Molecular biological approach for clarification of human spermatogenesis and its disorder

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1) Cloning of Azoospermy factor gene (AZF)For molecular cloning of AZF which is the candidate gene related human spermatogenesis located on the long arm of Y chromosome we made a deletion map of the long arm of Y chromosome from the results of the DNA analysis of male infertile patients with azoospermia or severe oligozoospermia and identified a specific location in which AZF should be included. Then, we found YAC in which AZF should be included and subcloned the cosmid contig mede from the YAC to a plasmid vector. AZF is now included within 30 Kb DNA.Another method using Exon trapping for the cloning of AZF is also on going.2) Cloning of genes which induce differentiation of human testis germ cellsFor molecular cloning of genes which induce differentiation of human testis germ cells we screened human testis cDNA library using mouse cDNAs as probes. We isolated human SCP-1 which appears in the phase before the miosis and determined sequences of the DNA and the protein.3) Establishment of in vitro culture of human germ cellsWe established in vitro culture system of human germ cells which has been very difficult. Using this system we will be able to do experiments in vitro differentiation human germ cells.