Chemotherapeutic Drugs Alter Functional Properties and Proteome of Mouse Testicular Germ Cells In Vitro

Chemotherapeutic Drugs Alter Functional Properties and Proteome of Mouse Testicular Germ Cells In... Abstract Many of the testicular cancer-survived patients, treated with chemotherapeutic drugs, show infertility, pre and postimplantation loss, and germ cell abnormality. Studies examining the negative effects of chemotherapeutic drugs on testicular germ cells are ongoing; however, information on the stemness properties and proteomic profiles of these cells are lacking. This study investigated the effects of chemotherapeutic drugs etoposide, cisplatin, bleomycin, and their combination (BEP) on the physiology and stem cell activity of mouse germ cells in vitro. Our results showed that treatment with the abovementioned drugs affected germ cell viability and decreased the number of proliferating germ cells significantly at specific concentrations (0.05 µM etoposide, 1 µM cisplatin, 10 µM bleomycin, and 0.1 µM BEP), which maintained a survival rate of >90%. We also observed a significantly higher percentage of apoptotic cells and alterations in the expression of undifferentiated and differentiated spermatogonia-related genes and marker proteins in germ cells exposed to abovementioned concentrations of the drugs. Next, we performed germ cell transplantation into recipient mice and observed a remarkable reduction in stemness properties of spermatogonial stem cells at these concentrations. Based on these results, we assessed the levels of differentially expressed proteins by performing proteomic analysis. We found that treatment with the abovementioned drugs induced cell damage, oxidative stress, metabolic disruption, and immune deficiency which may promote tumor regeneration, cytotoxicity, infertility, and transgenerational cellular function transmission. Thus, this study provides information about the chemotherapy-induced recurrent destruction and thereby can lead possible changes in medication. chemotherapeutic drugs, testicular germ cell culture, SSC functions, transplantation, proteomics Most of the testicular tumor patients suffer from the disease at reproductive age (15–40 years) (Chia et al., 2010). So, monotherapy or combination therapy with chemotherapeutic drugs such as etoposide, cisplatin, and bleomycin has become more popular over radiation and surgery to preserve fertility in these patients (Hanna and Einhorn, 2014). Chemotherapeutic drugs considerably decrease the uncontrolled growth of testicular germ cells along with spermatogonial stem cells (SSCs) by blocking their division at the gene level and/or by inducing programmed cell death (Huddart et al., 1995). SSCs, the foundation of male fertility (Oatley and Brinster, 2006), undergo self-renewal and differentiation into other cell types through mitosis and meiosis to produce spermatozoa as the final product. Therefore, exposure of chemotherapeutic drugs to SSCs can damage their characteristics, which may persist throughout the lifetime of an individual and may be transmitted to his progeny and subsequent generations. Most studies have used whole-animal models to investigate the toxic effects of chemotherapeutic drugs on spermatogenesis and stem/progenitor spermatogonia (Marcon et al., 2008, 2011; Meistrich et al., 1982). Although these studies provide important toxicological information on anticancer drugs in the physiological context, they do not indicate the degree of cell-specific effect of these drugs. In vivo studies have shown that bleomycin, a cytotoxic and genotoxic antibiotic, induces chromosomal aberrations in spermatocytes (van Buul and Goudzwaard, 1980) and DNA strand break in male germ cells (Coogan et al., 1986). Similarly, etoposide, a topoisomerase inhibitor, induces DNA double-stranded breaks and chromosomal deformity in spermatogonia and spermatocytes (Palo et al., 2005). Cisplatin induces DNA crosslinking and interferes with cell division, thus promoting the apoptosis of SSCs (Harman and Richburg, 2014). Studies on rats have shown that combination treatment with bleomycin, etoposide, and cisplatin (BEP) decreases sperm production and undifferentiated spermatogonia number (Marcon et al., 2011). Furthermore, cell culture-based studies have shown increased sensitivity of testicular germ cells to drugs in vitro (Kupeli et al., 1997) and to drug induced apoptosis (Huddart et al., 1995) and decreased cluster number and surface area of spermatogonia after monotherapy and combination therapy with chemotherapeutic drugs (Marcon et al., 2010), thus providing information on the survival and proliferation of testicular germ cells. Moreover, some studies have reported mutations in drug-treated germ cells (Parris et al., 1990), anticancer agent induced DNA and telomere damage due to disruption of telomerase activity of SSCs (Liu et al., 2014), and overexpression of heat shock proteins (Richards et al., 1996). Studies performed in rats indicated that BEP treatment affects progeny outcome and alters sperm chromatin integrity and sperm head proteins (Maselli et al., 2012). Therefore, it is important to assess the effects of chemotherapeutic drugs on the functions and stemness properties of SSCs and on the expression of proteins associated with the physiology and genetics of germ cells. In this study, we first determined the effects of BEP and combination therapy on the viability of recovered testicular germ cells and on the number of proliferating germ cells when cell viability was high (>90%). We also determined apoptotic rate among the recovered germ cells, drugs related effects on the expression of selfrenewal- and differentiation-related genes and marker proteins to determine the physiological states of cultured spermatogonia. Moreover, we determined the number of functional SSCs by transplanting germ cells into recipient mice. Finally, we determined differentially expressed proteins associated with the functions of germ cells by performing proteomic analysis to assess proteome alteration related to male fertility and health hazards. Materials and Methods Reagents All reagents were purchased from Sigma-Aldrich (St Louis), unless otherwise indicated. Minimum essential medium α (Gibco, New York) was used for testicular germ cell culture with SSC specified bovine serum albumin (BSA) and Dulbecco’s modified eagle medium (Gibco) was used for SIM mouse embryo-derived thioguanine- and ouabain-resistant feeder cells. Chemotherapeutic agents or drugs named as bleomycin sulfate, etoposide and cisplatin were collected from LKT Laboratories (St Paul, Minnesota). BEP represents the combination of bleomycin, etoposide, and cisplatin, and it was prepared by mixing at equimolar concentrations of these 3 drugs to give a combined concentration ranging from 0.025 to 0.8 µM; thus, for 0.1 µM BEP, each drug was added at a concentration of 0.033 µM. All of the drugs were dissolved dimethyl sulfoxide (DMSO). Experimental animals and ethical approval Testicular germ cells were collected from the C57BL/6-TG-EGFP mice (designated C57-GFP; Jackson Laboratory, Bar Harbor, Maine). Wild type C57BL/6 mice were used as recipient and were obtained from Harlan Laboratories (Indianapolis, Indiana). All animals were cared for according to guidelines established by Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, and all animal protocols were approved by the Animal Care and Use Committee of Chung-Ang University (IACUC number: 2016-00009). Isolation of testicular germ cells by magnetic-activated cell sorting The detailed procedure of magnetic activated cell sorting (MACS) methodology is described in Supplementary Material “Materials and Methods” section. Germ cell culture and treatment of chemotherapeutic drugs After MACS, testicular germ cells were maintained in vitro culture. The detailed culture procedure is described in Supplementary Material “Materials and Methods” section. Chemotherapeutic drugs, both single and combination, were mixed with culture media and were applied on day 4 of plating. Effects were assessed after 48 h of drugs administration. The concentrations of each drug’s stock solutions were prepared as mM, so that it reached to µM after adding to media as 0.1% (v/v). Several concentrations of each drug were administrated to the germ cell cultures with a 2-fold increase pattern. Control culture groups received 0.1% DMSO only. Cell viability and proliferation analysis After chemotherapeutic drugs treatment, cultured germ cells were harvested by 0.25% trypsin-EDTA digestion at 37°C. Percentage of germ cell viability percentage was measured by trypan blue exclusion test (Strober, 2001). Several doses of each drug were applied until the viability of cultured germ cells was observed as 50% of control (IC50). Number of proliferated germ cells was then examined among these concentrations. It was observed that drug administrated germ cell number was reduced at approximately 50% of control at a specific concentration of each drug, in which the cell viability was >90%. Proliferation of drug-treated germ cells was measured by counting GFP-positive cells and was normalized using the following equation: Normalized data (%) = Number of recovered cells after treatment × 100/Number of recovered cells from control. Apoptosis assay Chemotherapeutic drug treatment commonly aims to reduce cell proliferation and growth. Therefore, it is necessary to determine whether chemotherapeutic drugs induce cell death through apoptosis and to measure the percentage of apoptotic cells among recovered cells. Apoptosis assay was performed immediately after harvesting the germ cells by using annexin V-phycoerythrin apoptosis kit (BD Biosciences, San Diego, California), according to the manufacturer’s protocol, with a slight modification (Lee et al., 2013). Briefly, the harvested germ cells were washed twice with chilled Dulbecco's phosphate buffered saline (DPBS) and were resuspended in 1× binding buffer to obtain a density of 1 × 105 cells/200 μl cell suspension. Next, 5 μl annexin V was added to the cells, and the cells were incubated in the dark for 15 min at room temperature (RT). Next, propidium iodide buffer was added to the cells at a final concentration of 5 μg/ml. Percentages of apoptotic cells were determined using fluorescence-activated cell sorting (FACS) Aria II automated flow cytometer (BD Biosciences) and were analyzed using BD FACS Diva software (BD Biosciences). RNA extraction, quantitative real-time PCR, and reverse transcription PCR These procedures are described in detail in Supplementary Material “Materials and Methods” section. Immunocytochemistry Germ cell-specific marker protein Dead box protein 4 (DDX4) or Vasa homolog (VASA) (Toyooka et al., 2000), marker proteins related to undifferentiated spermatogonia such as glial cell line-derived neurotrophic factor family receptor alpha-1 (GFRα1) (Buageaw et al., 2005) and promyelocytic leukemia zinc-finger (PLZF) (Buaas et al., 2004) have already been identified. Similarly, differentiated spermatogonia-related marker protein, tyrosine-protein kinase or C-KIT (Morimoto et al., 2009) has been described as a negative marker of undifferentiated spermatogonia. Therefore, immunocytochemistry was performed to analyze those marker proteins’ expression in drugs treated germ cells by using a protocol described previously (Kim et al., 2013). In brief, drug treated cultured germ cells were fixed on slides with 4% paraformaldehyde for 30 min, and were permeabilized with 0.1% Triton X-100 (DPBS dissolved) at RT for 10 min. DPBS containing 5% (w/v) BSA was used to block the cells at RT for 1 h, and treated with a rabbit antihuman DDX4 (VASA, 1:200; Abcam, Cambridge, UK), rabbit antihuman GFRα 1 (1:200; Abcam), mouse antihuman PLZF (1:200; EDM Millipore, Billerica, Massachusetts), and goat antimouse C-KIT (1:200, Santa Cruz Biotechnology, Texas) primary antibodies for 12 h at 4°C. Cells were then washed 3 times with DPBS and incubated at RT for 1 h with secondary antibodies such as TRITC-conjugated goat antirabbit IgG (1:200, Jackson ImmunoResearch, West Grove, Pennsylvania), Alexafluor 568-conjugated goat antimouse IgG (1:200, Life Technologies, USA), and Alexafluor 568-conjugated donkey antigoat IgG (1:200, Invitrogen, USA). Cells were washed 3 times with DPBS, and then mounted with VectaShield mounting media including 4′,6′-diamidino-2-phenylindole (DAPI). Marked cells were analyzed using a Nikon TE2000-U microscope installed NIS Elements imaging software (Nikon, Chiyoda-ku, Tokyo, Japan). Cultured germ cells transplantation into recipient testes and analysis of donor germ cell-derived colonies Although SSC-related genes and marker proteins expression give us an overview about the functional and stemness properties of cultured germ cell enriched SSCs, transplantation analysis till today is the only way to clearly quantify the amount of functional SSCs in a germ cell population. Therefore, transplantation of germ cells cultured and treated with chemotherapeutic drugs was performed into recipient mice testes. The detailed procedure of transplantation is described in Supplementary Material “Materials and Methods section”. 2D gel electrophoresis analysis of chemotherapeutic drug induced proteome alterations in germ cells and pathway studio The detailed procedures are described in Supplementary Material “Materials and Methods” section. Statistical analysis SPSS software (version 23, IBM Inc., New York) and Prism software (version 5.03; GraphPad, La Jolla, California) were used to manage all statistical analysis. Each experiment was carried out at least 3 times. Duncan’s multiple range test was used to consider differences in 1-way analysis of variance in all data. For identification of differences of each drug group with control in transplantation study, student’s 2-tailed t test was used. The level of significant difference was identified by p < .05, and the values are showed as mean ± SEM. RESULTS Viability of Cultured Germ Cells After Exposure to Chemotherapeutic Drugs Percentages of viability of drugs treated cultured germ cells were evaluated by trypan blue exclusion assay, which stains dead cells in blue because of the disruption of their cell membrane. We assessed the effects of different concentrations of each drug because they exerted different levels of toxicity. The highest concentration of each drug was considered as the half maximum inhibitory concentration (IC50) (Figure 1). At each treatment, cells were treated with 2-fold higher concentration of the drug than the previous concentration so that the decrease in cell viability could be measured at the finest level. Concentration ranges of etoposide, cisplatin, bleomycin, and BEP used in this study are 0.0125–0.4, 0.25–8, 2.5–80, and 0.025–0.8 µM, respectively. Figure 1. View largeDownload slide Viability status of testicular germ cells treated with the chemotherapeutic drugs in vitro. Germ cells were cultured and exposed to different concentrations of each of the drug at fourth day of postplating for 48 h. After treatment, germ cells were counted to measure the effect of drugs on germ cells viability. (A–D) represents the viability of cultured germ cell treated with etoposide, cisplatin, bleomycin, and combination of these drugs (BEP) respectively. Data are presented as means ± SEM of 4 independent experiments. Different letters (a, b, and c) indicate significant difference (p < .05) compared with control. Figure 1. View largeDownload slide Viability status of testicular germ cells treated with the chemotherapeutic drugs in vitro. Germ cells were cultured and exposed to different concentrations of each of the drug at fourth day of postplating for 48 h. After treatment, germ cells were counted to measure the effect of drugs on germ cells viability. (A–D) represents the viability of cultured germ cell treated with etoposide, cisplatin, bleomycin, and combination of these drugs (BEP) respectively. Data are presented as means ± SEM of 4 independent experiments. Different letters (a, b, and c) indicate significant difference (p < .05) compared with control. Proliferation Rate of Testicular Germ Cells Was Significantly Decreased After Treated With Particular Drug Concentrations Effects of the chemotherapeutic drugs on the proliferation of cultured germ cells were measured based on the number of harvested cells after drug treatment. Relative proliferation rates were determined by comparing the number of drug-treated cells with that of control cells for each treatment. Proliferation strategies were determined by selecting drug concentrations that maintained cell viability up to 90% (Supplementary Table 1). Interestingly, results of fluorescence microscopy showed a sequential decrease in colony formation with an increase in the concentration of each drug (Figure 2A). These results indicated that treatment with each drug concentration significantly reduced the relative proliferation rate of germ cells at specific concentration of each drug, resulting in almost 50% decrease in cell number (Figs. 2B–E) and these concentration ranges were selected for further experiments. Figure 2. View largeDownload slide In vitro proliferation of testicular germ cells after drugs’ exposure. A, Fluorescent microscopic image of in vitro proliferated testicular germ cell of GFP mouse treated with etoposide, cisplatin, bleomycin, and their combination (BEP). After treatment, germ cells were counted to find out the percentages of relative proliferation rate of drugs treated germ cells with control. (B–E) represents relative proliferation rate of germ cells treated with etoposide, cisplatin, bleomycin and BEP respectively. Values are represented mean ± SEM of 4 independent experiments and different letters (a and b) indicate significant difference (p < 0.05) compared with control. Scale bars in (A) = 200 μm. Figure 2. View largeDownload slide In vitro proliferation of testicular germ cells after drugs’ exposure. A, Fluorescent microscopic image of in vitro proliferated testicular germ cell of GFP mouse treated with etoposide, cisplatin, bleomycin, and their combination (BEP). After treatment, germ cells were counted to find out the percentages of relative proliferation rate of drugs treated germ cells with control. (B–E) represents relative proliferation rate of germ cells treated with etoposide, cisplatin, bleomycin and BEP respectively. Values are represented mean ± SEM of 4 independent experiments and different letters (a and b) indicate significant difference (p < 0.05) compared with control. Scale bars in (A) = 200 μm. Chemotherapeutic Drugs Induce Apoptosis in Cultured Germ Cells We observed significant decreases in germ cell proliferation after administration of chemotherapeutic drugs which led us to scrutinize the percentage of apoptotic cells among the proliferated germ cells. We analyzed this study with FACS (Figure 3A) and detected significantly higher percentages of apoptotic cells for etoposide and cisplatin, at 0.05 and 1 µM concentrations, respectively (Figs. 3B and 3C). No differences were observed for bleomycin and BEP (Figs. 3D and 3E). Controls were tested individually for each of chemotherapeutic agents. Figure 3. View largeDownload slide Effects of chemotherapeutic drugs on the apoptotic rate of testicular germ cells. A, Flow cytometric determination of the apoptotic rate of drugs treated cultured germ cells after stained with Annexin V/PI. Bar graphs (B–E) represent the percentages of apoptotic germ cells due to administration of etoposide, cisplatin, bleomycin, and BEP respectively. Values are represented mean ± SEM of 6 independent experiments. Different letters (a and b) indicate significant difference (p < .05) compared with control. Figure 3. View largeDownload slide Effects of chemotherapeutic drugs on the apoptotic rate of testicular germ cells. A, Flow cytometric determination of the apoptotic rate of drugs treated cultured germ cells after stained with Annexin V/PI. Bar graphs (B–E) represent the percentages of apoptotic germ cells due to administration of etoposide, cisplatin, bleomycin, and BEP respectively. Values are represented mean ± SEM of 6 independent experiments. Different letters (a and b) indicate significant difference (p < .05) compared with control. Estimation of SSC-Related Genes Expression Using Quantitative Real-Time PCR After Chemotherapeutic Drugs Treatment Relative mRNA expression levels of Lhx1 (LIM homeobox 1), Bcl6b (B-cell chronic lymphocytic leukemia/lymphoma 6, member B), Etv5 (Ets [E-26] variant gene 5), and Sohlh1 (spermatogenesis and oogenesis specific basic helix-loop-helix transcription factor) were determined by performing quantitative reverse transcription-PCR (qRT-PCR). Undifferentiated spermatogonia-related gene Lhx1 was downregulated in etoposide-, cisplatin-, and BEP-treated germ cells. Another undifferentiated spermatogonia-related gene Bcl6b was downregulated in BEP-treated germ cells (Supplementary Figure 1). Differentiating germ cell-related gene Sohlh1 was downregulated in only etoposide-treated germ cells (Supplementary Figure 1). However, undifferentiated spermatogonia-related gene Etv5 did not show any significant differences in etoposide-, cisplatin-, bleomycin-, and BEP-treated germ cells (Supplementary Figure S1). Immunocytochemical Analysis of Cultured Germ Cells Exposed to the Chemotherapeutic Drugs Next, we assessed the expression of undifferentiated and differentiated spermatogonia-specific marker proteins in cultured cells exposed to the chemotherapeutic drugs by performing ICC analysis (Supplementary Figure S2). We analyzed the percentages of drugs treated germ cells expressing undifferentiated spermatogonia marker proteins such as PLZF and GFRα1, germ cell-specific marker protein VASA, and differentiated spermatogonia marker protein C-KIT. Only the percentages of C-KIT expressions were observed significantly high in case of etoposide-, cisplatin-, bleomycin-, and BEP-treated germ cells (Figure 4). We also evaluated a differentiated spermatogonia related gene, Stra8, by reverse transcription-polymerase chain reaction (RT-PCR) (Supplementary Figure 3). A very little expression (PCR bands) was visualized in the gel for Stra8 in case of the highest concentrations of drugs. Otherwise, expression percentages of PLZF, GFRα1, and VASA were observed unchanged among the chemotherapeutic drugs-treated germ cells. Figure 4. View largeDownload slide Immunocytochemistry assay of germ cells treated with chemotherapeutic drugs. Treated germ cells were immunostained with undifferentiated spermatogonia related marker proteins PLZF and GFRα1, germ cell-specific marker protein VASA, and differentiated spermatogonia related marker proteins C-KIT. The figure represents bar graph presentations of the percentage of undifferentiation and differentiation related marker proteins expressing cells after treated with etoposide, cisplatin, bleomycin, and BEP. Values are represented mean ± SEM of 5 independent experiments. Different letters (a, b) indicate significant difference (p < .05) compared with control. Figure 4. View largeDownload slide Immunocytochemistry assay of germ cells treated with chemotherapeutic drugs. Treated germ cells were immunostained with undifferentiated spermatogonia related marker proteins PLZF and GFRα1, germ cell-specific marker protein VASA, and differentiated spermatogonia related marker proteins C-KIT. The figure represents bar graph presentations of the percentage of undifferentiation and differentiation related marker proteins expressing cells after treated with etoposide, cisplatin, bleomycin, and BEP. Values are represented mean ± SEM of 5 independent experiments. Different letters (a, b) indicate significant difference (p < .05) compared with control. Chemotherapeutic Drug Treatment Affected Stemness Properties of SSCs Although results of ICC analysis and qRT-PCR indicated alterations in germ cell properties such as undifferentiation and differentiation after exposure to the chemotherapeutic drugs in vitro, these results could not provide an exact measure of changes in the functions of SSCs. Therefore, transplanted germ cells treated with the highest concentrations of each drug were injected into the testes of recipient mice and colonies were visualized under fluorescent microscope after 2 months of transplantation (Figure 5A). We observed significant reductions in both of the number of donor germ cell-derived colonies per 105 transplanted germ cells and the number of colonies per total number of cultured cells due to the treatment of the drugs (Figs. 5B and 5C). Moreover, germ cells treated with 10 µM bleomycin formed significantly less number of colonies than germ cells treated with 0.05 μM etoposide and 0.1 µM BEP. Thus, these results indicate that treatment of SSCs with high concentration of each drug (0.05 μΜ etoposide, 1 μΜ cisplatin, 10 μΜ bleomycin, and 0.1 μM BEP) affected their stemness properties. Figure 5. View largeDownload slide Effects of chemotherapeutic agents on the stemness properties of SSCs. Stemness properties of SSCs were evaluated by counting donor germ cell-derived colonies from recipient testes. Cultured germ cells treated with 0.05 µM etoposide, 1 µM cisplatin, 10 µM bleomycin, and 0.1 µM BEP were transplanted and colonies were counted after 2 months of transplantation. A, Testes of recipient showing GFP expressing colonies of donor SSCs. Scale bar = 2 mm. B, The number of colonies per 105 transplanted cells and (C) the number of colonies per total number of cultured germ cells. Values are represented mean ± SEM. Total numbers of mice/testes analyzed were 7/13, 9/18, 10/19, 11/20, and 9/17 for control, 0.05 μΜ etoposide, 1 μΜ cisplatin, 10 μΜ bleomycin, and 0.1 μM BEP, respectively. *compared with treatment groups where p < .05 and *** compared with control where p < .001. Figure 5. View largeDownload slide Effects of chemotherapeutic agents on the stemness properties of SSCs. Stemness properties of SSCs were evaluated by counting donor germ cell-derived colonies from recipient testes. Cultured germ cells treated with 0.05 µM etoposide, 1 µM cisplatin, 10 µM bleomycin, and 0.1 µM BEP were transplanted and colonies were counted after 2 months of transplantation. A, Testes of recipient showing GFP expressing colonies of donor SSCs. Scale bar = 2 mm. B, The number of colonies per 105 transplanted cells and (C) the number of colonies per total number of cultured germ cells. Values are represented mean ± SEM. Total numbers of mice/testes analyzed were 7/13, 9/18, 10/19, 11/20, and 9/17 for control, 0.05 μΜ etoposide, 1 μΜ cisplatin, 10 μΜ bleomycin, and 0.1 μM BEP, respectively. *compared with treatment groups where p < .05 and *** compared with control where p < .001. Differentially Expressed Proteins in Chemotherapeutic Drug-Treated Germ Cells Two-dimensional gel electrophoresis (2-DE) was performed to compare the proteomic profiles of germ cells treated with the chemotherapeutic drugs. For this, germ cells were treated with the highest concentration of each drug. Almost 314 protein spots were detected in all gels. Of these, 63 spots showed a significant difference in protein density compared with those obtained for control cells. We identified 47 differentially expressed proteins from these 63 spots, of which 15 proteins were differentially expressed in etoposide-treated cells, 11 proteins were differentially expressed in cisplatin-treated cells, 8 proteins were differentially expressed in bleomycin-treated cells, and 13 proteins were differentially expressed in BEP-treated cells. Moreover, differential expression of some proteins found common for 2 or 3 chemotherapeutic drugs. Thus, 38 different proteins were specified in total (Supplementary Figure 4). We identified several proteome changes with respect to cell proliferation, oxidative stress, energy metabolism, immune response, and sex development (Table 1). Etoposide and cisplatin decreased the expression of cell proliferation-related proteins and increased the expression of oxidative stress-related proteins (Table 1). Bleomycin and BEP treatment affected energy metabolism (Table 1). Table 1. Proteins With Significantly Lower or Higher Expressions in Chemotherapeutic Drugs Treated Germ Cells and Control Groups Symbol  Protein ID (swissprot/ncbi)  gi no.  MASCOT Scorea  Relative Intensity (Normalized)b   Control  Etoposide 0.05 μM  Cisplatin 1 μM  Bleomycin 10 μM  BEP 0.1 μM  Cell proliferation  NAP1L1  Nucleosome assembly protein 1  gi|148689782  79  1  2.22 ± 0.76  1.35 ± 0.34  0.47 ± 0.05*  0.59 ± 0.24  ANXA7  Annexin A7  gi|160707956  155  1  0.84 ± 0.27  0.75 ± 0.48  0.87 ± 0.29  0.48 ± 0.10*  HNRNPH2  Nuclear ribonucleoprotein H2  gi|922960031  69  1  0.36 ± 0.07*  0.67 ± 0.06  0.57 ± 0.20  0.56 ± 0.32  EFTU  Elongation factor Tu, mitochondrial  gi|27370092  295  1  0.43 ± 0.03*  0.54 ± 0.15  0.53 ± 0.20  1.48 ± 0.38  STIP1  Stress-induced-phosphoprotein 1  gi|14389431  205  1  3.65 ± 0.42*  3.63 ± 1.60  2.22 ± 0.17  1.77 ± 0.97  HNRNPA2B1  Heterogeneous nuclear ribonucleoproteins  gi|32880197  113  1  0.97 ± 0.19  0.46 ± 0.12*  0.63 ± 0.08  0.72 ± 0.21  PABPC1  Polyadenylate-binding protein 1  gi|53754  96  1  0.02 ± 0.01*  0.03 ± 0.02*  0.94 ± 0.93  0.56 ± 0.25  ROS metabolism/oxidative stress  LEG1  Galectin  gi|6678682  223  1  0.45 ± 0.06  0.65 ± 0.20*  0.76 ± 0.19  0.53 ± 0.10  TXNL1  Thioredoxin-like protein 1  gi|31543902  162  1  0.48 ± 0.04*  0.40 ± 0.15  0.52 ± 0.23  1.42 ± 0.16  HSPE1  10 kDa heat shock protein, mitochondrial  gi|6680309  132  1  4.53 ± 0.89  2.39 ± 0.76  2.75 ± 0.05*  1.74 ± 0.47  HSPE1  10 kDa heat shock protein, mitochondrial  gi|6680309  93  1  3.86 ± 0.63*  3.28 ± 0.49*  4.36 ± 0.91  4.75 ± 0.90  PRDX1  Peroxiredoxin-1  gi|6754976  107  1  1.60 ± 1.03  0.14 ± 0.08*  0.38 ± 0.23  1.27 ± 0.80  PPID  Peptidyl-prolyl cis-trans isomerase D  gi|13385854  77  1  0.62 ± 0.20  0.49 ± 0.08*  1.34 ± 0.36  0.88 ± 0.30  PRDX1  Peroxiredoxin-1  gi|6754976  299  1  0.25 ± 0.04*  0.71 ± 0.28  0.59 ± 0.18  0.97 ± 0.70  Immune system  PSMA1  Proteasome subunit alpha type  gi| 33563282  69  1  0.62 ± 0.22  0.30 ± 0.15*  0.65 ± 0.04  1.11 ± 0.22  PCBP2  Poly(rC)-binding protein 2  gi|157041229  46  1  0.70 ± 0.10  0.58 ± 0.13  1.24 ± 0.38  0.29 ± 0.08*  Sex development  FKBP4  Peptidyl-prolyl cis-trans isomerase  gi|6753882  214  1  0.59 ± 0.28  0.32 ± 0.03*  0.33 ± 0.17  0.36 ± 0.20  ANXA2  Annexin A2  gi|6996913  83  1  1.18 ± 0.65  1.20 ± 0.24  1.36 ± 0.66  0.24 ± 0.11*  DPYSL2  Dihydropyrimidinase-related protein 2  gi|40254595  166  1  0.29 ± 0.15*  0.47 ± 0.19  0.67 ± 0.31  0.97 ± 0.31  Energy metabolism  EEF1A1  Elongation factor 1  gi|148697551  100  1  0.53 ± 0.16  0.36 ± 0.10  1.10 ± 0.29*  0.53 ± 0.11  TCPQ  T-complex protein 1 subunit  gi|50510319  491  1  0.93 ± 0.27  0.34 ± 0.29  0.57 ± 0.31  0.19 ± 0.12*  ATP6V1A  V-type proton ATPase catalytic subunit A  gi|31560731  135  1  0.28 ± 0.02*  1.04 ± 0.31  0.19 ± 0.08*  0.12 ± 0.04*  TCP1  T-complex protein 1 subunit alpha  gi|201725  104  1  1.07 ± 0.27  1.23 ± 0.78  1.29 ± 0.65  0.35 ± 0.03*  ATP5I  ATP synthase subunit e, mitochondrial  gi|83715998  104  1  4.70 ± 1.17  2.44 ± 1.58  7.01 ± 3.73  6.20 ± 0.34*  ATP5O  ATP synthase subunit  gi|20070412  68  1  0.08 ± 0.08*  0.51 ± 0.45  0.06 ± 0.05*  2.24 ± 0.94  PSD13  26S proteasome  gi|6755210  163  1  1.20 ± 0.51  0.51 ± 0.05  0.89 ± 0.18  0.34 ± 0.15*  OAT  Ornithine aminotransferase  gi|8393866  136  1  0.33 ± 0.13  0.74 ± 0.36*  1.31 ± 0.44  0.25 ± 0.15*  DLAT  Dihydrolipoyllysine-residue acetyltransferase  gi|16580128  116  1  0.74 ± 0.02  0.71 ± 0.09  0.31 ± 0.07*  0.96 ± 0.22  THOP1  Thimet oligopeptidase  gi|239916005  303  1  0.61 ± 0.29  0.13 ± 0.01  0.02 ± 0.01*  0.31 ± 0.21  GANAB  Neutral alpha-glucosidase  gi|148701451  166  1  0.20 ± 0.07*  0.78 ± 0.33  1.16 ± 0.93  0.48 ± 0.10*  NIT2  Omega-amidase  gi|12963555  60  1  0.90 ± 0.15  0.18 ± 0.10*  0.60 ± 0.19  0.15 ± 0.01*  DLD  Dihydrolipoyl dehydrogenase, mitochondrial  gi|31982856  97  1  1.98 ± 1.34  2.21 ± 0.39  4.52 ± 0.57*  2.85 ± 1.43  ENO1  Alpha-enolase  gi|158853992  68  1  0.42 ± 0.06*  1.07 ± 0.64  0.67 ± 0.21  0.49 ± 0.24  ME2  NAD-dependent malic enzyme  gi|21703972  152  1  0.29 ± 0.10*  0.74 ± 0.10  1.38 ± 0.98  0.50 ± 0.29  ALDOC  Fructose-bisphosphate aldolase C  gi|742670581  98  1  0.66 ± 0.27  0.48 ± 0.19  0.91 ± 0.18  0.36 ± 0.04*  GAPDH  Glyceraldehyde-3-phosphate dehydrogenase  gi|309243  95  1  4.01 ± 0.50*  3.40 ± 2.31  2.34 ± 1.24  1.28 ± 0.32  MDH2  Malate dehydrogenase, mitochondrial  gi|89574115  133  1  0.37 ± 0.09*  0.53 ± 0.03  0.73 ± 0.26  0.42 ± 0.06*  EEF1A1  Elongation factor-1 alpha  gi|1220410  80  1  1.70 ± 0.34  3.47 ± 0.33*  1.51 ± 0.58  1.61 ± 0.33  Symbol  Protein ID (swissprot/ncbi)  gi no.  MASCOT Scorea  Relative Intensity (Normalized)b   Control  Etoposide 0.05 μM  Cisplatin 1 μM  Bleomycin 10 μM  BEP 0.1 μM  Cell proliferation  NAP1L1  Nucleosome assembly protein 1  gi|148689782  79  1  2.22 ± 0.76  1.35 ± 0.34  0.47 ± 0.05*  0.59 ± 0.24  ANXA7  Annexin A7  gi|160707956  155  1  0.84 ± 0.27  0.75 ± 0.48  0.87 ± 0.29  0.48 ± 0.10*  HNRNPH2  Nuclear ribonucleoprotein H2  gi|922960031  69  1  0.36 ± 0.07*  0.67 ± 0.06  0.57 ± 0.20  0.56 ± 0.32  EFTU  Elongation factor Tu, mitochondrial  gi|27370092  295  1  0.43 ± 0.03*  0.54 ± 0.15  0.53 ± 0.20  1.48 ± 0.38  STIP1  Stress-induced-phosphoprotein 1  gi|14389431  205  1  3.65 ± 0.42*  3.63 ± 1.60  2.22 ± 0.17  1.77 ± 0.97  HNRNPA2B1  Heterogeneous nuclear ribonucleoproteins  gi|32880197  113  1  0.97 ± 0.19  0.46 ± 0.12*  0.63 ± 0.08  0.72 ± 0.21  PABPC1  Polyadenylate-binding protein 1  gi|53754  96  1  0.02 ± 0.01*  0.03 ± 0.02*  0.94 ± 0.93  0.56 ± 0.25  ROS metabolism/oxidative stress  LEG1  Galectin  gi|6678682  223  1  0.45 ± 0.06  0.65 ± 0.20*  0.76 ± 0.19  0.53 ± 0.10  TXNL1  Thioredoxin-like protein 1  gi|31543902  162  1  0.48 ± 0.04*  0.40 ± 0.15  0.52 ± 0.23  1.42 ± 0.16  HSPE1  10 kDa heat shock protein, mitochondrial  gi|6680309  132  1  4.53 ± 0.89  2.39 ± 0.76  2.75 ± 0.05*  1.74 ± 0.47  HSPE1  10 kDa heat shock protein, mitochondrial  gi|6680309  93  1  3.86 ± 0.63*  3.28 ± 0.49*  4.36 ± 0.91  4.75 ± 0.90  PRDX1  Peroxiredoxin-1  gi|6754976  107  1  1.60 ± 1.03  0.14 ± 0.08*  0.38 ± 0.23  1.27 ± 0.80  PPID  Peptidyl-prolyl cis-trans isomerase D  gi|13385854  77  1  0.62 ± 0.20  0.49 ± 0.08*  1.34 ± 0.36  0.88 ± 0.30  PRDX1  Peroxiredoxin-1  gi|6754976  299  1  0.25 ± 0.04*  0.71 ± 0.28  0.59 ± 0.18  0.97 ± 0.70  Immune system  PSMA1  Proteasome subunit alpha type  gi| 33563282  69  1  0.62 ± 0.22  0.30 ± 0.15*  0.65 ± 0.04  1.11 ± 0.22  PCBP2  Poly(rC)-binding protein 2  gi|157041229  46  1  0.70 ± 0.10  0.58 ± 0.13  1.24 ± 0.38  0.29 ± 0.08*  Sex development  FKBP4  Peptidyl-prolyl cis-trans isomerase  gi|6753882  214  1  0.59 ± 0.28  0.32 ± 0.03*  0.33 ± 0.17  0.36 ± 0.20  ANXA2  Annexin A2  gi|6996913  83  1  1.18 ± 0.65  1.20 ± 0.24  1.36 ± 0.66  0.24 ± 0.11*  DPYSL2  Dihydropyrimidinase-related protein 2  gi|40254595  166  1  0.29 ± 0.15*  0.47 ± 0.19  0.67 ± 0.31  0.97 ± 0.31  Energy metabolism  EEF1A1  Elongation factor 1  gi|148697551  100  1  0.53 ± 0.16  0.36 ± 0.10  1.10 ± 0.29*  0.53 ± 0.11  TCPQ  T-complex protein 1 subunit  gi|50510319  491  1  0.93 ± 0.27  0.34 ± 0.29  0.57 ± 0.31  0.19 ± 0.12*  ATP6V1A  V-type proton ATPase catalytic subunit A  gi|31560731  135  1  0.28 ± 0.02*  1.04 ± 0.31  0.19 ± 0.08*  0.12 ± 0.04*  TCP1  T-complex protein 1 subunit alpha  gi|201725  104  1  1.07 ± 0.27  1.23 ± 0.78  1.29 ± 0.65  0.35 ± 0.03*  ATP5I  ATP synthase subunit e, mitochondrial  gi|83715998  104  1  4.70 ± 1.17  2.44 ± 1.58  7.01 ± 3.73  6.20 ± 0.34*  ATP5O  ATP synthase subunit  gi|20070412  68  1  0.08 ± 0.08*  0.51 ± 0.45  0.06 ± 0.05*  2.24 ± 0.94  PSD13  26S proteasome  gi|6755210  163  1  1.20 ± 0.51  0.51 ± 0.05  0.89 ± 0.18  0.34 ± 0.15*  OAT  Ornithine aminotransferase  gi|8393866  136  1  0.33 ± 0.13  0.74 ± 0.36*  1.31 ± 0.44  0.25 ± 0.15*  DLAT  Dihydrolipoyllysine-residue acetyltransferase  gi|16580128  116  1  0.74 ± 0.02  0.71 ± 0.09  0.31 ± 0.07*  0.96 ± 0.22  THOP1  Thimet oligopeptidase  gi|239916005  303  1  0.61 ± 0.29  0.13 ± 0.01  0.02 ± 0.01*  0.31 ± 0.21  GANAB  Neutral alpha-glucosidase  gi|148701451  166  1  0.20 ± 0.07*  0.78 ± 0.33  1.16 ± 0.93  0.48 ± 0.10*  NIT2  Omega-amidase  gi|12963555  60  1  0.90 ± 0.15  0.18 ± 0.10*  0.60 ± 0.19  0.15 ± 0.01*  DLD  Dihydrolipoyl dehydrogenase, mitochondrial  gi|31982856  97  1  1.98 ± 1.34  2.21 ± 0.39  4.52 ± 0.57*  2.85 ± 1.43  ENO1  Alpha-enolase  gi|158853992  68  1  0.42 ± 0.06*  1.07 ± 0.64  0.67 ± 0.21  0.49 ± 0.24  ME2  NAD-dependent malic enzyme  gi|21703972  152  1  0.29 ± 0.10*  0.74 ± 0.10  1.38 ± 0.98  0.50 ± 0.29  ALDOC  Fructose-bisphosphate aldolase C  gi|742670581  98  1  0.66 ± 0.27  0.48 ± 0.19  0.91 ± 0.18  0.36 ± 0.04*  GAPDH  Glyceraldehyde-3-phosphate dehydrogenase  gi|309243  95  1  4.01 ± 0.50*  3.40 ± 2.31  2.34 ± 1.24  1.28 ± 0.32  MDH2  Malate dehydrogenase, mitochondrial  gi|89574115  133  1  0.37 ± 0.09*  0.53 ± 0.03  0.73 ± 0.26  0.42 ± 0.06*  EEF1A1  Elongation factor-1 alpha  gi|1220410  80  1  1.70 ± 0.34  3.47 ± 0.33*  1.51 ± 0.58  1.61 ± 0.33  a MASCOT score means −10 log (p), where p represents the probability that the observed match is a random event. Individual scores > 30 indicate identity or extensive homology (p < .05). b Relative spots intensity between control and drugs treated cultured germ cells. Data are presented as mean ± SEM (3 replicates). Values with Asterisk (*) indicate significant differences between the control and each of the treatment groups as determined by t test (p < .05). Table 1. Proteins With Significantly Lower or Higher Expressions in Chemotherapeutic Drugs Treated Germ Cells and Control Groups Symbol  Protein ID (swissprot/ncbi)  gi no.  MASCOT Scorea  Relative Intensity (Normalized)b   Control  Etoposide 0.05 μM  Cisplatin 1 μM  Bleomycin 10 μM  BEP 0.1 μM  Cell proliferation  NAP1L1  Nucleosome assembly protein 1  gi|148689782  79  1  2.22 ± 0.76  1.35 ± 0.34  0.47 ± 0.05*  0.59 ± 0.24  ANXA7  Annexin A7  gi|160707956  155  1  0.84 ± 0.27  0.75 ± 0.48  0.87 ± 0.29  0.48 ± 0.10*  HNRNPH2  Nuclear ribonucleoprotein H2  gi|922960031  69  1  0.36 ± 0.07*  0.67 ± 0.06  0.57 ± 0.20  0.56 ± 0.32  EFTU  Elongation factor Tu, mitochondrial  gi|27370092  295  1  0.43 ± 0.03*  0.54 ± 0.15  0.53 ± 0.20  1.48 ± 0.38  STIP1  Stress-induced-phosphoprotein 1  gi|14389431  205  1  3.65 ± 0.42*  3.63 ± 1.60  2.22 ± 0.17  1.77 ± 0.97  HNRNPA2B1  Heterogeneous nuclear ribonucleoproteins  gi|32880197  113  1  0.97 ± 0.19  0.46 ± 0.12*  0.63 ± 0.08  0.72 ± 0.21  PABPC1  Polyadenylate-binding protein 1  gi|53754  96  1  0.02 ± 0.01*  0.03 ± 0.02*  0.94 ± 0.93  0.56 ± 0.25  ROS metabolism/oxidative stress  LEG1  Galectin  gi|6678682  223  1  0.45 ± 0.06  0.65 ± 0.20*  0.76 ± 0.19  0.53 ± 0.10  TXNL1  Thioredoxin-like protein 1  gi|31543902  162  1  0.48 ± 0.04*  0.40 ± 0.15  0.52 ± 0.23  1.42 ± 0.16  HSPE1  10 kDa heat shock protein, mitochondrial  gi|6680309  132  1  4.53 ± 0.89  2.39 ± 0.76  2.75 ± 0.05*  1.74 ± 0.47  HSPE1  10 kDa heat shock protein, mitochondrial  gi|6680309  93  1  3.86 ± 0.63*  3.28 ± 0.49*  4.36 ± 0.91  4.75 ± 0.90  PRDX1  Peroxiredoxin-1  gi|6754976  107  1  1.60 ± 1.03  0.14 ± 0.08*  0.38 ± 0.23  1.27 ± 0.80  PPID  Peptidyl-prolyl cis-trans isomerase D  gi|13385854  77  1  0.62 ± 0.20  0.49 ± 0.08*  1.34 ± 0.36  0.88 ± 0.30  PRDX1  Peroxiredoxin-1  gi|6754976  299  1  0.25 ± 0.04*  0.71 ± 0.28  0.59 ± 0.18  0.97 ± 0.70  Immune system  PSMA1  Proteasome subunit alpha type  gi| 33563282  69  1  0.62 ± 0.22  0.30 ± 0.15*  0.65 ± 0.04  1.11 ± 0.22  PCBP2  Poly(rC)-binding protein 2  gi|157041229  46  1  0.70 ± 0.10  0.58 ± 0.13  1.24 ± 0.38  0.29 ± 0.08*  Sex development  FKBP4  Peptidyl-prolyl cis-trans isomerase  gi|6753882  214  1  0.59 ± 0.28  0.32 ± 0.03*  0.33 ± 0.17  0.36 ± 0.20  ANXA2  Annexin A2  gi|6996913  83  1  1.18 ± 0.65  1.20 ± 0.24  1.36 ± 0.66  0.24 ± 0.11*  DPYSL2  Dihydropyrimidinase-related protein 2  gi|40254595  166  1  0.29 ± 0.15*  0.47 ± 0.19  0.67 ± 0.31  0.97 ± 0.31  Energy metabolism  EEF1A1  Elongation factor 1  gi|148697551  100  1  0.53 ± 0.16  0.36 ± 0.10  1.10 ± 0.29*  0.53 ± 0.11  TCPQ  T-complex protein 1 subunit  gi|50510319  491  1  0.93 ± 0.27  0.34 ± 0.29  0.57 ± 0.31  0.19 ± 0.12*  ATP6V1A  V-type proton ATPase catalytic subunit A  gi|31560731  135  1  0.28 ± 0.02*  1.04 ± 0.31  0.19 ± 0.08*  0.12 ± 0.04*  TCP1  T-complex protein 1 subunit alpha  gi|201725  104  1  1.07 ± 0.27  1.23 ± 0.78  1.29 ± 0.65  0.35 ± 0.03*  ATP5I  ATP synthase subunit e, mitochondrial  gi|83715998  104  1  4.70 ± 1.17  2.44 ± 1.58  7.01 ± 3.73  6.20 ± 0.34*  ATP5O  ATP synthase subunit  gi|20070412  68  1  0.08 ± 0.08*  0.51 ± 0.45  0.06 ± 0.05*  2.24 ± 0.94  PSD13  26S proteasome  gi|6755210  163  1  1.20 ± 0.51  0.51 ± 0.05  0.89 ± 0.18  0.34 ± 0.15*  OAT  Ornithine aminotransferase  gi|8393866  136  1  0.33 ± 0.13  0.74 ± 0.36*  1.31 ± 0.44  0.25 ± 0.15*  DLAT  Dihydrolipoyllysine-residue acetyltransferase  gi|16580128  116  1  0.74 ± 0.02  0.71 ± 0.09  0.31 ± 0.07*  0.96 ± 0.22  THOP1  Thimet oligopeptidase  gi|239916005  303  1  0.61 ± 0.29  0.13 ± 0.01  0.02 ± 0.01*  0.31 ± 0.21  GANAB  Neutral alpha-glucosidase  gi|148701451  166  1  0.20 ± 0.07*  0.78 ± 0.33  1.16 ± 0.93  0.48 ± 0.10*  NIT2  Omega-amidase  gi|12963555  60  1  0.90 ± 0.15  0.18 ± 0.10*  0.60 ± 0.19  0.15 ± 0.01*  DLD  Dihydrolipoyl dehydrogenase, mitochondrial  gi|31982856  97  1  1.98 ± 1.34  2.21 ± 0.39  4.52 ± 0.57*  2.85 ± 1.43  ENO1  Alpha-enolase  gi|158853992  68  1  0.42 ± 0.06*  1.07 ± 0.64  0.67 ± 0.21  0.49 ± 0.24  ME2  NAD-dependent malic enzyme  gi|21703972  152  1  0.29 ± 0.10*  0.74 ± 0.10  1.38 ± 0.98  0.50 ± 0.29  ALDOC  Fructose-bisphosphate aldolase C  gi|742670581  98  1  0.66 ± 0.27  0.48 ± 0.19  0.91 ± 0.18  0.36 ± 0.04*  GAPDH  Glyceraldehyde-3-phosphate dehydrogenase  gi|309243  95  1  4.01 ± 0.50*  3.40 ± 2.31  2.34 ± 1.24  1.28 ± 0.32  MDH2  Malate dehydrogenase, mitochondrial  gi|89574115  133  1  0.37 ± 0.09*  0.53 ± 0.03  0.73 ± 0.26  0.42 ± 0.06*  EEF1A1  Elongation factor-1 alpha  gi|1220410  80  1  1.70 ± 0.34  3.47 ± 0.33*  1.51 ± 0.58  1.61 ± 0.33  Symbol  Protein ID (swissprot/ncbi)  gi no.  MASCOT Scorea  Relative Intensity (Normalized)b   Control  Etoposide 0.05 μM  Cisplatin 1 μM  Bleomycin 10 μM  BEP 0.1 μM  Cell proliferation  NAP1L1  Nucleosome assembly protein 1  gi|148689782  79  1  2.22 ± 0.76  1.35 ± 0.34  0.47 ± 0.05*  0.59 ± 0.24  ANXA7  Annexin A7  gi|160707956  155  1  0.84 ± 0.27  0.75 ± 0.48  0.87 ± 0.29  0.48 ± 0.10*  HNRNPH2  Nuclear ribonucleoprotein H2  gi|922960031  69  1  0.36 ± 0.07*  0.67 ± 0.06  0.57 ± 0.20  0.56 ± 0.32  EFTU  Elongation factor Tu, mitochondrial  gi|27370092  295  1  0.43 ± 0.03*  0.54 ± 0.15  0.53 ± 0.20  1.48 ± 0.38  STIP1  Stress-induced-phosphoprotein 1  gi|14389431  205  1  3.65 ± 0.42*  3.63 ± 1.60  2.22 ± 0.17  1.77 ± 0.97  HNRNPA2B1  Heterogeneous nuclear ribonucleoproteins  gi|32880197  113  1  0.97 ± 0.19  0.46 ± 0.12*  0.63 ± 0.08  0.72 ± 0.21  PABPC1  Polyadenylate-binding protein 1  gi|53754  96  1  0.02 ± 0.01*  0.03 ± 0.02*  0.94 ± 0.93  0.56 ± 0.25  ROS metabolism/oxidative stress  LEG1  Galectin  gi|6678682  223  1  0.45 ± 0.06  0.65 ± 0.20*  0.76 ± 0.19  0.53 ± 0.10  TXNL1  Thioredoxin-like protein 1  gi|31543902  162  1  0.48 ± 0.04*  0.40 ± 0.15  0.52 ± 0.23  1.42 ± 0.16  HSPE1  10 kDa heat shock protein, mitochondrial  gi|6680309  132  1  4.53 ± 0.89  2.39 ± 0.76  2.75 ± 0.05*  1.74 ± 0.47  HSPE1  10 kDa heat shock protein, mitochondrial  gi|6680309  93  1  3.86 ± 0.63*  3.28 ± 0.49*  4.36 ± 0.91  4.75 ± 0.90  PRDX1  Peroxiredoxin-1  gi|6754976  107  1  1.60 ± 1.03  0.14 ± 0.08*  0.38 ± 0.23  1.27 ± 0.80  PPID  Peptidyl-prolyl cis-trans isomerase D  gi|13385854  77  1  0.62 ± 0.20  0.49 ± 0.08*  1.34 ± 0.36  0.88 ± 0.30  PRDX1  Peroxiredoxin-1  gi|6754976  299  1  0.25 ± 0.04*  0.71 ± 0.28  0.59 ± 0.18  0.97 ± 0.70  Immune system  PSMA1  Proteasome subunit alpha type  gi| 33563282  69  1  0.62 ± 0.22  0.30 ± 0.15*  0.65 ± 0.04  1.11 ± 0.22  PCBP2  Poly(rC)-binding protein 2  gi|157041229  46  1  0.70 ± 0.10  0.58 ± 0.13  1.24 ± 0.38  0.29 ± 0.08*  Sex development  FKBP4  Peptidyl-prolyl cis-trans isomerase  gi|6753882  214  1  0.59 ± 0.28  0.32 ± 0.03*  0.33 ± 0.17  0.36 ± 0.20  ANXA2  Annexin A2  gi|6996913  83  1  1.18 ± 0.65  1.20 ± 0.24  1.36 ± 0.66  0.24 ± 0.11*  DPYSL2  Dihydropyrimidinase-related protein 2  gi|40254595  166  1  0.29 ± 0.15*  0.47 ± 0.19  0.67 ± 0.31  0.97 ± 0.31  Energy metabolism  EEF1A1  Elongation factor 1  gi|148697551  100  1  0.53 ± 0.16  0.36 ± 0.10  1.10 ± 0.29*  0.53 ± 0.11  TCPQ  T-complex protein 1 subunit  gi|50510319  491  1  0.93 ± 0.27  0.34 ± 0.29  0.57 ± 0.31  0.19 ± 0.12*  ATP6V1A  V-type proton ATPase catalytic subunit A  gi|31560731  135  1  0.28 ± 0.02*  1.04 ± 0.31  0.19 ± 0.08*  0.12 ± 0.04*  TCP1  T-complex protein 1 subunit alpha  gi|201725  104  1  1.07 ± 0.27  1.23 ± 0.78  1.29 ± 0.65  0.35 ± 0.03*  ATP5I  ATP synthase subunit e, mitochondrial  gi|83715998  104  1  4.70 ± 1.17  2.44 ± 1.58  7.01 ± 3.73  6.20 ± 0.34*  ATP5O  ATP synthase subunit  gi|20070412  68  1  0.08 ± 0.08*  0.51 ± 0.45  0.06 ± 0.05*  2.24 ± 0.94  PSD13  26S proteasome  gi|6755210  163  1  1.20 ± 0.51  0.51 ± 0.05  0.89 ± 0.18  0.34 ± 0.15*  OAT  Ornithine aminotransferase  gi|8393866  136  1  0.33 ± 0.13  0.74 ± 0.36*  1.31 ± 0.44  0.25 ± 0.15*  DLAT  Dihydrolipoyllysine-residue acetyltransferase  gi|16580128  116  1  0.74 ± 0.02  0.71 ± 0.09  0.31 ± 0.07*  0.96 ± 0.22  THOP1  Thimet oligopeptidase  gi|239916005  303  1  0.61 ± 0.29  0.13 ± 0.01  0.02 ± 0.01*  0.31 ± 0.21  GANAB  Neutral alpha-glucosidase  gi|148701451  166  1  0.20 ± 0.07*  0.78 ± 0.33  1.16 ± 0.93  0.48 ± 0.10*  NIT2  Omega-amidase  gi|12963555  60  1  0.90 ± 0.15  0.18 ± 0.10*  0.60 ± 0.19  0.15 ± 0.01*  DLD  Dihydrolipoyl dehydrogenase, mitochondrial  gi|31982856  97  1  1.98 ± 1.34  2.21 ± 0.39  4.52 ± 0.57*  2.85 ± 1.43  ENO1  Alpha-enolase  gi|158853992  68  1  0.42 ± 0.06*  1.07 ± 0.64  0.67 ± 0.21  0.49 ± 0.24  ME2  NAD-dependent malic enzyme  gi|21703972  152  1  0.29 ± 0.10*  0.74 ± 0.10  1.38 ± 0.98  0.50 ± 0.29  ALDOC  Fructose-bisphosphate aldolase C  gi|742670581  98  1  0.66 ± 0.27  0.48 ± 0.19  0.91 ± 0.18  0.36 ± 0.04*  GAPDH  Glyceraldehyde-3-phosphate dehydrogenase  gi|309243  95  1  4.01 ± 0.50*  3.40 ± 2.31  2.34 ± 1.24  1.28 ± 0.32  MDH2  Malate dehydrogenase, mitochondrial  gi|89574115  133  1  0.37 ± 0.09*  0.53 ± 0.03  0.73 ± 0.26  0.42 ± 0.06*  EEF1A1  Elongation factor-1 alpha  gi|1220410  80  1  1.70 ± 0.34  3.47 ± 0.33*  1.51 ± 0.58  1.61 ± 0.33  a MASCOT score means −10 log (p), where p represents the probability that the observed match is a random event. Individual scores > 30 indicate identity or extensive homology (p < .05). b Relative spots intensity between control and drugs treated cultured germ cells. Data are presented as mean ± SEM (3 replicates). Values with Asterisk (*) indicate significant differences between the control and each of the treatment groups as determined by t test (p < .05). Based on the STRING system, functional protein association networks were also predicted among the identified proteins (Supplementary Figure 5). The protein-protein interactions were observed as gene cooccurrence, gene neighborhood, coexpression and gene fusions. It indicates that alteration in expression of any protein could be the reason of up- or down-regulation of connected proteins and these consequences may cause impact on cellular physiology. Some proteins that are directly linked with cell signaling, cell growth, apoptosis, and disease-related pathways were selected from each drug and were plotted using Pathway Studio to determine their coexpression and interrelationship with other cellular proteins (Supplementary Figs. 6A–D). Supplementary Figure 7 illustrates the accumulated consequences of the chemotherapeutic drugs derived from Pathway Studios and shows the drugs related effects on cellular processes and possible occurrences of diseases. Discussion As the main function of chemotherapeutic drugs is to reduce tumor growth and block activities associated with new carcinogenesis, exposure of germ cells to chemotherapeutic drug in vitro might decrease their viability. In present study, we treated germ cells with different concentrations of etoposide, cisplatin, and bleomycin and their combination (BEP) and observed almost 50% decreases in cell viability (IC50) after treatment with the highest concentrations of these drugs (Figure 1). Moreover, we observed a sharp dose-dependent decrease in the number of proliferating germ cells after treatment with each drug (Figure 2) and almost 50% proliferating germ cells compared with control were recovered after treatment with each drug at concentrations that maintained cell viability of >90% (Supplementary Table 1). High percentage of viable cells (>90%) among cultured cells indicates a good physiological state of the cells in vitro. Therefore, we considered those concentrations from each drug (0.0125–0.05 µM for etoposide, 0.25–1 µM for cisplatin, 2.5–10 µM for bleomycin, and 0.025–0.1 µM for BEP) where cell proliferation assessed up to half of control groups, and examined to measure the stem cell functions and proteome alteration of SSCs/progenitor spermatogonia. Percentages of apoptotic cells were measured among chemotherapeutic drugs treated proliferating germ cells to determine overall status of cell survival. Some studies indicate that etoposide induces apoptosis of seminiferous epithelial cells in rats (Stumpp et al., 2004) and cisplatin can decreases the proliferation of germ cells (Olive and Banath, 2009). Similarly, we observed significantly higher percentages of apoptotic cells in case of etoposide- and cisplatin-treated germ cell (Figs. 3B and 3C). Bleomycin and BEP did not induce significantly higher percentages of apoptotic cells. We performed qRT-PCR to determine the mRNA expression of undifferentiated spermatogonia-related genes in cells treated with the chemotherapeutic drugs in vitro. The mRNA expression of Lhx1, Bcl6b, and Etv5 in undifferentiated spermatogonia indicates their normal self-renewal in vitro (Oatley and Brinster, 2008; Song and Wilkinson, 2014). In this study, we observed that etoposide, cisplatin, and BEP treatment downregulated the mRNA expression of Lhx1 in germ cells compared with that in control cells, indicating that these drugs hampered the self-renewal characteristics of germ cells. Expression of Bcl6b and Etv5 was unchanged in germ cells treated with all the drugs, except BEP (Supplementary Figure 1), indicating that the combination treatment with the 3 drugs strongly inhibited the proliferation of germ cells in vitro. Sohlh1 encodes a germ cell-specific transcription factor necessary for spermatogonial differentiation (Ballow et al., 2006). Thus, Sohlh1 is a negative marker gene for in vitro proliferation of germ cells because growth factors of culture system always keep the cells in undifferentiated state. In this study, we observed that Sohlh1 expression decreased in germ cells treated with etoposide but remained unchanged in cells treated with the other drugs (Supplementary Figure 1). These findings indicate that chemotherapeutic drugs are able to alter the expression of essential genes in germ cells in vitro. Based on the results of gene expression analysis, we examined the levels of marker proteins associated with undifferentiated spermatogonia and differentiated germ cells. ICC analysis was performed to determine the levels of undifferentiated spermatogonia-specific marker proteins PLZF and GFRα1, and germ cell-specific marker VASA. Although results of qRT-PCR showed alterations in the expression of undifferentiated spermatogonia-associated genes after etoposide, cisplatin, and bleomycin treatment, results of ICC analysis did not show any changes in the levels of undifferentiated spermatogonia-associated marker proteins. Expression of some undifferentiated and germ cell-specific marker proteins decreased in a dose-dependent manner after chemotherapeutic drug treatment (Figure 4); however, this decrease was not statistically significant. These results indicated that treatment with the chemotherapeutic drugs did not alter the levels of major marker proteins in cultured germ cells. Next, we examined the expression pattern of differentiated germ cell-specific marker C-KIT and Stra8. As a result, very minute expression was observed for Stra8 in RT-PCR, it may be due to the exposure of the highest concentration of drugs (Oatley et al., 2006) (Supplementary Figure 3). Although only bleomycin and BEP at highest concentration showed little expression for Stra8, C-KIT showed a different scenario. C-KIT expression showed a significant dose-dependent increase in all drug-treated cells. Thus, our ICC data indicate that chemotherapeutic drug treatments may provoke cultured germ cells to in vitro differentiation. As the changes in gene and protein expression could not clarify the effect of the chemotherapeutic drugs on SSC functions and stemness properties, we transplanted drug-treated germ cells into recipient mice. To date, germ cell transplantation into recipient mice is the most reliable method to estimate the exact proportion of SSCs in a cell population. We observed alterations in the expressions of undifferentiated and differentiated spermatogonia related genes and marker proteins at the highest doses among the selected concentrations of these drugs (0.05 µM etoposide, 1 µM cisplatin, 10 µM bleomycin, and 0.1 µM BEP). So, we transplanted germ cells treated with abovementioned drugs’ doses into recipient mice. Our transplantation data showed that stem cell activity in SSCs was decreased significantly after in vitro treatment with chemotherapeutic drugs. Therefore, chemotherapeutic drugs treatment can alter stemness properties and functional abilities of SSCs. Finally, we examined the alterations in proteomic profiles of germ cells treated with the chemotherapeutic drugs. We observed that 0.05 µM etoposide, 1 µM cisplatin, 10 µM bleomycin, and 0.1 µM BEP altered the proteomic profiles of cultured germ cells. Therefore, we used the same drug concentrations for performing 2-DE. We examined 38 differentially expressed proteins, most of which performed special roles in the physiology of germ cells. We observed that expression of proteins associated with cell proliferation and energy metabolism decreased and that of proteins associated with reactive oxygen species increased in cells treated with the chemotherapeutic drugs (Table 1 and Supplementary Figure 6). Some of the identified proteins like polyadenylate-binding protein 1, annexin A7, heterogeneous nuclear ribonucleoproteins, peptidyl-prolyl cis-trans isomerase D, peroxiredoxin 1 exert crucial roles in cell physiology (Brownawell and Creutz, 1997; Choi et al., 2012; El Eter and Al-Masri, 2015; Gray et al., 2015; Yao et al., 2005). Moreover, expression of some proteins associated with sex-related development were down-regulated in drug-treated germ cells, indicating constraints in fetal development and infertility (Atchison and Means, 2004; Wang et al., 2015). Functional protein association networks were also predicted among the 2-DE identified proteins based on the STRING program which indicate the interrelationship of almost all of the drug- induced differentially expressed proteins (Supplementary Figure 5). The relationship reveals the occurrences of simultaneous up- and down-regulation of linked proteins due to chemotherapeutic administration that could be devastating for health. Finally, proteins related to cell functions and processes, and associated with disease development are summarized in Supplementary Figure 7. Although this illustrates enhancement of several important cellular activities like ROS generation, mRNA degradation, cell cycle, cell growth, autophagy, protein refolding, and Ca2+/glucose transport, these drug-related effects could ultimately trigger on reproductive health by blocking spermatogenesis, sperm motility, X chromosome inactivation and oocyte maturation (Supplementary Figure 7). To the best of our knowledge, this is the first comprehensive in vitro study to examine the effects of chemotherapeutic drugs on the survival, proliferation, and apoptosis of and alterations in the stemness properties and proteomic profiles of testicular germ cells. We selected etoposide, cisplatin, and bleomycin in this study because these drugs are frequently used for treating most patients with testicular cancers. We administered these drugs for a short period (48 h) and found that the lowest concentration of each drug induced minute changes in germ cells. Our results suggest that treatment with even low doses of these chemotherapeutic drugs alters the expression of SSC self-renewal- and differentiation-related genes and marker proteins. Moreover, these drug concentrations altered the stemness properties, characteristics, and proteomic profiles of germ cells. The cumulative effect of these drug concentrations may persist for a long period, resulting in cellular dysfunction and diseases, including infertility, and may be transmitted transgenerationally. Thus, the present study provides information on the effect of different concentrations of chemotherapeutic drugs on male germ cells and fertility status of patients with testicular cancers for developing new strategies for treating these patients. Furthermore, results of this in vitro study will help in evaluating and prescreening chemotherapeutic drugs before their clinical administrations. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. FUNDING This study was supported by grants from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM4251824), Republic of Korea. 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Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Toxicological Sciences Oxford University Press

Chemotherapeutic Drugs Alter Functional Properties and Proteome of Mouse Testicular Germ Cells In Vitro

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Abstract

Abstract Many of the testicular cancer-survived patients, treated with chemotherapeutic drugs, show infertility, pre and postimplantation loss, and germ cell abnormality. Studies examining the negative effects of chemotherapeutic drugs on testicular germ cells are ongoing; however, information on the stemness properties and proteomic profiles of these cells are lacking. This study investigated the effects of chemotherapeutic drugs etoposide, cisplatin, bleomycin, and their combination (BEP) on the physiology and stem cell activity of mouse germ cells in vitro. Our results showed that treatment with the abovementioned drugs affected germ cell viability and decreased the number of proliferating germ cells significantly at specific concentrations (0.05 µM etoposide, 1 µM cisplatin, 10 µM bleomycin, and 0.1 µM BEP), which maintained a survival rate of >90%. We also observed a significantly higher percentage of apoptotic cells and alterations in the expression of undifferentiated and differentiated spermatogonia-related genes and marker proteins in germ cells exposed to abovementioned concentrations of the drugs. Next, we performed germ cell transplantation into recipient mice and observed a remarkable reduction in stemness properties of spermatogonial stem cells at these concentrations. Based on these results, we assessed the levels of differentially expressed proteins by performing proteomic analysis. We found that treatment with the abovementioned drugs induced cell damage, oxidative stress, metabolic disruption, and immune deficiency which may promote tumor regeneration, cytotoxicity, infertility, and transgenerational cellular function transmission. Thus, this study provides information about the chemotherapy-induced recurrent destruction and thereby can lead possible changes in medication. chemotherapeutic drugs, testicular germ cell culture, SSC functions, transplantation, proteomics Most of the testicular tumor patients suffer from the disease at reproductive age (15–40 years) (Chia et al., 2010). So, monotherapy or combination therapy with chemotherapeutic drugs such as etoposide, cisplatin, and bleomycin has become more popular over radiation and surgery to preserve fertility in these patients (Hanna and Einhorn, 2014). Chemotherapeutic drugs considerably decrease the uncontrolled growth of testicular germ cells along with spermatogonial stem cells (SSCs) by blocking their division at the gene level and/or by inducing programmed cell death (Huddart et al., 1995). SSCs, the foundation of male fertility (Oatley and Brinster, 2006), undergo self-renewal and differentiation into other cell types through mitosis and meiosis to produce spermatozoa as the final product. Therefore, exposure of chemotherapeutic drugs to SSCs can damage their characteristics, which may persist throughout the lifetime of an individual and may be transmitted to his progeny and subsequent generations. Most studies have used whole-animal models to investigate the toxic effects of chemotherapeutic drugs on spermatogenesis and stem/progenitor spermatogonia (Marcon et al., 2008, 2011; Meistrich et al., 1982). Although these studies provide important toxicological information on anticancer drugs in the physiological context, they do not indicate the degree of cell-specific effect of these drugs. In vivo studies have shown that bleomycin, a cytotoxic and genotoxic antibiotic, induces chromosomal aberrations in spermatocytes (van Buul and Goudzwaard, 1980) and DNA strand break in male germ cells (Coogan et al., 1986). Similarly, etoposide, a topoisomerase inhibitor, induces DNA double-stranded breaks and chromosomal deformity in spermatogonia and spermatocytes (Palo et al., 2005). Cisplatin induces DNA crosslinking and interferes with cell division, thus promoting the apoptosis of SSCs (Harman and Richburg, 2014). Studies on rats have shown that combination treatment with bleomycin, etoposide, and cisplatin (BEP) decreases sperm production and undifferentiated spermatogonia number (Marcon et al., 2011). Furthermore, cell culture-based studies have shown increased sensitivity of testicular germ cells to drugs in vitro (Kupeli et al., 1997) and to drug induced apoptosis (Huddart et al., 1995) and decreased cluster number and surface area of spermatogonia after monotherapy and combination therapy with chemotherapeutic drugs (Marcon et al., 2010), thus providing information on the survival and proliferation of testicular germ cells. Moreover, some studies have reported mutations in drug-treated germ cells (Parris et al., 1990), anticancer agent induced DNA and telomere damage due to disruption of telomerase activity of SSCs (Liu et al., 2014), and overexpression of heat shock proteins (Richards et al., 1996). Studies performed in rats indicated that BEP treatment affects progeny outcome and alters sperm chromatin integrity and sperm head proteins (Maselli et al., 2012). Therefore, it is important to assess the effects of chemotherapeutic drugs on the functions and stemness properties of SSCs and on the expression of proteins associated with the physiology and genetics of germ cells. In this study, we first determined the effects of BEP and combination therapy on the viability of recovered testicular germ cells and on the number of proliferating germ cells when cell viability was high (>90%). We also determined apoptotic rate among the recovered germ cells, drugs related effects on the expression of selfrenewal- and differentiation-related genes and marker proteins to determine the physiological states of cultured spermatogonia. Moreover, we determined the number of functional SSCs by transplanting germ cells into recipient mice. Finally, we determined differentially expressed proteins associated with the functions of germ cells by performing proteomic analysis to assess proteome alteration related to male fertility and health hazards. Materials and Methods Reagents All reagents were purchased from Sigma-Aldrich (St Louis), unless otherwise indicated. Minimum essential medium α (Gibco, New York) was used for testicular germ cell culture with SSC specified bovine serum albumin (BSA) and Dulbecco’s modified eagle medium (Gibco) was used for SIM mouse embryo-derived thioguanine- and ouabain-resistant feeder cells. Chemotherapeutic agents or drugs named as bleomycin sulfate, etoposide and cisplatin were collected from LKT Laboratories (St Paul, Minnesota). BEP represents the combination of bleomycin, etoposide, and cisplatin, and it was prepared by mixing at equimolar concentrations of these 3 drugs to give a combined concentration ranging from 0.025 to 0.8 µM; thus, for 0.1 µM BEP, each drug was added at a concentration of 0.033 µM. All of the drugs were dissolved dimethyl sulfoxide (DMSO). Experimental animals and ethical approval Testicular germ cells were collected from the C57BL/6-TG-EGFP mice (designated C57-GFP; Jackson Laboratory, Bar Harbor, Maine). Wild type C57BL/6 mice were used as recipient and were obtained from Harlan Laboratories (Indianapolis, Indiana). All animals were cared for according to guidelines established by Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, and all animal protocols were approved by the Animal Care and Use Committee of Chung-Ang University (IACUC number: 2016-00009). Isolation of testicular germ cells by magnetic-activated cell sorting The detailed procedure of magnetic activated cell sorting (MACS) methodology is described in Supplementary Material “Materials and Methods” section. Germ cell culture and treatment of chemotherapeutic drugs After MACS, testicular germ cells were maintained in vitro culture. The detailed culture procedure is described in Supplementary Material “Materials and Methods” section. Chemotherapeutic drugs, both single and combination, were mixed with culture media and were applied on day 4 of plating. Effects were assessed after 48 h of drugs administration. The concentrations of each drug’s stock solutions were prepared as mM, so that it reached to µM after adding to media as 0.1% (v/v). Several concentrations of each drug were administrated to the germ cell cultures with a 2-fold increase pattern. Control culture groups received 0.1% DMSO only. Cell viability and proliferation analysis After chemotherapeutic drugs treatment, cultured germ cells were harvested by 0.25% trypsin-EDTA digestion at 37°C. Percentage of germ cell viability percentage was measured by trypan blue exclusion test (Strober, 2001). Several doses of each drug were applied until the viability of cultured germ cells was observed as 50% of control (IC50). Number of proliferated germ cells was then examined among these concentrations. It was observed that drug administrated germ cell number was reduced at approximately 50% of control at a specific concentration of each drug, in which the cell viability was >90%. Proliferation of drug-treated germ cells was measured by counting GFP-positive cells and was normalized using the following equation: Normalized data (%) = Number of recovered cells after treatment × 100/Number of recovered cells from control. Apoptosis assay Chemotherapeutic drug treatment commonly aims to reduce cell proliferation and growth. Therefore, it is necessary to determine whether chemotherapeutic drugs induce cell death through apoptosis and to measure the percentage of apoptotic cells among recovered cells. Apoptosis assay was performed immediately after harvesting the germ cells by using annexin V-phycoerythrin apoptosis kit (BD Biosciences, San Diego, California), according to the manufacturer’s protocol, with a slight modification (Lee et al., 2013). Briefly, the harvested germ cells were washed twice with chilled Dulbecco's phosphate buffered saline (DPBS) and were resuspended in 1× binding buffer to obtain a density of 1 × 105 cells/200 μl cell suspension. Next, 5 μl annexin V was added to the cells, and the cells were incubated in the dark for 15 min at room temperature (RT). Next, propidium iodide buffer was added to the cells at a final concentration of 5 μg/ml. Percentages of apoptotic cells were determined using fluorescence-activated cell sorting (FACS) Aria II automated flow cytometer (BD Biosciences) and were analyzed using BD FACS Diva software (BD Biosciences). RNA extraction, quantitative real-time PCR, and reverse transcription PCR These procedures are described in detail in Supplementary Material “Materials and Methods” section. Immunocytochemistry Germ cell-specific marker protein Dead box protein 4 (DDX4) or Vasa homolog (VASA) (Toyooka et al., 2000), marker proteins related to undifferentiated spermatogonia such as glial cell line-derived neurotrophic factor family receptor alpha-1 (GFRα1) (Buageaw et al., 2005) and promyelocytic leukemia zinc-finger (PLZF) (Buaas et al., 2004) have already been identified. Similarly, differentiated spermatogonia-related marker protein, tyrosine-protein kinase or C-KIT (Morimoto et al., 2009) has been described as a negative marker of undifferentiated spermatogonia. Therefore, immunocytochemistry was performed to analyze those marker proteins’ expression in drugs treated germ cells by using a protocol described previously (Kim et al., 2013). In brief, drug treated cultured germ cells were fixed on slides with 4% paraformaldehyde for 30 min, and were permeabilized with 0.1% Triton X-100 (DPBS dissolved) at RT for 10 min. DPBS containing 5% (w/v) BSA was used to block the cells at RT for 1 h, and treated with a rabbit antihuman DDX4 (VASA, 1:200; Abcam, Cambridge, UK), rabbit antihuman GFRα 1 (1:200; Abcam), mouse antihuman PLZF (1:200; EDM Millipore, Billerica, Massachusetts), and goat antimouse C-KIT (1:200, Santa Cruz Biotechnology, Texas) primary antibodies for 12 h at 4°C. Cells were then washed 3 times with DPBS and incubated at RT for 1 h with secondary antibodies such as TRITC-conjugated goat antirabbit IgG (1:200, Jackson ImmunoResearch, West Grove, Pennsylvania), Alexafluor 568-conjugated goat antimouse IgG (1:200, Life Technologies, USA), and Alexafluor 568-conjugated donkey antigoat IgG (1:200, Invitrogen, USA). Cells were washed 3 times with DPBS, and then mounted with VectaShield mounting media including 4′,6′-diamidino-2-phenylindole (DAPI). Marked cells were analyzed using a Nikon TE2000-U microscope installed NIS Elements imaging software (Nikon, Chiyoda-ku, Tokyo, Japan). Cultured germ cells transplantation into recipient testes and analysis of donor germ cell-derived colonies Although SSC-related genes and marker proteins expression give us an overview about the functional and stemness properties of cultured germ cell enriched SSCs, transplantation analysis till today is the only way to clearly quantify the amount of functional SSCs in a germ cell population. Therefore, transplantation of germ cells cultured and treated with chemotherapeutic drugs was performed into recipient mice testes. The detailed procedure of transplantation is described in Supplementary Material “Materials and Methods section”. 2D gel electrophoresis analysis of chemotherapeutic drug induced proteome alterations in germ cells and pathway studio The detailed procedures are described in Supplementary Material “Materials and Methods” section. Statistical analysis SPSS software (version 23, IBM Inc., New York) and Prism software (version 5.03; GraphPad, La Jolla, California) were used to manage all statistical analysis. Each experiment was carried out at least 3 times. Duncan’s multiple range test was used to consider differences in 1-way analysis of variance in all data. For identification of differences of each drug group with control in transplantation study, student’s 2-tailed t test was used. The level of significant difference was identified by p < .05, and the values are showed as mean ± SEM. RESULTS Viability of Cultured Germ Cells After Exposure to Chemotherapeutic Drugs Percentages of viability of drugs treated cultured germ cells were evaluated by trypan blue exclusion assay, which stains dead cells in blue because of the disruption of their cell membrane. We assessed the effects of different concentrations of each drug because they exerted different levels of toxicity. The highest concentration of each drug was considered as the half maximum inhibitory concentration (IC50) (Figure 1). At each treatment, cells were treated with 2-fold higher concentration of the drug than the previous concentration so that the decrease in cell viability could be measured at the finest level. Concentration ranges of etoposide, cisplatin, bleomycin, and BEP used in this study are 0.0125–0.4, 0.25–8, 2.5–80, and 0.025–0.8 µM, respectively. Figure 1. View largeDownload slide Viability status of testicular germ cells treated with the chemotherapeutic drugs in vitro. Germ cells were cultured and exposed to different concentrations of each of the drug at fourth day of postplating for 48 h. After treatment, germ cells were counted to measure the effect of drugs on germ cells viability. (A–D) represents the viability of cultured germ cell treated with etoposide, cisplatin, bleomycin, and combination of these drugs (BEP) respectively. Data are presented as means ± SEM of 4 independent experiments. Different letters (a, b, and c) indicate significant difference (p < .05) compared with control. Figure 1. View largeDownload slide Viability status of testicular germ cells treated with the chemotherapeutic drugs in vitro. Germ cells were cultured and exposed to different concentrations of each of the drug at fourth day of postplating for 48 h. After treatment, germ cells were counted to measure the effect of drugs on germ cells viability. (A–D) represents the viability of cultured germ cell treated with etoposide, cisplatin, bleomycin, and combination of these drugs (BEP) respectively. Data are presented as means ± SEM of 4 independent experiments. Different letters (a, b, and c) indicate significant difference (p < .05) compared with control. Proliferation Rate of Testicular Germ Cells Was Significantly Decreased After Treated With Particular Drug Concentrations Effects of the chemotherapeutic drugs on the proliferation of cultured germ cells were measured based on the number of harvested cells after drug treatment. Relative proliferation rates were determined by comparing the number of drug-treated cells with that of control cells for each treatment. Proliferation strategies were determined by selecting drug concentrations that maintained cell viability up to 90% (Supplementary Table 1). Interestingly, results of fluorescence microscopy showed a sequential decrease in colony formation with an increase in the concentration of each drug (Figure 2A). These results indicated that treatment with each drug concentration significantly reduced the relative proliferation rate of germ cells at specific concentration of each drug, resulting in almost 50% decrease in cell number (Figs. 2B–E) and these concentration ranges were selected for further experiments. Figure 2. View largeDownload slide In vitro proliferation of testicular germ cells after drugs’ exposure. A, Fluorescent microscopic image of in vitro proliferated testicular germ cell of GFP mouse treated with etoposide, cisplatin, bleomycin, and their combination (BEP). After treatment, germ cells were counted to find out the percentages of relative proliferation rate of drugs treated germ cells with control. (B–E) represents relative proliferation rate of germ cells treated with etoposide, cisplatin, bleomycin and BEP respectively. Values are represented mean ± SEM of 4 independent experiments and different letters (a and b) indicate significant difference (p < 0.05) compared with control. Scale bars in (A) = 200 μm. Figure 2. View largeDownload slide In vitro proliferation of testicular germ cells after drugs’ exposure. A, Fluorescent microscopic image of in vitro proliferated testicular germ cell of GFP mouse treated with etoposide, cisplatin, bleomycin, and their combination (BEP). After treatment, germ cells were counted to find out the percentages of relative proliferation rate of drugs treated germ cells with control. (B–E) represents relative proliferation rate of germ cells treated with etoposide, cisplatin, bleomycin and BEP respectively. Values are represented mean ± SEM of 4 independent experiments and different letters (a and b) indicate significant difference (p < 0.05) compared with control. Scale bars in (A) = 200 μm. Chemotherapeutic Drugs Induce Apoptosis in Cultured Germ Cells We observed significant decreases in germ cell proliferation after administration of chemotherapeutic drugs which led us to scrutinize the percentage of apoptotic cells among the proliferated germ cells. We analyzed this study with FACS (Figure 3A) and detected significantly higher percentages of apoptotic cells for etoposide and cisplatin, at 0.05 and 1 µM concentrations, respectively (Figs. 3B and 3C). No differences were observed for bleomycin and BEP (Figs. 3D and 3E). Controls were tested individually for each of chemotherapeutic agents. Figure 3. View largeDownload slide Effects of chemotherapeutic drugs on the apoptotic rate of testicular germ cells. A, Flow cytometric determination of the apoptotic rate of drugs treated cultured germ cells after stained with Annexin V/PI. Bar graphs (B–E) represent the percentages of apoptotic germ cells due to administration of etoposide, cisplatin, bleomycin, and BEP respectively. Values are represented mean ± SEM of 6 independent experiments. Different letters (a and b) indicate significant difference (p < .05) compared with control. Figure 3. View largeDownload slide Effects of chemotherapeutic drugs on the apoptotic rate of testicular germ cells. A, Flow cytometric determination of the apoptotic rate of drugs treated cultured germ cells after stained with Annexin V/PI. Bar graphs (B–E) represent the percentages of apoptotic germ cells due to administration of etoposide, cisplatin, bleomycin, and BEP respectively. Values are represented mean ± SEM of 6 independent experiments. Different letters (a and b) indicate significant difference (p < .05) compared with control. Estimation of SSC-Related Genes Expression Using Quantitative Real-Time PCR After Chemotherapeutic Drugs Treatment Relative mRNA expression levels of Lhx1 (LIM homeobox 1), Bcl6b (B-cell chronic lymphocytic leukemia/lymphoma 6, member B), Etv5 (Ets [E-26] variant gene 5), and Sohlh1 (spermatogenesis and oogenesis specific basic helix-loop-helix transcription factor) were determined by performing quantitative reverse transcription-PCR (qRT-PCR). Undifferentiated spermatogonia-related gene Lhx1 was downregulated in etoposide-, cisplatin-, and BEP-treated germ cells. Another undifferentiated spermatogonia-related gene Bcl6b was downregulated in BEP-treated germ cells (Supplementary Figure 1). Differentiating germ cell-related gene Sohlh1 was downregulated in only etoposide-treated germ cells (Supplementary Figure 1). However, undifferentiated spermatogonia-related gene Etv5 did not show any significant differences in etoposide-, cisplatin-, bleomycin-, and BEP-treated germ cells (Supplementary Figure S1). Immunocytochemical Analysis of Cultured Germ Cells Exposed to the Chemotherapeutic Drugs Next, we assessed the expression of undifferentiated and differentiated spermatogonia-specific marker proteins in cultured cells exposed to the chemotherapeutic drugs by performing ICC analysis (Supplementary Figure S2). We analyzed the percentages of drugs treated germ cells expressing undifferentiated spermatogonia marker proteins such as PLZF and GFRα1, germ cell-specific marker protein VASA, and differentiated spermatogonia marker protein C-KIT. Only the percentages of C-KIT expressions were observed significantly high in case of etoposide-, cisplatin-, bleomycin-, and BEP-treated germ cells (Figure 4). We also evaluated a differentiated spermatogonia related gene, Stra8, by reverse transcription-polymerase chain reaction (RT-PCR) (Supplementary Figure 3). A very little expression (PCR bands) was visualized in the gel for Stra8 in case of the highest concentrations of drugs. Otherwise, expression percentages of PLZF, GFRα1, and VASA were observed unchanged among the chemotherapeutic drugs-treated germ cells. Figure 4. View largeDownload slide Immunocytochemistry assay of germ cells treated with chemotherapeutic drugs. Treated germ cells were immunostained with undifferentiated spermatogonia related marker proteins PLZF and GFRα1, germ cell-specific marker protein VASA, and differentiated spermatogonia related marker proteins C-KIT. The figure represents bar graph presentations of the percentage of undifferentiation and differentiation related marker proteins expressing cells after treated with etoposide, cisplatin, bleomycin, and BEP. Values are represented mean ± SEM of 5 independent experiments. Different letters (a, b) indicate significant difference (p < .05) compared with control. Figure 4. View largeDownload slide Immunocytochemistry assay of germ cells treated with chemotherapeutic drugs. Treated germ cells were immunostained with undifferentiated spermatogonia related marker proteins PLZF and GFRα1, germ cell-specific marker protein VASA, and differentiated spermatogonia related marker proteins C-KIT. The figure represents bar graph presentations of the percentage of undifferentiation and differentiation related marker proteins expressing cells after treated with etoposide, cisplatin, bleomycin, and BEP. Values are represented mean ± SEM of 5 independent experiments. Different letters (a, b) indicate significant difference (p < .05) compared with control. Chemotherapeutic Drug Treatment Affected Stemness Properties of SSCs Although results of ICC analysis and qRT-PCR indicated alterations in germ cell properties such as undifferentiation and differentiation after exposure to the chemotherapeutic drugs in vitro, these results could not provide an exact measure of changes in the functions of SSCs. Therefore, transplanted germ cells treated with the highest concentrations of each drug were injected into the testes of recipient mice and colonies were visualized under fluorescent microscope after 2 months of transplantation (Figure 5A). We observed significant reductions in both of the number of donor germ cell-derived colonies per 105 transplanted germ cells and the number of colonies per total number of cultured cells due to the treatment of the drugs (Figs. 5B and 5C). Moreover, germ cells treated with 10 µM bleomycin formed significantly less number of colonies than germ cells treated with 0.05 μM etoposide and 0.1 µM BEP. Thus, these results indicate that treatment of SSCs with high concentration of each drug (0.05 μΜ etoposide, 1 μΜ cisplatin, 10 μΜ bleomycin, and 0.1 μM BEP) affected their stemness properties. Figure 5. View largeDownload slide Effects of chemotherapeutic agents on the stemness properties of SSCs. Stemness properties of SSCs were evaluated by counting donor germ cell-derived colonies from recipient testes. Cultured germ cells treated with 0.05 µM etoposide, 1 µM cisplatin, 10 µM bleomycin, and 0.1 µM BEP were transplanted and colonies were counted after 2 months of transplantation. A, Testes of recipient showing GFP expressing colonies of donor SSCs. Scale bar = 2 mm. B, The number of colonies per 105 transplanted cells and (C) the number of colonies per total number of cultured germ cells. Values are represented mean ± SEM. Total numbers of mice/testes analyzed were 7/13, 9/18, 10/19, 11/20, and 9/17 for control, 0.05 μΜ etoposide, 1 μΜ cisplatin, 10 μΜ bleomycin, and 0.1 μM BEP, respectively. *compared with treatment groups where p < .05 and *** compared with control where p < .001. Figure 5. View largeDownload slide Effects of chemotherapeutic agents on the stemness properties of SSCs. Stemness properties of SSCs were evaluated by counting donor germ cell-derived colonies from recipient testes. Cultured germ cells treated with 0.05 µM etoposide, 1 µM cisplatin, 10 µM bleomycin, and 0.1 µM BEP were transplanted and colonies were counted after 2 months of transplantation. A, Testes of recipient showing GFP expressing colonies of donor SSCs. Scale bar = 2 mm. B, The number of colonies per 105 transplanted cells and (C) the number of colonies per total number of cultured germ cells. Values are represented mean ± SEM. Total numbers of mice/testes analyzed were 7/13, 9/18, 10/19, 11/20, and 9/17 for control, 0.05 μΜ etoposide, 1 μΜ cisplatin, 10 μΜ bleomycin, and 0.1 μM BEP, respectively. *compared with treatment groups where p < .05 and *** compared with control where p < .001. Differentially Expressed Proteins in Chemotherapeutic Drug-Treated Germ Cells Two-dimensional gel electrophoresis (2-DE) was performed to compare the proteomic profiles of germ cells treated with the chemotherapeutic drugs. For this, germ cells were treated with the highest concentration of each drug. Almost 314 protein spots were detected in all gels. Of these, 63 spots showed a significant difference in protein density compared with those obtained for control cells. We identified 47 differentially expressed proteins from these 63 spots, of which 15 proteins were differentially expressed in etoposide-treated cells, 11 proteins were differentially expressed in cisplatin-treated cells, 8 proteins were differentially expressed in bleomycin-treated cells, and 13 proteins were differentially expressed in BEP-treated cells. Moreover, differential expression of some proteins found common for 2 or 3 chemotherapeutic drugs. Thus, 38 different proteins were specified in total (Supplementary Figure 4). We identified several proteome changes with respect to cell proliferation, oxidative stress, energy metabolism, immune response, and sex development (Table 1). Etoposide and cisplatin decreased the expression of cell proliferation-related proteins and increased the expression of oxidative stress-related proteins (Table 1). Bleomycin and BEP treatment affected energy metabolism (Table 1). Table 1. Proteins With Significantly Lower or Higher Expressions in Chemotherapeutic Drugs Treated Germ Cells and Control Groups Symbol  Protein ID (swissprot/ncbi)  gi no.  MASCOT Scorea  Relative Intensity (Normalized)b   Control  Etoposide 0.05 μM  Cisplatin 1 μM  Bleomycin 10 μM  BEP 0.1 μM  Cell proliferation  NAP1L1  Nucleosome assembly protein 1  gi|148689782  79  1  2.22 ± 0.76  1.35 ± 0.34  0.47 ± 0.05*  0.59 ± 0.24  ANXA7  Annexin A7  gi|160707956  155  1  0.84 ± 0.27  0.75 ± 0.48  0.87 ± 0.29  0.48 ± 0.10*  HNRNPH2  Nuclear ribonucleoprotein H2  gi|922960031  69  1  0.36 ± 0.07*  0.67 ± 0.06  0.57 ± 0.20  0.56 ± 0.32  EFTU  Elongation factor Tu, mitochondrial  gi|27370092  295  1  0.43 ± 0.03*  0.54 ± 0.15  0.53 ± 0.20  1.48 ± 0.38  STIP1  Stress-induced-phosphoprotein 1  gi|14389431  205  1  3.65 ± 0.42*  3.63 ± 1.60  2.22 ± 0.17  1.77 ± 0.97  HNRNPA2B1  Heterogeneous nuclear ribonucleoproteins  gi|32880197  113  1  0.97 ± 0.19  0.46 ± 0.12*  0.63 ± 0.08  0.72 ± 0.21  PABPC1  Polyadenylate-binding protein 1  gi|53754  96  1  0.02 ± 0.01*  0.03 ± 0.02*  0.94 ± 0.93  0.56 ± 0.25  ROS metabolism/oxidative stress  LEG1  Galectin  gi|6678682  223  1  0.45 ± 0.06  0.65 ± 0.20*  0.76 ± 0.19  0.53 ± 0.10  TXNL1  Thioredoxin-like protein 1  gi|31543902  162  1  0.48 ± 0.04*  0.40 ± 0.15  0.52 ± 0.23  1.42 ± 0.16  HSPE1  10 kDa heat shock protein, mitochondrial  gi|6680309  132  1  4.53 ± 0.89  2.39 ± 0.76  2.75 ± 0.05*  1.74 ± 0.47  HSPE1  10 kDa heat shock protein, mitochondrial  gi|6680309  93  1  3.86 ± 0.63*  3.28 ± 0.49*  4.36 ± 0.91  4.75 ± 0.90  PRDX1  Peroxiredoxin-1  gi|6754976  107  1  1.60 ± 1.03  0.14 ± 0.08*  0.38 ± 0.23  1.27 ± 0.80  PPID  Peptidyl-prolyl cis-trans isomerase D  gi|13385854  77  1  0.62 ± 0.20  0.49 ± 0.08*  1.34 ± 0.36  0.88 ± 0.30  PRDX1  Peroxiredoxin-1  gi|6754976  299  1  0.25 ± 0.04*  0.71 ± 0.28  0.59 ± 0.18  0.97 ± 0.70  Immune system  PSMA1  Proteasome subunit alpha type  gi| 33563282  69  1  0.62 ± 0.22  0.30 ± 0.15*  0.65 ± 0.04  1.11 ± 0.22  PCBP2  Poly(rC)-binding protein 2  gi|157041229  46  1  0.70 ± 0.10  0.58 ± 0.13  1.24 ± 0.38  0.29 ± 0.08*  Sex development  FKBP4  Peptidyl-prolyl cis-trans isomerase  gi|6753882  214  1  0.59 ± 0.28  0.32 ± 0.03*  0.33 ± 0.17  0.36 ± 0.20  ANXA2  Annexin A2  gi|6996913  83  1  1.18 ± 0.65  1.20 ± 0.24  1.36 ± 0.66  0.24 ± 0.11*  DPYSL2  Dihydropyrimidinase-related protein 2  gi|40254595  166  1  0.29 ± 0.15*  0.47 ± 0.19  0.67 ± 0.31  0.97 ± 0.31  Energy metabolism  EEF1A1  Elongation factor 1  gi|148697551  100  1  0.53 ± 0.16  0.36 ± 0.10  1.10 ± 0.29*  0.53 ± 0.11  TCPQ  T-complex protein 1 subunit  gi|50510319  491  1  0.93 ± 0.27  0.34 ± 0.29  0.57 ± 0.31  0.19 ± 0.12*  ATP6V1A  V-type proton ATPase catalytic subunit A  gi|31560731  135  1  0.28 ± 0.02*  1.04 ± 0.31  0.19 ± 0.08*  0.12 ± 0.04*  TCP1  T-complex protein 1 subunit alpha  gi|201725  104  1  1.07 ± 0.27  1.23 ± 0.78  1.29 ± 0.65  0.35 ± 0.03*  ATP5I  ATP synthase subunit e, mitochondrial  gi|83715998  104  1  4.70 ± 1.17  2.44 ± 1.58  7.01 ± 3.73  6.20 ± 0.34*  ATP5O  ATP synthase subunit  gi|20070412  68  1  0.08 ± 0.08*  0.51 ± 0.45  0.06 ± 0.05*  2.24 ± 0.94  PSD13  26S proteasome  gi|6755210  163  1  1.20 ± 0.51  0.51 ± 0.05  0.89 ± 0.18  0.34 ± 0.15*  OAT  Ornithine aminotransferase  gi|8393866  136  1  0.33 ± 0.13  0.74 ± 0.36*  1.31 ± 0.44  0.25 ± 0.15*  DLAT  Dihydrolipoyllysine-residue acetyltransferase  gi|16580128  116  1  0.74 ± 0.02  0.71 ± 0.09  0.31 ± 0.07*  0.96 ± 0.22  THOP1  Thimet oligopeptidase  gi|239916005  303  1  0.61 ± 0.29  0.13 ± 0.01  0.02 ± 0.01*  0.31 ± 0.21  GANAB  Neutral alpha-glucosidase  gi|148701451  166  1  0.20 ± 0.07*  0.78 ± 0.33  1.16 ± 0.93  0.48 ± 0.10*  NIT2  Omega-amidase  gi|12963555  60  1  0.90 ± 0.15  0.18 ± 0.10*  0.60 ± 0.19  0.15 ± 0.01*  DLD  Dihydrolipoyl dehydrogenase, mitochondrial  gi|31982856  97  1  1.98 ± 1.34  2.21 ± 0.39  4.52 ± 0.57*  2.85 ± 1.43  ENO1  Alpha-enolase  gi|158853992  68  1  0.42 ± 0.06*  1.07 ± 0.64  0.67 ± 0.21  0.49 ± 0.24  ME2  NAD-dependent malic enzyme  gi|21703972  152  1  0.29 ± 0.10*  0.74 ± 0.10  1.38 ± 0.98  0.50 ± 0.29  ALDOC  Fructose-bisphosphate aldolase C  gi|742670581  98  1  0.66 ± 0.27  0.48 ± 0.19  0.91 ± 0.18  0.36 ± 0.04*  GAPDH  Glyceraldehyde-3-phosphate dehydrogenase  gi|309243  95  1  4.01 ± 0.50*  3.40 ± 2.31  2.34 ± 1.24  1.28 ± 0.32  MDH2  Malate dehydrogenase, mitochondrial  gi|89574115  133  1  0.37 ± 0.09*  0.53 ± 0.03  0.73 ± 0.26  0.42 ± 0.06*  EEF1A1  Elongation factor-1 alpha  gi|1220410  80  1  1.70 ± 0.34  3.47 ± 0.33*  1.51 ± 0.58  1.61 ± 0.33  Symbol  Protein ID (swissprot/ncbi)  gi no.  MASCOT Scorea  Relative Intensity (Normalized)b   Control  Etoposide 0.05 μM  Cisplatin 1 μM  Bleomycin 10 μM  BEP 0.1 μM  Cell proliferation  NAP1L1  Nucleosome assembly protein 1  gi|148689782  79  1  2.22 ± 0.76  1.35 ± 0.34  0.47 ± 0.05*  0.59 ± 0.24  ANXA7  Annexin A7  gi|160707956  155  1  0.84 ± 0.27  0.75 ± 0.48  0.87 ± 0.29  0.48 ± 0.10*  HNRNPH2  Nuclear ribonucleoprotein H2  gi|922960031  69  1  0.36 ± 0.07*  0.67 ± 0.06  0.57 ± 0.20  0.56 ± 0.32  EFTU  Elongation factor Tu, mitochondrial  gi|27370092  295  1  0.43 ± 0.03*  0.54 ± 0.15  0.53 ± 0.20  1.48 ± 0.38  STIP1  Stress-induced-phosphoprotein 1  gi|14389431  205  1  3.65 ± 0.42*  3.63 ± 1.60  2.22 ± 0.17  1.77 ± 0.97  HNRNPA2B1  Heterogeneous nuclear ribonucleoproteins  gi|32880197  113  1  0.97 ± 0.19  0.46 ± 0.12*  0.63 ± 0.08  0.72 ± 0.21  PABPC1  Polyadenylate-binding protein 1  gi|53754  96  1  0.02 ± 0.01*  0.03 ± 0.02*  0.94 ± 0.93  0.56 ± 0.25  ROS metabolism/oxidative stress  LEG1  Galectin  gi|6678682  223  1  0.45 ± 0.06  0.65 ± 0.20*  0.76 ± 0.19  0.53 ± 0.10  TXNL1  Thioredoxin-like protein 1  gi|31543902  162  1  0.48 ± 0.04*  0.40 ± 0.15  0.52 ± 0.23  1.42 ± 0.16  HSPE1  10 kDa heat shock protein, mitochondrial  gi|6680309  132  1  4.53 ± 0.89  2.39 ± 0.76  2.75 ± 0.05*  1.74 ± 0.47  HSPE1  10 kDa heat shock protein, mitochondrial  gi|6680309  93  1  3.86 ± 0.63*  3.28 ± 0.49*  4.36 ± 0.91  4.75 ± 0.90  PRDX1  Peroxiredoxin-1  gi|6754976  107  1  1.60 ± 1.03  0.14 ± 0.08*  0.38 ± 0.23  1.27 ± 0.80  PPID  Peptidyl-prolyl cis-trans isomerase D  gi|13385854  77  1  0.62 ± 0.20  0.49 ± 0.08*  1.34 ± 0.36  0.88 ± 0.30  PRDX1  Peroxiredoxin-1  gi|6754976  299  1  0.25 ± 0.04*  0.71 ± 0.28  0.59 ± 0.18  0.97 ± 0.70  Immune system  PSMA1  Proteasome subunit alpha type  gi| 33563282  69  1  0.62 ± 0.22  0.30 ± 0.15*  0.65 ± 0.04  1.11 ± 0.22  PCBP2  Poly(rC)-binding protein 2  gi|157041229  46  1  0.70 ± 0.10  0.58 ± 0.13  1.24 ± 0.38  0.29 ± 0.08*  Sex development  FKBP4  Peptidyl-prolyl cis-trans isomerase  gi|6753882  214  1  0.59 ± 0.28  0.32 ± 0.03*  0.33 ± 0.17  0.36 ± 0.20  ANXA2  Annexin A2  gi|6996913  83  1  1.18 ± 0.65  1.20 ± 0.24  1.36 ± 0.66  0.24 ± 0.11*  DPYSL2  Dihydropyrimidinase-related protein 2  gi|40254595  166  1  0.29 ± 0.15*  0.47 ± 0.19  0.67 ± 0.31  0.97 ± 0.31  Energy metabolism  EEF1A1  Elongation factor 1  gi|148697551  100  1  0.53 ± 0.16  0.36 ± 0.10  1.10 ± 0.29*  0.53 ± 0.11  TCPQ  T-complex protein 1 subunit  gi|50510319  491  1  0.93 ± 0.27  0.34 ± 0.29  0.57 ± 0.31  0.19 ± 0.12*  ATP6V1A  V-type proton ATPase catalytic subunit A  gi|31560731  135  1  0.28 ± 0.02*  1.04 ± 0.31  0.19 ± 0.08*  0.12 ± 0.04*  TCP1  T-complex protein 1 subunit alpha  gi|201725  104  1  1.07 ± 0.27  1.23 ± 0.78  1.29 ± 0.65  0.35 ± 0.03*  ATP5I  ATP synthase subunit e, mitochondrial  gi|83715998  104  1  4.70 ± 1.17  2.44 ± 1.58  7.01 ± 3.73  6.20 ± 0.34*  ATP5O  ATP synthase subunit  gi|20070412  68  1  0.08 ± 0.08*  0.51 ± 0.45  0.06 ± 0.05*  2.24 ± 0.94  PSD13  26S proteasome  gi|6755210  163  1  1.20 ± 0.51  0.51 ± 0.05  0.89 ± 0.18  0.34 ± 0.15*  OAT  Ornithine aminotransferase  gi|8393866  136  1  0.33 ± 0.13  0.74 ± 0.36*  1.31 ± 0.44  0.25 ± 0.15*  DLAT  Dihydrolipoyllysine-residue acetyltransferase  gi|16580128  116  1  0.74 ± 0.02  0.71 ± 0.09  0.31 ± 0.07*  0.96 ± 0.22  THOP1  Thimet oligopeptidase  gi|239916005  303  1  0.61 ± 0.29  0.13 ± 0.01  0.02 ± 0.01*  0.31 ± 0.21  GANAB  Neutral alpha-glucosidase  gi|148701451  166  1  0.20 ± 0.07*  0.78 ± 0.33  1.16 ± 0.93  0.48 ± 0.10*  NIT2  Omega-amidase  gi|12963555  60  1  0.90 ± 0.15  0.18 ± 0.10*  0.60 ± 0.19  0.15 ± 0.01*  DLD  Dihydrolipoyl dehydrogenase, mitochondrial  gi|31982856  97  1  1.98 ± 1.34  2.21 ± 0.39  4.52 ± 0.57*  2.85 ± 1.43  ENO1  Alpha-enolase  gi|158853992  68  1  0.42 ± 0.06*  1.07 ± 0.64  0.67 ± 0.21  0.49 ± 0.24  ME2  NAD-dependent malic enzyme  gi|21703972  152  1  0.29 ± 0.10*  0.74 ± 0.10  1.38 ± 0.98  0.50 ± 0.29  ALDOC  Fructose-bisphosphate aldolase C  gi|742670581  98  1  0.66 ± 0.27  0.48 ± 0.19  0.91 ± 0.18  0.36 ± 0.04*  GAPDH  Glyceraldehyde-3-phosphate dehydrogenase  gi|309243  95  1  4.01 ± 0.50*  3.40 ± 2.31  2.34 ± 1.24  1.28 ± 0.32  MDH2  Malate dehydrogenase, mitochondrial  gi|89574115  133  1  0.37 ± 0.09*  0.53 ± 0.03  0.73 ± 0.26  0.42 ± 0.06*  EEF1A1  Elongation factor-1 alpha  gi|1220410  80  1  1.70 ± 0.34  3.47 ± 0.33*  1.51 ± 0.58  1.61 ± 0.33  a MASCOT score means −10 log (p), where p represents the probability that the observed match is a random event. Individual scores > 30 indicate identity or extensive homology (p < .05). b Relative spots intensity between control and drugs treated cultured germ cells. Data are presented as mean ± SEM (3 replicates). Values with Asterisk (*) indicate significant differences between the control and each of the treatment groups as determined by t test (p < .05). Table 1. Proteins With Significantly Lower or Higher Expressions in Chemotherapeutic Drugs Treated Germ Cells and Control Groups Symbol  Protein ID (swissprot/ncbi)  gi no.  MASCOT Scorea  Relative Intensity (Normalized)b   Control  Etoposide 0.05 μM  Cisplatin 1 μM  Bleomycin 10 μM  BEP 0.1 μM  Cell proliferation  NAP1L1  Nucleosome assembly protein 1  gi|148689782  79  1  2.22 ± 0.76  1.35 ± 0.34  0.47 ± 0.05*  0.59 ± 0.24  ANXA7  Annexin A7  gi|160707956  155  1  0.84 ± 0.27  0.75 ± 0.48  0.87 ± 0.29  0.48 ± 0.10*  HNRNPH2  Nuclear ribonucleoprotein H2  gi|922960031  69  1  0.36 ± 0.07*  0.67 ± 0.06  0.57 ± 0.20  0.56 ± 0.32  EFTU  Elongation factor Tu, mitochondrial  gi|27370092  295  1  0.43 ± 0.03*  0.54 ± 0.15  0.53 ± 0.20  1.48 ± 0.38  STIP1  Stress-induced-phosphoprotein 1  gi|14389431  205  1  3.65 ± 0.42*  3.63 ± 1.60  2.22 ± 0.17  1.77 ± 0.97  HNRNPA2B1  Heterogeneous nuclear ribonucleoproteins  gi|32880197  113  1  0.97 ± 0.19  0.46 ± 0.12*  0.63 ± 0.08  0.72 ± 0.21  PABPC1  Polyadenylate-binding protein 1  gi|53754  96  1  0.02 ± 0.01*  0.03 ± 0.02*  0.94 ± 0.93  0.56 ± 0.25  ROS metabolism/oxidative stress  LEG1  Galectin  gi|6678682  223  1  0.45 ± 0.06  0.65 ± 0.20*  0.76 ± 0.19  0.53 ± 0.10  TXNL1  Thioredoxin-like protein 1  gi|31543902  162  1  0.48 ± 0.04*  0.40 ± 0.15  0.52 ± 0.23  1.42 ± 0.16  HSPE1  10 kDa heat shock protein, mitochondrial  gi|6680309  132  1  4.53 ± 0.89  2.39 ± 0.76  2.75 ± 0.05*  1.74 ± 0.47  HSPE1  10 kDa heat shock protein, mitochondrial  gi|6680309  93  1  3.86 ± 0.63*  3.28 ± 0.49*  4.36 ± 0.91  4.75 ± 0.90  PRDX1  Peroxiredoxin-1  gi|6754976  107  1  1.60 ± 1.03  0.14 ± 0.08*  0.38 ± 0.23  1.27 ± 0.80  PPID  Peptidyl-prolyl cis-trans isomerase D  gi|13385854  77  1  0.62 ± 0.20  0.49 ± 0.08*  1.34 ± 0.36  0.88 ± 0.30  PRDX1  Peroxiredoxin-1  gi|6754976  299  1  0.25 ± 0.04*  0.71 ± 0.28  0.59 ± 0.18  0.97 ± 0.70  Immune system  PSMA1  Proteasome subunit alpha type  gi| 33563282  69  1  0.62 ± 0.22  0.30 ± 0.15*  0.65 ± 0.04  1.11 ± 0.22  PCBP2  Poly(rC)-binding protein 2  gi|157041229  46  1  0.70 ± 0.10  0.58 ± 0.13  1.24 ± 0.38  0.29 ± 0.08*  Sex development  FKBP4  Peptidyl-prolyl cis-trans isomerase  gi|6753882  214  1  0.59 ± 0.28  0.32 ± 0.03*  0.33 ± 0.17  0.36 ± 0.20  ANXA2  Annexin A2  gi|6996913  83  1  1.18 ± 0.65  1.20 ± 0.24  1.36 ± 0.66  0.24 ± 0.11*  DPYSL2  Dihydropyrimidinase-related protein 2  gi|40254595  166  1  0.29 ± 0.15*  0.47 ± 0.19  0.67 ± 0.31  0.97 ± 0.31  Energy metabolism  EEF1A1  Elongation factor 1  gi|148697551  100  1  0.53 ± 0.16  0.36 ± 0.10  1.10 ± 0.29*  0.53 ± 0.11  TCPQ  T-complex protein 1 subunit  gi|50510319  491  1  0.93 ± 0.27  0.34 ± 0.29  0.57 ± 0.31  0.19 ± 0.12*  ATP6V1A  V-type proton ATPase catalytic subunit A  gi|31560731  135  1  0.28 ± 0.02*  1.04 ± 0.31  0.19 ± 0.08*  0.12 ± 0.04*  TCP1  T-complex protein 1 subunit alpha  gi|201725  104  1  1.07 ± 0.27  1.23 ± 0.78  1.29 ± 0.65  0.35 ± 0.03*  ATP5I  ATP synthase subunit e, mitochondrial  gi|83715998  104  1  4.70 ± 1.17  2.44 ± 1.58  7.01 ± 3.73  6.20 ± 0.34*  ATP5O  ATP synthase subunit  gi|20070412  68  1  0.08 ± 0.08*  0.51 ± 0.45  0.06 ± 0.05*  2.24 ± 0.94  PSD13  26S proteasome  gi|6755210  163  1  1.20 ± 0.51  0.51 ± 0.05  0.89 ± 0.18  0.34 ± 0.15*  OAT  Ornithine aminotransferase  gi|8393866  136  1  0.33 ± 0.13  0.74 ± 0.36*  1.31 ± 0.44  0.25 ± 0.15*  DLAT  Dihydrolipoyllysine-residue acetyltransferase  gi|16580128  116  1  0.74 ± 0.02  0.71 ± 0.09  0.31 ± 0.07*  0.96 ± 0.22  THOP1  Thimet oligopeptidase  gi|239916005  303  1  0.61 ± 0.29  0.13 ± 0.01  0.02 ± 0.01*  0.31 ± 0.21  GANAB  Neutral alpha-glucosidase  gi|148701451  166  1  0.20 ± 0.07*  0.78 ± 0.33  1.16 ± 0.93  0.48 ± 0.10*  NIT2  Omega-amidase  gi|12963555  60  1  0.90 ± 0.15  0.18 ± 0.10*  0.60 ± 0.19  0.15 ± 0.01*  DLD  Dihydrolipoyl dehydrogenase, mitochondrial  gi|31982856  97  1  1.98 ± 1.34  2.21 ± 0.39  4.52 ± 0.57*  2.85 ± 1.43  ENO1  Alpha-enolase  gi|158853992  68  1  0.42 ± 0.06*  1.07 ± 0.64  0.67 ± 0.21  0.49 ± 0.24  ME2  NAD-dependent malic enzyme  gi|21703972  152  1  0.29 ± 0.10*  0.74 ± 0.10  1.38 ± 0.98  0.50 ± 0.29  ALDOC  Fructose-bisphosphate aldolase C  gi|742670581  98  1  0.66 ± 0.27  0.48 ± 0.19  0.91 ± 0.18  0.36 ± 0.04*  GAPDH  Glyceraldehyde-3-phosphate dehydrogenase  gi|309243  95  1  4.01 ± 0.50*  3.40 ± 2.31  2.34 ± 1.24  1.28 ± 0.32  MDH2  Malate dehydrogenase, mitochondrial  gi|89574115  133  1  0.37 ± 0.09*  0.53 ± 0.03  0.73 ± 0.26  0.42 ± 0.06*  EEF1A1  Elongation factor-1 alpha  gi|1220410  80  1  1.70 ± 0.34  3.47 ± 0.33*  1.51 ± 0.58  1.61 ± 0.33  Symbol  Protein ID (swissprot/ncbi)  gi no.  MASCOT Scorea  Relative Intensity (Normalized)b   Control  Etoposide 0.05 μM  Cisplatin 1 μM  Bleomycin 10 μM  BEP 0.1 μM  Cell proliferation  NAP1L1  Nucleosome assembly protein 1  gi|148689782  79  1  2.22 ± 0.76  1.35 ± 0.34  0.47 ± 0.05*  0.59 ± 0.24  ANXA7  Annexin A7  gi|160707956  155  1  0.84 ± 0.27  0.75 ± 0.48  0.87 ± 0.29  0.48 ± 0.10*  HNRNPH2  Nuclear ribonucleoprotein H2  gi|922960031  69  1  0.36 ± 0.07*  0.67 ± 0.06  0.57 ± 0.20  0.56 ± 0.32  EFTU  Elongation factor Tu, mitochondrial  gi|27370092  295  1  0.43 ± 0.03*  0.54 ± 0.15  0.53 ± 0.20  1.48 ± 0.38  STIP1  Stress-induced-phosphoprotein 1  gi|14389431  205  1  3.65 ± 0.42*  3.63 ± 1.60  2.22 ± 0.17  1.77 ± 0.97  HNRNPA2B1  Heterogeneous nuclear ribonucleoproteins  gi|32880197  113  1  0.97 ± 0.19  0.46 ± 0.12*  0.63 ± 0.08  0.72 ± 0.21  PABPC1  Polyadenylate-binding protein 1  gi|53754  96  1  0.02 ± 0.01*  0.03 ± 0.02*  0.94 ± 0.93  0.56 ± 0.25  ROS metabolism/oxidative stress  LEG1  Galectin  gi|6678682  223  1  0.45 ± 0.06  0.65 ± 0.20*  0.76 ± 0.19  0.53 ± 0.10  TXNL1  Thioredoxin-like protein 1  gi|31543902  162  1  0.48 ± 0.04*  0.40 ± 0.15  0.52 ± 0.23  1.42 ± 0.16  HSPE1  10 kDa heat shock protein, mitochondrial  gi|6680309  132  1  4.53 ± 0.89  2.39 ± 0.76  2.75 ± 0.05*  1.74 ± 0.47  HSPE1  10 kDa heat shock protein, mitochondrial  gi|6680309  93  1  3.86 ± 0.63*  3.28 ± 0.49*  4.36 ± 0.91  4.75 ± 0.90  PRDX1  Peroxiredoxin-1  gi|6754976  107  1  1.60 ± 1.03  0.14 ± 0.08*  0.38 ± 0.23  1.27 ± 0.80  PPID  Peptidyl-prolyl cis-trans isomerase D  gi|13385854  77  1  0.62 ± 0.20  0.49 ± 0.08*  1.34 ± 0.36  0.88 ± 0.30  PRDX1  Peroxiredoxin-1  gi|6754976  299  1  0.25 ± 0.04*  0.71 ± 0.28  0.59 ± 0.18  0.97 ± 0.70  Immune system  PSMA1  Proteasome subunit alpha type  gi| 33563282  69  1  0.62 ± 0.22  0.30 ± 0.15*  0.65 ± 0.04  1.11 ± 0.22  PCBP2  Poly(rC)-binding protein 2  gi|157041229  46  1  0.70 ± 0.10  0.58 ± 0.13  1.24 ± 0.38  0.29 ± 0.08*  Sex development  FKBP4  Peptidyl-prolyl cis-trans isomerase  gi|6753882  214  1  0.59 ± 0.28  0.32 ± 0.03*  0.33 ± 0.17  0.36 ± 0.20  ANXA2  Annexin A2  gi|6996913  83  1  1.18 ± 0.65  1.20 ± 0.24  1.36 ± 0.66  0.24 ± 0.11*  DPYSL2  Dihydropyrimidinase-related protein 2  gi|40254595  166  1  0.29 ± 0.15*  0.47 ± 0.19  0.67 ± 0.31  0.97 ± 0.31  Energy metabolism  EEF1A1  Elongation factor 1  gi|148697551  100  1  0.53 ± 0.16  0.36 ± 0.10  1.10 ± 0.29*  0.53 ± 0.11  TCPQ  T-complex protein 1 subunit  gi|50510319  491  1  0.93 ± 0.27  0.34 ± 0.29  0.57 ± 0.31  0.19 ± 0.12*  ATP6V1A  V-type proton ATPase catalytic subunit A  gi|31560731  135  1  0.28 ± 0.02*  1.04 ± 0.31  0.19 ± 0.08*  0.12 ± 0.04*  TCP1  T-complex protein 1 subunit alpha  gi|201725  104  1  1.07 ± 0.27  1.23 ± 0.78  1.29 ± 0.65  0.35 ± 0.03*  ATP5I  ATP synthase subunit e, mitochondrial  gi|83715998  104  1  4.70 ± 1.17  2.44 ± 1.58  7.01 ± 3.73  6.20 ± 0.34*  ATP5O  ATP synthase subunit  gi|20070412  68  1  0.08 ± 0.08*  0.51 ± 0.45  0.06 ± 0.05*  2.24 ± 0.94  PSD13  26S proteasome  gi|6755210  163  1  1.20 ± 0.51  0.51 ± 0.05  0.89 ± 0.18  0.34 ± 0.15*  OAT  Ornithine aminotransferase  gi|8393866  136  1  0.33 ± 0.13  0.74 ± 0.36*  1.31 ± 0.44  0.25 ± 0.15*  DLAT  Dihydrolipoyllysine-residue acetyltransferase  gi|16580128  116  1  0.74 ± 0.02  0.71 ± 0.09  0.31 ± 0.07*  0.96 ± 0.22  THOP1  Thimet oligopeptidase  gi|239916005  303  1  0.61 ± 0.29  0.13 ± 0.01  0.02 ± 0.01*  0.31 ± 0.21  GANAB  Neutral alpha-glucosidase  gi|148701451  166  1  0.20 ± 0.07*  0.78 ± 0.33  1.16 ± 0.93  0.48 ± 0.10*  NIT2  Omega-amidase  gi|12963555  60  1  0.90 ± 0.15  0.18 ± 0.10*  0.60 ± 0.19  0.15 ± 0.01*  DLD  Dihydrolipoyl dehydrogenase, mitochondrial  gi|31982856  97  1  1.98 ± 1.34  2.21 ± 0.39  4.52 ± 0.57*  2.85 ± 1.43  ENO1  Alpha-enolase  gi|158853992  68  1  0.42 ± 0.06*  1.07 ± 0.64  0.67 ± 0.21  0.49 ± 0.24  ME2  NAD-dependent malic enzyme  gi|21703972  152  1  0.29 ± 0.10*  0.74 ± 0.10  1.38 ± 0.98  0.50 ± 0.29  ALDOC  Fructose-bisphosphate aldolase C  gi|742670581  98  1  0.66 ± 0.27  0.48 ± 0.19  0.91 ± 0.18  0.36 ± 0.04*  GAPDH  Glyceraldehyde-3-phosphate dehydrogenase  gi|309243  95  1  4.01 ± 0.50*  3.40 ± 2.31  2.34 ± 1.24  1.28 ± 0.32  MDH2  Malate dehydrogenase, mitochondrial  gi|89574115  133  1  0.37 ± 0.09*  0.53 ± 0.03  0.73 ± 0.26  0.42 ± 0.06*  EEF1A1  Elongation factor-1 alpha  gi|1220410  80  1  1.70 ± 0.34  3.47 ± 0.33*  1.51 ± 0.58  1.61 ± 0.33  a MASCOT score means −10 log (p), where p represents the probability that the observed match is a random event. Individual scores > 30 indicate identity or extensive homology (p < .05). b Relative spots intensity between control and drugs treated cultured germ cells. Data are presented as mean ± SEM (3 replicates). Values with Asterisk (*) indicate significant differences between the control and each of the treatment groups as determined by t test (p < .05). Based on the STRING system, functional protein association networks were also predicted among the identified proteins (Supplementary Figure 5). The protein-protein interactions were observed as gene cooccurrence, gene neighborhood, coexpression and gene fusions. It indicates that alteration in expression of any protein could be the reason of up- or down-regulation of connected proteins and these consequences may cause impact on cellular physiology. Some proteins that are directly linked with cell signaling, cell growth, apoptosis, and disease-related pathways were selected from each drug and were plotted using Pathway Studio to determine their coexpression and interrelationship with other cellular proteins (Supplementary Figs. 6A–D). Supplementary Figure 7 illustrates the accumulated consequences of the chemotherapeutic drugs derived from Pathway Studios and shows the drugs related effects on cellular processes and possible occurrences of diseases. Discussion As the main function of chemotherapeutic drugs is to reduce tumor growth and block activities associated with new carcinogenesis, exposure of germ cells to chemotherapeutic drug in vitro might decrease their viability. In present study, we treated germ cells with different concentrations of etoposide, cisplatin, and bleomycin and their combination (BEP) and observed almost 50% decreases in cell viability (IC50) after treatment with the highest concentrations of these drugs (Figure 1). Moreover, we observed a sharp dose-dependent decrease in the number of proliferating germ cells after treatment with each drug (Figure 2) and almost 50% proliferating germ cells compared with control were recovered after treatment with each drug at concentrations that maintained cell viability of >90% (Supplementary Table 1). High percentage of viable cells (>90%) among cultured cells indicates a good physiological state of the cells in vitro. Therefore, we considered those concentrations from each drug (0.0125–0.05 µM for etoposide, 0.25–1 µM for cisplatin, 2.5–10 µM for bleomycin, and 0.025–0.1 µM for BEP) where cell proliferation assessed up to half of control groups, and examined to measure the stem cell functions and proteome alteration of SSCs/progenitor spermatogonia. Percentages of apoptotic cells were measured among chemotherapeutic drugs treated proliferating germ cells to determine overall status of cell survival. Some studies indicate that etoposide induces apoptosis of seminiferous epithelial cells in rats (Stumpp et al., 2004) and cisplatin can decreases the proliferation of germ cells (Olive and Banath, 2009). Similarly, we observed significantly higher percentages of apoptotic cells in case of etoposide- and cisplatin-treated germ cell (Figs. 3B and 3C). Bleomycin and BEP did not induce significantly higher percentages of apoptotic cells. We performed qRT-PCR to determine the mRNA expression of undifferentiated spermatogonia-related genes in cells treated with the chemotherapeutic drugs in vitro. The mRNA expression of Lhx1, Bcl6b, and Etv5 in undifferentiated spermatogonia indicates their normal self-renewal in vitro (Oatley and Brinster, 2008; Song and Wilkinson, 2014). In this study, we observed that etoposide, cisplatin, and BEP treatment downregulated the mRNA expression of Lhx1 in germ cells compared with that in control cells, indicating that these drugs hampered the self-renewal characteristics of germ cells. Expression of Bcl6b and Etv5 was unchanged in germ cells treated with all the drugs, except BEP (Supplementary Figure 1), indicating that the combination treatment with the 3 drugs strongly inhibited the proliferation of germ cells in vitro. Sohlh1 encodes a germ cell-specific transcription factor necessary for spermatogonial differentiation (Ballow et al., 2006). Thus, Sohlh1 is a negative marker gene for in vitro proliferation of germ cells because growth factors of culture system always keep the cells in undifferentiated state. In this study, we observed that Sohlh1 expression decreased in germ cells treated with etoposide but remained unchanged in cells treated with the other drugs (Supplementary Figure 1). These findings indicate that chemotherapeutic drugs are able to alter the expression of essential genes in germ cells in vitro. Based on the results of gene expression analysis, we examined the levels of marker proteins associated with undifferentiated spermatogonia and differentiated germ cells. ICC analysis was performed to determine the levels of undifferentiated spermatogonia-specific marker proteins PLZF and GFRα1, and germ cell-specific marker VASA. Although results of qRT-PCR showed alterations in the expression of undifferentiated spermatogonia-associated genes after etoposide, cisplatin, and bleomycin treatment, results of ICC analysis did not show any changes in the levels of undifferentiated spermatogonia-associated marker proteins. Expression of some undifferentiated and germ cell-specific marker proteins decreased in a dose-dependent manner after chemotherapeutic drug treatment (Figure 4); however, this decrease was not statistically significant. These results indicated that treatment with the chemotherapeutic drugs did not alter the levels of major marker proteins in cultured germ cells. Next, we examined the expression pattern of differentiated germ cell-specific marker C-KIT and Stra8. As a result, very minute expression was observed for Stra8 in RT-PCR, it may be due to the exposure of the highest concentration of drugs (Oatley et al., 2006) (Supplementary Figure 3). Although only bleomycin and BEP at highest concentration showed little expression for Stra8, C-KIT showed a different scenario. C-KIT expression showed a significant dose-dependent increase in all drug-treated cells. Thus, our ICC data indicate that chemotherapeutic drug treatments may provoke cultured germ cells to in vitro differentiation. As the changes in gene and protein expression could not clarify the effect of the chemotherapeutic drugs on SSC functions and stemness properties, we transplanted drug-treated germ cells into recipient mice. To date, germ cell transplantation into recipient mice is the most reliable method to estimate the exact proportion of SSCs in a cell population. We observed alterations in the expressions of undifferentiated and differentiated spermatogonia related genes and marker proteins at the highest doses among the selected concentrations of these drugs (0.05 µM etoposide, 1 µM cisplatin, 10 µM bleomycin, and 0.1 µM BEP). So, we transplanted germ cells treated with abovementioned drugs’ doses into recipient mice. Our transplantation data showed that stem cell activity in SSCs was decreased significantly after in vitro treatment with chemotherapeutic drugs. Therefore, chemotherapeutic drugs treatment can alter stemness properties and functional abilities of SSCs. Finally, we examined the alterations in proteomic profiles of germ cells treated with the chemotherapeutic drugs. We observed that 0.05 µM etoposide, 1 µM cisplatin, 10 µM bleomycin, and 0.1 µM BEP altered the proteomic profiles of cultured germ cells. Therefore, we used the same drug concentrations for performing 2-DE. We examined 38 differentially expressed proteins, most of which performed special roles in the physiology of germ cells. We observed that expression of proteins associated with cell proliferation and energy metabolism decreased and that of proteins associated with reactive oxygen species increased in cells treated with the chemotherapeutic drugs (Table 1 and Supplementary Figure 6). Some of the identified proteins like polyadenylate-binding protein 1, annexin A7, heterogeneous nuclear ribonucleoproteins, peptidyl-prolyl cis-trans isomerase D, peroxiredoxin 1 exert crucial roles in cell physiology (Brownawell and Creutz, 1997; Choi et al., 2012; El Eter and Al-Masri, 2015; Gray et al., 2015; Yao et al., 2005). Moreover, expression of some proteins associated with sex-related development were down-regulated in drug-treated germ cells, indicating constraints in fetal development and infertility (Atchison and Means, 2004; Wang et al., 2015). Functional protein association networks were also predicted among the 2-DE identified proteins based on the STRING program which indicate the interrelationship of almost all of the drug- induced differentially expressed proteins (Supplementary Figure 5). The relationship reveals the occurrences of simultaneous up- and down-regulation of linked proteins due to chemotherapeutic administration that could be devastating for health. Finally, proteins related to cell functions and processes, and associated with disease development are summarized in Supplementary Figure 7. Although this illustrates enhancement of several important cellular activities like ROS generation, mRNA degradation, cell cycle, cell growth, autophagy, protein refolding, and Ca2+/glucose transport, these drug-related effects could ultimately trigger on reproductive health by blocking spermatogenesis, sperm motility, X chromosome inactivation and oocyte maturation (Supplementary Figure 7). To the best of our knowledge, this is the first comprehensive in vitro study to examine the effects of chemotherapeutic drugs on the survival, proliferation, and apoptosis of and alterations in the stemness properties and proteomic profiles of testicular germ cells. We selected etoposide, cisplatin, and bleomycin in this study because these drugs are frequently used for treating most patients with testicular cancers. We administered these drugs for a short period (48 h) and found that the lowest concentration of each drug induced minute changes in germ cells. Our results suggest that treatment with even low doses of these chemotherapeutic drugs alters the expression of SSC self-renewal- and differentiation-related genes and marker proteins. Moreover, these drug concentrations altered the stemness properties, characteristics, and proteomic profiles of germ cells. The cumulative effect of these drug concentrations may persist for a long period, resulting in cellular dysfunction and diseases, including infertility, and may be transmitted transgenerationally. Thus, the present study provides information on the effect of different concentrations of chemotherapeutic drugs on male germ cells and fertility status of patients with testicular cancers for developing new strategies for treating these patients. Furthermore, results of this in vitro study will help in evaluating and prescreening chemotherapeutic drugs before their clinical administrations. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. FUNDING This study was supported by grants from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM4251824), Republic of Korea. 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Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Toxicological SciencesOxford University Press

Published: Apr 27, 2018

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