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Nuclear factor E2-related factor-2 (Nrf2) expression and regulation in male reproductive tract
Pharmacological Reports 68 (2016) 101–108
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Original research article
Nuclear factor E2-related factor-2 (Nrf2) expression and regulation in male reproductive tract Anna Wajda a, Joanna Łapczuk a, Marta Grabowska b, Marcin Słojewski c, Maria Laszczyn´ska b, Elz˙bieta Urasin´ska d, Marek Droz´dzik a,* a
Department of Experimental and Clinical Pharmacology, Pomeranian Medical University, Szczecin, Poland Department of Histology and Developmental Biology, Pomeranian Medical University, Szczecin, Poland Department of Urology and Urological Oncology, Pomeranian Medical University, Szczecin, Poland d Department of Pathology, Pomeranian Medical University, Szczecin, Poland b c
A R T I C L E I N F O
Article history: Received 20 May 2015 Received in revised form 25 June 2015 Accepted 8 July 2015 Available online 31 July 2015 Keywords: Nuclear factor E2-related factor-2 Male reproductive system
A B S T R A C T
Background: Nuclear factor E2-related factor-2 (Nrf2, Nfe2l2) plays an important, protective role in many tissues. However, information on molecular mechanisms of detoxification and drug metabolism regulated by Nrf2/NRF2 in testis and epididymis is scarce, but it may help to better characterize the function of blood-testis and epididymis barriers. Methods: Constitutive gene expression was analyzed by real time PCR with TaqMan Assay using DCTmethod. Additionally, gene expression after treatment with oltipraz- specific Nrf2 inducer was evaluated using DDCT-method. Cellular localization of the Nrf2 was visualized by immunohistochemical reaction. Results: The study showed that Nrf2 mRNA level in rat epididymis was higher than in testis. In human tissues, both testis and epididymis demonstrated similar expression levels of NRF2. Immunohistochemical analysis revealed NRF2/Nrf2 protein expression in testis and epididymis, which in the case of testis was dependant on spermatogenesis stage. Both in human and rat tissues constitutive expression of NQO1/Nqo1 was slightly higher in epididymis than in testis. Other Nrf2 regulated genes: GCLC/Gclc and UGT1A6/Ugt1a6 showed different ratios of testis/ epididymis/liver expression levels. Treatment with oltipraz (Nrf2 inducer) resulted in significant induction of Nrf2 expression solely in corpus of epididymis. Conclusions: Components of the Nrf2/NRF2 system along with coordinated genes are expressed in testis and epididymis. Moreover, some interspecies differences between rat and human were observed, which may impact extrapolation of experimental data into clinical findings. Studies on animal model showed that corpus of epididymis is the most responsive part of the male reproductive tract to oltipraz exposure at the gene expression level. ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
Introduction An increase in the number of studies on oxidative stress in the pathogenesis of idiopathic male infertility has been observed recently. The available data suggests that abnormal sperm morphology and function are associated with higher generation of reactive oxygen species (ROS) and reduced antioxidant status, i.e. oxidative stress [1]. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2, also called Nfe2I2) plays a key role in cellular antioxidant defence, and thus protects male reproductive tract against
* Corresponding author. E-mail address: [email protected] (M. Droz´dzik).
oxidative stress. Deficiency in Nrf2 function has deleterious effects in Sertoli and germ cells, and also in epididymal phase of sperm maturation [2]. In normal physiological conditions, Nrf2 is expressed at low level and resides mainly in cell cytoplasm, where it is promoted to degradation via ubiquitination by a repressor protein Keap1 [3]. Under oxidative stress or through Nrf2 activators such as xenobiotics, electrophiles or phytochemicals, Nrf2 dissociates from Keap1, translocates to the nucleus where heterodimer with Maf is formed, and then expression of genes with antioxidant response element (ARE) is activated [4]. Apart from antioxidative potential, Nrf2 is also an important regulator of both xenobiotic metabolism and transport [5]. It coordinates the function of genes coding for phase I and II
http://dx.doi.org/10.1016/j.pharep.2015.07.005 1734-1140/ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
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drug-metabolizing enzymes and drug transporters. ARE are located in promoter regions of many drug metabolizing genes such as CYP3A23, Nqo1, Ugt1a6 and drug transporters Mdr1a, Mdr1b, Mrp3, Mrp4 [6]. The present study focused on Nrf2/Keap1 system along with its coordinated enzymes Nqo1, Gsta2, Gclc, Ugt1a6 and transporter Mdr1 genes expression in testis and epididymis in rat and human. NQO1 (NAD(P)H:quinone acceptor oxidoreductase 1) is a twoelectron reductase considered as a chemoprotective enzyme, which deactivates many reactive species such as quinones, quinone-imines and azo-compounds [7]. In the 50 -flanking region of NQO1 gene two different response elements have been identified: ARE and XRE (xenobiotic response element). Hence, in many cellular systems activity of NQO1 may be regulated by NRF2 and aryl hydrocarbon receptor (AHR) [8]. Other Nrf2/Keap1 system controlled enzymes involved in drug metabolism and xenobiotic detoxification are glutathione transferases (GSTs), which cooperate with glutamate cystein ligase (GCLC). It was found that Gsta1 and Gsta2 isoforms are expressed in many human tissues, including male reproductive tract [9]. Observations indicated lower Gsta expression associated with an increase in apoptosis of germ cells in testis of adult rats exposed to androgen disruption in utero or transient mild testicular hyperthermia [10,11]. Another pathway of lipophilic xenobiotic and endobiotic elimination leads through glucuronidation, catalyzed by UGTs (uridine glucuronosyltransferases). UGT isoforms have been found not only in liver, but also in other tissues such as testis, kidney, gastrointestinal tract and brain [7,12]. Expression of UGT1A6, involved among others in metabolism of xenobiotics, may be controlled by many environmental factors through stress mediating receptors, including Nrf2, and also pregnane X receptor (PXR), constitutive androstane receptor (CAR) or AHR [7]. Just as in the case of enzymes, a group of drug transporter expression can be controlled by Nfr2/Keap system, including Mdr1a, Mdr1b, Mrp3, Mrp4 [6]. It is postulated that MDR1 (Pglycoprotein), the product of MDR1 gene in humans and Mdr1a and Mdr1b genes in rodents plays an important role in maintaining functional integrity of blood–testis and blood–epididymis barriers, limiting penetration of endo- and exogenous substrates (drugs, toxins). Higher testicular concentrations of Mdr1 substrates were found in Mdr1a/b/(knockout) mice in comparison with wild-type animals. P-glycoprotein is abundantly expressed in the capillary endothelium of human testis and in the myoid-cell layer around seminiferous tubules. Several agents are proposed to be transported and excreted by P glycoprotein, including carcinogens, xenobiotics, hormones and bilirubin [13,14]. Expression of proteins, controlling metabolism and transport constitute essential components of blood–testis and blood– epididymis barriers. The activity of metabolizing and transport systems within the barriers protects male reproductive tract against potential insults, and allows proper germ cell development, maturation and storage of functionally mature spermatozoa [6]. Therefore, it is important to define cellular localization and function factors providing protective actions within male reproductive tract, i.e. in testis and along epididymis. Up to now, efforts have been directed to testicular characteristics. Scarce data have been reported for epididymis, especially in view of segment specific functions within the organ. The protective mechanisms in epididymis seem to play an important role, as up to 40% of cases of male infertility are caused by disorders in sperm maturation, a process which occurs in testis as well as caput and corpus of epididymis [15]. The mRNA expression of nuclear receptors and factors does not provide information on their functional state due to presence of different modulators. Therefore, to characterize their activity in cells/tissues induction studies are mandatory. In the case of
Nrf2/Keap1 system, oltipraz is a model inducer. Therefore, the study aimed at determining the expression of Nrf2/Keap1 system components in testis and epididymis both in rat and human in order to better characterize detoxification mechanisms in male reproductive tract. Animal model provides an opportunity not only to characterize expression of the Nrf2/Keap1 system, but also to prove its responsiveness to external stimuli in the studied organs, i.e. testis and epididymis. What constitutes a novel aspect of the study is the description of Nrf2/Keap1 system function in the defined segments of epididymis, which play different roles in sperm maturation. Materials and methods Animals Adult male Sprague–Dawley rats (250 g) (Charles River Laboratories, Sulzfeld, Germany) were maintained in standard conditions, i.e. 12 h light–dark cycle, water and standard chow ad libidum. After 2 weeks of adaptation, animals were divided into 2 groups (n = 5) and administered for 4 consecutive days: Nrf2 group – oltipraz (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dose of 150 mg/kg bw in 1.5 ml/kg bw corn oil, ip once daily; and control animals – vehiculum only (corn oil 1.5 ml/kg bw ip). Afterwards, under ketamine (Ketanest, Pfizer) anesthesia at a dose of 60 mg/kg bw ip, liver, testis and epididymis (separately caput, corpus and cauda) were dissected and immediately transferred into RNAlater1 Solution (Life Technologies, Carlsbad, CA, USA). The adjacent part of the tissues was fixed in 10% neutral-buffered formalin for immunohistochemistry. The study protocol was approved by the local ethics committee for animal studies (decision number 8/2011, from 21 June 2011). Patients Human testis and epididymis were obtained from 8 patients, aged 52–73 years (58.9 12.4 years), diagnosed with testicular cancer or prostatic carcinoma. The surgical specimens were immediately fixed in RNAlater1 Solution (Life Technologies, Carlsbad, CA, USA) for RNA analysis. The adjacent tissue was fixed in 10% neutral-buffered formalin for immunohistochemistry. The study protocol was approved by the local ethics committee for human studies (decision number KB-0012/33/10) and the study participants gave an informed consent. Analysis of gene expression Total RNA was isolated using Ambion1 RiboPureTM Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s procedure. Concentration and quality were evaluated using NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), and the absorbance ratio of 260/280 nm was used as a marker of purity (threshold value for the study was set at >1.9). cDNA was prepared from 1.0 mg of total RNA using cDNA reverse transcription kit with random primers (High Capacity cDNA Reverse Transcription Kits, Applied Biosystems, Foster City, CA, USA), and 1 ml of cDNA for gene expression analysis was used (TaqMan Gene Expression Master Mix, Applied Biosystems, Foster City, CA, USA) with pre-validated TaqMan Gene Expression Assays: rat assays ID: Nrf2 Rn00477784_m1; Keap1 Rn0058292_m1; Ugt1a6 Rn00756113_m1; Nqo1 Rn00566528_m1; Gclc Rn00689046_m1; Gsta2 Rn00566636_m1; Mdr1a (Abcb1a) Rn01639253; Mdr1b (Abcb1b) Rn00561753_m1; human assays ID: NRF2 Hs00232352_ m1; KEAP1 Hs00202227_m1; UGT1A6 Hs01592477_m1; NQO1 Hs00168547; GCLC Hs00155249_m1; MDR1 (ABCB1) Hs00184500_ m1.
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Each sample was analyzed in two technical replications and mean cycle threshold (CT) values were used for further analysis. The relative constitutive gene expression was calculated by DCT method using 7500 Fast Real-Time PCR System (Life Technologies, Carlsbad, CA, USA). Changes in gene expression in the group treated with oltipraz were calculated by DDCT method. CT values for human samples were normalized to the mean value obtained for two house-keeping genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and CYC (cyclophilin). In the case of the rat samples, b-actin gene was used as the reference gene. Fold change between group and analyzed tissues was calculated from the means of the logarithmic expression values. Nrf-2 immunohistochemistry Formalin-fixed, paraffin-embedded 4 mm sections were fixed into poly-L-lysine coated slides (ThermoScientific, Karlsruhe, Germany). After deparaffinization and rehydration, the slides were immersed in buffer Target Retrieval Solution, pH 9.0 (Dako, Glostrup, Denmark). Peroxidase Blocking Solution (Dako, Glostrup, Denmark) was used to block activity of endogenous peroxidase. The slides were incubated with 1 mg/ml of Nrf2 Rabbit antiHuman Polyclonal Antibody (LS-C118543 – LSBio; LifeSpan Biosciences, Seattle, WA, USA) for 30 min at room temperature and immunostained with DAKO RealTM EnvisionTM Detection System Peroxidase/DAB+, HRP Rabbit/Mouse (Dako, Glostrup, Denmark). Hematoxylin was used as a counterstain. The staining intensity was classified in four categories: ‘‘’’ no staining, ‘‘+’’ weak, ‘‘++’’ strong, ‘‘+++’’ very strong. Statistics Data are shown as mean SEM. Differences in constitutive gene expression were determined using non-parametric Wilcoxon signedrank test, and expression changes after treatment with oltipraz were determined using non-parametric Mann–Whitney U-test. The level of statistical significance was set at p < 0.05. All calculations were performed using Statistica 10. Software Package (Statsoft, Cracow, Poland). Results Constitutive gene expression in rat and human testis and epididymis Fig. 1 shows constitutive expression level of the studied genes in rat liver, testis and epididymis (caput, corpus and cauda) in comparison to the reference gene Actb (Fig. 1A) and in human testis and epididymis in reference to the mean of GAPDH and CYC (Fig. 1B). Constitutive expression level of Nrf2 in rat testis was significantly lower (8-fold) than in liver. The highest expression of Nrf2 was observed in epididymis corpus (around 2-times higher in comparison to liver but not statistically significant). Generally, Nrf2 expression in each analyzed part of epididymis was significantly higher than in testis. Contrary, expression of Keap1 was markedly lower in all segments of epididymis as compared to liver and testis, where Keap1 expression was at the same level (Fig. 1A). In human samples the highest expression level of the analyzed genes was observed for NRF2. Expression of NRF2 and KEAP1 was at comparable levels (0.8 and 0.04, respectively) in all the analyzed parts of male reproductive system (Fig. 1B). In rat, expression of the analyzed Nrf2 controlled, phase II drug metabolizing genes, Nqo1, Gsta2, Gclc were lower both in testis and in the whole epididymis in comparison to liver. Expression of Ugt1a6 in epididymis corpus was the highest even in comparison to liver (but the difference was not significant).
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Additionally, the study revealed the highest expression (but not always statistically significant) of Nqo1, Ugt1a6, and Gclc in epididymis corpus, as compared to testis and other parts of epididymis. In the case of human tissue, NQO1 analysis revealed a significantly higher expression level in each epididymis segment (with the highest value in caput) as compared to testis. Constitutive expression of UGT1A6 was not detected in human testis. In all three segments of epididymis CT value of UGT1A6 was around 33–35. Expression of GCLC in epididymis was lower than in testis. The analysis also revealed variable expression of Mdr1a and Mdr1b in rat tissues. A significantly lower level of Mdr1a expression was noted in epididymis corpus (1.96 103) as compared to liver (2.6 103), testis (3.0 103) and epididymis caput (3.3 103). Contrary, expression of Mdr1b in epididymis corpus was similar to liver levels (1.33 103; 1.43 103 respectively). Expression of human MDR1 was 3–4-fold higher in all three segments of epididymis than in testis. Gene expression in rat testis and epididymis after exposure to oltipraz Under oltipraz induction expression of Nrf2 was significantly higher in corpus of epididymis (1536%) as compared to the control animals. In the remaining parts of the male reproductive tract no significant changes in Nrf2 expression were observed following oltipraz administration. Exposure to oltipraz also did not significantly affect the level of Keap1 expression. Significant induction was observed in the case of Nqo1 in rat testis and caput (2-fold change) and corpus of epididymis (21-fold change). Likewise, expression of Ugt1a6 was significantly higher in corpus epididymis (1643%) in rats administered with oltipraz. Higher expression of Gsta2 was observed in caput epididymis. Oltipraz decreased the expression of Gclc to 55% in testis, and Mdr1a in each part of the analyzed tissues to around 30%. No significant differences were observed in Mdr1b expression (Fig. 2). Immunohistochemistry of rat testis and epididymis Immunohistochemical analysis for Nrf2 expression in control rat testis revealed a weak or lack of cytoplasmic reaction in seminiferous tubules (excluding spermatocytes with strong positive staining) and strong reaction in Leydig cells (Fig. 3A). Testis of rats treated with oltipraz was characterized by more prominent reaction in spermatocytes and spermatids. Additionally, in some Leydig cells a very strong Nrf2 expression was observed (Fig. 3B) (Table 1). Immunohistochemistry staining revealed weak cytoplasmic reaction in caput and corpus of epididymis and weak or strong staining in cauda of epididymis in the control rats (Fig. 4A, C, E). Oltipraz administration resulted in more intensive Nrf2 expression observed in cauda and additionally in corpus of epididymis (Fig. 4B, D, F; Table 1). Immunohistochemistry of human testis and epididymis Positive nuclear and cytoplasmic immunohistochemical reaction for the presence of transcription factor NRF2 in human testis was observed (in seminiferous tubule and in Leydig cells) (Fig. 5, Table 2). In Sertoli cells only weak reaction was observed. Immunohistochemistry staining revealed strong reaction in spermatids and strong or very strong in spermatogonium and spermatocytes (Fig. 5A). In the case of epididymis very strong cytoplasmic and nuclear reaction was observed in epithelial cells and lack of or weak nuclear and cytoplasmic staining in connective tissue cells (Fig. 5B, C, D).
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Fig. 1. Constitutive gene expression in liver, testis and epididymis (caput, corpus and cauda) in rat (A) and testis and epididymis (caput, corpus and cauda) in human (B). Relative expression was normalized to reference genes (Actb in rat; mean of CYC and GAPDH in human). *Significance at p < 0.05 to testis (Wilcoxon signed-rank test).
Discussion There is a paucity of information on nuclear receptors and factors coordinating expression of genes coding for drug metabolizing enzymes and drug transporters in testis and, in particular, in epididymis. To the best of our knowledge, this is the first research conducted under the same conditions both in rat and human which aims at defining expression profile of genes coordinated by Nrf2/ Keap1 system, associated with oxidative stress and drug metabolism and transport in testis and all segments of epididymis. Additionally, functional regulation of genes controlled by Nrf2 in rat testis and epididymis after exposure to oltipraz, a reference Nrf2 inducer, has been evaluated.
Activity of enzymes and transporters controlled by Nrf2 in blood-testis and blood-epididymis barriers provides functional protection of male reproductive system. Some preliminary reports suggest that level of Nrf2 expression may be a new biomarker in male infertility [16]. Patients with astheno- and oligoashtenospermia were found to have lower levels of the transcription factor expression. Moreover, mouse Nrf2/ are characterized by age decreasing sperm count and lower sperm motility [2]. The present study revealed different profile of Nrf2/NRF2 expression in rat and human testis and epididymis. In rat, significantly higher level of Nrf2 and markedly lower of Keap1 was observed in epididymis as compared to testis. In comparison with liver, lower expression was observed in testis and epididymis
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Fig. 2. Relative expression of Nrf2, Nqo1, Ugt1a6, Gclc, Gsta2, Keap1 and transporters Mdr1a, Mdr1b in rat testis and epididymis (caput, corpus and cauda) after oltipraz exposure to the respective control tissue (control tissue expression was taken as 100%). *Significance at p < 0.05 (Mann–Whitney U-test).
(excluding insignificant value in epididymis corpus). These findings suggest that rat epididymis is better protected by Nrf2 system or oxidative processes are more intensive in epididymis than in testis (resulting in higher Nrf2 expression). Treatment with oltipraz resulted in significant induction of Nrf2 only in corpus of epididymis with insignificant changes in Keap1 expression, pointing to epididymal corpus as a part of male reproductive tract which is the most responsive and has the highest adaptation potential. Animal model studies showed that knockout of Nrf2 led to lack of adaptive response to oltipraz via phase II drug metabolizing enzymes expression. Available data also demonstrate that chemoprotective mechanism of dithiotethiones results from activation of Nrf2 and induction of phase II enzymes [17]. Our study demonstrates that expression of the evaluated phase II drug metabolizing enzymes was lower in testis and epididymis than in liver, suggesting less efficient protection of these parts of male reproductive tract. The expression of phase II genes was variable within rat male reproductive tract. The present study revealed that constitutive expression of Nqo1, Ugt1a6 and Gclc was higher in epididymis corpus as compared to testis and other segments of epididymis, reflecting functional differences of the defined parts of male reproductive tract, including the defined segments of epididymis,
also documented in our study within Nrf2/Keap1 system coordinated genes [18–20]. As with constitutive expression, differential adaptive induction of different parts of male reproductive tract in rat was revealed, with corpus of epididymis being the most responsive segment to oltipraz exposure, as evidenced by significantly higher Nqo1 expression. Just as in the case of rats, human NQO1 expression was the highest in epididymal corpus and caput. These observations are in line with reports proposing epididymal basal cells to play an active role in detoxification [21] and protection of epididymal endothelium [19]. Our observations in both rat and human testis on higher expression level of Nrf2/NRF2 in Leydig cells in comparison to Sertoli cells (immunohistochemistry study) may suggest better protection of Leydig cells in comparison to Sertoli cells. These findings are in line with a report of Robaire and Viger, who revealed that protein expression of phase II enzymes such as GST-P, or subunit Yp is higher in Leydig cells than in Sertoli cells [19]. The present study also demonstrates interspecies differences in expression of Nrf2/NRF2 and its regulators, as well as the controlled genes. Contrary to findings regarding rats, constitutive expression in human reproductive tract revealed no differences in NRF2 and KEAP1 expression levels in each part of epididymis in comparison to testis. That observation is limited to a certain extent
Fig. 3. Immunohistochemical localization and immunoexpression of Nrf2 in testis of control rats (A) and in animals treated with oltipraz (B). Control group (A): lack and weak cytoplasmic expression in Sertoli cells (black arrow); lack and weak reaction in spermatogonia (green arrow); lack, weak and strong staining in spermatocytes (red arrow) and weak reaction in spermatids (yellow arrow); strong expression in Leydig cells (black arrowhead); oltipraz group (B): lack and weak cytoplasmic expression in Sertoli cells (black arrow); lack and weak reaction in spermatogonia (green arrow); strong and very strong expression in spermatocytes (red arrow); strong staining in spermatids (yellow arrow); very strong or strong expression in Leydig cells (black arrowhead). Magnification 400, – vessels. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Table 1 Immunolocalization and immunoexpression of Nrf2 in rat testis and epididymis (staining classification: no staining, +weak staining, ++ strong staining, +++ very strong staining). Testis
Control Oltipraz
Type of reaction
Sertoli cells
Spermatogonia
Spermatocytes
Spermatids
Leydig cells
Cytoplasmic Cytoplasmic
/+ /+
/+ /+
/+/++ ++/+++
+ ++
++ ++
Epididymal epithelial cells
Control Oltipraz
Cytoplasmic Cytoplasmic
Caput
Corpus
Principal cells
Principal cells
Clear cells
Principal cells
Clear cells
+ +
+ ++/+++
+/++ ++/+++
by specimen sampling from unhealthy subjects. Immunohistochemical analysis showed generally strong reaction in seminiferous tubules but weak in Sertoli cells. Unlike weak reaction in rat spermatogonia, human spermatogonia demonstrated very strong
Cauda
staining. The rat-human differences in cellular Nrf2 localization, i.e. mainly cytoplasmic in rat and cytoplasmic and nuclear humans may result from Nrf2/Keap1 system activation by drugs (as specimens were sampled from patients). Another reason for the
Fig. 4. Immunohistochemical localization and immunoexpression of transcription factor Nrf2 in rat epididymis of control animals (A, C, E) and oltipraz group (B, D, F). Control group: weak cytoplasmic reaction in epithelial principal cells of caput of epididymis (A), weak cytoplasmic reaction in epithelial principal cells of corpus (C) and weak or strong in cauda of epididymis (E). Oltipraz group: weak cytoplasmic reaction in epithelial principal cells of caput (B), strong and very strong in epithelial principal cells of caput (D), strong and very strong in epithelial cells of cauda (F). Epididymal epithelial principal cells (black arrow); epithelial clear cells (blue arrow). Magnification 400. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 5. Positive nuclear and cytoplasmic immunohistochemical reaction for the presence of transcription factor NRF2 in human testis (A) and epididymis (B, C, D). Testis: very strong cytoplasmic and nuclear reaction in seminiferous tubule; weak expression in Sertoli cells (not shown); strong cytoplasmic and nuclear expression in Leydig cells (black arrow); strong and very strong in spermatogonium (green arrow); strong and very strong reaction in spermatocytes (red arrow) and spermatids (yellow arrow) (A). Epididymis: lack or weak nuclear and cytoplasmic staining in connective tissue cells; epithelial cells of epididymis (black arrow); very strong cytoplasmic and nuclear reaction in epithelial cells of caput (B); strong and very strong expression in epithelial cells of corpus (C) and cauda of epididymis (D). Magnification 200. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
observed differential expression can be life-stage differences, as rats at reproductive age together with relatively aged humans were subjected for analysis. In each segment of epididymis a very strong and strong expression was observed. Likewise, NQO1 and UGT1A6 mRNA level was higher in epididymis than in testis (CT value of UGT1A6 in testis was below quantification level), and expression of GCLC in epididymis was lower than in testis. In rat tissue there was no statistically significant difference in Ugt1a6 expression in epididymis compared to testis, and higher level of Gclc mRNA in epididymis than in testis. To some extent these variability may be explained by interspecies differences in physiological processes and molecular mechanisms or by health Table 2 Immunolocalization and immunoexpression of NRF2 in human testis and epididymis (staining classification: no staining, + weak staining, ++ strong staining, +++ very strong staining). Type of reaction
Tissue Testis
Cytoplasmic Nuclear
Sertoli cells
Spermatogonia
Spermatocytes
Spermatids
Leydig cells
+
++/+++ +++
++/+++ +++
++ +++
++/+++ ++
Epididymis
Cytoplasmic Nuclear
Caput epithelial cells
Corpus epithelial cells
Cauda epithelial cells
+++ +++
++/+++ +++
++/+++ +++
condition. Some authors single out only two regions in human epididymis: caput and corpus, whereas cauda is absent [22] probably because of rapid sperm transit through epididymis (2–6 days in human and 10–13 days in rodents) [23], and rapid sperm maturation. Nevertheless, it is assumed that mechanisms of sperm maturation in human epididymis are similar as in rodent species. Transporters expressed within blood–testis barrier and blood– epididymis barrier regulate xenobiotic penetration through the barriers. The protective mechanism activated by NRF2 involves induction of ABC-transporters expression, including MDR1 [24,25]. Our study revealed that constitutive expression of Mdr1a in testis and epididymis was similar and at comparable level to liver. Expression of Mdr1b in the male reproductive tract was lower than in liver (excluding epididymis corpus), and higher in epididymis than in testis. The observations are in keeping with data of Augustine et al. [15], who reported that constitutive Mdr1a expression was at the same level in rat testis as compared to liver. However, in the case of Mdr1b, expression of this gene was similar both in liver and in testis. Jones and Cyr evaluated the expression of Mdr1a and Mdr1b in caput, corpus and cauda of epididymis (but not in testis), and found that expression of Mdr1a in rat epididymis was higher than Mdr1b. The highest level of Mdr1a was observed in cauda of epididymis, and Mdr1b expression was comparable in each analyzed epididymis segment [26]. The present rat study revealed levels of Mdr1a expression in each part of epididymis similar to liver; and, except for epididymis corpus, lower in the case of Mdr1b. The same pattern on Mdr1a expression in epididymis was observed by Klein et al. [27]. However, Klein’s report did not include any analysis of epididymis corpus. Our human data revealed MDR1 gene expression in testis and epididymis, at higher level in the latter organ. In normal testis,
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MDR1 is expressed in myoid cells, wall of endothelial cells of luminal capillaries and in Leydig cells. This pattern of drug transporter expression asserts optimal protection of spermatogenesis. The profile of MDR1 expression in epididymis and its ratio to testis in human tissues has not been reported. According to the present study, constitutive MDR1 expression was found to be higher in epididymis (significantly in caput and cauda) than in testis. Our functional observations revealed that oltipraz exposure did not result in elevated expression levels of both Mdr1a (even a decrease was observed) and Mdr1b. However, those findings are consistent with reports on other cells and tissues, where no effects of Nrf2 inducers on Mdr1a and Mdr1b levels were seen, i.e. despite nuclear Nrf2 translocation, no effects were noted of GCDCA (glycochenodeoxycholic acid) and paracetamol-induced ROS production on Mdr1 expression in Rho cells [28]. The authors proposed that Nrf2 is insufficient to up-regulate Mdr1 (also Mrp1 and Mrp4), which requires the participation of other regulatory element(s). Conclusions This study revealed that the expression of functional Nrf2/NRF2 system in male reproductive tract can play a potentially protective role. However, the components of the system, i.e. Nrf2/NRF2 along with coordinated genes, are expressed at lower levels than in liver. Moreover, some interspecies differences between rat and human were observed, which may impact extrapolation of experimental data into clinical findings. Nevertheless, it should be noted that analyzed human tissues were taken from relatively aged unhealthy patients, which could affect the study outcomes. Studies on the animal model showed that corpus of epididymis is the most responsive part of the male reproductive tract to oltipraz exposure at the gene expression level. Conflict of interest The authors declare that they have no conflicts of interest. Funding This study was supported by the National Science Centre, Cracow, Poland grant no. UMO-2011/03/N/NZ7/04683. References [1] Durairajanauagam D, Agarwal A, Ong C, Prashast P. Lycopen male infertility. Asian J Androl 2014;16(3):420–5. [2] Nakamura BN, Lawson G, Chan JY, Banuelos J, Corte´s MM, Hoang YD, et al. Knockout of the transcription factor NRF2 disrupts spermatogenesis in an agedependent manner. Free Radic Biol Med 2010;49(9):1368–79. [3] Hayes JD, Dinkova-Kostova AT. The Nrf2 regulatory network provides an inrface between redox and intermediary metabolism. Trends Biochem Sci 2014;39(4):199–218. [4] Zhan DD. Mechanistic studies of the Nrf2–KeaP1 signaling pathway. Drug Metab Rev 2006;38(4):769–89.
[5] Petrick JS, Klaassen CD. Importance of hepatic induction of constitutive androstane receptor and other transcription factors that regulate xenobiotic metabolism and transport. Drug Metab Dispos 2007;35(10):1806–15. [6] Cornwall A. New insights into epididymal biology function. Hum Reprod Update 2009;15(2):213–27. [7] Siegel D, Gustafson DL, Dehn DL, Han JY, Boonchoong P, Berliner LJ, et al. NAD(P)H:quinone oxidoreductase 1: role as a superoxide scavenger. Mol Pharmacol 2004;65(5):1238–47. [8] Ross D, Kepa JK, Winski SL, Beall HD, Anwar A, Siegel D. NAD(P)H: quinone oxidoreductase 1 (NQO1): chemoprotection, bioactivation, gene regulation and genetic polymorphisms. Chem Biol Interact 2000;129(1–2):77–97. [9] Silva SN, Azevedo AP, Teixeira V, Pina JE, Rueff J, Gaspar JF. The role of GSTA2 polymorphisms and haplotypes in breast cancer susceptibility: a case–control study in the Portuguese population. Oncol Rep 2009;22(3):593–8. [10] Benbrahim-Tallaa L, Siddeek B, Bozec A, Tronchon V, Florin A, Friry C, et al. Alterations of Sertoli cell activity in the long-term testicular germ cell death process induced by fetal androgen disruption. J Endocrinol 2008;196(1):21–31. [11] Paul C, Teng S, Saunders PT. A single, mild, transient scrotal heat stress causes hypoxia and oxidative stress in mouse testes, which induces germ cell death. Biol Reprod 2009;80(5):913–9. [12] Bock KW, Ko¨hle C. UDP-glucuronosyltransferase 1A6: structural, functional, andregulatory aspects. Methods Enzymol 2005;400:57–75. [13] Su L, Mruk DD, Lee WM, Cheng CY. Drug transporters and blood–testis barrier function. J Endocrinol 2011;209(3):337–51. [14] Su L, Smit JJ, van Tellingen O, Beijnen JH, Wagenaar E, van Deemter L, et al. Drug transporter, P-glycoprotein (MDR1), is an integrated component of the mammalian blood–testis barrier. Int J Biochem Cell Biol 2009;41(12): 2578–87. [15] Augustine LM, Markelewicz RJ, Boekelheide K, Cherinton NJ. Xenobiotic and endobiotic transporter mRNA expression in the blood–testis barrier. Drug Metab Dispos 2005;33(1):182–9. [16] Chen K, Mai Z, Zhou Y, Gao X, Yu B. Low NRF2 mRNA expression in spermatozoa from men with low sperm motility. Tohoku J Exp Med 2012;228(3): 259–66. [17] Zhang Y, Munday R. Dithiolethiones for cancer chemoprevention: where do we stand? Mol Cancer Ther 2008;7(11):3470–9. [18] Dube´ E, Chan PT, Hermo L, Cyr DG. Gene expression profiling and its relevance to the blood-epididymal barrier in the human epididymis. Biol Reprod 2007;76(6):1034–44. [19] Robaire B, Viger RS. Regulation of epididymal epithelial cell functions. Biol Reprod 1995;52(2):226–36. [20] Cornwall GA, Hsia N. Cres (cystatin-related epididymal spermatogenic) gene regulation and function. Zhonghua Nan Ke Xue 2002;8(5):313–8. [21] Veri J-P, Hermo L, Robaire B. Immunocytochemical localization of the Yf subunit of glutathione S-transferase P shows regional variation in the staining of epithelial cells of the testis, efferent ducts, and epididymis of the male rat. J Androl 1993;14(1):23–44. [22] Bedford JM. The status and the state of the human epididymis. Hum Reprod 1994;9(11):2187–99. [23] Amann RP, Howards SS. Daily spermatozoal production and epididymal spermatozoal reserves of the human male. J Urol 1980;124(2):211–5. [24] Hayashi A, Suzuki H, Itoh K, Yamamoto M, Sugiyama Y. Transcription factor Nrf2 is required for the constitutive and inducible expression of multidrug resistance-associated protein 1 in mouse embryo fibroblasts. Biochem Biophys Res Commun 2003;310(3):824–9. [25] Gonzalez-Sanchez E, Marin JJ, Perez MJ. The expression of genes involved in hepatocellular carcinoma chemoresistance is affected by mitochondrial genome depletion. Mol Pharm 2014;11(6):1856–68. [26] Jones SR, Cyr DG. Regulation and characterization of the ATP-binding cassette transporter-B1 in the epididymis and epididymal spermatozoa of the rat. Toxicol Sci 2011;119(2):369–79. [27] Klein DM, Wright SH, Cherrington NJ. Xenobiotic transporter expression along the male genital tract. Reprod Toxicol 2014;47:1–8. [28] Perez MJ, Gonzalez-Sanchez E, Gonzalez-Loyola A, Gonzalez-Buitrago JM, Marin JJ. Mitochondrial genome depletion dysregulates bile acid- and paracetamol-induced expression of the transporters Mdr1, Mrp1 and Mrp4 in liver cells. Br J Pharmacol 2011;162(8):1686–99.
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Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Nuclear factor E2-related factor-2 (Nrf2) expression and regulation in male reproductive tract Anna Wajda a, Joanna Łapczuk a, Marta Grabowska b, Marcin Słojewski c, Maria Laszczyn´ska b, Elz˙bieta Urasin´ska d, Marek Droz´dzik a,* a
Department of Experimental and Clinical Pharmacology, Pomeranian Medical University, Szczecin, Poland Department of Histology and Developmental Biology, Pomeranian Medical University, Szczecin, Poland Department of Urology and Urological Oncology, Pomeranian Medical University, Szczecin, Poland d Department of Pathology, Pomeranian Medical University, Szczecin, Poland b c
A R T I C L E I N F O
Article history: Received 20 May 2015 Received in revised form 25 June 2015 Accepted 8 July 2015 Available online 31 July 2015 Keywords: Nuclear factor E2-related factor-2 Male reproductive system
A B S T R A C T
Background: Nuclear factor E2-related factor-2 (Nrf2, Nfe2l2) plays an important, protective role in many tissues. However, information on molecular mechanisms of detoxification and drug metabolism regulated by Nrf2/NRF2 in testis and epididymis is scarce, but it may help to better characterize the function of blood-testis and epididymis barriers. Methods: Constitutive gene expression was analyzed by real time PCR with TaqMan Assay using DCTmethod. Additionally, gene expression after treatment with oltipraz- specific Nrf2 inducer was evaluated using DDCT-method. Cellular localization of the Nrf2 was visualized by immunohistochemical reaction. Results: The study showed that Nrf2 mRNA level in rat epididymis was higher than in testis. In human tissues, both testis and epididymis demonstrated similar expression levels of NRF2. Immunohistochemical analysis revealed NRF2/Nrf2 protein expression in testis and epididymis, which in the case of testis was dependant on spermatogenesis stage. Both in human and rat tissues constitutive expression of NQO1/Nqo1 was slightly higher in epididymis than in testis. Other Nrf2 regulated genes: GCLC/Gclc and UGT1A6/Ugt1a6 showed different ratios of testis/ epididymis/liver expression levels. Treatment with oltipraz (Nrf2 inducer) resulted in significant induction of Nrf2 expression solely in corpus of epididymis. Conclusions: Components of the Nrf2/NRF2 system along with coordinated genes are expressed in testis and epididymis. Moreover, some interspecies differences between rat and human were observed, which may impact extrapolation of experimental data into clinical findings. Studies on animal model showed that corpus of epididymis is the most responsive part of the male reproductive tract to oltipraz exposure at the gene expression level. ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
Introduction An increase in the number of studies on oxidative stress in the pathogenesis of idiopathic male infertility has been observed recently. The available data suggests that abnormal sperm morphology and function are associated with higher generation of reactive oxygen species (ROS) and reduced antioxidant status, i.e. oxidative stress [1]. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2, also called Nfe2I2) plays a key role in cellular antioxidant defence, and thus protects male reproductive tract against
* Corresponding author. E-mail address: [email protected] (M. Droz´dzik).
oxidative stress. Deficiency in Nrf2 function has deleterious effects in Sertoli and germ cells, and also in epididymal phase of sperm maturation [2]. In normal physiological conditions, Nrf2 is expressed at low level and resides mainly in cell cytoplasm, where it is promoted to degradation via ubiquitination by a repressor protein Keap1 [3]. Under oxidative stress or through Nrf2 activators such as xenobiotics, electrophiles or phytochemicals, Nrf2 dissociates from Keap1, translocates to the nucleus where heterodimer with Maf is formed, and then expression of genes with antioxidant response element (ARE) is activated [4]. Apart from antioxidative potential, Nrf2 is also an important regulator of both xenobiotic metabolism and transport [5]. It coordinates the function of genes coding for phase I and II
http://dx.doi.org/10.1016/j.pharep.2015.07.005 1734-1140/ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
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drug-metabolizing enzymes and drug transporters. ARE are located in promoter regions of many drug metabolizing genes such as CYP3A23, Nqo1, Ugt1a6 and drug transporters Mdr1a, Mdr1b, Mrp3, Mrp4 [6]. The present study focused on Nrf2/Keap1 system along with its coordinated enzymes Nqo1, Gsta2, Gclc, Ugt1a6 and transporter Mdr1 genes expression in testis and epididymis in rat and human. NQO1 (NAD(P)H:quinone acceptor oxidoreductase 1) is a twoelectron reductase considered as a chemoprotective enzyme, which deactivates many reactive species such as quinones, quinone-imines and azo-compounds [7]. In the 50 -flanking region of NQO1 gene two different response elements have been identified: ARE and XRE (xenobiotic response element). Hence, in many cellular systems activity of NQO1 may be regulated by NRF2 and aryl hydrocarbon receptor (AHR) [8]. Other Nrf2/Keap1 system controlled enzymes involved in drug metabolism and xenobiotic detoxification are glutathione transferases (GSTs), which cooperate with glutamate cystein ligase (GCLC). It was found that Gsta1 and Gsta2 isoforms are expressed in many human tissues, including male reproductive tract [9]. Observations indicated lower Gsta expression associated with an increase in apoptosis of germ cells in testis of adult rats exposed to androgen disruption in utero or transient mild testicular hyperthermia [10,11]. Another pathway of lipophilic xenobiotic and endobiotic elimination leads through glucuronidation, catalyzed by UGTs (uridine glucuronosyltransferases). UGT isoforms have been found not only in liver, but also in other tissues such as testis, kidney, gastrointestinal tract and brain [7,12]. Expression of UGT1A6, involved among others in metabolism of xenobiotics, may be controlled by many environmental factors through stress mediating receptors, including Nrf2, and also pregnane X receptor (PXR), constitutive androstane receptor (CAR) or AHR [7]. Just as in the case of enzymes, a group of drug transporter expression can be controlled by Nfr2/Keap system, including Mdr1a, Mdr1b, Mrp3, Mrp4 [6]. It is postulated that MDR1 (Pglycoprotein), the product of MDR1 gene in humans and Mdr1a and Mdr1b genes in rodents plays an important role in maintaining functional integrity of blood–testis and blood–epididymis barriers, limiting penetration of endo- and exogenous substrates (drugs, toxins). Higher testicular concentrations of Mdr1 substrates were found in Mdr1a/b/(knockout) mice in comparison with wild-type animals. P-glycoprotein is abundantly expressed in the capillary endothelium of human testis and in the myoid-cell layer around seminiferous tubules. Several agents are proposed to be transported and excreted by P glycoprotein, including carcinogens, xenobiotics, hormones and bilirubin [13,14]. Expression of proteins, controlling metabolism and transport constitute essential components of blood–testis and blood– epididymis barriers. The activity of metabolizing and transport systems within the barriers protects male reproductive tract against potential insults, and allows proper germ cell development, maturation and storage of functionally mature spermatozoa [6]. Therefore, it is important to define cellular localization and function factors providing protective actions within male reproductive tract, i.e. in testis and along epididymis. Up to now, efforts have been directed to testicular characteristics. Scarce data have been reported for epididymis, especially in view of segment specific functions within the organ. The protective mechanisms in epididymis seem to play an important role, as up to 40% of cases of male infertility are caused by disorders in sperm maturation, a process which occurs in testis as well as caput and corpus of epididymis [15]. The mRNA expression of nuclear receptors and factors does not provide information on their functional state due to presence of different modulators. Therefore, to characterize their activity in cells/tissues induction studies are mandatory. In the case of
Nrf2/Keap1 system, oltipraz is a model inducer. Therefore, the study aimed at determining the expression of Nrf2/Keap1 system components in testis and epididymis both in rat and human in order to better characterize detoxification mechanisms in male reproductive tract. Animal model provides an opportunity not only to characterize expression of the Nrf2/Keap1 system, but also to prove its responsiveness to external stimuli in the studied organs, i.e. testis and epididymis. What constitutes a novel aspect of the study is the description of Nrf2/Keap1 system function in the defined segments of epididymis, which play different roles in sperm maturation. Materials and methods Animals Adult male Sprague–Dawley rats (250 g) (Charles River Laboratories, Sulzfeld, Germany) were maintained in standard conditions, i.e. 12 h light–dark cycle, water and standard chow ad libidum. After 2 weeks of adaptation, animals were divided into 2 groups (n = 5) and administered for 4 consecutive days: Nrf2 group – oltipraz (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dose of 150 mg/kg bw in 1.5 ml/kg bw corn oil, ip once daily; and control animals – vehiculum only (corn oil 1.5 ml/kg bw ip). Afterwards, under ketamine (Ketanest, Pfizer) anesthesia at a dose of 60 mg/kg bw ip, liver, testis and epididymis (separately caput, corpus and cauda) were dissected and immediately transferred into RNAlater1 Solution (Life Technologies, Carlsbad, CA, USA). The adjacent part of the tissues was fixed in 10% neutral-buffered formalin for immunohistochemistry. The study protocol was approved by the local ethics committee for animal studies (decision number 8/2011, from 21 June 2011). Patients Human testis and epididymis were obtained from 8 patients, aged 52–73 years (58.9 12.4 years), diagnosed with testicular cancer or prostatic carcinoma. The surgical specimens were immediately fixed in RNAlater1 Solution (Life Technologies, Carlsbad, CA, USA) for RNA analysis. The adjacent tissue was fixed in 10% neutral-buffered formalin for immunohistochemistry. The study protocol was approved by the local ethics committee for human studies (decision number KB-0012/33/10) and the study participants gave an informed consent. Analysis of gene expression Total RNA was isolated using Ambion1 RiboPureTM Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s procedure. Concentration and quality were evaluated using NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), and the absorbance ratio of 260/280 nm was used as a marker of purity (threshold value for the study was set at >1.9). cDNA was prepared from 1.0 mg of total RNA using cDNA reverse transcription kit with random primers (High Capacity cDNA Reverse Transcription Kits, Applied Biosystems, Foster City, CA, USA), and 1 ml of cDNA for gene expression analysis was used (TaqMan Gene Expression Master Mix, Applied Biosystems, Foster City, CA, USA) with pre-validated TaqMan Gene Expression Assays: rat assays ID: Nrf2 Rn00477784_m1; Keap1 Rn0058292_m1; Ugt1a6 Rn00756113_m1; Nqo1 Rn00566528_m1; Gclc Rn00689046_m1; Gsta2 Rn00566636_m1; Mdr1a (Abcb1a) Rn01639253; Mdr1b (Abcb1b) Rn00561753_m1; human assays ID: NRF2 Hs00232352_ m1; KEAP1 Hs00202227_m1; UGT1A6 Hs01592477_m1; NQO1 Hs00168547; GCLC Hs00155249_m1; MDR1 (ABCB1) Hs00184500_ m1.
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Each sample was analyzed in two technical replications and mean cycle threshold (CT) values were used for further analysis. The relative constitutive gene expression was calculated by DCT method using 7500 Fast Real-Time PCR System (Life Technologies, Carlsbad, CA, USA). Changes in gene expression in the group treated with oltipraz were calculated by DDCT method. CT values for human samples were normalized to the mean value obtained for two house-keeping genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and CYC (cyclophilin). In the case of the rat samples, b-actin gene was used as the reference gene. Fold change between group and analyzed tissues was calculated from the means of the logarithmic expression values. Nrf-2 immunohistochemistry Formalin-fixed, paraffin-embedded 4 mm sections were fixed into poly-L-lysine coated slides (ThermoScientific, Karlsruhe, Germany). After deparaffinization and rehydration, the slides were immersed in buffer Target Retrieval Solution, pH 9.0 (Dako, Glostrup, Denmark). Peroxidase Blocking Solution (Dako, Glostrup, Denmark) was used to block activity of endogenous peroxidase. The slides were incubated with 1 mg/ml of Nrf2 Rabbit antiHuman Polyclonal Antibody (LS-C118543 – LSBio; LifeSpan Biosciences, Seattle, WA, USA) for 30 min at room temperature and immunostained with DAKO RealTM EnvisionTM Detection System Peroxidase/DAB+, HRP Rabbit/Mouse (Dako, Glostrup, Denmark). Hematoxylin was used as a counterstain. The staining intensity was classified in four categories: ‘‘’’ no staining, ‘‘+’’ weak, ‘‘++’’ strong, ‘‘+++’’ very strong. Statistics Data are shown as mean SEM. Differences in constitutive gene expression were determined using non-parametric Wilcoxon signedrank test, and expression changes after treatment with oltipraz were determined using non-parametric Mann–Whitney U-test. The level of statistical significance was set at p < 0.05. All calculations were performed using Statistica 10. Software Package (Statsoft, Cracow, Poland). Results Constitutive gene expression in rat and human testis and epididymis Fig. 1 shows constitutive expression level of the studied genes in rat liver, testis and epididymis (caput, corpus and cauda) in comparison to the reference gene Actb (Fig. 1A) and in human testis and epididymis in reference to the mean of GAPDH and CYC (Fig. 1B). Constitutive expression level of Nrf2 in rat testis was significantly lower (8-fold) than in liver. The highest expression of Nrf2 was observed in epididymis corpus (around 2-times higher in comparison to liver but not statistically significant). Generally, Nrf2 expression in each analyzed part of epididymis was significantly higher than in testis. Contrary, expression of Keap1 was markedly lower in all segments of epididymis as compared to liver and testis, where Keap1 expression was at the same level (Fig. 1A). In human samples the highest expression level of the analyzed genes was observed for NRF2. Expression of NRF2 and KEAP1 was at comparable levels (0.8 and 0.04, respectively) in all the analyzed parts of male reproductive system (Fig. 1B). In rat, expression of the analyzed Nrf2 controlled, phase II drug metabolizing genes, Nqo1, Gsta2, Gclc were lower both in testis and in the whole epididymis in comparison to liver. Expression of Ugt1a6 in epididymis corpus was the highest even in comparison to liver (but the difference was not significant).
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Additionally, the study revealed the highest expression (but not always statistically significant) of Nqo1, Ugt1a6, and Gclc in epididymis corpus, as compared to testis and other parts of epididymis. In the case of human tissue, NQO1 analysis revealed a significantly higher expression level in each epididymis segment (with the highest value in caput) as compared to testis. Constitutive expression of UGT1A6 was not detected in human testis. In all three segments of epididymis CT value of UGT1A6 was around 33–35. Expression of GCLC in epididymis was lower than in testis. The analysis also revealed variable expression of Mdr1a and Mdr1b in rat tissues. A significantly lower level of Mdr1a expression was noted in epididymis corpus (1.96 103) as compared to liver (2.6 103), testis (3.0 103) and epididymis caput (3.3 103). Contrary, expression of Mdr1b in epididymis corpus was similar to liver levels (1.33 103; 1.43 103 respectively). Expression of human MDR1 was 3–4-fold higher in all three segments of epididymis than in testis. Gene expression in rat testis and epididymis after exposure to oltipraz Under oltipraz induction expression of Nrf2 was significantly higher in corpus of epididymis (1536%) as compared to the control animals. In the remaining parts of the male reproductive tract no significant changes in Nrf2 expression were observed following oltipraz administration. Exposure to oltipraz also did not significantly affect the level of Keap1 expression. Significant induction was observed in the case of Nqo1 in rat testis and caput (2-fold change) and corpus of epididymis (21-fold change). Likewise, expression of Ugt1a6 was significantly higher in corpus epididymis (1643%) in rats administered with oltipraz. Higher expression of Gsta2 was observed in caput epididymis. Oltipraz decreased the expression of Gclc to 55% in testis, and Mdr1a in each part of the analyzed tissues to around 30%. No significant differences were observed in Mdr1b expression (Fig. 2). Immunohistochemistry of rat testis and epididymis Immunohistochemical analysis for Nrf2 expression in control rat testis revealed a weak or lack of cytoplasmic reaction in seminiferous tubules (excluding spermatocytes with strong positive staining) and strong reaction in Leydig cells (Fig. 3A). Testis of rats treated with oltipraz was characterized by more prominent reaction in spermatocytes and spermatids. Additionally, in some Leydig cells a very strong Nrf2 expression was observed (Fig. 3B) (Table 1). Immunohistochemistry staining revealed weak cytoplasmic reaction in caput and corpus of epididymis and weak or strong staining in cauda of epididymis in the control rats (Fig. 4A, C, E). Oltipraz administration resulted in more intensive Nrf2 expression observed in cauda and additionally in corpus of epididymis (Fig. 4B, D, F; Table 1). Immunohistochemistry of human testis and epididymis Positive nuclear and cytoplasmic immunohistochemical reaction for the presence of transcription factor NRF2 in human testis was observed (in seminiferous tubule and in Leydig cells) (Fig. 5, Table 2). In Sertoli cells only weak reaction was observed. Immunohistochemistry staining revealed strong reaction in spermatids and strong or very strong in spermatogonium and spermatocytes (Fig. 5A). In the case of epididymis very strong cytoplasmic and nuclear reaction was observed in epithelial cells and lack of or weak nuclear and cytoplasmic staining in connective tissue cells (Fig. 5B, C, D).
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Fig. 1. Constitutive gene expression in liver, testis and epididymis (caput, corpus and cauda) in rat (A) and testis and epididymis (caput, corpus and cauda) in human (B). Relative expression was normalized to reference genes (Actb in rat; mean of CYC and GAPDH in human). *Significance at p < 0.05 to testis (Wilcoxon signed-rank test).
Discussion There is a paucity of information on nuclear receptors and factors coordinating expression of genes coding for drug metabolizing enzymes and drug transporters in testis and, in particular, in epididymis. To the best of our knowledge, this is the first research conducted under the same conditions both in rat and human which aims at defining expression profile of genes coordinated by Nrf2/ Keap1 system, associated with oxidative stress and drug metabolism and transport in testis and all segments of epididymis. Additionally, functional regulation of genes controlled by Nrf2 in rat testis and epididymis after exposure to oltipraz, a reference Nrf2 inducer, has been evaluated.
Activity of enzymes and transporters controlled by Nrf2 in blood-testis and blood-epididymis barriers provides functional protection of male reproductive system. Some preliminary reports suggest that level of Nrf2 expression may be a new biomarker in male infertility [16]. Patients with astheno- and oligoashtenospermia were found to have lower levels of the transcription factor expression. Moreover, mouse Nrf2/ are characterized by age decreasing sperm count and lower sperm motility [2]. The present study revealed different profile of Nrf2/NRF2 expression in rat and human testis and epididymis. In rat, significantly higher level of Nrf2 and markedly lower of Keap1 was observed in epididymis as compared to testis. In comparison with liver, lower expression was observed in testis and epididymis
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Fig. 2. Relative expression of Nrf2, Nqo1, Ugt1a6, Gclc, Gsta2, Keap1 and transporters Mdr1a, Mdr1b in rat testis and epididymis (caput, corpus and cauda) after oltipraz exposure to the respective control tissue (control tissue expression was taken as 100%). *Significance at p < 0.05 (Mann–Whitney U-test).
(excluding insignificant value in epididymis corpus). These findings suggest that rat epididymis is better protected by Nrf2 system or oxidative processes are more intensive in epididymis than in testis (resulting in higher Nrf2 expression). Treatment with oltipraz resulted in significant induction of Nrf2 only in corpus of epididymis with insignificant changes in Keap1 expression, pointing to epididymal corpus as a part of male reproductive tract which is the most responsive and has the highest adaptation potential. Animal model studies showed that knockout of Nrf2 led to lack of adaptive response to oltipraz via phase II drug metabolizing enzymes expression. Available data also demonstrate that chemoprotective mechanism of dithiotethiones results from activation of Nrf2 and induction of phase II enzymes [17]. Our study demonstrates that expression of the evaluated phase II drug metabolizing enzymes was lower in testis and epididymis than in liver, suggesting less efficient protection of these parts of male reproductive tract. The expression of phase II genes was variable within rat male reproductive tract. The present study revealed that constitutive expression of Nqo1, Ugt1a6 and Gclc was higher in epididymis corpus as compared to testis and other segments of epididymis, reflecting functional differences of the defined parts of male reproductive tract, including the defined segments of epididymis,
also documented in our study within Nrf2/Keap1 system coordinated genes [18–20]. As with constitutive expression, differential adaptive induction of different parts of male reproductive tract in rat was revealed, with corpus of epididymis being the most responsive segment to oltipraz exposure, as evidenced by significantly higher Nqo1 expression. Just as in the case of rats, human NQO1 expression was the highest in epididymal corpus and caput. These observations are in line with reports proposing epididymal basal cells to play an active role in detoxification [21] and protection of epididymal endothelium [19]. Our observations in both rat and human testis on higher expression level of Nrf2/NRF2 in Leydig cells in comparison to Sertoli cells (immunohistochemistry study) may suggest better protection of Leydig cells in comparison to Sertoli cells. These findings are in line with a report of Robaire and Viger, who revealed that protein expression of phase II enzymes such as GST-P, or subunit Yp is higher in Leydig cells than in Sertoli cells [19]. The present study also demonstrates interspecies differences in expression of Nrf2/NRF2 and its regulators, as well as the controlled genes. Contrary to findings regarding rats, constitutive expression in human reproductive tract revealed no differences in NRF2 and KEAP1 expression levels in each part of epididymis in comparison to testis. That observation is limited to a certain extent
Fig. 3. Immunohistochemical localization and immunoexpression of Nrf2 in testis of control rats (A) and in animals treated with oltipraz (B). Control group (A): lack and weak cytoplasmic expression in Sertoli cells (black arrow); lack and weak reaction in spermatogonia (green arrow); lack, weak and strong staining in spermatocytes (red arrow) and weak reaction in spermatids (yellow arrow); strong expression in Leydig cells (black arrowhead); oltipraz group (B): lack and weak cytoplasmic expression in Sertoli cells (black arrow); lack and weak reaction in spermatogonia (green arrow); strong and very strong expression in spermatocytes (red arrow); strong staining in spermatids (yellow arrow); very strong or strong expression in Leydig cells (black arrowhead). Magnification 400, – vessels. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Table 1 Immunolocalization and immunoexpression of Nrf2 in rat testis and epididymis (staining classification: no staining, +weak staining, ++ strong staining, +++ very strong staining). Testis
Control Oltipraz
Type of reaction
Sertoli cells
Spermatogonia
Spermatocytes
Spermatids
Leydig cells
Cytoplasmic Cytoplasmic
/+ /+
/+ /+
/+/++ ++/+++
+ ++
++ ++
Epididymal epithelial cells
Control Oltipraz
Cytoplasmic Cytoplasmic
Caput
Corpus
Principal cells
Principal cells
Clear cells
Principal cells
Clear cells
+ +
+ ++/+++
+/++ ++/+++
by specimen sampling from unhealthy subjects. Immunohistochemical analysis showed generally strong reaction in seminiferous tubules but weak in Sertoli cells. Unlike weak reaction in rat spermatogonia, human spermatogonia demonstrated very strong
Cauda
staining. The rat-human differences in cellular Nrf2 localization, i.e. mainly cytoplasmic in rat and cytoplasmic and nuclear humans may result from Nrf2/Keap1 system activation by drugs (as specimens were sampled from patients). Another reason for the
Fig. 4. Immunohistochemical localization and immunoexpression of transcription factor Nrf2 in rat epididymis of control animals (A, C, E) and oltipraz group (B, D, F). Control group: weak cytoplasmic reaction in epithelial principal cells of caput of epididymis (A), weak cytoplasmic reaction in epithelial principal cells of corpus (C) and weak or strong in cauda of epididymis (E). Oltipraz group: weak cytoplasmic reaction in epithelial principal cells of caput (B), strong and very strong in epithelial principal cells of caput (D), strong and very strong in epithelial cells of cauda (F). Epididymal epithelial principal cells (black arrow); epithelial clear cells (blue arrow). Magnification 400. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 5. Positive nuclear and cytoplasmic immunohistochemical reaction for the presence of transcription factor NRF2 in human testis (A) and epididymis (B, C, D). Testis: very strong cytoplasmic and nuclear reaction in seminiferous tubule; weak expression in Sertoli cells (not shown); strong cytoplasmic and nuclear expression in Leydig cells (black arrow); strong and very strong in spermatogonium (green arrow); strong and very strong reaction in spermatocytes (red arrow) and spermatids (yellow arrow) (A). Epididymis: lack or weak nuclear and cytoplasmic staining in connective tissue cells; epithelial cells of epididymis (black arrow); very strong cytoplasmic and nuclear reaction in epithelial cells of caput (B); strong and very strong expression in epithelial cells of corpus (C) and cauda of epididymis (D). Magnification 200. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
observed differential expression can be life-stage differences, as rats at reproductive age together with relatively aged humans were subjected for analysis. In each segment of epididymis a very strong and strong expression was observed. Likewise, NQO1 and UGT1A6 mRNA level was higher in epididymis than in testis (CT value of UGT1A6 in testis was below quantification level), and expression of GCLC in epididymis was lower than in testis. In rat tissue there was no statistically significant difference in Ugt1a6 expression in epididymis compared to testis, and higher level of Gclc mRNA in epididymis than in testis. To some extent these variability may be explained by interspecies differences in physiological processes and molecular mechanisms or by health Table 2 Immunolocalization and immunoexpression of NRF2 in human testis and epididymis (staining classification: no staining, + weak staining, ++ strong staining, +++ very strong staining). Type of reaction
Tissue Testis
Cytoplasmic Nuclear
Sertoli cells
Spermatogonia
Spermatocytes
Spermatids
Leydig cells
+
++/+++ +++
++/+++ +++
++ +++
++/+++ ++
Epididymis
Cytoplasmic Nuclear
Caput epithelial cells
Corpus epithelial cells
Cauda epithelial cells
+++ +++
++/+++ +++
++/+++ +++
condition. Some authors single out only two regions in human epididymis: caput and corpus, whereas cauda is absent [22] probably because of rapid sperm transit through epididymis (2–6 days in human and 10–13 days in rodents) [23], and rapid sperm maturation. Nevertheless, it is assumed that mechanisms of sperm maturation in human epididymis are similar as in rodent species. Transporters expressed within blood–testis barrier and blood– epididymis barrier regulate xenobiotic penetration through the barriers. The protective mechanism activated by NRF2 involves induction of ABC-transporters expression, including MDR1 [24,25]. Our study revealed that constitutive expression of Mdr1a in testis and epididymis was similar and at comparable level to liver. Expression of Mdr1b in the male reproductive tract was lower than in liver (excluding epididymis corpus), and higher in epididymis than in testis. The observations are in keeping with data of Augustine et al. [15], who reported that constitutive Mdr1a expression was at the same level in rat testis as compared to liver. However, in the case of Mdr1b, expression of this gene was similar both in liver and in testis. Jones and Cyr evaluated the expression of Mdr1a and Mdr1b in caput, corpus and cauda of epididymis (but not in testis), and found that expression of Mdr1a in rat epididymis was higher than Mdr1b. The highest level of Mdr1a was observed in cauda of epididymis, and Mdr1b expression was comparable in each analyzed epididymis segment [26]. The present rat study revealed levels of Mdr1a expression in each part of epididymis similar to liver; and, except for epididymis corpus, lower in the case of Mdr1b. The same pattern on Mdr1a expression in epididymis was observed by Klein et al. [27]. However, Klein’s report did not include any analysis of epididymis corpus. Our human data revealed MDR1 gene expression in testis and epididymis, at higher level in the latter organ. In normal testis,
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MDR1 is expressed in myoid cells, wall of endothelial cells of luminal capillaries and in Leydig cells. This pattern of drug transporter expression asserts optimal protection of spermatogenesis. The profile of MDR1 expression in epididymis and its ratio to testis in human tissues has not been reported. According to the present study, constitutive MDR1 expression was found to be higher in epididymis (significantly in caput and cauda) than in testis. Our functional observations revealed that oltipraz exposure did not result in elevated expression levels of both Mdr1a (even a decrease was observed) and Mdr1b. However, those findings are consistent with reports on other cells and tissues, where no effects of Nrf2 inducers on Mdr1a and Mdr1b levels were seen, i.e. despite nuclear Nrf2 translocation, no effects were noted of GCDCA (glycochenodeoxycholic acid) and paracetamol-induced ROS production on Mdr1 expression in Rho cells [28]. The authors proposed that Nrf2 is insufficient to up-regulate Mdr1 (also Mrp1 and Mrp4), which requires the participation of other regulatory element(s). Conclusions This study revealed that the expression of functional Nrf2/NRF2 system in male reproductive tract can play a potentially protective role. However, the components of the system, i.e. Nrf2/NRF2 along with coordinated genes, are expressed at lower levels than in liver. Moreover, some interspecies differences between rat and human were observed, which may impact extrapolation of experimental data into clinical findings. Nevertheless, it should be noted that analyzed human tissues were taken from relatively aged unhealthy patients, which could affect the study outcomes. Studies on the animal model showed that corpus of epididymis is the most responsive part of the male reproductive tract to oltipraz exposure at the gene expression level. Conflict of interest The authors declare that they have no conflicts of interest. Funding This study was supported by the National Science Centre, Cracow, Poland grant no. UMO-2011/03/N/NZ7/04683. References [1] Durairajanauagam D, Agarwal A, Ong C, Prashast P. Lycopen male infertility. Asian J Androl 2014;16(3):420–5. [2] Nakamura BN, Lawson G, Chan JY, Banuelos J, Corte´s MM, Hoang YD, et al. Knockout of the transcription factor NRF2 disrupts spermatogenesis in an agedependent manner. Free Radic Biol Med 2010;49(9):1368–79. [3] Hayes JD, Dinkova-Kostova AT. The Nrf2 regulatory network provides an inrface between redox and intermediary metabolism. Trends Biochem Sci 2014;39(4):199–218. [4] Zhan DD. Mechanistic studies of the Nrf2–KeaP1 signaling pathway. Drug Metab Rev 2006;38(4):769–89.
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