It has been demonstrated that mouse blastocysts in delay, either lactational or following ovariectomy, are metabolically quite inactive (see McLaren, 1972). Although cell division continues in the first few days following the onset of delay (McLaren, 1968), mitotic figures are not seen in blastocysts held in delay for several days (McLaren, 1968; see Table 1). Just as trophoblast outgrowth characteristic of the implantation event may be reproduced in the appropriate culture media (Mintz, 1964; Cole & Paul, 1965), so too can delay of this event be achieved in vitro. Gwatkin (1966) has shown that omission of arginine and leucine from Eagle's medium can prevent trophoblast outgrowth from blastocysts for several days. Viability is apparently unaffected, as restoration of the missing amino acids to the medium after
M. I. SHERMAN and P. W. BARLOW
M. H. Sellens, S. Stein and M. I. Sherman
Summary. By using a rapid and sensitive method for determination of total protein and free amino acid contents we have shown that mouse preimplantation embryos developing in vivo and in vitro contain decreasing amounts of protein until the late blastocyst stage. Free amino acid contents in embryos developing in vivo drop following fertilization and then rise gradually until the late blastocyst stage. Embryos developing in vitro from the 2-cell stage have much lower levels of free amino acids than those in vivo. Embryos placed in culture at the blastocyst stage show a decrease in protein content within 24 h, but protein levels rise rapidly thereafter in medium containing serum and amino acids. In the same medium, total amino acid pools increase from the time the blastocysts are placed in culture. Blastocysts cultured in the absence of amino acids show a very slight net increase in protein content and free amino acid pools are eventually depleted. In the presence of amino acids but the absence of serum, there is only a modest increase in the protein content of cultured blastocysts, suggesting that, in addition to an exogenous supply of amino acids, other factors are required for optimal growth.