Thomas F. Ogle

Regression of the Decidualized Mesometrium and Decidual Cell Apoptosis Are Associated with a Shift in Expression of Bcl2 Family Members

Abstract 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-action in the decidual mesometrium of pregnancy☆

The mesometrial decidua is absolutely dependent on progesterone action for its maintenance and growth. Hormone action is mediated by intranuclear progesterone receptors (PR) that regulate target cell gene transcription. In early pregnancy of the rat gene expression is particularly enhanced for regulators of cell cycle progression, growth factors and their cognate receptors; cell cycle arrest proteins are suppressed. Cell survival proteins such as Bcl2 are also up-regulated. These events are associated with abundant expression of PR-A and PR-B isoforms and STAT (signal transducers and activators of transcription) family members. Proliferation of decidual cells no longer occurs after mid-pregnancy despite high levels of circulating progesterone and the decidua begins a slow process of regression, which continues to term. Regression is characterized by an increase in abundance of proteins that promote apoptosis such as p27, Bax and Caspase-3. These late pregnancy changes are associated with a relative increase in PR-C, a third form of the PR molecule, that binds progesterone but probably has limited transcriptional activity. Protein kinase C, which is suppressed by progesterone in early pregnancy, may be a key mediator of these processes.

Signal Transducer and Activator of Transcription 3 Is Expressed in the Decidualized Mesometrium of Pregnancy and Associates with the Progesterone Receptor Through Protein-Protein Interactions

Abstract Progesterone is known to enhance epidermal growth factor (EGF)-mediated cellular responses by up-regulating EGF-receptor (EGF-R) expression. Ligand activation of EGF-R in turn has been shown to activate cytoplasmic stores of the STAT3 (signal transducer and activator of transcription) transcription factor, whereupon it translocates to the nucleus. The aim of this study was to examine the ontogeny of STAT3 protein expression in the decidualized mesometrium (i.e., decidua basalis) of the rat during pregnancy and its interactions with the progesterone-progesterone receptor (PR) system. STAT3 was abundantly expressed in the cytosolic fraction of decidual homogenates throughout pregnancy; however, expression in the particulate fraction (assumed to reflect primarily nuclear accumulation) was reduced more than 75% on Days 12–17 than it was on Days 8 and 10. This pattern of expression parallels the decline in EGF-R and coincides with decidual regression. Treatment of pregnant rats with antiprogestin (RU486) in early pregnancy resulted in an 80% reduction in cytosolic abundance of STAT3 within 12 h, but it had no influence on STAT3 abundance in the particulate fraction. Immunoprecipitation of decidual lysates with PR or STAT3 antibodies resulted in coprecipitation of STAT3 and PR, respectively. These observations suggest that STAT3 expression is a prevalent feature of progesterone action, and that STAT3 and PR interactions represent a convergence of diverse signal transduction pathways in the decidualized mesometrium during pregnancy.

Steroid-binding specificity of the progesterone receptor from rat placenta☆

We have attempted to delineate some salient features of the progesterone binding site of the cytosol progesterone receptor (Rp) in rat placenta by studying the binding profile of various chemical modifications of the progesterone molecule (P). The relative competition ratio (RCR) was used to calculate the relative affinity of P and the modified ligand for receptor (Kaprog/Kainh). Cortisol exhibited no appreciable binding. Other corticoids (corticosterone, deoxycorticosterone, 11 beta-hydroxyprogesterone) had relative affinities 10-30-fold lower than P. Alterations in the structure of P which caused extensive declines in relative binding affinity (i.e. greater than or equal to 100-fold) include: reduction of A-ring to the 5 alpha-stereoisomere (A/B trans), introduction of a 17 alpha-hydroxyl group greater than removal of C17 side chain greater than reduction of C20 carbonyl. The binding profile of the rat placental Rp was similar to that described for uterine Rp from other species indicating a high degree of conservation of molecular structure for the progesterone receptor binding site from species to species.

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.

Effects of pregnancy on steady-state kinetics of 21-hydroxylase and 11β-hydroxylase in the rat adrenal gland

The effects of pregnancy on the steady-state kinetic constants of 21-hydroxylase and 11β-hydroxylase were studied in rat adrenal microsomes and mitochondria, respectively. Progesterone and 11-deoxycorticosterone (DOC) served as substrate for the respective assays. The apparent KM values for 21-hydroxylase were lower on the mornings of proestrus (12 ± 1 μM), day 22 (D22) of pregnancy (10 ± 2 μM) and on the first day post-partum (12 ± 2 μM) than on the mornings of days 5 (D5) and 12 (D12) of pregnancy, 31 ± 6 and 28 ± 6μM, respectively (P < 0.01). Enzyme activity was not lower at D5 or D12 than at proestrus or post-partum. Vmax attained highest value at post-partum. No significant differences were observed in the apparent Km for 11β-hydroxylase (1.9 to 5.4 μM). However, Vmax was higher on proestrus and post-partum than during pregnancy (P < 0.05). S.A. of the enzyme was not altered, although total adrenal content of enzyme activity was greatest post-partum (P < 0.01). 21-Hydroxylase at D5 and D12 shows characteristics of mixed inhibition relative to the post-partum enzyme. On the other hand the proestrus and D22 enzyme exhibits characteristics of non-competitive inhibition relative to the post-partum enzyme, 11β-Hydroxylase exhibits characteristics of non-competitive inhibition at D5, D12, and D22 relative to the proestrus and post-partum enzyme. These findings suggest the presence of either a single effector which may change in concentration at D5 and D12 or multiple effectors which act simultaneously to decrease apparent affinity of enzymes for substrate and decrease Vmax. These changes seem to account for the 80–85% decline in plasma corticosterone levels observed in resting rats during mid-pregnancy.

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.

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.

Characteristics of an Endogenous Inhibitor of Progesterone Binding in Rat Trophoblast

This study investigates the existence of an endogenous inhibitory substance (I) in the cytosol of rat trophoblast that acts to decrease the affinity of progesterone (P) for the progesterone receptor (PR). The kinetic behavior of I at several reproductive stages and its separation from the cytosol PR are reported.

The effects of enzyme concentration on the kinetic behavior of adrenal 21-hydroxylase and 11β-hydroxylase in the pregnant rat

Experiments were designed to study the kinetic behavior of 21-hydroxylase and 11beta-hydroxylase as a function of enzyme concentration (Et) during proestrus, dasy 5 (D5), 12 (D12), and 22 (D22) of pregnancy, and within 24 h post-partum. The enzymes were prepared from rat adrenal microsomes and mitochondria, respectively. The experiments consisted of measuring the initial velocity of each reaction for a series of substrate concentrations at three fixed Et. Double reciprocal plots were constructed and the slope (Km/Vmax) of each line estimated. Variation in the value of the slope as a function of enzyme dilution would predict the presence of an endogenous effector. The kinetic behavior of 21-hydroxylase was not altered throughout the range of Et (10-100 microgram protein) at any of the reproductive stages. In contrast, kinetic behavior of 11beta-hydroxylase was clearly dependent upon Et. Dilution of the enzyme preparation (25-200 microgram of protein) increased the slope of the double reciprocal plot at all reproductive stages, thus suggesting that an activator substance may be present within the mitochondrial preparation. A secondary plot of the slope (Km/Vmax) versus Et described a power function (Km/Vmax = a [Et]b) with the greatest rate of change in Km/Vmax occurring at low values of Et. The rate of change in Km/Vmax per mg rise in mitochondrial protein at all dilutions of enzyme was greatest for proestrus and post-partum, followed by D22 greater than D12 greater than D5. In addition, repeated washing of the enzyme preparation at 4 degrees C increased Km/Vmax to a greater extent at all Et than did the control preparation. These findings suggest the presence of a diffusible endogenous activator of 11beta-hydroxylase whose influence decreases markedly at D5 and D12. On the other hand, there is no evidence to suggest the presence of a diffusible endogenous effector for 21-hydroxylase.