231 STAGE-DEPENDENT CHANGES OF IP₃R1 PHOSPHORYLATION DURING IN VITRO MATURATION AND DEVELOPMENT IN PIG OOCYTES

Abstract

During fertilization in mammalian species, a sperm-induced intracellular Ca²⁺ signal [Ca²⁺] is well suited to mediate the highly specialized spatiotemporal patterns of [Ca²⁺]i responses that underlie fertilization. Recently, we demonstrated that the expression pattern of inositol 1,4,5- triphosphate receptor type 1 (IP₃R1) did not change during in vitro maturation and parthenogenetic activation in mouse oocytes; however, the phosphorylation status of IP₃R1 depended on the cell cycle during meiosis. Moreover, it was shown that IP₃R1 phosphorylation played a crucial role in the induction of [Ca²⁺]i oscillations (Lee et al. 2006 Development 133, 4355–4365). In other species, expression of IP₃R1, especially phosphorylation levels of IP3R1 during meiosis, has not been examined. The aim of this study was to examine the kinetics of IP₃R1 expression and phosphorylation during in vitro maturation and activation in pig oocytes. Immature oocytes at the germinal vesicle (GV) stage were collected from ovaries and cultured in modified NCSU37 up to 48 h. After culture, cumulus cells were removed and oocytes were parthenogenetically activated by 25 µm Ca²⁺ ionophore for 3 min and 2 mm 6-DMAP for 6 h. After activation, oocytes were further cultured up to the 2-cell stage. Groups of 30 oocytes were collected at each culture period for detection of IP₃R1. According to our previous report in the mouse, IP₃R1s were detected by western blotting using MPM-2 and Rbt03 antibody for detecting IP₃R1 phosphorylation and total IP₃R1 expression, respectively (Lee et al. 2006). In pig oocytes, IP₃R1 was abundantly expressed at the GV stage. The total level of IP₃R1 expression did not change during in vitro maturation or after activation. However, phosphorylated IP₃R1 levels increased by 24 h although they were undetectable at the start of culture. Phosphorylation of IP₃R1 reached maximal levels at 36 h. After activation, phosphorylation levels decreased progressively until the pronuclear (PN) stage. Phosphorylation of IP₃R1 was observed at mitosis I to some extent. From these results, we detected for the first time IP₃R1 expression and phosphorylation in pig oocytes. Moreover, our data suggest that phosphorylation of IP₃R1 is dependent on cell cycle at least during meiosis, especially M-phase, as already shown for mouse oocytes. In vitro kinase assays for p34^cdc2 kinase and MAPK will be carried out to clarify the relationship between IP₃R1 phosphorylation and M-phase kinase(s).