Research Project
2889164
Flawless sperm functioning is essential for the successful completion of fertilization in humans. This functioning includes activities of the sperm prior to meeting the oocyte, sperm-oocyte plasma membrane fusion, and the cytoplasmic events mediated by the sperm nucleus and centrosome, which lead to the culmination of the fertilization process -- the merging of the parental genomes. Defects in sperm behavior at any stage result in fertilization failure. This application investigates the microtubule organizing capacity of the human sperm centrosome as causes of idiopathic male infertility . Eighteen questions are posed in four specific aims using sperm from fertile and infertile men. Aim #1: Are centrosomal proteins found in human sperm, and do their concentrations vary predictably in sperm from infertile donors? Aim #2. Does the binding of maternal gamma-tubulin vary predictably in sperm from infertile donors? Aim #3. Are there variations in the ability of sperm from infertile donors to nucleate microtubules in egg extracts? Aim #4. Will the sperm asters formed in pronucleate-stage mammalian oocytes, inseminated with sperm from men of varying fertility, predictably vary in their size and microtubule density? These hypotheses are tested: i. human sperm centrosomes contain centrin; ii. sperm centrosomes from some infertile men are deficient in centrin; iii. human sperm do not contain appreciable amounts of gamma- tubulin, a microtubule nucleating protein; iv. human sperm bind gamma- tubulin after exposure to egg extracts, but the centrosome must be primed before it acquires this capability; v. sperm from some infertile men are unable to bind maternal gamma-tubulin; vi. human sperm centrosomes can become heavily phosphorylated; vii. human sperm are unable to nucleate microtubule assembly when exposed to egg extracts, until they have been 'primed'; viii. after priming, the centrosomes from fertile men nucleate radial microtubule arrays, resembling sperm asters; ix. sperm from some infertile men are unable to nucleate microtubules; x. human sperm organize sperm asters after incorporation into zona-free oocytes; xi. the size of these sperm asters is correlated with the donor's fertility; xii. sperm with defective centrosomes from infertile men are unable to complete fertilization in vitro; and xiii. these approaches might be developed into reliable assays for male infertility. This investigation characterizes the molecules present in human sperm centrosomes, the molecules bound to the sperm centrosome after exposure to egg cytoplasm, and the microtubule organizing abilities of the sperm centrosome in vitro and in vivo. These approaches, which explore defects in interactions of the sperm and oocyte, as well as subtle aspects of sperm function not currently detected with existing methods, may translate into clinical applications for the diagnosis and possible treatment of idiopathic male infertility.
