Orexin 1 Receptor Messenger Ribonucleic AcidExpression and Stimulation of Testosterone Secretion byOrexin-A in Rat Testis

M. L. BARREIRO, R. PINEDA, V. M. NAVARRO, M. LOPEZ, J. S. SUOMINEN, L. PINILLA,R. SENARIS, J. TOPPARI, E. AGUILAR, C. DIEGUEZ, AND M. TENA-SEMPERE

Department of Cell Biology, Physiology and Immunology (M.L.B., R.P., V.M.N., L.P. E.A., M.T.-S.), University of Cordoba,14004 Cordoba, Spain; Department of Physiology (M.L., R.S., C.D.), University of Santiago de Compostela, 15705 Santiagode Compostela, Spain; and Departments of Physiology and Pediatrics (J.S.S., J.T.), University of Turku, 20520 Turku,Finland

Orexins are hypothalamic neuropeptides primarily involvedin the regulation of food intake and arousal states. In addition,a role for orexins as central neuroendocrine modulators ofreproductive function has recently emerged. Prepro-orexinand orexin type-1 receptor mRNAs have been detected in therat testis. This raises the possibility of additional peripheralactions of orexins in the control of reproductive axis, whichremains so far unexplored. To analyze the biological effectsand mechanisms of action of orexins in the male gonad, weevaluated testicular expression of orexin receptor 1 (OX1R)and orexin receptor 2 (OX2R) mRNAs in different experimen-tal settings and the effect of orexin-A on testicular testoster-one (T) secretion. Persistent expression of OX1R mRNA wasdemonstrated in the rat testis throughout postnatal develop-ment. In contrast, OX2R transcript was not detected at anydevelopmental stage. Expression of OX1R mRNA persisted af-ter selective elimination of mature Leydig cells and was de-

tected in isolated seminiferous tubules at defined stages of theseminiferous epithelial cycle. In addition, testicular OX1RmRNA expression appeared to be under hormonal regulation;it was reduced by long-term hypophysectomy and partiallyrestored by FSH replacement, whereas down-regulation wasobserved after exposure to increasing doses of the ligand invitro. Moreover, OX1R mRNA expression was sensitive to neo-natal imprinting by estrogen. Finally, orexin-A, in a dose-dependent manner, significantly increased basal, but not hu-man choriogonadotropin-stimulated, T secretion in vitro. Asimilar stimulatory effect was observed in vivo after intrates-ticular administration of orexin-A. In conclusion, our presentresults provide the first evidence for the regulated expressionof OX1R mRNA and functional role of orexin-A in the rat testis.Overall, our data are suggestive of a novel site of action oforexins in the control of male reproductive axis. (Endocrinol-ogy 145: 2297–2306, 2004)

OREXINS, ALSO TERMED hypocretins, are recentlycloned hypothalamic neuropeptides initially involved

in the control of feeding behavior (1, 2). Two different orexinmolecules have been identified, orexin-A and orexin-B, whichderive from the proteolysis of a common 130-amino acid pre-cursor, the prepro-orexin (1, 2). Orexins are primarily expressedin the lateral hypothalamic area, a region with a key role in thecontrol of food intake, and prepro-orexin levels are regulated bya number of metabolic and nutritional signals, such as glucose,leptin, and fasting (2–4). Moreover, orexigenic fibers are widelydistributed and project to multiple brain regions (5), thus sug-gesting the involvement of orexins in the central control ofadditional biological functions, such as the regulation of thesleep-wake cycle (6), stress reactions (7), arterial blood pressureand heart rate (8), the autonomic nervous system (8, 9), andseveral neuroendocrine axes, including corticotrope, lactotrope,somatotrope, and gonadotrope systems (10–13). The biologicalactions of orexins are conducted through interaction with two

closely related G protein-coupled receptors, termed orexin re-ceptor 1 (OX1R) and orexin receptor 2 (OX2R). The bindingproperties of these receptors are partially different; OX1R ishighly selective for orexin-A, whereas OX2R is a nonselectivereceptor that binds both orexin-A and orexin-B (2).

Among other neuroendocrine functions, a role for orexinsin the central control of the reproductive axis has been re-cently demonstrated. Orexin-A stimulates GnRH releasefrom rat hypothalamic explants of male and female rats (13),and hypothalamic GnRH neurons express OX1R and receivedirect contacts from orexin fibers (14, 15). Moreover, de-pending on the prevailing sex steroid background, orexin-Ais able to stimulate or inhibit LH secretion. Thus, orexin-Aelicited LH release in steroid-primed ovariectomized rats,but it decreased LH secretion in the absence of ovarian ste-roids (12, 16, 17). Such a bimodal mode of action is apparentlylinked with site-specific effects of orexin-A within differenthypothalamic nuclei; orexin is stimulatory at the rostral pre-optic area and inhibitory at the medial preoptic area or thearcuate nucleus/median eminence (15). Potential actions oforexins at other sites of the hypothalamo-pituitary-gonadalaxis remain largely unknown, although it has been reportedthat orexin-A is able to inhibit GnRH-induced LH release bydispersed pituitary cells (13).

Despite the initial contention that orexins are exclusivelyproduced and acting in the brain, emerging data have dem-

Abbreviations: CT, Cycle threshold; EDS, ethylene dimethane sulfo-nate; hCG, human chorionic gonadotropin; HPX, hypophysectomized;OX1R, orexin receptor 1; OX2R, orexin receptor 2; RT, reverse transcrip-tion; T, testosterone.Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.

0013-7227/04/$15.00/0 Endocrinology 145(5):2297–2306Printed in U.S.A. Copyright © 2004 by The Endocrine Society

doi: 10.1210/en.2003-1405

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onstrated peripheral endocrine actions of orexins, includingdirect modulation of adrenal steroidogenic function (18–21).In addition, expression of the mRNAs encoding prepro-orexin and the cognate receptors (OX1R and OX2R) has beenreported in an array of endocrine and nonendocrine periph-eral tissues (22). Moreover, orexin-A has been found in hu-man plasma, and its expression has been detected in the gutand pancreas (23–26). To note, significant amounts of prepro-orexin mRNA have been demonstrated in the rat testis; atissue that also expressed low levels of OX1R transcript (22).However, the potential involvement of orexins in the directcontrol of testicular function remains so far unexplored. Inthis context, the present experimental work was undertakento analyze the biological effects and mechanisms of action oforexins in the male gonad. To this end, we evaluated testic-ular expression of the mRNAs encoding OX1R and OX2R indifferent experimental settings, and we assayed the effect oforexin-A on testicular testosterone (T) secretion. On the lat-ter, we limited our studies to orexin-A because it is providedwith a higher biopotency than orexin-B in different physio-logical systems (2, 16), and preliminary results evidencedlack of expression of OX2R mRNA in rat testis tissue.

Materials and MethodsAnimals and drugs

Wistar male rats bred in the vivarium of the University of Cordoba wereused. The day the litters were born was considered as d 1 of age. Theanimals were maintained under constant conditions of light (14 h of light,from 0700) and temperature (22 C) and were weaned at d 21 of age in groupsof five rats per cage with free access to pelleted food and tap water. Ex-perimental procedures were approved by the Cordoba University EthicalCommittee for animal experimentation and were conducted in accordancewith the European Union normative for care and use of experimentalanimals. Special attention was taken to minimize the number of animals perexperimental group. In all experiments, the animals were killed by decap-itation, and for those experiments involving mRNA analysis (experiments1–6), testes were immediately removed, frozen in liquid nitrogen, andstored at �80 C until processing. In addition, in specific settings, trunkblood was collected, and samples of testicular tissue were taken and pro-cessed for histological evaluation (experiments 2 and 4). Rat orexin-A wasobtained from Bachem AG (Bubendorf, Switzerland). Highly purified hu-man chorionic gonadotropin (hCG; Profasi) and human recombinant FSH(Gonal-F) were purchased from Serono (Madrid, Spain). Estradiol benzoatewas obtained from Sigma (St. Louis, MO), and the GnRH antagonistOrg 30276 (Ac-d-pClPhe-d-pClPhe-d-Trp-Ser-Tyr-d-Arg-Leu-Arg-Pro-d-Ala-NH2CH3COOH) was generously supplied by Organon (Oss, Nether-lands). Ethylene dimethane sulfonate (EDS) was synthesized in our labo-ratory and dissolved in dimethylsulfoxide-water (1:3, vol/vol).

Experimental designs

In experiment 1, analysis of testicular expression of OX1R and OX2RmRNAs was conducted at different stages of postnatal development.Thus, testicular samples were obtained from 1- (n � 10), 5- (n � 10), 10-(n � 10), 15- (n � 5), 20- (n � 5), 30- (n � 5), 45- (n � 5), and 75-d-oldrats (n � 5 per group), corresponding to the neonatal-infantile (1 d, 5 d,and 10 d), prepubertal (15 d, 20 d), pubertal (30 d), early adult (45 d),and adult (75 d) stages of postnatal maturation.

In experiment 2, testicular expression of OX1R mRNA was studiedafter selective Leydig cell elimination by systemic administration of thecytotoxic drug EDS (single dose of 75 mg/kg, ip). This in vivo modelprovides an optimal experimental background in which to test Leydigcell-specific expression and/or regulation of testicular factors (for ex-ample, see Refs. 27 and 28). Testicular samples were obtained from adult75-d-old rats (n � 5 per group) before (0) and 5, 15, 20, 30, and 40 d afterEDS administration and assayed for OX1R mRNA expression. In addi-tion, to monitor elimination of Leydig cells in EDS-treated rats, trunk

blood samples were collected upon decapitation of the animals, andtestis specimens were taken and processed for histological examination,as described in detail elsewhere (29).

In experiment 3, expression of OX1R mRNA was assessed in semi-niferous tubule preparations at different stages of the epithelial cycle.Microdissection of seminiferous tubule segments was carried out asdescribed in detail elsewhere (30). Briefly, testes from adult rats weredecapsulated, and 5-mm seminiferous tubule segments were isolatedunder a transilluminating stereomicroscope. Specific stages of the sem-iniferous epithelial cycle were identified and pooled in four majorgroups corresponding to stages II–VI, stages VII–VIII, stages IX–XII, andstages XIII–I of the cycle. After exhaustive washing, tubular tissuewas processed for RNA analysis as described in RNA analysis by semi-quantitative RT-PCR. Quadruplicate pools per stage of the seminiferousepithelium were processed.

In experiment 4, the ability of pituitary gonadotropins to regulatetesticular expression of OX1R gene was monitored. To this end, testicularOX1R mRNA levels were analyzed in groups (n � 5) of adult (75-d-old)control and long-term hypophysectomized (HPX) rats (i.e. 4 wk afterpituitary removal) with or without gonadotropin replacement: hCG (10IU/rat�24 h) or recombinant FSH (7.5 IU/rat�24 h) for 7 d before sam-pling. In addition, in experiment 5, the regulation of testicular OX1RmRNA levels by its cognate ligand was assayed in vitro. As experimentalsetting, slices of testicular tissue were prepared from 75-d-old rats andincubated for 180 min in the presence of increasing doses (10�10 to 10�6

m) of rat orexin-A (n � 8 samples per group), as described in detailelsewhere (28).

In experiment 6, the effects of neonatal exposure to estrogen on theexpression levels of OX1R mRNA in rat testis were evaluated. In thissetting, 1-d-old male rats were injected sc with a single dose of estradiolbenzoate (500 �g/rat; dissolved in 100 �l of olive oil), a regimen that hasbeen reported to induce complete estrogenization in the male rat, with-out major systemic toxicity (31, 32). Groups of males (n � 5) were killedon d 15, 30, and 75 of age. Vehicle (oil)-injected animals served ascontrols. In this experiment, the potential mechanism(s) for the effectsof neonatal estrogen exposure on testicular OX1R gene expression wasalso assessed. In this sense, because the effect of neonatal estrogen on thedeveloping testis may be direct or mediated by postnatal suppression ofgonadotropins (31), the level of OX1R mRNA was determined also intestes of rats (n � 5) treated neonatally with a potent GnRH antagonist,as described previously (33). Groups of male rats were injected sc withGnRH antagonist (5 mg/kg body weight) on d 1, 4, 7, 10, 13, and 15postpartum, and tissue sampling was conducted on d 15 (4 h after thelast injection), 30, and 75.

Finally, in experiment 7, the effects of orexin-A on basal and stim-ulated testosterone (T) secretion in vitro were assessed using static in-cubations of adult rat tissue, as described in detail elsewhere (28, 34).Tissue samples (n � 10–12 per group) were incubated in the presenceof increasing doses of orexin-A (10�10–10�6 m), under basal or stimu-lated (co-incubation with 10 IU/ml hCG) conditions, and T concentra-tions in the incubation media were determined at 90 and 180 min. Inaddition, in experiment 8, the effects of orexin-A on T secretion wereevaluated in vivo. To this end, bilateral intratesticular injection oforexin-A (10�6 m; 50 �l/testis) or vehicle was performed (n � 12 rats pergroup), and systemic blood samples were obtained by jugular veni-puncture before (0) and at 30, 60, 120, 180, and 240 min after orexin-Aadministration.

RNA analysis by semiquantitative RT-PCR

Total RNA was isolated from testicular samples using the single-step,acid guanidinium thiocyanate-phenol-chloroform extraction method(35). Testicular expression of OX1R and OX2R mRNAs was assessed byRT-PCR, optimized for semiquantitative detection, using the followingspecific primer pairs: OX1 forward (5�-TGGGCTGTGTCGCTGGCTG-3�)and OX1 reverse (5�-GTTGGGGCTCTGTACACAGG-3�), flanking a328-bp coding area of rat OX1R cDNA (GenBank accession no.AF041244); OX2 forward (5�-TTGGGGTTCATCGTCGTC AAG-3�) andOX2 reverse (5�-AGCCAGGTGGACAGGAGTGA-3�), flanking a 226-bpcoding area of rat OX2R cDNA (GenBank accession no. AF041246). Thesesets of primers were selected on the basis of previous references (36, 37).As internal control for reverse transcription (RT) and reaction efficiency,amplification of a 290-bp fragment of L19 ribosomal protein mRNA was

2298 Endocrinology, May 2004, 145(5):2297–2306 Barreiro et al. • Orexin Actions in Rat Testis

carried out in parallel in each sample, using the primer pair: L19 forward(5�-GAAATCGCCAATGCCAACTC-3�) and L19 reverse (5�-ACCT-TCAGGTACAGGCTGTG-3�), as described in detail elsewhere (28, 33).Given its constitutive expression and rather constant mRNA levels alongpostnatal development and under different experimental conditions, theRP-L19 gene has been widely used by our group as internal control forRT-PCR assessment of testicular gene expression (for examples, see Refs.28, 33).

For amplification of the targets, 4 �g of total RNA was used toperform RT-PCR in two consecutive separate steps. In addition, to en-able appropriate amplification in the exponential phase for each target,PCR amplification of specific signals (OX1R and OX2R) and L19 ribo-somal protein transcript was carried out in separate reactions withdifferent numbers of cycles but using similar amounts of the corre-sponding cDNA templates, generated in single RT reactions, as previ-ously described (28, 33). PCRs consisted of a first denaturing cycle at 97C for 5 min, followed by a variable number of cycles of amplificationdefined by denaturation at 96 C for 30 sec, annealing for 30 sec, andextension at 72 C for 1:30 min. A final extension cycle of 72 C for 15 minwas included. Annealing temperature was adjusted for each target: 66C for OX1R, 63 C for OX2R, and 56 C for RP-L19 transcripts. For OX1Rand RP-L19, different numbers of cycles were tested to optimize am-plification in the exponential phase of PCR. In detail, analysis of intensityof PCR signals as function of the number of amplification cycles revealeda strong linear relationship between cycles 25–38 in the case of OX1R(r2 � 0.97) and cycles 19 –29 in the case of RP-L19 (r2 � 0.992). On thisbasis, 33 and 23 PCR cycles were chosen for further analysis of theOX1R and L19 species, respectively.

PCR-generated DNA fragments were resolved in Tris-borate-buff-ered 1.5% agarose gels and visualized by ethidium bromide staining.Specificity of PCR products was confirmed by direct sequencing usinga fluorescent dye termination reaction and an automated sequencer(Central Sequencing Service; University of Cordoba, Cordoba, Spain).Quantification of intensity of RT-PCR signals was carried out by den-sitometric scanning using an image analysis system (1-D Manager; TDILtd., Madrid, Spain), and values of the specific targets were normalizedto those of internal controls to express arbitrary units of relative ex-pression. In all assays, liquid controls and reactions without RT resultedin negative amplification.

RNA analysis by real-time RT-PCR

To verify changes in gene expression observed by final-time RT-PCR,real-time RT-PCR was performed in selected experimental samples us-ing the iCycler iQ Real-Time PCR detection system (Bio-Rad Labora-tories, Hercules, CA). In detail, OX1R mRNA levels were assayed inrepresentative testicular samples of different stages of postnatal devel-opment (15-, 45-, and 75-d-old rats) and after EDS treatment (at 0 and5 and 40 d after EDS) and from HXP rats with or without gonadotropinreplacement. RT of total RNA was conducted as described earlier. Thesynthesized cDNAs were further amplified (1/10th) in triplicate by PCRusing SYBR green I (Bio-Rad) as fluorescent dye and 1 � iQ Supermixcontaining 50 mm KCl, 20 mm Tris-HCl, 0.2 mm deoxynucleotidetriphosphates, 3 mm MgCl2, and 2.5 U iTaq DNA polymerase (Bio-Rad),in a final volume of 25 �l. The PCR cycling conditions were as follows:initial denaturation and enzyme activation at 95 C for 5 min, followedby 40 cycles of denaturation at 95 C for 15 sec, annealing at 66 C (OX1R,)or 56 C (RP-L19) for 15 sec, and extension at 72 C for 1 min. Productpurity was confirmed by dissociation curves and random agarose gelelectrophoresis. No-template controls were included in all assays, yield-ing no consistent amplification. Calculation of relative expression levelsof OX1R mRNA was conducted based on the cycle threshold (CT)method (38). The CT for each sample was calculated using the iCycleriQ Real-Time PCR detection system software with an automatic fluo-rescence threshold setting. Accordingly, fold expression of OX1R mRNAover reference values was calculated by the equation 2���CT, where �CTis determined by subtracting the corresponding RP-L19 CT value (in-ternal control) from the specific CT of the target (OX1R), and ��CT isobtained by subtracting the �CT of each experimental sample from thatof the reference sample (taken as reference value 100). For the experi-mental groups assayed, reference samples were arbitrarily taken from15-d-old testis (postnatal development), control testis before EDS ad-ministration (EDS model), and control adult testis (HPX model). To note,

no significant differences in CT values were observed for RP-L19 be-tween the treatment groups.

T measurement by specific RIA

T levels in static incubation media and serum samples were measuredusing a commercial kit from ICN Biomedicals (Costa Mesa, CA), fol-lowing the instructions of the manufacturer. All medium samples weremeasured in the same assay. The sensitivity of the assay was 0.1 ng/tube,and the intraassay coefficient of variation was 4.5%. Accuracy of hor-mone determination was confirmed by assessment of rat serum samplesof known T concentrations used as external controls.

Presentation of data and statistics

Semiquantitative RT-PCR analyses were carried out in duplicate fromat least four independent RNA samples of each experimental group. Forgeneration of RNA samples, individual testis specimens were used,except for 1-, 5-, and 10-d-old rat testes, which were pooled (n � 2–4)before RNA isolation. Final-time RT-PCR analyses were conducted intriplicate using independent RNA samples. Tissue incubations for Tmeasurement were conducted in duplicate, with a total number of 10–12samples/determinations per group. In addition, integrated T secretoryresponses in vivo were expressed as the area under the curve, calculatedfollowing the trapezoidal rule, over a 240-min period. Data are presentedas mean � sem. Results were analyzed for statistically significant dif-ferences using ANOVA, followed by the Student-Newman-Keuls mul-tiple range test. P � 0.05 was considered significant.

ResultsTesticular expression of OX1R mRNA along postnataldevelopment, after selective Leydig cell elimination, and instaged seminiferous tubule preparations

Expression of the genes encoding OX1R and OX2R wasevaluated in the rat testis at different stages of postnatalmaturation. Our semiquantitative RT-PCR assays demon-strated persistent expression of OX1R mRNA throughoutpostnatal development (Fig. 1). In detail, representativestages of development, corresponding to neonatal (1 and 5 dold), infantile (10 d old), prepubertal (15 and 20 d old),pubertal (30 d old), early adult (45 d old), and adult (75 d old)periods, were explored. Quantitative analysis of the RT-PCRsignals demonstrated rather constant relative expression lev-els of OX1R mRNA along the study period, with peak valuesin neonatal and pubertal to early adult samples. Such anexpression profile was confirmed by real-time RT-PCR anal-ysis of representative testicular samples that showed maxi-mum expression levels in 45-d-old rat testis (Table 1). Con-trary to OX1R transcript, OX2R mRNA was not detectable intesticular samples at any developmental stage assayed, fromthe neonatal period to adulthood. However, using similarRT-PCR conditions, OX2R mRNA was detected in adult hy-pothalamic samples, used as positive controls (data notshown). Consequently, further studies were focused in theanalysis of testicular expression of OX1R transcript in dif-ferent experimental settings.

In the next step, semiquantitative RT-PCR assays of OX1RmRNA expression were conducted in testis samples at dif-ferent time points after selective Leydig cell elimination invivo by administration of the cytotoxic compound EDS. Ef-ficiency of Leydig cell destruction was monitored by histo-logical examination of testicular tissue sections and mea-surement of serum T concentrations in EDS-treated groups.In line with previous references (29), serum T levels dropped

Barreiro et al. • Orexin Actions in Rat Testis Endocrinology, May 2004, 145(5):2297–2306 2299

to nearly undetectable values, and no mature Leydig cellswere detected in testicular interstitium 5 and 15 d after EDS(data not shown). In this setting, persistent testicular expres-sion of specific OX1R mRNA was demonstrated throughouta 40-d period after EDS administration (Fig. 2). In detail, 5and 15 d after EDS administration, amplification of OX1Rsignal was clearly obtained at levels similar to those of con-trol animals. However, although persistent expression ofOX1R message was detected, the relative levels of the signalprogressively declined from d 20 onwards and remainedlower than controls at d 40 after EDS treatment. Such apattern of expression, with persistent levels of OX1R mRNA5 d after EDS and decreased levels at d 40, was confirmed byreal-time RT-PCR assays (Table 1).

Because these data pointed out that OX1R gene is ex-

pressed in the tubular compartment of the testis, analysis ofmRNA expression was undertaken in preparations of sem-iniferous tubule fragments isolated at different stages of theseminiferous epithelial cycle. Tubule segments were sepa-rated in four major groups corresponding to stages II–VI,VII–VIII, IX–XII, and XIII–I of the cycle. Positive amplifica-tion of OX1R mRNA was obtained in seminiferous tubulepreparations at all stages of the cycle. However, the relativelevels of OX1R mRNA varied throughout the cycle, withmaximum values in stages VII-VIII and minimum expressionlevels in stages XIII-I (Fig. 3).

Hormonal regulation of testicular expression ofOX1R mRNA

In the first step, hormonal regulation of testicular expres-sion of OX1R mRNA by pituitary gonadotropins was as-sessed using the HPX rat as experimental model. Long-term(4-wk) HPX resulted in a clear-cut decrease of serum T levelsand the atrophy of all testicular compartments, with atrophicLeydig cells in the interstitial space and regressing seminif-erous epithelium with apparent arrest of spermatogenesis atearly meiosis phase within the tubules (data not shown).Semiquantitative RT-PCR analyses demonstrated that 4-wkHPX induced a significant decrease in OX1R mRNA expres-sion levels; a response that was partially prevented by re-placement with recombinant FSH (7.5 IU/rat�24 h), but notwith hCG (10 IU/rat�24 h), for 7 d before sampling (Fig. 4).Similar responses to HPX and gonadotropin replacementwere detected for testicular OX1R mRNA levels using real-time RT-PCR assays (Table 1).

FIG. 1. Developmental profile of expression of OX1R gene in rat testisthroughout postnatal maturation. In the upper panel, a representa-tive RT-PCR assay is presented of expression levels of OX1R mRNAin testicular samples from 1-, 5-, 10-, 15-, 20-, 30-, 45-, and 75-d-oldrats. Parallel amplification of L19 ribosomal protein mRNA served asinternal control. In the lower panel, semiquantitative values are themean � SEM of at least four independent determinations. For pre-sentation of data, the level of expression of OX1R mRNA on d 1postpartum was taken as 100%, and the other values were normalizedaccordingly, thus allowing semiquantitative comparison between thedifferent age points. Groups with different lowercase letters are sta-tistically different (P � 0.05; ANOVA followed by Student-Newman-Keuls multiple range test).

TABLE 1. Compilation of real-time RT-PCR data from triplicateanalyses of testicular OX1R mRNA levels in representativeexperimental groups

GroupsCT OX1-R mRNA levels

(% over reference)OX1R RP-L19

Group AT 15 d 24.3 � 0.2 19.0 � 0.3 107.0 � 6.0T 45 d 23.3 � 0.15 19.3 � 0.2 199.0 � 4.5a

T 75 d 23.5 � 0.25 19.2 � 0.15 168.5 � 16.0a

Group BControl 75 d 23.2 � 0.2 19.4 � 0.3 104.5 � 6.55 d post-EDS 23.0 � 0.25 19.3 � 0.25 108.0 � 8.040 d post-EDS 25.35 � 0.35 19.3 � 0.2 27.5 � 5.0a

Group CControl 75 d 23.7 � 0.2a 19.4 � 0.3 102.0 � 5.5HXP 24.6 � 0.25 19.5 � 0.35 53.5 � 5.0a

HPX � FSH 23.5 � 0.25a 19.3 � 0.2 107.0 � 12.5HPX � hCG 24.85 � 0.3a 20.1 � 0.2 41.5 � 6.5a

Mean CT values were calculated using an automatic baseline set-ting, as described in Materials and Methods. Representative samplesfrom three settings were assayed: group A, different stages of post-natal development; group B, control (0) and 5 and 40 d after EDS; andgroup C, HPX rats, with or without gonadotropin replacement. In theright column, relative mRNA levels in the experimental groups areexpressed as percentage over reference values. Within each experi-mental setting, reference values were arbitrarily set at T 15 d (groupA) and control 75 d groups (groups B and C) and assigned to a levelof 100%; the other values were normalized accordingly. Percentageexpression over reference values was calculated for each group by theequation 2���CT.

a P � 0.05 vs. corresponding reference values (ANOVA followed byStudent-Newman-Keuls multiple range test).

2300 Endocrinology, May 2004, 145(5):2297–2306 Barreiro et al. • Orexin Actions in Rat Testis

In addition, the ability of the cognate ligand (orexin-A) tomodulate testicular levels of OX1R mRNA was evaluated invitro. Challenge of testicular tissue for 180 min with increas-ing doses of orexin-A (10�10, 10�8, and 10�6 m) induced asignificant decrease in OX1R mRNA levels. Of note, doseresponsiveness to orexin-A exhibited a biphasic pattern, witha progressive decline in OX1R mRNA levels between 10�10

and 10�8 m, and a trend to normalization of expression val-ues for doses 10�6 m (Fig. 5).

Finally, the effects of neonatal estrogen exposure on theexpression levels of OX1R mRNA in rat testis were moni-tored. Neonatal administration of estradiol benzoate (500�g/rat, on d 1 postpartum) did not induce significantchanges in OX1R mRNA levels in 15-d-old animals, whereasa significant decline to nearly undetectable levels was de-

tected on d 30 and 75 postpartum. In addition, expressionlevels of OX1R mRNA were assayed in testes of rats treatedneonatally with a potent GnRH antagonist. This treatmenthas been previously reported to induce a significant sup-pression in serum LH, FSH, and T levels at the end of the 15-dperiod of treatment, which is similar in magnitude to thatinduced by neonatal estrogenization in pair-aged animals

FIG. 2. Expression of OX1R mRNA in rat testis after selective with-drawal of mature adult-type Leydig cells. In the upper panel, a rep-resentative RT-PCR assay is presented of the expression levels ofOX1R transcript in testicular samples from adult rats before (0) and5, 15, 20, 30, and 40 d after administration of the Leydig cell-specifictoxicant EDS. Parallel amplification of L19 ribosomal protein mRNAserved as internal control. In the lower panel, semiquantitative valuesare the mean � SEM of at least four independent determinations. Forpresentation of data, the level of expression of OX1R mRNA in control(0) samples (solid bar) was taken as 100%, and the other values werenormalized accordingly, thus allowing semiquantitative comparisonbetween the different time points after EDS. Groups with differentlowercase letters are statistically different (P � 0.05; ANOVA followedby Student-Newman-Keuls multiple range test).

FIG. 3. Expression profile of OX1R gene in seminiferous tubule seg-ments at different stages of the seminiferous epithelial cycle. In theupper panel, a representative RT-PCR assay is presented of the rel-ative levels of OX1R mRNA in tubule preparations isolated at stagesII–VI, VII–VIII, IX–XII, and XIII–I of the cycle, as described in Ma-terials and Methods. Parallel amplification of L19 ribosomal proteinmRNA served as internal control. In the lower panel, semiquantita-tive values are the mean � SEM of at least four independent deter-minations. For presentation of data, the level of expression of OX1RmRNA in stages II–VI (solid bar) was taken as 100%, and the othervalues were normalized accordingly, thus allowing semiquantitativecomparison between different stages of the seminiferous epithelialcycle. Groups with different lowercase letters are statistically different(P � 0.05; ANOVA followed by Student-Newman-Keuls multiplerange test).

Barreiro et al. • Orexin Actions in Rat Testis Endocrinology, May 2004, 145(5):2297–2306 2301

(39). In contrast to neonatal estrogen exposure, treatmentwith a potent GnRH antagonist did not induce significantchanges in OX1R mRNA levels at any age point studied(Fig. 6).

Effects of orexin-A stimulation on testicular T secretion invivo and in vitro

Finally, the ability of orexin-A, the cognate ligand of OX1R,to modulate testicular steroidogenesis was evaluated using

in vitro and in vivo settings. In the first approach, T responsesto different doses of orexin-A (10�10–10�6 m) were assayedat 90 and 180 min after incubation in basal and hCG-stimulated conditions. Orexin-A, in a dose-dependent man-ner, was able to significantly stimulate basal T secretion byincubated testicular tissue; an increase in T levels in themedia was detected after challenge with 10�8 and 10�6 mdoses but not with 10�10 m orexin-A (Fig. 7). However, theresponse to 10�8 m orexin-A at 180 min was shortly below thelimit of statistical significance (P � 0.06). In contrast to basalconditions, hCG-stimulated T secretion was not significantlymodified by co-incubation with orexin-A at any of the dosestested (data not shown).

In addition, the effects of orexin-A on testicular T secretionwere monitored in vivo. Bilateral intratesticular injection oforexin-A (10�6 m; 50 �l per testis) resulted in a significant

FIG. 4. Effects of HPX and gonadotropin replacement on testicularexpression of OX1R gene. In the upper panel, a representative RT-PCR assay is presented of OX1R mRNA levels in testes from long-term(4-wk) HPX rats, with or without replacement with hCG (10 IU/rat�24h for 7 d) or FSH (7.5 IU/rat�24 h for 7 d). Parallel amplification of L19ribosomal protein mRNA served as internal control. In the lowerpanel, semiquantitative values are the mean � SEM of at least fourindependent determinations. For presentation of data, the level ofexpression of OX1R mRNA in control (Co; solid bar) samples wastaken as 100%, and the other values were normalized accordingly,thus allowing semiquantitative comparison between the differentexperimental groups. Groups with different lowercase letters are sta-tistically different (P � 0.05; ANOVA followed by Student-Newman-Keuls multiple range test).

FIG. 5. Homologous regulation of OX1R mRNA expression in rat tes-tis by the cognate ligand, orexin-A. In the upper panel, a represen-tative RT-PCR assay is presented of OX1R mRNA levels in testes afterin vitro exposure to increasing concentrations of orexin-A (10�10–10�6

M). Parallel amplification of L19 ribosomal protein mRNA served asinternal control. In the lower panel, semiquantitative values are themean � SEM of at least four independent determinations. For pre-sentation of data, the level of expression of OX1R mRNA in control (0)samples, incubated in the presence of medium alone, was taken as100%, and the other values were normalized accordingly, thus allow-ing semiquantitative comparison between the different doses. Groupswith different lowercase letters are statistically different (P � 0.05;ANOVA followed by Student-Newman-Keuls multiple range test).

2302 Endocrinology, May 2004, 145(5):2297–2306 Barreiro et al. • Orexin Actions in Rat Testis

increase in serum T levels over basal (0 min) values at 30 minafter injection. Moreover, serum T concentrations in orexin-A-injected animals were higher than those of paired vehicle-injected animals at 120 min after drug administration. Ac-cordingly, integrated T secretion during the study period(240 min), as estimated by the area under the curve, wassignificantly increased by intratesticular injection of orexin-A(Fig. 8). Of note, a trend to decline in serum T levels wasobserved for both vehicle- and orexin-A-injected groups atadvanced time points of the study.

Discussion

In our study, assessment of testicular OX1R mRNA ex-pression was conducted by semiquantitative RT-PCR and, inselected representative samples, by real-time RT-PCR. Such

analyses demonstrated that OX1R gene is expressed in rattestis throughout postnatal development, with peak valuesbeing detected in the neonatal and pubertal to early adultperiods. Although direct evaluation of its cellular locationwithin the rat testis was not conducted, persistent testicularexpression of OX1R message was also detected after selectiveelimination of mature Leydig cells. In this model, matureLeydig cells are completely and selectively eliminated fromthe testicular interstitium within 24–48 h after administra-tion of the toxicant in vivo (40). This suggests that this celltype is not the major source for the testicular expression ofOX1R gene. Conversely, our results point out a predominantlocation of this transcript in the seminiferous tubules withinrat testis. It has to be stressed, however, that our experimentalapproach does not rule out the possibility of low expressionof OX1R signal in Leydig cells. Of note, despite persistentexpression, a progressive decline in testicular expression lev-els of OX1R mRNA was observed from d 15 after EDS on-wards. Different possibilities may account for such a finding,such as regulation of OX1R mRNA levels by T. The fact,however, that OX1R mRNA expression remained unalteredafter short-term hCG replacement to HPX rats strongly sug-gests that the potential role of T in the acute regulation ofOX1R expression, if any, might be mostly permissive andlikely related with its ability to maintain the spermatogenicepithelium in the adult rat.

Assessment of the tubular expression of OX1R gene wascarried out using preparations of seminiferous tubules iso-lated at different stages of the epithelial cycle. Positive am-plification of OX1R was obtained in tubule preparations at allstages of the spermatogenic cycle, but relative levels of ex-pression of OX1R transcript varied throughout the cycle, withmaximum values in stages VII–VIII and minimum expres-sion levels in stages XIII–I. Of note, stages VII–VIII are themost androgen-dependent phases of spermatogenesis (41,42), and, as reported herein for OX1R mRNA, androgen re-ceptor expression is the highest at stages VII–VIII (43). Thisobservation is in keeping with the potential involvement ofT in the regulation of testicular OX1R mRNA levels. Overall,the staged profile of expression of OX1R gene in rat semi-niferous epithelium is highly suggestive of direct actions oforexin-A (on Sertoli and/or germ cells) in the control ofspermatogenesis. This possibility is presently under evalu-ation in our laboratory.

Among others, a candidate for the hormonal control ofOX1R mRNA in rat testis is pituitary FSH because treatmentof HPX rats with FSH, but not with hCG (as super-agonist ofLH), was able to restore testicular OX1R mRNA levels. Ofnote, FSH receptors are solely expressed in Sertoli cellswithin the tubular compartment of the testis (44). Thus, it islikely that OX1R gene is expressed in Sertoli cells, and theabove stimulatory effect may be a direct action of FSH onSertoli cells. This contention is indirectly supported also byour observation of high expression levels of OX1R mRNA inthe neonatal testis, when Sertoli cells actively proliferate (44).Alternatively, an indirect action of FSH on other cell typeswithin the testis (either at the interstitium or the tubularcompartment) cannot be ruled out. An additional signal reg-ulating testicular expression levels of OX1R mRNA is likelythe cognate ligand, orexin-A. Thus, orexin may participate in

FIG. 6. Regulation of OX1R gene expression in rat testis by neonatalexposure to estrogen. In the upper panel, a representative RT-PCRassay is presented of OX1R mRNA levels in testes from controls (C),neonatally estrogenized (EB) rats, and rats treated neonatally witha potent GnRH antagonist (A). The following three age points wereanalyzed: d 15, d 30, and d 75 postpartum. Parallel amplification ofL19 ribosomal protein mRNA served as internal control. In the lowerpanel, semiquantitative values are the mean � SEM of at least fourindependent determinations. For presentation of data, the level ofexpression of OX1R mRNA in control samples was taken as 100%, andthe other values were normalized accordingly, thus allowing semi-quantitative comparison between the different experimental groups.Groups with different lowercase letters are statistically different (P �0.05; ANOVA followed by Student-Newman-Keuls multiple rangetest).

Barreiro et al. • Orexin Actions in Rat Testis Endocrinology, May 2004, 145(5):2297–2306 2303

the autolimitation of its biological effects at the testis throughligand-induced down-regulation of OX1R gene expression.Notably, homologous regulation has also been demonstratedby our group for the testicular expression of leptin andghrelin receptors (33, 45). From a functional standpoint, thehormonally regulated pattern of expression of OX1R genereported herein strongly suggests a finely tuned, direct roleof orexins in the control of testicular function. This contentionis supported by our data on the effects of orexin-A in thecontrol of T secretion in vivo and in vitro.

In addition to acute regulation by FSH and orexin-A, tes-ticular expression of OX1R mRNA appears to be sensitivealso to the organizing effects of neonatal estrogen. A phys-iological role for estrogen in the regulation of testicular de-velopment and function has recently emerged (46). Indeed,key events in testicular maturation, such as Sertoli cell de-velopment (47), Leydig cell differentiation and function (32),and expression of estrogen receptor � and � genes (39), arelikely imprinted by neonatal estrogen. Our current data pointout that the neonatal endocrine (estrogen) milieu can imprintalso the pattern of expression of OX1R gene in rat testis. In

fact, relative OX1R mRNA levels were persistently sup-pressed by neonatal estrogenization up to adulthood. Suchan effect cannot be attributed to estrogen-induced suppres-sion of serum gonadotropin levels during the neonatal pe-riod because it was not mimicked by postnatal administra-tion of a potent GnRH antagonist. This suggests a directaction of estrogen, during early developmental stages, in thecontrol of testicular OX1R gene expression. It is worthy ofnote that neonatal estrogen exposure induces a strikinglyopposite effect upon testicular expression levels of leptinreceptors (33). The contribution of persistently decreasedOX1R expression to the plethora of functional defects in rattestis after neonatal exposure to supraphysiological doses ofestrogen remains to be elucidated.

Further evidence for the presence of functional OX1R in therat testis and for a role of orexin-A in the control of testicularfunction is provided by its ability to moderately but signif-icantly elicit basal T secretion both in vitro and in vivo. Thephysiological relevance of this phenomenon, as well as thesite of action of orexin-A for the direct control of testicularsteroidogenesis, has yet to be established. On the latter, our

FIG. 7. Regulation of testicular T secretion byorexin-A in vitro. Testicular slices were incubatedwith increasing doses of orexin-A (10�10–10�6 M),and concentration of T in the media was moni-tored after 90 and 180 min of incubation. Valuesare normalized per gram of incubated tissue.Data are expressed as mean � SEM (n � 10–12samples/group). **, P � 0.01 vs. correspondingcontrols (ANOVA followed by Student-Newman-Keuls multiple range test).

FIG. 8. Regulation of testicular T secretion byorexin-A in vivo. Bilateral intratesticular injec-tions of orexin-A (10�6 M; 50 �l per testis) wereperformed. Rats intratesticularly injected withvehicle served as controls. Blood sampling wasconducted by jugular venipuncture before (0) andat 30, 60, 120, 180, and 240 min after drug ad-ministration. Profiles of T secretion along thestudy period in the experimental groups are pre-sented. In addition, integrated secretory re-sponses over the study period (240 min) areshown as area under the curve (AUC) in the inset.**, P � 0.01 vs. corresponding controls; a, P � 0.01vs. basal (0) values (ANOVA followed by Student-Newman-Keuls multiple range test).

2304 Endocrinology, May 2004, 145(5):2297–2306 Barreiro et al. • Orexin Actions in Rat Testis

results are suggestive of a primary action of orexin-A on theseminiferous tubules (likely on Sertoli cells) to modulatetesticular T secretion, although direct effects of this moleculeon steroidogenic Leydig cells cannot be completely ruled outon the basis of our present data. Of note, evidence for astimulatory role of orexin-A in the control of adrenal steroi-dogenesis has been presented in rats, pigs, and humans (20,21, 48). Thus, orexin may operate as a common secretagoguein different steroidogenic tissues (such as the adrenal andtestis). Under physiological conditions, the source oforexin-A for the stimulation of testicular T secretion may beeither systemic, because orexin has been detected in plasma(23), or locally produced, because significant levels of ex-pression of prepro-orexin gene have been reported in the rattestis (22). In contrast to the stimulatory effect of orexin, wehave recently demonstrated that leptin and ghrelin, otherrelevant signals in the control of energy homeostasis, aredirect inhibitory factors in the regulation of testicular ste-roidogenic function (28, 49). Interactions between leptin,ghrelin, and orexin systems in the control of food intake havebeen demonstrated at central levels (3, 50). Whether an anal-ogous crosstalk between stimulatory (orexin) and inhibitory(leptin and ghrelin) signals of steroidogenesis operateswithin the rat testis merits further investigation.

Over the last years, compelling evidence has demon-strated a close link between energy status and reproductivefunction (51, 52). The integrated control of these pivotal phys-iological systems is probably conducted by a plethora ofsignals acting at different levels of the neuroendocrine axesgoverning food intake, energy homeostasis, metabolism, andfertility. Among others, the adipocyte-derived hormone, lep-tin, and the gut-derived peptide, ghrelin, have been identi-fied as putative neuroendocrine integrators, mainly throughactions at central hypothalamic levels (51–52). However, ex-pression of their cognate receptors and direct actions of leptinand ghrelin in the rat testis have also been reported (28, 32,33). In this context, our present data demonstrate that thegene encoding the putative receptor for orexin-A, OX1R, butnot the OX2R gene, is expressed in rat testis along postnataldevelopment, under the regulation of an array of hormonesand mediators that include pituitary FSH, the cognate ligand(orexin-A), and the perinatal endocrine milieu. In addition,we provide the first evidence for a potential role of orexin-Ain the direct control of testicular steroidogenic function inrats. Overall, our data point out a novel site of action of orexinin the control of male reproductive axis, thus suggesting acomplex, multifaceted role of this molecule in the integratedcontrol of energy homeostasis and reproductive function.

Acknowledgments

We are indebted to R. Fernandez-Fernandez and A. Mayen for out-standing assistance during conduction of some of the experimentalstudies.

Received October 20, 2003. Accepted January 26, 2004.Address all correspondence and requests for reprints to: Manuel

Tena-Sempere, Physiology Section, Department of Cell Biology,Physiology and Immunology, Faculty of Medicine, University ofCordoba, Avenida Menendez Pidal s/n, 14004 Cordoba, Spain.E-mail: fi1tesem@uco.es.

This work was supported by Grants BFI 2000-0419-CO3-03 and BFI2002-00176 from DGESIC (Ministerio de Ciencia y Tecnologıa, Spain),the Academy of Finland, and Turku University Central Hospital.

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