The deliberate creation of human zygotes for biomedical research is unlikely to ever be free of religious, ethical, moral, political and financial complexities. Nevertheless, a detailed understanding of the cellular and molecular events during humans fertilization is essential for diagnosing and treating infertility, as well as for developing new approaches for managing reproduction. To address these issues, this renewal application consolidated from two grants [R01 HD12913 and R01 HD22902] poses fifteen questions in three specific aims to understand egg-mediated motility during fertilization in clinically relevant systems. I. Is the recruitment of maternal molecules to the sperm centrosome essential for the completion of fertilization, and does paternal centrin sever the sperm tail from the head and midpiece in a Ca plus plus minus dependent manner? II. What is the molecular basis of the motility which unites the male and female pronuclei? III. How do centrosomal and microtubule molecules behave during ICSI and ROSI/ROSNI, and are there clinically useful diagnostic probes for assessing and/or predicting the normalcy of fertilization? Tested hypotheses include that: Maternal gamma-tubulin is attracted to, but not retained at, the sperm centrosome by microtubules and is itself responsible for microtubule nucleation; NuMA is also attracted to the zygote's centrosome by microtubules and is responsible for organizing the gamma-tubulin nucleated microtubules into the radially symmetric sperm aster; Pericentrin, PCM-1 and PCM-2 are components of the zygote centrosome's substructure; Paternal centrin severs the sperm tail microtubules; Pronuclear motility in a cell-free model will demonstrate motor protein and/or microtubule dynamics in effecting genomic union; Microtubule and centrosome dynamics differ during ICSI and ROSI/ROSNI; and some oocytes discarded by infertility clinics as either unfertilized or fertilization failures display centrosome defects. Together these experiments will advance clinically-relevant knowledge about genomic union during fertilization, providing information on the biological events occurring during the new and well accepted, but poorly understood, methods of ART. By exploring defects in interactions of the gametes, as well as subtle aspects of sperm function not detected with existing methods, the results may translate into applications for the diagnosis and treatment of infertility.
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