Proteomic Analysis of Effect of Hyperthermia on Spermatogenesis in Adult Male Mice Ye-Fei Zhu,† Yu-Gui Cui,† Xue-Jiang Guo, Lei Wang, Ye Bi, Yan-Qiu Hu, Xin Zhao, Quan Liu, Ran Huo, Min Lin, Zuo-Min Zhou, and Jia-Hao Sha* Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, P. R. China Received March 1, 2006
We characterized cellular and molecular mechanisms involved in spermatogenesis following shortterm heat exposure of murine testis. For these studies, we utilized a proteomic approach with twodimensional gel electrophoresis (2DE) analyses and mass spectroscopic identification of proteins with altered expression in mouse testes at different times after heat shock. We established a proteome reference map from 7-wk-old mouse testis linked to a federated proteome database. We used these tools to analyze quantitative variations in the tissue over a time course of 0.5, 2, 6, and 12 h following heat exposure. We separated 108 protein spots expressed differentially between the heat shock tissues and the control mouse testes. Of these spots, we identified 36 by comparing with the control reference map. We then focused on the heterogeneous nuclear ribonucleoproteins (hnRNPs) and the chaperonins containing t-complex polypeptide-1 (CCT). Further analysis in this heat-shocked model suggests numerous potential mechanisms for heat shock-induced spermatogenic disorder. Keywords: proteomics • mouse testis • heat shock
Introduction In most animals, including humans, the scrotal location of the testes keeps them cooler than the core body temperature. This lower temperature is important for germ cell viability; exposure of the testis to temperatures at or above body temperature results in increased germ cell death.1 Mild testicular heating safely and reversibly suppresses spermatogenesis in several mammalian species, including mice,2 rats,3 cows,4 pigs,5 sheep,6 and humans.1 Although the physiological and cellular responses to testicular heat treatment are welldocumented,7 and gene expression following increased scrotal temperature has been described,8 only limited proteomic information is available regarding testicular heat shock. The proteomics field is becoming increasingly important because proteins are directly related to cellular function.9 Proteome analysis provides key information about posttranscriptional control of gene expression, changes in protein expression levels, protein synthesis and degradation rates and protein post-translational modifications.10 Furthermore, the availability of the entire mouse genome facilitates global gene expression analysis on parallel samples under various conditions. Two-dimensional gel electrophoresis (2DE) and matrixassisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) were used to characterize hyperthermal disruption of spermatogenesis. On the basis of peptide mass fingerprinting (PMF), we mapped 504 proteins on the * To whom correspondence should be addressed. E-mail: shajh@ njmu.edu.cn; Phone: 86-25-86862908; Fax: 86-25-86862908. † These authors contributed equally to this work. 10.1021/pr0600733 CCC: $33.50
2006 American Chemical Society
reference gel and constructed a federated mouse testis proteome database. Moreover, differential proteome analysis findings over four time points following the heat treatments were reported in the present study. Several of these proteins were selected for immunohistochemistry to characterize the mechanisms of known and suspected toxicants.
Experimental Procedures Animals. Twenty-four adult (7-wk-old) male ICR mice from the lab animal center of Nanjing Medical University (Nanjing, China) were used and maintained under a controlled environment of 20-22 °C, 12/12 h light/dark cycle, and 50-70% humidity, with food and water ad libitum. Three ICR mice were used to construct a reference map as control for the heatinduced differential expression analysis. We immersed the tails and the scrotums containing the testes of the remaining animals for 15 min in a thermostatically controlled water bath at 42 °C, as previously described.8 We sacrificed these animals at 0.5, 2, 6, and 12 h after heat exposure (3 animals per time phase). We monitored the protein expression changes against mild changes in the spermatogenic epithelial structure. For these experiments, we fixed one testis from each animal in Bouin’s solution for 48 h at 4 °C. We then paraffin-embedded, sectioned, and stained the fixed tissues with hematoxylin and eosin for histological examination. Two-Dimensional Electrophoresis. Proteins from single testis from each experimental animal were extracted and analyzed. The protein concentration in each sample was determined by the Bradford method11 using BSA as the Journal of Proteome Research 2006, 5, 2217-2225
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research articles standard. We rehydrated IPG strips (24 cm, pH 3-10, NL; Amersham Bioscience, Uppsala, Sweden) with 80 µg of testis proteins (for silver staining), as described previously.12 We loaded each IPG strip with 800 µg of the control mouse testicular protein sample for the Coomassie Brilliant Blue staining, and focused the protein samples of the normal adult mouse testis simultaneously. After isoelectric focusing, we equilibrated the IPG strips, ran them in a Ettan-Dalt six electrophoresis system (Amersham Bioscience, Uppsala, Sweden) and visualized them as described previously.12 Statistical Analysis. We scanned the stained gels and used ImageMasterTM 2D Platinum Software (Version 5.0, Amersham Bioscience, Swiss Institute of Bioinformatics, Geneva, Switzerland) for spot detection, quantification, as well as comparative and statistical analyses. The amount of each protein spot was expressed as its volume, which was calculated as the volume above the spot border and situated at 75% of the spot height (measured from the peak of the spot). To reflect the quantitative variations in the protein spot volumes, we normalized the spot volumes as a percentage of the total volume of all the spots present in a gel. Each 2DE pattern was repeated three times from three individual mouse testis samples. Within each experiment, we compared the protein expression profile of heat-treated mice to that of untreated control ones. Protein samples from the separate experiments were not pooled for the 2DE analysis, because combining them would have obscured the biological variability of the experimental system. Instead, we averaged the values from the three independent experiments, calculated the means and standard deviations and assessed statistical significance between nontreated and heattreated conditions over time using independent t-tests using ImageMasterTM 2D Platinum Software. p values less than 0.05 were considered statistically significant. In-Gel Tryptic Digestion and MALDI-TOF MS. We cut out off the 2DE gels of the matched Coomassie-stained protein spots. We digested and extracted the peptide mixtures and analyzed them on a Biflex IV (Bruker Daltonics, Germany), as described previously.13 Database Searches. The monoisotopic masses of the tryptic peptides observed in the MALDI-TOF MS spectra were used to query the Swiss-Prot/TrEMBL or NCBInr sequence databases using the Mascot search programs (http://www.matrixscience.com). The peptide masses were compared with theoretical peptide masses of all available proteins from all species. We used the following search conditions: 100 ppm for external calibration, one missed cleavage allowed, modification of cysteines by iodoacetamide, methionine oxidation and Nterminal pyroglutamylation allowed as variable modifications. Four matching peptides were the minimal requirement for an identity assignment, and only considered mouse proteins were selected for further studies. The algorithm used for determining the probability of a false positive match with a given MS spectrum has already been reported previously in the literature.14 Construction of an Online Database. To construct a mouse testis proteome database, we used a web-based system, the Make2D-DB II Package (ver.1.0), an HTML generator for 2D images through ExPASy (http://www.expasy.org/ch2d/ make2ddb.html). We stored the identified protein spots in a relational database that was made accessible online via a common gateway interface (cgi) script on a webserver (http:// reprod.njmu.edu.cn/2d). We hyperlinked the individual protein 2218
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entries to the relevant spots on an image map created from the reference gel. Western Blotting. Samples containing 50 µg of protein from normal and heat-treated mouse testes were electrophoresed on 12% SDS polyacrylamide gels and transferred to nitrocellulose membranes (Amersham Biosciences AB, Uppsala, Sweden). The membranes were blocked in phosphate-buffered saline (PBS) containing 5% nonfat milk powder for 1 h and then incubated overnight with a 1:100-diluted anti-hnRNP-E1, hnRNP A2/B1, CCT, and CCTθ goat polyclonal antibodies (sc-16504, sc-10035, sc-13886, and sc-13891, respectively, Santa Cruz Technology, Inc., Santa Cruz, CA) or a 1:1000-diluted anti-βtubulin rabbit polyclonal antibody (ab6046, Abcam, Cambridge, United Kingdom) in PBS containing 5% nonfat milk powder. They were washed three times (10 min each) with PBS. The filters were then incubated for 1 h with horseradish peroxidase (HRP)-conjugated anti-goat IgG (Beijing ZhongShan Biotechnology CO., LTD, Beijing, China). Specific proteins were detected using an ECL kit (Amersham Biosciences, Buckinghamshire, England) and AlphaImager (FluorChem5500, Alpha Innotech, San Leandro, CA). The protein expression level was analyzed by AlphaEaseFC software (Alpha Innotech). Immunohistochemistry. We dewaxed and rehydrated the unstained sections through a series of descending grades of alcohol to distilled water. We performed immunohistochemistry on the sections as described previously.15 Briefly, sections were incubated in 1% hydrogen peroxide and washed in phosphate buffered saline (PBS). We blocked nonspecific protein binding with rabbit serum (Beijing ZhongShan Biotechnology CO., LTD, Beijing, China). Sections were then incubated overnight at 4 °C with primary antibodies. Following three PBS washes, sections were incubated with horseradish peroxidase (HRP) conjugated secondary antibody. Immunoreactive sites were visualized brown with diaminobenzidine and mounted for bright field microscopy (Axioskop 2 plus, ZEISS, Germany). Negative controls were incubated with a dilution absent of primary antibody, and otherwise subjected to all immunohistochemical procedures.
Results Construction of a Web-Accessible Protein Database. We analyzed the control mouse testis proteome initially to define a protein profile as groundwork for parallel comparisons. To this foundation profile we compared expression changes following hyperthermal treatment. We also established future mouse testis proteome analysis strategies. We observed the 2D protein map reproducibly in three silver-stained gels from three individual untreated mouse testis samples. Nearly all spots detected in the Coomassie-stained gel (supplementary Figure 1, http://reprod.njmu.edu.cn/data/index.htm) can be found in the silver-stained gels. Moreover, we established an online federated 2DE database16 containing 504 identified proteins (full data sets are available as Supporting Information on http:// reprod.njmu.edu.cn/data/index.htm). The online reference map (Figure 1) hyperlinks the identified protein spots to the individual protein entries. Each protein entry contains identification information including accession number, name, description, spot ID number, organism, pI, molecular weight, and mass spectrum data, as well as links to relevant entries in other online databases. This database could be accessed by WORLD2DPAGE (http://cn.expasy.org/ch2d/2d-index.html). Morphological Examination. Histological examination of testes harvested 30 min after a 42 °C heat shock treatment
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Proteomics of Mouse Testis following Heat Shock
Figure 1. A reference 2DE map of mouse testis proteins. Identified spots are indicated by spot numbers.
(Figure 2) revealed no substantial morphologic differences from the normal testes (Figure 2). However, 2 h after a 42 °C heat shock treatment, apoptotic cells were visible in the spermatogenic epithelium with intensely eosinophilic cytoplasm and small, dense nuclei (Figure 2). Six hours after a 42 °C heat shock treatment, the spermatocytes showed intracytoplasmic hyalinosis and many spermatocyte nuclei contained highly condensed chromatin (pyknotic nuclei) (Figure 2). Twelve hours after a 42 °C heat shock treatment we noted acidophilic, coagulated spermatids in these samples (Figure 2). Identification of Proteins Expressed Differentially After Heat Treatment. To detect proteome changes following heat exposure, we constructed triplicate 2D maps of the three individual mouse testis from multiple time points following heat treatment (supplementary Figure 2a-e on http://reprod.njmu.edu.cn/data/index.htm). We identified 108 protein spots with significant differences in expression levels relative to the untreated control groups during the 12 h following heat treatment (p < 0.05). After comparing them to the 2D reference map, we retrieved 36 spots from the 2DE database. One spot (ID number 3232) possibly contains two different proteins, according to the Mascot search results. We identified several
proteins as the same protein with different molecular weights and pIs in the 2D map, which were expressed differentially after heat-treatment. These included hnRNP K, CCT, 60 kDa heat shock protein, and splice isoform 1 from the P57776 elongation factor 1-delta. Thirty-seven proteins showed a translation regulational peak between 0.5 h and 12 h. An overview of these proteins is presented in Table 1, which also includes information accession numbers, protein names, mean normalized volumes (% volume) and standard deviations of the protein spots in the control and heat-treated mouse testis gels. Gel spot 3271 (hnRNP A2/B1) decreased in expression intensity from the 12 h phase. Gel spots 1455 (hnRNP K) and 1456 decreased significantly beyond the 12 h phase, and spot 1467 decreased significantly beyond the 0.5 h phase following heat shock treatment (p < 0.05; Table 1). Spot 1778, also identified as hnRNP K on the reference map, was not significantly expressed following the heat-treatment (p > 0.05; data not shown). FatiGO, a web-based (http://fatigo.bioinfo.cnio.es) procedure,17 was used to extract relevant molecular functions for significantly regulated proteins following heat treatment. The four categories listed above are purine nucleotide binding, unfolded protein binding, RNA binding and DNA binding Journal of Proteome Research • Vol. 5, No. 9, 2006 2219
2220
P48722-00-00-00 gi|51708124 Q99KI0 P61979-0200-00 P61979 P61979-0200-00 Q9JHU9 P80317 P80316 P11983 P80316 P63038
P63038
P42932 P30416 gi|56270548 P56480 Q9WTM5 Q9JHX6 Q9Z2I9
Q921F2 Q922R8 Q61990-0200-00 O88544 P07724 Q9CQM9 P60335 Q9R1T2 Q9Z1Z2 P57776-0000-00 gi|31543902 P57776-0000-00 O88569 P17751 O35723 Q9JHI5 P19157
551 1052 1178 1455
1499 1516 1560 1586 1594 1603
1620
1624 1825 1909 2067 2125 2170 2553
2559 2681 2699
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COP9 signalosome complex subunit 4 serum albumin precursor thioredoxin-like protein 2 poly(rC)-binding protein 1 (hnRNP-E1) ubiquitin-like 1 activating enzyme E1A serine-threonine kinase receptor-associated protein splice isoform 1 from P57776 elongation factor 1-delta thioredoxin-like 1 splice isoform 1 from P57776 elongation factor 1-delta heterogeneous nuclear ribonucleoproteins A2/B1 triosephosphate isomerase DnaJ homologue subfamily B member 3 isovaleryl dehydrogenase precursor glutathione S-transferase P 1
splice isoform 1 P48722 Heat shock 70 kDa protein 4L PREDICTED: similar to GMP synthase aconitate hydratase, mitochondrial precursor splice isoform 3 from P61979 Heterogeneous nuclear ribonucleoprotein K heterogeneous nuclear ribonucleoprotein K splice isoform 2 from P61979 heterogeneous nuclear ribonucleoprotein K myo-inositol 1-phosphate synthase A1 CCT-zeta-1 CCT-epsilon CCT-alpha CCT-epsilon 60 kDa heat shock protein, mitochondrial precursor (Hsp60) 60 kDa heat shock protein, mitochondrial precursor (Hsp60) CCT-theta FK506-binding protein 4 aldehyde dehydrogenase 1 family, member B1 ATP synthase beta chain, mitochondrial precursor ruvB-like 2 allantoicase succinyl-CoA ligase [ADP-forming] beta-chain, mitochondrial precursor TAR DNA-binding protein-43 txndc7 protein putative heterogeneous nuclear ribonucleoprotein X
protein
0.5 h (n ) 3)
0.097 ( 0.013 0.072 ( 0.009 0.004 ( 0.001 0.086 ( 0.012 0.047 ( 0.009 0.065 ( 0.019* 0.044 ( 0.021 0.146 ( 0.009 0.215 ( 0.024 0.139 ( 0.008 0.065 ( 0.007* 0.113 ( 0.044 0.020 ( 0.010 0.052 ( 0.022 0.046 ( 0.024 0.093 ( 0.011 0.209 ( 0.027* 0.088 ( 0.010 0.020 ( 0.004* 0.133 ( 0.067 0.047 ( 0.002 0.022 ( 0.005 0.115 ( 0.014 0.077 ( 0.006 0.031 ( 0.011 0.132 ( 0.006 0.167 ( 0.048 0.058 ( 0.002 0.098 ( 0.005 0.077 ( 0.039 0.077 ( 0.039 0.041 ( 0.021 0.060 ( 0.021 0.021 ( 0.005 0.035 ( 0.008 0.033 ( 0.005 0.032 ( 0.009
control (n ) 3)
0.113 ( 0.019 0.085 ( 0.002 0.003 ( 0.001 0.106 ( 0.021 0.058 ( 0.009 0.100 ( 0.006 0.025 ( 0.005 0.162 ( 0.023 0.231 ( 0.013 0.186 ( 0.033 0.047 ( 0.008 0.153 ( 0.007 0.037 ( 0.010 0.110 ( 0.043 0.054 ( 0.005 0.091 ( 0.001 0.310 ( 0.050 0.126 ( 0.012 0.013 ( 0.002 0.217 ( 0.021 0.047 ( 0.002 0.044 ( 0.013 0.129 ( 0.006 0.069 ( 0.007 0.029 ( 0.006 0.160 ( 0.017 0.182 ( 0.008 0.072 ( 0.013 0.086 ( 0.021 0.103 ( 0.007 0.103 ( 0.007 0.025 ( 0.008 0.083 ( 0.029 0.019 ( 0.004 0.027 ( 0.006 0.036 ( 0.003 0.013 ( 0.011
0.059 ( 0.026 0.026 ( 0.005 0.045 ( 0.016* 0.052 ( 0.005* 0.058 ( 0.028
0.063 ( 0.037 0.054 ( 0.031
0.071 ( 0.009 0.032 ( 0.018 0.154 ( 0.012 0.163 ( 0.036 0.065 ( 0.030 0.086 ( 0.002 0.063 ( 0.037
0.037 ( 0.007 0.021 ( 0.008 0.128 ( 0.006
0.034 ( 0.009 0.032 ( 0.014 0.099 ( 0.019 0.151 ( 0.073* 0.101 ( 0.021 0.025 ( 0.007* 0.199 ( 0.059
0.020 ( 0.005
0.041 ( 0.003* 0.151 ( 0.021 0.179 ( 0.037 0.136 ( 0.007 0.063 ( 0.006* 0.108 ( 0.033
0.036 ( 0.014 0.052 ( 0.015*
0.062 ( 0.048 0.077 ( 0.013 0.005 ( 0.002 0.053 ( 0.034
2h (n ) 3)
0.051 ( 0.008 0.029 ( 0.018 0.044 ( 0.002* 0.038 ( 0.005 0.042 ( 0.008*
0.067 ( 0.042 0.035 ( 0.007
0.074 ( 0.006 0.034 ( 0.005 0.128 ( 0.028 0.123 ( 0.022* 0.049 ( 0.003 0.088 ( 0.000 0.067 ( 0.042
0.032 ( 0.009* 0.013 ( 0.010* 0.086 ( 0.025*
0.028 ( 0.010 0.032 ( 0.004 0.085 ( 0.019 0.201 ( 0.039* 0.070 ( 0.003 0.022 ( 0.001* 0.140 ( 0.037*
0.023 ( 0.008
0.035 ( 0.003* 0.142 ( 0.005 0.156 ( 0.064 0.124 ( 0.053 0.066 ( 0.008* 0.109 ( 0.034
0.040 ( 0.009 0.067 ( 0.031*
0.087 ( 0.003 0.060 ( 0.004* 0.003 ( 0.001 0.079 ( 0.031
6h (n ) 3)
0.018 ( 0.002* 0.036 ( 0.002* 0.074 ( 0.026* 0.040 ( 0.001 0.050 ( 0.005*
0.046 ( 0.010* 0.049 ( 0.004*
0.043 ( 0.010* 0.067 ( 0.023* 0.114 ( 0.014* 0.116 ( 0.009* 0.036 ( 0.007* 0.050 ( 0.012* 0.046 ( 0.010*
0.030 ( 0.017 0.040 ( 0.024 0.094 ( 0.014*
0.018 ( 0.007* 0.034 ( 0.017* 0.069 ( 0.012* 0.109 ( 0.067* 0.051 ( 0.010* 0.028 ( 0.001* 0.129 ( 0.079
0.014 ( 0.006*
0.032 ( 0.008 0.109 ( 0.016* 0.139 ( 0.006* 0.114 ( 0.009* 0.053 ( 0.009 0.091 ( 0.020*
0.018 ( 0.010* 0.036 ( 0.008*
0.052 ( 0.007* 0.062 ( 0.004* 0.006 ( 0.001* 0.051 ( 0.018*
12 h (n ) 3)
down up up up up
down up
down up down down down down down
down down/upd down
down down down down down up down
down
up down down down down down
down down
down down up down
overall trendc
a To reflect quantitative variations in the volume of protein spots, spot volumes were normalized as a percentage of the total volume of all spots present in a gel. The direction of protein regulation between control and 12 h is indicated by overall trend. Values are presented as mean ( SEM, *p < 0.05 compared to the normal group. b The nos. refer to the spot numbers as given in Figure 1. c The direction of protein regulation between control and 12 h is indicated by overall trend. d The overall trend of spot 2681 “down/up” means that this protein spots were down regulated between the control and 6 h following heat treatment and then up regulated.
3271 3380 3750 3944 3966
3232 3258
2752 2778 2787 2794 2893 2946 3232
1456 1467
accession no.
spot IDb
Table 1. Summary of Significantly Regulated Proteins Following Heat Treatmenta
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Proteomics of Mouse Testis following Heat Shock
Figure 2. Morphology of normal and heat shock (42 °C) testes was revealed by hematoxylin & eosin staining. The name of the cell type was denoted by its abbreviation. Sg, spermatogonia; Sc, spermatocyte; Sd, spermatid; Se, Sertoli cell; Le, Leydig cell. Morphological examination of testes harvested 0.5h after a 42 °C heat shock treatment revealed no substantial morphologic differences from the normal testis. Tubules contained apoptotic cells (white arrows) 2 h after heat shock. Spermatocytes showed intracytoplasmic hyalinosis (white arrows) and many spermatocyte nuclei contained highly condensed chromatin (pynotic nuclei) (black arrows) 6h after heat shock. Tubules contained degenerated spermatocytes (white arrows) and coagulated spermatids 12 h after heat shock. Bar ) 50 µm.
Figure 3. Molecular functions were assigned to the above categories. Differentially expressed proteins were classified by FatiGO procedure according to proteins’ molecular functions. Framed are the same proteins between two categories. Table 2. “RNA Binding”-Associated Proteins That Were Significantly Regulated During the 12 h Following Heat-Treatment and Classified by FatiGoa According to Their Molecular Functions accession no.
O88569 P60335 Q61990 Q921F2 P61979 a
proteins
heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1) poly(rC)-binding protein 1 (hnRNP-E1) heterogeneous nuclear ribonucleoprotein X (hnRNP X) TAR DNA-binding protein-43 heterogeneous nuclear ribonucleoprotein K (hnRNP K)
Table 3. “Unfolded Protein Binding”-Associated Proteins Which Were Significantly Regulated During the 12 h Following Heat-Treatment and Classified by FatiGoa According to Their Molecular Functions accession no.
P42932 P11983 P48722 O35723 P80316 P80317 P63038 a
proteins
CCT-theta CCT-alpha Heat shock 70 kDa protein 4L DnaJ homologue subfamily B member 3 CCT-epsilon CCT-zeta-1 60 kDa heat shock protein
http://fatigo.bioinfo.cnio.es.
http://fatigo.bioinfo.cnio.es.
(Figure 3). In fact, there are five same proteins between the groups of purine nucleotide binding and unfolded protein binding, four same proteins between the groups of RNA binding and DNA binding. Because “unfolded protein binding” proteins18 and “RNA binding” proteins19 play important roles during the process of spermatogenesis and spermiogenesis, they were further assessed in the present study. Five downregulated proteins were associated with mRNA binding, including four members of hnRNPs (hnRNP A2/B1, hnRNP-E1,
hnRNP X, and hnRNP K) and TAR DNA-binding protein-43 (TDP43, Table 2). TDP43, also a highly conserved heterogeneous nuclear ribonucleoprotein,20 was down-regulated from 6 h after heat shock. Seven proteins involved in unfolded protein binding, including four members of CCT family (CCTtheta, CCT-alpha, CCT-epsilon (spot 1560), and CCT-zeta-1), heat shock 70 kDa protein 4L, DnaJ homologue subfamily B member 3, and 60 kDa heat shock protein (Table 3) were downregulated in response to hyperthermia as well. However, another spot 1594, representing CCT-epsilon, was up-regulated Journal of Proteome Research • Vol. 5, No. 9, 2006 2221
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Figure 4. Typical kinetics of regulated proteins following heat treatment. The kinetics of expression change over time was depicted for selected proteins. (A) Regulation of hnRNPs members. (B) Regulation of CCT subunits. Lines connected the average of normalized abundance values. *Significant difference from control (P
