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.
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.
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.
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.
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.
E. A. Moon and C. O'Neill
Cytidine 5′-triphosphate (CTP):phosphocholine cytidylyltransferase (EC 184.108.40.206) catalyses the synthesis of the active metabolic intermediate cytidine diphosphocholine (which is mainly used in the synthesis of choline-containing phospholipids). It is a rate-limiting reaction in choline phospholipid biosynthesis in many cells. In this study, a microassay is reported for the detection of this enzyme in small numbers of cells. This enzyme was present in mouse oocytes and at all stages during preimplantation development. Enzyme activity was destroyed by boiling but increased with time and number of embryos in the reaction. Activity in two-cell embryos was dependent on Mg2+ but independent of Ca2+ and was enhanced by the addition of 1 μg lysophosphatidylethanolamine ml−1 to the reaction mixture. Activity was apparently dependent upon the phosphorylation status of the enzyme since the absence of the phosphatase inhibitor NaF caused a significant inhibition of activity. The enzyme in oocytes had a specific activity of 2.8 ± 0.3 fmol cytidine diphosphocholine (CDP-choline) per oocyte min−1 (mean ± sem). The specific activity in two-cell and eight-cell embryos and blastocysts was not different from that of oocytes. Fertilized one-cell embryos had significantly less activity (1.4 ± 0.05 fmol CDP-choline produced per embryo min−1) than other stages studied. Furthermore, the enzyme present in one-cell embryos was not capable of being further activated by the addition of exogenous lysophosphatidylethanolamine to the reaction. The increase in activity from the one-cell to the two-cell stage was not inhibited by α-amanitin (an inhibitor of RNA polymerase II), cycloheximide (a protein synthesis inhibitor) [1-(5-isoquinolinesulfonyl)-2-methylpiperazine, HCl]dihydrochloride (H-7; a protein kinase inhibitor) and was independent of cell-cycle progression; these results suggest that enzyme activity is independent of transcription, protein synthesis and the action of some kinases, including cell-cycle-dependent kinases. This study provides the first description of cytidylyltransferase in the early mammalian embryo.
X L Jin and C O’Neill
Gene expression from the new embryonic genome is required for normal preimplantation embryo development. Two members of the cAMP-responsive element-binding protein (Creb) family of transcription factors, Creb1 and activating transcription factor 1 (Atf1), are essential for normal preimplantation development. These transcription factors are activated by phosphorylation. Creb1 mRNA was expressed throughout the preimplantation phase. Cytoplasmic immunolocalization of Creb1 was detected in all preimplantation embryo stages. The antigen was largely excluded from the pronuclei/nuclei at embryonic stages except in the mid-cycle two-cell and compacted eight-cell embryo. Activation-state-specific antibodies showed serine 133 phosphorylated Creb1 localization was similar to Creb1 staining, except that there was no increase in staining at the eight-cell stage. Increased staining of phosphorylated Creb1 was observed in the nucleus of mid-cycle two-cell embryos. Increased expression of phosphorylated Creb1 in the two-cell embryo was induced by brief exposure of embryos to ionomycin, but not by a dibutyryl cAMP. This was blocked by buffering intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester), but not by a cAMP antagonist, Rp-cyclic 3′,5′-hydrogen phosphorothioate adenosine. Calmodulin is an intracellular receptor for calcium. Calmodulin mRNA was expressed throughout the preimplantation phase of development. The calmodulin antagonist, W-7, inhibited the ionomycin-induced localization of phosphorylated Creb1 in the nucleus. Treatment of embryos with W-7 caused a dose-dependent inhibition of normal development of zygotes to the blastocysts stage. The study shows Creb1 expression and nuclear localization was dynamically regulated in the early embryo. The marked nuclear accumulation and phosphorylation of Creb1 at the two-cell stage occurred at the time of transcription from the embryonic genome and was regulated in a calcium- and calmodulin-dependent manner.
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
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. 220.127.116.11) 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.