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

Abstract

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.