In vitro cultivation of mammalian eggs

Current knowledge of the factors influencing early mammalian development from the stage of the zygote to the stage of implantation is limited. The fertilized ovum is, during this phase of its life, a free floating and rapidly differentiating cell, depending entirely on the fluids in the Fallopian tube and uterus for its nutrition and growth. While attempts at isolating the fertilized ovum in drops of serum or in flasks containing serum and physiologic salt solutions have been successful, the exact requirements for normal growth and development of the egg from the unicellular to the blastocyst stage are not yet determined. This is so because of the difficulty in keeping alive mammalian eggs in completely defined chemical media such as those that have been evolved for other tissue cells. However, it has been established that mammalian eggs and early embryos will tolerate periods of external cultivation outside the maternal reproductive organs and are capable of survival as well as a certain amount of development in vitro, provided serum of the species is present in the surrounding media. Lewis and Gregory ('29) described the growth and development of rabbit eggs in hanging drop cultures, and Long and Mark ('1 l ) carefully recorded the maturational phases of mouse eggs in serum as seen through a microscope over periods of 72 to 96 hours. Pincus and Werthessen ('38) attempted to study the factors controlling the growth of rabbit blastocysts and described three types of culture and conditions: ( 1 ) in a watch glass placed in a moist chamber, (2) in regular Carrel flasks, and (3) in modified Carrel flasks through which serum could be circulated constantly, thus imitating blood circulation. They obtained best results in a circulating medium, or perfusion culture, although the apparatus was very complex in design. After studying the cleavage rates of rabbit ova in serum at different levels and after addition of a number of chemical compounds with a view to influencing development or clarifying metabolic pathways, Pincus ('42) concluded that only when a chemically defined artificial medium free from serum or other unknown components is evolved, will nutrition of early mammalian eggs be clarified. During the past three years there have been reports of successful storage and growth under in vitro conditions of rabbit and mouse eggs from the 8 cell stage to that of early blastocyst. Whitten ('56) has recovered and cultivated mouse ova in saline, glucose and egg albumin for a period of 48 hours, securing development up to the blastocyst stage, provided the initial development had progressed beyond the 8-cell stage. Adams ('56) showed that the rabbit egg could be stored for short periods of time such as 21Yz hours, and later would grow in a recipient mother on transfer. McLaren and Biggers ('58) have successfully transferred mouse eggs that were cultivated for a period of 48 hours in simple, chemically defined media. These investigators observed full and normal development within recipient mothers from such cultured eggs, and presumably normal mice were born at term. The eggs in culture did not develop beyond the early blastocyst stage, and the rate of this development was slower than that observed in vivo. The following is a report on the study of rabbit and mouse eggs cultivated in certain synthetic media that were supple. mented with small amounts of serum