Differentiation of neonate mouse spermatogonia on two-dimensional and three-dimensional culture systems supplemented with d-Serine and Dizocilpine (MK-801)

Abstract

N-methyl-d-aspartate (NMDA) modulates the spermatogenesis process through stimulating the steroid hormone biosynthesis. The aim of this study was to evaluate the effects of NMDA receptors agonists (d-Serine) and antagonists (MK801) on spermatogonia differentiation on decellularization testicular matrix (DTM) hydrogel scaffold. Four treatment groups were planned: 2D + D-Serine, 3D + D-Serine, 2D + MK801, and 3D + MK801. Results showed that cell viability was significantly decreased after 48 h in the 3D + D-Serine group and after 24 and 48 h in the 3D + MK801 group compared to the controls. The spermatogonia proliferation after two, four, and eight weeks was significantly increased in the 3D + D-Serine culture, while it was significantly reduced in the 2D + MK801 and 3D + MK801 groups after four and eight weeks. Real-time PCR results demonstrated that pre-meiotic gene (Plzf) expression was significantly increased only in the 3D + D-Serine culture compared to the control groups after four weeks of culture. The meiotic gene (Sycp3) expression was significantly increased in the 2D + D-Serine and 3D + D-Serine compared to the 2D controls after four and eight weeks. The post-meiotic gene (Tnp1) level in the 3D + D-Serine was significantly higher than the other groups. Flow-cytometry results indicated that the protein expression of Plzf (after four and eight weeks), Sycp3 (after eight weeks), and Tnp1 (after eight weeks) in the d-Serine-treated groups was significantly increased compared with the 2D control groups. There were not any significant changes in the gene expression of spermatogenic-related markers in MK801 culture media. However, a significant decrease in the protein levels of Plzf after eight weeks and Sycp3 after four and eight weeks was observed. In conclusion, the addition of NMDARs agonists (d-Serine) could be used to regulate the differentiation of spermatogonia in the 3D culture system.

Keywords: Differentiation; MK801; Spermatogonia; Three-dimensional culture; Two-dimensional culture; d-Serine.