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Imaging Sea Urchin Fertilization

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Confocal Microscopy Methods and Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 122))

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Abstract

Imaging animal fertilization presents a number of unique challenges that arise from the unusual nature of the cells involved. Eggs are among the largest cells produced by animals, and sperm are frequently the smallest. Observation of the initial steps of fertilization, including sperm-egg binding and fusion, can be challenging owing to the size discrepancy between these two cell types and, in many species, the rapid time course over which they occur. Subsequent events in fertilization, including the remarkable activities of the microtubular cytoskeleton during the first cell cycle, are usually obscured by the mass of the egg cytoplasm, and only the most obvious features, such as pronuclear formation, are apparent by routine microscopic methods. The application of confocal microscopy methods to the demands of imaging studies in fertilization has proven to be an extremely valuable approach, as the morphological features of most types of animal eggs are extremely well suited to the specific advantages this technology offers. Confocal microscopy is a powerful and flexible tool, and has been skillfully used and developed by a number of researchers not only to study the structural features of fertilization at high resolution, but also to examine dynamic events in living gametes, including imaging signaling events that occur during and after gamete fusion.

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References

  1. Zalensky, A. O., Allen, M. J., Kobayashi, A., Zalenskaya, I. A., Balhorn, R., and Bradbury, E. M. (1995) Well-defined genome architecture in the human sperm nucleus. Chromosoma 103, 577–590.

    Article  PubMed  CAS  Google Scholar 

  2. Blerkom, J. V., Davis, P. W., and Merriam, J. (1994) A retrospective analysis of unfertilized and presumed parthenogenetically activated human oocytes demonstrates a high frequency of sperm penetration. Hum. Reprod. 9, 2381–2388.

    PubMed  Google Scholar 

  3. Blerkom, J. V., Davis, P., Merriam, J., and Sinclair, J. (1995) Nuclear and cytoplasmic dynamics of sperm penetration, pronuclear formation, and microtubule organization during fertilization and early preimplantation development in the human. Hum. Reprod. Update 1, 429–461.

    Article  PubMed  Google Scholar 

  4. Simerly, C., Wu, G., Zoran, S., Ord, T., Rawlins, R., Jones, J., Navara, C., Gerrity, M., Rinehart, J., Binor, Z., Asch, R., and Schatten, G. (1995) The paternal inheritance of the centrosome, the cell’s microtubule organizing center, in humans and the implications for infertility. Nature Med. 1, 47–53.

    Article  PubMed  Google Scholar 

  5. Wu, G.-J., Simerly, C., Zoran, S. S., Funte, L. R., and Schatten, G. (1996) Microtubule and chromatin dynamics during fertilization and early development in rhesus monkeys and regulation by intracellular calcium ions. Biol. Reprod. 55, 260–270.

    Article  PubMed  CAS  Google Scholar 

  6. Kim, N. H., Funahashi, H., Abeydeera, L. R., Moon, S. J., Prather, R. S., and Day, B. N. (1996) Effects of oviductal fluid on sperm penetration and cortical granule expcytosis during fertilization of pig oocytes in vitro. J. Reprod. Fertil. 107, 79–86.

    Article  PubMed  CAS  Google Scholar 

  7. Navara, C. S., First, N. L., and Schatten, G. (1996) Phenotypic variations among paternal centrosomes are expressed within the zygote as disparate microtubule lengths and sperm aster organization: correlations between centrosome activity and development success. Proc. Natl. Acad. Sci. USA 93, 5384–5388.

    Article  PubMed  CAS  Google Scholar 

  8. Coonrod, S. A., Westhusin, M. E., and Naz, R. K. (1994) Monoclonal antibody to human fertilization antigen-1 (FA-1) inhibits bovine fertilization in vitro: application in immunocontraception. Biol. Reprod. 51, 14–23.

    Article  PubMed  CAS  Google Scholar 

  9. Shiraishi, K., Okada, A., Shirakawa, H., Nakanishi, S., Mikoshiba, K., and Miyazaki, S. (1995) Developmental changes in the distribution of the endoplasmic reticulum and inositol 1,4,5-triphosphate receptors and the spatial pattern of Ca2+ release during maturation of hamster oocytes. Dev. Biol. 170, 594–606.

    Article  PubMed  CAS  Google Scholar 

  10. Mehlman, L. M., Terasaki, M., Jaffe, L. A., and Kline, D. (1995) Reorganization of the endoplasmic reticulum during meiotic maturation of the mouse oocyte. Dev. Biol. 170, 607–615.

    Article  Google Scholar 

  11. Ayabe, T., Kopf, G. S., and Schultz, R. M. (1995) Regulation of mouse egg activation: presence of ryanodine receptors and effects of microinjected ryanodine and cyclic ADP ribose on uninseminated and inseminated eggs. Development 121, 2233–2244.

    PubMed  CAS  Google Scholar 

  12. House, C. R. (1994) Confocal ratio-imaging of intracellular pH in unfertilized mouse oocytes. Zygote 1, 37–45.

    Google Scholar 

  13. Simerly, C. R., Hecht, N. B., Goldberg, E., and Schatten, G. (1993) Tracing the incorporation of the sperm tail in the mouse zygote and early embryo using an anti-testicular alpha-tubulin antibody. Dev. Biol. 158, 536–548.

    Article  PubMed  CAS  Google Scholar 

  14. Ducibella, T., Duffy, P., and Buetow, J. (1994) Quantification and localization of cortical granules during oogenesis in the mouse. Biol. Reprod. 50, 467–473.

    Article  PubMed  CAS  Google Scholar 

  15. Carroll, D. J., Dikegoros, E., Koppel, D. E., and Cowan, A. E. (1995) Surface expression of the pre-beta subunit of fertilin is regulated at a post-translational level in guinea pig spermatids. Dev. Biol. 168, 429–437.

    Article  PubMed  CAS  Google Scholar 

  16. Gard, D. L. (1993) Confocal immunofluorescence microscopy of microtubules in amphibian oocytes and eggs. Methods Cell Biol. 38, 241–264.

    Article  PubMed  CAS  Google Scholar 

  17. Gard, D. L. (1991) Organization, nucleation, and acetylation of microtubules in Xenopus laevis oocytes: a study by confocal immunofluorescence microscopy. Dev. Biol. 143, 346–362.

    Article  PubMed  CAS  Google Scholar 

  18. Gard, D. L. (1992) Microtubule organization during maturation of Xenopus oocytes: assembly and rotation of the meiotic spindles. Dev. Biol. 151, 516–530.

    Article  PubMed  CAS  Google Scholar 

  19. Gard, D. L. (1993) Ectopic spindle assembly during maturation of Xenopus oocytes: evidence for functional polarization of the oocyte. Dev. Biol. 159, 298–310.

    Article  PubMed  CAS  Google Scholar 

  20. Gard, D. L. (1994) Gamma-tubulin is asymmetrically distributed in the cortex of Xenopus oocytes. Dev. Biol. 161, 131–140.

    Article  PubMed  Google Scholar 

  21. Gard, D. L., Cha, B. J., and Schroeder, M. M. (1995) Confocal immunofluorescence microscopy of microtubules, microtubule-associated proteins, and microtubule-organizing centers during amphibian oogenesis and early development. Curr. Top. Dev. Biol. 31, 383–431.

    Article  PubMed  CAS  Google Scholar 

  22. Gard, D. L., Cha, B. J., and Roeder, A. D. (1995) F-actin is required for spindle anchoring and rotation in Xenopus oocytes; a re-examination of the effects of cytochalasin B on oocyte maturation. Zygote 3, 17–26.

    Article  PubMed  CAS  Google Scholar 

  23. Gard, D. L., Affleck, D., and Error, B. M. (1995) Microtubule organization, acetylation, and nucleation in Xenopus laevis oocytes: II. A developmental transition in microtubule organization during early diplotene. Dev. Biol. 168, 189–201.

    Article  PubMed  CAS  Google Scholar 

  24. Roeder, A. D. and Gard, D. L. (1994) Confocal microscopy of F-actin distribution in Xenopus oocytes. Zygote 2, 111–124.

    Article  PubMed  CAS  Google Scholar 

  25. Schroeder, M. M. and Gard, D. L. (1992) Organization and regulation of cortical microtubules during the first cell cycle of Xenopus eggs. Development 114, 699–709.

    PubMed  CAS  Google Scholar 

  26. Fagotto, F. and Maxfield, F. R. (1994) Changes in yolk platelent pH during Xenopus laevis development correlate with yolk utilization. A quantitative confocal microscopy study. J. Cell Sci. 107, 3325–3337.

    PubMed  CAS  Google Scholar 

  27. Olds, J. L., Favit, A., Nelson, T., Ascoli, G., Gerstein, A., Cameron, M., Cameron, L., Lester, D. S., Rakow, T., and DeBarry, J. (1995) Imaging protein kinase C activation in living sea urchin eggs after fertilization. Dev. Biol. 172, 675–682.

    Article  PubMed  CAS  Google Scholar 

  28. Terasaki, M. (1995) Visualization of exocytosis during sea urchin egg fertilization using confocal microscopy. J. Cell Sci. 108, 2293–2300.

    PubMed  CAS  Google Scholar 

  29. Stricker, S. A., Centonze, V. E., Paddock, S. W., and Schatten, G. (1992) Confocal microscopy of fertilization-induced calcium dynamics in sea urchin eggs. Dev. Biol. 149, 370–380.

    Article  PubMed  CAS  Google Scholar 

  30. Terasaki, M. and Jaffe, L. A. (1991) Organization of the sea urchin egg endoplasmic reticulum and its reorganization at fertilization. J. Cell Biol. 114, 929–940.

    Article  PubMed  CAS  Google Scholar 

  31. Holy, J. and Schatten, G. (1991) Spindle pole centrosomes of sea urchin embryos are partially composed of material recruited from maternal stores. Dev. Biol. 147, 343–353.

    Article  PubMed  CAS  Google Scholar 

  32. Holy, J. and Schatten, G. (1997) Recruitment of maternal material during assembly of the zygote centrosome in fertilized sea urchin eggs. Cell Tiss. Res., 289, 285–297.

    Article  CAS  Google Scholar 

  33. Stricker, S. A. (1995) Time-lapse confocal imaging of calcium dynamics in starfish embryos. Dev. Biol. 170, 496–518.

    Article  PubMed  CAS  Google Scholar 

  34. Jaffe, L. A. and Terasaki, M. (1994) Structural changes in the endoplasmic reticulum of starfish oocytes during meiotic maturation and fertilization. Dev. Biol. 164, 579–587.

    Article  PubMed  CAS  Google Scholar 

  35. Speksnijder, J. E., Terasaki, M., Hage, W. J., Jaffe, L. F., and Sardet, C. (1993) Polarity and reorganization of the endoplasmic reticulum during fertilization and ooplasmic segregation in the ascidian egg. J. Cell Biol. 120, 1337–1346.

    Article  PubMed  CAS  Google Scholar 

  36. Snook, R. R., Markow, T. A., and Karr, T. L. (1994) Functional nonequivalence of sperm in Drosophila. Proc. Natl. Acad. Sci. USA 91, 11,222–11,226.

    Article  PubMed  CAS  Google Scholar 

  37. Theurkauf, W. E., Smiley, S., Wong, M. L. and Alberts, B. M. (1992) Reorganization of the cytoskeleton during Drosophila oogenesis: implications for axis specification and intercellular transport. Development 115, 923–936.

    PubMed  CAS  Google Scholar 

  38. Watson, C. A., Sauman, I. and Berry, S. J. (1993) Actin is a major structural and functional element of the egg cortex of giant silkmoths during oogenesis. Dev. Biol. 155, 315–323.

    Article  PubMed  CAS  Google Scholar 

  39. Holy, J. (1997) Chlorpropham [N-(3-chlorophenyl)carbamate] disrupts microtubule organization, cell division, and early development of sea urchin embryos. J. Toxicol. Env. Health 54, 319–333.

    Article  Google Scholar 

  40. Showman, R. M. and Foerder, C. A. (1979) Removal of the fertilization membrane of sea urchin embryos employing aminotriazole. Exp. Cell Res. 120, 253–255.

    Article  PubMed  CAS  Google Scholar 

  41. Balczon, R. and Schatten, G. (1983) Microtubule-containing detergent-extracted cytoskeletons in sea urchin eggs from fertilization through cell division: antitubulin immunofluorescence microscopy. Cell Motil. Cytoskel. 3, 213–226.

    Article  Google Scholar 

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© 1999 Humana Press Inc., Totowa, NJ

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Holy, J.M. (1999). Imaging Sea Urchin Fertilization. In: Paddock, S.W. (eds) Confocal Microscopy Methods and Protocols. Methods in Molecular Biology™, vol 122. Humana Press. https://doi.org/10.1385/1-59259-722-X:153

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  • DOI: https://doi.org/10.1385/1-59259-722-X:153

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-526-3

  • Online ISBN: 978-1-59259-722-2

  • eBook Packages: Springer Protocols

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