48 Effect of Dimethyl Sulfoxide- or Glycerol-Based Vitrification Protocols on Meiotic Spindle of In Vitro-Matured Bovine Oocytes

Abstract

There is evidence suggesting that high concentrations of cryoprotectants (CPA) and very low temperatures during vitrification cause disruption of the meiotic spindle, resulting in poor post-warming meiotic resumption and other abnormalities at fertilization. This study sought to determine the damage caused by CPA and freezing upon the meiotic spindle of bovine oocytes vitrified at the metaphase II stage, and whether a subsequent incubation could promote recovery from this damage. Bovine cumulus–oocyte complexes were purchased from a commercial vendor (n = 154). Oocytes were removed from in vitro maturation media at 22 h, denuded by vortexing in hyaluronidase, and divided into 4 groups according to CPA exposure and whether they were incubated or not. The resulting groups were DMSO I (n = 36), DMSO NI (n = 41), GLY I (n = 39), GLY NI (n = 38). Two repetitions were carried out for each protocol evaluated, which included a combination of ethylene glycol (EG) with either dimethyl sulfoxide (DMSO) or glycerol (GLY). Oocytes were exposed to equilibration solution consisting of 7.5% EG and 7.5% DMSO or GLY for 9 min at room temperature (RT) and then placed into vitrification solution (VS) that contained 15% EG, 15% DMSO or GLY, and 0.5 M sucrose. While in VS, 3 to 4 denuded oocytes were loaded onto a Cryolock^® (Biotech Inc., Alpharetta, GA, USA) and plunged into liquid nitrogen within 1 min. For warming, oocytes were exposed to previously warmed (37°C) dilution solution 1 (DS1) consisting of 0.5 M sucrose for 1 and 2 min at RT, for a total of 3 min in DS1, and then placed in dilution solution 2 containing 0.25 M sucrose for 3 min. Finally, oocytes were washed in base media. Base media for all solutions was PBS supplemented with 20% fetal bovine serum. After warming, half of the oocytes were fixed and the rest were submitted to a 2-h incubation period in maturation media at 37°C and 5.5% CO₂, and then fixed. To examine microtubule distribution and chromosome arrangement, fixed oocytes were submitted to an immunofluorescence protocol using α-tubulin antibody (1:100) as primary antibody, Alexa Fluor 488 (1:1000) as secondary antibody, and counterstained with propidium iodide (10 mg mL⁻¹). Oocytes were observed under a fluorescence microscope. The effects of CPA and incubation on the incidence of abnormal spindles measured with both microtubules distribution and chromosome arrangement were evaluated using logistic regression with a binomial response variable (normal/abnormal). For microtubule distribution, results showed that oocytes treated with DMSO presented significantly lower normality (31.17%) than those treated with glycerol (54.55%; P < 0.003). The most common abnormality observed in oocytes treated with DMSO was that the spindle was smaller and more faintly stained than those treated with glycerol. For chromosome arrangement, there was no significant difference between treatments (P = 0.7093). Additionally, there was no sign of improvement when submitting the oocytes to an incubation period for any of the components examined.