Progesterone withdrawal and RU-486 treatment stimulate apoptosis in specific uterine decidual cells

Progesterone secretion is required for the growth and differentiation of endometrial stromal cells to form decidual cells. For many cells where a growth factor supports cell growth and proliferation, withdrawal of the growth factor initiates apoptosis. This study determined the time course and tissue location of apoptosis in deciduomal tissue after withdrawal of progesterone or injection of the antiprogestin, RU-486. Total DNA was isolated from decidual tissues at intervals after experimental treatments and separated electrophoretically. Internucleosomal DNA fragmentation characteristic of apoptosis was measured by quantitating levels of the 200 bp fragment. Apoptotic cells in tissue sections were detected by direct immunoperoxidase detection of digoxigenin-labeled DNA. Decidual apoptosis reached maximal levels at 12 h after withdrawal of progesterone or injection of RU-486. Increased concentrations of apoptotic cells were observed at the periphery of the growing deciduoma and in the antimesometrial deciduoma near the luminal epithelium after both treatments. These results suggest the withdrawal of progestin initiates apoptosis in cells at the early stages of decidualization.     

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.     

Temporal relationships among uterine pituitary adenylate cyclase-activating polypeptide, decidual prolactin-related protein and progesterone receptor mRNAs expressions during decidualization and gestation in rats

Pituitary adenylate cyclase-activating peptide (PACAP), a novel compound with vasoactive intestinal polypeptide-like activity, was recently shown to be localized in the neuronal endings of the human uterus. The purpose of the present study was to assess the functional presence of PACAP mRNA in the decidual endometrium and its relationship to the expression levels of decidual prolactin-related protein (dPRP) and the progesterone receptor mRNAs during decidualization and pregnancy in Sprague-Dawley rats. PACAP was constitutively and temporally expressed in the decidual endometrium and gravid uterus. The time-dependent correlated expression levels of PACAP, dPRP and the progesterone receptor were induced by the neurogenic reproductive signals, i.e. the vagino-cervical/deciduogenic stimuli of decidualization and by the normal equivalent stimuli of mating/blastocyst implantation of gestation. Correlation among the mRNA expression levels of PACAP, dPRP and the progesterone receptor and the coordinated inhibitory actions of the anti-progesterone (RU-486) suggest that there is also correlated time-dependent steroid regulation of the mRNA levels of PACAP, dPRP and the progesterone receptor in the decidual and pregnant uteri. One possible functional meaning for the time-related localization of endometrial/uterine PACAP could be to facilitate endometrial blood flow and increase the availability of metabolic substrates to the developing deciduoma or embryo. The study demonstrates the potential importance of PACAP expression in the regulation of the maternal feto-placental component and suggests a prominent reproductive role for the neuropeptide in mammalian pregnancy.     

Progesterone and DNA damage encourage uterine cell proliferation and decidualization through up-regulating ribonucleotide reductase 2 expression during early pregnancy in mice

Embryo implantation into the maternal uterus is a crucial step for the successful establishment of mammalian pregnancy. Following the attachment of embryo to the uterine luminal epithelium, uterine stromal cells undergo steroid hormone-dependent decidualization, which is characterized by stromal cell proliferation and differentiation. The mechanisms underlying steroid hormone-induced stromal cell proliferation and differentiation during decidualization are still poorly understood. Ribonucleotide reductase, consisting of two subunits (RRM1 and RRM2), is a rate-limiting enzyme in deoxynucleotide production for DNA synthesis and plays an important role in cell proliferation and tumorgenicity. Based on our microarray analysis, Rrm2 expression was significantly higher at implantation sites compared with interimplantation sites in mouse uterus. However, the expression, regulation, and function of RRM2 in mouse uterus during embryo implantation and decidualization are still unknown. Here we show that although both RRM1 and RRM2 expression are markedly induced in mouse uterine stromal cells undergoing decidualization, only RRM2 is regulated by progesterone, a key regulator of decidualization. Further studies showed that the induction of progesterone on RRM2 expression in stromal cells is mediated by the AKT/c-MYC pathway. RRM2 can also be induced by replication stress and DNA damage during decidualization through the ATR/ATM-CHK1-E2F1 pathway. The weight of implantation sites and deciduoma was effectively reduced by specific inhibitors for RRM2. The expression of decidual/trophoblast prolactin-related protein (Dtprp), a reliable marker for decidualization in mice, was significantly reduced in deciduoma and steroid-induced decidual cells after HU treatment. Therefore, RRM2 may be an important effector of progesterone signaling to induce cell proliferation and decidualization in mouse uterus.     

Effects of ethanol on the decidual cell reaction in rats.

The effects of ethanol on uterine sensitivity to induction of decidualization and deciduoma growth were determined. Rats were ovariectomized, given an oestrogen-progesterone regimen to optimize induction and growth of deciduoma and randomly assigned to one of three ethanol treatment groups: (i) days 1-4 (pre-induction/period of sensitivity), (ii) days 5-9 (post-induction/period of growth), (iii) days 1-9 (periods of sensitivity and growth); or to a control group not treated with ethanol (pair-fed to treated groups). Ethanol (0, 1, 2, or 4 g kg-1) diluted in water was administered by stomach tube on the days prescribed. Decidualization was induced in one uterine horn by intraluminal injection of sodium phosphate buffer. Uterine sensitivity and decidual growth were assessed as cornu weight. Blood alcohol concentrations were measured by gas chromatography. Alcohol treatment reduced uterine sensitivity, but increased deciduoma growth. Blood alcohol concentrations rose to 133 mg% at 30 min, remained high for 90 min and declined to 82 mg% at 120 min. Thus, blood alcohol concentrations sufficient to induce mild intoxication in humans suppressed uterine sensitivity to decidualization and enhanced deciduoma growth in rats. As all ovarian steroid hormone support was exogenous, the effects of ethanol on deciduoma induction and growth were not due to alterations in the hypothalamic-pituitary-ovarian axis.     

Prostaglandin synthesis in decidual tissue of the rat uterus

Prostaglandin (PG) synthetase activity and tissue concentration were measured in unilateral deciduomata induced by traumatization of the pseudopregnant rat uterus and in the decidua of pregnancy. PG synthetase activity per unit weight of deciduoma tissue was 7–10 fold higher, throughout the life-span of the deciduoma, than that in the untraumatized control horn. The concentration of prostaglandins of the E-type in the deciduoma exceeded that found in the control uterine horn by a factor of 10–20 on days 3–4 after decidual induction, and about five-fold on days 9–10. The concentration of prostaglandins of the F-type in the deciduoma measured on days 4 and 8 did not differ significantly from that in the control horn.

In the decidua of pregnant rats, both PG synthetase activity and PGE content were 20–40 times higher than the corresponding values for the myometrium of the same horn. The physiological role of the high level of prostaglandin production in decidual tissue requires further investigation.     

Involvement of apoptosis during deciduomal regression in pseudopregnant hamsters effect of progesterone

We determined whether fragmentation of genomic DNA, apoptosis, occurs during deciduomal regression in pseudopregnant hamsters and the effect of progesterone on the apoptotic processes. Artificially induced deciduoma were obtained on different days of pseudopregnancy and separated into mesometrial and antimesometrial tissues. The deciduomal cell cycle progression and population profiles of both sides were compared by flow cytometry. The proportion of sub-G1 peak, which was correlated with the apoptotic cells, were about 10% on day 8 and reached to 40% in both tissues on day 10. Exogenous progesterone treatment by daily injection (2 mg; s.c.) on and after day 8 reduced the percentage of low molecular weight DNA in both tissues on day 10 and day 12 as compared to the nontreated control one, respectively. The appearance of DNA ladder was also delayed at least 24 h by progesterone administration. The intensity of DNA fragmentation was more pronounced in antimesometrial deciduoma. In situ 3'-end labeling of apoptotic cells further substantiated the apoptotic process. The apoptotic cells first appeared in the luminal region in antimesometrial deciduoma on day 8 and spreaded all over the entire deciduomal tissue on day 10. Progesterone treatment stimulated deciduomal proliferating cell nuclear antigen (PCNA) expression, maintained deciduoma until day 14 and retarded the differentiation and regeneration of the uterine epithelium.     

Decidual cell reaction: relative response to traumatization in uterus of rat, hamster and guinea pig

Relative decidual cell response to traumatization was studied in the uterus of immature rat, hamster and guinea pig. A single dose of progesterone (1.0 mg/animal) caused 285 +/- 41 and 459 +/- 58% uterotrophy in traumatized horn of rat and hamster respectively, while in guinea pig increasing doses of progesterone (1, 2 and 5 mg/animal), several combinations of estrogen and progesterone or alteration in treatment schedule of the two hormones as well as extending the day of traumatization failed to elicit comparable uterotrophic response. Uterus of immature hamster was found to be most responsive to traumatization and that of guinea pig the least.     

Progesterone potentiating effect of Dipsacus mitis D. Don for its contraceptive action in hamster

A pure compound, isolated from ethyl acetate extract (root) of D. mitis D. Don, prevented pregnancy by 100% in adult female hamster but partially in rat when administered orally on Days 1-7 and 1-10 post-coitum respectively. The effective dose in both species was 150 mg/kg. Using uterine wet weight in ovariectomized immature rat as bioassay method, the compound was found to be devoid of estrogenic and antiestrogenic property. On examination for progestational and antiprogestational activity, using trauma-induced deciduoma formation in immature rat uterus as end points, the compound (per se) did not show the former activity but in a conjoint treatment with progesterone it augmented the action of latter. The compound was assumed to act by potentiating progesterone biosynthesis, the excess of which might be the cause for interruption of pregnancy in hamster. This is the first study to report contraceptive efficacy and mode of its action at the uterine level.     

Tissue spaces during development and regression of the decidual cell reaction in ovariectomized, steroid-treated mice

Changes in the extracellular and blood spaces of the uterus were assessed from the distribution volumes of 51Cr-EDTA and 51Cr-labelled red blood cells during the development and regression of the artificially induced decidual cell reaction in ovariectomized, steroid-treated mice. The normally high values for uterine extracellular space (0.35-0.40 microliter/mg) fell to less than 0.20 microliter/mg in association with decidual growth. Uterine blood space increased from around 0.02 microliter/mg to 0.03-0.05 microliter/mg with decidual development. Induction of decidual regression by removal of s.c. progesterone implants caused a rapid decline in tissue blood volume to reach control values (0.01-0.02 microliter/mg) within 24 h and preceded any reduction in uterine weight. Uterine vascular permeability, as determined from the tissue accumulation of 125I-labelled human serum albumin, fell with a similar time course. Tissue extracellular space returned to the higher control values within 48 h of initiating decidual regression.     

Serotonin-induced disruption of implantation in the rat: II. Suppression of decidualization

Subcutaneous injection of serotonin (20 mg/kg) on Day 5 of pregnancy disrupts implantation in the rat as indicated by the reduction in number of live fetuses/cornu present on Day 19 (0.9 vs. 6.1, treated vs. control). Such disruption of implantation possibly results from impaired decidualization. To test for suppression of decidualization, serotonin was administered to pseudopregnant rats on the day before, on (Day 4) or after artificial induction of the decidual cell reaction. Relative to saline-treated controls (C), serotonin (S) reduced decidualization when injected either before [C: 1987 +/- 130 vs. S: 1085 +/- 155 mg (Day 3); P less than 0.005] or after [C: 1987 +/- 130 vs. S: 173 +/- 8 mg (Day 5); P less than 0.001] administration of the deciduogenic stimulus. In addition, serotonin markedly decreased uterine blood flow (C: 0.47 +/- 0.05 vs. S: 0.25 +/- 0.06 ml/min per g; P less than 0.01) during pseudopregnancy. However, serotonin altered neither the duration of luteal function in pseudopregnant rats (C: 15.3 vs. S: 14.3 days) nor serum progesterone levels (C: 74-91 vs. S: 53-82 ng/ml) in pregnant animals. It is concluded that serotonin may disrupt implantation, in part, by suppression of decidualization. The loss of endometrial competence to undergo decidualization appears to be a consequence of serotonin-induced uterine ischemia rather than impaired corpus luteum activity.     

Replenishment of uterine estrogen receptor in chronically estrogenized rats

Replenishment of uterine estrogen receptor (ER) following a single injection of estradiol-17 beta (E2) was examined in chronically estrogenized rats. Subcutaneous implantation of E2-pellet for 7 days in ovariectomized rats resulted in a significant stimulation of uteri with regard to wet tissue weight, DNA content and progesterone receptor content, with a shift of ER distribution. An intraperitoneal injection of 5 micrograms E2 in the E2-implanted rats induced a significant decrease in soluble ER (from 141.1 +/- 12.6 to 69.2 +/- 8.8 fmol/mg protein) with a concomitant increase in nuclear ER (from 58.2 +/- 8.6 to 129.2 +/- 11.6 fmol/100 micrograms DNA) 1 h after the injection. However, soluble ER was rapidly replenished, which was accompanied by nuclear ER reduction, and both values returned to the pre-injection levels at 4 h after the injection. An administration of 150 micrograms cycloheximide, that effectively blocked protein synthesis in the uterus of the E2-implanted rats, completely inhibited the replenishment of soluble ER induced by 5 micrograms E2. These findings, combined with our previous findings that replenishment of ER following a single E2 administration in the pituitary of chronically estrogenized rats was inhibited by cycloheximide, suggest that replenishment of ER is entirely dependent on protein synthesis in chronically estrogenized rats.     

Estradiol binding components in intestinal mucosa of female rats.

The presence of estrogen binding components (EBC) in intestinal mucosa of female rats was investigated by competitive-binding assay using radiolabelled and nonlabelled estradiol 17 beta (E2). EBC were found exclusively in the nuclear fraction and were absent from the cytosolic and from the microsomal fractions. Two types of nuclear EBC with different binding characteristics and capacities were found: Kd1 = 4.8 +/- 0.8 nM, n1 = 18.4 +/- 4.2 fmol/mg protein (n1 = 83.4 +/- 12.5 fmol/mg DNA) and Kd2 = 31.1 +/- 6.8 nM, n2 = 91.1 +/- 18.5 fmol/mg protein (412.7 +/- 80.0 fmol/mg DNA). Type 1 component showed slightly greater affinity for estrogens as compared to progesterone and dexamethasone whereas type 2 component bound other competitors with even greater affinity than E2.     

Involvement of Adenosine in the Acute Action of Progesterone on Glucose Metabolism In Rat Adipocytes

Abstract

Progesterone and synthetic steroids (RU-5020, progestomimetic; RU-38486, anti-progesterone and anti-glucocorticoid) inhibit glucose netabolism in female rat adipocytes. The inhibitory effect, which is dose-related, occured after 20-min of incubation and is sustained during 2-hours of incubation. This effect of progesterone is similar to that observed during pregnancy and after treatment of ovariectomized rats with progesterone (5 mg/rat/day-15 days).     

Endogenous concentration of progesterone and 2 slpha -pregnane-3,20-dione in rat decidula tissue.

The endogenous concentration of progesterone (P) and 5 alpha-pregnane-3,20-dione (5AP) were determined in the dedicual tissue of mature rats by radiommunoassay. On the 4th day of pseudopregnancy the uterine concentration was 281 +/- 28 ng/g for P and 266 +/- 41 ng/g for 5AP. Horns were traumatized on the 4th day of pseudopregnancy. On the 4th day following trauma the endometrial concentration of P was 100 +/- 7 ng/g and for 5AP was 104 +/- 10 ng/g. Plasma concentrations were 35.5 +/- 4.1 ng/g. For P and 16.1 +/- 4.9 ng/ml for 5AP. When one horn was removed and the contralateral horn was traumatized, there was direct correlation between the endogenous concentrations of P and 5AP in the previously removed utraumatized horn and the amount of decidual tissue in the contralateral horn 4 days following trauma (r2 = .8949). These experiments indicate the endometrial response to progesterone is a complex process determined in part by local metabolsm and the ability of the uterus to concentrate P.     

Androgen binding by skeletal muscle nuclei

A study was made of 3H-19-nortestosterone binding by isolated nuclei and 0.4 M KCl nuclear extract of the rat skeletal muscle. Binding specificity was ascertained by incubation in the presence of various unlabeled steroids. The Kd values were measured for nuclei and 0.4 M KCl nuclear extract (11.6 +/- 2.5 nM and 9.9 +/- 1.6 nM, respectively). The amount of binding sites was 24.1 +/- 1.7 fmol/mg DNA or 13.7 +/- 1.0 fmol/g tissue. Enzymatic treatment with pronase and DNase shows that nuclear androgen receptors are proteins. DNA was noted to have a stabilizing effect. DNase treatment of nuclei during extraction with 0.4 M KCl was shown to significantly increase the amount of specifically bound radioactivity in the extract.     

Expression of adenosine deaminase and 5'-nucleotidase in artificially induced deciduoma in rat and hamster

Enzymes adenosine deaminase (ADA) and 5-nucleotidase (5-'NT) are known to play active role in tissue/cell proliferation and differentiation. To validate this the two enzymes were studied in artificially induced deciduoma of rat and hamster. The deciduoma was induced by traumatizing one of the uterine horns of progesterone primed animals. Non traumatized horn served as control. The animals were later maintained on progesterone, given alone (Gr.I) or conjointly with estrogen (Gr.II). The weight of each uterine horn was recorded to determine the formation of deciduoma. There was no marked difference between the weights of traumatized and control horn on day 2 post-traumatization (PT), but a progressive rise was noticed after this day in both species. The ADA activity however differed, day and species wise. While in the rats of Gr.I it was low in the traumatized horn on all the days, in the hamsters it was remarkably high from day 2 to 6 PT. In the rats of Gr.II also the activity though was low in the traumatized horn, but on day 2 and 4 only; on day 6 and 7 PT it increased markedly. In hamster, on the contrary, again the enzyme activity was remarkably high on all the three days. The 5'-NT activity, however, did not show any marked difference between the two horns under Gr.I and II in both species. It was rather high in the control horn of each group. The results suggest: (I) the progesterone alone though produces a significant rise in the uterine weight of traumatized horn in both species, the ADA activity increases only in hamster, (2) under the conjoint treatment also the enzyme activity remains high in hamster; and (3) the activity of enzyme 5'-NT does not alter during the deciduoma formation in both the species.     

Tamoxifen decreases progesterone and nuclear androgen receptors in the human prostate

Androgen (AR) and progesterone (PR) receptors were measured in resected prostate tissues of patients with benign prostatic hypertrophy. One group of patients received an anti-estrogen, tamoxifen (Tm 20 mg b.i.d.) for 10 days prior to prostate resection; a second group served as controls and were untreated. Plasma levels of Tm were 200-500 pmol/ml at the time of surgery. Statistically significant decreases (P less than 0.05) were found in cytosol PR (154 fmol/mg DNA +/- 33 SE in 14 Tm-patients vs 266 +/- 40 SE in 13 untreated patients) and in nuclear AR (103 fmol/mg DNA +/- 70 SE in 18 Tm-patients vs 257 +/- 62 SE in 17 controls). Cytosol AR was not significantly different in Tm-treated patients (257 fmol/mg DNA +/- 79 SE in 15 Tm-patients vs 346 +/- 130 SE in 17 controls, P greater than 0.6). Although receptor recycling is one of several possible explanations, these decreases in progesterone and nuclear androgen receptors in Tm-treated patients suggest that estrogen has a role in the biological regulation of steroid receptors in the human prostate.