Lee K H, Lee W Y, Kim J H, Yoon M J, Kim N H, Kim J H, Uhm S J, Kim D H, Chung H J, Song H
Department of Animal and Food Bioscience, College of Biomedical and Health Science, Konkuk University, Chung-ju, Korea, Korea.
Reprod Domest Anim. 2013 Dec;48(6):954-60. doi: 10.1111/rda.12193. Epub 2013 Jun 28.
Type A spermatogonia, including spermatogonial stem cells, are primary cells that maintain spermatogenesis and produce spermatozoa. Many spermatogonial markers have been reported in rodents. However, few markers have been identified in pig spermatogonia. Despite the lack of information, it is necessary to separate pure spermatogonial cells from whole testicular cells to understand the mechanism of spermatogenic meiosis and to establish spermatogonial stem cells for further biotechnological studies. The purpose of this study was to identify glial cell-derived neurotrophic factor receptor alpha-1 (GFRα-1) as a surface marker for early spermatogonia in neonatal pig testes. Histological analysis of 3-day-old pig testes revealed that type A spermatogonia, which lack heterochromatin, could be distinguished in neonatal pig testes. Immunohistochemistry of neonatal pig testes with GFRα-1 antibody identified that some of the spermatogonial cells expressed GFRα-1 on the cell membrane. Co-immunostaining with both GFRα-1 and protein gene product 9.5 (PGP 9.5) detected PGP 9.5 in all spermatogonia of neonatal pig testes, whereas GFRα-1 was not detected on the surface of some PGP 9.5-positive cells, indicating that some of the spermatogonial cells were PGP 9.5 positive and GFRα-1 negative. After immunomagnetic cell sorting using a GFRα-1 antibody, both GFRα-1-positive and GFRα-1-negative cells expressed PGP 9.5. Identifying the differential mRNA expression of both GFRα-1-positive and GFRα-1-negative cells using reverse transcription-polymerase chain reaction analysis revealed the expression of promyelocytic leukaemia zinc finger, octamer-binding protein 4 and homeobox transcription factor in both cell types. These results suggest that GFRα-1-positive and GFRα-1-negative spermatogonia exist in PGP 9.5-positive spermatogonia during the early stage of pig testes spermatogenesis, and that GFRα-1 can be used for sorting PGP 9.5-expressing spermatogonia.
A型精原细胞，包括精原干细胞，是维持精子发生并产生精子的原始细胞。在啮齿动物中已报道了许多精原细胞标志物。然而，在猪精原细胞中鉴定出的标志物很少。尽管缺乏相关信息，但有必要从整个睾丸细胞中分离出纯精原细胞，以了解精子发生减数分裂的机制，并建立精原干细胞用于进一步的生物技术研究。本研究的目的是鉴定胶质细胞源性神经营养因子受体α-1（GFRα-1）作为新生猪睾丸早期精原细胞的表面标志物。对3日龄猪睾丸的组织学分析表明，在新生猪睾丸中可以区分出缺乏异染色质的A型精原细胞。用GFRα-1抗体对新生猪睾丸进行免疫组织化学分析发现，一些精原细胞在细胞膜上表达GFRα-1。用GFRα-1和蛋白基因产物9.5（PGP 9.5）进行共免疫染色，在新生猪睾丸的所有精原细胞中均检测到PGP 9.5，而在一些PGP 9.5阳性细胞表面未检测到GFRα-1，这表明一些精原细胞是PGP 9.5阳性而GFRα-1阴性。使用GFRα-1抗体进行免疫磁珠细胞分选后，GFRα-1阳性和GFRα-1阴性细胞均表达PGP 9.5。用逆转录-聚合酶链反应分析鉴定GFRα-1阳性和GFRα-1阴性细胞的差异mRNA表达，结果显示两种细胞类型中均有早幼粒细胞白血病锌指蛋白、八聚体结合蛋白4和同源盒转录因子的表达。这些结果表明，在猪睾丸精子发生早期，PGP 9.5阳性的精原细胞中存在GFRα-1阳性和GFRα-1阴性的精原细胞，并且GFRα-1可用于分选表达PGP 9.5的精原细胞。
