Role of transcription factors Ad4BP/SF-1 and DAX-1 in spermatogenesis and future prospects of gene therapy for male infertility

转录因子Ad4BP/SF-1和DAX-1在精子发生中的作用及男性不育基因治疗的前景

• 批准号: 17591693
• 负责人: IKEUCHI Takahito
• 金额: $ 2.31万
• 依托单位: Nagoya City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17591693/
• 关键词: transcription factors; DAX-1; gene therapy; 遺伝子治療; 造精機能; 男性不妊症; Sertoli細胞

Background: Ad4BP/SF-1 and DAX-1 are orphan members of the nuclear hormone receptor superfamily of transcription factors. In order to obtain better understandings of human testicular steroidogenesis and spermatogenesis, we examined the expression levels of both factors in human normal and idiopathic azoospermic testes and investigated their physical meaning. In addition, we identify the most appreciate gene transfer method to testis for future clinical application.Methods : (1)We examined the expression level of Ad4BP/SF-1 and DAX-1 by quantitative RT-PCR, immunohistochemistry and western blotting analysis using 8 normal human testicular tissues from infants to adults. Next, we performed quantitiative RT-PCR using testicular biopsy samples obtained from 22 idiopathic azoospermic patients to examine the expression of Ad4BP/SF-1 and DAX-1, and analyzed the correlation between the expression levels of both factors and the serum hormone levels or histological evaluation to study their pote … More ntial correlation with steroidogenesis and spermatogenesis on idiopathic azoospermia. (2) We directly injected DNA into mouse testes in vivo using three methods, liposome, electroporation and adenovirus vector, to transfect testicular cells using the same exogenous DNA, and compared the three methods for transfection efficiency and effects of gene transfer on spermatogenesis to identify the most appreciate method for future clinical application.Results : (1)The expression levels of both factors in the normal testes increased with testicular development. Ad4BP/SF-1 was abundantly expressed in Leydig cell, whereas DAX-1 was expressed in Sertoli cell. The expression level of Ad4BP/SF-1 in idiopathic azoospermic patients testes positively correlated with serum testosterone (p<0.05). The average expression levels of DAX-1 mRNA for patients with maturation arrest (0.39+/-0.19)and Sertoli cell-only syndrome (0.13+/-0.08) were lower than that with hypospermatogenesis (1.60+/-1.32) and normal spermatogenesis (1.30+/-1.41). (2)Although liposome and electroporation methods were simple, they had low efficiency, short duration of the physiological effect and high spermatogenic damage in comparison with adenovirus-mediated gene transfer. The disadvantage of the liposome and electroporation methods for clinical application was that they showed potential for gene expression in germ cells and strong adverse effects on spermatogenesis. Our results suggest that adenovirus-mediated gene transfer may be a more effective and superior method for transfecting testicular cells among these three methods and may be more applicable for in vivo gene therapy for male infertility in the future.Conclusion : (1)Ad4BP/SF-1 is essential for the maintenance of steroidogenesis in the human testis. DAX-1 plays a critical role in spermatogenesis in the human testis, and Sertoli cell-only syndrome and maturation arrest may result from abnormal Sertoli cell function that disrupts the normal progression of spermatogenesis. (2)Although more investigations on the mechanism of spermatogenesis and male infertility and the establishment of techniques for more efficient and safer gene transfer to the sperm and testis will be needed, gene therapy will enable a revolutionary advance for reproductive treatment and provide great benefit for patient with male infertility in the future. Less

背景：Ad4BP/SF-1和DAX-1是核激素受体超家族转录因子的孤儿成员。为了更好地了解人类睾丸类固醇激素的发生和精子的发生，我们检测了这两种因子在人类正常和特发性无精子症睾丸中的表达水平，并探讨了它们的物理意义。方法：(1)采用定量RT-PCR、免疫组织化学和免疫印迹等方法，检测了8例正常成人睾丸组织中Ad4BP/SF-1和DAX-1的表达水平。接下来，我们对22例特发性无精子症患者的睾丸活检标本进行了定量RT-pr检测Ad4BP/sf-1和dax-1的表达，并分析了这两个因子的表达水平与血清激素水平或组织学评价的相关性，以研究它们的POTE…。特发性无精子症与类固醇激素和精子发生的相关性。(2)采用脂质体、电穿孔和腺病毒载体三种方法将DNA直接注射到小鼠体内，用相同的外源DNA导入睾丸细胞，比较三种方法的转染率和基因转移对生精的影响，以确定最适合临床应用的方法。结果：(1)随着睾丸发育，两种因子在正常睾丸中的表达水平均逐渐升高。Ad4BP/SF-1在间质细胞中大量表达，而DAX-1在支持细胞中大量表达。Ad4BP/SF-1在特发性无精子症患者睾丸中的表达水平与血清睾酮水平呈正相关(p&lt；0.05)。成熟停滞组(0.39+/-0.19)和仅支持细胞综合征组(0.13+/-0.08)DAX-1mRNA的平均表达水平低于生精功能低下组(1.60+/-1.32)和生精正常组(1.30+/-1.41)。(2)脂质体法和电穿孔法虽然简单，但与腺病毒介导的基因转移相比，效率低，生理作用持续时间短，生精损伤大。临床应用的脂质体和电穿孔方法的缺点是它们具有在生殖细胞中表达基因的潜力，并且对精子发生有强烈的不良影响。结论：(1)Ad4BP/SF-1在维持睾丸类固醇激素合成中起着至关重要的作用。DAX-1在人类睾丸的精子发生中起关键作用，仅支持细胞综合征和成熟停滞可能是由于支持细胞功能异常而扰乱精子发生的正常进行。(2)虽然精子发生和男性不育的发生机制还需要更多的研究，还需要建立更有效和更安全的精子和睾丸基因转移技术，但基因治疗将使生殖治疗取得革命性的进步，并为未来的男性不育患者提供巨大的好处。较少

项目成果

Assessment of serum FSH level and testicular volume for prediction of future paternity in pubertal boys who had bilateral orchiopexy in childhood. J Urol, 175, 2290-2294, 2006.

评估血清 FSH 水平和睾丸体积，以预测儿童期双侧睾丸固定术的青春期男孩未来的亲子关系。

• 发表时间: 2006
• 期刊: Journal of Urology 175
• 作者: Kaneko;T.;Sasaki;S.;Umemoto;Y.;Koiima;Y.;Ikeuchi;T.;Kohri;K;Kojima et al.;Kojima et al.
• 通讯作者: Kojima et al.

Simulated conditions of microgravity increases progesterone production in I-10 cells o fLeydig tumor cell line

模拟微重力条件增加 Leydig 肿瘤细胞系 I-10 细胞中孕酮的产生

• 发表时间: 2008
• 期刊: Int J Urol 15
• 作者: Kaneko T;Kojima Y;Ikeuchi T;Kohri K;et. al.
• 通讯作者: et. al.

Simulated conditions of microgravity increases progesterone production in I-10 cells of Leydig tumor cell line

模拟微重力条件增加 Leydig 肿瘤细胞系 I-10 细胞中孕酮的产生

• 发表时间: 2008
• 期刊: Int J Urol 15
• 作者: Kaneko;T.;Sasaki;S.;Umemoto;Y.;Koiima;Y.;Ikeuchi;T.;Kohri;K
• 通讯作者: K

Role of transcription factors Ad4BP/SF-1 and DAX-1 in steroidogenesis and spermatogenesis in human testicular development and idiopathic azoospermia. Int J Urol,13 : 783-791,2006.

转录因子 Ad4BP/SF-1 和 DAX-1 在人类睾丸发育和特发性无精子症的类固醇生成和精子发生中的作用。

• 发表时间: 2006
• 期刊: International Journal of Urology 13
• 作者: Kaneko;T.;Sasaki;S.;Umemoto;Y.;Koiima;Y.;Ikeuchi;T.;Kohri;K;Kojima et al.
• 通讯作者: Kojima et al.