Mammalian fertilization requires structural changes in the participating nuclei and chromosomes. This research will investigate these structural alterations during mouse fertilization and explore their interactions with kinetochores, centrosomes and microtubules, which are responsible for the intracellular positioning of the pronuclei and chromosomes.
Six aims are proposed, three of which explore the appearance and distribution of nuclear architectural proteins while the remainder study their interactions with kinetochores, centrosomes and microtubules. I. Pronuclear development and nuclear lamin acquisition, as assayed by immunofluorescence and video microscopy, is hypothesized to require microtubule assembly. II. Changes in exposed lamins and the new appearance of nuclear matrix proteins are proposed to be developmentally regulated during early embryogenesis. III. Centrosomes specify the organization of the microtubules required for the union of the pronuclei and the alignment and separation of the chromosomes. The distribution of centrosomes and microtubules will be determined through the entire course of fertilization. Furthermore unlike lower animals, the centrosome in this mammal is hypothesized to be maternally inherited and this will be tested during parthenogenesis and polyspermy. In both cases the maternal contribution remains about the same while the paternal contribution is absent in the former case and is multiplied in the latter. IV. Kinetochores, which attach microtubules to chromosomes and might even participate during pronuclear apposition, will be traced throughout fertilization and their interactions with interphase microtubules determined. V. The cytoskeletal elements which position centrosomes and kinetochores will be explored by the use of microtubule and microfilament inhibitors to affect their configurations. VI. The effects of DNA, RNA, and protein synthesis inhibitors on the pattern of these changes in nuclear architecture will be explored. Since centrosomes and kinetochores must duplicate each cell cycle the effects of these inhibitors on these processes will be also determined. By addressing fundamental questions regarding the union of the parental genomes during fertilization, this research may contribute to new approaches for treating infertility, designing contraceptive approaches and for the avoidance or screening of birth defects.
