PGF2α Synthesis Research Articles

High α-linolenic acid and fish oil ingestion promotes ovulation to the same extent in rats

Prostaglandins (PG) have a regulatory influence on ovulation. α-Linolenic acid (ALA) vs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) differently influence PG biosynthesis. Whereas high EPA/DHA reduces PGE₂, enhancing ovulation, we hypothesized that ALA would not affect ovulation. Our objective was to determine the effect of low and high ALA intake vs EPA/DHA on ovarian phospholipids, ovulation, and PG synthesis in rats. Following 27 days on diet and ovulation induction, ovaries were isolated and analyzed in 22 pups per diet. Ovarian phospholipid (n-3) polyunsaturated fatty acid (PUFA) incorporation increased with EPA/DHA ingestion. With significant ovarian (n-3) PUFA or EPA (P < .05) enrichment in the high-n-3 PUFA diets, ova release increased. Although high ALA did not enrich total (n-3), it increased ova release and tissue EPA over low ALA or control. Dietary EPA/DHA more effectively reduced ovarian arachidonic acid levels than dietary ALA. Dietary ALA increased PGF and very high intake reduced PGE, whereas EPA/DHA did not alter PGE or PGF. Enhanced ova release with high (n-3) PUFA intake may be induced via multiple mechanisms including reduced ovarian arachidonic acid. Significant ovarian retention of EPA and DHA enhanced ovulation with unchanged total PGE and PGF. Lack of change in PGE may have resulted from reduced PGE₂ combined with increased PGE₃. When EPA alone was elevated, PGE was reduced, whereas PGF was increased. Results indicate that very high ALA intake enhances ovulation similar to very high EPA/DHA ingestion, an effect potentially mediated via similar patterns of PGF₂α and PGE₂ synthesis.

ChemInform Abstract: Organopalladium Approaches to Prostaglandins. Part 11. Synthesis of PGF2α and 12-epi-PGF2α by the Controlled, One-Step, Palladium-Promoted, Intermolecular Coupling of Three Different Alkenes.

ChemInform Abstract: Organopalladium Approaches to Prostaglandins. Part 11. Synthesis of PGF2α and 12-epi-PGF2α by the Controlled, One-Step, Palladium-Promoted, Intermolecular Coupling of Three Different Alkenes.

Distinction of microsomal prostaglandin E synthase-1 (mPGES-1) inhibition from cyclooxygenase-2 inhibition in cells using a novel, selective mPGES-1 inhibitor

Inflammation-induced microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme that synthesizes prostaglandin E 2 (PGE 2) downstream of cyclooxygenase-2 (COX-2). The efficacy of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors in the treatment of the signs and symptoms of osteoarthritis, rheumatoid arthritis and inflammatory pain, largely attributed to the inhibition of PGE 2 synthesis, provides a rationale for exploring mPGES-1 inhibition as a potential novel therapy for these diseases. Toward this aim, we identified PF-9184 as a novel mPGES-1 inhibitor. PF-9184 potently inhibited recombinant human (rh) mPGES-1 (IC 50 = 16.5 ± 3.8 nM), and had no effect against rhCOX-1 and rhCOX-2 (>6500-fold selectivity). In inflammation and clinically relevant biological systems, mPGES-1 expression, like COX-2 expression was induced in cell context- and time-dependent manner, consistent with the kinetics of PGE 2 synthesis. In rationally designed cell systems ideal for determining direct effects of the inhibitors on mPGES-1 function, but not its expression, PF-9184 inhibited PGE 2 synthesis (IC 50 in the range of 0.5–5 μM in serum-free cell and human whole blood cultures, respectively) while sparing the synthesis of 6-keto-PGF 1α (PGF 1α) and PGF 2α. In contrast, as expected, the selective COX-2 inhibitor, SC-236, inhibited PGE 2, PGF 1α and PGF 2α synthesis. This profile of mPGES-1 inhibition, distinct from COX-2 inhibition in cells, validates mPGES-1 as an attractive target for therapeutic intervention.

The relationship between the production and the anti-gonadotrophic action of prostaglandin F 2α in luteal cells from the marmoset monkey ( Callithrix jacchus) in the early and mid-luteal phase

To address the potential luteolytic role for prostaglandin F 2α (PGF 2α) in the corpus luteum of the common marmoset monkey ( Callithrix jacchus), the ability of marmoset luteal cells, maintained in monolayer culture, to produce PGF 2α was determined in vitro in the presence and absence of human chorionic gonadotrophin (hCG) and other established pharmacological modulators of PGF 2α synthesis. We also assessed the effects of the PGF 2α analogue, cloprostenol, on progesterone output from luteal cells isolated in the early luteal phase versus the mid-luteal phase (days 3 and 14 post ovulation, respectively). Cloprostenol had no effect on progesterone output from luteal cells isolated on day 3 of the luteal phase, whereas it significantly inhibited both basal and hCG-stimulated progesterone synthesis by day 14 luteal cells during the culture period 48–72 h ( P < 0.001). Intra-luteal PGF 2α concentrations were 5-fold higher in luteal cells isolated in the early luteal phase than in mid-luteal phase cells (16.5 ± 3.5 versus 3.5 ± 0.6 pmol/10 5 cells). While PGF 2α production was unaffected by hCG in vitro, it was decreased by indomethacin (1000 ng/ml) ( P < 0.05) and stimulated by the calcium ionophore A23187 (10 μmol/l) ( P < 0.05) in luteal cells from both stages of the luteal phase. Phospholipase A 2 did not influence PGF 2α production by day 3 luteal cells whereas at 10 IU/ml, it significantly stimulated PGF 2α production by day 14 luteal cells ( P < 0.05). Hence, the timing of luteolysis in the common marmoset monkey appears to involve changes in both the luteal cell response to and production of PGF 2α.

The effect of ovarian steroids on oxytocin-stimulated secretion and synthesis of prostaglandins in bovine myometrial cells

The aim was to study whether bovine myometrial cells have an enzymatic system able to produce prostaglandins (PGs) and whether PGs synthesis is regulated by steroids in a similar manner as endometrial cells. In Experiment 1, immunohistochemical studies localized proteins for cyclooxygenase 2 (COX2), PGE synthase (PGES) and PGF2α synthase (PGFS) in myometrial and endometrial (as positive control) slices from days 14 to 16 of the estrous cycle. Enzymatically isolated myometrial cells (2.5 × 10 5/ml) were cultured for 96 h to attach them to the bottom of the culture well. In Experiment 2, cells were preincubated for 30 min with progesterone (P4; 10 −5 M), and thereafter incubated for 4 or 6 h with arachidonic acid (AA; 10 −5 M, as positive control), oxytocin (OT; 10 −7 M), and OT + P4. In medium, PGE and PGFM (PGF2α metabolite) were increased ( P < 0.05) by AA treatment after 4 and 6 h, but by OT only after 6 h of incubation. Progesterone did not affect ( P > 0.05) basal secretion of both PGs, but it diminished ( P < 0.05) the stimulatory effect of OT on PGF2α and PGE secretion after 6 h of incubation. The amount of enzyme protein for COX2, PGES and PGFS analyzed by Western Blot was affected ( P > 0.05) by any of the factors added to the culture medium. In Experiment 3, myometrial cells were preincubated with P4 (10 −5 M) and pregnenolone (P5; 10 −5 M) for 30 min, and then incubated for 6 h with OT (10 −7 M) and OT plus each of these steroids used. Expression of mRNA for COX2, but not PGFS and PGES, was found in the cells stimulated with OT. Neither P4 nor P5 affected expression of the studied genes; however, both steroids diminished ( P < 0.05) OT-stimulated mRNA expression of COX2. The data suggest that: (a) myometrial cells are able to synthesize both PGF2α and PGE and (b) synthesis of these PGs may be regulated by steroids through a transcription-independent manner, which modulated the effect of OT on COX2 mRNA expression.

Involvement of prostaglandin F2α in the adverse effect of PCB 77 on the force of contractions of bovine myometrium

Polychlorinated biphenyls (PCBs) stimulate in vitro both the force of myometrial contractions and endometrial secretion of PGF2α in cattle. Therefore, the goal of this study was to investigate the participation of PGF2α in the effect of PCBs on uterine contractility. For this aim, the myometrial strips were incubated (48 h) with PCB 77 at the dose of 1, 10 and 100 ng/ml (i.e., 0.0034, 0.034 and 0.34 nmol/ml) separately or jointly with indomethacin (INDO, 10 −4 M), which blocks the PGF2α synthesis. Next, the force of myometrial strips contractions was measured. Further, the influence of PCB 77 (0.1, 1 and 10 ng/ml) on the PGF2α secretion from myometrial cells after 6, 24, and 48 h and PCB 77 (1 and 10 ng/ml) on the mRNA expression of cyclooxygenase 2 (COX-2) and PGF2α synthase (PGFS) in myometrial cells after 6 and 24 h, was investigated. The increase ( P < 0.05–0.001) of the contractions force of myometrial strips evoked by each dose of PCB 77, was markedly reduced ( P < 0.05–001) by INDO. There was an increase ( P < 0.05–0.001) of both PGF2α secretion after all studied periods of cell incubation and mRNA expression for COX-2 and PGFS after 6 h treatment of myometrial cells with PCB 77. It can be concluded that myometrial synthesis of PGF2α and its further secretion is a part of the mechanism by means of which PCB 77 may affect the force of myometrial contractions in cattle.

Effects of RU486 on cyclooxygenase-2 gene expression, prostaglandin F2α synthesis and ovulation in Xenopus laevis

RU486 is a synthetic analog of progesterone and functions as a progesterone receptor antagonist. It binds to the progesterone receptor to prevent progesterone from occupying its receptor in many cellular systems. Early studies from our laboratory have shown that in Xenopus laevis ovarian follicles progesterone stimulates the expression of cyclooxygenase-2 (COX-2) gene which leads to a rapid increase in the production of prostaglandin F2α (PGF2α) and subsequent ovulation. In this study, we examined the effect of RU486 on the synthesis of COX-2 mRNA, production of PGF2α and ovulation in X. laevis. Ovarian tissue fragments were primed with human chorionic gonadotropin (hCG) and then incubated with progesterone (P4) alone or in the presence of varying concentrations of RU486 over a period of 12 h. After the incubation ovulated oocytes were counted, COX-2 expression and synthesis of PGF2α were measured. Results demonstrated that RU486 attenuated the expression of COX-2 gene, reduced the synthesis of PGF2α, and inhibited ovulation in a dose-dependent manner. This finding suggests that progesterone receptor is an important regulator in the progesterone–cyclooxygenase–prostaglandin-mediated ovulation in amphibians.

Lysophosphatic Acid Modulates Prostaglandin Secretion in the Bovine Endometrial Cells Differently on Days 8-10 of the Estrous Cycle and Early Pregnancy

Lysophosphatidic acid (LPA) is becoming a new player in regulation of the reproductive processes of domestic animals. In the present study, we examined whether LPA modulates prostaglandin (PG) synthesis in the bovine endometrium at the time of the early maternal pregnancy recognition compared with the respective days of the estrous cycle and the enzymatic mechanism of this action. Bovine epithelial and stromal endometrial cells isolated from the uteri on days 8-10 of the estrous cycle and pregnancy were cultured with LPA for 24 h. LPA increased PGE(2) production in stromal cells during the estrous cycle and early pregnancy. On days 8-10 of pregnancy, LPA inhibited PGF(2alpha) production in epithelial cells. LPA stimulated PGES mRNA expression in stromal cells during both examined phases and inhibited PGFS mRNA expression in epithelial cells on days 8-10 of pregnancy. The overall results indicate that LPA may serve as a luteotropic factor during the luteal phase of the estrous cycle and early pregnancy stimulating PGE(2) synthesis and mRNA expression for PGES in stromal cells. Moreover, during early pregnancy, LPA might protect bovine CL and early embryo development by decreasing PGF(2alpha) synthesis and mRNA expression for PGFS in the epithelial cells of the bovine endometrium.

Acetylsalicylic acid regulates MMP-2 activity and inhibits colorectal invasion of murine B16F0 melanoma cells in C57BL/6J mice: Effects of prostaglandin F 2α

Epidemiological studies indicate that acetylsalicylic acid may reduce the risk of mortality due to colon cancers. Metastasis is the major cause of cancer death. Matrix metalloproteinases (MMPs) play important roles in tumor invasion regulation, and prostaglandin F 2α (PGF 2α) is a key stimulator of MMP production. Thus, we investigated whether acetylsalicylic acid regulated MMP activity and the invasion of cancer cells and whether PGF 2α attenuated acetylsalicylic acid-inhibited invasion of cancer cells. Gelatin-based zymography assays showed that acetylsalicylic acid inhibited the MMP-2 activity of B16F0 melanoma cells. Matrigel-based chemoinvasion assays showed that acetylsalicylic acid inhibited the invasion of B16F0 cells. Acetylsalicylic acid can inhibit PGF 2α synthesis and PGF 2α is a key stimulator of MMP-2 production. Our data showed that PGF 2α treatment attenuated the acetylsalicylic acid-inhibited invasion of B16F0 cells. In animal experiments, acetylsalicylic acid reduced colorectal metastasis of B16F0 cells in C57BL/6J mice by 44%. Our results suggest that PGF 2α is a therapeutic target for metastasis inhibition and acetylsalicylic acid may possess anti-metastasis ability.

Expression of cyclooxygenase genes and production of prostaglandins during ovulation in the ovarian follicles of Xenopus laevis

Involvement of cyclooxygenase (COX) and prostaglandin (PG) synthesis during ovulation in Xenopus laevis ovarian follicles was investigated. X. laevis COX cDNAs were isolated from ovarian tissues by using reverse transcription-polymerase chain reaction (RT-PCR) followed by rapid amplification of cDNA ends (RACE). In X. laevis ovary, expression of COX-2 but not COX-1 mRNA was up-regulated during gonadotropin-induced oocyte maturation and ovulation in vitro. Elevation of PGF 2α synthesis was directly correlated with the up-regulation of COX-2 mRNA. Synthesis of PGE 2 also increased during periovulatory period, however, the concentrations were much lower than those of PGF 2α. Progesterone (P4) also induced up-regulation of COX-2 mRNA as well as ovulation. Actinomycin D (ActD) blocked P4-induced ovulation. The inhibition of ovulation by Act D was rescued by co-treatment with exogenous PGF 2α in a dose dependent manner. A non-selective COX inhibitor (indomethacin) and selective COX-2 inhibitor (NS398) strongly inhibited the hCG- and P4-dependent production of PGF 2α. Inhibitory effects of selective COX-1 inhibitor (SC560) on PGF 2α production were lower than that of other inhibitors. Indomethacin and NS398 blocked P4-induced ovulation. NS398 did not block hCG-induced ovulation although it strongly suppressed PGF 2α production. These results suggest that (1) in Xenopus ovarian follicles, PGF 2α is synthesized during periovulatory period, similar to that in mammals, (2) PGF 2α synthesis is regulated by de novo transcription of COX-2 but not COX-1, (3) PGF 2α is necessary for P4-induced ovulation but may not be essential for hCG-induced ovulation, and other factor(s) may be involved in the hCG-induced ovulation.

Two pathways for prostaglandin F2α synthesis by the primate periovulatory follicle

Prostaglandin E2 (PGE2) has been identified as a PG necessary for ovulation, but the ovulatory gonadotropin surge also increases PGF2 alpha levels in primate periovulatory follicles. To better understand the role of PGF2 alpha in ovulation, pathways utilized for PGF2 alpha synthesis by the primate follicle were examined. Monkeys were treated with gonadotropins to stimulate multiple follicular development; follicular aspirates and whole ovaries were removed before and at specific times after administration of an ovulatory dose of hCG to span the 40 h periovulatory interval. Human granulosa cells were also obtained (typically 34-36 h after hCG) from in vitro fertilization patients. PGF2 alpha can be synthesized from PGH2 via the aldo-keto reductase (AKR) 1C3. AKR1C3 mRNA and protein levels in monkey granulosa cells were low before hCG and peaked 24-36 h after hCG administration. Human granulosa cells converted PGD2 into 11 beta-PGF2 alpha, confirming that these cells possess AKR1C3 activity. PGF2 alpha can also be synthesized from PGE2 via the enzymes AKR1C1 and AKR1C2. Monkey granulosa cell levels of AKR1C1/AKR1C2 mRNA was low 0-12 h, peaked at 24 h, and returned to low levels by 36 h after hCG administration. Human granulosa cell conversion of [(3)H]PGE2 into [(3)H]PGF2 alpha was reduced by an AKR1C2-selective inhibitor, supporting the concept that granulosa cells preferentially express AKR1C2 over AKR1C1. In summary, the ovulatory gonadotropin surge increases granulosa cell expression of AKR1C1/AKR1C2 and AKR1C3. Both of these enzyme activities are present in periovulatory granulosa cells. These data support the concept that follicular PGF2 alpha can be synthesized via two pathways during the periovulatory interval.

Effect of luteinizing hormone (LH), pregnancy-specific protein B (PSPB), or arachidonic acid (AA) on secretion of progesterone and prostaglandins (PG) E (PGE; PGE 1 and PGE 2) and F 2α (PGF 2α) by ovine corpora lutea of the estrous cycle or pregnancy in vitro

LH regulates luteal progesterone secretion during the estrous cycle in ewes and cows [1]. However, PGE, not LH, stimulated ovine luteal progesterone secretion in vitro at day 90 of pregnancy [2] and at day 200 in cows [3–5]. The hypophysis is not obligatory after day 50 [6] nor the ovaries [7] after day 55 to maintain pregnancy in ewes. LH has been reported to regulate ovine placental PGE secretion up to day 50 of pregnancy and by pregnancy-specific protein B (PSPB) after day 50 of pregnancy [4,6,8]. The objective of this experiment was to determine if and when a switch from LH to PGE occurred as the luteotropin regulating luteal progesterone secretion during pregnancy in ewes. Ovine luteal tissue slices of the estrous cycle (days 8, 11, 13, and 15) or pregnancy (days 8, 11, 13, 15, 20, 30, 40, 50, 60, and 90) were incubated in vitro with vehicle, LH, AA (precursor to PGE 2 and PGF 2α synthesis; [9]), or PSPB in M199 for 4 h and 8 h. Concentrations of progesterone in jugular venous plasma of bred ewes increased ( P ≤ 0.05) after day 50 and continued to increase through day 90. Secretion of progesterone by luteal tissue of non-bred ewes on days 8, 11, 13 and 15 and by bred ewes on days 8, 11, 13, 15, 20, 30, 40, and 50 was increased ( P ≤ 0.05) by LH, but not by luteal tissue from pregnant ewes after day 50 ( P ≥ 0.05). LH-stimulated progesterone secretion by luteal tissue from day 15 bred ewes was greater ( P ≤ 0.05) than day 15 luteal tissue from non-bred ewes. Concentrations of progesterone in media were increased ( P ≤ 0.05) when luteal tissue from pregnant ewes on day 50, 60, or 90 were incubated with AA or PSPB. Concentrations of PGE in media of non-bred ewes on days 8, 11, 13, or 15 and bred ewes on days 8 and 11 did not differ ( P ≥ 0.05). Concentrations of PGE were increased ( P ≤ 0.05) in media by luteal slices from bred ewes on days 13, 15, 20, 30, 40, 50, 60, and 90 of vehicle, LH, AA or PSPB-treated ewes. In addition, PSPB increased ( P ≤ 0.05) PGE in media by luteal slices from pregnant ewes only on days 40, 50, 60, and 90. Concentrations of PGF 2α were increased in media ( P < 0.05) of vehicle, AA, LH, or PSPB-treated luteal tissue from non-bred ewes and bred ewes on day 15 and by luteal tissue from bred ewes on days 20 and 30 after which concentrations of PGF 2α in media declined ( P ≤ 0.05) and did not differ ( P ≥ 0.05) from non-bred or bred ewes on days 8, 11, or 13. It is concluded that LH regulates luteal progesterone secretion during the estrous cycle of non-bred ewes and up to day 50 of pregnancy, while only PGE regulates luteal progresterone secretion by ovine corpora lutea from days 50 to 90 of pregnancy. In addition, PSPB appears to regulate luteal secretion of progesterone from days 50 to 90 of pregnancy through stimulation of PGE secretion by ovine luteal tissue.

Progesterone metabolism in bovine endometrial cells and the effect of metabolites on the responsiveness of the cells to OT-stimulation of PGF 2α

Oxytocin receptor (OTR) expression is suppressed by progesterone (P4) during the luteal phase of the estrous cycle and then it increases at the time of luteolysis, but its regulation is still not completely understood. The objective of this work was to characterize P4 metabolism by endometrial cells in vitro and determine if metabolites were able to modify prostaglandin secretion in response to oxytocin (OT). Endometrial epithelial and stromal cells were incubated with 3H-P4 or 3H-pregnenolone (P5) for 6 or 24 h. Metabolites in the medium were separated by HPLC. The results showed that P4 and P5 were converted to two major polar metabolites and a less polar metabolite that was identified as 5α- or 5β-pregnanedione by LC/MS. Progesterone metabolism was similar in both stromal and epithelial cells. To determine if 5α- or 5β-pregnanedione were able to modify PGF 2α synthesis, cells were cultured with P4, 5α- or 5β-pregnanedione (100 ng ml −1) for 48 h and then each group of cells was incubated for a further 4–6 h with or without OT (200 ng ml −1). Results showed that only P4 caused significant ( P < 0.001) increase in basal, but not OT-stimulated, PGF 2α synthesis. OT binding assays showed no significant effect of progesterone or its metabolites on OTR concentration. In conclusion, bovine endometrial cells are able to metabolize pregnenolone and progesterone but neither 5α- nor 5β-pregnanedione altered prostaglandin synthesis or OTR number in endometrial epithelial cells. These data suggest that 5-pregnanediones do not play a role in the regulation OT-stimulated PGF 2α secretion during the bovine estrous cycle.

Intraluteal regulation of prostaglandin F2α-induced prostaglandin biosynthesis in pseudopregnant rabbits

The objective of the present study was to investigate in rabbit corpora lutea (CL), at both the cellular and molecular level, intraluteal cyclooxygenase (COX)-1, COX-2 and prostaglandin (PG) E2-9-ketoreductase (PGE2-9-K) enzymatic activities as well as in vitro PGE2 and PGF2alpha synthesis following PGF2alpha treatment at either early- (day-4) or mid-luteal (day-9) stage of pseudopregnancy. By immunohistochemistry, positive staining for COX-2 was localized in luteal and endothelial cells of stromal arteries at both the stages. In CL of both stages, basal COX-2 mRNA levels were poorly expressed, but rose (P < 0.01) 4- to 10-fold 1.5-6 h after treatment and then gradually decreased within 24 h. Compared to mid-stage, day-4 CL had lower (P < 0.01) COX-2 and PGE2-9-K basal activities, and PGF2alpha synthesis rate, but higher (P < 0.01) PGE2 production. Independent of luteal stage, PGF2alpha treatment did not affect COX-1 activity. In day-4 CL, PGF2alpha induced an increase (P < 0.01) in both COX-2 activity and PGF2alpha synthesis, whereas that of PGE2 remained unchanged. In day-9 CL, PGF2alpha up-regulated (P < 0.01) both COX-2 and PGE-9-K activities, and PGF2alpha production, but decreased (P < 0.01) PGE2 synthesis. All changes in gene expression and enzymatic activities occurred within 1.5 h after PGF2alpha challenge and were more marked in day-9 CL. Our data suggest that PGF2alpha directs intraluteal PG biosynthesis in mature CL, by affecting the CL biosynthetic machinery to increase the PGF2alpha synthesis in an auto-amplifying manner, with the activation of COX-2 and PGE-9-K; this may partly explain their differentially, age-dependent, luteolytic capacity to exogenous PGF2alpha in rabbits.

Regulation of luteal prostaglandin F 2α production and its relevance to cell death: An in vitro study using rat dispersed luteal cells

We investigated the mechanism by which rat luteal cells produce prostaglandin F 2α (PGF 2α) and its relevance to cell death in vitro. Treatment with progesterone (P4) of dispersed luteal cells prepared from rats on day 9 of pseudopregnancy caused dose-dependent inhibition of PGF 2α secretion. Cytokines, tumor necrosis factor α (TNFα) or interferon γ (IFNγ) alone had no or modest regulatory effects. Arachidonyl trifluoromethyl ketone (AACOCF 3), a specific group IVA phospholipase A 2 inhibitor, depressed both basal and cytokine-regulated PGF 2α production. A combination of TNFα and IFNγ stimulated PGF 2α synthesis and cytotoxicity (both, P < 0.05). Agonistic anti-Fas antibody challenge caused a significant cytotoxic effect but without affecting PGF 2α production. The present data suggest that P4 inhibits and TNFα and IFNγ cooperatively stimulate PGF 2α release by rat luteal cells. They also suggest that luteal cell death induced by TNFα/IFNγ and Fas stimulation seems to occur via distinct signaling pathways involving PGF 2α production.

Regulation of uterine function by cytokines in cows: Possible actions of tumor necrosis factor‐α, interleukin‐1α and interferon‐τ

ABSTRACTWhen animals do not become pregnant, regression of the corpus luteum (CL) is essential for normal cyclicity because it allows the development of a new ovulatory follicle. Luteal regression is caused by a pulsatile release of prostaglandin (PG) F2α from the uterus in the late luteal phase in most mammals including cattle. Although it has been proposed in ruminants that pulsatile PGF2α secretion is generated by a positive feedback loop between luteal and/or hypophyseal oxytocin and uterine PGF2α, the bovine endometrium may possess other mechanisms for initiation of luteolytic PGF2α secretion. There is increasing evidence that several cytokines mainly produced by immune cells modulate CL and uterine function in many species. Tumor necrosis factor‐α (TNF‐α) stimulates PGF2α output from bovine endometrium not only at the follicular phase but also at the late luteal phase. Administration of TNF‐α at a high concentration prolongs luteal lifespan, whereas administration of a low concentration of TNF‐α accelerates luteal regression in cows. The data obtained from the authors’ previous in vitro and in vivo studies strongly suggest that TNF‐α is a crucial factor in regulating luteolysis in cows. The authors’ recent study has shown that interleukin‐1α mediates PG secretion from bovine endometrium as a local regulator. Furthermore, interferon‐τ (IFN‐τ) suppresses the action of TNF‐α on PGF2α synthesis by the bovine endometrium in vitro, suggesting that IFN‐τ plays a luteoprotective role by inhibiting TNF‐α‐induced PGF2α production in early pregnancy. The purpose of the present review is to summarize current understanding of the endocrine mechanisms that regulate uterine function by cytokines during the estrous cycle and early pregnancy in cows.

Nitric oxide (NO) inhibits prostaglandin E2 9-ketoreductase (9-KPR) activity in human fetal membranes

Nitric oxide (NO) synthesized by fetal membranes may act either directly inhibiting myometrium contractility or indirectly interacting with tocolytic agents as prostaglandins (PGs). Here we examined if NO could modulate prostaglandin E 2 9-ketoreductase (9-KPR) activity in human fetal membranes (HFM). 9-KPR is the enzyme that converts PGE 2 into PGF 2α, the main PGs known to induce uterine contractility at term. Chorioamnion explants obtained from elective caesareans were incubated with aminoguanidine (AG), an iNOS inhibitor, or NOC-18, a NO donor. NOC-18 (2 mM) increased PGE 2 production and diminished PGF 2α synthesis in HFM. AG presented the opposite effect. When we evaluated the activity of 9-KPR by the conversion of [ 3H]-PGE 2 into [ 3H]-PGF 2α and 13,14-dihidro-15-keto prostaglandin F 2α (the PGF 2α metabolite), we found that NOC-18 inhibited 9-KPR activity. Interestingly, AG did not elicit any effect on 9-KPR but l-NAME, a non-selective NOS inhibitor, significantly increased its activity. Our data suggests that exogenous NO inhibits 9-KPR activity in HFM, thus modulating the synthesis of important labor mediators as PGF 2α.

Gene expression of arachidonate cyclooxygenase pathway leading to the delayed synthesis of prostaglandins E2 and F2α in response to phorbol 12-myristate 13-acetate and action of these prostanoids during life cycle of adipocytes

Several types of prostaglandin (PG)s are synthesized in adipocytes and involved differently in the control of adipogenesis. To elucidate how the PG synthesis is regulated at different stages in the life cycle of adipocytes, we examined the gene expression of arachidonate cyclooxygenase (COX) pathway leading to the delayed synthesis of PGE2 and PGF2α and their roles in adipogenesis after exposure of cultured cells to phorbol 12-myristate 13-acetate (PMA), which is a useful system for monitoring mitogen-induced changes. While the expression of COX-1 remained constitutive, mRNA and protein levels of COX-2 were up-regulated by treatment with PMA. Preadipocytes exhibited higher gene expression of cytosolic phospholipase A2α (cPLA2α) and PGF synthase. In contrast, three isoforms of PGE synthase are expressed constitutively during all phases. The delayed synthesis of PGE2 and PGF2α following the stimulation for 24 with a mixture of PMA and calcium ionophore A23187 was the highest in preadipocytes, reflecting the increased expression levels of cPLA2α and COX-2. Cultured cells treated with PMA during the differentiation phase and then exposed to the maturation medium, or cells treated with PMA in the maturation medium after the differentiation phase showed the suppression of adipogenesis in adipocytes. The attenuating effect of PMA was additionally enhanced when the cell were treated along with A32187 during the differentiation phase, suggesting the involvement of endogenous PGs. The cells at the stages of the differentiation and maturation phases were highly sensitive to exogenous PGE2 and PGF2α, respectively, resulting in the marked suppression of the stored fats in adipocytes. Taken together, these results provided the evidence for the distinct gene expression of isoformic enzymes in the COX pathway leading to the synthesis of PGE2 and PGF2α and the specific action of these prostanoids at different cycle stages of adipocytes.

463. COX-2-Based Gene Therapy: Reduction of Intraocular Pressure in a Glaucoma Gene Therapy Animal Model with Lentiviral Vectors Expressing COX-2, a Prostaglandin Synthase, or a Prostaglandin Receptor

BACKGROUND: Cyclooxygenase-2 (COX-2), an inducible enzyme in the prostaglandin (PG) biosynthesis cascade, is important in medically significant disease states, including glaucoma and cancer. Most recently, COX-2 inhibitors have been implicated in adverse cardiovascular outcomes. Durable COX-2 expression by exogenous genes has not been previously achieved. We hypothesized that the inducible PG biosynthetic and response pathways could be manipulated via a gene therapy approach with lentiviral vectors. Since PGF2α analogs are the most effective glaucoma drugs, we further hypothesized that these vectors could be used to durably reduce intraocular pressure (IOP) in a large animal model we developed. Glaucoma is an appealing candidate for lentiviral vector therapy, since the causative IOP elevation results from pathophysiology that is (i) chronic; (ii) frequently refractory to conventional therapies; (iii) anatomically confined to a discrete anterior chamber region that is targetable by transcorneal injection. Therapeutic transgenes have not been described for glaucoma, which remains the leading cause of irreversible blindness. METHODS: We constructed lentiviral vectors expressing cDNAs for human COX- 2 or other relevant proteins (PGF synthase, and the PGF receptor). mRNA and protein levels were measured by Northern & Western blotting. PGF2α production was assayed. Anterior chambers of 15 domestic cats were injected with lentiviral vectors. Left eyes were injected with 107–108 TU of a control eGFP vector, while right eyes received equivalent doses of one or more therapeutic vectors. Animals were monitored serially for IOP and clinical effects. RESULTS: COX-2 mRNA proved highly unstable even with a minimal cDNA lacking the known destabilizing 3′ UTR. We then observed that the human COX-2 cDNA has highly skewed codon use very similar in composition to that of lentiviral structural genes. We therefore codon-optimized the cDNA, which enabled robust, sustained expression for the first time. Increased synthesis of PGF2α up to 0.9 × 104 –fold greater than control, was observed. In the animals, vectors were well-tolerated and produced marked, sustained (2 months at present, with all animals under ongoing observation), and highly significant IOP decreases (mean over entire two months = 4.2 mm Hg, p < 0.002) compared to control eyes were achieved by this gene therapy manipulation of prostaglandin production or receptor response. A combination of the COX-2 and PGF receptor vectors produced the largest IOP decrease (mean = 5.6 mm Hg, 38% reduction, p < 3 × 10-9). CONCLUSIONS: Major prostaglandin biosynthetic and response pathways can be manipulated by gene therapy. Codon optimization of the COX-2 coding region profoundly augments mRNA stability. Sustained, substantial, highly statistically significant decreases in IOP were achieved in a large animal model developed for glaucoma studies. This is the first gene therapy study to demonstrate sustained IOP reduction, defining a first therapeutic transgene possibility. Investigations of other COX-2-and PG-related processes and diseases will also be enabled by these findings.

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

Phytoestrogens have recently been suggested to be the cause of infertility by stimulating luteolytic prostaglandin (PG) F 2α 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 2α 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 24 h. Prostaglandin F 2α and PGE 2 were stimulated by phytoestrogens in both stromal and epithelial cells, with a preference for PGF 2α 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 PGE 2 synthase (PGES) in stromal and epithelial cells ( P > 0.05), the stimulative effects of equol and p-ethyl phenol on PGF 2α 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 2α synthesis, is caused by increasing PGFSL2 in epithelial cells, and by decreasing stromal cell viability, which are the main source of luteotropic PGE 2 production.