Effects of hypotonic stress on the survival of mouse oocytes and embryos at various stages.

To examine the sensitivity of mammalian oocytes and embryos to osmotic swelling, which can occur during the removal of cryoprotectant from cryopreserved cells, the effect of hypotonic stress on the survival of fresh and vitrified mouse oocytes/embryos at various stages was examined. Oocytes and embryos were suspended in phosphate-buffered saline (PBS) media of various hypotonicities for 30 min at 25 degrees C. They were then returned to isotonic PBS medium, and the survival was assessed by the apparent integrity of the blastomeres and/or the developmental potential during culture. The survival of stressed embryos at one- to eight-cell stages assessed by the appearance was close to that assessed by the developmental ability, suggesting that hypotonic stress causes physical damage in the cell membrane. Fresh oocytes and embryos were almost totally unaffected by exposure to a 0.5x isotonic solution at all developmental stages examined. However, the extent of injury resulting from exposure to 0.3 to 0.2x isotonic solutions varied and depended on the developmental stage of the embryos. For example, zygotes were the least sensitive and morulae were the most sensitive to the hypotonic stresses. Except for morulae, vitrified cells were more sensitive to hypotonic stresses than were fresh ones. However, in many cases, the sensitivity was reduced or eliminated when the oocytes and embryos were cultured for a short period before exposure to the hypotonic stress. Furthermore, the survival rate of some stressed embryos which had been equilibrated in vitrification solution without cooling was higher than the survival of embryos stressed immediately following vitrification. These results show that sensitivity to osmotic swelling is variable among oocytes and embryos. The results also show that cryopreserved cells just after warming are more sensitive to osmotic swelling than are fresh ones, and even swelling corresponding to that in 0.5x solution may decrease survival in some stages. Copyright 1997 Academic Press.