85 VITRIFICATION OF IN VITRO-MATURED PORCINE OOCYTES AT THE METAPHASE-II STAGE

Abstract

Cryopreservation of mammalian metaphase-II (M-II) oocytes is still impractical compared to that of early stage embryos. In this study we examined the effects of delipation and mitotic spindle stabilization in order to improve the post-vitrification survival rate of in vitro-matured (IVM) porcine oocytes at the M-II stage. Cumulus–oocyte complexes that had been collected from slaughterhouse ovaries were matured in vitro in NCSU23 supplemented with 0.6 mm cysteine, 10 ng mL^–1 epidermal growth factor (EGF), 10% porcine follcular fluid (PFF), and 10 IU mL^–1 eCG and hCG. The denuded M-II oocytes were vitrified in the presence of 30% ethylene glycol and 0.5 m sucrose using the minimum volume cooling (MVC) method with a MVC plate (Cryotop^®; Kitazato Supply, Tokyo, Japan). Vitrified embryos were rewarmed by immersing the MVC plate directly into rewarming solution containing 1 m sucrose and 20% calf serum at 39°C for 1 min, followed by stepwise dilution of the cryoprotectants. We compared the effects of previtrification treatments, namely, (1) delipation, (2) mitotic spindle stabilization, (3) delipation + mitotic spindle stabilization, and (4) no treatment. For delipation, we used a noninvasive method (Esaki et al. 2004 Biol. Reprod. 71, 432–437) that we had published previously with slight modification. The embryos were treated with 4% trypsin at 38°C for approximately two min to expand the zona pellucida, and then centrifuged (12 000g, 38°C 23 min) with 7.5 µg mL^–1 cytochalasin B to polarize cytoplasmic lipid droplets within the perivitelline space. For mitotic spindle stabilization, M-II oocytes were vitrified in the presence of 1 µm paclitaxel. After the oocytes were rewarmed, electrical activation of the oocytes (150 V mm^–1, 100 µs, one time) was carried out to induce parthenogenesis. These parthenogenetic embryos were cultured in PZM-5 for 7 days, and the number of vitrified embryos that developed into blastocysts with respect to each treatment was determined. The blastcyst formation rate and mean cell numbers of the blastcysts were compared among the treatment groups (chi-square test, Tukey's test). Of the 50 M-II oocytes that had been vitrified without pretreatment, only one oocyte (2.0%) developed into a blastocyst with 20 cells. By contrast, the number of vitrified embryos that developed into blastocysts was significantly high when they were delipated prior to vitrification (37.8%, 14/37, 64.0 ± 9.6; P < 0.01). Mitotic spindle stabilization also improved the survival rate of vitrified oocytes (18.6%, 21/113, 56.7 ± 9.6; P < 0.01). The combination of delipation and mitotic spindle stabilization produced the highest number of vitrified oocytes that developed into blastocysts (43.8%, 35/80, 69.4 ± 6.4), although the difference between the combination group and the delipation group was not significant. These results indicate that blastocysts can be produced very efficiently from IVM porcine oocytes that have been vitrified at the M-II stage using both noninvasive delipation and mitotic spindle stabilization procedures.