In this study localization of the SSC population

In this study, localization of the SSC population was determined by PGP 9.5 expression. PGP 9.5, also called ubiquitin C-terminal hydrolase L-1, is expressed in neuronal erk pathway and testes, and specific expression of PGP9.5 was identified in spermatogonia of day 8 and 16 mice, and PGP9.5 expression has also been identified in Sertoli cells of adult mice (Kon et al., 1999). PGP9.5 expression has also been reported in cattle (Herrid et al., 2007; Wrobel et al., 1996), monkeys (Tokunaga et al., 1999), and porcine (Luo et al., 2006), whereas its expression was localized to gonocytes and spermatogonia. Based on these findings, PGP9.5 was used as a marker for identifying porcine spermatogonia in 5-day-old porcine testes; only spermatogonia were successfully identified, suggesting that PGP9.5 can be used as a marker of SSCs in porcine. Mammalian testicular temperature is lower than body temperatures to protect spermatogonial cells from heat damage. In this study, three different culture temperatures of 31, 34, and 37°C were applied to derive pSGCs. The different culture temperatures showed comparable pSGC colony formation, and mRNA and protein expression (Figs. 3–5). Among the three conditions, AP strongly stained cells at 31°C rather than at 34 and 37°C, and more intensive PGP9.5 mRNA expression was also detected at 31°C than that at other temperatures. Interestingly, the pre-meiotic marker, PLZF, and the pluripotency markers, OCT4 and NANOG mRNA, were also strongly expressed at 31°C than those at 34 and 37°C. These data suggest that the lower temperature for pSGC derivation and culture may provide an optimal environment to maintain spermatogonial characteristics. In support of this hypothesis, when mice spermatogenic cells were cultured under a 37°C condition, the expression of many proliferation and differentiation-related genes was inhibited and c-kit gene mutation was identified, whereas mRNA and protein expression levels of c-kit and PI3-kinase in spermatogonial cells increased at a 32°C culture temperature compared to those at 37°C (Zhu et al., 2012). In addition, cultures of human testicular tissue fragments also showed decreased DNA synthesis at 37°C compared to that at 31°C (Nakamura et al., 1987). Besides the molecular characteristics, morphological differences were also compared among the cultures at different temperatures. Round and embryonic body shaped colonies that appeared on day 5, and their morphology were maintained for a longer period at 31°C. However, the morphology of the round-shaped colonies on day 3 changed dramatically at 37°C and the degree of morphological changes in the colonies at 34°C was intermediate between that at 31 and 37°C. In addition, the number of colonies increased under the 31 and 34°C culture conditions, but no increase in colony number was observed at 37°C. These data clearly demonstrate that an appropriate culture temperature is important to maintain pSGC colony formation and to increase the number of colonies. A cell cycle analysis was performed with both pSGC and feeder cells from cells cultured at the three different temperatures to determine the reason for the different gene expression levels and morphological changes in pSGC. At the time initial colonies appeared, a significant number of pSGCs at 31 and 34°C were in the G2/M phase than those at 37°C; however, a significant number of pSGCs were in the G2/M phase at 31°C than that at 37°C at the subculture stage. These results indicate that pSGCs grew slowly at 31°C but that the ability of DNA synthesis for proliferation was maintained over the long term. Culturing at a reduced temperature during the early stage of mesenchymal stem cell culture is more beneficial than culturing at 37°C, as anti-apoptotic heat shock proteins are upregulated, pro-apoptotic proteins are downregulated, and the levels of reactive oxide species, nitric oxide, and lipofuscin decrease (Stolzing and Scutt, 2006; Stolzing et al., 2006). In MEB5 neural stem cell cultures, the apoptotic cell population is significantly lower at 32°C than that at 37°C in the absence of EGF (Saito et al., 2010). In Chinese hamster ovary cells, culture temperatures <37°C suppress cell growth but cell viability remains high for long-term culture (Yoon et al., 2003). Presently, cell cycle arrest was prevented under the 31°C culture condition, suggesting that a low temperature is useful and beneficial to derive and maintain pSGCs in vitro.