Isolation and characterization of asymmetrically localized antigens from Xenopus oocytes and eggs

The asymmetric localization of cytoplasmic components can be the basis for the cell fate decisions in embryonic patterning. The Xenopus egg has a single axis of asymmetry prior to fertilization, the animal-vegetal axis. Molecules that show animal-vegetal asymmetry in Xenopus oocytes and/or eggs include cytoskeletal components and maternal RNAs. However, there is still much to be learned about asymmetries in other types of cytoplasmic components that can contribute to the development of morphological asymmetries. Previously, monoclonal antibodies against antigens that are highly localized to the vegetal hemisphere of Xenopus eggs have been raised in our laboratory. As seen by immunocytochemistry, the epitopes of these antibodies are restricted to the vegetal cortex of Xenopus eggs and matured oocytes. We have focused on two of these antibodies, which recognize 67 and 48 kDa protein antigens, named Vp67 and Vp48 respectively. Anti-Vp67 and anti-Vp48 antibodies allowed us to purify Vp67 and Vp48 proteins to determine their molecular identity. Vp67 protein has been identified as a Xenopus homolog of dyhydrolipoamide acetyltransferase, an inner mitochondrial membrane-bound E2 component of pyruvate dehydrogenase complex (PDC-E2). Vp48 protein was purified and identified also as a mitochondrial protein (beta subunit of ATP-F1 synthase). Thus, mitochondrial components appear by immunocytochemistry to be asymmetrically localized to the vegetal cortex of Xenopus eggs and matured oocytes. As visualized by immunocytochemistry, the localization occurs during oocyte maturation. However, Western blot analysis of animal and vegetal halves of Xenopus eggs showed that the amount of Vp67 and Vp48 proteins in the vegetal hemisphere is not greater than that in the animal hemisphere. We have performed a differential detergent extraction of Vp67 protein to explain this discrepancy. Detergent extraction of Vp67 protein shows that, in Xenopus eggs, Vp67 appears to be extracted more efficiently from the vegetal hemisphere than from the animal hemisphere. Such differential antigen accessibility and staining with these and other mitochondrial markers suggest repatterning of mitochondria during oocyte maturation that leads to asymmetries in the distribution of different populations of mitochondria. Possible roles of mitochondria in association with maternal determinants, such as germ plasm (localized to the vegetal cortex in Xenopus eggs and embryos), are discussed.