Lee J H, Lee D R, Yoon S J, Chai Y G, Roh S I, Yoon H S
Infertility Research Center, Jeil Women's Hospital, Seoul, Korea.
Mol Hum Reprod. 1998 Sep;4(9):827-34. doi: 10.1093/molehr/4.9.827.
Spermatogenesis is regulated by hormones, local regulatory factors in the testes and specific gene expression of spermatogenic cells in humans. In this study, we have detected the expression of the deleted in azoospermia (DAZ), the DAZ-like autosome (DAZL1), and the protamine-2 genes in spermatogenic cells. Spermatogenesis in 38 male infertility patients was evaluated by the semen analysis and histological examination. Patients were diagnosed as Sertoli cell-only syndrome (n = 20), maturation arrest (n = 6), hypospermatogenesis (n = 6), and obstructive azoospermic patients with normal spermatogenesis (n = 6). After microscopic observation of the wet preparation of the testis tissues, seminiferous tubule contents were used for reverse transcription-polymerase chain reaction (RT-PCR) analysis of DAZ, DAZL1 and protamine-2. In cases with Sertoli-cell only syndrome, we found spermatogenic cells in 30% of patients (6/20) by the wet preparation method. There was no difference between the histology and the wet preparation results in maturation arrest and obstructive azoospermia; however, in one case of hypospermatogenesis, spermatozoa were not detectable by the wet preparation method. Using in-situ hybridization with DAZ and protamine-2 ribonuclear probes, we confirmed spermatogenic cell-specific expression of DAZ (spermatogonia/early spermatocyte) and protamine-2 (spermatid/spermatozoon). DAZ and protamine-2 expression can therefore be considered spermatogenic cell markers and could be useful in molecular diagnosis of spermatogenesis. In 13 patients with spermatozoa under the wet preparation, the expression of DAZ, DAZL1 and protamine-2 was detected in all the preparations. In one wet preparation showing only spermatogonia/spermatocyte, only DAZ and DAZL1 RNA were detected. In 14 wet preparations showing no spermatogenic cells, DAZ, DAZL1 and protamine-2 were not detected except in one preparation where DAZL1 expression was detected. In 10 wet preparations representing spermatogonia/spermatocyte to spermatids, but showing no spermaozoa, DAZ and DAZL1 were detected in eight and nine preparations respectively, and protamine-2 was detected in six preparations. These results of gene expression were similar to the wet preparation results. RT-PCR for DAZ, DAZL1 and protamine-2 was informative for the existence of germ cells, germ cell physiology and differentiation. From these results, we suggest that the analysis of DAZ, DAZL1 and protamine-2 expression by RT-PCR and wet preparation might offer a better method for finding the spermatogenic cells compared to the histological method.
精子发生受激素、睾丸局部调节因子以及人类生精细胞特定基因表达的调控。在本研究中，我们检测了无精子症缺失基因（DAZ）、常染色体DAZ样基因（DAZL1）和鱼精蛋白-2基因在生精细胞中的表达。通过精液分析和组织学检查对38例男性不育患者的精子发生情况进行了评估。患者被诊断为唯支持细胞综合征（n = 20）、成熟障碍（n = 6）、精子发生低下（n = 6）以及生精正常的梗阻性无精子症患者（n = 6）。在对睾丸组织湿片进行显微镜观察后，将曲细精管内容物用于DAZ、DAZL1和鱼精蛋白-2的逆转录-聚合酶链反应（RT-PCR）分析。在唯支持细胞综合征病例中，通过湿片法在30%的患者（6/20）中发现了生精细胞。成熟障碍和梗阻性无精子症患者的组织学检查结果与湿片检查结果无差异；然而，在1例精子发生低下的病例中，湿片法未检测到精子。使用DAZ和鱼精蛋白-2核糖核酸探针进行原位杂交，我们证实了DAZ（精原细胞/早期精母细胞）和鱼精蛋白-2（精子细胞/精子）在生精细胞中的特异性表达。因此，DAZ和鱼精蛋白-2的表达可被视为生精细胞标志物，可能有助于精子发生的分子诊断。在13例湿片中可见精子的患者中，所有样本均检测到了DAZ、DAZL1和鱼精蛋白-2的表达。在1例仅显示精原细胞/精母细胞的湿片中，仅检测到了DAZ和DAZL1 RNA。在14例未检测到生精细胞的湿片中，除1例检测到DAZL1表达外，未检测到DAZ、DAZL1和鱼精蛋白-2。在10例代表精原细胞/精母细胞到精子细胞但未见精子的湿片中，分别在8例和9例样本中检测到了DAZ和DAZL1，在6例样本中检测到了鱼精蛋白-2。这些基因表达结果与湿片检查结果相似。DAZ、DAZL1和鱼精蛋白-2的RT-PCR对于生殖细胞的存在、生殖细胞生理和分化具有参考价值。基于这些结果，我们认为与组织学方法相比，通过RT-PCR和湿片检查分析DAZ、DAZL1和鱼精蛋白-2的表达可能为寻找生精细胞提供更好的方法。
