title: Cathepsin activities and membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 degrees C using controlled slow cooling.
abstract: This study investigated enzymatic activity of cathepsins and the membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 degrees C using controlled slow cooling. Stage III oocytes (>0.5mm), obtained through dissection of anaesthetised female fish and desegregation of ovarian cumulus, were exposed to 2M methanol or 2M DMSO (both prepared in Hank's medium) for 30min at 22 degrees C before being loaded into 0.5ml plastic straws and placed into a programmable cooler. After controlled slow freezing, samples were plunged into liquid nitrogen (LN) and held for at least 10min, and thawed by immersing straws into a 27 degrees C water bath for 10s. Thawed oocytes were washed twice in Hank's medium. Cathepsin activity and membrane integrity of oocytes were assessed both after cryoprotectant treatment at 22 degrees C and after freezing in LN. Cathepsin B and L colorimetric analyses were performed using substrates Z-Arg-ArgNNap and Z-Phe-Arg-4MbetaNA-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 5min. Analysis of cathepsin activities showed that whilst the activity of cathepsin B and D was not affected by 2M DMSO treatment, their activity was lowered when treated with 2M methanol. Following freezing to -196 degrees C, the activity of all cathepsins (B, D and L) was significantly decreased in both 2M DMSO and 2M 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 30min at 22 degrees C, respectively, whilst 14.9+/-2.6% and 1.4+/-0.8% stayed intact after freezing in DMSO and methanol to -196 degrees C. The results indicate that cryoprotectant treatment and freezing modified the activities of lysosomal enzymes involved in oocyte maturation and yolk mobilisation.
authors.affiliation: Institute of Research in the Applied Natural Sciences, University of Bedfordshire, The Spires, 2 Adelaide Street, Luton, Bedfordshire LU1 5DU, UK. tiantian.zhang@luton.ac.uk
authors.forename: T
authors.initials: T
authors.lastname: Zhang
authors.forename: D M
authors.initials: DM
authors.lastname: Rawson
authors.forename: L
authors.initials: L
authors.lastname: Tosti
authors.forename: O
authors.initials: O
authors.lastname: Carnevali
mesh_terms: D000818:Animals; D002401:Cathepsin B / Q000378:metabolism; D002402:Cathepsin D / Q000378:metabolism; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology*; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005260:Female; D005615:Freezing; D000432:Methanol / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D015027:Zebrafish
chemical_list: D003451:Cryoprotective Agents; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; D056668:Cathepsin L; D002402:Cathepsin D; D000432:Methanol; D004121:Dimethyl Sulfoxide