The type and extent of injuries in vitrified mouse oocytes

Abstract

To improve the vitrification of mouse oocytes using straws, we attempted to estimate the type and extent of injuries during vitrification with a vitrification solution EAFS10/10. Injuries in oocytes were assessed based on cellular viability, the integrity of the plasma membrane, the status of the meiotic spindle/chromosomes, and morphological appearance. For morphologically normal oocytes, the ability to be fertilized and to develop into blastocysts was examined. Morphological assessment revealed 15% of oocytes to be injured by intracellular ice formed during vitrification, and 10% by osmotic swelling during removal of the cryoprotectant. When assessed by the status of spindles/chromosomes, the most sensitive criterion, damage was found in 16% of oocytes without any treatment. This value was similar to the proportion of fresh oocytes that did not cleave after insemination (13%). On exposure to EAFS10/10, the spindles/chromosomes were affected in 33% of oocytes. The exposure reduced the rate of cleavage by 18% points and the rate of development into blastocysts by 19 points. Vitrification reduced these rates by 15% and 36% points, respectively. Although the mechanism responsible for this moderate toxic effect on developmental ability is not known, information obtained in the present study will be useful to develop a practical method for the vitrification of mouse oocytes using straws.

Introduction

The cryopreservation of mammalian oocytes and embryos is useful for the preservation of genetic materials. In mice, cattle, and humans, cryopreservation of embryos has been used to preserve genetic variants, for breeding/reproduction, and to treat infertility, respectively. On the other hand, mammalian oocytes are less tolerant to cryopreservation and so less practical than embryos even in the mouse, although successful production of progeny from cryopreserved oocytes was first reported in the mouse over 30 years ago [35]. For the cryopreservation of cells, higher permeability is preferable, but oocytes are less permeable than embryos [6], [23].

In the 1980s, vitrification was developed as an innovative means of cryopreserving mammalian embryos [21], [25]. With this method, embryos can be cryopreserved simply and rapidly, with high survival rates [13], [24]. However, vitrification solutions contain much higher concentrations of cryoprotectants and so are much more toxic than solutions used for slow-freezing. Therefore, excessive exposure to the vitrification solution causes injury by the toxicity while insufficient exposure causes damage from the formation of intracellular ice. Thus, the most suitable condition is a compromise between the two injuries.

Ultrarapid vitrification has often been used to cryopreserve oocytes [15], [16], [18], [31], [32]. Rapid cooling and especially rapid warming suppress the recrystallization of intracellular ice [19], [28], which enables oocytes to survive even when less well dehydrated and less well permeated by the cryoprotectant. This method would therefore be effective for cells having low membrane-permeability, such as oocytes [6], [23]. However, it requires skill to obtain high survival consistently, and limits the number of cells to be cryopreserved. If oocytes could be cryopreserved by conventional vitrification using straws, the availability would increase, especially in the mouse, where numerous oocytes are cryopreserved at one time.

To improve conventional vitrification, it is important to know the type and extent of the injuries sustained at each stage of the process. Oocytes would be at risk of damage from the toxicity of cryoprotectant, chilling, the formation of intracellular ice, and osmotic swelling [12]. Even if they appear normal under a stereo-microscope, their cellular components, such as the plasma membrane and the meiotic spindle/chromosomes, and their ability to be fertilized and develop, may be reduced. In addition, the zona pellucida of oocytes can be hardened by cryopreservation [4], [9], [33], which prevents penetration by sperm. To our knowledge, however, no study has analyzed the type and extent of injuries during vitrification.

In the present study, we examined the normality of vitrified mouse oocytes based on various criteria.