Cathepsin activities and membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 degrees C using controlled slow cooling

This study investigated enzymatic activity of cathepsins and the membrane integrity of zebrafish (Danio rerio) oocytes after freezing to −196 °C using controlled slow cooling. Stage III oocytes (>0.5 mm), obtained through dissection of anaesthetised female fish and desegregation of ovarian cumulus, were exposed to 2M methanol or 2 M DMSO (both prepared in Hank’s medium) for 30 min at 22 °C before being loaded into 0.5 ml plastic straws and placed into a programmable cooler. After controlled slow freezing, samples were plunged into liquid nitrogen (LN) and held for at least 10 min, and thawed by immersing straws into a 27 °C water bath for 10 s. Thawed oocytes were washed twice in Hank’s medium. Cathepsin activity and membrane integrity of oocytes were assessed both after cryoprotectant treatment at 22 °C and after freezing in LN. Cathepsin B and L colorimetric analyses were performed using substrates Z-Arg-ArgNNap and Z-Phe-Arg-4 MβNA-HCl, respectively, and 2-naphthylamine and 4-methoxy-2-naphthylamine were used as standards. Cathepsin D activity was performed by analysing the level of hydrolytic action on haemoglobin. Oocytes membrane integrity was assessed using 0.2% Trypan blue staining for 5 min. Analysis of cathepsin activities showed that whilst the activity of cathepsin B and D was not affected by 2 M DMSO treatment, their activity was lowered when treated with 2M methanol. Following freezing to −196 °C, the activity of all cathepsins (B, D and L) was significantly decreased in both 2 M DMSO and 2 M methanol. Trypan blue staining showed that 63.0 ± 11.3% and 72.7 ± 5.2% oocytes membrane stayed intact after DMSO and methanol treatment for 30 min at 22 °C, respectively, whilst 14.9 ± 2.6% and 1.4 ± 0.8% stayed intact after freezing in DMSO and methanol to −196 °C. The results indicate that cryoprotectant treatment and freezing modified the activities of lysosomal enzymes involved in oocyte maturation and yolk mobilisation.