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

背景：腺病毒4 BP/SF-1和DAX-1是核激素受体转录因子超家族的孤儿成员。为了更好地了解人类睾丸类固醇生成和精子发生，我们检测了这两个因子在人类正常和特发性无精子症睾丸中的表达水平，并探讨其物理意义。方法：（1）采用定量RT-PCR、免疫组化和western blotting方法检测8例正常人睾丸组织中Ad 4 BP/SF-1和DAX-1的表达水平。其次，我们对22例原发性无精子症患者的睾丸活检标本进行了定量RT-PCR，检测了Ad 4 BP/SF-1和DAX-1的表达，并分析了这两种因子的表达水平与血清激素水平或组织学评价之间的相关性，以研究它们在原发性无精子症中的作用。 关于我们 特发性无精子症患者类固醇激素生成与精子发生的初步相关性(2)本研究采用脂质体法、电穿孔法和腺病毒载体法三种方法将外源DNA直接注射到小鼠睾丸内，以相同的外源DNA转染睾丸细胞，比较三种方法的转染效率和转基因对精子发生的影响，以确定最适合临床应用的方法。（1）两种因子在正常睾丸中的表达水平随睾丸发育而增加。Ad 4 BP/SF-1在Leydig细胞中表达量较高，DAX-1在Sertoli细胞中表达量较高。Ad 4 BP/SF-1在特发性无精子症患者睾丸组织中的表达水平与血清睾酮水平呈正相关（p<0.05）。成熟停滞（0.39+/-0.19）和仅有支持细胞综合征（0.13+/-0.08）患者的DAX-1 mRNA平均表达水平低于精子发生低下（1.60+/-1.32）和正常精子发生（1.30+/-1.41）患者。（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，生理效应持续时间短，对生精细胞的损伤大。脂质体法和电穿孔法在临床应用中的缺点是它们在生殖细胞中显示出基因表达的潜力，并且对精子发生有强烈的不利影响。结论：（1）腺病毒介导的SF-1/4 BP在维持人睾丸激素合成中起重要作用。DAX-1在人类睾丸中的精子发生中起着关键作用，并且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破坏精子发生的正常进展而导致的。（2）尽管对精子发生和男性不育的机制还需要更多的研究，并需要建立更有效和更安全的基因转移到精子和睾丸的技术，但基因治疗将使生殖治疗取得革命性的进展，并为未来的男性不育患者提供巨大的利益。少

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