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.     

Characteristics of pulsatile luteinizing hormone secretion in middle-aged ovariectomized rats

Experiments were performed to characterize the pulsatile patterns of circulating luteinizing hormone (LH) in the middle-aged ovariectomized (OVX) rat. Frequent blood samples were taken from OVX rats at 6, 7-8, and 9-10 mo of age, and LH was measured by radioimmunoassay. Rats had been OVX either 2 wk (STO) or 10-20 wk (LTO) previously. Mean LH levels were significantly lower with increasing age, reflecting effects on both pulse amplitude and pulse frequency. Mean LH levels were higher in LTO than STO groups, reflecting primarily an increase in pulse amplitude, but there was also a small, significant decrease in pulse frequency with increased time following OVX. In a second experiment, a random selection of the rats in the STO groups was tested again 10 wk after OVX. A significantly higher number of 9- to 10-mo-old rats had pulsatile patterns at 10 wk than at 2 wk following OVX. Furthermore, mean plasma LH concentrations were higher in all three groups. We conclude that decreases in several parameters of LH secretion are seen in middle-aged OVX rats, at the time when irregularities are first seen in the estrous cycle in the intact rat.     

Effect of adrenergic blocking drugs on secretion of luteinizing hormone in the ovariectomized ewe.

The effects of the adrenergic blocking drugs phenoxybenzamine, phentolamine, and pimozide on basal luteinizing hormone (LH) levels and on estrogen-induced LH release were tested in ovariectomized ewes. Phentolamine was given at a dose of 10 mg/kg; phenoxybenzamine was given at a dose of 8 mg/kg; and pimozide was given at a dose of 800 mcg/kg. Estradiol benzoate (EB) was given at a dose of 50 mcg/animal. Sera were assayed for LH levels by double-antibody radioimmunoassay. Phenoxybenzamine given as a single dose significantly reduced basal LH levels. Given at extremenly high doses, phenoxybenzamine was unable to consistently block estrogen-induced LH release. Pimozide significantly reduced basal LH levels in the ewes and blocked or greatly reduced estrogen-induced LH release in 9 of 10 treated animals. Reduced basal LH levels were seen with phentolamine injection but were of short duration of action. It is concluded that normal function of noradrenergic neurons is required for maintenance of normal basal LH release but unnecessary for estrogen-induced LH release. Dopaminergic neurons appear to facilitate basal and estrogen-induced LH release.     

Inhibition of cell-associated herpes simplex virus type 2 glycoproteins by delta 9-tetrahydrocannabinol

This study was conducted to define the effect of micromolar concentrations of delta 9-tetrahydrocannabinol (delta 9-THC) on the biosynthesis and expression of herpes simplex virus type 2 (HSV2)-specified glycoproteins. Dose-related reductions in all species of virus glycoproteins were recorded by one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of [14C]glucosamine-labeled infected Vero cells treated with 10(-7) to 10(-5) M delta 9-THC. A drug dose-related depletion of the mature HSV2 major envelope glycoprotein complex (119-kDa average molecular weight), accompanied by accumulation of immature unglycosylated species, was demonstrated by two-dimensional SDS-PAGE in concert with Western immunoblotting or autoradiography. Light and electron microscopy immunoperoxidase staining revealed that delta 9-THC effected depletion of 119-kDa determinants from the infected cell surface. This depletion occurred concomitantly with accumulation of 119-kDa components at the perinucleus. However, the expression of 119-kDa glycoproteins on the virion envelope was not affected. These results indicate that delta 9-THC inhibits the synthesis, maturation, and cellular transport of HSV2-specified glycoproteins. Decreased expression of virus glycoproteins on the infected cell surface may affect host immune responsiveness to HSV2.     

Delta-9-tetrahydrocannabinol attenuates luteinizing hormone release induced by electrochemical stimulation of the medial preoptic area.

Despite diverse pharmacological actions, drugs commonly used for blocking ovulation in the rat have not been observed to exert differential effects on the LH response to preoptic stimulation, thus suggesting blocking action above the final hypothalamic GnRH pathway. To determine if ovulatory blockade by delta-9-tetrahydrocannabinol (THC) is consistent with that scheme, LH surges evoked by preoptic stimulation were contrasted with those elicited during blockade by atropine (ATR), a classic ovulation-blocking agent with which other drugs have been compared. THC (10 mg/kg) or ATR (350 mg/kg) treatment before the proestrous critical period uniformly blocked LH release and ovulation in sham-stimulated rats. Preoptic stimulation evoked LH surges after both drug treatments (p less than 0.001), peak levels increasing with the intensity of stimulation (p less than 0.05). However, both maximum LH concentration (p less than 0.05) and total integrated LH release (p less than 0.01) were lower in THC-blocked rats. Inspection of the oviducts revealed no difference in the incidence of ovulation or the number of ova discharged. The reduced LH response during THC blockade was not attributable to variation in the extent or locus of histologically determined stimulation sites. These results distinguish THC from ATR and, by extension, other blocking drugs that do not overtly affect the LH response to preoptic stimulation. Thus, ovulatory blockade by THC may involve a different mechanism, which likely includes inhibitory action within the preoptic-to-tuberal GnRH pathway.     

Intracerebroventricular administration of ghrelin rapidly suppresses pulsatile luteinizing hormone secretion in ovariectomized rats.

Ghrelin, an endogenous growth hormone (GH) secretagogue, is shown to increase food intake, which action is similar to that of orexin, also a hypothalamic peptide. Since orexin suppresses pulsatile LH secretion in ovariectomized (OVX) rats, the present study was undertaken to investigate whether ghrelin also suppresses LH secretion. Effects of intracerebroventricularly injected ghrelin (0.1 nmol/0.3 microl) were examined in OVX rats treated with a small dose of 17beta-estradiol (E(2)). After ghrelin injection, pulsatile LH secretions which were ongoing in these E(2)-treated OVX rats were significantly suppressed for about 1 h, whereas GH secretion increased, peaking at 30 min. The main parameter suppressed by ghrelin was the pulse frequency, not the pulse amplitude, suggesting the hypothalamus as the site of ghrelin action. This study provides evidence that ghrelin acts not only in the control of food intake but also in the control of LH secretion.     

Inhibition of testicular luteinizing hormone receptor level by treatment with a potent luteinizing hormone-releasing hormone agonist of human chorionic gonadotropin.

Two proteins larger than proinsulin (estimated molecular weight 11,000 and 10,000 daltons), were observed when labeled rat islet proteins were electrophoresed on sodium dodecyl sulfate gels. The proteins are synthesized before proinsulin, turn over more rapidly than proinsulin, their synthesis is stimulated by glucose, and they are specifically bound by anti-insulin antibodies.     

Effects of intraventricular injection of delta receptor antagonist ICI 154, 129 on the secretion of luteinizing hormone and prolactin in the proestrous rat.

In order to ascertain the role of delta receptors in the control of gonadotropin secretion, a preferential delta receptor antagonist ICI 143,129 was microinjected into the third ventricle through chronically implanted cannulae and the effects on the serum concentration of luteinizing hormone (LH) and prolactin (PRL) were determined in female rats in proestrus. When the injection was given at 1030 h, ICI 154,129 (50 micrograms) exerted no significant effects on either LH or PRL. However, in the rat given a microinjection of ICI 154,129 at 1300 h, an afternoon rise in LH occurred in advance and was of greater magnitude, with the peak time more than 1 h earlier and the peak amplitude approximately 100% greater than that in the control rat, respectively. The injection also suppressed the PRL rise during the plateau phase. The results indicate that delta receptors are involved in the mediation of the inhibitory influence of endogenous opioids on the surge of LH in proestrus, and that delta receptors mediate the facilitatory influence of opioids on the PRL surge during the plateau phase.     

Orchidectomy unleashes pulsatile luteinizing hormone secretion in the rat

We charted the development of pulsatile luteinizing hormone (LH) secretion as a function of the time elapsed after removal of the testes. On seven occasions between the moment of castration and 80 days afterwards, we obtained consecutive blood samples at frequent (2.5- to 5-min) intervals from cannulated male rats. Orchidectomy increased both the amplitude and frequency of LH release within 1 day after surgery. Amplitude: From 19 h through 80 days postcastration, peak LH levels rose steadily, and LH pulses grew progressively more pronounced in nadir-to-peak amplitude. Frequency: Our findings offer new evidence establishing an increase in LH pulse frequency from less than 1 per h to 2-3 per h within 1 day after orchidectomy. Once deprived of testicular influences, the frequency of pulsatile LH discharges remained static through 80 days. The sudden onset (less than 1 day after castration) and temporal uniformity of high-frequency LH pulses demonstrate that LH release is governed by an intrinsic, 20- to 30-min neural periodicity in castrate rats. Most important, these findings imply that the testes mask or modulate the expression of an intrinsic, 20- to 30-min neural generator directing the periodic discharge of LH in the intact male rat.     

Suppressive effect of delta 9-tetrahydrocannabinol in vitro on phagocytosis by murine macrophages

Incubation of normal mouse peritoneal cells consisting of over 90% phagocytizing macrophages with delta 9-tetrahydrocannabinol (THC) resulted in a inhibition of phagocytic function. The THC in a dose-related manner suppressed the percentage of macrophages per culture which ingested yeast and the average number of yeast particles ingested by the phagocytizing macrophages. The vehicle used to suspend the THC in vitro, i.e., DMSO, had no detectable effect on macrophage function. Suppression of phagocytosis with no effects on viability or cell number occurred with doses of 10 micrograms or less THC per milliliter culture medium. Measurable suppression also occurred after 24- to 48-hr treatment of the macrophages with the THC. This compound had little if any detectable effect on phagocytosis when added directly to the cultures shortly before testing for phagocytosis. Further studies concerning the effects of THC on macrophage function appear warranted.     

Opioid regulation of luteinizing hormone secretion in the male rat

Current evidence suggests that endogenous opioid peptides (EOPs) tonically inhibit secretion of luteinizing hormone (LH) by modulating the release of gonadotropin-releasing hormone (GnRH). Because of their apparent inhibitory actions, EOPs have been assumed to alter both pulse frequency and amplitude of LH in the rat; and it has been hypothesized that EOP pathways mediate the negative feedback actions of steroids on secretion of GnRH. In order to better delineate the role of EOPs in regulating secretion of LH in the male rat, we assessed the effects of a sustained blockade of opiate receptors by naloxone on pulsatile LH release in four groups: intact male rats, acutely castrated male rats implanted for 20 h with a 30-mm capsule made from Silastic and filled with testosterone, acutely castrated male rats implanted for 20 h with an osmotic minipump dispensing 10 mg morphine/24 h, and male rats castrated approximately 20 h before treatment with naloxone. We hypothesized that if EOPs tonically inhibited pulsatile LH secretion, a sustained blockade of opiate receptors should result in a sustained increase in LH release. We found that treatment with naloxone resulted in an immediate but transient increase in LH levels in intact males compared to controls treated with saline. Even though mean levels of LH increased from 0.15 +/- 0.04 to a high of 0.57 +/- 0.14 ng/ml, no significant difference was observed between the groups in either frequency or amplitude of LH pulses across the 4-h treatment period. The transient increase in LH did result in a 3- to 4-fold elevation in levels of plasma testosterone over baseline. This increase in testosterone appeared to correspond with the waning of the LH response to naloxone. The LH response to naloxone was eliminated in acutely castrated rats implanted with testosterone. Likewise, acutely castrated rats treated with morphine also failed to respond to naloxone with an increase in LH. These observations suggest that chronic morphine and chronic testosterone may act through the same mechanism to modulate secretion of LH, or once shut down, the GnRH pulse-generating system becomes refractory to stimulation by naloxone. In acutely castrated male rats, levels of LH were significantly increased above baseline throughout the period of naloxone treatment; this finding supports the hypothesis that the acute elevation in testosterone acting through mechanism independent of opioid is responsible for the transient response of LH to naloxone in the intact rat.     

Influence of season and estradiol on secretion of luteinizing hormone in ovariectomized cows

The hypotheses that secretion of luteinizing hormone (LH) varies with season and that estradiol may modulate the seasonal fluctuation in secretion of LH in cows were investigated. Seven mature cows were ovariectomized approximately 30 days before initiation of the experiment. Three of the ovariectomized cows (OVX-E2) were administered a subcutaneous estradiol implant that provided low circulating levels of 17 beta-estradiol. The remaining 4 cows (OVX) were not implanted. From December 21, 1982, to September 20, 1984, blood samples were collected sequentially (at 10-min intervals for 6 h) at each summer and winter solstice, and each spring and autumn equinox. In addition, from March 17, 1983, to March 17, 1984, sequential samples were collected midway between each solstice and equinox. Concentration of LH was measured in all samples, and concentration of estradiol was measured in pools of samples. An annual cycle in mean serum concentration of LH and amplitude of LH pulses was detected in both groups of cows. The seasonal pattern did not differ in the two treatment groups. Serum concentration of LH and amplitude of LH pulses were highest around the spring equinox, decreased gradually to the autumn equinox, and then increased and peaked again during the following spring equinox. Frequency of LH pulses and concentration of estradiol in serum did not vary with season. Circulating concentrations of LH and amplitude of pulses tended to be higher in OVX-E2 than OVX cows throughout the experimental period. Frequency of pulses of LH was lower in OVX-E2 than OVX cows throughout the experiment. Concentrations of estradiol were higher in the implanted cows.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prolactin does not mediate the suppressive effect of the suckling stimulus on luteinizing hormone secretion in ovariectomized lactating rats

The plasma LH concentration in ovariectomized lactating rats is low for 14 days postpartum, while the prolactin concentration is high during this period. We examined the effect of the inhibition of increased prolactin secretion with bromocriptine (CB-154) on the LH secretion in lactating rats ovariectomized on day 2 (day 0 = day of parturition). Blood samples were collected through an indwelling atrial cannula every day. LH levels were kept low until day 9 in lactating rats injected daily with CB-154 (0.6 mg/day, s.c.). The duration of the period during which LH secretion was suppressed was shorter in lactating rats treated with CB-154 than in saline-injected controls. The replacement with ovine prolactin by means of a mini-osmotic pump (0.3 mg/day, s.c.) in CB-154-treated lactating rats restored the duration of LH suppression. In rats deprived of their pups on day 2, the LH concentration rose immediately after removal of the pups and the LH level was not significantly different between rats treated with CB-154, ovine prolactin and saline, indicating that neither the CB-154 treatment nor the high level of prolactin alone has any effect on LH secretion in rats deprived of their pups. The present results clearly demonstrate that prolactin does not mediate the suppressing effect of the suckling stimulus on LH secretion in early lactation and support our theory that the suckling stimulus controls the LH and prolactin secretion independently at the hypothalamic level.     

Effect of acute intravenous administration of delta-9-tetrahydrocannabinol on the episodic secretion of immunoassayable growth hormone in the rat.

Blood samples from unrestrained, unanesthetized, male rats (300-350 g) were obtained every 15 min. for 9 consecutive hrs. (1000-1900 h). Each rat received, intravenously, a vehicle injection (controls) or a 2.0 mg/kg dose of delta-9-tetrahydrocannabinol (THC) at 1300 h to determine the effect of this drug on the spontaneous episodic secretion of plasma immunoassayable rat growth hormone (rGH). Acute administration of THC suppressed the secretion of rGH, as is evident from mean plasma level (p less than .01), peak height (p less than .02), and integrated peak amplitude (p less than .02) analyses. Episodic secretion was inhibited in all animals (n = 7) receiving THC. Although further investigation is needed to define clearly the physiological mechanisms involved in this response, these data indicate that THC can inhibit the hypothalamic-pituitary control of normal episodic growth hormone secretion in the rat.     

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

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

Teratologic evaluation of synthetic delta 9-tetrahydrocannabinol in rabbits.

Synthetic delta 9-tetrahydrocannabinol (THC) was dissolved in undiluted propylene glycol and administered in daily subcutaneous doses of 15.0, 30.0 or 60.0 mg/kg to pregnant New Zealand white rabbits on days 7--19 of gestation. Maternal food consumption and weight gain were markedly reduced at all dose levels. Embryotoxicity and embryocidal effects were observed in the form of reduced litter weight and number of viable fetuses, respectively, in offspring from pregnant mothers treated with THC. However, on the basis of extensive external, visceral and skeletal examination of all fetuses it may be concluded that THC is not teratogenic in the New Zealand white strain rabbit following subcutaneous administration of doses as high as 60.0 mg/kg/day during the critical period of organogenesis (days 7--19 of gestation). On the other hand, an oral dose of thalidomide (200.0 mg/kg/day), the positive control used in this study, was both embryocidal and teratogenic.