Figure 4

Quantitative RT-PCR data from two independent cultures on D7 compared to cells used for seeding (D0). An upregulation was noted for various transcripts but apparently the response in the cultures differed. An upregulation (> 40 folds) of Sca-1 in both the cultures was suggestive of self-renewal of VSELs in culture as Sca-1 is a marker for mouse VSELs [11]. An increase in Gfra (marker specific for spermatogonial stem cells) suggested proliferation of spermatogonial stem cells. Increased proliferative activity in VSELs as well as SSCs was confirmed even by upregulation of Pcna in both the cultures. Cells in the culture underwent meiosis as Scp3 and prohibitin (spermatocytes specific marker) transcript were upregulated in vitro. Protamine (post-meiotic marker for spermatid and sperm) transcript was also found upregulated suggesting differentiation of sperm in both the cultures (>1200 folds in one culture and > 40 folds in another). Variability in extent of upregulation of various transcripts during spontaneous differentiation of stem cells into sperm in the two cultures reflects biological variability and different stage of differentiation when cells were harvested for RNA extraction. However, in both cultures proliferation of cells and differentiation into sperm occurred.