Identification of a Novel and Functional membrane-associated Spermatogonial Stem Cell (SSC) Marker

Ali Abumadighem 1,2,5 Ehab Matanis 1,2,5 Areej Jorban 1,2,5 Eitan Rubin 1,2,5 Eitan Lunenfeld 2,3,4,5 Mahmoud Huleihel 1,2,5
1The Shraga Segal Dept. Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, Israel
2(CARER), The Center of Advanced Research and Education in Reproduction, Israel
3Ariel University, Adelson Faculty of Medicine, Israel
4Soroka, University Medical Center, Israel
5Faculty of Health Sciences, Ben-Gurion University of the Negev

Introduction:
Spermatogonial stem cell (SSC) transplantation is successful to restore fertility in sterile animal models. It is not safe in human since there is no precise methodology to isolate pure SSCs from cancer cells.

Aim of the Study:
Identification of a functional membrane-associate specific SSC markers.

Methodology:
Sexually immature mice were intraperitoneally injected with busulfan (45 mg/kg) or DMSO (as control). Ten days later testes were removed, cells were enzymatically isolated from the seminiferous tubules (STs) and spermatogonial cells (Thy1, alpha-6-intigren and C-KIT) were sorted by FACS. RNA was extracted and used for RNAseq analysis. Immunofluorescence staining was used to localize melanocortin receptor 2 (MC2R)-positive cells in testes of immature and adult human and mice. The effect of adrenocorticotropic hormone (ACTH) – the ligand of MC2R- on the capacity of spermatogonial cells to develop spermatogenesis was performed in vitro using methylcellulose culture system (MCS).

Results:
RNAseq analysis showed a high expression of MC2R in Thy1-positive cells compared to alpha-6-intigren and C-KIT-positive cells. MC2R-positive cells were localized in the periphery of the STs of human and mice at immature and adult ages. MC2R was doubled stained with known SSC markers. Addition of ACTH to isolated cells from STs in MCS significantly increased the pre-meiotic and meiotic/post-meiotic cells after 4 weeks of culture.

Conclusions:
We identified, for the first time, using RNAseq analysis of FACS sorted spermatogonial cells, a novel membrane-associated and functional SSCs marker. This marker can be used in future fertility preservation strategies for perpubertal cancer boys.