Search Results

You are looking at 1 - 10 of 24 items for

  • Author: C. O'Neill x
Clear All Modify Search
Free access

C. O'Neill

Summary. The platelet-activating factor (PAF) produced by mouse embryos showed similar kinetics of action and dose—response curve, in a bioassay, as did 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine (PAF-acether). The activity of the embryo-derived PAF was not affected by inhibitors of the ADP (pyruvate kinase with phosphoenol pyruvate) or cyclo-oxygenase (indomethacin) pathways of platelet activation. Chlorpromazine, an inhibitor of the PAF-acether pathway of platelet activation, caused a significant inhibition of the effects of embryo-derived PAF. Phospholipases A2, C and D significantly inhibited the activity while lipase had no effect, suggesting a phospholipid structure. All the embryo-derived PAF was found in the chloroform fraction after chloroform:methanol (2:1 v/v) extraction, as was PAF-acether. Both factors migrated at a similar rate (R f0·10–0·12) on silica thin-layer chromatography (chloroform:methanol:water; 65:35:4 by vol.). The embryo-derived PAF therefore displays chemical, biochemical and physiological properties similar to those of PAF-acether.

Free access

C. O'Neill

Summary. There was an increase in weight of the spleens of pregnant and pseudopregnant mice in the first week after mating, but the increase occurred on Day 4 in pregnant mice and Day 2 in pseudopregnant mice. The retardation of the presumed hormonally induced increase in spleen weight during pregnancy corresponded with a significant reduction in the splenic platelet pool. This response by the spleen to early pregnancy suggested that platelets were being supplied to the vascular pool. There was a significant reduction in the platelet count by 10:30 h on the day of mating in pregnant mice and persisted until Day 7 of pregnancy, then returning to normal levels. This response did not occur in pseudopregnant mice. The decrease in platelet count was dependent upon the presence of fertilized eggs. It did not occur in mice sterilized by bilateral ligations of the oviducts and mated with fertile males. Thrombocytopenia did occur within 3 h of transfer of fertilized eggs to pseudopregnant recipients and the magnitude of the response was significantly correlated (b = − 0·86) with the number of embryos present in the reproductive tract. An initial systemic response to pregnancy in mice was therefore an increased vascular demand for blood platelets, resulting in a significant reduction in the splenic and peripheral blood platelet concentration.

Free access

C. O'Neill

Summary. In mice, neither the bleeding time nor the clotting time of whole blood was different on Day 2 of pregnancy compared with pseudopregnancy. Standardization of the platelet concentration to 106/μl plasma resulted in a significant reduction in the clotting time of plasma from pregnant animals. This reduction was not due to an increase in the intrinsic or extrinsic pathways of the coagulation cascade but to enhanced platelet factor III activity, indicating increased platelet activation and consumption. Increased activation was not due to immunological recognition of the embryo because thrombocytopenia occurred after syngeneic and allogeneic matings of inbred strains of mice and also after parthenogenetic activation of ova in situ. Injection of embryo culture medium into splenectomized mice induced a significant dose-dependent thrombocytopenia. It occurred within 10 min after injection and persisted for up to 2 h. There was no reduction in platelet count when animals were injected with culture media in which unfertilized ova had been incubated. Early pregnancy-associated thrombocytopenia was caused by the production of platelet-activating factors by the fertilized eggs. The induction of thrombocytopenia by embryo culture media displayed a dose—response curve that was parallel to that of the platelet-activating factor, 1-0-alkyl-2-acetyl-sn-glycero(3)phosphocholine.

Free access

C. O'Neill and P. Quinn

Summary. Uterine flushings from artificially 'pseudopregnant', pseudopregnant and pregnant mice and those with 'diapausing' embryos were tested for their effect on [3H]uridine incorporation by mouse blastocysts. An inhibitor of [3H]uridine incorporation was detected in the uterine fluid of the mice with diapausing embryos and the activity of the inhibitor was significantly reduced 6·25 h after an injection of oestrogen. This reduction of the inhibitory activity was dependent on the presence of blastocysts in utero, since a similar reduction did not occur in uterine fluids of pseudopregnant mice. The results support the suggestion that 'delayed' implantation in mice is caused by the presence of inhibitors of blastocyst metabolism and that activation, after an increase in oestradiol, is due to an embryo-dependent loss of activity of the inhibitors.

Free access

C. O'Neill and P. Quinn

Summary. Culture of mouse blastocysts in medium supplemented with uterine flushings from mice at random stages of the oestrous cycle resulted in a depression of [3H]uridine incorporation. This depression was maintained for up to 12 h, but by 24 h of culture, inhibition of uterine incorporation was no longer apparent. The loss of inhibition was due to a change in the activity of the flushings and not to a change in the ability of blastocysts to respond to the inhibitory influence. The inhibition of [3H]uridine incorporation was maintained for at least 24 h when blastocysts were transferred every 6 h to fresh uterine flushings.

Free access

X. E. Wells and C. O'Neill

Summary. Incubation of two-cell mouse embryos with a range of radiolabelled compounds resulted in the incorporation of label into platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in the culture media. The demonstration that known precursors ([1-14C]hexadecanol, [1-3H]hexadecanol, 1-O-[alkyl-1′2′-3H]lyso-PAF, 1-O-[alkyl-1′2′-3H]acetyl-glycerol and [methyl-3H]choline chloride) were incorporated into PAF showed that embryo-derived PAF biosynthesis occurred via pathways present in other PAF-producing cells. The enzyme responsible for the formation of the ether linkage of the PAF molecule, alkyl-dihydroxyacetone-phosphate synthase, was present in the preimplantation embryo as [1-3H]hexadecanol was incorporated into PAF. Incorporation of label from alkylacetylglycerol and choline chloride into lyso-PAF was also observed, suggesting a role for lyso-PAF in the metabolism of embryo-derived PAF. Incubation of embryos with each of three [14C]carbohydrate energy substrates resulted in the incorporation of label into PAF in culture media, indicating that the composition of embryo culture media is important in the synthesis of PAF precursors. Incorporation of label from [2-14C]pyruvate was greatest and is consistent with the suggestion that pyruvate is the major energy source at the two-cell stage of development. l-[U-14C]Lactate was also incorporated into embryo-derived PAF, but the mean amount incorporated relative to the concentration of labelled substrate in the medium was 40 times less. The incorporation of d-[U-14C]glucose into PAF was 2405 times less than that from pyruvate, relative to the concentration in the medium.

Keywords: embryo; platelet-activating factor; mouse

Free access

N. R. Spinks and C. O'Neill

Summary. Inhibitors of platelet activation, alprazolam, iloprost and SRI 63-441, were used to demonstrate the necessity of embryo-derived platelet-activating factor (PAF) activity for the establishment of pregnancy in mice. In a splenectomized mouse bioassay 6 μg alprazolam inhibited, for 3 h, the thrombocytopenia induced by 0·1 μg PAF: 4 μg iloprost and 0·5 μg SRI 63-441 were effective for 6 and 12 h respectively. The administration of 2 μg iloprost/30 g body weight on Days 1 and 4 of pregnancy and twice daily on Days 2 and 3 caused a 50% reduction (P < 0·0005) in the number of implantation sites in the uterus at Day 8 of pregnancy, without affecting (P > 0·05) the number of corpora lutea. A similar reduction in the number of implantation sites was achieved with 20 μg SRI 63-441/30 g body weight/day. The reduction in implantation rate was evident on Day 5 of pregnancy by visualizing the implantation sites with pontamine sky blue. SRI 63-441 had no effect on peripheral blood progesterone concentrations from Day 1 to Day 9 of pregnancy, and did not appear to inhibit implantation by blocking the preimplantation surge of oestradiol. The number and morphology of blastocysts flushed from the uterus of Day 4 inhibitor-treated mice was not different (P > 0·05) from the controls. The cleavage rate and morphology of embryos cultured from the 2-cell to blastocyst stage in media containing SRI 63-441 or iloprost (10 μg/ml) were normal, precluding a gross toxic effect. Simultaneous administration of 1 μg PAF-acether to treated animals re-established pregnancy rates to levels not significantly different (P > 0·05) from the controls.

Keywords: mouse; implantation; PAF; iloprost; alprazolam; SRI 63-441

Free access

A. J. Ammit and C. O'Neill

Platelet-activating factor (PAF) produced by embryos remained associated with mouse four-cell embryos after culture in vitro and was also released into the medium. The release of PAF into medium required albumin as a media supplement and the amount of PAF released increased (P < 0.05) with increasing albumin concentration. There was a trend for the amount of PAF remaining associated with embryos to decrease as the extracellular albumin concentration increased. The association of released PAF with albumin was confirmed by size fractionation with size exclusion membranes and high performance gel filtration, and by affinity chromatography (Cibacron blue and anti-BSA) and native PAGE. PAF released from embryos was not degraded by serum PAF:acetylhydrolase (PAF:AH; E.C. 3.1.1.47) after exposure for 24 h to the serum in vitro, while an equivalent concentration of synthetic PAF added to identical media was readily degraded, suggesting that PAF released from the embryo was protected from PAF:AH action. However, when the medium was subjected to organic extraction by the Bligh–Dyer (methanol/chloroform) method and the resulting extract added to equivalent media, embryo-derived PAF was readily degraded by PAF:AH. Furthermore, PAF in embryo-conditioned medium (30 two-cell embryos for 24 h) could not be detected after direct assay of the culture medium by radioimmunoassay or bioassay (platelet aggregation in vitro), yet after extraction, purification and addition to medium with BSA, the embryo-derived PAF was readily detected in either assay. To determine whether the different behaviour of synthetic PAF and embryo-derived PAF resulted from differences in the nature of their binding to albumin, the location to which PAF bound was assessed by limited proteolytic digestion of albumin. Digestion with pepsin or trypsin showed that embryo-derived PAF was located exclusively between amino acids 240 and 386 (domain II) of albumin. Most synthetic PAF added to equivalent medium not exposed to embryos was not at this location, suggesting that PAF released from embryos bound to a site on albumin not generally accessible to synthetic PAF added to similar media.

Free access

X L Jin and C O'Neill

The co-expression of the CREB and ATF1 transcription factors is required for the development of preimplantation embryos. Embryotropin-mediated, calcium/calmodulin-dependent signalling activates CREB-induced transcription in the two-cell embryo, but the regulation of ATF1 in the embryo is not known. This study demonstrates that ATF1 begins to accumulate within both pronuclei of the mouse zygote by 20 h post-human chorionic gonadotrophin. This did not require new transcription (not blocked by α-amanitin), but was dependent upon protein synthesis (blocked by puromycin) and the activity of P38 MAP kinase. ATF1 becomes an active transcription factor upon being phosphorylated. A marked accumulation of phosphorylated ATF1 was evident in two-cell embryos and this persisted in subsequent stages of development. This phosphorylation was enhanced by the actions of autocrine embryotropic mediators (including Paf) and required the mutual actions of P38 MAP kinase and calmodulin-dependent pathways for maximum levels of phosphorylation. The combined inhibition of these two pathways blocked embryonic genome activation (EGA) and caused embryos to enter a developmental block at the two-cell stage. The members of the CREB family of transcription factors can generate one of the most diverse transcriptomes of any transcription factor. The demonstration of the presence of activated CREB and ATF1 within the embryonic nucleus at the time of EGA places these transcription factors as priority targets as key regulators of EGA.