Figure 5

Evaluation of biological activity of pGDNF on the proliferation of porcine gonocytes/undifferentiated spermatogonia. (A) Enrichment of gonocytes/undifferentiated spermatogonia by differential plating. The first non-adherent (Enriched, 1^st), the second non-adherent (Enriched, 2^nd), and the first adherent cell fractions in addition to the unselected cell fraction (Unenriched) from porcine testicular cells were analysed by western blot analysis. DDX4 and UCHL1 were strongly expressed in the enriched fractions. (B) Immunofluorescence analysis of porcine gonocytes/undifferentiated spermatogonia cultured with pGDNF. A DDX4⁺ Ki67⁺ cell (arrow head) and tightly attached DDX4⁺ cells (asterisk) are indicated. Nuclei were counterstained with DAPI. Bar = 50 µm. (C) Effect of pGDNF on porcine gonocytes/undifferentiated spermatogonia. No significant difference was found between the number of DDX4⁺ Ki67⁺ or tightly attached DDX4⁺ cells in the presence and absence of pGDNF. Three independent experiments were performed. More than 100 DDX4⁺ cells were analysed in each experiment (mean ± SEM, n = 3). (D) EdU incorporation analysis for DNA replication in porcine gonocytes/undifferentiated spermatogonia cultured with pGDNF. A DDX4⁺ EdU⁺ cell (arrow head) is indicated. Nuclei were counterstained with DAPI. Bar = 50 µm. (E) Effect of pGDNF on enriched or unenriched porcine gonocytes/undifferentiated spermatogonia. No significant difference was found between the number of DDX4⁺ EdU⁺ cells in the presence and absence of pGDNF in either cell fraction. Four independent experiments were performed. More than 100 DDX4⁺ cells were analysed in each experiment (mean ± SEM, n = 4).