FIG. 4.

Characterization of Bhlhe40, Hoxc4, and Tec transcript and protein expression in vivo and in vitro. A) BHLHE40, HOXC4, and TEC proteins were expressed in cultures of EPCAM⁺ clump-forming germ cells. Column 1 = bright field; column 2 = dark field, gene-specific primary antibody (GFP); column 3 = DAPI; column 4 = merged, overlay of columns 2 and 3. Control immunofluorescence images had rabbit IgG as primary antibody. Bars = 100 μm. B) Quantitative RT-PCR of gene expression in testes from Day 0 (0), Day 6 (6), Day 15 (15), and adult (Ad) testes, fresh EPCAM⁺ cells (Ep), and cultured EPCAM⁺ cells (Cu) indicated that Bhlhe40, Hoxc4, and Tec transcripts were present in vivo and enriched in EPCAM⁺ clump-forming germ cells. Bars with different letters are significantly different; error bars represent the SEM. C) BHLHE40, HOXC4, and TEC proteins appeared to stain at low levels in spermatogonia in adult testis tissue (arrows). Staining was more intense in other differentiated germ cells. Insets are representative images of negative control sections in which specific primary antibodies were replaced with IgG controls. Bars = 100 μm.