Endometrial hyperplasia and apoptosis following neonatal diethylstilbestrol exposure and subsequent estrogen stimulation in both host and transplanted hamster uteri.

Prenatal exposure to the synthetic estrogen diethylstilbestrol (DES) causes morphogenetic alterations and neoplasia in the human reproductive tract. In the hamster, neonatal DES exposure alters early uterine morphogenesis and induces endometrial adenocarcinomas in adults. We now demonstrate that the preneoplastic stages of this phenomenon in the hamster reflect an abnormal uterotropic response to estrogen that is characterized by hyperplastic lesions in the endometrial epithelium and includes an immune and/or inflammatory component. Interestingly, biochemical and in situ analysis revealed that the hyperplastic epithelium is also an active site of cell death by apoptosis. To further probe the mechanism of this phenomenon, uteri from 7-day-old control or DES-exposed donors were transplanted into the cheek pouches of control or neonatally DES-exposed adult hosts, and both host groups were treated to provide high circulating levels of estradiol. Among the four ectopic scenarios, histopathological lesions (epithelial hyperplasia, dysplasia, and apoptosis), segregated almost exclusively to the two that consisted of neonatally DES-exposed uteri. The virtual absence of lesions in control uteri transplanted to DES hosts eliminated host systemic factors as causative agents. Therefore, we conclude that DES or its metabolites alter the cellular physiology and/or composition of the developing uterus (initiating event) in such a way that it thereafter responds abnormally to estrogenic stimulation (promoting event). These observations serve to further define a unique experimental system for probing: (a) various aspects of the clinical "DES Syndrome"; (b) how estrogen regulates normal uterine growth and morphogenesis; and (c) how this process can degenerate to the unregulated neoplastic state.

Altered morphogenesis of the immature hamster uterus following neonatal exposure to diethylstilbestrol.

In previous studies, we found that a single neonatal exposure to diethylstilbestrol (DES) resulted in severe hyperplasia and a high incidence of endometrial adenocarcinoma in the uterus of adult hamsters. These observations prompted us to investigate the consequences of DES exposure on earlier stages of uterine morphogenesis. After neonates were treated within 6 h of birth (day 1) with 100 micrograms of DES or oil vehicle, uterine tissue morphometry plus cell labelling indices following in vivo pulse labeling with [3H]thymidine were determined on days 3-21 of life. The sequential findings were: (1) a precocious (day 3) burst of cellular proliferation throughout the uterus, (2) an early period (days 3-9) of hypertrophy and increased cell density in the luminal epithelium, (3) an extreme acceleration of uterine growth resulting in a persistent increase in total uterine mass (> threefold enhancement on days 5-21), (4) precocious development of endometrial glands (day 9) that were sites of intense but transient proliferative activity, (5) a middle period (days 9-15) when the percentage of stromal cells engaged in proliferative activity was reduced, (6) a second wave (days 15-21) of enhanced proliferative activity in the luminal epithelium, and (7) later development (day 21) of reduced cell density in the uterine stroma, apparently due to increased intercellular collagen accumulation. These results support our working hypothesis that the acute uterotropic response to neonatal DES treatment initiates a change in the developing hamster uterus, and later estrogenic stimulation promotes neoplastic progression in the DES-altered adult organ, perhaps due to disruption of stromal-epithelial interactions.

Progesterone downregulates progesterone receptor, but not estrogen receptor, in the estrogen-primed oviduct of a turtle (Trachemys scripta).

Progesterone downregulates nuclear progesterone receptor (Rp) and estrogen receptor (Re) in the estrogen-primed mammalian uterus and chick oviduct. We sought to determine if this downregulation mechanism is operative in the turtle oviduct. Female turtles were primed for 4 days with 17-beta-estradiol, after which progesterone (5 mg) was administered by injection every 24 h. Re and Rp levels in progesterone-treated and control turtle oviducts were measured by [3H]steroid-binding assays (pyridoxal 5' phosphate method) at 12, 24, 48 and 72 hr after initial progesterone treatment. Serum progesterone levels of progesterone-treated turtles increased only slightly from 0 hr (0.3 ng/ml) to 12 hr (0.6 ng/ml) after progesterone administration, increased considerably by 24 hr (5.3 ng/ml), and remained elevated (6-8 ng/ml) through 72 hr. Cytosol and nuclear Rp levels of estrogen-primed turtle oviducts showed distinct seasonal variation, with Rp levels higher in spring and summer months than in winter months. There was no seasonal variation in Re levels. Both cytosol and nuclear Rp responded to progesterone treatment. Cytosol Rp levels of progesterone-treated oviducts were significantly reduced below control levels by 12 hr after progesterone administration and remained low through 72 hr. Nuclear Rp levels of progesterone-treated oviducts showed no change at 12 hr, increased at 24 hr and then dropped at 48 and 72 hr. However, progesterone did not downregulate Re in the turtle oviduct.(ABSTRACT TRUNCATED AT 250 WORDS)

Serum corticosteroid-binding globulin (CBG) and hepatic CBG mRNA relationships during hamster pregnancy: contribution of decidualization.

We measured serum corticosteroid-binding globulin (CBG) and hepatic CBG mRNA from individual hamsters throughout pregnancy and during decidualization. Serum CBG levels were determined by 3H-cortisol binding assay, and hepatic CBG mRNA levels were measured by Northern blots and solution hybridization assays, using a 32P-labeled cRNA probe derived from a rat CBG cDNA. There was a positive relationship between hepatic CBG mRNA levels and serum CBG levels during pregnancy. Both parameters increased significantly from the time of mating (cycle Day 4) to pregnancy Day 4, showing that CBG synthesis and secretion increased prior to implantation (Day 4). After implantation, serum CBG and hepatic CBG mRNA rose further from pregnancy Day 4 to a peak on Day 14, then decreased before parturition on Day 16. The prepartum decline in hepatic CBG mRNA preceded the fall in serum CBG. Decidualization on pseudopregnancy Day 4 resulted in an increase in serum CBG and hepatic CBG mRNA. Hepatic CBG mRNA increased from Day 5 to Day 7, and serum CBG increased progressively from Day 5 through Day 9 after uterine decidualization in the hamster. The present results demonstrate that the pattern of serum CBG observed in the pregnant hamster follows closely that of hepatic CBG mRNA. A signal emanating from uterine decidual tissue appears to be important in the regulation of hepatic CBG synthesis and secretion during midpregnancy, but other unknown factors appear to be involved in controlling CBG during the early and late stages of pregnancy.

Characterization of the corticosteroid-binding globulin messenger ribonucleic acid response in the pregnant hamster.

In this study we measured corticosteroid-binding globulin (CBG) mRNA levels in liver and various nonhepatic tissues of pregnant and nonpregnant hamsters. The N-terminal amino acid sequence (37 residues) of hamster CBG was determined and compared with published cDNA-deduced sequence information for rat and human CBG. Hamster CBG showed considerable sequence homology with both rat (70%) and human (59%) CBG. Because of the high level of homology, we were able to use a cRNA prepared from a rat CBG cDNA as a probe in Northern blot and solution hybridization analyses. Northern blots of hamster and rat liver RNA extracts revealed that the rat CBG cDNA probe hybridized to RNAs that were the same size in rats and hamsters. Further, the Northern blot showed that pregnant hamster liver contained substantially more CBG mRNA than nonpregnant hamster liver. The relative amounts of CBG mRNA in pregnant and nonpregnant hamster livers were compared using a solution hybridization assay. Slope-ratio analysis of the hybridization data revealed that pregnant hamster liver (day 14) contained 40-fold more CBG mRNA than nonpregnant hamster liver. When other tissues (kidney, spleen, small intestine, and decidual tissue) were assayed for CBG mRNA, a small amount of hybridization was detected by solution hybridization. However, Northern blot analysis of RNA extracts from nonhepatic tissues showed that the hybridizable sequences did not migrate at the same position as mature CBG mRNA. These results indicate that the observed increase in serum CBG during hamster pregnancy is largely attributable to an increase in hepatic CBG mRNA.

Characterization of the corticosteroid-binding globulin response to decidualization in the hamster.

We sought to characterize the relationship between the decidualized uterus and circulating corticosteroid-binding globulin (CBG) levels during pseudopregnancy in the hamster. Blood CBG levels (as measured by [3H] cortisol binding) averaged 11-fold greater for decidualized hamsters than for sham-operated controls on day 8 of pseudopregnancy (PSP). We used sequential blood sampling from individual hamsters to monitor changes in CBG content after decidualization. The increase in blood CBG after decidualization was rapid, as evidenced by a significant difference in CBG content between sham-operated and decidualized groups within 24 h after surgery. We varied the extent of uterine traumatization to induce differing amounts of decidual tissue. Regardless of the amount of decidual tissue, all decidualized hamsters showed a significant increase in blood CBG; however, there was a dose-dependent relationship between the amount of decidual tissue formed and the magnitude of the CBG response. We used hysterectomy (on PSP day 6, after decidualization on PSP day 4) to determine if removal of the decidualized uterus influenced the duration of the CBG response. Bilateral hysterectomy blocked further increases in blood CBG, whereas in control animals (unilateral hysterectomy) blood CBG continued to increase after surgery. Thus, once the CBG response is initiated, the decidualized uterus is necessary to maintain elevated CBG levels. The findings in this study provide further evidence for the presence of a decidua-hepatic endocrine axis that appears to be responsible for elevation of circulating CBG levels during early pregnancy in the hamster.

Progesterone down-regulation of nuclear estrogen receptor: a fundamental mechanism in birds and mammals.

Progesterone is known to selectively down-regulate nuclear estrogen receptor (Re) in the mammalian uterus, and this process is functionally related to embryo retention. It is unclear if this mechanism is operative in the chick oviduct, where egg retention does not occur. We investigated the regulation of Re by progesterone in a mammalian model (proestrous hamster uterus) and an avian model (DES-primed chick oviduct), under the same assay conditions, in an effort to compare progesterone action in viviparous and oviparous species. Nuclear and cytosol estrogen receptor were measured with an assay employing pyridoxal 5'-phosphate (PLP). The PLP assay has the advantage of allowing exchange at low temperature, which results in improved receptor recovery, especially from the nuclear fraction. Parallel studies were done under two different hormonal settings, estrogen primed and estrogen + progesterone primed. Experiments were: (1) response of Re to acute progesterone treatment (5 mg progesterone, 4 hr) in estrogen-primed preparation, (2) time course of the Re down-regulation response (4, 8, and 12 hr after progesterone treatment), and (3) recovery of Re after progesterone withdrawal in estrogen + progesterone-primed preparation. Chick oviduct contained little cytosol Re (0.96 +/- 0.32 pmol/g tissue) compared to hamster uterus (4.27 +/- 0.15 pmol/g tissue), and progesterone treatment had no effect on cytosol Re levels in either species. Nuclear Re levels were similar for chick oviduct (2.68 +/- 0.14 pmol/g tissue) and hamster uterus (2.64 +/- 0.14 pmol/g tissue). Progesterone treatment reduced nuclear Re levels in both the hamster uterus and chick oviduct to about 50% of control levels. In the chick oviduct, down-regulation was transient, as evidenced by complete recovery of nuclear Re to control levels by 12 hr after progesterone administration. In the estrogen + progesterone-primed chick oviduct, nuclear Re increased within 6 hr after progesterone withdrawal and approached maximal levels by 12 hr. These data indicate that progesterone rapidly and selectively down-regulates the nuclear form of Re in the chick oviduct as in the hamster uterus. Thus, the regulation of Re by progesterone appears to be similar in the mammalian uterus and the chick oviduct, despite the basic differences in reproductive strategy between birds and mammals.(ABSTRACT TRUNCATED AT 400 WORDS)

Hamster uterine tissues accumulate corticosteroid-binding globulin during decidualization.

Although corticosteroid-binding globulin (CBG) is known to be a serum steroid-binding protein, its function outside of the vascular space is not well understood. To prove an extravascular role for CBG, it must first be established that CBG occurs in steroid target tissues. We sought information on the occurrence of CBG in the cytosol, nuclear, and membrane fractions of 6 tissues during decidualization in the hamster. Our objectives were to determine if CBG is distributed in a tissue-specific manner, and to investigate the relationship between serum CBG and tissue CBG. Hamsters were given progesterone pellets s.c. on cycle Day 1 and decidualization was induced on Day 4. A 3H-cortisol-binding assay, which distinguished between CBG and glucocorticoid receptor, was used to determine CGB levels in the serum and in the cytosol, nuclear, and membrane fractions of deciduoma, myometrium, liver, kidney, muscle, and small intestine. Cytosol CBG accounted for greater than 97% of the total CBG detected in all tissues except liver, where nuclei contained 11% of the measurable CBG. For all cell fractions, CBG levels showed consistent tissue-specific differences. Cytosol CBG was highest in deciduoma and myometrium, 2-fold less in liver and kidney, and 5-fold less in muscle and small intestine. Nuclear CBG concentration was greatest in liver and approximately 10-fold less in other tissues, except for small intestine, where nuclear CBG was undetectable. Membrane CBG was highest in liver, 5-fold less in deciduoma, 10-fold less in myometrium, and about 20-fold less in other tissues. Serum CBG increased 7-fold from Day 4 to Day 9 in decidualized hamsters, but not in nondecidualized sham-operated hamsters. In all tissues, serum CBG was correlated with cytosol CBG. The high levels of CBG in uterine tissues were not the result of serum contamination because whole-body perfusion with buffered saline failed to remove the majority of cytosol CBG under conditions where over 70% of 51Cr-labeled red blood cells were removed. The identity of uterine cytosol CBG with serum CBG was established by ion-exchange chromatography (O-(diethylaminoethyl)-cellulose) and by immunoprecipitation with an antibody generated against serum CBG. These data demonstrate that uterine tissues accumulate substantial amounts of CBG during decidualization, thus raising the possibility of a functional role of CBG in uterine tissues during early pregnancy.

Binding and biologic activity of diethylstilbestrol in the hamster: influence of a serum component on estrogen receptor binding and estrogenic activity.

The purpose of this study was to compare the biological activity and estrogen receptor (Re) binding affinity of diethylstilbestrol (DES) and estradiol (E2). Uterine weight response and cytosolic progesterone receptor (Rp) induction were equivalent following daily (3 days) injections of DES or E2 to ovariectomized animals. The biological equivalence of DES and E2 was not reflected by competition assays done with either uterine cytosolic or nuclear Re: the relative binding affinity (RBA) of DES to cytosolic Re was 46 +/- 5.3 and to nuclear Re was 380 +/- 42 compared to E2 (100). The RBAs of estrone, estriol and enclomiphene with cytosolic or nuclear Re were not significantly different. Further studies showed that this discrepancy in RBA of DES between cytosolic and nuclear Re could not be attributed to salt concentration but could be mimicked by addition of serum to nuclear Re preparations. The RBA of DES done with ammonium sulfate precipitated cytosolic Re approached that observed for nuclear Re. Gel filtration chromatography (Sephacryl S-300) of serum bound tritiated DES was shown to coelute with bovine serum albumin. These results suggest that a serum component (tentatively identified as albumin) can bind DES and cause a decrease in in vitro binding affinity and a reduction in biological activity in vivo.

Pattern of ovarian progesterone secretion during the luteal phase of the ovine estrous cycle.

Pulsatile secretion of progesterone has been observed during the late luteal phase of the menstrual cycle in the rhesus monkey and human. As the luteal phase progresses in each of these species, there is a pattern of decreased frequency and increased amplitude of progesterone pulses. The present study was designed to determine the pattern of progesterone secretion during the late luteal phase (Days 10-16) of the normal ovine estrous cycle. Five unanesthetized ewes, each bearing an indwelling cannula in the utero-ovarian vein, were bled every 15 min from 0800 h on Day 10 through 0800 h on Day 16 of the estrous cycle. With the computer program PULSAR, it was determined that progesterone secretion was episodic, with pulsations observed on all days. Analysis of variance was used to determine differences in frequency, amplitude, and interpeak interval (IPI) of progesterone pulses among ewes and days. The ewes averaged 8.0 +/- 0.63 pulses of progesterone per 24 h. Mean frequency of pulses was not different between days but showed differences between ewes. Mean amplitude of progesterone pulses was 7.0 +/- 0.27 ng/ml, with no differences observed either between days or between ewes. Mean IPI was 197 +/- 7.1 min, and, like frequency, the IPI was not different between days, but varied between ewes. No consistent temporal relationship was found between progesterone pulses and luteinizing hormone (LH), as determined by bioassay and radioimmunoassay, on Day 14 of the cycle in one ewe. The results indicate that progesterone secretion is episodic during the luteal phase of the ovine estrous cycle and is independent of LH pulses.(ABSTRACT TRUNCATED AT 250 WORDS)

RU486 is not an antiprogestin in the hamster.

The biological activity and progestin receptor binding activity of the synthetic steroid RU486 (RU38486; 17-beta-hydroxy-11-beta-(4-dimethylaminophenyl)-17-alpha-(1-propynl++ +)- estra-4,9-diene-3-one) were investigated in the hamster. RU486 demonstrated no antiprogestational activity in the female hamster in that it was ineffective in blocking decidualization or interrupting early pregnancy. Competitive binding assays showed RU486 did not compete from hamster uterine progestin receptor. It is concluded that hamster uterine progestin receptor has unique steroid binding specificity.

Immunocytochemical localization of relaxin in the golden hamster (Mesocricetus auratus) during the last half of gestation.

The objective of this study was to determine the tissue source of relaxin in pregnant hamsters by immunocytochemical techniques. Ovarian, uterine, and placental tissues were recovered from hamsters on Days 8, 10, 12, 14, and 15 of gestation and processed for light microscopy. Relaxin immunoreactivity was localized in tissue sections by the avidin-biotin-peroxidase technique using antiserum to porcine relaxin. On Day 8 of gestation, relaxin immunoreactivity was localized in primary giant trophoblast cells (GTC-1s) adjacent to the uterine decidua. On Day 10, relaxin immunoreactivity was localized in GTC-1s, secondary giant trophoblast cells (GTC-2s) adjacent to the ectoplacental cone, and endometrial granulocytes in the wall of sheathed arteries. On Day 12, relaxin immunoreactivity was observed primarily in GTC-2s interspersed among cells of the placental trophospongium but not in cells of the placental labyrinth. The intensity of staining and number of relaxin immunoreactive GTCs increased between Days 12 and 14 but was decreased by Day 15 PM. Relaxin was not localized in uterine glands or corpora lutea. These observations suggest that the placenta is the tissue source of relaxin in pregnant hamsters.

Purification and partial characterization of a corticosteroid-binding globulin from hamster serum.

Our objective was to characterize and purify the corticosteroid-binding proteins in hamster pregnancy serum. When [3H]cortisol-labeled pregnancy and proestrous serum were subjected to native polyacrylamide gel electrophoresis, a single peak of specific steroid-binding activity was detected in each, with identical electrophoretic mobility. The steroid-binding affinity (Ka = 1.07.10(8) M-1 for cortisol) is typical of corticosteroid-binding globulin from other species, but the steroid-binding specificity (cortisol greater than testosterone greater than progesterone) is not. An ultraviolet photoaffinity-labeling protocol was developed using 17 beta-hydroxy-4,6-[1,2-3H]androstadiene-3-one ([3H]androstadienolone), permitting analysis of ultraviolet photoaffinity-labeled proestrous and pregnancy serum by two-dimensional polyacrylamide gel electrophoresis and fluorography. Both sera contained the same labeled protein species. Corticosteroid-binding globulin was purified from pregnancy serum by DEAE-cellulose chromatography followed by steroid affinity chromatography on androstadienolone-17 beta-hemisuccinate-ethylenediamine-AffiGel 10. The purified protein (Mr = 62,250; pI = 3.95; n = 1; Stokes radius = 3.5; S = 4-5) was determined to be a glycoprotein. When analyzed by gel filtration and two-dimensional polyacrylamide gel electrophoresis, purified corticosteroid-binding globulin behaved the same as in unfractionated serum, and when ultraviolet photoaffinity-labeled with [3H]androstadienolone, purified corticosteroid-binding globulin produced the same fluorogram spot pattern seen in unfractionated serum. A specific corticosteroid-binding globulin antiserum was raised in rabbits, and this antiserum reacted with a single spot in Western blots of unfractionated serum. Thus, hamster pregnancy serum was determined to have one corticosteroid-binding protein. This protein is identical to the corticosteroid-binding globulin found in proestrous serum, with a higher titer in pregnancy serum. No other steroid-binding component is observed in hamster serum.

Characterization of nuclear acceptor sites for mammalian progesterone receptor: comparison with the chick oviduct system.

We performed binding studies with hamster uterine progesterone receptor (Rp) and various chromatin-cellulose preparations to determine whether Rp acceptor sites exist in mammalian uterus analogous to those observed in the chick oviduct. Chick and hamster Rp acceptor site assays were done according to the method of Spelsberg et al. [(1983) Recent Prog. Horm. Res. 39, 463-513]. Hamster Rp bound to hamster uterine chromatin-cellulose, NAP-cellulose (a 4 M guanidine hydrochloride-extracted fraction) and DNA-cellulose in a manner similar to that observed in the chick oviduct. Hamster Rp binding was tissue specific as evidenced by higher Rp binding to target-tissue vs non-target-tissue chromatin. The greatest degree of Rp binding occurred in the NAP fraction, and the higher level of binding seen in NAP-chromatin as compared with that in crude chromatin may be attributed to extraction of "masking proteins" which inhibit Rp-chromosomal protein interactions. When guanidine hydrochloride at greater than 4 M was used to extract crude chromatin, Rp binding decreased, indicating that the Rp acceptor sites were removed or denatured. These findings demonstrate the existence of Rp acceptor sites in the mammalian uterus which are similar to avian oviduct acceptor sites, suggesting that such sites may play a role in mediating Rp-induced gene expression.

Characterization of chromatin binding sites for different forms of uterine progesterone receptor.

We tested hamster uterine progesterone receptor (Rp) forms for binding to different chromatin preparations. Similar forms of chick oviduct Rp were used for comparison. Hamster Rp elutes from DEAE-Sephacel in the two peaks, peak I at 115 mM KCl and peak II at 205 mM KCl. Chick Rp peaks I and II elute at 125 mM and 300 mM KCl, respectively. Both chick and hamster peak I displayed a higher level of binding to SDS-stripped chromatin (DNA) than to crude chromatin or 4 M guanidine hydrochloride (GuHCl)-extracted (nucleoacidic protein, NAP) chromatin while peak II bound 50% better to the NAP chromatin than to crude chromatin or DNA. 10 mM molybdate was used to stabilize Rp and to increase Rp recovery. Molybdate-stabilized hamster Rp elutes from DEAE at the peak II position and like peak II, binds poorly to DNA. Since molybdate prevents receptor activation, DNA-Rp interactions require activated Rp. Because molybdate did not prevent Rp binding to NAP chromatin, we conclude that both activated and unactivated Rp bind well to that matrix. Activated hamster Rp could be extracted from crude chromatin, NAP chromatin and DNA with 200 mM KCl. Unactivated Rp was extracted from NAP only with 6 M GuHCl or NaSCN, whereas KCl, glycerol or pyridoxal 5'-phosphate were not able to remove unactivated Rp from NAP. Various Rp forms did not compete with [3H]ORG 2058-Rp for binding to NAP but BSA did compete. Thus a large portion of Rp binding to NAP may represent nonspecific binding rather than binding to a finite number of Rp acceptor sites. These results suggest that the binding of activated Rp to crude chromatin may represent the actual acceptor sites in target cell nuclei. Since the high level of Rp binding sites in NAP chromatin may be an extraction artifact, the involvement of proposed masking proteins in regulating the availability of acceptor sites should be reconsidered. As an alternative to acceptor site regulation, changes in the Rp molecule itself may be important. Rp isolated from hamster uteri on days 1-4 of the estrous cycle was incubated with crude chromatin, NAP chromatin and DNA. The apparent level of Rp binding to chromatin and NAP chromatin increased 2.5-fold from day 1 to day 4, but Rp binding to DNA remained constant. This suggests that ovarian cycle-dependent changes occur in the unactivated Rp which affect its interactions with chromatin, and these changes disappear when receptor is activated.(ABSTRACT TRUNCATED AT 400 WORDS)

Ligand-receptor dissociation: a potential mechanism for the attenuation of estrogen action in the juvenile rabbit uterus.

Estradiol binding kinetics and receptor activation were investigated using cytosol estrogen receptor from adult rabbit uterine endometrium and from the undifferentiated uteri of 2-week-old rabbits. The cytosol estrogen receptor from juvenile compared to that from adult rabbit uteri was lower (P less than 0.01) in concentration, was associated with reduced (P less than 0.01) titers of serum estradiol, and had a lower affinity for estradiol (Ka = 10(7) M-1). The equilibrium association constant (Ka) for the estrogen receptor from juvenile uteri was reduced by an increase in the dissociation rate constant (kd), as measured by [3H]E2 dissociation from the receptor. Enhanced steroid-receptor dissociation in juvenile uteri was correlated with a reduced rate (P less than 0.01) of receptor activation, as measured by the binding of steroid-receptor complex to DNA-cellulose. Because receptor activation was limited at elevated temperature (30 C), activation studies were performed at low temperature (0 C), and under these optimum conditions, the change in binding kinetics observed in the juvenile was correlated with a reduced rate of receptor activation. Equilibrium binding of [3H]E2 to the estrogen receptor exhibited positive cooperativity, as indicated by Hill coefficients of 3.39 +/- 0.12 and 3.44 +/- 0.11 for juveniles and adults, respectively. The ratio of bound to free steroid was decreased in cytosol from juvenile compared to adult uteri. Collectively, these results support the hypothesis that the increased off rate and decreased activation rate of estrogen receptor in immature rabbit uteri may represent a mechanism for the attenuation of estrogen action before sexual maturation.

Decidual cell function: evidence for a role in the regulation of serum CBG and a 60 kDa protein during early pregnancy in the hamster.

Several serum proteins increase in titer during pregnancy. We tested the hypothesis that decidual cells may signal the production of certain serum proteins in the hamster. Measurement of serum CBG by equilibrium binding using either [3H]-progesterone or [3H]-cortisol in conjection with ion exchange chromatography showed that decidualization increased CBG levels. Two-dimensional gel electrophoresis revealed that a 60 kDa++ protein increases markedly in the serum of the hormonally pseudopregnant (PSP) animal soon after artificial induction of decidualization on PSP day 4. The 60 kDa serum protein remains low in the nondecidualized PSP animal, but it increases in the pregnant animal. A photoaffinity labeling procedure was used to covalently bind [3H]-androstadienolone to CBG. Fluorography of 2D gels run under denaturing conditions established that the 60 kDa protein did not bind steroid as did CBG (69 kDa). To determine whether decidual cells could induce the 60 kDa and CBG proteins, different numbers of decidual cells were injected IP into PSP recipients. A single injection of 50 x 10(6) decidual cells induced both serum proteins within 48h, whereas the same number of hamster fetal cells was ineffective. Thus, these results demonstrate that hamster decidual cells induce a 60 kDa protein of unknown function and serum CBG. Since the decidual cell itself does not appear to be the source of either protein, it follows that the decidual cell signals the synthesis and secretion of these proteins elsewhere in the body, most likely in the liver. To our knowledge, this is the first demonstration that the decidual cell regulates serum CBG and other proteins in this manner.

Progesterone-modulation of estrogen action: rapid down regulation of nuclear acceptor sites for the estrogen receptor.

Our previous studies demonstrated that progesterone down regulates the occupied form of nuclear estrogen receptor (Re). Using the density shift method, we discovered that progestins stimulate the turnover of nuclear Re within 3 h of treatment, and Re synthesis is suppressed subsequently. Thus, the primary site of progestin action in down-regulating Re is the stimulation of nuclear Re turnover followed by the inhibition of Re replenishment. A major breakthrough in our understanding of how progestin controls Re turnover was made by studying nuclear acceptor sites for Re that were found to decrease markedly within 2 h of progestin treatment. These and other results indicate that progestin induces a factor called the Re regulatory factor (ReRF) which acts to block nuclear Re acceptor sites, and this in turn decreases nuclear Re retention on chromatin acceptor sites, leading to an enhanced turnover (or processing) of nuclear Re.

Hormonal regulation of estrogen and progestin receptors in decidual cells.

Total estrogen receptor (Re) and total progestin receptor (Rp) were measured in the cytosol and nuclear fractions from hamster deciduomal tissue and decidual cell cultures. Correlation of serum steroid (estradiol and progesterone) and deciduomal receptor profiles revealed a significant loss of Re during the first four days of decidualization that was not attributable to changes in serum steroid levels. A decidual cell-tissue culture system was used to study the receptor's recovery response to progesterone withdrawal. Decidual cells were plated and grown in Ham's F12/Dulbecco's modified Eagle's medium with 5% horse serum supplemented with insulin, transferrin, selenium and progesterone (10 ng/ml). Within 48 h of culture large, multinucleate decidual cells were observed by phase microscopy. At 72 h of culture in medium containing progesterone, only Rp was detectable in decidual cells. Re was not detectable (less than 200 fmol/mg DNA) in either cytosol or nuclei from cells maintained in the presence of progesterone. However, when progesterone was deleted from the medium, cytosol Re recovered progressively from 8 h to 16 h of culture. Progesterone withdrawal also caused parallel increases in cytosol and nuclear Rp, and estradiol treatment (2 ng/ml) in combination with progesterone withdrawal further enhanced Rp levels in decidual cell cultures. These results with cultured decidual cells demonstrate that progesterone down-regulates Re and Rp, Re recovers rapidly upon progesterone withdrawal, and the Re system is competent to respond to estrogen action in terms of Rp induction. We used the density-shift method to determine that progestin increases the turnover of nuclear Re in hamster decidual cells within 3 h. Hamster decidual cells were isolated from the endometrium and cultured in progesterone-free medium containing normal amino acids (1H, 12C, 14N) for 2 days. Confluent monolayers of cells were exposed to 1 nM estradiol (E2) for 1 h to maximize the amount of occupied Re in the nuclear fraction. Then, at time 0, cells were transferred to medium supplemented with dense (2H, 13C, 15N) amino acids and either 1 nM E2 or E2 plus 100 nM progesterone. After Re was labeled with dense amino acids for 1, 3, 6 and 9 h, nuclear Re was extracted with 10 mM pyridoxal -5' phosphate and labeled with 125I-iodoestradiol (5 nM). Two radioactive peaks representing preexisting and newly synthesized Re were separated by sucrose density-gradient centrifugation. The halflife of nuclear Re in decidual cells was 3.7 h when cells were treated with E2 alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Temporal effects of progesterone domination on estrogen and oxytocin receptors in hamster uterus.

The purpose of this study was to determine whether progesterone (P)-induced down regulation of estrogen receptors (Re) and oxytocin receptors (ROT) changes with the time of P exposure. Ovariectomized hamsters were given s.c. Silastic implants of estradiol (E2) and P for 4, 8 and 16 days. Cytosol and nuclear Re were measured at low temperature with the pyridoxal phosphate exchange assay, and ROT was assayed in the membrane fraction by [3H] oxytocin binding. Nuclear Re and ROT were down regulated throughout the 16-day P exposure period, but cytosol Re (and total Re) increased progressively from 4 to 16 days indicating that the down regulation of cytosol Re escapes P control with time. This conclusion was supported by P withdrawal studies in which P implants were removed for 6 or 12 h. P withdrawal resulted in equivalent recovery responses of nuclear Re and ROT after 4, 8 and 16 days of P exposure. Although cytosol Re recovery to P withdrawal occurred at 4 and 8 days, no response was obtained after 16 days of P exposure. Uterine weight increased during steroid treatment, and morphometric analysis of the P-dominated uterus revealed significant increases in the cross sectional area of the endometrium and myometrium with time of P exposure. Cytological examination of the uterus showed prominent secretory changes in the epithelial compartment on day 16 with accumulation of secretion in the uterine lumen. These results demonstrate that P can chronically down regulate nuclear Re and ROT. However, the control of cytosol Re varies with the time of P exposure, and cytosol Re levels become refractory to P domination by 16 days. The present observations indicate that the escape of cytosol Re from P control may be associated with the proliferation of one of more uterine cell populations such as glandular and luminal epithelial cells.