The use of isoflurane to cause multiple large Ca2+ transients in mouse eggs

Abstract

Mammalian eggs are activated at fertilization by a series of sperm-induced increases in cytosolic Ca2+. The Ca2+ ionophores, A23187 and ionomycin, are used to stimulate a single large Ca2+ transient to induce mammalian egg activation after intracytoplasmic sperm injection (ICSI) but this response is of unclear effectiveness. Some protocols involve two or more incubations in Ca2+ ionophore, but we now report that repeated application of either A23187 or ionomycin in mouse eggs leads to Ca2+ transients of decreasing amplitude, or else egg lysis. In contrast we show that the halogenated anaesthetic isoflurane causes a large Ca2+ increase in mouse eggs, but when washed out and re-applied to eggs, it repeatedly generates another large Ca2+ transient. We also found that, after fertilization or PLCz1 injection, brief exposure to Ca2+ ionophores caused one Ca2+ rise but then inhibited further Ca2+ oscillations in eggs. In contrast, brief application of isoflurane to fertilizing or PLCz1-injected eggs enhanced ongoing induced Ca2+ oscillations. The triple application of isoflurane triggered mouse egg activation more effectively than A23187 or ionomycin. Isoflurane is volatile and rapidly evaporates from culture medium, but we demonstrate that isoflurane can be reliably used to cause Ca2+ increases and egg activation when it is added to the mineral oil that covers the drops of medium into which eggs are placed. Our data suggest that isoflurane can be used to induce multiple large amplitude Ca2+ transients and promote efficient egg activation with a simple and adjustable protocol.