Differential suppression of progesterone receptors by progesterone in the reproductive tract of female macaques

Ovariectomized cynomolgus macaques were treated with implants of estradiol (E2) for 14 days. Some animals then received an additional implant of progesterone (P) for 7-14 more days. After treatment with either E2 alone or with E2 plus P we removed the reproductive tracts and measured nuclear and cytosolic P receptors by exchange assay. In addition we used steroid radioimmunoassays(RIA) to measure levels of E2 and P in parallel aliquots of the nuclear and cytosolic fractions. P treatment reduced the concentrations of E2 in nuclear and cytosolic fractions in the cervix, endometrium, myometrium and oviduct compared to the amounts present after 14 days of E2; these data are consistent with many reports that P treatment significantly lowers the amount of nuclear and cytosolic estrogen receptors in all of these tissues. In the oviduct, myometrium and cervix both cytosolic and nuclear P receptor levels were lowered during P action. In the endometrium, however, P treatment reduced the amount of P receptor only in the cytosolic but not the nuclear fraction. RIA determinations of the amount of P retained in nuclear fractions of the P-treated animals indicate that P levels were significantly elevated only in the nuclei obtained from endometrium. This specific increase in the retention of P by endometrial nuclei during P action is consistent with the specific retention of P receptor by endometrial nuclei. These results lead to the unexpected conclusion that the stimulatory effects of P as expressed in the maintenance of the progestational state in the primate endometrium may require higher levels of occupied nuclear P receptor than do the suppressive effects of P as expressed in oviductal atrophy, diminished cervical secretion and myometrial quieting.     

Effect of ovarian steroids on nitric oxide synthase in the rat uterus, cervix and vagina

The effects of estrogen (E2), progesterone (P) and E2 and P (E2 + P) were examined on nitric oxide synthase (NOS) activity in both cytosolic and particulate fractions isolated from the rat uterus, vagina, cervix and cerebral cortex. Additionally plasma nitrate + nitrite (NO3 + NO2) levels were measured in control and hormone treated rats. Cytosolic NOS was the predominant form being approximately 80% of the total in all four tissues. NOS activity in both fractions from all tissues was highly Ca-dependent (> 90%). Among the reproductive tract tissues, the highest activity was found in the cervix, which was nearly 5- and 2-fold higher than the uterus and vagina, respectively. NOS activity in the cerebral cortex was by far the highest being 5-fold higher than in the cervix. In contrast to the cortex, E2 treatment downregulated cytosolic NOS in all reproductive tract tissue, but this was statistically significant in only uterus. When compared with E2 treated rats, P increased cytosolic NOS in uterus, vagina, and particulate NOS in the cervix. The data do not give any indication whatsoever of differential effects of P in the uterus and cervix.     

Estrogen and progesterone receptors in the ovine cervix during the postpartum period

Cervical estrogen (E) and progesterone (P) receptors were characterized and quantified during the postpartum period in Corriedale ewes lambing in the late breeding season. Cervices and uteri were collected after ovariohysterectomy at 1 d (n = 2), 5 d (n = 4), 17 d (n = 2) or 30 d (n = 2) post partum. The estrogen and progesterone receptors were measured using binding assays with tritiated hormones, dextran charcoal separation and inverse Scatchard analysis. Similar kinetic parameters in cytosolic binding sites for both hormones were found in all cervical and uterine samples, indicating that the binding protein in both tissues is of the same nature. Receptor concentrations (fmol/mg cytosolic protein) in the cervix of early (1 to 5 d, n = 6) and late (17 to 30 d, n = 4) postpartum ewes were 348 +/- 66 vs 994 +/- 145 (P < 0.05) for E and 618 +/- 126 vs 1170 +/- 201 (P < 0.05) for P, respectively. These data suggest an increased synthesis of receptors, probably due to the presence of ovarian estrogen-active follicles. Cervical E and P receptor concentrations were similar or higher than those in the uterus (1.40 +/- 0.15, n = 10 and 1.51 +/- 0.19, n = 10; for E and P respectively), and these receptor ratios did not differ between the early and late postpartum period. The high ratio between cervical/uterine receptors suggests that the ovine cervix may be a very sensitive to steroid action. In conclusion, it was shown that restoration of steroid receptors during the postpartum period in the ovine cervix is similar to receptor dynamics in the uterus, and is probably associated with the recovery of ovarian cyclicity.     

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.     

Effect of the antiprogestin RU-486 on progesterone inhibition of occupied nuclear estrogen receptor in the uterus

The ability of the antiprogestin, RU-486, to reverse progesterone (P) antagonism of occupied nuclear E receptor retention was studied in the rat and hamster uterus. RU-486 was shown to effectively displace [3H]P binding from rat uterine cytosolic P receptor in in vitro competition assay. In contrast, no competition by RU-486 for [3H]P binding was observed for uterine cytosolic P receptor from the hamster uterus. In the presence of sustained serum levels (silastic implants) of P and estradiol (E), occupied nuclear E receptor was significantly inhibited in the rat uterus. At 6, 12 and 24h after RU-486 treatment (5 mg/animal, s.c.) uterine receptors for E and P were determined. No significant differences in cytosolic E and P receptors were observed between treated (E + P, + RU-486) and control (E + P alone) animals. However, by 6 h following RU-486 treatment, occupied nuclear E receptor retention increased significantly (0.30 +/- 0.05 vs 0.60 +/- 0.09, pmol/uterus) and reached a peak between 12 h (1.32 +/- 0.09) and 24 h (0.83 +/- 0.09). The increase in nuclear E receptor approached the level observed in animals with an E implant alone (1.55 +/- 0.15). Measurement of uterine fluid accumulation following RU-486 treatment showed an increase which paralleled that observed for occupied nuclear E receptor retention. A similar in vivo experiment in the hamster showed no reversal of P inhibition of occupied nuclear E receptor. These results show that: 1. RU-486 is an effective competitor for rat uterine P receptor but not hamster P receptor; 2. RU-486 can rapidly reverse P inhibition of uterine occupied nuclear E receptor in the presence of sustained serum levels of E and P; 3. The recovery of occupied nuclear E receptor is coincident with a resumption of E action (uterine fluid accumulation). The studies also provide a novel means by which antiprogestin activity can be assessed in vivo in the presence of sustained E and P serum levels, e.g. the reversal of P inhibition of uterine nuclear E receptor retention.     

Localization of relaxin binding sites in the rat uterus and cervix by autoradiography

125I-labeled porcine relaxin was injected into 27-day-old rats treated with pregnant mare's serum gonadotropin (PMSG) and known target tissues for relaxin, the myometrium, endometrium and cervix, and putative control tissues, heart, thigh muscle and duodenum, examined for binding by autoradiography. Specific binding in the target tissues was demonstrated by simultaneous injection of excess unlabeled relaxin. Radioactivity was located and quantified by grain counts predominantly over the inner, circular muscle layer of the myometrium and the cervix and to a lesser extent over the outer longitudinal muscle layer of the myometrium and the endometrium. The route of injection, the circulation time, or counting grains in transverse or longitudinal sections of myometrium made little difference in these results. Ovariectomy decreased, but not significantly, the grain count in all of the target tissues studied and estrogen treatment of ovariectomized animals restored the numbers of grains to approximately that of intact PMSG-treated rats. The degree of binding of the cervix was approximately that of the circular myometrial muscle. This work confirms the presence of specific receptors for relaxin in the rat uterus and cervix of primed rats and it also suggests that the inhibitory action of relaxin upon the myometrium is primarily on the inner circular muscle layer.     

Influence of neonatal diethylstilbestrol treatment on androgen and estrogen receptor levels in the mouse anterior prostate, ventral prostate and seminal vesicle

Perinatal exposure to the synthetic estrogen, diethylstilbestrol (DES), affects the structure of both male and female reproductive systems. Changes may also occur in the levels of steroid hormone receptors. Cytosolic and nuclear androgen and estrogen receptor levels (expressed per mg DNA) from the sex accessory glands of male BALB/c mice exposed neonatally to DES were analyzed by exchange assays. Neonatal DES exposure caused significant decreases in: (1) cytosolic androgen and cytosolic and nuclear estrogen receptor levels in the anterior prostate and (2) cytosolic estrogen receptor levels in the ventral prostate. A significant increase was seen in the cytosolic estrogen receptor levels in the seminal vesicle. Significant decreases in cytosolic protein levels occurred in all DES-exposed glands.     

Quantification of receptors for estradiol-17 beta and progesterone in the pituitary and hypothalamus of gilts during the estrous cycle and early pregnancy

Nuclear and cytoplasmic exchange assays were utilized to quantify receptors for estradiol-17 beta (E2) and progesterone (P4) in hypothalamic and pituitary tissues from 4-6 gilts each on Days 1, 5, 10, 15 and 18 of the estrous cycle and from 4-5 gilts each on Days 5, 10, 15, 21 and 30 of pregnancy. No differences in the number of cytoplasmic E2 or P4 receptors in the pituitary were found from Days 1 to 15 of the estrous cycle (P greater than 0.05). However, on Day 18, the quantities of E2 and P4 receptors were 64-fold and 25-fold lower (P less than 0.01) than those found during Days 1 to 15 of the estrous cycle. No differences in the number of nuclear receptors for E2 in the pituitary were observed from Days 1 to 18 of the estrous cycle, but nuclear receptors for P4 were 2-fold higher (P less than 0.01) on Day 1 than Days 5 to 18. In hypothalamic tissue, the numbers of cytoplasmic and nuclear receptors for E2 and P4 were lower (P less than 0.05) on Day 18 than Day 10 of the cycle. The quantity of most steroid receptors decreased between Days 15 and 18 in nonpregnant gilts as luteolysis occurred and a new follicular phase was initiated. Pregnant pigs on Days 5, 10 and 15 had decreased pituitary receptors for E2 and P4 when compared with cycling animals on these days. In general, numbers of receptors in hypothalamic tissue did not differ between pregnant and nonpregnant pigs except for decreased (P less than 0.01) nuclear P4 receptors on Day 15.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucocorticoids inhibit estradiol-mediated uterine growth: possible role of the uterine estradiol receptor

Stress-related activation of the hypothalamic-pituitary-adrenal axis (HPA) is associated with suppression of the reproductive axis. This effect has been explained by findings indicating that corticotropin-releasing hormone suppresses hypothalamic gonadotropin-releasing hormone (GnRH) secretion via an opioid peptide-mediated mechanism, and that glucocorticoids suppress both GnRH and gonadotropin secretion and inhibit testosterone and estradiol production by the testis and ovary, respectively. To evaluate whether glucocorticoids suppress the effects of estradiol on its target tissues, we examined the ability of dexamethasone to inhibit estradiol-stimulated uterine and thymic growth in ovariectomized rats. Estradiol alone, given daily for 5 days, caused dose-dependent uterine and thymic growth. Dexamethasone alone, given daily for 5 days, caused a dose-dependent decrease in body weight gain and in thymic growth. When estradiol and dexamethasone were administered simultaneously, however, body weight gain and thymic growth were also inhibited (p less than 0.05). Dexamethasone decreased estradiol-induced uterine cytosolic and nuclear estrogen receptor concentrations (E2 R0, p less than 0.05; E2nR0, respectively), but had no effect on estradiol-induced progesterone receptor concentrations (P4R0, p greater than 0.05). Levels of uterine glucocorticoid receptors were not affected by estrogen and/or dexamethasone treatment. These findings suggest that stress levels of glucocorticoids, administered over a 5-day interval, block the estradiol-stimulated growth of female sex hormone target tissues. This effect may be partially mediated by a glucocorticoid-induced decrease of the estradiol receptor concentration. Thus, another mechanism by which the HPA may influence reproductive function during stress is by a direct effect of glucocorticoids on the target tissues of sex steroids.     

Oxytocin enhances the basal release of uterine prostaglandin F2 alpha, but not that of PGE1, or of PGE2, and changes the metabolism of exogenous arachidonate, favouring the formation of prostaglandin F2 alpha and 5-HETE. Relationships with its uterotonic action and modulation by estradiol

We attempted to explore possible mechanism(s) subserving the influence of oxytocin on uterine motility by studying the action of the hormone on: 1) the contractile activity of isolated rat uteri in the presence or absence of indomethacin; 2) the synthesis and release of prostaglandins (PGs) into the solution incubating the uterine tissue as well as the metabolism of labelled arachidonic acid; 3) the uptake of 45Ca2+ by uterine strips. The experiments were bone with uterine preparations isolated from spayed rats treated or not with 17-beta-estradiol. The values of isometric developed tension (IDT) and of frequency of contractions (FC) induced by oxytocin in uterine strips isolated from spayed and spayed-estrogenized rats, were not modified by indomethacin at 10(-6) M. On the other hand, uterine strips from untreated spayed rats, release into the incubating medium approximately equal amounts of PGE1, PGE2 and PGF2 alpha. The in vitro presence of oxytocin (50 mU/ml) increased significantly (p 0.05) the output of PGF 2 alpha without changing the release of PGE1 or PGE2. Uteri from spayed rats injected prior to sacrifice with 17-beta-estradiol released significantly less PGE1 and PGE2 (p less than 0.005) than preparations from non-injected animals, whereas the output of PGF2 alpha in the suspending solution remained unchanged. Following estrogenization the addition of oxytocin to preparations obtained from spayed-estrogenized rats also increased the output of uterine PGF2 alpha (p less than 0.001) without changing that of PGs E1 or E2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of estrogen-dependent and estrogen-independent levels of progesterone receptor in hamster vagina and uterus

It is generally accepted that progesterone action is mediated in target cells through a specific, intracellular receptor protein. Since various progesterone target tissues respond differently to progesterone action, it may be postulated that such differences result from variations in: (1) the physicochemical properties; (2) the regulation, and/or (3) the intracellular response of the progesterone receptor (Rp) complex. A previous report demonstrated similar physicochemical properties of hamster vaginal and uterine Rp [1]. Our objective in this report was to analyze the regulation of estrogen-independent (ID-Rp) and estrogen-dependent (D-Rp) populations of receptor in different tissues of the lower reproductive tract of the golden hamster. In untreated ovariectomized animals, a basal level of (ID-Rp) was detected in endometrium, myometrium and vagina. Thus, each compartment contained a significant quantity of Rp which was maintained in the absence of continued estrogen support. Following 3 days of estradiol (E2) administration, the level of nuclear estrogen receptor increased and was related quantitatively to the amount of cytoplasmic Rp produced in these tissues. Maximal weight and D-Rp responses in endometrium, myometrium and vagina were obtained with 10-100 micrograms E2 per 100 g BW. The weight response of these tissues was due primarily to cellular proliferation in the endometrium; cellular hypertrophy in the myometrium; and cellular proliferation with concomitant nuclear pyknosis in the vagina. Although the morphological response of these tissues to estrogen action is quite different, the present study reveals no differences in the regulation of ID-Rp and D-Rp levels in these particular compartments. Furthermore, our results demonstrate a relationship between DNA content and ID-Rp and D-Rp levels in target tissues of the lower reproductive tract.     

Characterization of uterine sex steroid receptors in the pig and their variation during the oestrous cycle

The present study establishes and validates an in vitro binding and exchange assay for tissue receptors for oestradiol (E) and progesterone (P) in pig uterus. Both hormones bound to specific cytoplasmic (Rc) and nuclear (Rn) receptor proteins with high affinity. The relative concentrations of the receptors were measured in dissected samples from endometrium and myometrium obtained at late prooestrus, oestrus, and luteal phases of the oestrous cycle. The Scatchard analysis of the oestradiol and R 5020-receptor complex displayed linearity and indicated a single class of high affinity, low capacity binding sites. Significant variations were seen in the binding of E and P to their cytosolic and nuclear receptors, following the changes in the circulating levels of the hormones in blood plasma during the oestrous cycle. Both tissue components, i.e. endometrium and myometrium followed a similar pattern when related to the stage of the oestrous cycle considered. The ERc increased from prooestrus, reaching a maximum at standing oestrus, thereafter decreasing. The concentration of ERn increased from prooestrus towards the early luteal phase, with a significant reduction by day 8 of the cycle. The amounts of PRc were maximal at standing oestrus, remaining high during the early luteal phase, while the PRn showed a linear increase from oestrus onwards throughout the luteal phase.     

TRAIL mediates apoptosis in cancerous but not normal primary cultured cells of the human reproductive tract

Cancer of the reproductive tract encompasses malignancies of the uterine corpus, cervix, ovary, Fallopian tube, among others and accounts for 15% of female cancer mortalities. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates apoptosis by binding to death receptors and offers a promising cancer treatment. The goal of this study was to investigate and characterize the effect of TRAIL in endometrial cancer cell lines and normal (non-cancerous) epithelial cells of endometrial origin. We also examined the effect of TRAIL in other primary cultured cancers and normal cells of the human female reproductive tract and evaluated if TRAIL mediated apoptosis correlated with death receptors and decoy receptors 1 and 2. Herein, we demonstrate that TRAIL at concentrations which kill cancerous cells, does not mediate apoptosis or alter cell viability in normal human endometrium, ovary, cervix or Fallopian tube. The partial inhibition by a caspase 9 inhibitor and the total inhibition by a caspase 8 inhibitor demonstrates the dependency on the extrinsic apoptotic pathway. The selective mortality does not correlate with the presence of death or decoy receptors. These results suggest that TRAIL may be an effective treatment for endometrial cancer and other female reproductive cancers, with minimal secondary effects on healthy tissue. This work was supported by a grant from the Wellcome Trust GR071469 (GIO) and the Chilean national science grants FONDECYT 1060495 (GIO) and 1020675 (MC). An erratum to this article is available at .     

Effects of estradiol and progesterone on the reproductive tract and on uterine sex steroid receptors in female lambs

The effects of estradiol-17 beta (E(2)) and progesterone (P) on the reproductive tract and on uterine estrogen receptors and P receptors were studied in 2-mo-old female lambs (n = 11). On Days 0, 1 and 2, E(2) (1 ug/kg, Group E, n = 4), P (0.3 mg/kg, Group P, n = 4) or corn oil (control) vehicle (Group C, n = 3) were administered, and in Day 3 all lambs were slaughtered. Group E (n = 12) had E(2) serum concentrations (mean +/- SEM) of 43.8 +/- 2.2 pmol L , similar to that of the follicular phase; while P concentrations in Group P (n = 12) were similar (2.8 +/- 0.18 nmol L ) to those of the luteal phase of the ewe estrous cycle. The E(2) treatment increased the reproductive tract weight, while P treatment increased only the uterine weight. Both E(2) and P receptors from upper and middle uterine zones (including the myometrium, endometrium and caruncles) were determined by binding assays with tritiated hormones, dextran-charcoal separation and inverse Scatchard analysis. Both the E(2) and P treatments decreased E(2) and P receptor concentrations in upper and middle zones, although the upper zone had higher receptor concentrations than the middle zone (P < 0.01). E(2) receptor concentrations in the upper zone (mean +/- SEM, fmol mg prot) were 1236 +/- 34, 667 +/- 80 and 444 +/- 103 for Groups C, P and E, respectively. The P receptor concentrations were 2434 +/- 135, 1273 +/- 102 and 1536 +/- 213 for the same groups. The high uterine P receptor concentrations allowed P action without prior estrogen priming of female lambs. The present results suggest that E(2) and P might down-regulate their own and each other's receptors during development. The biological responses induced by E(2) and P, as measured by the reproductive tract weight, demonstrated that at an early stage of development uterine receptors are physiologically active.     

Growth hormone-binding proteins in high-speed cytosols of multiple tissues of the rabbit

Soluble, specific binding protein(s) for growth hormone (GH) have been identified and partially characterized in high-speed cytosolic preparations from a number of rabbit tissues. The binding of 125I-labelled human GH to proteins in liver, heart, adipose tissue, skeletal muscle and kidney cytosols was dependent on time and cytosolic protein concentration. By Scatchard analysis, the binding affinities (KA: (2-7) X 10(9) M-1) were somewhat higher than those generally reported for membrane GH receptors. The binding proteins had a greater specificity for somatotrophic hormones than lactogenic hormones, although the kidney appeared to have, in addition, a lactogen-binding protein. By gel filtration, the Mr of the cytosolic GH-binding protein was approximately 100 000 in all tissues. None of the binding proteins was detectable by the poly(ethylene glycol) precipitation method used widely for soluble hormone receptors. The cytosolic GH-binding proteins also cross-reacted with a monoclonal antibody to the rabbit liver membrane GH receptor. These results indicate the ubiquitous presence of apparently naturally soluble GH-binding proteins in the cytosolic fractions of several tissues in the rabbit. Of great interest is their presence in muscle, where GH receptors or binding proteins have not previously been detected, despite muscle being recognized as a classical GH target tissue.     

Variations in hepatic estradiol-17beta receptor concentrations during the annual reproductive cycle of diploid and triploid female catfish, Heteropneustes fossilis (Bloch)

Concentrations of hepatic estradiol-17beta (E2) receptors (ER) in cytosolic and nuclear fractions were evaluated in diploid and triploid female catfish Heteropneustes fossilis (Bloch) during four different reproductive periods of a complete reproductive cycle. Basal level of ER concentration was noted in the resting period of both diploids and triploids. Receptor level gradually elevated through the preparatory period and reached a peak in the pre-spawning period in both diploids and triploids. However, ER concentrations were overall reduced in triploid to that of diploid females. In a single point assay, in diploids, ER concentration showed about a 3-fold rise (p<0.001) in the cytosolic and a 4-fold rise (p<0.001) in the nuclear extracts from resting to the pre-spawning period. In triploids, only a 2-fold rise was observed both in cytosolic (p<0.01) and nuclear (p<0.05) ER concentration during the same span. Finally, a sudden fall of receptor level was observed in the spawning period in both the ploidy groups with a lower concentration in the triploids. The K(d) value did not differ between the females of diploids (cytosolic 1.12+/-0.21 nM and nuclear 6.9+/-0.9 nM) and triploids (cytosolic--1.13+/-0.17 nM, nuclear--6.8+/-2 nM). However, B(max) of the diploid showed about double the value than triploid females both in the cytosolic (diploid--367.4+/-33.24 pmol/mg protein, triploid--187.3+/-13.20 pmol/mg protein, p<0.001) and nuclear extracts (diploid--946+/-66 pmol/mg DNA, triploid-558+/-98 pmol/mg DNA, p<0.01) of liver. Lower E2 binding capacity and lower amount of E2 receptors of triploid catfish liver with a stunted vitellogenic status could be one of the major causes for reduced gonadal development and sterility in female triploids.     

Binding of estradiol-17 beta and estriol in cytosolic and nuclear fractions from urogenital tissues

This report describes the measurement and partial characterization of the specific binding of estradiol (E2) and estriol (E3) in the cytosols and nuclear fractions from uterus, vagina, urethra and urinary bladder of the rabbit. Fractions from uterine and vaginal tissues showed the highest binding with both E2 and E3 approx 300 fmol/mg protein. The levels of specifically bound E3 were about 70% of the corresponding values for E2. In the sedimentation analysis the cytosolic receptors for both E2 and E3 exhibited 2 major forms, 3 S and 8 S, in addition to the heavy aggregates. Sephacryl S-200 chromatography showed major and minor components corresponding to mol. wt of 250.000 and 20.000-40.000 respectively. No noteworthy differences in the cytosolic receptors from different tissues could be observed in the above analyses. Whereas the uterine nuclear E2 receptor sedimented at 4.3 S the vaginal receptor distributed in two peaks, 2.5 S and 4.3 S. The receptor from both urethra and urinary bladder sedimented at 2.5 S. Nuclear E3 receptor, however, showed a major peak at 3.5 S in all cases. Some differences in tissue nuclear receptor were also observed in their chromatographic behaviour. The results confirm the existence of estrogen receptor in both urethra and urinary bladder, and the data of the limited physiochemical analysis indicate that the receptors in these tissues are essentially similar to those in the uterus or vagina.     

Receptors for estrogens and progesterone in the porcine cervix

In vitro binding and exchange methods were used to determine the levels of estradiol and progesterone receptors in cytosolic and nuclear fractions of cells obtained from the porcine cervix at different stages of the estrous cycle. The concentration of estradiol cytosolic receptors was about 4500 sites/cell during the luteal phase and increased to a maximum of approximately 7600 sites/cell on day 1 of the cycle, decreasing to a level of 2700 sites/cell on days 3-4. The estradiol nuclear receptor level increased between the end of the luteal phase and the onset of heat from 300 to 1200 sites/cell. No reduction in the number of nuclear sites was seen between day 1 and 3-4. The level of the progesterone cytosolic receptor and its cycle profile was very similar to that of the estradiol receptor. The nuclear receptor, however, reached its lowest level of 760 sites/cell on day 1 of the cycle, increased to a value of 4700 sites on days 3-4 and showed a steady level of about 1000 sites/cell during the luteal phase. The data obtained agree with present theories on the endocrine mechanisms regulating receptor levels in the uterus. Furthermore, these data support a concept in which the constriction of the cervix occurring in response to increased concentrations of circulating estradiol is mediated via steroid receptors.     

Interaction of ring B unsaturated estrogens with estrogen receptors of human endometrium and rat uterus

The present investigation was undertaken to compare the binding affinities (Ka) of the ring B unsaturated equine estrogens (equilin [Eq], equilenin [Eqn], 17 beta-dihydroequilin [17 beta-Eq], 17 beta-dihydroequilenin [17 beta-Eqn], 17 alpha-dihydroequilin [17 alpha-Eq], and17 alpha-dihydroequilenin [17 alpha-Eqn]) and the classic estrogens (estrone [E1], 17 beta-estradiol [17 beta-E2], and 17 alpha-estradiol [17 alpha-E2]) for estrogen receptors in human endometrium and rat uterus. In both species, the ring B unsaturated estrogens bind with cytosol and nuclear receptors with high affinity (Ka x 10(9) M-1). The relative binding affinities of these estrogens were measured by determining the amount of unlabeled estrogen required to reduce by 50% the specific binding of [3H]17 beta-Eq to endometrial cytosol receptors. The order of activity found was 17 beta-Eq greater than 17 beta-E2 greater than 17 beta-Eqn greater than E1 greater than Eq greater than 17 alpha-Eq greater than 17 alpha-E2 greater than 17 alpha-Eqn greater than Eqn. Essentially the same order of activity was observed when the apparent affinity constants of these estrogens for human and rat cytosol and nuclear receptors were determined by a competitive (inhibition) binding assay. Sucrose density gradient analysis indicated that these estrogens form protein complexes with cytosol and nuclear preparation that sediment at approximately 8S and 4S, respectively. The affinity constants for 17 beta-Eq were approximately two- to six-fold higher than E2 in both species. In a rat uterotropic assay, all nine estrogens were uterotropic. These data indicate that all ring B unsaturated estrogens present in conjugated equine estrogen preparations are biologically active and they express their biologic effects in the human endometrium by mechanisms similar to those described for the classic estrogens.