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

通过操纵基因表达鉴定负责雄性减数分裂停滞的突变基因并进行功能分析

• 批准号: 15380204
• 负责人: NOGUCHI Junko
• 金额: $ 8.58万
• 依托单位: Notional Institute of Agrobiological Sciences
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15380204/
• 关键词: spermatogenesis; meiosis; Fkbp6; synaptonemal complex; chromosome pairing; blood-testis barrier; 性染色体不活性化

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.

为了鉴定导致精子生成受阻的突变大鼠基因AS，我们进行了连锁分析。结果表明，Fkbp6基因是一个较强的候选基因，该基因定位于大鼠Cl.12中。对该突变体进行基因组DNA分析，检测到包括Fkbp6外显子8在内的约1kb的缺失。在突变的睾丸中，观察到蛋白质和mRNA的缺失。综上所述，我们得出的结论是，AS是一个突变的Fkbp6基因。由于突变的雄性在粗线期表现出减数分裂停滞，我们分析了染色体配对，这是粗线期精母细胞的特征事件。观察到同源对的非同源配对和部分突触。免疫细胞化学显示FKBP6定位于联会复合体。根据这些结果，我们推测FKBP6的缺失触发了联会复合体的不稳定，从而导致了异常的染色体配对。虽然FKBP6在雄性和雌性减数分裂中都有表达，但只有雄性表现出异常的表型。XY染色体失活是减数分裂中雄性特有的事件之一，因此我们研究了FHBP6缺乏对失活的影响。在类似野生的突变生殖细胞中，γH 2AX定位于XY染色体周围区域，这表明失活是正常发生的。我们的初步研究表明，突变大鼠的血-睾丸屏障功能障碍。我们在Fkbp6基因敲除小鼠身上进行了屏障的示踪测试，并证实了它的故障。我们还检测了药物诱导的生殖细胞缺陷睾丸的屏障功能，以阐明生精不足是否会影响屏障功能。我们认识到，尽管缺乏生殖细胞，但屏障功能正常，这表明Fkbp6在血-睾丸屏障的建立中起着至关重要的作用。

项目成果

野口 純子: "遺伝的組換え機構解明に貢献する実験動物モデル"J.Animal Genetics. 31. 13-19 (2003)

Junko Noguchi：“有助于阐明基因重组机制的实验动物模型”J.Animal Genetics。31. 13-19 (2003)

Fine mapping of a region of rat chr.12 close to the aspermia (as) locus and comparison with the human orthologous region.

对靠近无精 (as) 基因座的大鼠第 12 号染色体区域进行精细定位，并与人类直系同源区域进行比较。

• 发表时间: 2004
• 期刊: Experimental Animals 53
• 作者: Noguchi J;et al.
• 通讯作者: et al.

Linkage mapping of the locus responsible for male pseudohermaphroditism (mp9 on rat chromosome 7.

负责雄性假两性畸形的基因座的连锁图谱（大鼠 7 号染色体上的 mp9）。

• 发表时间: 2004
• 期刊: Experimental Animals 53(4)
• 作者: Noguchi J;et al.;Kawai et al.
• 通讯作者: Kawai et al.

遺伝的組換えに重要なシナプトネマ構造の形成に関わる因子

参与对基因重组重要的联会结构形成的因素

• 发表时间: 2004
• 期刊: 化学と生物 42
• 作者: Noguchi J;et al.;Kawai et al.;野口 純子
• 通讯作者: 野口 純子

遺伝的組換え機構解明に貢献する実験動物モデル

有助于阐明基因重组机制的实验动物模型

• 发表时间: 2003
• 期刊: J. Animal Genetics 31
• 作者: Nagahata;H.;Higuchi;H.;Teraoka;H.;Takahashi;K.;Inanami;O.;Kuwabara;M.;Noguchi J et al.;桑原 幹典;Crackower M et al.;野口 純子
• 通讯作者: 野口 純子