Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced maturation of spermatogonial cells from prepubertal mice in vitro is enhanced by testosterone

Spermatogenesis is the complicated process of sperm generation. During this process, spermatogonial cells proliferate and differentiate via meiotic and post-meiotic stages to produce mature sperm. This process is under the regulation of testicular autocrine/paracrine factors. In addition, endocrine factors are crucial to complete spermatogenesis. We aimed to localize granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor (GM-CSFR) in testicular cells and further evaluate its involvement in the development of spermatogenesis in vitro. We isolated cells from seminiferous tubule cells of seven-day-old mice and cultured them in vitro using a methylcellulose culture system (MCS), in the presence of GM-CSF and/or testosterone for four weeks. The cells were then examined for markers of different stages of spermatogenesis by immunofluorescence staining and/or qPCR analyses. Our results revealed the presence of GM-CSF and GM-CSFR in testicular cells (premeiotic and meiotic cells as well as somatic cells; Leydig and Sertoli cells). We further demonstrated the development of colonies/ spheroids in the MCS which contained pre-meiotic, meiotic, and post-meiotic cells. The addition of GM-CSF to the MCS significantly increased the percentage of pre-meiotic and meiotic cells compared to control. Furthermore, the addition of GM-CSF and testosterone together significantly increased the percentage of cells in the post-meiotic stage compared to the addition of each separately. In conclusion, our results indicate that testicular cells express GM-CSF/GM-CSFR, and that GM-CSF is involved in the development of different stages of spermatogenesis in vitro. Furthermore, testosterone enhances the development of spermatogenic cells and potentiates the effect of GMCSF on the development of post-meiotic cells. These findings provide evidence that GM-CSF and testosterone are involved in the development of spermatogenesis in vitro and in vivo.