Elsevier

Cryobiology

Volume 56, Issue 2, April 2008, Pages 138-143
Cryobiology

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

https://doi.org/10.1016/j.cryobiol.2008.01.002Get rights and content

Abstract

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.

Section snippets

Collection of oocytes

Zebrafish (D. rerio) were kept in filtered and aerated 20 L tanks at 28 °C with a light/dark cycle of 12/12 h. Fish were fed two times a day with ‘TetraMin’ dry fish food and once a day with live brine shrimps. Five zebrafish oocyte developmental stages were classified: stage I (primary growth stage); stage II (cortical alveoli stage); stage III (vitellogenic stage); stage IV oocytes (maturation stage) and stage V (mature eggs) after Selman et al. [26]. Stage III oocytes were used in this study (

Cathepsin activities of stage III zebrafish oocytes after cryoprotectant treatment and freezing

Cathepsins B, D and L activities of stage III zebrafish oocytes after 2 M methanol and 2 M DMSO treatment for 30 min at 22 °C are shown in Fig. 2A–C. The results showed that whilst oocyte cathepsin B and D (Fig. 2A and B) were not affected by 2 M DMSO treatment, 2 M methanol treatments significantly decreased cathepsin B and D activity to 4.13 ± 0.3 U (1U = 1 nmol/min/mg/ml) of 2-naphthylamine and 0.3 ± 0.1 U when compared to their controls of 8.25 ± 1 U (nmol/min/mg/ml 2-naphthylamine) and 0.7 ± 0.1 U,

Discussions

The vitellogenic growth of follicles occurs by accumulating vitellogenin (VTG) in oocytes and this represents one of the key events in the process of ovarian maturation. Once synthesised in the liver, the VTG protein is released into the blood stream and transported to the ovary. A specific cell-surface receptor involving coated pits and coated vesicles is present on the oocyte surface. The VTG is selectively sequestered through a specific receptor mediated endocytosis by growing oocytes,

Acknowledgments

This work is funded by European Commission, DG-Fishers, #Q5RS-2002-00784 CRYOCYTE. The authors wish to thank Dr. Kageyama, Kyoto University, Japan, for providing the Ascaris pepsin inhibitor.

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    Statement of funding: This work is funded by European Commission, DG-Fishers, #Q5RS-2002-00784 CRYOCYTE.

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