RESUMO
Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF), a potent signaling phospholipid, has a significant role in preimplantation embryo development. CFW mouse embryos respond to PAF with improved development and implantation rates. PAF's signal transduction mechanism in other cell types is receptor mediated. However, embryonic mRNA for the PAF receptor has not been detected. The study objectives were to determine the presence of PAF receptor mRNA in CFW mouse two-cell embryos by reverse transcription (RT)-polymerase chain reaction and Northern blot analysis and to ascertain the effect of PAF on intracellular calcium levels (a receptor-mediated event). Total RNA was purified by acid-phenol extraction and ethanol precipitation. Complementary DNA was synthesized by RT. RNA was primed with oligo-dT plus PAF receptor-specific primer (3' to 5') at 42 degrees C for 60 min, 95 degrees C for 10 min, and 5 degrees C for 5 min. The RT product was amplified with Taq polymerase and PAF receptor-specific primer (5' to 3') at 94 degrees C for 5 min and 54 degrees C for 5 min for one cycle, and at 72 degrees C for 3 min, 93 degrees C for 90 sec, and 61 degrees C for 150 sec for 30 cycles followed by 72 degrees C for 10 min and then holding at 4 degrees C. The product was analyzed by agarose gel electrophoresis, producing a single band (610 base pairs [bp]), thus demonstrating the presence of PAF-receptor mRNA. Sequence analysis of the cloned 610-bp fragment confirmed that it is the PAF receptor. Northern blot analysis also confirmed the expression of the PAF receptor in the CFW mouse preimplantation two-cell-stage embryo. PAF treatment of the two-cell-stage CFW mouse embryo resulted in a fourfold increase in intracellular calcium over background levels.