Elsevier

Pharmacological Reports

Volume 68, Issue 1, February 2016, Pages 101-108
Pharmacological Reports

Original research article
Nuclear factor E2-related factor-2 (Nrf2) expression and regulation in male reproductive tract

https://doi.org/10.1016/j.pharep.2015.07.005Get rights and content

Abstract

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 ΔCT-method. Additionally, gene expression after treatment with oltipraz- specific Nrf2 inducer was evaluated using ΔΔCT-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.

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 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 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 two-electron reductase considered as a chemoprotective enzyme, which deactivates many reactive species such as quinones, quinone-imines and azo-compounds [7]. In the 5′-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 (P-glycoprotein), 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.

Section snippets

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).

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

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

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

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.

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