Flow cytometric method to isolate round spermatids from mouse testis

The purpose of this study was to isolate pure populations of round spermati ds from mouse testis by how cytometry followed by cell sorting. Cell suspen sions from mouse testis were enriched in germ cells by centrifugation on a discontinuous Percoll gradient, then analysed using a FACScalibur how cytom eter measuring the cell size and density. A large and well-delimited popula tion of cells (R1) expected to contain round spermatids was observed on the dot plot diagram. Sorted R1 cells were very homogeneous in size (similar t o 11 mu m) and displayed the characteristic cytological aspect of round spe rmatids. Spermatid-specific gene expression was confirmed by reverse transc riptase-polymerase chain reaction (RT-PCR) analysis of R1 cells using prime rs for protamine 2 gene (PRM2) and SP-10. A positive signal for SP-10 was o btained 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-P CR, excluding a contamination with spermatogonia. Our results clearly estab lished that flow cytometry followed by cell sorting allows the isolation of a highly homogeneous population of round spermatids from the testis.