Differential PGE2 and cAMP production by allogeneic and syngeneic pregnant mice uteri.

Prostaglandin E2 (PGE2) and cAMP production in allogeneic and syngeneic pregnant mice uteri, were measured in relation to the ratio of plasma estrogen/progesterone levels. PGE2 generation by allopregnant uteri varied with the days of pregnancy. The increment of the prostanoid coincided with the increase in plasma estrogen concentration, whereas the decrement of its production was in parallel with the increment of plasma progesterone. The syngeneic pregnant uterus was unable to increase the release of PGE2 above basal values during the whole pregnancy. The rise of PGE2 production by the allogeneic pregnant uterus was correlated with an increase in cAMP levels. It is proposed that the pregnant mouse uterus increases its ability to release PGE2 in response to an ovarian steroids.     

Prostaglandin production by the pregnant and non-pregnant rabbit uterus

We have studied the prostaglandin synthesis of the pregnant and non-pregnant rabbit uterus in a microsomal membrane preparation, and in an perfused uterus preparation which retains agonist stimulated prostaglandin production. In both the microsomal and isolated perfused system, prostacyclin was the major arachidonic acid metabolite produced; PGE₂ was also produced in substantial quantities while TxB₂ and PGF_2α were not detectable. Moreover, oxytocin was a specific stimulus of PGE₂ release. the steroid hormone milieu influenced the level of agonist stimulated prostaglandin release; in general, ovariectomized, estrogen treated animals were more responsive to agonist stimulation than those treated with estrogen followed by progesterone. The microsomal studies indicated that the pregnant animal had a greatly enhanced capacity to metabolize arachidonic acid when compared with the non-pregnant animal. However, this was not reflected in the ability of agonists to stimulate prostaglandin release in the perfused preparation.     

Estrogen induced changes in uterine sensitivity for the decidual cell reaction: Interactions between prostaglandin E2 and histamine or bradykinin

In rats receiving high doses of estrogen along with progesterone, the uterus is desensitized and does not respond to artificial stimuli with increased endometrial vascular permeability or decidualization. In addition, prostaglandin E₂ (PGE₂), the putative mediator of endometrial vascular permeability changes in sensitized uteri, is ineffective when given into the uterine lumen. The possibility that this inability of PGE₂ to increase endometrial vascular permeability may be related to the unavailability of hitamine of bradykinin was investigated. Rats were differentially sensitized for the decidual cell reaction by the daily injection of 2 mg progesterone with either 0.5 of 10 μg estrone for the 3 days preceding the unilateral intra-uterine injection of 50 μl phosphate buffered saline containing gelatin with or without 10 μg PGE₂ and with or without 1 mg histamine or 1 μg bradykinin. Prior to the intrauterine injection, all rats were treated with indomethacin to inhibit endogenous prostaglandin production. Endometrial vascular permeability changes were determined 8 h later by determining radioactivity levels in injected and non-injected uterine horns 15 min after the i.v. injection of ¹²⁵I-labelled bovine serum albumin. PGE₂ increased endometrial vascular permeability in rats receiving 0.5 μg estrone, but not in those receiving 10 μg. Histamine or bradykinin, alone or with PGE₂, did not affect endometrial vascular permeability in rats receiving either estrogen dose. The data suggest that the unresponsiveness of uteri from rats treated with high doses of estrogen is not simply due to the unavailability of bradykinin or histamine.     

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.     

Correlation between cyclic AMP and LH release following prostaglandin E2 administration

Five min following a single iv injection of PGE₂ into ovariectomized mature rats pretreated with estrogen and progesterone, plasma LH and plasma and pituitary cyclic AMP levels were raised significantly. A close correlation was observed between increased pituitary cyclic AMP contents and release of plasma LH. The average level of cyclic AMP in the anterior pituitary and plasma cyclic AMP increased significantly, while the circulating plasma LH level was not changed at 1 min after PGE₂ injection. Plasma LH level increased at 2 min after PGE₂ and reached a maximum level at the above-mentioned time. This is consistent with hypothesis that increased release of hormone is a consequence of increased pituitary cyclic AMP content.     

Prostaglandins and oxytocin: Their effects on uterine smooth muscle

A subcellular fraction was isolated from uteri of non-pregnant and pregnant cows. ATP-dependent calcium binding was shown to take place in this fraction. This calcium binding was inhibited in a dose related fashion when increasing amounts of prostaglandin (PG) E₂ or F_2α were added to the in vitro experimental medium. The physiologically inactive PGF_1β had no inhibitory effect. Oxytocin caused inhibition of calcium binding in preparations from both pregnant and non-pregnant cows. The response to PGE₂ and PGF_2α was somewhat greater in preparations from pregnant uteri than from non-pregnant uteri. The response to oxytocin was very much greater in pregnant uteri. Because of the high PG sensitivity of calcium binding in preparations from the non-pregnant uterus, it is concluded that the PGs may be the more suitable agent in the control of reproduction.     

Plasma levels of 13,14-dihydro-15-keto PGE2 after vaginal application of a new PGE2 film

To determine the release and absorption profile of prostaglandin E₂ from a new vaginal film formulation containing 850 μg PGE₂, serial plasma levels of 13,14-dihydro-15-keto PGE₂ were measured by radioimmunoassay in pregnant women between 16 and 18 weeks gestation. A control group, using placebo vaginal film was included in the study. There was a somewhat uniform increase in the plasma levels of the PGE₂ metabolite, reaching peak levels between 4 and 6 hours after application of the film. The findings suggest that this drug formulation could be used clinically when slow constant release of the prostaglandin is required over a period of hours such as in pre-induction cervical ripening of term pregnancy.     

Cyclic AMP formation and release by cultured bone cells stimulated with prostaglandin E2

PGE₂ produced a marked and dose-related increase in cAMP content of cultured bone cells and in the release of cAMP into the incubation medium. The amount of cAMP released from the cells by PGE₂ was proportional to the cellular concentration, and was dependent upon the time of incubation with PGE₂. The cAMP levels released into the media increased slowly at a linear rate during a 60 min treatment with PGE₂. This release was blocked by theophylline, probenecid, ouabain and dinitrophenol, suggesting that the release of cAMP was not a simple diffusive process and required energy. SC-19220 reduced the formation of cAMP more than the release, suggesting that the formation and the release may arise from separate events. Inability of D600 to inhibit PGE₂-induced release of cAMP indicates that the release does not require calcium.     

Absorption of prostaglandin E2 and uterine sensitivity of the non-pregnant and pregnant monkey in vivo

Radioactive (11-³H) prostaglandin E₂(PGE₂) levels in plasma of non-pregnant Rhesus and Japanese monkeys were determined by radioimmunoassay. The amounts of PGE₂ in plasma increased gradually and reached a peak 90 minutes after oral administration. Comparatively low levels were detected 24 hours after oral administration. Plasma PGE₂ levels increased rapidly and disappeared within 5 minutes when 5 μg/kg of PGE₂ was administered intravenously.Uterine contractile sensitivity to PGE₂ and F_2α was measured by the threshold of a venous dosage required to evoke an elevation of uterine contractility in non-pregnant and pre- and post-labor Japanese monkeys. Uterine sensitivity to PGE₂ in the non-pregnant monkey appear to vary in accordance with the sexual life span. At term of pregnancy, PGE₂ was much more potent in causing uterine contraction than PGF_2α. During labor and at postpartum period with lactation, effectiveness of PGE₂ appear to be less than that of PGF_2α. The non-pregnant and pregnant uterus of the third trimester are more sensitive to PGE₂ than the laboring and postpartum uterus.The long latency of the elevation of uterine contractility induced by the intravenous administration of PG suggests that the PG compounds have potent actions on the central nervous system.     

Leukocyte Population Dynamics and Detection of IL-9 as a Major Cytokine at the Mouse Fetal-Maternal Interface

Despite much interest in the mechanisms regulating fetal-maternal interactions, information on leukocyte populations and major cytokines present in uterus and placenta remains fragmentary. This report presents a detailed and quantitative study of leukocyte populations at the mouse fetal-maternal interface, including a comparison between pregnancies from syngeneic and allogeneic crosses. Our results provide evidence for drastic differences not only in the composition of leukocyte populations in the uterus during pregnancy, but also between uterine and placental tissues. Interestingly, we have observed a significant decrease in the number of myeloid Gr1+ cells including monocytes, and myeloid CD11c+ cells including DCs in placenta from an allogeneic pregnancy. In addition, we have compared the expression levels of a panel of cytokines in non-pregnant (NP) or pregnant mouse uterus, in placenta, or in their isolated resident leukocytes. Qualitative and quantitative differences have emerged between NP, pregnant uterus and placenta. Unexpectedly, IL-9 was the major cytokine in NP uterus, and was maintained at high levels during pregnancy both in uterus and placenta. Moreover, we have found that pregnancy is associated with an increase in uterine IL-1a and a significant decrease in uterine G-CSF and GM-CSF. Comparing allogeneic versus syngeneic pregnancy, less allogeneic placental pro-inflammatory cytokines CCL2 (MCP-1), CXCL10 (IP-10) and more IL1-α in whole uterus was reproducibly observed. To our knowledge, this is the first report showing a detailed overview of the leukocyte and cytokine repertoire in the uterus of virgin females and at the fetal-maternal interface, including a comparison between syngeneic and allogeneic pregnancy. This is also the first evidence for the presence of IL-9 in NP uterus and at the maternal-fetal interface, suggesting a major role in the regulation of local inflammatory or immune responses potentially detrimental to the conceptus.     

Macrophage prostaglandin E2 and oxidative responses to endotoxin during immunosuppression associated with anaesthesia and transfusion

The widespread use of blood transfusion in major surgical procedures has led to concern about the immunosuppressive effect of transfusion on patients with underlying malignancy. Transfusion may also suppress the host response to infection. The cellular mechanisms of transfusion-associated immunosuppression may involve macrophage prostaglandin E₂ (PGE₂) in modulating the host response to cancer and infection. We previously observed that the transfusion of blood increased PGE₂ production by unstimulated macrophages. To investigate this PGE₂ associated immunosuppression, we studied the effect of transfusion of rats using a physiological stimulus of macrophage PGE₂ production, bacterial endotoxin. In the same macrophages, we analysed intracellular oxidative activity. Both allogeneic and syngeneic blood transfusion were associated with increased PGE₂ release by macrophages. This stimulation of PGE₂ increased with duration of storage of blood. A similar effect of serum indicated that a humoral factor was involved. Endotoxin (50 ng/ml–500 μg/ml) stimulated PGE₂ production in all transfused subjects. The lowest endotoxin concentration gave proportionately the greatest stimulation. Oxidative activity was down-regulated in macrophages of transfused rats, supporting an immunosuppressive role of PGE₂ within the macrophage. An effect of surgery on the oxidative response was also detected.     

Alteration in the reactivity of hamster cheek pouch arterioles to prostaglandin E2 and noradrenaline during pregnancy or sex steroid treatment

Dioestrous and pregnant, or ovariectomized hamsters treated with sunflower oil, oestradiol or progesterone were anesthetized with pentobarbital and the arterioles of the cheek pouch membrane were prepared for microcirculatory study. Prostaglandin E₂ (PGE₂) or noradrenaline (NA) were topically applied and changes of the arteriolar diameter were measured on the screen of a closed-circuit television.PGE₂ induced arteriolar dilatation in dioestrous or ovariectomized hamsters, but induced vasoconstriction in pregnant or in oestrogen-treated animals. Vasoconstriction induced by PGE₂ in oestrogen-treated animals disappeared after administration of alpha-adrenergic blockers. In this situation, PGE₂ induced vasodilatation once again. NA elicited arteriolar constriction in each experimental group. In ovariectomized hamsters treated with oestrogen the constriction was more pronounced than in progesterone treated animals. In pregnant animals it was significantly greater on day 14 than in dioestrous animals.Progesterone treatment blunted the vascular effect of both PGE₂ and NA.It was concluded that the reverse effects of PGE₂ and the increased sensitivity to NA induced by high oestradiol levels may have roles in the regulation of local blood flow during the ovarian cycle and pregnancy.     

Production of leukotrienes and prostaglandins in the rat uterus during periimplantation period

We have measured by radioimmunoassay the production of leukotrienes (LTC₄ and LTB₄) and prostaglandins (PGE₂ and PGF_2α) in the rat uterus on Days 1 through 6 of pregnancy. The production is defined as the synthesis minus the degradation for a defined period. The production of LTC₄ or LTB₄ remained unaltered on days 1–3, but exhibited a marked increase on Day 4 showing a peak at noon. This was then followed by a sharp decline on Day-5 morning. A small but consistent peak in uterine LT production was also noticed on Day-5 noon prior to implantation and this was followed by a decline on Day-6 morning i.e. after initiation of implantation. The production profile of PGE₂ and PGE_2α showed a striking resemblance to that of LTs; one exception being that maximal PG production was noticed on Day-4 morning and preceded the peak production of LTs. These vasoactive arachidonate derivatives reached their peak production rates at around the time when a surge in estrogen level is noticed in the uterus on Day 4. Implantation is a local proinflammatory type of reaction that is associated with increased uterine vascular permeability. Vascular changes in inflammatory reactions are provoked by two kinds of chemical mediators: (1) vasodilators and (2) agents that increase vascular permeability. PGs (especially of the E series) are known as vasodilators, while LTs and histamine mediate increases in vascular permeability. Therefore, an interaction between LTs, PGs, and histamine could be important for uterine preparation for implantation and/or implantation .     

Estrogen metabolites in the release of inflammatory mediators from human amnion-derived cells

AimsHuman amnion-derived cells have been used as in vitro models to test the release of inflammatory mediators, such as arachidonic acid (AA) and prostaglandin E₂ (PGE₂). We compared estrogen metabolites for their ability to induce AA release, to influence PGE₂ production and to interact toward intracellular estrogen receptors (ERs).Main methodsMetabolite effects on AA and PGE₂ release were examined by radiolabelled substrate incorporation and by colorimetric enzyme immunoassays, respectively. [³H]17-β-estradiol binding displacements were performed on Ro-20-1724 treated whole cells.Key findingsIn WISH cells, estrone, 2-hydroxy-estrone and estriol induced a rapid dose dependent release of AA that was not inhibited by cycloheximide. Estrone and 2-hydroxy-estrone showed biphasic dose–response curves of PGE₂, whereas estriol and 16-α-hydroxy-estrone increased PGE₂ levels at high concentrations. 2-methoxy-estrone, 4-hydroxy-estradiol and 4-hydroxy-estrone did not significantly affect PGE₂ release. 2-methoxy-estradiol and 2-hydroxy-estradiol decreased the PGE₂ release. Effects of metabolites on PGE₂ were inhibited by cycloheximide and by the ER antagonist tamoxifen. In AV3 cells PGE₂ production was poorly detectable. On Ro-20-1724 treated WISH cells the K_i of 17-β-estradiol was 29.2 ± 5.4 nM. Estrone, 2-methoxy-estrone and 2-methoxy-estradiol showed similar affinity values. The hydroxyl substituent at position 2, 4 and 16 decreased or markedly increased the affinity for estradiol or estrone derivatives, respectively.SignificanceThe estrogen metabolites induced nongenomic effects on AA release from WISH cells. The influence on PGE₂ release was detectable only on WISH cells. These effects appeared genomic and mediated by intracellular ERs, whose properties seemed strongly dependent on intracellular cAMP levels.     

Metabolism of progesterone in rat uterus

The in vitro metabolism of progesterone was studied in uteri of untreated and estrogen stimulated immature rats. In intact uteri the rate of metabolism varied with the hormonal status of the animal in a concentration dependent manner. At a low (3 × 10^?9M) progesterone concentration the rate of ring A reduction was decreased in estrogen stimulated uteri. At a high progesterone concentration (3 × 10^?6M) the rate of ring A reduction was increased after estrogen treatment. The rate of reduction of the C20 ketone was increased after estrogen treatment at all concentrations of incubated progesterone. In dilute homogenates of uterus, estrogen stimulation always increased the rate of progesterone metabolism.Estrogen stimulation results in increased concentration of progesterone receptor in the uterus. It is proposed that increased activity of ring A reductases also occurs. The relative influence of these two factors on the metabolism of progesterone is dependent on the progesterone concentration in the incubation medium.     

Murine peritoneal macrophages in syngeneic and allogeneic pregnancies

We have previously observed that some functional characteristics of peritoneal macrophages (MOp) are altered during syngeneic murine pregnancy. To determine if these alterations are related to the immunological stimulation that the embryo produces on the mother, we evaluated MOp activity in allogeneic pregnancy. We also compared expression of the la antigen, ability to phagocyte and reduce nitro blue tetrazolium (NBT) and to produce interleukin-1 (IL-1) in allogeneic and syngeneic pregnancies. We observed that at Day 7 of pregnancy the increment in MOpIa(+) percentages was more evident in allogeneic (P < 0.05) than syngeneic pregnancies, and that these values remained high during the second week of gestation. We also observed a significant decrease in the macrophages that reduced NAT during the first week both in allogeneic and syngeneic pregnancies. Yet, in the former, the percentages of MOpNBT(+) were still low in the last week of pregnancy (P < 0.05). No differences were found in IL-1 production or in estradiol and progesterone levels between the 2 types of pregnancies. Thus, it is possible to postulate that during the first week of pregnancy the strong antigenic challenge that the embryo represents may activate MOp and that this activation could be augmented when major antigenic differences between mother and embryo are present.     

Interacting effects of estrogen, progesterone and catecholamines on rat uterine cyclic AMP and glycogen phosphorylase.

Ovariectomized rats were treated with estrogen, progesterone or a combination of estrogen plus progesterone. Rats were anesthetized with sodium pentobarbital and uteri were frozen $\text{in situ}$, uterine extracts were prepared and assayed for cyclic AMP and phosphorylase. Uterine cyclic AMP levels were highest in estrogen treated uteri and were significantly reduced when estrogen was withdrawn for two days. Addition of progesterone to the estrogen regimen for two days or changing from estrogen to progesterone for two days produced results comparable to those obtained when estrogen was withdrawn. Similar experiments were done except that 30 seconds before tissue freezing epinephrine was injected intravenously. Both cyclic AMP and glycogen phosphorylase increased markedly in response to epinephrine. The magnitude of the responses were greatest in the uteri pretreated with estrogen. The magnitudes of both the cyclic AMP and phosphorylase responses were significantly reduced by withdrawing estrogen for two days, by adding progesterone to the estrogen treatment or by changing to progesterone from estrogen. Isoproterenol-stimulated cyclic AMP responses were affected by the steroid state of the uterus in the same way as the epinephrine responses.     

Dienogest,a synthetic progestin,inhibits prostaglandin E2 production and aromatase expression by human endometrial epithelial cells in a spheroid culture system

Prostaglandin E₂ (PGE₂) is a major mediator in the pathophysiology, and pathogenesis of gynecological diseases associated with abnormal endometrial disease with proliferation and inflammation, such as endometriosis. In this study, we investigated the effect of dienogest, a selective progesterone receptor agonist, on PGE₂ production and the expression of aromatase, an estrogen synthase, in human immortalized endometrial epithelial cells. Compared with monolayer culture, the cells showed enhanced PGE₂ production and expression of the PGE₂ synthases cyclooxygenase-2 (COX-2), and microsomal prostaglandin E₂ synthase-1 (mPGES-1) in a spheroid culture system. Dienogest inhibited PGE₂ production and this effect was reversed by RU486, a progesterone receptor antagonist. Dienogest inhibited the PGE₂ synthases mRNA and protein expression, and the nuclear factor-κB activation. Moreover, the suppressive effect of dienogest on PGE₂ production was sustained 24 h after the drug was withdrawn. Dienogest but not COX inhibitors inhibited aromatase expression. These results suggest that progesterone receptor activation reduces the gene expressions of COX-2, mPGES-1, and aromatase. Our findings suggest that the pharmacological mechanism of dienogest includes the direct inhibition of PGE₂ synthase and aromatase expression and may contribute to the therapeutic effect on the progression of endometriosis.     

Tissue- and hormone- dependent progesterone receptor distribution in the rat uterus

Background  

Estradiol (E2) and progesterone (P) are well known regulators of progesterone receptor (PR) expression in the rat uterus. However, it is not known which receptor subtypes are involved. Little knowledge exist about possible differences in PR regulation through ERalpha or ERbeta, and whether the PR subtypes are differently regulated depending on ER type bound. Thus, in the present study PR immunostaining has been examined in uteri of ovariectomized (ovx) rats after different treatments of estrogen and P, in comparison with that in immature, cycling, and pregnant animals.     

Cyclic AMP inhibits synthesis of prostaglandin endoperoxide (PGG2) in human platelets.

Dibutyryl-cAMP but not dibutyryl-cGMP inhibited platelet aggregation and release of ¹⁴C-serotonin and ADP when induced by collagen and arachidonate but not when induced by the endoperoxide PGG₂^* (TXB₂) induced by addition of collagen to platelet rich plasma (PRP) was decreased by dibutyryl-cAMP and agents known to increase the concentration of cAMP (PGE₁, PGD₂, theophylline and acetyl choline).PGE₂ in concentrations known to decrease cAMP levels increased the formation of TXB₂ whereas concentrations of PGE₂ known to increase cAMP levels decreased the amount of TXB₂ formed. That this was due to an effect on the cyclooxygenase was indicated by inhibition of the transformation of arachidonic acid by DB-cAMP and by high concentrations of PGE₂. Additional support for regulation of the cyclo-oxygenase by cAMP and its relevance to platelet aggregation was obtained by demonstrating stimulation of PGG₂ induced aggregation by low concentrations of PGE₂ and the absence of this effect in the presence of a cyclo-oxygenase inhibitor.