Available evidence indicates that protein incorporation by amphibian oocytes is mediated through a process of micropinocytosis. Oocytes incubated under our standard conditions sequester protein at a constant rate for a t least 24 hours and appear to maintain approximately the same level of micropinocytosis as that observed in situ. Protein uptake is markedly sensitive to temperature and the ionic composition of the medium, with calcium being a particularly important component. Magnesium, phosphate, and additional energy sources are of only minor significance. Based on these observations, several slightly improved reformulations of the standard incubation medium are provided

Female toads given 1000 units of human chorionic gonadotropin ovulated within 24 h and began to synthesize a phosphoprotein which appeared, but did not accumulate, in the serum (physiological half-life, 2 days). The period of synthesis lasted about 30 days, after which mature oocytes were once again observed in the ovary. Male toads given a single dose of 0.1–1.0 mg estradiol-17β also immediately began to synthesize a phosphoprotein. The rate of maximum synthesis and the length of time for the maximum to be reached were directly proportional to the amount of estrogen administered. The serum phosphoprotein in male toads, however, had a physiological half-life of approximately 40 days. Ovariectomized females produced a serum phosphoprotein with a physiological half-life similar to that of males. We therefore concluded that under normal conditions the circulating phosphoprotein produced by the liver of the female as a response to estrogen is accumulated by the vitellogenic ovary. The serum phosphoprotein is apparently a lipophosphoprotein complex from which at least one of the yolk proteins, phosvitin, may be derived.

Developing Xenopus oocytes removed from the ovary and divested of their outermost cellular layers (theca and surface epithelium) will incorporate protein continuously over a period of at least two to six days when cultured in uitro. The rate of protein incorporation depends upon:(a) the extent to which the donor has been stimulated by gonadotropin; (b) the size of the oocyte; and (c) the protein composition of the medium, since a mechanism exists to sequester selected protein. Protein uptake is negligible when oocytes are cultured either with all their cellular layers intact or with all their cellular layers removed. These results imply that the outer surface epithelium is relatively impermeable to protein and that the integrity of the investing follicular epithelium is essential for vigorous protein incorporation by the oocyte.

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A general method has been developed for the isolation and purification of phosvitin from vertebrate eggs. The method is detailed in three steps consisting of (i) isolation of a phosvitin–lipovitellin complex; (ii) ammonium sulfate precipitation of the lipovitellin; and (iii) DEAE-cellulose chromatography of the remaining phosvitin. Phosvitin was isolated from the eggs of five representative vertebrates (lamprey, trout, frog, turtle, and chicken), and chemical analyses together with sedimentation studies were performed on the samples. Preparations were obtained with the lowest N/P ratios reported to date. The analytical results also suggested that trout phosvitin has approximately half the molecular weight (24,000) of the other proteins and that "purified" phosvitin may still be heterogeneous.