Effect of NMDA Receptor Agonist and Antagonist on Spermatogonia Stem Cells Proliferation in 2- and 3- Dimensional Culture Systems

Abstract

The main purpose of this study was to investigate the effect of D-serine (DS) and Dizocilpine (MK-801or Mk) on the proliferation of SSCs in two-dimensional (2D) and three-dimensional (3D) culture systems. The SSCs of male NMRI mice were isolated by enzymatic digestion and cultured for two weeks. Then, the identity of SSCs was confirmed by anti-Plzf and anti-GFR-α1 antibodies via immunocytochemistry (ICC). The proliferation capacity of SSCs was evaluated by their culture on a layer of the decellularized testicular matrix (DTM) prepared from mouse testis, as well as two-dimensional (2D) with different mediums. After two weeks of the initiation of proliferation culture on 3D and 2D medium, the pre-meiotic at the mRNA and protein levels were evaluated via qRT-PCR and flow cytometry methods, respectively. The results showed that the proliferation rate of SSCs in three-dimensional culture with 50 mM glutamic acid and 20 mM D-serine was significantly different from other groups after 14 days treatment. mRNA expression levels of Plzf in 3D-cultures supplemented by 20 mM D-serine and 50 mM glutamic acid were considerably higher than the 3D control group (p<0.001). The flow cytometry analysis revealed that the amount of Plzf in the 2D-culture groups of SSCs with 20mM MK-801 was considerably lower compared to the 2D-culture control group (p<0.001). This study indicated that decellularized testicular matrix supplemented with D-serine and glutamic acid could be considered a promising vehicle to support cells and provide an appropriate niche for the proliferation of SSCs.