Effect of prostaglandins on in vitro synthesis of progesterone and oestradiol-17β in placenta from early human pregnancy

In vitro synthesis of progesterone and estradiol-17β from endogenous precursors was studied in the placenta from women in early stage of gestation (< 7 weeks). Radioimmunoassay techniques were used to measure progesterone and estradiol-17β.It was shown that placental tissue from as early as six weeks of gestation can synthesize both progesterone and estradiol-17β in vitro. Prostaglandins F_2α and E₂ in concentration of 100 μg/ml of the incubation media did not have any significant effect on the in vitro synthesis of progesterone and estradiol-17β in the placental tissue.It seems unlikely that the abortifacient effect of natural prostaglandins PGE₂ and PGF_2α is due to their direct action on the synthesis of progesterone and estradiol-17β in the placenta.     

Differential hormonal regulation of leukemia inhibitory factor (LIF) in rabbit and mouse uterus

Leukemia inhibitory factor (LIF) has been shown to be essential for the implantation of mouse blastocysts. The present study was designed to determine how LIF protein was hormonally regulated in rabbit and mouse uterus using immunohistochemistry. In unmated rabbits, LIF protein was at a low level in the uterine epithelium and glands, and up-regulated by progesterone alone or estradiol-17β and progesterone combined. Estradiol-17β alone had no apparent effect. In ovariectomized mice, the level of LIF protein was very low in the uterine epithelium and glands, and was up-regulated by estradiol-17β alone or estradiol-17β and progesterone combined. Progesterone alone had no apparent effect. These results suggest that LIF protein is differentially regulated in rabbit and mouse uterus by progesterone and estrogen, respectively. This would explain the high level of LIF protein observed in uterine epithelium and glands prior to blastocyst implantation in the two species with different hormonal requirements for implantation. © 1996 Wiley-Liss, Inc.     

The effect of estradiol-17β and actinomycin D on the release of PGF and PGE from separated cells of human endometrium

Estradiol-17β selestively stimulated the release of PGF from separated glandular but not stromal cells of human secretory endometrium (p<0.025) but had no effect on PGF release from either type of cells obtained from proliferative endometrium. PGE release was not affected by estradiol-17β. Actinomycin D did not antagonise the effect of estradiol-17β on PGF release from secretory, glandular cells. Basal release of PGF from these cells was stimulated by actinomycin D alone (100 ng/ml) (p<0.025) and PGE release stimulated in the presence of estradiol-17β. Actinomycin D had no effect on PGF or PGE release from proliferative endometrium. These findings suggest that estradiol-17β stimulates PGF release by a mechanism that does not affect PGE release and which is not dependent on the synthesis of new protein. The basal release of PGF and PGE by glandular cells of secretory endometrium in vitro is regulated by protein/proteins which reduce PG release.     

Release of prostaglandin F, regression of corpora lutea and induction of premature parturition in goats treated with estradiol-17β

Premature parturition was induced in five pregnant goats infused intravenously with 4.65–8.4 mg estradiol-17β but not in one treated with 5.85 mg estradiol-17α. A single intramuscular injection of 12 mg estradiol benzoate (8.8 mg estradiol-17β equivalents) was also effective. These doses were estimated to provide plasma concentrations of estradiol-17β in the physiological range for animals at spontaneous parturition. Circulating plasma concentrations of progesterone decreased and lactogenesis occurred before all instances of induced parturition but no such changes resulted from infusion of estradiol-17α. Placental delivery was normal in all animals but neonates of more than 10 days prematurity were non-viable.In three of the five goats infused with estradiol-17β, evidence was obtained for release of F-group prostaglandins from the uterus at the time of onset of the decline in progesterone.     

Considerations into the mechanism of estrogen-stimulated uterine prostaglandin synthesis

Progesterone priming of the ovariectomized rat, followed by a single injection of estradiol-17β (10 μg) is followed by an increased uterine synthesis of both PGF and PGE. The administration of an estrogen antagonist (MER-25; 10 mg) concomitantly with estradiol had no effect on uterine prostaglandin synthesis. Similarly, the administration of either Actinomycin D or cycloheximide, antibiotics demonstrated to inhibit mRNA and protein synthesis, respectively, is without effect on estrogen-stimulated uterine prostaglandin synthesis. These results are considered with regard to the classic receptor theory of estrogen action and are a preliminary indication that estrogen-stimulated uterine prostaglandin synthesis may not require those receptor mediated events.     

Uterine growth responses of the mature castrate rat to estradiol-17B

To examine estrogen-stimulated uterine growth we have monitored changes in uterine DNA synthesis, ornithine decarboxylase (ODC) activity and protein content as well as luminal epithelial (LE) cell mitotic index and ultrastructural changes. We have utilized this model to examine castrate mature rat uterine growth as a function of time between 18 and 40 hours following a single injection of 25.0 ug of estradiol-17B. LE cell mitotic index and protein content increases were maximally elevated as early as 18 hours postinjection while uterine ODC activity was maximal at 28 hours; uterine DNA synthesis increases continued throughout the experiment. In addition, the infusion of either 1 or 2 ug E2 plus progesterone over a 24 hour period, stimulated elevated ODC activity under both treatment regimens and LE cell mitotic index which was inversely related to E2 dose.     

Effect of 15(S)15-methyl-PGF2α-methyl ester vaginal suppositories on circulating hormone levels in early pregnancy

Intravaginal administration of 15-methyl-PGF_2α-methyl ester in the form of suppositories terminated pregnancy in 70 percent of the cases whose last menstrual periods ranged from 35 to 56 days. The use of these suppositories in 49 patients, between 57 to 80 days of gestation, dilated the cervix by 10 mm or more, in one hundred percent of the cases. A decrease in circulating levels of estradiol-17β and progesterone was observed following 15-methyl-PGF_2α administration. The mean estradiol-17β levels declined by about 55.9 percent at 9 hours whereas, the corresponding fall in progesterone was 32.7 percent. This was indicative of a direct action of 15-methyl-PGF_2α on the corpus luteum. The vaginal use of 15-methyl-PGF_2α-methyl ester suppositories thus appears to be a promising method for the termination of early pregnancy and for pre-operative cervical dilatation. The termination of early pregnancy appears to be partly due to the luteolytic effect of 15-methyl-PGF_2α besides stimulating uterine contractions.     

Early effects of oestradiol-17β on the chromatin and activity of the deoxyribonucleic acid-dependent ribonucleic acid polymerases (I and II) of the rat uterus

Oestradiol-17β (1.0μg) was injected intravenously into ovariectomized rats. The earliest detectable hormonal response in isolated uterine nuclei was an increase (10–15min) in RNA polymerase II activity (DNA-like RNA synthesis), which reached a peak at 30min and then decreased to control values (by 1–2h) before displaying a second increase over control activity from 2 to 12h. The next response to oestradiol-17β was an increase (30–60min) in polymerase I activity (rRNA synthesis) and template capacity of the chromatin. The concentrations of acidic chromatin proteins did not begin to increase until 1h after injection of oestradiol-17β and histone concentrations showed no significant changes during the 8h period after administration. The early (15min) increase in RNA synthesis in `high-salt conditions' can be completely eliminated by α-amanitin, an inhibitor of the RNA polymerase II. The exact nature of this early increase in endogenous polymerase II activity remains to be determined, e.g. whether it is caused by the increased availability of transcribable DNA of the chromatin or via direct hormonal activation of the enzyme per se.     

Oxytocin-stimulated production of prostaglandin F2α by isolated endometrium of rabbit: Modulation by ovarian steroids

To test the hypothesis that ovarian steroid hormones modulate oxytocin-induced release of prostaglandin F_2α (PGF_2α) from uterine endometrium, 2 ovariectomized rabbits were pretreated with progesterone (5 mg/day for 10 days), 2 with estradiol-17β (25 μg/day for 10 days), 2 with both steroids, and one with sesame oil only. On the last day of treatment, endometrial fragments were excised and incubated in vitro with or without oxytocin (100 μU/ml). Although endometrium from rabbits pretreated with combined steroids released more PGF_2α immediately after excision than did tissue from animals pretreated with either steroid by itself, endometrium from animals pretreated with estrabdiol-17β alone released the most PGF_2α during sustained incubation in vitro. Moreover, only this tissue exhibited significant oxytocin-dependent release of PGF_2α. At the dosages used, progesterone completely antagonized both of these effects of estradiol-17β. The results support the hypothesis that ovarian steroid hormones regulate oxytocindependent release of PGF_2α from endometrial cells. A possible mechanism of action is suggested.     

Hormone changes occurring during second trimester abortion induced with 15(S)-15-methyl prostaglandin F2α

Changes in progesterone, human placental lactogen (HPL), cortisol and estradiol-17β were measured during second trimester abortion induced by I.M. 15-methyl PGF2α. A rapid decline in progesterone and HPL was found, indicating perhaps an initial effect on the placenta. A rapid rise in cortisol was found, but it is not clear if this is due to stress or part of the termination mechanism. The changes of estradiol were not as distinct and may reflect opposite effects of the prostaglandin on the placenta and adrenals. Similar hormonal changes were observed regardless of the duration of gestation.     

Prostaglandin E2 in human gingiva in health and disease and its stimulation by female sex steroids

The levels of prostaglandin E₂ were measured by means of radioimmunoassay in 12 clinically normal and 24 chronically inflamed human gingival tissue homogenates. The average values were found to be 16 and 285 pmol/gm wet weight of the normal and inflamed samples, respectively. Addition of estradiol-17β alone or a mixture of estradiol-17β and progesterone to incubation media containing normal or inflamed gingiva caused a significant increase in the synthesis of endogenous PGE₂. Increased levels of female sex steroids during pregnancy may enhance gingival inflammation.     

Immunohistochemical detection of progesterone receptors in the canine uterus and their relation to sex steroid hormone levels

The aim of this immunohistochemical study is to describe the normal distribution of progesterone receptors in the various cell types of the canine uterine horns, body and cervix. The results can be used for research on uterine and endocrinological pathology, since the impact of progesterone on different uterine cell types is partly determined by the receptor availability. Nuclear staining for progesterone receptors was observed in epithelial cells of the surface epithelium, glandular ducts and basal glands of the endometrium, in endometrial stroma cells and in myometrial smooth muscle cells. This staining was positively correlated with the estradiol-17 beta:progesterone ratio, and reflects the positive effect of estradiol-17 beta and the negative influence of progesterone on the receptors. Staining scores were high during proestrus and decreased through estrus to early metestrus. In late metestrus, staining scores of the stromal and smooth muscle cells increased again. In anestrus, high scores of the surface-epithelial cells contrasted with minimal scores of the basal glands. This finding suggests a different hormonal regulation of the progesterone receptor expression in both epithelial cell groups. The higher staining intensities for progesterone receptors in stromal cells compared with epithelial cells might be explained by the fact that stromal cells mediate some effects of steroid hormones on the epithelial cells in the genital tract. Therefore, the role of stromal cells in regulation of the cyclic endometrial changes and in pathologic changes of uterine tissue should not be underestimated.     

Estrogens and cell death in murine uterine luminal epithelium

Summary The luminal epithelium of adult ovariectomized mice responds to estradiol-17 with a synchronised wave of DNA synthesis and mitosis. Estriol, however, although producing a similar DNA-synthetic and mitotic response fails to cause an increase in cell number owing to a wave of cell death occurring at mitosis. In the present study it was shown that cells died by two different routes. The majority died by apoptosis but, unusually, a minority also died by necrosis. In the apoptotic cells the cytoplasm became dense, the endoplasmic reticulum and nuclear cisternae dilated; chromatin became marginated the nucleus shrank and became deeply infolded and contorted. Apoptosis, however, was uncharacteristic in that the nucleus failed to fragment, form caps or show disruption before the cells died by membrane rupture. Furthermore, the cells were frequently lost in sheets from the epithelium into the lumen. Part of the biochemical explanation for this onset of cell death comes from the accelerated loss from the tissue of estriol when compared to estradiol-17. This resulted in a decline in protein and rRNA biosynthesis and a failure to complete ribosomal maturation. Evidence in favour of this explanation came from experiments that showed a return to the estradiol-17 level of response and an inhibition of cell death when the occupancy of the estriol receptor was maintained.     

Endogenous peroxidase: specific marker enzyme for tissues displaying growth dependency on estrogen

Data derived from a correlated morphological and biochemical study suggest the following: (a) estradiol-17beta, diethylstilbestrol, the estrogen antagonists nafoxidine (Upjohn 11,000), and Parke Davis C1628 induce synthesis of an endogenous peroxidase in the epithelium of target tissues like the vagina, the cervix, the uterus, and in the acinar cells of the estrogen-dependent rat mammary tumor; (b) peroxidase is a "specific" secretory protein of the estrogen-sensitized uterine endometrium; (c) peroxidase synthesis is not a nonspecific response to steroid hormone action, since progesterone and testosterone do not induce its synthesis; (d) endogenous peroxidase is a possible diagnositc protein for the detection of estrogen-dependent growing tissues, including breast cancer; (e) movement of exogenous horseradish peroxidase from the interstitium to the uterine lumina is restricted by tight junctions located at the apices of epithelial cells. Estrogen and antagonists do not appear to influence the transepithelial movement of exogenous peroxidase into the lumen.     

Role of prostaglandins in development of porcine blastocysts

Rapid elongation of porcine blastocysts between Days 11 to 12 of pregnancy coincides with an increase in uterine luminal content of prostaglandins. The present study evaluated the effect of two prostaglandin synthesis inhibitors (indomethacin and flunixin meglumine) on elongation of porcine blastocysts from spherical to filamentous forms between Day 11 to 12 of pregnancy. Gilts were hemi-hysterectomized on Day 11 of prenancy. The excised uterine horn was flushed with 0.9% saline and diameter of blastocysts recovered were measured. Immediately following surgery, pregnant gilts were assigned to receive either: 1) vehicle every 4 h, 2) flunixin meglumine (banamine) every 4 h, or 3) indomethacin every 12 h. The remaining uterine horn was removed and flushed after the time of blastocyst elongation estimated for each gilt on basis of blastocyst development in the first horn. Uterine flushings were analyzed for total calcium, protein, acid phosphatase activity, estrone, estradiol-17β and prostaglandin F. Pretreatment blastocyst diameter was similar for all groups and ranged from 1 mm to 20 mm. Treatment of gilts with either banamine or indomethacin effectively inhibited (P<0.001) the increase in uterine luminal content of PGF. Total calcium, estrone and estradiol-17β were not influenced by treatment. Total uterine luminal protein and acid phosphatase activity were reduced (P<0.05) in banamine treated gilts compared to those receiving vehicle or indomethacin treatments. Although total PGF recovered in uterine flushings was reduced during the period of blastocyst elongation, treatment with PGF synthetase inhibitors failed to block rapid elongation of blastocysts from the spherical to filamentous forms.     

Inhibition of uterine prostaglandin-F2α production by bovine conceptus secretory proteins

The effect of bovine conceptus secretory proteins (CSP) on uterine prostaglandin (PG)-F_2α production was evaluated in dairy cattle following injection of estradiol-17β. Intrauterine injections of dialyzed serum proteins (Control, n=5) or CSP (n=5) were administered from days 15 through 18 post-estrus. Following intrauterine treatments on day 18, all cows were injected with E₂ (3 mg) to stimulate uterine PGF_2α production. Plasma concentrations of progesterone (P₄) and 15-keto-13,14-dihydro-PGF_2α (PGFM) were determined by RIA. The PGFM responses following E₂ challenge were decreased (p<0.01) for cows receiving CSP versus serum proteins into the uterine lumen. Individual PGFM, P₄ and cycle length responses are discussed. Data suggest that proteins secreted by the bovine conceptus suppress uterine PGF_2α production during pregnancy recognition in the cow.     

Influence of sex hormones on prostaglandin dehydrogenase activity in the rat uterus

The present experiments report the effects of estradiol or of progesterone on the activity of 15-prostaglandin-dehydrogenase (PGDH) in the uterus of spayed rats. When the substrate was PGF_2α the treatment with progesterone (4 mg.day⁻¹, two days) or with estradiol-17-beta (0.5 ug + 1 ug) did not show any effect on the activity of the enzyme. On the contrary, uteri from ovariectomized rats injected with a higher dose of estradiol- 17-beta (0.5 ug + 50 ug) exhibited a significant increment. When the substrate was PGE₂, progesterone failed again to modify the enzyme activity, whereas estradiol, both at a low and at a high doses, enhanced significantly the uterine PGDH activity. The possibility of two different PGDHs for each PG and the role of estradiol in enhancing PGE₂ catabolism into 15-keto- PGE₂ as a mechanism subserving the effect of estrogens on the output of this PG in the rat uterus, are discussed.     

Steroidal control of rat uterine 17β-hydroxysteroid dehydrogenase activity

The interconversion of estradiol-17β and estrone in the rat uterus is due to the action of 17β-hydroxysteroid dehydrogenase. Whole uteri or 800 × g supernatant fractions of the uteri were incubated in the presence of [³H] estradiol-17β and NAD at 37°C for 3 h or 1 h, respectively. In the mature rat uterus the oxidation of estradiol-17β and estrone was dependent on the stage of the estrous cycle, suggesting hormonal control. The 17β-hydroxysteroid dehydrogenase activity was highest at estrus (200 fmol estrone) and lowest at diestrus (80 fmol estrone). An enhancement of activity occurred when adult rats at each stage of the estrous cycle were administered estradiol-17β, while progesterone administration at each stage resulted in decreased enzyme activity. The uterine 17β-hydroxysteroid dehydrogenase activity of estradiol-17β treated ovariectomized rats was time and dose dependent but decreased when progesterone was administered with or without estradiol-17β administration. These results suggest that estradiol-17β caused an increase in enzyme activity that was inhibitable by progesterone in the rat uterus. The increased 17β -hydroxysteroid dehydrogenase activity may reflect a specific response of the rat uterus to estradiol-17β.     

Steroidal control mechanism of cell proliferation in mouse uterine epithelium

The percentage of labeled cells in the uterine luminal epithelium of cycling mice showed the different zonal distributions at each stage of estrous cycle after cumulative labeling with 3H-thymidine for 36 hr. It was estimated that the proliferating fraction in the epithelium at proestrus, estrus, metestrus, and diestrus was 100%, 100%, 40% and 5%, respectively. The percentage of labeled cells in the uterine luminal epithelium of cycling mice treated with progesterone remained below 10% level for at least 20 hr after injections of progesterone. Total labeling was attained in the uterine epithelium of castrated mice by the administration of estradiol-17beta. On the other hand, the cell proliferation in the uterine epithelium of castrated mice treated with estradiol and progesterone was markedly suppressed and the percentage of labeled cells remained approximately at 35%. The remaining cell population, however, still showed the mitotic potency when mice received estradiol. It is suggested from this study that the effect of progesterone is to suppress the epithelial cell proliferation and transfer cells into resting cell fraction which is still evoked to proliferate as the effect of estradiol and that a key factor controlling epithelial proliferation in mouse uterus during the estrous cycle is proliferating fraction rather than cell cycle time.     

The synthesis and study of some potential affinity labeling reagents for estrogen receptors

The influence of the following affinity labeling reagents on the binding of tritiated estradiol-17β(E) by human and calf uterine cytosols was studied: 11β-chloromethylestradiol (ORG4333), 2-azidoestradiol (2A-E), 4-azidoestradiol (4A-E), 3-azidohexestrol (3A-H), estradiol-17β 17-bromoacetate (E-17BrAc), 6-[o-carbo-(2'-chloroethoxy)methyl]oximinoestradiol (6-CMOEtCl), 17-[o-carbo-(2'-chloroethoxy)methyl]oximinoestrone (17-CMOEtCl), 2-di (2'-hydroxy-3'-chloropropyl)aminoestradiol (E-Mustard). For the human uterine estrogen receptor the relative binding affinity decreased in the order E > ORG 4333 > E-17BrAc > 3A-H > 2A-E > 4A-E > 6-CMOEtCl > E-Mustard > 17-CMOEtCl. The binding characteristics of the calf uterine estrogen receptor were qualitatively similar, but quantitatively different. ORG 4333 appeared to form a highly stable association with the receptors, but alkylation of the protein could not be conclusively demonstrated.