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