These studies will identify and characterize key components of the intrinsic genetic program that controls development and function of male germ cells and that ultimately define the conditions responsible for male fertility. The approaches currently 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, employ yeast two-hybrid assays and deletion mutagenesis to identify protein-protein interactions essential for development of the male gamete, and prepare antisera to determine the temporal-spatial distribution of specific gene products. Many genes are expressed only in male germ cells and selected genes are being studied that encode proteins whose functions are essential for novel aspects of gamete development and/or function. (1) Role of peritubular myoid cells in spermatogonial stem cell self-renewal in the testis niche: We hypothesized that T-regulated GDNF expression in PM cells is required for SSC renewal. This hypothesis was tested using an adult mouse PM cell primary culture system. We found that T induced GDNF expression at the mRNA and protein levels in PM cells. Furthermore, when SSCs were isolated and co-cultured with PM cells with or without T and transplanted to the testes of germ cell-depleted mice, the number of SSC-derived colonies was increased significantly by in vitro T treatment. These results strongly suggest that T-dependent regulation of GDNF expression in PM cells has a significant role in defining the microenvironment of the niche and influencing SSC self-renewal. (2) Regulation of meiotic progression in spermatogenesis: The mechanisms regulating transition from prophase I to metaphase I of meiosis during spermatogenesis are uncertain. Previous studies have suggested that proteins other than or in addition to CDK1 and Cyclin B1 may regulate this process during spermatogenesis. Conditional gene targeting approaches are being used to determine if these proteins are essential for the progression of meiosis in male germ cells. (3) The role of novel scaffold proteins and glycolytic enzymes in sperm motility: The flagellum is one of the most complex of all cell organelles and many of its proteins are encoded by genes expressed during post-meiotic haploid phase of spermatogenesis. We have used a variety of approaches to identify components of the fibrous sheath, a key cytoskeletal component of the sperm flagellum. These included structural proteins, signal transduction anchoring proteins, and glycolytic enzymes, most of which are encoded by genes only expressed in male germ cells. This led us to hypothesize thatmammalian sperm require ATP produced by glycolysis for motility and the fibrous sheath is an essential scaffold for the glycolytic enzymes. This hypothesis has been confirmed using gene targeting and current studies are determining how the different glycolytic enzymes and structural components of the fibrous sheath are assembled and how their functions are regulated. (4) Strain-specific modifier gene for male fertility in mice heterozyogus for a targeted mutation in protamine 2 (Prm2) gene: Chromatin condensation following meiosis in the male germ line involves removal of most histones and their replacement with transition proteins and then protamines. Mice and humans have two protamines (PRM1 and PRM2), while most other mammals have only PRM1. These genes are expressed only in post-meiotic male germ cells. Cell cleavage is incomplete in male germ cells and they share the mRNAs and proteins for PRM1 and PRM2. We previously generated chimeras with ES cells derived from 129 strain mice with a mutation in the Prm2 gene and were unable to achieve germ line transmission, even though half of the sperm contained an intact Prm2 allele. Using ICSI, we found that sperm from mice heterozygous for a mutation in Prm2 were unable to produce viable embryos beyond early cleavage stages and comet assays indicated that the sperm DNA was fragile and prone to fragmentation. We hypothesized that disruption of chromatin integrity in heterozygous mice was due to haploinsufficiency in PRM1 and PRM2 levels in spermatids with either a mutant or an intact allele. To allow further studies, mice were generated with a floxed Prm2 gene, the gene was disrupted in oocytes, and colonies of C57BL/6 and 129S6 strain mice were generated and maintained by transmitting the mutation through females. While heterozygous C57BL/6 males were fertile, heterozygous 129S6 males were infertile. By qPCR analysis, the Prm2 mRNA levels in the testes of heterozygous C57BL/6 and 129S6 mice were half the levels of their WT littermates. The morphology and motility of sperm from C57BL/6 males were indistinguishable from WT sperm, but sperm from 129S6 males had reduced motility and many were seen by SEM to be abnormal in shape and organization. This suggested that a 129 strain-specific modifier gene (or genes) causes the infertility. To identify the modifier gene, F2 male mice were generated by crossing WT C57BL/6 male mice with heterozygous 129S6 females and intercrossing their offspring to maximize meiotic cross-over events. The F2 males were test-mated for three months and total pups sired, average pups per litter, sperm numbers, and sperm percent motility were determined for each male. DNA samples from these male were subjected to QTL analysis by JAX Services, using a low-resolution SNP panel (6-8 strain-specific SNPs per chromosome). No significant QTLs were detected for sperm numbers or sperm motility, but significant QTLs for average pups per sire and number of pups per sire were identified on chr4 and chr11. The stronger and more promising of the QTLs was in the interval between 42.44 and 83.48 Mb from the kinetochore on chr11. The candidate QTL interval on chr11 contains around 600 genes. Significant differences were detected by eQTL microarray analysis between the levels of mRNA from genes in this interval in the testes of heterozygous C57BL/6 and 129S6 mice.