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

背景：Ad4BP/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激素和精子发生，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。此外，我们确定了最有价值的基因转移到睾丸的方法，以供将来的临床应用。方法：(1)采用定量RT-PCR、免疫组织化学和免疫印迹法检测8例婴幼儿至成人正常人睾丸组织中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样本进行定量RT-PCR检测Ad4BP/SF-1和DAX-1的表达，并分析这两种因子的表达水平与血清激素水平或组织学评价的相关性，以研究其与特发性无精子症的甾体发生和精子发生的初步相关性。(2)采用脂质体、电穿孔和腺病毒载体三种方法在体内直接将DNA注射到小鼠睾丸中，用相同的外源DNA转染睾丸细胞，比较三种方法的转染效率和基因转移对精子发生的影响，以确定未来临床应用中最有价值的方法。结果：(1)正常睾丸中这两种因子的表达水平随睾丸发育而升高。Ad4BP/SF-1在Leydig细胞中大量表达，而DAX-1在Sertoli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<0.05）。成熟阻滞患者DAX-1 mRNA的平均表达水平（0.39+/-0.19）和仅支持细胞综合征患者（0.13+/-0.08）低于精子发生不足患者（1.60+/-1.32）和精子发生正常患者（1.30+/-1.41）。(2)脂质体和电穿孔方法虽然简单，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、致精损伤大。脂质体法和电穿孔法在临床应用的缺点是在生殖细胞中有基因表达的潜力，对精子发生有较强的不良影响。我们的研究结果表明，在这三种方法中，腺病毒介导的基因转移可能是一种更有效、更优越的转染睾丸细胞的方法，未来可能更适用于男性不育的体内基因治疗。结论：(1)Ad4BP/SF-1对维持人睾丸激素生成至关重要。DAX-1在人类睾丸的精子发生中起着关键作用，而支持细胞功能异常可能导致精子发生的正常进程被破坏，从而导致仅支持细胞综合征和成熟停滞。(2)虽然还需要对精子发生与男性不育的机制进行更多的研究，并建立更高效、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益处。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