Oocyte cryopreservation an analysis of cyclin B and map kinase protein levels as an indicator for oocyte activation following cryopreservation

Abstract

This study evaluated cell cycle protein dynamics in oocytes following cryopreservation by either vitrification in calcium containing and calcium free mediums or controlled rate freezing. Using a murine model, the effect on oocyte survival and activation was assessed using western blot analysis to evaluate changes in cell cycle proteins. Prospective comparative study. Oocyte-cumulus complexes isolated from superovulated FVB mice were denuded with 80 U/ml Cumulase™. Metaphase II oocytes (n = 830) were alternately allocated into four groups: 1) Control – not cryopreserved, 2) Controlled-rate, 3) Vitrification with calcium (Vit with Ca+), and 4) Vitrification without calcium (Vit without Ca+). Western Blot analysis was performed on groups of five oocytes per treatment for quantitative assessment of cyclin B and MAPK protein levels. Comparisons between cryopreservation protocols are presented in the table; individual variables represent means ± SEM after normalization to non-cryopreserved oocytes. Vitrified oocytes had a significantly higher survival rate compared to oocytes frozen by controlled rate (P<0.01a,b). Vitrification without calcium resulted in a decrease in cyclin B at 0 hours post thaw compared to vitrification with calcium and controls (P=0.06c,d). MAPK levels did not differ statistically between treatments (P=0.24). Tabled 1Table 1TreatmentOocytes ThawedOocytes Survived (%)0 hr post-thaw Cyclin B4 hr post-thaw Cyclin B0 hr post-thaw MAPK4 hr post-thaw MAPKControls (Fresh)—238 (100)100100100100Vit with Ca+182174 (96)a106 ± 38c65 ± 40132 ± 26172 ± 63Vit without Ca+178168 (94)a29 ± 7d231 ± 138163 ± 40132 ± 74Controlled Rate23274 (32)bn/an/a69 ± 6103 ± 22 Open table in a new tab Results show that vitrification without calcium caused a reduction in cell cycle proteins 0 hours after cryopreservation, indicating cell cycle activation. A trend for higher levels of cyclin B is seen in the same treatment four hours post-warming, suggesting that the oocytes were able to restore prior protein levels. This indicates that oocyte activation was minimized, as a high level of cyclin B would prevent premature activation. MAPK levels were unaffected by these cryopreservation protocols. The novel approach of measuring changes in cell cycle proteins following cryopreservation can be used to determine cryodamage and degree of oocyte activation.