Abstract
Metastasis-associated protein 3 (MTA3) functions as a versatile coregulator in cancers and in physiological contexts. A predominant expression of MTA3 in interstitial Leydig cells (LCs) and its role as a local modulator of testicular steroidogenesis have recently emerged. Incubation with insulin decreased MTA3 expression in a concentration- and exposure time-dependent manner in LCs. This raises the possibility of additional endocrine actions of insulin in the direct control of MTA3 expression, which remains so far unexplored. Herein, we reported that type 2 diabetes mellitus (T2DM)-mediated inhibition of MTA3 was associated with an increase in testicular oxidative stress. In contrast, a gavage of the strong antioxidant melatonin effectively ameliorated oxidative stress and restored the expression of MTA3, but failed to change serum insulin levels in the diabetic mice with testosterone deficiency (TD). Using multiple biochemical approaches, we demonstrated that oxidative stress suppressed MTA3 expression via repression of nuclear receptor subfamily 4 group A member 1 (NR4A1)-mediated transactivation of MTA3 in mouse LCs. By contrast, ectopic expression of NR4A1 ameliorated oxidative stress-impaired MTA3 expression in LCs. By employing an effective in vivo gene transfer method with microinjection of lentiviral plasmids, we showed that replenishment of MTA3 expression in vivo partially restored testicular steroidogenesis and improved male fertility in diabetic mice with TD. Thus, we have unveiled a central regulatory hub, involving oxidative stress-impaired NR4A1-driven transactivation of MTA3 in stimulated LCs, as a potential mechanism regulating crosstalk between hyperinsulinemia and male infertility associated with TD.
Introduction
Globally, the prevalence of type 2 diabetes mellitus (T2DM) is increasing rapidly. It is estimated that approximately 4.3% of the world population will have a diagnosis of T2DM by 2030 (Ezzati et al. 2017). Recent reports documented that T2DM is tightly linked to an increased risk of isolated male hypogonadotropic hypogonadism, also called testosterone deficiency (TD)/late-onset hypogonadism that is different from hypogonadism secondary to distinct hypothalamopituitary pathology (Gibb & Strachan 2014). Indeed, 30–50% of men with T2DM suffer from TD and more than 70% of these patients eventually have sexual dysfunction, including erectile dysfunction (ED), premature ejaculation and low libido, as reported by epidemiological studies (Bebb et al. 2018, Malipatil et al. 2019). With the known limitations of testosterone measurement and lack of a valid symptom score, however, diagnosis of TD status is not straightforward, depending largely on biochemical threshold or syndromic approaches employed (Hackett 2015). This phenomenon reflects the complexity of pathogenesis (Gibb & Strachan 2014). Although the underlying molecular basis remains ill defined, insulin signaling has been shown to directly inhibit testicular steroidogenesis via modulation of certain transcription factors in Leydig cells (LCs) (Ahn et al. 2013). In turn, TD predisposes males to visceral obesity, insulin resistance and T2DM (Zitzmann 2009). These associations warrant a further investigation of testicular steroidogenesis in diabetic patients.
As the latest member of the metastasis-associated proteins (MTAs) family being identified, metastasis-associated protein 3 (MTA3) has started to attract much attention during recent decades (You et al. 2021). Unlike the other family members (e.g. MTA1 and MTA2), MTA3, functioning as a tumor suppressor as well as an oncogene, plays more complicated roles in cancers depending on contexts (Ma et al. 2016). MTA3 regulates cancer initiation, progression, recurrence and metastasis through epigenetic regulation of gene expression by chromatin remodeling or interaction with other crucial factors (Yao et al. 2019). Despite the initial recognition that MTA3 is exclusively expressed and acts in malignant cells, emerging data point to the notion that MTA3 is a rather pleiotropic regulator of a large set of physiological functions, ranging from B lymphocyte differentiation (Fujita et al. 2004), mammary gland development (Fujita et al. 2003) to primitive hematopoiesis (Li et al. 2009) and granulosa cell proliferation (Kwintkiewicz et al. 2012). This notion is further supported by the recently published work from this lab, in which MTA3 is observed to be exclusively expressed in interstitial LCs from the testis. MTA3 expression is regulated by testicular development and pituitary gonadotropins, as well as many metabolic cues, including insulin and T4 (He et al. 2016). From a functional standpoint, MTA3 regulates human chorionic gonadotrophin/cAMP-stimulated steroidogenesis in LCs. Importantly, MTA3 deficiency is involved in insulin-mediated inhibition of testosterone synthesis in diabetic mice (He et al. 2016, Gao et al. 2018).
The prominent role of insulin in the regulation of testicular steroidogenesis and spermatogenesis has been well recognized over the past decades (Ahn et al. 2013). However, the potential involvement of insulin in the direct control of testicular expression of MTA3 remains ill defined. On the above basis, the current work was undertaken to evaluate the biological effects and the underlying molecular basis of insulin control of MTA3 in murine LCs. Our initial identification of the indirect regulation of testicular expression of MTA3 by insulin prompted us to further evaluate the direct effects of manipulation of MTA3 expression at the whole animal level.
Materials and methods
Ethics statement
All procedures involved in animal work were conducted in conformity with the criteria outlined in the ‘Guide for Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1996)’, and were approved by the Ethics Committee for Animal Experiments of the Air Force Medical University (Approval no.: KY20183306-1). All surgeries were performed under sodium pentobarbital anesthesia (0.04–0.05 mg/g body weight, i.p.), and all efforts were made to minimize suffering (Zhang et al. 2013).
Animal model
Male C57BL/6 mice, at the age of 2 months, were obtained from the Animal Research Center of Air Force Medical University. Mice were housed under a strictly controlled condition (n = 5 per cage, 12-h daylight cycle, lights on at 8:00 h), and were acclimatized for 7 days prior to experiments (Zhang et al. 2018). The mice were randomly allocated into four groups: Group 1, Ctrl mice; Group 2, T2DM without TD; Group 3, diabetic mice with TD were treated with PBS; Group 3, diabetic mice with TD were treated with melatonin (n = 5/group). To induce T2DM, mice were provided with a high-fat feeding diet (HFD) with 60 kcal% fat (Research Diets Inc., Shanghai, China). At 8 weeks after HFD feeding, mice were injected intraperitonially with one dose of streptozotocin (STZ, 80 mg/kg in 0.1 M citrate buffer, Sigma-Aldrich, Beijing, China). The HFD/STZ-treated mice with distinct hyperglycemia concomitant with insulin resistance (as determined by insulin tolerance test as described below) were considered of having T2DM (Zhao et al. 2017). At 4 months after T2DM induction, mice were anesthetized, blood samples were collected from the orbital sinus and subjected to the measurement of serum testosterone concentrations using a testosterone ELISA kit (DRG, Marburg, Germany), as described below. This experiment was used to determine whether or not these animals displayed deficiency in testosterone production (below 1–2 s.d. values from the Ctrl mice mean). Two months following STZ injection, mice were treated either with a gavage of melatonin (Sigma-Aldrich,. 200 mg/kg/body weight, dissolved in saline and administrated at 1800) or with a gavage of saline (Ctrl) on a daily basis, for consecutive 14 days. The dosage and duration were chosen based on the average antioxidative activity of melatonin that has been reported in rodents (Jung et al. 2004). Following sodium pentobarbital anesthesia, the animals were finally euthanized by cervical dislocation.
In vivo overexpression of MTA3 was carried out according to a previously validated protocol (Ikawa et al. 2002, Park et al. 2013). Following anesthesia, both testes from T2DM mice with TD were exposed. Twenty microliters of viral vector solution (0.9% saline containing 8 μg/mL of polybrene) containing either pLV-EGFP-Mta3 (7 ng/μL) or empty vector (GeneCopoeia, Guangzhou, China) was injected into the testes by gentle syringe pressure, using a fiber optics probe (diameter = 1.65 mm) (n = 7/group). The pLV vector used cytomegalovirus (CMV) as its promoter. The incisions were then sutured and mice were returned to cages. Seventy days after microinjection (corresponding to two cycles of murine spermatogenesis), mice were subjected to analysis of epididymal sperm parameters (Dong et al. 2016), serum testosterone concentrations and histological changes as indicated. Sham and T2DM mice receiving only microinjection with viral vector solution (0.9% saline containing 8 μg/mL of polybrene) were used as negative controls.
Metabolic testing
After 2.5-h of fasting, blood samples from mouse lateral tail veins were subjected to the measurement of serum glucose and insulin using a Mouse Glucose kit and a Mouse Insulin ELISA kit (both from Crystal Chem, Elk Grove, IL, USA), respectively (n = 5/group). To perform an insulin tolerance test (ITT), mice were fasted for 6 h, and were then injected intraperitoneally with insulin (0.75 U/kg/body weight, Sigma-Aldrich), followed by the determination of serum glucose as described above. The blood glucose excursion, reflected as a percentile of the baseline levels over a 2-h duration, was presented as an area under the curve (AUC0–2 h, % min, n = 5/group) (Kim et al. 2016).
Following anesthetization, mouse blood was collected from the orbital sinus between 9:00 and 10:00 from the same mice as mentioned above. Blood samples were stored at 4°C overnight. On the next day, the serum was isolated by means of centrifugation at room temperature at 500 g for 10 min, aliquoted and stored at −80°C until further use. Serum samples were then subjected to measurement of serum testosterone with the aid of an ELISA kit purchased from Cayman Chemical (Shenzhen, China) (Qin et al. 2020). Absorbance was measured finally using an xMark™ plate reader (Bio-Rad, Shanghai, China), and measuring A450 values. A630 values were utilized for background subtraction. The sensitivity of the testosterone assay was 6.0 pg/mL, the intra- and inter-assay coefficient of variations was 3.0–10.2 and 5.8–11.4%, respectively. Serum levels of luteinizing hormone (LH) were evaluated using a Mouse Luteinizing Hormone ELISA Kit (Novus Biologicals, Shanghai, China). The sensitivity of the LH assay was 4.8 pmol/L and the intra- and inter-assay coefficient of variations was less than 5.17 or 5.4%, respectively.
Determination of intratesticular oxidant and antioxidant statuses
Testicular samples from the same mice employed in metabolic testing were lysed in pre-cold NaCl solution. Following centrifugation at 1700 g at 4°C for 15 min, the supernatants were collected. Levels of malondialdehyde (MDA) and total superoxide dismutase (T-SOD) were then measured using commercial kits from Jiancheng Bioengineering (Nanjing, China), with absorbance read at 532 nm for MDA and 560 nm for T-SOD, respectively.
Cell treatment
Mouse TM3 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and were maintained in DMEM/F-12 supplemented with 10% FBS (Thermo Fisher Scientific) at 37°C with 5% CO2. Primary LCs were isolated from 10-week-old C57BL/6 mice as described elsewhere (He et al. 2016). Briefly, the mice were euthanized and immersed in 75% alcohol for 5 min. After careful removal of fat and other connective tissues and tunica albuginea, dissociated seminiferous tubules were transferred into a 50-mL centrifuge tube containing 5 mL of 0.03% collagenase NB4 (SERVA Electrophoresis, Heidelberg, Baden-Württemberg Land, Germany), followed by digestion at 37°C for 15 min with constant shaking at 150 rpm. The resultant pellets received another round of collagenase digestion as described above, except that the vibration speed was changed to 130 rpm. The cells in the supernatants were finally resuspended in fresh DMEM/F-12 supplemented with 10% FBS (Thermo Fisher Scientific). After a 7-day culture, the purity of isolated LCs was >90% as revealed by 3β-HSD staining (Zhao et al. 2008). To study whether MTA3 expression was regulated by moderate oxidative stress, primary LCs or TM3 cells were challenged with different doses of H2O2 or diamide, in the presence or absence of 20 μmol/L diphenyleneiodonium chloride/DPI (Diamide and DPI were both obtained from Sigma-Aldrich. DPI is an inducer as well as a potential suppressor of oxidative stress due to its general inhibitory action on flavoproteins (Kucera et al. 2016)), for 2 h as indicated. To stimulate in vitro steroidogenesis, the cells were treated for 5 h with 0.2 mmol/L of dibutyryl-cAMP (db-cAMP, Tocris Bioscience, Shanghai, China). To investigate the negative regulation of MTA3 expression by insulin signaling, cells were incubated for 4 h with 40 nM of insulin (Sigma-Aldrich).
Cell transfection
A growing body of evidence points to the correlation between hyperinsulinemia, TD and infertility, but there is scarce information about the mechanisms (Radovic Pletikosic et al. 2021). To generate the TM3 cells that were stably deprived of endogenous Insr, TM3 cells were transfected with Insr shRNA (Sigma-Aldrich) using Lipofectamine 2000 (Thermo Fisher Scientific), followed by selection with 0.5 μg/mL G418 (Thermo Fisher Scientific). Moreover, the orphan nuclear receptor nuclear receptor subfamily 4 group A member 1 (NR4A1, also called NUR77) is one of the major transcription factors that regulate the transactivation of key steroidogenic enzyme genes in LCs (Lee et al. 2009). To study the potential NR4A1-dependent regulation of MTA3 expression, TM3 cells were transfected for 48 h with NR4A1 siRNA or Ctrl siRNA (Sigma-Aldrich) using Lipofectamine® RNAiMAX. TM3/NR4A1 cells were generated by the transfection of pCMV6-NR4A1 (OriGene, Rockville, MD, USA) followed by selection with 0.5 μg/mL G418.
Quantitative RT-PCR
Total RNA from testicular samples was isolated and purified using RNeasy Mini kit (Qiagen Inc.) as per the manufacturer’s instructions. After a routine DNase treatment (Applied Biosystems/Ambion), RT was performed with aid of SuperScript III system (Thermo Fisher Scientific). Subsequent qPCR analysis using ~10 ng cDNA/reaction was carried out according to Promega’s protocol on the MiniOpticon system (Bio-Rad). Relative expression levels of target genes were calculated by SYBR green intercalation using the 2−ΔΔCt method, with amplification of 18S as the internal control (Zhang et al. 2012). Primers used were described in our previous work (He et al. 2016).
Western blotting
Western blotting was carried out according to our previous work (He et al. 2016). Briefly, total protein samples were prepared in ice-cold RIPA buffer (Tris–HCl 50 mM, NaCl 150 mM, Triton X-100 1% (v/v), sodium deoxycholate 1% (w/v) and SDS 0.1% (w/v), pH 7.5) containing complete proteinase inhibitor cocktail tablets (Roche). About 30 µg of protein sample were separated on SDS/PAGE and transferred to nitrocellulose membrane (Millipore), followed by incubation with primary antibodies at 4°C overnight. The primary antibodies used were anti-MTA3 (dilution 1: 1000; Santa Cruz Biotechnology), anti-TUBULIN (dilution 1: 2000; Santa Cruz Biotechnology), anti-INSR (dilution 1: 500; Sigma-Aldrich), anti-p-Akt (dilution 1: 1000; Cell Signaling), anti-Akt (dilution 1: 2000; Cell Signaling), anti-AR (dilution 1: 1000; Abcam), anti-LHCGR (dilution 1: 2000; Thermo Fisher Scientific) and anti-NR4A1 (dilution 1: 1000; Santa Cruz Biotechnology). The next day, following a thorough rinse, the blots were incubated with peroxidase-conjugated secondary antibodies at room temperature for 2 h, followed by signal development using an enhanced ECL kit (Amersham Biosciences) according to the manufacturer’s instructions. Densitometric scanning of immunoblots was performed using the Image J software (National Institutes of Health) and normalized for the TUBULIN staining (Zhang et al. 2014a).
Histological examination and measurement of testicular apoptosis
Following routine deparaffinization and rehydration, 5-μm-thick testicular sections were subjected to hematoxylin and eosin (H&E) staining using a H&E Staining Kit (Beyotime, Shanghai, China). Localization of MTA3 in the testis was revealed by immunostaining (Zhang et al. 2012). Following routine deparaffinization and rehydration, 5-μm-thick testicular sections were subjected to antigen retrieval with the aid of a Microwave Antigen Retrieval Accessory Kit (BioGenex, Fremont, CA, USA). Subsequently, sections were incubated with 0.5% (v/v) H2O2/methanol for 20 min at room temperature to eliminate endogenous peroxidase activity. Following a thorough rinse, the sections were incubated with anti-MTA3 (dilution 1: 200; Santa Cruz Biotechnology) at 4°C overnight. Subsequent hybridization with second antibody and avidin–biotin complex, along with the development of peroxidase reaction, were carried out using the VECTASTAIN ELITE® Kit (Vector Lab, Burlingame, CA, USA).
We employed an apoptosis ELISA kit (Roche) to assess testicular apoptosis, as described in our previous report (Liang et al. 2013). Final spectrophotometry was performed at 405 nm by a microplate reader (Bio-Rad).
Luciferase reporter assay
The genomic fragments of mouse Mta3 promoter (−1,384 to +348) were PCR amplified from genomic DNA from mouse testis and were then cloned into pGL3-Basic vector (Promega) using CloneJET PCR Cloning Kit (Thermo Fisher Scientific). For reporter assay, 0.5 μg reporter plasmid and pRL-TK Renilla reporter plasmid were co-transfected into TM3 cells with different plasmids as indicated; 48 h later, the cells were treated for 5 h with 0.2 mmol/L of db-cAMP, followed by the measurement of luciferase activities using a dual-luciferase reporter assay kit (Promega).
Non-radioactive electrophoretic mobility shift assay
TM3/NR4A1 cells were treated for 5 h with 0.2 mmol/L of db-cAMP in the presence or absence of co-incubation with 1 mM of diamide. Subsequently, the cells were collected and nuclear protein extracts were prepared using the Nuclear Extraction Kit (Abcam) following the vendor’s protocol. To test the direct binding of NR4A1 to the Mta3 chromatin in vitro, 1 μg of NR4A1-enriched nuclear extracts was incubated with 30 mM of customized DIG-labeled probes corresponding to the putative NR4A1 binding motifs. Following gel electrophoresis and nylon membrane transfer, protein-bound DIG-labeled probes were immunologically detected with anti-DIG–alkaline phosphatase conjugate and CSPD chemiluminescent substrate (Roche), as per the manufacturer’s instructions. The oligonucleotide probe used was AGTTTTGGCACAGCCACGACTGACCTGCCAGTGTGGTC. Competitive inhibition consisted of incubation with a 50-fold molar excess of unlabeled/cold probe (Jin et al. 2020) or with 1 mM of diamide for 2 h.
Chromatin immunoprecipitation
TM3/NR4A1 cells were treated for 5 h with different doses of db-cAMP, in the presence or absence of co-incubation with 1 mM of diamide. Cells were then harvested and subjected to chromatin immunoprecipitation (ChIP) assay, as described in detail in our previous work (Zhang et al. 2014a). Recovery and preparation of DNA were performed and followed by PCR using primers for the Mta3 promoter (NCBI reference sequence: NC_000083.7), 5’-CAAAGCCTGTTTAGAGGCA-3’ and 5’-TGGAAGCTGACTTTCTCA-3’.
Statistical analysis
Quantitative data that were expressed as mean ± s.d. were analyzed for statistical difference using Student’s t-test or one-way ANOVA followed by Tukey’s test, wherever appropriate. Statistical analysis was performed with the aid of SPSS 19.0 software and P< 0.05 was considered statistically significant.
Results
T2DM-mediated inhibition of MTA3 is associated with increase in testicular oxidative stress
Our previous work has shown that in vitro treatment with insulin inhibits the expression levels of MTA3 in LCs in a dose- and time-dependent manner (He et al. 2016). To validate this observation in vivo, we established the murine model of T2DM according to a reported protocol (Zhao et al. 2017) (Fig. 1A). Four months after T2DM induction, we performed ITT, and measured levels of blood glucose and serum insulin to confirm the successful establishment of the T2DM model. As expected, T2DM significantly prolonged the excursion of blood glucose in mice, as reflected by a more than two-fold increase in AUC0–2 h, % min (Fig. 1B). Consequently, T2DM resulted in a higher level of blood glucose (Fig. 1C), as well as a lower level of serum insulin (Fig. 1D), relative to Ctrl mice. These findings collectively suggest a severe glucose intolerance in the HFD/STZ-treated mice. Notably, disruption in testosterone synthesis in some diabetic mice failed to change levels of ITT AUC, blood glucose and serum insulin (Fig. 1B, C and D), reemphasizing the point that insulin may function upstream of androgen synthesis in LCs. In addition, the effects of melatonin supplement on testis were also tested in the diabetic mice with TD, as this circulating indoleamine is known to suppress the progression of T2DM (NaveenKumar et al. 2020, Park et al. 2020). In accordance with a previous report (Shieh et al. 2009), a gavage of melatonin for consecutive 2 weeks successfully reversed ITT AUC and ameliorated blood glucose levels, but had no impact on serum insulin levels in diabetic mice (Fig. 1B, C and D). To examine the relationship between MTA3 expression and diabetes, we performed qPCR, western blotting and immunohistochemistry in testicular samples. Interestingly, testicular expression of MTA3 was only attenuated in diabetic mice with TD, but not in diabetic mice without TD. Importantly, treatment with melatonin gavage successfully rescued the expression levels of MTA3 in testicular samples from diabetic mice with TD (Fig. 1E, F and G). These findings collectively raised a possibility that insulin might not be the direct signaling module upstream of MTA3 expression in testis. Because hyperinsulinemia is a leading cause of oxidative stress (Nakamura et al. 2015), and because oxidative stress is a common factor leading to eventual declines in testicular steroidogenesis (Turner & Lysiak 2008, Nna et al. 2020), we next studied the oxidative stress status in our diabetic mouse model. T2DM induction caused a significant increase of oxidative stress in murine testes, evidenced by the elevated generation of MDA (Fig. 1H) and impaired enzymatic antioxidant activities of T-SOD (Fig. 1I) in testicular samples. Furthermore, the supplementation of exogenous melatonin could noticeably reduce T2DM-elicited oxidative stress in mouse testes (Fig. 1H and I). Thus, increased oxidative stress was clearly associated with impaired MTA3 expression in diabetic testis.
T2DM inhibition of testicular MTA3 expression correlates to oxidative stress. (A) Schematic experimental design for in vivo analysis of T2DM inhibition of testicular MTA3 expression. Group 1, Ctrl mice; Group 2, T2DM without TD; Group 3, diabetic mice with TD were treated with PBS; Group 3, diabetic mice with TD were treated with melatonin. (B) For ITT, 6 h-fasted mice were intraperitoneally injected with insulin (0.75 U/kg), followed by measurement of serum glucose using a Mouse Glucose kit. The excursion of blood glucose, reflected as a percentile of the baseline levels over a 2-h duration, was presented as area under the curve (AUC0–2 h, % min) (one-way ANOVA followed by Tukey’s post hoc tests, n = 5, *P< 0.05 and **P< 0.01). (C and D) After 2.5-h fasting, the blood samples from mouse lateral tail veins were subjected to measurement of serum glucose and insulin using commercial ELISA kits (one-way ANOVA followed by Tukey’s post hoc tests, n = 5, *P< 0.05 and **P< 0.01). (E) Four months following T2DM induction, mice were treated either with a gavage of melatonin (200 mg/kg/body weight, dissolved in saline and administrated at 18:00 h) on a daily basis or with a gavage of saline (Ctrl), for consecutive 14 days. Expression levels of Mta3 in different experimental groups were assayed using quantitative RT-PCR (one-way ANOVA followed by Tukey’s post hoc tests, n = 5, *P< 0.05 and **P< 0.01). (F) Mice were treated as described in Panel E. Expression levels of MTA3 in different experimental groups were assayed using western blotting. TUBULIN served as a loading control (n =5). Densitometric scanning of immunoblots was performed in which the level of a target protein w as normalized against the protein level in Group 1, which was arbitrarily set at 1. (G) Localization of MTA3 protein in testicular sections from different experimental groups was evaluated using immunohistochemistry (arrows denote the positive staining of MTA3 protein). Scale bar, 20 μm. (H and I) Levels of MDA and T-SOD from different experimental groups were determined using commercial kits, with absorbance read at 532 nm for MDA and 560 nm for T-SOD, respectively (one-way ANOVA followed by Tukey’s post hoc tests, n = 5, *P< 0.05 and **P< 0.01). HFD, high-fat diet; ITT, insulin tolerance test; MDA, malondialdehyde; MTA3, metastasis-associated protein 3; STZ, streptozotocin; TD, testosterone deficiency; T2DM, type 2 diabetes mellitus; T-SOD, total superoxide dismutase.
Citation: Reproduction 163, 5; 10.1530/REP-21-0413
T2DM inhibition of testicular MTA3 expression correlates to oxidative stress. (A) Schematic experimental design for in vivo analysis of T2DM inhibition of testicular MTA3 expression. Group 1, Ctrl mice; Group 2, T2DM without TD; Group 3, diabetic mice with TD were treated with PBS; Group 3, diabetic mice with TD were treated with melatonin. (B) For ITT, 6 h-fasted mice were intraperitoneally injected with insulin (0.75 U/kg), followed by measurement of serum glucose using a Mouse Glucose kit. The excursion of blood glucose, reflected as a percentile of the baseline levels over a 2-h duration, was presented as area under the curve (AUC0–2 h, % min) (one-way ANOVA followed by Tukey’s post hoc tests, n = 5, *P< 0.05 and **P< 0.01). (C and D) After 2.5-h fasting, the blood samples from mouse lateral tail veins were subjected to measurement of serum glucose and insulin using commercial ELISA kits (one-way ANOVA followed by Tukey’s post hoc tests, n = 5, *P< 0.05 and **P< 0.01). (E) Four months following T2DM induction, mice were treated either with a gavage of melatonin (200 mg/kg/body weight, dissolved in saline and administrated at 18:00 h) on a daily basis or with a gavage of saline (Ctrl), for consecutive 14 days. Expression levels of Mta3 in different experimental groups were assayed using quantitative RT-PCR (one-way ANOVA followed by Tukey’s post hoc tests, n = 5, *P< 0.05 and **P< 0.01). (F) Mice were treated as described in Panel E. Expression levels of MTA3 in different experimental groups were assayed using western blotting. TUBULIN served as a loading control (n =5). Densitometric scanning of immunoblots was performed in which the level of a target protein w as normalized against the protein level in Group 1, which was arbitrarily set at 1. (G) Localization of MTA3 protein in testicular sections from different experimental groups was evaluated using immunohistochemistry (arrows denote the positive staining of MTA3 protein). Scale bar, 20 μm. (H and I) Levels of MDA and T-SOD from different experimental groups were determined using commercial kits, with absorbance read at 532 nm for MDA and 560 nm for T-SOD, respectively (one-way ANOVA followed by Tukey’s post hoc tests, n = 5, *P< 0.05 and **P< 0.01). HFD, high-fat diet; ITT, insulin tolerance test; MDA, malondialdehyde; MTA3, metastasis-associated protein 3; STZ, streptozotocin; TD, testosterone deficiency; T2DM, type 2 diabetes mellitus; T-SOD, total superoxide dismutase.
Citation: Reproduction 163, 5; 10.1530/REP-21-0413
T2DM inhibition of testicular MTA3 expression correlates to oxidative stress. (A) Schematic experimental design for in vivo analysis of T2DM inhibition of testicular MTA3 expression. Group 1, Ctrl mice; Group 2, T2DM without TD; Group 3, diabetic mice with TD were treated with PBS; Group 3, diabetic mice with TD were treated with melatonin. (B) For ITT, 6 h-fasted mice were intraperitoneally injected with insulin (0.75 U/kg), followed by measurement of serum glucose using a Mouse Glucose kit. The excursion of blood glucose, reflected as a percentile of the baseline levels over a 2-h duration, was presented as area under the curve (AUC0–2 h, % min) (one-way ANOVA followed by Tukey’s post hoc tests, n = 5, *P< 0.05 and **P< 0.01). (C and D) After 2.5-h fasting, the blood samples from mouse lateral tail veins were subjected to measurement of serum glucose and insulin using commercial ELISA kits (one-way ANOVA followed by Tukey’s post hoc tests, n = 5, *P< 0.05 and **P< 0.01). (E) Four months following T2DM induction, mice were treated either with a gavage of melatonin (200 mg/kg/body weight, dissolved in saline and administrated at 18:00 h) on a daily basis or with a gavage of saline (Ctrl), for consecutive 14 days. Expression levels of Mta3 in different experimental groups were assayed using quantitative RT-PCR (one-way ANOVA followed by Tukey’s post hoc tests, n = 5, *P< 0.05 and **P< 0.01). (F) Mice were treated as described in Panel E. Expression levels of MTA3 in different experimental groups were assayed using western blotting. TUBULIN served as a loading control (n =5). Densitometric scanning of immunoblots was performed in which the level of a target protein w as normalized against the protein level in Group 1, which was arbitrarily set at 1. (G) Localization of MTA3 protein in testicular sections from different experimental groups was evaluated using immunohistochemistry (arrows denote the positive staining of MTA3 protein). Scale bar, 20 μm. (H and I) Levels of MDA and T-SOD from different experimental groups were determined using commercial kits, with absorbance read at 532 nm for MDA and 560 nm for T-SOD, respectively (one-way ANOVA followed by Tukey’s post hoc tests, n = 5, *P< 0.05 and **P< 0.01). HFD, high-fat diet; ITT, insulin tolerance test; MDA, malondialdehyde; MTA3, metastasis-associated protein 3; STZ, streptozotocin; TD, testosterone deficiency; T2DM, type 2 diabetes mellitus; T-SOD, total superoxide dismutase.
Citation: Reproduction 163, 5; 10.1530/REP-21-0413
Repression of testicular MTA3 expression in LCs by oxidative stress
Previous studies of our group evidenced a prominent negative regulation of MTA3 expression by insulin signaling in LCs (He et al. 2016). As hyperinsulinemia favors the accumulation of oxidized proteins by reducing their degradation (Facchini et al. 2000), and treatment with hydrogen peroxide (H2O2) can partially mimic the oxidative stress-induced cellular events (Zhang et al. 2014b), the ability of oxidative stress to modulate MTA3 expression was then explored using in vitro settings. Exposure to H2O2 for 2 h significantly inhibited the relative expression levels of MTA3 in a dose-dependent manner in primary-cultured LCs, and this inhibition effect was substantially reversed by co-incubation with the oxidative stress inhibitor diphenyleneiodonium chloride (Fig. 2A). To ask whether oxidative stress other than H2O2 can cause MTA3 downregulation, we treated primary LCs with another oxidative stress inducer diamide (Ning et al. 2016). Challenge with diamide for 2 h resulted in a dose-dependent downregulation of MTA3 expression in LCs. This inhibition was completely neutralized by supplementing with diphenyleneiodonium chloride (Fig. 2B). We were then curious whether oxidative stress could directly suppress the MTA3 activation at the transcriptional level. Mouse Mta3 promoter–reporter constructs were transfected into TM3 cells. Forty-eight hours after transfection, the cells were challenged for 5 h with 0.2 mM of db-cAMP. db-cAMP evoked a significant approximately seven-fold increase in luciferase activities vs control values, whereas co-incubation with H2O2 significantly inhibited, in a dose-dependent manner, relative luciferase activities. In keeping with the above-mentioned data, concomitant treatment with diphenyleneiodonium chloride successfully rescued the H2O2-impaired luciferase activities in db-cAMP-challenged TM3 cells (Fig. 2C). Previous studies suggest that a reciprocal feedback regulatory loop between insulin and oxidative stress is implemented in various cell types to govern cell metabolism and dictate cell fate (Laurent et al. 2008, Akhtar & Sah 2020). To this end, we assessed the possibility that oxidative stress may mediate, at least in part, the inhibitory effects of insulin on MTA3 expression by generating the TM3 cells that were stably deprived of endogenous Insr. Treatment for 4 h with 40 nM of insulin resulted in the binding of insulin to its receptor INSR, leading to subsequent activation of downstream signaling inter-mediators (e.g.Akt), along with significant inhibition of MTA3 expression (by ~90%) in LCs. By contrast, the insulin activation-mediated inhibition of MTA3 expression was totally abolished in the TM3 cells that were stably deprived of endogenous INSR. Noteworthy, co-incubation with diamide alone could noticeably suppress the MTA3 expression, even in the presence of ablation of INSR (Fig. 2D). In line with these findings, co-treatment for 4 h with diphenyleneiodonium chloride successfully rescued insulin-impaired MTA3 expression in primary-cultured LCs (Fig. 2E). Thus, upregulation oxidative stress was important for the suppression of MTA3 expression in insulin-challenged LCs.
Oxidative stress mediates the inhibitory effects of insulin on MTA3 in LCs. (A) Primary LCs were challenged with different doses of H2O2 for 2 h, in the presence or absence of 20 μmol/L diphenyleneiodonium chloride, followed by western blotting analysis of MTA3 expression. Densitometric scanning of immunoblots was performed in which the level of a target protein was normalized against the protein level at 0 mM of H2O2, which was arbitrarily set at 1. (B) Primary LCs were challenged with different doses of diamide for 2 h, in the presence or absence of 20 μmol/L diphenyleneiodonium chloride, followed by western blotting analysis of MTA3 expression. Densitometric scanning of immunoblots was performed in which the level of a target protein was normalized against the protein level at 0 mM of diamide, which was arbitrarily set at 1. (C) The pGL3-Mta3 reporter plasmid and pRL-TK Renilla reporter plasmid were co-transfected into TM3 cells; 48 h later, the cells were treated for 5 h with 0.2 mmol/L of db-cAMP, in the presence or absence of a combined treatment with H2O2and 20 μmol/L diphenyleneiodonium chloride, followed by measurement of luciferase activities using the Promega dual reporter assay system. (D) The TM3 cells that were stably deprived of endogenous Insr were incubated for 4 h with 40 nM of insulin, in the presence or absence of 1 mM of diamide, followed by western blotting analysis. Densitometric scanning of immunoblots was performed in which the level of a target protein was normalized against the protein level in TM3 cells transfected with Ctrl shRNA, which was arbitrarily set at 1. (E) Primary LCs were treated for 4 h with 40 nM of insulin, in the presence or absence of 20 μmol/L diphenyleneiodonium chloride, followed by western blotting analysis. Densitometric scanning of immunoblots was performed in which the level of a target protein was normalized against the protein level in non-treated LCs, which was arbitrarily set at 1. LCs, Leydig cells; MTA3, metastasis-associated protein 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0413
Oxidative stress mediates the inhibitory effects of insulin on MTA3 in LCs. (A) Primary LCs were challenged with different doses of H2O2 for 2 h, in the presence or absence of 20 μmol/L diphenyleneiodonium chloride, followed by western blotting analysis of MTA3 expression. Densitometric scanning of immunoblots was performed in which the level of a target protein was normalized against the protein level at 0 mM of H2O2, which was arbitrarily set at 1. (B) Primary LCs were challenged with different doses of diamide for 2 h, in the presence or absence of 20 μmol/L diphenyleneiodonium chloride, followed by western blotting analysis of MTA3 expression. Densitometric scanning of immunoblots was performed in which the level of a target protein was normalized against the protein level at 0 mM of diamide, which was arbitrarily set at 1. (C) The pGL3-Mta3 reporter plasmid and pRL-TK Renilla reporter plasmid were co-transfected into TM3 cells; 48 h later, the cells were treated for 5 h with 0.2 mmol/L of db-cAMP, in the presence or absence of a combined treatment with H2O2and 20 μmol/L diphenyleneiodonium chloride, followed by measurement of luciferase activities using the Promega dual reporter assay system. (D) The TM3 cells that were stably deprived of endogenous Insr were incubated for 4 h with 40 nM of insulin, in the presence or absence of 1 mM of diamide, followed by western blotting analysis. Densitometric scanning of immunoblots was performed in which the level of a target protein was normalized against the protein level in TM3 cells transfected with Ctrl shRNA, which was arbitrarily set at 1. (E) Primary LCs were treated for 4 h with 40 nM of insulin, in the presence or absence of 20 μmol/L diphenyleneiodonium chloride, followed by western blotting analysis. Densitometric scanning of immunoblots was performed in which the level of a target protein was normalized against the protein level in non-treated LCs, which was arbitrarily set at 1. LCs, Leydig cells; MTA3, metastasis-associated protein 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0413
Oxidative stress mediates the inhibitory effects of insulin on MTA3 in LCs. (A) Primary LCs were challenged with different doses of H2O2 for 2 h, in the presence or absence of 20 μmol/L diphenyleneiodonium chloride, followed by western blotting analysis of MTA3 expression. Densitometric scanning of immunoblots was performed in which the level of a target protein was normalized against the protein level at 0 mM of H2O2, which was arbitrarily set at 1. (B) Primary LCs were challenged with different doses of diamide for 2 h, in the presence or absence of 20 μmol/L diphenyleneiodonium chloride, followed by western blotting analysis of MTA3 expression. Densitometric scanning of immunoblots was performed in which the level of a target protein was normalized against the protein level at 0 mM of diamide, which was arbitrarily set at 1. (C) The pGL3-Mta3 reporter plasmid and pRL-TK Renilla reporter plasmid were co-transfected into TM3 cells; 48 h later, the cells were treated for 5 h with 0.2 mmol/L of db-cAMP, in the presence or absence of a combined treatment with H2O2and 20 μmol/L diphenyleneiodonium chloride, followed by measurement of luciferase activities using the Promega dual reporter assay system. (D) The TM3 cells that were stably deprived of endogenous Insr were incubated for 4 h with 40 nM of insulin, in the presence or absence of 1 mM of diamide, followed by western blotting analysis. Densitometric scanning of immunoblots was performed in which the level of a target protein was normalized against the protein level in TM3 cells transfected with Ctrl shRNA, which was arbitrarily set at 1. (E) Primary LCs were treated for 4 h with 40 nM of insulin, in the presence or absence of 20 μmol/L diphenyleneiodonium chloride, followed by western blotting analysis. Densitometric scanning of immunoblots was performed in which the level of a target protein was normalized against the protein level in non-treated LCs, which was arbitrarily set at 1. LCs, Leydig cells; MTA3, metastasis-associated protein 3.
Citation: Reproduction 163, 5; 10.1530/REP-21-0413
Oxidative stress inhibits MTA3 expression via suppression of NR4A1-mediated transactivation
Both luciferase reporter assay and western blotting analysis revealed a tight association between MTA3 transactivation/upregulation and elevated steroidogenic activity in LCs (Fig. 3A and B). Intriguingly, a dose-dependent increase in the expression levels of NR4A1, one of the major transcription factors that function along the testicular steroidogenesis (Song et al. 2012), was also observed in the dbcAMP-stimulated TM3 cells, well correlated to MTA3 expression (Fig. 3B). Considering that NR4A1 regulates the expression of a majority of the steroidogenic enzyme genes (Park et al. 2014), we sought to determine whether the positive correlation that exists between MTA3 and NR4A1 involves a causal relationship. We transiently knocked down the expression of NR4A1 in TM3 cells. Subsequent promoter assay showed that TM3/MTA3 siRNA cells exhibited a significant reduction in dbcAMP-stimulated Mta3 promoter activities, compared to TM3/Ctrl siRNA cells (Fig. 3C). Conversely, overexpression of the exogenous NR4A1 increased the Mta3 promoter activities by ~53.1% in dbcAMP-stimulated TM3 cells (Fig. 3D). To further provide the molecular evidence for the transcriptional regulation of Mta3 by NR4A1, we evaluated the direct binding of NR4A1 onto the Mta3 chromatin using an electrophoretic mobility shift assay. NR4A1 in the nuclear extracts from db-cAMP-treated TM3 cells clearly formed a shifted DIG-labeled band (Fig. 3E, lane 3), which was supershifted by anti-NR4A1 antibody (Fig. 3E, lane 5). The band was completely abrogated by competition with a 50-fold excess of cold probe (Fig. 3E, lane 2) or con-incubation with 1 mM of diamide for 2 h. To demonstrate the occupancy of NR4A1 at the Mta3 promoter in vivo, we carried out the ChIP assay. dbcAMP treatment induced the recruitment of NR4A1 onto the Mta3 promoter in a dose-dependent manner. This recruitment was totally abrogated in the presence of co-incubation with 1 mM of diamide (Fig. 3F). Together, we have identified Mta3 as the direct downstream target of NR4A1 in stimulated LCs.
Oxidative stress suppresses NR4A1-mediated transactivation of Mta3 in stimulated LCs. (A) The pGL3-Mta3 reporter plasmid and pRL-TK Renilla reporter plasmid were co-transfected into TM3 cells; 48 h later, the cells were treated for 5 h with different doses of db-cAMP, followed by measurement of luciferase activities (paired Student’s t-test, n = 4, *P< 0.05). (B) TM3 cells were treated for 5 h with different doses of db-cAMP, followed by western blotting analysis. Densitometric scanning of immunoblots was performed in which the level of a target protein was normalized against the protein level in non-treated TM3, which was arbitrarily set at 1. (C) TM3 cells were transfected for 48 h with NR4A1 siRNA or Ctrl siRNA. Cells were then subjected to db-cAMP challenge as described in Panel B, followed by measurement of luciferase activities (paired Student’s t-test, n = 4, *P<0.05). Effects of siRNA treatment on NR4A1 expression were evaluated by western blotting analysis. (D) TM3/NR4A1 cells were subjected to db-cAMP challenge as described in Panel B, followed by measurement of luciferase activities (paired Student’s t-test, n = 4, *P< 0.05). NR4A1 overexpression was confirmed by western blotting analysis. (E) TM3/NR4A1 cells were treated for 5 h with 0.2 mmol/L of db-cAMP, in the presence or absence of co-incubation with 1 mM of diamide. Subsequently, the cells were collected and subjected to nonradioactive EMSA. Competitive inhibition consisted of incubation with a 50-fold molar excess of unlabeled/cold probe or with 1 mM of diamide for 2 h. Anti-NR4A1 antibody was also used to confirm the specificity of the band. (F) ChIP-qPCR analysis showing recruitment of NR4A1 onto a specific region of the Mta3 promoter. This recruitment was further enhanced by treatment with db-cAMP in a dose-dependent manner and was totally abolished by co-treatment with 1 mM of diamide (one-way ANOVA followed by Tukey’s post hoc tests, n = 4, *P< 0.05 and **P< 0.01). db-cAMP, dibutyryl-cAMP; EMSA, electrophoretic mobility shift assay; LCs, Leydig cells; MTA3, metastasis-associated protein 3; NR4A1, nuclear receptor subfamily 4 group A member 1.
Citation: Reproduction 163, 5; 10.1530/REP-21-0413
Oxidative stress suppresses NR4A1-mediated transactivation of Mta3 in stimulated LCs. (A) The pGL3-Mta3 reporter plasmid and pRL-TK Renilla reporter plasmid were co-transfected into TM3 cells; 48 h later, the cells were treated for 5 h with different doses of db-cAMP, followed by measurement of luciferase activities (paired Student’s t-test, n = 4, *P< 0.05). (B) TM3 cells were treated for 5 h with different doses of db-cAMP, followed by western blotting analysis. Densitometric scanning of immunoblots was performed in which the level of a target protein was normalized against the protein level in non-treated TM3, which was arbitrarily set at 1. (C) TM3 cells were transfected for 48 h with NR4A1 siRNA or Ctrl siRNA. Cells were then subjected to db-cAMP challenge as described in Panel B, followed by measurement of luciferase activities (paired Student’s t-test, n = 4, *P<0.05). Effects of siRNA treatment on NR4A1 expression were evaluated by western blotting analysis. (D) TM3/NR4A1 cells were subjected to db-cAMP challenge as described in Panel B, followed by measurement of luciferase activities (paired Student’s t-test, n = 4, *P< 0.05). NR4A1 overexpression was confirmed by western blotting analysis. (E) TM3/NR4A1 cells were treated for 5 h with 0.2 mmol/L of db-cAMP, in the presence or absence of co-incubation with 1 mM of diamide. Subsequently, the cells were collected and subjected to nonradioactive EMSA. Competitive inhibition consisted of incubation with a 50-fold molar excess of unlabeled/cold probe or with 1 mM of diamide for 2 h. Anti-NR4A1 antibody was also used to confirm the specificity of the band. (F) ChIP-qPCR analysis showing recruitment of NR4A1 onto a specific region of the Mta3 promoter. This recruitment was further enhanced by treatment with db-cAMP in a dose-dependent manner and was totally abolished by co-treatment with 1 mM of diamide (one-way ANOVA followed by Tukey’s post hoc tests, n = 4, *P< 0.05 and **P< 0.01). db-cAMP, dibutyryl-cAMP; EMSA, electrophoretic mobility shift assay; LCs, Leydig cells; MTA3, metastasis-associated protein 3; NR4A1, nuclear receptor subfamily 4 group A member 1.
Citation: Reproduction 163, 5; 10.1530/REP-21-0413
Oxidative stress suppresses NR4A1-mediated transactivation of Mta3 in stimulated LCs. (A) The pGL3-Mta3 reporter plasmid and pRL-TK Renilla reporter plasmid were co-transfected into TM3 cells; 48 h later, the cells were treated for 5 h with different doses of db-cAMP, followed by measurement of luciferase activities (paired Student’s t-test, n = 4, *P< 0.05). (B) TM3 cells were treated for 5 h with different doses of db-cAMP, followed by western blotting analysis. Densitometric scanning of immunoblots was performed in which the level of a target protein was normalized against the protein level in non-treated TM3, which was arbitrarily set at 1. (C) TM3 cells were transfected for 48 h with NR4A1 siRNA or Ctrl siRNA. Cells were then subjected to db-cAMP challenge as described in Panel B, followed by measurement of luciferase activities (paired Student’s t-test, n = 4, *P<0.05). Effects of siRNA treatment on NR4A1 expression were evaluated by western blotting analysis. (D) TM3/NR4A1 cells were subjected to db-cAMP challenge as described in Panel B, followed by measurement of luciferase activities (paired Student’s t-test, n = 4, *P< 0.05). NR4A1 overexpression was confirmed by western blotting analysis. (E) TM3/NR4A1 cells were treated for 5 h with 0.2 mmol/L of db-cAMP, in the presence or absence of co-incubation with 1 mM of diamide. Subsequently, the cells were collected and subjected to nonradioactive EMSA. Competitive inhibition consisted of incubation with a 50-fold molar excess of unlabeled/cold probe or with 1 mM of diamide for 2 h. Anti-NR4A1 antibody was also used to confirm the specificity of the band. (F) ChIP-qPCR analysis showing recruitment of NR4A1 onto a specific region of the Mta3 promoter. This recruitment was further enhanced by treatment with db-cAMP in a dose-dependent manner and was totally abolished by co-treatment with 1 mM of diamide (one-way ANOVA followed by Tukey’s post hoc tests, n = 4, *P< 0.05 and **P< 0.01). db-cAMP, dibutyryl-cAMP; EMSA, electrophoretic mobility shift assay; LCs, Leydig cells; MTA3, metastasis-associated protein 3; NR4A1, nuclear receptor subfamily 4 group A member 1.
Citation: Reproduction 163, 5; 10.1530/REP-21-0413
Ectopic NR4A1 expression ameliorates oxidative stress-impaired MTA3 expression in LCs
To unequivocally elucidate whether oxidative stress inhibits MTA3 expression by disrupting the NR4A1-mediated transactivation, we stably overexpressed the exogenous pCMV6-NR4A1 in TM3 cells, as NR4A1 activity requires no ligand stimulation and is constitutively active when overexpressed (Suzuki et al. 2003). Challenge with diamide significantly attenuated the expression levels of MTA3 in stimulated TM3 cells, but did not alter the dbcAMP-elicited upregulation of MTA3 in TM3/NR4A1 cells (Fig. 4A). Consistently, NR4A1 overexpression effectively restored the oxidative stress-impaired Mta3 promoter activities in stimulated TM3 cells (Fig. 4B).
Constitutive activation of NR4A1 ameliorates oxidative stress-impaired MTA3 expression in LCs. (A) TM3/NR4A1 cells were treated for 5 h with 0.2 mM of db-cAMP, in the presence or absence of 1 mM of diamide, followed by western blotting analysis. (B) The pGL3-Mta3 reporter plasmid and pRL-TK Renilla reporter plasmid were co-transfected into TM3/NR4A1 cells; 48 h later, the cells were treated for 5 h with different 0.2 mM of db-cAMP, in the presence or absence of 1 mM of diamide, followed by the measurement of luciferase activities (n = 5). db-cAMP, dibutyryl-cAMP; LCs, Leydig cells; MTA3, metastasis-associated protein 3; NR4A1, nuclear receptor subfamily 4 group A member 1.
Citation: Reproduction 163, 5; 10.1530/REP-21-0413
Constitutive activation of NR4A1 ameliorates oxidative stress-impaired MTA3 expression in LCs. (A) TM3/NR4A1 cells were treated for 5 h with 0.2 mM of db-cAMP, in the presence or absence of 1 mM of diamide, followed by western blotting analysis. (B) The pGL3-Mta3 reporter plasmid and pRL-TK Renilla reporter plasmid were co-transfected into TM3/NR4A1 cells; 48 h later, the cells were treated for 5 h with different 0.2 mM of db-cAMP, in the presence or absence of 1 mM of diamide, followed by the measurement of luciferase activities (n = 5). db-cAMP, dibutyryl-cAMP; LCs, Leydig cells; MTA3, metastasis-associated protein 3; NR4A1, nuclear receptor subfamily 4 group A member 1.
Citation: Reproduction 163, 5; 10.1530/REP-21-0413
Constitutive activation of NR4A1 ameliorates oxidative stress-impaired MTA3 expression in LCs. (A) TM3/NR4A1 cells were treated for 5 h with 0.2 mM of db-cAMP, in the presence or absence of 1 mM of diamide, followed by western blotting analysis. (B) The pGL3-Mta3 reporter plasmid and pRL-TK Renilla reporter plasmid were co-transfected into TM3/NR4A1 cells; 48 h later, the cells were treated for 5 h with different 0.2 mM of db-cAMP, in the presence or absence of 1 mM of diamide, followed by the measurement of luciferase activities (n = 5). db-cAMP, dibutyryl-cAMP; LCs, Leydig cells; MTA3, metastasis-associated protein 3; NR4A1, nuclear receptor subfamily 4 group A member 1.
Citation: Reproduction 163, 5; 10.1530/REP-21-0413
Replenishment of MTA3 expression in vivo partially restores testicular steroidogenesis and improves male fertility in diabetic mice
Having unveiled the molecular events underpinning the oxidative stress-impaired MTA3 expression in LCs, we sought to determine the biological importance of MTA3 signaling in steroidogenesis and spermatogenesis. Recent advances in this field have demonstrated that microinjection of lentiviral plasmids could achieve an efficient in vivo gene transfer in LCs, thus providing a valuable alternative approach for studying the function of steroidogenesis related to the testis (Park et al. 2013). In this regard, pLV-EGFP-Mta3 or empty vector was microinjected into seminiferous tubules of diabetic mice with TD. Seventy days later (this duration corresponds to two cycles of murine spermatogenesis), mice were subjected to epididymal analysis, testosterone assay and histological examination (Fig. 5A). We first noticed that lentivirus-mediated bioluminescence was clearly enriched in the testicular interstitium (7.21 ± 1.66 vs 0.96 ± 0.11 relative green fluorescent protein (GFP) intensity for pLV-EGFP-Mta3 vs Vector, P< 0.01), confirming the efficiency and specificity of the assay (Fig. 5B). Consequently, Mta3/MTA3 expression was increased by ~2.7-fold in the diabetic testes injected with pLV-EGFP-Mta3 compared with Ctrl testes or diabetic testes injected with empty vector, as revealed by RT-qPCR (Fig. 5C) and western blotting (Fig. 5D), respectively. Mimicking the phenotype of MTA3-overexpressing LCs (He et al. 2016), Ctrl testes displayed normal testicular architecture while diabetic testes with TD had signs of degenerative changes, including germ cells desquamation, decrease of germinal epithelium height and enlarged interstitial space. These morphological changes in diabetic testes with TD were noticeably ameliorated following microinjection of pLV-EGFP-Mta3 (Fig. 5E). We further confirmed the therapeutic effects of the microinjection of pLV-EGFP-Mta3 on male reproductive function by assessing several quantitative endpoints (T2DM with TD + Mta3 vs T2DM with TD + vector), including testicular apoptosis (P< 0.05, Fig. 5F), serum testosterone levels (P< 0.01, Fig. 5F), epididymal sperm density (P< 0.01, Fig. 5G) and percentage of progressive motility in epididymal sperms (P< 0.01, Fig. 5H). Surprisingly, ectopic overexpression of MTA3 also effectively ameliorated T2DM-elicited oxidative stress in diabetic testes with TD (Fig. 5I and J). In addition, we assessed the serum levels of LH, as well as expression levels of luteinizing hormone/choriogonadotropin receptor (LHCGR) and androgen receptor (AR) in primary LCs, from our lentiviral plasmid-treated mice, given that these are all key markers of Leydig cell health and function. Serum LH levels appeared to be comparable among different experimental groups. In accordance with the serological results, the expression of LHCGR and NR4A1 were unaffected in different groups (Fig. 5L). By contrast, TD resulted in a significant decrease in the expression levels of AR in primary LCs from diabetic testis, and this compromised expression of AR was totally reversed upon ectopic overexpression of MTA3 (Fig. 5M). These findings, consistent with a recent report in which LH plasma concentration is observed to be unaltered in T2DM patients with TD (Schianca et al. 2017), strengthen the notion that insulin may impair testicular steroidogenesis without affecting hypothalamic–pituitary axis directly. As for AR, it is an autoregulated protein (Lu et al. 1998), so it is a logical observation that its expression correlated well to changes in circulated testosterone in our in vivo model. Altogether, these data support the concept that manipulation of MTA3 expression in the testis can affect testicular steroidogenesis, as well as spermatogenesis.
Effects of MTA3 replenishment on testicular steroidogenesis and male fertility in diabetic mice. (A) Schematic representation of the experimental procedures used in the in vivo microinjection of lentiviral plasmids. (B) Enrichment of lentiviral plasmids in the testicular interstitium following microinjection was demonstrated by observing frozen sections under a fluorescence microscope. Scale bar, 50 μm. Right panel, GFP fluorescence (λex488/9/λem525/20 nm) of pLV-EGFP-Mta3-treated testis and vector-treated testis were measured using a fluorimeter. The fluorescence increase was calculated using the non-treated control of each group as reference (paired Student’s t-test, n = 5, **P< 0.01). (C) Augmentation of Mta3 expression in vivo was verified using RT-qPCR at 70 days after microinjection. Data were presented as the mean ± s.d. of at least three determinations (one-way ANOVA followed by Tukey’s post hoc tests, n = 7, *P < 0.05 and **P < 0.01). (D) Western blotting analysis of MTA3 expression in mouse testis 70 days after microinjection. (E) Representative H&E-stained transverse testis sections showing morphological changes 70 days following microinjection. Scale bar, 50 μm. (F) Effects of in vivo overexpression of MTA3 on testicular apoptosis 70 days after microinjection were determined using an ELISA kit (one-way ANOVA followed by Tukey’s post hoc tests, n = 7, *P< 0.05 and **P< 0.01). (G, H and I) Effects of in vivo overexpression of MTA3 on serum testosterone, sperm concentrations in caudal epididymis and progressive motility of sperm in caudal epididymis were assessed as described in the ‘Materials and methods’ section (one-way ANOVA followed by Tukey’s post hoc tests, n = 7, *P< 0.05 and **P<0.01). (J and K) Effects of in vivo overexpression of MTA3 on levels of MDA and T-SOD from different experimental groups were determined using commercial kits, with absorbance read at 532 nm for MDA and 560 nm for T-SOD, respectively (one-way ANOVA followed by Tukey’s post hoc tests, n = 7, *P< 0.05 and **P< 0.01). (L) Effects of in vivooverexpression of MTA3 on serum luteinizing hormone were determined using an ELISA kit (one-way ANOVA followed by Tukey’s post hoc tests, n = 7). (M) Effects of in vivo overexpression of MTA3 on expression levels of LHCGR, AR and NR4A1 were assessed by western blotting in primary LCs from different groups 70 days after microinjection. AR, androgen receptor; H&E, hematoxylin and eosin; LHCGR, luteinizing hormone/choriogonadotropin receptor; MTA3, metastasis-associated protein 3; NR4A1, nuclear receptor subfamily 4 group A member 1.
Citation: Reproduction 163, 5; 10.1530/REP-21-0413
Effects of MTA3 replenishment on testicular steroidogenesis and male fertility in diabetic mice. (A) Schematic representation of the experimental procedures used in the in vivo microinjection of lentiviral plasmids. (B) Enrichment of lentiviral plasmids in the testicular interstitium following microinjection was demonstrated by observing frozen sections under a fluorescence microscope. Scale bar, 50 μm. Right panel, GFP fluorescence (λex488/9/λem525/20 nm) of pLV-EGFP-Mta3-treated testis and vector-treated testis were measured using a fluorimeter. The fluorescence increase was calculated using the non-treated control of each group as reference (paired Student’s t-test, n = 5, **P< 0.01). (C) Augmentation of Mta3 expression in vivo was verified using RT-qPCR at 70 days after microinjection. Data were presented as the mean ± s.d. of at least three determinations (one-way ANOVA followed by Tukey’s post hoc tests, n = 7, *P < 0.05 and **P < 0.01). (D) Western blotting analysis of MTA3 expression in mouse testis 70 days after microinjection. (E) Representative H&E-stained transverse testis sections showing morphological changes 70 days following microinjection. Scale bar, 50 μm. (F) Effects of in vivo overexpression of MTA3 on testicular apoptosis 70 days after microinjection were determined using an ELISA kit (one-way ANOVA followed by Tukey’s post hoc tests, n = 7, *P< 0.05 and **P< 0.01). (G, H and I) Effects of in vivo overexpression of MTA3 on serum testosterone, sperm concentrations in caudal epididymis and progressive motility of sperm in caudal epididymis were assessed as described in the ‘Materials and methods’ section (one-way ANOVA followed by Tukey’s post hoc tests, n = 7, *P< 0.05 and **P<0.01). (J and K) Effects of in vivo overexpression of MTA3 on levels of MDA and T-SOD from different experimental groups were determined using commercial kits, with absorbance read at 532 nm for MDA and 560 nm for T-SOD, respectively (one-way ANOVA followed by Tukey’s post hoc tests, n = 7, *P< 0.05 and **P< 0.01). (L) Effects of in vivooverexpression of MTA3 on serum luteinizing hormone were determined using an ELISA kit (one-way ANOVA followed by Tukey’s post hoc tests, n = 7). (M) Effects of in vivo overexpression of MTA3 on expression levels of LHCGR, AR and NR4A1 were assessed by western blotting in primary LCs from different groups 70 days after microinjection. AR, androgen receptor; H&E, hematoxylin and eosin; LHCGR, luteinizing hormone/choriogonadotropin receptor; MTA3, metastasis-associated protein 3; NR4A1, nuclear receptor subfamily 4 group A member 1.
Citation: Reproduction 163, 5; 10.1530/REP-21-0413
Effects of MTA3 replenishment on testicular steroidogenesis and male fertility in diabetic mice. (A) Schematic representation of the experimental procedures used in the in vivo microinjection of lentiviral plasmids. (B) Enrichment of lentiviral plasmids in the testicular interstitium following microinjection was demonstrated by observing frozen sections under a fluorescence microscope. Scale bar, 50 μm. Right panel, GFP fluorescence (λex488/9/λem525/20 nm) of pLV-EGFP-Mta3-treated testis and vector-treated testis were measured using a fluorimeter. The fluorescence increase was calculated using the non-treated control of each group as reference (paired Student’s t-test, n = 5, **P< 0.01). (C) Augmentation of Mta3 expression in vivo was verified using RT-qPCR at 70 days after microinjection. Data were presented as the mean ± s.d. of at least three determinations (one-way ANOVA followed by Tukey’s post hoc tests, n = 7, *P < 0.05 and **P < 0.01). (D) Western blotting analysis of MTA3 expression in mouse testis 70 days after microinjection. (E) Representative H&E-stained transverse testis sections showing morphological changes 70 days following microinjection. Scale bar, 50 μm. (F) Effects of in vivo overexpression of MTA3 on testicular apoptosis 70 days after microinjection were determined using an ELISA kit (one-way ANOVA followed by Tukey’s post hoc tests, n = 7, *P< 0.05 and **P< 0.01). (G, H and I) Effects of in vivo overexpression of MTA3 on serum testosterone, sperm concentrations in caudal epididymis and progressive motility of sperm in caudal epididymis were assessed as described in the ‘Materials and methods’ section (one-way ANOVA followed by Tukey’s post hoc tests, n = 7, *P< 0.05 and **P<0.01). (J and K) Effects of in vivo overexpression of MTA3 on levels of MDA and T-SOD from different experimental groups were determined using commercial kits, with absorbance read at 532 nm for MDA and 560 nm for T-SOD, respectively (one-way ANOVA followed by Tukey’s post hoc tests, n = 7, *P< 0.05 and **P< 0.01). (L) Effects of in vivooverexpression of MTA3 on serum luteinizing hormone were determined using an ELISA kit (one-way ANOVA followed by Tukey’s post hoc tests, n = 7). (M) Effects of in vivo overexpression of MTA3 on expression levels of LHCGR, AR and NR4A1 were assessed by western blotting in primary LCs from different groups 70 days after microinjection. AR, androgen receptor; H&E, hematoxylin and eosin; LHCGR, luteinizing hormone/choriogonadotropin receptor; MTA3, metastasis-associated protein 3; NR4A1, nuclear receptor subfamily 4 group A member 1.
Citation: Reproduction 163, 5; 10.1530/REP-21-0413
Discussion
It is well documented that neural, endocrine, paracrine and autocrine inputs form a complicated signaling network governing the testicular homeostasis and steroidogenesis in males (Zirkin & Papadopoulos 2018). However, characterization of the repertoire of the core regulatory factors, especially as they pertain to the transcriptional control of steroidogenic gene expression and their deregulation in conditions of metabolic stress (e.g. diabetes or obesity), is extremely incomplete. Together with our previous findings (He et al. 2016), the current study has uncovered a novel mechanism of T2DM-induced testosterone deficiency that involves the repurposing of a pivotal chromatin remodeling transcription factor in a new role as a positive effector of testicular steroidogenesis. In particular, we observe that T2DM-elicited oxidative stress inhibits MTA3 expression, which involves dose-dependent alterations in the repression of NR4A1-mediated transactivation of MTA1 in LCs (Fig. 3). Our results thereby pave the way for the identification of upstream regulators of testicular MTA3 that may modulate androgen synthesis in diabetes.
The deleterious effect of oxidative stress on the testicular expression of MTA3 was disclosed by a combination of pathological and molecular approaches. Both T2DM induction in mice and treatment of murine LCs with oxidative stress inducers (H2O2 or diamide) resulted in a significant decrease in the expression levels of MTA3 (Figs 1E, F, G and 2A, B). The causal nature of these associations is further displayed by our reporter assay at the transcriptional level. Thus, co-incubation with H2O2 significantly inhibited, in a dose-dependent manner, the Mta3 promoter activities, whereas concomitant treatment with the oxidative stress inhibitor, diphenyleneiodonium chloride, effectively rescued the H2O2-impaired luciferase activities in db-cAMP-challenged TM3 cells (Fig. 2C). Testicular oxidative stress is generally believed to be the principal underlying cause for defective steroidogenesis and spermatogenesis (Turner & Lysiak 2008). Indeed, intervention with antioxidants (e.g. tert-butylhydroquinone and resveratrol) has been shown to be effective in the quest to dampen oxidative stress-induced testicular dysfunction (Faid et al. 2015, Nna et al. 2020). Together with our findings, the available data strongly suggest a key role for MTA3 as a putative conduit for the regulatory actions of oxidative stress on testicular steroidogenesis.
Despite our initial effort elucidating the dose and time dependency of insulin-inhibited MTA3 expression in LCs (He et al. 2016), the current study showed that oxidative stress mediates the inhibitory effects of insulin on MTA3 expression. This contention is supported by three demonstrations: (i) supplement with the strong antioxidant melatonin successfully rescued the expression levels of MTA3 in LCs, but failed to restore serum insulin levels in diabetic mice (Fig. 1D, E, F and G); (ii) co-incubation with diamide alone inhibited MTA3 expression in the TM3 cells that were stably deprived of endogenous Insr (Fig. 2D), indicating a direct inhibitory effect of oxidative stress on MTA3 expression; (iii) co-treatment with the oxidative stress inhibitor diphenyleneiodonium chloride substantially rescued insulin-impaired MTA3 expression in primary cultured LCs (Fig. 2E). Notwithstanding the potential possibility that insulin might regulate the expression of MTA3 through other indirect signaling pathways in LCs, our results conclusively identify oxidative stress as a bona fide upstream signal that transduces metabolic stress information into changes of MTA3 expression during androgen production. Additionally, it is well known that mammalian testis contains comparably higher levels of unsaturated fatty acids as well as high rates of mitochondrial oxygen consumption than in other tissues due to its active cell replication and weakness of the testicular artery, which makes testis prone to oxidative stress-induced tissue damage (Asadi et al. 2017). On the other hand, inflammation triggers peroxidase-positive leukocytes (50–60%) as well as macrophages (20–30%) to generate reactive oxygen species (ROS) about 100 times more than its production under normal conditions. So these two cell types located in prostate and seminal vesicles may serve as another source of testicular ROS (Darbandi et al. 2018). In summary, cell types responsible for the generation of testicular ROS are heterogeneous and consist of different germ cell (GC) types such as spermatozoa and various microenvironmental cell types such as Sertoli cells (SCs) and LCs. The exact roles of different cell types in the production of oxidative stress within testis merit further investigation.
The molecular underpinnings of the above-mentioned phenotype have been also disclosed in the present study. The expression of steroidogenic genes is regulated fundamentally at the transcriptional level (Lavoie & King 2009, Roumaud & Martin 2015), while NR4A1 has long been recognized as a master transcription factor modulating the expression of steroidogenic enzyme genes, including steroid 21-hydroxylase, P450c17, 3β-HSD and StAR (Lee et al. 2009). It is therefore a logical observation that NR4A1 potentiates the transactivation of Mta3 in stimulated LCs (Fig. 3C and D). Moreover, our molecular analysis further demonstrated that dbcAMP treatment induced, in a dose-dependent manner, the direct recruitment of NR4A1 onto the Mta3 promoter, whereas the upregulation of oxidative stress substantially abrogated this recruitment (Fig. 3E and F). Thus, oxidative stress appears to operate as a critical signaling link between metabolic stress (e.g. hyperinsulinemia and hyperglycemia) and steroidogenic activity in LCs, by changing the association between NR4A1 and the Mta3 promoter.
Testosterone deficiency, or male hypogonadism, is a condition where the serum testosterone levels are below normal. It can be caused by many pathophysiological conditions, including obesity, old age, diabetes mellitus, hyper- and hypothyroidism, as well as in men taking certain medications (Bhasin et al. 2010). Patients with TD are frequently associated with an increased risk of sexual dysfunction, fatigue, fractures, depressed mood, anemia and loss of muscle. These patients are therefore recommended to have testosterone replacement therapy to improve symptoms and signs and to augment the benefits of lifestyle interventions (Barbonetti et al. 2020). However, there are mounting evidence that exogenous testosterone therapy may cause certain adverse outcomes, such as an increased risk of prostate cancer, poorly controlled congestive heart failure or severe lower urinary tract symptoms (Weikert et al. 2010). In this regard, our study has practical and potential therapeutic relevance because we have shown that the replenishment of MTA3 expression in vivo partially restored androgen synthesis, alleviated oxidative stress and improved male fertility in diabetic mice (Fig. 5). These results suggest that the ectopic expression of MTA3 using pharmacogenetic approaches may serve as an alternative therapeutic option for the treatment of TD, thus emphasizing the translational significance of such a local oxidative stress/NR4A1/MTA3 pathway in the pathological control of androgen production upon metabolic stress.
In summary, our results have unveiled a central regulatory hub, involving oxidative stress-impaired NR4A1-driven transactivation of MTA3 in stimulated LCs, as a pivotal mechanism regulating diabetes and TD (Fig. 6). These findings expand our current understanding of the molecular basis for the upstream regulation of MTA3 in LCs, and pave the way for the identification of novel transcription factors in TD-related disorders.
Proposed model of the insulin/oxidative stress signaling pathway in the negative regulation of NR4A1-mediated transactivation of Mta3 in stimulated LCs. LCs, Leydig cells; MTA3, metastasis-associated protein 3; NR4A1, nuclear receptor subfamily 4 group A member 1.
Citation: Reproduction 163, 5; 10.1530/REP-21-0413
Proposed model of the insulin/oxidative stress signaling pathway in the negative regulation of NR4A1-mediated transactivation of Mta3 in stimulated LCs. LCs, Leydig cells; MTA3, metastasis-associated protein 3; NR4A1, nuclear receptor subfamily 4 group A member 1.
Citation: Reproduction 163, 5; 10.1530/REP-21-0413
Proposed model of the insulin/oxidative stress signaling pathway in the negative regulation of NR4A1-mediated transactivation of Mta3 in stimulated LCs. LCs, Leydig cells; MTA3, metastasis-associated protein 3; NR4A1, nuclear receptor subfamily 4 group A member 1.
Citation: Reproduction 163, 5; 10.1530/REP-21-0413
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
This work was supported by the National Natural Science Foundation of China (NSFC) 31971070, 31671198 and 81270844.
Author contribution statement
Study design: L W and M J. Study conduct: L F, C Z and Z J. Data collection: L F, C Z and Z J. Data analysis: L F, C Z, L W and M J. Data interpretation: L W, Z Y and M J. Contribution of reagents: L W, Z Y and M J. Drafting and submission of manuscript: L W and L F. Research supervision: L W, M J and Z Y.
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