High Developmental Rates of Mouse Oocytes Cryopreserved by an Optimized Vitrification Protocol: The Effects of Cryoprotectants, Calcium and Cumulus Cells

Abstract

Unfertilized oocytes are one of the most desired germ cell stages for cryopreservation because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, in general, the fertility and developmental ability of cryopreserved oocytes are still low. The aim of the present study was to improve vitrification of mouse oocytes. First, the effects of calcium and cryoprotectants, dimethyl sulfoxide and ethylene glycol (EG), in vitrification medium on survival and developmental ability of vitrified oocytes were evaluated. Oocytes were vitrified by a minimal volume cooling procedure using different cryoprotectants. Most of the vitrified oocytes were morphologically normal after warming, but their fertility and development were low independently of calcium and cryoprotectants. Second, the effect of cumulus cells on ability of oocytes to be fertilized and develop in vitro was examined. The fertility and developmental ability of denuded oocytes (DOs) after IVF were reduced compared with cumulus-oocyte complexes (COCs) both in fresh and cryopreserved groups. Vitrified COCs showed significantly (P<0.05) higher fertility and ability to develop to the 2-cell and blastocyst stages than those of vitrified DOs with cumulus cells and vitrified DOs alone. The vitrified COCs developed to term at a high success rate equivalent to the rate obtained with IVF using fresh COCs. Taken together, the current results clearly demonstrate that, in the presence of surrounding cumulus cells, matured mouse oocytes vitrified using calcium-free media and EG retain their developmental competence. These findings will contribute to improve oocyte vitrification in not only experimental animals but also clinical application for human infertility.

Applications of cryopreserved unfertilized mouse oocytes for in vitro fertilization

“…There are some reports about the vitrification of mouse oocytes [13][14][15]28,37]. Seki and Mazur yields high survival of vitrifiedwarmed ICR mouse oocytes using the Cryotop with ultra-rapid warming (117,000°C/min) [28].…”

“…However, although many papers concerning the cryopreservation of mouse oocytes have been published since the first successful reports of oocyte freezing [24,36], a simple and practical freezing method for unfertilized C57BL/6 mouse oocytes, and an IVF system using these cryopreserved oocytes have yet to be established [13,14,18,20,21,29]. There is a strong need to develop a technology of oocyte cryopreservation of C57BL/6 mouse and IVF using the oocytes for efficiently producing GEM in the animal facility.…”

Effect of vitrification on mitochondrial membrane potential in human metaphase II oocytes

“…2 The red fluorescence images on LSCM of human MII oocyte stained by JC-1. 2A, 2B, 2C and 2D represents the typical distribution of red fluorescence in fresh, cultured for 0 h, 2 h and 4 h oocytes after vitrification/thawing, respectively be the ideal cryoprotectant for oocytes and embryo vitrification [24], and it could retain oocytes developmental competence better than DMSO [25]. Moreover, it has been reported that cryoprotectant mixtures may have some advantage over solutions containing only one permeable cryoprotectant, since PROH and sugar exert major influences on the vitrification properties of ethylene glycol-based solutions and have low toxicity to embryos and oocytes [26].…”

Successful vitrification of pronuclear-stage pig embryos with a novel cryoprotective agent, carboxylated ε-poly-L-lysine

“…Our previous study demonstrated that compared to DMSO, EG had a lesser toxic effect on the vitrification of unfertilized oocytes in mice [17]. It was also suggested that the current CPAs, even EG, have toxic effects on cell viability in a dose-dependent manner [18], indicating that the development of a new CPA showing high efficiency and low toxicity is necessary for further improvements in vitrification.…”