Glycolysis-Optimized Conditions Enhance Maintenance of Regenerative Integrity in Mouse Spermatogonial Stem Cells during Long-Term Culture

Abstract

SummaryThe application of spermatogonial stem cell (SSC) transplantation for regenerating male fertility requires amplification of SSC number in vitro during which the integrity to re-establish spermatogenesis must be preserved. Conventional conditions supporting proliferation of SSCs from mouse pups have been the basis for developing methodology with adult human cells but are unrefined. We found that the integrity to regenerate spermatogenesis after transplantation declines with advancing time in primary cultures of pup SSCs and that the efficacy of deriving cultures from adult SSCs is limited with conventional conditions. To address these deficiencies, we optimized the culture environment to favor glycolysis as the primary bioenergetics process. In these conditions, regenerative integrity of pup and adult SSCs was significantly improved and the efficiency of establishing primary cultures was 100%. Collectively, these findings suggest that SSCs are primed for conditions favoring glycolytic activity, and matching culture environments to their bioenergetics is critical for maintaining functional integrity.

Enhanced stress tolerance through reduction of G3BP and suppression of stress granules

“…Spermatogonial cultures were maintained on mitotically inactivated SIM mouse embryo-derived thioguanine-and ouabain-resistant feeder monolayers (STOs) in mouse serum-free medium (mSFM), devoid of fatty acids and supplemented with the growth factors GDNF (20 ng/mL; Peprotech, NJ, USA) and FGF2 (1 ng/mL; Peprotech). Cultures were kept in glycolysis-optimized conditions in humidified incubators at 37 °C, 10% O2 and 5% CO2 in air (Helsel et al, 2017a). Culture media was replaced every other day, and passaging onto fresh feeders was performed every 6-8 days.…”

Astrogliopathology in the infectious insults of the brain

“…Indeed, it was reported that ZIKV is present in the testes of mice in cells that are highly active in cell division including spermatocytes, Sertoli cells and spermatogonia [33]. For survival, spermatogonial stem cells, exhibiting dynamic proliferation, require an environment that favours glycolysis [39]. Why is glycolysis important for proliferation?…”

Spermatogenesis and Its Significance in Reproductive Medicine

“…In terms of the SSC niche, one recent report found that the reduction in O 2 tension during in vitro conditions enhanced the in vivo maintenance of the SSCs' regenerative integrity. SSCs cultured long term in hypoxic conditions (10% O 2 tension rather than the standard 21% O 2 tension), had the capacity to continue spermatogenesis following transplantation in recipient tubules devoid of germ cells [48]. Previous work from the same group had also shown that key glycolysis regulating enzymes were elevated in cultured undifferentiated germ cells [49].…”

“…The excess of free fatty acids, however, promotes fatty acid catabolism (i.e. β-oxidation and oxidative phosphorylation) rather than glycolytic activity [48]. Together, there are possible explanations and takeaways from such discrepancies regarding hypoxia and metabolism.…”

“…Furthermore, reactive oxygen species (ROS) were shown to also influence the outcome of in vitro SSC maintenance. Moreover, modulating ROS levels demonstrated that moderate concentrations may promote SSC self-renewal and that achieving this may be possible by manipulating or reducing the use of β-oxidation as the primary bioenergetics pathway [48,54]. Such speculations, however, only point to the need for further investigations of the three-dimensional relationship of the SSC and its niche.…”