The effect of pregnancy on the release of prostaglandin F2 alpha (PGF2 alpha) in response to oxytocin (OT) has been examined. Fourteen cyclic heifers received one intravenous injection of 1 IU OT (n = 6) or 100 IU OT (n = 8) 17, 18, or 19 days (Day 17-19) after the onset of estrus (Day 0). Five of these animals also received 100 IU OT at Days 6 and 13 to determine the effect of OT at different times of the cycle. Frequent blood samples were taken for 60 min before and for 90 min after OT injection for the measurement of 15-keto-13,14-dihydro-PGF2 alpha (PGFM) by radioimmunoassay. The experiment was then repeated using the same animals at Day 17-19 of pregnancy (confirmed by the recovery of an embryo the day after OT injection). Following the injection of 1 IU OT, plasma PGFM reached its peak within 30 min with the increase significantly lower (P less than 0.05) in pregnant (1.13 +/- 0.10-fold) than in nonpregnant animals (2.07 +/- 0.27-fold). However, because only 3 of the 6 cyclic animals showed a response to 1 IU OT, the dose was increased to 100 IU in subsequent experiments. The animals that received 100 IU at Days 6 and 13 had no significant increase in PGFM concentrations (1.18 +/- 0.05-fold and 1.01 +/- 0.04-fold, respectively). At Day 17-19 the increase in plasma PGFM reached its peak 5-15 min after 100 IU OT and the increase was significantly greater in nonpregnant (3.23 +/- 0.17-fold) than in pregnant (1.21 +/- 0.02-fold; P = 0.003) heifers. Six of 11 animals injected at Day 17-19 of the cycle showed a decrease in progesterone (P4) the day after OT administration. These data show that the release of PGF2 alpha in response to OT is suppressed in pregnant animals in vivo, suggesting an antiluteolytic role for the embryo in luteostasis.

Granulosa cells from the first (F1), third (F3) and fifth and sixth (F5-6) preovulatory follicles and the small yellow follicles (SYFs; diameter 6-8 mm) were cultured for 21 h in the absence and presence of murine and human epidermal growth factors, fibroblast growth factor, transforming growth factors alpha and beta-I (TGF alpha, TGF beta), platelet-derived growth factor and insulin-like growth factor-I at concentrations of 0.1-100 ng/ml. Plasminogen activator (PA) activities in the cell (PAc) and in the medium (PAm) were measured by fibrinolysis and fibrin overlay methods. Basal PAc and PAm activities were highest in cell cultures from the less mature follicles (F5-6 and SYF) and decreased as the follicles matured (F3 > F1). PAc activity was greater than PAm activity, irrespective of the stage of follicular development. All growth factors examined at the 100 ng/ml level were effective in increasing PAc and PAm activities in cultures of granulosa cells from F1 follicles. However, only TGF alpha was able to increase PA activities at lower concentrations. The stimulation of the PA activities of granulosa cells from F3 follicles was inconsistent. None of the growth factors significantly increased PA activities in granulosa cells from F5-6 follicles and SYFs, as determined by fibrinolysis. The major PAc and PAm species (characterized by fibrin overlay) had a molecular mass of about 35 kDa, which is characteristic of the urokinase type. Both assay methods detected a stimulatory effect of the growth factors on PA activities in the granulosa cells from F1 follicles. However, an increase in PA activities in cells from F3 and F5-6 follicles and SYFs was indicated only after fibrin overlay analysis. Tritiated thymidine was incorporated into the DNA of granulosa cells at all stages of follicular development and was enhanced by all growth factors, although TGF alpha and TGF beta were the most effective and had a ranked order of activity: F3, F5-6 > F1, SYF. The present findings show that, of the growth factors examined, TGF alpha may be an effective regulator of PA activity in avian granulosa cells during follicular development, in addition to its observed mitogenic action.

This study examined the influence of transforming growth factor-alpha (TGF alpha), TGF beta, and LH on progesterone (P4) secretion and plasminogen activator (PA) activity in cultured avian granulosa cells from the first (F1), third (F3), and fifth and sixth (F5-6) preovulatory follicles during a 21-h incubation period. PA activity in the cell (PAc) and the medium (PAm) fractions was measured by fibrinolysis and fibrin overlay methods. P4 was determined by RIA. Basal PAc and PAm activities were highest in cell cultures from the less mature (F5-6) follicles and decreased as follicles matured to the F1 stage of development. PAc activity was greater than PAm activity regardless of the stage of follicular maturation. TGF alpha (0.1-10 ng/ml) increased PA activity in cultures of granulosa cells from F1, F3, and F5-6 follicles in a concentration-dependent manner. TGF alpha-induced PAc and PAm activities were observed by 6 and 15 h of incubation, respectively, and increased rapidly between 15-21 h. LH (100 ng/ml) attenuated TGF alpha-induced PA activity by 15 h in cultures of granulosa cells from F1 and F3, but not F5-6, follicles. Basal PA activities were unaffected by the gonadotropin. TGF beta (2-100 ng/ml) stimulated PAc activity in a dose-dependent manner only in cultures of granulosa cells from F5-6 follicles and significantly enhanced TGF alpha-induced PAc and PAm activities in cell cultures from F3 and F5-6, but not F1, follicles. Basal and growth factor-induced PAc and PAm activities corresponded to a mol wt of about 35 kDa, a value consistent with that of the low mol wt uPA species. TGF alpha and TGF beta, alone or in combination, had no effect on basal P4 secretion at all stages of follicular development. TGF alpha, however, decreased LH-induced P4 secretion in F1 and F3 cultures. These results demonstrate a tightly controlled interaction of TGF alpha, TGF beta, and LH in regulating PA activity and P4 secretion during follicular development in the domestic hen.

Summary. Seven bilaterally ovariectomized heifers were used in 4 experiments and received: (1) saline injections, as control; (2) one injection of oestradiol (3 mg; i.v.); (3) two i.v. injections of oxytocin (100 i.u.) 6 h apart; or (4) one oestradiol injection 30 min after the first oxytocin injection and a second oxytocin injection 6 h later. All experiments were performed without progesterone and then after 7, 14 and 21 days of progesterone treatment. Frequent blood samples were taken for 1 h before and 7 h after the first injection of oxytocin or oestradiol for the measurement of 13,14-dihydro-15-keto-PGF-2\g=a\ (PGFM) by radioimmunoassay. After 7, 14 and 21 days of progesterone priming, oestradiol caused a significant increase (P < 0\m=.\001) in plasma PGFM after 6 h but not before. After 7, 14 and 21 days of progesterone, there was a significant increase (P < 0\m=.\005) in PGFM after the first oxytocin injection and a similar increase following the second. The oxytocin-induced increase in PGFM after 14 and 21 days of progesterone was significantly higher (P < 0\m=.\001) 6h after oestradiol injection than before the oestradiol injection. There was no significant effect of oestradiol on the response to oxytocin in animals that received no progesterone or in those animals that received progesterone for only 7 days. These results show that, under the influence of progesterone, oestradiol enhances the oxytocin-induced release of PGF-2\g=a\, and suggest a possible synergistic action of these hormones for the induction of luteolysis in heifers.

Prostaglandin F2 alpha (PGF2 alpha) release from the uterus causes luteolysis in ruminants, and oxytocin is thought to be a regulator of this release. In the present study, we have examined the mechanisms involved in oxytocin stimulation of PGF2 alpha secretion by bovine endometrium in vitro. Endometrial tissue explants, obtained from heifers at Day 19 or 20 (n = 3) and Day 0 (estrus, n = 5) of the estrous cycle, were incubated for 2 h and 6 h, and PGF2 alpha concentration in the medium was determined by radioimmunoassay (RIA). Basal PGF2 alpha release increased for up to 6 h and was significantly stimulated after 2 h of incubation with 100 microU and 1000 microU of oxytocin at Day 0 but not at Day 19 or 20. Secretion of PGF2 alpha was not affected by cholera toxin (10 ng/ml) or the cyclic nucleotide analogs dibutyryl cyclic adenosine 3',5'-monophosphate and dibutyryl cyclic guanosine 3',5'-monophosphate at a concentration of 1 mM. A protein kinase A inhibitor (500 microM) had no effect on the oxytocin-induced release of PGF2 alpha. Both the phorbol ester, 12-myristate-13-acetate (100 mM), and the non-phorbol stimulator of protein kinase C, 1-octanoyl-2-acetylglycerol (500 microM), significantly stimulated PGF2 alpha secretion to the same extent as oxytocin. Neither basal nor stimulated PGF2 alpha release was affected by the calcium ionophore A23187 (0.1-5.0 microM). However, PGF2 alpha secretion was sensitive to cycloheximide (1 microgram/ml) suggesting that protein synthesis may be involved. In conclusion, these data suggest that the stimulation of PGF2 alpha by oxytocin is via the protein kinase C effector pathway.