FIG. 4.

Functional analysis of RSGLs derived in SG medium. A) Germline transmission from RSGL-GCS9 following its development into functional spermatozoa in recipient rat testes. Recipient/founder rats were transplanted with cells from line GCS9 after proliferating for 120 days in SG medium. Recipients were then paired with wild-type female rats 75 days after transplantation. Transmission of the donor cell haplotype to progeny was based on inheritance of the GCS-EGFP transgene (Table 1). B) Transmission of donor cell haplotypes from wild-type recipient/founders (F0) R883 and R901, which were transplanted with lines GCS9 and GCS10, respectively. Spermatogonial lines GCS9 and GCS10 were derived from siblings that were each homozygous for the GCS-EGFP transgene. Thus, F1 progeny represent cousins, some of which were crossed to rederive transgenic F2 progeny homozygous for the TgGCS-EGFP allele. C) Stable transmission of donor stem cell transgene to F2 progeny. Expression of the GCS-EGFP transgene (green fluorescence) in testes (top) and ovaries (bottom) of wild-type (−/−) and hemizygous (−/+) F2 progeny derived from a cross between hemizygous F1 progeny from recipients R883 and R901. For testes, the image shown in the left panel represents brightfield microscopy of the same specimen shown in the right panel by green fluorescent microscopy. Bars = 1 cm in images of testes and 100 μm in images of ovaries. D) Spermatogonial lines derived in SG medium show long-term spermatogenesis colony-forming potential. Left: Expression of the GCS-EGFP transgene in the testes of wild-type recipient R881 at 263 days following transplantation with RSGL-GCS9. Bar = 1 cm. Right: Histological testis sections from recipient R881 at 263 days after transplantation (TP) with line GCS9. Representative seminiferous tubules show normal spermatogenesis regenerated from the donor cells (green [EGFP]), which express markers for different generations of spermatogenic cells (red [immunolabeling for SYCP3, DAZL, and CREM tau]). Bar = 50 μm.