Relative activity, plasma elimination and tissue degradation of synthetic luteinizing hormone releasing hormone and certain of its analogues.

The abilities of three nonapeptide analogues of synthetic luteinizing hormone releasing hormone (LH-RH) to release luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in anoestrous and cyclic ewes were examined, as were their elimination from the plasma in vivo and degradation by extracts of the hypothalamus, anterior pituitary gland, lung, kidney, liver and plasma in vitro. In all cases, comparisons were made with synthetic LH-RH. When injected i.v. into mature ewes as a single dose, the potencies of the analogues were graded and Des Gly-NH2(10) LH-RH ethylamide was found to be the least potent. It was not possible to demonstrate any significant increase in the potency of this analogue over LH-RH, although a trend was apparent with each parameter examined. [D-Ser(But)6] Des Gly-NH2(10) LH-RH ethylamide had the greatest potency. There were no differences between the responses of anoestrous ewes and those of ewes treated on day 10 of the oestrous cycle. None of the analogues had a rate of elimination from the plasma different from that of LH-RH during either the first or the second components of the biphasic disappearance curve. The incubation of LH-RH with tissue extracts showed that extracts of the hypothalamus and anterior pituitary gland degraded LH-RH to a similar extent. Both the hypothalamic and anterior pituitary gland extracts degraded more LH-RH than did lung extract, which in turn destroyed more LH-RH than did extracts of kidney or liver tissue. The degradative abilities of kidney and liver extracts did not differ from each other. Plasma failed to degrade LH-RH or the analogues. Although LH-RH was rapidly destroyed by hypothalamic extract in vitro, of the analogues, only Des Gly-NH2(10) LH-RH ethylamide was degraded. The anterior pituitary gland and kidney extracts failed to degrade [D-Ser6] Des Gly-NH2(10) LH-RH ethylamide and [D-Ser(But)6] Des Gly-NH2(10) LH-RH ethylamide as rapidly as LH-RH. Extracts of liver and lung were incapable of catabolizing any of the analogues. There was an inverse correlation between the LH- and FSH-releasing potency of an analogue and its rate of degradation by anterior pituitary gland extract. The slower rates of catabolism of certain analogues of LH-RH by the anterior pituitary gland may explain their increased LH- and FSH-releasing potency.     

Possible priming effect of luteinizing hormone releasing hormone on the anterior pituitary gland in the Japanese quail and the stimulation of secretion of follicle-stimulating hormone.

After the i.m. injection of 10 micrograms synthetic LH releasing hormone (LH-RH) into Japanese quail the levels of LH and FSH in plasma rose significantly within 2 min. The increased level of LH declined rapidly but that of FSH was maintained for the duration of the experiment. To determine whether the anterior pituitary gland is primed by LH-RH a double injection schedule was adopted. It would appear that, while endogenous LH-RH may prime the avian pituitary gland slightly, synthetic LH-RH is ineffective.     

Studies with fragments of a highly active analogue of luteinizing hormone releasing hormone.

The minimal structural requirements for gonadotrophin releasing activity were studied with fragments of a highly active analogue of luteinizing hormone releasing hormone (LH-RH), [D-Ser(But)6]LH-RH(1-9)nonapeptide-ethylamide (Hoe 766). All fragments are related to the C-terminal structure of LH-RH and have increased enzyme stability. Ovulation in phenobarbitone-blocked rats was induced with a median effective dose/rat, of 1.9 microgram of the (3-9)-heptapeptide, Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-ethylamide and 6.8, 18.0 and 38.3 microgram for the (4-9), (5-9) and (6-9) fragments respectively. The (3-9)- heptapeptide and (4-9)-hexapeptide induced release of LH and FSH in phenobarbitone-blocked rats with a ratio similar to that of LH-RH. Degradation of LH-RH by enzyme preparations of liver, kidney and hypothalamic or anterior pituitary tissue was not modified by addition of the (3-9)-heptapeptide fragment. The organ distribution of the 125I-labelled (3-9)-heptapeptide fragments was similar to LH-RH, but not to Hoe 766. The peptide accumulated in liver and kidney, but was eliminated from the anterior pituitary gland 15 min after i.v. injection, whereas Hoe 766 showed progressive accumulation in the pituitary gland (tissue:plasma ratio = 6.6 after 60 min). In contrast to C-terminal fragments of LH-RH, the corresponding fragments of nonapeptide analogues retained significant biological activity, and the minimal structural requirements for LH release may be related to the C-terminal sequence of LH-RH.     

Studies on the feedback regulation of gonadotropin concentrations in male rats. (III). The effects of testosterone and its metabolites on gonadotropin secretion from rat pituitary cells in culture

To determine whether dihydrotestosterone (DHT) or estradiol (E2) exerts negative or positive feedback effects on rat pituitary gland, Testosterone (T) metabolite (T, DHT, 5 alpha-androstane-3 alpha, 17 beta-diol:3 alpha-diol or E2) was added to the cultured pituitary cells. Anterior pituitary glands were obtained from 6-week-old male rats. Pituitary cells were prepared by trypsin digestion and incubated with various concentrations of steroid hormones for 72 h to determine the effects of steroid hormones on basal secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) after 48 h preculture without steroids. Then 10 nM luteinizing hormone-releasing hormone (LH-RH) with appropriate concentrations of these steroid hormones was added to the pituitary cells in culture and incubated for another 6h to determine the effects of steroid hormones on LH-RH induced gonadotropin release. After the incubation, pituitary cells were lysed with 0.1% Triton X100 to measure the intracellular gonadotropin content. The concentration of LH and FSH was determined by radioimmunoassay. T, DHT and 3 alpha-diol stimulated basal FSH but not basal LH secretion, and inhibited both the release of FSH and LH from cultured pituitary cells during incubations with LH-RH in a dose-dependent fashion. Intracellular content of both FSH and LH were increased, and total FSH and not LH was also increased by the addition of DHT in a dose-dependent manner. E2 did not exert any of such effects on pituitary cells in culture. These studies suggest that 5 alpha-reduced metabolites but not aromatized metabolite of T play an important role on feedback regulation of gonadotropin secretion at pituitary level. DHT directly acts on pituitary gland not only to stimulate the production of FSH but also to suppress FSH and LH secretion induced by LH-RH.     

Gonadal steroid-mediated alteration of luteinizing hormone secretion by anterior pituitary cells of young turkeys

The magnitude of luteinizing hormone (LH) release during a 3-hr test incubation was diminished (P less than 0.05) when anterior pituitary cells from young turkeys were cultured for 24 to 120 hr. This trend was evident with basal LH release and with LH release induced by luteinizing hormone-releasing hormone (LH-RH) or hypothalamic extract. Anterior pituitary cells were cultured with various concentrations (10(-14) to 10(-6) M) of estradiol (E2), progesterone (P4), or testosterone (T) for 24 hr and then exposed to LH-RH or control medium for 3 hr, still in the presence of steroids. Basal LH release was potentiated (P less than 0.05) when cells were cultured with 10(-8) or 10(-6) M T, but not with E2 or P4. When cells were cultured with E2, LH release in the presence of 10(-8) M LH-RH was enhanced (P less than 0.05) in a dose-dependent fashion. LH-RH mediated LH release was also enhanced (P less than 0.05) when cells were cultured with 10(-8) M P4 or 10(-6) M T. Gonadal steroids can act directly on the anterior pituitary of the young domestic turkey to modulate LH release, with T enhancing basal LH release and E2 potentiating LH-RH-mediated LH release.     

Prostaglandin D2 induces release of luteinizing hormone from the rat pituitary gland without the modulation of hypothalamic luteinizing hormone releasing hormone

The effect of prostaglandin D2 (PGD2) on release of LH and LH releasing hormone (LHRH) was studied in a sequential double-chamber superfusion system using the medial basal hypothalamus (MBH) and the pituitary gland from female rats at dioestrus. Infusion of PGD2 (5.7 or 57 mumol/l) caused a significant (P less than 0.05) increase in LH release to values 40-60% above the preinjection values from the pituitary gland superfused either alone or in series with the MBH. No release of LHRH in response to PGD2 was observed from the superfused MBH. These data demonstrate that PGD2 causes LH release from the pituitary gland not by inducing release of hypothalamic LHRH but by a direct action on the gland.     

In vitro gonadotropin-releasing hormone release from hypothalamic tissues of ovariectomized estrogen-treated cynomolgus macaques

The effects of in vivo 17 beta-estradiol (E2) treatment on in vitro GnRH release and serum LH levels were studied to determine the loci of E2 feedback actions and to examine the hypothalamic mechanisms by which this steroid may regulate LH secretion in monkeys. Ovariectomized cynomolgus macaques received sc Silastic capsule implants containing E2 and were killed 12, 36, 42, or 48 h later. At least one control (CTL) animal received a blank implant and was killed concurrently with each E2-treated monkey. Three untreated animals were used in validation experiments. Before death, each animal was anesthetized with ketamine (15 mg/kg, im), and blood samples were drawn for subsequent LH analysis by Leydig cell bioassay. A diencephalic tissue block was obtained at autopsy and immediately immersed in Krebs-Ringer-phosphate medium (KRP). Mediobasal hypothalamic (MBH) and anterior hypothalamic/preoptic (AH/POA) fragments were quickly dissected from the block and placed in separate superfusion chambers maintained at 37 C. Tissues were superfused at 50 microliter/min with KRP, and 10-min fractions were collected, acidified, and stored at -20 C for subsequent GnRH RIA. Basal immunoreactive GnRH (IR-GnRH) release was measurable from MBH (0.367 +/- 0.063 pg/min) and AH/POA (0.176 +/- 0.065 pg/min) fragments from CTL monkeys. In validation experiments, IR-GnRH release was increased 3- to 7-fold by superfusion with 60 mM K+-KRP only in the presence of Ca+2. Superfusate IR-GnRH coeluted with synthetic GnRH from a Sephadex G-25 chromatographic column, and superfusate and tissue extract GnRH showed appropriate LH-releasing capacities, as determined by rat pituitary cell culture assay. IR-GnRH release rates from MBH or AH/POA tissues varied as a function of in vivo estrogen treatment. GnRH release from both tissues was increased in the E2-treated group killed at 12 h when LH levels were suppressed. Thirty-six hours after E2 treatment, in vitro GnRH release was not significantly different from CTL values. GnRH release rates from MBH and AH/POA tissues obtained 42 h after E2 treatment were significantly greater than CTL release rates (P less than 0.01). This increased in vitro GnRH release at 42 h occurred during the apparent rising phase of the LH surge. Elevated GnRH release was not sustained at 48 h, when surge levels of LH were apparent.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rapid augmentation by progesterone of agonist-stimulated luteinizing hormone secretion by cultured pituitary cells

Progesterone acts bimodally at the hypothalamus and at the pituitary gland, the sequelae in vivo being either stimulation or inhibition of gonadotropin secretion depending on a host of preconditions. Pituitary cells in culture were studied to characterize the acute action of progesterone on LH secretion. Preliminary studies established that anterior pituitary cells from adult female rats cultured for three days in 10% charcoal treated fetal bovine serum (c/t FBS) resulted in LH secretory responses to GnRH pulses which were half that for cells cultured in untreated FBS or c/t FBS + 0.2 nM 17 beta-estradiol (E2). Under standardized culture conditions (c/t FBS + E2), GnRH self-potentiation was evident. With this system, 90 min exposure to 200 nM progesterone resulted in a 3-fold augmentation of GnRH-stimulated LH secretion without affecting baseline LH. This action was manifested by 45 but not 15 min of progesterone exposure and was inhibited by simultaneous addition of cycloheximide. The augmentation of agonist-stimulated LH release could be elicited up to 4-5 h after progesterone addition. The estimated half-maximal effect was 10(-9) M, and this concentration of progesterone required E2-pretreatment of the cultured cells. In summary, addition of progesterone to cultured anterior pituitary cells pretreated with E2 leads to a concentration-, time-, and protein synthesis-dependent augmentation of pulsatile GnRH-stimulated LH secretion within 45 min of progesterone exposure. This rapid and unambiguous progesterone action in pituitary cells could function in vivo to define the final magnitude of the preovulatory LH surge.     

Comparison of steroid and LH-RH effects on the responsiveness of hemipituitary glands and dispersed pituitary cells

We have carried out further in vitro studies on the priming effect of LH-RH and the effect of steroids on pituitary responsiveness to LH-RH. In hemipituitary glands, the priming effect could be elicited only once within an 11-h period and was found to dimmish significantly with time after the first exposure to LH-RH. Incubation with oestradiol-17β (E₂) had no significant effect on the responsiveness of hemipituitary glands to LH—RH. By contrast, E₂ increased the responsiveness of dispersed pituitary cells to LH-RH, but the priming effect could not be elicited in the dispersed cell system. The presence of hypothalamus or synthetic LH-RH did not facilitate the effects of e₂. Testosterone significantly reduced the spontaneous and LH-RH-induced release of LH while progesterone had no effect. Exposure to E₂ alone in either of the systems did not produce a consistent increase in the total amount of LH in the system. Synthesis of LH was, however, stimulated by exposure to LH—RH for 48 h but not 12 h. These results demonstrate that there is a marked difference between the mechanisms by which LH-RH and steroids affect the responsiveness of the anterior pituitary gland to LH-RH.     

Superfusion of rat anterior pituitary cells attached to Cytodex beads: validation of a technique

A superfusion method consisting of fully recovered, dissociated pituitary cells adhering to Cytodex beads has proved useful in monitoring the dynamics of hormone secretion over time. Male rat anterior pituitaries were dissociated with collagenase and Viokase, then cultured in the presence of Cytodex beads for 3-5 days, during which time the cells attached firmly to the surface of the beads. The bead-attached cells were stable and could be transferred to any vessel without the need for centrifugation or further trypsinization. For this application, the bead-attached cells were packed in a column and superfused with a low bicarbonate buffer requiring no CO2 gassing. Viability was more than 95% after 48 h in the column. The cells responded in a normal physiological manner to hypothalamic releasing and inhibitory peptides. The ED50 was 0.3 nM for somatostatin and 1.2 nM for gonadotropin-releasing hormone. A postinhibitory rebound of GH secretion was observed after the discontinuation of large doses of somatostatin. LH secretion reached maximal levels within 6 min after 10 nM gonadotropin-releasing hormone, but started declining after 2 h of continuous stimulation and dropped close to baseline within 18 h. GH release was significantly increased by prostaglandin E2, 3-isobutyl-1-methylxanthine, and 8-bromo-cAMP. LH secretion increased 5-fold in response to 1 mM 8-bromo-cAMP, but showed little increase during prostaglandin E2 or 3-isobutyl-1-methylxanthine stimulation. The cocarcinogen phorbol myristate acetate (12-O-tetradecanoyl-phorbol-13-acetate) induced secretion of all pituitary hormones and continued to do so for hours after a short pulse. The superfusion system is simple to operate and has proven effective in studying transient phenomena, desensitization, and short term kinetics of secretagogues.     

Inhibition by prolactin of post-castration rise in LH.

Castration of male and female rats resulted in a marked rise in serum LH. The rise in serum LH was partially or completely prevented by injection of prolactin (Prl), by implantation of a small amount of Prl in the median eminence (ME), by grafting 2 anterior pituitaries (APs) underneath the kidney capsule, or by transplantation of a Prl-secreting pituitary tumor underneath the skin. The larger pituitary tumor transplants secreted more Prl and were more effective in reducing LH release than the smaller tumors which secreted less Prl. Suppression of LH release generally was greater during the earlier than in the later phases of the different treatments. The pituitary LH response to synthetic LH-RH was the same in ovariectomized rats with or without pituitary grafts, and the decrease in hypothalamic LH-RH after orchidectomy was prevented by pituitary grafts. These results indicate that Prl can depress LH release after castration and that these effects are mediated via the hypothalamus.     

LH-RH and dopamine levels in hypophysial stalk plasma and their relationship to plasma gonadotrophins and prolactin levels in male rats bearing a prolactin- and adrenocorticotrophin-secreting pituitary tumor

The present study was concerned with the effects of a transplantable pituitary tumor secreting prolactin (PRL) and adrenocorticotrophin (ACTH) on the levels of LH and FSH in peripheral plasma and on the hypothalamic release of LH-RH and dopamine in the male rat. Male rats of the same age not inoculated with the tumor served as controls. Hypophysial stalk blood was collected from urethane-anesthetized rats 4-5 weeks after tumor inoculation to measure their LH-RH and dopamine content. A peripheral blood sample was withdrawn from the animals just before sectioning the hypophysial stalk to measure their content of LH, FSH and PRL. It was found that in the tumor-bearing rats the levels of PRL increased 17-fold, whereas plasma levels of LH and FSH decreased by 45 and 70% respectively, when compared with the control rats. In the tumor-bearing rats, the secretion rate of dopamine in hypophysial stalk plasma increased from 1.4 to 4.1 ng/h, whereas the secretion rate of LH-RH decreased from 122 to 61 pg/h. However, when at the time of tumor inoculation adrenalectomy was performed, the tumor did not decrease plasma levels of LH and FSH and the secretion of LH-RH into hypophysial stalk blood any longer. The effect of the tumor on hypothalamic dopamine secretion was, however, still present in the adrenalectomized rats. It is concluded that the effect of the PRL- and ACTH-secreting pituitary tumor on plasma levels of LH and FSH requires the presence of the adrenal gland and that this effect is mediated through an inhibition of the hypothalamic release of LH-RH. Furthermore, this tumor increases the hypothalamic release of dopamine independent of the presence of the adrenal gland.     

Effects of anterior hypothalamic deafferentation on plasma LH response to LH-RH in the baboon (author's transl)]

We reported previously that a biphasic LH release (within 30 min and 90 approximately 150 min after LH-RH injection) was observed in normal menstruating baboons (non-human primates). Plasma immunoassayable LH-RH reached a maximum within 4 min after injection and was undetectable within 60 min. Plasma estrogen and progestin were elevated within 45 min. No plasma LH release with increment of plasma estrogen and progestin was observed in saline injected baboons. This study was performed to investigate the mechanism of biphasic LH release by LH-RH injection. Synthetic LH-RH (100 microgram) was injected sc into four female retrochiasmatic deafferented baboons. Blood samples collected 30 and 2 min before injection and 5, 10, 15, 30, 45, 60, 90, 120, 150 and 180 min after injection were assayed for LH, estrogen and progestin. In four baboons plasma LH peak was observed within 30 min after injection and plasma estrogen and progestin were elevated within 45 min. However, plasma LH peak within 90 approximately 150 min was not observed. These results infer that exogenous LH-RH exerts an effect on the pituitary to release LH within 30 min after injection. Increased estrogen and progestin exert the effect on the higher brain area (extrahypothalamic area) and the anterior hypothalamus to facilitate release of endogenous LH-RH, which subsequently release LH within 90 approximately 150 min in cooperation with exogenous LH-RH which may be considered to participate LH synthesis in the pituitary. Thus, it seems likely that the extrahypothalamic area (for example; limbic system) and the anterior hypothalamus have an important role in regulating LH-RH secretion and subsequent LH release in the baboon.     

Estradiol potentiation of hypothalamic uptake of LH-RH from the CSF.

Ovariectomized (Ovx) rats were treated (s.c.) with estradiol benzoate (E2B) for 7 days, and then 10, 25 or 100 ng LH-RH were microinjected into the 3rd ventricle. Intraventricular LH-RH elevated plasma LH at 10 and 30 min in a dose-response manner. In experiment 2, Ovx rats were treated with E2B or oil for 7 days and then intraventricularly injected with 25 ng LH-RH. Two days later, 100 ng LH-RH were systemically administered. E2B treatment resulted in a greater release of LH in response to the intraventricular administration of LH-RH but not to the systemic injection. A direct measure of median eminence (ME) uptake of LH-RH was used in experiment 3. Ovx-E2B or oil-treated rats were decapitated 10 min after intraventricular injection of 125I-LH-RH, 125I or 3H-glycine. The ME region of the hypothalamus, cortex, anterior pituitary (AP) and plasma were solubilized and their radioactivity determined. E2B increased the radioactivity in the ME following injection of 125I-LH-RH but not in other tissues. Tissue uptake of 125I and 3H-glycine were similar in E2B or oil-treated rats. These data indicate E2B facilitates the incorporation of LH-RH into the ME from the cerebrospinal fluid (CSF).     

Parallel stimulation of cyclic AMP accumulation and LH and FSH release by analogs of LH-RH in vitro

Dose-response studies with des-(pyro) Glu-LH-RH, [Phe³] LH-RH, [Phe⁵] LH-RH [0-methyl-Tyr⁵] LH-RH, [Ile⁷] LH-RH and des-Gly¹⁰-LH-RH-ethylamide show concomitant stimulation of adenohypophyseal adenosine 3',5'-monophosphate (cyclic AMP) concentration, and LH and FSH release in rat anterior pituitary gland in vitro.Des-His²-des-Gly¹⁰-LH-RH-ethylamide, and inhibitor of the response to LH-RH in vivo, had no effect on cyclic AMP accumulation or on LH release in vitro. The present finding of parallel changes of intracellular cyclic AMP levels and hormonal release with LH-RH analogs having a wide spectrum of biological activity adds strong support for a role of the cyclic nucleotide as mediator of the action of LH-RH in the anterior pituitary gland.     

Clomiphene citrate induces pituitary GnRH receptors in ovariectomized rats: its possible role in induction of ovulation

Since our previous studies have shown that clomiphene citrate (clomiphene) acts directly on the pituitary gland and exerts a facilitatory role on oestradiol-17 beta (E2)-induced LH surge in chronically ovariectomized rats, the effect of clomiphene on pituitary GnRH receptors was investigated. A single ip injection of either 5 micrograms E2 or 200 micrograms clomiphene did not induce LH release in adult rats ovariectomized 1-2 weeks before the injection. However, a significant increase in serum LH was noted 24 h after a single injection of E2 in the ovariectomized rats, if clomiphene was pre-injected 48 h before the E2 injection. The content of pituitary GnRH receptors in the ovariectomized rats (62 +/- 9 fmol/pituitary) remained almost unchanged until 24 h after a single injection of clomiphene but significantly increased 48 h after the injection (105 +/- 13 fmol/pituitary) without any alterations in the affinity for GnRH. To determine steroid specificity for the increase in pituitary GnRH receptors, other classes of steroids were injected in the ovariectomized rats. A single dose of E2 increased GnRH receptors, but either progesterone or 5 alpha-dihydrotestosterone failed to show any effect on the level of GnRH receptors. These results suggest that clomiphene may augment oestrogen-induced pre-ovulatory LH surge in anovulatory women, at least in part by increasing the number of pituitary GnRH receptors.     

Changes in the hypothalamic contents of LHRH-I and -II and in pituitary responsiveness to synthetic chicken LHRH-I and -II during the progesterone-induced surge of LH in the laying hen

The contents of LHRH-I and -II in the anterior hypothalamus and posterior hypothalamus (including the mediobasal hypothalamus and median eminence) were measured at 90, 180 and 360 min after the i.m. injection of laying hens with progesterone. Whilst no changes were observed in the content of LHRH-I in the anterior hypothalamus, LHRH-I in the posterior hypothalamus tended to fall at 90 and 180 min after injection of progesterone in hens maintained on 16 h light:8 h darkness (16L:8D) and 8L:16D respectively. Pretreatment of laying hens with tamoxifen significantly increased the hypothalamic contents of LHRH-I and -II, raised the basal plasma concentration of LH and modified the LH response to progesterone injection. In hens in which tamoxifen prevented an increase in the plasma concentration of LH after progesterone injection, the content of LHRH-I in the posterior hypothalamus remained unchanged. In contrast, in hens in which progesterone stimulated a steep increase in LH within 90 min, there was a pronounced and significant fall in LHRH-I content of the posterior hypothalamus. No change in the hypothalamic content of LHRH-II was observed during the progesterone-induced surge of LH until plasma concentrations had attained maximal values or started to decline. Then, in hens maintained on 16L:8D, a significant fall in the content of LHRH-II in the anterior hypothalamus was found at both 180 and 360 min after injection with progesterone. Tests in vitro and in vivo of the responsiveness of the pituitary gland to synthetic LHRH-I and -II revealed no change at 90 min after injection of laying hens with progesterone, when plasma concentrations of LH were increasing, but a pronounced reduction when plasma LH concentrations were maximal or falling. These results suggest that LHRH-I mediates in the progesterone-induced increase in the plasma concentration of LH. Although the subsequent decline in plasma LH was associated with a reduced responsiveness of the pituitary gland to LHRH, a significant correlation between the contents of LHRH-I and -II in the anterior hypothalamus and a fall in the hypothalamic content of LHRH-II when plasma LH was maximal or declining allows the possibility of an involvement of this peptide in the neuroendocrine events preceding ovulation.     

A possible role of clomiphene citrate in the control of pre-ovulatory LH surge during induction of ovulation

A possible role of clomiphene citrate (clomiphene) in the control of ovulation in anovulatory women was investigated. Since a single ip administration of 5 micrograms oestradiol-17 beta (E2) to long-term ovariectomized rats did not induce LH surge, the following studies were designed to determine whether pretreatment with clomiphene followed by administration of E2 could induce LH surge in the ovariectomized rats. Changes in cytoplasmic and nuclear oestrogen receptors (ER) were also examined in the pituitaries of these animals. An ip injection of 200 micrograms clomiphene suppressed serum LH levels significantly for 72 h. The clomiphene injection rapidly caused an elevation of nuclear ER with a concomitant depletion of cytoplasmic ER level in the pituitary and the ER levels remained almost unchanged for 72 h. An administration of E2 12 or 24 h after the clomiphene injection had no significant effects on either the serum LH levels or the cytoplasmic and nuclear ER levels, compared with those induced by clomiphene alone. However, LH surge and the depletion of nuclear ER in the pituitary occurred 24 h later when E2 was injected 48 h after the clomiphene administration. The E2-induced LH release seems to be induced by a replacement of clomiphene by E2 on the nuclear receptor complex. These results suggest that clomiphene may exert actions directly on the pituitary gland to augment oestrogen-induced LH release.     

Acute inhibitory effects of 17 beta-estradiol are observed on gonadotropin secretion from perifused pituitary fragments of metestrous, but not proestrous, rats

The in vivo suppression of LH by 17 beta-estradiol (E2) has been documented frequently. However, the demonstration of a direct inhibitory action of E2, in contrast to a stimulatory action, on the secretion of LH from the anterior pituitary has been inconsistent. The aim of this study was to determine if E2 can suppress either basal (unstimulated) or GnRH-stimulated gonadotropin secretion directly at the level of the anterior pituitary gland. Anterior pituitaries were obtained from metestrous and proestrous females rats at 0900 h, and trunk blood was collected for serum measurements of LH, FSH, E2, and progesterone (P). Each anterior pituitary was cut into eighths and placed into a microchamber for perifusion. Pituitary fragments were perifused at a rate of 10 ml/h using medium 199 (without phenol red) that contained E2 (1 nM) or ethanol as a control. Six pulses of GnRH (peak amplitude, 50 ng/ml; duration, 2 min) were administered one per h starting at 60 min. Fractions of perfusate were collected every 5 min for measurement of LH and FSH. The total amounts of LH and FSH secreted during the 1-h interval after each GnRH pulse or corresponding basal hour were calculated. Both basal and LH and FSH responses to GnRH were significantly greater from pituitaries of proestrous compared to metestrous rats. The selective suppression of LH secretion by in vitro treatment with E2 was demonstrated using pituitaries from metestrous rats receiving GnRH pulses, but not using pituitaries from proestrous rats. Thus, a negative feedback effect of E2 on LH secretion was observed only in pituitaries from donors with low serum levels of E2 and high P, but not from donors with high serum levels of E2 and low P. We believe that the in vivo steroid environment determined the subsequent responses to in vitro treatment with E2 on GnRH-stimulated gonadotropin secretion from the isolated pituitary gland.     

Gonadotrophin release by a highly active analogue of luteinizing hormone releasing hormone in rats immunized against luteinizing hormone releasing hormone.

Immunization against luteinizing hormone releasing hormone (LH-RH) in adult male rats produced a progressive decline in LH and FSH in the circulation to low or non-detectable levels. D-Serine-tertiary-butyl6,des-glycine-NH210 LH-RH ethylamide is an analogue of LH-RH having highly active LH-RH properties in the normal rat. Because it is also immunologically different from LH-RH it can stimulate gonadotrophin release from the anterior pituitary gland of rats immunized against LH-RH without interference from the antibody. The analogue stimulated LH and FSH release in rats 15 weeks after immunization against LH-RH when antibody titre was highest, and after long-term (35 weeks) immunization against LH-RH. D-Serine-tertiary-butyl6,des-glycine-NH210 LH-RH ethylamide and related analogues are therefore potentially useful for reversing the effects of immunization against LH-RH.