Pituitary receptors for gonadotropin-releasing hormone in relation to changes in pituitary and plasma luteinizing hormone in ovariectomized-hypothalamo pituitary disconnected ewes. I. Effect of changing frequency of gonadotropin-releasing hormone pulses

Studies were undertaken to determine if changes in the amplitude of luteinizing hormone (LH) pulses that occur in response to changes in the frequency of gonadotropin-releasing hormone (GnRH) pulses are due to an alteration in the number of GnRH receptors. Ewes were ovariectomized (OVX) and the hypothalamus was disconnected from the pituitary (HPD). Ewes were then given pulses of GnRH at a frequency of 1/h or 1/3 h. Two control groups were included: OVX ewes not subjected to HPD, and HPD ewes that were not OVX. At the end of one week of treatment, blood samples were collected to determine the amplitude of LH pulses. The treated ewes were killed just before the next scheduled pulse of GnRH, and the content of LH and number of GnRH receptors were measured in each pituitary. The amplitude of LH pulses was highly correlated with the amount of LH in the pituitary gland (r = 0.71, p less than 0.01), and both LH content and pulse amplitude (mean + SEM) were higher in ewes receiving GnRH once per 3 h (189.7 +/- 39.3 microgram/pituitary, 10.3 +/- 1.1 ng/ml, respectively) than in ewes receiving GnRH once per h (77.8 +/- 11.4 microgram/pituitary, 5.2 +/- 1.3 ng/ml). The pituitary content of LH was highest in the OVX ewes (260.2 +/- 57.4 micrograms/pituitary) and lowest in the nonpulsed HPD ewes (61.7 +/- 51.2 micrograms/pituitary). The number of GnRH receptors was similar in all groups, and was not correlated with any other variable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct effects of estradiol-17 beta on the number of gonadotropin-releasing hormone receptors in the ovine pituitary

Concentrations of pituitary receptors for gonadotropin-releasing hormone (GnRH) are affected by GnRH and gonadal steroids. To test the hypothesis that estradiol-17 beta (E2) directly affects the number of GnRH receptors in the pituitary, independent of GnRH secretion, ovariectomized ewes with hypothalamic-pituitary disconnections (HPD) were given 25 micrograms (i.m.) of E2 (HPD + E2, n = 5) or oil (HPD + OIL, n = 5). Ovariectomized control ewes, with intact hypothalamic-pituitary axes (INT), also received either E2 or oil (INT + E2, n = 6; INT + OIL, n = 6). Blood samples were taken hourly for analysis of serum concentrations of luteinizing hormone (LH) from 4 h prior to until 16 h after treatment. Pituitaries were collected 16 h after treatment for analysis of GnRH receptors. Treatment with E2 increased concentrations of LH in serum beginning 12.7 +/- 0.6 h after injection in INT ewes but not in HPD ewes. Compared to INT + OIL ewes, E2 treatment increased (p less than 0.001) the number of GnRH receptors by 2.5-fold in INT ewes and by 2.0-fold in HPD ewes. These results suggest that although GnRH is necessary for secretion of gonadotropins, E2 alone can directly increase the number of GnRH receptors in the pituitary.     

Early pituitary follicle stimulating hormone response to gonadotrophin releasing hormone in ewes

The object of our experiments was to characterize the response of plasma follicle stimulating hormone (FSH) within minutes of an i.v. injection of high or low doses of gonadotrophin releasing hormone (GnRH), especially in relation to contemporary changes in luteinizing hormone (LH) concentrations. In the deep anoestrous period (June), three intact ewes and two ovariectomized ewes were injected with 1 mug synthetic GnRH followed 2 h later by a second identical injection. A week later, the same regimen was repeated with the same sheep but with 50 mug GnRH after an interval of 5 h 20 min. Blood samples were collected every 15 sec for 15 min after each injection (early release), then at longer intervals (main release) till the next treatment, followed by sampling for a further 6-h period after the second treatment. FSH was released as soon as the second minute after GnRH injection in all ewes. The mean pituitary FSH response, during this early release, in intact and ovariectomized ewes was similar after either 1 or 50 mug GnRH. However, the main release was less pronounced in the ovariectomized sheep and was not stimulated after the second treatment in all sheep. Three other ewes were injected with 40 mug GnRH and sampled every 15 sec for seven, 6-min periods during the period of release to compare FSH and LH secretion. The profiles reflected a similarity in sensitivity and responsiveness to GnRH, especially soon after GnRH injection. Increases in both hormones were formed by several grouped associated spikes. It is suggested that a readily releasable pool of FSH exists in the ewe. There are probably differences in the mechanisms of synthesis and/or release between pituitary FSH and LH.     

Role of estradiol in inducing an ovulatory-like surge of luteinizing hormone in sheep

Two experiments were performed to examine the effect of estradiol on secretion of luteinizing hormone (LH) and on the number of receptors for gonadotropin-releasing hormone (GnRH) after down regulation of GnRH receptors in ovariectomized ewes. In the first experiment, ovariectomized ewes were administered one of four treatments: Group 1) infusion of GnRH i.v. for 40 h; Group 2) injection of 100 micrograms estradiol i.m.; Group 3) infusion of GnRH i.v. for 16 h followed immediately by an injection of 100 micrograms estradiol i.m.; and Group 4) infusion of GnRH i.v. for 40 h plus injection of 100 micrograms estradiol i.m. after the 16th h of infusion. Ewes in Groups 1, 3 and 4 responded to the infusion of GnRH with an immediate increase in serum concentrations of LH, with maximum values occurring between 2 and 4 h after the start of infusion; serum concentrations of LH then began to decline and were approaching the pretreatment baseline within 16 h. Administration of estradiol resulted in a surge of LH regardless of whether the pituitary had been desensitized by infusion of GnRH or not. In all cases the magnitude of the surge was similar to that induced by the initial infusion of GnRH. In Groups 2 and 3 the surge of LH began at 12.3 +/- 0.1 and 11.9 +/- 0.1 h after administration of estradiol. In contrast, the ewes in Group 4 had a surge of LH beginning 3.7 +/- 0.1 h after administration of estradiol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of long-term administration of a superactive agonistic and an antagonistic GnRH analog on the pituitary-gonad system

A powerful GnRH antagonist: [Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10]-GnRH (MI-1544) and a superactive GnRH agonist: [D-Phe6,desGly10]-GnRH(1-9)EA (OVURELIN) were used in long-term administration to compare their effects on the inhibition of ovulation, LH and progesterone (P) release, LH content of pituitaries as well as on the recovery period. Both analogs showed 100% inhibitory effects on ovulation in very low doses during the daily treatment for 21 days. The antagonist prevented LH release already after the first injection, decreased the serum P level to 40%, and increased the LH content of the pituitary up to 180%, inhibiting only the release but not the synthesis of LH. The agonist showed marked LH-releasing effects on the first day of the treatment, which were reduced to 12% on the 7th day. Serum P concentration was dropped to 68% by the end of the treatment. No change was found in the LH content of pituitaries in the group treated with the agonist. Ovaries showed polifollicular pictures in the antagonist-treated group, and persistent corpora lutea were seen in the ovaries from the agonist-treated group. Regular estrous cycles returned 13-15 days after ceasing the treatment with the antagonist and 3-5 days after ceasing the treatment with the agonist. No edema-inducing effect was observed after the injections of the antagonist in doses of 100 times higher than the single antiovulatory dose.     

Phorbol ester-induced LH release in pituitary gonadotrophs: effects of antagonists of calmodulin and GnRH.

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of Ca(2+)- and phospholipid-dependent protein kinase (C kinase), stimulates luteinizing hormone (LH) release from rat pituitary cells. The actions of TPA upon LH release were compared with those of the GnRH superagonist [D-Ala6] des-Gly10-GnRH N-ethylamide (GnRHa) in cultured pituitary cells. LH release was stimulated by 0.1 nM TPA and the maximum response at 10 nM TPA was 50% of the LH response to GnRHa. The ED50 values for TPA and GnRHa were 1.2 and 0.037 nM, respectively, and the maximum stimulatory effects of TPA and GnRHa on LH release were not additive. GnRHa-stimulated LH release was decreased by calmodulin (CaM) antagonists including pimozide, trifluoperazine, W5 and W7, being most effectively reduced (by 70%) by 10 microM pimozide. In contrast to their inhibition of GnRH action, these antagonists enhanced TPA-stimulated LH release, so that 10 microM pimozide and W7 doubled the maximum LH response. The potent GnRH antagonist [Ac-D-p-Cl-Phe1.2, D-Trp3, D-Lys6, D-Ala10]GnRH, which completely inhibited GnRHa-stimulated LH release with ID50 of 6.8 nM, also reduced maximum TPA-stimulated LH release by about 50%. These results suggest that both Ca2+/CaM and C kinase pathways are involved in the LH release mechanism, and indicate that C kinase plays a major role in the action of GnRH upon gonadotropin secretion. The synergism between CaM antagonists and TPA suggests that blockade of CaM-mediated processes leads to enhanced activation of the C kinase pathway, possibly by removal of an inhibitory influence. Furthermore, the partial inhibition of TPA-stimulated LH release by a GnRH antagonist suggests that the pathway(s), specifically connected with LH release in the diverse effects of C kinase, might be locked by the continuous receptor inactivation by antagonist and indicates the complicated pathways which diverge from the receptor and converge into specific cellular response.     

Pituitary gonadotropin-releasing hormone receptor content in rats with polycystic ovaries

A single injection of estradiol valerate (EV) induces, after a lag period of 4-6 wk, a chronic anovulatory polycystic ovarian (PCO) condition in adult rats. This condition is associated with a selective compromise of luteinizing hormone (LH) release and/or synthesis reflected in low basal serum LH concentrations, decreased pituitary content of LH, and decreased gonadotropin-releasing hormone (GnRH)-stimulated LH secretion. The present study was undertaken to determine to what extent the aberrant LH release in rats with PCO could be related to alterations in pituitary content of GnRH receptors. Pituitary GnRH-receptor content was assessed by the evaluation of saturation binding of a GnRH analog, [125I]-D-Ala6-des-Gly10-GnRH, to pituitary membrane preparations. The receptor content of pituitaries from rats with PCO was compared to that obtained from intact animals at estrus and diestrus. Receptor levels in ovariectomized normal rats and rats with PCO were also assessed. The pituitary GnRH receptor content in PCO rats was similar to that observed in normal controls at estrus and was significantly lower than that for rats at diestrus. Although a twofold increase in pituitary GnRH receptor content was observed at 28 days following the castration of control rats, GnRH receptor content in the pituitaries of PCO rats, at 28 days following ovariectomy, remained unchanged. Although, castration-induced elevations in mean serum LH and follicle-stimulating hormone (FSH) concentrations were observed in both the PCO and control animals, the rise in both gonadotropins was significantly attenuated in the PCO-castrates when compared to the ovariectomized controls. Since GnRH is a major factor in the regulation of pituitary GnRH receptor content, these findings suggest that hypothalamic GnRH release is impaired in rats with PCO and that this impairment is independent of any influences from the polycystic ovaries.     

Inhibition of pituitary-gonadal function in male rats by a potent GnRH antagonist

The inhibitory effects of the potent GnRH antagonist, [Ac-D-pCl-Phe1,2,D-Trp3,D-Arg6,DAla10]GnRH (GnRHant) upon pituitary-gonadal function were investigated in normal and castrated male rats. The antagonist was given a single subcutaneous (s.c.) injections of 1-500 micrograms to 40-60 day old rats which were killed from 1 to 7 days later for assay of pituitary GnRH receptors, gonadal receptors for LH, FSH, and PRL, and plasma gonadotropins, PRL, and testosterone (T). In intact rats treated with low doses of the antagonist (1, 5 or 10 micrograms), available pituitary GnRH receptors were reduced to 40, 30 and 15% of the control values, respectively, with no change in serum gonadotropin, PRL, and T levels. Higher antagonist doses (50, 100 or 500 micrograms) caused more marked decreases in free GnRH receptors, to 8, 4 and 1% of the control values, which were accompanied by dose-related reductions in serum LH and T concentrations. After the highest dose of GnRHant (500 micrograms), serum LH and T levels were completely suppressed at 24 h, and serum levels of the GnRH antagonist were detectable for up to 3 days by radioimmunoassay. The 500 micrograms dose of GnRHant also reduced testicular LH and PRL receptors by 30 and 50% respectively, at 24 h; by 72 h, PRL receptors and LH receptors were still slightly below control values. In castrate rats, treatment with GnRHant reduced pituitary GnRH receptors by 90% and suppressed serum LH and FSH to hypophysectomized levels. Such responses in castrate animals were observed following injection of relatively low doses of GnRHant (100 micrograms), after which the antagonist was detectable in serum for up to 24 h. These data suggest that extensive or complete occupancy of the pituitary receptor population by a GnRH antagonist is necessary to reduce plasma gonadotropin and testosterone levels in intact rats. In castrate animals, partial occupancy of the available GnRH receptor sites appears to be sufficient to inhibit the elevated rate of gonadotropin secretion.     

Pituitary receptors for gonadotropin-releasing hormone in relation to changes in pituitary and plasma gonadotropins in ovariectomized hypothalamo/pituitary-disconnected ewes. II. A marked rise in receptor number during the acute feedback effects of estradiol

Ovariectomized (OVX), hypothalamo/pituitary-disconnected (HPD) ewes were used to ascertain the short-term effects of estradiol on the number of gonadotropin-releasing hormone (GnRH) receptors in the pituitary gland. The time course of the study was such that measurements were made during the period of short-term negative feedback and positive feedback. Groups of 4 OVX-HPD ewes were given 250-ng pulses of GnRH each hour and an i.m. injection of oil (Group 1) or 50 micrograms estradiol benzoate in oil (Groups 2-4). Blood samples were collected from each ewe prior to treatment with estradiol or oil and again immediately before slaughter. Groups 2, 3, and 4 were killed 6, 16, and 20 h, respectively, after administration of estradiol. Amplitudes of luteinizing hormone (LH) pulses and average plasma concentrations of LH were reduced 6 h after estradiol treatment. Sixteen and 20 h after injection, the average plasma LH levels were elevated, but pulse amplitudes were similar to preinjection values. The number of GnRH receptors was significantly (p less than 0.01) increased within 6 h of estrogen treatment and further increased 16 and 20 h after treatment. Pituitary content of LH was similar in all groups. These data indicate that the number of GnRH receptors in the pituitary gland of ewes can be acutely influenced by a direct effect of estradiol. However, the magnitude and direction of the change in receptors number does not account for the changes in pituitary responsiveness to GnRH, suggesting estradiol also modifies post-receptor mechanisms that influence secretion of LH.     

Identification of a gonadotropin-releasing hormone-like factor in the rabbit fetal placenta

The biological and immunological gonadotropin-releasing hormone (GnRH)-like activities in rabbit fetal placentas collected at Day 18 of gestation were investigated. Both crude and partially purified acid extracts of placental tissue were tested. A similarly prepared liver extract served as a control. Immunological GnRH-like activity, determined through a GnRH radioimmunoassay was 1.3-2.0 pg/mg protein for the crude placental extract, 7.1-9.2 pg/mg protein for the partially purified placental extract and was nondetectable for liver extract. Both the crude and partially purified placental extracts increased (P less than 0.01) luteinizing hormone (LH) release by dispersed rabbit pituitary cells, whereas the liver extract had no effect. The (Ac-D-p-Cl-Phe1,2, D-Trp3, D-Arg6, D-Ala10)-GnRH antagonist was used to determine whether the biological GnRH-like activity in the placental extract was mediated through GnRH receptors. All three doses of antagonist (10, 100 and 1000 ng) suppressed the biological GnRH-like activity in the placental extracts. Molecular sieve chromatography of the partially purified placental extract showed that the immunoreactive GnRH-like factor eluted in the same fractions as the GnRH standard. These data indicate that the rabbit fetal placenta has both immunological and biological GnRH-like activity.     

Separate neural systems mediate the steroid-dependent and steroid-independent suppression of tonic luteinizing hormone secretion in the anestrous ewe

In the ewe, two types of seasonal fluctuations in secretion of tonic luteinizing hormone (LH) have been described: a steroid-dependent change whereby estradiol gains the capacity to suppress LH pulse frequency in anestrus, and a steroid-independent decrease in pulse frequency in ovariectomized animals during anestrus. We have proposed that the former reflects activation, in anestrus, of estradiol-sensitive catecholaminergic neurons that inhibit gonadotropin-releasing hormone (GnRH). Three results reported here support this hypothesis: dopaminergic (pimozide) and alpha-adrenergic (phenoxybenzamine) antagonists increased LH in intact anestrous ewes without altering pituitary responses to GnRH; other dopaminergic (fluphenazine) and alpha-adrenergic (dibenamine) antagonists also increased LH in anestrus; agonists for dopaminergic (apomorphine) and alpha-adrenergic (clonidine) receptors suppressed LH secretion in both seasons, suggesting that the appropriate receptors are present in breeding-season ewes. In contrast, catecholamines do not appear to mediate the steroid-independent suppression of pulse frequency; neither pimozide nor phenoxybenzamine increased LH pulse frequency in ovariectomized ewes during anestrus. When antagonists for 6 other neurotransmitter receptors (muscarinic and nicotinic cholinergic, GABAnergic, serotonergic, opioid, and beta-adrenergic) were tested in anestrus, only cyproheptadine, the serotonergic antagonist, increased pulse frequency in ovariectomized ewes. Cyproheptadine had no effect on frequency during the breeding season. On the basis of these results, we propose that the steroid-dependent and -independent actions of anestrous photoperiod occur via catecholaminergic and serotonergic neurons, respectively.     

Early pituitary LH response to injections of GnRH in ewes

In the deep anoestrous period (June), five intact ewes and five ovariectomized ewes received 50 ug synthetic gonadotrophin-releasing hormone (GnRH). In the mid-breeding season (October), the GnRH administrations were repeated in five intact and four ovariectomized ewes; the former were in the luteal phase of the cycle. Blood samples were collected every 30 sec for 15 min, then at 15-min intervals. Release of luteinizing hormone (LH) occurred as soon as the second minute after injection in all ewes. This early response was earlier and more abrupt in the ovariectomized ewes than in the intact animals. In a second experiment three intact ewes that were in deep anoestrus received 50 ug GnRH followed 5 h 20 min later by a second identical injection. Another three intact ewes in deep anoestrus received two injections of 1 ug GnRH. Blood samples were taken every 15 sec for 15 min, then every 20 min until the next injection, and for a further 5 h after the second injection. This regimen was repeated in mid-breeding season during the luteal phase. There was again a very early release of LH; the magnitude of response was similar after the first injection of either 50 ug or 1 ug GnRH to intact ewes either in the breeding season or during deep anoestrus. However, a greater early release of LH was obtained at the lower dose only after the second injection of GnRH. Apart from this exception, the similar early release of LH occurred in spite of different amounts of LH released thereafter in response to the two doses of GnRH. It is suggested that the early response to GnRH consists of LH stored in a "readily releasable" pool in the pituitary, whereas the main release of LH may be a result of increased synthesis and/or release of a more stable pool.     

Effects of the intensity of the suckling stimulus and ovarian steroids on pituitary gonadotropin-releasing hormone receptors during lactation

These studies examined whether the decrease in pituitary responsiveness to gonadotropin-releasing hormone (GnRH) observed during lactation in the rat results from a change in pituitary GnRH receptors. GnRH binding capacity was determined by saturation analysis using D-Ala6 as both ligand and tracer. During the estrous cycle, the number of GnRH binding sites increased from 199 +/- 38 fmol/mg protein on estrus to 527 +/- 31 fmol/mg protein on the morning of proestrus, whereas there was no change in receptor affinity (Ka, 6-10 X 10(9) M-1), During lactation, females nursing 8 pups on Days 5 or 10 postpartum had 50% fewer GnRH receptors (109-120 fmol/mg protein) than observed during estrus or diestrus 1 (199-242 fmol/mg protein) although receptor affinity was similar among all the groups. No deficits in pituitary GnRH receptors were observed in females nursing 2 pups on Day 10 postpartum. Removal of the 8-pup suckling stimulus for 24 or 48 h resulted in a dramatic increase in GnRH receptor capacity by 24 h from 120 +/- 16 to 355 +/- 39 fmol/mg protein. The rise in GnRH receptors after pup removal was accompanied by an increase in serum luteinizing hormone (LH) and estradiol concentrations. To assess the role of ovarian steroids in determining GnRH receptor capacity during lactation, females were ovariectomized (OVX) on Day 2 postpartum. Suckling of a large litter (8 pups) completely blocked the postcastration rise in serum LH and in pituitary GnRH receptors on Day 10 postpartum (OVX+ 8, 77 +/- 12 fmol/mg protein; OVX+ 0, 442 +/- 38 fmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian steroid hormone involvement in endogenous opioid modulation of LH secretion in mature ewes during the breeding and non-breeding seasons

The opioid antagonist WIN-44441-3 (WIN-3, Sterling-Winthrop) caused significant increases in LH secretion in ovariectomized ewes treated with progesterone but not in ovariectomized animals treated with oestradiol-17 beta. In the non-breeding season, plasma LH concentrations in ovariectomized ewes without steroid therapy, given oestradiol-17 beta or oestradiol-17 beta and progesterone together were not affected by treatment with WIN-3 on Day 6 after ovariectomy (there was a significant increase in LH as a result of WIN-3 treatment 13 days after ovariectomy in sheep given no steroid therapy). However, WIN-3 treatment of ovariectomized sheep given progesterone resulted in a significant increase in plasma LH. WIN-3 was ineffective when given to intact ewes treated with progesterone during the non-breeding season. With ovariectomized sheep during the breeding season there was again no response to WIN-3 at 6 days after ovariectomy in sheep given oestradiol-17 beta, but significant LH elevations in animals given no steroid, those given progesterone and those given progesterone + oestradiol-17 beta. The lack of an LH response to WIN-3 in ovariectomized sheep treated with oestradiol-17 beta did not result from a reduced pituitary response to GnRH since such animals responded normally to exogenous GnRH treatment. Overall, these results are consistent with the idea that, irrespective of the time of year, progesterone exerts negative feedback upon LH release at least in part through an opioidergic mechanism, whereas oestradiol-17 beta exerts negative feedback through steps unlikely to involve opioids. Progesterone can override the effect of oestradiol-17 beta during the breeding season only. Further, there appears to be a steroid-independent opioid involvement in LH suppression, operating at both times of year.     

Comparison of the effect of several gonadotropin releasing hormone antagonists on luteinizing hormone secretion, receptor binding and ovulation

The biological activity of three gonadotropin releasing hormone (GnRH) antagonists was evaluated in the following assays: suppression of GnRH-mediated luteinizing hormone (LH) secretion by cultured pituitary cells, suppression of the spontaneous LH release by ovariectomized rats, blockade of ovulation in regularly cycling females and inhibition of binding of a potent radiolabeled agonist to rat pituitary membrane homogenates. The peptides were: [Ac-delta 3Pro1,4FDPhe2, DTrp3,6]-GnRH (Antagonist 1); [Ac-delta 3Pro1,4FDPhe2,DNAL(2)3,6]-GnRH (Antagonist 2); and [Ac-DNAL(2)2,4FDPhe2,DTrp3,DArg6]-GnRH (Antagonist 3). All three antagonists exhibited similarly high potency in suppressing LH secretion in vitro, while Antagonist 1 was the most active peptide in the radioreceptor assay. When administered by gavage, Antagonist 3 exhibited the highest potency to inhibit LH secretion in gonadectomized rats and to block ovulation. Comparison of the oral versus the subcutaneous mode of administration of these analogs indicates that less than 1% is absorbed after gavage. However, these data demonstrate that the intragastric administration of GnRH antagonists can lower gonadotropin secretion and interfere with reproductive functions.     

Independent actions of gonadotropin releasing hormone upon cyclic GMP production and luteinizing hormone release

Gonadotropin releasing hormone (GnRH) and its potent analog [D-Ser(tBu)6]des-Gly10-GnRH N-ethylamide elevate pituitary cyclic GMP levels while stimulating gonadotropin release in cultured pituitary cells. Addition of mycophenolic acid to pituitary cell cultures decreased basal and GnRH-induced cGMP production to undetectable levels, but did not reduce basal or GnRH-stimulated luteinizing hormone (LH) release. Elevation of endogenous cGMP levels by sodium nitroprusside, or addition of cGMP or its potent derivatives, was also without effect on basal or GnRH-stimulated LH release. These findings demonstrate that the elevation of intracellular cGMP during GnRH action does not mediate the release of LH by pituitary cells.     

Alterations in gonadotropin-releasing hormone-dependent gonadotropin secretion in rats with polycystic ovaries.

A single injection of estradiol valerate (EV) to adult female rats induces a persistent anovulatory polycystic ovarian (PCO) condition. During the 8-20-wk interval following EV treatment, this condition is associated with a selective compromise of LH release, decreased pituitary content of LH, and decreased GnRH-stimulated LH secretion. A marked increase in mean plasma concentrations of LH and enhanced LH response to GnRH occur after 20 wk post-EV treatment. Despite this apparent improvement, the PCO condition remains unchanged. The present study was undertaken to elucidate the underlying causes for these spontaneous improvements in LH parameters. We reasoned that these changes may be the result of alterations in 1) pituitary GnRH receptor levels; or 2) the mode of LH secretion, i.e. GnRH-dependent versus GnRH-independent; or 3) post-GnRH receptor events. Hence, we assessed pituitary GnRH receptor concentration as well as the pituitary content of LH and FSH in rats with PCO of 9 wk and 22 wk duration. To examine the possibility of a change in the mode of LH secretion, we examined the effects of in vivo suppression of LH secretion by treatment with a GnRH antagonist [N-Ac-D-Nal1, D-Phe2,3, D-Arg6, Phe7, D-Ala10]-GnRH (GnRH-ANTAG) in the same groups of animals. Mean pituitary weights were greater in the 9-wk-PCO than in the 22-wk-PCO animals. The pituitary concentration of GnRH receptors (on either a weight or milligram pituitary-membrane protein basis) was similar in the 9-wk- and 22-wk-PCO animals. Pituitary LH and FSH contents, however, were significantly higher (5-fold and 2-fold, respectively) in 22-wk-PCO rats compared to the 9-wk-PCO animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation and characterization of photoreactive derivatives of GnRH. Persistent activation of function by photoaffinity labeling

A photoreactive derivative of the highly potent gonadotropin releasing hormone (GnRH) agonist, D-Lys6-GnRH(1-9)-ethylamide, was prepared by selective modification of the epsilon-amino group with 2-nitro-4-azidophenyl sulfenyl chloride (2,4-NAPS C1). The modified peptide [D-Lys(NAPS)]6-GnRH-(1-9)-ethylamide was found to be a full agonist of LH release from rat pituitary cells with a relative potency 23 compared to GnRH. Covalent attachment of the photoreactive analog to rat pituitary cells resulted in prolonged activation of LH secretion which could not be inhibited by a potent GnRH antagonist. Persistent stimulation of pituitary gonadotrophs caused by covalently bound hormone led to desensitization of the LH releasing mechanism.     

GnRH-induced gonadotrophin secretion in ovariectomized Booroola ewes with hypothalamic-pituitary disconnection

To test whether the F gene-specific differences in the plasma concentrations of FSH and LH are due to differences in the pituitary responsiveness to exogenous GnRH, ovariectomized Booroola ewes with hypothalamic-pituitary disconnection (HPD-ovx) were treated with GnRH (250 ng i.v.) once every 2 h for up to 5 weeks. In Exp. 1, jugular venous blood was collected once weekly from 13 FF and 14 ++ HPD-ovx ewes for 6 weeks before GnRH treatment and every 2nd, 3rd or 6th day for 5 weeks during treatment. In Exp. 2, jugular venous blood was collected from another 8 FF and 7 ++ HPD-ovx ewes at 5- or 10-min intervals over 4 GnRH pulses (250 ng i.v. once every 2 h) on 3 separate occasions after the animals had been subjected to the GnRH pulse regimen for approximately 7 days beforehand. Also in Exp. 2, the animals were extensively sampled around a larger (10 micrograms) i.v. injection of GnRH and the pituitary FSH and LH contents assessed after the animals had been re-exposed to the once every 2 h GnRH (250 ng i.v.) pulse regimen for several days following the larger GnRH bolus. In Exp. 3 the distributions of mean plasma concentrations of FSH and LH in individual GnRH-treated HPD-ovx ewes were compared with those in ovariectomized and ovary-intact FF and ++ ewes. During the 6 weeks before GnRH treatment (Exp. 1), the plasma concentrations of FSH (approximately 1 ng/ml) and LH (less than or equal to 0.8 ng/ml) were not different between the genotypes. After GnRH treatment both the mean FSH and LH concentrations increased significantly (P less than 0.01) above basal values after 2 days with F gene-specific differences being noted for FSH but not LH (FSH; FF greater than ++; P less than 0.05). Thereafter, the mean FSH but not LH concentrations increased at a faster rate in FF than in ++ ewes with the overall mean FSH concentrations between the genotypes being significantly different (P less than 0.05). In Exp. 2 considerable between-animal variation in the pulsatile pattern of FSH but not LH concentrations was seen in ewes of both genotypes during GnRH treatment. The overall mean FSH concentrations were higher in FF than in ++ ewes (P less than 0.05) and the mean FSH response to each GnRH pulse was significantly higher in FF than in ++ ewes (P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)     

Luteinizing hormone release in intact and castrate rams is altered with immunoneutralization of endogenous estradiol

Changes in the dynamics of luteinizing hormone (LH) release in the adult ram following immunoneutralization of endogenous estradiol were investigated. Castrate rams were actively immunized against estradiol-6-bovine serum albumin for 7 months and then their patterns of episodic LH release and LH response to multiple injections of gonadotropin-releasing hormone (GnRH, two 5-micrograms doses given iv 2 h apart) were assessed (April). In comparison with control rams immunized against rabbit gamma globulin, estradiol-immunized rams (antibody titre approximately 1:5000) exhibited more frequent LH releases (11.7 +/- 0.3 vs. 9.3 +/- 0.8 pulses/8 h, P less than 0.05) and a greater LH response to the first GnRH injection (peak delta value 190 +/- 8 vs. 130 +/- 25 ng/mL, P less than 0.01). Estradiol antiserum collected from the castrate rams was used in the passive immunization of intact rams (antibody titre approximately 1:200) for 1 month (beginning mid-July). Although episodic LH release was always similar for control and immunized rams, testosterone levels in the latter group increased approximately 150%. In contrast with the castrate ram response, GnRH treatment (two 5-micrograms doses given iv 80 min apart) produced a "self-priming" effect on LH release in the intact rams, an effect that was dampened with estradiol immunoneutralization. Consequently, peak 2:peak 1 ratios for delta value and 80-min mean incremental increase were much smaller (P less than 0.01) for the immunized rams (approximately 2:1 vs. 4:1 for the control rams).(ABSTRACT TRUNCATED AT 250 WORDS)