In vitro viability and ultrastructural changes in bovine oocytes treated with a vitrification solution

Abstract

Abattoir-derived oocytes were exposed to a concentrated cryoprotectant solution (DAP213: 2 M DMSO, 1 M acetamide, 3 M propanediol, and 10% FCS in TCM199) for 1.5 or 5 min at the germinal vesicle (GV) stage or after maturation in vitro (IVM). Their viability was assessed by in vitro fertilization (IVF) and culture (IVC) to blastocysts. To investigate the effect of DAP213 on the ultrastructure, GV and IVM oocytes were processed for transmission electron microscopy (TEM) before (control) or after exposure to the cryoprotectant. DAP213 induced profound ultrastructural modifications to the microvilli and mitochondria, resulted in large vesicle formation, and, most significantly, caused the premature release of the cortical granules (CG). In IVM oocytes exposed to the cryoprotectant for 5 min, exocytosis of CG into the perivitelline space was common and the IVF rate was reduced (P < .05). After exposure for 5 min, GV oocytes displayed clusters of CG comparable to controls, but after IVM-IVF, polyspermy rate was increased (P < .05). Furthermore, treated GV oocytes showed a reduced rate of cleavage and blastocyst formation and an increased percentage of oocytes exhibiting alterations in organelles, whereas the viability and ultrastructure of IVM oocytes treated for 1.5 min was not different from controls. These observations demonstrate that 1) cortical granule kinetics is one of the key elements controlling fertilizability of bovine oocytes treated with cryoprotectant, and 2) GV oocytes are more sensitive to the cryoprotectant than those that have already been matured in vitro.

Bovine oocyte vitrification before or after meiotic arrest: effects on ultrastructure and developmental ability

“…These alterations are consistent with previous findings [11,36]. Interestingly, the cytoplasmic structure of the vitrified oocyte was not altered, as evidenced by location and morphology of cellular membrane, organelle, CG, lipid droplets, mitochondria and cytoplasmic vesicles, resembling structures in fresh oocytes.…”

“…Degeneration percentages are lower and cleavage rates are higher that those obtained by other authors [15,32,35], although development up to the morula and blastocyst stage is compromised. Such as observed in our work, a low correlation between cleavage and further embryonic development has been previously stated [9,36]. Nevertheless, cleavage rates higher than 45% for oocytes vitrified both as immature and matured have been reported by other researchers [9,16].…”

“…In the mature oocyte, however, close coupling is no longer essential, and the oocyte may be less vulnerable to functional damage by exposure to cryoprotectant or cryopreservation. Therefore, immature oocytes seem to be more vulnerable to cryodamage than matured oocytes [36], which contributes to explain our results. As previously demonstrated in mouse [29], exposure to high CP concentrations may trigger premature release of CG, altering their distribution after warming and IVM [49] and resulting in hardening of the ZP with decreased fertilization outcomes [50,51].…”

“…Apoptosis [10,26,36,52] and disorganization of the MII spindle as a cause of chromosomal aberrations [31,52,53], have been observed in oocytes after vitrification procedures. In addition, ZP may be either broken or at least modified by the premature release of CG, as seen in frozen-thawed human and mouse oocytes [44,54].…”

Effect of Braun-Collins and Saline solutions at different temperatures and incubation times on the quality of goat preantral follicles preserved in situ

“…These vacuoles represent endoplasmic reticulum swelling. The presence of vacuoles was also observed in equine oocytes matured in vitro (Willis et al, 1994) and bovine oocytes after exposure to vitrification solution (Fuku et al, 1995).…”