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Testosterone and Follicle Stimulating Hormone–Dependent Glyoxalase 1 Up-Regulation Sustains the Viability of Porcine Sertoli Cells through the Control of Hydroimidazolone– and Argpyrimidine-Mediated NF-κB Pathway

2018; Elsevier BV; Volume: 188; Issue: 11 Linguagem: Inglês

10.1016/j.ajpath.2018.07.013

ISSN

1525-2191

Autores

Cinzia Antognelli, Francesca Mancuso, Roberta Frosini, Iva Arato, Mario Calvitti, Riccardo Calafiore, Vincenzo Nicola Talesa, Giovanni Luca,

Tópico(s)

Advanced Glycation End Products research

Resumo

Because Sertoli cells (SCs) play a central role in germ cell survival, their death may result in marked germ cell loss and infertility. SCs are the only somatic cells within the seminiferous tubules and are essential for regulating spermatogenesis. Factors that enhance or diminish the viability of SCs may have profound effects on spermatogenesis. Yet the mechanisms underlying the maintenance of SC viability remain largely unknown. Glyoxalase 1 (Glo1) detoxifies methylglyoxal (MG), a highly reactive carbonyl species mainly formed during glycolysis, which is a potent precursor of cytotoxic advanced glycation end products (AGEs). Hydroimidazolone (MG-H1) and argpyrimidine (ArgPyr) are AGEs resulting from MG-mediated post-translational modification of arginine residues in various proteins. The role of Glo1 and MG-derived AGEs in regulating the fate of SCs has never been investigated. By using gene silencing and the specific MG scavenger, aminoguanidine, the authors demonstrate that Glo1, under testosterone and follicle-stimulating hormone control, sustains viability of porcine neonatal SCs through a mechanism involving the NF-κB pathway. Glo1 knockdown induces a mitochondrial apoptotic pathway driven by the intracellular accumulation of MG-H1 and ArgPyr that desensitizes NF-κB signaling by modifying the inhibitor of NF-κB kinase, IKKß. This is the first report describing a role for Glo1 and MG-derived AGEs in SC biology, providing valuable new insights into the potential involvement of this metabolic axis into spermatogenesis. Because Sertoli cells (SCs) play a central role in germ cell survival, their death may result in marked germ cell loss and infertility. SCs are the only somatic cells within the seminiferous tubules and are essential for regulating spermatogenesis. Factors that enhance or diminish the viability of SCs may have profound effects on spermatogenesis. Yet the mechanisms underlying the maintenance of SC viability remain largely unknown. Glyoxalase 1 (Glo1) detoxifies methylglyoxal (MG), a highly reactive carbonyl species mainly formed during glycolysis, which is a potent precursor of cytotoxic advanced glycation end products (AGEs). Hydroimidazolone (MG-H1) and argpyrimidine (ArgPyr) are AGEs resulting from MG-mediated post-translational modification of arginine residues in various proteins. The role of Glo1 and MG-derived AGEs in regulating the fate of SCs has never been investigated. By using gene silencing and the specific MG scavenger, aminoguanidine, the authors demonstrate that Glo1, under testosterone and follicle-stimulating hormone control, sustains viability of porcine neonatal SCs through a mechanism involving the NF-κB pathway. Glo1 knockdown induces a mitochondrial apoptotic pathway driven by the intracellular accumulation of MG-H1 and ArgPyr that desensitizes NF-κB signaling by modifying the inhibitor of NF-κB kinase, IKKß. This is the first report describing a role for Glo1 and MG-derived AGEs in SC biology, providing valuable new insights into the potential involvement of this metabolic axis into spermatogenesis. Sertoli cells (SCs) are the only somatic cells within the seminiferous tubules and play a pivotal role in the maintenance and control of spermatogenesis.1Griswold M.D. The central role of Sertoli cells in spermatogenesis.Semin Cell Dev Biol. 1998; 9: 411-416Crossref PubMed Scopus (630) Google Scholar They create tight junctions that make up the blood–testis barrier, forming a peculiar morphological and chemical microenvironment to provide nutrients and essential regulatory factors for developing germ cells while protecting seminiferous tubules from autoantigens and invading pathogens.2Luca G. Baroni T. Arato I. Hansen B.C. Cameron D.F. Calafiore R. Role of Sertoli cell proteins in immunomodulation.Protein Pept Lett. 2018; 25: 440-445Crossref PubMed Scopus (9) Google Scholar For all these functions, SCs, traditionally considered merely "sustentacular cells",3Kaur G. Thompson L.A. Dufour J.M. Sertoli cells: immunological sentinels of spermatogenesis.Semin Cell Dev Biol. 2014; 30: 36-44Crossref PubMed Scopus (149) Google Scholar have been recently re-evaluated and elevated to the rank of a "sentinel"3Kaur G. Thompson L.A. Dufour J.M. Sertoli cells: immunological sentinels of spermatogenesis.Semin Cell Dev Biol. 2014; 30: 36-44Crossref PubMed Scopus (149) Google Scholar in spermatogenesis.4França L.R. Hess R.A. Dufour J.M. Hofmann M.C. Griswold M.D. The Sertoli cell: one hundred fifty years of beauty and plasticity.Andrology. 2016; 4: 189-212Crossref PubMed Scopus (231) Google Scholar In line with this pivotal role of SCs in spermatogenesis is the fact that the number of SCs present at puberty sets the upper limit for potential sperm production in adulthood, each SC being able to support a fixed number of germ cells,5Wang H. Yuan Q. Sun M. Niu M. Wen L. Fu H. Zhou F. Chen Z. Yao C. Hou J. Shen R. Lin Q. Liu W. Jia R. Li Z. He Z. BMP6 regulates proliferation and apoptosis of human Sertoli cells via Smad2/3 and Cyclin D1 pathway and DACH1 and TFAP2A activation.Sci Rep. 2017; 7: 45298Crossref PubMed Scopus (29) Google Scholar consequently controlling male reproductive capacity.6Rato L. Meneses M.J. Silva B.M. Sousa M. Alves M.G. Oliveira P.F. New insights on hormones and factors that modulate Sertoli cell metabolism.Histol Histopathol. 2016; 31: 499-513PubMed Google Scholar, 7Sharpe R.M. McKinnell C. Kivlin C. Fisher J.S. Proliferation and functional maturation of Sertoli cells, and their relevance to disorders of testis function in adulthood.Reproduction. 2003; 125: 769-784Crossref PubMed Scopus (943) Google Scholar, 8Menegazzo M. Zuccarello D. Luca G. Ferlin A. Calvitti M. Mancuso F. Calafiore R. Foresta C. Improvements in human sperm quality by long-term in vitro co-culture with isolated porcine Sertoli cells.Hum Reprod. 2011; 26: 2598-2605Crossref PubMed Scopus (13) Google Scholar In addition to the number of SCs, defects in their quality can lead to the imperfect formation of spermatozoa.7Sharpe R.M. McKinnell C. Kivlin C. Fisher J.S. Proliferation and functional maturation of Sertoli cells, and their relevance to disorders of testis function in adulthood.Reproduction. 2003; 125: 769-784Crossref PubMed Scopus (943) Google Scholar In this ambit, SCs metabolic performance, especially glucose metabolism, which is under a tight endocrine control in these cells, is essential for the success of spermatogenesis and fertility.9Cardoso A.M. Alves M.G. Sousa A.C. Jarak I. Carvalho R.A. Oliveira P.F. Cavaco J.E. Rato L. The effects of the obesogen tributyltin on the metabolism of Sertoli cells cultured ex vivo.Arch Toxicol. 2018; 92: 601-610Crossref PubMed Scopus (11) Google Scholar In fact, a close metabolic cooperation between SCs and developing germ cells has long been postulated. In particular, developing germ cells are highly dependent on the lactate produced by SCs as metabolic fuel,10Alves M.G. Dias T.R. Silva B.M. Oliveira P.F. Metabolic cooperation in testis as a pharmacological target: from disease to contraception.Curr Mol Pharmacol. 2014; 7: 83-95Crossref PubMed Scopus (28) Google Scholar, 11Dimitriadis F. Tsiampali C. Chaliasos N. Tsounapi P. Takenaka A. Sofikitis N. The Sertoli cell as the orchestra conductor of spermatogenesis: spermatogenic cells dance to the tune of testosterone.Hormones (Athens). 2015; 14: 479-503PubMed Google Scholar whereas high lactate production in SCs is in turn underpinned by their unusual carbohydrate metabolism; they present a high glycolytic flux where the majority of glucose is converted into lactate and not oxidized via the citric acid cycle.11Dimitriadis F. Tsiampali C. Chaliasos N. Tsounapi P. Takenaka A. Sofikitis N. The Sertoli cell as the orchestra conductor of spermatogenesis: spermatogenic cells dance to the tune of testosterone.Hormones (Athens). 2015; 14: 479-503PubMed Google Scholar This metabolic behavior of SCs is consistent with Otto Warburg's observations in cancer cells.6Rato L. Meneses M.J. Silva B.M. Sousa M. Alves M.G. Oliveira P.F. New insights on hormones and factors that modulate Sertoli cell metabolism.Histol Histopathol. 2016; 31: 499-513PubMed Google Scholar, 11Dimitriadis F. Tsiampali C. Chaliasos N. Tsounapi P. Takenaka A. Sofikitis N. The Sertoli cell as the orchestra conductor of spermatogenesis: spermatogenic cells dance to the tune of testosterone.Hormones (Athens). 2015; 14: 479-503PubMed Google Scholar, 12Oliveira P.F. Martins A.D. Moreira A.C. Cheng C.Y. Alves M.G. The Warburg effect revisited--lesson from the Sertoli cell.Med Res Rev. 2015; 35: 126-151Crossref PubMed Scopus (116) Google Scholar Any factor that impairs or supports SC viability (and hence, SC number), and/or functionality, may have profound effects on spermatogenesis.13Syed V. Hecht N.B. Disruption of germ cell-Sertoli cell interactions leads to spermatogenic defects.Mol Cell Endocrinol. 2002; 186: 155-157Crossref PubMed Scopus (29) Google Scholar Glyoxalase 1 (Glo1) is the major detoxifying enzyme of methylglyoxal (MG),14Sousa Silva M. Gomes R.A. Ferreira A.E. Ponces Freire A. Cordeiro C. 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High glucose increases angiopoietin-2 transcription in microvascular endothelial cells through methylglyoxal modification of mSin3A.J Biol Chem. 2007; 282: 31038-31045Crossref PubMed Scopus (195) Google Scholar Hydroimidazolone (MG-H1) and argpyrimidine (ArgPyr) are two such AGEs derived from the spontaneous reaction of MG with protein arginine residues.15Antognelli C. Talesa V.N. Glyoxalases in urological malignancies.Int J Mol Sci. 2018; 19: E415Crossref PubMed Scopus (38) Google Scholar, 20Rabbani N. Thornalley P.J. Methlylgyloxal, glyoxalase 1 and the dicarbonyl proteome.Amino Acids. 2012; 42: 1133-1142Crossref PubMed Scopus (298) Google Scholar, 21Shipanova I.N. Glomb M.A. Nagaraj R.H. Protein modification by methylglyoxal: chemical nature and synthetic mechanism of a major fluorescent adduct.Arch Biochem Biophys. 1997; 344: 29-36Crossref PubMed Scopus (243) Google Scholar Several studies have demonstrated that MG-induced carbonyl stress can affect many biological processes in human cells,22Choi E.M. Suh K.S. Rhee S.Y. Kim Y.S. Sciadopitysin alleviates methylglyoxal-mediated glycation in osteoblastic MC3T3-E1 cells by enhancing glyoxalase system and mitochondrial biogenesis.Free Radic Res. 2014; 48: 729-739Crossref PubMed Scopus (13) Google Scholar, 23Antognelli C. Cecchetti R. Riuzzi F. Peirce M.J. Talesa V.N. Glyoxalase 1 sustains the metastatic phenotype of prostate cancer cells via EMT control.J Cell Mol Med. 2018; 22: 2865-2883Crossref PubMed Scopus (35) Google Scholar, 24Rabbani N. Thornalley P.J. Glyoxalase in diabetes, obesity and related disorders.Semin Cell Dev Biol. 2011; 22: 309-317Crossref PubMed Scopus (188) Google Scholar including apoptosis.25Antognelli C. Mezzasoma L. Fettucciari K. Mearini E. Talesa V.N. Role of glyoxalase I in the proliferation and apoptosis control of human LNCaP and PC3 prostate cancer cells.Prostate. 2013; 73: 121-132Crossref PubMed Scopus (41) Google Scholar, 26Antognelli C. Mezzasoma L. Fettucciari K. Talesa V.N. A novel mechanism of methylglyoxal cytotoxicity in prostate cancer cells.Int J Biochem Cell Biol. 2013; 45: 836-844Crossref PubMed Scopus (59) Google Scholar, 27Antognelli C. Palumbo I. Aristei C. Talesa V.N. Glyoxalase I inhibition induces apoptosis in irradiated MCF-7 cells via a novel mechanism involving Hsp27, p53 and NF-κB.Br J Cancer. 2014; 111: 395-406Crossref PubMed Scopus (48) Google Scholar, 28Antognelli C. Gambelunghe A. Talesa V.N. Muzi G. Reactive oxygen species induce apoptosis in bronchial epithelial BEAS-2B cells by inhibiting the antiglycation glyoxalase I defence: involvement of superoxide anion, hydrogen peroxide and NF-κB.Apoptosis. 2014; 19: 102-116Crossref PubMed Scopus (37) Google Scholar, 29Antognelli C. Gambelunghe A. Muzi G. Talesa V.N. Peroxynitrite-mediated glyoxalase I epigenetic inhibition drives apoptosis in airway epithelial cells exposed to crystalline silica via a novel mechanism involving argpyrimidine-modified Hsp70, JNK, and NF-κB.Free Radic Biol Med. 2015; 84: 128-141Crossref PubMed Scopus (28) Google Scholar, 30Antognelli C. Trapani E. Delle Monache S. Perrelli A. Daga M. Pizzimenti S. Barrera G. Cassoni P. Angelucci A. Trabalzini L. Talesa V.N. Goitre L. Retta S.F. KRIT1 loss-of-function induces a chronic Nrf2-mediated adaptive homeostasis that sensitizes cells to oxidative stress: implication for Cerebral Cavernous Malformation disease.Free Radic Biol Med. 2018; 115: 202-218Crossref PubMed Scopus (58) Google Scholar, 31Antognelli C. Trapani E. Delle Monache S. Perrelli A. Fornelli C. Retta F. Cassoni P. Talesa V.N. Retta S.F. Data in support of sustained upregulation of adaptive redox homeostasis mechanisms caused by KRIT1 loss-of-function.Data Brief. 2017; 16: 929-938Crossref PubMed Scopus (29) Google Scholar, 32Marinucci L. Balloni S. Fettucciari K. Bodo M. Talesa V.N. Antognelli C. Nicotine induces apoptosis in human osteoblasts via a novel mechanism driven by H2O2 and entailing Glyoxalase 1-dependent MG-H1 accumulation leading to TG2-mediated NF-κB desensitization: implication for smokers-related osteoporosis.Free Radic Biol Med. 2018; 117: 6-17Crossref PubMed Scopus (56) Google Scholar By using the cofactor glutathione (GSH), Glo1 prevents the accumulation of MG, thereby suppressing dicarbonyl-mediated glycation reactions. More importantly, Glo1 overexpression decreases accumulation of AGEs in Caenorhabditis elegans,33Morcos M. Du X. Pfisterer F. Hutter H. Sayed A.A. Thornalley P. Ahmed N. Baynes J. Thorpe S. Kukudov G. Schlotterer A. Bozorgmehr F. El Baki R.A. Stern D. Moehrlen F. Ibrahim Y. Oikonomou D. Hamann A. Becker C. Zeier M. Schwenger V. Miftari N. Humpert Hammes H.P. Buechler M. Bierhaus A. Brownlee M. Nawroth P.P. Glyoxalase-1 prevents mitochondrial protein modification and enhances lifespan in Caenorhabditis elegans.Aging Cell. 2008; 7: 260-269Crossref PubMed Scopus (238) Google Scholar diabetic rats,34Brouwers O. Niessen P.M. Ferreira I. Miyata T. Scheffer P.G. Teerlink T. Schrauwen P. Brownlee M. Stehouwer C.D. Schalkwijk C.G. Overexpression of glyoxalase-I reduces hyperglycemia-induced levels of advanced glycation end products and oxidative stress in diabetic rats.J Biol Chem. 2011; 286: 1374-1380Crossref PubMed Scopus (177) Google Scholar and human cells,29Antognelli C. Gambelunghe A. Muzi G. Talesa V.N. Peroxynitrite-mediated glyoxalase I epigenetic inhibition drives apoptosis in airway epithelial cells exposed to crystalline silica via a novel mechanism involving argpyrimidine-modified Hsp70, JNK, and NF-κB.Free Radic Biol Med. 2015; 84: 128-141Crossref PubMed Scopus (28) Google Scholar, 32Marinucci L. Balloni S. Fettucciari K. Bodo M. Talesa V.N. Antognelli C. Nicotine induces apoptosis in human osteoblasts via a novel mechanism driven by H2O2 and entailing Glyoxalase 1-dependent MG-H1 accumulation leading to TG2-mediated NF-κB desensitization: implication for smokers-related osteoporosis.Free Radic Biol Med. 2018; 117: 6-17Crossref PubMed Scopus (56) Google Scholar and Glo1 knockdown induces the intracellular accumulation of MG-derived AGEs.25Antognelli C. Mezzasoma L. Fettucciari K. Mearini E. Talesa V.N. Role of glyoxalase I in the proliferation and apoptosis control of human LNCaP and PC3 prostate cancer cells.Prostate. 2013; 73: 121-132Crossref PubMed Scopus (41) Google Scholar, 26Antognelli C. Mezzasoma L. Fettucciari K. Talesa V.N. A novel mechanism of methylglyoxal cytotoxicity in prostate cancer cells.Int J Biochem Cell Biol. 2013; 45: 836-844Crossref PubMed Scopus (59) Google Scholar, 27Antognelli C. Palumbo I. Aristei C. Talesa V.N. Glyoxalase I inhibition induces apoptosis in irradiated MCF-7 cells via a novel mechanism involving Hsp27, p53 and NF-κB.Br J Cancer. 2014; 111: 395-406Crossref PubMed Scopus (48) Google Scholar, 32Marinucci L. Balloni S. Fettucciari K. Bodo M. Talesa V.N. Antognelli C. Nicotine induces apoptosis in human osteoblasts via a novel mechanism driven by H2O2 and entailing Glyoxalase 1-dependent MG-H1 accumulation leading to TG2-mediated NF-κB desensitization: implication for smokers-related osteoporosis.Free Radic Biol Med. 2018; 117: 6-17Crossref PubMed Scopus (56) Google Scholar, 35Antognelli C. Gambelunghe A. Muzi G. Talesa V.N. Glyoxalase I drives epithelial-to-mesenchymal transition via argpyrimidine-modified Hsp70, miR-21 and SMAD signalling in human bronchial cells BEAS-2B chronically exposed to crystalline silica Min-U-Sil 5: transformation into a neoplastic-like phenotype.Free Radic Biol Med. 2016; 92: 110-125Crossref PubMed Scopus (27) Google Scholar Therefore, Glo1, by preventing the onset of MG-dependent carbonyl stress leading to apoptosis, is a key antiglycation defense that sustains cell viability. Despite this, the role of Glo1 in the context of infertility remains underinvestigated, and those studies that have been done have been mainly limited to female infertility.36Tatone C. Carbone M.C. Campanella G. Festuccia C. Artini P.G. Talesa V. Focarelli R. Amicarelli F. Female reproductive dysfunction during ageing: role of methylglyoxal in the formation of advanced glycation endproducts in ovaries of reproductively-aged mice.J Biol Regul Homeost Agents. 2010; 24: 63-72PubMed Google Scholar, 37Tatone C. Eichenlaub-Ritter U. Amicarelli F. Dicarbonyl stress and glyoxalases in ovarian function.Biochem Soc Trans. 2014; 42: 433-438Crossref PubMed Scopus (27) Google Scholar, 38Tatone C. Heizenrieder T. Di Emidio G. Treffon P. Amicarelli F. Seidel T. Eichenlaub-Ritter U. Evidence that carbonyl stress by methylglyoxal exposure induces DNA damage and spindle aberrations, affects mitochondrial integrity in mammalian oocytes and contributes to oocyte ageing.Hum Reprod. 2011; 26: 1843-1859Crossref PubMed Scopus (61) Google Scholar However, one proteomic study, more than 10 years ago, identified Glo1 among the antigens expressed in prostasomes of infertile men, suggesting Glo1 as a target for naturally occurring sperm agglutinating antibodies, a well-known cause of infertility.39Carlsson L. Ronquist G. Nilsson B.O. Larsson A. Dominant prostasome immunogens for sperm-agglutinating autoantibodies of infertile men.J Androl. 2004; 25: 699-705Crossref PubMed Scopus (39) Google Scholar In the present study, the authors attempted to shed further light on the role of Glo1 in male infertility, focusing on SCs because their biology remains poorly understood and because they have a primary role in spermatogenesis.3Kaur G. Thompson L.A. Dufour J.M. Sertoli cells: immunological sentinels of spermatogenesis.Semin Cell Dev Biol. 2014; 30: 36-44Crossref PubMed Scopus (149) Google Scholar, 4França L.R. Hess R.A. Dufour J.M. Hofmann M.C. Griswold M.D. The Sertoli cell: one hundred fifty years of beauty and plasticity.Andrology. 2016; 4: 189-212Crossref PubMed Scopus (231) Google Scholar In particular, whether and how Glo1 might be involved in sustaining the viability of SCs isolated from porcine neonatal pre-pubertal pig testes cotreated with testosterone (T) and follicle-stimulating hormone (FSH) was investigated, focusing on MG-derived AGEs and NF-κB signaling. The reagents included: T (Sigma-Aldrich, St. Louis, MO); FSH (GONAL-f; Merck Serono, Rome, Italy); Laemmli buffer (Invitrogen, Milan, Italy); Roti-Block (Carl Roth, Karsruhe, Germany); BCA Assay kit (Pierce; Thermo Fisher Scientific, Waltham, MA), and aminoguanidine bicarbonate (AG) (Sigma-Aldrich, Milan, Italy). The antibodies used in this study included: mouse anti-Glo1 (D6), rabbit anti–Bcl-XL, rabbit anti-Bax (N20), mouse anti–β-actin (DBA Italia, Milan, Italy); mouse anti-ArgPyr (Antibodies-online, Aachen, Germany); mouse anti–MG-H1 (STA-011; Cell Biolabs, DBA Italia); rabbit anti–caspase-3, rabbit anti–phospho-IkBα (Ser32) (14D4) (Cell Signaling Technology, Milan, Italy); mouse anti–Bcl-2 (Dako, Milan, Italy); mouse anti-cytochrome c (Cyt c) (BD Pharmingen, Milan, Italy); mouse anti-Cyt c oxidase subunit IV (Cox IV) (Molecular Probes, Monza, Italy). Studies with animals were conducted in agreement with national (Italian Approved Animal Welfare Assurance A-3143-01) and local (University of Perugia Animal Care and Use Committee) guidelines. Large white neonatal pigs (15 to 20 days old) underwent bilateral orchidectomy after general anesthesia with ketamine (Ketavet 100; Intervet, Milan, Italy), at a dose of 40 mg/kg, and dexmedetomidine (Dexdomitor, Orion Corporation, Finland), at a dose of 40 g/kg, and were used as SC donors. Specifically, pure porcine pre-pubertal SCs were isolated, exhaustively characterized, and tested for functional competence according to previously established methods.40Luca G. Mancuso F. Calvitti M. Arato I. Falabella G. Bufalari A. De Monte V. Tresoldi E. Nastruzzi C. Basta G. Fallarino F. Lilli C. Bellucci C. Baroni T. Aglietti M.C. Giovagnoli S. Cameron D.F. Bodo M. Calafiore R. Long-term stability, functional competence, and safety of microencapsulated specific pathogen-free neonatal porcine Sertoli cells: a potential product for cell transplant therapy.Xenotransplantation. 2015; 22: 273-283Crossref PubMed Scopus (23) Google Scholar, 41Mancuso F. Arato I. Lilli C. Bellucci C. Bodo M. Calvitti M. Aglietti M.C. dell'Omo M. Nastruzzi C. Calafiore R. Luca G. Marinucci L. Acute effects of lead on porcine neonatal Sertoli cells in vitro.Toxicol in Vitro. 2018; 48: 45-52Crossref PubMed Scopus (27) Google Scholar, 42Arato I. Luca G. Mancuso F. Bellucci C. Lilli C. Calvitti M. Hansen B.C. Milardi D. Grande G. Calafiore R. An in vitro prototype of a porcine biomimetic testis-like cell culture system: a novel tool for the study of reassembled Sertoli and Leydig cells.Asian J Androl. 2018; 20: 160-165Crossref PubMed Google Scholar SCs were stimulated with FSH (1 mg/mL of α-follitropin) for 48 hours; thereafter, 0.2 mg/mL of testosterone enanthate was added for 8 hours, as previously described.41Mancuso F. Arato I. Lilli C. Bellucci C. Bodo M. Calvitti M. Aglietti M.C. dell'Omo M. Nastruzzi C. Calafiore R. Luca G. Marinucci L. Acute effects of lead on porcine neonatal Sertoli cells in vitro.Toxicol in Vitro. 2018; 48: 45-52Crossref PubMed Scopus (27) Google Scholar, 42Arato I. Luca G. Mancuso F. Bellucci C. Lilli C. Calvitti M. Hansen B.C. Milardi D. Grande G. Calafiore R. An in vitro prototype of a porcine biomimetic testis-like cell culture system: a novel tool for the study of reassembled Sertoli and Leydig cells.Asian J Androl. 2018; 20: 160-165Crossref PubMed Google Scholar Exposure of SCs to FSH and T was deemed necessary to mimic the physiological condition whereby both hormones are essential for adequate spermatogenesis.11Dimitriadis F. Tsiampali C. Chaliasos N. Tsounapi P. Takenaka A. Sofikitis N. The Sertoli cell as the orchestra conductor of spermatogenesis: spermatogenic cells dance to the tune of testosterone.Hormones (Athens). 2015; 14: 479-503PubMed Google Scholar, 42Arato I. Luca G. Mancuso F. Bellucci C. Lilli C. Calvitti M. Hansen B.C. Milardi D. Grande G. Calafiore R. An in vitro prototype of a porcine biomimetic testis-like cell culture system: a novel tool for the study of reassembled Sertoli and Leydig cells.Asian J Androl. 2018; 20: 160-165Crossref PubMed Google Scholar Untreated cells, as well cells treated with FSH or T singly, were used as controls. RNA isolation, reverse transcription, and real-time quantitative PCR analyses were performed as previously described.25Antognelli C. Mezzasoma L. Fettucciari K. Mearini E. Talesa V.N. Role of glyoxalase I in the proliferation and apoptosis control of human LNCaP and PC3 prostate cancer cells.Prostate. 2013; 73: 121-132Crossref PubMed Scopus (41) Google Scholar, 26Antognelli C. Mezzasoma L. Fettucciari K. Talesa V.N. A novel mechanism of methylglyoxal cytotoxicity in prostate cancer cells.Int J Biochem Cell Biol. 2013; 45: 836-844Crossref PubMed Scopus (59) Google Scholar, 27Antognelli C. Palumbo I. Aristei C. Talesa V.N. Glyoxalase I inhibition induces apoptosis in irradiated MCF-7 cells via a novel mechanism involving Hsp27, p53 and NF-κB.Br J Cancer. 2014; 111: 395-406Crossref PubMed Scopus (48) Google Scholar, 28Antognelli C. Gambelunghe A. Talesa V.N. Muzi G. Reactive oxygen species induce apoptosis in bronchial epithelial BEAS-2B cells by inhibiting the antiglycation glyoxalase I defence: involvement of superoxide anion, hydrogen peroxide and NF-κB.Apoptosis. 2014; 19: 102-116Crossref PubMed Scopus (37) Google Scholar, 29Antognelli C. Gambelunghe A. Muzi G. Talesa V.N. Peroxynitrite-mediated glyoxalase I epigenetic inhibition drives apoptosis in airway epithelial cells exposed to crystalline silica via a novel mechanism involving argpyrimidine-modified Hsp70, JNK, and NF-κB.Free Radic Biol Med. 2015; 84: 128-141Crossref PubMed Scopus (28) Google Scholar Briefly, total cellular RNA was isolated using TRIzol Reagent (Life Technologies Italia, Milan, Italy). Using 1 μg of total RNA, cDNA was then synthesized using the RevertAid H Minus First Strand cDNA Synthesis Kit (Life Technologies Italia). The expression of target genes relative to a reference gene (Actb) was evaluated by RT-qPCR, using SYBR Green biochemistry, on a MX3000P Real-Time PCR System (Agilent Technology, Milan, Italy). The sequences of oligonucleotide primers used were as follows: Glo1: 5′-CTCTCCAGAAAAGCTACACTTTGAG-3′ (sense, 400 nmol/L), 5′-CGAGGGTCTGAATTGCCATTG-3′ (antisense, 400 nmol/L); Actb: 5′-CACTCTTCCAGCCTTCCTTCC-3′ (sense, 600 nmol/L), 5′-ACAGCACTGTGTTGGCGTAC-3′ (antisense, 600 nmol/L). The thermal cycling conditions were as follows: 1 cycle at 95°C for 10 minutes, followed by 45 cycles at 95°C for 20 seconds and 60°C for 1 minute. Data for comparative analysis of gene expression were obtained using the 2(−ΔΔCT) method.43Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real time quantitative PCR and the 2_ΔΔCT method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (123265) Google Scholar Total protein extracts were prepared by lysing the cells (4°C) in radioimmunoprecipitation assay lysis buffer.29Antognelli C. Gambelunghe A. Muzi G. Talesa V.N. Peroxynitrite-mediated glyoxalase I epigenetic inhibition drives apoptosis in airway epithelial cells exposed to crystalline silica via a novel mechanism involving argpyrimidine-modified Hsp70, JNK, and NF-κB.Free Radic Biol Med. 2015; 84: 128-141Crossref PubMed Scopus (28) Google Scholar For subcellular fractionation, cells were resuspended in Mitobuffer.29Antognelli C. Gambelunghe A. Muzi G. Talesa V.N. Peroxynitrite-mediated glyoxalase I epigenetic inhibition drives apoptosis in airway epithelial cells exposed to crystalline silica via a novel mechanism involving argpyrimidine-modified Hsp70, JNK, and NF-κB.Free Radic Biol Med. 2015; 84: 128-141Crossref PubMed Scopus (28) Google Scholar Protein concentration in cell extracts was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), by reference to a standard curve prepared using dilutions of bovine serum albumin. For Western blot analysis, samples (containing 30 μg total protein), in Laemmli buffer, were boiled for 5 minutes, resolved on 10%, 12%, or 15% SDS-PAGE, and then transferred (iBlot Dry Blotting System; Invitrogen) to nitrocellulose membranes. Membranes were blocked in Roti-Block for 1 hour at room temperature, incubated overnight at 4°C with an appropriate dilution of the indicated primary antibody, washed with Tris-buffered saline/Tween, before being incubated (1 hour at room temperature) with the appropriate horseradish peroxidase-conjugated secondary antibody and visualized using the ECL system (Microtech, Naples, Italy). As internal loading controls, all membranes were subsequently stripped of the first antibody in a stripping buffer [100 mmol/L 2-mercaptoethanol, 2% SDS, and 62.5 mmol/L Tris-HCl (pH 6.8)] and reprobed with the appropriate housekeeping antibody. The activity of Glo1 was assessed as previously described in lysates from cell lines.28Antognelli C. Gambelunghe A. Talesa V.N. Muzi G. Reactive oxygen species induce apoptosis in bronchial epithelial BEAS-2B cells by inhibiting the antiglycation glyoxalase I defence: in

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