168 IMPROVED MITOCHONDRIAL MEMBRANE POTENTIAL AND PRE-IMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS BY DIBUTYRYL cAMP TREATMENT

Abstract

Mitochondria play a pivotal role in energy metabolism and apoptosis during embryo development. In general, cAMP that exists at high level in GV oocytes inhibits germinal vesicle breakdown (GVBD), and the amount of cAMP in oocyte cytoplasm is gradually decreased for meiotic resumption. We first examined the effects of dibutyryl cAMP (dbcAMP) on nuclear maturation, fertilization, and early embryonic development. To determine whether mitocondrial activity is related to embryonic development, mitochondrial membrane potential (ΔΨm) was measured in porcine embryos. Porcine oocytes were cultured in NCSU-23 medium supplemented with 1 mM dbcAMP for 22 h and further cultured without dbcAMP for 22 h. After in vitro fertilization, porcine eggs were cultured in NCSU-23 medium with 4% BSA at 39°C, 5% CO₂ in air for 6 d. Porcine embryos were obtained at various developmental stages and stained with the mitochondrial membrane potential-sensitive dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide). The mitochondrial membrane potential of porcine embryos was quantitatively evaluated by the ratio of green (presumptively alive mitochondria) to red fluorescent signal (presumptively dead mitochondria) using a fluorescence microscope. Acquired images were analyzed using DeltaVision Software (Applied Precision, Inc., Hsin-Chu City, Taiwan, ROC). After completion of IVM, dbcAMP-treated oocytes (91.3 ± 0.9%) showed a higher proportion of the metaphase II stage than nontreated ones (72.8 ± 2.6%) (P < 0.05). In the dbcAMP-treated group, sperm penetration rate was increased and polyspermic rate was reduced as compared to the nontreated group. Furthermore, the rate (37.3%, 47/126) of blastocyst formation in dbcAMP-treated group was higher than that (19.2%, 28/146) of the nontreated group (P < 0.05). After JC-1 staining, the number of blastomeres having live mitochondria per embryo increased in the dbcAMP-treated group at various developmental stages, whereas the number of blastomeres having dead mitochondria per embryo was enhanced in the nontreated group (43.3% vs. 30.2%). Our results indicate that in vitro maturation of porcine oocytes may affect mitochondrial membrane potential, apoptosis, and embryonic quality during pre-implantation development.