43 EFFECT OF VITRIFICATION AT GERMINAL VESICLE STAGE ON THE MITOCHONDRIAL AND CYTOSKELETAL INTEGRITY IN BOVINE OOCYTES

Abstract

In the previous report, we demonstrated that bovine germinal vesicle (GV) stage oocytes vitrified using a nylon mesh holder showed an in vitro maturation rate to the metaphase II (MII) stage similar to that of fresh ones. However, cleavage and developmental rates of vitrified oocytes were low. Because mitochondria and the cytoskeleton are thought to have a central role in energy supply and cellular division in mammalian embryogenesis, it seems possible that alternation in their function in vitrified GV oocytes may contribute to subsequent lower cleavage and developmental rates. The oxygen consumption rate reflects the mitochondrial activity and its measurement may be an effective way for non-invasive evaluation of oocyte quality. In this study, to ascertain that altered mitochondrial functions and cytoskeleton may contribute to reduce the quality of oocytes after vitrification, we evaluated the distribution of active mitochondria and the cytoskeleton in vitrified oocytes. We also examined the relationship between oxygen consumption rate and the distribution of active mitochondria in vitrified oocytes. Bovine GV oocytes connected with cumulus cells were exposed to the cryoprotectant (EFS40) in a stepwise way and transferred onto a nylon mesh holder, followed by plunging them directly into liquid nitrogen. After warming, vitrified oocytes were allowed in vitro maturation. After denuding, matured oocytes were stained with a mitochondria-specific probe, rhodamine-123 and then oxygen consumption rate using an embryo respirometer (HV-403; Research Institute for Functional Peptides, Yamagata, Japan) was measured in each oocyte. According to morphological distribution of mitochondria, oocytes were classified as follows: type 1, uniform distribution; type 2, spotted distribution; and type 3, a weak fluorescence. The oxygen consumption rate of the fresh oocytes at the MII stage was significantly (P < 0.05) higher than that of vitrified oocytes (5.24 and 4 × 10¹⁵ mol^–1 s^–1, respectively), although there was no difference between the fresh and vitrified groups at the GV stage (5.02 and 5.06 × 10¹⁵ mol^–1 s^–1, respectively). The oxygen consumption rates of type 1 oocytes in fresh and vitrified groups at the MII stage tended to be higher than those of type 2 and 3 oocytes (type 1, 5.29 and 5.27; type 2, 4.99 and 4.52; type 3, 4.77 and 4.48 × 10¹⁵ mol^–1 s^–1, respectively). In addition, the percentage of type 1 oocytes in the fresh group was significantly (P < 0.05) higher than that in the vitrified group (59.4 and 34.3%, respectively). The matured oocytes also were stained with α-tubulin monoclonal antibody or F-phalloidin independently to examine the morphological status of microtubules or microfilament. The rates of oocytes with abnormal microtubules and microfilament in the vitrified group were 29.7 and 43.5%, respectively, showing higher rates compared with corresponding fresh oocytes (9.8%; P < 0.05 and 25.0%; P = 0.21, respectively). These results suggested that the reduction of quality and subsequent developmental competence in vitrified oocytes might be related to damages of mitochondria and cytoskeleton.