Regression of the decidualized mesometrium and decidual cell apoptosis are associated with a shift in expression of Bcl2 family members.

The purpose of this study was to determine whether regression of the decidua basalis (DB), which begins on Day 14 of pregnancy in the rat, results from an intrinsic program of apoptosis regulated by Bax and Bcl2. Expression of Bax and Bcl2 and the incidence of apoptosis were evaluated throughout gestation by Western blot analysis and detection of DNA fragments. Antiprogestin (RU486) was also administered during proliferation of DB to study progesterone regulation of Bax/Bcl2 balance. Bax, the pro-apoptotic protein, was expressed at a low level throughout pregnancy, whereas Bcl2, the pro-survival partner, was most abundantly expressed on Days 8 and 10, which are a time of proliferation and decidualization, and declined to barely detectable levels thereafter. These changes resulted in a 12-fold increase in the Bax:Bcl2 ratio on Day 17 as compared with Day 8 of pregnancy (P < 0.05). DNA laddering and in situ staining of DNA fragments first became visible on Day 14 and involved 2% of cells by Days 17 and 21 (P < 0.05). Treatment with RU486 on Day 9 enhanced Bax and suppressed Bcl2 within 6 h, increasing the Bax:Bcl2 ratio sixfold (P < 0.05). Apoptosis was minimal at 6 h and increased to 9% of cells by 24 h (P < 0.05). Thus, progesterone appears to regulate the apoptotic threshold of stromal cells by modulating Bax and Bcl2 expression.

Progesterone-regulated determinants of stromal cell survival and death in uterine decidua are linked to protein kinase C activity.

This study examined the role of protein kinase C enzymatic activity as a physiologic determinant of stromal cell death in decidua basalis (DB) during pregnancy. The expression of epidermal growth factor receptor (EGF-R) and Bcl2 was used as an indicator of stromal cell proliferation/survival, whereas Bax and the occurrence of apoptosis provided an index of cell death. Stromal cell cycle progression during pregnancy and after in vivo administration of phorbol esters was analyzed by flow cytometry. DB were isolated from pregnant rats between Days 8 and 21 of pregnancy and prepared for immunohistochemistry, Western blotting procedures, or flow cytometry. The results showed that stromal cells were actively proliferating on Days 8 and 10, whereas the frequency of cell death by apoptosis increased progressively between Days 14 and 21 (Day 22 is term). The proliferative stage was characterized by low PKC activity and high levels of EGF-R and Bcl2 expression. On the other hand, DB regression (Days 14-21) was marked by an elevation in endogenous PKC activity and Bax expression; EGF-R and Bcl2 were suppressed. Administration of phorbol 12-myristate, 13-acetate (0.4 micromole/kg) induced apoptosis on Day 10. Additionally, antiprogestin (RU-486) given on Day 9 induced PKC activity and Bax expression within 6 h and suppressed Bcl2 and EGF-R. By 12 h, RU-486 enhanced percent apoptotic cells. Thus, enhanced levels of PKC activity were closely linked to stromal cell apoptosis.

Progesterone regulation of epidermal growth factor receptor in rat decidua basalis during pregnancy.

Ovarian steroid hormones and epidermal growth factor (EGF) play important interactive roles in proliferation and decidualization of mesometrial stromal cells during pregnancy. This study determined the ontogeny of EGF receptor (EGF-R) expression in the decidua basalis (DB) throughout pregnancy and its regulation by estrogen and progesterone (P4). DB were isolated from rats between Days 8-21 of pregnancy and prepared for immunohistochemistry or Western analysis. In one study, rats were ovariectomized (Ovx) on Day 8 or 9 and given estradiol-17beta, P4, or both. In another study, the antiprogestin, mifepristone (RU-486), was administered on Day 9. During normal pregnancy, total EGF-R (phosphorylated and unphosphorylated forms) increased from Day 8 to a maximum level on Days 10 and 12. Tyrosine-phosphorylated EGF-R (pEGF-R), the bioactive form, was also highest on Days 10 and 12. Both forms of receptor decreased to almost undetectable levels during DB regression on Days 17-21. Immunohistochemistry of DB from Ovx rats revealed that only P4 was able to maintain normal expression of EGF-R; RU-486 decreased EGF-R expression within 6 h, and by 24 h EGF-R and pEGF-R were 15% of the Day 10 control group levels. These findings show that EGF-R is a P4-dependent protein associated with stromal cell proliferation and decidualization.

Progesterone and estrogen regulation of rat decidual cell expression of proliferating cell nuclear antigen.

This study was an examination of the role of progesterone (P4) and estradiol-17beta (E2) as stromal cell mitogens in the decidua basalis (DB) of the rat during pregnancy. Pregnant rats were ovariectomized (Ovx) on Days 8 and 12 of pregnancy, treated with P4, E2, or both, and killed on Days 10 and 14, which correspond to times of stromal cell proliferation and regression, respectively. In some experiments, rats received pellets of the anti-progestin RU-486 on Day 9 and were killed 6, 12, and 24 h later. The mitotic index (MI) and in situ image analysis of expression of proliferating cell nuclear antigen (PCNA) were used to assess cell cycle progression. Highest expression of PCNA occurred on Days 8-12 of pregnancy, and MI was maximum; MI became zero and PCNA expression decreased dramatically thereafter (i.e., Days 14, 17, 21). Percentage of cells expressing intense PCNA on Day 10 (40%) declined to 5% after Ovx and Ovx + E2 (p < 0.05), whereas Ovx + P4 maintained PCNA. By Day 14, only 1% of stromal cells expressed intense PCNA, which was not significantly altered by Ovx, Ovx + E2, or Ovx + P4 but increased after Ovx + P4 and E2 (p < 0.05). By 6 h of RU-486, MI declined 3-fold, and intense PCNA expression was essentially lost. These changes preceded loss of histological integrity of the DB. Cells with undetectable PCNA steadily increased from 8% at 6 h to 28% by 24 h (p < 0.05). Thus RU-486 appeared to block cell cycle progression and enhanced PCNA turnover. P4 was essential for stromal cell proliferation during early pregnancy (Days 8-10), but this action was lost by Day 14.

Regulation of the progesterone receptor and estrogen receptor in decidua basalis by progesterone and estradiol during pregnancy.

In this study we examined the roles of progesterone (P4) and estradiol-17beta (E2) in regulation of the P4 receptor (PR) and estrogen receptor (ER) in the decidua basalis (DB) during stromal cell proliferation and regression (Days 10 and 14 of pregnancy, respectively). Pregnant rats were ovariectomized (Ovx) on Day 8 or 12 and killed on Day 10 or 14, respectively, following treatment with P4, E2, or both. In some experiments, rats received pellets of the anti-progestin RU-486 on Day 9 and were killed 3, 6, 12, and 24 h later. Immunolocalization of PR and ER showed that both receptors decreased from Day 10 to Day 14. Histologic integrity of the placenta and DB were maintained only when P4 was present. Control and hormone-treated groups expressed established isoforms of PR and ER: PR-B, 110 kDa; PR-A, 80-90 kDa; PR-C, 64-60 kDa; ER-66, 66 kDa; and ER-49, 49 kDa. On Day 10, expression of PR-A, PR-B, and ER-66 decreased 50-99% (p < 0.05) after Ovx or RU-486 treatment but was restored to control levels after Ovx by exogenous P4. On Day 14, PR-B and ER-66 declined 66-75% (p < 0.05) after Ovx and could not be restored by P4 treatment. Estrogen could not substitute for P4, and co-administration of E2 with P4 did not enhance the response over P4 alone. In contrast, PR-C was abundantly expressed on Days 10 and 14 in all treatment groups after Ovx and RU-486. P4 maintained PR mRNA and ER mRNA after Ovx. Thus, regression of the DB may be initiated via changes in relative expression of PR isoforms, which result in impaired stromal cell response to P4 action.

Stromal cell progesterone and estrogen receptors during proliferation and regression of the decidua basalis in the pregnant rat.

This study examines the distribution and abundance of progesterone receptors (PR) and estrogen receptors (ER) in the decidua basalis (DB) during proliferation (Days 8-12 of gestation) and regression (Days 14-21) in the rat. Stromal cells of the DB and metrial gland exhibited strong nuclear immunostaining for PR throughout gestation. Nuclear localization of ER was detectable only between Days 8-12. The heavily granulated natural killer cells were always negative for PR and ER. DB were dissected between Days 8 and 17 to measure progesterone (P4)-binding sites and receptor proteins by Western blotting. The latter revealed four specific PR isoforms: B (110 kDa), A1 (90 kDa), A2 (76-82 kDa), and C (60-64 kDa). Stromal cell nuclei contained more than 50% of P4-binding sites during DB proliferation but less than 22% during regression (p < 0.05). PR-A and PR-B expression was greatest at proliferative stages (p < 0.05). PR-C increased in relative abundance during DB regression. Two ER isoforms of 66 kDa and 49 kDa were revealed. The 66-kDa ER, the most abundant form, was maximally expressed during proliferation, declining 71% by Day 12 (p < 0.01), whereas the 49-kDa form accounted for up to 90% of ER during regression. Northern blot analysis revealed three prominent transcripts of approximately 11, 7, and 4 kilobases (kb) for PR mRNA, which declined markedly at Days 14 to 17 (p < 0.05), and one of 6.0 kb for ER mRNA, which declined markedly on Day 17 (p < 0.05). Our study establishes that the DB expresses heterogeneity of receptor message and proteins. We propose that preferential expression of receptor isoforms in late pregnancy limits P4 action and promotes DB regression in spite of invariant levels of serum P4, P4-binding sites, and total receptor protein.

Ovine interferon-tau inhibits estrogen receptor up-regulation and estrogen-induced luteolysis in cyclic ewes.

This study determined whether intrauterine injection of interferon-tau (IFN tau) could block luteolysis in cyclic ewes treated with a luteolytic dose of 17 beta-estradiol benzoate (E) on day 12 of the estrous cycle. Thirty-two ewes were fitted with uterine catheters on day 5 of the estrous cycle and treated with recombinant ovine IFN tau (2 x 10(7) antiviral units/ewe/day) or control proteins (6 mg/day) by intrauterine injection from day 10 until hysterectomy. At 1900 h on day 12, all ewes received 750 micrograms E, im, and were hysterectomized 12, 24, 36, or 48 h post-E administration. Plasma concentrations of progesterone declined in control animals but increased in IFN tau-treated ewes after E injection (P < 0.01, treatment x day interaction). Likewise, total corpus luteum weight decreased in control but not IFN tau-treated ewes after E administration (P < 0.02, treatment x time interaction). In control ewes, endometrial estrogen receptor (ER) messenger RNA (mRNA; P < 0.03) and progesterone receptor (PR) mRNA (P < 0.10) increased after 12 h, whereas concentrations of ER protein (P < 0.02) and PR protein (P < 0.04) increased after 24 h. In situ hybridization and immunohistochemical analyses indicated that ER gene expression increased first in the epithelium at 12 h and then in the stroma by 48 h, whereas PR gene expression first increased in the stroma and then in the epithelium. In control ewes, endometrial oxytocin receptor (OTR) density increased (P < 0.10) after 12 h, with the largest increase occurring between 36-48 h. In IFN tau-treated ewes, endometrial ER mRNA and protein and OTR density did not increase after E administration. Levels of PR mRNA increased (P < 0.01) between 12-36 h, but decreased after 36 h. PR mRNA abundance increased between 12-36 h in the stroma, but not in the epithelium. Concentrations of PR protein were low and did not change in IFN tau-treated ewes. Immunoreactive PR protein was present at low levels in the stroma of all IFN tau-treated ewes. The results indicate that induction of luteolysis by E in control ewes involved sequential increases in endometrial ER mRNA and ER protein in the epithelium that preceded maximal increases in OTR density. Intrauterine injection of recombinant ovine IFN tau prevented luteolysis by inhibiting estrogen-induced increases in endometrial ER and OTR gene expression.

Ovine interferon-tau regulates expression of endometrial receptors for estrogen and oxytocin but not progesterone.

Ovine interferon-tau (oIFN-tau) may stabilize endometrial progesterone receptor (PR) and/or inhibit estrogen receptor (ER) gene expression during pregnancy recognition to suppress endometrial oxytocin receptor (OTR) formation and production of luteolytic prostaglandin (PG) F2 alpha pulses. This study determined whether or not oIFN-tau stabilized PR expression in the endometrium during PR down-regulation by continuous exposure to progesterone. Twenty cyclic ewes were bilaterally ovariectomized and fitted with uterine catheters on Day 2 of the estrous cycle (Day 0 = estrus). Ewes were then assigned randomly to be treated, in a 2 x 2 factorial arrangement, with recombinant oIFN-tau (roIFN-tau; 2 x 10(7) antiviral units per ewe per day) or control proteins (6 mg/day) by intrauterine injection from Days 10 to 14, and with daily i.m. injections of 20 mg progesterone from Days 2 to 14 (P) or progesterone from Days 2 to 14 plus 50 micrograms estradiol-17 beta from Days 12 to 14 (P+E). All ewes were hysterectomized on Day 15. Endometrial PR mRNA (p < 0.01) and protein (p < 0.03) were higher in ewes receiving P+E than in those receiving P alone. However, the increase in PR mRNA and protein was not as great in the endometrium of roIFN-tau-treated ewes as compared to controls (p < 0.08, treatment x steroid). In ewes receiving P alone, PR mRNA and immunoreactive PR were localized to stroma and deep glandular epithelium and were not present in endometrial luminal and shallow glandular epithelium. Values for endometrial ER mRNA (p < 0.02) and ER protein (p < 0.01) were greater in controls than in roIFN-tau-treated ewes regardless of steroid treatment. Among controls, ER mRNA and immunoreactive ER protein were present in the luminal and glandular epithelium and were increased in the epithelium and stroma in ewes receiving estrogen. In contrast, endometrial ER mRNA and immunoreactive ER protein were very low or absent in the endometrium of roIFN-tau-treated ewes and were not increased by estrogen. Among controls, endometrial OTR density was greater (p < 0.09) in ewes treated with P+E than in those treated with P alone. In roIFN-tau-treated ewes, endometrial OTR density was lower (p < 0.01) than in the controls. Results indicate that roIFN-tau did not stabilize or prevent autologous down-regulation of PR mRNA or protein expression in the endometrium. However, roIFN-tau did suppress endometrial ER expression and OTR formation in ewes regardless of steroid treatment. The results support the hypothesis that the antiluteolytic effects of oIFN-tau are to suppress endometrial ER gene expression in the endometrial epithelium, thereby inhibiting formation of OTR and production of luteolytic PGF2 alpha pulses.

Regulation of the estrogen receptor in the decidua basalis of the pregnant rat.

This study examines the distribution of the estrogen receptor (ER) in the mesometrial decidua basalis (DB) and the chorioallantoic placenta between Days 8 and 21 of pregnancy (Day 1 = presence of vaginal sperm) and its regulation by estradiol and progesterone. Immunocytochemistry revealed that ER was localized within nuclei of cells of the DB but not in trophoblastic cells (Day 10) or in cells of the junctional zone (JZ) or labyrinth zone (LZ). Western blot analysis and estradiol binding assays of DB, JZ, and LZ also revealed that only DB expressed ER. Native ER (66 kDa) was most abundant on Days 8 and 9, and declined 67% on Day 11 (p < 0.01), becoming barely detectable by Day 17. A truncated ER moiety (49 kDa) gradually increased, becoming the dominant form on Day 13. The effects of estradiol and progesterone on ER were studied during periods of growth and decline of DB (i.e., Days 8-10 and 12-14, respectively). Rats were ovariectomized on Day 8 or 12 and treated with estradiol daily (0.2, 0.75, or 2 micrograms, s.c.), with a progesterone pellet, or with both, for 48 h. Progesterone, but not estradiol, stimulated the 66-kDa ER moiety and ER binding activity (p < 0.01). Estradiol administered with progesterone antagonized progesterone action, at least in part, by enhancing expression of the 49-kDa ER at the expense of the native form (p < 0.01). Thus, progesterone up-regulated ER whereas estradiol down-regulated ER in rat DB.

Regulation of estrogen receptor protein and messenger ribonucleic acid by estradiol and progesterone in rat uterus.

The objective of this study was to examine the mechanisms of estrogen receptor (ER) processing and replenishment in the uterus of ovariectomized rats after estradiol and progesterone treatment. Uterine ER binding activity, ER protein and ER mRNA were measured by receptor binding exchange assay, Western blot and slot blot, respectively. The regulation of ER levels in rat uterus by estradiol and progesterone was very dramatic. Changes in ER protein were faithfully reflected by changes in binding activity. Estradiol caused receptor "processing" within 4 h of administration followed by recovery or "replenishment" of ER levels to the initial level by 20 h. The term "processing" has previously been used to describe the loss of ER binding activity in the early phase of estradiol-action, but it was never clear whether the ligand binding site was inactivated by processing or if the receptor molecule actually disappeared. This study shows that receptor "processing" constitutes disappearance of receptor protein and the later "replenishment" phase represents new ER protein rather than recycling of "processed" receptor. Progesterone-action, on the other hand, influenced only the "replenishment" phase by blocking recovery of ER protein. ER mRNA was suppressed by estradiol at 8 h, after the receptor was "processed" and "replenishment" already initiated. Progesterone, on the other hand, did not alter the steady state level of the message. Other mechanisms, such as regulation of translation rate of existing mRNA and changes in the rate of degradation of ER proteins are more likely involved in acute regulation of ER by these ovarian steroid hormones.

Changes in progesterone and oestrogen receptor mRNA and protein during maternal recognition of pregnancy and luteolysis in ewes.

This study characterized changes in levels of mRNA and protein for endometrial oestrogen receptors (ERs) and progesterone receptors (PRs) during luteolysis and maternal recognition of pregnancy. For cyclic and pregnant ewes, endometrium was collected on days 10, 12, 14, or 16 post-oestrus (4 ewes/day for each status) for the measurement of ER and PR mRNA and protein. The amount of receptor mRNA is expressed in relative units above background, measured from radiographs of dot-blot hybridization of total endometrial RNA with ER and PR cDNAs. At hysterectomy, jugular vein blood samples were collected and assayed for progesterone, total corpus luteum weight was recorded and, in vitro, endometrial oxytocin-stimulated inositol phosphate formation was estimated. In pregnant ewes, plasma progesterone increased gradually between days 10 and 16 (P < 0.01), corpus luteum weight was stable at approximately 0.8 g and oxytocin did not stimulate endometrial formation of inositol phosphates in vitro. In contrast, in cyclic ewes, plasma progesterone decreased from day 10 to day 16 (P < 0.01), corpus luteum weight decreased after day 14 to approximately 0.48 g (P = 0.05) and oxytocin stimulated an increase of approximately 1300% in the endometrial formation of inositol phosphates on day 16. cDNAs specifically hybridized with 1.6 and 3.1 kb transcripts for PR mRNA and a 6.5 kb transcript for ER mRNA. In cyclic ewes, the amount of PR mRNA increased from day 10 to maximum levels on days 14-16. The number of PRs decreased from day 10 (2.25 pmol/mg DNA) to day 12 (0.98 pmol/mg DNA) and then increased from day 14 to day 16 (2.8 pmol/mg DNA). In pregnant ewes, PR mRNA levels were greatest on days 10-12 and decreased by approximately 50% by day 16. In contrast, the number of PRs was relatively unchanged from day 10 to day 16 (1.53 to 1.03 pmol/mg DNA). In cyclic ewes, the amount of ER mRNA was lowest at day 10 and increased fivefold by day 16. The number of ERs remained relatively unchanged from day 10 to day 14 (6.07 pmol/mg DNA) and increased by day 16 (16.12 pmol/mg DNA). In pregnant ewes, ER mRNA decreased by approximately 80% from day 12 to day 16. Similarly, the number of ERs decreased from day 10 to day 16 (5.41 to 2.05 pmol/mg DNA). Correlations between ER mRNA and PR mRNA (r = 0.68), ERs and PRs (r = 0.50) and ER mRNA and ERs (r = 0.50) were high (P < 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in progesterone and oestrogen receptor mRNA and protein and oxytocin receptors in endometrium of ewes after intrauterine injection of ovine trophoblast interferon.

This study determined whether twice-daily intrauterine injections of ovine conceptus secretory proteins (oCSP) containing type-I trophoblast interferon (25 micrograms/uterine horn) from day 11 to day 15 post-oestrus (oestrus = day 0) could alter the binding capacities of endometrial receptors for oxytocin, progesterone and oestrogen in cyclic ewes when compared with control ewes receiving serum protein (SP) injections. Injections of oCSP on days 11-15 post-oestrus decreased concentrations of oestrogen receptors (P < 0.06), oestrogen receptor mRNA (P < 0.05) and progesterone receptors (P < 0.08) in endometrium on day 16 when compared with SP-infused control ewes, which were undergoing corpus luteum regression on days 14-16. Injection of oCSP also decreased the number (P < 0.10) and affinity (P < 0.06) of oxytocin receptors. Inositol phosphate formation induced in the endometrium on day 16 by 100 nM oxytocin in vitro was highly correlated with the concentration (r > or = 0.93, P < 0.001) and Kd (r = -0.91, P < 0.01) of oxytocin receptors in SP-infused ewes, but was not as highly correlated with concentration (r < or = 0.83, P < 0.06) and Kd (r < or = 0.40, P > 0.40) of oxytocin receptors in oCSP-infused ewes. This indicates that oCSP disrupted the relationship between oxytocin receptor binding and oxytocin-induced activation of its second messenger system. These results indicate that antiluteolytic type-I trophoblast interferon may prevent oxytocin-induced luteolytic pulsatile secretion of prostaglandin F2 alpha during maternal recognition of pregnancy in sheep, by reducing the synthesis and affinity of endometrial oxytocin receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Progesterone maintenance of the placental progesterone receptor and placental growth in ovariectomized rats.

These studies examine the trophic effects of progesterone (P) on the progesterone receptor (Rp) and growth of the decidua basalis (DB) and junctional zone (JZ) in the rat placenta. Pregnant rats were ovariectomized (Ovx) in mid-pregnancy and received steroid replacement therapy consisting of implantation of P pellets (25 mg) and injections of estradiol (E), 2 micrograms s.c., daily. Placental protein synthesis, measured by 3H-leucine incorporation in vitro, decreased more than 99% within 24 h of Ovx. However, treatment with P immediately after castration maintained control levels of synthesis. Delay of P treatment for 4 h caused a 60% decline in protein production measured 20 h later (p less than 0.01). Intraperitoneal implantation of a 50-mg pellet of the antiprogestin, RU-38486, in intact pregnant rats decreased protein synthesis by 50% within 6 h and by more than 90% 12 h and 24 h post-implantation (p less than 0.01). Growth of DB and JZ in Ovx rats treated for 48 h with P and/or E was studied both histologically and by changes in protein and DNA content. Rp binding activity was also measured by exchange assay under equilibrium conditions. Only P was able to reverse the effects of Ovx on growth of the DB and JZ. P also maintained Rp levels in the DB above those observed in Ovx and Ovx + E-treated groups (p less than 0.01). The Rp may be a constitutive product in the JZ since binding activity was not altered by Ovx or by steroid treatments. This study shows that P is clearly a trophic hormone of the maternal and chorioallantoic placenta and is essential for placental growth, cellular differentiation, and histological integrity.

Changes in cytosolic and nuclear progesterone receptors during pregnancy in rat placenta.

These studies examine changes in placental growth and the abundance of progesterone receptors (Rp) in whole placentas between Days 9 and 22 of pregnancy. In addition, some placentas were dissected into decidual basalis, junctional zone, and labyrinth zone before assay of Rp. High affinity binding of 3H-progesterone to Rp was detected at all stages of pregnancy in whole placentas and in decidua basalis and the junctional zone of the placenta. Cytosolic and nuclear receptors exhibited similar affinity for progesterone in both tissues (Kd = 3.1 +/- 0.3 and 4.4 +/- 0.7 nM, respectively). Receptor binding in whole placentas increased from Day 9 to Day 12 (p less than 0.05), declined markedly at Day 16 (p less than 0.05), and returned to former levels on Days 19 and 22 (p less than 0.05). Decidua basalis contained 84% of total Rp on Day 14, which declined to 67% on Day 17 (p less than 0.05). The junctional zone contained 16% of total Rp on Day 14 and 33% on Day 17. After Day 17, junctional zone was the only source of Rp. The decline in Rp on Day 16 followed regression of decidua basalis; recovery of Rp thereafter was due to growth of the junctional zone. The labyrinth zone did not express significant amounts of Rp at any stage despite a 4-fold increase in growth in late pregnancy. Although the biologic role of the Rp in maintenance of pregnancy is poorly understood, these studies suggest that the maternal decidua basalis and fetal junctional zone are targets of progesterone action.

Nuclear acceptor sites for progesterone-receptor complexes in rat placenta.

A cell-free system was used to characterize the binding reaction between the progesterone receptor and nuclear acceptor sites prepared from rat placenta. Two forms of receptor-acceptor complex were examined. One was extracted from nuclei by exposure to 0.6 M KCl; the other type was resistant to salt extraction. Kinetic analysis indicated that the binding reactions were saturable (3-4 pmol binding sites/mg DNA) and of high affinity (Kd = 3-6 nM). Acceptor binding was specific for placental nuclei and did not occur with nuclei prepared from spleen or with denatured nuclei from placenta. Acceptor sites were further characterized by their sensitivity to RNase, DNase I, and protease. RNase treatment had no influence on receptor-acceptor binding. However, DNase I reduced the number of KCl-resistant acceptor sites by 41%, but only a 19% reduction occurred in KCl-extractable acceptor sites (P less than 0.05). Protease removed 34% and 48% of the KCl-resistant and -extractable acceptor sites, respectively, and combined treatment with DNase and protease eliminated 76% of acceptor-binding activity. The endogenous inhibitor previously described from rat placental cytosol blocked acceptor-binding sites in a concentration-dependent manner, a decrease of 1.15 pmol sites/mg inhibitor protein for resistant sites and 0.76 pmol/mg inhibitor protein for extractable sites. However, receptor-acceptor binding was not altered by treating nuclei with actinomycin D or chloroquine. Mercurial reagents reduced receptor-acceptor interaction by 80% and 94% in KCl-resistant and -extractable sites, respectively, whereas sulfhydryl alkylating agents reduced binding 35% and 76%. Pyridoxal phosphate destroyed 88-93% of acceptor binding. The results of these studies suggest that the progesterone receptor acceptor sites are composed of a complex of chromatin protein and DNA in rat placenta. Furthermore, the binding reaction requires the participation of sulfhydryl and terminal amino groups.

Evidence for nuclear processing of progesterone receptors in rat placenta.

The distribution of the progesterone receptor (Rp) in cytosolic and nuclear compartments of placenta has been studied in intact and ovariectomized (Ovx) rats on the 14th day of pregnancy. Removal of estradiol (E) and progesterone (P) by Ovx caused a 50% decrease in progesterone receptors from cytosolic and nuclear compartments. Estradiol replacement restored binding to intact levels. Progesterone, given 19 h after E, induced an additional 3-fold increment in the number of cytosolic and nuclear binding sites 1 h later. Four hours after progesterone the number of receptor sites in the placenta fell 60%, signifying processing. This was followed 4 h later by reversal of processing mechanisms leading to full recovery of nuclear and cytoplasmic binding sites. Actinomycin D (0.6 mg/ani) was found to have no influence on these events. On the other hand cycloheximide (0.5 mg/ani) completely prevented processing of binding sites when administered at the same time as progesterone or 2 h before, but did not influence the unmasking of nuclear sites which occurred 1 h after a progesterone challenge. The cycloheximide block to processing was partial when given 2 or 3 h after progesterone (61 and 43% complete, respectively). The full complement of receptors was processed when cycloheximide treatment was delayed 3.75 h after progesterone administration. These findings have led to the view that processing represents rapid and reversible changes in binding properties of the receptor rather than a gain or loss of receptor protein per se. The findings of this study suggest that a hypothetical substance, "processin", whose production is blocked by cycloheximide binds to the receptor and in some undefined manner inhibits ligand-receptor interaction within 4 h after an in vivo progesterone challenge. Nuclear accumulation of receptor induced by progesterone was not accompanied by cytoplasmic depletion of receptor nor was the apparent loss of processed nuclear receptor due to recycling of receptor to cytoplasm. We propose that nuclear receptors continually recycle within the nucleus in masked and unmasked states regulated by delicate interplay between progesterone and processin.

Characterization of progesterone binding to nuclear receptors in rat placenta.

Exchange assays have been validated to study several forms of the progesterone receptor found to occur in nuclei of rat placenta after extraction with high salt. One form was solubilized by the extraction procedure (KCl extractable Rpn) and another form remained attached to nuclear structures (KCl resistant Rpn). Specific binding of progesterone was optimized in both forms using buffered media containing 0.01 M Tris, 30%-glycerol (v/v), 0.2 mM leupeptin, and 1 mM dithiothreitol (TDGL), pH 7.8, at 0-4 degrees C for 18-24 h. At 0-4 degrees C the nuclear receptors were stable and degradation was negligible even after 44 h of in vitro incubation. The binding reaction between progesterone and receptor demonstrated mass action principles of ligand exchange throughout this interval. Saturation analysis indicated the presence of a single binding moiety of high affinity (app Kd = 2.9-3.2 nM) for both forms of the receptor. However, the nuclear progesterone receptor was thermolabile and after a 10 min exposure to 30 degrees C no longer complexed ligand. At an intermediate incubation temperature of 22 degrees C the binding reaction was stable for about 30 min. The KCl resistant binding sites were markedly more thermolabile. Addition of 10 mM Na molybdate protected all forms of the nuclear progesterone receptor from thermal denaturation and extended the life of the complex 3-4-fold. The dissociation rate constant of progesterone-nuclear receptor complex in each preparation was 6-8 X 10(5) s-1 resulting in a half-life of about 3 h. The KCl resistant and extractable binding sites were sensitive to blockade by 1 mM N-ethylmaleimide which was reversed by co-incubation with a 2-fold molar excess of dithiothreitol. This suggested that reduced sulfhydryl groups located on or near the surface of the ligand binding domain of the receptor were necessary to bind hormone. These studies showed that the interactions between ligand and the KCl resistant and extractable receptor sites found in rat placenta were of high affinity, saturable, and heat sensitive. Thus, these binding moieties exhibited physicochemical behavior very similar to each other and to the placental receptor which has previously been partially purified from the cytosol. The conclusion is made that all of the nuclear receptor binding sites for progesterone are structurally identical. Thus, the distinctive physicochemical properties responsible for KCl resistant and extractable forms of the nuclear progesterone receptor must reside in other domains of the receptor molecule.

Estimation of molecular parameters of the endogenous inhibitor to progesterone--receptor binding in rat trophoblast.

This study was undertaken to investigate molecular properties of an endogenous substance which inhibits progesterone--receptor binding. The inhibitor was isolated from the cytosol of rat trophoblast by ammonium sulfate precipitation. Further purification was achieved by gel filtration and sucrose density gradient centrifugation. These techniques revealed that the inhibitor had a Stokes radius of 2.8 +/- 0.1 nm and an s of 4.9 +/- 0.2. For a protein exhibiting normal density and solvation, these parameters indicated that the inhibitor molecule has a mol. wt of 56,000 +/- 2000 g/mol and a frictional coefficient of 1.11 +/- 0.01. In order to obtain an additional independent estimate of molecular weight, the migratory pattern of the inhibitor was studied on SDS-polyacrylamide gels after identification and purification by polyacrylamide gel electrophoresis. The inhibitor-active fraction was resolved into two principle bands (Band 1 and 2) by SDS-gel electrophoresis having mol. wt of 59,000 +/- 700 g/mol and 51,000 +/- 300 g/mol (n = 6), respectively. The molecular weight thus determined was in excellent agreement with the value obtained by calculation. Thus, molecular parameters of the inhibitor indicate that it is a very symmetrical molecule of approx. 56,000 mol. wt. Characterization of the molecular properties of the inhibitor substance should facilitate future studies concerning the biological significance of this molecule and its role in progesterone--receptor binding interactions.

Action of glycerol and sodium molybdate in stabilization of the progesterone receptor from rat trophoblast.

This study compares the stabilizing effects of glycerol and sodium molybdate on the progesterone receptor in vitro. Trophoblastic tissue from day 12 embryos was homogenized in TD buffer (10 mM Tris, 1 mM dithiothreitol at pH 7.8, 4 degrees C) with 30% glycerol (v/v) (TDG-30%) and/or 10 mM sodium molybdate (TDG-30% + Mo and TD + Mo, respectively). Receptor was partially purified from cytosol by ammonium sulfate precipitation and incubated overnight (4 degrees C) with [3H]P +/- unlabeled progesterone in appropriate buffer to measure binding properties of the receptor under various buffer conditions. The effects of glycerol and sodium molybdate on sedimentation behavior of receptor in 5-20% sucrose gradients was studied in other experiments. Binding parameters were indeterminate when the receptor was incubated in the basic TD buffer only. However, in the presence of molybdate and/or glycerol high affinity binding was maintained at 0 and 15 degrees C (Kd congruent to 2 nM). In TD + Mo media high affinity binding was lost (Kd = 14 and 35 nM at 0 and 15 degrees C, respectively). The receptor always sedimented as 4-5 S forms unless molybdate was present whereupon they were replaced by 7-8 S moieties. Under conditions of high salt (0.3 M KCl) high affinity was preserved by glycerol, as in low salt conditions, and a new 6-7 S moiety occurred; in addition to this form, the 7-8 S aggregate was retained in the presence of molybdate. Thermodynamic studies showed that the addition of molybdate to glycerol media did not alter the energy of activation for progesterone-receptor interaction measured at 0 and 15 degrees C; however, at 30 degrees C molybdate was necessary to prevent denaturation of receptor. Glycerol and molybdate must be present from the time of tissue extirpation for maximum stabilization. Denaturation begins immediately and is only partially reversible. It is concluded that glycerol interacts with the surface of the receptor molecule in such a way as to favor the folded native state which preserves high affinity interactions whereas molybdate interacts directly with receptor to maintain 7-8 S aggregates which increases the availability of binding sites.