Bacterial lipopolysaccharide differently modulates steroidogenic enzymes gene expressions in the brain and testis in rats

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acterial lipopolysaccharide differently modulates steroidogenicnzymes gene expressions in the brain and testis in rats

ohanraj Sadasivam, Balamurugan Ramatchandirin, Ananth Ayyanar,hidambaram Prahalathan ∗

epartment of Biochemistry, Bharathidasan University, Tiruchirappalli 620 024, India

r t i c l e i n f o

rticle history:eceived 7 November 2013eceived in revised form 30 January 2014ccepted 12 February 2014vailable online xxx

eywords:ipopolysaccharide�-HSD

a b s t r a c t

Bacterial lipopolysaccharide (LPS) is a major component of the cell wall of gram negative bacteria con-tributing to the pathogenesis of bacterial infection, in particular in those diseases affecting central nervoussystem and reproductive tissues. The present work is an attempt to study the regulation of steroidogenicenzymes gene expression in the brain and testis in LPS induced rats. Adult male albino rats were admin-istered LPS (5 mg/kg BW) to induce acute inflammation. LPS administration induced severe oxidativedamage in the brain and testicular tissue which was evident from decreased activities of enzymic antioxi-dants and increased lipid peroxidation levels. The mRNA expression of 3�-hydroxysteroid dehydrogenase(3�-HSD), 17�-hydroxysteroid dehydrogenase (17�-HSD) and androgen receptor corepressor-19 kDa

7�-HSDRR19teroidogenesis

(ARR19) in the brain and testis were determined. The mRNA expression of 3�-HSD and 17�-HSD wasincreased in the brain with significant decrease in the testis at 24 h and 48 h in LPS treated animals.The results also demonstrated an interesting finding that LPS treatment completely represses ARR19 inthe brain, while not in the testis. These findings show ARR19 might play a crucial role in regulation ofneuronal and testicular steroidogenesis in inflammatory diseases.

© 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

. Introduction

Steroid hormones are synthesized in steroidogenic cells of thedrenal, testis, ovary and brain and are required for normal repro-uctive function and bodily homeostasis (Stocco et al., 2005). Therain and the peripheral nerves synthesize steroids which thenct as neuromodulators in a paracrine or autocrine mode. Recenttudies have demonstrated the role of sex steroids in regula-ion of gene expression, neuronal survival, synaptic transmission,euronal and glial differentiation in many brain areas (Saldanhat al., 2009; Garcia-Segura, 2008; Stoffel-Wagner et al., 1999). Con-ersely, androgens produced in testicular Leydig cells are essentialor male sexual differentiation, maintenance of spermatogenesisnd expression of male secondary sex characteristics (Song et al.,012).

Proinflammatory cytokines are important pathogenic media-

Please cite this article in press as: Sadasivam, M., et al., Bacterial lipopexpressions in the brain and testis in rats. Neurosci. Res. (2014), http:

ors in infectious and inflammatory diseases, in particular diseasesffecting central nervous system and reproductive tissues. Infec-ion and inflammation can be reproduced in vivo by administration

∗ Corresponding author. Tel.: +91 431 2407071x484.E-mail addresses: [email protected], [email protected] (C. Prahalathan).

ttp://dx.doi.org/10.1016/j.neures.2014.02.011168-0102/© 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights res

of bacterial lipopolysaccharide (LPS), and several studies haveobserved inhibition of testicular steroidogenesis in animals treatedwith LPS (O’Bryan et al., 2000; Allen et al., 2004) or with septicagents that generate LPS (Sharma et al., 1996). It has also beenshown that proinflammatory cytokines generated by LPS havean inhibitory role in steroidogenesis through the production ofincreased reactive oxygen species (Reddy et al., 2006). A numerousstudies have shown that LPS plays a pivotal role in the regulationof steroidogenic enzymes gene expression including 3�-HSD and17�-HSD (Magata et al., 2014; Gottfried-Blackmore et al., 2008).

It has been demonstrated that proinflammatory cytokines suchas tumor necrosis factor alpha (TNF-�) and interleukin 1 (IL-1) havean inhibitory role on gonadal functions particularly in the steroido-genesis of Leydig cells (Hong et al., 2004; Herrmann et al., 2002).Elevated levels of TNF-� and IL-1 have been observed in humanpatients with critical illness, burns and sepsis (Calandra et al., 1990;Cannon et al., 1990; Damas et al., 1989), who experience impairedgonadal functions with low serum testosterone levels (Vogel et al.,1985; Woolf et al., 1985). Moreover, administration of TNF-� to

olysaccharide differently modulates steroidogenic enzymes gene//dx.doi.org/10.1016/j.neures.2014.02.011

healthy men and rats causes a decline in serum testosterone levels(Mealy et al., 1990; van der Poll et al., 1993), while treatment withTNF-� or IL-1 causes an inhibition of steroidogenesis in culturedLeydig cells (Hales, 2002). In contrast, Ghezzi et al. (2000) have

erved.

dx.doi.org/10.1016/j.neures.2014.02.011


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hown that neurosteroid levels are increased in vivo after LPS treat-ent and also negatively regulate LPS-induced TNF production. It

as also been demonstrated that LPS increases the expression andctivity of 17�-HSD in brain microglia (Gottfried-Blackmore et al.,008). With this background, the present work was designed totudy the mechanism behind the differential regulation of neuronalnd testicular steroidogenesis in LPS administered rats.

. Materials and methods

.1. Chemicals

Lipopolysaccharide from Escherichia coli (serotype 055:B5),regnenolone and androsten-3.17-dione were purchased fromigma Chemicals Company, Saint Louis, MO, USA. All other chem-cals used were of analytical grade and were obtained from Siscoesearch Laboratories Pvt. Ltd., Mumbai, India and HiMedia Labo-atories Pvt. Ltd., Mumbai, India.

.2. Experimental animals

Adult male albino rats of Wistar strain weighing 200 ± 10 g10–12 weeks old) were used in the study. The animals were

aintained under standard conditions of humidity, temperature25 ± 2 ◦C), and light (12 h light/12 h dark). The animals wereoused in large spacious cages bedded with husk. They were

ed with a standard rat pelleted diet and had free access toater throughout the experimental period. Experimental animalsere handled according to the University and Institutional Leg-

slation, regulated by the Committee for the Purpose of Controlnd Supervision of Experiments on Animals (CPCSEA), Min-stry of Social Justice and Empowerment, Government of IndiaBDU/IAEC/2011/28/29.03.2011).

.3. Study design

The animals were randomly divided into four groups consistingf six rats each. Saline treated animals served as controls (Group I).he animals of other groups were injected with LPS (dissolved in.5 ml of sterile saline) at a dose of 5 mg/kg body weight intraperi-oneally (i.p.) to induce acute inflammation. The animals wereacrificed at different time intervals i.e., 12 h (Group II), 24 h (GroupII), 48 h (Group IV) after LPS injection. Both the testes and brain

ere excised immediately and used for further analyses.

.4. Preparation of tissue homogenate

Briefly, the tissue was chopped and minced in 6–8 ml of 0.25 Mucrose/TKM buffer (50 mM Tris–HCl, 25 mM KCl and 5 mM MgCl2).he tissue was then homogenized gently by using Potter–Elvehjemomogenizer. The homogenate was centrifuged at 1500 rpm for

min. The supernatant was transferred and used as whole tissueomogenate for further analysis.

.5. Estimation of protein

Protein content of the tissue fractions was estimated by theethod of Lowry et al. (1951). Briefly, 0.1 ml of the diluted tis-

ue homogenate was made up to 1 ml with water. To this, 4.5 mlf alkaline copper reagent (2% Na2CO3 in 0.1 N NaOH was mixedith 0.5% CuSO4 containing 1% sodium potassium tartrate in the


atio of 50:1) was added, mixed and allowed to stand at room tem-erature for 20 min. Later, 0.5 ml of Folin’s Ciocalteau reagent wasdded and shaken well. The blank and bovine serum albumin (BSA)tandards were also treated in a similar manner. The blue color

PRESSResearch xxx (2014) xxx–xxx

complex formed was measured at 640 nm after 15 min against theblank.

2.6. Assay of enzymic antioxidants

2.6.1. Superoxide dismutase (SOD)The enzyme was assayed according to the method of Marklund

and Marklund (1974). The degree of inhibition of auto-oxidationof pyrogallol, in an alkaline pH by SOD was used as a measure ofthe enzyme activity. The enzyme activity is defined as units/mgprotein, where one unit is equal to the amount of enzyme requiredto inhibit auto oxidation of pyrogallol by 50% per minute.

2.6.2. Catalase (CAT)The activity of catalase was assayed by the method of Sinha

(1972). The method is based on the fact that dichromate in aceticacid is reduced to chromic acetate when heated in the presenceof H2O2 with the formation of perchloric acid as an unstableintermediate. The chromic acetate thus formed was measured at610 nm. The activity of catalase was expressed as �mol of H2O2consumed/min/mg protein.

2.7. Determination of reactive oxygen species (ROS) levels

Generation of ROS was determined by using 2′,7′-dichlorofluorescin diacetate (DCFH-DA) as a probe, accordingto the method of LeBel et al. (1992). In brief, the assay buffercontained 20 mM Tris–HCl, 130 mM KCl, 5 mM MgCl2, 20 mMNaH2PO4, 30 mM glucose and 5 �M DCFH-DA. The assay mediumwas incubated at 37 ◦C for 15 min and 1 �mol of H2O2 was addedto the mixture at the end of assay. Dichlorofluorescein (DCF)formation was measured in a fluorescence spectrophotometerwith 488 and 525 nm as excitation and emission wavelengths,respectively.

2.8. Assay of lipid peroxidation (LPO)

Estimation of LPO was carried out following the procedure ofHogberg et al. (1974) using thiobarbituric acid (TBA). Malondi-aldehyde (MDA), formed as an end product of peroxidation oflipids, served as an index of the intensity of oxidative stress.MDA reacts with TBA to generate a colored product that absorbsat 532 nm. The ferrous sulphate and ascorbate induced LPO sys-tem contained 10 mM ferrous sulphate and 0.2 mM ascorbate asinducers (Devasagayam, 1986). The level of lipid peroxides wasexpressed as �mol of MDA formed/mg protein.

2.9. Assay of steroidogenic enzymes

2.9.1. 3ˇ-Hydroxysteroid dehydrogenase (3ˇ-HSD)The activity of 3�-HSD was measured by the method of

Shivanandappa and Venkatesh (1997). The enzyme was assayed in0.1 M Tris–HCl buffer (pH 7.8) containing 500 �M NAD+, 0.5 ml ofcolor reagent (1% Tween 20 containing 0.08% iodonitrotetrazoliumchloride) and the substrate (100 �M pregnenolone) in a total vol-ume of 3 ml. The reaction was started by adding the enzyme (50 �l)and incubated at 37 ◦C for 60 min. The reaction was stopped bythe addition of 2 ml of phthalate buffer (2.55 g of potassium hydro-gen phthalate dissolved in a mixture of 51 ml 0.1 N HCl and 2.5 mlTween 20; pH was adjusted to 3.0 and the volume made up to250 ml). The turbidity was removed by centrifugation at 3000 rpmfor 20 min and the supernatant was read at 490 nm in a spectropho-


tometer. For standard curve, aliquots of graded concentrations offreshly prepared NADH (0–150 nM) were reacted with 0.5 ml ofcolor reagent (1% Tween 20 containing 0.08% iodonitrotetrazoliumchloride and 0.02% phenazinemethosulfate) and after color formed,


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Table 1Primer sequences for PCR for each target gene.

Gene Primer

3�-HSD F: 5′-AACTGGTCTTCAGGTCACCAGAA-3′

R: 5′-GTCCCCTGCACCTTGTTCA-3′

17�-HSD F: 5′-CCTTTGGCTTTGCCATGAGA-3′

R: 5′-CAATCCATCCTGCTCCAACCT-3′

ARR19 F: 5′-TGCTGCAATCTCTTGTTTCC-3′

R: 5′-TGTGCCATGATGAAAAAGGT-3′

Nur77 F: 5′-AAAGGCGGACTCTAGCAACA-3′

R: 5′-GGCATGGTGAAGGAAGTTGT-3′

2acp

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�-Actin F: 5′-TTCAACACCCCAGCCATGT-3′

R: 5′-TGGTACGACCAGAGGCATACAG-3′

ml of phthalate buffer was added and the absorbance was readt 490 nm. The enzyme activity was calculated from the standardurve of NADH and expressed as nmol of NADH formed/min/mgrotein.


.9.2. 17ˇ-Hydroxysteroid dehydrogenase (17ˇ-HSD)The activity of 17�-HSD was determined by the method as

escribed previously (Bergmeyer, 1974). 17�-HSD catalyses theeversible reaction of androstenedione into testosterone using

ig. 1. (A) Effect of LPS on SOD activity in rat brain and testis. Enzyme activity is expresso inhibit auto oxidation of pyrogallol by 50% per minute. (B) Effect of LPS on CAT activitynit is equal to �moles of H2O2 consumed per minute. Values represent mean ± S.D. Valu

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NADPH as a coenzyme. The activity is determined by the opticalmeasurement of the rate of conversion of NADPH to NADP. In brief,the reaction mixture contained 100 �l of testicular supernatant,200 �l of 0.5 �M NADPH, 100 �l of 0.8 �M androsten-3,17-dionein a final volume of 3 ml 100 �M phosphate buffer solution (pH7.4). The reaction was initiated by the addition of the substrate andthe decrease in absorbance of NADPH was followed at 340 nm for5 min at 20 s interval. The enzyme activity was expressed as nmolof NADPH oxidized/min/mg protein.

2.10. Analysis of the gene expression by reverse transcriptasepolymerase chain reaction (RT-PCR)

Briefly, total RNA from testicular tissue and whole brain was iso-lated according to the RNA isolation kit instructions (One step RNATrizol Reagent; Biobasic Inc., Canada). RT-PCR was performed with4 �g of total RNA isolated from tissue by using AMV-one step RT-PCR kit (GeNei, India). The specific sets of primers for target genesare shown in Table 1. PCR amplification was carried out accord-


ing to a protocol for the initial denaturing step at 95 C for 10 min;then 30 cycles at 95 ◦C for 1 min (denaturing), at 55 ◦C for 1 min(annealing) and 72 ◦C for 1.5 (extension); and a further extensionat 72 ◦C for 10 min. To compare the amount of steady state mRNA,

ed as unit/mg protein; where one unit is equal to the amount of enzyme required in rat brain and testis. Enzyme activity is expressed as unit/mg protein; where onees are statistically significant at *P < 0.05 (n = 6).


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ig. 2. (A) Effect of LPS on levels of ROS in rat brain and testis. (B) Effect of LPS on liphere one unit is equal to nmoles of MDA formed. Values represent mean ± S.D. Va

�l of each PCR product was resolved onto 1.4% agarose gel usingBE buffer. After electrophoresis, the gels were viewed under UVight and digital images were captured on Gelstan gel documenta-ion system. The densitometric analyses were carried out with labmage platform ver 2.1 software by Kapelan Bio-Imaging GmbH.he expression of each target gene was normalized with internalontrol and represented as a ratio.

.11. Data analysis

The values are expressed as mean ± standard deviation (SD). Dif-erences between groups were assessed by Kruskal–Wallis test andunn’s multiple comparisons test using the GraphPad Prism 6.0

oftware package for Windows. A P-value < 0.05 was consideredignificant.

. Results

.1. LPS induces oxidative stress

There were no recorded deaths in any of the experimentalroups during the study period. To test LPS induced oxidative dam-ge, we determined the levels of ROS, antioxidant enzymes andPO in the brain and testis. In the present study, LPS administrationnduced severe oxidative damage in the brain and testicular tissuen rats. The activities of the enzymic antioxidants SOD and CAT were


ignificantly decreased in LPS treated animals when compared toontrols (Fig. 1A and B). Fig. 2A represents abnormal elevation ineuronal and testicular ROS levels (measured by using DCF as arobe) in LPS administered rats; which exhibited a 3 fold increase

oxidation in rat brain and testis. Lipid peroxidation is expressed as unit/mg protein;re statistically significant at *P < 0.05 (n = 6).

as compared to those of control animals. Moreover, the LPO levels inthe brain and testicular tissue were markedly increased after 48 hof LPS treatment. Addition of exogenous inducers such as H2O2,ascorbate and FeSO4 further increased LPO levels than basal in LPSadministered animals (Fig. 2B).

3.2. Effect of LPS on steroidogenesis

We observed significant increase in the activities of 3�-HSDand 17�-HSD in the brain of LPS treated animals after 24 h and48 h than control animals. Interestingly, activities of these steroido-genic enzymes were decreased in the testis of LPS treated animalswhen compared to controls (Fig. 3A and B). Further, the deter-mination of the expression of 3�-HSD and 17�-HSD mRNAs inthe brain revealed that LPS treatment increases the mRNA expres-sion of these enzymes at 24 h and 48 h. In contrast, LPS treatmentsignificantly down regulated the expression of 3�-HSD and 17�-HSD mRNAs in the testis. The expression levels of 17�-HSD mRNAwere altered about 10-fold by LPS treatment in the brain and testis(Figs. 4 and 5); suggest its key role in regulation of steroidogenesisin infections.

3.3. Effect of LPS on ARR19 expression

To test whether ARR19 involved in the regulation of steroido-genic enzymes gene expression, we determined the mRNA levels


of ARR19 in the brain and the testis of control and LPS treatedanimals. RT-PCR analysis of ARR19 in the brain showed completerepression, while the expression pattern of ARR19 mRNA in thetestis was not much altered by LPS (Fig. 6). These results suggest


Please cite this article in press as: Sadasivam, M., et al., Bacterial lipopolysaccharide differently modulates steroidogenic enzymes geneexpressions in the brain and testis in rats. Neurosci. Res. (2014), http://dx.doi.org/10.1016/j.neures.2014.02.011


M. Sadasivam et al. / Neuroscience Research xxx (2014) xxx–xxx 5

Fig. 3. (A) Effect of LPS on 3�-HSD enzyme activity in rat brain and testis. Enzyme activity is expressed as unit/mg protein; where one unit is equal to nmoles of NADHformed per minute. (B) Effect of LPS on 17�-HSD enzyme activity in rat brain and testis. Enzyme activity is expressed as unit/mg protein; where one unit is equal to nmolesof NADPH oxidized per minute. Values represent mean ± S.D. Values are statistically significant at *P < 0.05 (n = 6).

Fig. 4. Effect of LPS on 3�-HSD enzyme gene expression in rat brain and testis. Values represent mean ± S.D. Values are statistically significant at *P < 0.05 (n = 6).



6 M. Sadasivam et al. / Neuroscience Research xxx (2014) xxx–xxx

s. Valu

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Fig. 5. Effect of LPS on 17�-HSD enzyme gene expression in rat brain and testi

hat the brain and the testis respond differently in steroidogenesisnder LPS treatment; at least through modulating the expressionf 3�-HSD, 17�-HSD and ARR19.

. Discussion


LPS is a major component of the cell wall of gram negativeacteria contributing to the pathogenesis of bacterial infection, inarticular in those diseases affecting central nervous system andeproductive tissues (Van Amersfoort et al., 2003). The harmful

Fig. 6. Effect of LPS on ARR19 gene expression in rat bra

es represent mean ± S.D. Values are statistically significant at *P < 0.05 (n = 6).

effects of this endotoxin are mainly mediated through inductionof inflammatory cytokines. It has been demonstrated that proin-flammatory cytokines generated by LPS have an inhibitory role insteroidogenesis through the production of increased reactive oxy-gen species (Reddy et al., 2006). The present findings shows thatLPS exposure had a marked oxidative impact as evidenced by the


significant increase in ROS levels in brain and testis. Furthermore,the results also suggest that LPS treated rats were more susceptibleto LPO in the presence of exogenous promoters like ascorbate andferrous sulphate. The present study also shows that the changes

in and testis. Values represent mean ± S.D. (n = 6).


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n LPO are accompanied by concomitant decrease in the activitiesf antioxidant enzymes namely SOD and CAT. SOD acts as first lineefense against deleterious effects of oxyradicals in cells by catalyz-

ng the dismutation of superoxide radicals to H2O2 and molecularxygen. The decreased activities of SOD in LPS treated rats may leado the continuous production of superoxide anions. Similarly, theeduction in the activities of CAT may reflect the inability of brainnd testis to eliminate the H2O2 which in turn magnifies LPO in LPSreated rats. From the present study, it is clear that systemic inflam-

ation induced by LPS in rats results in the generation of ROS inhe brain and testis probably as a result of impaired antioxidantefenses.

The steroidogenesis is mainly controlled by two rate limitingnzymes, namely 3�-HSD and 17�-HSD. Both the enzymesre directly involved in the biosynthesis of testosterone fromregnenolone. The steroidogenic enzyme 3�-HSD catalyses theehydrogenation and isomerization of pregnenolone to pro-esterone (Leers-Sucheta et al., 1997). Progesterone in turns converted to 17�-hydroxy progesterone, then androstene-ione by the action of 17�-hydroxylase/C17–20 lyase (P450c17).ndrostenedione is then converted to testosterone via the actionf 17�-HSD (Hales, 2002). Any alteration in the activities of thesenzymes reflects in the production of steroid hormones. The resultsf this investigation demonstrate significant increase in the activi-ies of 3�-HSD and 17�-HSD in the brain; while activities of theseteroidogenic enzymes were decreased in the testis of LPS treatednimals when compared to controls. The activity of any enzymen different cells or tissues under different treatment conditionss adjusted to reflect the metabolic needs of the cell. The activ-ties of steroidogenic enzymes in neuronal and testicular tissueomogenate from control and LPS treated animals in our studyeflect the effect of LPS on these enzymes, even though their expres-ions may vary in distinct cell types.

Further, the determination of the expression of 3�-HSD and7�-HSD mRNAs in the brain revealed that LPS treatment increaseshe mRNA expression of these enzymes at 24 h and 48 h. In con-rast, LPS treatment significantly down regulated the expressionf 3�-HSD and 17�-HSD mRNAs in the testis. It has been shownhat ROS play a key role in regulation of the expression of severalenes involved in steroidogenesis (Lee et al., 2009). The alteration inhe expression of the steroidogenic enzymes mRNAs in brain andestis of LPS treated rats may be as a result of the increased ROSroduction.

The increase in the expression of 3�-HSD and 17�-HSD mRNAsnd their enzymic activities may result in increased productionf sex steroids under inflammation in the brain. Ghezzi et al.2000) have already shown that neurosteroid levels are increasedn vivo after LPS treatment and negatively regulate LPS-inducedNF production and our results also support their findings. Fur-her, several studies have demonstrated the role of sex steroidsn neuroprotection (Saldanha et al., 2009; Garcia-Segura, 2008;toffel-Wagner et al., 1999) and the response of brain to LPS injec-ion in our study may also be a neuroprotective function of therain under acute inflammation. The results of the present studylso well corroborate with the findings of the previous studieshich observed decreased steroidogenesis in LPS treated Leydig

ells (Allen et al., 2004). Similar mechanisms may be responsible forhe male infertility associated with the local/systemic pathogenicnfections.

ARR19 is leucine-rich repressor protein differentially expresseduring the development of Leydig cells (Qamar et al., 2009) and

nhibits testicular steroidogenesis by reducing the expression of


teroidogenic enzymes through the suppression of the transacti-ation of Nur77 (Qamar et al., 2010). The results of the currenttudy also demonstrated an interesting finding that LPS treatmentompletely represses ARR19 in the brain, while not much altering

PRESSResearch xxx (2014) xxx–xxx 7

the expression pattern of ARR19 mRNA in the testis. These find-ings give a novel and valuable information that ARR19 might play acrucial role in regulation of testicular and brain steroidogenesis ininflammatory diseases. These results collectively suggest that thebrain and the testis respond differently in steroidogenesis underinfection. However, further studies are required to understandthe molecular mechanisms behind ARR19 mediated regulation ofsteroidogenesis in the brain and the testis.

Acknowledgements

The authors gratefully acknowledge Department of Biotechnol-ogy (DBT) and Indian Council of Medical Research (ICMR), NewDelhi, India for the financial assistance. The infrastructure providedby DST-FIST is also gratefully acknowledged.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.neures.2014.02.011.

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Bacterial lipopolysaccharide differently modulates steroidogenic enzymes gene expressions in the brain and testis in rats