The effect of poly-L-lysine as a new cryoprotectant for ovine oocyte vitrification.

摘要

The objective of this study was to evaluate polyampholyte---Poly--L--lysine (PLL) as a new cryoprotectant for ovine oocyte vitrification, alternative to the traditionally used cryoprotectant dimethyl sulfoxide (DMSO). Recently, Matsumura et al. (2009) demonstrated that PLL effectively protects L929 mouse stem cells during cryopreservation. Oocytes were screened and collected from fresh ovine ovarian tissues, then randomly classed into two developmental stages, namely, germinal vesicle (GV) stage oocytes (immature) and metaphase 2 (M2) stage oocytes (mature). GV stage oocytes were vitrified immediately upon collection, while M2 stage oocytes were vitrified after 24 h in vitro culture for oocyte maturation. Each developmental group was subjected to two different vitrification solutions: (1) PLL based formula (5%, 10% PLL); (2) and DMSO based formula (10%, 20% DMSO, ethylene glycol, and holding medium), resulting in four groups: PLL: GV, PLL-M2, DMSO-GV, and DMSO-M2. In the first experiment, there are six replicated batches (block) for each group, and each batch was processed on the same day under the same condition. The normality of developing oocytes was examined under microscope. Normal oocytes showed the characteristics of homogeneous cytoplasm and intact zona pellucid, with abundant organelles uniformly dispersed within the plasma; while abnormal oocytes displayed a reduced amount of organelles and low density of granular cells. The data were analyzed with proc Glimmix procedure of SAS. The results showed that the oocyte survival rate (%) of PLL-M2 (80.13) was significantly (P0.05) higher than PLL-GV (62.28); DMSO-M2 (63.99) was significantly (P0.05) higher than DMSO-GV (40.50), whereas PLL-GV was significantly (P0.05) higher than DMSO-GV, and PLL-M2 was in turn significantly (P0.05) higher than DMSO-M2. There was no difference in survival rate after vitrification between group PLL-GV and DMSO-M2. For the trypan blue staining comparison, the number of PLL-M2 (25+/-2.89) was significantly (P0.05) higher than PLL-GV (16+2.31). There was no significant difference between the density of the PLL based formula and DMSO based formula, at the same time, there were no different injuries found among layer 1, 2 and 3. However, more frequent injuries were found in vitrified groups than the control groups. In the process of vitrification, the removal of granular cell after 24 h in vitro maturation appeared to be important to the success of cryopreserved oocytes and more disrupted spindle with dispersion of chromosomes were found in GV stage vitrified groups than M2 stage ones. Furthermore, M2 stage oocytes have shown a high viability. In conclusion, PLL can be used as an alternative reagent for ovine oocytes vitrification, with a high efficiency in a lower concentration compared with the traditional DMSO protocol.