Tito T Jesus1, Pedro F Oliveira2, Joaquina Silva3, Alberto Barros4, Rita Ferreira5, Mário Sousa6, C Yan Cheng7, Branca M Silva8, Marco G Alves9. 1. Department of Microscopy, Laboratory of Cell Biology and Unit for Multidisciplinary Research in Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal; CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Covilhã. 2. Department of Microscopy, Laboratory of Cell Biology and Unit for Multidisciplinary Research in Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal; Institute of Health Research an Innovation, Portugal. 3. Centre for Reproductive Genetics Prof. Alberto Barros, Porto, Portugal. 4. Institute of Health Research an Innovation, Portugal; Centre for Reproductive Genetics Prof. Alberto Barros, Porto, Portugal; Department of Genetics, Faculty of Medicine, University of Porto, Porto, Portugal. 5. Organic Chemistry, Natural and Agrofood Products Centre, Department of Chemistry, University of Aveiro, Aveiro, Portugal. 6. Department of Microscopy, Laboratory of Cell Biology and Unit for Multidisciplinary Research in Biomedicine (UMIB), Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal; Centre for Reproductive Genetics Prof. Alberto Barros, Porto, Portugal. 7. Center for Biomedical Research, Population Council, New York, New York. 8. CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Covilhã. 9. CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Covilhã. Electronic address: alvesmarc@gmail.com.
Abstract
OBJECTIVE: To study the role of mammalian target of rapamycin (mTOR) in the regulation of human Sertoli cell (hSC) metabolism, mitochondrial activity, and oxidative stress. DESIGN: Experimental study. SETTING: University research center and private assisted reproductive technology centers. PATIENT(S): Six men with anejaculation (psychological, vascular, neurologic) and conserved spermatogenesis. INTERVENTION(S): Testicular biopsies were used from patients under treatment for recovery of male gametes. Primary hSCs cultures were established from each biopsy and divided into a control group and one treated with rapamycin, the inhibitor of mTOR, for 24 hours. MAIN OUTCOME MEASURE(S): Cytotoxicity of hSCs to rapamycin was evaluated by sulforhodamine B assay. The glycolytic profile of hSCs was assessed by proton nuclear magnetic resonance and by studying protein expression of key glycolysis-related transporters and enzymes. Expression of mitochondrial complexes and citrate synthase activity were determined. Protein carbonylation, nitration, lipid peroxidation, and sulfhydryl protein group contents were quantified. The mTOR signaling pathway was studied. RESULT(S): Rapamycin increased glucose consumption by hSCs, maintaining lactate production. Alanine production by rapamycin-exposed hSCs was affected, resulting in an unbalanced intracellular redox state. Rapamycin-exposed hSCs had decreased expression of mitochondrial complex III and increased lipid peroxidation, whereas other oxidative stress markers were unaltered. Treatment of hSCs with rapamycin down-regulated phospho-mTOR (Ser-2448) levels, illustrating an effective partial inhibition of mTORC1. Protein levels of downstream signaling molecule p-4E-BP1 were not altered, suggesting that during treatment it became rephosphorylated. CONCLUSION(S): We show that mTOR regulates the nutritional support of spermatogenesis by hSCs and redox balance in these cells.
OBJECTIVE: To study the role of mammalian target of rapamycin (mTOR) in the regulation of human Sertoli cell (hSC) metabolism, mitochondrial activity, and oxidative stress. DESIGN: Experimental study. SETTING: University research center and private assisted reproductive technology centers. PATIENT(S): Six men with anejaculation (psychological, vascular, neurologic) and conserved spermatogenesis. INTERVENTION(S): Testicular biopsies were used from patients under treatment for recovery of male gametes. Primary hSCs cultures were established from each biopsy and divided into a control group and one treated with rapamycin, the inhibitor of mTOR, for 24 hours. MAIN OUTCOME MEASURE(S): Cytotoxicity of hSCs to rapamycin was evaluated by sulforhodamine B assay. The glycolytic profile of hSCs was assessed by proton nuclear magnetic resonance and by studying protein expression of key glycolysis-related transporters and enzymes. Expression of mitochondrial complexes and citrate synthase activity were determined. Protein carbonylation, nitration, lipid peroxidation, and sulfhydryl protein group contents were quantified. The mTOR signaling pathway was studied. RESULT(S): Rapamycin increased glucose consumption by hSCs, maintaining lactate production. Alanine production by rapamycin-exposed hSCs was affected, resulting in an unbalanced intracellular redox state. Rapamycin-exposed hSCs had decreased expression of mitochondrial complex III and increased lipid peroxidation, whereas other oxidative stress markers were unaltered. Treatment of hSCs with rapamycin down-regulated phospho-mTOR (Ser-2448) levels, illustrating an effective partial inhibition of mTORC1. Protein levels of downstream signaling molecule p-4E-BP1 were not altered, suggesting that during treatment it became rephosphorylated. CONCLUSION(S): We show that mTOR regulates the nutritional support of spermatogenesis by hSCs and redox balance in these cells.
Authors: Aimee L Edinger; Corinne M Linardic; Gary G Chiang; Craig B Thompson; Robert T Abraham Journal: Cancer Res Date: 2003-12-01 Impact factor: 12.701
Authors: David K Finlay; Ella Rosenzweig; Linda V Sinclair; Carmen Feijoo-Carnero; Jens L Hukelmann; Julia Rolf; Andrey A Panteleyev; Klaus Okkenhaug; Doreen A Cantrell Journal: J Exp Med Date: 2012-11-26 Impact factor: 14.307
Authors: Tito T Jesus; Pedro F Oliveira; Mário Sousa; C Yan Cheng; Marco G Alves Journal: Crit Rev Biochem Mol Biol Date: 2017-01-26 Impact factor: 8.250