Lassalle B, Ziyyat A, Testart J, Finaz C, Lefèvre A
Unité Maturation Gamètique et Fécondation, INSERM unité 355 (Maturation gamètique et fécondation), Clamart, France.
Hum Reprod. 1999 Feb;14(2):388-94. doi: 10.1093/humrep/14.2.388.
The purpose of this study was to isolate pure populations of round spermatids from mouse testis by flow cytometry followed by cell sorting. Cell suspensions from mouse testis were enriched in germ cells by centrifugation on a discontinuous Percoll gradient, then analysed using a FACScalibur flow cytometer measuring the cell size and density. A large and well-delimited population of cells (R1) expected to contain round spermatids was observed on the dot plot diagram. Sorted R1 cells were very homogeneous in size (approximately 11 microns) and displayed the characteristic cytological aspect of round spermatids. Spermatid-specific gene expression was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of R1 cells using primers for protamine 2 gene (PRM2) and SP-10. A positive signal for SP-10 was obtained with a single cell using nested primers. The 5.5 kb transcript of c-kit, which is not expressed in spermatids, was not detected by nested RT-PCR, excluding a contamination with spermatogonia. Our results clearly established that flow cytometry followed by cell sorting allows the isolation of a highly homogeneous population of round spermatids from the testis.
本研究的目的是通过流式细胞术及后续的细胞分选，从小鼠睾丸中分离出纯的圆形精子细胞群体。通过在不连续的Percoll梯度上离心，从小鼠睾丸获得的细胞悬液得以富集生殖细胞，然后使用FACScalibur流式细胞仪分析，测量细胞大小和密度。在点图上观察到一个大的、界限分明的细胞群体（R1），预计其包含圆形精子细胞。分选后的R1细胞大小非常均匀（约11微米），并呈现出圆形精子细胞特有的细胞学特征。通过使用鱼精蛋白2基因（PRM2）和SP-10的引物对R1细胞进行逆转录聚合酶链反应（RT-PCR）分析，证实了精子细胞特异性基因表达。使用巢式引物对单个细胞获得了SP-10的阳性信号。通过巢式RT-PCR未检测到精子细胞中不表达的c-kit的5.5 kb转录本，排除了精原细胞污染的可能性。我们的结果清楚地表明，通过流式细胞术及后续的细胞分选能够从小鼠睾丸中分离出高度均匀的圆形精子细胞群体。
