Vitrifying equine oocytes at the germinal vesicle stage disturbs spindle morphology and chromosome alignment

Cryopreserving horse oocytes compromises the ability of immature oocytes to complete meiosis, and reduces the post-fertilization developmental competence of mature oocytes. Previous studies suggest that this reduced competence is, at least in part, related to a compromised ability to form a normal microtubular spindle and complete the meiotic divisions. To better understand the effects of cryopreservation on microtubule spindle function, we examined the effects of vitrifying germinal vesicle stage oocytes on meiotic spindle organization and chromosome alignment after maturation in vitro. Cumulus oocyte complexes from slaughtered mares were either matured in vitro for 26 h (n = 303) or vitrified, warmed and then matured in vitro (n = 260). Before vitrification, the cumulus investment was de-bulked until only the innermost corona radiata remained. The partially denuded, immature oocytes were then equilibrated in 3 steps with vitrifying solution, the last step of which was for 45 s at 37 °C in TCM-199 containing 20 % ethylene glycol, 20 % dimethyl sulphoxide and 0.5 M sucrose, before plunging into liquid nitrogen. Oocytes were warmed in TCM-199 supplemented with 0.5 M sucrose, and subsequently matured in vitro for 26 h. After maturation, oocytes were denuded and evaluated for first polar body extrusion, as an indicator of successfully reaching the metaphase II stage (MII). Spindle morphology and chromosome alignment in MII oocytes (n = 63 vitrified; n = 53 control) were then evaluated by confocal microscopy after fixation and immunofluorescent staining for DNA (Hoechst 33342) and α-tubulin (mouse monoclonal anti-α-tubulin antibody T5168 conjugated to Alexa Fluor 488). Spindle morphology (shape and dimensions), metaphase plate thickness and chromosome alignment were evaluated using 3D-image analysis software (Imaris 8.2). Spindle morphology was scored as normal (fusiform, bipolar) or abnormal (tri- or tetra-polar; absence of spindle formation), chromosome misalignment was scored as absent, mild (1 - 5 misaligned chromosomes) or severe (> 5 misaligned chromosomes). Vitrification reduced the proportion of oocytes that reached MII compared to the control group (30 % vs. 44 %; P < 0.05). Vitrification also resulted in a reduced length (16.2 μm ± 0.9 μm vs. 21.1 μm ± 0.7 μm; mean ± SE; P < 0.001) and volume (751 μm3 ± 67 μm3 vs. 1255 μm3 ± 102 μm3; P < 0.001) of the spindle, and in an increased metaphase plate thickness (6.2 μm ± 0.2 μm vs. 5.6 μm ± 0.2 μm; P < 0.05). The results demonstrate that oocyte vitrification induces irreversible ultrastructural changes to the microtubular spindle, impairing correct chromosome alignment. This presumably interferes with normal chromosome segregation and predisposes to aneuploidy. These phenomena are likely to contribute to the reduced developmental competence of vitrified, immature equine oocytes