Kühholzer B, Baguisi A, Overström E W
Department of Biomedical Sciences, Tufts University, School of Veterinary Medicine, North Grafton, MA 01536, USA.
Theriogenology. 2000 Mar 15;53(5):1071-9. doi: 10.1016/S0093-691X(00)00253-3.
While the culture and identification of primordial germ cells (PGCs) in mice is established, only limited investigations on PGCs in livestock have been reported. This study was performed to characterize goat PGCs after culture and cryopreservation. Goat PGCs were isolated from Day 32 fetuses and cultured on a continuous cell line of murine embryonal fibroblasts (STO) as feeder-cells in the presence of leukemia inhibitory factor (LIF). The PGCs proliferated slowly and showed colony formation in early passages. Frozen-thawed PGCs continued to proliferate when stem cell factor (SCF) was added to the culture medium. However, differentiation into epithelial-like polygonal cells or neuronal cells was observed after 1 or 2 passages. The PGCs of 1 female and 1 male cell line were characterized by immunocytochemistry. The PGCs showed positive staining for anti stage-specific embryonic antigen-1 (SSEA-1) and FMA-1 (monoclonal antibody produced against a glycoprotein cell surface antigen of the embryonal carcinoma Nulli SCC1), whereas the reactivity to alkaline phosphatase (AP), an established marker for PGCs in mice, was inconsistent. After differentiation, PGCs lost their positive reaction to SSEA-1, EMA-1 and AP. In conclusion, SSEA-1 and EMA-1 can be used as reliable markers for identifying goat PGCs in addition to morphological criteria. The results indicate that goat PGCs can be kept in long-term culture without losing their morphological characteristics and their positive reaction to SSEA-1 and EMA-1, thus providing a promising source of donor-karyoplasts for nuclear transfer procedures.
虽然小鼠原始生殖细胞（PGCs）的培养和鉴定方法已经确立，但关于家畜PGCs的研究报道却很有限。本研究旨在对培养和冷冻保存后的山羊PGCs进行特性分析。从32日龄山羊胎儿中分离出PGCs，并在白血病抑制因子（LIF）存在的情况下，将其培养在小鼠胚胎成纤维细胞（STO）的连续细胞系上作为饲养层细胞。PGCs增殖缓慢，在早期传代时形成集落。当向培养基中添加干细胞因子（SCF）时，冻融后的PGCs继续增殖。然而，在传代1或2次后观察到其分化为上皮样多边形细胞或神经细胞。通过免疫细胞化学对1个雌性和1个雄性细胞系的PGCs进行了特性分析。PGCs对阶段特异性胚胎抗原-1（SSEA-1）和FMA-1（针对胚胎癌Nulli SCC1糖蛋白细胞表面抗原产生的单克隆抗体）呈阳性染色，而对碱性磷酸酶（AP）（小鼠PGCs的既定标志物）的反应则不一致。分化后，PGCs对SSEA-1、EMA-1和AP失去阳性反应。总之，除形态学标准外，SSEA-1和EMA-1可作为鉴定山羊PGCs的可靠标志物。结果表明，山羊PGCs可以长期培养而不丧失其形态特征以及对SSEA-1和EMA-1的阳性反应，从而为核移植程序提供了一个有前景的供体核质体来源。
