238 CAFFEINE PREVENTS A LOSS OF CALCIUM OSCILLATORY RESPONSE ASSOCIATED WITH POSTOVULATORY AGING OF MOUSE OOCYTES

Abstract

Numerous studies have demonstrated that postovulatory aging of oocytes prior to fertilization has detrimental effects on oocyte quality and developmental competence. Oocyte aging is accompanied by abnormal oocyte activation and subsequent development, suggesting a disruption of Ca₂⁺ oscillations after fertilization. The inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) in mammals is responsible for the majority of Ca₂⁺ release during fertilization (Miyazaki S et al. 1993 Dev. Biol.). Previously, we reported that phosphorylation of IP3R1 at an MPM-2 epitope may play an important role in facilitating the induction of Ca₂⁺ oscillations at the MII stage (Lee B et al. 2006 Development), indicating that IP3R1 phosphorylation may be a good indicator of the health of the oocyte. However, few studies have investigated the alteration of the Ca₂⁺ signaling and IP3R1 function associated with oocyte aging. On the other hand, a previous report showed that caffeine increased MPF activity and suppressed fragmentation after parthenogenetic activation of aged oocytes (Kikuchi K et al. 2000 Biol. Reprod.). Therefore, the purpose of the present study was to examine whether and how Ca₂⁺ oscillatory activity changes during oocyte aging and to test if caffeine prevents the negative effects of oocyte aging. MII mouse oocytes were collected 14 h after hCG injection and cultured in vitro for 8, 24 or 48 h with or without caffeine (5 or 10 mm). Oocyte quality was assessed by the occurrence of spontaneous fragmentation, monitoring of Ca₂⁺ oscillations after exposure to 10 mm strontium chloride, Western blot analysis of IP3R1 phosphorylation and immunostaining of IP3R1. In oocytes in vitro aged for 8 h, the duration of the first Ca₂⁺ rise was significantly decreased compared with fresh MII oocytes, although this reduction was not observed in MII oocytes treated with 5 mm caffeine. The phosphorylation of IP3R1 at the MPM-2 epitope was slightly decreased during oocyte aging in both caffeine and noncaffeine treatment. Importantly, whereas IP3R1 in MII oocytes treated for 8 h with 5 mm caffeine displayed the typical cortical cluster organization, IP3R1 in aged oocytes without caffeine became dispersed in the cytoplasm. In addition, caffeine significantly suppressed the spontaneous fragmentation that is normally observed by 48 h of in vitro culture. These results suggest that the Ca₂⁺ oscillatory activity is compromised during oocyte aging and caffeine prevents the loss of integrity of Ca₂⁺ signaling possibly by keeping the cortical distribution of IP3R1.