FIGURE 1. Expression of the GDNF-regulated transcription factors Bcl6b, Erm, and Lhx1 in self-renewing SSCs in vitro and undifferentiated spermatogonia in vivo.

A, cultured germ cell clumps (arrows) established from 6–8-dpp mouse pup Thy1+ germ cells and maintained in a serum-free environment with GDNF, Gfrα1, and basic fibroblast growth factor. Self-renewing SSCs grow within these clumps and are loosely attached to an underlying STO feeder (arrowhead). B, recipient mouse testis transplanted with cultured clump-forming Rosa donor germ cells, demonstrating the SSC content of these cultures. Each blue colony of spermatogenesis is derived from a single donor SSC that was present in the injected cell population (17). C, immunofluorescent detection of Bcl6b, Erm, and Lhx1 protein expression in SSC-enriched cultured germ cell clumps. Negative control was omission of primary antibody. D, immunoblot detection of Bcl6b, Erm, and Lhx1 expression in two independent SSC enriched germ cell clump cultures. Glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) was used as a loading control. E, examination of Bcl6b, Erm, and Lhx1 expression in cross-sections of 8-dpp mouse pup testes using immunohistochemistry. Expression of all three molecules was detected in undifferentiated spermatogonia. Both Bcl6b and Lhx1 expression was observed in individual spermatogonia (arrows), whereas Erm expression was detected in most spermatogonia (arrows) as well as Sertoli cell nuclei (arrowhead). Negative control was omission of primary antibody. Scale bars, 100 µm (A), 2 mm (B), 50 µm (C and E).