[A modified method for spermatogonial cell sorting].

ABSTRACT

OBJECTIVE: To improve the method of sorting undifferentiated and differentiated spermatogonial cells by magnetic bead sorting with specific antibodies. METHODS: Using the magnetic bead sorting technique combined with Thy1 and c-Kit specific antibodies, we sorted Thy1+ and c-Kit+ cells in the testis of 7-postnatal-day male mice as undifferentiated and differentiated spermatogonia, respectively. We determined the purities of the two types of spermatogonial cells by immunofluorescence and flow cytometry, identified them via the differential expressions of Gfrα1, Plzf, c-Kit and Sohlh2 by real-time quantitative PCR, and cultured the Thy1+ cells primarily. RESULTS: The purities of the Thy1+ and c-kit+ cells were as high as (85.65 ± 8.35)% and (89.40 ± 2.77)%, respectively (P < 0.01). The relative expressions of the Gfrα1 and Plzf genes were 9.47 ± 1.29 and 4.40 ± 0.59 times higher in the Thy1+ than in the c-Kit+ cells, and those of the kit and sohlh2 genes 7.38 ± 1.07 and 3.88 ± 0.28 times lower in the former than in the latter (P < 0.01). After primary culture, the cells were seen in a normal state, proliferating smoothly with the characteristics of the proliferation of spermatogonial stem cells. CONCLUSIONS: The magnetic bead sorting technique with Thy1 and c-Kit specific antibodies can be used to effectively identify undifferentiated and differentiated spermatogonia and culture undifferentiated Thy1+ cells in vitro.