O-10: A Marked Animal-Vegetal Polarity in The Localization of Na+,K+-ATPase Activity and Its Down-Regulation Following Progesterone-Induced Maturation

Background: Polarized cells are key to the process of differentiation. Xenopus oocyte is a polarized cell that has complete blue-print to differentiate 3 germ layers following fertilization, as key determinant molecules (Proteins and RNAs) are asymmetrically localized. The objective of this work was to localize Na+, K+-ATPase activity along animal-vegetal axis of polarized Xenopus oocyte and following their progesterone-induced maturation (M-phase of the cell cycle) and their role in polarity. Materials and Methods: Enzyme-specific electron microscopy phosphatase histochemistry techniques were used. Results: The Na+, K+-ATPase is the most critical and important protein. The stage-VI Xenopus oocyte has a very distinct animal-vegetal polarity with structural and functional asymmetry. This polarity begins to develop in late stage III with accumulation of pigment granules towards the animal hemisphere. In this study, we have shown the expression and distribution pattern of Na+,K+-ATPase activity in stage-VI oocytes, and following progesteroneinduced maturation. Using enzyme-specific electron microscopy phosphatase histochemistry, [3H]-ouabain autoradiography, and immunofluorescence cytochemistry, we have shown that Na+,K+-ATPase activity is mainly confined to the entire animal hemisphere as bell (the Nucleus/Germinal Vesicle is present in the animal hemisphere). The polarized distribution of Na+,K+-ATPase activity begins to develop in late stage III and continues through to stage VI. Electron microscopy histochemical results also show this polarized distribution persists following progesterone-induced maturation (leading to M-phase), and it becomes gradually more polarized towards the animal pole. The time course following progesterone- induced maturation suggests that there is gradual down-regulation of Na+, K+-ATPase activity leading to germinal vesicle breakdown (GVBD). By GVBD, the Na+, K+-ATPase activity is completely down-regulated due to endocytotic removal of pump molecules from the plasma membrane into the sub-cortical region of the oocyte. This study provides the first direct evidence for a marked asymmetric localization of Na+, K+-ATPase activity in any vertebrate oocyte. Here, we propose that such asymmetry in Na+,K+-ATPase activity establishes and maintains polarity in Xenopus oocytes due to extracellular and intracellular ionic and electrical gradients. Also such asymmetry in stage VI oocyte and their down regulation following progesterone-induced maturation plays a key role in the active state of the germinal vesicle in stage-VI oocytes and chromosomal condensation after GVBD. This work will entail 1. What are the key Na+ dependent genes expressed in cell? 2. What role Na+,K+-ATPase plays in cell cycle? 3. Does down-regulation of Na+,K+-ATPase in MPhase phosphorelate key enzymes involved in cell cycle? 4. What is the level of Na+,K+-ATPase activity in uterine cancer and if alpha subunit of the protein acts as receptor for progesterone? Conclusion: At the resolution of electron microscopy and light microscopy, Na+,K+-ATPase activity is found to be localized in the animal hemispheres of polarized Xenopus oocytes and it is completely down regulated following progesterone induced maturation.