(T674) MORPHOLOGICAL STATUS OF ACTIVE MITOCHONDRIA IN VITRIFIED BOVINE GV OOCYTES AFFECTS SUBSEQUENT DEVELOPMENT.
Abe, Yasuyuki1, Aono, Nobuya2, Hara, Kenshiro, Matsumoto, Hiromichi1, Sasada, Hiroshi1, Sato, Eimei1, 1 Tohoku University, Sendai, Japan2 Yoshida Lady’s Clinic, Sendai, Japan
ABSTRACT- In the previous report, we demonstrated that using a nylon mesh container bovine germinal stage (GV) oocytes vitrified showed in vitro maturation rate to MII stage as same as that in non-vitrified ones but low cleavage and developmental rates after in vitro fertilization and culture. Since mitochondria are thought to have a central role in mammalian embryogenesis, it seems possible that alternation in mitochondrial function in vitrified GV oocytes may be a cause in subsequent lower cleavage and developmental rates. In this study, to ascertain this possibility, we evaluated status of active mitochondria in vitrified oocytes, and then examined the relationship between it and developmental capacity. Bovine GV oocytes were collected as cumulus-oocytes complexes (COCs) from follicles of 2-5 mm in diameter in ovaries, and were subjected to the experiments. Before vitrification, COCs were exposed to the cryoprotectant (EFS40) by step-wise way, and 40 of them each were transferred onto a nylon mesh container, followed by plunging them directly into liquid nitrogen. After thawing vitrified COCs, they were allowed for in vitro maturation. After denuding cumulus cells, matured oocytes having 1st polar body were immunocytochemically stained with a mitochondria-specific probe, rhodamine 123, and then were observed by a confocal laser scanning microscopy. According to morphological status of mitochondria, they were classified as follows: type 1; uniform distribution, type 2; spotted distribution, and type 3; a weak fluorescence. In the non-vitrified, the percentages of type 1, 2 and 3 were 59.4, 31.3 and 9.4%, respectively, while in the vitrified oocyte the corresponding percentages were 34.3, 31.4 and 34.3%, respectively. There was a significant difference between them showing type 1. After IVFC, only the matured oocyte that had been classified into type 1 developed to blastocysts in both non-vitrified and vitrified oocytes (10.3% and 1.4%, respectively). These results indicate that the morphological status of active mitochondria in matured oocytes may be responsible for their cleavage and developmental capacity, and that vitrification may affect the intercellular distribution of mitochondria in bovine GV oocytes even though they can be matured in vitro.
KEY WORDS: vitrification, GV oocyte, active mitochondria, bovine