While all of the original and refined conventional condition

While all of the original and refined conventional conditions appear to support primary germ cell populations derived from testes of neonatal or prepubertal pups in which at least some SSCs persist for various periods of time, the impacts of the conditions on derivation and long-term maintenance of SSC containing cultures from adults have not been thoroughly explored. For the methodology reported by Kubota et al. (2004b), a single study succeeded in culturing adult SSCs (Schmidt et al., 2011), but the efficiency by which the cultures could be established was not assessed and the relative SSC content was examined at only a single time point. To our knowledge, only two other groups have demonstrated success with culture of adult mouse SSCs. Previous studies by Ogawa et al. (2004) reported a success rate of 50% for adult DBA/2 mice in deriving cultures of ghrelin receptor with the morphological appearance of spermatogonia when utilizing the complex StemPro-based medium reported by Kanatsu-Shinohara et al. (2003). Unfortunately, as transplantation analyses were not conducted with the cultures, the actual stem cell content of the cultures was undefined. Similarly, studies by Seandel et al. (2007) reported a culture method using testicular stromal cells as feeders and a variation of the complex StemPro-based medium originally reported by Kanatsu-Shinohara et al. (2003) that supports long-term expansion of a primary population of spermatogonia isolated from adult testes. Although data were not provided, the efficiency of establishing cultures from adult mice with various genetic backgrounds was reported to be high. Importantly, colonies of spermatogenesis were generated in recipient testes following transplantation of the cells, thus demonstrating that a portion of the population possessed SSC capacity. However, the number of colonies generated from the adult cultures was not reported, nor were transplantation analyses conducted at multiple points throughout the culture period. Therefore, assessing effectiveness of the culture method for long-term maintenance of the SSC pool is not possible at this time. Consequently, a void exists for robust and reproducible methodology to establish and maintain long-term primary cultures of adult SSCs. For all of the culture conditions that support long-term maintenance of a primary germ cell population reported to date, the regenerative integrity of SSCs to re-establish complete colonies of spermatogenesis has not been thoroughly evaluated. It is essential to address this gap in knowledge if the intent of cultures is to re-establish fertility following transplantation. In the current study, we discovered that the percentage of SSCs that could regenerate complete colonies of spermatogenesis declines rapidly in cultures established from prepubertal pup spermatogonia and maintained in conventional conditions (Figure 1). A potential major problem is the accumulation of DNA damage in cultured SSCs, which could lead to induction of apoptosis or maturation arrest in descendant germ cells that arise in colonies following transplantation, thereby eliminating the development of complete spermatogenesis. In this regard, generation of low levels of reactive oxygen species (ROS) during long-term culture would be critical. Indeed, outcomes of previous studies suggest that SSC maintenance in vitro is influenced by ROS (Morimoto et al., 2013, 2015). Although those studies were conducted with primary cultures derived from prospermatogonia, results of manipulating ROS levels revealed that a moderate concentration promotes self-renewal. Thus, modulating ROS levels in cultures of primary spermatogonia is likely important for maintenance of SSC regenerative integrity, and a possible means of achieving this is to maintain cells in conditions that reduce the utilization of β-oxidation as the primary bioenergetics process. Our previous finding that expression of Eno1/2 is upregulated in cells that make up the SSC pool in primary cultures of pup undifferentiated spermatogonia indicated a priming to thrive in conditions that favor glycolysis as the primary bioenergetics process. All of the culture conditions reported in previous studies have maintained cells in lipid-rich or free fatty acid-supplemented media and an atmospheric O2 tension of 21%, which likely promotes the utilization of β-oxidation.