Kojima Yoshiyuki, Sasaki Shoichi, Umemoto Yukihiro, Hashimoto Yoshihiro, Hayashi Yutaro, Kohri Kenjiro
Department of Nephro-urology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan.
J Urol. 2003 Nov;170(5):2109-14. doi: 10.1097/01.ju.0000092898.91658.08.
We directly injected DNA into mouse testes in vivo using an adenovirus vector to transfect testicular cells. We then analyzed the transfection efficiency, immunological problems and effects of gene transfer on spermatogenesis and the next generation. In this study we discuss the potential of gene therapy for male infertility.
A replication incompetent human adenovirus serotype 5 contained 2 deletions (E1 and E3 deletions) and was constructed such that the transgene was driven by the chicken beta-actin promoter to promote over expression of the downstream target gene (Lac Z). This adenovirus vector or control solution was injected into the interstitial space (intratesticular injection) or seminiferous tubules (intratubular injection) of the mouse testis. We investigated beta-galactosidase gene expression by X-gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside) staining, the effects of gene transfer on spermatogenesis by evaluating the frequency of apoptotic cells by the TUNEL method, the inflammatory response on testes by detecting CD4 and CD8 positive cells immunohistochemically, and interleukin (IL)-6 and IL-8 by immunoblot analysis, epididymides sperm motility and the reproductive response of each mouse 3, 7, 14 and 28 days after injection.
Intratesticular injection of adenovirus vector resulted in strong transgene expression in Leydig cells. In contrast, intratubular injection resulted in strong expression in Sertoli cells. Transgene expression was not detected in germ cells by either method. The peak of beta-galactosidase activity was on day 7, ie 0.674 +/- 0.20 (intratesticular) and 0.534 +/- 0.22 U (intratubular), and it decreased with time thereafter. The apoptosis index on day 7 was significantly higher in adenovirus injected groups than in noninjected groups, ie 0.46 +/- 0.20 vs 0.10 +/- 0.11 (intratesticular) and 0.78 +/- 0.31 vs 0.24 +/- 0.10 (intratubular). Transfected animals showed a slight mononuclear inflammatory response in the testes composed of CD4 and CD8 positive cells. Adenovirus vector stimulation resulted in the induction of IL-6 and IL-8 secretion in the testis. These immune responses subsided after day 7. There were no significant differences in the percent of motile sperm or the rate of abnormal sperm between the groups on any day after injection. Reproductive ability remained almost normal even after adenovirus mediated gene transfer with no effect observed in offspring.
Our results suggest that although slight spermatogenic damage and inflammatory response caused by these methods may present problems, adenovirus mediated gene transfer may be effective for transfecting testicular somatic cells and applicable for in vivo gene therapy for male infertility in the future.
我们使用腺病毒载体将DNA直接注入小鼠睾丸内，以转染睾丸细胞。然后我们分析了转染效率、免疫问题以及基因转移对精子发生和下一代的影响。在本研究中，我们探讨了基因治疗男性不育的潜力。
构建了一种无复制能力的人5型腺病毒，其包含2个缺失（E1和E3缺失），使得转基因由鸡β-肌动蛋白启动子驱动，以促进下游靶基因（Lac Z）的过表达。将该腺病毒载体或对照溶液注入小鼠睾丸的间质间隙（睾丸内注射）或生精小管（管内注射）。我们通过X-gal（5-溴-4-氯-3-吲哚基-β-D-吡喃半乳糖苷）染色研究β-半乳糖苷酶基因表达，通过TUNEL法评估凋亡细胞频率来研究基因转移对精子发生的影响，通过免疫组织化学检测CD4和CD8阳性细胞以及通过免疫印迹分析检测白细胞介素（IL）-6和IL-8来研究睾丸的炎症反应，在注射后3、7、14和28天分析附睾精子活力和每只小鼠的生殖反应。
睾丸内注射腺病毒载体导致Leydig细胞中强烈的转基因表达。相比之下，管内注射导致支持细胞中强烈表达。两种方法均未在生殖细胞中检测到转基因表达。β-半乳糖苷酶活性在第7天达到峰值，即0.674±0.20（睾丸内注射）和0.534±0.22 U（管内注射），此后随时间下降。注射腺病毒组在第7天的凋亡指数显著高于未注射组，即0.46±0.20对0.10±0.11（睾丸内注射）和0.78±0.31对0.24±0.10（管内注射）。转染动物的睾丸中出现由CD4和CD8阳性细胞组成的轻微单核炎症反应。腺病毒载体刺激导致睾丸中IL-6和IL-8分泌的诱导。这些免疫反应在第7天后消退。注射后任何一天，各组之间活动精子百分比或异常精子率均无显著差异。即使经过腺病毒介导的基因转移，生殖能力仍几乎正常，且未观察到对后代有影响。
我们的结果表明，尽管这些方法引起的轻微生精损伤和炎症反应可能存在问题，但腺病毒介导的基因转移可能有效地转染睾丸体细胞，并可能适用于未来男性不育的体内基因治疗。
