243 NUCLEOSIDES REDUCE THE POTENTIAL OF THE MITOCHONDRIAL INNER MEMBRANE AND THE DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES DURING IN VITRO MATURATION

Abstract

Although nucleosides such as adenosine exist in follicular fluid and decrease during oocyte maturation (Eppig et al. 1985 Biol. Reprod. 33, 1041–1049), the role of nucleosides is still unclear. The present study was undertaken to determine the effect of nucleoside on nuclear and cytoplasmic maturation of mouse oocytes. Oocyte–cumulus complexes (OCCs)were collected from the large antral follicles of C57BL/6J female mice (3–5 weeks old) 4 h after a combination injection of equine chorionic gonadotropin (eCG) and hCG with a 48-h interval, and cultured in maturation medium (αMEM containing 3 mg mL^–1 BSA, 0.23 mm Na pyruvate and antibiotics) with or without ribo- and deoxyribo-nucleosides (10–11 µg mL^–1) for 12 h. Statistical analyses in this study were carried out by ANOVA and Bonferroni/Dunn's post hoc test. Regardless of the presence of nucleosides, a majority of oocytes developed to the metaphase-II stage in vitro, and the incidence was not different with in vivo-matured oocytes. However, the mitochondrial membrane potential (MMP) of oocytes was significantly lower when the oocytes were matured in the presence of nucleosides, as compared with nucleoside-free controls. In the scanning experiment of MMP level during in vitro maturation (IVM), the MMP of oocytes maturing in vivo increased between 8 and 12 h after hCG injection, whereas no rises of MMP was observed in oocytes matured in vitro in the presence of nucleosides. To examine the affecting period of nucleosides, OCCs were cultured for the first 4 h or the latter 8 h of IVM in the presence of nucleosides. MMP of the oocytes was significantly lower only when the OCCs were exposed to nucleosides for the latter 8 h of IVM. To determine if adenosine in the nucleosides (10 µg mL^–1) affects the MMP of oocytes, OCCs were exposed to adenosine or a mixture of guanosine, cytidine, and uridine during the latter 8 h of IVM. The MMP of oocytes exposed to adenosine was lower than that of in vivo-matured oocytes and of oocytes exposed to a mixture of guanosine, cytidine, and uridine. To determine the effect of nucleosides on the developmental competence, oocytes exposed to adenosine during the latter 8 h of IVM were cultured in kSOMaa containing 1 mg mL^–1 BSA after activation in the presence of 10 mm SrCl₂ and 5 µg mL^–1 cytochalasin B for 6 h. Pronuclear formation and early development of those were compared with artificially activated oocytes matured in vivo or in the absence of nucleosides. The incidences of pronuclear formation and cleavage of oocytes matured in the presence of adenosine following activation were extremely decreased, as compared with control oocytes matured in vivo or in the absence of adenosine. These observations indicate that nucleosides, at least adenosine, inhibit an increase of MMP of oocytes during the latter half of meiotic maturation, and detrimentally affect the developmental competence of murine oocytes.