MICROINJECTION OF ANTI-ALPHA-TUBULIN ANTIBODY (DM1A) INHIBITS PROGESTERONE-INDUCED MEIOTIC MATURATION AND DERANGES THE MICROTUBULE ARRAY INFOLLICLE-ENCLOSED OOCYTES OF THE FROG, RANA-PIPIENS

Microinjection of anti-alpha-tubulin (Dm1A) inhibited progesterone-ind uced meiotic maturation in large follicle-enclosed oocytes of the frog , Rana pipiens. DM1A (46 nl; 10 mg/ml) injection significantly increas ed the ED50 value for progesterone as determined by germinal vesicle d issolution (GVD) bioassay. By contrast, low doses of microinjected DM1 A (46 nl; 2.5 mg/ml), anti-actin (clone KJ43A), anti-cytokeratin (C-11 ), anti-intermediate filament antibody (IFA), generic IgG (46 nl; 20 m g/ml) or sodium azide (46 nl; 1 mg/ml), an antibody preservative, were without inhibitory effect in this bioassay. Microinjected, affinity-p urified DM1A (46 nl; 7.5 mg/ml) was also inhibitory, but preabsorption with pure tubulin prior to injection significantly reduced the inhibi tory effect. DM1A injection had no effect on centrifugation-induced ge rminal vesicle migration (GVM). Previous work indicated that drugs (e. g. demecolcine and nocodazole), which destabilise microtubules, enhanc e both centrifugation-induced GVM and progesterone-induced GVD in Rana oocytes. Taking these results together, it is suggested that DM1A inj ection may have differential effects on microtubules in this cell. Thu s, while the majority of microtubules were apparently depolymerised by DM1A (46 nl; 10 mg/ml) injection, a small subpopulation appeared to b e stabilised as bundles. Confocal immunofluorescence microscopy of fol licle-enclosed oocytes after DM1A injection revealed a major loss of m icrotubules throughout the cell; however, apparent sparse bundles of m icrotubules arranged in an approximately 600 mu m shell were associate d with the injectate region 24h post-injection. By contrast, control f ollicle-enclosed oocytes topically labelled with DM1A post-fixation ha d extensive microtubule arrays similar to those previously reported in Xenopus oocytes. Intracellular recording after DM1A injection and pro gesterone treatment yielded an intermediate membrane potential (V-m = -31.8 mV) compared with control (immature) DM1A-injected cells (V-m = -44.7mV) or potassium balanced salt solution (KBS)-injected cells matu red with progesterone (V-m = -13.9mV). These results suggest that DM1A injection does not completely inhibit electrophysiological changes in itiated by progesterone. Working hypotheses are proposed that suggest a role for microtubules in the action of progesterone which normally l ifts the prophase I block in the Rana follicle-enclosed oocyte.