Theodore J. Brown

Inhibition of sexual behavior in female guinea pigs by a progestin receptor antagonist

The new steroidal progestin receptor antagonist, RU 38486, was used to determine if progesterone-facilitation of sexual behavior in female guinea pigs requires interaction of the hormone with neural progestin receptors. Five milligrams but not 0.5 mg RU 38486 inhibits the expression of sexual behavior in ovariectomized, estrogen-primed guinea pigs treated with 0.1 mg progesterone. This inhibition can be overcome by administration of a large dose of progesterone, suggesting that the drug-effect is specific to the progestin receptor system. RU 38486 binds, in vitro, to progestin receptors and decreases the availability of hypothalamic progestin receptors in estrogen-treated guinea pigs. These studies provide strong evidence that progesterone interaction with intracellular neural progestin receptors mediates the facilitation of sexual behavior by progesterone in female guinea pigs.

Progesterone decreases the concentration of hypothalamic and anterior pituitary estrogen receptors in ovariectomized rats

In a study of cellular mechanisms of progesterones antiestrogenic action on behavior and neuroendocrine responses, we investigated the influence of progesterone on the concentration of estrogen receptors in the hypothalamus-preoptic area (HP), anterior pituitary gland (AP), and uterus of chronically estradiol-treated ovariectomized rats. Ovariectomized (OVX) rats were implanted s.c. with 15 mm silastic capsules of estradiol. One week later, they were injected with progesterone or oil vehicle and killed 6 h or 24 h later. Confirming previous reports, progesterone caused a decrease in the concentration of uterine cytosol and nuclear estrogen receptors at both times. Less consistent results were obtained in HP and AP; a decrease in the concentration of HP cytosol estrogen receptors was detected at 6 h, as was a small decrease in the concentration of HP nuclear estrogen receptors at 24 h. More consistent results were seen when a low priming dose of estradiol was used. Although progesterone was without effect on the concentration of nuclear estrogen receptors in HP and AP at 6 h, cytosol receptor levels were depressed by 25% in HP and 14% in AP. At 24 h after progesterone injection, nuclear estrogen receptor levels were decreased in all tissues, while cytosol estrogen receptor levels remained depressed. A study of the time course of progesterones suppression of cytosol estrogen receptor concentration revealed that the effect is transient, occurring by 6 h after progesterone injection, but returning to baseline by 48 h after injection. Scatchard analysis confirmed that the decreased concentration of cytosol binding in HP was due to a decrease in the concentration of binding sites. As with nearly all of progesterones neuroendocrine effects, the suppression of estrogen receptor levels requires estrogen priming. HP and AP cytosol from progesterone-treated rats did not seem to contain an estrogen receptor-regulatory factor as do uterine cell nuclei; loss of binding sites at 37 degrees C was no faster in cytosol from progesterone-treated rats. These results demonstrate that, under some conditions, progesterone decreases HP and AP estrogen receptor concentrations. Unlike progesterones action in the uterus, the primary effect in the brain and pituitary gland seems to be on the cytosol receptor.

Dopamine-β-hydroxylase Inhibitors Modulate the Concentration of Functional Estrogen Receptors in Female Rat Hypothalamus and Pituitary Gland

In order to study further the regulation of steroid hormone receptors in the brain and anterior pituitary gland of ovariectomized rats by catecholamines, the influence of two dopamine-β-hydroxylase in

Abbreviation of the period of sexual behavior in female guinea pigs by the progesterone antagonist RU 486

In previous studies we have tested the hypothesis that the termination of the period of sexual behavior in female guinea pigs results from the loss of progestin receptors from hypothalamic cell nuclei. We have shown that hormonal manipulations that delay heat termination also delay loss of hypothalamic nuclear progestin receptors. In order to determine if accelerated nuclear receptor loss results in abbreviation of the period of sexual behavior, we tested the effect of 17 beta-hydroxy-11 beta-(4-dimethylaminophenyl)-17 alpha-(1-propyl)-estra-4,9-diene-3-one (RU 486), a progesterone antagonist, on heat termination. Ovariectomized guinea pigs were treated with estradiol benzoate. Forty hours later, they received progesterone followed 4 h later by injection of RU 486 or vehicle. RU 486 injected 4 h after progesterone caused heat abbreviation. We have found that RU 486 administration to estradiol-treated guinea pigs causes accumulation of progestin receptors in cell nuclear extract. Because this accumulation can be detected only when assay conditions are used that promote exchange of RU 486 progestin receptor complexes (15 degrees C incubation rather than 0 degree C); our routine assay conditions (at 0 degree C) can be used to measure primarily receptors that are occupied by progesterone. In order to confirm that RU 486 decreased progesterone-occupied nuclear progestin receptor levels when injected 4 h after progesterone, animals treated as in the behavioral experiment were killed 6 or 10 h after progesterone injection (2 or 6 h after RU 486), and nuclear progestin receptor levels were measured. RU 486 treatment resulted in lowered nuclear concentrations of hypothalamic progestin receptors at both times. These results support our hypothesis that the termination of the period of sexual receptivity in female guinea pigs is the result of loss of progestin receptors from hypothalamic cell nuclei.

Some Catecholamine Inhibitors Do Not Cause Accumulation of Nuclear Estrogen Receptors in Rat Hypothalamus and Anterior Pituitary Gland

We have recently shown that the dopamine-beta-hydroxylase inhibitor, U-14,624, decreases the concentration of cytosol estrogen receptors in the mediobasal hypothalamus (MBH) and anterior pituitary gland (AP) in ovariectomized rats, but that it also causes cell nuclear accumulation of estrogen receptors. We tried to determine if this is the mechanism by which other catecholaminergic inhibitors decrease the concentration of cytosol estrogen receptors in either the MBH or AP. The previously reported decrease in the concentration of cytosol estrogen receptors in AP by the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine was confirmed. Also, the decrease in the concentration of cytosol estrogen receptors in MBH after treatment with the dopamine-beta-hydroxylase inhibitors, diethyldithiocarbamate and FLA 63 was demonstrated. In no case was an increase in the concentration of nuclear estrogen receptor accumulation detected after treatment with the drugs. Results of assays of norepinephrine and dopamine levels in MBH after the various treatments suggest that, at the dosage used, U-14,624 has a greater effect on norepinephrine and dopamine levels that the other dopamine-beta-hydroxylase inhibitors. The results of these experiments suggest that inhibitors of dopamine-beta-hydroxylase and tyrosine hydroxylase cause decreases in the concentration of cytosol estrogen receptors in either the MBH or AP that are not referable to increased cell nuclear accumulation of estrogen receptors.

Loss of hypothalamic nuclear-bound progestin receptors: factors involved and the relationship to heat termination in female guinea pigs.

The hypothesis that the termination of sexual receptivity (heat) in female guinea pigs results from loss of progestin receptors from hypothalamic cell nuclei was tested. First, we attempted to find an optimal dose of progesterone that would result in a prolonged period of sexual receptivity. Ovariectomized guinea pigs were implanted with 10% estradiol capsules. Forty hours later, each received one of several sized progesterone capsules before being tested hourly for lordosis. Surprisingly, none of the progesterone doses resulted in delayed heat termination. In order to determine whether elevated levels of estradiol and progesterone maintain elevated levels of nuclear progestin receptors despite the lack of effect on heat duration, animals were treated as described above except that only one size progesterone capsule (3.0 cm) or an empty capsule was implanted. Despite elevated serum progesterone concentrations, nuclear progestin receptor levels decreased gradually and approached control levels at about the same time as heat termination had occurred in similarly-treated animals. Cytosol progestin receptor levels decreased following progesterone treatment and remained lowered at all times measured. In order to further investigate the relationship between blood progesterone concentrations and retention of nuclear progestin receptors we decreased blood progesterone concentrations by removing progesterone capsules 2 h following insertion. Nuclear progestin receptor levels declined gradually concurrent with a decline in serum progesterone levels in animals exposed to progesterone capsules for only 2 h. In animals exposed to capsules continuously, nuclear progestin receptor levels again decreased but at a slightly slower rate. In order to test the effect of progesterone capsule removal on female sexual behavior, ovariectomized guinea pigs were treated as described and tested hourly for lordosis. Fewer animals in the group exposed to progesterone capsules for 2 h became sexually receptive as compared to animals continuously-exposed to progesterone capsules. Of those animals that did respond, heat termination had occurred by the same time that nuclear progestin receptor levels had returned to control levels in similarly-treated animals. These experiments support the hypothesis that heat termination results from the loss of progestin receptors from hypothalamic cell nuclei. In addition, they demonstrate that circulating progesterone levels play a role in regulating nuclear progestin receptor retention.

Supplemental progesterone delays heat termination and the loss of progestin receptors from hypothalamic cell nuclei in female guinea pigs

Previous studies indicate that the retention of hypothalamic nuclear progestin receptors parallels the expression of sexual receptivity in ovariectomized, hormonally treated guinea pigs. In this study, the effect of a supplemental progesterone injection on heat termination and retention of nuclear progestin receptors was examined. Ovariectomized guinea pigs were injected with 50 micrograms progesterone 48 h after receiving 4 micrograms estradiol benzoate and were tested hourly for lordosis. 8 h after progesterone treatment, animals received either 500 micrograms progesterone or the oil vehicle. The supplemental progesterone injection delayed the termination of heat by more than 2 h compared to oil-injected animals. The effect of supplemental progesterone on the retention of nuclear progestin receptors was then determined. Animals were killed 8, 10, or 14 h after the initial injection of progesterone, and cytosol and nuclear progestin receptor concentrations in mediobasal hypothalamus-preoptic area were determined. Supplemental progesterone further increased the level of nuclear progestin receptors measured at 10 h, and at 14 h these levels were still elevated above baseline. In contrast, nuclear progestin receptor levels in animals which received only the initial progesterone injection had returned to baseline by 14 h, a time when similarly treated animals were no longer receptive. These results suggest that the mechanism by which heat termination occurs may involve loss of nuclear progestin receptors.

Failure of protein synthesis inhibition to block progesterone desensitization of lordosis in female rats

Progesterones desensitization effect on lordosis has been shown to correlate with a decreased concentration of hypothalamic progestin receptors after progesterone injection. In a recent study, one group of investigators found that the protein synthesis inhibitor anisomycin appeared to block progesterones desensitization effect. Despite decreased levels of cytoplasmic progestin receptors, progesterone + anisomycin-treated rats exhibited a high level of lordosis four hr after a second progesterone injection. Because this finding conflicts with a progestin receptor model of progesterones desensitization effect, we investigated it further. In the first experiments, ovariectomized rats were injected with estradiol benzoate followed 24 hr later by either progesterone or vehicle. Anisomycin injected 3 hr after progesterone, at a dose that causes inhibition of hypothalamic protein synthesis for at least 4 hr, was without effect on progesterone desensitization a day later. In other experiments silastic implants containing estradiol were inserted into ovariectomized rats. Forty-five hr later, rats received progesterone or vehicle, followed by injections of anisomycin or saline. Rats receiving anisomycin + progesterone were still highly receptive at 30 hr while saline + progesterone controls were not. Furthermore, the results were similar 4 hr after a second injection of progesterone at 30 hr. In a related experiment, we confirmed that anisomycin delayed dramatically termination of the period of sexual receptivity. In this laboratory anisomycin does not seem to block progesterones desensitization effect. However, with certain procedures anisomycin delays the termination of sexual receptivity. Thus it is important in investigations of the mechanism of progesterones desensitization effect that animals be tested prior to the second progesterone injection to determine if they are actually responding to the progesterone.

Hypothalamic nuclear progestin receptors and the duration of sexual receptivity in ovariectomized and ovariectomized-hysterectomized rats

In order to determine if the enhancement of sexual behavior in hysterectomized rats is associated with an increased level of hypothalamic nuclear progestin receptors, ovariectomized (OVX) and ovariectomized-hysterectomized (OVHX) rats were injected with 2 micrograms estradiol benzoate. Twenty-four hr later, animals were injected with 0.5 mg progesterone and were tested for sexual receptivity every 4 hr. Hysterectomy had an overall facilitatory effect on lordosis and increased the duration of the period of sexual receptivity by about 4 hr. In a second experiment, similarly-treated animals were killed 4, 8, or 12 hr after progesterone injection, and hypothalamic nuclear progestin receptor levels were measured. In contrast to what has been reported for guinea pigs, nuclear progestin receptor levels decreased to baseline 8-12 hr before the termination of sexual receptivity. Nuclear progestin receptor concentrations were higher in OVHX rats than in OVX rats at 4 hr after progesterone injection, and there was a trend toward higher receptor concentrations in OVHX rats at 8 hr. These results demonstrate that hysterectomy-induced facilitation of sexual receptivity is associated with an increased level of hypothalamic nuclear progestin receptors. Furthermore, they suggest a fundamental difference in the regulation of nuclear progestin receptor retention between rats and guinea pigs.