Original research articleNuclear factor E2-related factor-2 (Nrf2) expression and regulation in male reproductive tract
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.
References (28)
- et al.
Knockout of the transcription factor NRF2 disrupts spermatogenesis in an age-dependent manner
Free Radic Biol Med
(2010) - et al.
The Nrf2 regulatory network provides an inrface between redox and intermediary metabolism
Trends Biochem Sci
(2014) - et al.
NAD(P)H: quinone oxidoreductase 1 (NQO1): chemoprotection, bioactivation, gene regulation and genetic polymorphisms
Chem Biol Interact
(2000) - et al.
Drug transporter, P-glycoprotein (MDR1), is an integrated component of the mammalian blood–testis barrier
Int J Biochem Cell Biol
(2009) - et al.
Daily spermatozoal production and epididymal spermatozoal reserves of the human male
J Urol
(1980) - et al.
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) - et al.
Xenobiotic transporter expression along the male genital tract
Reprod Toxicol
(2014) - et al.
Lycopen male infertility
Asian J Androl
(2014) Mechanistic studies of the Nrf2–KeaP1 signaling pathway
Drug Metab Rev
(2006)- et al.
Importance of hepatic induction of constitutive androstane receptor and other transcription factors that regulate xenobiotic metabolism and transport
Drug Metab Dispos
(2007)