262 ISOBUTYLMETHYLXANTHINE REVERSIBLY SUPPRESSES SPONTANEOUS AND EQUINE CHORIONIC GONADOTROPIN/EPIDERMAL GROWTH FACTOR-STIMULATED MEIOSIS IN FELINE OOCYTES

Abstract

Recent studies have shown that short-term exposure to phosphodiesterase inhibitors (decreased cAMP degradation) or adenylate cyclase stimulators (increased cAMP synthesis) inhibits spontaneous oocyte maturation and improves the developmental competence of oocytes after gonadotropin-induced maturation. A similar approach may improve the developmental competence of in vitro matured feline oocytes. The objectives of this study were to 1) determine if a nonspecific phosphodiesterase inhibitor [isobutylmethylxanthine (IBMX); 100 μM] or an adenylate cyclase stimulator (forskolin; 100 μM) would suppress spontaneous or eCG (1 IU mL^–1)/epidermal growth factor (EGF; 25 ng mL^–1)-induced maturation of feline oocytes in vitro; 2) evaluate the reversibility of chemically induced meiotic arrest; and 3) assess the developmental competence of meiotically arrested oocytes. Cumulus–oocyte complexes were cultured in modified feline optimized culture medium [6 mM glucose, 0.1 mM cysteamine, 0.6 mM cysteine, 0.5× minimal essential medium (MEM) essential amino acids, 1× MEM vitamins, and 1× insulin-transferrin-selenium; Herrick et al. 2010 Biol. Reprod. 82, 552–562] supplemented with (+) or without (–) IBMX, forskolin, or eCG/EGF (eE). The IBMX decreased (P < 0.05; mixed model ANOVA) the incidence of spontaneous maturation (–eE) after 24 h of culture (84.6%, germinal vesicle, GV; 6.7% metaphase II, MII) compared with control (no supplements) oocytes (18.8% GV, 42.0% MII). Forskolin (–eE) stimulated (P < 0.05) meiosis (6.1% GV, 81.7% MII), so it was not tested in subsequent experiments. The IBMX also inhibited (P < 0.05) eE-induced meiosis compared with control (–IBMX +eE) oocytes after 18 h (87.6 v. 27.1% GV, 2.5 v. 11.0% MII), 24 h (68.0 v. 11.9% GV, 5.6 v. 66.1% MII), and 30 h (58.1 v. 9.4% GV, 14.6 v. 64.4% MII) of culture. To evaluate the reversibility of IBMX, oocytes were cultured +IBMX –eE for 12 h and then transferred to medium –IBMX +eE. After 12 h of culture –IBMX +eE (24 h total), fewer (P < 0.05) IBMX-exposed oocytes were MII (6.6%) compared with control oocytes cultured for 24 h +eE –IBMX (70.2%). The proportion of IBMX-exposed oocytes reaching MII increased (P < 0.05) following 18 h of culture –IBMX +eE (30 h total; 49.4% MII), and by 24 h of culture –IBMX +eE (36 h total; 66.1% MII) was similar (P > 0.05) to the proportion of MII oocytes (78.3%) that had been cultured continuously for 36 h –IBMX +eE. Finally, oocytes were cultured for 0 h or 12 h +IBMX –eE followed by 24 h of culture –IBMX +eE, coincubated with frozen–thawed spermatozoa (5 × 10⁵ sperm mL^–1, 22 h), and the resulting embryos cultured (6% CO₂, 5% O₂) until day 7 post-insemination (Herrick et al. 2007 Biol. Reprod. 76, 858–870). The proportion of oocytes cleaving (83.0 v. 79.9%) and the proportions of oocytes (20.8 v. 18.7%) or embryos (25.2 v. 23.6%) developing to the blastocyst stage were not affected (P > 0.05) by 12 h of IBMX exposure during oocyte maturation. These results demonstrate that IBMX reversibly inhibits both spontaneous and eCG/EGF-induced meiosis in feline oocytes without compromising the oocyte’s developmental competence.