Identification of a mutant gene responsible for male meiotic arrest and functional analysis by manipulation of the gene expression

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In order to identify a mutant rat gene ‘as', which causes arrested spermatogenesis, we performed linkage analysis. The results indicated that Fkbp6,a gene localized in rat Chr.12,was a strong candidate gene. When analyzed genomic DNA of the mutant, approximately 1 Kb deletion including exon 8 of Fkbp6 was detected. In the mutant testis, deficiency of the protein as well as mRNA was observed. Taken together, we concluded that ‘as' is a mutated Fkbp6 gene.Since the mutant male displays meiotic arrest at the pachytene stage, we analyzed chromosome pairing, a characteristic event of pachytene spermatocytes by electlon microscopy. Non-homologous pairing as well as partial synapsis of homologous pairs was observed. Iimmucytochemistry in the wild-type males revealed that FKBP6 localized at a synaptonemal complex. With these results, we hypothesed that lack of FKBP6 triggers instability of synaptonemal complex, which resulting in abnormal chromosome pairing. Although FKBP6 expresses in both male and female meiosis, only males show abnormal phenotype. Inactivation of XY chromosomes is one of the male specific events in meiosis, so that we investigated whether inactivation was affected in FHBP6 deficiency. Localization of γH2AX was observed in the region surround XY chromosomes in the mutant germ cell like the wild, which indicating that inactivation occurred normally.Our preliminary study had shown dysfunction of the blood-testis barrier in the mutant rat. We performed the tracer test of the barrier in the Fkbp6 knockout mice and certified its malfunction. We also examined the barrier function of the drug-induced germ cell deficient testis in order to clarify whether lack of spermatogenesis could influence the barrier function. We recognized that the barrier functions normally despite lack of germ cells, which showing an essential role of Fkbp6 in the establishment of the blood-testis barrier.