25 Effect of taxol and epothilone B on meiotic spindle stabilization in vitrified bovine oocytes

Abstract

Vitrification has the potential to be a valuable technique for preservation of bovine oocytes; however, this method often results in abnormal microtubule and chromosome arrangement. The aim of this experiment was to evaluate taxol and epothilone B as meiotic spindle stabilising pretreatments in a vitrification protocol. Bovine oocytes were purchased and matured in vitro during shipment. At 18 h of maturation, oocytes were divided randomly into control, taxol, and epothilone B treatments (Table 1). All treatments were prepared in in vitro maturation (IVM) medium (IVF Biosciences). Partially denuded oocytes were incubated in either control or treatment medium for 15 min at 38.5°C before vitrification. Oocytes were incubated in an equilibration solution (10% dimethyl sulfoxide, 10% ethylene glycol) for 5 min, transferred to a vitrification solution (20% dimethyl sulfoxide, 20% ethylene glycol, 0.5 M sucrose), loaded onto a Cryolock, and plunged into liquid nitrogen within 45 s. For warming, a Cryolock was placed directly into a 0.5 M sucrose solution and incubated for 3 min. Oocytes were transferred to a 0.25 M solution for 3 min and washed in the basal solution used for vitrification and warming media (Dulbecco’s phosphate-buffered saline, 20% fetal bovine serum). Once warmed, oocytes were transferred to IVM medium for a 4-h recovery period and completely denuded before staining. Staining to evaluate spindle morphology was performed with anti α-tubulin primary antibody and secondary antibody Alexa Fluor 488. Oocytes were also stained with Hoechst to evaluate chromosome arrangement. Both spindle morphology and chromosome arrangement data were analysed using a logistic regression with a binomial response variable (normal/abnormal). Both 0.5 μM and 1.0 μM Taxol treatments had no effect on either meiotic spindle or chromosome arrangement compared with the control group (P > 0.05). The 2.0 μM taxol treatment improved chromosome configuration (P < 0.05) with no effect on microtubule distribution compared with the control group (P > 0.05). All epothilone B treatments resulted in disruption of microtubule distribution and chromosome arrangement compared with control (P < 0.001) and resulted in a consistent abnormality hypothesised to be tubulin polymerization. These results indicate that taxol is capable of increasing the occurrence of normal chromosome arrangement in vitrified bovine oocytes and that epothilone B may cause additional harm to the oocyte that is not associated with the metaphase plate.

Table 1.  Effect of stabilisation agents on meiotic spindle of in vitro-matured bovine oocytes