Molecular biological approach for clarification of human spermatogenesis and its disorder

1)无精子因子基因（AzoospermyFactor，AZF）的克隆为了对位于Y染色体长臂上的人类精子发生相关候选基因AZF进行分子克隆，我们根据无精子症和严重少精子症男性不育患者的DNA分析结果，绘制了Y染色体长臂的缺失图谱，并确定了AZF的特异性位点。然后，我们找到了含有AZF的YAC，并将YAC上的粘粒重叠群亚克隆到质粒载体上。AZF已被包含在30 Kb的DNA中，另一种利用外显子捕获技术克隆AZF的方法也在进行中。2）人睾丸生殖细胞分化诱导基因的克隆为了克隆人睾丸生殖细胞分化诱导基因，我们以小鼠cDNA为探针，筛选人睾丸cDNA文库。我们分离了减数分裂前出现的SCP-1，并测定了其DNA和蛋白质的序列。3）人生殖细胞体外培养体系的建立我们建立了人生殖细胞体外培养体系。利用该系统，我们将能够进行体外分化人类生殖细胞的实验。

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.