Microinjection of anti-α-tubulin antibody (DM1A) inhibits progesterone-induced meiotic maturation and deranges the microtubule array in follicle-enclosed oocytes of the frog,Rana pipiens

Abstract

SummaryMicroinjection of anti-α-tubulin (Dm1A) inhibited progesterone-induced meiotic maturation in large follicle-enclosed oocytes of the frog,Rana pipiens. DM1A (46nl; 10mg/ml) injection significantly increased the ED50value for progesterone as determined by germinal vesicle dissolution (GVD) bioassay. By contrast, low doses of microinjected DM1A (46nl; 2.5mg/ml), anti-actin (clone KJ43A), anti-cytokeratin (C-11), anti-intermediate filament antibody (IFA), generic IgG (46 nl; 20 mg/ml) or sodium azide (46 nl; 1 mg/ml), an antibody preservative, were without inhibitory effect in this bioassay. Microinjected, affinity-purified DM1A (46n1; 7.5mg/ml) was also inhibitory, but preabsorption with pure tubulin prior to injection significantly reduced the inhibitory effect. DM1A injection had no effect on centrifugation-induced germinal vesicle migration (GVM). Previous work indicated that drugs (e.g. demecolcine and nocodazole), which destabilise microtubules, enhance both centrifugation-induced GVM and progesterone-induced GVD inRanaoocytes. Taking these results together, it is suggested that DM1A injection may have differential effects on microtubules in this cell. Thus, while the majority of microtubules were apparentl depolymerised by DM1A (46nl; 10mg/ml) injection, a small subpopulation appeared to be stabilised as bundles. Confocal immunofluorescence microscopy of follicle-enclosed oocytes after DM1A injection revealed a major loss of microtubules throughout the cell however, apparent sparse bundles of nucrotubules arranged in an approximately 600 μm shell were associated with the injectate region 24h post-injection. By contrast, control follicle-enclosed oocytes topically labelled with DM1A post-fixation had extensive microtubule arrays similar to those previously reported inXenopusoocytes. Intracellular recording after DM1A injection and progesterone treatment yielded an intermediate membrane potential (Vm= −31.8mV) compared with control (immature) DMIA-injected cells (Vm= −44.7 mV) or potassium balanced salt solution (KBS)-injected cells matured with progesterone (Vm= −13.9mV). These results suggest that DM1A injection does not completely inhibit electrophysiological changes initiated by progesterone. Working hypotheses are proposed that suggest a role for microtubules in the action of progesterone which normally lifts the prophase I block in theRanafollicle-enclosed oocyte.