There has been considerable discussion as to whether the developing mouse ova requires a n exogenous protein supply. A series of experiments were performed to determine the effect of various fixed-nitrogen sources on the development in vitro of two-cell mouse ova into blastocysts. It was found that two-cell mouse ova would not develop into blastocysts in medium that did not contain a hed-nitrogen source. Crystalline bovine serum albumin (BSA) at 1 mg/ml or, alternatively, the constituent amino acids of BSA could supply this requirement for fixed-nitrogen. In the presence of single amino acids at M, only an occasional twocell ova developed into a blastocyst.
Single amino acids were omitted from the medium containing the constituent amino acids of BSA in a n attempt to demonstrate an essential amino acid requirement of two-cell mouse ova. The omission of no singIe amino acid from the medium, completely prevented the development of some two-cell ova into blastocysts. The only amino acid whose omission resulted in a significant decrease in development was cystine. In the medium where amino acids were used in place of BSA, a non-protein polymer was used to replace the physical properties of albumin. The non-protein polymers which appeared best were Polyvinylpyrrolidone (MW = 150,000), Acacia, Dextran, and Ficoll.
M or 8 X
Only a limited amount of work has been performed in the field of nitrogen metabolism of the developing mammalian ovum. In the past, media used in the in vitro cultivation of rabbit ova have usually contained 50% or more serum (Austin, '61). Purshottom and Pincus ('61) suggest that cleavage to the morula stage will occur in simple solutions without serum and that the rabbit ovum carries within itself the nutrients necessary for the early growth. However, for blastocyst formation they feel that serum is necessary. Whitten ('57) observed the development of eight-cell mouse ova into blastocysts when the medium contained no protein supplement, and the only nitrogen source was glycine ( 3 X M ) or other simple amino acids. He concluded that the requirements of the developing ova were simple and compared them to the developmental stages of invertebrates in this respect. Some support for this view was obtained by Greenwald and Everett ('59) when they observed that F5 methionine injected into pregnant female mice was not incorporated into the developing ova between the two-cell and morula stage. However, Mintz ('62) has demonstrated that HR-leucine and HR-uri-