Figure 4.
Impact of Direct Exposure to RA on Regenerative Capacity of the SSC Population
(A) Experimental scheme for assessing changes in SSC content of primary undifferentiated spermatogonial cultures following direct exposure to RA. Note that all spermatogonia from the culture well (ID4-eGFP+ and ID4-eGFP–) were subjected to DMSO or RA treatment.
(B) Representative scatterplot for the ID4-eGFP+ population in primary cultures of undifferentiated spermatogonia. The population can be subdivided based on Bright, Mid, Dim, and Negative EGFP intensity and the SSC population is mostly contained in the ID4-eGFPBright subset.
(C) Representative image of western blot analysis for RARγ expression in ID4-eGFP Bright, Dim, and Negative spermatogonial pools from primary cultures. Tubulin (TUBB1) expression was used as a loading control.
(D) Quantification of KIT+ cells in the ID4-eGFP subpopulations following direct exposure to RA. Data are presented mean ± SEM for n = 3 different cultures established from 3 independent P6–8 pups or adult mice.
(E) Quantification of the percentage of cells that could be classified as ID4-eGFP+ in pup- or adult-derived primary cultures at 2–6 days after treatment with DMSO (control) or RA. Data were generated from flow cytometric analyses (representative histogram plot is presented) and are presented as mean ± SEM for n = 3 different cultures (biological replicates).
(F) Quantification of the percentage of cells in primary cultures established from pup or adult mice that were ID4-eGFPBright at 4 and 6 days after treatment with DMSO (control) or RA. Data were generated by flow cytometric analysis and are presented as mean ± SEM for n = 3 different cultures (biological replicates).
(G and H) Quantification of SSC number (donor-derived colonies of spermatogenesis/105 cells transplanted) in primary cultures established from (G) pup or (H) adult mice, transplanted 16 hr after treatment with DMSO (control) or RA. Data were generated by functional transplantation analyses (representative images of recipient testes with LacZ-stained donor-derived spermatogenic colonies are presented) and are mean ± SEM for n = 3 different cultures (biological replicates).
∗Significantly different at p < 0.05.