An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro

Abstract

Summary. One-cell CF-1 \m=x\ B6SJLF1/J embryos, which usually exhibit a 2-cell block to development in vitro, have been cultured to the blastocyst stage using CZB medium and a glucose washing procedure. CZB medium is a further modification of modified BMOC-2 containing an increased lactate/pyruvate ratio of 116, 1 mM-glutamine and 0\m=.\1mM-EDTA but lacking glucose. Continuous culture of one-cell embryos in CZB medium allowed 83% of embryos to develop beyond the 2-cell stage of which 63% were morulae at 72 h of culture, but blastocysts did not develop. However, washing embryos into CZB medium containing glucose after 48 h of culture (3-4-cell stage) was sufficient to allow development to proceed, with 48% of embryos reaching the blastocyst stage by 96 h of culture. Exposure of embryos to glucose was only necessary from the 3\p=n-\4-cell stage through the early morula stage since washing back into medium CZB without glucose at 72 h of culture still promoted the development of 50% of embryos to the blastocyst stage. The presence of glucose in this medium for the first 48 h of culture (1-cell to 4-cell stage) was detrimental to embryo development. Glutamine, however, exerted a beneficial effect on embryo development from the 1-cell to the 4-cell stage although its presence was not required for development to proceed during the final 48 h of culture. Blastocysts which developed under optimum conditions contained an average of 33\m=.\7total cells. The in-vitro development of 1-cell embryos beyond the 2-cell stage in response to the removal of glucose and the addition of glutamine to the culture medium suggests that glucose may block some essential metabolic process, and that glutamine may be a preferred energy substrate during early development for these mouse embryos.

Chromosome analysis of mouse zygotes produced by intracytoplasmic injection of spermatozoa exposed to acrosome reaction inducing agents methyl-β-cyclodextrin and calcium ionophore A23187

“…Hepes-buffered H-mCZB [18] was used for preparing oocytes where appropriate. Temporary storage of oocytes and cultivation of fertilized ova were performed using bicarbonate-buffered CZB [19] with modification by addition of 5.56 mM glucose. This was designated as mCZB.…”

Transition from maternal to embryonic control in early mammalian development: A comparison of several species

“…The reasons for this in vitro phenomenon are not clear. The two-cell block can be rescued by transfer of cytoplasm from the oocytes of nonblocking strains (Muggleton-Harris et al, 1982) and can be circumvented by improved culture media (Chatot et al, 1989). Nonetheless, the block in culture coincides with the time of transition from ma-ternal to embryonic control and embryos appear to be particularly sensitive to culture conditions during this critical phase of their developmental program.…”

Chromosome analysis of mouse one-cell androgenones derived from a sperm nucleus exposed to topoisomerase II inhibitors at pre- and post-fertilization stages

“…This was referred to as Hepes-TYH medium. The medium used for culturing oocytes and androgenones under 5 % CO2 in air was CZB [25] temporarily kept in CZB medium at 37 °C and then transferred into a droplet (10 µl) of Hepes-CZB medium containing 5 µg/ml cytochalasin B (Sigma-Aldrich) and enucleated as described elsewhere [26]. The enucleation 7 was performed at room temperature.…”