Cell-type specificity of interleukins 1alpha and 1beta on prostaglandin and plasminogen activator production in bovine endometrial cells

Interleukin (IL)-1alpha is a potent stimulator of prostaglandin production in bovine endometrium, and IL-1 affects plasminogen activator (PA) activity in several types of cells. In this study, we determined the effects of IL-1alpha and IL-1beta on production of the prostaglandins PGF(2alpha) and PGE(2) and on PA activity in cultured bovine endometrial epithelial and stromal cells. We also determined the effects of PGE(2) and PGF(2alpha) on PA activity in these cells. Finally, we used RT-PCR to examine the expression of IL-1alpha, IL-1beta, and IL-1 receptor type 1 (IL-1R) mRNA in cultured bovine endometrial cells. This analysis revealed that IL-1alpha mRNA was present only in the stromal cells, whereas IL-1beta and IL-1R mRNAs were present in both cell types. When cultured cells were exposed to IL-1alpha and IL-1beta at concentrations ranging from 0.006 to 3 nM for 24h, IL-1alpha and IL-1beta were found to dose-dependently stimulate PGE(2) and PGF(2alpha) production in stromal cells (P<0.05) but not in epithelial cells. On the other hand, exposure to IL-1alpha and IL-1beta dose-dependently increased PA activity in the epithelial cells, whereas neither stimulated PA production in the stromal cells. When cells were exposed to IL-1alpha and IL-1beta at concentrations ranging from 0.06 to 3 nM for 24h, the two IL-1s differed in their effects on both PGE(2) and PGF(2alpha) production in stromal cells and had significantly differed in their effects on PA activity in epithelial cells. Exposure to PGE(2) and PGF(2alpha) did not affect PA activity in either stromal or epithelial cells (P>0.05). Taken together, these results suggest the possibility that both IL-1alpha and IL-1beta are produced by the stromal cells, that IL-1beta is produced by the epithelial cells, and that IL-1alpha is a far more potent stimulator than IL-1beta of prostaglandin and PA production in cultured bovine endometrial epithelial and stromal cells.     

Phytoestrogen metabolites are much more active than phytoestrogens themselves in increasing prostaglandin F(2alpha) synthesis via prostaglanin F(2alpha) synthase-like 2 stimulation in bovine endometrium

Phytoestrogens have recently been suggested to be the cause of infertility by stimulating luteolytic prostaglandin (PG) F(2alpha) secretion from endometrium in cattle. The purpose of this study was to examine the enzymatic and molecular mechanisms involved in the preferential induction of PGF(2alpha) synthesis by phytoestrogens, and whether phytoestrogens influence endometrial cell viability. Cultured bovine endometrial epithelial and stromal cells were exposed to phytoestrogens (daidzein and genistein) and their metabolites (equol and p-ethyl phenol) for 24h. Prostaglandin F(2alpha) and PGE2 were stimulated by phytoestrogens in both stromal and epithelial cells, with a preference for PGF(2alpha) synthesis in epithelial cells (P<0.001). Although RT-PCR and Western Blot analyses did not reveal the influence of phytoestrogens on either gene expression or protein level of cyclooxygenase-2 (COX-2) and PGE2 synthase (PGES) in stromal and epithelial cells (P>0.05), the stimulative effects of equol and p-ethyl phenol on PGF(2alpha) synthase-like 2 (PGFSL2) gene expression and protein level were observed only in epithelial cells (P<0.05). The same compounds did not affect PGFSL2 gene expression and protein in stromal cells (P>0.05). Exposure to phytoestrogens and their metabolites decreased cell viability in both stromal and epithelial cells. Stromal cell viability decreased to 50% of the control and was more evident than that in epithelial cells (P<0.001). The overall results suggest that infertility in cattle, caused by phytoestrogen-dependent preferential stimulation of luteolytic PGF(2alpha) synthesis, is caused by increasing PGFSL2 in epithelial cells, and by decreasing stromal cell viability, which are the main source of luteotropic PGE2 production.     

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.     

Anti-apoptotic roles of prostaglandin E2 and F2alpha in bovine luteal steroidogenic cells

Production of prostaglandins (PGs) and expression of their receptors have been demonstrated in bovine corpus luteum (CL). The aim of the present study was to determine whether PGE2 and PGF2alpha have roles in bovine luteal steroidogenic cell (LSC) apoptosis. Cultured bovine LSCs obtained at the midluteal stage (Days 8-12 of the cycle) were treated for 24 h with PGE2 (0.001-1 microM) and PGF2alpha (0.001-1 microM). Prostaglandin E2 (1 microM) and PGF2alpha (1 microM) significantly stimulated progesterone (P4) production and reduced the levels of cell death in the cells cultured with or without tumor necrosis factor alpha (TNF)/interferon gamma (IFNG), in the presence and absence of FAS ligand (P < 0.05). Furthermore, DNA fragmentation induced by TNF/IFNG was observed to be suppressed by PGE2 and PGF2alpha. Prostaglandin E2 and PGF2alpha also attenuated mRNA expression of caspase 3 and caspase 8, as well as caspase 3 activity (P < 0.05) in TNF/IFNG-treated cells. FAS mRNA and protein expression were decreased only by PGF2alpha (P < 0.05). A specific P4 receptor antagonist (onapristone) attenuated the apoptosis-inhibitory effects of PGE2 and PGF2alpha in the absence of TNF/IFNG (P < 0.05). A PG synthesis inhibitor (indomethacin) reduced cell viability in PGE2- and PGF2alpha-treated cells (P < 0.05). A specific inhibitor of cyclooxygenase (PTGS), PTGS2 (NS-398), also reduced cell viability, whereas an inhibitor of PTGS1 (FR122047) did not affect it. The overall results suggest that PGE2 and PGF2alpha locally play luteoprotective roles in bovine CL by suppressing apoptosis of LSCs.     

Effects of indomethacin, luteinizing hormone (LH), prostaglandin E2 (PGE2), trilostane, mifepristone, ethamoxytriphetol (MER-25) on secretion of prostaglandin E (PGE), prostaglandin F2alpha (PGF2alpha) and progesterone by ovine corpora lutea of pregnancy or the estrous cycle

Two experiments were conducted to determine the luteotropin of pregnancy in sheep and to examine autocrine and paracrine roles of progesterone and estradiol-17 beta on progesterone secretion by the ovine corpus luteum (CL). Secretion of progesterone per unit mass by day-8 or day-11 CL of the estrous cycle was similar to day-90 CL of pregnancy (P > or = 0.05). In experiment 1, secretion of progesterone in vitro by slices of CL from ewes on day-8 of the estrous cycle was increased (P < or = 0.05) by LH or PGE2. Secretion of progesterone in vitro by CL slices from day-90 pregnant ewes was not affected by LH (P > or = 0.05) while PGE2 increased (P < or = 0.05) secretion of progesterone. Day 8 ovine CL of the estrous cycle did not secrete (P > or = 0.05) detectable quantities of PGF2alpha or PGE while day-90 ovine CL of pregnancy secreted PGE (P < or = 0.05) but not PGF2alpha. Secretion of progesterone and PGE in vitro by day-90 CL of pregnancy was decreased (P < or = 0.05) by indomethacin. The addition of PGE2, but not LH, in combination with indomethacin overcame the decreases in progesterone by indomethacin (P < or = 0.05). In experiment 2, secretion of progesterone in vitro by day-11 CL of the estrous cycle was increased at 4-h (P < or = 0.05) in the absence of treatments. Both day-11 CL of the estrous cycle and day-90 CL of pregnancy secreted detectable quantities of PGE and PGF2alpha (P < or = 0.05). In experiment 1, PGF2alpha secretion by day-8 CL of the estrous cycle and day-90 ovine CL of pregnancy was undetectable, but was detectable in experiment 2 by day-90 CL. Day 90 ovine CL of pregnancy also secreted more PGE than day-11 CL of the estrous cycle (P < or = 0.05), whereas day-8 CL of the estrous cycle did not secrete detectable quantities of PGE (P > or = 0.05). Trilostane, mifepristone, or MER-25 did not affect secretion of progesterone, PGE, or PGF2alpha by day- 11 CL of the estrous cycle or day-90 CL of pregnancy (P > or = 0.05). It is concluded that PGE2, not LH, is the luteotropin at day-90 of pregnancy in sheep and that progesterone does not modify the response to luteotropins. Thus, we found no evidence for an autocrine or paracrine role for progesterone or estradiol-17 36 on luteal secretion of progesterone, PGE or PGF2alpha.     

Effects of indomethacin, luteinizing hormone (LH), prostaglandin E(2) (PGE(2)), trilostane, mifepristone, ethamoxytriphetol (MER-25) on secretion of prostaglandin E (PGE), prostaglandin F(2alpha) (PGF(2alpha)) and progesterone by ovine corpora lutea of pregnancy or the estrous cycle

Two experiments were conducted to determine the luteotropin of pregnancy in sheep and to examine autocrine and paracrine roles of progesterone and estradiol-17 beta on progesterone secretion by the ovine corpus luteum (CL). Secretion of progesterone per unit mass by day-8 or day-11 CL of the estrous cycle was similar to day-90 CL of pregnancy (P >/= 0.05). In experiment 1, secretion of progesterone in vitro by slices of CL from ewes on day-8 of the estrous cycle was increased (P /= 0.05) while PGE(2) increased (P /= 0.05) detectable quantities of PGF(2alpha) or PGE while day-90 ovine CL of pregnancy secreted PGE (P /= 0.05). Trilostane, mifepristone, or MER-25 did not affect secretion of progesterone, PGE, or PGF(2alpha) by day-11 CL of the estrous cycle or day-90 CL of pregnancy (P >/= 0.05). It is concluded that PGE(2), not LH, is the luteotropin at day-90 of pregnancy in sheep and that progesterone does not modify the response to luteotropins. Thus, we found no evidence for an autocrine or paracrine role for progesterone or estradiol-17 36 on luteal secretion of progesterone, PGE or PGF(2alpha).     

Placental lactogen as a regulator of luteal cells function during pregnancy in sheep

The luteotropic activity of ovine placental lactogen (oPL) on different days of gestation in ewes was assessed using in vitro methods. Corpora lutea (CL) harvested on Days 45, 70, 95, 120 and 135 of gestation and during parturition were enzymatically dispersed and plated on multiwell plates. After 48 h of incubation, all cultures were terminated and media were frozen for further steroid analysis. Cells were cultured in control medium, with addition of oPL alone, or in combination with PGE2 or PGF2alpha. Supplementation of culture media with oPL increased basal progesterone secretion by cells isolated on Days 45 and 70 of gestation. There was no effect on progesterone secretion by cells isolated on other days of gestation; PGE2 added to the culture media increased progesterone production only by cells isolated on Day 70 of pregnancy. Simultaneous oPL treatment with PGE2 had a statistically significant and stimulatory effect on progesterone production by luteal cells collected on Days 70 and 95 of pregnancy. In contrast, PGF2alpha alone in culture media decreased progesterone secretion by cells isolated on Days 45, 70 and 95 of gestation, while oPL plus PGF2alpha on Days 70 and 95 of gestation protected against luteolytic action of PGF2alpha. The results showed 1) a direct effect of the oPL on luteal cells isolated on Days 45 and 70 of gestation; 2) synergism between PL and PGE2 in progesterone production; by cells isolated on Day 70; 3) and a luteoprotective effect of oPL against the luteolytic action of prostaglandin F (PGF2alpha) observed on Days 70 and 95 of gestation.     

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.     

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.     

Effect of mifepristone on pregnancy,pregnancy-specific protein B (PSPB), progesterone,estradiol-17beta,prostaglandin F2alpha (PGF2alpha) and prostaglandin E (PGE) in ovariectomized 90-day pregnant ewes

The objective of this experiment was to determine the effect of mifepristone, a progesterone receptor antagonist, on pregnancy and secretion of steroids, pregnancy-specific protein B (PSPB) and prostaglandins at mid-pregnancy in ewes. Ninety-day pregnant ewes were ovariectomized (OVX) and treatments were initiated 72 h post-OVX. Ewes received (1) vehicle, (2) prostaglandin F2alpha (PGF2alpha, 8 mg/58 kg/bw, i.m.) 84 h post-OVX, (3) mifepristone (50 mg intrajugular at 72, 84, 96, and 108 h post-OVX), (4) mifepristone (50mg) + PGF2alpha, (5) mifepristone (100 mg intrajugular at 72, 84, 96, and 108 h), and (6) mifepristone (100 mg) + PGF2alpha. Ewes treated with vehicle or PGF2alpha alone did not abort (P > or = 0.05). But, 60, 80, 60, and 100% of ewes treated with mifepristone (50 mg), mifepristone (50 mg) + PGF2alpha, mifepristone (100 mg), and mifepristone (100 mg) + PGF2alpha, respectively, aborted (P < or = 0.05). Profiles of progesterone, estradiol-17beta, prostaglandin E (PGE), or PSPB did not differ (P > or = 0.05) among treatment groups. Profiles of PGF2alpha of treatment groups receiving mifepristone with or without PGF2alpha differed (P < 0.05) from vehicle or PGF2alpha alone-treated ewes. It is concluded that progesterone actions are necessary to suppress uterine/placental secretion of PGF2alpha and that maintenance of critical progesterone: estradiol-17beta and PGE:PGF2alpha ratios are necessary for maintenance of pregnancy.     

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.     

In vitro prostaglandin release from and platelet-activating factor accumulation in isolated endometrial cells from pregnant and pseudopregnant rabbits.

Prostaglandin (PG) release from and platelet-activating factor (PAF) accumulation by enzymatically isolated endometrial epithelial and stromal cells from Day 6 pregnant and Day 6 pseudopregnant rabbits were studied in vitro, using RIA for PG measurement and a platelet aggregation assay for PAF measurement. On the first day of culture in serum-free media, PGF release into the medium was significantly higher from epithelial cells from Day 6 of pregnancy than from stromal cells from Day 6 of pregnancy or pseudopregnancy. PGE release did not differ significantly among these cell types. The addition of indomethacin (10(-5) M) to similar cultures inhibited release of both PGs from both cell types, but to a much greater extent from stromal than from epithelial cells. Significant stimulation of PG release by A23187 was achieved under all conditions on the fifth day of culture; PGE release was significantly greater than PGF release from stromal cells from Day 6 of pregnancy and pseudopregnancy, and release of both PGs from stromal cells was significantly greater from Day 6 of pregnancy than from Day 6 of pseudopregnancy. PG release from similar cells, cultured in medium containing 10% calf serum, was highest on the first or second day of culture and then, especially for PGF, declined with continued culture. PGE release was significantly higher than PGF release from stromal cells on the third and fourth days of culture. The ratios of PGF/PGE release from epithelial cells were significantly higher than those from stromal cells over the 5-day culture period for both reproductive stages. These ratios indicate the differential release of PGE and PGF from rabbit endometrial cell subpopulations and indicate a preferential release of PGE from stromal and of PGF from epithelial cells. Under basal conditions, PAF was not detected in epithelial or stromal cells cultured for 2 or 4 days, or in the associated culture media. If PAF had been released into the medium, it would have rapidly metabolized. Short exposure to calcium ionophore A23187 (10(-5) M) was able to stimulate PAF accumulation in epithelial and stroma cells in serum-free media, probably via the remodeling pathway. PAF was not detected in the medium. Intracellular PAF accumulation after exposure to A23187 (10(-5) M) for 5 min was significantly greater on the second day of culture than on the fourth day in epithelial and stromal cells from Day 6 of pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)     

The influence of embryo presence on prostaglandins synthesis and prostaglandin E2 and F2alpha content in corpora lutea during periimplantation period in the pig

We determined the expression of PGE2 synthase (mPGES-1), PGF synthase (PGFS), carbonyl reductase/prostaglandin 9-ketoreductase (CBR1) genes and the content of PGE2, PGF2alpha in porcine corpora lutea on Days 12-14 of pregnancy and Days 12-14 of the estrous cycle. For this study we used a surgically-generated model in which one of the uterine horns was cut transversely and a part of this horn was detached from the uterine corpus. The expression of mPGES-1, PGFS, and CBR1 genes and mPGES-1/PGFS ratio were significantly higher in corpora lutea of the pregnant gilts compared to the corpora lutea from the parallel ovaries of the cyclic gilts. There was no difference in mPGES-1, PGFS, CBR1 genes expression and mPGES-1/PGFS ratio between corpora lutea ipsi-(CL1) and contralateral (CL2) to the uterine horn with the developing embryos. The highest content of PGE2 was found in CL1 of the pregnant gilts. The PGE2/PGF2alpha ratio was significantly higher in CL1 of the pregnant gilts compared to corpora lutea from parallel ovary of the cyclic gilts. We suggest that the activity of the investigated genes is induced by compounds of embryonic origin which are not distributed only to the ipsilateral ovary but are transported within the mesometrium to both ovaries in a more systemic manner.     

Phytoestrogens modulate prostaglandin production in bovine endometrium: cell type specificity and intracellular mechanisms

Prostaglandins (PGs) are known to modulate the proper cyclicity of bovine reproductive organs. The main luteolytic agent in ruminants is PGF2alpha, whereas PGE2 has luteotropic actions. Estradiol 17beta (E2) regulates uterus function by influencing PG synthesis. Phytoestrogens structurally resemble E2 and possess estrogenic activity; therefore, they may mimic the effects of E2 on PG synthesis and influence the reproductive system. Using a cell-culture system of bovine epithelial and stromal cells, we determined cell-specific effects of phytoestrogens (i.e., daidzein, genistein), their metabolites (i.e., equol and para-ethyl-phenol, respectively), and E2 on PGF2alpha and PGE2 synthesis and examined the intracellular mechanisms of their actions. Both PGs produced by stromal and epithelial cells were significantly stimulated by phytoestrogens and their metabolites. However, PGF2alpha synthesis by both kinds of cells was greater stimulated than PGE2 synthesis. Moreover, epithelial cells treated with phytoestrogens synthesized more PGF2alpha than stromal cells, increasing the PGF2alpha to PGE2 ratio. The epithelial and stromal cells were preincubated with an estrogen-receptor (ER) antagonist (i.e., ICI), a translation inhibitor (i.e., actinomycin D), a protein kinase A inhibitor (i.e., staurosporin), and a phospholipase C inhibitor (i.e., U73122) for 0.5 hrs and then stimulated with equol, para-ethyl-phenol, or E2. Although the action of E2 on PGF2alpha synthesis was blocked by all reagents, the stimulatory effect of phytoestrogens was blocked only by ICI and actinomycin D in both cell types. Moreover, in contrast to E2 action, phytoestrogens did not cause intracellular calcium mobilization in either epithelial or stromal cells. Phytoestrogens stimulate both PGF2alpha and PGE2 in both cell types of bovine endometrium via an ER-dependent genomic pathway. However, because phytoestrogens preferentially stimulated PGF2alpha synthesis in epithelial cells of bovine endometrium, they may disrupt uterus function by altering the PGF2alpha to PGE2 ratio.     

Progesterone and estradiol secretion by porcine luteal cells is influenced by individual and combined treatment with prostaglandins E2 and F2alpha throughout the estrus cycle.

The present experiments were conducted to test whether the ratio of PGE2:PGF2alpha affects steroid secretion by porcine luteal cells. We examined the effect of separate and combined treatment with PGE2 and PGF2alpha on progesterone and estradiol secretion. Luteal cells were collected at three different stages of the luteal phase (1-3 days after ovulation; 10-12 days after ovulation and 14-16 days after ovulation). PGE2 alone in a dose dependent manner increased progesterone production by cells collected from mature corpora lutea. On the other hand, PGF2alpha in a dose dependent manner decreased progesterone secretion by cells of the same origin. Progesterone secretion by cells isolated from mature and regressing corpora lutea and treated with both prostaglandins increased in comparison to PGF2alpha-treated cultures. However, in cells collected from regressing corpora lutea PGE2 and PGF2alpha in a ratio of 2:1 and 4:1 increased estradiol production when compared to control and both ratios increased estradiol secretion in comparison to PGF2alpha-treated cells. These data 1) confirm the luteotropic effect of PGE2 and the luteolytic effect of PGF2alpha; 2) demonstrate that when the ratio of PGE2 to PGF2alpha changed from 1:1 to 2:1 or 4:1 cells were protected against the inhibitory effects of PGF2alpha on progesterone secretion by cells collected during the mid- and late luteal phase; and 3) suggest that elevated estradiol production by luteal cells, isolated during late luteal phase, under the influence of increased doses of PGE2 may serve as an additional source of estradiol to blastocysts, during early pregnancy in the pig.     

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.     

Regulation of prostaglandin secretion from epithelial and stromal cells of the bovine endometrium by interleukin-1 beta, interleukin-2, granulocyte-macrophage colony stimulating factor and tumor necrosis factor-alpha.

Bovine endometrium was obtained on day 16 of pregnancy (estrus = 0) and separated into epithelial and stromal cell populations. When confluent, the two cell populations were treated for 24 h with cytokines at 1, 10 and 100 ng/ml. Prostaglandin (PG) E2 was the major prostaglandin produced by both cell types. For control cultures, more PGE2 was secreted into medium by stromal cells than by epithelial cells, whereas secretion of PGF was similar for epithelial and stromal cells. Interleukin-1 beta had no effect on prostaglandin production by stromal cell cultures but increased epithelial production of PGE2 and, to a lesser extent, PGF. Conversely, granulocyte-macrophage colony stimulating factor had no effect on epithelial cells but reduced secretion of PGE2 and PGF from stromal cells. There were no effects of interleukin-2 or tumor necrosis factor-alpha on prostaglandin secretion. Results indicate that certain cytokines can regulate endometrial prostaglandin secretion in a cell type-restricted manner.     

In vivo levels of prostaglandin F2 alpha, E2 and prostacyclin in the corpus luteum of pregnant and pseudopregnant rats

Prostaglandin F2 alpha (PGF2 alpha) is a well-known luteolytic factor in the rat corpus luteum. To investigate a possible luteal origin of PGF2 alpha, measurements of this prostaglandin were performed in different luteal tissues in vivo. Prostaglandin E2 (PGE2) and the stable metabolite of prostacyclin, 6-keto-PGF1 alpha, were assayed simultaneously. Corpora lutea of different ages from 57 pregnant and pseudopregnant rats (mated with sterile males) were rapidly excised, dissected in 0 degree C indomethacin solution, homogenized, and extracted for prostaglandins with solid-phase extraction cartridges. Prostaglandins were determined by radioimmunoassay. Plasma levels of progesterone and 20 alpha-dihydroprogesterone were also monitored. In the adult pseudopregnant rat model, luteolysis occurs at Day 13 +/- 1, and maximal levels of all three prostaglandins were detected on Day 13 of pseudopregnancy: 0.40 +/- 0.02, 2.6 +/- 0.29, and 1.76 +/- 0.24 pmol/mg protein (mean +/- SEM, n=7) for PGF2 alpha, PGE2, and 6-keto-PGF1 alpha respectively. In pregnant rats, on the corresponding day, levels were considerably lower: 0.15 +/- 0.02, 0.90 +/- 0.13, and 0.50 +/- 0.06 pmol/mg protein (mean +/- SEM, n=9, p less than 0.0001), respectively. Luteal levels in pregnant rats showed a continuous decline on Days 13 and 19 for all prostaglandins measured, whereas in pseudopregnant rats an increment of PGF2 alpha was noted between Days 7 and 13 and remained high on Day 19. PGE2 closely followed levels of PGF2 alpha, but at a 5- to 10-fold higher level. The coefficient of correlation between PGF2 alpha and PGE2 in the luteal compartment of both models was 0.87 (p less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of prostaglandins E2 and F2alpha (PGE2; PGF2alpha), trilostane,mifepristone, palmitic acid (PA), indomethacin (INDO), ethamoxytriphetol (MER-25), PGE2 + PA,or PGF2alpha + PA on PGE2, PGF2alpha,and progesterone secretion by bovine corpora lutea of mid-pregnancy in vitro

The effects of PGE2, PGF2alpha, trilostane, RU-486, PA, INDO, MER-25, PGE2, or PGF2alpha + PA on secretion of progesterone, PGE2, or PGF2alpha by bovine corpora lutea (CL) of mid-pregnancy in vitro for 4 and 8 hr was examined. Secretion of PGE2 and PGF2alpha increased with time in culture (P < or = 0.05). PGE2 and PGE2 + PA increased (P < or = 0.05) secretion of progesterone at 4 and 8 h, progesterone secretion was increased (P < or = 0.05) at 4 h; but not at 8 h (P > or = 0.05) by trilostane, mifepristone, PGF2alpha and PGF2alpha + PA, and was decreased at 8 h by PGF2alpha and PGF2alpha + PA. Indomethacin decreased (P < or = 0.05) secretion of PGE2, PGF2alpha, and progesterone at 4 and 8 h. Trilostane, PA, PGF2alpha, RU-486 and PGF2alpha + PA increased (P < or = 0.05) PGE2 at 4 h only. Palmitic acid decreased (P < or = 0.05) PGF2alpha at 4 h, while trilostane, RU-486, or MER-25 did not affect (P < or = 0.05) PGE2 of PGF2alpha secretion. It is concluded that PGE2 of luteal tissue origin is the luteotropin at mid-pregnancy in cows. Also, it is suggested that PA may alter progesterone secretion by affecting the inter conversion of PGE2 and PGF2alpha.     

The effect of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta and IL-6 on chorioamnion secretion of prostaglandins (PG)F 2 alpha and E2 in pigs

The aim of the present study was to determine the effect of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) on prostaglandin (PG)F(2 alpha) and PGE(2) secretion as well as cyclooxygenase-2 (COX-2) protein expression in chorioamnion collected on days 25, 30 and 40 of pregnancy in pigs. Fetal membrane slices were incubated for 16 h with TNF-alpha, IL-1 beta, IL-6 (1 or 10 ng/ml of medium) or two combinations of the three cytokines (1 or 10 ng/ml of each cytokine per combination). We demonstrated the stimulatory effect of TNF-alpha, IL-1 beta and/or IL-6 on PGF(2 alpha) and PGE(2) secretion by the porcine fetal membranes. The medium content of these PGs depended on the cytokine type, treatment dose and day of pregnancy. Cytokine stimulation of PGE(2) was more pronounced than that of PGF(2 alpha). In addition, an increase in PGF(2 alpha) and/or PGE(2) secretion was usually associated with an augmentation of COX-2 protein expression. Our results support the notion concerning the possible role of cytokines in modulating production of PGs by fetal membranes during the first trimester of gestation.