Applied Research Section

University Of California At Davis, Davis CA

Investigators

Linked publications, trials & patents

Abstract

ABSTRACT: Applied Research Section Development of Mouse Oocyte Vitrification Methodology in a Closed System For the Mutant Mouse Resource and Research Center (MMRRC) Consortium, highly effective and efficient cryopreservation methods using slow freezing have been established to preserve genetically modified mouse lines as frozen sperm and embryos that can then be recovered using in vitro fertilization (IVF) and/or surgical embryo transfer (ET) to live mice for sharing and distribution. On the other hand, not all mutant mouse lines can be safely and securely preserved by cryopreservation of embryos and/or sperm, such as those exhibiting male infertility or poor embryo development. Even fertile and viable mouse lines are not always amenable to cryopreservation and/or cryorecovery of sperm and embryos, often due to the vagaries of genetic background, fecundity, in vitro manipulation, embryo culture factors, etc. In these cases, the ability to preserve mouse oocytes could provide a suitable alternative and added flexibility to ensure the safe and secure preservation of mutant mouse lines. A reliable and reproducible method for oocyte preservation would complement, and in some case, replace existing procedures used on sperm and embryos. Unfortunately, oocytes are notably vulnerable to slow freezing, which causes intracellular ice formation, premature hardening of the zona pellucida (ZP), and significantly reduced oocyte fertility by IVF. On the other hand, rapid freezing via vitrification overcomes the biophysical deficiencies of slow freezing and can increase the survivability of oocytes. We have identified three components of current protocols that can be readily modified to significantly improve the success of vitrification of mouse oocytes: 1) inhibition of ovastacin protease activity, 2) reduction in reactive oxygen species (ROS), and 3) chelation of free calcium and zinc ions. In addition, a closed system will be needed to avoid the risk of microorganism contamination and pathogen cross contamination of oocytes during vitrification. Therefore, we hypothesize that inhibition of ovastacin protease activity, reduction in reactive oxygen species, and chelation of free calcium and zinc under closed conditions will preserve the integrity of the zona pellucida and dramatically improve the viability and fertility of vitrified mouse oocytes. This proposal will have 3 Specific Aims to develop this new vitrification and recovery protocol on the C57BL/6N genetic background, which is the same as that for many mutant mouse lines distributed by the MMRRC, including from the KOMP2 mutant mouse collection. After verifying the new protocol on wildtype mice, we will validate its applicability to reliably preserving several MMRRC mutant mouse lines on various genetic backgrounds in which traditional sperm and/or embryo cryopreservation methods are inappropriate or ineffective. Ultimately, funding of this project will enable a systematic approach for optimizing vitrification in order to establish a dependable, reproducible, and effective vitrification protocol that will be published and shared with all 4 Centers in the MMRRC consortium and can be broadly adopted across the biomedical research community, including and especially for the preservation of mutant mouse lines by the MMRRC. Doing so will dramatically improve the protection of valuable genetic material, enhance repository operations, reduce overall costs, and contribute to scientific rigor and reproducibility.