Excision and disassembly of sperm tail microtubules during sea urchin fertilization: requirements for microtubule...

To determine the fate of the sperm tail during fertilization, the microtubules of the incorporated axoneme are measured using a monoclonal antibody against acetylated alpha-tubulin in zygotes from the sea urchin Strongylocentrotus purpuratus. This antibody recognizes axonemal microtubules, but does not recognize egg cytoplasmic tubulin or microtubules. 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, detected at 25 min, continues with only a few micrometers remaining at 85 min post-insemination. The fluorescence intensity of the axonemal fragments diminishes over time as compared to intact axonemes. At 100 min post-insemination, the sperm axoneme is no longer detected. Alternative imaging approaches using brief cold or elevated calcium extraction to disrupt the labile cytoplasmic, but not axonemal, microtubules, indicate that these observations are not due to changes in the post-translational modifications of alpha-tubulin. In the presence of nocodazole, a microtubule assembly inhibitor, a large portion of the tail remains visible at 100 min post-insemination; this suggests that microtubule dynamics are required for the disassembly of the sperm tail. Furthermore, the detachment of the axoneme from the male pronucleus requires the formation of the sperm aster. This suggests that the sperm aster microtubules both detach the axoneme from the male pronucleus, and also cause the translocation of the tail towards the female pronucleus after pronuclear union. In summary, the sperm tail is excised from the male pronucleus and the tail microtubules disassembled during the first cell cycle of sea urchin fertilization, and these events require new microtubule assembly within the zygote.