NOTCH PATHWAY IS REQUIRED TO SPERMATOGONIAL STEM CELLS HEAT-STRESS PROTECTION

MORENO ACOSTA O.D.; BOAN, A.; HATTORI, R.S.; FERNANDINO, J.I.

CABA

Congreso; XX JORNADAS ANUALES MULTIDISCIPLINARIAS DE LA SAB; 2018

Sociedad Argentina de Biología

Stress causes degeneration and disappearance of germ cells in testis of mammals, and also fish, producing sterile males in extreme conditions. It is known that under thermal stress, spermatogonial stem cells (SSCs), cell type crucial to the production of male gametes, remain quiescent to protect themselves from apoptosis. Moreover, Notch is a well-studied pathway as a regulator of cell proliferation and differentiation; however, the molecular process involvement in the stress-induced protection of SSC is not well clarified. In the present work we investigated the role of psen2, a protein of the gamma-secretase complex signals that transduced the Notch pathway, in the heat-induced SSCs protection. In order to clarify this stress-induced protection, two groups of 10 adult males of medaka fish Oryzias latipes were reared at 33°C (warm temperature group, HT) and 26°C (control group, NT) up to 30 days. At 0, 3, 10 and 30 days, 5 individual per group were sampled for testes dissection. After 3 days of heat-treatment the transcript abundance (RT-qPCR) of p53 (pro-apoptotic) was up-regulated at HT. Moreover, the number of apoptotic cells (positive TUNEL assay) was increased at HT from 10 days of treatment, showing spermatogenic cell-dead. Additionally, the Notch pathway was analyzed, showing that psen2, Notch receptor (notch1), ligands (jag1a, jag1b, jag2a, jag2b and dll4) and hes-1 (Notch signaling downstream target) were up-regulated in HT at 10 days of treatment. Moreover, the localization of psen2 was characterized (antibody anti-psen2) by immunofluorescence, presenting an ubicuos expression in the testes exposed to NT, but restricting around the SSCs, at the end of spermatogenic tubules, in the HT. Finally, the inhibition of the Notch signaling pathway was analyzed at HT in an ex vivo experiment with DAPT (gamma-secretase inhibitor) for 24 hours. The number of SSCs (oct-4 positive cells) analyzed by RT-qPCR and in situ hybridization was decreased in HT testes exposed with DAPT (12.5 and 25 nM), but not in the NT treatment, showing that the Notch pathway is crucial to the quiescence of SSCs, and then, to their survival. Taken together, these results revealed for the first time the participation of Notch pathway in the protection of SSCs in thermal stress.