Cryopreservation has become in recent years an important method for the extended storage of preimplantation mammalian embryos, both from commercially important species, such as the bovine, as well as the human. Owing to evidence in the human and bovine species that placement of multiple embryos in the uterus can be detrimental to the establishment of pregnancy with a single embryo, cryopreservation has emerged as a means of maintaining normal cleavage-stage embryos for subsequent uterine placement. The foremost problem in the application of cryopreservation in mammalian embryology is the inability to determine objectively and quantitatively the developmental potential of a thawed embryo prior to uterine placement. The proposed research is designed to develop noninvasive, quantitative criteria that would assess the developmental potential of individual cryopreserved embryos prior to placement in the uterus. Preliminary studies demonstrate that mouse and bovine embryos liberate specific cytoplasmic proteins into cryopreservative solutions during dehydration, freezing or rehydration. Because these proteins have been shown not to be secreted or liberated by normally developing embryos, their presence in cryopreservation solutions can be considered to be diagnostic of cellular damage. The proposed research is designed to determine by fluorescent probes (organelle, membrane- and DNA-specific) and light and electron microscopy the nature and extent of cellular damage that occurs during cryopreservation, and to correlate this damage with (1) the quantity of specific cytoplasmic proteins liberated into cryopreservative fluids by the embryos of three mammalian species (mouse, bovine and human) as a function of freezing protocol, and (2) developmental potential of individual embryos in vivo and in vitro. The findings will be used to develop noninvasive, immunologic means (radioimmunoassay) to identify and quantify specific embryonic proteins in cryopreservation fluids. The level of protein(s) present is expected to be proportional to the extent of cellular damage and therefore correlated with embryo viability and developmental potential after thawing.
