Development and differentiation of mouse blastocysts in serum-free medium

Numerous studies have shown that the in vitro development and differentiation of mouse blastocysts require serum, but the number and nature of serum factors involved remains unclear. In this article, we describe a culture medium, EM-2, containing as a source of protein only commercially purified bovine serum albumin (BSA) and fetuin. This medium supports hatching, attachment and outgrowth of mouse blastocysts. Although attachment and outgrowth are delayed in EM-2 medium 12-15 and 5-8 h, respectively, these events occur at frequencies comparable to those observed in serum-containing media. Trophoblast cells are capable of differentiating in this medium: they synthesize Δ⁵,3β-hydroxysteroid dehydrogenase and their nuclei become polyploid. The inner cell mass also appears to differentiate to some extent in EM-2 medium as evidenced by the appearance of cells with characteristics of parietal endoderm. The fetuin factor is necessary at least for trophoblast outgrowth and the albumin factor is required for the survival and/or growth of the inner cell mass. It is, however, not evident from these studies whether the serum fractions used are actually involved in the induction of differentiation, or whether the early differentiative steps in the mouse blastocyst are preprogrammed and require for expression only a normal cellular metabolic rate.