Eagle's basal medium, modified to contain essential amino acids at the concentrations optimal for mouse blastocyst hatching, attachment, and outgrowth, supported in vitro development of the mouse blastocyst better than other tissue culture media tested. This medium was improved for growth and differentiation of the inner cell mass by doubling the concentration of amino acids and glucose and by adding uridine (10(-5) M) and beta-mercaptoethanol (10(-5) M). In this improved medium nearly all blastocysts grown from the two-cell stage hatched and formed trophoblast outgrowths, and 62% developed into two-layer egg cylinders.

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 (1OX-20X BME) concentrations of cystine, histidine, or lysine increases the extent of outgrowth, but addition of 10-~-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 Preimplantation mouse embryos develop from the two-cell stage to the early blastocyst stage in the absence of an exogenous fixed nitrogen source (Cholewa and Whitten, '70). The 26% decrease in protein content of the mouse embryo that is observed during the first three days of embryonic development (Brinster, '67) suggests that the embryo uses endogenous fixed nitrogen reserves during this period. However, the mouse embryo must eventually rely on exogenous sources of fixed nitrogen to support the marked increase in the rate of protein synthesis between morula and early blastocyst stages (Brinster, '71; Epstein and Smith, '73), and for growth and differentiation following cleavage. Our study was undertaken to determine the specific fixed nitrogen requirements for blastocyst hatching, attachment, trophoblastic outgrowth, and inner cell mass growth in vitro.When mouse blastocysts are cultured in J. EXP. ZOOL., I S G . 3 0 5 3 1 8

The developmental potential of the inner cell mass (ICM) of the cultured mouse embryo was determined by testing the ability of the ICM to regenerate trophoblast in vitro. ICM's isolated by immunosurgery from either single or chimeric embryos were able to regenerate trophoblast when they were isolated at 69 hours of culture from the 2-cell stage, but they had lost this capacity by 93 hours of culture. Trophoblast regeneration by isolated ICM's did not appear to require either a critical cell mass at the time of isolation or cell proliferation during regeneration.

No abstract

During implantation the embryo attaches to the endometrial surface and trophoblast traverses the uterine epithelium, anchoring in the uterine connective tissue. To determine whether trophoblast can facilitate invasion of the uterus by degrading components of normal uterine extracellular matrix, mouse blastocysts were cultured on a radio-labeled extracellular matrix that contained glycoproteins, elastin, and collagen. The embryos attached to the matrix, and trophoblast spread over the surface. Starting on day 5 of culture there was a release of labeled peptides into the medium. The radioactive peptides released from the matrix by the embryos had molecular weights ranging from >25,000 to <200. By day 7 there were areas where individual trophoblast cells had separated from one another, revealing the underlying substratum that was cleared of matrix. When trophoblast cells were lysed with NH4OH on day 8, it was apparent that the area underneath the trophoblast outgrowth had been cleared of matrix. Scanning electron microscopy and time-lapse cinemicrography confirmed that the digestion of matrix was highly localized, taking place only underneath the trophoblast, with no evidence of digestion of the matrix beyond the periphery of the trophoblast outgrowth. The sharp boundaries of degradation observed may be due to localized proteinase secretion by trophoblast, to membrane proteinases on the surface of trophoblast, or to endocytosis. Digestion of the matrix was not dependent on plasminogen, thus ruling out a role for plasminogen activator. Digestion was not inhibited by a variety of hormones and inhibitors, including progesterone, 17fl-estradiol, leupeptin, EDTA, colchicine, NH4CI, or E-aminocaproic acid. This system of culturing embryos on extracellular matrix may be useful in determining the processes that regulate trophoblast migration and invasion into the maternal tissues during implantation.Implantation is an invasive process that is limited in both time and space. In the mouse it begins on day 5 of gestation, with the initial apposition of the embryo to the endometrial surface, and extends to the time that the invading trophoblast taps into the maternal blood vessels. During this period the trophoblast traverses a uterine epithelium composed of cells and connective tissue matrix and penetrates the basement membrane before entering the uterine stroma (26). Although the actual mechanisms have not been elucidated, morpliologic study (26) suggests that entry of trophoblast into the endometrium does not depend on the release of a cytolytic enzyme because maternal cells surrounding the implanting embryo remain largely intact.Attachment of blastocysts onto glass or plastic and subsequent trophoblast outgrowth has been used as a model to study proteinase activity and the interaction of the embryo with other cell types in culture (10,20,30). Studies in culture with timelapse cinemicrography have shown that there is contact inhibition between trophoblast and co-cultured dispersed ceils (10). Trophoblast displaces ...