The initiation of protein synthesis in eggs of rana pipiens.

During amphibian o6genesis, small o6cytes in the first meiotic prophase undergo a prolonged growth period resulting in the formation of fully developed o6cytes which have greatly increased in size. The injection of a pituitary suspension into mature females stimulates the release of the full-grown o6cytes from the ovary and the breakdown of their large nuclei (germinal vesicles). The ovulated eggs then continue maturation to the second meiotic metaphase. It is only in the latter condition that the eggs will respond to an activating stimulus, provided either by sperm or parthenogenetic agents, before they complete the second meiotic division and continue their development.1 The period of o6genesis is an interval of intense metabolic activity,2 and it is during the later phases of o6cyte growth that the large yolk reserve is laid down.3 However, it has been heretofore claimed that the full-grown ovarian o6cyte is metabolically inactive: studies on amphibian eggs, based on autoradiographic evidence, have failed to demonstrate protein synthesis in either the full-grown o6cytes or in ovulated eggs prior to fertilization.4' Similar studies have demonstrated only small amounts of protein synthesis during cleavage stages.6 In earlier experiments, we measured protein synthesis after fertilization or artificial activation of Rana pipiens eggs by injecting tritiated leucine directly into the eggs and measuring its incorporation into acid-precipitable proteins. These experiments showed that significant protein synthesis occurs in the first six hours after fertilization or artificial activation, and that the rate of this synthesis remains essentially constant over the time studied.8 Furthermore, our observation that protein synthesis assumes its definitive rate with virtually no time lag after activation suggests that protein synthesis may already be in progress at the time of activation. In the present investigation, we have measured the incorporation of labeled leucine into the proteins of full-grown ovarian o6cytes and ovulated eggs during the whole period of maturation, up to and including fertilization or artificial activation. On the basis of these experiments, we have attempted to determine the time when protein synthesis begins prior to activation and the stimulus responsible for its initiation. Materials and Methods.-Rana pipiens which were collected in the fall were used in these studies. Ovulation was induced in mature females by the intraperitoneal injection of a pituitary suspension. This was prepared by mincing two female and one male R. pipiens pituitaries in 1.5 ml of Steinberg's solution.9 After injection of the suspension, the frogs were maintained at a temperature of 18 ± 0.50C. Prior to ovulation, individual eggs could be obtained only by dissecting them from their ovarian sheath and follicular membranes with watchmaker's forceps. Release of eggs from the ovary into the coelom usually began about 20 hours after injection of the pituitary suspension and continued over a period of several hours. During this time, free body-cavity eggs, surrounded only by a thin vitelline membrane, could be obtained from the exposed coelom with a wide-mouth pipette. After the completion of ovulation, uterine eggs were obtained by stripping the frogs.