The role of albumin in the release of platelet-activating factor by mouse preimplantation embryos in vitro

Abstract

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.

Characterization and Functional Significance of Calcium Transients in the 2-Cell Mouse Embryo Induced by an Autocrine Growth Factor

“…Mobilization of this PAF to elicit the calcium response was dependent upon extracellular protein. This is consistent with previous observations that albumin has high affinity binding sites for PAF (37), being located on domain II of albumin (27), and that extracellular albumin was essential for the release of PAF from the 2-cell embryo (26,27). Since PAF is a phospholipid it is hydrophobic, only readily entering the aqueous phase by binding to a hydrophilic carrier such as albumin.…”

Studies of the Nature of the Binding by Albumin of Platelet-activating Factor Released from Cells

“…Murine Embryo Culture-Two-cell mouse embryos were collected from superovulated Swiss outbred albino mice (Department of Veterinary Physiology, University of Sydney, NSW, Australia) as described (21). Thirty two-cell embryos were cultured for 24 h at 37°C in 5% CO 2 in air in 1 ml of embryo culture medium (HTFM: Ref.…”

“…Half was left untreated and the remainder acid-treated (pH 3.0), with 1 M HCl (BDH Chemicals, Poole, United Kingdom) for 20 min at 37°C and then returned to pH 7.4 by 1 M NaOH (BDH), which deactivates PAF:AH (28). The PAF:AH specific activity was measured as described previously (21). Untreated or acidtreated sera (100 l) were added to 900 l of the ECCM pool or corresponding synthetic PAF pool, for 24 h at 37°C, in 5% CO 2 phenylethylchloromethyl ketone-treated, from bovine pancreas; Sigma).…”

“…A recent study (21) showed that this method of extracting media was required to detect PAF released from preimplantation embryos, due apparently to PAF's exclusive binding to domain II of albumin. Binding of PAF at that site on albumin resulted in PAF being protected from the actions of PAF-acetylhydrolase (PAF:AH) and also resulted in PAF being undetectable in bioassays without prior extraction.…”

“…Binding of PAF at that site on albumin resulted in PAF being protected from the actions of PAF-acetylhydrolase (PAF:AH) and also resulted in PAF being undetectable in bioassays without prior extraction. It was suggested that binding of PAF to domain II of albumin resulted in it being presented in a protected form (21). PAF added to medium also bound to albumin but was not found on domain II and was not in a protected form.…”