Besides the production of transgenic

Besides the production of transgenic spermatozoa and animal production, studies using in vitro maintained spermatogonial stem AGI-5198 cost are important to identify the mechanism of spermatogenesis in mammals, facilitate research on male infertility, and disseminate the genetics of animal production. To reach these goals using SSCs, a large number of SSCs must be maintained under in vitro culture conditions. SSCs in mice have been cultured on mitomycin C-treated mouse embryonic fibroblasts (MEFs) and long-term culture (4–5months) and self-renewal of SSCs have been reported in the presence of added glial cell line-derived neurotrophic factor (GDNF), epidermal growth factor (EGF), and basic fibroblast growth factor 2 (FGF2) in the presence of fetal bovine serum (FBS) (Kanatsu-Shinohara et al., 2003). Furthermore, the same group showed the successful culture of mice SSCs under serum and feeder-free conditions using Stempro-34 medium containing GDNF, FGF2, and EGF (Kanatsu-Shinohara et al., 2011). In addition, porcine SSC-like cells have been isolated and cultured using the same Stempro-34 medium with additional growth factors, although proliferation stopped after nine passages (Kuijk et al., 2009). Mammalian testicular temperature is maintained constantly lower than that of body temperature. Hyperthermia studies of rat testes have revealed the stage-specific nature of germ cell degeneration (Chowdhury and Steinberger, 1970) and apoptosis of pachytene spermatocytes (Lue et al., 1999; Yin et al., 1997). In addition, culture of human testis tissue fragments at 31 and 37°C for 22h demonstrated that DNA synthesis is significantly lower at 37°C than at 31°C, although the number of spermatogonia and resting primary spermatocytes is not significantly different (Nakamura et al., 1987). In view of the extended functional changes due to heat stress of germ cells, we hypothesized that the optimal temperature for porcine SSC culture might be an important factor to control proliferation and maintain porcine SSC characteristics in vitro. Although the specific and accurate species-specific markers for SSC have been disputed, identifying SSC-specific markers is very important to isolate and characterize SSCs. In addition, cell type-specific markers can be used to monitor whether SSCs maintain their characteristics during in vitro culture or whether they have initiated cellular differentiation into functional spermatozoa. Mouse SSCs express Stra8, Neurog3, promyelocytic leukemia zinc finger (PLZF), Ret, and OCT4, but NANOG and teratocarcinoma derived growth factor 1 (TDGF1) are not expressed (Kanatsu-Shinohara et al., 2011). Esr3 is expressed in rat SSCs (Hamra et al., 2004). The lectin Dolichos biflorus agglutinin (DBA) and PGP9.5 (ubiquitin C-terminal hydrolase L-1) are strongly expressed in bull SSCs (Herrid et al., 2007). PGP9.5 has been reported as a marker of porcine spermatogonial cells (Luo et al., 2006), and NANOG is expressed in most DBA and ZBTB16-positive gonocytes in porcine testes, while POU5F1 is not expressed in the same cells (Goel et al., 2008). Although markers for SSCs have been developed by many researchers, the disputations for species-dependent marker genes still remain. However, if porcine spermatogonial cells express these putative marker proteins, these cells could be putative porcine spermatogonial stem cells. In the present study, we examined the effect of various culture temperatures on porcine SSC colony formation, SSC characteristics, and marker expression to establish the porcine spermatogonial germ cells in vitro culture condition.
Materials and methods
Results
Discussion Among testicular germ cells, A single (As) spermatogonia can be differentiated as A paired (Apr) and A aligned (Aal) spermatogonia, and these cells are referred to as SSCs (de Rooij and Russell, 2000). The SSC population accounts for <4% of all spermatogenic cells in adult testes (Meachem et al., 2001). Furthermore, the very few SSCs in adult testes and the lack of information on specific markers for SSCs in different species have made it difficult to prepare pure populations of SSCs (Tegelenbosch and de Rooij, 1993). However, prior to onset of spermatogenic differentiation in neonatal and prepubertal testes, the seminiferous tubules contain a relatively larger number of SSCs. Therefore, enrichment of SSCs from immature testes is more efficient than that from adult testes. The histological comparison between 5- and 180-day-old testes showed that spermatogonia were the unique germ cells in 5-day-old porcine testes, whereas various stages of differentiating spermatogenic germ cells were identified in 180-day-old porcine testes (Fig. 1A). In addition, expression of the porcine spermatogonial cell marker, PGP9.5, was also observed in 5-day-old porcine testes with high frequency (Fig. 1B). Therefore, 5-day-old porcine testes should be an optimal source for preparing SGCs to study these cells and spermatogenesis.