Uptake and release of H prostaglandins E2 and F2α in uterin strips isolated from ovariectomized rats. Influence of progesterone

The accumulation and output of ³H -prostaglandins (PGs), E₂ and F₂α, into and from uterine strips isolated from ovariectomized rats, either in presence or in absence of exogenous progesterone, were explored. Tissue-to-medium ratio of ³H - counts (T/M-ratio), was determined. The same was done in solutions containing ¹⁴C-sucrose. During a 60 min incubation period in a solution containing ³H -PGF₂α, a net accumulation of radioactivity was evident in control (no progesterone) uterine slices. The T/M-ratio for ³H-PGF₂α, increased with time, reaching maximal values at 45 min. Progesterone (100 ug.ml⁻¹) attenuated the uptake process, as evidenced by stable values of T/M-ratio, as time progressed. On the other hand, control T/M-ratio for fluenced by the presence of exogenous progesterone. Regarding labelled PG release from the tissue, it was observed that, during an experimental period of 60 min, most tritium from control slices was released within the first 30 min after incubation with ³H -PGF₂α, whereas, following the presence and subsequent removal of exogenous progesterone, the bulk of ³H -released took place at 6–70 min. On the other hand, the release of ³H after an incubation with ³H -PGE₂, was also maximal as that for ³H -PGF₂, α within the first 30 min and resulted not altered after a period of exposure and removal of progesterone. The foregoing results suggest an specific pharmacological effect of progesterone, attenuating the uptake and retarding the outflow of PGF₂α, but not that of PGE₂, into and from uterine slices of ovariectomized rats. Findings reported herein are discussed in terms of progesterone priming and withdrawal, in relation to PGF₂α fluxes in the rat uterus during the sex cycle, as well as in relation to PG binding to tissue receptors.     

Estradiol -17-beta enhances the formation of (3H)-PGF2 alpha from (3H)-PGE2 in the uterus isolated from ovariectomized rats

Formation of (3H)-PGF2 alpha and (3H)-13, 14,dihydro-15-keto-PGF2 alpha from (3H)-PGE2 by the supernatant of uterine homogenates from estrous and ovariectomized rats, was studied, using the reaction system PGE2 + NADPH + (3H)-PGE2 + supernatant. Enzymatic conversion was lower in uterine supernatants from spayed rats than in uterine homogenates of rats at natural estrus. Spayed animals were injected with progesterone (P) or with estradiol-17-beta (Eo) at a dose of 1.0 or 50.0 micrograms. Conversion of (3H)-PGF2 alpha to (3H)-PGE2 or to (3H)-13, 14,dihydro-15-keto-PGF2 alpha did not differ in control ovariectomized or ovariectomized rats receiving P or 1.0 micrograms Eo. However, 50.0 micrograms Eo induced a significant conversion after 30 (P less than 0.01) and 60 (P less than 0.001) min of incubation. It is concluded that Eo, at the 50.0 micrograms dose, but not the 1.0 microgram dose of Eo, nor progesterone, stimulated conversion of (3H)-PGE2 into (3H)-PGF2 alpha or (3H)-13, 14,dihydro-15-keto-PGF2 alpha, presumably through the activity of the enzyme PGE2-9-keto-reductase.     

Uterine production of prostaglandins F2 alpha and 6-keto-F1 alpha by ovariectomized pregnant rats receiving antiprogesterone steroid or in which progesterone has been withdrawn.

Ovariectomized early pregnant rats given continuous steroid replacement therapy have been treated with antiprogesterone steroid, ZK98299 or RU38486. At 24 h following treatment, uterine explants in culture were found to produce significantly greater amounts of PGF2 alpha, but not of 6-keto-PGF1 alpha, when compared to controls. ZK98299 and RU38486 gave almost identical levels of uterine PG production. The 6-keto-PGF1 alpha/PGF2 alpha production ratio for uteri of treated rats was decreased by 45% relative to controls. Similar changes in uterine PGF2 alpha production and 6-keto-PGF1 alpha/PGF2 alpha ratio have been shown for ovariectomized early pregnant rats in which progesterone has been withdrawn when compared to control animals. It has been suggested that inhibiting or withdrawing progesterone in rat uteri exposed to estradiol and progesterone may lead to a stimulation of endoperoxide F-reductase and/or E2 9-ketoreductase activities. The presence of luminal fluid in the uteri was observed for animals treated with antiprogesterone steroid or in which progesterone had been withdrawn. This was associated with a decrease in % dry weight for the uteri of these animals.     

Role of prostaglandin F2alpha in ovulation.

Using radioimmunoassay procedures, the levels of plasma, uterine and ovarian prostaglandin (PG) F2alpha, and those of plasma estradiol and progesterone were measured in intact, hysterectomized or ovariectomized immature female rats pretreated with PMS and subsequent HCG. Occurrence of ovulation was confirmed at 8 hours after the HCG administration not only in the intact rats but also in the hysterectomzied rats. The levels of plasma estradiol and progesterone, and of uterine and ovarian PGF2alpha rose with the PMS injection alone, but they did not reach the peaks before the HCG administration. Both plasma estradiol and uterine PGF2alpha showed a peak at 2 hours after the HCG injection. These peaks were antecedent 2 or 6 hours before the peaks of ovarian and plasma PGF2alpha, respectively. However, such increase of uterine PGF2alpha does not seem to be indispensable for ovulation, because ovulation could occur in the hysterectomized rats. The levels of ovarian PGF2alpha showed a high plateau from 4 to 8 hours after the HCG injection, and then rapidly decreased after ovulation. The levels of plasma PGF2alpha peaked not only in the intact rats but also in the hysterectomized rats at 8 hours after the HCG treatment. But in the ovariectomized rats, this plasma PGF2alpha peak at 8 hours disappeared and there was no statistical change of plasma PGF2alpha throughout the PMS-HCG treatment. Plasma progesterone gradually increased and reached the maximum at 10 hours after the HCG injection. These results conclude that the main source of increased plasma PGF2alpha during the ovulatory process induced with the PMS-HCG treatment is the ovary, and it is strongly suggested that a rapid increase of PGF2alpha in the ovary may play some important role(s) in the ovulatory process.     

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

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

Effect of prostaglandin F2 alpha (PGF2 alpha) on sources of progesterone and pregnancy in intact, ovariectomized and hysterectomized 90-100 day pregnant ewes.

A single dose of 8 or 16 mg of PGF2 alpha per 58 kg body weight was injected intramuscular into intact, ovariectomized or hysterectomized 90-100 day pregnant sheep in three separate experiments. Both doses of PGF2 alpha decreased the weights of the corpora lutea (P less than or equal to 0.05) and the concentration of progesterone in ovarian venous plasma at 72 hr (P less than or equal to 0.05) compared to the 0 hr sample within treatment groups and to control ewes at 72 hr in intact and hysterectomized pregnant ewes. In hysterectomized pregnant ewes, progesterone in jugular plasma declined (P less than or equal to 0.05) from 0 to 72 hr but never fell below 4 mg/ml and this decrease in progesterone after 8 or 16 mg PGF2 alpha was greater than in control hysterectomized ewes (P less than or equal to 0.05). There was a significant decrease in progesterone over time in jugular or uterine venous plasma in the presence of absence of the ovaries in 90-100 day pregnant ewes (P less than or equal to 0.05) but the profiles of progesterone were not different between vehicle and PGF2 alpha-treated ewes (P greater than or equal to 0.05). Uterine venous progesterone never declined below 30 ng/ml in the presence or absence of the ovaries and there was a significant quadratic increase (P less than or equal to 0.05) in uterine venous progesterone toward the end of the 72 hr sampling period indicating an increase in steroidogenic activity of the placenta. PGF2 alpha did not affect the number of abortions in intact or ovariectomized pregnant ewes (P greater than 0.05). Thus, the corpus luteum of sheep at 90-100 days of pregnancy is functional and responsive to PGF2 alpha, placentomes are functional but do not appear to be responsive to the doses of PGF2 alpha tested and PGF2 alpha was not an abortifacient over the 72 hr treatment period.     

Role of progesterone in regulating uteroovarian venous concentrations of PGF2 alpha and PGE2 during the estrous cycle and early pregnancy in ewes

The role of progesterone in regulation of uteroovarian venous concentrations of prostaglandins F2 alpha(PGF2 alpha) and E2 (PGE2) during days 13 to 16 of the ovine estrous cycle or early pregnancy was examined. At estrus, ewes were either mated to a fertile ram or unmated. On day 12 postestrus, ewes were laparotomized and a catheter was inserted into a uteroovarian vein. Six mated and 7 unmated ewes received no further treatment. Fifteen mated and 13 unmated ewes were ovariectomized on day 12 and of these, 7 mated and 5 unmated ewes were given 10 mg progesterone sc and an intravaginal pessary containing 30 mg of progesterone. Uteroovarian venous samples were collected every 15 min for 3 h on days 13 to 16 postestrus. Mating resulted in higher mean daily concentrations of PGE2 in the uteroovarian vein than in unmated ewes. Ovariectomy prevented the rise in PGE2 with day in mated ewes but had no effect in unmated ewes. Progesterone treatment restored PGE2 in ovariectomized, mated ewes with intact embryos. Mating had no effect on mean daily concentrations of PGE2 alpha or the patterns of the natural logarithm (1n) of the variance of PGF2 alpha. Ovariectomy resulted in higher mean concentrations and 1n variances of PGF2 alpha on day 13 and lower mean concentrations and 1n variances of PGF2 alpha on days 15 and 16. Replacement with progesterone prevented these changes in patterns of mean concentrations and 1n variances of PGF2 alpha following ovariectomy. It is concluded that progesterone regulates the release of PGF2 alpha from the uterus, maintaining high concentrations while also preventing the occurrence of the final peaks of PGF2 alpha which are seen with falling concentrations of progesterone. This occurs in both pregnant and non-pregnant ewes. Progesterone is also needed to maintain increasing concentrations of PGE2 in mated ewes.     

Prostaglandin translocation from the lumen of the rabbit uterus in vitro in relation to day of pregnancy or pseudopregnancy

Transport of 3H-labeled prostaglandins (PGs) E2 and F2 alpha from the uterine lumen across the uterine wall has been studied in rabbit uteri in vitro in incubations lasting up to 180 min, in relation to sexual state of the rabbit, incubation temperature, intraluminal PG concentration, addition of metabolic inhibitors and time of incubation. PG accumulation by the tissue increased rapidly up to 30 min and then remained relatively constant. By 30 min, radioactivity was found in the external incubation medium, and this increased linearly with time. The translocation of PGF2 alpha was significantly greater in pseudopregnant than in pregnant animals on Day 6, whereas that of PGE2 was significantly higher in pregnant than in pseudopregnant animals on Day 6.8. In pregnant animals, both PGF2 alpha and PGE2 were translocated to the exterior more rapidly on Day 6.8 than on Days 5 or 6. Transport of PGs was reduced by low temperature, unaffected by metabolic inhibitors and only that of PGE2 increased with increased (5 microM) intraluminal concentrations. During incubation, the tissue remained viable as judged by T/M ratios (dpm tissue/dpm medium) for 204 thallium. Transport of [14C] sucrose was much slower than that of [14C] urea, which was greater than the fastest rates exhibited by the PGs. In general, amounts of radioactivity found in antimesometrial, mesometrial and lateral portions of the uterine wall, or in implantation and interimplantation areas did not differ, but more was found in the endometrium than the myometrium. PGF2 alpha was translocated unmetabolized to the external medium, while only two-thirds of the PGE2 was translocated unchanged, and one-third converted to PGF2 alpha. It is concluded that the rabbit uterus shows some selectivity in handling PGs in relation to stage of pregnancy.     

Histamine alters prostaglandin output from diestrous rat uteri. Involvement of H2-receptors and 9-keto-reductase

The effects of exogenous histamine (H) on prostaglandin (PG) generation and release in uteri isolated from diestrous rats and the influences of H2-receptors blockers (cimetidine and metiamide) on the output of uterine PGs, were explored. Moreover, the action of H on the uterine 9-keto-reductase, was also studied. Histamine (10(-4) M) failed to alter the basal output of PGE1 but reduced significantly the generation and release of PGE2 and augmented the output of PGF2 alpha. On the other hand, cimetidine (10(-5) M) enhanced the basal release of PGE2 but had no action on the outputs of PGs E1 or F2 alpha. The enhancing effect of H on the production and release of PGF2 alpha was abolished in the presence of cimetidine. Also, the antagonist reversed the influence of H on the output of PGE2. Metiamide, another H2-receptor antagonist, did not alter the basal control generation and release of uterine PGs, but antagonized the augmenting influence of H on PGF2 alpha uterine output, as much as cimetidine did, and prevented the depressive action of H on the release of PGE2 from uteri. Histamine (10(-4) M) significantly stimulated uterine formation of cyclic-adenosine monophosphate, an action which was antagonized by the presence of cimetidine (10(-5) M), a blocker of H2 receptors. Also, histamine (10(-5) M) and dibutyrylcyclic-adenosine monophosphate (DB-cAMP) at 10(-3) M, enhanced significantly the formation 3H-PGF2 alpha from 3H-PGE2. Results presented herein demonstrate that H is able to diminish the generation of PGE2 in uteri from rats at diestrus augmenting the synthesis of PGF2 alpha, apparently via the activation of H2-receptors, enhancing adenylate-cyclase. These effects appear to increase uterine 9-keto-reductase activity which transforms PGE2 into PGF2 alpha. Relationships between the foregoing results and those evoked by estradiol, are also discussed.     

Activity of phospholipase C in ovine luteal tissue in response to PGF2 alpha, PGE2 and luteinizing hormone.

Four ewes were utilized to determine the effects of prostaglandin (PG) F2 alpha, PGE2 and luteinizing hormone (LH) on activity of phospholipase C (PLC) in ovine luteal tissue. Corpora lutea were collected on d 10 post-estrus and six slices from one corpus luteum from each ewe were pre-incubated with [3H]-inositol prior to incubation with one of 6 treatments. Treatments were 1) control, 2) PGF2 alpha (100 ng/ml), 3) PGE2 (10 ng/ml), 4) LH (10 ng/ml), 5) PGF2 alpha + PGE2 and 6) PGF2 alpha + LH. Phospholipase C was determined indirectly by measuring the accumulation of [3H]-inositol mono-, bis- and tris-phosphates (IP, IP2, IP3). Effects of PGF2 alpha (0 vs. PGF2 alpha) and luteotropic treatment (0 vs. PGE2 vs. LH) and their interactions were determined by analysis of variance. There was a significant main effect of PGF2 alpha (P less than 0.01) as concentrations of IP, IP2, IP3 and total [3H]-inositol phosphates were greater in tissue slices treated with PGF2 alpha, regardless of luteotropic treatment. Within groups receiving no PGF2 alpha (1,3,4), no effect of luteotropic treatment was observed. Within groups receiving PGF2 alpha (2,5,6), LH caused a significant (P less than .05) increase in the accumulation of total [3H]-inositol phosphates. Thus, PGF2 alpha can stimulate the activity of PLC in ovine luteal tissue and LH can potentiate this effect.     

Inhibition of (3H) prostaglandin F2alpha binding to its receptors by progesterone.

The specific binding of [3H] prostaglandin (PG) F2alpha to bovine corpus luteum cell membranes was inhibited by progesterone. Progesterone inhibition of binding was dependent on membrane protein and independent of [3H] PGF2alpha concentrations in the medium. The lower inhibition of binding at high protein concentrations can be overcome by increasing the amounts of progesterone added. Progesterone inhibition of binding appears to be due to a decrease in the receptor number rather than a decrease in the receptor affinities. The kinetic properties (association and dissociation rates) of the remaining receptors were unchanged. The inhibition of [3H] PGF2alpha binding was observed by preincubating the membranes with progesterone or by adding at the beginning but not during incubation. The concentrations of progesterone that inhibited binding by about 50% do occur in bovine corpora lutea of estrous cycle and pregnancy.     

The effect of progesterone on oxytocin-stimulated intracellular mobilization of Ca2+ and prostaglandin E2 and F2alpha secretion from porcine myometrial cells

Our past studies have shown that porcine myometrium produce prostaglandins (PG) during luteolysis and early pregnancy and that oxytocin (OT) and its receptor (OTr) support myometrial secretion of prostaglandins E2 and F2alpha (PGE2 and PGF2alpha) during luteolysis. This study investigates the role of intracellular Ca2+ [Ca2+]i as a mediator of OT effects on PG secretion from isolated myometrial cells in the presence or absence of progesterone (P4). Basal [Ca2+]i was similar in myometrial cells from cyclic and pregnant pigs (days 14-16). OT (10(-7)M) increased [Ca2+]i in myometrial cells of cyclic and pregnant pigs, although this effect was delayed in myometrium from pregnant females. After pre-incubation of the myocytes with P4 (10(-5)M) the influence of OT on [Ca2+]i)was delayed during luteolysis and inhibited during pregnancy. Myometrial cells in culture produce more PGE2 than PGF2alpha regardless of reproductive state of the female. OT (10(-7)M) increased PGE2 secretion after 6 and 12 h incubation for the tissue harvested during luteolysis and after 12 h incubation when myometrium from gravid females was used. In the presence of P4 (10(-5)M), the stimulatory effect of OT on PG secretion was diminished. In conclusion: (1) porcine myometrial cells in culture secrete PG preferentially during early pregnancy and produce more PGE2 than PGF2alpha, (2) OT controls myometrial PGF2alpha secretion during luteolysis, (3) release of [Ca2+]i is associated with the influence of OT on PG secretion, and (4) the effects of OT on PG secretion and Ca2+ accumulation are delayed by P4 during luteolysis and completely inhibited by P4 during pregnancy.     

Effect of exogenous gonadal steroids and pregnancy on uterine luminal prostaglandin F in mares

Two experiments were conducted to assess the effect of exogenous hormone treatment on uterine luminal prostaglandin F (PGF). In the first experiment ovariectomized pony mares received either corn oil (21 days, n = 3), estradiol valerate (21 days, n = 3), progesterone (21 days, n = 3) or estradiol valerate (7 days) followed by progesterone (14 days, n = 4). Progesterone treated mares had higher (P<.01) uterine luminal PGF compared with all other groups, and no differences were detected between other treatment comparisons. In Experiment II, uterine fluid was collected from 4 ovariectomized horse mares before and after treatment with estradiol valerate (7 days) followed by progesterone (50 days). Pretreatment uterine luminal PGF levels were lower (P<.001) than post-treatment levels (.03 vs 76.80 ng/ml). In a third experiment PGF was measured in uterine fluid of pony mares on days 8, 12, 14, 16, 18 and 20 of the estrous cycle and pregnancy. In nonpregnant mares a day effect P<.03) was observed in which uterine fluid PGF increased during the late luteal phase and declined thereafter. In contrast, no day effect was observed in pregnant animals and uterine luminal PGF was lower (P<.001) than in cycling animals. These studies indicate that exogenous progesterone administration results in a large increase in uterine luminal PGF, whereas, pregnancy results in suppression. Taken collectively with previous work from our laboratory, these results suggest that while the endometrium of pregnant mares is capable of producing large amounts of PGF, the presence of a conceptus impedes its synthesis and/or release which allows for luteal maintenance.     

In vitro release of PGF(2alpha) from the caprine uterus and the effect of ovarian steroids

The purpose of the study was to determine the in vitro release of prostaglandin F(2)alpha (PGF(2alpha)) from the uterine tissues of ovariectomized goats after steroid treatment. Strips of endometrial tissues (separated by trypsin treatment) and intact uterine tissues (with endometrium and myometrium) were maintained in organ culture and exposed to estradiol-17beta(E) at 50 ng/ml and to progesterone (P) at 250 pg/ml alone or in combination. The endometrial tissues, in general, continued to release PGF(2alpha) without steroids treatment throughout the 96-h incubation period; their output was consistently higher (based on ng/gm of wet tissue) than the corresponding intact uterine tissues. However, exposure to E reversed this trend and produced a three-fold increase from the intact uterine strips at 24 h. A combined E-plus-P treatment blocked the stimulatory response, whereas P treatment alone evoked a delayed stimulatory response at 48 h. Endometrial tissue reaction was similar to that of P treatment, but it exhibited a more moderated response to E, which could not be blocked by a combined E-plus-P treatment. The results suggest that both endometrial and myometrial tissues release PGF(2alpha) and that this release is regulated by ovarian steroid hormones.     

Activity of phospholipase C and release of prostaglandin F2 alpha by endometrial tissue from ovariectomized ewes receiving progesterone and estradiol

Progesterone and estradiol interact to regulate secretion of prostaglandin (PG) F2 alpha from the ovine endometrium in response to oxytocin. Two experiments were conducted to determine if these effects were due to changes in activity of phospholipase C or in the second messenger responsive pathways that regulate production of PGF2 alpha. In both experiments, ovariectomized ewes were assigned to one of four treatment groups (control, estradiol, progesterone, progesterone and estradiol). Steroids were administered, in vivo, to mimic the changes that occur during the estrous cycle. On Day 16 of steroid treatment, endometrial tissue was collected and incubated, in vitro, to measure activity of phospholipase C and release of PGF2 alpha. Treatment with progesterone, in vivo, enhanced basal and oxytocin-induced activity of phospholipase C and release of PGF2 alpha, in vitro. Estradiol suppressed oxytocin-induced activity of phospholipase C, both in the presence and absence of progesterone. In contrast to its effects on phospholipase C, estradiol inhibited basal and oxytocin-induced release of PGF2 alpha when administered alone, but not when administered with progesterone. Steroids had similar effects on the release of PGF2 alpha induced by phorbol 12-myristate 13-acetate and A23187. It was concluded that progesterone and estradiol regulate endometrial release of PGF2 alpha by affecting both the activity of phospholipase C and its associated second messenger responsive pathways that may regulate production of PGF2 alpha.     

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

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

The effects of ovarian steroids in controlling rat uterine prostaglandin F2-alpha during the peri-implantation period

Rats with delayed implantation, induced by ovariectomy or hypophysectomy, as well as those with normal pregnancy were used to examine the changes in uterine prostaglandin F2 alpha (PGF2 alpha) associated with implantation. In normal pregnant rats, while maximal uterine production of PGF2 alpha was found at 09:00, maximal catabolic enzyme activity (CEA) was seen at 17:00 of day 4. Uterine content of PGF2 alpha was high at 17:00 of day 4, but decreased by 80% within the next 24 h. There was no change in PGF2 alpha production during the first 6 h after injection of estradiol to hypophysectomized animals. There was, however, a dramatic decrease in production within the next 6 h. In contrast, CEA was not different in animals treated with estrogen than in those receiving only progesterone. In ovariectomized animals, uterine PGF2 alpha production also was lowered by estrogen but in these animals CEA was significantly elevated 18 h after injection of estradiol. Estrogen caused a greater increase in PGF2 alpha content in the hypophysectomized, compared to the ovariectomized, rats. The results are consistent with the view that ovarian steroids play an important role in controlling the changes in uterine PGF2 alpha around the time of implantation in rat.     

Synthesis of prostaglandin F2 alpha, E2 and prostacyclin in isolated corpora lutea of adult pseudopregnant rats throughout the luteal life-span.

The ability of de novo biosynthesis of prostaglandins (PGs) in individual whole corpora lutea (CL) obtained from sterile-mated adult pseudopregnant rats on different days of the luteal phase and the post-luteolytic period was evaluated. Production of PGs, progesterone and 20 alpha-dihydroprogesterone were determined after in vitro incubation of CL extirpated from Day 2 to Day 19 after mating. A time-relationship with increased accumulation of PGs in the medium was demonstrated from 18 s to 5 h, with large increments during the first 30 min. Basal accumulation of PGs in the incubation medium was highest for 6-keto-PGF1 alpha (the stable metabolite of prostacyclin) greater than PGE2 greater than PGF2 alpha greater than thromboxane B2 (TXB2) and basal accumulation of PGF2 alpha and PGE2 measured in the medium was maximal on Day 10-11 of pseudopregnancy, concomitantly with a decline in secretion of progesterone. Addition of arachidonic acid (AA) dose-dependently increased synthesis of PGs, with absolute amounts of PGE2 greater than 6-keto-PGF1 alpha greater than PGF2 alpha greater than TXB2 and addition of 14 microM indomethacin markedly inhibited accumulation of all PGs measured. Luteinizing hormone (LH, 10 micrograms/ml) stimulated progesterone secretion on all days during pseudopregnancy, but not on the post-luteolytic Day 19. LH increased PGF2 alpha, PGE2 and 6-keto-PGF1 alpha secretion on Day 13 of pseudopregnancy by 76%, 91% and 28%, respectively, but not on the other days tested. Furthermore, stimulation of PG-synthesis by addition of AA abrogated the LH-induced progesterone accumulation markedly, but only on Day 13 of pseudopregnancy. Epinephrine (5 micrograms/ml) increased production of progesterone and also PGs, but only on Day 2 of pseudopregnancy, whereas oxytocin (100 mIU/ml) was found to be without effect on progesterone as well as PG secretion on all days tested. The results of the present study demonstrates the independent ability of the rat CL to synthesize PGG/PGH2-derived prostaglandins, including the putative luteolysin PGF2 alpha. Secondly, we demonstrate that LH and AA-induced increases in PGF2 alpha and PGE2 production during the luteolytic period, may be an autocrine or paracrine mechanism involved in luteolysis.     

Concentrations of endogenous prostaglandin F2alpha in boar semen and effect of a 72-h incubation period on exogenous prostaglandin F2alpha concentration in extended boar semen

PGF2alpha in semen has been shown to induce uterine contractions, thereby, facilitating sperm transport during fertilization. Previously, we demonstrated that extended boar semen used in artificial insemination does not increase myometrial contractility, but PGF2alpha supplementation did. In this study, we determined the concentrations of endogenous PGF2alpha in pre-sperm and sperm-rich fractions of the boar ejaculate and examined whether changes in the concentration of exogenous PGF2alpha occurred when added to extended boar semen after 72-h incubation at a 17 degrees C storage temperature. Concentrations of endogenous PGF2alpha (n = 10 boars) in pre-sperm and sperm-rich fractions were 69.6 +/- 7.6 and 58.9 +/- 4.4 pg/ml, respectively. No differences were observed in the concentrations of exogenous PGF2alpha in the extended boar semen at 0 h (59.3 +/- 3.3 microg/ml) and after a 72-h incubation period (52.0 +/- 2.1 microg/ml). These results suggest that the concentration of endogenous PGF2alpha in boar semen used for artificial insemination is < 100 pg/ml. The concentration of exogenous PGF2alpha in the extended boar semen did not differ after 72 h, which indicates that it is not metabolized during this period of time.     

Changes in steady-state concentrations of messenger ribonucleic acids in luteal tissue during prostaglandin F2alpha induced luteolysis in mares

Transvaginal ultrasound-guided luteal biopsy was used to evaluate the effects of prostaglandin (PG)F2alpha on steady-state concentrations of mRNA for specific genes that may be involved in regression of the corpus luteum (CL). Eight days after ovulation (Hour 0), mares (n=8/group) were randomized into three groups: control (no treatment or biopsy), saline+biopsy (saline treatment at Hour 0 and luteal biopsy at Hour 12), or PGF2alpha+biopsy (5mg PGF2alpha at Hour 0 and luteal biopsy at Hour 12). The effects of biopsy on CL were compared between the controls (no biopsy) and saline+biopsy group. At Hour 24 (12h after biopsy) there was a decrease in circulating progesterone in saline group to 56% of pre-biopsy values, indicating an effect of biopsy on luteal function. Mean plasma progesterone concentrations were lower (P<0.001) at Hour 12 in the PG group compared to the other two groups. The relative concentrations of mRNA for different genes in luteal tissue at Hour 12 was quantified by real time PCR. Compared to saline-treated mares, treatment with PGF2alpha increased mRNA for cyclooxygenase-2 (Cox-2, 310%, P<0.006), but decreased mRNA for LH receptor to 44% (P<0.05), steroidogenic acute regulatory protein to 22% (P<0.001), and aromatase to 43% (P<0.1) of controls. There was no difference in mRNA levels for PGF2alpha receptor between PG and saline-treated groups. Results indicated that luteal biopsy alters subsequent luteal function. However, the biopsy approach was effective for collecting CL tissue for demonstrating dynamic changes in steady-state levels of mRNAs during PGF2alpha-induced luteolysis. Increased Cox-2 mRNA concentrations suggested that exogenous PGF2alpha induced the synthesis of intraluteal PGF2alpha. Thus, the findings are consistent with the concept that an intraluteal autocrine loop augments the luteolytic effect of uterine PGF2alpha in mares.