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)     

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.     

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.     

Induction of the synthesis of the pregnancy-specific protein p70 in the endometrium by intramuscular injection of recombinant bovine interferon-alpha I1 in nonpregnant ewes.

We examined the effect of recombinant bovine interferon-alpha I1 (rboIFN-alpha I1) or recombinant bovine trophoblast protein-1 (rbTP-1) on protein synthesis by endometrial explants from Day-13 cyclic ewes and studied the ability of rboIFN-alpha I1 injected i.m. to influence subsequent protein secretion by endometrial tissue explants. In Expt 1, ewes were injected with either 2 mg rboIFN-alpha I1 or vehicle alone at 12 h intervals beginning on Day 11 of the oestrous cycle and ending on the morning of Day 13; 8 h after the last injection, ewes were hysterectomized and endometrial explant cultures were prepared. Explants were cultured for 24 h in leucine-deficient medium supplemented with 250 microCi L-[3H]leucine per culture. For Expt 2, additional explants were prepared from Expt 1 controls. Explants were cultured in the presence of 0, 20 or 200 ng/ml of either rboIFN-alpha I1 or rbTP-1 for 24 h in leucine-deficient medium supplemented with 250 microCi L-[3H]leucine per culture. Secreted proteins were analysed by two-dimensional electrophoresis and fluorography. There was a marked enhancement of a 70 kDa acidic protein, p70, in explants cultured in the presence of rboIFN-alpha I1 or rbTP-1. This polypeptide is a product of the gravid uterine horn from Day 14 to Day 20 of pregnancy and is a useful marker of the action of interferon-alpha (IFN-alpha) on endometrium. Enhanced production of p70 also occurred in ewes injected i.m. with rboIFN-alpha I1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Continuous infusion of oxytocin prevents induction of uterine oxytocin receptor and blocks luteal regression in cyclic ewes

Continuous intravenous infusion of oxytocin (3 micrograms/h) between Days 13 and 21 after oestrus delayed return to oestrus by 7 days (length of cycle 23.3 +/- 0.6 days compared to 16.6 +/- 0.2 days in control ewes). At a lower infusion rate (0.3 micrograms/h) oxytocin delayed luteolysis in only 2 of 5 ewes. Treatment from Day 14, when luteolysis had already begun, was ineffective. Delay of luteal regression by oxytocin had no effect on the length of subsequent cycles. Measurement of circulating progesterone concentrations and luteal weight showed that prolongation of the oestrous cycle was due to prevention of luteal regression. Luteal regression and behavioural oestrus were induced during continuous oxytocin administration begun on Day 13 when cloprostenol was given on Day 15 (mean cycle length, 17.3 +/- 0.21 days). Continuous oxytocin infusion from Day 13 blocked the rise in uterine oxytocin receptor concentrations which normally precedes oestrus. Mean receptor concentrations in caruncular and intercaruncular endometrium and in myometrium were 76, 36 and 9 fmol/mg protein on Day 17 in ewes receiving continuous oxytocin (3 micrograms/h); in control ewes these values were 675, 638 and 130 fmol/mg protein respectively at oestrus. Receptor concentrations on the day of oestrus in ewes receiving oxytocin and cloprostenol were not significantly different from those in control ewes (649, 852, and 109 fmol/mg protein respectively). Since cloprostenol, a PGF-2 alpha analogue, overcame the antiluteolytic action of oxytocin, it is suggested that continuous oxytocin treatment may inhibit uterine production of PGF-2 alpha, possibly by down regulating the uterine oxytocin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Oxytocin-stimulated inositol phosphate turnover in endometrium of ewes is influenced by stage of the estrous cycle, pregnancy, and intrauterine infusion of ovine conceptus secretory proteins

Three experiments (Exp) assessed the influence of stage of the estrous cycle, pregnancy, and intrauterine infusion of ovine conceptus secretory proteins (oCSP) on turnover of inositol trisphosphate (the putative second-messenger for oxytocin-stimulated secretion of prostaglandin F2 alpha) in ovine endometrium during luteolysis and maternal recognition of pregnancy. In Exp 1, endometrium was collected from 5 cyclic (Cy) and 6 pregnant (P) ewes on Day 16 after onset of estrus. In Exp 2, endometrium was collected from Day 12 Cy (n = 5), Day 12 P (n = 3), Day 16 Cy (n = 4), and Day 16 P (n = 3) ewes. In Exp 3, 12 Cy ewes were allotted randomly, in a 2 x 2 factorial arrangement, to receive serum protein (SP), or oCSP and estradiol-17 beta (E2), or vehicle treatments. Ewes were injected i.v. with 0.5 mg E2 or vehicle on Day 12 and received twice-daily infusions of 1.5 mg SP or oCSP (containing 25 micrograms ovine trophoblast protein-1 by radioimmunoassay [RIA]) + SP (1.5 mg total protein) into each uterine horn on Days 12, 13, and 14. Blood samples for RIA of plasma progesterone were collected on Days 10-15 (before treatment on each day) and endometrium was collected on Day 15. For each Exp, 100 mg endometrium was incubated, in duplicate, for 2 h with 10 microCi [3H] inositol and treated with 0 or 100 nM oxytocin (OT) for 20 min, then [3H]inositol mono-, bis-, and trisphosphates (IP1, IP2, and IP3, respectively) were quantified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between variation in conceptus development and differences in estrous cycle duration in ewes

The objective of this study was to examine conceptus development on Day 13 in ewes with estrous cycles of different durations. Ewes (n = 80) were screened according to the length of their estrous cycles. Subsequently, ewes that had either SHORT or LONG cycles were utilized (15.9 +/- 0.1 or 18.6 +/- 0.4 days; mean +/- SEM, p less than 0.01; 10 ewes per group). Jugular blood samples were collected twice daily from Days 0-6 after mating and then once a day until slaughter on Day 13. Concentrations of progesterone in plasma and amounts of ovine trophoblast protein-1 (oTP-1), protein, and prostaglandins (PG) E2 and F2 alpha (PGF2 alpha) in uterine flushings were determined. Concentrations of progesterone were greater (Day by treatment interaction, p less than 0.01) on Days 2-4 for ewes in the SHORT group. On Day 5 and thereafter, progesterone concentrations were not different between groups. More (p less than 0.05) oTP-1 and protein (8.1 +/- 1.3 micrograms and 1.8 +/- 0.3 micrograms versus 2.4 +/- 1.3 micrograms and 0.8 +/- 0.3 mg) were recovered from uterine flushings from ewes in the SHORT versus LONG groups, respectively. The ratio of PGE2:PGF2 alpha was higher (p less than 0.06) in flushings from ewes in the SHORT versus LONG group (1.4 +/- 0.2 versus 0.9 +/- 0.2, respectively). Conceptuses were classified by stage of morphological development. Conceptus development was accelerated (p less than 0.01) in ewes of the SHORT group, as shown by filamentous conceptuses recovered from 78% versus 0% of SHORT versus LONG ewes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovine trophoblast protein-1 and bovine trophoblast protein-1 are present as specific components of uterine flushings of pregnant ewes and cows

A rabbit antiserum raised against ovine trophoblast protein-1 (oTP-1) was used to stain Western blots of the protein components from the uterine flushings of pregnant ewes (n = 61), non-bred cyclic ewes (n = 22), bred-but-nonpregnant ewes (n = 36), pregnant cows (n = 34), and bred-but-nonpregnant cows (n = 15). Nonpregnant animals were defined as ones from which no embryo was recovered. Uterine flushings of pregnant ewes contained oTP-1 between Days 14 and 24 of pregnancy, but not at Day 12. All of the cyclic ewes and 34 of 36 bred ewes, judged as nonpregnant, tested negatively for the presence of oTP-1. With one exception, oTP-1 was not detected in the nongravid uterine horns of pregnant ewes in which the conceptus had been confined to one uterine horn. Bovine trophoblast protein-1 (bTP-1), which cross-reacts immunologically with oTP-1, was also detectable specifically in the uterine flushings of pregnant cows when anti-oTP-1 antiserum was used. The urine (n = 14) and certival mucus (n = 20) samples of all the pregnant ewes tested were free of any detectable oTP-1. Thus, a useful pregnancy test for ewes based on oTP-1 release into these fluids seems unlikely. Results of this study show that oTP-1 and bTP-1 are pregnancy-specific proteins that are secreted into the uterine lumen where they possibly exert a local response.     

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)     

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)     

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 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.     

Administration of recombinant bovine interferon-alpha I at the time of maternal recognition of pregnancy inhibits prostaglandin F2 alpha secretion and causes luteal maintenance in cyclic ewes.

The antiluteolytic protein, ovine trophoblast protein-1, which is secreted by sheep embryos at about the time of the maternal recognition of pregnancy, exhibits significant structural homology with alpha interferons. Experiments were conducted to examine the effects of intra-uterine and systemic administration of a recombinant bovine interferon-alpha I (rboIFN-alpha I) upon the interoestrus interval, endometrial oxytocin receptor concentrations and secretion of prostaglandin (PG) F2 alpha in cyclic ewes. In Expt 1, each ewe had a cannula placed in the tip of a uterine horn ipsilateral to a corpus luteum, 7 days after an induced oestrus. From day 9 after oestrus until day 19, ewes received either 200 (n = 4), 667 (n = 5) or 2000 (n = 9) micrograms/24 h of rboIFN-alpha I, meclofenamic acid (n = 4) or vehicle (n = 11). Other ewes received 2000 micrograms rboIFN-alpha I/24 h (n = 5) between days 12 and 15 only. All ewes were killed on day 19. Mean luteal phase, as determined by daily plasma progesterone measurements, was significantly longer (P less than 0.01) and mean concentrations of 13,14-dihydro-15-keto PGF 2 alpha (PGFM) in plasma were lower (P less than 0.05) in ewes receiving 667 or 2000 micrograms rboIFN-alpha I between days 9 and 19, or 2000 micrograms between days 12 and 15, than in animals from other treatment or control groups. A similar protocol was used in Expt 2, in which further ewes received either 2000 micrograms rboIFN-alpha I/24 h (n = 5) or vehicle (n = 5) by bolus infusions twice a day into one uterine horn. Mean luteal phase was significantly (P less than 0.05) longer in treated than in control animals, but differences in PGFM concentrations were not significant. In Expt 3, after a synchronized oestrus, ewes received either 2.5 mg rboIFN-alpha I by i.m. injection twice a day between days 12 and 15 (n = 10), 2.5 mg rboIFN-alpha I by i.m. injection twice a day between days 9 and 15 (n = 11), i.m. injection of vehicle alone twice a day (n = 20), or continual intra-uterine infusion of 2 mg rboIFN-alpha I/day between days 12 and 15 (n = 7). The mean luteal phase of ewes receiving rboIFN-alpha I by intrauterine infusion or i.m. injection between days 9 and 15 was significantly longer than for animals from the other two groups (P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)     

Correlation between the release of ovine trophoblast protein-1 by the conceptus and the production of polypeptides by the maternal endometrium of ewes

We studied the biosynthesis of two proteins, p70 (Mr 70,000; pI 4.0) and p15 (Mr 15,000; pI 5.7), by endometrial tissues from ewes between Days 12 and 24 of pregnancy and between Days 12 and 16 of the oestrous cycle to determine whether production of the two was correlated with the period of biosynthesis of ovine trophoblast protein-1 (oTP-1) by the conceptus. We also compared the protein synthetic activities of endometrium from gravid and non-gravid horns of pregnant ewes at Days 14, 16 and 18 in which the conceptus had been confined to one uterine horn. Proteins p70 and p15 were produced maximally between Days 14 and 20 of pregnancy, but synthesis by endometrial cultures from cyclic ewes was low or absent. Furthermore, synthesis of Protein p70 in particular was much greater by the gravid than non-gravid horn of unilaterally pregnant ewes. We conclude that synthesis of Proteins p70 and p15 by the uterus of sheep coincides with the time of oTP-1 production by the conceptus.     

Proteins secreted by the sheep conceptus suppress induction of uterine prostaglandin F-2 alpha release by oestradiol and oxytocin

In Exp. I, 0.5 mg oestradiol or vehicle (0.5 ml absolute ethanol + 0.5 ml 0.9% NaCl) was injected i.v. at 08:00 h on Day 14 (onset of oestrus = Day 0). Blood samples were obtained via a jugular catheter at 30 and 1 min before oestradiol and every 30 min for 10 h afterwards. Plasma was obtained and assayed for 15-keto-13,14-dihydro-PGF-2 alpha (PGFM) by radioimmunoassay. Before oestradiol, PGFM basal values were higher (P less than 0.01) in pregnant (N = 10) than nonpregnant (N = 6) ewes (193 +/- 30 vs 67 +/- 8 pg/ml). However, at 4-10 h after oestradiol, pregnant ewes (N = 5) had less variable (P less than 0.01) PGFM values than did nonpregnant ewes (N = 5). In Exp II, conceptus secretory proteins (CSP) were obtained by pooling medium from cultures of Day-16 sheep conceptuses (N = 40). Ewes received 750 micrograms CSP + 750 micrograms plasma protein (N = 6) or 1500 micrograms plasma protein (N = 6) per uterine horn at 08:00 h and 18:00 h on Days 12-14. All ewes received 0.5 mg oestradiol at 08:00 h on Day 14 and blood samples were collected as in Exp. I and assayed for PGFM. On Day 15, 3 ewes in each group received 10 i.u. oxytocin and 3 received saline i.v. at 08:00 h and blood samples were taken continuously from 10 min before to 60 min after treatment. Mean PGFM response to oestradiol was suppressed (P = 0.05) in CSP- vs plasma protein-treated ewes (371 +/- 129 vs 1188 +/- 139 pg/ml).(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.     

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)     

Effect of systemic or intrauterine injections of indomethacin on plasma oxytocin-neurophysin concentrations in ewes over the time of normal corpus luteum regression

This study was undertaken to investigate the effect of systemic or intrauterine injections of indomethacin, a known prostaglandin (PG) synthetase inhibitor, on peripheral plasma oxytocin-associated neurophysin (OT-N) concentrations in ewes over the time of expected luteolysis. In the first experiment, 9 ewes were given i.m. injections of indomethacin (4 mg/kg live weight, n = 4) or vehicle (n = 5) 3 times/day over Days 13-15 of the estrous cycle. Blood samples were collected at hourly intervals from 0700 h on Day 13 to 1800 h on Day 15 post-estrus. In the second experiment, indomethacin (20 mg, n = 5) or the injection vehicle (n = 4) was given twice daily into the uterine horn over Days 12-14 post-estrus. Blood samples were collected at hourly intervals from Day 12 to 14. In the third experiment, 4 additional ewes were bled at 5-min intervals from 1200 to 1600 h on Day 13 of the estrous cycle. Plasma samples were analyzed for OT-N and 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) to provide an indirect index for ovarian oxytocin and uterine prostaglandin F2 alpha release, respectively. Results from the first experiment indicated that surges in plasma OT-N concentrations occurred in the vehicle-treated ewes but were suppressed in ewes given systemic injections of indomethacin. Intrauterine indomethacin injections did not cause a significant reduction in the maximum peak height or number of peaks when compared with the control ewes. In the third experiment, there was a marked increase in plasma OT-N concentrations, but no significant rise in plasma PGFM concentrations in one ewe.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intrauterine injection of recombinant ovine interferon-tau extends the interestrous interval in sheep

The ability of recombinant ovine interferon-tau (roIFNtau) to extend the interestrous interval (IEI) in sheep was studied. Ewes were fitted with bilateral uterine catheters 7 or 8 days post estrus and were assigned to receive either 10 or 20 million antiviral (AV) units/day i.u. ( approximately 100 or 200 ug) of roIFNtau or ovine conceptus secretory proteins containing equivalent AV units of native oIFNtau (noIFNtau; 4 ewes/treatment). Four control ewes received ovine serum proteins (SP). Total protein injected was 6 mg per day, half at 0700 hours and half at 1730 hours. The treatments were administered from Day 11.5 (estrus=Day 0) to Day 16. Blood samples were collected by jugular vienipuncture daily from Day 11 until ewes returned to estrus. Concentrations of progesterone (P) in plasma were determined by RIA. Treatment with either noIFNtau or roIFNtau extended IEI beyond that of SP-treated ewes (19.1 vs 31.2+/-3.4 days P<0.03). Of the ewes receiving 100 mug/day of oIFNtau, 2 of 4 receiving noIFNtau (23.6+/-5.2 days) and 3 of 4 receiving roIFNtau (34.2+/-5.2 days) had an extended IEI. All ewes receiving 200 mug/day of noIFNtau or roIFNtau had an extended IEI (28.8 and 38.5+/-5.2 days. respectively). Ewes receiving roIFNtau had a longer IEI than those receiving noIFNtau (36.7 vs 26.2+/-3.4 days; P=0.07). Ewes with an extended IEI had functional corpora lutea, as assessed by P production. The results demonstrate that 10 or 20 million AV units ( approximately 100 or 200 ug) of roIFNtau extends the IEI and that the length of the IEI is longer for ewes receiving roIFNtau than noIFNtau following injection of equivalent AV units.