PUBLICATION
Cathepsin activities and membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 degrees C using controlled slow cooling
- Authors
- Zhang, T., Rawson, D.M., Tosti, L., and Carnevali, O.
- ID
- ZDB-PUB-080306-13
- Date
- 2008
- Source
- Cryobiology 56(2): 138-143 (Journal)
- Registered Authors
- Rawson, David M., Zhang, Tiantian
- Keywords
- none
- MeSH Terms
-
- Animals
- Cathepsin B/metabolism
- Cathepsin D/metabolism
- Cathepsin L
- Cathepsins/metabolism*
- Cell Membrane Permeability/drug effects
- Cell Membrane Permeability/physiology*
- Cryopreservation/methods*
- Cryoprotective Agents/pharmacology
- Cysteine Endopeptidases/metabolism
- Dimethyl Sulfoxide/pharmacology
- Female
- Freezing*
- Methanol/pharmacology
- Oocytes/drug effects
- Oocytes/physiology*
- Zebrafish
- PubMed
- 18289520 Full text @ Cryobiology
Citation
Zhang, T., Rawson, D.M., Tosti, L., and Carnevali, O. (2008) Cathepsin activities and membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 degrees C using controlled slow cooling. Cryobiology. 56(2):138-143.
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping