Effects of pregnancy, oxytocin, ovine trophoblast protein-1 and their interactions on endometrial production of prostaglandin F2α in vitro in perifusion chambers

Pregnancy and intrauterine infusion of ovine trophoblast protein one (oTP-1) decrease oxytocin-induced secretion of prostaglandin F2α (PGF) from the uterus. In the present study, effects of oTP-1 and pregnancy on endometrial secretion of PHF 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. Endormetrium pregnant ewes secreted more PGF fro both lumenal and myotrial sides than endometrium from cyclic ewes (P<0.05). Oxytocin stimulated secretion of PGF was greater from the lumenal surface of endometrium compared to myometrium 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 alon. Oxytocin stimulated greater release of PGF from oTP-1-treated than BSA-treated endometrium. Pretreament of endometrium with oTP-1 has the same effect on oxytocin-induced PGF section was 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) preganancy 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 ovine trophoblast protein-1, oestrogen and progesterone on oxytocin-induced phosphatidylinositol turnover in endometrium of sheep

In Exp. 1, endometrium was collected from Day-15 cyclic ewes and effects of oTP-1, oxytocin and oTP-1 + oxytocin, in various temporal relationships, on phosphatidylinositol (PI) turnover were determined. Co-treatment of endometrium with oTP-1 and oxytocin inhibited stimulatory effects of oxytocin, while treatment with oTP-1 before and during oxytocin administration had no effect. Turnover of PI was unaffected by oTP-1 alone. In Exp. 2, ovariectomized ewes were treated with progesterone (50 mg/day) for 10 days and then oestrogen (100 micrograms/day) for 2 days and endometrium was collected. Oxytocin stimulated PI turnover in endometrium, but oTP-1 had no effect alone or in combination with oxytocin. In Exp. 3, ovariectomized ewes were treated with corn oil (1 ml/day), oestrogen (50 micrograms/day), progesterone (50 mg/day) or progesterone + oestrogen for 10 days and endometrium was collected. Oxytocin stimulated PI turnover only in ewes that received progesterone. oTP-1 alone had no effect on PI turnover, while co-treatment of endometrium with oxytocin and oTP-1 stimulated PI turnover in ewes treated with progesterone, but not progesterone and oestrogen. Pretreatment of endometrium with oTP-1 stimulated PI turnover when ewes were treated with progesterone or progesterone + oestrogen. Pretreatment of endometrium with oxytocin and then treatment with oTP-1 inhibited PI turnover compared to treatment with oxytocin alone. In Exp. 4, ovariectomized ewes were treated as in Exp. 2. Catheters were placed into the uterine horns and ewes received oTP-1 into one horn and serum into the other twice daily on Days 10-12 of steroid treatment. Endometrium collected on Day 13 was used to measure PI turnover and received either no treatment or oxytocin. Oxytocin stimulated PI turnover in endometrium of these ewes and in-vivo treatment of the ewes with oTP-1 had no effect on PI turnover. These results indicate that antiluteolytic effects of oTP-1 are not mediated by inhibiting effects of oxytocin on phosphatidylinositol turnover if oxytocin receptors are present and that uterine responsiveness to oxytocin is progesterone dependent.     

Prostaglandin secretion by endometrium of pregnant and cyclic cattle at day 17 after oestrus in response to in-vitro heat stress

Bilateral perifusion devices were utilized to measure prostaglandin F-2 alpha (PGF) secretion by bovine endometrium in response to in-vitro heat stress. Tissues were collected at Day 17 after oestrus from cyclic (N = 4) and pregnant (N = 5) cows, placed into 3 perifusion devices, perifused (3 ml/10 min, Krebs-Ringer-bicarbonate [KRB]) for 5 h, and fractions were collected every 10 min. Endometrial tissues within each device were subjected to a different temperature and oxytocin (1 i.u./ml KRB) treatment sequence: (1) control-oxytocin: 1 h at 39 degrees C; 2 h at 39 degrees C, 0.5 h at 39 degrees C with oxytocin, 0.5 h at 39 degrees C and 1 h at 39 degrees C; (2) heat-oxytocin: 1 h at 39 degrees C, 2 h at 42 degrees C, 0.5 h at 42 degrees C with oxytocin, 0.5 h at 42 degrees C and 1 h at 39 degrees C; (3) heat-KRB: 1 h at 39 degrees C, 2 h at 42 degrees C, 0.5 h at 42 degrees C, 0.5 h at 42 degrees C and 1 h at 39 degrees C. Regardless of reproductive status, heat stress induced a rapid increase (P less than 0.01) in PGF secretion rates. Oxytocin induced an increase (P less than 0.01) in PGF secretion for endometrium from cyclic cows regardless of temperature. Endometria from pregnant cows did not respond to oxytocin when perifused at 39 degrees C. However, PGF secretion rates from endometrium of pregnant cows increased (P less than 0.01) in response to oxytocin when perifused under heat stress conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of porcine conceptus secretory proteins on in vitro secretion of prostaglandins-F2 alpha and -E2 from luminal and myometrial surfaces of endometrium from cyclic and pseudopregnant gilts

Uterine endometrium collected from pseudopregnant (PP) and cyclic gilts on day (D) 15 after estrus were perifused in vitro with 10 ug/ml of porcine conceptus secretory proteins (pCSP) or serum proteins (SP) in Krebs ringer bicarbonate (KRB) buffer. In Experiment 1, samples were collected from luminal and myometrial surfaces of endometrium and concentrations of prostaglandin F2 alpha (PGF) determined by radioimmunoassay (RIA). Secretion of PGF by endometrium from cyclic gilts was stimulated (P less than .05) by pCSP. In Experiment 2, endometrium from D 14 cyclic and PP gilts was perifused and concentrations of PGF and prostaglandin E2 (PGE) in perfusate were determined by RIA. Across both statuses, luminal surface secretion of PGF was stimulated (P less than .05) by pCSP. Treatment with pCSP decreased secretion of PGE from myometrial surface of endometrium from cyclic gilts and increased (P less than .01) secretion of PGE from the myometrial surface of endometrium from PP gilts. In Experiment 3, pCSP were separated into acidic and basic fractions by anion exchange chromatography and each fraction was perifused separately over the luminal surface of endometrium from cyclic and PP gilts. Perifusion with acidic pCSP suppressed secretion of PGF by endometrium from cyclic or PP gilts; while basic pCSP did not influence secretion of PGF. These results demonstrated that products secreted by Day 15 pig conceptuses stimulate release of PGF and PGE from porcine uterine endometrium.     

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)     

Ovine trophoblast protein-one inhibits development of endometrial responsiveness to oxytocin in ewes

In experiment (Exp) 1, 12 cyclic ewes had catheters placed into each uterine horn on Day 7 (estrus = Day 0). On Days 11-15, 6 ewes received twice-daily intrauterine infusions of 1.5 mg serum protein (SP) into each uterine horn and 6 ewes received infusions of 1.08 mg SP + 0.42 mg ovine conceptus secretory proteins (oCSP) containing 25 micrograms ovine trophoblast protein-one (oTP-1) as determined by radioimmunoassay (25-35% bioactive by antiviral assay). SP-infused and oCSP-infused ewes had similar plasma 13,14-dihydro-15-keto prostaglandin F2 alpha (PGF2 alpha) profiles in response to oxytocin on Day 11, but SP ewes became more responsive (p less than 0.01) to oxytocin on Days 13 and 15 than oCSP ewes. SP ewes also had greater incorporation of [3H]inositol into inositol trisphosphate (IP3) (+3449%, p less than 0.01) and total inositol phosphate (IP) (+760%, p less than 0.08), in response to oxytocin, than did oCSP ewes (+553 and +168% for IP3 and total IP, respectively) in endometrium collected at ovariectomy/hysterectomy on Day 16. Mean CL weights on Day 16 and mean concentrations of progesterone in plasma collected at 12-h intervals on Days 6-16 were not different for SP and oCSP ewes, but concentrations of progesterone were lower (p less than 0.05) in SP ewes on Days 15-16 than for oCSP ewes. These results indicate that oTP-1 may prevent luteolysis by inhibiting development of endometrial responsiveness to oxytocin and, therefore, reduce oxytocin-induced synthesis of IP3 and PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin secretion by perifused bovine endometrium: secretion towards the myometrial and luminal sides at day 17 post-estrus as altered by pregnancy

Bilateral perifusion devices were utilized to measure prostaglandin secretion towards luminal and myometrial sides of bovine endometria. Tissues were collected at Day 17 post-estrus from cyclic (n = 4), pregnant (n = 5) and bred but subsequently non-pregnant (n = 6) cows. Tissue from each cow was placed into two perifusion devices, perifused with Krebs-Ringer Bicarbonate solution (3 ml/10 min) for 2.5 h and fractions collected every 10 min. Oxytocin (1 IU/ml) was perifused during fractions 7-12 to the luminal side of one device and to the myometrial side of the other device. Regardless of status, prostaglandin secretion rates (PGF and PGE2) were higher (P less than 0.01) from the luminal side than the myometrial side. Secretion rates of PGF were lower (P less than 0.01) for endometria from pregnant cows than for endometria from cyclic or bred/non-pregnant cows, whereas secretion rates of PGE2 were not affected by pregnancy status. Regardless of the side of perifusion, secretion rates of PGF and PGE2 from endometria of cyclic and bred/non-pregnant cows were elevated (P less than 0.01) throughout the period of oxytocin treatment, whereas prostaglandin secretion by endometria from pregnant cows was not stimulated by oxytocin. Decreased secretion of PGF from endometria of pregnant cows suggests that the corpus luteum and pregnancy are maintained because of an inhibition of endometrial prostaglandin synthesis or an inability to respond to stimulators of prostaglandin synthesis (i.e. oxytocin).     

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)     

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)     

Effects of ovine conceptus secretory proteins and progesterone on oxytocin-stimulated endometrial production of prostaglandin and turnover of inositol phosphate in ovariectomized ewes.

This study examined the effects of progesterone and intrauterine injection of ovine conceptus secretory proteins (oCSP) on endometrial responsiveness to oxytocin. Twelve ewes were ovariectomized on day 4 of the cycle (oestrus = day 0) and assigned in a 2 x 2 factorial arrangement, to receive either 1.5 mg ovine serum proteins (SP) or oCSP containing 25 micrograms ovine trophoblast protein 1 (oTP-1) (by radioimmunoassay) in 1.5 mg total protein into each uterine horn, via catheters, twice a day on days 11, 12, 13 and 14. Ewes received 200 mg progesterone per day (i.m.) from day 4 to day 10 or 15. Oxytocin-induced prostaglandin F2 alpha was measured as 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) on days 11, 12, 13 and 14 in plasma from three integrated, 10 min (10 ml) blood samples (0-10, 10-20, 20-30 min) obtained after intravenous injection of 20 iu oxytocin, and in a pre-oxytocin (-10 to 0 min) sample collected via an indwelling jugular catheter. The pre-oxytocin samples were also assayed for progesterone. Oxytocin-induced turnover of inositol phosphate was determined in endometrium on day 15 after hysterectomy. In ewes receiving progesterone to day 10, plasma progesterone decreased from about 12 to 2 ng ml-1 (SEM +/- 2.6) during the treatment period (days 11-14), but remained high (12-20 +/- 2.6 ng ml-1) in ewes that received progesterone to day 15. Intrauterine injection of oCSP resulted in high basal concentrations of PGFM on days 12 and 13 compared with SP-treated ewes (P less than 0.01). Treatments with progesterone did not affect basal PGFM concentrations. Treatment with oCSP abolished oxytocin-induced endometrial secretion of prostaglandin only if progesterone was maintained to day 15 (P less than 0.01); in ewes receiving such treatment, oCSP inhibited (P less than 0.01), but SP did not inhibit, oxytocin-induced endometrial turnover of inositol phosphate (P less than 0.06), which was greater in ewes treated with progesterone to day 10 than in those treated to day 15 (P less than 0.05). Ewes that responded to oxytocin with increased PGFM exhibited increased oxytocin-stimulated turnover of inositol phosphate on day 15. These results indicate that the antiluteolytic action oTP-1 exerts on the endometrium requires progesterone and that this mechanism involves inhibition of oxytocin-stimulated turnover of inositol phosphate.     

Prostaglandin secretion by perifused bovine endometrium: Secretion towards the myometrial and luminal sides at day 17 post-estrus as altered by pregnancy

Bilateral perifusion devices were utilized to measure prostaglandin secretion towards luminal and myometrial sides of bovine endometria. Tissues were collected at Day 17 post-estrus from cyclic (n=4), pregnant (n=5) and bred but subsequently non-pregnant (n=6) cows. Tissue from each cow was placed into two perifusion devices, perifused with Krebs-Ringer Bicarbonate solution (3 ml/10 min) for 2.5h and fractions collected every 10 min. Oxytocin (1 IU/ml) was perifused during fractions 7–12 to the luminal side of one device and to the myometrial side of the other device. Regardless of stratus, prostaglandin secretion rates (PGF and PGE₂) were higher (P< 0.01) from the luminal side than the myometrial side. Secretion rates of PGF were lower (P< 0.01) for endometria from pregnant cows than for endometria from cyclic or bred/non-pregnant cows, whereas secretion rates of PGE₂ were not affected by pregnancy status. Regardless of the side of perifusion, secretion rates of PGF and PGE₂ from endometria of cyclic and bred/non-pregnant cows were elevated (P< 0.01) throughout the period of oxytocin treatment, whereas prostaglandin secretion by endometria from pregnant cows wasnot stimulated by oxytocin. Decreased secretion of PGF from endometria of pregnant cows suggests that the corpus luteum and pregnancy are maintained because of an inhibition of endometrial prostaglandin synthesis or an inability to responsd to stimulators of prostaglandin synthesis (i.e. oxytocin).     

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

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

Effect of exogenous melatonin on in vivo and in vitro prostaglandin secretion in Rasa Aragonesa ewes

The effect of exogenous melatonin on prostaglandin secretion was measured on Rasa Aragonesa ewes. Fourteen ewes received an 18 mg melatonin implant (M+) on 10 April and were compared with 13 control animals (without implants M-). Twenty days later, intravaginal pessaries were inserted in all animals to induce a synchronized oestrus (day 0). On day 14, ewes were injected, i.v., with 0.5 IU oxytocin. Plasma 15-ketodihydro-PGF(2alpha) (PGFM) concentrations were measured to assess uterine secretory responsiveness to oxytocin. After euthanasia, pieces of endometrium were collected to determine progesterone content and PGE(2) and PGF(2alpha) secretion in vitro, in the presence or absence of either 20 microg/ml recombinant ovine interferon-tau (roIFNt) or 1 nmol/l oxytocin in the medium. Endometrial progesterone content was similar in the two treatments (M+: 50.25+/-17.34 ng/mg tissue, M-: 43.08+/-11.21 ng/mg tissue). M+ ewes that responded to oxytocin had significantly higher plasma PGFM concentrations between 10 and 80 min after oxytocin administration, a higher mean PGFM peak (P<0.001), higher plasma PGFM levels after the challenge (P<0.05) and higher plasma progesterone concentrations (P<0.01) than control ewes. In the in vitro experiment, M+ and M- control samples secreted similar amounts of PGE(2). The presence of roIFNtau and oxytocin only stimulated PGE(2) production (P<0.05) in M- tissues. Control M+ tissues secreted higher amounts of PGF(2alpha) (P=0.07) and PGF(2alpha) secretion was significantly (P<0.01) stimulated by roIFNtau. Oxytocin produced this effect only in M- samples (P<0.01). In conclusion, although previous studies have demonstrated a positive effect of melatonin on lamb production, PGF(2alpha) secretion is higher in vitro and the PGE(2):PGF(2alpha) ratio is unfavourable in response to IFNtau, which could affect embryo survival. Whether or not these mechanisms are similar in pregnant ewes remains to be elucidated.     

Characterization of endometrial receptors for ovine trophoblast protein-1 during the estrous cycle and early pregnancy in sheep

Scatchard analysis was used to determine the distribution, number, and affinity of unoccupied receptors for ovine trophoblast protein-1 (oTP-1) in endometrium of sheep throughout the estrous cycle and early pregnancy. In Experiment I, oTP-1 receptor characteristics were determined in membrane preparations of caruncular and intercaruncular regions of endometrium collected from uterine horns ipsilateral and contralateral to the ovary bearing the corpus luteum. Receptor concentrations and affinity constants for oTP-1 were not different (p greater than 0.1) between the four endometrial regions examined, suggesting that the expression of receptors for oTP-1 occurs uniformly throughout the endometrium. Endometrial receptor characteristics for oTP-1, luteal wet weights, and progesterone contents were determined throughout the estrous cycle and early pregnancy in Experiment II. Concentration of receptors and affinity constants for oTP-1 varied throughout the estrous cycle and early pregnancy (p less than 0.01), with the pattern of change differing between cyclic and pregnant ewes (p less than 0.01). Numbers of receptors for oTP-1 were maximal on Day 4 of the estrous cycle and declined progressively to Day 12 (p less than 0.05) in both cyclic and pregnant ewes. After Day 12, the quantity of unoccupied receptors for oTP-1 increased (p less than 0.05) gradually to Day 16 in cyclic ewes, but declined (p less than 0.05) further in the endometrium of pregnant ewes. The affinity constants of endometrial receptors for oTP-1 were similar in cyclic and pregnant ewes prior to Day 12, increasing threefold from Days 4 to 12 (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of endometrial prostaglandin synthesis during early pregnancy in cattle: Effects of phospholipases and calcium In vitro

In experiment 1, endometrial explants from 3 cyclic (Day 17) cows were incubated with arachidonic acid (AA), phospholipase A-2 (PLA-2) and calcium ionophore A23187 (CaI) or control. AA (0.2 mg), PLA-2 (1 U/ml) and Cal (4 μg/ml) increased PGF and PGE secretion. In experiment 2, endometrial explants from cyclic (n = 4) and pregnant (n = 3) cows were incubated +/- Ca⁺⁺ and with either: control, AA, PLA-2, CaI, PLA-2 + CaI, or AA + CaI. PG secretion was higher in cultures with Ca⁺⁺. In presence of Ca⁺⁺, PGF secretion was lower for pregnant than cyclic endometrium. AA with Ca⁺⁺ stimulated PGF and PGE secretion, indicating that AA availability may limit PG secretion. The stimulatory effect of PLA-2 on PGF and PGE secretion was greater in pregnant than cyclic Endometrium. However, CaI inhibited the PLA-2 response of pregnant, but not cyclic endometrium. In experiment 3, endometrium (4 cyclic cows) failed to convert ³H-PGF₂ to PGE₂ or ³H-PGE₂ to PGF₂ Responsiveness of PG secretion to PLA-2, and CaI is altered by reproductive status suggesting that these factors may be involved in the differential regulation of PG production during early pregnancy in cattle.     

Stimulation of 2',5'-oligoadenylate synthetase activity in sheep endometrium during pregnancy, by intrauterine infusion of ovine trophoblast protein-1, and by intramuscular administration of recombinant bovine interferon-alpha I1.

In Expt 1, activity of 2',5'-oligoadenylate (2',5'-A) synthetase in endometrium collected on Day 16 (oestrus is Day 0) from the uterine horn ipsilateral to the corpus luteum was greater (P less than 0.001) for pregnant (135.5 +/- 1.72 nmol/mg protein/h) than for cyclic ewes (58.5 +/- 0.99 nmol/mg protein/h). In pregnant ewes, activity of 2',5'-A synthetase in endometrium collected from the contralateral uterine horn (119.5 +/- 1.72 nmol/mg protein/h) did not differ from that of the ipsilateral horn. In Expt 2, three ovariectomized ewes were treated with progesterone for 10 days and then with oestrogen for 2 days. Activity of 2',5'-A synthetase on Day 13 was 18% greater (P less than 0.10) in endometrium collected from the uterine horn receiving infusions of 30 micrograms ovine trophoblast protein-1 (oTP-1) twice a day on Days 10, 11 and 12(57.7 +/- 0.22 nmol/mg protein/h) than from the uterine horn receiving control infusions of serum protein (SP; 48.8 +/- 0.22 nmol/mg protein/h). In Expt 3, activity of 2',5'-A synthetase on Day 15 was not significantly greater in endometrium collected from the uterine horn of cyclic ewes receiving infusions of 30 micrograms oTP-1 twice a day on Days 12, 13 and 14 (46.5 +/- 0.37 nmol/mg protein/h) than in endometrium from the uterine horn receiving infusions of SP (38.2 +/- 0.37 nmol/mg protein/h). When results of Expt 2 and Expt 3 were combined, intrauterine infusion of oTP-1 increased (P less than 0.05) activity of 2',5'-A synthetase in endometrium by 20%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of oxytocin on concentration of PGF2 alpha in the uterine lumen and subsequent endometrial responsiveness to oxytocin in pigs

The aim of this study was to determine the effect of oxytocin on PGF2 alpha secretion into the uterine lumen of pigs and subsequent endometrial responsiveness to oxytocin in vitro. Cyclic, pregnant and oestradiol-induced pseudopregnant gilts were injected i.v. with vehicle or 20 iu oxytocin 10 min before hysterectomy on day 16 after oestrus. Concentrations of PGF2 alpha and 13,14-dihydro-15-keto PGF2 alpha (PGFM) were significantly increased in uterine flushings collected at hysterectomy (P < 0.05) in pregnant oxytocin-injected gilts. Concentrations of PGF2 alpha and PGFM were greater (P < 0.001) in pregnant than in pseudopregnant and cyclic gilts, and greater (P < 0.01) in pseudopregnant than in cyclic gilts. The ratio of PGFM:PGF2 alpha tended to be greater in cyclic (P < 0.06) and pseudopregnant gilts (P < 0.1) than in pregnant gilts. At 85 +/- 5 min after oxytocin injection, endometrium from each gilt was incubated for 3 h for determination of phosphoinositide hydrolysis and PGF2 alpha secretion in response to treatment with 0 or 100 nmol oxytocin l-1. Endometrial phosphoinositide hydrolysis in response to 100 nmol oxytocin l-1 in vitro was greater (P < 0.05) in cyclic oxytocin-injected gilts than in cyclic vehicle-injected gilts. Treatment with oxytocin in vitro did not stimulate phosphoinositide hydrolysis significantly in vehicle- or oxytocin-injected pregnant gilts or pseudopregnant gilts. Endometrial PGF2 alpha secretion increased after treatment with 100 nmol oxytocin l-1 in vitro in cyclic vehicle-injected (P < 0.01), cyclic oxytocin-injected (P < 0.01), pregnant vehicle-injected (P = 0.06), pseudopregnant vehicle-injected (P < 0.05) and pseudopregnant oxytocin-injected (P < 0.05) gilts, but not in pregnant oxytocin-injected gilts. The increase in PGF2 alpha in pseudopregnant oxytocin-injected gilts was less (P < 0.05) than that in cyclic oxytocin-injected gilts. These results indicate that oxytocin increases the concentration of PGF2 alpha and PGFM in the uterine lumen during pregnancy and may upregulate endometrial responsiveness to oxytocin during late dioestrus in pigs, but does not have the latter effect during early pregnancy or oestradiol-induced pseudopregnancy.     

What regulates placental steroidogenesis in 90-day pregnant ewes?

By day-90, the placenta secretes half of the circulating progesterone and 85% of the circulating estradiol-17beta [Weems YS, Vincent D, Tanaka Y, et al. Effects of prostaglandin F(2alpha) on sources of progesterone and pregnancy in intact, ovariectomized, and hysterectomized 90-100 day pregnant ewes. Prostaglandins 1992;43:203-22; Weems YS, Vincent DL, Nusser K, et al. Effects of prostaglandin F(2alpha) (PGF(2alpha)) on secretion of estradiol-17beta and cortisol in 90-100 day hysterectomized, intact, or ovariectomized pregnant ewes. Prostaglandins 1994;48:139-57]. Ovariectomy (OVX) or prostaglandin (PG) F(2alpha) (PGF(2alpha)) does not abort intact or OVX 90-day pregnant ewes and PGF(2alpha) regresses the corpus luteum, but does not affect placental progesterone secretion in vivo [Weems YS, Vincent D, Tanaka Y, et al. Effects of prostaglandin F(2alpha) on sources of progesterone and pregnancy in intact, ovariectomized, and hysterectomized 90-100 day pregnant ewes. Prostaglandins 1992;43:203-22]. Luteal progesterone secretion in vitro at day-90 of pregnancy in ewes is regulated by PGE(1)and/or PGE(2), not by ovine luteinizing hormone (LH; 3). Concentrations of PGE in uterine or ovarian venous plasma averaged 6 ng/ml at 90-100 days of pregnancy in ewes [Weems YS, Vincent DL, Tanaka Y, Nusser K, Ledgerwood KS, Weems CW. Effect of prostaglandin F(2alpha) on uterine or ovarian secretion of prostaglandins E and F(2alpha) (PGE; PGF(2alpha)) in vivo in 90-100 day hysterectomized, intact or ovariectomized pregnant ewes. Prostaglandins. 1993;46:277-96]. Ovine placental PGE secretion is regulated by LH up to day-50 and by pregnancy specific protein B (PSPB) after day-50 of pregnancy [Weems YS, Kim L, Humphreys V, Tsuda V, Weems CW. Effect of luteinizing hormone (LH), pregnancy specific protein B (PSPB), or arachidonic acid (AA) on ovine endometrium of the estrous cycle or placental secretion of prostaglandins E(2) (PGE(2)) and F(2alpha) (PGF(2alpha)), and progesterone in vitro. Prostaglandins Other Lipid Mediators 2003;71:55-73]. Indomethacin (INDO), a prostaglandin synthesis inhibitor [Lands WEM. The biosynthesis and metabolism of prostaglandins. Annu Rev Physiol 1979;41:633-46], lowers jugular venous progesterone [Bridges PJ, Weems YS, Kim L, et al. Effect of prostaglandin F(2alpha) (PGF(2alpha)), indomethacin, tamoxifen or estradiol-17beta on pregnancy, progesterone and pregnancy specific protein B (PSPB) secretion in 88-90 day pregnant ewes. Prostaglandins Other Lipid Mediators 1999;58:113-24] and inferior vena cava PGE of pregnant ewes with ovaries by half at day-90 [Bridges PJ, Weems YS, Kim L, LeaMaster BR, Vincent DL, Weems CW. Effect of prostaglandin F(2alpha) (PGF(2alpha)), indomethacin, tamoxifen or estradiol-17beta on prostaglandin E (PGE), PGF(2alpha) and estradiol-17beta secretion in 88-90 day pregnant sheep. Prostaglandins Other Lipid Mediators 1999;58:167-78]. In addition, treatment of 90 day ovine diced placental slices with androstenedione in vitro increased placental estradiol-17beta, but treatment with PGF(2alpha)in vitro did not decrease placental progesterone secretion, which indicates that ovine placenta progesterone secretion is resistant to the luteolytic action of PGF(2alpha) [Weems YS, Bridges PJ, LeaMaster BR, Sasser RG, Vincent DL, Weems CW. Secretion of progesterone, estradiol-17beta, prostaglandins (PG) E (PGE), F(2alpha) (PGF(2alpha)), and pregnancy specific protein B (PSPB) by day 90 intact or ovariectomized pregnant ewes. Prostaglandins Other Lipid Mediators 1999;58:139-48]. This also explains why ovine uterine secretion of decreased around day-50 [Weems YS, Kim L, Humphreys V, Tsuda V, Weems CW. Effect of luteinizing hormone (LH), pregnancy specific protein B (PSPB), or arachidonic acid (AA) on ovine endometrium of the estrous cycle or placental secretion of prostaglandins E(2) (PGE(2)) and F(2alpha) (PGF(2alpha)), and progesterone in vitro. Prostaglandins Other Lipid Mediators 2003;71:55-73], when placental estradiol-17beta secretion is increasing [Weems C, Weems Y, Vincent D. Maternal recognition of pregnancy and maintenance of gestation in sheep. In: Reproduction and animal breeding: advances and strategies. Enne G, Greppi G, Lauria A, editors, Elsevier Pub., Amsterdam 1995. p. 277-93]. Treatment of 90 day pregnant ewes with estradiol-17beta+ PGF(2alpha), but not either treatment alone, caused a linear increase in both estradiol-17beta and PGF(2alpha) and ewes were aborting [Bridges PJ, Weems YS, Kim L, Sasser RG, LeaMaster BR, Vincent DL, Weems CW. Effect of prostaglandin F(2alpha) (PGF(2alpha)), indomethacin, tamoxifen or estradiol-17beta on pregnancy, progesterone and pregnancy specific protein B (PSPB) secretion in 88-90 day pregnant ewes. Prostaglandins Other Lipid Mediators 1999;58:113-24; Bridges PJ, Weems YS, Kim L, LeaMaster BR, Vincent DL, Weems CW. Effect of prostaglandin F(2alpha) (PGF(2alpha)), indomethacin, tamoxifen or estradiol-17beta on prostaglandin E (PGE), PGF(2alpha) and estradiol-17beta secretion in 88-90 day pregnant sheep. Prostaglandins Other Lipid Mediators 1999;58:167-78]. Pregnant ewes OVX on day 83 of pregnancy and placental slices cultured in vitro secretes 2-3-fold more estradiol-17beta, PSPB, PGE, and progesterone than placental slices from 90 day intact pregnant ewes, but placental PGF(2alpha) secretion by placental slices from intact or OVX ewes did not change [Denamur R, Kann G, Short R V. How does the corpus luteum of the sheep know that there is an embryo in the uterus? In: Pierrepont G, editor. Endocrinology of pregnancy and parturition, vol. 2. Cardiff, Wales, UK: Alpha Omega Pub Co.; 1973. p. 4-38]. The objective of these experiments was to determine what regulates ovine placental progesterone and estradiol-17beta secretion at day-90 of pregnancy, since the hypophysis [Casida LE, Warwick J. The necessity of the corpus luteum for maintenance of pregnancy in the ewe. J Anim Sci 1945;4:34-9] or ovaries [Weems CW, Weems YS, Randel RD. Prostaglandins and reproduction in female farm animals. Vet J 2006;171:206-28] are not necessary after day-55 to maintain pregnancy. In Experiment 1, diced placental slices from day-90 intact or OVX pregnant ewes that were ovariectomized or laparotomized and ovaries were not removed on day 83 were collected on day-90 and incubated in vitro in M-199 with Vehicle, ovine luteinizing hormone (oLH), ovine follicle stimulating hormone (oFSH), ovine placental lactogen (oPL), PGE(l), PGE(2), PGD(2), PGI(2), insulin-like growth factor (IGF) 1 or 2 (IGF(l); IGF(2)), leukotriene C(4) (LTC(4)), platelet activating factor (PAF) 16 or 18 (PAF-16; PAF-18) at doses of 0, 1, 10, or 100ng/ml for 4h. In Experiment 2, placental slices from day-90 intact and OVX (intact or OVX laporotomized 7 days earlier) pregnant ewes were incubated in vitro with vehicle, INDO, Meclofenamate (MECLO), PGE(l), PGE(2), INDO+PGE(1), MECLO+PGE(l), INDO+PGE(2), or MECLO+PGE(2) for 4h. Media were analyzed for progesterone, estradiol-17beta, PGE, or PGF(2alpha) by RIA. Hormone data in media were analyzed in Experiment 1 by a 2x3x13 and in Experiment 2 by a 2x9 Factorial Design for ANOVA. In Experiment 1, placental progesterone, PGE, or estradiol-17beta secretion were increased (P< or =0.05) two-fold by OVX. Progesterone was not increased (P> or =0.05) by any treatment other than OVX and only FSH increased (P< or =0.05) estradiol-17beta secretion by placental slices in both OVX and intact ewes 90-day pregnant ewes. In Experiment 2, INDO or MECLO decreased (P< or =0.05) placental progesterone secretion by 88% but did not decrease (P> or =0.05) placental estradiol-17beta secretion from intact or OVX ewes. PGE(l) or PGE(2) increased (P< or =0.05) progesterone secretion only in ewes treated with INDO or MECLO. It is concluded that FSH probably regulates day-90 ovine placental estradiol-17beta secretion, while PGE(l) or PGE(2) regulates day-90 placental progesterone secretion.     

Uterine secretion of prostaglandin F2 alpha in response to oxytocin in ewes: changes during the estrous cycle and early pregnancy.

Experiment 1 was conducted to determine when the ovine uterus develops the ability to secrete prostaglandin F2 alpha (PGF2 alpha) in response to oxytocin and how development is affected by pregnancy. Pregnant and nonpregnant ewes received an injection of oxytocin (10 IU, i.v.) on Day 10, 13, or 16 postestrus. Jugular venous blood samples were collected for 2 h after injection for quantification of 13,14-dihydro-15-keto-PGF2 alpha (PGFM). In nonpregnant ewes, concentrations of PGFM increased following oxytocin on Day 16 but not on Day 10 or 13. Concentrations of PGFM did not increase following treatment on Day 10, 13, or 16 in pregnant ewes. Therefore, the ability of oxytocin to induce uterine secretion of PGF2 alpha develops after Day 13 in nonpregnant but not in pregnant ewes. Experiment 2 was conducted to precisely define when uterine secretory responsiveness to oxytocin develops. Pregnant and nonpregnant ewes received oxytocin on Day 12, 13, 14, or 15. In nonpregnant ewes, concentrations of PGFM increased following treatment on Days 14 and 15, but not earlier. Peripheral concentrations of progesterone showed that uterine secretory responsiveness to oxytocin developed prior to the onset of luteal regression. As in experiment 1, the increase in concentrations of PGFM following administration of oxytocin was much lower in pregnant than in nonpregnant ewes; however, some pregnant ewes did respond to oxytocin with an increase in PGFM. In experiment 3, pregnant ewes received an injection of oxytocin on Day 18, 24, or 30 postmating. Concentrations of PGFM increased following oxytocin on Days 18 and 24. The conceptus appears to delay and attenuate the development of uterine secretory responsiveness to oxytocin.