title: Resistance to apoptosis and autophagy leads to enhanced survival in Sertoli cells.
abstract: What is the underlying mechanism of Sertoli cell (SC) resistance to cell death?
  
  
  
  High expression of prosurvival B-cell lymphoma-2 (BCL2) proteins and inhibition of apoptosis and autophagy prolongs SC survival upon exposure to stress stimuli.
  
  
  
  In human and in experimental models of orchitis, tolerogenic SC survive stress conditions, while germ cells undergo massive apoptosis. In general, non-dividing highly differentiated cells tend to resist stress conditions for a longer time by favoring activation of prosurvival mechanisms and inhibition of cell death pathways.
  
  
  
  In this cross sectional study, conditions stimulating apoptosis and autophagy were used to induce cell death in primary rat SC. Primary rat peritubular cells (PTC) and immortalized rat 93RS2 SC were used as controls. Each cell isolation was counted as one experiment (n = 1), and each experiment was repeated three to six times.
  
  
  
  Testis biopsy samples from infertile or subfertile patients and testis samples from rats with experimental autoimmune orchitis were used for immunohistological analysis. Primary SC were isolated from 19-day-old male Wistar rats. To maintain cell purity, cells were cultured in serum-free medium for apoptosis experiments and in medium supplemented with 1% serum for autophagy analyses. To induce apoptosis, cells were stimulated with staurosporine, borrelidin, cisplatin and etoposide for 4 or 24 h. Caspase three activation was examined by immunoblotting and enzymatic activity assay. Mitochondrial membrane potential was measured using tetramethylrhodamine methyl ester followed by flow cytometric analysis. Cytochrome c release was monitored by immunofluorescence. Cell viability was determined using the methylthiazole tetrazolium assay. To monitor autophagy flux, cells were deprived of nutrients using Hank's balanced salt solution for 1, 2 and 3 h. Formation of autophagosomes was analyzed by using immunoblotting, immunofluorescence labeling and ultrastructural analyses. Relative mRNA levels of genes involved in the regulation of apoptosis and autophagy were evaluated. Extracellular high mobility group box protein one was measured as a marker of necrosis using ELISA.
  
  
  
  SC survive the inflammatory conditions in vivo in human testis and in experimental autoimmune orchitis. Treatment with apoptosis inducing chemotherapeutics did not cause caspase three activation in isolated rat SC. Moreover, mitochondrial membrane potential and mitochondrial localization of cytochrome c were not changed by treatment with staurosporine, suggesting a premitochondrial blockade of apoptosis in SC. Expression levels of prosurvival BCL2 family members were significantly higher in SC compared to PTC at both mRNA and protein levels. Furthermore, after nutrient starvation, autophagy signaling was initiated in SC as observed by decreased levels of phosphorylated UNC- 51-like kinase -1 (ULK1). However, levels of light chain 3 II (LC3 II) and sequestosome1 (SQSTM1) remained unchanged, indicating blockade of the autophagy flux. Lysosomal activity was intact in SC as shown by accumulation of LC3 II following administration of lysosomal protease inhibitors, indicating that inhibition of autophagy flux occurs at a preceding stage.
  
  
  
  N/A.
  
  
  
  In this study, we have used primary SC from prepubertal rats. Caution should be taken when translating our results to adult animals, where crosstalk with other testicular cells and hormonal factors may also play a role in regulating survival of SC.
  
  
  
  Our results suggest that inhibition of autophagy and apoptosis following exposure to extrinsic stress stimuli promotes SC survival, and is a possible mechanism to explain the robustness of SC in response to stress. Cell death resistance in SC is crucial for the recovery of spermatogenesis after chemotherapy treatment in cancer patients. Additionally, understanding the molecular mechanisms of SC survival unravels valuable target proteins, such as BCL2, that may be manipulated therapeutically to control cell viability depending on the context of the disease.
  
  
  
  This study was funded by the Deutsche Forschungsgemeinschaft (DFG) Grant BH93/1-1, and by the International Research Training Group between Justus Liebig University of Giessen and Monash University, Melbourne (GRK 1871/1) funded by the DFG and Monash University. The support of the Medical Faculty of Justus-Liebig University of Giessen is gratefully acknowledged. The authors declare no conflict of interest.
authors.affiliation: Department of Anatomy and Cell Biology, Justus-Liebig University, Aulweg 123, Giessen 35392, Germany.
authors.forename: Ferial
authors.initials: F
authors.lastname: Aslani
authors.affiliation: Department of Anatomy and Cell Biology, Justus-Liebig University, Aulweg 123, Giessen 35392, Germany.
authors.forename: Tim
authors.initials: T
authors.lastname: Sebastian
authors.affiliation: Department of Anatomy and Cell Biology, Justus-Liebig University, Aulweg 123, Giessen 35392, Germany.
authors.forename: Miguel
authors.initials: M
authors.lastname: Keidel
authors.affiliation: Department of Anatomy and Cell Biology, Justus-Liebig University, Aulweg 123, Giessen 35392, Germany.
authors.forename: Suada
authors.initials: S
authors.lastname: Fröhlich
authors.affiliation: Department of Cell Biology and Cytopathology, Philipps University of Marburg, Germany.
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Elsässer
authors.affiliation: Department of Urology, Pediatric Urology and Andrology, Justus-Liebig University, Giessen, Germany.
authors.forename: Hans-Christian
authors.initials: HC
authors.lastname: Schuppe
authors.affiliation: Department of Anatomy and Cell Biology, Justus-Liebig University, Aulweg 123, Giessen 35392, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Klug
authors.affiliation: Department of Internal Medicine, Justus-Liebig University, Universities of Giessen and Marburg Lung Center, Member of the German Center for Lung Research (DZL), Giessen, Germany.
authors.forename: Poornima
authors.initials: P
authors.lastname: Mahavadi
authors.affiliation: Department of Anatomy and Cell Biology, Justus-Liebig University, Aulweg 123, Giessen 35392, Germany.
authors.forename: Monika
authors.initials: M
authors.lastname: Fijak
authors.affiliation: Institute of Veterinary Anatomy, Histology, and Embryology, Justus-Liebig-University, Giessen, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Bergmann
authors.affiliation: Department of Anatomy and Cell Biology, Justus-Liebig University, Aulweg 123, Giessen 35392, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Meinhardt
authors.affiliation: Department of Anatomy and Cell Biology, Justus-Liebig University, Aulweg 123, Giessen 35392, Germany.
authors.forename: Sudhanshu
authors.initials: S
authors.lastname: Bhushan
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000378:metabolism; D001327:Autoimmune Diseases / Q000235:genetics / Q000276:immunology / Q000473:pathology; D001343:Autophagy / Q000187:drug effects; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival / Q000235:genetics; D002945:Cisplatin / Q000494:pharmacology; D003430:Cross-Sectional Studies; D045304:Cytochromes c / Q000378:metabolism; D004195:Disease Models, Animal; D005047:Etoposide / Q000494:pharmacology; D005233:Fatty Alcohols / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D007248:Infertility, Male / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D009920:Orchitis / Q000235:genetics* / Q000276:immunology / Q000473:pathology; D061251:Primary Cell Culture; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012708:Sertoli Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D013091:Spermatogenesis / Q000235:genetics; D019311:Staurosporine / Q000494:pharmacology
chemical_list: D051017:Apoptosis Regulatory Proteins; C000595707:BCL2 protein, human; D005233:Fatty Alcohols; D019253:Proto-Oncogene Proteins c-bcl-2; D005047:Etoposide; C005066:borrelidin; D045304:Cytochromes c; C505407:CASP3 protein, human; D053148:Caspase 3; D019311:Staurosporine; D002945:Cisplatin
keywords: BCL2 family; Sertoli cell; apoptosis; autophagy; cell death; cell survival; fertility; peritubular cell; testis