EXCISION AND DISASSEMBLY OF SPERM TAIL MICROTUBULES DURING SEA-URCHINFERTILIZATION - REQUIREMENTS FOR MICROTUBULE DYNAMICS

To determine the fate of the sperm tail during fertilization, the micr otubules of the incorporated axoneme are measured using a monoclonal a ntibody against acetylated alpha-tubulin in zygotes from the sea urchi n Strongylocentrotus purpuratus. This antibody recognizes axonemal mic rotubules, but does not recognize egg cytoplasmic tubulin or microtubu les. The detachment of the axoneme from the male pronucleus occurs as early as 15 min post-insemination. Following excision, the axoneme is often found in close association with the female pronucleus during its migration to the male pronucleus. Fragmentation of the sperm tail, de tected at 25 min, continues with only a few micrometers remaining at 8 5 min post-insemination. The fluorescence intensity of the axonemal fr agments diminishes over time as compared to intact axonemes. At 100 mi n post-insemination, the sperm axoneme is no longer detected. Alternat ive imaging approaches using brief cold or elevated calcium extraction to disrupt the labile cytoplasmic, but not axonemal, microtubules, in dicate that these observations are not due to changes in the posttrans lational modifications of alpha-tubulin. In the presence of nocodazole , a microtubule assembly inhibitor, a large portion of the tail remain s visible at 100 min post-insemination; this suggests that microtubule dynamics are required for the disassembly of the sperm tail. Furtherm ore, the detachment of the axoneme from the male pronucleus requires t he formation of the sperm aster. This suggests that the sperm aster mi crotubules both detach the axoneme from the male pronucleus, and also cause the translocation of the tail towards the female pronucleus afte r pronuclear union. In summary, the sperm tail is excised from the mal e pronucleus and the tail microtubules disassembled during the first c ell cycle of sea urchin fertilization, and these events require new mi crotubule assembly within the zygote. (C) 1996 Wiley-Liss, Inc.