Effect of platelet activating factor on prostaglandin release from ovine endometrial cells in culture

Platelet activating factor (PAF) added in vitro to ovine endometrial cells in primary culture caused a dose-dependent increase in the release of prostaglandin (PG) E into the medium compared with release from untreated cells. At a concentration of 1000 ng/ml of PAF, PGE levels in treatment dishes were significantly higher (P less than 0.05) than those in control dishes [130 +/- 8% vs 100% (mean +/- SEM, N = 5 ewes)]. PAF did not alter the release of PGF2 alpha by the same cells. By contrast, the ovine trophoblast interferon, ovine trophoblast protein-1 (oTP-1, 1 ng/ml) attenuated the release of both PGE and PGF2 alpha and this was not overcome by the presence of PAF (100 ng/ml). Thus does not appear that PAF contributes to the antiluteolytic signal in sheep by a direct action on release of PGF2 alpha although it could influence implantation via stimulation of PGE.     

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.     

In vitro action of PG F2 alpha on progesterone and cAMP synthesis in small bovine luteal cells

The action of prostaglandin F2 alpha (PG F2 alpha) on incubated small bovine luteal cells in the presence or in the absence of bovine luteinizing hormone (LH) or dibutyryl cyclic adenosine monophosphate (db cAMP) was investigated. In the absence of LH and db cAMP, PG F2 alpha stimulated progesterone synthesis at concentrations of 10 ng/ml and 100 ng/ml but had no effects at concentrations below 1 ng/ml. PG F2 alpha partially inhibited the LH or db cAMP stimulated progesterone synthesis. This inhibition was maximal for PG F2 alpha concentrations around 100 pg/ml whereas distinctly higher or lower concentrations were without effect. At the concentration of 100 pg/ml, PG F2 alpha partially inhibited the LH induced cAMP accumulation. These results demonstrate an "in vitro" action of PG F2 alpha on bovine luteal cells. They indicate that the luteolytic action of PG F2 alpha in the bovine species could involve, as already suggested for the rat, both an inhibition of the LH induced synthesis of cAMP and an inhibition of the action of cAMP.     

Proteins secreted from the early ovine conceptus block the action of prostaglandin F2 alpha on large luteal cells.

In this study we evaluated whether the early conceptus secretes a factor that blocks the action of prostaglandin (PG) F2 alpha on cultured ovine large luteal cells. PGF2 alpha inhibited progesterone production by lipoprotein-stimulated large luteal cells and this anti-steroidogenic action was blocked in a dose-dependent manner by conceptus proteins secreted from Day 15 embryos. Purified ovine trophoblast protein-1 (oTP-1) did not exhibit the anti-PGF2 alpha activity, but secreted conceptus proteins devoid of oTP-1 did prevent the anti-steroidogenic effects of PGF2 alpha. This activity does not appear to be a nonspecific effect of protein since neither serum albumin nor thyroglobulin, gamma globulin, insulin, LH, secreted ovine endometrial proteins, or heat-inactivated secreted conceptus proteins had this action. After molecular-sizing chromatography we found a high- and a low-molecular weight fraction with luteal protective activity. Neither of the secreted conceptus protein fractions blocked the binding of 3H-PGF2 alpha to large luteal cells. However, conceptus proteins did block the anti-steroidogenic action of phorbol ester and calcium ionophore on large luteal cells, suggesting that secreted conceptus proteins act after activation of the free calcium/protein kinase C intracellular effector pathways. Thus, the early ovine conceptus secretes a luteal protective protein(s) that may be important for maintaining the corpus luteum during early pregnancy; however, the physiologic significance of this luteal protective protein(s) cannot be stated without further investigation.     

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.     

Production of prostaglandins by dispersed cells and fragments from bovine parathyroid glands

We studied the production of prostaglandins by fragments and dispersed cells from bovine parathyroid glands. Fragments released 138 +/- 19 (SE), 132 +/- 21, 4.3 +/- 0.5, and 13 +/- 6.6 pg/mg/h of 6-keto-PGF1 alpha, PGF2 alpha, PGE2, and thromboxane B2, respectively (n = 7 - 26), while dispersed cells released 414 +/- 110, 22 +/- 7.3, 27 +/- 3.8, and 29 +/- 11 pg/10(6) cells/h, respectively, of the same compounds (n = 6 - 25). Indomethacin (1 microgram/ml) inhibited the release of 6-keto-PGF1 alpha by 80-90% in fragments and cells, while mellitin stimulated release of this prostaglandin, suggesting de novo synthesis of prostaglandins in these preparations. Calcium stimulated production of 6-keto-PGF1 alpha by 1.3-fold in cells and 2.6-fold in fragments and also enhanced production of PGF2 alpha by 1.9-fold in fragments. Isoproterenol, on the other hand, had no effect on production of 6-keto-PGF1 alpha in either preparation. These results demonstrate that parathyroid tissue as well as parathyroid cells per se produce a variety of prostaglandins. We have previously shown that PGE2 and PGF2 alpha modulate cAMP accumulation and PTH release in dispersed bovine parathyroid cells. The role of the endogenous production of prostaglandins by the parathyroid gland in the acute or chronic regulation of parathyroid function, however, remains to be determined.     

Site of action for endotoxin-induced cortisol release in the suppression of preovulatory luteinizing hormone surges

The study was conducted to identify the mechanisms of endotoxin/cortisol action in the suppression of preovulatory LH surges in heifers infused with Escherichia coli (E. coli ) endotoxin. The hypotheses tested were that 1) endotoxin stimulates the release of progesterone, possibly from the adrenal leading to the LH blockade; 2) cortisol released in response to endotoxin infusion blocks the synthesis of estradiol at the ovarian level, culminating in a failure of the LH surge. Eight Holstein heifers were given two injections of prostaglandin F(2alpha) (PG), 11 d apart, to synchronize estrus. Starting from 25 h after the second injection of PG (PG-2), the uterus of each heifer was infused either with 5 ml of pyrogen-free water (control, n = 3) or with E. coli endotoxin (5 mug/kg of body weight) in 5 ml of pyrogen-free water (treated, n = 5), once every 6 h for 10 treatments. Blood samples were obtained every 15 min for 1 h before infusion and again 2 h after each infusion, then hourly until 1 h before the next infusion. After the tenth infusion, blood was collected daily until estrus. Serum progesterone concentrations remained at baseline values (< 1 ng/ml) in control and treated heifers. The total amount of progesterone measured starting 24 to 84 h after PG-2 injection was not different between control and treated heifers (P 0.05). In the control heifers, serum estradiol concentrations remained basal (< 10 pg/ml) until 4 h before the LH surge. Serum estradiol concentrations increased to 20 +/- 5.6 pg/ml, 4 h before the LH surge in control heifers (LH surge occurred 60 to 66 h after the PG-2 injection). There were no changes in serum estradiol concentrations in treated heifers during the sampling period, and the concentrations remained < 10 pg/ml. The total amount of estradiol measured in control heifers was higher (P < 0.05) than in treated heifers. The results if this study suggest that increases in cortisol concentrations after the infusion of endotoxin might block the synthesis of estradiol at the ovarian level, resulting in the failure of a preovulatory LH surge to occur.     

Bovine placental prostaglandin synthesis: principal cell synthesis as modulated by the binucleate cell

Bovine placentomes were collected during late gestation, prepartum, and immediately postpartum. Postpartum tissues were collected prior to fetal placental release. A procedure for separating fetal placental principal cells from fetal binucleate cells (BNC) was developed. Dispersed fetal placental cells (mixed types), principal cells, and BNC were each examined for their ability to produce prostaglandins (PGs) from arachidonic acid (AA) and to metabolize prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha) in vitro. Dispersed fetal placental cells obtained prepartum produced predominantly PGs of the E series (PGEs) from AA (p less than 0.05). PGE synthesis predominated (p less than .05) in cells from postpartum tissue if the fetal placental membranes were subsequently retained, whereas synthesis of PGs of the F series (PGFs) predominated (p less than 0.05) if the fetal membranes were subsequently released. Principal cells were the primary source of fetal placental PG synthesis from AA (p less than 0.05). BNC exhibited a lesser ability to synthesize PGs from AA (p less than 0.05), but were able to convert PGF2 alpha to PGE2. Dispersed fetal placental cells exhibited greater ability to convert PGF2 alpha to PGE2 (p less than 0.05) than did the separated cells. These data suggest the function of a two-cell system within the fetal villi such that the BNC modulate the output of principal cell PG synthesis and/or metabolism.     

Effect of ovine conceptus secretory proteins and purified ovine trophoblast protein-1 on interoestrous interval and plasma concentrations of prostaglandins F-2 alpha and E and of 13,14-dihydro-15-keto prostaglandin F-2 alpha in cyclic ewes

Conceptus secretory proteins (oCSP) were obtained from medium in which sheep conceptuses, collected on Day 16 of pregnancy, were cultured for 30 h. A portion of the culture medium (500 ml) was prepared for intrauterine infusion by concentrating the proteins by Amicon ultrafiltration (Mr 500 cutoff). A second portion (500 ml medium) was used to purify sheep trophoblast protein one (oTP-1). Proteins remaining after oTP-1 purification were concentrated and then passed through an anti-oTP-1 sepharose CL-4B affinity column to remove any remaining oTP-1 (oCSP-oTP-1). Serum proteins (oSP) were collected from a Day-16 pregnant ewe and diluted for infusion. Catheters were placed in the uterus of cyclic (Day 10) ewes. The following combinations of proteins were infused: 0.75 mg oCSP + 0.75 mg oSP (5 ewes), 0.75 mg oCSP - oTP-1 + 0.75 mg oSP (4 ewes), 0.05 mg oTP-1 + 1.45 mg oSP (5 ewes) and 1.5 mg oSP only (5 ewes). Infusions were twice daily on Days 12 and 13 (08:00 and 17:00 h) and once on Day 14 (08:00 h). On Day 14, ewes were injected intravenously at 08:00 h with 0.5 mg oestradiol-17 beta. Blood sampling began 30 min before oestradiol injection and continued every 30 min for 10 h. On Day 15 ewes received 10 i.u. oxytocin intravenously (08:00 h). Blood samples were collected 10 min before oxytocin and every 10 min for 1 h after oxytocin injection. Concentrations of prostaglandin (PG) F, PGE-2/PGE-1 (PGE) and 13,14-dihydro-15-keto-PGF-2 alpha (PGFM) were measured by specific radioimmunoassay. Ewes treated with oTP-1 and oCSP had longer (P less than 0.05) interoestrous intervals (27 and 25 days, respectively) compared to ewes treated with oSP and oCSP--oTP-1 (19 and 19 days, respectively) (s.e.m. = 1.56 days). These results indicate that oTP-1 alone is as potent as total conceptus secretory proteins in extending luteal maintenance. Ewes treated with oTP-1 and oCSP had no increase in PGF after oestradiol injection while production of PGF did increase 6-10 h after oestradiol in ewes treated with oSP and oCSP--oTP-1. PGFM was correlated with PGF concentrations (r = 0.57, P less than 0.01) although presence or absence of increases in production of PGFM for the treatment groups were not the same as those for PGF. No effects of treatment on PGE were detected.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of pregnancy, oxytocin, ovine trophoblast protein-1 and their interactions on endometrial production of prostaglandin F2 alpha in vitro in perifusion chambers

Pregnancy and intrauterine infusion of ovine trophoblast protein one (oTP-1) decrease oxytocin-induced secretion of prostaglandin F2 alpha (PGF) from the uterus. In the present study, effects of oTP-1 and pregnancy on endometrial secretion of PGF were examined in an in vitro perifusion system. In Experiment 1, endometrium from day 14 pregnant and cyclic ewes was perifused sequentially on both the lumenal and myometrial sides with Krebs Ringers Bicorbonate solution (KRB), KRB plus oxytocin (1 IU/ml) and KRB alone. Endometrium from pregnant ewes secreted more PGF from both lumenal and myometrial sides than endometrium from cyclic ewes (P less than 0.05). Oxytocin stimulated secretion of PGF from both sides of endometrium regardless of status. Secretion of PGF was greater from the lumenal surface of endometrium compared to myometrium (P less than 0.05) for pregnant and cyclic ewes. For Experiment 2, endometrium was collected from day 15 cyclic ewes and perifused sequentially with KRB, KRB plus 300 ng/ml of either Bovine Serum Albumin (BSA) or oTP-1, KRB with or without BSA or oTP-1 plus oxytocin (1 IU/ml) and then KRB alone. Oxytocin stimulated greater release of PGF from oTP-1-treated than BSA-treated endometrium. Pretreatment of endometrium with oTP-1 had the same effect on oxytocin-induced PGF secretion as cotreatment with oTP-1 and oxytocin. In Experiment 3, uterine horns of cyclic ewes were catheterized on day 10 of the estrous cycle, and infused with either oTP-1 or day 16 pregnant sheep serum proteins on days 12, 13 and 14. Endometrium was collected on day 15 and perifused sequentially with KRB, KRB plus oxytocin (1 IU/ml) and then KRB alone. Treatment of ewes with oTP-1 attenuated endometrial secretion of PGF in response to oxytocin. Results of this study indicate that: (1) pregnancy stimulates basal secretion of PGF from endometrium and has no effect on oxytocin-induced secretion of PGF in vitro; (2) short-term oTP-1 treatment enhances oxytocin-induced PGF secretion from day 15 cyclic endometrium and (3) long-term oTP-1 treatment in vivo inhibits oxytocin-induced PGF secretion in ewes.     

Effect of oxytocin and estradiol on uterine prostaglandin release in nonpregnant and early-pregnant ewes

The effects of exogenous oxytocin (OT) and estradiol-17 beta (E2) on plasma concentrations of prostaglandin (PG) E2 and 13, 14-dihydro-15-keto-PGF2 alpha (PGFM) were investigated on Day 14-15 (NP) of the estrous cycle and Days 14-16 (PI) and 21-25 (EP) of pregnancy in the ewe. Basal concentrations of PGFM were significantly elevated in utero-ovarian venous (UOV) plasma on Day 14 of pregnancy (4.05 +/- 0.81 nM, mean +/- SEM) compared to that observed on Day 14 of the cycle or Days 21-25 of pregnancy (2.29 +/- 1.3 nM and 1.06 +/- 0.56 nM, respectively). PGFM release increased significantly following intera-arterial bolus injections of 50, 500, and 5000 mU OT at 2-h intervals in all experimental groups. There was no significant difference in area and peak height of the PGFM response between the 3 groups studied. The time to peak PGFM response was, however, significantly longer in the PI group. No significant changes in concentration of PGFM were observed in any experimental group following 1-h infusions of E2 at 5, 50, and 500 pmol/min. Long-term (15-18 h) infusion of E2 at 83 pmol/min increased the peak height of the OT-induced PGFM response at both stages of gestation studied. PGE2 concentrations in UOV plasma were less than 0.05 nM in all samples studied. These results demonstrate that PG release can be induced in response to OT during the period in which ovine trophoblastic protein-1 (oTP-1) is released by the conceptus. During pregnancy, oTP-1 does not appear to inhibit the E2 induction of uterine OT receptors.     

Relaxin, oxytocin, and prostaglandin effects on progesterone secretion from bovine luteal cells during different stages of gestation

To determine the effects of relaxin, oxytocin, and prostaglandin F2 alpha on progesterone secretion, bovine luteal cells from different stages of gestation were dispersed in Medium 199 with 200 units/ml penicillin, 1.0% kanamycin, 0.5% bovine serum albumin, and 400 units/ml collagenase. Cells (10(5) were cultured in 400 microliters of Dulbecco's modified Eagle's medium and Ham's F-12 medium containing fetal bovine serum and antibiotics, in Falcon multiwell plates, in a humidified environment of 95% O2 and 5% CO2 at 37 degrees C. Cells were cultured for 24 hr without treatment and thereafter with medium-hormone replacement every 24 hr. Progesterone was quantified from unextracted media by radioimmunoassay. Basal progesterone secretion after 24 hr was 1.81 +/- 0.14, 1.76 +/- 0.17, 0.54 +/- 0.49, and 0.57 +/- 0.21 pg/ml per viable luteal cell from 145-, 165-, 185-, and 240-day-old corpora lutea, respectively. Basal progesterone secretion increased (P less than 0.05) with time in culture. Relaxin induced a dose-dependent (greater than 100 ng/ml) increase in progesterone release, compared with the controls. Oxytocin and prostaglandin F2 alpha induced greater release (P less than 0.05) of progesterone than relaxin at all stages of gestation, but progesterone release was dependent on the stage of gestation and the duration in culture. Luteinizing hormone (100 ng/ml) stimulated whereas 17 beta-estradiol (50 ng/ml) inhibited progesterone secretion by luteal cells at all stages of gestation examined. Relaxin obliterated the prostaglandin- and oxytocin-induced progesterone secretion by bovine luteal cells from 145 to 214 days of gestation. Thus, relaxin, cloprostenol, and oxytocin regulate progesterone production by cultured bovine luteal cells, but hormone secretion was dependent on the stage of gestation.     

Gonadotropic regulation of prostaglandin production by ovarian follicular cells of the pig

Prepubertal gilts were treated with 750 IU pregnant mare's serum gonadotropin (PMSG) and 72 h later with 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. Dispersed granulosa cells (GC) and theca interna cells (TC) from follicles of gilts 72 h (GC-72 and TC-72, respectively) and 108 h (GC-108 and TC-108 h, respectively) after PMSG treatment were cultured for 0, 12, 24, and 36 h in medium with or without luteinizing hormone (LH), dibutyryl cyclic adenosine 3',5'-monophosphate [Bu)2cAMP), calcium ionophore (A23187), and/or arachidonic acid (AA), and the production of prostaglandin E2 (PGE) and prostaglandin F2 alpha (PGF) was measured by radioimmunoassay. TC-72 was the principal source of PGs 72 h after PMSG. At 108 h, the production of PGE and PGF by GC was increased 10- and 30-fold, respectively, whereas corresponding increases by TC were 2-fold. LH and A23187 significantly stimulated PGE and PGF production by both GC-72 and TC-72, but only thecal PG production was stimulated by (Bu)2cAMP. LH had minimal or no effect on PG production by GC-108 and TC-108, but A23187 (GC-108, TC-108) and (Bu)2cAMP (TC-108) were stimulatory. Basal PG production by GC-72, GC-108, and TC-108 was stimulated by AA. However, production by GC and TC cultured in medium containing AA and LH, A23187, or (Bu)2cAMP was not different from that produced by AA alone. These findings suggested that GC and TC can synthesize PGs in vitro, but AA availability is rate-limiting in GC. After exposure to hCG in vivo, the capacity of both cell types to produce PGs is increased but is limited by AA availability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a two-step process in prostaglandin secretion. Intracellular accumulation of prostacyclin precedes its release from human endothelial cells in culture

Cultured endothelial cells (EC) from human umbilical vein were incubated with [U-14C]arachidonic acid (AA) followed by a challenge with thrombin (2 units/ml) or calcium ionophore A23187 (5 microM) for 0.5-10 min at 37 degrees C. In both cases, AA was rapidly liberated from phospholipids and converted into prostaglandin I2 (PGI2), as determined by the radioactivity of the stable derivative 6-keto-PGF1 alpha. Maximal liberation of AA and synthesis of PGI2 were achieved within 2 min, but the two compounds first accumulated in EC prior to their release into supernatants. This finding, which was never reported before, raises the question of the mechanism of AA and PG release through the cell membranes and offers a convenient model to investigate this still obscure process.     

Arachidonic acid release and prostaglandin biosynthesis in synchronized rat embryo fibroblasts

Arachidonic acid (AA) release and prostaglandin (PG) biosynthesis were studied in rat embryo fibroblasts (R 129) synchronized by double thymidine-excess block. Whatever the culture medium was (medium 199 supplemented with 10% fetal calf serum (FCS) or 1% FCS plus 0.1% bovine serum albumin (BSA], AA release rapidly increased until the 4th hour of the cell cycle (S phase), remained on a plateau in G2M and G1 phases and did not increase again in the S phase of the following cell cycle. Time course and amplitude of AA release in synchronized cells did not differ from what it was observed after the simple renewal of the culture medium in asynchronous cells. So AA release seemed to be independent of the cell cycle. By contrast, PGE2 and PGF2 alpha biosynthesis clearly increased in the S phase of two consecutive cell cycles, indicating that cyclooxygenase activity and not phospholipase A2 activity vary according to the cell cycle.     

Bradykinin synergistically potentiates interleukin-1 induced bone resorption and prostanoid biosynthesis in neonatal mouse calvarial bones

Interleukin-1 (IL-1) alpha and beta dose-dependently stimulated the release of 45Ca and the formation of prostaglandin E2 (PGE2) and PGI2 in cultured mouse calvarial bones, with IL-1 beta being the most potent agonist. Bradykinin (BK; 10 nmol/l) synergistically potentiated the effect of IL-1 alpha (10 pg/ml) and IL-1 beta (5 pg/ml) both on 45Ca release and on biosynthesis of PGE2 and PGI2. The capacity of BK to potentiate IL-1 beta induced 45Ca release and PGE2 formation was seen at concentrations of BK from 1-1000 nmol/l. These data indicate that BK and IL-1, which are formed in inflammatory processes, may act in concert to stimulate bone resorption in the vicinity of inflammatory lesions.     

Down-regulation of oxytocin-induced cyclooxygenase-2 and prostaglandin F synthase expression by interferon-tau in bovine endometrial cells

Oxytocin (OT) is responsible for the episodic release of luteolytic prostaglandin (PG) F2alpha from the uterus in ruminants. The attenuation of OT-stimulated uterine PGF2alpha secretion by interferon-tau (IFN-tau) is essential for prevention of luteolysis during pregnancy in cows. To better understand the mechanisms involved, the effect of recombinant bovine IFN-tau (rbIFN-tau) on OT-induced PG production and cyclooxygenase-2 (COX-2) and PGF synthase (PGFS) expression in cultured endometrial epithelial cells was investigated. Cells were obtained from cows at Days 1-3 of the estrous cycle and cultured to confluence in RPMI medium supplemented with 5% steroid-free fetal calf serum. The cells were then incubated in the presence or absence of either 100 ng/ml OT or OT+100 ng/ml rbIFN-tau for 3, 6, 12, and 24 h. OT significantly increased PGF2alpha and PGE2 secretion at all time points (p < 0.01), while rbIFN-tau inhibited the OT-induced PG production and reduced OT receptor binding in a time-dependent manner. OT increased the steady-state level of COX-2 mRNA, measured by Northern blot, which was maximal at 3 h (9-fold increase) and then decreased with time (p < 0.01). OT also caused an increase in COX-2 protein, which peaked at 12 h (11-fold increase), as measured by Western blot. Addition of rbIFN-tau suppressed the induction of COX-2 mRNA (89%, p < 0.01) and COX-2 protein (50%, p < 0.01) by OT. OT also increased PGFS mRNA, and this stimulation was attenuated by rbIFN-tau (p < 0.01). To ensure that the decrease in COX-2 was not solely due to down-regulation of the OT receptor, cells were stimulated with a phorbol ester (phorbol 12-myristate 13-acetate; PMA) in the presence and absence of rbIFN-tau. The results showed that rbIFN-tau also decreased PMA-stimulated PG production and COX-2 protein. It can be concluded that rbIFN-tau inhibition of OT-stimulated PG production is due to down-regulation of OT receptor, COX-2, and PGFS.     

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.     

The effect of ovine trophoblast protein-one on endometrial protein secretion and cyclic nucleotides

The effect of ovine trophoblast protein-one (oTP-1) on endometrial protein secretion was examined by using a dual radioisotope technique in which 3H- and 35S-methionine were employed to measure relative rates of protein release into the medium by endometrial explant cultures (Exp. I). Endometrium (200 mg) from Day (D) 12 of the cycle was cultured with either 5 micrograms/ml oTP-1, 5 micrograms/ml bovine serum albumin (BSA) or 1 mM dibutyryl cyclic adenosine 3',5'-monophosphate (DbcAMP). Culture media from control BSA and treated explant cultures were mixed. Proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and detected by fluorography. Individual protein spots were punched from gels, extracted, and their radioactive content measured. Ratios of 3H:35S were used to determine treatment effects. In Experiment II, 3H- and 14C-leucine were used for the dual radiolabel, and the DbcAMP treatment was omitted. In both experiments, a protein having a molecular weight (Mr) of about 70,000 and a pI approximately equal to 4 was increased (p less than 0.01) 200-400% by oTP-1. Secretion of several other endometrial proteins was also amplified in the presence of oTP-1. The polypeptides that increased in response to oTP-1 were inhibited by DbcAMP, and vica versa. In Experiment III, endometrial explants from D12 cyclic ewes were cultured for 4 h with either 5 micrograms/ml oTP-1 or 5 micrograms/ml BSA to determine whether oTP-1 influenced concentrations of 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP). Concentrations of cAMP in oTP-1-treated endometrium were lower (p less than 0.1) than in BSA-treated endometrium (0.29 vs. 0.41 pmoles/mg tissue, respectively). Levels of cGMP were unaffected by oTP-1. In Experiment IV, endometrium from D14 of the cycle was incubated in medium alone or in medium containing either 2 micrograms/ml oTP-1, 1 microgram/ml oxytocin (OXY), or oTP-1-plus-OXY. None of the treatments significantly affected cAMP levels. In Experiment V, D16 endometrium was collected from pregnant and nonpregnant ewes that had received either 0 or 10 IU OXY i.v. cAMP was higher (p less than 0.01) in endometrium from pregnant ewes compared to nonpregnant ewes (27.9 vs. 13.0 pmoles/mg tissue, respectively), but OXY had no detectable effect on endometrial content of cAMP in either nonpregnant or pregnant ewes.(ABSTRACT TRUNCATED AT 400 WORDS)     

The relationship between the antiviral action of interferon and prostaglandins in virus-infected murine cells

The relationship between prostaglandins (PG) and interferon (IFN) was investigated. IFN induced the synthesis of immunoreactive PGE and PGA at early and late stages, respectively, of vaccinia virus infection in mouse L fibroblasts. Only species-specific IFN possessed this activity and PG synthesis was stimulated in virus-infected cells, while normal L cells were not affected. The vaccinia virus infection did not significantly alter PG synthesis in the absence of IFN. Indomethacin increased the rate of vaccinia virus replication and partially inhibited the IFN-induced protection of L cells. The addition of exogenous PGA1 only partially reversed this effect. Finally, short-term PGA treatment induced the synthesis of two enzymes (protein kinase and 2,5A synthetase) thought to be partially responsible for the antiviral action of interferon. These findings suggest that a prostaglandin or PG-related compound seems to mediate at least one aspect of IFN action.