244 AMPHIREGULIN CAN ASSIST IMMATURE PORCINE OOCYTES TO DEVELOP IN AN IN VITRO SYSTEM

Abstract

Porcine IVM-IVF technique has been used and improved. However, polyspermic penetration, low rate of blastocyst (BL) formation, and poor quality of BLs are induced by imperfect nuclear and cytoplasmic maturation. It has been reported that epidermal growth factor (EGF) is beneficial for oocyte maturation to improve the IVM system, and amphiregulin (AR) is a growth factor containing an EGF-like domain. Consequently, the present study was performed to investigate whether TCM199 supplemented with EGF and/or AR is profitable for improving oocyte maturation and embryo development. The experimental groups were control (A), EGF 15 ng mL^–1 (B), AR 15 ng mL^–1 (C), and EGF 15 ng mL + AR 15 ng mL^–1 (D) in TCM199. In Experiment 1, oocytes were selected and maturated in each group. After 44 h, oocytes were stained with Hoechst, and metaphase II and development rate were evaluated. In Experiment 2, maturated oocytes were fertilized with proven sperm and stained with Hoechst; the penetration rate after 10 h of post-insemination was evaluated. In Experiment 3, after 168 h of in vitro culture, cleavage rate and BL formation rate were recorded. BLs were stained with differential stain and the cell numbers calculated. In Experiment 4, porcine oocytes were maturated for 44 h and denuded by hyaluronidase. Thereafter, oocytes were stained with fluorescein isothiocyanate-labeled peanut agglutinin and the distribution of cortical granules (CGs) was evaluated by laser confocal microscopy. All data was analyzed by a Duncan test using SPSS (SPSS, Inc., Chicago, IL, USA). In Experiment 1, the maturation rate into metaphase II stage in groups B, C, and D (71.0 ± 15.5%, 75.8 ± 9.8%, and 71.5 ± 10.8%, respectively) was significantly different (P < 0.05) from that of group A (51.4 ± 18.3%). In Experiment 2, there was no difference among all groups in relation to penetration rate. But polyspermy was decreased (P < 0.05) in group D (26.2 ± 11.3%) compared with group A (42.3 ± 13.0%). In Experiment 3, there was no significant difference among all groups regarding the cleavage rate. However, BL developmental rates in group D (17.3 ± 11.5%) were increased (P < 0.05) compared with groups A and B (9.6 ± 3.2% and 13.3 ± 4.6%, respectively). In groups B, C, and D, the cell number in BL increased significantly (P < 0.05) compared with that of group A(81.4 ± 32.0%, 79.7 ± 27.3%, and 80.3 ± 28.5% v. 56.3 ± 25.5%). In Experiment 4, group D had the largest CG area among the groups. The results of this study indicate that the addition of EGF or AR or both to TCM199 promotes oocyte maturation and increases the total cell number. EGF plus AR increases development to the BL stage and decreases polyspermy. It is suggested that AR can assist immature porcine oocytes to the metaphase II stage, that AR may enhance developmental ability in the in vitro system, and that there is a synergistic effect between EGF and AR during the maturation period.