The long-term goals are to identify and characterize key components of the intrinsic genetic program that controls development and function of male germ cells. The approaches being applied are to identify genes expressed specifically in male germ cells, use the gene knockout approach to define the roles of the proteins they encode, use yeast two-hybrid assays and deletion mutagenesis to identify protein-protein interactions essention for development of the male gamete, and preparation and use of antisera to determine the temporal-spatial distribution of specific gene products. Many genes are expressed only in male germ cells and we are focusing on a few encoding proteins whose functions can be assayed and are likely to be important in gamete development or function. (1) One project involves a key enzyme in the glycolytic pathway. The gene for glyceraldehyde 3-phosphate dehydrogenase (GAPD) expressed throughout the body is silent in male germ cells and instead GAPDS, a spermatogenic cell-specific homolog, is expressed. We cloned the genes for mouse and human GAPDS, prepared antibodies to peptide sequences specific to mouse GAPDS, and localized GAPDS to the fibrous sheath of the sperm flagellum. We hypothesized that the enzyme has a key role in generating the ATP required for sperm motility. GAPDS knockout mice were generated and we found that sperm motility was severely compromised, causing the males to be infertile. Recombinant GAPDS was produced to study the interaction of the enzyme with its natural substrate and cofactor. Reproductive toxicology studies by others suggested that GAPD and GAPDS have different substrate -binding characteristics. Certain compounds act as competitive inhibitors of substrate binding to GAPDS at a lower concentration than GAPD. Our molecular modeling studies indicate that residue differences between GAPDS and GAPD in the vicinity of the substrate-binding pocket probably account for effects of the toxicants on sperm. This also suggests that GAPDS is a potential target for development of a highly specific male contraceptive. (2)Another project involves the assembly and function of the fibrous sheath, a major cytoskeletal structure in the sperm flagellum. Yeast two-hybrid screens identified a WW-domain protein (FBP3) which binds to a proline-rich region of mouse GAPDS. We are mapping the binding domains on each protein and testing the hypothesis that FBP3 anchors GAPDS to the fibrous sheath. We demonstrated that GAPDS binds to the fibrous sheath, a cytoskeletal structure in the sperm flagellum. The major structural protein of the fibrous sheath is a protein kinase A (PKA) anchoring protein (AKAP) encoded by the AKAP4 gene. We used yeast two-hybrid assays to identify proteins that associate with AKAP4 and confirmed that PKA binds to AKAP4 and may regulate cAMP-dependent protein-phosphorylation essential for activation of sperm motility. Yeast two-hybrid assays, alanine and valine scanning-mutagenesis, and pull-down assays were used to define the amino acids of AKAP4 responsible for the binding of regulatory (R) subunits of the PKA tetramer. RI-alpha-specific and dual RI-alpha/RII-alpha-specific binding motifs were identified. It was found that hydrophobic amino acids at three consensus positions are required for PKA binding and that specificity of PKA binding is determined by the size of the aliphatic side-chain on the amino acid in the middle position. This was verified by introducing point mutations into these motifs to switch between RI-alpha-specific, RII-alpha-specific, and dual RI-alpha/RII-alpha-specific binding. These findings advance our understanding of the relationship between the primary sequence and the three-dimensional spatial distribution of residues within the amphipathic alpha-helix of AKAP anchoring domains that determine PKA binding specificity. They are also significant for understanding the molecular mechanisms involved in PKA subtype localization within cells. In addition, yeast two-hybrid screens were used to identify other proteins that bind to AKAP4 in the fibrous sheath. We found that AKAP3 and two novel proteins expressed only in spermatogenic cells also bind to AKAP4. Cre/loxP-mediated gene mutation was used to produce Akap4 gene knockout mice and the males were found to be infertile due to disruption of flagellar structure and function. (3) Another project used gene targeting to disrupt expression of the genes encoding protamines 1 and 2, highly basic nuclear proteins that replace histones following meiosis. They are thought essential for DNA compaction in spermatids, whose nuclei are haploid and lack nucleosomes. Spermatids are connected by cytoplasmic bridges, through which they share mRNA and protein. We found that disruption of one copy of a gene for either protamine 1 or 2 led to altered sperm structure and function and failure to transmit the mutant allele through the germ line of male chimeras. We found that protein sharing leads to reduction in amount of protein by one-half in all spermatids, resulting in defective nuclear compaction during spermiogenesis. This appears to be the first observation of haplo-insufficiency leading to disruption of genetic inheritance in mammals. (4) An addition project is based on findings by others that p55CDC is the mammalian homologue of yeast Cdc20, a protein which associates with the cyclosome/anaphase-promoting complex (APC) and is essential for APC-dependent proteolysis. In cultured cells, p55CDC is located at the kinetochores at the beginning of mitosis, at the spindle poles through the metaphase/anaphase transition, and at the spindle equator from anaphase to telophase. We found that point mutations altering the conserved seventh WD40 motif of p55CDC abolished these dynamics and causes the protein to accumulate in the perinuclear endoplasmic reticulum. This suggested that a protein that binds to the WD40 motif is involved in localizing p55CDC. By using the seventh WD40 motif as bait in a yeast two-hybrid screen, WDAP1 (WD40 adapter protein 1) was identified as a p55CDC-binding factor. Current studies are determing if ADAP1 regulates the function of p55CDC during meiosis in the male.
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