Antibody to hog brain tubulin was raised in rabbits and used to stain microtubules within fertilized eggs of the sea urchins Arbacia punctulata and Lytechinus variegatus. The unfertilized egg was found to be devoid of microtubules until 3 min post-insemination, at which time numerous microtubules appear, all of which emanate from the sperm centioles. These microtubules radiate throughout much of the egg's volume by 8 min and appear to bind to and apply a pulling force to the egg nucleus. As a result the egg nucleus moves to the sperm aster in a fashion very reminiscent of mitotic chromosome motion. These observations may help to resolve the long-standing controversy over the mechanism of pronuclear movement. / Since the sea urchin egg contains no microtubules until after sperm-egg fusion it seemed that this might be an excellent system for studying the control of microtubule assembly. Eggs can be artificially activated in the absence of sperm at a number of control points and it was hoped that comparison of the microtubule configurations of eggs activated by different treatments might allow the identification of the cause of microtubule assembly. By this method it was found that neither the Ca('++) transient nor the presence of centrioles can be said to control the number of microtubules developed by the egg, although Ca('++) and centrioles profoundly effect the behavior of microtubules in vitro. Surprisingly, blocking the shift in cytoplasmic pH which accompanies fertilization prevented the appearance of microtubules, even though the direction of the pH shift is away from the optimum pH for microtubule assembly in vitro. To assist in these experiments a spectroscopic method of intracellular pH determination was adapted to sea urchin eggs and found to work extremely well with these cells. This method was used to show that pH can be clamped at 6.8-6.9 either by incubation in choline-substituted sea water (to block Na('+)-H('+) antiport) or by incubation in 10('-2) M Na acetate. Either treatment prevented the formation of microtubules after fertilization. / There are recent suggestions that microtubules in animal cells are associated at all times with centrioles. In the sea urchin egg centrioles are not present and yet the cell is capable of assembling large numbers of microtubules after metabolic derepression. However, the behavior of the microtubule-containing structures in activated eggs is quite different from that seen in fertilized eggs (which contain centrioles), suggesting that centrioles have a minimal effect on the extent of microtubule assembly but are important in regulating the behavior of microtubule-containing structures. / Briefly, the major contributions of the work reported in this monograph are: (i) The establishment of the structure of the apparatus which moves the pronuclei at fertilization and the identification of this apparatus as a favorable system for studies on the mechanism by which microtubule-containing structures generate force, one of the major unsolved problems in cell biology, (ii) The finding that prior to fertilization the egg contains no microtubules, and the identification of the change in cytoplasmic pH as being important in making egg cytoplasm conducive to microtubule assembly, and (iii) The use of artificially activated eggs to show that at least in these cells centrioles do not play a major role in the control of microtubule assembly. / Source: Dissertation Abstracts International, Volume: 42-06, Section: B, page: 2179. / Thesis (Ph.D.)--The Florida State University, 1981.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_74557 |
| Contributors | BESTOR, TIMOTHY HENRY., Florida State University |
| Source Sets | Florida State University |
| Detected Language | English |
| Type | Text |
| Format | 148 p. |
| Rights | On campus use only. |
| Relation | Dissertation Abstracts International |
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