Growth of preimplantation embryos is influenced byThe cells of the mammalian preimplantation embryo (from the time of fertilization until the implantation of the blastocyst into the uterus) form the progenitor cells for all other cell lineages. The regulation of the growth and survival of the cells of the early embryo is, however, poorly understood. Mammalian preimplantation embryos develop in vitro with simple medium requirements and have no absolute requirement for exogenous vitamins, hormones, or growth factors. This contrasts with the absolute requirement of normal somatic cells for exogenous mitogens and survival factors. 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 (3-8); 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).Experimental partial deprivation of released autocrine trophic factors did not arrest the cell-cycle at given checkpoints (9). Rather, there was progressive loss of viability with increased cell death as embryos progressed past the 8-cell stage. This finding suggests that the autocrine factors may act as survival factors rather than classical growth factors (triggering progression through specific cell-cycle checkpoints). While several autocrine factors have been implicated, platelet-activating factor (PAF) 1 seems to be one of the first produced, being synthesized de novo by the embryo soon after fertilization (10, 11). Its actions are required by the mid-2-cell stage for normal rates of embryo survival (9).Despite this range of supportive data, there is limited direct evidence for the action of autocrine trophic factors in early embryo development. Transgenic and recombinant knock-out models have not generally been informative of the growth requirements of the early embryo prior to implantation. This may be due to extensive redundancy of regulatory pathways.

The growth and survival of the preimplantation mammalian embryo may be regulated by several autocrine trophic factors that have redundant or overlapping actions. One of the earliest trophic factors to be produced is embryo-derived platelet-activating factor (1-O-alky-2-acetyl-sn-glyceryl-3-phosphocholine). The addition of platelet-activating factor to embryo culture media exerted a trophic effect, but structurally related lipids (3-O-alky-2-acetyl-sn-glyceryl-1-phosphocholine, 1-O-alky-sn-glyceryl-3-phosphocholine, octadecyl-phosphocholine) had no effect. Platelet-activating factor induced a pertussis toxin-sensitive [Ca2+]i transient in two-cell embryos that did not occur in platelet-activating factor-receptor null (Pafr–/–) genotype embryos. Fewer Pafr–/– mouse zygotes developed to the blastocyst stage in vitro compared with Pafr+/+ zygotes (P<0.02), those that developed to blastocysts had fewer cells (P<0.001) and more cells with fragmented nuclei (P<0.001). The inhibition of 1-O-phosphatidylinositol 3-kinase (LY294002 (3 μM and 15 μM) and wortmannin (10 nM and 50 nM)) caused a dose-dependent inhibition of platelet-activating factor-induced [Ca2+]i transients (P<0.001). The two-cell embryo expressed 1-O-phosphatidylinositol 3-kinase catalytic subunits p110α, β, γ and δ, and regulatory subunits p85α and β. LY294002 and wortmannin each caused a significant reduction in the proportion of embryos developing to the morula and blastocyst stages in vitro, reduced the number of cells within each blastocyst, and significantly increased the proportion of cells in blastocysts with fragmented nuclei. The results indicate that embryo-derived platelet-activating factor (and other embryotrophic factors) act through its membrane receptor to enhance embryo survival through a 1-O-phosphatidylinositol 3-kinase-dependent survival pathway.

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Platelet-activating factor (PAF) is an autocrine trophic/survival factor for the preimplantation embryo. PAF induced an increase in intracellular calcium concentration ([Ca2+]i) in the 2-cell embryo that had an absolute requirement for external calcium. L-type calcium channel blockers (diltiazem, verapamil, and nimodipine) significantly inhibited PAF-induced Ca2+ transients, but inhibitors of P/Q type (omega-agatoxin; omega-conotoxin MVIIC), N-type (omega-conotoxin GVIA), T-type (pimozide), and store-operated channels (SKF 96365 and econazole) did not block the transient. mRNA and protein for the alpha1-C subunit of L-type channels was expressed in the 2-cell embryo. The L-type calcium channel agonist (+/-) BAY K 8644 induced [Ca2+]i transients and, PAF and BAY K 8644 each caused mutual heterologous desensitization of each other's responses. Depolarization of the embryo (75 mM KCl) induced a [Ca2+]i transient that was inhibited by diltiazem and verapamil. Whole-cell patch-clamp measurements detected a voltage-gated channel (blocked by diltiazem, verapamil, and nifedipine) that was desensitized by prior responses of embryos to exogenous or embryo-derived PAF. Replacement of media Ca2+ with Mn2+ allowed Mn2+ influx to be observed directly; activation of a diltiazem-sensitive influx channel was an early response to PAF. The activation of a voltage-gated L-type calcium channel in the 2-cell embryo is required for normal signal transduction to an embryonic trophic factor.