Autoradiography of progesterone and model compound entry and distribution in Xenopus laevis oocytes.
Xenopus laevis oocytes resume meiosis in response to progesterone. The initial interaction involves surface binding to numerous low-affinity receptor proteins. The mechanism of entry and functions of intracellular steroid are unknown. Because the latter are important for understanding progesterone-induced maturation, a dry-mount autoradiographic technique for analyzing entry and intracellular distribution of radiolabeled steroids was developed and tested. The distinguishing feature of this cryo-technique is sample preparation directly in incubation media using uncross-linked polyacrylamide for inert support. The external ligand functions as an internal standard, so quantitation is by simple ratio (bound/free). The entry kinetics and subcellular binding patterns in large oocytes were studied using this method at nM levels of radiolabeled steroids and model compounds. Progesterone, estradiol, corticosterone, and 1,25-dihydroxycholecalciferol all showed rapid entry (P approximately 10(-6) cm/sec). Entry rates were not saturable with unlabeled steroid. Intracellular patterns of these steroids were highly specific and negatively associated with yolk protein and lipid. Intracellular binding in animal hemisphere ooplasm was 10x that of the yolk-rich vegetative ooplasm. In contrast, dexamethasone, ponasterone-A, and ecdysone displayed entry rates 20-60x slower than progesterone with little compartmentalization. Glycerol, glucose, and leucine entered over 10x slower than progesterone. Cholesterol and Ca++ had entry rates below detection. Evidence for mediated entry of progesterone included the rapid saturation of a cortical compartment equivalent in magnitude to reported receptor numbers. The kinetics and specificity of cortical uptake were consistent with low-affinity, high capacity protein binding. Intracellular binding was seen to correlate with rhodamine 123 patterns, suggesting involvement of mitochondrial or other microtubule-associated structures in steroid responses. Mitochondrial binding is consistent with the limited steroid metabolism seen in oocytes. Since several maturation events are consistent with respiratory uncoupling, reported by others for steroids and isolated organelles, and since mitochondria contain nearly all of the oocyte DNA, a role for these organelles in steroid-induced oocyte maturation was proposed.
Bronson
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Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill 27599-7090.
Stumpf
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WE
D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D005260:Female; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism* / Q000648:ultrastructure; D011374:Progesterone / Q000378:metabolism*; D013256:Steroids / Q000378:metabolism*; D014982:Xenopus laevis
D013256:Steroids; D011374:Progesterone; D007930:Leucine; D005947:Glucose; D005990:Glycerol; D002118:Calcium