Ontogeny of Expression of a Receptor for Platelet-Activating Factor in Mouse Preimplantation Embryos and the Effects of Fertilization and Culture In Vitro on Its Expression1

Abstract

Platelet-activating factor (PAF; 1-o-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent ether phospholipid. It is one of the preimplantation embryo's autocrine growth/survival factors. It may act via a G protein-linked receptor on the embryo; however, the evidence for this is conflicting. The recent description of the intracellular form of the PAF:acetlyhydrolase enzyme as having structural homology with G proteins and Ras also suggests this as a potential intracellular receptor/transducer for PAF. This study used reverse transcription-polymerase chain reaction to examine the ontogeny of expression of the genes for these proteins in the oocyte and preimplantation-stage embryo. Transcripts for the G protein-linked PAF receptor were detected in the late 2-cell-stage embryo and in all stages from the 4-cell stage to blastocysts. They were also present in unfertilized oocytes and newly fertilized zygotes but only at relatively low levels. The incidence of expression was generally low and variable in late zygotes and early 2-cell embryos. Expression past the 2-cell stage was alpha-amanitin sensitive. The results indicated that mRNA for this receptor is a maternal transcript that was degraded during the zygote-2-cell stage. New expression of the receptor transcript required activation of the zygotic genome. Fertilization of embryos in vitro caused this transcript not to be expressed in the zygote. Culture of zygotes (irrespective of their method of fertilization) caused expression from the zygotic genome to be retarded by more than 24 h. This retardation did not occur if culture commenced at the 2-cell stage. The transcripts for the subunits of intracellular PAF:acetylhydrolase were not detected in oocytes or at any stage of embryo development examined, despite their being readily detected in control tissue. This study confirms the presence of the G protein-linked PAF receptor in the 2-cell embryo and describes for the first time its normal pattern of expression during early development. The adverse effects of in vitro fertilization (IVF) and embryo culture on the expression of this transcript may be a contributing factor for the poor viability of embryos produced in this manner. The reduced expression of PAF-receptor mRNA following IVF predicts that such embryos may have a deficiency in autocrine stimulation and also suggests that supplementation of growth media with exogenous PAF would be only partially beneficial. The effect of IVF and culture may also explain the conflicting literature.

The effect of paracrine/autocrine interactions on the in vitro culture of bovine preimplantation embryos

“…It is possible that this is a consequence of PAF receptor expression; Roudebush et al (2002) found a decrease in murine receptor expression during preimplantation development, suggesting that elevated PAF concentrations are likely to saturate functional receptors. In addition, Stojanov and O'Neill (1999) reported a reduction in PAF-receptor mRNA expression after IVF, and suggested that this would lead to a deficiency of this particular autocrine signal, thereby accounting for the limited benefits of exogenous PAF supplementation. Continuous culture of bovine embryos throughout preimplantation development in the presence of SOFaaBSA supplemented with 10 -20 mM PAF led to a significant increase in the utilisation of pyruvate and glucose but did not alter lactate production.…”

Trophic signals acting via phosphatidylinositol-3 kinase are required for normal pre-implantation mouse embryo development

“…The receptor has wide tissue distribution (Ishii and Shimizu, 2000) and is expressed in the early mouse embryo (Roudebush et al, 1997;Stojanov and O'Neill, 1999). A single receptor subtype is currently thought to mediate all the actions of PAF described (Ishii and Shimizu, 2000).…”

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

“…The continued mitoses of preimplantation embryo cells in the absence of exogenous growth factors implicates a role for endogenous, autocrine trophic factors, or the constitutive activation of signaling pathways in the early embryo. Several lines of evidence support a role for the former: (i) the rate of embryo development in vitro is density-dependent, with embryos growing in relatively small volumes (or in large groups) developing more successfully than those grown in large volumes (or individually) (1, 2); (ii) the synthesis by the preimplantation embryo of a number of growth factor ligands and their receptors (3)(4)(5)(6)(7)(8); and (iii) the capacity of some exogenous growth factors to enhance embryo metabolism in vitro and to compensate for the adverse effects of culture in large medium volumes (1,2,9).…”