Abstract
Although chronic ethanol consumption results in Sertoli cell vacuolization and augmented testicular germ cell apoptosis via death receptor and mitochondrial pathways, Sertoli cells are resistant to apoptosis. The aim of this study was to examine whether the activation of autophagy in the Sertoli cells of ethanol-treated rats (ETR) may have a role in their survival. Adult Wistar rats were fed either 5% ethanol in Lieber–DeCarli liquid diet or an isocaloric control diet for 12 weeks. The TUNEL method demonstrated that Sertoli cells were always TUNEL-negative despite the presence of many apoptotic germ cells in ETR, supporting our previous studies. Electron microscopy revealed the presence of large numbers of autophagic vacuoles (AVs) in Sertoli cells of ETR compared to few AVs in control testes. Most of the AVs in Sertoli cells of ETR enveloped and sequestered damaged and abnormally shaped mitochondria, without cytoplasm, indicating mitochondrial autophagy (mitophagy). Immuno-electron microscopy showed the localization of LC3, a specific marker of early AVs (autophagosomes), around AVs sequestering mitochondria in Sertoli cells of ETR. Immunohistochemical staining of LC3 demonstrated a punctate pattern in Sertoli cells of ETR, confirming the formation of autophagosomes, while LC3 puncta were almost absent in control testes. Moreover, increased immunoreactivity of LAMP-2, a lysosomal membrane protein and marker of late AVs (autolysosomes), was mainly observed in Sertoli cells of ETR, with weaker expression in control testes. Via the deletion of pro-apoptotic damaged mitochondria, enhanced Sertoli cell mitophagy in ETR may be an anti-apoptotic mechanism that is essential for spermatogenesis.
Similar content being viewed by others
References
Anderson RA Jr, Berryman SH, Phillips JF, Feathergill KA, Zaneveld LJ, Russell LD (1989) Biochemical and structural evidence for ethanol-induced impairment of testicular development: apparent lack of Leydig cell involvement. Toxic Appl Pharmacol 100:62–85
Aumüller G, Renneberg H, Hasilik A (1997) Distribution and subcellular localization of a lysosome-associated protein in human genital organs. Cell Tissue Res 287:335–342
Barth S, Glick D, Macleod KF (2010) Autophagy: assays and artifacts. J Pathol 221:117–124
Bozec A, Chuzel F, Chater S, Paulin C, Bars R, Benahmed M, Mauduit C (2004) The mitochondrial-dependent pathway is chronically affected in testicular germ cell death in adult rats exposed in utero to anti-androgens. J Endocrinol 183:79–90
Chemes H (1986) The phagocytic function of Sertoli cells: a morphological, biochemical, and endocrinological study of lysosomes and acid phosphatase localization in the rat testis. Endocrinol 119:1673–1681
Chetty-Raju N, Cook R, Erber WN (2004) Vacuolated neutrophils in ethanol toxicity. Br J Haematol 127:478
Cordero MD, De Miguel M, Moreno Fernández AM, Carmona López IM, Garrido Maraver J, Cotán D, Gómez Izquierdo L, Bonal P, Campa F, Bullon P, Navas P, Sánchez Alcázar JA (2010) Mitochondrial dysfunction and mitophagy activation in blood mononuclear cells of fibromyalgia patients: implications in the pathogenesis of the disease. Arthritis Res Ther 12:R17. doi:10.1186/ar2918
Czaja MJ (2011) Functions of autophagy in hepatic and pancreatic physiology and disease. Gastroenterol 140:1895–1908
Dagda RK, Zhu J, Kulich SM, Chu CT (2008) Mitochondrially localized ERK2 regulates mitophagy and autophagic cell stress: implications for Parkinson’s disease. Autophagy 4:770–782
Ding WX, Li M, Chen X, Ni HM, Lin CW, Gao W, Lu B, Stolz DB, Clemens DL, Yin XM (2010) Autophagy reduces acute ethanol-induced hepatotoxicity and steatosis in mice. Gastroenterol 139:1740–1752
Ding WX, Li M, Yin XM (2011a) Selective taste of ethanol-induced autophagy for mitochondria and lipid droplets. Autophagy 7:248–249
Ding WX, Manley S, Ni HM (2011b) The emerging role of autophagy in alcoholic liver disease. Exp Biol Med 236:546–556
Eid NAS, Ito Y, Shibata MA, Kusakabe K, Abe H, Li Z, Otsuki Y (2001) Sertoli cell injury in ethanol-treated rats: novel roles for apoptosis-related genes. J Androl 176 (May/June Suppl) (Abstract)
Eid NA, Shibata MA, Ito Y, Kusakabe K, Hammad H, Otsuki Y (2002) Involvement of Fas system and active caspases in apoptotic signalling in testicular germ cells of ethanol-treated rats. Int J Androl 25:159–167
Eid N, Ito Y, Otsuki Y (2011) Involvement of inducible nitric oxide synthase in DNA fragmentation in various testicular germ cells of ethanol-treated rats. Journal of Men’s Health 8:s36–s40
Eskelinen EL, Illert AL, Tanaka Y, Schwarzmann G, Blanz J, Von Figura K, Saftig P (2002) Role of LAMP-2 in lysosome biogenesis and autophagy. Mol Biol Cell 13:3355–3368
Fortunato F, Burgers H, Bergmann F, Rieger P, Büchler MW, Kroemer G, Werner J (2009) Impaired autolysosome formation correlates with Lamp-2 depletion: role of apoptosis, autophagy, and necrosis in pancreatitis. Gastroenterol 137:350–360
Fujii S, Mitsunaga S, Yamazaki M, Hasebe T, Ishii G, Kojima M, Kinoshita T, Ueno T, Esumi H, Ochiai A (2008) Autophagy is activated in pancreatic cancer cells and correlates with poor patient outcome. Cancer Sci 99:1813–1819
Ge W, Ren J (2011) MTOR-STAT3-notch signaling contributes to ALDH2-induced protection against cardiac contractile dysfunction and autophagy under alcoholism. J Cell Mol Med. doi:10.1111/j.1582-4934.2011.01347.x
Gorbunov NV, Kiang JG (2009) Up-regulation of autophagy in small intestine Paneth cells in response to total-body gamma-irradiation. J Pathol 219:242–245
Hoek JB, Cahill A, Pastorino JG (2002) Alcohol and mitochondria: a dysfunctional relationship. Gastroenterol 122:2049–2063
Holt SV, Wyspianska B, Randall KJ, James D, Foster JR, Wilkinson RW (2011) The development of an immunohistochemical method to detect the autophagy-associated protein LC3-II in human tumor xenografts. Toxicol Pathol 39:516–523
Houwerzijl EJ, Pol HW, Blom NR, van der Want JJ, de Wolf JT, Vellenga E (2009) Erythroid precursors from patients with low-risk myelodysplasia demonstrate ultrastructural features of enhanced autophagy of mitochondria. Leukemia 23:886–889
Hu JH, Jiang J, Ma YH, Yang N, Zhang MH, Wu M, Fei J, Guo LH (2003) Enhancement of germ cell apoptosis induced by ethanol in transgenic mice over expressing Fas-Ligand. Cell Res 13:361–367
Jana K, Jana N, De DK, Guha SK (2010) Ethanol induces mouse spermatogenic cell apoptosis in vivo through over-expression of Fas/Fas-L, p53, and caspase-3 along with cytochrome c translocation and glutathione depletion. Mol Reprod Dev 77:820–833
Jia G, Cheng G, Gangahar DM, Agrawal DK (2006) Insulin-like growth factor-1 and TNF-alpha regulate autophagy through c-jun N-terminal kinase and Akt pathways in human atherosclerotic vascular smooth cells. Immunol Cell Biol 84:448–454
Kanter M, Topcu-Tarladacalisir Y, Parlar S (2010) Antiapoptotic effect of L-carnitine on testicular irradiation in rats. J Mol Histol 41:121–128
Kanthasamy A, Anantharam V, Ali SF, Kanthasamy AG (2006) Methamphetamine induces autophagy and apoptosis in a mesencephalic dopaminergic neuronal culture model: role of cathepsin-D in methamphetamine-induced apoptotic cell death. Ann N Y Acad Sci 1074:234–244
Kim J-M, Luo L, Zirkin BR (2000) Caspase-3 activation is required for Leydig cell apoptosis induced by ethane dimethanesulfonate. Endocrinol 141:1846–1853
Kim I, Rodriguez-Enriquez S, Lemasters JJ (2007a) Selective degradation of mitochondria by mitophagy. Arch Biochem Biophys 462:245–253
Kim SK, Yoon YD, Park YS, Seo JT, Kim JH (2007b) Involvement of the Fas–Fas ligand system and active caspase-3 in abnormal apoptosis in human testes with maturation arrest and Sertoli cell-only syndrome. Fertil Steril 87:547–553
Klionsky DJ, Abeliovich H, Agostinis P et al (2008) Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4:151–175
Kroemer G, Mariño G, Levine B (2010) Autophagy and the integrated stress response. Mol Cell 40:280–293
Lee J, Richburg JH, Shipp EB, Meistrich ML, Boekelheide K (1999) The Fas system, a regulator of testicular germ cell apoptosis, is differentially up-regulated in Sertoli cell versus germ cell injury of the testis. Endocrinology 140:852–858
Lieber CS, De Carli LM, Sorrell MF (1989) Experimental methods of ethanol administration. Hepatol 10:501–510
Mitsuhashi S, Hatakeyama H, Karahashi M, Koumura T, Nonaka I, Hayashi YK, Noguchi S, Sher RB, Nakagawa Y, Manfredi G, Goto Y, Cox GA, Nishino I (2011) Muscle choline kinase beta defect causes mitochondrial dysfunction and increased mitophagy. Hum Mol Genet 20:3841–3845
Mizushima N, Yamamoto A, Matsui M, Yoshimori T, Ohsumi Y (2004) In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell 15:1101–1111
Mizushima N, Yoshimori T, Levine B (2010) Methods in mammalian autophagy research. Cell 140:313–326
Nahdi A, Hammami I, Kouidhi W, Chargui A, Ben Ammar A, Hamdaoui MH, El May A, El May M (2010) Protective effects of crude garlic by reducing iron-mediated oxidative stress, proliferation and autophagy in rats. J Mol Histol 41:233–245
Obert LA, Sobocinski GP, Bobrowski WF, Metz AL, Rolsma MD, Altrogge DM, Dunstan RW (2007) An immunohistochemical approach to differentiate hepatic lipidosis from hepatic phospholipidosis in rats. Toxicol Pathol 35:728–734
Osna NA, Thomes PG, Jr TM (2011) Involvement of autophagy in alcoholic liver injury and hepatitis C pathogenesis. World J Gastroenterol 17:2507–2514
Pajarinen JT, Karhunen PJ (1994) Spermatogenic arrest and ‘Sertoli cell-only syndrome’–common alcohol-induced disorders of the human testis. Int J Androl 17:292–299
Pajarinen JT, Karhunen PJ, Savolainen V, Lalu K, Penttilä A, Laippala P (1996) Moderate alcohol consumption and disorders of human spermatogenesis. Alcohol Clin Exp Res 20:332–337
Pan T, Rawal P, Wu Y, Xie W, Jankovic J, Le W (2009) Rapamycin protects against rotenone-induced apoptosis through autophagy induction. Neuroscience 164:541–551
Pflieger-Bruss S, Schuppe HC, Schill WB (2004) The male reproductive system and its susceptibility to endocrine disrupting chemicals. Andrologia 36:337–345
Rao AV, Shaha C (2000) Role of glutathione S-transferase in oxidative stress-induced male germ cell apoptosis. Free Radic Biol Med 29:1015–1027
Rosenblum E, Gavaler JS, Van Thiel DH (1985) Lipid peroxidation: a mechanism for ethanol-associated testicular injury in rats. Endocrinol 116:311–318
Russell LD, Ettlin RA, Sinha Hikim AP, Clegg ED (1990) Histological and histopathological evaluation of the testis. Cache River Press, Clearwater
Sansanwal P, Yen B, Gahl WA, Ma Y, Ying L, Wong LJ, Sarwal MM (2010) Mitochondrial autophagy promotes cellular injury in nephropathic cystinosis. J Am Soc Nephrol 21:272–283
Shintaku M (2011) Immunohistochemical localization of autophagosomal membrane-associated protein LC3 in granular cell tumor and schwannoma. Virchows Arch doi:10.1007/s00428-011-1104-z
Shirai T, Ikemoto I (1992) Mechanism of alcoholic testicular damage. Nippon Hinyoukika Gakkai Zasshi 83:305–314
Stiblar-Martincic D (2009) Morphometrical evaluation of germ cell apoptosis in infertile men. Folia Biol 55:233–237
Teckman JH, An JK, Blomenkamp K, Schmidt B, Perlmutter D (2004) Mitochondrial autophagy and injury in the liver in alpha 1-antitrypsin deficiency. Am J Physiol Gastrointest Liver Physiol 286:G851–G862
Tolkovsky AM (2009) Mitophagy. Biochim Biophys Acta 1793:1508–1515
Van Thiel DH (1983) Ethanol: its adverse effects upon the hypothalamic-pituitary-gonadal axis. J Lab Clin Med 101:21–33
Vives-Bauza C, Zhou C, Huang Y, Cui M, de Vries RL, Kim J, May J, Tocilescu MA, Liu W, Ko HS, Magrané J, Moore DJ, Dawson VL, Grailhe R, Dawson TM, Li C, Tieu K, Przedborski S (2010) PINK1-dependent recruitment of Parkin to mitochondria in mitophagy. Proc Natl Acad Sci 107:378–383
Watanabe E, Muenzer JT, Hawkins WG, Davis CG, Dixon DJ, McDunn JE, Brackett DJ, Lerner MR, Swanson PE, Hotchkiss RS (2009) Sepsis induces extensive autophagic vacuolization in hepatocytes: a clinical and laboratory-based study. Lab Invest 89:549–561
Wong TW, Strauss FH, Foster LV (1988) Cytoplasmic granular change of Sertoli cells in two cases of Sertoli-cell-only syndrome. Arch Pathol Lab Med 112:200–205
Yan W, Samson M, Jegou B, Toppari J (2000a) Bcl-w forms complexes with Bax and Bak, and elevated ratios of Bax/Bcl-w and Bak/Bcl-w correspond to spermatogonial and spermatocyte apoptosis in the testis. Mol Endocrinol 14:682–699
Yan W, Suominen J, Samson M, Jegou B, Toppari J (2000b) Involvement of Bcl-2 family proteins in germ cell apoptosis during testicular development in the rat and pro-survival effect of stem cell factor on germ cells in vitro. Molecular Cellular Endocrinol 165:115–129
Yang DS, Lee JH, Nixon RA (2009) Monitoring autophagy in Alzheimer’s disease and related neurodegenerative diseases. Methods Enzymol 453:111–114
Yla-Anttila P, Vihinen H, Jokitalo E, Eskelinen EL (2009) Monitoring autophagy by electron microscopy in Mammalian cells. Methods Enzymol 452:143–164
Youle RJ, Narendra DP (2011) Mechanisms of mitophagy. Nat Rev Mol Cell Biol 12:9–14
Zhu Q, Van Thiel DH, Gavaler JS (1997) Effects of ethanol on rat Sertoli cell function: studies in vitro and in vivo. Alc Clin Exp Res 21:1409–1417
Zhu Q, Emanuele MA, LaPaglia N, Kovacs EJ, Emanuele NV (2007) Vitamin E prevents ethanol-induced inflammatory, hormonal, and cytotoxic changes in reproductive tissues. Endocrine 32:59–68
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
10735_2011_9372_MOESM1_ESM.tif
Supplementary Fig. 1 Enhanced mitophagy in Sertoli cell of ETR. The short arrows indicate isolation membranes enveloping damaged mitochondria (M) in Sertoli cell. Long arrow shows a large AV sequestrating a cluster of damaged mitochondria (white arrows). Note the peri-nuclear aggregation of the mitochondria. AV, autophgagic vacuole. S, nucleus of Sertoli cell. ×5,000 (magnification) (TIFF 1,183 kb)
Rights and permissions
About this article
Cite this article
Eid, N., Ito, Y. & Otsuki, Y. Enhanced mitophagy in Sertoli cells of ethanol-treated rats: morphological evidence and clinical relevance. J Mol Hist 43, 71–80 (2012). https://doi.org/10.1007/s10735-011-9372-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10735-011-9372-0