Oral Presentation The Annual Scientific Meeting of the Endocrine Society of Australia and the Society for Reproductive Biology 2014

Calcium activated chloride channel activity induces changes in membrane potential at fertilisation in the mouse oocyte (#35)

Sarah Dalati 1 , Margot Day 1
  1. University of Sydney, Camperdown, NSW, Australia

Fertilisation is characterised by [Ca2+]i oscillations and accompanying membrane potential (Em) hyperpolarisations. [Ca2+]i oscillations have been shown to be important in initiating egg activation and for subsequent embryo development, whereas the role that Em changes play in fertilisation remains unknown. Patch clamp analysis of unfertilised mouse oocytes has demonstrated that the sulphydryl reagent thimerosal elicits simultaneous Em changes and [Ca2+]i oscillations mimicking those induced at fertilisation. 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. It has been recently discovered that the TMEM16A protein forms CaCC’s in Xenopus oocytes where it induces changes in Em. The present study aims to determine the expression of these ion channels in mouse oocytes and embryos, assess their role in fertilisation and identify whether TMEM16A maybe a possible candidate for CaCC’s. The CaCC channel inhibitor T16Ainh-A01 (10 μM) had no effect on the number of [Ca2+]i oscillations induced by thimerosal. Perforated whole-cell patch clamping of single oocytes in the presence of T16Ainh-A01 (10 μM) significantly reduced the number of Em hyperpolarisations induced by thimerosal by 65% when compared to controls. The culture of zygotes in the presence of T16Ainh-A01 (10 μM) completely prevented their cleavage to the 2-cell stage. Western blot analyses have also shown that TMEM16A is expressed at high levels from the oocyte to 8-cell stage mouse embryo, but reduced in the morula and blastocyst stages. Collectively these data show for the first time that the CaCC may be formed by TMEM16A and that the activation of this CaCC channel may play an important role in initiating the cellular events that occur during fertilisation and early cell division.