Mammalian target of rapamycin modulates Sertoli cells metabolism and oxidative profile T.T. Jesus†, P.F. Oliveira‡, J. Silva§, A. Barros‡§⊥, R. Ferreira¶,

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Mammalian target of rapamycin modulates Sertoli cells metabolism and oxidative profile T.T. Jesus*†, P.F. Oliveira*‡, J. Silva§, A. Barros‡§⊥, R. Ferreira¶, M. Sousa*§, C.Y. Cheng ○, B.M. Silva† & M.G. Alves† * Laboratory of Cell Biology, Department of Microscopy, and Unit for Multidisciplinary Research in Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Portugal; † CICS-UBI, Health Sciences Research Centre, University of Beira Interior, Portugal; ‡ I3S,Institute of Health Research an Innovation, University of Porto, Portugal; § Center for Reproductive Genetics Professor Alberto Barros, Porto, Portugal; ⊥ FMUP, Faculty of Medicine, University of Porto, Portugal; ¶ QOPNA, Organic Chemistry, Natural and Agrofood Products, Department of Chemistry, University of Aveiro, Portugal; ○ Population Council’s Center for Biomedical Research, New York, USA  alvesmarc@gmail.com INTRODUCTION Sertoli cells (SCs) provide physical and nutritional support to germ cells in different stages during spermatogenesis. The metabolic cooperation between these somatic cells and developing germ cells is crucial for the normal occurrence of spermatogenesis. The mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase implicated in the regulation of several cellular functions, including cellular metabolism. To study the role of mTOR in the regulation of human Sertoli cell (hSC): metabolism, mitochondrial activity and oxidative stress. OBJECTIVES MATERIALS AND METHODS 1. mTOR pathway related proteins expression: Western Blot 2. Glucose metabolism related proteins expression: Western Blot 3. Oxidative stress indicators: Slot Blot 4. Mitochondrial function indicators: Western Blot Control Group Cellular pellet Human testicular biopsies (n=6) SC primary culture Rapamycin Group (100ng/mL) Extracellular medium 5. Metabolites analysis 1H-NMR spectroscopy RESULTS Rapamycin decreased mTOR’s phosphorylation at Ser2448 Rapamycin increased glucose uptake and lactate/alanine ratio Rapamycin decreased mitochondrial complex III protein levels Rapamycin increased lipid peroxidation CONCLUSIONS Our data provides clear evidence that mTOR signaling, particularly mTORC1, alters the glycolytic and oxidative profile of hSCs. The unraveling of these biochemical cascades may open novel molecular paradigms to new pharmacological targets to counteract male subfertility/infertility or to design new male contraceptives. ACKNOWLEDGEMENTS This work was supported by the Portuguese “Fundação para a Ciência e a Tecnologia” - FCT: TT Jesus (SFRH/BD/103518/2014); MG Alves (SFRH/BPD/80451/2011); PF Oliveira (SFRH/BPD/108837/2014); UMIB (Pest-OE/SAU/UI0215/2014) and co-funded by FEDER funds through the POCI - COMPETE 2020 - Operational Programme Competitiveness and Internationalization in Axis I - Strengthening research, technological development and innovation (Project POCI-01-0145-FEDER-007491) and National Funds by FCT (Project UID/Multi /00709/2013).