235.PAF induced changes in intracellular Ca2+ and membrane potential in the 2-cell mouse embryo

Abstract

Platelet-activating factor (PAF) is an autocrine survival factor for the preimplantation embryo. PAF induces a transient increase in intracellular Ca2+ ([Ca2+]i) in 2-cell embryos that is caused by the interdependent influx of external calcium and release of calcium from internal stores. A membrane current with L-type calcium channel properties is activated during PAF-induced calcium signalling. Since the L-type channel in many cell types is primarily voltage-gated we were interested to learn whether this was also the case in the 2-cell embryo. The present study investigated the relationship between the PAF-induced Ca2+ transient and changes in membrane potential (Em) in the 2-cell embryo. The perforated whole-cell patch-clamp technique was used to detect changes in Em and standard calcium imaging techniques were used to measure changes in [Ca2+]i in 2-cell embryos from QS mice. Embryos were first loaded with Fluo-3 and then pretreated with PAF:acetylhydrolase to degrade the embryo derived PAF before patch clamping. Whole-cell perforated patch-clamping was performed by inclusion of 240mg/ml Nystatin in the pipette solution. Changes in Em and [Ca2+]i were recorded simultaneously after treatment of the embryo with PAF. In 2-cell embryos PAF induced a change in Em, consisting of an initial small depolarisation of 2.4 � 0.2 mV (42 � 4 sec after addition of PAF) followed by one or more transient hyperpolarisations of -8 � 1 mV (100 � 9 sec after addition of PAF). Transient increases in [Ca2+]i paralleled the membrane hyperpolaristions and were initiated at 84 � 8 sec after addition of PAF. These responses to PAF were seen in 58% of 2-cell embryos (n = 52). It is not yet clear whether these changes in Em account for the activation of calcium influx through the L-type channel. The results show for the first time that the 2-cell embryo is an electrically active organism.