efficiency of stimulation (release of gonadotrophin/unit stimulatory GnRH) decreased (was desensitized) with increasing pulse duration in the same way for both hormones. Thus, varying the dose, interval and duration of GnRH pulses did not alter the proportions of LH and FSH released in the short-term from freshly dissociated cells. However, the same cell preparations released more LH relative to FSH when treated with maximally stimulating levels of GnRH for 3 h in the presence of 10% serum from a sheep in the follicular phase of its ovulatory cycle compared with charcoal-treated serum. Because there was no gonadotrophin synthesis under the conditions used in vitro these results suggest that changes in the LH/FSH ratio seen in whole animals are more likely to result from differential clearance from the circulation, ovarian feedback at the pituitary, differential synthesis in intact tissue or another hormone influencing FSH secretion, rather than from differences in the mechanism of acute release controlled by GnRH. J. Endocr. (1986) 109, 155–161

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