This was further confirmed and quantified by studying CAP induced DNA fragmentation using flow cytometry and by determining hypodiploid DNA content stained with PI. As illustrated by the dot blot histograms, CAP induced a significant shift in the number of apoptotic until cells with hypodiploid DNA content in comparison to control cultures. The percentage of apoptotic cells from quadru plicate cultures was quantified and was found to signifi cantly increase from about 8% and 14,6% respectively in the untreated Gc 5spg and Gc 6spg cell lines to 17,8% and 26,8% in respective cell lines with 200 M CAP after 24 h. With increasing CAP concentrations, the effect was even more pronounced with both cell lines, and after either both 24 and 48 h. Staurosporine induced 52. 3% and 56.

2% underwent apoptosis after 24 and 48 hours respectively. Statistical analysis of the data demon strated that the response of Gc 6spg was dependent on the incubation time, i. e. the shorter the incubation time the stronger the effect, while this was not the case for Gc 5spg. This might reflect intrinsic differences between these two cell lines. The transient receptor potential vanilloid receptor 1 is e pressed by premeiotic germ cells Since CAP is a TRPV1 agonist and no information was available on the e pression of this receptor in germ cells, we determined the e pression of TRPV1 on the spermato gonial stem cells and also on germ cells in vivo. TRPV1 was localized on the Gc 5spg and Gc 6spg rat sper matogonial cell lines as determined by immuno labeling and confocal microscopy.

Batimastat TRPV 1 reactivity was predomi nantly observed on the plasma membrane of both cell lines. The protein was also detected in both the positive control and the Gc 5spg and Gc 6spg cell lines by a band migrating to 90 kD, the e pected molecular weight. TRPV1 was also e pressed in vivo by premeiotic germ cells including both undifferentiated and differentiated sper matogonia independent of the stage of the epithelial cycle. Early spermatocytes only weakly e pressed TRPV1 whereas no e pression was detected in spermatids. Discussion Herein, we demonstrate that CAP can induce apoptosis in two different spermatogonial stem cell lines in vitro. In addition we show that the cell lines used and the germ cells from which the cell lines originated e press the CAP receptor, TRPV1. An increase in apoptosis following CAP treatment was demonstrated by using two independent methods, detec tion of activated caspase 3 by immuno cytochemistry and quantification of DNA fragmentation by flow cytometry. Our observations are in accordance with previously reported findings and add to the list of cell types that respond to CAP by undergoing apoptosis.