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)     

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)     

Luteal function and blastocyst development in ewes following treatment with PGF(2)alpha and GnRH

Luteal function and blastocyst development were compared in ewes treated with GnRH (100 mug) on Day 1 (Day 0 = day of estrus) or in ewes previously induced into estrus with PGF(2)alpha. In Experiment 1, the duration of estrous cycles of ewes previously treated with PGF(2)alpha were longer (P<0.06) than those that received PGF(2)alpha plus GnRH, GnRH alone, or remained untreated (control) ewes. Progesterone concentrations were lower (P<0.07) on Day 1 and higher (P<0.01) on Days 16 and 17 of the estrous cycles following PGF(2)alpha treatment relative to those of the natural (control) cycles. In Experiment 2, blastocysts of ewes treated with PGF(2)alpha were less developed (P<0.06) by Day 13 of pregnancy than those of the control ewes. The GnRH treatment did not influence any of these characteristics. Treatment with PGF(2)alpha delayed luteal formation during the subsequent estrous cycle, increased the duration of the estrous cycle and slowed the rate of blastocyst development relative to GnRH-treated and untreated ewes.     

Receptors for prostaglandins F2 alpha and E2 in ovine corpora lutea during maternal recognition of pregnancy.

Plasma membrane receptors for prostaglandins (PG) F2 alpha and E2 were quantified in ovine corpora lutea obtained from nonpregnant and pregnant ewes on Days 10, 13, and 15 post-estrus, and from additional ewes on Days 25 and 40 of pregnancy. Regardless of reproductive status or day post-estrus, concentrations of luteal receptors for PGF2 alpha were 7- to 10-fold greater than those for PGE2. In pregnant ewes the concentration of receptors for PGF2 alpha was highest on Day 10 (35.4 +/- 2.8 fmol/mg) and lowest on Day 25 (22.3 +/- 2.5 fmol/mg). A difference in the concentration of luteal receptors for PGF2 alpha between pregnant and nonpregnant ewes was apparent only on Day 15 post-estrus, at which time the concentration of receptors for PGF2 alpha was higher in pregnant ewes than in nonpregnant ewes (27.1 +/- 2.7 vs. 17.7 +/- 2.7 fmol/mg). Concentrations of receptors for PGE2 in pregnant ewes were similar (p > 0.05; 2.8 +/- 0.3 to 3.7 +/- 0.2 fmol/mg) between Days 13 and 40 but were higher (p < 0.05) than in corpora lutea obtained from nonpregnant ewes on Days 10 (5.0 +/- 0.4 vs. 4.1 +/- 0.2 fmol/mg) and 15 (3.7 +/- 0.2 vs. 2.0 +/- 0.4 fmol/mg) post-estrus. Although concentrations of receptors for both PGF2 alpha and PGE2 were lowest in corpora lutea obtained from nonpregnant ewes on Day 15, this was not due to luteal regression since the weights and concentrations of progesterone in corpora lutea on Day 15 were not lower than those for corpora lutea obtained on Days 10 and 13.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of recombinant alpha interferons on fertility and interestrous interval in sheep

In Experiment 1, 12 unmated cyclic ewes received twice-daily intrauterine injections on Days 12 to 14 of one of the following treatments: 1) ovine conceptus secretory proteins (oCSP) containing 25 mug of ovine trophoblast protein-1 (oTP-1) as determined by RIA; 2) 25 or 50 mug recombinant human interferon alpha1 (rhlFN); or 3) 1500 ug of serum proteins (oSP) from a Day-16 pregnant ewe (estrus = Day 0) per uterine horn. Ewes receiving oCSP had longer interestrous intervals (27 +/- 2 days; P<0.05) than ewes receiving oSP (17 +/- 2 days). Ewes receiving either dose of rhlFN had an interestrous interval of 16 +/- 2 days which did not differ (P>0.10) from that of oSP-treated ewes. In Experiment 2, 59 normally cycling ewes, mated on Day 0, received twice-daily intramuscular injections of either 2 mg recombinant bovine interferon alpha1 (rblFN) or placebo on Days 12 to 15 post estrus. On Day 16, pregnancy was confirmed by flushing a morphologically normal conceptus from the uterus. Pregnancy rates for rblFN-treated (80%) and placebo-treated (62%) ewes were not different (P>0.10). Uterine flushings and conceptus-conditioned medium were assayed for oTP-1. Total oTP-1 in conceptus-conditioned culture medium was higher (P<0.02) when conceptuses were from placebo-treated (104 +/- 14 mug/conceptus) than from rblFN-treated (56 +/- 12 mug/conceptus) ewes; while total oTP-1 in uterine flushings was similar (P>0.10) for placebo-treated (132 +/- 15 mug/conceptus) and rblFN-treated (147 +/- 17 mug/conceptus) ewes. The interval from mating to subsequent estrus following conceptus removal was 31 +/- 1 and 28 +/- 1 days for pregnant ewes treated with rblFN and placebo, respectively. Interestrous intervals for nonpregnant ewes were longer (P<0.02) for rblFN-treated (27 +/- 3 days) than for placebo-treated (18 +/- 2 days) ewes.     

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)     

Prostaglandin F2 alpha, oxytocin and progesterone secretion by bovine luteal cells at several stages of luteal development: effects of oxytocin, luteinizing hormone, prostaglandin F2 alpha and estradiol-17 beta

Bovine luteal cells from Days 4, 8, 14 and 18 of the estrous cycle were incubated for 2 h (1 x 10(5) cells/ml) in serum-free media with one or a combination of treatments [control (no hormone), prostaglandin F2 alpha (PGF), oxytocin (OT), estradiol-17 beta (E) or luteinizing hormone (LH)]. Luteal cell conditioned media were then assayed by RIA for progesterone (P), PGF, and OT. Basal secretion of PGF on Days 4, 8, 14 and 18 was 173.8 +/- 66.2, 111.1 +/- 37.8, 57.7 +/- 15.4 and 124.3 +/- 29.9 pg/ml, respectively. Basal release of OT and P was greater on Day 4 (P less than 0.01) than on Day 8, 14 and 18 (OT: 17.5 +/- 2.6 versus 5.6 +/- 0.7, 6.0 +/- 1.4 and 3.1 +/- 0.4 pg/ml; P: 138.9 +/- 19.5 versus 23.2 +/- 7.5, 35.4 +/- 6.5 and 43.6 +/- 8.1 ng/ml, respectively). Oxytocin increased (P less than 0.01) PGF release by luteal cells compared with control cultures irrespective of day of estrous cycle. Estradiol-17 beta stimulated (P less than 0.05) PGF secretion on Days 8, 14 and 18, and LH increased (P less than 0.01) PGF production only on Day 14. Prostaglandin F2 alpha, E and LH had no effect on OT release by luteal cells from any day. Luteinizing hormone alone or in combination with PGF, OT or E increased (P less than 0.01) P secretion by cells from Days 8, 14 and 18. However on Day 8, a combination of PGF + OT and PGF + E decreased (P less than 0.05) LH-stimulated P secretion. These data demonstrate that OT stimulates PGF secretion by bovine luteal cells in vitro. In addition, LH and E also stimulate PGF release but effects may vary with stage of estrous cycle.     

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)     

Prostaglandin F2alpha-induced estrus in ewes exhibiting estrous cycles of different duration

Effects of prostaglandin F(2alpha) (PGF(2alpha)), administered during the mid-luteal phase of the estrous cycle, were examined in ewes exhibiting estrous cycles classified as short (< or =16.5 days, short-cycle ewes, n = 10) or long (> or =18 days, long-cycle ewes, n = 9) based on the durations of two estrous cycles (cycles -2 and -1) before treatment. The ewes received (i.m.) 20mg of PGF(2alpha) on day 10 of the third estrous cycle (cycle 0) followed, 36 h later, by 25 microg of gonadotropin releasing hormone (GnRH) to time the events of ovulation. Duration of subsequent estrous cycles +1 and +2 were recorded, and then the ewes were treated with the same combination of PGF(2alpha) and GnRH beginning on day 10 of estrous cycle +3. Ovaries were recovered 6h after GnRH administration to assess development of pre-ovulatory follicles. The proportion of ewes that exhibited estrus after PGF(2alpha) and GnRH treatment on cycle 0 was not different (P > 0.05) between short- and long-cycle ewes. Onset of estrus occurred sooner (P < 0.05) after PGF(2alpha) injection in short-cycle ewes than in long-cycle ewes (1.9 +/- 0.1 days and 2.3 +/- 0.1 days, duration of cycle 0 was 11.9 and 12.3 days, respectively). Duration of estrous cycle +1 was 1.2 days longer (P < 0.01) than cycle -1 in short-cycle ewes. However, duration of estrous cycle +1 did not change (P > 0.05) after PGF(2alpha) and GnRH administration in ewes having long cycles. Pre-ovulatory follicles did not differ (P > 0.05) in numbers, diameter, layers of granulosa cells nor concentrations of progesterone and estradiol-17beta in follicular fluid between short- and long-cycle ewes after PGF(2alpha) and GnRH treatment. In conclusion, ewes having short or long estrous cycles responded differently to PGF(2alpha) and GnRH treatment with respect to the interval to onset of estrus and duration of the subsequent estrous cycle.     

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)     

Failure of human chorionic gonadotropin injections to sustain gonadotropin-releasing hormone-induced corpora lutea in postpartum beef cows

Anestrous postpartum (PP) Hereford cows (n =20) were used to determine the effects of repeated injections of human chorionic gonadotropin (hCG) on the progesterone (P4) secretion and functional lifespan of gonadotropin-releasing hormone (GnRH)-induced corpora lutea (CL). Suckling was reduced to once a day from Day 21 to Day 25 PP, and all cows received injections of 200 micrograms GnRH at 1500 h on Day 24 PP to induce ovulation. Treated cows (HCG, n = 10) received 200 IU hCG b.i.d. from 1900 h on Day 27 PP to 1900 h on Day 33 PP; control cows (CTRL, n=10) were not injected. Blood was collected on Days 21, 23, 25, and 27 to 33, 35, 37, and 39 PP. Serum P4 concentration was measured by radioimmunoassay and used to classify luteal lifespan and the associated estrous cycle as short (SHORT) or normal (NORM) in duration. Treatment with hCG resulted in more (p less than 0.01) cows with SHORT cycles (7 of 9 vs. 4 of 9). Serum P4 concentrations were similar (p greater than 0.20) between groups from 4 days before until 6 days after GnRH injection. Cows with NORM cycles (n = 7) had greater serum P4 concentrations (p less than 0.05) on Days 7 to 11 after GnRH than cows with SHORT cycles (n = 11). By Day 39 PP, all cows with SHORT cycles appeared to have undergone a second ovulation. Charcoal-stripped serum pools from before (PRE) and during hCG injection (INJ) were assayed for total luteinizing hormone-like bioactivity (LH-BA) using a dispersed mouse-Leydig cell bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction by human chorionic gonadotropin of the luteolytic effect of prostaglandin F2 alpha in ewes

The ability of human chorionic gonadotropin (HCG) to reduce the luteolytic effect of prostaglandin (PGF2 alpha) was demonstrated in cycling ewes. As expected, treatment with 10 mg of PGF2 alpha alone on Day 10 of the estrous cycle exerted a potent negative effect on the function and structure of corpus luteum (CL) as indicated by reduced plasma progesterone, CL progesterone, and CL weight. However, the identical PGF2 alpha treatment failed to significantly reduce either luteal function or luteal weight when administered to ewes that were also treated with HCG on Days 9 and 10 of the estrous cycle. Treatment with HCG alone had a positive effect on CL as indicated by increased plasma progesterone, CL progesterone, and CL weight. Treatment with HCG did not render the CL totally insensitive to the negative effects of PGF2 alpha because plasma progesterone was reduced when the dose of PGF2 alpha was doubled. Whether CL regressed or continued to function after treatment with both HCG and PGF2 alpha appeared to depend upon a balance between the positive and negative effects of the two hormones.     

Effect of xylazine on interovulatory interval and serum progesterone concentrations in the horse mare

The objective of this study was to determine the effect of the alpha(2)-adrenergic agonist, xylazine, on interovulatory interval and progesterone concentrations in the horse mare. Mares were assigned to one of four treatments: Group 1 (controls) received an intramuscular injection (i.m.) of 5 cc saline (n=6), Group 2 received 10 mg prostaglandin F(2alpha) (PGF(2alpha)) i.m. (n=5), Group 3 received 500 mg xylazine i.m. (n=6) and group 4 received an intravenous injection (i.v) of 350 mg xylazine (n=6). Treatment was administered on Day 10 of the estrous cycle (Day 0 = Day of detected ovulation). There was no difference in length of interovulatory interval between PGF(2alpha)-treated mares and control mares (mean +/- SEM; 18.8 +/- 1.0 versus 21.7 +/- 1.6 d). When compared with either xylazine-treated group, PGF(2alpha)-treated mares had a shorter interovulatory interval (18.3 +/- 1.0 d versus 22.2 +/- 0.6 and 22.8 +/- 1.3 d, respectively; P < 0.05). There was no difference in the length of interovulatory interval between control mares and either xylazine-treated group. At the time of treatment all mares had progesterone concentrations > 10 ng/ml, therefore the onset of luteolysis was defined as the day of the estrous cycle when progesterone concentrations decreased below 10 ng/ml. In PGF(2alpha)-treated mares, this event occurred earlier than in any other group (Day 11.2 +/- 0.2 of the estrous cycle versus 16.0 +/- 1.3 for control, Day 15.7 +/- 0.2 for Group 3 and Day 15.2 +/- 0.6 for Group 4; P < 0.002). It was concluded that a single treatment with xylazine, either by an intramuscular or intravenous route, had no significant effect on interovulatory interval or progesterone concentrations in horse mares.     

Prostacyclin, prostaglandin F2 alpha and progesterone production by bovine luteal cells during the estrous cycle

Corpora lutea (CL) were collected from Holstein heifers on Days 5, 10, 15 and 18 (5/day) of the estrous cycle. Dispersed luteal cell preparations were made and 10(6) viable luteal cells were incubated with bovine luteinizing hormone (LH) and different amounts of arachidonic acid in the presence and absence of the prostaglandin (PG) synthetase inhibitor indomethacin. The concentrations of progesterone, PGF2 alpha and 6-keto-PGF1 alpha, the stable inactive metabolite of prostacyclin (PGI2), were measured. Day 5 CL had the greatest initial content of 6-keto-PGF1 alpha (1.01 +/- 0.16 ng/10(6) cells), and synthesized more 6-keto-PGF1 alpha (2.55 +/- 0.43) than CL collected on Days 10 (0.57 +/- 0.11), 15 (0.08 +/- 0.05) and 18 (0.19 +/- 0.03) during a 2-h incubation period. Arachidonic acid stimulated the production of 6-keto-PGF1 alpha by Days 10, 15 and 18 luteal tissue. PGF2 alpha was produced at a greater rate on Day 5 (0.69 +/- 0.17 ng/10(6) cells) than on Days 10 (0.06 +/- 0.01), 15 (0.04 +/- 0.02) and 18 (0.08 +/- 0.01). Arachidonic acid stimulated and indomethacin inhibited the production of PGF2 alpha, in most cases. The initial content of 6-keto-PGF1 alpha was higher than that of PGF2 alpha on all days of the cycle and more 6-keto-PGF1 alpha was synthesized in response to arachidonic acid addition. The ratio of 6-keto-PGF1 alpha content to PGF2 alpha content was 4.39, 2.30, 1.25 and 1.13 on Days 5, 10, 15 and 18, respectively.(ABSTRACT TRUNCATED AT 250 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.     

Effects of TPA, A23187, and prostaglandin F2 alpha on progesterone synthesis by dispersed ovine luteal cells

The possible roles of protein kinase C, intracellular calcium, and oxygen environment in luteal progesterone (P4) production and their interaction with prostaglandin (PGF2 alpha) were investigated in dispersed ovine luteal cells. The following experiments were performed: 1) dose response to TPA and A23187, 2) interactions between the phorbol ester TPA and PGF2 alpha at 5% or 18% O2, 3) effect of TPA and PGF2 alpha on basal and luteinizing hormone (LH)-stimulated P4 secretion, 4) interaction of submaximal inhibitory concentrations of TPA with PGF2 alpha and the effect of indomethacin (IN) on the TPA response. Day 9 (Day 0 = first day of estrus) corpora lutea (CL) from ewes exhibiting estrous cycles of normal duration (15-17 days) were dispersed and 50,000-150,000 cells were cultured for 4 h in Dulbecco's Modified Eagle Medium. The proportion of luteal cells greater than 22 microns in diameter in these preparations averaged 17.8 +/- 2.1%. P4 in medium and cells was measured by radioimmunoassay. Both TPA and A23187 inhibited basal P4 accumulation in a dose-dependent manner. Maximum inhibition (500 nM) by TPA was greater than by A23187 at the same concentration (66.4 +/- 3.4 and 83.2 +/- 7.2% of controls, respectively; p less than 0.05), and the two were not additive in their effects. Reducing O2 did not affect P4 accumulation with or without TPA, PGF2 alpha, or both. Basal P4 accumulation was inhibited 30% by TPA and 10% by PGF2 alpha, but no additivity was seen.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Modification of prostaglandin F-2 alpha synthesis and release in the ewe during the initial establishment of pregnancy

Pregnant (N = 10) and non-pregnant (N = 10) ewes were bled every 2 h from Days 12 to 17 after oestrus (oestrus = Day 0). Plasma concentrations of progesterone, 15-keto-13,14-dihydro-PGF-2 alpha and 11-ketotetranor-PGF metabolites were determined in all samples. The number of PGF-2 alpha pulses in non-pregnant ewes was 8.2 +/- 0.4 (mean +/- s.e.m.) with an interpulse interval of 10.7 +/- 0.7 h. Two or 3 pulses of low frequency (interpulse interval = 13.4 +/- 1.6 h) occurred in most non-pregnant ewes before the onset of luteolysis; the interpulse interval then decreased to 7.9 +/- 0.4 h for the 6.0 +/- 0.3 pulses temporally associated with luteolysis. In contrast, the number of PGF-2 alpha pulses in pregnant ewes was lower (2.5 +/- 0.7, 0-8) and the interpulse intervals longer (18.9 +/- 6.1 h). Most pulses occurred on Days 14 and 15 in the pregnant and non-pregnant ewes. The mean concentrations of both PGF-2 alpha metabolites in non-pregnant ewes were highest on Day 15 while basal levels of both metabolites remained constant at all times. In pregnant ewes, the mean concentrations of both metabolites were highest on Day 14; basal concentrations of both metabolites were also highest on Day 14. The mean concentrations of 15-keto-13,14-dihydro-PGF-2 alpha were higher in pregnant than in non-pregnant ewes on Days 13 and 14 (P less than 0.05) and higher in non-pregnant than pregnant ewes on Day 15 (P less than 0.05). The basal concentrations of the 15-keto metabolite were higher in pregnant than non-pregnant ewes at Days 13, 14, 15, 16 and 17 (P less than 0.05). Both the mean and the basal concentrations of 11-ketotetranor-PGF metabolites were higher in pregnant than in non-pregnant ewes on Day 14 (P less than 0.05). It is concluded that uterine production of PGF-2 alpha peaks at Days 14-15 after oestrus in pregnant and non-pregnant ewes. Patterns of release differ, however, in that non-pregnant ewes have a pulsatile PGF-2 alpha pattern superimposed on a constant baseline, while pregnant ewes have an increasing basal secretory pattern which is more nearly continuous, i.e. not pulsatile in form. Modification of pulsatile PGF-2 alpha synthesis and release is therefore a key aspect of prolongation of luteal function at the beginning of pregnancy in the ewe.     

Changes in ovine conceptus and endometrial function following asynchronous embryo transfer or administration of progesterone

The secretory protein profile from conceptuses collected from naturally mated ewes on Days 10, 12, 14, and 16 was characterized by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. The presence of the anti-luteolysin ovine trophoblast protein-1 (oTP-1) in culture medium from Day 10 conceptuses was confirmed by fluorography, Western blotting, and radioimmunoassay (RIA). On each of the days studied, oTP-1 was the dominant secretory protein, and was secreted in increasing quantities as pregnancy progressed. In a second experiment, Day 6 embryos were transferred to either Day 6 (SR) or Day 4 (AR) recipients. Three mated ewes (P) received daily injections of 50 mg progesterone on Days 4-9. Controls consisted of 2 groups of pregnant ewes (D8 and D10). Conceptuses and ipsilateral endometrium were collected 4 days following transfer of conceptuses to SR and AR ewes, on Day 10 in P and D10 ewes, and on Day 8 in D8 ewes. Conceptus volume was estimated upon recovery from the uterus. Tissues were cultured with 35S-methionine, and the medium was analyzed for total and trichloroacetic acid-precipitable radiolabeled proteins. Levels of specific endometrial secretory proteins were determined after protein separation by 2D-PAGE and estimation of the radioactivity associated with discrete radiolabeled proteins on fluorographs. The concentration of oTP-1 in conceptus culture medium was estimated by RIA. Thirty endometrial proteins were investigated. All 30 proteins were present in endometrial cultures from SR, AR, D10, and P ewes, but 13 proteins were absent from D8 ewes. Levels of three proteins were higher in AR compared to D8 (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin metabolism in the ovine corpus luteum: catabolism of prostaglandin F(2alpha) (PGF(2alpha)) coincides with resistance of the corpus luteum to PGF(2alpha)

To examine possible mechanisms involved in resistance of the ovine corpus luteum to the luteolytic activity of prostaglandin (PG)F(2alpha), the enzymatic activity of 15-hydroxyprostaglandin dehydrogenase (PGDH) and the quantity of mRNA encoding PGDH and cyclooxygenase (COX-2) were determined in ovine corpora lutea on Days 4 and 13 of the estrous cycle and Day 13 of pregnancy. The corpus luteum is resistant to the action of PGF(2alpha) on Days 4 of the estrous cycle and 13 of pregnancy while on Day 13 of the estrous cycle the corpus luteum is sensitive to the actions PGF(2alpha). Enzymatic activity of PGDH, measured by rate of conversion of PGF(2alpha) to PGFM, was greater in corpora lutea on Day 4 of the estrous cycle (P < 0.05) and Day 13 of pregnancy (P < 0.05) than on Day 13 of the estrous cycle. Levels of mRNA encoding PGDH were also greater in corpora lutea on Day 4 of the estrous cycle (P < 0. 01) and Day 13 of pregnancy (P < 0.01) than on Day 13 of the estrous cycle. Thus, during the early estrous cycle and early pregnancy, the corpus luteum has a greater capacity to catabolize PGF, which may play a role in the resistance of the corpus luteum to the actions of this hormone. Levels of mRNA encoding COX-2 were undetectable in corpora lutea collected on Day 13 of the estrous cycle but were 11 +/- 4 and 44 +/- 28 amol/microgram poly(A)(+) RNA in corpora lutea collected on Day 4 of the estrous cycle and Day 13 of pregnancy, respectively. These data suggest that there is a greater capacity to synthesize PGF(2alpha), early in the estrous cycle and early in pregnancy than on Day 13 of the estrous cycle. In conclusion, enzymatic activity of PGDH may play an important role in the mechanism involved in luteal resistance to the luteolytic effects of PGF(2alpha).