Two Closely Related Ubiquitin C-Terminal Hydrolase Isozymes Function as Reciprocal Modulators of Germ Cell Apoptosis in Cryptorchid Testis
2004; Elsevier BV; Volume: 165; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63394-9
ISSN1525-2191
AutoresJungkee Kwon, Yu-Lai Wang, Rieko Setsuie, Satoshi Sekiguchi, Yae Sato, Mikako Sakurai, Mami Noda, Shunsuke Aoki, Yasuhiro Yoshikawa, Keiji Wada,
Tópico(s)Cell death mechanisms and regulation
ResumoThe experimentally induced cryptorchid mouse model is useful for elucidating the in vivo molecular mechanism of germ cell apoptosis. Apoptosis, in general, is thought to be partly regulated by the ubiquitin-proteasome system. Here, we analyzed the function of two closely related members of the ubiquitin C-terminal hydrolase (UCH) family in testicular germ cell apoptosis experimentally induced by cryptorchidism. The two enzymes, UCH-L1 and UCH-L3, deubiquitinate ubiquitin-protein conjugates and control the cellular balance of ubiquitin. The testes of gracile axonal dystrophy (gad) mice, which lack UCH-L1, were resistant to cryptorchid stress-related injury and had reduced ubiquitin levels. The level of both anti-apoptotic (Bcl-2 family and XIAP) and prosurvival (pCREB and BDNF) proteins was significantly higher in gad mice after cryptorchid stress. In contrast, Uchl3 knockout mice showed profound testicular atrophy and apoptotic germ cell loss after cryptorchid injury. Ubiquitin level was not significantly different between wild-type and Uchl3 knockout mice, whereas the levels of Nedd8 and the apoptotic proteins p53, Bax, and caspase3 were elevated in Uchl3 knockout mice. These results demonstrate that UCH-L1 and UCH-L3 function differentially to regulate the cellular levels of anti-apoptotic, prosurvival, and apoptotic proteins during testicular germ cell apoptosis. The experimentally induced cryptorchid mouse model is useful for elucidating the in vivo molecular mechanism of germ cell apoptosis. Apoptosis, in general, is thought to be partly regulated by the ubiquitin-proteasome system. Here, we analyzed the function of two closely related members of the ubiquitin C-terminal hydrolase (UCH) family in testicular germ cell apoptosis experimentally induced by cryptorchidism. The two enzymes, UCH-L1 and UCH-L3, deubiquitinate ubiquitin-protein conjugates and control the cellular balance of ubiquitin. The testes of gracile axonal dystrophy (gad) mice, which lack UCH-L1, were resistant to cryptorchid stress-related injury and had reduced ubiquitin levels. The level of both anti-apoptotic (Bcl-2 family and XIAP) and prosurvival (pCREB and BDNF) proteins was significantly higher in gad mice after cryptorchid stress. In contrast, Uchl3 knockout mice showed profound testicular atrophy and apoptotic germ cell loss after cryptorchid injury. Ubiquitin level was not significantly different between wild-type and Uchl3 knockout mice, whereas the levels of Nedd8 and the apoptotic proteins p53, Bax, and caspase3 were elevated in Uchl3 knockout mice. These results demonstrate that UCH-L1 and UCH-L3 function differentially to regulate the cellular levels of anti-apoptotic, prosurvival, and apoptotic proteins during testicular germ cell apoptosis. In the ubiquitin-proteasome system, the levels of poly- and monoubiquitin are strictly controlled by the balance of two groups of specific enzymes: ubiquitinating enzymes (E1, E2, and E3) and deubiquitinating enzymes (DUBs).1Weissman AM Themes and variations on ubiquitylation.Nat Rev Mol Cell Biol. 2001; 2: 169-178Crossref PubMed Scopus (1270) Google Scholar, 2Ciechanover A The ubiquitin-proteasome pathway: on protein death and cell life.EMBO J. 1998; 17: 7151-7160Crossref PubMed Scopus (1215) Google Scholar DUBs are subdivided into ubiquitin C-terminal hydrolases (UCHs) and ubiquitin-specific proteases (UBPs).3Pickart CM Rose IA Ubiquitin carboxyl-terminal hydrolase acts on ubiquitin carboxyl-terminal amides.J Biol Chem. 1985; 260: 7903-7910Abstract Full Text PDF PubMed Google Scholar, 4Baker RT Tobias JW Varshavsky A Ubiquitin-specific proteases of Saccharomyces cerevisiae. 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The activity (Kcat) of UCH-L3 is more than 200-fold higher than UCH-L1 when a fluorogenic ubiquitin substrate is used.11Liu Y Fallon L Lashuel HA Liu Z Lansbury Jr, PT The UCH-L1 gene encodes two opposing enzymatic activities that affect alpha-synuclein degradation and Parkinson's disease susceptibility.Cell. 2002; 111: 209-218Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar In addition to its relatively weak hydrolase activity, UCH-L1 exhibits dimerization-dependent ubiquityl ligase activity.11Liu Y Fallon L Lashuel HA Liu Z Lansbury Jr, PT The UCH-L1 gene encodes two opposing enzymatic activities that affect alpha-synuclein degradation and Parkinson's disease susceptibility.Cell. 2002; 111: 209-218Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar In contrast, UCH-L3 has little or no ligase activity compared with UCH-L1.11Liu Y Fallon L Lashuel HA Liu Z Lansbury Jr, PT The UCH-L1 gene encodes two opposing enzymatic activities that affect alpha-synuclein degradation and Parkinson's disease susceptibility.Cell. 2002; 111: 209-218Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar It was recently suggested that UCH-L1 has anti-proliferative activity in tumor cells, and that its expression is induced in response to tumor growth.12Liu Y Lashuel HA Choi S Xing X Case A Ni J Yeh LA Cuny GD Stein RL Lansbury Jr, PT Discovery of inhibitors that elucidate the role of UCH-L1 activity in the H1299 lung cancer cell line.Chem Biol. 2003; 10: 837-846Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar Furthermore, UCH-L1 associates with monoubiquitin and prolongs ubiquitin half-life in neurons.13Osaka H Wang YL Takada K Takizawa S Setsuie R Li H Sato Y Nishikawa K Sun YJ Sakurai M Harada T Hara Y Kimura I Chiba S Namikawa K Kiyama H Noda M Aoki S Wada K Ubiquitin carboxy-terminal hydrolase L1 binds to and stabilizes monoubiquitin in neuron.Hum Mol Genet. 2003; 12: 1945-1958Crossref PubMed Scopus (330) Google Scholar Other work demonstrated that UCH-L3 binds to Nedd8 and subsequently processes its C-terminus.14Wada H Kito K Caskey LS Yeh ET Kamitani T Cleavage of the C-terminus of NEDD8 by UCH-L3.Biochem Biophys Res Commun. 1998; 251: 688-692Crossref PubMed Scopus (136) Google Scholar Nedd8 is a small ubiquitin-like protein that shares with ubiquitin the ability to be conjugated to a lysine residue in a substrate protein.15Hemelaar J Borodovsky A Kessler BM Reverter D Cook J Kolli N Gan-Erdene T Wilkinson KD Gill G Lima CD Ploegh HL Ovaa H Specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins.Mol Cell Biol. 2004; 24: 84-95Crossref PubMed Scopus (173) Google Scholar Covalent conjugation of proteins by Nedd8 is an important form of the posttranscriptional modification and plays a critical role in many cellular processes.16Gong L Kamitani T Millas S Yeh ET Identification of a novel isopeptidase with dual specificity for ubiquitin- and NEDD8-conjugated proteins.J Biol Chem. 2000; 275: 14212-14216Crossref PubMed Scopus (95) Google Scholar These conjugates are regulated by a large number of deconjugating enzymes. This activity is unique to UCH-L3 because UCH-L1 is relatively weak to cleave the C terminus of Nedd8.14Wada H Kito K Caskey LS Yeh ET Kamitani T Cleavage of the C-terminus of NEDD8 by UCH-L3.Biochem Biophys Res Commun. 1998; 251: 688-692Crossref PubMed Scopus (136) Google Scholar, 15Hemelaar J Borodovsky A Kessler BM Reverter D Cook J Kolli N Gan-Erdene T Wilkinson KD Gill G Lima CD Ploegh HL Ovaa H Specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins.Mol Cell Biol. 2004; 24: 84-95Crossref PubMed Scopus (173) Google Scholar, 16Gong L Kamitani T Millas S Yeh ET Identification of a novel isopeptidase with dual specificity for ubiquitin- and NEDD8-conjugated proteins.J Biol Chem. 2000; 275: 14212-14216Crossref PubMed Scopus (95) Google Scholar Collectively, these data suggest that the two mouse isozymes, UCH-L1 and UCH-L3, have distinct but overlapping functions. In addition, we recently found that gad mice, which lack UCH-L1 expression, show reduced retinal cell apoptosis in response to ischemia, suggesting that UCH-L1 may promote apoptosis.17Harada T Harada C Wang YL Osaka H Amanai K Tanaka K Takizawa S Setsuie R Sakurai M Sato Y Noda M Wada K Role of ubiquitin carboxy terminal hydrolase-L1 in neural cell apoptosis induced by ischemic retinal injury in vivo.Am J Pathol. 2004; 164: 59-64Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar Our previous work focused on the possibility that UCH-L1 and UCH-L3 exhibit functional diversity during spermatogenesis. We showed that both UCH-L1 and UCH-L3 are strongly but reciprocally expressed in the testis during spermatogenesis,18Kwon J Wang YL Setsuie R Sekiguchi S Sakurai M Sato Y Lee WW Ishii Y Kyuwa S Noda M Wada K Yoshikawa Y Developmental regulation of ubiquitin C-terminal hydrolase isozyme expression during spermatogenesis in mice.Biol Reprod. 2004; 71: 515-521Crossref PubMed Scopus (91) Google Scholar suggesting that each isozyme may have a distinct function in the testis. To elucidate the pathophysiological roles of these two isozymes in the testis, our present work examines the extent of heat-induced stress using experimentally induced cryptorchidism in Uchl3 knockout7Kurihara LJ Semenova E Levorse JM Tilghman SM Expression and functional analysis of Uch-L3 during mouse development.Mol Cell Biol. 2000; 20: 2498-2504Crossref PubMed Scopus (67) Google Scholar and gad mice.8Saigoh K Wang YL Suh JG Yamanishi T Sakai Y Kiyosawa H Harada T Ichihara N Wakana S Kikuchi T Wada K Intragenic deletion in the gene encoding ubiquitin carboxy-terminal hydrolase in gad mice.Nat Genet. 1999; 23: 47-51PubMed Scopus (0) Google Scholar Normally, the testes are maintained in the scrotum at a temperature lower than that of the abdomen. Exposure of a testis to higher body temperature via experimentally induced cryptorchidism results in rapid degeneration of testicular germ cells.19Boekelheide K Hall SJ 2,5-Hexanedione exposure in the rat results in long-term testicular atrophy despite the presence of residual spermatogonia.J Androl. 1991; 12: 18-26PubMed Google Scholar, 20Ohta Y Nishikawa A Fukazawa Y Urushitani H Matsuzawa A Nishina Y Iguchi T Apoptosis in adult mouse testis induced by experimental cryptorchidism.Acta Anat (Basel). 1996; 157: 195-204Crossref PubMed Scopus (61) Google Scholar, 21Yin Y Hawkins KL DeWolf WC Morgentaler A Heat stress causes testicular germ cell apoptosis in adult mice.J Androl. 1997; 18: 159-165PubMed Google Scholar, 22Rasoulpour RJ Schoenfeld HA Gray DA Boekelheide K Expression of a K48R mutant ubiquitin protects mouse testis from cryptorchid injury and aging.Am J Pathol. 2003; 163: 2595-2603Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar Recent studies show that testicular germ cell degeneration in cryptorchid testes occurs via apoptosis, and that protein and lipid oxidation, along with p53 promote germ cell death.23Yin Y DeWolf WC Morgentaler A Experimental cryptorchidism induces testicular germ cell apoptosis by p53-dependent and -independent pathways in mice.Biol Reprod. 1998; 58: 492-496Crossref PubMed Scopus (140) Google Scholar, 24Peltola V Huhtaniemi I Ahotupa M Abdominal position of the rat testis is associated with high level of lipid peroxidation.Biol Reprod. 1995; 53: 1146-1150Crossref PubMed Scopus (67) Google Scholar, 25Ahotupa M Huhtaniemi I Impaired detoxification of reactive oxygen and consequent oxidative stress in experimentally cryptorchid rat testis.Biol Reprod. 1992; 46: 1114-1118Crossref PubMed Scopus (105) Google Scholar The ubiquitin-proteasome system is required for the subsequent degradation of the damaged testicular germ cells.26Morimoto RI Santoro MG Stress-inducible responses and heat shock proteins: new pharmacologic targets for cytoprotection.Nature Biotechnol. 1998; 16: 833-838Crossref Scopus (531) Google Scholar, 27Wojcik C Proteasomes in apoptosis: villains or guardians?.Cell Mol Life Sci. 1999; 56: 908-917Crossref PubMed Scopus (75) Google Scholar, 28Yang Y Yu X Regulation of apoptosis: the ubiquitous way.EMBO J. 2003; 17: 790-799Google Scholar Here, we show that both UCH-L1 and UCH-L3 have reciprocal functions in testicular germ cells during cryptorchid-induced apoptosis. Our data show that the absence of UCH-L1 causes resistance to cryptorchid-induced testicular germ cell apoptosis, and that the knockout of UCH-L3 promotes germ cell apoptosis after cryptorchid injury. We used 8-week-old Uchl3 knockout (C57BL/6J)7Kurihara LJ Semenova E Levorse JM Tilghman SM Expression and functional analysis of Uch-L3 during mouse development.Mol Cell Biol. 2000; 20: 2498-2504Crossref PubMed Scopus (67) Google Scholar, 18Kwon J Wang YL Setsuie R Sekiguchi S Sakurai M Sato Y Lee WW Ishii Y Kyuwa S Noda M Wada K Yoshikawa Y Developmental regulation of ubiquitin C-terminal hydrolase isozyme expression during spermatogenesis in mice.Biol Reprod. 2004; 71: 515-521Crossref PubMed Scopus (91) Google Scholar and gad8Saigoh K Wang YL Suh JG Yamanishi T Sakai Y Kiyosawa H Harada T Ichihara N Wakana S Kikuchi T Wada K Intragenic deletion in the gene encoding ubiquitin carboxy-terminal hydrolase in gad mice.Nat Genet. 1999; 23: 47-51PubMed Scopus (0) Google Scholar, 18Kwon J Wang YL Setsuie R Sekiguchi S Sakurai M Sato Y Lee WW Ishii Y Kyuwa S Noda M Wada K Yoshikawa Y Developmental regulation of ubiquitin C-terminal hydrolase isozyme expression during spermatogenesis in mice.Biol Reprod. 2004; 71: 515-521Crossref PubMed Scopus (91) Google Scholar, 29Kwon J Kikuchi T Setsuie R Ishii Y Kyuwa S Yoshikawa Y Characterization of the testis in congenitally ubiquitin carboxy-terminal hydrolase-1 (Uch-L1) defective (gad) mice.Exp Anim. 2003; 52: 1-9Crossref PubMed Scopus (45) Google Scholar (CBA/RFM) male mice. Uchl3 knockout mice were generated by the standard method using homologously recombinant ES cells, and the knockout line was back-crossed several times to C57BL/6J mice.7Kurihara LJ Semenova E Levorse JM Tilghman SM Expression and functional analysis of Uch-L3 during mouse development.Mol Cell Biol. 2000; 20: 2498-2504Crossref PubMed Scopus (67) Google Scholar The gad mouse is an autosomal recessive mutant that was obtained by crossing CBA and RFM mice.8Saigoh K Wang YL Suh JG Yamanishi T Sakai Y Kiyosawa H Harada T Ichihara N Wakana S Kikuchi T Wada K Intragenic deletion in the gene encoding ubiquitin carboxy-terminal hydrolase in gad mice.Nat Genet. 1999; 23: 47-51PubMed Scopus (0) Google Scholar The gad line was maintained by intercrossing for more than 20 generations.8Saigoh K Wang YL Suh JG Yamanishi T Sakai Y Kiyosawa H Harada T Ichihara N Wakana S Kikuchi T Wada K Intragenic deletion in the gene encoding ubiquitin carboxy-terminal hydrolase in gad mice.Nat Genet. 1999; 23: 47-51PubMed Scopus (0) Google Scholar, 29Kwon J Kikuchi T Setsuie R Ishii Y Kyuwa S Yoshikawa Y Characterization of the testis in congenitally ubiquitin carboxy-terminal hydrolase-1 (Uch-L1) defective (gad) mice.Exp Anim. 2003; 52: 1-9Crossref PubMed Scopus (45) Google Scholar Both strains were maintained at our institute. Animal care and handling were in accordance with institutional regulations for animal care and were approved by the Animal Investigation Committee of the National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan. Unilateral cryptorchidism was experimentally induced under pentobarbital anesthesia (Abbott Laboratories, North Chicago, IL).20Ohta Y Nishikawa A Fukazawa Y Urushitani H Matsuzawa A Nishina Y Iguchi T Apoptosis in adult mouse testis induced by experimental cryptorchidism.Acta Anat (Basel). 1996; 157: 195-204Crossref PubMed Scopus (61) Google Scholar, 22Rasoulpour RJ Schoenfeld HA Gray DA Boekelheide K Expression of a K48R mutant ubiquitin protects mouse testis from cryptorchid injury and aging.Am J Pathol. 2003; 163: 2595-2603Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar Briefly, a midline abdominal incision was made, and the left testis was displaced from scrotum and fixed to the upper abdominal wall. The right testis remained in the scrotum as an intact control within the same animal. At 0, 4, 7, and 14 days after the operation, four control and four cryptorchid testes were harvested to determine testis weight. Testes were embedded in paraffin wax after fixation in 4% paraformaldehyde, sectioned at 4-μm thickness, and stained with hematoxylin and eosin.29Kwon J Kikuchi T Setsuie R Ishii Y Kyuwa S Yoshikawa Y Characterization of the testis in congenitally ubiquitin carboxy-terminal hydrolase-1 (Uch-L1) defective (gad) mice.Exp Anim. 2003; 52: 1-9Crossref PubMed Scopus (45) Google Scholar Light microscopy was used for routine observations. For immunohistochemical staining, the sections were incubated with 10% goat serum for 1 hour at room temperature, followed by incubation overnight at 4°C with a rabbit polyclonal antibody against ubiquitin (1:500; DakoCytomation, Glostrup, Denmark) or Nedd8 (1:500; Alexis Biochemicals, San Diego, CA) diluted in phosphate-buffered saline (PBS) containing 1% bovine serum albumin. Sections were then incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:200; Jackson ImmunoResearch, West Grove, PA) for 1 hour at room temperature and examined by confocal laser-scanning microscopy (Olympus, Tokyo, Japan). Apoptotic cells in testicular tissues were identified by terminal deoxynucleotidyl transferase (TdT)-mediated nick-end labeling (TUNEL) using the DeadEnd Fluorimetric TUNEL system kit (Promega, Madison, WI) and the anti-PARP p85 fragment pAb (Promega) according to the manufacturer's instructions. The number of apoptotic cells was determined by counting the positively stained nuclei in 30 circular seminiferous tubule cross-sections per testis section.23Yin Y DeWolf WC Morgentaler A Experimental cryptorchidism induces testicular germ cell apoptosis by p53-dependent and -independent pathways in mice.Biol Reprod. 1998; 58: 492-496Crossref PubMed Scopus (140) Google Scholar, 29Kwon J Kikuchi T Setsuie R Ishii Y Kyuwa S Yoshikawa Y Characterization of the testis in congenitally ubiquitin carboxy-terminal hydrolase-1 (Uch-L1) defective (gad) mice.Exp Anim. 2003; 52: 1-9Crossref PubMed Scopus (45) Google Scholar The proportion of seminiferous tubules containing apoptotic germ cells was calculated by dividing the number of seminiferous tubules containing apoptotic cells by the total number of seminiferous tubules. The incidence of apoptotic cells per apoptotic cell-containing seminiferous tubule was categorized into three groups, defined as 1 to 5, 6 to 10, and >11 positive cells. Western blots were performed as previously reported.8Saigoh K Wang YL Suh JG Yamanishi T Sakai Y Kiyosawa H Harada T Ichihara N Wakana S Kikuchi T Wada K Intragenic deletion in the gene encoding ubiquitin carboxy-terminal hydrolase in gad mice.Nat Genet. 1999; 23: 47-51PubMed Scopus (0) Google Scholar, 18Kwon J Wang YL Setsuie R Sekiguchi S Sakurai M Sato Y Lee WW Ishii Y Kyuwa S Noda M Wada K Yoshikawa Y Developmental regulation of ubiquitin C-terminal hydrolase isozyme expression during spermatogenesis in mice.Biol Reprod. 2004; 71: 515-521Crossref PubMed Scopus (91) Google Scholar, 29Kwon J Kikuchi T Setsuie R Ishii Y Kyuwa S Yoshikawa Y Characterization of the testis in congenitally ubiquitin carboxy-terminal hydrolase-1 (Uch-L1) defective (gad) mice.Exp Anim. 2003; 52: 1-9Crossref PubMed Scopus (45) Google Scholar Total protein (5 μg/lane) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 15% gels (Perfect NT Gel; DRC, Japan). Proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA) and blocked with 5% nonfat milk in TBS-T [50 mmol/L Tris base, pH 7.5, 150 mmol/L NaCl, 0.1% (w/v) Tween-20]. The membranes were incubated individually with one or more primary antibodies to UCH-L1 and UCH-L3 (1:1000; peptide antibodies18Kwon J Wang YL Setsuie R Sekiguchi S Sakurai M Sato Y Lee WW Ishii Y Kyuwa S Noda M Wada K Yoshikawa Y Developmental regulation of ubiquitin C-terminal hydrolase isozyme expression during spermatogenesis in mice.Biol Reprod. 2004; 71: 515-521Crossref PubMed Scopus (91) Google Scholar), Bcl-2, Bcl-xL, Bax, p53, and caspase-3, (1:1000; all from Cell Signaling Technology, Beverly, MA), phosphorylated cyclic AMP response element-binding protein (pCREB, 1:500; Upstate Biotechnology, Waltham, MA), brain-derived neurotrophic factor (BDNF, 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), XIAP (1:500; Transduction Laboratories, Franklin Lakes, NJ), polyubiquitin (1:1000, clone FK-2; Medical & Biological Laboratories, Nagoya, Japan), monoubiquitin (1:1000, u5379; Sigma-Aldrich, St. Louis, MO), and Nedd8 (1:1000; Alexis Biochemicals, San Diego, CA). Blots were further incubated with peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (1:5000; Pierce, Rockford, IL) for 1 hour at room temperature. Immunoreactions were visualized using the SuperSignal West Dura extended duration substrate (Pierce) and analyzed with a ChemiImager (Alpha Innotech, San Leandro, CA). Each protein level was relatively quantificated after analysis with a ChemiImager using AlphaEase software. The mean and SD were calculated for all data (presented as mean ± SD). One-way analysis of variance was used for all statistical analyses. We first confirmed the lack of UCH-L3 protein in the testes from Uchl3 knockout mice by Western blotting (Figure 1). Similarly, we did not detect UCH-L1 protein in the testes of gad mice (Figure 1), as we previously observed.13Osaka H Wang YL Takada K Takizawa S Setsuie R Li H Sato Y Nishikawa K Sun YJ Sakurai M Harada T Hara Y Kimura I Chiba S Namikawa K Kiyama H Noda M Aoki S Wada K Ubiquitin carboxy-terminal hydrolase L1 binds to and stabilizes monoubiquitin in neuron.Hum Mol Genet. 2003; 12: 1945-1958Crossref PubMed Scopus (330) Google Scholar Thus, in a biochemical sense, gad mice are analogous to Uchl1-null mice.8Saigoh K Wang YL Suh JG Yamanishi T Sakai Y Kiyosawa H Harada T Ichihara N Wakana S Kikuchi T Wada K Intragenic deletion in the gene encoding ubiquitin carboxy-terminal hydrolase in gad mice.Nat Genet. 1999; 23: 47-51PubMed Scopus (0) Google Scholar, 13Osaka H Wang YL Takada K Takizawa S Setsuie R Li H Sato Y Nishikawa K Sun YJ Sakurai M Harada T Hara Y Kimura I Chiba S Namikawa K Kiyama H Noda M Aoki S Wada K Ubiquitin carboxy-terminal hydrolase L1 binds to and stabilizes monoubiquitin in neuron.Hum Mol Genet. 2003; 12: 1945-1958Crossref PubMed Scopus (330) Google Scholar Compensatory level of UCH-L3 and UCH-L1 in gad and Uchl3 knockout mice, respectively, was not observed (Figure 1; compare UCH-L3/UCH-L1 level with that of wild-type control mice). Experimental cryptorchidism did not affect UCH-L3 level in gad or wild-type control mice. Similarly, cryptorchidism did not affect UCH-L1 level in Uchl3 knockout and wild-type control mice (Figure 1). Quantitative reverse transcriptase-polymerase chain reaction analysis showed that transcription from the Uchl3 and Uchl1 in both scrotal and cryptorchid testes from gad and Uchl3 knockout mice was not significantly different from that measured in the corresponding wild-type control mice (data not shown). These results suggest that the level of UCH-L3 is regulated independently of UCH-L1 in the mouse testis, and that cryptorchid injury does not affect the level of either protein. Unilateral cryptorchidism was surgically induced in Uchl3 knockout and gad mice, and testes were evaluated on days 0, 4, 7, and 14 after the operation (Figure 2). Nonoperated (scrotal) testes served as controls for the evaluation of testicular weight and histochemistry. Cryptorchid testes from Uchl3 knockout mice appeared smaller than the nonoperated controls at each time point, whereas the size of the cryptorchid testes from gad mice was similar to the controls (Figure 2A). Figure 2B shows the time course of the ratio of testicular weight of cryptorchid testes to scrotal testes. In wild-type mice (C57BL/6J and CBA/RFM), the ratio transiently increased 4 days after cryptorchid injury, most likely a consequence of inflammation-induced fluid accumulation22Rasoulpour RJ Schoenfeld HA Gray DA Boekelheide K Expression of a K48R mutant ubiquitin protects mouse testis from cryptorchid injury and aging.Am J Pathol. 2003; 163: 2595-2603Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 23Yin Y DeWolf WC Morgentaler A Experimental cryptorchidism induces testicular germ cell apoptosis by p53-dependent and -independent pathways in mice.Biol Reprod. 1998; 58: 492-496Crossref PubMed Scopus (140) Google Scholar and biochemical changes observed. The ratio for these mice subsequently decreased below 1.0 by day 7. The ratio remained ∼1.0 in gad mice (range, 1.15 ∼ 0.85), whereas it decreased significantly in Uchl3 knockout mice compared with wild-type mice (Figure 2B). These results demonstrate that testes from Uchl3 knockout and gad mice differ in their response to experimental cryptorchidism. To explore the mechanism underlying the observed differences between Uchl3 knockout and gad cryptorchid testes, we prepared histological cross-sections on day 7 after testicular injury (Figure 3, A and C). The presence of nuclear pyknosis, multinucleated giant cells, and Sertoli cell vacuolization with germ cell loss in the germinal epithelium is indicative of cryptorchid testes.22Rasoulpour RJ Schoenfeld HA Gray DA Boekelheide K Expression of a K48R mutant ubiquitin protects mouse testis from cryptorchid injury and aging.Am J Pathol. 2003; 163: 2595-2603Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 23Yin Y DeWolf WC Morgentaler A Experimental cryptorchidism induces testicular germ cell apoptosis by p53-dependent and -independent pathways in mice.Biol Reprod. 1998; 58: 492-496Crossref PubMed Scopus (140) Google Scholar These hallmarks of testicular injury were the most remarkable characteristics of cryptorchid testes from Uchl3 knockout mice, demonstrating profound testicular atrophy and germ cell loss compared with wild-type mice (Figure 3, A and C). In contrast, no nuclear pyknosis, cellular shrinkage, or germ cell loss was observed in cryptorchid testes from gad mice. Spermatocytes and early spermatids comprised the majority of affected cell types in cryptorchid testes (Figure 3, A and C). Germ cell apoptosis was further examined by TUNEL and PARP assays in tissue sections from postoperative day 7 mice (Figure 3, B and D). All but the gad cryptorchid testes showed a time-dependent increase in germ cell apoptosis during experimental cryptorchidism; germ cell apoptosis was always found in tubules that had germ cell loss on days 4, 7, and 14 (Figure 3, B and D, and Figure 4). Compared to wild-type mice, the cryptorchid testes of Uchl3 knockout mice showed a marked increase in apoptotic germ cells in response to testicular injury, whereas gad mice lacked cryptorchid-induced germ cell apoptosis (Figure 3B and Figure 4). By postoperative days 4 and 7, the percentage of seminiferous tubules containing apoptotic germ cells increased with statistical significance (n = 4) (P < 0.05) in cryptorchid testes of Uchl3 knockout mice as compared with wild-type mice (Figure 4A). In addition, cryptorchid testes of Uchl3 knockout mice showed a high incidence of seminiferous tubules containing >11 apoptotic germ cells on days 4 and 7 days as compared with wild-type mice (Figure 4B); however, germ cell apoptosis did not increase in cryptorchid testes of gad mice during postoperative days 4 to 14 (P < 0.01) (Figure 4, A and B). Ubiquitin is required for energy-dependent degradation of structurally altered proteins.26Morimoto RI Santoro MG Stress-inducible responses and heat shock proteins: new pharmacologic targets for cytoprotection.Nature Biotechnol. 1998; 16: 833-838Crossr
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