Ca2+ activated CI- channel activity at fertilisation in the mouse oocyte

Abstract

Sperm entry into the oocyte triggers a signal transduction pathway that results in intracellular calcium ([Ca2+]i) oscillations and membrane potential (Em) changes known as hyperpolarisations. [Ca2+]i oscillations have been widely studied and are important for embryo development. The role of Em hyperpolarisations and their importance at fertilisation remains unclear. Thimerosal, a sulfhydryl reagent, has been shown to mimic the physiological changes caused by sperm following fertilisation. Previous patch clamp analysis of unfertilised mouse oocytes has shown that thimerosal elicits simultaneous Em hyperpolarisations and [Ca2+]i oscillations. It has been recently discovered that the TMEM16A protein forms CaCC’s in Xenopus oocytes where it induces changes in Em. We hypothesise that changes in Em following fertilisation may be due to the activation of Ca2+ activated Cl- channels (CaCC) present in the membrane of mouse oocytes. This study aims to determine the expression of these ion channels in mouse oocytes and embryos, to assess their role in fertilisation and identify whether TMEM16A maybe a possible candidate for CaCC’s. The thimerosal induced Em and [Ca2+]i changes were inhibited by the CaCC blockers, DIDS and NFA and the specific CaCC inhibitor T16Ainh-A01. In vitro fertilisation studies showed that the KCa channel blocker tetraethylammonium (TEA), and voltage gated Cl- channel blocker 9AC had no effect on fertilisation rates but there was a significant reduction in the percentage of oocytes that fertilsed in the presence of the CaCC inhibitors DIDS and T16Ainh-A01 Culturing zygotes in the presence of T16Ainh-A01 (10 μM) completely inhibited their cleavage to the 2-cell stage. Furthermore, western blot analyses showed that TMEM16A is expressed at higher levels from the oocyte to 8-cell stage mouse embryo, but reduced in the morula and blastocyst stages. In conclusion these data show that the activation of a CaCC channel may play an important role in initiating the cellular events that occur during fertilisation and early cell division and that TMEM16A may be a possible candidate in forming this channel.