Egg culture: the foundation

Abstract

Ralph Brinster began his classic work on egg culture more than 35 years ago. His interest in mammalian egg culture had developed, in part, as a consequence of his experiences with animal breeding and reproduction that he gained while growing up on a farm. Ralph decided early in his career that an in vitro approach to culturing eggs would provide a powerful tool with which to study the development of these cells. Beginning at the close of the 19th century, a number of investigators had performed in vitro studies on egg culture and the related area of egg transfer; however, the ability to recover and transplant eggs had reached a much higher level of perfection than had culture. Eggs of many species could be successfully transferred, but there was no reliable technique for egg culture. In 1963, Ralph reported a method for culturing eggs in microdrops of medium under oil (Brinster, 1963), which has become universally used. Two years later, he identified pyruvate as the central and essential energy source for early stages of mouse eggs (Brinster, 1965b). These two developments revolutionized in vitro studies of mammalian eggs and issued in an era of intense research activity concerning egg culture and egg manipulation. Effective formulations of culture media could now be developed to allow routine in vitro maintenance of eggs, and important parameters for these recipes were soon determined. It was quickly established that the requirement for pyruvate as an energy source exists at ovulation in many species and is already present in germ cells of the mouse fetus. The metabolic activity of the fertilized mouse egg was shown to be low and comparable to bone; however, four days later, at the blastocyst stage of development, the metabolic activity was comparable to brain. Thus, a foundation of understanding about the biology of early mammalian eggs was established between 1960 and 1970, and subsequent studies have broadened this understanding. However, the greatest impact of a simple, reliable egg culture method has been to provide the ability to perform complicated manipulative procedures on preimplantation stages of mammalian embryos. In no area has this been more important than in development of transgenic animals. All methods for generating germ line genetic modifications rely on the ability to maintain and manipulate eggs and early developmental stages in vitro without loss of developmental competence. The importance of efficient egg culture to manipulation and transgenesis is fundamental and enabling.