Cryopreservation of murine zygotes for use in testing culture environments.

Developing one-cell mouse zygotes are more sensitive to in vitro environmental conditions than are cleavage-stage embryos. However, for convenience and reproducibility, cryopreserved two-cell zygotes are routinely used for such assays. Concern over the possibility of inducing damage by exposing one-cell zygotes to cryoprotective agents and freeze-thaw procedures during syngamy led us to examine one-cell zygotes, with and without visible pronuclei, in an effort to minimize or avoid these effects and obtain the highest possible developmental rate. In vivo fertilized mouse zygotes were collected 21 to 43 h after administration of human chorionic gonadotropin (hCG). Suspensions of zygotes in 2M ethylene glycol were aspirated into 0.25-ml plastic insemination straws and slowly cooled at -0.5 degree C/min to -40 degrees C before being plunged into liquid nitrogen for storage. Zygotes were thawed, rinsed, and placed in culture. Zygotes were examined initially for damage from the freeze-thaw procedure. Daily in vitro development was recorded. In this group of zygotes, no damage was apparent immediately after thawing, and a high degree of development in vitro was observed. Thus, usefulness of a cryopreservation method for one-cell murine zygotes has been confirmed.

Absence of a direct effect of recombinant tumor necrosis factor-alpha on human sperm function and murine preimplantation development.

OBJECTIVE: This study was designed to test the in vitro effects of human recombinant tumor necrosis factor (rTNF) on sperm motility, fertilization, and preimplantation development. DESIGN: A sensitive enzyme immunoassay was used to determine half-lives of rTNF and confirm concentrations of cytokine throughout experimental conditions. Effect of rTNF on human sperm survival was measured by computer-assisted methodology, and effect on human sperm penetration was assessed by hamster ova penetration. Cytokine effect on murine gamete interaction was determined by in vitro fertilization (IVF). Murine preimplantation development was assessed by in vitro development of cryopreserved-thawed one-cell zygotes. RESULTS: The half-life of rTNF was reduced by the addition of sperm to culture media (P less than 0.001). Sperm motility (P = 0.245) and hamster ova penetration (P = 0.62) were not altered by incubations in the presence of concentrations of rTNF up to 10,000 U/mL. Mouse IVF (P = 0.60) and preimplantation development (P = 0.56) were not altered by rTNF in concentrations up to 5,000 U/mL. CONCLUSIONS: These results demonstrate rTNF by itself does not interfere with gamete function or early embryo development.

The efficacy of cryopreserved hamster ova in the sperm penetration assay.

To solve the logistical problems of the sperm penetration assay (SPA) to provide just a sufficient number of hamster ova exactly when they are needed, a new method to cryopreserve the ova has been devised. The ova, suspended in a 1.5 M solution of propylene glycol as a cryoprotectant in an isotonic salt solution, were frozen in 1/4 mL plastic straws. Included in each straw was a sucrose solution, isosmotic to the propylene glycol solution, to serve as an osmotic buffer during dilution of the cryoprotectant out of the ova. This one-step method of dilution permitted the ova to be recovered and diluted out of the cryoprotectant within the straw in which they had been originally frozen. A total of 547 cryopreserved ova were thawed, 504 (92.1%) of which were morphologically normal after they had been incubated at 37 degrees C for 3 hours. After removal of the zonae, the frozen-thawed ova were compared with fresh, control ova in SPAs of donor and patient semen that had been capacitated in TEST-yolk buffer. The percent penetration and penetration index of fresh versus cryopreserved ova did not differ significantly for either donor or patient semen.

Zona fracture damage and its avoidance during the cryopreservation of mammalian embryos.

Although fracture damage to the zonae pellucidae and blastomeres is frequently observed after the cryopreservation of mammalian embryos, little is known of the mechanism by which this occurs. The incidence of damage to zonae was measured when bovine ova with normal zonae were frozen in straws or glass test tubes by standard embryo cryopreservation procedures that yield high rates of survival. Ova were examined for zona damage after warming by procedures that ought to produce little or no thermal stress (slow warming in 20 degrees C air) or high levels of stress (rapid warming in liquid baths). Ova frozen in straws exhibited no zona damage after slow warming at 150 degrees C/min in air (n = 206). However, the incidence of zona damage increased when the straws were warmed rapidly in 20 degrees C (n = 157) or 36 degrees C (n = 159) water (17 and 24%, respectively). Ova in straws warmed rapidly in nonaqueous liquids (ethylene glycol, or silicone oil) exhibited lower rates of zona damage (2 to 5%). Ova frozen in glass tubes exhibited a much higher incidence of zona damage than those frozen in straws, regardless of the warming conditions. Thus, 30% of 114 ova exhibited damage when tubes were warmed slowly at 25 degrees C/min in air, while 54% of 98 ova showed zona damage when tubes were warmed rapidly at 500 degrees C/min in 36 degrees C water. These results are consistent with the view that zona damage is associated with thermally-induced fracturing of the suspension during rapid changes of temperature.