349 DEVELOPMENTAL COMPETENCE AFTER IVF OF CUMULUS-ENCLOSED OOCYTES FROM A ICR MOUSE STRAIN CULTURED WITH A LOW CONCENTRATION OF HYPOXANTHINE DURING IVM

Abstract

Hypoxanthine (HX, 4 mM) is known to contribute to meiotic arrest at the germinal vesicle (GV) stage of cumulus-enclosed oocytes (CEOs) cultured in vitro by causing accumulation of cAMP within the oocytes. Recently, it was found that when CEOs at the GV stage from the ICR mouse strain were treated with a small amount of HX (200 µM) during in vitro maturation (IVM) culture, cortical granule migration was much improved. The aim of this study was to examine the effects of treatment of ICR mouse CEOs with 200 µM HX during IVM culture on the in vitro development of oocytes after in vitro fertilization (IVF). CEOs were isolated from eCG-primed 3-week-old ICR mice by rupturing preovulatory follicles with needles in M16 medium containing 5% FCS, 1 IU mL^-1 FSH, and essential and nonessential amino acids (basal medium). IVM media were basal medium without (control) or with 200 µM HX, 100 µM dibutyryl cyclic adenosine monophosphate (dbcAMP), or 10 IU mL^-1 FSH. Denuded oocytes (DOs) were prepared by treating CEOs for 30 min with 0.1% hyaluronidase and 0.1 mM EDTA, followed by repeated pipetting. CEOs and DOs were cultured in IVM media at 37°C under 5% CO₂ in air for 16.5 h. After IVM, CEOs and DOs were transferred to TYH medium (modified Krebs-Ringer bicarbonate) containing 0.4% BSA, and inseminated with capacitated sperm. After 6 h of IVF, zygotes were freed from cumulus cells by gently pipetting, and cultured in KSOM medium with 0.3% BSA. Development to the 2-cell and blastocyst stages was determined at 24 h and 120 h after IVF, respectively. All experiments included 3 replicates, and the statistical analysis was carried out by ANOVA and Fisher's PLSD test. When CEOs matured in basal medium (controls), although almost all of the oocytes reached the MII stage, the rates of their post-fertilization development to the 2-cell and blastocyst stages were very low (21.8% and 8.9%, respectively), whereas those of CEOs matured with 200 µM HX were significantly increased (63.4% and 52.2%, respectively; P < 0.05). Next, CEOs were cultured with 100 µM dbcAMP or a high concentration (10 IU mL^-1) of FSH during IVM. The rates of the 2-cell and blastocyst stages of CEOs cultured with 100 µM dbcAMP were 53.3% and 41.3%, respectively, and those of CEOs cultured with 10 IU mL^-1 FSH were 55.5% and 27.6%, respectively. Although the blastocyst rate was significantly lower in FSH-treated oocytes than in HX- or dbcAMP-treated oocytes (P < 0.05), this result suggests that increasing concentration of intracellular cAMP is important for improving the developmental competence of mouse CEOs after IVF. Finally, when DOs were cultured with 200 µM HX, although most of the oocytes were arrested at the MI stage, 6.3% of the oocytes developed into 2-cell embryos after IVF but not into blastocysts. Therefore, it is suggested that some accumulation of cAMP in cumulus cells by 200 µM HX enhances fertility and post-fertilization development of oocytes in the ICR mouse strain.