Cytoplasmic Progestin Receptors Research Articles

Progestin Receptor Subtypes in the Brain: The Known and the Unknown

Progesterone (P), the most biologically active progestin of ovarian origin, modulates numerous cellular functions in the central nervous system to coordinate physiology and reproduction. The neurobiological activity of P is mediated not by a single form of the progestin receptor (PR), but by two neural isoforms of PRs, PR-A and PR-B. Classical model of P action assumes that these neural effects are primarily mediated via their intracellular PRs, acting as transcriptional regulators, in steroid-sensitive neurons, modulating genes and genomic networks. Evidence has emerged, however, that activation of neural PRs is much more diverse; four distinct classes of molecules, neurotransmitters, peptide growth factors, cyclic nucleotides, and neurosteroids have been shown to activate the PRs via cross-talk and pathway convergence. In addition, rapid signaling events associated with membrane receptors and/or subpopulations of cytoplasmic PRs, via activation of protein kinase cascades, regulate PR gene expression in the cytoplasm independent of PR nuclear action. The increasing in vitro and in vivo evidence of differential transcriptional activities and coregulator interactions between PR-A and PR-B predict that these isoforms could have distinct roles in mediating additional and/or alternate signaling pathways within steroid-sensitive neurons. In this minireview, we evaluate the available data and discuss the possible roles of the isoforms in the regulation of neurobiological processes.

Estrogenic feminization of the LH response to orchidectomy: Association with prolonged nuclear estradiol receptor retention and induction of cytoplasmic progestin receptors in brain and pituitary

A sex difference in the LH rise after gonadectomy is clearly observable in the rat. While male rats respond with an early (10–12 h) increase in LH after orchidectomy, a delayed response (2–3 days) is recorded after ovariectomy. In this study we tested the hypothesis that the delayed response to gonadectomy in the E 2-treated males is due to a more prolonged retention of E 2 (when compared with the corresponding male feedback signal, testosterone) within specific central nuclear receptor sites. Orchidectomized (ORDX) animals implanted with either empty or E 2-filled Silastic capsules were sacrificed at 0, 24. 48 and 72 h after ORDX or E 2-capsule removal. LH levels in ORDX rats rose several-fold by 24 h, whereas E 2treated, ORDX rats. showed no changes in peripheral LH levels until 72 h after E 2-capsule removal. At the time of E 2-capsule removal (0 h) large increases in nuclear estradiol receptor (NER) levels were seen in anterior pituitary, preoptic area, and hypothalamus (HYP). Twenty-four hours after E 2-capsule removal. NER levels were still high in the 3 areas, and by 48 h NER values had returned to control (ORDX) levels, with the exception of HYP where they were slightly but significantly elevated. The increase in NER, as well as the subsequent decline after E 2-capsule removal was paralleled by similar changes in cytosolic progestin receptor (CPR) levels in all three regions. Cytosolic testosterone levels were not changed by the E 2-treatment. The results indicate that the feminized response to orchidectomy observed in E 2-implanted males is related to a prolonged retention of the E 2-receptor in nuclear sites. Further, they indicate that E 2-treatment in males, as is the case in females, can induce a marked increase in progestin receptor levels within specific brain regions as well as in the pituitary. The reduction in NER and CPR levels to castrate values precedes the first detectable increase in peripheral LH levels. In conclusion, the pattern of LH rise after gonadectomy in the rat is dependent upon the steroidal milieu at the time of removal of the gonads.

Tamoxifen alone or in combination with estradiol-17β inhibits the growth and malignant transformation of hepatic hyperplastic nodules

Hepatic hyperplastic nodules (HHNs) induced by the ‘resistant hepatocyte method’ of Solt et al. were studied as an experimental prototype of oral contraceptive-related tumors. Cytoplasmic estrogen receptors were present in all HHNs harvested and their concentration was always less than that in normal liver. No specific cytoplasmic progestin receptors could be measured in the above tumor or liver specimens. The long-term administration of estradiol-17β ( 4.8–24.0 μg/day) resulted in the death of all but one of 20 animals prior to termination at 10 months. Tamoxifen ( 0.25–2.5 mg biweekly), which did not lead to excess mortality, decreased HHN grade (proportion of liver slice occupied by HHN) and inhibited malignant transformation. Combination therapy with single-dose estradiol-17β ( 4.8 μg/day) and various doses of tamoxifen ( 0.25–2.5 mg biweekly) in most cases reduced mortality, HHN grade and malignant transformation. Cytoplasmic progestin receptors were absent and estrogen receptors were either undetectable or present in low concentrations in hepatic tumors harvested at the time of termination. Our results indicated that HHNs are hormone-dependent and that malignant transformation can be inhibited by tamoxifen alone or in combination with estradiol-17β.

Tamoxifen alone or in combination with estradiol-17β inhibits the growth and malignant transformation of hepatic hyperplastic nodules

Hepatic hyperplastic nodules (HHNs) induced by the ‘resistant hepatocyte method’ of Solt and Farber were studied as an experimental prototype of oral contraceptive-related tumors. Cytoplasmic estrogen receptors were present in all HHNs harvested and their concentration was always less than that in normal liver. No specific cytoplasmic progestin receptors could be measured in the above tumor or liver specimens. The long-term administration of estradiol-17β (4.8–24.0 μg/day) resulted in the death of all but one of 20 animals prior to termination at 10 months. Tamoxifen (0.25–2.5 mg biweekly) which did not lead to excess mortality, decreased HHN grade (proportion of liver slice occupied by HHN) and inhihited malignant transformation. Combination therapy with single-dose estradiol-17β (4.8 μg/day) and various doses of tamoxifen (0.25–2.5 mg biweekly) in most cases reduced mortality, HHN grade and malignant transformation. Cytoplasmic progestin receptors were absent and estrogen receptors were either undetectable or present in low concentration in hepatic tumors harvested at the time of termination. Our results indicate that HHNs are hormone-dependent and that malignant transformation can be inhibited by tamoxifen alone or in combination with estradiol-17β.

1-(o-Chlorophenyl)-1 (p-chlorophenyl)2,2,2-trichloroethane induces functional progestin receptors in the rat hypothalamus and pituitary gland.

A recent report suggests that estrogen induction of hypothalamic progestin receptors is not a prerequisite for the facilitation by progesterone of female sexual behavior in rats. Progesterone was found to facilitate sexual behavior despite no apparent induction of progestin receptors by 1-(o-chlorophenyl) 1-(p-chlorophenyl)2,2,2-trichloroethane (o,p'-DDT). To investigate these findings further, ovariectomized rats were treated with daily injections of o,p'-DDT for 3 days, followed by progesterone on day 4. Daily injections of 400 mg o,p'-DDT/kg resulted in activation of lordosis in 50% of the animals; injections of 200 mg/kg were ineffective. Cytoplasmic and nuclear progestin receptor levels in the mediobasal hypothalamus-preoptic area and pituitary gland were then determined in similarly treated animals. Because competition assays revealed that o,p'DDT interacts with progestin-binding sites in vitro, residual o,p'-DDT was removed from the cytosol fraction before assay. o,p'-DDT treatment increased the level of cytoplasmic progestin receptors by 43% compared with that in oil-injected controls. Progesterone administration to DDT-treated rats resulted in a 137% increase in the level of nuclear progestin receptors relative to levels observed in animals receiving no progesterone. These findings contradict a previous report using o,p'-DDT and support the hypothesis that estrogen-induced progestin receptors are required for the facilitation of sexual receptivity by progesterone.

Development of steroid-receptor systems in guinea pig brain. II. Cytoplasmic progestin receptors

The development of the cytoplasmic progestin receptor (CPR) system in male and female guinea pigs was examined to determine if insensitivity to the lordosis promoting activity of estrogen-progestin treatment in adult males and neonatal guinea pigs is correlated with alterations in this receptor system. Gonadectomized neonatal (4–6 days old) and adult (50–65 days old) guinea pigs of both sexes were injected with estradiol benzoate (EB) and killed 40 h later. CPRs were measured in the hypothalamus (HYPO), preoptic area (POA) and cortex (CORT). Neonatal brain contained both high ( K d ⋍ 0.1 nM ) and low ( K d > 10 nM) affinity binders for [ 3H]R5020 similar to those found in adult brain. In the absence of EB-priming the concentration of CPR in HYPO, POA and CORT was lower in neonatal animals than adults. In both males and females, neither a low (1.6 μg) nor high (10 μg) dose of EB resulted in a concentration of estrogen inducible CPR in neonatal HYPO greater than that found in adult HYPO after 1.6 μg EB. Even after treatment with 50 μg EB the concentration of CPR in neonatal female HYPO was not different from adult female HYPO after 1.6 μg EB. In neonatal POA, 10 μg EB resulted in a concentration of CPR greater than that found in adult POA after 1.6 μg EB. Finally, there was a sex difference in the concentration of estrogen inducible CPR in HYPO (female > male) but not POA of adult guinea pigs. No sex difference in CPR concentration was found in any neonatal brain area.

Development of steroid-receptor systems in guinea pig brain. III. Nuclear progestin receptors

Nuclear progestin receptors (NPR) were measured, with an exchange assay, in neonatal (4–6 days old) and adult (50–60 days old) guinea pigs of both sexes in order to further examine deficits in the neonatal hypothalamic progestin receptor system. Both neonatal and adult hypothalamus (HYPO) and cortex (CORT) contained a KCl extractable high affinity ( K d ⋍ 0.1 nM ) binder for R5020. In neonatal guinea pigs, given 10 μg estradiol benzoate (EB), NPR accumulation in HYPO and CORT peaked 2–4 h after a 0.5 mg injection of progesterone (P). There was no dose response of NPR to EB in neonatal HYPO, CORT or adult CORT. In adult HYPO, 10 μg EB-priming resulted in a higher concentration of NPR than 1.6 μg EB-priming. Neonatal HYPO (1.6 or 10 μg EB) did not differ in concentration of NPR from adult HYPO (1.6 μg EB). Neonatal CORT (1.6 or 10 μg EB) showed a lower NPR concentration tha adult CORT (1.6 μg EB). There was a sex difference (females > males) in NPR concentration in both neonatal and adult HYPO, but not CORT. These results are discussed with regard to deficits in cytoplasmic progestin receptors and behavioral insensitivity to estrogen-progestin treatment found in neonatal guinea pigs.

Administration of estradiol-17β in pulses to female guinea pigs: Self-priming effects of estrogen on brain tissues mediating lordosis

Lordosis behavior in ovariectomized guinea pigs is facilitated by the sequential action of estradiol-17β (E) and progesterone (P). The present study was designed to explore the possibility that administration of E in a pulsatile manner is more efficacious than a single injection of E with respect to lordosis facilitation in ovariectomized guinea pigs. The data indicate that pulse administration of unesterified E is more effective than a single large dose of E for the facilitation of lordosis behavior. Three injections of as little as 0.5 μg E at 0, 19 and 28 hr followed by 0.5 mg P at 39 hr was more effective (63.6% responding with lordosis) than a single injection of as much as 15 μg E at hr 0 followed by vehicle injections at 19 and 28 hr and 0.5 mg P at 39 hr (0% responding). We also demonstrated that the 19 hr E injection could be eliminated and that two injections of E (0.5 μg at hr 0 and 1.0 μg at hr 28 followed by 0.5 mg P at hr 39) was at least as effective (69.0% responding) as three split injections in facilitating lordosis. This behaviorally effective pulse administration of E also resulted in significant induction of cytoplasmic progestin receptors in hypothalamic tissue. Further experiments indicated that a 28 hr interval between E pulses was optimal in terms of percent animals desplaying lordosis. The data suggest that pulsatile E stimulation of brain tissues mediating lordosis is a highly effective mode of stimulation, and that an initial pulse of E sensitizes neural tissues to subsequent E administration.

Cytoplasmic progesterone receptors in the hypothalamus-preoptic area of the mouse: Effect of estrogen priming

A synthetic progestin, R5020, was used to identify cytoplasmic progestin receptors in the hypothalamuspreoptic area (HPOA) of ovariectomized mice. These high-affinity receptors exhibited an apparent dissociation constant of approx. 1 nM. The receptors were specific for progestins. [ 3H]R5020 binding was inhibited by more than 50% with a 50-fold excess of either radioinert R5020 or progesterone. 5α-Dihydroprogesterone inhibited binding to a lesser extent. 3α-Hydroxy-5α-pregnane 20-one and cortisol did not compete for [ 3H]R5020 binding. Administration of estradiol benzoate (10 μg), 48 h prior to death, resulted in a 54% increase in the HPOA progestin receptor concentration when compared to oil-injected controls. These data demonstrate that there are specific and saturable cytoplasmic progestin receptors in the mouse HPOA and that the concentration of these receptors is increased after estrogen treatment.

Progesterone-induced sequential inhibition of copulatory behavior in hysterectomized rats: Relationship to neural cytoplasmic progestin receptors

The inhibitory effects of progesterone on the copulatory behavior and on neural cytoplasmic progestin receptors were examined in ovariectomized (DV) and ovariectomized-hysterectomized (OH) rats. Ovariectomized and OH rats were given 2.0 μg of estradiol benzoate (EB) followed 24 hr later by 0.1, 0.5, 0.1 or 2.0 mg of progesterone (P) and were tested for lordosis 6 hr later (30 hr). Twenty four hr after the first P treatment, all animals received 0.5 mg of P and were tested again (54 hr). The initial doses of 1.0 and 2.0 mg of P significantly reduced lordosis quotients in response to the subsequent P treatment in OV animals but only the 2.0 mg dose of P effectively suppressed lordosis in OH animals. In order to determine whether the priming dose of EB influences the inhibitory effects of progesterone, OV and OH rats were injected with 1.0 μg of EB followed by 1.0 mg of P, or 6.0 μg of EB followed by 2.0 mg of P and were tested for receptivity under the same schedule. Treatment with 1.0 μg of EB permitted, and 6.0 μg of EB prevented sequential inhibition of sexual behavior by progesterone in both OV and OH rats. Hypothalamic cytoplasmic progestin ([ 3H]R5020) receptors were then measured 48 hr after EB injection in OV or OH rats given the same treatments with EB and P. Hypothalamic progestin receptors were significantly lower in OV animals given 2.0 μg of EB followed by 1.0 or 2.0 mg of P than oil-treated controls, but only the 2.0 mg dose of P significantly reduced receptors in OH rats. The 1.0 mg dose of P significantly depleted hypothalamic progestin receptors in OV and OH animals given just 1.0 μg EB. The result of these experiments indicate a close correlation between the progesterone-induced inhibition of copulatory behavior and the ability of the steroid to deplete its own receptors and suggest that the higher dose of progesterone required to inhibit receptivity in OH animals may be due to the enhanced induction of hypothalamic progestin receptors by EB following hysterectomy. The latter finding is consistant with the generally increased effectiveness of estradiol in OH rats.

Copulatory behavior and hypothalamic estrogen and progestin receptors in chronically insulin-deficient female rats

The effects of relatively short- or long-term diabetes on sexual receptivity and proceptivity and on hypothalamic estrogen and progestin receptors were examined in rats fed regular chow or high fat diet. Ovariectomized, streptozotocin-induced chronically insulin deficient and normal rats received sequential treatments with 2 μg estradiol benzoate (EB) and 1 mg progesterone (P) 10 days following the induction of diabetes and were tested for lordosis and soliciting behaviors. Nondiabetic rats fed either diet displayed significantly higher lordosis and solicitation behaviors than chow-fed diabetics, and fat-fed diabetic animals displayed behavior levels intermediate to those of nondiabetic and chow-fed rats. Ten days after the induction of diabetes, the levels of hypothalamic estrogen receptors of chow-fed diabetics were significantly lower than nondiabetics with the fat-fed diabetic group showing intermediate levels. However, 70 days after streptozotocin treatment diabetic rats had significantly lower estrogen receptors than nondiabetics regardless of the diet. Treatment of long-term (70 days) diabetic rats with 1–2 U of U-100 Lente insulin for 24 hr or 7 days was ineffective in restoring the hypothalamic estrogen receptor concentrations to those of nondiabetics. Three weeks following induction of diabetes, induction of cytoplasmic progestin receptors by EB treatment was significantly impaired in diabetic animals fed either chow or high fat diet. Although the reproductive dysfunctions present in short-term diabetic female rats may be due in part to chronic fuel deprivation, it appears that the long-term maintenance of cytosol estrogen receptor level is dependent on other actions of insulin. Furthermore, restoration of hypothalamic estrogen receptors in long-term diabetic rats may require either longer than 7 days of insulin therapy or another mode of treatment (e.g., sex hormones). The behavioral improvement of diabetic animals on HF diet could be accounted for by mechanisms other than enhancement of EB-induced cytoplasmic progestin receptors.

Effects of serotonin agonists on lordosis, myoclonus, and cytoplasmic progestin receptors in guinea pigs

Peripheral treatment with the serotonin releaser fenfluramine or the serotonin agonist quipazine abolished lordosis behavior in ovariectomized estradiol and progesterone-primed female guinea pigs. Quipazine was also effective when administered into a lateral cerebroventricle. The lowest dose of fenfluramine that induced myoclonus (10 mg/kg) was higher than the dose needed to inhibit lordosis (5 mg/kg). Therefore, it appears that myoclonus and lordosis are differentially sensitive to serotonin agonists. The effects of quipazine on lordosis were time dependent. Quipazine had no effect on lordosis when given prior to the onset of sexual receptivity. These data suggest that serotonin agonists might be effective only when progesterone has had sufficient time to induce sexual receptivity. Quipazine did not affect cytoplasmic progestin receptors in brain areas involved in steroid hormone effects on lordosis. This finding, and the finding that quipazine had no effect on lordosis when given prior to the onset of sexual receptivity, suggest increased serotonin transmission does not interfere with estrogen priming or sensitivity of hypothalamic cells to progesterone.

Long-term priming with a low dosage of estradiol benzoate or an antiestrogen (enclomiphene) increases nuclear progestin receptor levels in brain

Previous work from our laboratory showed that pretreatment with the antiestrogen enclomiphene (ENC) or with subthreshold doses of estradiol benzoate (EB) facilitates the action of subsequent EB plus progesterone injections on lordosis behavior in ovariectomized guinea pigs. The present study was performed in order to explore possible neurochemical correlates of this phenomenon. We found that facilitative behavioral actions of ENC or subthreshold EB pretreatment were correlated with significant increases in KCl extractable nuclear receptor for progestin (NRP) in hypothalamus-preoptic area-septum (HPS) samples (ENC pretreatment group= 54.8 ± 10.6, behaviorally subthresholdEB pretreatment group= 70.5 ± 11.2, vs contraol group given only oil vehicle17.6 ± 4.8fmol [ 3H]R5020 bound/mgDNA in the nuclear pellet. On the other hand, females given an injection of EB 11 h prior to progesterone but not given pretreatment with ENC or behaviorally subthreshold EB failed to show significant increases in HPS-NRP concentration compared to the uninjected control group (28.6 ± 3.3vs17.6 ± 4.88, respectively). These data suggest that: (1) ENC has some degree of estrogenic activity in brain; and (2) ENC or behaviorally subthreshold EB pretreatment facilitates subsequent EB plus progesterone action by increasing the induction of cytoplasmic progestin receptor and/or altering NRP levels in the HPS.

Behavioral and neuroendocrine effects of long-term progesterone treatment in the rat.

Progesterone and estradiol, alone or in combination, were administered to ovariectomized rats for 2 weeks. Progesterone alone had no effect on body weight, luteinizing hormone (LH) secretion, receptivity or brain cytoplasmic progestin receptors (CPR). Progesterone in combination with estradiol significantly attenuated estrogen suppression of weight gain and estrogen stimulation of receptivity, the LH afternoon surge and induction of CPR, but did not affect the negative feedback of estrogen on morning LH levels. The decrease in CPR after 2 weeks of progesterone is very similar in magnitude to the decrease observed following acute treatment, suggesting that, unlike neurotransmitter agonists and glucocorticoids, progesterone does not cause down regulation of its receptors in the brain following chronic treatment.

Cytoplasmic progestin receptors in myomal and myometrial tissues. Concentrations and hormonal dependency.

Cytoplasmic progestin receptors in human myometrium and myomata of uteri, both with and without tumors, were determined. The same concentration was found in all tissues when expressed in terms of tissue weight or DNA content. However, tumors had slightly more receptors when calculated per mg of soluble protein. This was due to the fact that myomata contained considerably less soluble protein than the normal myometrium. The receptors present were dependent upon the serum concentration of estradiol. Increasing values were accompanied by an increase in the number of receptors. At estradiol levels exceeding 500 pmoles/l their number reached a plateau and became independent of the hormone concentration. Progesterone had no influence except at very high levels where it depressed the number of receptors significantly. The investigation did not reveal any difference in the number of or the hormonal regulation of cytoplasmic progesterone receptors present in the various tissues. However, it showed that tumors contained considerably less soluble protein than normal myometrium.

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

Progesterone's 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 progesterone's 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 progesterone's 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 progesterone's desensitization effect. However, with certain procedures anisomycin delays the termination of sexual receptivity. Thus it is important in investigations of the mechanism of progesterone's desensitization effect that animals be tested prior to the second progesterone injection to determine if they are actually responding to the progesterone.

Alteration of sensitivity to progesterone facilitation of lordosis in guinea pigs by modulation of hypothalamic progestin receptors

In order to delineate further the role of the progestin receptor system in the hypothalamus-preoptic area in the regulation of lordisis in female guinea pigs, we injected estradiol benzoate and progesterone in various regimens that result in sensitivity to, desensitization to, or restoration of sensitivity to progesterone-facilitation of lordosis. We attempted to correlate the effectiveness of progesterone to facilitate lordosis with the effectiveness of progesterone to cause accumulation of nuclear progestin receptors measured by an in vitro [ 3H]R 5020 binding assay. A progesterone injection 40 h after an estradiol benzoate injection in ovariectomized guinea pigs resulted in lordosis in 83% of the animals, caused a 30% depletion of cytoplasmic progestin receptors and a 200% increase in the concentration of nuclear progestin receptors 4 h after injection compared with control animals. By 24 h after the injection, after the period of sexual receptivity had terminated, the concentration of nuclear progestin receptors was no longer elevated, and the concentration of cytoplasmic progestin receptors was decreased to a level 55% lower than control animals. This depression was due to a decrease in the concentration of binding sites, not to in vitro competition of the injected progesterone with the [ 3H]R 5020 because similar results were obtained with cytosol in which progesterone had been removed by gel filtration prior to incubation. These progesterone-injected guinea pigs did not respond to a second progesterone injection 24 h after the first (64 h), and the injection resulted in 70% fewer nuclear progestin receptors than in oil-injected controls. This decreased accumulation appears to be due to the decreased level of cytoplasmic progestin receptors brought about by the first progesterone injection. Estradiol benzoate injected concurrently with the first progesterone injection restored behavioral sensitivity to the second progesterone injection and caused 160% greater accumulation of nuclear progestin receptors in response to the second progesterone injection than control animals that had received only progesterone. The increased concentration of nuclear progestin receptors in the estradiol-injected animals appear to be due to increased availability of cytoplasmic progestin receptors. It seems that estradiol increases and progesterone decreases the concentration of cytoplasmic progesterone receptors in hypothalamus-preoptic area such that a subsequent injection of progesterone results in a high or low concentration of nuclear progestin receptors. The accumulation of sufficient nuclear progestin receptors in response to the progesterone injection may be a requirement in the process by which progesterone facilitates lordosis in estradiol-primed rodents.

Progesterone in high doses may overcome progesterone's desensitization effect on lordosis by translocation of hypothalamic progestin receptors

The cellular mechanism by which progesterone desensitizes the central nervous sytem to further facilitation of lordosis by progesterone was studied. In the first experiment guinea pigs were injected with doses of estradiol benzoate and progesterone that result in refractoriness to further stimulation of lordosis by moderate doses of progesterone. Confirming previous reports, a large dose of progesterone was effective in overcoming this desensitization, resulting in lordosis responses. A lower dose of progesterone was ineffective in this regard. In subsequent experiments, cytosol and nuclear progestin receptors were measured in the hypothalamus-preoptic area and cerebral cortex with an in vitro exchange assay using [ 3H]R 5020 as the ligand. A high, effective dose of progesterone resulted in 46–48% higher levels of nuclear progestin receptors assayed in the hypothalamus-preoptic area than did a lower, ineffective dose. These results are consistent with the idea that progesterone injection in estradiol-primed rodents causes a subsequent desensitization to itself by decreasing the concentration of cytoplasmic progestin receptors in hypothalamus-preoptic area. A subsequent progesterone injection then results in a lower level of nuclear progestin receptors. Large doses of progesterone may overcome this desensitization by causing accumulation of levels of nuclear progestin receptors sufficient to facilitate lordosis.

Progestin receptors in prolactin and growth hormone producing tumours in rats.

Progesterone and corticosterone have a similar effect on the production of growth hormone (GH) and prolactin (Prl) by pituitary tumour cells (GH3 cells) in culture. Previously we have shown that progesterone has a high affinity for the glucocorticoid receptors in these cells. Progesterone may therefore exert its effects through binding to the glucocorticoid receptor. The aim of the present study was to investigate if the GH3 tumour cells and an oestrogen induced pituitary tumours, which also produce GH and Prl, possess specific receptors for progesterone. Both the GH3 tumours and the oestrogen induced pituitary tumour were in fact found to possess cytoplasmatic receptor molecules for progesterone by using the potent progestin R5020 as a marker. Isoelectric focusing revealed one binding component (pH 5.9), which was of protein nature. The binding was of high affinity (KD 2 X 10(-9) mol/l). In the oestrogen induced tumour, the maximal binding was 70 fmol/mg cytosol protein. In female rats with GH3 tumours the binding was 55 fmol/mg cytosol protein. Priming of the animals with 1 mg oestradiol-valerate increased the binding to 116 fmol/mg cytosol protein, whereas very little binding was found in GH3 tumours from rats castrated 7 days before sacrifice. The receptors in the oestrogen induced pituitary tumour and the GH3 tumours exhibited high affinity for R5020 and progesterone, whereas corticosterone had no significant affinity for the receptors. Using exchange assay, it was demonstrated that the cytoplasmic progestin receptors could be translocated to the nucleus after administration of progesterone to the animals. Thus, the presence of specific progesterone receptors, different from the glucocorticoid receptors, strongly indicates that athe effects of progesterone on GH and Prl production are mediated through the progesterone receptors.

Effects of nafoxidine on the luteinizing hormone surge: temporal distribution of estrogen receptors and induction of cytoplasmic progestin receptors in the hypothalamus-preoptic area, pituitary, and uterus of the immature rat.

The antiestrogen nafoxidine 1-(2-[P-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy]pyrrolidine hydrochloride; 2 mg/rat), blocked LH surges induced by Silastic implants containing estradiol (E2) in oil (150 micrograms/ml) in the immature female rat and had no stimulatory effect on gonadotropin secretion by itself. Furthermore, the administration of progesterone (P) to rats primed for 24 h with nafoxidine alone or nafoxidine plus E2 did not lead to premature and enhanced LH surges (facilitation) as it does in E2-primed animals. Pituitary LH content was depleted by 30-50% after E2-induced or P-facilitated LH surges, but was unchanged after the administration of nafoxidine or nafoxidine plus E2. To investigate the cellular mechanisms involved, levels of total cytoplasmic and nuclear estrogen receptors and cytoplasmic progestin receptors in the hypothalamus-preoptic area (HPOA), pituitary, and uterus were measured by [3H]E2 or [3H]R5020 (3H-labeled 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) exchange assays at various times after the administration of E2 and /or nafoxidine. The ability of nafoxidine to bind in vitro to cytoplasmic or nuclear estrogen receptors was, respectively, 4% and 2% (HPOA), 5% and 6% (pituitary), and 1% and 2% (uterus) relative to E2 (100%). After the injection of nafoxidine or the insertion of E2 implants, cytoplasmic estrogen receptors were depleted with a similar time course and remained depressed for at least 48 h in both the HPOA and pituitary. In the uterus, the antiestrogen prevented the replenishment of cytoplasmic estrogen receptors observed in response to E2. Nuclear estrogen receptor levels in the HPOA and pituitary peaked 1 h after E2 and subsequently declined to a plateau from 24-48 h (at 2-4 times the levels found in untreated rats). After nafoxidine, accumulation of these receptors in the nucleus was more gradual and prolonged. Absolute levels of estrogen receptors translocated to the nucleus by E2 or nafoxidine were comparable. E2 treatment led to a substantial induction of cytoplasmic progestin receptors (approximately 2-fold in the HPOA, approximately 4-fold in the pituitary, and approximately 7-fold in the uterus after 48 h), and this process was considerably inhibited by nafoxidine. These results support the notions that antagonism by nafoxidine of estrogen action may be due to a defective association of the antiestrogen-receptor complex with nuclear sites, ad progestin receptor induction may be a prerequisite for the facilitation of gonadotropin surges by P.