33 Assessment of spindle morphology and reactive oxygen species production after vitrification of bovine oocytes following invitro maturation in the presence of glutathione ethyl ester
Authors: García-Martínez T. , Vendrell-Flotats M. , Martínez-Rodero I. , Álvarez-Rodríguez M. , López-Béjar M. , Mogas T. 
Published: December 2019

Oocyte cryopreservation by vitrification affects spindle integrity and causes oxidative stress induced by reactive oxygen species (ROS). Glutathione (GSH) ensures the correct assembly of microtubules, prevents ROS from attacking tubulin assembly, and ensures normal spindle function during meiosis. We hypothesised that addition of GSH ethyl ester (GSH-OEt), a cell-permeable GSH donor, to IVM medium before vitrification may improve oocyte cryotolerance by maintaining spindle morphology and preventing ROS production. For the IVM, viable cumulus-oocyte complexes were randomly distributed into two groups: 1) control: conventional IVM media with tissue culture medium-199 + 20% fetal calf serum + epidermal growth factor and 2) GSH-OEt: IVM medium supplemented with 5mM GSH-OEt at 38.5°C in a 5% CO2 humidified air atmosphere. After 22h of IVM, half of the oocytes from each group were vitrified/warmed using the Cryotop method (Kitazato Corp.). After 24h of IVM, oocytes were denuded, fixed, and microtubule and chromosome distribution were analysed by immunofluorescence. To measure ROS, oocytes were denuded and incubated in 5μM 2′,7′-dichlorodihydrofluorescein diacetate. Fluorescence was acquired, and resulting images were quantified with ImageJ software. A linear mixed effect followed by a pairwise comparison test (Tukey adjustment) were performed to analyse differences in meiotic spindle configuration and ROS production (P&lt;0.05). No significant differences in percentages of MII or normal spindle configuration were observed among treatments. Treatment with GSH-OEt before vitrification resulted in similar percentages of abnormal spindle configuration to control oocytes, whereas vitrified oocytes showed significantly higher percentages of abnormal spindle configuration relative with control oocytes (Table 1). When the content of ROS was measured, vitrification resulted in significantly higher levels (15.1E+06±1.2E+06; P&lt;0.05) when compared with other treatments. However, oocytes vitrified after IVM with GSH-OEt (11.8E+06±0.6E+06) showed similar ROS levels to control (10.7E+06±0.2E+06) and GSH-OEt (10.0E+06±0.1E+06) groups. In conclusion, addition of GSH-OEt to IVM before vitrification did not have detrimental effects on spindle morphology and was able to reduce ROS content. Further experiments are warranted to assess whether the addition of GSH-OEt to IVM medium can improve oocyte development competence after vitrification.

Table 1.Assessment of spindle morphology after oocyte vitrification (VIT) following IVM in the presence of glutathione ethyl ester (GSH-OEt)

Item
n
MII (%)
Spindle configuration (%)
Chromosome distribution (%)

Normal
Abnormal
Absent
Dispersed
Decondensed
Absent

Control
127
79.4±92
74.1±4.4
16.2±5.6a
9.7±1.1
16.2±3.8
9.7±1.1
0

GSH-OEt
113
76.5±6.5
65.6±5.7
26.2±9.0ab
8.1±4.3
19.5±2.6
13.7±3.0
1.1±1.1

VIT
71
60.0±5.6
54.8±7.8
32.7±10.8b
12.4±1.4
34.4±8.7
10.7±6.4
0

GSH-OEt VIT
53
57.4±10.7
58.5±8.8
31.1±9.3ab
10.4±5.8
34.8±5.6
6.7±6.7
0

a,bDifferent superscripts indicate significant differences (P&lt;0.05). *Percentage referred to the total number of oocytes at MII.

This work was supported by MCIU, Spain (AGL2016-79802-P) and Generalitat de Catalunya (García-Martínez, 2017_FI_00451).