Abstract
Recent data suggest that the DNA damage response (DDR) is altered in the eutopic endometrium (EE) of women with endometriosis and this probably ensues in response to higher DNA damage encountered by the EE in endometriosis. DDR operates in a tissue-specific manner and involves different pathways depending on the type of DNA lesions. Among these pathways, the non-homologous end joining (NHEJ) pathway plays a critical role in the repair of dsDNA breaks. The present study was undertaken to explore whether NHEJ is affected in the EE of women with endometriosis. Toward this, we focused on the X-ray repair cross-complementing 4 (XRCC4) protein, one of the core components of the NHEJ pathway. Endometrial XRCC4 protein levels in the mid-proliferative phase were found significantly (P < 0.05) downregulated in women with endometriosis, compared to control women. Investigation of a microarray-based largest dataset in the Gene Expression Omnibus database (GSE51981) revealed a similar trend at the transcript level in the EE of women with endometriosis, compared to control women. Further in vitro studies were undertaken to explore the effects of H2O2-induced oxidative stress on DNA damage, as assessed by γ-H2AX and 8-hydroxy-2’-deoxyguanosine (8-OHdG) immunolocalization, and XRCC4 protein levels in endometrial stromal (hTERT immortalized human endometrial stromal cell line (ThESCs)) and epithelial (Ishikawa) cells. A significant decrease in XRCC4 protein levels and significantly higher localization of γ-H2AX and 8-OHdG were evident in ThESCs and Ishikawa cells experiencing oxidative stress. Overall, the study demonstrates that the endometrial XRCC4 expression is dysregulated in women with endometriosis and this could be due to higher oxidative stress in endometriosis.
Introduction
Endometriosis, a disease caused by the survival and proliferation of endometrium-like tissue outside the uterus, is clinically characterized by pelvic pain, dysmenorrhea, dyspareunia, and dyschezia. This disease, though not fatal, severely affects the quality of life in about one-tenth of the global women population of the reproductive age group (Zondervan et al. 2020) and also contributes to infertility in 30–40% of affected women (Practice Committee of the American Society for Reproductive 2012, Carvalho et al. 2013).
An ample body of evidence implicates oxidative stress, characterized by an imbalance in the levels of anti-oxidants and oxidants, as a major pathobiological event in the sequelae of events involved in the progression of endometriosis (Scutiero et al. 2017). Various oxidative stress markers have been assessed for their levels in peripheral blood, follicular fluid, peritoneal fluid, and ectopic lesions in women with endometriosis. Collectively, these investigations revealed higher levels or activity of reactive oxygen species and lipid hydroperoxides in endometriotic lesions; higher concentrations of malondialdehyde (MDA) (Nasiri et al. 2017) and reduced levels/activity of anti-oxidants such as paraoxonase (Verit et al. 2008); superoxide dismutase (Prieto et al. 2012); total anti-oxidant system and native thiols (Turgut et al. 2013, Turkyilmaz et al. 2016) in the serum of women with endometriosis. The peritoneal fluid of women with endometriosis also showed higher levels of advanced oxidation protein products (Santulli et al. 2015), oxidized low-density lipoproteins (Murphy et al. 1998, Polak et al. 2013), nitrates and nitrites (Santulli et al. 2015), and higher activity of nitric oxide synthase (Osborn et al. 2002), compared to women without endometriosis. Likewise, the levels of various oxidants were also high in the follicular fluid of women with endometriosis (Singh et al. 2013). Interestingly, alterations in the redox status were not restricted to the environment surrounding endometriotic lesions. Eutopic endometrium (EE) also showed signs of oxidative stress in endometriosis. Higher levels of superoxide anions were detected in the EE samples from women with endometriosis, compared to those without the disease (Ngo et al. 2009). In addition to this, higher levels of lipid peroxides such as MDA and 8-hydroxy-1-deoxyguanosine (8-OHdG) were detected in the EE of women with endometriosis (Kao et al. 2005, Dai et al. 2019).
Higher oxidative stress is known to cause single-strand breaks (SSBs), double-strand breaks (DSBs), oxidation of bases, and intrastrand crosslinks in the genome (Karanjawala et al. 2002, Markkanen 2017). However, these assaults are efficiently handled by healthy mammalian cells. Various types of genomic insults such as SSBs, DSBs, oxidized or modified bases are sensed and repaired by DNA repair pathways like base excision repair (BER), mismatch repair, nucleotide excision repair, homologous recombination (HR), and non-homologous end joining (NHEJ) pathways. Among different types of DNA lesions, DSBs are the most deleterious DNA lesions leading to cell death or accumulation of mutations. DSBs are repaired by HR and NHEJ pathways (Ingram et al. 2019). While HR is active in the S and G2/M phases of the cell cycle, NHEJ is active throughout the cell cycle.
NHEJ is reported to be one of the major DSB repair pathways in mammalian cells (Rothkamm et al. 2003, Mao et al. 2008). The core components of the NHEJ pathway are X-ray repair cross-complementing 4 (XRCC4), XRCC5 (Ku80), XRCC6 (Ku70), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), DNA ligase IV, Artemis, and XRCC4-like factor (XLF) (Richter et al. 2019). In addition, other factors such as PAralog of XRCC4 and XLF (PAXX) and modulator of retrovirus infection or cell cycle regulator of NHEJ have recently been identified for their role in the canonical NHEJ pathway (Ochi et al. 2015, Hung et al. 2018, Ghosh & Raghavan 2021). DNA ends created by DSBs are loaded by the heterodimer of XRCC5 (Ku80) and XRCC6 (Ku70), followed by recruitment of DNA-PKcs to DNA ends. PAXX interacts with the Ku heterodimer to provide stability to the core assembly of the NHEJ machinery (Ochi et al. 2015). The ends of DNA breaks are processed by nucleases and polymerases such as POLL and POLM. Artemis also has exo- and endonuclease activity which is dependent upon DNA-PKcs autophosphorylation (Chang et al. 2017). Finally, the ends are ligated by XRCC4/DNA ligase IV (Chatterjee & Walker 2017). Thus, XRCC4 is one of the most critical components of DSB repair machinery and its aberrant levels and/or function may influence the repair of damaged DNA (Murray et al. 2015, Lu et al. 2017, Ruis et al. 2020).
Our recent study demonstrated the differential expression of genes associated with DNA damage response (DDR) signaling and DNA repair in the EE of women with endometriosis (Bane et al. 2021). Interestingly, the levels of transcripts encoding various factors involved in the NHEJ pathway such as POLL, POLM, PNKP, Artemis, and XRCC4 showed a trend toward higher expression in the EE of women with endometriosis, compared to those without the disease, in the mid-proliferative phase of the menstrual cycle. These observations were suggestive of a probability of the NHEJ pathway being upregulated in the EE of women with endometriosis. In contrast, other reports indicating G-1394T (rs6869366) and codon 247 (rs3734091) polymorphisms in the XRCC4 gene hinted at a reduction in the capacity of the NHEJ pathway in women with endometriosis (Hsieh et al. 2008, Saliminejad et al. 2015). The present study was therefore undertaken to investigate whether the protein levels of XRCC4, a critical component of the NHEJ pathway, are altered in the EE of women with endometriosis, compared to those without the disease and whether oxidative stress modulates the level of XRCC4 protein in endometrial cells.
Materials and methods
Participant recruitment and tissue collection
National Institute for Research in Reproductive Health Ethics Committee for Clinical Studies (258/2014) approved the study. Written informed consent was obtained from the participants for the use of their samples. Women having leiomyomas, adenomyosis, polyps, polycystic ovary syndrome, or gynecological and non-gynecological malignancies were excluded from the study. All the study participants have not had any hormonal medication in the past 3 months. EE samples were collected using a pipelle endometrial sampler from women assigned either to the ‘endometriosis group’ or the ‘control group’. Women included in the ‘endometriosis group’ (n = 20) aged (32.15 ± 1.36 years) were undergoing diagnostic laparoscopy for endometriosis. Ectopic tissues excised from ovaries, peritoneum, Pouch of Douglas, or uterosacral sites were histologically confirmed for their endometrium-like features. Women with endometriosis were categorized into stage I–IV following revised American Society for Reproductive Medicine (1997) criteria. Two women presented with stage II (1 each in mid-proliferative and mid-secretory phase), 5 with stage III (2 in mid-proliferative and 3 in mid-secretory phase), and 13 with stage IV (5 in mid-proliferative and 8 in mid-secretory phase). Women in the ‘control group’ (n = 14) aged (32.07 ± 1.45 years) were either undergoing interval tubal ligation or experiencing infertility due to male factors. Women included in this group showed an absence of endometriosis on diagnostic laparoscopy. Endometrial tissues were histologically dated according to the Noyes criteria (Noyes et al. 1975). The days elapsed since the last menstrual period were also taken into account to determine the phase of the menstrual cycle. In the endometriosis group, 8 EE samples showed the histological features typical of the mid-proliferative phase and 12 had ‘mid-secretory’ phase characteristics. In the control group, eight samples were in the mid-proliferative phase and six were in the mid-secretory phase of the cycle.
Endometrial tissues were thoroughly rinsed with saline to get rid of red blood cell contamination. Tissues were then divided into three parts. A solution of 10% neutral buffered formalin and 10% (v/v) formaldehyde prepared in PBS (46 mM disodium hydrogen phosphate, 29 mM sodium dihydrogen phosphate, and pH 7.4) was used to fix one part of the tissue for histological dating and another part was suspended in cell lysis buffer (9.0 M urea, 4% w/v CHAPS, and 40 mM Tris base) and stored at −20°C until total protein extraction.
Maintenance of cell lines
Ishikawa (human endometrial adenocarcinoma) cell line was procured from Sigma-Aldrich and maintained in DMEM/F-12 medium supplemented with 10% (v/v) fetal bovine serum (FBS) (Cat. No. 10082147, Thermo Fisher Scientific), 100 units/mL penicillin, and 100 μg/mL streptomycin (Cat. No. 15140122, Gibco). hTERT immortalized human endometrial stromal cell line (ThESC) (CRL-4003; ATCC) was purchased from American Type Culture Collection and maintained in DMEM/F-12 medium supplemented with 1.5 g/L sodium bicarbonate, 1% (v/v) ITS + Premix (Cat. No. 354352, BD Biosciences, San Jose, CA, USA), 500 ng/mL puromycin (Cat. No. P8833, Sigma-Aldrich), and 10% (v/v) charcoal-stripped FBS (Cat. No. 12676029, Thermo Fisher Scientific).
Histological dating
After fixation for 16–18 h at room temperature (RT), EE tissues were transferred to 70% (v/v) alcohol for 24 h. Next, tissues were passed through ascending grades of alcohol (70–100% v/v) for 30 min each and then transferred to xylol (1:1 v/v xylene and alcohol). This was followed by incubation of tissues in 100% xylene with a change given every 15 min till the clearing of the tissues. Cleared tissues were then transferred to molten paraffin wax at 56°C for 15 min and then solidified overnight at RT. The next day, the tissues were transferred to fresh molten wax and tissue blocks were made. Tissues were then sectioned at 5 µm thickness using a microtome (Leica Biosystems, Buffalo Grove, IL, USA) on poly-l-lysine (Cat. No. P8920, Sigma-Aldrich)-coated glass slides for histological dating.
Tissue sections were deparaffinized in xylene twice for 15 min each. This was followed by the passage of sections in descending grades of alcohol (100–30% v/v) solution for 5 min each and then incubated in water for 5 min. Sections were kept in hematoxylin for 1 min and then in water for 10 min. Sections were then passed through ascending grades of alcohol (30–70% v/v) and then dipped in eosin Y solution. This was followed by dehydration in 90% and 100% (v/v) alcohol for 5 min each and 100% xylene overnight before mounting using distyrene, a plasticizer, and xylene (DPX) mountant (Cat. No. 18404, Fisher Scientific, India).
Protein extraction and western blotting
Endometrial tissues suspended in lysis buffer were homogenized using a sample grinding kit (Cat. No. GE80-6483-37, Merck Millipore) for the extraction of total proteins. Total protein extract (6–10 μg) was resolved by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes (Cat. No. IPVH00010, Merck Millipore). The blots were blocked for 1 h at RT using non-fat dried milk (NFDM) powder (5% w/v) made in 1× TBS containing 0.05% (v/v) Tween-20 (Cat. No. 3601181001730, Bangalore GeNei, India) (TBST) and then incubated overnight at 4°C with mouse monoclonal anti-XRCC4 antibody (0.4 ng/μL; Cat. No. sc-271087, Santa Cruz Biotechnology). After 16 h of incubation, the blots were washed with 0.05% TBST and probed with a secondary anti-mouse antibody conjugated to horseradish peroxidase (0.13 ng/μL; Cat. No. P0161, Dako) for 1 h at RT. All the antibody solutions were prepared in 2% (w/v) NFDM made in 0.05% TBST. Subsequently, the blots were rigorously washed with 0.05% TBST before developing using SuperSignalTM West Femto Maximum Sensitivity Substrate (Cat. No. 34095, Thermo Fisher Scientific). The chemiluminescent signals were captured using ChemiDocTM MP Imaging System (Bio-rad). While one study proposed GAPDH as the most appropriate housekeeping protein for the endometrium tissue in the control and endometriosis patients (Vestergaard et al. 2011), another report demonstrated differential expression of GAPDH in the EE of women with and without endometriosis (Joseph & Mahale 2019). Our investigations also revealed differential expression of GAPDH protein in the endometrial samples from women with and without endometriosis (Supplementary Fig. 1, see section on supplementary materials given at the end of this article). There is evidence emerging that supports total protein load as a better loading control (Eaton et al. 2013, Fosang & Colbran 2015, Moritz 2017). Therefore, XRCC4 protein levels were normalized to the total protein load for each sample. Toward this, the blots post-chemiluminescent detection were stained with Coomassie brilliant blue (CBB) R-250 for densitometric analysis using Image Quant TL software (GE Healthcare Biosciences) to compare the total protein load across different samples. CBB staining of the blots was done after performing all the steps (primary antibody probing, washings, secondary antibody probing, washings, and chemiluminescent detection) and hence staining of the blots truly reflected the total protein load for each sample. The band corresponding to XRCC4 was quantified using Image Lab software (Bio-rad). The ratio of the XRCC4 immunoreactive band intensity to the total protein intensity (summed up intensities of all the bands in the total protein load of each sample, visualized by CBB staining of the blot post-chemiluminescent detection of XRCC4 and assessed by densitometric analysis) for each sample was used to determine the relative change in XRCC4 levels between the control and endometriosis groups.
Analysis of GSE51981 dataset
GSE51981 (Tamaresis et al. 2014), largest dataset available in the Gene Expression Omnibus (GEO) database, was screened for the expression of select oxidant and anti-oxidant genes, and XRCC4 gene at the transcript level in women with (n = 57) and without (n = 28) endometriosis during the proliferative ((control, (n = 20); endometriosis, (n = 29)) and mid-secretory ((control, (n = 8); endometriosis, (n = 28)) phases of the menstrual cycle. Log2 GC-RMA (normalized signal intensity) values for each probe ID in the platform for each sample were available in the dataset. For multiple probes spanning different regions of the same gene, the probe with the maximum signal intensity was chosen for comparative analyses between the two groups.
Alamar blue assay
ThESCs and Ishikawa cells were seeded at a density of 5.5 × 103 and 7.2 × 103cells, respectively, per well in a 96-well plate. After overnight incubation, cells were treated with 125, 250, and 500 µM of H2O2 for 2, 4, and 8 h (Kim et al. 2010, Choi et al. 2018), respectively. Post-treatment, cells were given a wash before the addition of 10% (v/v) Alamar blue (Cat. No. DAL1025, Thermo Fisher Scientific) in a complete medium. Absorbance readings at 570 and 600 nm were taken on Epoch2 microplate reader (Biotek) and the percentage reduction of Alamar blue was determined (Demitri et al. 2014).
Immunofluorescence
ThESCs or Ishikawa cells were seeded on a coverslip in a 12-well microplate. The next day, cells were treated with H2O2. Post-treatment, cells were fixed in 4% paraformaldehyde for 20 min at RT. ThESCs and Ishikawa cells were permeabilized using 0.1% (v/v) Triton in 1× TBS for 10 and 20 min, respectively, for γ-H2AX (a phosphorylated variant of histone H2AX) immunostaining. For 8-OHdG immunostaining, ThESCs and Ishikawa cells were permeabilized for 20 min in 1× PBS containing 0.2% (v/v) Triton and 0.1% (v/v) Triton, respectively. Blocking was performed in 1% (w/v) BSA for 30 min at RT. Cells were incubated for 16 h at 4°C with mouse monoclonals against human γ-H2AX (4 ng/µL) (Cat. No. 05–636, Merck Millipore) or 8-OHdG (40 ng/µL) (Cat. No. ab48508, Abcam). After three washes with TBS or PBS, cells were incubated with Alexa Fluor 488 goat anti-mouse IgG (20 ng/µL) (Cat. No. A11001, Thermo Fisher Scientific) for 1 h at 37°C. Cells were counterstained with DAPI (1 ng/µL) (Cat. No. 10236276001, Roche) and mounted in Vectashield (Cat. No. H-1000, Vector Laboratories, Burlingame, CA, USA). Immunostained cells were scanned using Fluoview 3000 Olympus confocal microscope (Germany). ImageJ software was used to measure the intensity of immunopositivity.
Dose- and time-dependent effect of H2O2 treatment on XRCC4 protein expression
Ishikawa cells and ThESCs were seeded at a density of 9 × 104 and 6 × 104 cells, respectively, per well in a 12-well culture plate. Cells were treated with different concentrations of H2O2 (125, 250, and 500 µM) for varying lengths of duration (2, 4, and 8 h). Treated and untreated cells were harvested post-treatment with H2O2 and suspended in lysis buffer for protein extraction. As H2O2 treatment altered the levels of GAPDH protein (Supplementary Fig. 2), XRCC4 protein levels were normalized to the total protein load for each sample.
Statistical analysis
Statistical Package for the Social Sciences software (v25.0) was used for all statistical analyses. Data are presented as mean± s.e.m. The normality of the data was tested using Shapiro–Wilk test. For immunoblotting, the difference in the mean values across groups was evaluated using either the Student’s unpaired t-test or one-way ANOVA with Tukey’s multiple comparison test. The difference in the transcript levels of the select oxidant and anti-oxidant genes between women with and without endometriosis was determined using the Student's unpaired t-test. Wherever unequal variance was observed, Welch correction was applied. P-value < 0.05 was considered significant for all comparisons.
Results
Endometrial XRCC4 is differentially expressed in women with endometriosis
Endometrial protein lysates were evaluated for their XRCC4 protein levels in women with and without endometriosis during the mid-proliferative (Fig. 1A, B and C) and mid-secretory (Fig. 1D, E, and F ) phases of the menstrual cycle. Endometrial XRCC4 protein levels in the mid-proliferative phase were significantly (P < 0.05) lower in women with endometriosis (EE), compared to women without endometriosis (CE) (Fig. 1C). This pattern was also evident in the mid-secretory phase, although the difference between the two groups failed to reach statistical significance (Fig. 1F).
Endometrial expression of XRCC4 in women with and without endometriosis. (A) and (D) Representative images indicating chemiluminescent detection of endometrial XRCC4 protein in mid-proliferative (A, B, and C) and mid-secretory (D, E, and F) phases of the menstrual cycle, respectively, in women with (EE) and without (CE) endometriosis. (B) and (E) Coomassie blue-stained images of the respective blots indicative of the total protein load of each sample. (C) and (F) The quantitative analyses of normalized XRCC4 protein in EE (n = 8 in mid-proliferative phase; 12 in mid-secretory phase) and CE (n = 8 in mid-proliferative phase; 6 in mid-secretory phase) samples, respectively. The significance of difference in XRCC4 protein levels between EE and CE groups was determined using Student’s unpaired t-test. *P < 0.05.
Citation: Reproduction 163, 2; 10.1530/REP-21-0436
Endometrial expression of XRCC4 in women with and without endometriosis. (A) and (D) Representative images indicating chemiluminescent detection of endometrial XRCC4 protein in mid-proliferative (A, B, and C) and mid-secretory (D, E, and F) phases of the menstrual cycle, respectively, in women with (EE) and without (CE) endometriosis. (B) and (E) Coomassie blue-stained images of the respective blots indicative of the total protein load of each sample. (C) and (F) The quantitative analyses of normalized XRCC4 protein in EE (n = 8 in mid-proliferative phase; 12 in mid-secretory phase) and CE (n = 8 in mid-proliferative phase; 6 in mid-secretory phase) samples, respectively. The significance of difference in XRCC4 protein levels between EE and CE groups was determined using Student’s unpaired t-test. *P < 0.05.
Citation: Reproduction 163, 2; 10.1530/REP-21-0436
Endometrial expression of XRCC4 in women with and without endometriosis. (A) and (D) Representative images indicating chemiluminescent detection of endometrial XRCC4 protein in mid-proliferative (A, B, and C) and mid-secretory (D, E, and F) phases of the menstrual cycle, respectively, in women with (EE) and without (CE) endometriosis. (B) and (E) Coomassie blue-stained images of the respective blots indicative of the total protein load of each sample. (C) and (F) The quantitative analyses of normalized XRCC4 protein in EE (n = 8 in mid-proliferative phase; 12 in mid-secretory phase) and CE (n = 8 in mid-proliferative phase; 6 in mid-secretory phase) samples, respectively. The significance of difference in XRCC4 protein levels between EE and CE groups was determined using Student’s unpaired t-test. *P < 0.05.
Citation: Reproduction 163, 2; 10.1530/REP-21-0436
Analysis of the largest GSE51981 dataset available in the GEO database revealed significantly reduced XRCC4 transcript levels in the proliferative phase EE samples of women with endometriosis (n = 29), compared to those from women without the disease (n = 20), as shown in Supplementary Fig. 3A. The trend of downregulation in the endometrial XRCC4 expression at the transcript level was also observed in the mid-secretory phase women with endometriosis (n = 28), compared to women without endometriosis (n = 8) (Supplementary Fig. 3B). Overall, these results indicated that XRCC4 is significantly reduced at the protein as well as the transcript level in women with endometriosis, compared to those without the disease.
Dysregulation of oxidant and anti-oxidant genes in women with and without endometriosis
GSE51981 dataset was mined to compare the expression pattern of transcripts encoding various oxidant and anti-oxidant factors in the proliferative and mid-secretory phases of the menstrual cycle in women with and without endometriosis. A majority of oxidant genes such as NADPH oxidase (NOX)1 (P < 0.001), NOX5 (P < 0.001), xanthine oxidase (XDH) (P < 0.05), nitric oxide synthase (NOS)3 (P < 0.01), cyclooxygenase (COX) 2 (P < 0.001), and cytochrome P450 oxidoreductase (POR) (P < 0.001) were significantly upregulated in endometriosis, compared to women without endometriosis. On the other hand, the level of transcripts encoding anti-oxidant factors such as superoxide dismutase (SOD)1 (P < 0.001), SOD2 (P < 0.001), glutathione peroxidase (GPX)8 (P < 0.001), catalase (CAT) (P < 0.001), thioredoxin (TXN) (P < 0.001), NAD(P)H: quinone oxidoreductase (NQO)1 (P < 0.001), and glutamate-cysteine ligase (GCLC) (P < 0.001) were found downregulated in the EE of women with endometriosis, compared to those without the disease (Supplementary Fig. 4). This was implicative of a possibility of proliferating EE experiencing higher oxidative stress in women with endometriosis, compared to those without the disease. Interestingly, these expression trends for oxidant and anti-oxidant genes were also observed in the mid-secretory phase of the menstrual cycle (Supplementary Fig. 5). This suggests that the EE in women with endometriosis experiences oxidative stress in mid-proliferative as well as mid-secretory phases of the menstrual cycle.
Effect of oxidative stress on cellular morphology, proliferation, and immunolocalization of γ-H2AX, 8-OHdG, and the expression of XRCC4 protein in ThESC cells
Next, to investigate whether oxidative stress has an impact on the levels of XRCC4 protein in endometrial cells, stromal (ThESCs) and epithelial (Ishikawa) cells were subjected to in vitro oxidative stress using H2O2 and checked for XRCC4 protein levels. ThESCs were treated with 125, 250, and 500 μM of H2O2for 2–8 h (Kim et al. 2010, Choi et al. 2018). Morphology of ThESCs treated with 125 μM of H2O2appeared unaffected, irrespective of the duration of treatment. At higher doses (250–500 μM), however, the morphology of cells treated appeared adversely affected (Fig. 2A).
Effect of oxidative stress on morphology, proliferation, and DNA damage marker (γ-H2AX and 8-OHdG) localization in ThESCs. (A) Representative phase-contrast images of ThESCs treated with H2O2 (125–500 μM) for varying periods of exposure (2, 4, and 8 h). (B) Percentage reduction in Alamar blue by ThESCs. At least four technical replicates for each condition were used to calculate the percentage of Alamar blue reduced, compared to respective time-matched untreated cells. Panels (C) and (D) represent immunolocalization of γ-H2AX and 8-OHdG, respectively, in untreated (control), 125 and 250 μM H2O2-treated ThESCs. Cells stained with the secondary antibody only are provided as insets. Scale bar = 20 μm. Panels (E) and (F) represent mean fluorescence intensity for γ-H2AX and 8-OHdG immunostaining, respectively. Differences in the mean intensities between different groups were determined using one-way ANOVA with Tukey’s post hoc test (*P < 0.05, ***P < 0.001).
Citation: Reproduction 163, 2; 10.1530/REP-21-0436
Effect of oxidative stress on morphology, proliferation, and DNA damage marker (γ-H2AX and 8-OHdG) localization in ThESCs. (A) Representative phase-contrast images of ThESCs treated with H2O2 (125–500 μM) for varying periods of exposure (2, 4, and 8 h). (B) Percentage reduction in Alamar blue by ThESCs. At least four technical replicates for each condition were used to calculate the percentage of Alamar blue reduced, compared to respective time-matched untreated cells. Panels (C) and (D) represent immunolocalization of γ-H2AX and 8-OHdG, respectively, in untreated (control), 125 and 250 μM H2O2-treated ThESCs. Cells stained with the secondary antibody only are provided as insets. Scale bar = 20 μm. Panels (E) and (F) represent mean fluorescence intensity for γ-H2AX and 8-OHdG immunostaining, respectively. Differences in the mean intensities between different groups were determined using one-way ANOVA with Tukey’s post hoc test (*P < 0.05, ***P < 0.001).
Citation: Reproduction 163, 2; 10.1530/REP-21-0436
Effect of oxidative stress on morphology, proliferation, and DNA damage marker (γ-H2AX and 8-OHdG) localization in ThESCs. (A) Representative phase-contrast images of ThESCs treated with H2O2 (125–500 μM) for varying periods of exposure (2, 4, and 8 h). (B) Percentage reduction in Alamar blue by ThESCs. At least four technical replicates for each condition were used to calculate the percentage of Alamar blue reduced, compared to respective time-matched untreated cells. Panels (C) and (D) represent immunolocalization of γ-H2AX and 8-OHdG, respectively, in untreated (control), 125 and 250 μM H2O2-treated ThESCs. Cells stained with the secondary antibody only are provided as insets. Scale bar = 20 μm. Panels (E) and (F) represent mean fluorescence intensity for γ-H2AX and 8-OHdG immunostaining, respectively. Differences in the mean intensities between different groups were determined using one-way ANOVA with Tukey’s post hoc test (*P < 0.05, ***P < 0.001).
Citation: Reproduction 163, 2; 10.1530/REP-21-0436
The proliferation of ThESCs treated with 125 μM of H2O2for 2, 4, and 8 h was not severely affected, compared to time-matched untreated cells, as indicated by Alamar blue assay (Fig. 2B). ThESCs treated with 125 μM maintained their proliferative status after the removal of stress, as shown in Supplementary Fig. 6. This again reaffirmed that H2O2at 125 μM does not severely affect the viability of cells.
Further, to check whether oxidative stress causes DNA damage, markers of DNA damage (γ-H2AX) and oxidized base (8-OHdG) were checked in ThESCs treated with 125 and 250 μM of H2O2. A significant increase (P < 0.05) in the mean fluorescence intensity for γ-H2AX immunostaining was observed in ThESCs treated with 125 μM of H2O2, compared to untreated cells (Fig. 2C and E ). Also, immunolocalization of 8-OHdG was found to be significantly higher (P < 0.05) in 125 μM-treated ThESCs (Fig. 2D and F ). These patterns were also evident in the cells treated with 250 μM H2O2 (Fig. 2C, D, E, and F ). This set of experiments revealed induction of higher DNA damage in cells exposed to higher oxidative stress.
ThESCs exposed to oxidative stress (at 125 μM H2O2) showed significantly reduced expression of XRCC4, compared to untreated cells (Fig. 3F and I ). This pattern was evident in cells treated with H2O2for 4 h as well as 8 h. A trend, though non-significant, toward a downregulation in the XRCC4 protein levels, was also observed at 2 h in ThESCs exposed to 125 μM of H2O2 (Fig. 3C). Thus, oxidative stress leads to a downregulation in the expression of XRCC4 protein in endometrial stromal cells.
Effect of oxidative stress on XRCC4 expression in ThESCs. ThESCs were treated with 125–500 μM H2O2 for varying lengths of duration, that is 2, 4, and 8 h. (A, D, and G) Representative chemiluminescent images to depict XRCC4 protein levels in ThESCs. (B, E, and H) Coomassie blue-stained images of the respective blots indicative of the total protein load in each cell lysate. (C, F, and I) The quantitative analyses of XRCC4 normalized to the total protein load of each sample in at least triplicates. The significance of the difference in XRCC4 protein levels between untreated and treated cells was determined using one-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001).
Citation: Reproduction 163, 2; 10.1530/REP-21-0436
Effect of oxidative stress on XRCC4 expression in ThESCs. ThESCs were treated with 125–500 μM H2O2 for varying lengths of duration, that is 2, 4, and 8 h. (A, D, and G) Representative chemiluminescent images to depict XRCC4 protein levels in ThESCs. (B, E, and H) Coomassie blue-stained images of the respective blots indicative of the total protein load in each cell lysate. (C, F, and I) The quantitative analyses of XRCC4 normalized to the total protein load of each sample in at least triplicates. The significance of the difference in XRCC4 protein levels between untreated and treated cells was determined using one-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001).
Citation: Reproduction 163, 2; 10.1530/REP-21-0436
Effect of oxidative stress on XRCC4 expression in ThESCs. ThESCs were treated with 125–500 μM H2O2 for varying lengths of duration, that is 2, 4, and 8 h. (A, D, and G) Representative chemiluminescent images to depict XRCC4 protein levels in ThESCs. (B, E, and H) Coomassie blue-stained images of the respective blots indicative of the total protein load in each cell lysate. (C, F, and I) The quantitative analyses of XRCC4 normalized to the total protein load of each sample in at least triplicates. The significance of the difference in XRCC4 protein levels between untreated and treated cells was determined using one-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001).
Citation: Reproduction 163, 2; 10.1530/REP-21-0436
Effect of oxidative stress on cellular morphology, proliferation, and immunolocalization of γ-H2AX and 8-OHdG and XRCC4 protein levels in Ishikawa cells
Akin to ThESCs, Ishikawa cells also displayed signs of DNA damage (γ-H2AX and 8-OHdG) in response to oxidative stress (Fig. 4C, D, E, and F ). However, a significant increase in the localization of γ-H2AX and 8-OHdG was evident only in the cells exposed to 250 μM, not in the cells exposed to 125 μM H2O2 (Fig. 4E and F ). A trend toward XRCC4 downregulation was also evident in Ishikawa cells exposed to higher oxidative stress (250 and 500 μM for 8 h) (Fig. 5I). Thus, Ishikawa cells appeared to be more resistant to oxidative stress-induced DNA damage. This could be one of the reasons underlying XRCC4 downregulation occurring only in very high-stress conditions (250 μM H2O2 for 8 h) in Ishikawa cells.
Effect of oxidative stress on morphology, proliferation, and DNA damage marker (γ-H2AX and 8-OHdG) localization in Ishikawa cells. (A) Representative phase-contrast images of Ishikawa cells treated with H2O2(125–500 μM) for varying periods of exposure (2, 4, and 8 h). (B) Percentage reduction in Alamar blue by Ishikawa cells. At least four technical replicates for each condition were used to calculate the percentage of Alamar blue reduced, compared to respective time-matched untreated cells. Panels (C) and (D) represent immunolocalization of γ-H2AX and 8-OHdG, respectively, in untreated (control), 125 and 250 μM H2O2-treated Ishikawa cells. Cells stained with the secondary antibody only are provided as insets. Scale bar = 20 μm. Panels (E) and (F) represent mean fluorescence intensity for γ-H2AX and 8-OHdG immunostaining, respectively. Differences in the mean intensities between different groups were determined using one-way ANOVA with Tukey’s post hoc test (***P < 0.001).
Citation: Reproduction 163, 2; 10.1530/REP-21-0436
Effect of oxidative stress on morphology, proliferation, and DNA damage marker (γ-H2AX and 8-OHdG) localization in Ishikawa cells. (A) Representative phase-contrast images of Ishikawa cells treated with H2O2(125–500 μM) for varying periods of exposure (2, 4, and 8 h). (B) Percentage reduction in Alamar blue by Ishikawa cells. At least four technical replicates for each condition were used to calculate the percentage of Alamar blue reduced, compared to respective time-matched untreated cells. Panels (C) and (D) represent immunolocalization of γ-H2AX and 8-OHdG, respectively, in untreated (control), 125 and 250 μM H2O2-treated Ishikawa cells. Cells stained with the secondary antibody only are provided as insets. Scale bar = 20 μm. Panels (E) and (F) represent mean fluorescence intensity for γ-H2AX and 8-OHdG immunostaining, respectively. Differences in the mean intensities between different groups were determined using one-way ANOVA with Tukey’s post hoc test (***P < 0.001).
Citation: Reproduction 163, 2; 10.1530/REP-21-0436
Effect of oxidative stress on morphology, proliferation, and DNA damage marker (γ-H2AX and 8-OHdG) localization in Ishikawa cells. (A) Representative phase-contrast images of Ishikawa cells treated with H2O2(125–500 μM) for varying periods of exposure (2, 4, and 8 h). (B) Percentage reduction in Alamar blue by Ishikawa cells. At least four technical replicates for each condition were used to calculate the percentage of Alamar blue reduced, compared to respective time-matched untreated cells. Panels (C) and (D) represent immunolocalization of γ-H2AX and 8-OHdG, respectively, in untreated (control), 125 and 250 μM H2O2-treated Ishikawa cells. Cells stained with the secondary antibody only are provided as insets. Scale bar = 20 μm. Panels (E) and (F) represent mean fluorescence intensity for γ-H2AX and 8-OHdG immunostaining, respectively. Differences in the mean intensities between different groups were determined using one-way ANOVA with Tukey’s post hoc test (***P < 0.001).
Citation: Reproduction 163, 2; 10.1530/REP-21-0436
Effect of oxidative stress on XRCC4 expression in Ishikawa cells. Ishikawa cells were treated with 125–500 μM H2O2for varying lengths of duration, that is 2, 4, and 8 h. (A, D, and G) Representative chemiluminescent images to depict XRCC4 protein levels in Ishikawa cells. (B, E, and H) Coomassie blue-stained images of the respective blots indicative of the total protein load in each cell lysate. (C, F, and I) The quantitative analyses of XRCC4 normalized to the total protein load of each sample in triplicates. The significance of the difference in XRCC4 protein levels between untreated and treated cells was determined using one-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01).
Citation: Reproduction 163, 2; 10.1530/REP-21-0436
Effect of oxidative stress on XRCC4 expression in Ishikawa cells. Ishikawa cells were treated with 125–500 μM H2O2for varying lengths of duration, that is 2, 4, and 8 h. (A, D, and G) Representative chemiluminescent images to depict XRCC4 protein levels in Ishikawa cells. (B, E, and H) Coomassie blue-stained images of the respective blots indicative of the total protein load in each cell lysate. (C, F, and I) The quantitative analyses of XRCC4 normalized to the total protein load of each sample in triplicates. The significance of the difference in XRCC4 protein levels between untreated and treated cells was determined using one-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01).
Citation: Reproduction 163, 2; 10.1530/REP-21-0436
Effect of oxidative stress on XRCC4 expression in Ishikawa cells. Ishikawa cells were treated with 125–500 μM H2O2for varying lengths of duration, that is 2, 4, and 8 h. (A, D, and G) Representative chemiluminescent images to depict XRCC4 protein levels in Ishikawa cells. (B, E, and H) Coomassie blue-stained images of the respective blots indicative of the total protein load in each cell lysate. (C, F, and I) The quantitative analyses of XRCC4 normalized to the total protein load of each sample in triplicates. The significance of the difference in XRCC4 protein levels between untreated and treated cells was determined using one-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01).
Citation: Reproduction 163, 2; 10.1530/REP-21-0436
Discussion
A few strides have been made in recent years to investigate whether the EE in women with endometriosis encounters higher DNA damage compared to those without endometriosis. Studies by our group and others indicated higher DNA damage as revealed by higher localization of γ-H2AX (indicative of dsDNA breaks) positive foci in the EE of women with endometriosis (Choi et al. 2018, Bane et al. 2021). Also, the levels of 8-hydroxy-2′-deoxyguanosine – the oxidized form of deoxyguanosine – in the EE were found to be higher in endometriosis (Dai et al. 2019, Bane et al. 2021). Collectively, these investigations indirectly suggest the presence of stimuli that induce endometrial DNA damage in endometriosis. At present, the nature of stimuli that induce DNA damage in the EE of women with endometriosis is precisely not known. These stimuli could be estrogenic or associated with certain environmental toxicants. Normally, estrogen is catabolized through the action of catechol-o-methyltransferase. However, if present in excess, estrogen is oxidized into quinone radicals which lead to DNA damage (Bolton & Thatcher 2008). Indeed, there are reports that hint at an estrogenic milieu in the EE of women with endometriosis, as evident by higher levels/activity of aromatase and steroid sulphatase enzymes, leading to a higher level of estrogens (Dassen et al. 2007, Purohit et al. 2008). It is likely that this excess of estrogen (or their metabolites) contributes to higher DNA damage in women with endometriosis. Also, the EE of women with endometriosis, compared to those without the disease, is reported to have a higher proliferative capacity (Hapangama et al. 2009, Bane et al. 2021). This high proliferation may contribute to high oxidative stress, which may lead to the oxidation of nitrogenous bases and ssDNA breaks (Trzeciak et al. 2012) and subsequently more DNA damage.
Various genomic insults induce the activation of different DNA repair pathways. Oxidative stress-induced DNA damage is often repaired by the BER pathway (Webster et al. 1992). However, excessive damage due to oxidative stress may lead to SSBs in DNA and accumulation of SSB lesions. SSB lesions are converted into DSBs during DNA replication. DSBs are also generated during BER if two SSBs are located close to each other in the cDNA strands (Jackson & Bartek 2009). These DSBs are often repaired by the classical NHEJ pathway. There exists data to demonstrate the activation of error-prone NHEJ repair processes in response to H2O2-induced DSBs (Sharma et al. 2016). In mouse embryonic fibroblasts also, the NHEJ pathway was found critical in tolerating oxidative DNA damage (Arrington et al. 2000).
Typically, the classical NHEJ pathway is initiated by the recognition of DSBs by Ku70/Ku80 heterodimer, followed by recruitment of other factors such as DNAPKcs, XRCC4, LIG4, XLF (XRCC4-like factor), and APLF (Aprataxin-and-PNK-like factor) to the site of the break. XRCC4 tethers to the ends of the breaks, stabilizes the repair complex, and serves as a scaffold to facilitate the functional interaction among different NHEJ factors. This is followed by filling in the residual gaps by POLL and POLM and ligation of the ends by LIG4 (Chatterjee & Walker 2017).
Our recent study indicated an upregulation in the DDR in the EE, probably elicited in response to higher DNA damage especially in the proliferative phase of the menstrual cycle, in women with endometriosis, compared to those without the disease (Bane et al. 2021). However, other studies hinted at the reduced competence of EE cells to repair their DNA. Women with endometriosis, compared to those without the disease, were found to have reduced levels of endometrial BARD1 and BRCA1 proteins, involved in the repair of dsDNA breaks (Dai et al. 2019). Further, BRCA1, BRCA2, ATM, and RAD51 transcript levels were also found reduced in the endometrial tissues of women with endometriosis, compared to those without the disease (Choi et al. 2018), thereby indicating a dysregulation in the HR pathway in the EE of women with endometriosis. These aberrations may contribute to the accumulation of somatic mutations. Indeed, there exist reports suggesting somatic mutations in genes such as KRAS, PIK3CA in the EE of women with endometriosis (Suda et al. 2018). However, more investigations are warranted to establish if DNA repair pathways are altered in the EE of women with endometriosis. The present study was undertaken to investigate whether the expression of XRCC4 – a critical component involved in the repair of dsDNA breaks – is altered in the EE of women with endometriosis, compared to those without the disease. The study demonstrated that endometrial XRCC4 expression is significantly downregulated in women with endometriosis, and this downregulation was evident at both the protein and the transcript levels. As the majority of women with endometriosis included in the study presented the late stage of disease (stage III or stage IV), it was not possible to conclude whether a dysregulation in the XRCC4 expression is associated with the origin of endometriosis or acquired during the progression of endometriosis. Nonetheless, reduced XRCC4 protein expression implies the possible derangements in the NHEJ pathway in the EE of women with endometriosis. These observations were not in line with our recent RNA-Seq-based study hinting at the upregulation of several components of the NHEJ pathway such as XRCC4, Artemis, POLL, POLM, and PNKP at the transcript level in the mid-proliferative phase endometrium of women with endometriosis, compared to those without the disease (Bane et al. 2021). It may be attributed to a discordance in the expression patterns at RNA and protein levels. On the other hand, a downregulation in the levels of XRCC4 does not negate a possibility of higher expression/activation of alternate-NHEJ (DNA ligase 4/XRCC4-independent pathway) components in the EE of women with endometriosis.
Further, it was intended to probe whether XRCC4 expression is modulated due to higher oxidative stress in endometrial stromal cells. There exists sufficient data to suggest that the EE tissues experience higher oxidative stress (Scutiero et al. 2017). Also, our analysis of the largest well-characterized microarray-based dataset of the endometrial transcriptomes of women with and without endometriosis (Tamaresis et al. 2014) revealed significant alterations in the expression of several genes encoding oxidant or anti-oxidant proteins. Further, we detected a significant downregulation in the expression of XRCC4 in response to oxidative stress in ThESCs endometrial stromal cells.
To create varying levels of oxidative stress, cells were treated with 125, 250, and 500 μM H2O2for 2, 4, and 8 h. In polymorphonuclear cells, total H2O2production is reported to be up to 160 μM over an hour (Liu & Zweier 2001). However, there exists no information on the physiological levels of intracellular H2O2in endometrial stromal or epithelial cells. Nonetheless, the concentrations of H2O2used in this study are likely to be sufficiently high to create oxidative stress in ThESCs and Ishikawa cells. We were interested in creating an in vitro condition that induces high oxidative stress and higher DNA damage with no adverse effect on proliferation, considering that the EE maintains its proliferative status, despite experiencing high oxidative stress in endometriosis. Excessive death was observed in cells exposed to 500 μM H2O2 for 8 h in ThESCs. However, in cells treated with 125 μM H2O2, proliferation was not severely affected, though DNA damage (as indicated by higher immunolocalization of γ-H2AX and 8-OHdG) was significantly higher compared to untreated cells. This was paralleled by a significant decrease in the expression of XRCC4 protein in endometrial stromal cells. In contrast to endometrial stromal cells, Ishikawa cells exposed to 125 μM H2O2did not show significant DNA damage and XRCC4 downregulation. It is likely that Ishikawa cells, because of their tumorigenic nature, are more resistant to oxidative stress-induced DNA damage compared to primary stromal cells. Also, these differences in differential response to oxidative stress maybe because of the different origin of cells (epithelium vs stroma).
Overall, our study demonstrates reduced levels of XRCC4 in the EE of women with endometriosis, compared to women without the disease. Also, our study hints at the possibility of XRCC4 expression being regulated by oxidative stress. A major limitation of the study is that it does not reveal whether reduced levels of XRCC4, a critical component of the classical NHEJ pathway, have an adverse effect on the DNA repair capacity in endometrial cells experiencing high oxidative stress in endometriosis. Detailed studies in this direction will help researchers gain more insights into the pathophysiology of endometriosis.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/REP-21-0436.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
The study was funded by Department of Biotechnology, Government of India (Grant Nos. BT/PR11828/MED/97/233/2014 and BT/PR30794/MED/97/439/2018 to G S) and Indian Council of Medical Research. K B was financially supported by Research Fellowships awarded by the University Grants Commission and Indian Council of Medical Research.
Author contribution statement
K B carried out immunoblotting of endometrial tissues, in vitro experiments, and analysis of data, data compilation, and preparation of the manuscript. J D was involved in collection and post-sample processing. A R carried out immunoblotting using Ishikawa cell lysates. R K provided his help in histological analysis. G F performed the dating of endometrial samples. R S, U D, N W, and A C were involved in the recruitment of clinically categorized study participants, administration of consent, and the collection of endometrial samples. U C provided his assistance in statistical analyses and manuscript editing. R G provided scientific inputs and helped in the coordination of the study. G S was involved in study design, experimental planning and execution, interpretation of data, and manuscript preparation.
Acknowledgements
K B is thankful to University Grants Commission and Indian Council of Medical Research for providing Research Fellowships. S Mandavkar is acknowledged for his help in microtomy. S Jadhav and S Sonawane are thanked for their help in confocal imaging. B Mayekar and Sameer are acknowledged for their technical support toward the study.
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