Mammalian germ cell development is a fundamental yet poorly understood part of the reproductive process. Only a small number of genes specific to germ cell development in mammals has been identified and characterized, the exact function of most of these genes is unknown, and many genes remain to be discovered. Identification and characterization of pure fertility genes is a key step towards the understanding of germ cell development. The existence of pure male sterile phenotypes in the human population offers a good starting point to search for the pure fertility genes, especially the genes for male germ cell development. It has been estimated that approximately 2 percent of the human male population is infertile due to nonobstructive azoospermia or oligospermia (no or few sperm). Most of these patients are otherwise healthy, suggesting that the sperm deficiency is caused by defects in germ cell development. Study of Y chromosome deficiencies associated with infertility offers an unparalled opportunity to identify genes that function in male germ cell development. It is known that the Y chromosome plays a critical role in somatic sex differentiation and male germ cell development. Results from several experiments have indicated that interstitial deletions of certain Y chromosome regions may cause male infertility in humans, suggesting that the Y chromosome contains genes required for male germ cell development. The goal of this proposed research is to achieve a better understanding of the mechanism of male germ cell development and differentiation in mammals, and to explore the roles of the Y chromosome in these processes. Specifically, the following issues will be addressed: 1). Construction of a precise map of the human Y chromosome loci involved in male germ cell differentiation and development. The Y chromosome of azoospermia and oligospermia patients will be screened for interstitial deletions by PCR analysis with a large number of densely spaced sequence-specific sites (STS). It is expected that novel loci as well as those previously known loci will be identified and precisely mapped by this approach. 2). Identification and cloning of the male germ cell development genes in the mapped loci. BAC (Bacteria Artificial Chromosome) clones of DNA in the deleted Y chromosome region will be isolated from a human BAC library by screening with STSs, and BAC clone contigs that encompass the deletions will be constructed. Genes in each locus will be identified and cloned with a combination of several approaches, including the candidate gene approach, direct sequencing of the BAC clones and cDNA selection with the BAC clones. The candidate male fertility genes will be identified based on sequence analysis, expression patterns, evolutionary conservation, and ideally, by finding internal mutations that correspond with infertile phenotypes. The strong candidate gene(s) will then be studied further. 3). Elucidation of the functions of the male fertility gene(s) in transgenic mice. The mouse homologue gene will be identified and mapped by several methods, and the cDNA clones will be isolated from a mouse testes cDNA library. The expression pattern of the gene will be examined by Northern blot and in situ hybridization on RNAs or tissues prepared from animals at different developmental stages. A gene knock-out transgenic mouse strain will be constructed. The in vivo functions of the gene will be studied in the transgenic mice at cellular and biochemical levels. The completion of this project will help us to better understand mammalian male germ cell development and differentiation, and may provide a genetic basis for an effective diagnosis and treatment of human male infertility. Furthermore, it will offer insight into the function, structure, and evolution of the mammalian Y chromosome.
