Meiotic maturation in amphibian oocytes involves a sequence of cell division events (e.g., breakdown of the nuclear membrane, condensation of chromosomes, spindle formation, and cytokinesis) as well as activation of metabolic pathways in the cytoplasm and alterations of cell surface properties. The product of maturation is the unfertilized egg. Full-growth Xenopus oocytes can be removed from their follicles and matured in vitro in simple salt solutions, provided they are exposed to steroid hormones such as progesterone. Cytoplasmic intermediates in the progesterone stimulation of maturation have been detected: a small aliquot (2 to 5%) of cytoplasm from a maturing oocyte is withdrawn in a glass micropipet and injected into an unstimulated recipient oocyte, with the result that the recipient itself matured. This "maturation promoting factor" does not attenuate on 10 fold repeated serial transfer, indicating it does not contain progesterone or a progesterone product, and is formed by an autoactivation mechanism. Thus, this system is particularly favorable to study triggers of cell division and metabolic activation. We propose to investigate the phosphorylation of proteins during maturation, since this biochemical mechanism underlies the control circuits for large-scale metabolic shifts in muscle and liver. We inject radioactive phosphate into maturing oocytes and achieve such high specific activities that we can analyze the variety and time of appearance of phosphoprotein targets, using just 2 to 5 oocytes for a gel. We are isolating classes of phosphoproteins (non-yolk) and reinjecting them into maturing oocytes to assay in vivo when specific phosphoprotein phosphatases are active. We are testing the causal role of protein kinases in triggering maturation by injecting them into oocytes: preparations of phosphorylase kinase, Ca ions requiring and autoactivating enzyme, have promoted maturation, but irregularly. We are isolating and testing endogenous kinases for their time of appearance and ability to promote maturation when injected back into oocytes. BIBLIOGRAPHIC REFERENCES: Tokunaga, C. and J.C. Gerhart. The effect of growth and joint formation on bristle pattern in D. Melanogaster. J. Exp. Zool. 198:79-96, 1976. Maller, J., M. Wu and J.C. Gerhart. Changes in protein phosphorylation accompanying maturation of Xenopus laevis oocytes. Dev. Biol. 1977, in press.
NIH ExPorter
NIH ExPorter