Hatching, attachment, and outgrowth of mouse blastocysts in vitro: Fixed nitrogen requirements

Abstract

Abstract Hatching, attachment and outgrowth of mouse blastocysts in vitro are dependent upon the presence of specific free amino acids in the medium. When grown in Eagle's Basal Medium (BME) containing 1% dialyzed fetal calf serum, 67% of blastocysts hatch. Omission of histidine, methionine, threonine, tryptophan, tyrosine, or valine from BME significantly reduces the incidence of hatching. For attachment to occur, cystine and lysine are also required, and for trophoblastic outgrowth, every essential amino acid except isoleucine is required. Omission of glutamine reduces the extent of outgrowth. Addition of higher (10X–20X BME) concentrations of cystine, histidine, or lysine increases the extent of outgrowth, but addition of 10 −5 –10 −2 M concentrations of non‐essential amino acids does not stimulate either hatching, attachment, or outgrowth. When all essential amino acids are at optimal concentrations, nearly 100% hatching occurs in a chemically defined medium in the absence of a macromolecular fixed nitrogen source. Extensive trophoblastic outgrowth and inner cell mass growth occur at optimal amino acid concentrations, but only in the presence of serum. Under these conditions, a collagen substrate is not necessary for growth and differentiation of the inner cell mass into endoderm and ectoderm in vitro. These requirements indicate that during post‐blastocyst development in vitro the mouse embryo gradually becomes dependent upon specific exogenous fixed nitrogen sources, including essential amino acids and a non‐dialyzable component from serum.