124. THE ROLE OF CALCIUM ACTIVATED CHLORIDE CHANNELS AT FERTILISATION

Abstract

Sperm entry into the oocyte triggers a signal transduction pathway resulting in intracellular calcium [Ca²⁺] oscillations that coincide with hyperpolarisations in membrane potential (Em). Ca²⁺ oscillations have been previously described and found to be important for embryo development, yet Em hyperpolarisations and their importance at fertilisation still remains unclear. Thimerosal, a sulfhydryl reagent, has been shown to mimic the physiological changes caused by sperm following fertilisation. It does this by direct sensitisation of the inositol 1,4,5-triphosphate receptor-1 to basal levels of inositol 1,4,5-triphopshate. Previous patch clamp analysis of unfertilised mouse oocytes has shown that thimerosal elicits simultaneous Em hyperpolarisations and Ca²⁺ oscillations. These results have lead us to hypothesise that hyperpolarisations in Em may be due to the activation of a Ca²⁺ activated Cl^- channel (CaCC) present in the membrane of mouse oocytes. The present study aims to identify this CaCC and assess its role in early development following fertilisation. Hyperpolarisations induced by thimerosal were inhibited by niflumic acid, a selective blocker of CaCCs. The inhibition of Em hyperpolarisations suggests that a CaCC is present and plays an active role in initiating hyperpolarisations. To identify the function of the CaCC at fertilisation, in vitro fertilisation was performed in the presence of niflumic acid. Niflumic acid inhibited polar body extrusion and pronuclei formation; two events that are indicators of fertilisation. Furthermore, Ca²⁺ imaging experiments with the calcium sensitive dye fura 2-AM, demonstrated that in the presence of niflumic acid, Ca²⁺ oscillations induced by thimerosal are reduced in size, number and duration. Taken together these data suggest that the activation of a Ca²⁺ activated Cl^– channel in the mouse oocyte may play an important role in the events occurring at fertilisation.