Storage of Two-Cell Mouse Embryos in Vitro

Abstract

There are reports of the storage at O-lOoC of fertilized rabbit (Chang 1947(Chang , 1948Hafez 1965) and sheep ova (Averill 1956; Kardymowicz et al. 1963; Kardymowicz, Kardymowicz, and Grochowalski 1966; Kardymowicz, Kardymowicz, and Kremer 1966). However, the storage of fertilized mouse ova in vitro does not appear to have been studied. Wales (1969) found that glycolysis at 37°C by cultured blastocysts was not impaired if the two-cell embryo was stored at 5°C for a short period prior to culture, and the author suggested that storage of two-cell embryos at this temperature may be successful. In the present study the viability of mouse embryos after various periods at low temperature was investigated. Method8Two·cell mouse embryos were obtained as described by Brinster (1963). In the initial experiments, Krebs-Ringer bicarbonate (Ringer medium) was the basic medium but, in the last experiment, Dulbecco's phosphate·buffered saline was used (Dulbecco and Vogt 1954). All media contained 25 mM sodium DL·lactate, 0·25 mM sodium pyruvate, 1 mgfml bovine serum albumin, 60 f.Lgfml penicillin, and 50 f.Lgfml streptomycin. When glycerol or dimethylsulphoxide were included, they replaced an equivalent volume of water in the medium.For the cooling experiments, the embryos (usually 20 per treatment) were washed through two changes of Ringer medium before transfer to glass culture tubes containing 1 ml of medium. Preliminary tests had indicated that embryos did not survive for 24 hr when cooled and stored in either embryological watch glasses containing 0·5 ml of Ringer medium under 1 ml of paraffin oil or micro droplets under paraffin oil in Petri dishes. The culture tubes were surrounded by 100 ml of water and placed in a refrigerator, the rate of cooling being regulated by the initial temperature of the water jacket.The formation of blastocysts from stored embryos following subsequent culture for 72 hr in Ringer medium (Brinster 1963) was used as a measure of viability. In most instances each experiment was repeated on four separate occasions and the results presented are the mean percentage of blastocysts cultured from the stored embryos. Prior to statistical analysis, all results were transformed to angles. Re8ult8 and Di8cu88ionPreliminary results indicated that the inclusion of either glycerol or dimethylsulphoxide (5% v/v) in Ringer medium during storage of two-cell mouse embryos at 4-5°C for 24 hr did not improve survival irrespective of whether the embryos were transferred through stepwise increments in concentration of the compounds before cooling to 5°C. Therefore glycerol and dimethylsulphoxide were excluded from subsequent experiments examining the effects of low-temperature storage. In * Manuscript

Eomes::GFP—a tool for live imaging cells of the trophoblast, primitive streak, and telencephalon in the mouse embryo

“…P35G-1.5-14C) at 378C, 5% CO 2 . Postimplantation embryos and organs were dissected in modified PB-1 (Papaioannou and West, 1981;Whittingham and Wales, 1969) medium containing 10% fetal bovine serum (FBS). Live embryos were counterstained with 7.5 mM DRAQ5 (DAKO, Carpinteria, CA).…”

Alpha-fetoprotein, the major fetal serum protein, is not essential for embryonic development but is required for female fertility

“…Blastocysts were flushed from uteri 3.5 days after fertilization into phosphatebuffered medium 1 (24) and were immediately transferred individually into uteri of pseudopregnant recipients at 2.5 days postcoitum, as described (23). Twelve recipients were used, and nine pups were obtained.…”

Survival of Rat Embryos After Freezing and Thawing

“…The embryos were collected in a standard culture medium for mouse embryos containing 94-59 mM-NaCl, 4-78 mM-KCl, 1-71 mM-CaCl2, 1-19 mM-MgS04.7H20, 1-19 mM-KH2P04, 25-07 raMNaHC03, 23-28 mM-Na lactate, 0-33 mM-Na pyruvate, 5-56 mM-glucose, 4 mg bovine serum albumin/ml, 100 Units penicillin G (potassium salt)/ml and 50 µg streptomycin sulphate/ml (Whittingham, 1971). They were washed in two changes of medium and transferred to 0-1 ml phosphate-buffered medium (PB1 : Whittingham & Wales, 1969) contained in a glass culture tube (12 x 75 mm). The embryos were equilibrated with 1-5 M-dimethylsulphoxide (DMSO) at 0°C for 15 min by the addition of a further 0-1 ml PB1 containing 3 m-DMSO.…”