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 invitro during shipment. At 18h of maturation, oocytes were divided randomly into control, taxol, and epothilone B treatments (Table 1). All treatments were prepared in invitro maturation (IVM) medium (IVF Biosciences). Partially denuded oocytes were incubated in either control or treatment medium for 15min at 38.5°C before vitrification. Oocytes were incubated in an equilibration solution (10% dimethyl sulfoxide, 10% ethylene glycol) for 5min, transferred to a vitrification solution (20% dimethyl sulfoxide, 20% ethylene glycol, 0.5M sucrose), loaded onto a Cryolock, and plunged into liquid nitrogen within 45s. For warming, a Cryolock was placed directly into a 0.5M sucrose solution and incubated for 3min. Oocytes were transferred to a 0.25M solution for 3min 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 0.5μM and 1.0μM Taxol treatments had no effect on either meiotic spindle or chromosome arrangement compared with the control group (P>0.05). The 2.0μM 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 invitro-matured bovine oocytes Treatment n Normal microtubule distribution (%) Normal chromosome arrangement (%) Control 100 44 47 0.5μM Taxol 104 44 37 1.0μM Taxol 98 43 56 2.0μM Taxol 102 49 62a 0.5μM Epothilone B 103 11b 11b 1.0μM Epothilone B 97 6b 8b 2.0μM Epothilone B 100 2b 1b aP<0.05;. bP<0.001: Different superscripts within a column indicate a significant difference.