Effects of cryopreservation on the developmental competence, ultrastructure and cytoskeletal structure of porcine oocytes

The purpose of this study was to determine ultrastructural and cytoskeletal changes that result from vitrification of porcine germinal vesicle- (GV-) and meiosis II- (MII-) stage oocytes. To investigate the effects of vitrification on developmental competence, oocytes were divided into three groups: fresh GV-oocytes (control), vitrified GV-oocytes, and vitrified MII-oocytes. In both GV- and MII-oocytes, vitrification resulted in a high proportion with normal morphology (92.4 vs. 94.2%, P > 0.05), while vitrified GV-oocytes yielded a higher survival rate than did vitrified MII-oocytes (56.8 vs. 41.9%, P < 0.05). In vitrified GV-oocytes, 12 of 154 oocytes underwent cleavage after fertilization in vitro, and 6 of these developed to the 8-cell stage, 3 developed to the 16-cell stage, and 3 developed into morulae. No cleavage was obtained from vitrified MII-oocytes. For ultrastructural analysis of oocytes, fresh and vitrified-warmed GV- and MII-oocytes were randomly selected for transmission electron microscopy (TEM). Results showed that vitrification caused various degrees of cryodamage in GV-oocytes. Cumulus cells of some oocytes were separated from the cumulus-oocyte complex (COC), and the zona pellucida adjacent to cumulus cells was fractured. The gap junctions between cumulus cells were ruptured, and many microvilli were disrupted or disappeared. Only homogeneous lipid droplets were observed. After vitrification, cortical granules still lined the oolemma of MII-oocytes. Only morphologically irregular, nonhomogeneous lipid droplets surrounding large vacuoles were found. To examine cytoskeletal structures, fresh and vitrified-warmed MII-oocytes were analyzed by laser-scanning confocal microscopy (LSCM); vitrified-warmed GV-oocytes were cultured for 42-44 hr before LSCM. Of 58 control oocytes, 79.5% displayed normal spindles with chromosomes aligned along the equatorial plate. In vitrified oocytes the percentage with normal spindle organization was decreased significantly in both vitrified GV-oocytes and MII-oocytes (10.1 and 12.9%, respectively, P < 0.05). The proportion of oocytes with normal distribution of F-actin was lower for vitrified GV- and MII-oocytes than for controls (16.9 and 37.2% vs. 72.3%). Results of this experiment suggest that irreversible damage to the cytoskeleton of porcine GV- and MII-oocytes after vitrification could be an important factor affecting developmental competence.