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Towards the development of technologies for cryopreservation of fish cocytes

Deliverables

Protocols were developed for induction of ovulation in in vitro incubated gilthead seabream and zebrafish ovarian follicles. These protocols need further implementation in future R & D projects to obtain in vitro fertilization and thus could be used in conjunction with oocyte cryorpeservation methods.
1. Methods were developed for evaluating viability for zebrafish and gilthead seabream oocytes at all stages of oogenesis, by vital stains such as trypan blue and thiazolyl blue tetrazoliumbromide. 2. A method was developed for evaluating functional viability of maturing GSB oocytes by promoting germinal vesicle breakdown (GVBD) and hydration during in vitro incubation.
1. Studies on the effect of cryoprotective agents on cathepsin mRNA localization in zebrafish oocytes. 2. Studies on the effect of cryopreservation and thawing on zebrafish ovarian follicles. 3. Studies on the effect of cryopreservation procedures on cathepsin activity in gilthead seabream oocytes.
1. Studies on chilling sensitivity of zebrafish oocytes. 2. Studies on cryo-protectant toxicity to zebrafish oocytes. 3. Studies on zebrafish oocyte membrane permeability to water and cryo-protectants. 4. Developing cryo-preservation protocols for zebrafish oocytes.
We have developed an Oocyte Viability Molecular Signature (OVMS) assay for monitoring cryo-preservation and oocyte manipulations. The data obtained demonstrated that the polarization of maternal transcripts inside the oocyte could be used as a new and original method to monitor oocyte viability after manipulation and/or cryo-preservation and will help in cryo-preservation protocol refinement. OVMS assay is therefore a nice complementary approach for the evaluation of oocyte viability both before and after a cryo-preservation procedure, and may substitute visual and staining methods, and to heavier functional evaluations.
1. Identification of a large set of proteins from ZF and GSB during different developmental stages. 2. Assigning some of the proteins as more or less abundant during specific developmental stages. 3. Defining some of the proteins changes induced by cryo-preservation of oocytes. 4. Comparison between the transciptome and the proteome of the same samples obtained from stage specific zebrafish oocytes.
1. A novel subfamily of molecular water channels in teleosts, related to mammalian aquaporin-1(AQP1), named AQP1o, was discovered and characterized. These channels are highly expressed in the ovary of marine fish. 2. The sub-cellular localization of AQP1o in gilthead seabream oocyte was elucidated, demonstrating its role during hydration of the oocyte that occurs during oocyte maturation. 3. Amino acid domains located in the C-termini of gilthead seabream AQP1o involved in the regulation of the trafficking of the proteins into the plasma membrane of the oocyte were identified. 4. Protocols were developed for the induction of ovulation in vitro of gilthead seanream and zebrafish ovarian follicles.
1. A recombinat GSB-LH (rGSB-LH) was produced in the methylotrophic yeast Pichia pastoris expression system. 2. The rGSB-LH induced E2 release from in vitro incubated GSB ovarian follicles. 3. The rGSB-LH promoted final ovarian maturation in in vitro incubated GSB follicles and attenuated the occurrence of atresia among these cultured oocytes.
1. Isolation of zona pellucida (ZP) proteins constituting the vitelline envelope of gilthead seabream oocytes. 3 new isoforms of zona pellucida (ZP) proteins were identified in gilthead seabream and found to have dual expression in liver and early oocytes. Further, the ZP proteins were found to be under estrogenic control in liver. 2. Determination of timing of gene expression and ZP deposition within the VE. Two Zp proteins were found to be translated in the early oocytes and the four ZP proteins were found to be unevenly distributed in the early VE. 3. Composition of VE from gilthead seabream and zebrafish. The relative amount of each ZP protein in the VE was quantified and we found that they were not present in equal amounts.
This study provides a complete sequence data set of maternal mRNA stored in zebrafish germ cells at the end of oogenesis. This catalogue contains highly expressed transcripts that are part of a vertebrate ovarian expressed gene signature. The molecular phenotype described provides groundwork for future experimental approaches aimed at identifying functionally important maternal transcripts and proteins involved in oogenesis and early stages of embryo development. Two manuscripts were already published on important identified genes (Tingaud-Sequeira et al., 2004 Gene Expression Patterns 4:561-568, 2004; Marza et al., Developmental Dynamics 232: 506-518, 2005). A third manuscript has been published describing the molecular phenotype of zebrafish ovarian follicle by serial analysis of gene expression (SAGE) and proteomic profiling, and comparison with the transcriptomes of other animals (Knoll-Gellida et al., BMC Genomics 7:46, 2006). This publication is available at: http://www.biomedcentral.com/1471-2164/7/46 and has been highly accessed and downloaded since its online publication on the BioMed Central website (Total accesses to this article: 1,648, from March 9, 2006 to November 30, 2006) demonstrating the high scientific impact of this research.
1. Cryoprotectant toxicity was determined for GSB oocytes. 2. Accumulation of radiolabelled methanol in ZF and GSB oocytes was determined as an indication for permeability of permeable cryoprotective agents. 3. A method was developed for successful cryopreservation of small size GSB oocytes as determined by MTT staining.
1. Cloning and expression studies of ovarian cathepsin mRNA by Real Time PCR. 2. Optimisation of ovarian cathepsin enzymatic assays. 3. Determination of cathepsin mRNA expression during ovarian maturation. 4. Determination of cathepsin enzymatic activity during oocyte maturation. 5. Studies on the relationship between in vitro oocyte maturation, cathepsin activity and hydration.