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Summary. Day-6 rabbit blastocysts were recovered from superovulated donor animals, washed in ice-cold Krebs–Ringer–bicarbonate (KRB) buffer, pooled and randomly allocated to polypropylene incubation tubes, usually 10 blastocysts in 1 ml KRB. The blastocysts were ruptured with a dissecting needle and incubated at 37°C for periods of 1–3 h with 10 μCi [3H]arachidonic acid/tube. A control tube without blastocysts was run in each experiment. At the end of the incubation, the samples were acidified, extracted with ethyl acetate, dried down and resuspended in h.p.l.c. column solvent. The radioactivity from the control tube eluting from the h.p.l.c., using a solvent system for prostaglandins (PGs), was subtracted from each experimental run in the same experiment. The remaining radioactivity constituted 0·14% of the original [3H]arachidonic acid added to each incubation tube. This was considered to have been the result of conversion of the radiolabelled arachidonic acid to prostanoids. In the absence of 10 mm-EDTA no conversion occurred, whereas in its presence peaks of radioactivity co-eluting with [3H]PGF-2α and [3H]PGE-2 were seen. A third peak that eluted was either 15-keto metabolites of these PGs or PGD-2. These 3 peaks were always significantly above background, and usually did not differ from each other. No differences in amount of conversion could be related to incubation time. Addition of indomethacin (100 μg/ml) or radioinert arachidonic acid (10 μg/ml) inhibited production of [3H]PG, even in the presence of EDTA. Removal of calcium from the incubation medium was per se without effect. Addition of atropine (0·15 mm) or carbachol (0·15 mm) in the presence or absence of EDTA did not change the pattern of conversion of [3H]arachidonic acid to [3H]PGs. These experiments demonstrate that rabbit blastocysts have the capacity for de-novo synthesis of PGs from exogenous substrate, when utilization of endogenous substrate is inhibited. The extent of conversion observed may not be a true reflection of the capacity for conversion of endogenous substrate.
Keywords: rabbit; blastocysts; prostaglandins
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Summary. Blastocysts recovered from control or indomethacin-treated (10 mg/kg s.c. twice daily starting on Day 4·5 of pregnancy) donor rabbits were transferred to the uteri of Day-6 or 6·5 pseudopregnant recipients. The minimal time required to cause an increase in capillary permeability in the endometrium underlying control blastocysts was ∼9 h. Blastocysts derived from the indomethacin-treated donors were depleted of PGE and PGF (determined by RIA) and were unable to produce any increase in capillary permeability during the same time period, although after 46 h in vivo the diameters of the implantation swellings related to control or indomethacin-treated blastocysts were not different. This suggests that, in the untreated recipients, blastocysts depleted of PGs can become replenished and then release these PGs in a site-directed manner. Indomethacin thus causes a delay rather than a complete inhibition of implantation. Incubation of the indomethacin-treated blastocysts in vitro led to replenishment with PGs, but such replenished blastocysts failed to induce an increase in capillary permeability within the same time-frame as control blastocysts. Evidence is presented that indomethacin is probably not the cyclooxygenase inhibitor of choice, since it interferes with PG uptake and efflux. Such an action could explain the failure of the replenished blastocysts to induce a normal increase in capillary permeability.
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In an initial experiment, rabbits were injected i.v. with a platelet-activating factor (PAF) antagonist CV-3988 twice a day on days 5 and 6 of pregnancy. Some inhibition of implantation was observed. This effect could not be reproduced in subsequent experiments at the same or at larger or smaller doses. The non-metabolized analogue of PAF, N-carbamyl-PAF (C-PAF) had an inhibitory effect on implantation only when given at toxic concentrations. When CV-3988 and C-PAF were given together on days 5 and 6, there was no effect on implantation. None of the other PAF antagonists tested – BN52021, SRI63,441, WEB2086 or TCV-309 – at various doses could inhibit implantation when given on the same days of pregnancy. TCV-309, at 0.1 mg kg−1 i.v. given on days 2–4 of pregnancy, was also ineffective. These results provide no clear support for a role of PAF in implantation in rabbits.
