Embryos were collected from superovulated ewes on Day 2 (2-8 cell), Day 4 (8-16 cell) and Day 6 (morula/early blastocyst). Two embryos were cultured in 1 ml of one of four media: (i) Ham's F10 + 4 mg bovine serum albumin (BSA)/ml, (ii) synthetic oviduct fluid medium + 20% human serum, (iii) Quinn's human tubal fluid medium (HTF) + 3 mg BSA/ml or (iv) HTF + 10% acid-treated fetal calf serum for 24 h. They were transferred to fresh media of the same type and their further development was monitored. A quantitative bioassay and radioimmunoassay was used to measure the concentration of platelet-activating factor (PAF, 1-o-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine) produced. Following extraction and partial purification, 21/95 (22.1%) of the embryo-conditioned media samples had PAF concentrations greater than that measured in corresponding control media. This was designated as embryo-derived PAF and the corresponding cultures were termed 'PAF-positive'. PAF was produced by embryos at all three developmental stages examined and in each of the four media used, and the average amount of PAF produced was 60.9 +/- 9.8 pmol/embryo/24 h. However, neither the developmental stage of the embryo, nor the type of media affected the proportion of PAF-positive cultures nor the amount of PAF produced during culture. Thus, it is demonstrated for the first time that early ovine embryos can secrete PAF in vitro, and that there is considerable variability in their capacity for PAF secretion.

This study was conducted to determine whether platelet-activating factor (PAF) (1) attenuated oxytocin-induced secretion of the prostaglandin (PG) F2 alpha metabolite, PGFM, by the ovine uterus in situ and (2) inhibited the generation of the inositol phosphate secondary messengers by endometrial tissue in response to oxytocin challenge in vitro. Ovariectomized ewes received steroid replacement to mimic the luteal phase. Six ewes received intrauterine injections of 200 micrograms PAF/uterine horn/day on Days 11-15, and 6 ewes were treated with vehicle. All ewes received 1 microgram oxytocin i.v. on Days 13-16. Pretreatment of ewes with PAF significantly suppressed PGFM release in response to oxytocin on Days 14 and 15 (p less than 0.005) compared to vehicle-treated ewes. PAF was not administered on Day 16, and the PGFM response to oxytocin was not different between groups. In a second experiment, ewes were given intrauterine injections of 200 micrograms PAF/uterine horn/day (n = 8) or vehicle (n = 7) on Days 11-15, and all ewes received 1 microgram oxytocin i.v. on Days 13 and 14. On Day 15 the uterus was removed, and the incorporation of 3H-inositol into inositol phosphates was determined in caruncular endometrium. Treatment of ewes with PAF in vivo reduced inositol monophosphate (IP1) generated by oxytocin (10(-6) M) by 56.4%, compared to that in endometrium from vehicle-treated controls, and also inhibited the incorporation of 3H-inositol into glycerophosphoinositol (GPI). If PAF was added to the endometrium during the incubation in vitro, the attenuation of inositol phosphate generation did not occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Explanted endometrial tissue from ovariectomized ewes that had received hormone replacement to mimic the luteal phase released platelet-activating factor (PAF) into medium in vitro. On Day 14, 310.1 (261-2185), and on Day 15, 424.4 (14.1-590.7) pg PAF (median 25th-75th percentile; p > 0.05) was released per 100 mg endometrial tissue after a 20-min incubation. A regulatory and final enzyme in the PAF biosynthetic pathway, lysoPAF:acetyltransferase, was also present with a specific activity of 0.533 +/- 0.124 pmol acetate/50 micrograms protein/30 min in Day 14 endometrial tissue and 0.810 +/- 0.468 in Day 15 tissue (mean +/- SEM; p > 0.05). PAF:acetylhydrolase, the metabolic enzyme regulating PAF's half-life, was present in uterine luminal washings, with a specific activity of 3.78 +/- 1.37 nmol acetate released/min/mg protein on Day 14 and 3.41 +/- 0.34 on Day 15 (mean +/- SEM; p > 0.05). Intrauterine infusion of 50-400 micrograms PAF caused a dose-dependent release of prostaglandin (PG) F2 alpha (measured as venous 14-dihydro-15-keto-prostaglandin F2 alpha, PGFM) within 10 min on Days 13-15. The enantiomeric form of PAF was significantly less effective in inducing a rise in venous PGFM. WEB 2086 is reported to be a competitive receptor antagonist for PAF, but a 10-fold excess of WEB 2086 failed to inhibit PAF-induced release of PGFM. It was observed that this dose of WEB alone induced PGFM release, suggesting that in this model this agent may not work as a true competitive antagonist. Repeat challenges at intervals of 0, 90, and 120 min with either 200 micrograms PAF or micrograms oxytocin resulted in a marked tachyphylaxis of response by the third challenge. This desensitization after repeated PAF challenge suggests a specificity of its actions. Infusion of 200 micrograms PAF on Day 14 or 15 at five 60-min intervals resulted in a tachyphylaxis such that by the fourth and fifth challenges, essentially no response was observed. The tachyphylaxis that was induced by four repeat challenges with PAF had no apparent effect on the capacity of the uterus to respond to a fifth challenge with oxytocin. Thus, PAF and oxytocin both caused homologous desensitization of their own capacity to mobilize uterine PGF2 alpha, but PAF did not cause heterologous desensitization of the response to oxytocin. This failure of heterologous desensitization suggests differences in the mechanism of action of the two ligands. Kinetic binding studies showed that PAF did not compete for the oxytocin receptor in vitro. This result demonstrated that the ovine endometrium produces PAF and responds to it by the release of PGF2 alpha in situ. The dynamics of the response elicited by PAF were similar to those of oxytocin, yet the two mediators apparently act separately. PAF may be a modulator of PGF2 alpha release by the ovine uterus during the luteal phase.

Summary. The greater in goats treated with GnRH (12\m=.\7 \m=+-\ 1\m=.\3) than in those that received PMSG only (9\m=.\7\ m=+-\1\m=.\1; P < 0\m=.\05). Out of 47 of the females in Exp. 1, 43 had one or more corpora lutea at laparoscopy 24 h after withdrawal of progestagen. These early corpora lutea were associated with an increased concentration of plasma progesterone during the periovulatory period. Experiment 3 provided evidence that these corpora lutea arose before the withdrawal of progestagen-impregnated sponges.

Feral does of various ages were treated with intravaginal progestagen sponges for 16 days to synchronize oestrus. On Day 2 before sponge removal the goats were given 1200 i.u. PMSG to induce superovulation: 6 of the goats were also injected every 12 h with flunixin meglumine, a prostaglandin (PG) synthetase inhibitor, from Day 3 to 7 of the synchronized oestrous cycle. Jugular blood samples were collected from all females into heparinized syringes at daily intervals over the 2 days before sponge removal, twice daily for the next 2 days, then at hourly intervals from 09:00 to 17:00 h for 2 days and then twice daily for a further 2 days, for measurement of plasma progesterone and the PGF metabolite 13,14-dihydro-15-keto-PGF (PGFM) by radioimmunoassay. Intermittent surges in plasma PGFM concentrations were observed in hourly samples collected from 4/4 untreated females but in only 2/6 of the inhibitor-treated females (P less than 0.05), and the peak plasma PGFM concentrations were reduced in these 2 inhibitor-treated goats compared with the control goats. The corpora lutea (CL) of the inhibitor-treated females appeared to be functional as indicated by the plasma progesterone profile and endoscopic examination of CL. In the control females, however, there was evidence of premature regression of CL. These results suggest that the premature release of PGF-2 alpha may be the cause of premature regression of CL in nanny goats induced to superovulate.