Nuclear Estrogen Receptor Concentrations Research Articles

Correlation in isolated nuclei of template-engaged RNA polymerase II, ovalbumin mRNA synthesis, and estrogen receptor concentrations.

Template-engaged and total RNA polymerase II molecules were quantitated in isolated nuclei at various stages of estrogen withdrawal and secondary stimulation by using [3H]amanitin titration assays. Estrogen receptors, RNA transcriptional activity, and ovalbumin mRNA were also measured, and comparisons were made between these parameters to determine whether any significant correlations exist. In isolated nuclei, the highest positive correlations existed between template-engaged RNA polymerase II, ovalbumin mRNA synthesis in vitro, and estrogen receptor concentration. Interestingly, restimulation of estrogen-withdrawn chicks results in replenishment of RNA polymerase II activity to prewithdrawal levels within 4 h; however, the recovery of the numbers of template-engaged polymerase II molecules, ovalbumin gene transcription, and nuclear receptor binding lags behind. These findings suggest that the estrogen effect on RNA polymerase activity is more rapid than the increase in template-engaged RNA polymerase II and ovalbumin-specific gene transcription. The excellent correlation that exists between nuclear estrogen receptor concentrations, template-engaged RNA polymerase II, and ovalbumin gene transcription strongly supports the hypothesis that estrogen receptors mediate RNA polymerase II binding to sequences associated with preferential transcription of the ovalbumin gene.

Estrogen stimulates the transient association of calmodulin and myosin light chain kinase with the chicken liver nuclear matrix.

Previous work has demonstrated that estrogen administration to immature chickens results in a rapid but transient increase in nuclear estrogen receptor content, a large portion of which is associated with the nuclear matrix. The present studies were undertaken to determine whether estrogen produced a more generalized change in the protein composition of the nuclear matrix. High-resolution two-dimensional gel analysis of the matrix revealed a very complex protein pattern, but several major qualitative differences were observed after estrogen treatment. To simplify the number of proteins evaluated, we examined the effects of estrogen on a subset of matrix proteins, namely, calmodulin and its binding proteins. Calmodulin was measured by radioimmunoassay and the binding proteins were detected by interaction of 125I-calmodulin with matrix proteins distributed on one-dimensional polyacrylamide gels. Calmodulin and two specific Ca2+-dependent calmodulin-binding proteins were found to be associated with matrix preparations. The two binding proteins exhibited apparent Mr of 200,000 and 130,000. The Mr 130,000 protein was identified as myosin light chain kinase on the basis of enzymatic activity and immunoreactivity with a specific antibody to this enzyme. Estrogen treatment of immature chickens did not alter the hepatic content of calmodulin. However, the steroid did result in an enrichment of the proportion of calmodulin and its two binding proteins associated with the nuclear matrix within 4 h after injection. The time course of these changes paralleled those previously documented for estrogen receptor. Taken together, these data are compatible with a role for calmodulin and myosin light chain kinase in the response of chicken liver cells to steroid hormones.

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

In a study of cellular mechanisms of progesterone's 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 progesterone's suppression of cytosol estrogen receptor concentration revealed that the effect is transient, occuring 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 progesterone's 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 °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 progesterone's action in the uterus, the primary effect in the brain and pituitary gland seems to be on the cytosol receptor.

Effects of fasting on the distribution of cytoplasmic and nuclear oestrogen receptors in rat liver, uterus, pituitary and hypothalamus before and after exogenous oestrogen administration.

The accumulation of oestrogen receptors in the liver cell nuclei of intact female rats 45 min after administration of 100 micrograms 17 alpha-ethynyloestradiol-17 beta i.p., decreased progressively during a 72-h fast from 2550 +/- 860 to 257 +/- 67 fmol/mg DNA, a level not significantly different from that in uninjected animals. Cytoplasmic oestrogen receptor concentrations also decreased, but only to about 60% of the original level (from 84.1 +/- 27.5 to 50.3 +/- 2.09 fmol/mg protein during the fast). Similar differences were found when these parameters were examined in normally fed and 72-h-fasted ovariectomized rats. On the other hand these parameters were unaffected in uterus, pituitary and hypothalamus. Uterine cytoplasmic receptor concentrations remained at about 500 fmol/mg protein during the fasting period, those in the pituitary and hypothalamus at about 230 and 30 fmol/mg protein, respectively. Nor was in vivo translocation in these organs affected by fasting. Regardless of nutritional status, the nuclear oestrogen receptor concentrations in uterus rose from about 500 to 2000 fmol/mg DNA after ethynyloestradiol administration, those in the pituitary and hypothalamus from approximately 250 to 2000 and from 250 to 500 fmol/mg DNA respectively.

Estrogen receptor levels in the oviducts and endometria of cynomolgus macaques during the menstrual cycle.

We sampled oviducts and endometria of 27 cynomolgus macaques during the menstrual cycle and measured the concentration of nuclear and cytoplasmic estrogen receptors in these tissues by exchange assay. We assessed the stage of the cycle by correlating serum estradiol (E2) and progesterone (P), as measured by radioimmunoassay, with the morphological condition of the ovaries, oviducts and endometrium of each animal. We have previously identified a series of oviductal stages that reflected the orderly sequence of cytological changes in the oviduct during the cycle, and we normalized receptor measurements to these stages. The amounts of nuclear and cytoplasmic estrogen receptor in both the oviduct and the endometrium were approximately twofold greater in the follicular phase than in the luteal phase. In the follicular phase, elevated receptor levels were associated with oviductal proliferation and differentiation, as well as with endometrial proliferation. During the luteal phase, lowered levels were correlated with atrophy and dedifferentiation in the oviduct, but with hypertrophy and progestational development in the endometrium. When the luteal phase of one cycle ends and the follicular phase of the next begins, it is a decline in serum P, not a rise in serum E2, that precedes the elevation in estrogen receptor level and the onset of proliferation in the oviduct and endometrium. Proliferation of the reproductive tract and elevations in nuclear estrogen receptor levels during the early follicular phase can therefore be viewed as consequences of the release of the system from antagonism by P.

Hepatic nuclear estrogen receptor concentrations in the rat—influence of age, sex, gestation, lactation and estrous cycle

Nuclear estrogen receptor concentrations in rat liver were determined by exchange assay. The concentration of estrogen receptors in nuclei from vehicle-treated male and female rats show agedependent, but not sex-dependent variations in the course of life. Levels are highest during the perinatal period (~ 1500 binding sites/nucleus), whereafter they decrease towards the onset of puberty (~ 300 binding sites/nucleus) before rising again to reach the postpuberal maximum (~ 800 binding sites/nucleus). Pregnancy further raised receptor concentrations in the last week of gestation when they reach ~ 1200 binding sites/nucleus. Studies with ethynylestradiol-treated rats demonstrated that virtually no translocation can be detected before the onset of puberty; thereafter the number of translocated receptors increases dramatically reaching a maximum (9000 binding sites/nucleus) between day 80 and 87 of life. The extractability of the nuclear receptors with 0.4 M KC1 varies during the course of life. Extractability is very high (~90%) up to about day 12 of life, but then decreases markedly (to ~70% in vehicle-treated and to ~ 50% in ethynylestradiol-treated rats) before the onset of puberty.

Correlation between oxytocin receptor concentration and responsiveness to oxytocin in pregnant rat myometrium: effects of ovarian steroids.

Marked changes in the uterine binding of oxytocin (OT) occur in rats at the time of parturition or after treatment of ovariectomized rats with estrogen or progesterone. To ascertain that these binding sites represent the biological receptors for OT, we measured the uterine response to OT in various groups of rats in which specific OT binding was also determined. Intact pregnant rats and rats ovariectomized on day 20 of gestation and treated thereafter with oil, estradiol benzoate (5 micrograms/24 h), progesterone (5 mg/24 h), or estradiol and progesterone together had indwelling balloons inserted on day 20 for the recording of uterine response to either iv bolus injections or iv infusions of OT. The uterus was removed 24-48 h after balloon insertion, and OT binding to the particulate fraction as well as nuclear estrogen and cytosolic estrogen receptor concentrations were determined. An inverse correlation (r2 = 0.758) was found between the concentration of OT-binding sites and the threshold dose of OT, and a linear correlation was found between the concentration of binding sites and the uterine activity induced by OT infusion (r2 = 0.852). We conclude, therefore, that the high affinity (Kd, 1-2 nM) binding sites for OT represent the physiological receptors. The concentration of these sites increased progressively during estrogen treatment. Progesterone completely inhibited this estrogen-induced rise. After ovariectomy, there was a modest, but significant, increase in OT receptor concentration which also was prevented by progesterone. The increase in OT receptor concentration was correlated with the estrogen receptor concentration in intact pregnant and estrogen-treated ovariectomized animals, but not in the other groups of animals. The apparent affinity of the receptors for OT was not significantly affected by hormone treatment. We conclude that the concentration of receptors is a major factor controlling the uterine responsiveness to OT, and that the receptor concentrations are regulated by ovarian hormones in a manner related to estrogen receptor activation. In addition, estrogen appeared to enhance the coupling of OT receptor occupancy to the tissue response to OT.

Systemic and local regulation of oxytocin receptors in the rat uterus, and their functional significance.

Rats were made unilaterally pregnant by tying the right oviduct on the day after mating, to compare the oxytocin receptor concentrations in a nondistended, nonpregnant uterine horn with those in a distended, pregnant horn. On day 20, they were subjected to bilateral ovariectomy and indwelling balloons were inserted into both uterine horns. Following ovariectomy, the rats were injected im with either oil, estradiol benzoate (5 micrograms/rat per 24 h), or estradiol and progesterone together. For comparison, intact rats were studied on days 21 and 22, 24 and 48 h after insertion of the indwelling balloons. Spontaneous uterine activity and the response to increasing amounts of oxytocin were recorded 20-24 h and 44-48 h after surgery, following which the uteri were excised and assayed for oxytocin and estrogen receptors. The oxytocin receptor concentrations in the two horns were different on day 20 before the treatments were begun, the distended pregnant horn having a higher concentration per milligram DNA than the nonpregnant horn. The various treatments always changed the oxytocin receptor concentrations in the same direction; estrogen increased and progesterone inhibited the estrogen-induced rise in oxytocin receptor concentrations. In intact rats, the distention-induced increase in oxytocin receptor concentrations present on day 20 disappeared near term, but in the absence of the ovaries distention of the uterus had a significant influence on the myometrial oxytocin receptor concentrations, potentiating the effect of estrogen. Progesterone selectively inhibited the distention-induced increase in oxytocin receptor concentrations without inhibiting the hypertrophic effect of distention in general. A good correlation between oxytocin receptor numbers and tissue responsiveness was observed in all instances. The changes in spontaneous activity induced by the various treatments were distinct from the changes in oxytocin responsiveness. Estrogen exerted a strong inhibitory action on the activity stimulated by hormone withdrawal, while progesterone had no inhibitory effect. The pregnant distended horn always showed more spontaneous activity than the nonpregnant horn. There was an overall significant correlation between nuclear estrogen receptor and oxytocin receptor concentrations per milligram DNA, although the partial correlations were not significant in all groups (oil and progesterone).(ABSTRACT TRUNCATED AT 400 WORDS)

Progestin-like effects of danazol on rabbit uterus.

The action of danazol on cytosol and nuclear progestin and estrogen receptor concentrations and on the induction of uteroglobin synthesis were studied in the rabbit uterus in vivo. In addition, the relative binding affinity of danazol for the uterine progestin receptor was measured in vitro. Administration of increasing doses of danazol (10, 50, or 100 mg/kg BW daily) for 5 days to adult rabbits tended to decrease the concentrations of cytosol and nuclear progestin and estrogen receptors, whereas the uterine uteroglobin content increased with increasing doses of danazol. Progesterone (1 mg/kg BW daily) caused changes which were greater than those caused by the largest dose of danazol. Danazol was found to bind to the uterine progestin receptor in vitro with an affinity approximately 3.3% of that of progesterone. These findings suggest that danazol has a profound progestin-like activity in the rabbit uterus which may be mediated through binding to cytosol progestin receptors.

Effects of tamoxifen, medroxyprogesterone acetate, and their combination on human endometrial estrogen and progestin receptor concentrations, 17β-hydroxysteroid dehydrogenase activity, and serum hormone concentrations

Short-term and long-term progestin exposures elicit secretory and atrophic changes, respectively, in the human endometrium. To understand the mechanisms for these diverse effects of progestins, we administered medroxyprogesterone acetate (100 mg daily orally) alone or together with the antiestrogen tamoxifen (20 mg daily) for 3 weeks (from the third through twenty-third day of the cycle) to healthy, normally cycling women. Medroxyprogesterone acetate administration led to atrophic changes in the endometrium and reduced the activity of endometrial 17β-hydroxysteroid dehydrogenase on the twenty-third day of the cycle below the values measured on the same day of the preceding control cycle. These changes were accompanied by decreased cytosol estrogen and progestin receptor concentrations, while the respective nuclear receptor levels remained unchanged. Since the diminished cytosol progestin receptor content could be the primary reason for the aforementioned results, the 3-week progestin treatment was supplemented with the antiestrogen tamoxifen, also known to be a partial estrogen agonist. This combination elicited, however, endometrial atrophy and an even more pronounced reduction in the 17β-hydroxysteroid dehydrogenase activity, although it increased cytosol progestin and nuclear estrogen receptor concentrations. Tamoxifen alone did not alter endometrial receptor concentrations or 17β-hydroxysteroid dehydrogenase activity. However, tamoxifen markedly increased circulating sex steroid concentrations, which changes were abolished by a concomitant medroxyprogesterone acetate administration. It thus appears that the initial and stimulatory effect of progestins on the endometrial function is relatively short-lived and that the later depressive actions of these steroids are achieved as soon as 3 weeks. Therapeutic implications of these findings are discussed.

A direct effect of LHRH on anterior pituitary estrogen receptors in the female rat

An analysis has been made of the effects of LHRH on anterior pituitary estrogen receptors. Dispersed pituitary cells from ovariectomized, estrogen-primed rats were incubated in the presence or absence of 1–10 ng of LHRH per pituitary equivalent for 30 min at 37°C. Following lysis, nuclear estrogen receptor concentration was observed to be significantly elevated in a dose-related fashion, while extranuclear binding was depressed by a constant factor. At 5 or 10 ng LHRH levels, the increased nuclear binding could not be explained by translocation of cytoplasmic receptor, and total cellular receptor activity was significantly enhanced. Isolated cytosol and nuclei were not subject to significant fluctuations in the presence of 1–10 ng of LHRH, although 100 or 1000 ng of LHRH did significantly stimulate nuclear binding. In separate experiments, 15 min incubations with 1 or 10 ng of LHRH were not sufficient to cause altered receptor binding, under conditions where 30 min incubation effected the above results. At 120 min of incubation, binding in the extranuclear compartment was increased dramatically over the 30-min interval, and this was entirely at the expense of the nuclear binding capacity. In vivo administration of LHRH caused an increase in nuclear anterior pituitary receptor activity at 1 h, without concomitant alteration in cytosol content. At 10 h, the intracellular distribution of receptors had shifted in a manner consistent with nuclear→cytoplasmic recycling of receptor, and the distribution mirrored that of the untreated control animals. The results indicate that LHRH has a direct stimulatory effect on nuclear estrogen receptor activity which cannot be explained by stimulation of cytoplasmic receptor depletion. Such an action could account for the self-priming effect of LHRH on its own activity, since estrogens are known to modulate (presumably through a receptor-mediated mechanism of action) the responsiveness of pituitary cells to LHRH.

Antiestrogenic and antifertility actions of anordrin (2α,17ga-diethynyl-A-nor-5α-androstane-2β,17β-diol 2,17-dipropionate)

Anordrin, administered in a single s.c. dose of 62.5 μg in sesame oil, stimulated sustained uterine growth (wet weight) when measured at 24 and 72 hr, but total soluble protein and total DNA per uterus was not increased. By comparison, 3 μg of estradiol-17β under the same conditions significantly increased all three parameters of uterine growth. Both of the above steroid treatments significantly increased nuclear estrogen receptor content of the uterus, but only the estradi-ol-17β treatment resulted in significantly elevated cytosol receptor content per uterus. Anordrin binds to the 8S estrogen receptor with an affinity of about 2 × 10 5 M -1 as determined by competition with [ 3H]estradiol-17β. The abortifacient activity of Anordrin when given orally (8 mg/kg b.w.) to mice on the 7th day of pregnancy was almost completely blocked by simultaneous oral administration of estradiol-17β (0.8 mg/kg b.w.). It is concluded that the actions of Anordrin on the uterus can be attributed to its antiestrogenic activities.

Estradiol receptor levels in spontaneous leydig cell tumors of fischer rats

Spontaneous Leydig cell tumors were found in a high proportion of Fischer rats after the age of 1 year. At 2 years of age, virtually all the testes were affected to various degrees. Cytosolic estradiol receptors with properties identical to those of normal testes were increased 2- to 10-fold in Leydig cell tumors. Nuclear receptors were found to be lower in normal Fischer rat testes than in Sprague-Dawley rats [75 ± 12 (SD) fmol/mg DNA vs 104 ± 3 (SD) fmol/mg DNA]. However, 1 h after a single injection of 25 μg of estradiol, the nuclear receptor levels were increased 1.4-fold in Sprague-Dawley rats and 2.9-fold in normal Fischer rats. The unstimulated nuclear receptor levels in testicular tumors were higher than in normal Fischer rat testes and similar to Sprague-Dawley rat testes. After estradiol injection, normal testes and testicular tumors from Fischer rats had similar estradiol receptor levels [218 ± 49 (SD) and 211 ± 106 (SD) fmol/mg DNA]. These subtle changes in nuclear estrogen receptor concentrations do not support nor disprove the hypothesis that estrogens are implicated in the development of testicular tumors in Fischer rats.

Steroid receptors and effects of oestradiol and progesterone on chick oviduct proteins.

After a single injection of oestradiol benzoate (1.5 mg/kg) to oestrogen-withdrawn chickens, there was an increase in magnum wet weight, DNA polymerase alpha activity, adenosine-3',5'-monophosphate-dependent protein-kinase activity and estrogen-receptor concentration, as measured over 36 h. Besides these intracellular proteins, the secretory proteins ovalbumin and conalbumin were also augmented, and detailed time-course studies were performed. Early induction kinetics for ovalbumin and conalbumin synthesis, which differed for each protein, were independent of the dose of oestradiol benzoate injected if it exceeded 0.1 mg/kg. After 6 h for ovalbumin and 2 h for conalbumin, the induction curves diverged according to the dose of hormone administered and in correlation with the persistence of elevated nuclear oestrogen-receptor concentrations, a result confirmed with 11 beta-methoxy-17 alpha-ethynyloestradiol (R 2858), a powerful synthetic oestrogen. When oestradiol benzoate (1 mg/kg) and progesterone (3 mg/kg) were injected simultaneously, the rate of conalbumin sythesis, during the first 6-8 h, was lower than that observed in animals injected with oestradiol benzoate alone. However at later times conalbumin synthesis was greater in animals receiving both hormones than with oestradiol alone. In contrast, the rate of ovalbumin synthesis after the combined injection was higher than that induced by either hormone alone throughout the entire experimental period. In order to study further the synergistic and antagonistic activities of these two hormones, a single injection of progesterone (3 mg/kg) was administered 6, 12 or 18 h after 1.5 mg/kg oestradiol benzoate. Progesterone administration resulted in a reduction in cytoplasmic, nuclear and total oestrogen receptor concentration for at least 6 h when compared with the values in birds treated with oestrogen alone. DNA polymerase and protein kinase activities were also reduced during this period. Subsequently, all parameters increased, and by 18-24 h after progesterone treatment, reached values higher than those observed in animals receiving oestrogen alone.

Differential response of individual uterine cell types from immature rats treated with estradiol.

The separation of viable epithelial, stromal, and myometrial cells from immature rat uteri is described. The integrity of the separated cells was established by their ability to grow in culture and to exclude trypan blue and by transmission and scanning microscopy. Epithelial preparations contain insignificant contamination by other cells. Stromal fractions are estimated to contain 10% epithelial contamination. Myometrial fractions probably contain significant contamination by stroma, but the amount is difficult to quantify. The three cell types separated from untreated rats or from rats treated with 17 beta-estradiol (E2) differ from each other physiologically. Control epithelial cells incorporated more tritiated cytidine per microgram DNA than stromal or myometrial cells. 17 beta-Estradiol enhanced incorporation of tritiated cytidine into RNA of epithelial cells but not stromal or myometrial cells. RNA to DNA ratios of cells from untreated rats were 1.0 (epithelial), 0.5 (stromal), and 0.6 (myometrial). RNA to DNA ratios rose approximately 30% in all cell types after E2. Estrogen receptor (ER) concentrations, determined by Scatchard analysis under exchange conditions, also differed in the three cell types. Nuclear ER concentrations (as measured in crude nuclear fractions) were significantly higher (P less than 0.01) in epithelial and stromal cell fractions from untreated animals than in the myometrial fraction, i.e. 5.5, 3.8, and 1.2 fmol/microgram DNA, respectively. The chief effect of E2 administration was a significant increase (P less than 0.05) in cytosol ER of all cell types. Epithelial cells had the highest cytosol ER concentration, increasing from 0.5 to 3.1 fmol/microgram DNA 48 h after E2 administration. Although the myometrial cells had the lowest ER concentration, they contained 84-89% of the entire uterine ER by virtue of the proponderance of myometrium in the whole uterus. The differing responses of epithelial and stromal cells, therefore, are diluted or masked by the ER content of the myometrium in assays of whole uterus. Although the total number of ER sites in the cytosol and nucleus (16,900-32,100 sites/cell) in separated stromal cells at various times after E2 stimulation was similar to ER concentrations (20,000-30,000 sites/cell) previously reported for the whole uterus, the partition of ER between cytosol and nucleus (10:90) was different, favoring the nucleus even before E2 administration. ER began to appear in increased amounts in the cytosol only 24 h post E2 administration. When tritiated E2 was given in vivo and its specific binding was assessed in the uterine cells subsequently separated, ER partition between cytosol and nucleus was also heavily in favor of the nucleus. However, when whole uterus was assayed, the partition of ER between cytosol and nucleus (90:10) was exactly the opposite. Possible reasons for this unusual finding are discussed.

Intratumoral variation of cytoplasmic and nuclear estrogen receptor concentrations in human mammary carcinoma

This study demonstrates that the cellular content of the cytoplasmic specific estradiol receptor varies in mammary carcinomas in a reproducible way; the central parts of the tumor contained the lowest and the peripheral parts the highest level of the receptor. These intratumoral differences in receptor content, which varied between 3- and 20-fold, were found in tumors from both pre- and postmenopausal women. In contrast to the cytoplasmic receptor the intranuclear receptor level was highest in the central parts of mammary carcinomas. The finding of large intratumoral variations in receptor content strongly suggest that tumor samples for receptor analysis should be collected in a standardized way.

Inhibition of estrous behavior by progesterone in rats: role of neural estrogen and progestin receptors.

Sexual receptivity can be induced in ovariectomized rats with sequential injections of 17 beta-estradiol-3-benzoate (EB; 2 micrograms sc) and progesterone given 24 h apart. If a high dose of progesterone is used, estrous behavior is followed by a period during which sexual receptivity cannot be reinduced with a second progesterone injection (sequential inhibition). We have examined the effects of a hormone treatment which causes a sequential inhibition on levels of estrogen and progestin receptors in the hypothalamus-preoptic area-septum of ovariectomized rats. In a preliminary experiment, a single injection of 2 micrograms EB caused a significant increase in the concentration of brain cell nuclear estrogen receptors, Peak levels were seen at 12-24 h and remained significantly elevated as long as 72 h after EB. In a second experiment, we found that 48 h after EB, brain cytoplasmic estrogen receptors were significantly depressed and nuclear estrogen receptors were significantly elevated. However, a single progesterone injection (5 mg, sc) 24 h after EB had no significant effect on either cytoplasmic or nuclear estrogen receptor levels. Forty-eight hours after EB, brain cytoplasmic progestin receptors were significantly elevated compared with oil-injected controls. In a third experiment, progesterone (5 mg, sc) given 24 h after EB reduced neural cytoplasmic progestin receptors to levels significantly below those of oil-injected controls at 48 h. These results are consistent with the suggestion that progesterone does not interact with neural estrogen receptors to inhibit lordosis in rats. Rather, we suggest that progesterone may induce a behavioral refractoriness to subsequent progesterone treatments, at least in part, by reducing the concentration of neural cytoplasmic progestin receptors.

The Relevance of Interaction Kinetics in Determining Biological Response to Estrogens

The study of the kinetics of the interaction between two ethynyl estradiol derivatives (llβ-methoxy-17-ethynyl-l,3,5(10)-estratriene-3,17β-diol and llα-methoxy-17-ethynyl-l,3,5(10)-estratriene-3,17β-diol), differing in the configuration of the 11-methoxy substituent, and the cytoplasmic estrogen receptor in mouse uterus and the study of their ability to induce short and long term estrogen responses (uterine weight increase, estrogen receptor replenishment, and progestin receptor induction) in the mouse have led to the following conclusions. Potent agonists are compounds which form slowly dissociating cytoplasmic receptor complexes. Low doses of weak agonists are able to induce short term responses to the same extent as potent agonists but are unable to maintain these responses; higher doses or frequent administration are required to maintain an effect. The degree of agonist activity is related to the nuclear estrogen receptor concentration, which is in turn related to the ability of the ligand to form a stable cytoplasmic receptor complex. Low doses of weak agonists can effectively compete with endogenous hormone for cytoplasmic receptor sites and thus prevent the endogenous hormone from inducing its full biological response. Since they are themselves unable to induce a full response, this competition results in antihormonal activity.

Peroxidase as a marker enzyme in estrogen-responsive tissues

Peroxidase, an enzyme that can be induced readily in immature rat uteri by physiological doses of estrogen and is also present in some DMBA-induced mammary tumors, has proved to be a useful marker for the study of estrogen action. The induction of peroxidase in the rat uterus under various endocrine conditions has been investigated together with the effect of progesterone and a number of estrogens and related compounds. The nature of the steroids present in the uteri of immature rats and in those of animals pretreated with estrogen which contain peroxidase was examined after the in vivo administration of [ 3H]estradiol. Only unchanged estradiol and some estrone were detected in the uteri of both groups of rats and most of the radioactivity was released into the medium after incubation with p-hydroxymercuribenzoate or diethylstilbestrol, indicating an absence of covalent binding. Peroxidase is therefore unlikely to limit the duration of estrogen action in the intact animal as had been suggested earlier. In DMBA-induced mammary tumors, a moderate correlation was obtained between peroxidase activity and the concentration of nuclear estrogen receptors. It is proposed that the enzyme peroxidase which is dependent on estrogen for its biosynthesis provides a better indication of the hormone responsiveness of tumors than the concentration of estrogen receptor.

Studies on the mechanism of action of the nonsteroidal antioestrogen tamoxifen (I.C.I. 46,474) in the rat

The interaction of tamoxifen with the rat uterine oestrogen receptor protein has been investigated. The administration of tamoxifen to immature female rats produced a dose-dependent decrease in cytoplasmic oestrogen receptor concentrations and exchange methods were used to demonstrate a rise in nuclear oestrogen receptor concentrations. In immature rat uterine weight tests tamoxifen produced a partial uterotrophic response over a dose range of 0.5–8.0 μg/day and the same doses, administered simultaneously with oestradiol, produced a dose-related inhibition of oestrogen-stimulated uterine wet weight increases and uterine DNA content. Measurement of cytoplasmic oestrogen receptor concentrations during a uterine weight test demonstrated that 4 μg tamoxifen produced significant antiuterotrophic effects without a complete depletion of cytoplasmic oestrogen receptors. A single administration of tamoxifen (4 μg) produced a slow but prolonged rise in uterine wet weight associated with a slow decrease in cytoplasmic oestrogen receptors and a prolonged rise in oestrogen receptor levels in the nucleus. By 24 h cytoplasmic oestrogen receptor concentrations had returned to control levels. After a single dose of oestradiol (0.08 μg) there was a rapid decrease in cytoplasmic oestrogen receptors associated with a rapid rise in uterine wet weight but only a small rise in nuclear oestrogen receptor concentrations. In oestradiol-treated animals neither rises in uterine weight nor nuclear oestrogen receptor concentrations were maintained after 24 h. Oestrogen receptors which were translocated to the nucleus after a large dose of oestradiol (0.9 μg) were in the main salt (0.4 M KC1) extraetable although a small but significant proportion were salt resistant. By comparison oestrogen receptors translocated after tamoxifen were completely salt extractable. It is suggested that the change in the properties of the oestrogen receptor is responsible for the partial agonistic effects of tamoxifen. Since tamoxifen does not have to deny oestrogen binding completely to produce antioestrogenic effects in the uterus, a competition between tamoxifen-oestrogen receptor complexes and oestradiol-oestrogen receptor complexes for nuclear acceptor sites may be the primary antioestrogenic mechanism.