The effect of supplementation with n-6 polyunsaturated fatty acids on 1-, 2- and 3-series prostaglandin F production by ovine uterine epithelial cells

Linoleic acid (LA, 18:2n-6) has variously been found to increase or inhibit synthesis of 2-series prostaglandins (PGs), derived from arachidonic acid (AA, 20:4n-6). gamma-linolenic acid (GLA, 18:3n-6) containing oils are promoted to women for a variety of reproductive problems. Little is known concerning their actual effects on reproduction. We investigated the effects of LA, GLA and AA supplementation (25-100 microM) on basal and oxytocin (OT) stimulated production of 1-, 2- and-3 series PGs by uterine epithelial cells isolated from non-pregnant ewes, used as a model system to study endometrial PG production. PGF isomers were measured using radioimmunoassays following separation by high performance chromatography (HPLC). OT challenge increased the proportion of PGF2alpha in relation to PGF1alpha and PGF3alpha in control medium. LA supplementation decreased all PGF isomer production and reduced responsiveness to OT. GLA increased both absolute and proportional PGF1alpha production and slightly enhanced PGF2alpha generation. AA increased PGF2alpha generation and raised its isometric proportion. Both GLA and AA increased overall PGF output significantly but prevented the cells from responding to OT. These results suggest that consumption of LA and GLA are likely to differentially alter both uterine PG metabolism and responsiveness to OT. This may have implications for the control of a variety of reproductive processes.     

Progesterone reduces erectile dysfunction in sleep-deprived spontaneously hypertensive rats

Background

The rate-limiting step in prostaglandin (PG) biosynthesis is catalyzed by phospholipase A2 (PLA2) enzymes which hydrolyze arachidonic acid from membrane phospholipids. Despite their importance in uterine PG production, little is known concerning the specific PLA2 enzymes that regulate arachidonic acid liberation in the uterine endometrium. The objectives of this study were to evaluate the expression and activities of calcium-independent Group VI and Group IVC PLA2 (PLA2G6 and PLA2G4C) and calcium-dependent Group IVA PLA2 (PLA2G4A) enzymes in the regulation of bovine uterine endometrial epithelial cell PG production.

Methods

Bovine endometrial epithelial cells in culture were treated with oxytocin, interferon-tau and the PLA2G6 inhibitor bromoenol lactone, alone and in combination. Concentrations of PGF2alpha and PGE2 released into the medium were analyzed. Western blot analysis was performed on cellular protein to determine the effects of treatments on expression of PLA2G4A, PLA2G6 and PLA2G4C. Group-specific PLA2 activity assays were performed on cell lysates following treatment with oxytocin, interferon-tau or vehicle (control), alone and in combination. To further evaluate the role of specific PLA2 enzymes in uterine cell PG biosynthesis, cells were transfected with cDNAs encoding human PLA2G6 and PLA24C, treated as described above and PG assays performed.

Results

Constitutive cell production of PGF2alpha was about two-fold higher than PGE2. Oxytocin stimulated production of both PGs but the increase of PGF2alpha was significantly greater. Interferon-tau diminished oxytocin stimulation of both PGs. The PLA2G6 inhibitor, bromoenol lactone, abolished oxytocin-stimulated production of PGF2alpha. Treatments had little effect on PLA2G4A protein expression. In contrast, oxytocin enhanced expression of PLA2G6 and this effect was diminished in the presence of interferon-tau. Expression of PLA2G4C was barely detectable in control and oxytocin treated cells but it was enhanced in cells treated with interferon-tau. Oxytocin stimulated PLA2 activity in assays designed to evaluate PLA2G6 activity and interferon-tau inhibited this response. In assays designed to measure PLA2G4C activity, only interferon-tau was stimulatory. Cells overexpressing PLA2G6 produced similar quantities of the two PGs and these values were significantly higher than PG production by non-transfected cells. Oxytocin stimulated production of both PGs and this response was inhibited by interferon-tau. Bromoenol lactone inhibited oxtocin stimulation of PGF2alpha production but stimulated PGE2 production, both in the absence and presence of oxytocin. Cells over-expressing PLA2G4C produced more PGE2 than PGF2alpha and interferon-tau stimulated PGE2 production.

Conclusion

Results from these studies indicate that oxytocin stimulation of uterine PGF2alpha production is mediated, at least in part, by up-regulation of PLA2G6 expression and activity. In addition to its known inhibitory effect on oxytocin receptor expression, interferon-tau represses oxytocin-stimulated PLA2G6 expression and activity and this contributes to diminished PGF2alpha production. Furthermore, endometrial cell PGE2 biosynthesis was associated with PLA2G4C expression and activity and interferon-tau was stimulatory to this process.     

Effects of oestradiol, progesterone, hydrocortisone and oxytocin on prostaglandin output from the guinea-pig endometrium maintained in tissue culture

The effects of oestradiol, oxytocin, progesterone and hydrocortisone in vitro on prostaglandin (PG) output from guinea-pig endometrium, removed on days 7 and 15 of the oestrous cycle and maintained in tissue culture for 3 days, have been investigated. Oestradiol (3.7 to 3700 nM) and oxytocin (2 to 200 pM) did not stimulate endometrial PGF2 alpha output, thus not confirming the findings of a previous report (Leaver & Seawright, 1982), nor did they stimulate the outputs of PGE2 and 6-keto-PGF1 alpha. In fact, oestradiol (3700 nM) inhibited the outputs of PGF2 alpha, PGE2 and, to a lesser extent, 6-keto-PGF1 alpha. Progesterone (3.2 to 3200 nM) inhibited the outputs of PGF2 alpha and PGE2; hydrocortisone (2.8 to 2800 nM) had no effect on endometrial PG output. These findings indicate that the inhibitory effect of progesterone on endometrial PG synthesis and release in the guinea-pig is not due to progesterone having a glucocorticoid-like action. Furthermore, progesterone had no effect on 6-keto-PGF1 alpha output, suggesting that the mechanisms controlling endometrial PGI2 synthesis (as reflected by measuring 6-keto-PGF1 alpha) are different from those controlling endometrial PGF2 alpha and PGE2 synthesis.     

The effect of progesterone and human interferon alpha-2 on the release of PGF2 alpha and PGE from epithelial cells of human proliferative endometrium.

Progesterone and interferon-like trophoblastic proteins modulate prostaglandin (PG) synthesis from endometrium in early ovine and bovine pregnancy. Enriched epithelial cells were prepared from human endometrium removed in the proliferative phase of menstrual cycle (n = 8). Progesterone at a concentration of 1 microM suppressed PGE release from the cells during the first 24 hours in culture. After 48 hours in culture progesterone at a dose of 100 nM and 1 microM suppressed both the release of PGF2 alpha and PGE from the cells and this suppression was maintained for a further two days. Addition of exogenous 30 microM arachidonic acid (AA) abolished this effect of progesterone on both PGF2 alpha and PGE release. Interferon alpha-2 did not suppress the basal release of PGF2 alpha nor PGE. In the presence of progesterone, interferon alpha-2 attenuated the progesterone mediated suppression of PGF2 alpha but not PGE release from endometrial cells. These findings suggest that progesterone suppresses the basal release of PGs from human endometrium, but unlike the sheep, interferon alpha-2 does not exert this action on human endometrium.     

The effects of danazol, mefenamic acid, norethisterone and a progesterone-impregnated coil on endometrial prostaglandin concentrations in women with menorrhagia

The effects of four medical treatments have been assessed on menstrual blood loss (MBL) and endometrial prostaglandin (PG) concentrations in 30 women with objectively confirmed menorrhagia. Patients were randomly treated with danazol, 200 mg daily (n = 6), mefenamic acid, 500 mg three times daily during menses (n = 8), norethisterone, 5 mg twice daily from day 15-25 of the cycle (n = 8) or a progesterone-impregnated coil releasing 65 micrograms progesterone daily (n = 8). Endometrial biopsies were obtained in the mid-luteal phase before and after treatment in 23 cases, and assayed for PG content using radioimmunoassay. Treatment with norethisterone had no effect on either MBL or the concentration of PGs in the endometrium. MBL was significantly reduced after treatment with mefenamic acid (P = 0.05, n = 6) and the progesterone coil (P less than 0.05, n = 6), and was reduced in each of 4 cases treated with danazol in whom endometrial biopsies were available. Although there was no consistent change in endometrial PG concentrations in either the mefenamic acid or danazol groups, the lower MBL after insertion of the progesterone coil was associated with a reduced endometrial content of PGE, PGF2 alpha and "total" PG (6oxo PGF1 alpha + PGE + PGF2 alpha)-P = 0.05. Whereas the cyclooxygenase inhibitor mefenamic acid is likely to exert its effect on endometrial PGs at the time of menstruation itself, the continuous administration of progesterone throughout the menstrual cycle could result in both an impairment in estrogen receptor generation leading to reduced estrogen-mediated cyclooxygenase activity, and an increase in endometrial PG metabolism.     

Differential effects of progesterone treatment on the oxytocin-induced prostaglandin F2 alpha response and the levels of endometrial oxytocin receptors in ovariectomized ewes.

The oxytocin-induced uterine prostaglandin (PG) F2 alpha response and the levels of endometrial oxytocin receptors were measured in ovariectomized ewes after they had been given steroid pretreatment (SP) with progesterone and estrogen to induce estrus (day of expected estrus = Day 0) and had subsequently been treated with progesterone over Days 1-12 and/or PGF2 alpha over Days 10-12 postestrus. The uterine PGF2 alpha response was measured after an i.v. injection of 10 IU oxytocin on Days 13 and 14, using the PGF2 alpha metabolite, 13,14-dihydro-15-keto-PGF2 alpha (PGFM), as an indicator for PGF2 alpha release. The levels of oxytocin receptors in the endometrium were measured on Day 14. During the treatment with progesterone, the peripheral progesterone concentrations were elevated and remained above 1.8 ng/ml until the morning of Day 14. The PGFM responses to oxytocin in untreated controls and SP controls were low on both Days 13 and 14 whereas the levels of endometrial oxytocin receptors in the same ewes were high. Treatment with progesterone either alone or in combination with PGF2 alpha significantly (p less than 0.04) increased the PGFM response on Day 14 and reduced the levels of endometrial oxytocin receptors; treatment with PGF2 alpha alone had no effect. It is concluded that progesterone promotes the PGFM response to oxytocin while simultaneously suppressing the levels of endometrial oxytocin receptors. PGF2 alpha treatment had no effect on either the uterine secretory response to oxytocin or the levels of oxytocin receptors in the endometrium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell-type specific responses in prostaglandin secretion by glandular and stromal cells from pig endometrium treated with catecholestrogens, methoxyestrogens and progesterone.

The pig conceptus and endometrium possess the ability to convert estrogens into catecholestrogens and catecholestrogens into methoxyestrogens. Experiments were carried out to evaluate the effect of catecholestrogens, methoxyestrogens and progesterone on the secretion of prostaglandin (PG) E and F2 alpha by porcine endometrial glandular and stromal cells in vitro. Both 2-hydroxyestradiol (2-OH-E2, 0-20 microM) and 4-hydroxyestradiol (4-OH-E2, 0-20 microM) increased (P less than .05) PGE and PGF2 alpha secretion by stromal cells in a dose response manner. Two-hydroxyestradiol tended (P less than .1) to decrease PGF2 alpha production by glandular cells. Two-methoxyestradiol (20 microM) suppressed (P less than .05) PGF2 alpha secretion by glandular and stromal cells. Four-methoxyestradiol (20 microM) stimulated (P less than .05) PGE production and PGE:PGF2 alpha ratio. Progesterone (.1 microM) suppressed (P less than .05) PG secretion in both cell types. We conclude that catecholestrogens, methoxyestrogens, and progesterone may participate in the establishment of pregnancy by modulating PG production in the endometrium.     

The release of PGF2 alpha and PGE2 from separated cells of human endometrium and decidua

The capacity of separated glandular and stromal cells from endometrium and first trimester decidua to release prostaglandins (PGs) was studied over 48 hours in culture. Glandular preparations released more PGs than stromal preparations in all tissues. Stromal release of PGs did not alter throughout the cycle or in early pregnancy but the capacity of glandular preparations to release PGs varied considerably. Proliferative glands released most PGF2 alpha and PGE2 followed by secretory glands and decidua. Histamine (10(-5)) stimulated PG release from endometrial and decidual glands but the response of proliferative glands was greatest. Actinomycin D stimulated release of PGF2 alpha and PGE2 from glandular cells of secretory endometrium and decidua. These results suggest that in vitro release of PGs is suppressed after ovulation and is in part due to inhibition of PG release by a protein or proteins synthesized in the glandular fraction of secretory endometrium or decidua.     

Polyunsaturated fatty acids differentially alter PGF(2alpha) and PGE(2) release from bovine trophoblast and endometrial tissues during short-term culture

Two studies tested the hypothesis that eicosapentaenoic (20:5omega3; EPA), docosahexaenoic acids (22:6omega3; DHA) or linoleic acid (C18:2omega6; LIN) reduced bovine endometrial and trophoblast prostaglandin F(2alpha) (PGF(2alpha)) and prostaglandin E(2) (PGE(2)) release during short-term culture. In Study 1, endometrial tissues were collected from non-lactating, non-pregnant cows and endometrial plus trophoblast tissues from pregnant cows 16 days post-insemination. In Study 2, endometrial and trophoblast tissues were collected on day 17 of pregnancy, from cows synchronised using a double prostaglandin (PG) or Ovagentrade mark synchronisation. Tissues were incubated in medium only (M) or media supplemented with fatty acids: eicosapentaenoic (20:5omega3; EPA), docosahexaenoic acids (22:6omega3; DHA) or linoleic acid (C18:2omega6; LIN). In Study 1, PGE(2) release from 'pregnant' endometria was higher (P=0.094) than from 'non-pregnant' endometria, while PGF(2alpha) concentrations were similar. Fatty acids treatment had no effect on PGF(2alpha) or PGE(2) release from either pregnant or non-pregnant endometria. Individual fatty acid treatments had no effect on the ratio of PGF(2alpha) to PGE(2) from trophoblast tissues, but when the data from the 3 fatty acid treatments were combined (EPA, DHA and LIN treatment groups) the ratio of PGF(2alpha) to PGE(2) was reduced (P=0.026) when compared to medium only. In Study 2, PGE(2) concentrations were higher (P=0.013) from the trophoblast collected from Ovagentrade mark cows as compared to that of the PG synchrony group. When the data from the 3-omega fatty acids were combined (DHA and EPA treatment groups), the 3-omega treatments decreased (P<0.05) PGE(2) biosynthesis from both endometrial and trophoblast tissues from animals synchronised following PG synchrony but not Ovagentrade mark synchrony. Short-term culture with low concentrations of 3-omega fatty acids tended to reduce prostaglandin release from trophoblast collected 16 days after insemination, with the type of synchrony modifying PGE(2) production from the trophoblast tissues collected 17 days after insemination. The ability of exogenous fatty acids to modify embryonic prostaglandin release needs to be examined in the context of supplementing dairy cows with different sources of fats. Synchronisation method altered trophoblast PGE(2) release, highlighting the importance of the hormonal environment in modifying embryonic prostaglandin synthesis and release.     

Effects of tumor necrosis factor-alpha on secretion of prostaglandins E2 and F2alpha in bovine endometrium throughout the estrous cycle

To determine the physiological significance of tumor necrosis factor-alpha (TNFalpha) in the regulation of endometrial prostaglandin (PG) release in cattle, we investigated the effects of TNFalpha on the secretion of PGE2 and PGF2alpha by bovine endometrium during the estrous cycle. Bovine uteri were classified into six stages (estrus: Day 0, early luteal 1: Days 2 to 3, early luteal 11: Days 5 to 6, mid-luteal: Days 8 to 12, late luteal: Days 15 to 17 and follicular: Days 19 to 21). After 1 h of pre-incubation, endometrial tissues (20 to 30 mg) were exposed to 0 or 0.6 nM TNFalpha for 4 h. The PGE2 concentrations in the medium were higher in the luteal stages than in the follicular stage and in estrus. In contrast, PGF2alpha concentrations were higher in the follicular stage and in estrus than in the luteal stages. The ratio of the basal concentrations of PGE2 and PGF2alpha (PGE2/PGF2alpha ratio) was higher in the luteal stages than in the follicular stage and in estrus. Although TNFalpha stimulated both PGE2 and PGF2alpha secretion during the entire period of the estrous cycle, the level of stimulation of TNFalpha on PGE2 output by the bovine endometrium does not show the same cyclical changes as that shown on PGF2alpha output. The stimulation of TNFalpha resulted in a decrease in the PGE2/PGF2alpha ratio only in the late luteal stage. Furthermore, TNFalpha stimulated PGE2 secretion in stromal, but not epithelial cells. The overall results suggest that TNFalpha is a potent regulator of endometrial PGE2 secretion as well as PGF2alpha secretion during the entire period of estrous cycle, and that TNFalpha plays different roles in the regulation of secretory function of bovine endometrium at different phases of the estrous cycle.     

Prostaglandin E and E2 alpha secretion by glandular and stromal cells of the pig endometrium in vitro: effects of estradiol-17 beta, progesterone, and day of pregnancy.

Prostaglandins (PGs) are believed to play important roles in the establishment of pregnancy. Glandular and stromal cells were isolated from pig endometrium on days 11 through 19 of pregnancy and cultured in the presence of estradiol-17 beta (E2) and progesterone (P4) to determine the effect of day of pregnancy and steroids on the secretion of PGE and PGF2 alpha. Estradiol at concentrations between .01 and 1 microM did not affect PGE and PGF2 alpha secretion into the medium by glandular and stromal cells. Progesterone (.1 microM) suppressed (P less than .001) PGE and PGF2 alpha production from both cell types. Glandular cells secreted more (P less than .01) PGF2 alpha than PGE, whereas stromal cells collected on days 11, 12, 13, and 19 secreted more (P less than .05) PGE than PGF2 alpha. Stromal cells isolated from tissues collected on day 13 of pregnancy produced PGs with higher (P less than .01) PGE:PGF2 alpha ratio than those from tissues harvested on other days of pregnancy. Glandular cells isolated from tissues collected on days 13 and 19 and stromal cells isolated from tissue collected on day 13 of pregnancy secreted more (P less than .05) PGE and PGF2 alpha than cells isolated on other days of pregnancy. We conclude that: 1) P4 has a suppressing effect on PG secretion; 2) endometrial glandular and stromal cells each produce a unique profile of PGs; and 3) endometrial cells harvested on different days of pregnancy secrete different amounts of PGE and PGF2 alpha.     

Regulation of heat shock-induced alterations in the release of prostaglandins by the uterine endometrium of cows

Maternal heat stress in cattle may disrupt pregnancy by elevating uterine prostaglandin F(2alpha) (PGF(2alpha)) secretion. The objectives of this study were to determine the effects of elevated temperature (42 degrees C) in vitro upon 1) prostaglandin secretion by endometrial tissue; 2) the actions of extracellular regulators of uterine PGF [conceptus secretory proteins (bCSPs) and platelet-activating factor, (PAF)]; 3) the activity of the cyclooxygenase-endoperoxidase enzyme complex (PG synthetase); and 4) the activity of the endometrial PG synthesis inhibitor present in the endometrium from pregnant cattle. Endometrial explants at Day 17 of the estrous cycle produced more PGF than PGE(2) while elevated temperature caused increased PGF secretion but did not affect PGE(2) secretion. Elevated temperature did not reduce the ability of bCSPs or PAF to suppress release of PGF. The heat shock-induced increase in PGF at Day 17 was not due to the direct effects on PG synthetase, because PGF production from a cell-free cotyledonary microsomal enzyme preparation was reduced at elevated temperature. The activity of the cytosolic inhibitor of cyclooxygenase present in the endometrium of Day-17 pregnant cows could be reduced but not eliminated at 42 degrees C. We conclude that in vitro heat stress induces PGF secretion from the bovine uterine endometrium at Day 17 after estrus. This increase is not accompanied by the loss of regulatory capacity of conceptus products or increased activity of PG synthetase.     

Effects of conjugated linoleic acids and docosahexaenoic acid on rat liver and reproductive tissue fatty acids, prostaglandins and matrix metalloproteinase production

Long chain n-6 and n-3 fatty acids play important roles in labor and delivery. These effects may be mediated by prostaglandin (PG) synthesis and by regulation of matrix metalloproteinases (MMPs), both of which play roles in uterine contraction, cervical ripening and rupture of fetal membranes. The effects of altering dietary n-6:n-3 long chain fatty acid ratios, and the addition of dietary conjugated linoleic acids (CLA) and docosahexaenoic acid (DHA) on fatty acid composition of reproductive tissues, PG synthesis in liver and reproductive tissue and serum MMP levels were examined in pregnant rats. Modified AIN-96G diets with n-6:n-3 ratios of 7:1 and 34:1 with and without added 1.1% (by weight) conjugated linoleic acid (CLA) and/or 0.3% (by weight) DHA were fed through day 20 of gestation. Reproductive tissues readily incorporated both DHA and CLA. CLA significantly (P<0.05) depressed PGF(2 alpha)synthesis in placenta, uterus and liver by 50% when the n-6:n-3 ratio was 7:1 and by 66% at 34:1 ratio. Significant differences (P<0.05) in PGE(2)synthesis in uterus and liver were seen only between groups fed the high ratio of n-6:n-3 without CLA, and the low ratio with CLA. Addition of CLA to DHA containing diets depressed PGF(2alpha) by one-third in uterus and liver (P<0.05). Serum MMP-9 and active MMP-2 were suppressed (P<0.05) by addition of either CLA or DHA.     

Production of prostaglandin f(2alpha) by cultured bovine endometrial cells in response to tumor necrosis factor alpha: cell type specificity and intracellular mechanisms

Tumor necrosis factor alpha (TNFalpha) has been shown to be a potent stimulator of prostaglandin (PG) F(2alpha) secretion in the bovine endometrium. The aims of the present study were to determine the cell types in the endometrium (epithelial or stromal cells) responsible for the secretion of PGF(2alpha) in response to TNFalpha, and the intracellular mechanisms of TNFalpha action. Cultured bovine epithelial and stromal cells were exposed to TNFalpha (0.006-6 nM) or oxytocin (100 nM) for 4 h. TNFalpha resulted in a dose-dependent increase of PGF(2alpha) production in the stromal cells (P < 0.001) but not in the epithelial cells. On the other hand, oxytocin stimulated PGF(2alpha) output in the epithelial cells but not in the stromal cells. When the stromal cells were incubated for 24 h with TNFalpha and inhibitors of phospholipase (PL) C or PLA(2), only PLA(2) inhibitor completely stopped the actions of TNFalpha (P < 0.001). When the stromal cells were exposed to TNFalpha and arachidonic acid, the action of TNFalpha was augmented (P < 0.001). When the stromal cells were incubated for 24 h with a nitric oxide (NO) donor (S-NAP), S-NAP stimulated the PGF(2alpha) production dose-dependently. Although an NO synthase (NOS) inhibitor (L-NAME) reduced TNFalpha-stimulated PGF(2alpha) production, an inhibitor of phosphodiesterase augmented the actions of TNFalpha and S-NAP (P < 0. 05). The overall results indicate that the target of TNFalpha for stimulation of PGF(2alpha) production in cattle is the endometrial stromal cells, and that the actions of TNFalpha are mediated via the activation of PLA(2) and arachidonic acid conversion. Moreover, TNFalpha may exert a stimulatory effect on PGF(2alpha) production via the induction of NOS and the subsequent NO-cGMP formation.     

Cellular mechanisms by which oxytocin mediates ovine endometrial prostaglandin F2alpha synthesis: role of G(i) proteins and mitogen-activated protein kinases

Oxytocin stimulates a rapid increase in ovine endometrial prostaglandin (PG) F2alpha synthesis. The overall objective of these experiments was to investigate the cellular mechanisms by which oxytocin induces endometrial PGF2alpha synthesis. The objective of experiment 1 was to determine whether G(i) proteins mediate oxytocin-induced PGF2alpha synthesis. Uteri were collected from four ovary-intact ewes on Day 14 postestrus. Caruncular endometrial explants were dissected and subjected to in vitro incubation. Pertussis toxin, an inhibitor of G(i) proteins, had no effect on the ability of oxytocin to induce PGF2alpha synthesis (P > 0.10). The objective of experiment 2 was to determine whether any of the three mitogen-activated protein kinases (MAPKs), extracellular signal regulated protein kinase (ERK1/2), c-Jun N-terminal/stress-activated protein kinase (JNK/SAPK), or p38 MAPK, mediate oxytocin-induced PGF(2alpha) synthesis. Eleven ovary-intact ewes were given an injection of oxytocin (10 IU; i.v.; n = 5) or physiological saline (i.v.; n = 6) on Day 15 postestrus. Uteri were collected 15 min after injection and caruncular endometrium was dissected. Endometrial homogenates were prepared and subjected to Western blotting. Membranes were probed for both total and phosphorylated forms of all three classes of MAPK. All classes of MAPK were detected in ovine endometrium, but oxytocin treatment had no effect on the expression of these proteins (P > 0.10). ERK1/2 was the only phosphorylated MAPK detected and its concentrations were higher in oxytocin-treated ewes (P < 0.01). The objective of experiment 3 was to further investigate the role of ERK1/2 during oxytocin-induced PGF2alpha synthesis. Uteri were collected from four ovary-intact ewes on Day 14 postestrus. Caruncular endometrial explants were dissected and subjected to in vitro incubation. PD98059, a specific inhibitor of ERK1/2 activity, blocked the ability of oxytocin to stimulate PGF(2alpha synthesis in a dose-dependent manner (P < 0.05). These results indicate that the ovine oxytocin receptor is not coupled to G(i) proteins. These results indicate that oxytocin induces phosphorylation of ERK1/2 and that this MAPK appears to mediate oxytocin-induced PGF2alpha synthesis in ovine endometrium.     

Comparison of the inhibitory effect of polyunsaturated fatty acids on prostaglandin synthesis I oral squamous carcinoma cells

Polyunsaturated fatty acids (PUFAs) have been shown to suppress the growth rate of human osteogenic sarcoma cells and to have selective cytotoxic activity against human cancer cells. The purpose of this study was to investigate the efficacy of various PUFAs on inhibition of prostaglandin (PG) synthesis by oral squamous carcinoma cells (SCC-25). A significant inhibition of PGE2 and PGF2 alpha synthesis in SCC-25 was observed by all PUFAs tested except in the case of linoleic acid (LA) at 10 microM level. At 10 microM level the rank order of inhibition of PG synthesis by PUFAs was docosahexaenoic (DHA) greater than eicosapentaenoic (EPA) + DHA greater than dihomogamma-linolenic (DGLA) greater than EPA greater than alpha-linolenic (ALA) greater than linoleic (LA). At 50, 75, 100 microM the rank order of inhibition was DGLA greater than EPA greater than EPA + DHA greater than DHA greater than ALA greater than LA.     

Fatty acid and prostaglandin metabolism in children with diabetes mellitus. II. The effect of evening primrose oil supplementation on serum fatty acid and plasma prostaglandin levels

Our previous study demonstrated that levels of dihomo-gamma-linolenic acid (DGLA) and arachidonic acid in serum total lipids decreased in association with increased plasma levels of prostaglandins E2 (PGE2) and F2 alpha (PGF2 alpha) in patients with insulin-dependent diabetes mellitus. In this study, 11 children with insulin-dependent diabetes mellitus completed a double-blind, placebo-controlled study to assess the effect of dietary supplementation with gamma-linolenic acid (GLA) on serum essential fatty acid and plasma PGE2 and PGF2 alpha levels. GLA was given as the seed oil from the evening primrose (EPO) and all patients received either EPO capsules (containing 45 mg of GLA and 360 mg of linoleic acid) or indistinguishable placebo capsules for 8 months. Initially patients took 2 capsules daily for 4 months then 4 capsules daily for a further 4 months. All patients were assessed at the start of the study, after 4 months and at the end of the study, by measuring serum essential fatty acid and plasma PGE2 and PGF2 alpha levels. After administration of 4 capsules daily the DGLA levels increased and PGE2 levels decreased significantly (p less than 0.01) in the EPO compared with the placebo group. Neither fatty acid nor PGE2 and PGF2 alpha levels were altered by administration of 2 EPO capsules daily. This suggests that the altered essential fatty acid and PG metabolism in diabetes may be reversed by direct GLA supplementation.     

Autocrine/paracrine action of oxytocin in pig endometrium

Luminal epithelial cells of porcine endometrium are unresponsive to oxytocin (OT) in vitro although they express the greatest quantity of OT and receptors for OT in vivo. Therefore, the objective of this study was to determine if oxytocin acted in an autocrine manner on luminal epithelial cells to stimulate prostaglandin (PG)F(2alpha) secretion. Treatment of endometrial explants or enriched luminal epithelial cells with OT antagonist L-366,948 decreased (P < 0.05) basal secretion of PGF(2alpha). Oxytocin increased (P < 0.01) PGF(2alpha) secretion from luminal epithelial cells that were pretreated with 1:5000 or 1:500 OT antiserum for 3 h to immunoneutralize endogenously secreted OT. However, OT only increased (P < 0.05) PGF(2alpha) secretion from glandular epithelial cells when pretreated with 1:500 OT antiserum. Pretreatment with OT antiserum did not alter the ability of OT to induce PGF(2alpha) secretion from stromal cells. Medium conditioned by culture of luminal epithelial cells stimulated (P < 0.05) phospholipase C activity in stromal cells, indicative of the presence of bioactive OT. Oxytocin was secreted by luminal epithelial cells and 33% was released from the apical surface. These results indicate that luminal epithelial cells secrete OT that acts in an autocrine and/or paracrine manner in pig endometrium to stimulate PGF(2alpha) secretion.     

Endothelin-1 stimulates prostaglandin F2 alpha release from human endometrium

Despite a key role in the pathogenesis of menorrhagia, the factors controlling the uterine vascular bed are poorly understood. This study has assessed the effects of the potent vasoconstrictor endothelin (ET)-1 on prostaglandin (PG) release from human endometrial explants in short-term culture. There was no significant difference between the production of PGF2 alpha in proliferative and secretory tissue (1709 and 2434 pg/mg/h--median values, range 70,3745 and 219,6700 pg/mg/h). Less PGE was released than PGF2 alpha, and the amount did not vary with the phase of the menstrual cycle (308 and 296 pg/mg/h (range 65,387 and 105,429) for proliferative and secretory tissue). ET-1 (10 and 100 nM) and arachidonic acid (AA, 30 microM), stimulated PGF2 alpha release from proliferative, but not secretory endometrium, by 78%, 86% (P less than 0.01) and 80% respectively, compared with control tissue. No effect was seen on PGE release. ET-1 may play a role in the local control of the endometrial vascular bed either directly, or via the release of PGF2 alpha.     

Effect of steroids on basal and LH-stimulated prostaglandins F(2alpha) and E(2) release and cyclooxygenase-2 expression in cultured porcine endometrial stromal cells

Ovarian steroids modulate uterine receptivity in domestic species. Luteinizing hormone (LH) stimulates prostaglandin (PG)F(2alpha) release from the porcine endometrium. However, the combined action of LH and steroids on PGs secretion has not yet been studied in pigs. The aim of the present study was to examine the effect of estradiol (E(2)) and progesterone (P(4)) on basal and LH-stimulated PGF(2alpha) and PGE(2) secretion and cyclooxygenase-2 (COX-2) protein expression in porcine endometrial stromal cells obtained on days 12-13 of the estrous cycle. Cells were cultured for 48 h in a medium containing charcoal-stripped newborn calf serum alone or supplemented with 10 nM E(2) and/or 50 nM P(4). Then, the cells were incubated for 6 h in the presence or absence of LH (20 ng/ml). Long exposure of stromal cells to steroids had no effect on PGF(2alpha) secretion, but PGE(2) release increased in the presence of E(2) plus P(4) (p<0.05). Pre-incubation of cells with E(2) plus P(4) resulted in enhanced PGF(2alpha) (p<0.05) and PGE(2) (p<0.001) secretion. Moreover, LH increased PG(2alpha) secretion in control (p<0.05) and E(2)-treated stromal cells (p<0.01). LH tended (p=0.07) to elevate PGE(2) release only in cells pre-exposed to E(2) plus P(4). The expression of COX-2 protein was increased by LH (p<0.05), but not by steroids. These results confirm the stimulatory effect of LH on PGF(2alpha) secretion and COX-2 expression in porcine stromal cells before luteolysis. PG release from porcine endometrium seems to be controlled by ovarian steroids, however only E(2)-treated-treated cells responded to LH.