Abstract
In mammals, unfertilized oocytes are one of the most available stages for cryopreservation because the cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has generally been reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. C57BL/6J mice, an inbred strain, are used extensively for the production of transgenic and knockout mice. If the oocytes from C57BL/6J mice can be successfully cryopreserved, the cryopreservation protocol used will contribute to the high-speed production of not only gene-modified mice but also hybrid mice. Very recently, we succeeded in the vitrification of mouse oocytes derived from ICR (outbred) mice. However, our protocol can be applied to the vitrification of oocytes from an inbred strain. The aim of the present study was to establish the vitrification of oocytes from C57BL/6J mice. First, the effect of cumulus cells on the ability of C57BL/6J mouse oocytes to fertilize and develop in vitro was examined. The fertility and developmental ability of oocyte-removed cumulus cells (i.e., denuded oocytes, or DOs) after IVF were reduced compared to cumulus oocyte complexes (COCs) in both fresh and cryopreserved groups. Vitrified COCs showed significantly (P<0.05) higher fertility and ability to develop into the 2-cell and blastocyst stages compared to the 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, our results demonstrate that we succeeded for the first time in the vitrification of mouse oocytes from C57BL/6J mice. Our findings will also contribute to the improvement of oocyte vitrification not only in animals but also in clinical applications for human infertility.
Highlights
As one of the most informative experimental animals, mice have been used in a broad range of research fields
The successful cryopreservation of oocytes is highly desirable because it leads to a high efficiency of oocyte cryopreservation, making large numbers of viable oocytes available for the generation of offspring via in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI)
If the oocytes from C57BL/6J mice can be cryopreserved, the cryopreservation protocol will be useful for the high-speed production of gene-modified mice and hybrid mice from different gene-modified mice
Summary
As one of the most informative experimental animals, mice have been used in a broad range of research fields. ’gene-targeted’ knockout mice have been generated via embryonic stem (ES) cells, spermatogonial stem cells [5], and DNA-nicking nucleases [6]. To manage this ever-increasing number of mouse lines, animal facilities will benefit greatly from cryopreservation of the germ cells. Whittingham and his colleagues [7] first succeeded in the cryopreservation of mammalian embryos at 2196uC by iceseeding and a cryopreservation method that has been referred to as ’slow-freezing.’. Vitrification can supplant slow-freezing as the optimal method for embryo cryopreservation in mammals
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