Adrenocorticotropic hormone and its receptor as a novel testicular system involves in the development of spermatogenesis

Aims: To identify functional membrane-associate-specific SSC markers and examine the development of these cells under in vitro conditions. Materials and methods: Cells were enzymatically isolated from seminiferous tubules (STs) of immature mice. Spermatogonial cells (Thy1, alpha-6-integrin, and C-KIT) were sorted by FACS. RNA was extracted from these cells for RNAseq analysis. The effect of adrenocorticotropic hormone (ACTH) – the ligand of MC2R- on the development of mouse spermatogonial cells was performed in vitro using a methylcellulose culture system (MCS). Immunofluorescence staining was used to localize MC2R-positive cells in the testes of immature and adult humans and mice and testes of busulfan-treated immature mice. Key findings: Our RNAseq analysis revealed a high expression of melanocortin receptor 2 (MC2R) in Thy1-positive sorted cells. MC2R-positive cells were localized in the periphery of the STs of humans (prepubertal and adults) and mice at immature and adult ages (normal and busulfan-treated mice). MC2R was doubled stained with PLZF and CDH1 (SSC markers). ACTH was localized in mouse testicular germ cells (pre-meiotic, meiotic, and post-meiotic cells) and somatic cells (Sertoli, Leydig, and peritubular cells). The addition of ACTH to isolated cells from mouse STs in MCS significantly increased the development of pre-meiotic and meiotic/post-meiotic cells in vitro. Significance: We were able to identify, for the first time, a novel membrane-associated and functional SSC marker (MC2R) with relation to ACTH. This marker can be used in future male fertility preservation strategies. Furthermore, we explored a novel testicular system (ACTH system) that regulates the development of spermatogenesis.