O-264 Testosterone potentiates the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the maturation of spermatogonial cells in vitro from prepubertal mice

Abstract

Abstract Study question Can granulocyte-macrophage colony-stimulating factor (GM-CSF) and testosterone induce spermatogonial cells to develop spermatogenesis in vitro using methylcellulose culture system (MCS)? Summary answer GM-CSF and testosterone induced the development of premeiotic and meiotic cells in vitro. Testosterone also induced and potentiate the development of post-meiotic cells by GM-CSF. What is known already Spermatogenesis is a complicated process of sperm generation. During this process spermatogonial cells proliferate and differentiate to meiotic, postmeiotic stages that continue in spermiogenesis to generate mature sperm. This process is under regulation of autocrine and paracrine factors provided by developed germ cells and somatic cells such as Sertoli, peritubular and Leydig cells. In addition, endocrine factors such as FSH, LH and testosterone are crucial for complete spermatogenesis. Different in vitro culture systems were used to induce the development of complete spermatogenesis in vitro, however this was not yet achieved. Study design, size, duration Sexually immature mice (7-day-old) were used to isolated cells from the seminiferous tubules. These isolated ells were cultured in methylcellulose culture system (MCS) as a 3-dimensinal in vitro system. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and testosterone were added in the beginning of the cultures and after two weeks. The cultures were determined after 4 weeks. Participants/materials, setting, methods Cells were enzymatically isolated from the seminiferous tubules and cultured (2x105/well/0.5ml) in MCS that contained StemPro-34 medium, KSR, rEGF, rGDNF, rLIF, and r-bFGF. The cultures were grown in the presence/absence of GM-CSF (0.1 or 1ng/ml) and/or testosterone (10-7M) and incubated for four weeks in a CO2 incubator at 37°C. The developed cells and colonies/spheroids were examined microscopically, and quantified the developing cells of the different stages of spermatogenesis by immunofluorescence staining (IF) and/or qPCR analyses Main results and the role of chance Our results show the presence of GM-CSF and its receptor (GM-CSFR) in different testicular cells. We demonstrate the development of colonies/spheroids which contain premeiotic, meiotic and post-meiotic cells in MCS, as examined by IF and qPCR analyses. The percentage of pre-meiotic and miotic stage in the cultures with GM-CSF were significantly higher than those in the control group (p < 0.001), and the percentage of cells of the post-meiotic stage were significantly higher in the cultures with GM-CSF and testosterone together compared to each one separately (p < 0.001). In conclusion, testicular cells express GM-CSF/GM-CSFR, and GM-CSF affects only the development of premeiotic and meiotic cells in vitro. In addition, testosterone not only increased the development of cells from the premeiotic, meiotic and post-meiotic stages, but it also potentiated the effect of GM-CSF on the development of post-meiotic cells in vitro. Limitations, reasons for caution no limitation Wider implications of the findings These findings providing evidence that GM-CSF and testosterone could be used to improve the maturation of spermatogonial cells in vitro and that they might play a role in the maturation process of spermatogonial cells in vivo. Trial registration number not applicable