Successful fertilization, completed when parental genomes unite within an activated oocyte, is critically important for efficient reproduction. Investigations on the microtubule configurations in bovine and porcine zygotes have lead to the discovery that the sperm contributes the centrosome during fertilization in these domestic species. In addition, we have demonstrated that sperm from breeding bulls vary predictably in the ability to organize microtubules after sperm incorporation, and that variations in sperm centrosomes affect the outcome of fertilization. The goals of this project are to characterize, at the molecular level, the relative parental contributions to the bovine zygote's centrosome during fertilization. We have posed eight questions in two aims.
Aim 1. Is the recruitment of maternal molecules to the sperm centrosome essential for the completion of fertilization? 1.1. Do sperm asters in inseminated oocytes, but not in unincorporated sperm, contain maternal centrosomal molecules? 1.2. Does nuclear mitotic apparatus (NuMA) behave atypically during the first cell cycle by being accumulated at the centrosome during interphase? 1.3. Are microtubules essential for their recruitment to, or retention at, the zygote's centrosome? 1.4. Is recruitment and/or retention of maternal molecules essential for the nucleation, organization and/or functioning of the sperm astral microtubules? 1.5. Are -tubulin, NuMA, pericentrin, and PCM-1 and PCM-2 found at the meiotic spindle poles in unfertilized oocytes? Aim 2. What is the molecular basis of the motility which unites the male and female pronuclei? 2.1. Will sperm astral microtubules, nucleated by Xenopus and/or bovine sperm centrosomes and assembling in cell-free extracts, capture female pronuclei and effect the pronuclear motions? 2.2. Can these cell-free sperm asters capture and translocate female pronuclei isolated from fertilized or activated bovine oocytes? 2.3. Are microtubule dynamics required for pronuclear migration in this cell-free model, and will antibodies to motors, centrosomal molecules, or nuclear envelope proteins interfere with this motility? The information obtained during these experiments will add to our fundamental knowledge about the molecular mechanisms leading to pronuclear apposition and genomic union during fertilization in cows, a key step in the reproductive process of all animals. Furthermore, it holds promise for novel assays to test male reproductive function in potential breeding studs. This research may also uncover a new site at which to assay for compounds affecting fertility, with applications for fertility assessment, reproductive management, and reproductive toxicology, as well as aid in developing new approaches for improving reproductive efficiency in domestic species.
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