87 EFFECT OF CRYOPROTECTANT CONCENTRATION IN THE VITRIFICATION SOLUTION ON THE ZONA PELLUCIDA HARDENING AND SPERMATOZOA PENETRATION OF BOVINE OOCYTES

Abstract

In the murine model, it has been shown that the high concentration of cryoprotectants required for vitrification can activate the oocytes through a process mediated by calcium influx. This activation induces the zona pellucida (ZP) hardening and affects the sperm penetration. This study aimed to evaluate the effect of exposure of bovine oocytes to the vitrification solutions (VS1 and VS2) in calcium-free medium with 3 concentrations of etilenglycol (EG) and dimetylsulfoxide (DMSO) on the oocyte activation. Cumulus oocyte complexes (COC) were matured in vitro (22 h), partially denuded through pipetting in medium with hyaluronidase, and subject to four treatments: T1, untreated (control); T2, exposed to 20% EG+0% DMSO (VS1) and then 40% EG+0% DMSO (VS2); T3, 10% EG+10% DMSO (VS1) and then 20% EG+20% DMSO (VS2); and T4, 0% EG+20% DMSO (VS1) and then 0% EG+40% DMSO (VS2). The contact with each VS was 3 min and 30 s, respectively. After this, the COC were matured up to 24 h. In Expt. 1, COC were denuded and placed in a solution of pronase E in PBS (1 mg mL^–1) to determine the number of oocytes with ZP digested after 9 min of exposure to the enzyme. In Expt. 2, COC were fertilized in TALP medium with 50 mg mL^–1 heparin and 1 million mL^–1 sperm. After 12 h, COC were denuded and stained with bisbenzimide (Hoechst 33342) and examined under epi-fluorescence. The number of oocytes indicating spermatic penetration was determined by presence of intact sperm heads, spermatic pro-nucleus, or 2 polar bodies. Data were analysed by the PROC GENMOD (SAS; see Table 1). In Expt. 1, there were no differences in the percentage of oocytes without ZP after pronase treatment in groups T1, T2, and T3. The T4 group had the lowest percentage of digestion, and T3 was not different from T4. In Expt. 2 there were no differences in the percentage of sperm penetration between T2, T3, and T4. All treatments had lower values than T1. In conclusion, bovine oocytes undergo hardening of the ZP when put in contact with the cryoprotectants, and this effect was significantly increased with the use of DMSO. Moreover, there was a decrease in sperm penetration in all treated groups, indicating that the natural blocking of polyspermy depends not only on the hardening of the ZP, but another process that could act at the plasma membrane. It is possible that cryoprotectants, regardless of their concentration, may trigger this early block through a mechanism that would be independent of calcium.

Table 1.  Effect of EG and DMSO concentration in the VS on the ZP hardening and sperm penetration of bovine oocytes exposed to these solutions