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Endocrinology Vol. 145, No. 5 2297-2306
Copyright © 2004 by The Endocrine Society

Orexin 1 Receptor Messenger Ribonucleic Acid Expression and Stimulation of Testosterone Secretion by Orexin-A in Rat Testis

M. L. Barreiro, R. Pineda, V. M. Navarro, M. Lopez, J. S. Suominen, L. Pinilla, R. Señaris, J. Toppari, E. Aguilar, C. Diéguez 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 Córdoba, 14004 Córdoba, Spain; Department of Physiology (M.L., R.S., C.D.), University of Santiago de Compostela, 15705 Santiago de Compostela, Spain; and Departments of Physiology and Pediatrics (J.S.S., J.T.), University of Turku, 20520 Turku, Finland

Address all correspondence and requests for reprints to: Manuel Tena-Sempere, Physiology Section, Department of Cell Biology, Physiology and Immunology, Faculty of Medicine, University of Córdoba, Avenida Menéndez Pidal s/n, 14004 Córdoba, Spain. E-mail: fi1tesem{at}uco.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Orexins are hypothalamic neuropeptides primarily involved in the regulation of food intake and arousal states. In addition, a role for orexins as central neuroendocrine modulators of reproductive function has recently emerged. Prepro-orexin and orexin type-1 receptor mRNAs have been detected in the rat testis. This raises the possibility of additional peripheral actions of orexins in the control of reproductive axis, which remains so far unexplored. To analyze the biological effects and mechanisms of action of orexins in the male gonad, we evaluated testicular expression of orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R) mRNAs in different experimental settings and the effect of orexin-A on testicular testosterone (T) secretion. Persistent expression of OX1R mRNA was demonstrated in the rat testis throughout postnatal development. In contrast, OX2R transcript was not detected at any developmental stage. Expression of OX1R mRNA persisted after selective elimination of mature Leydig cells and was detected in isolated seminiferous tubules at defined stages of the seminiferous epithelial cycle. In addition, testicular OX1R mRNA expression appeared to be under hormonal regulation; it was reduced by long-term hypophysectomy and partially restored by FSH replacement, whereas down-regulation was observed after exposure to increasing doses of the ligand in vitro. Moreover, OX1R mRNA expression was sensitive to neonatal imprinting by estrogen. Finally, orexin-A, in a dosedependent manner, significantly increased basal, but not human choriogonadotropin-stimulated, T secretion in vitro. A similar stimulatory effect was observed in vivo after intratesticular administration of orexin-A. In conclusion, our present results provide the first evidence for the regulated expression of OX1R mRNA and functional role of orexin-A in the rat testis. Overall, our data are suggestive of a novel site of action of orexins in the control of male reproductive axis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OREXINS, ALSO TERMED hypocretins, are recently cloned hypothalamic neuropeptides initially involved in the control of feeding behavior (1, 2). Two different orexin molecules have been identified, orexin-A and orexin-B, which derive from the proteolysis of a common 130-amino acid precursor, the prepro-orexin (1, 2). Orexins are primarily expressed in the lateral hypothalamic area, a region with a key role in the control of food intake, and prepro-orexin levels are regulated by a number of metabolic and nutritional signals, such as glucose, leptin, and fasting (2, 3, 4). Moreover, orexigenic fibers are widely distributed and project to multiple brain regions (5), thus suggesting the involvement of orexins in the central control of additional biological functions, such as the regulation of the sleep-wake cycle (6), stress reactions (7), arterial blood pressure and heart rate (8), the autonomic nervous system (8, 9), and several neuroendocrine axes, including corticotrope, lactotrope, somatotrope, and gonadotrope systems (10, 11, 12, 13). The biological actions of orexins are conducted through interaction with two closely related G protein-coupled receptors, termed orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). The binding properties of these receptors are partially different; OX1R is highly selective for orexin-A, whereas OX2R is a nonselective receptor that binds both orexin-A and orexin-B (2).

Among other neuroendocrine functions, a role for orexins in the central control of the reproductive axis has been recently demonstrated. Orexin-A stimulates GnRH release from rat hypothalamic explants of male and female rats (13), and hypothalamic GnRH neurons express OX1R and receive direct contacts from orexin fibers (14, 15). Moreover, depending on the prevailing sex steroid background, orexin-A is able to stimulate or inhibit LH secretion. Thus, orexin-A elicited LH release in steroid-primed ovariectomized rats, but it decreased LH secretion in the absence of ovarian steroids (12, 16, 17). Such a bimodal mode of action is apparently linked with site-specific effects of orexin-A within different hypothalamic nuclei; orexin is stimulatory at the rostral preoptic area and inhibitory at the medial preoptic area or the arcuate nucleus/median eminence (15). Potential actions of orexins at other sites of the hypothalamo-pituitary-gonadal axis remain largely unknown, although it has been reported that orexin-A is able to inhibit GnRH-induced LH release by dispersed pituitary cells (13).

Despite the initial contention that orexins are exclusively produced and acting in the brain, emerging data have demonstrated peripheral endocrine actions of orexins, including direct modulation of adrenal steroidogenic function (18, 19, 20, 21). In addition, expression of the mRNAs encoding prepro-orexin and the cognate receptors (OX1R and OX2R) has been reported in an array of endocrine and nonendocrine peripheral tissues (22). Moreover, orexin-A has been found in human plasma, and its expression has been detected in the gut and pancreas (23, 24, 25, 26). To note, significant amounts of prepro-orexin mRNA have been demonstrated in the rat testis; a tissue that also expressed low levels of OX1R transcript (22). However, the potential involvement of orexins in the direct control of testicular function remains so far unexplored. In this context, the present experimental work was undertaken to analyze the biological effects and mechanisms of action of orexins in the male gonad. To this end, we evaluated testicular expression of the mRNAs encoding OX1R and OX2R in different experimental settings, and we assayed the effect of orexin-A on testicular testosterone (T) secretion. On the latter, we limited our studies to orexin-A because it is provided with a higher biopotency than orexin-B in different physiological systems (2, 16), and preliminary results evidenced lack of expression of OX2R mRNA in rat testis tissue.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and drugs
Wistar male rats bred in the vivarium of the University of Córdoba were used. The day the litters were born was considered as d 1 of age. The animals 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 groups of five rats per cage with free access to pelleted food and tap water. Experimental procedures were approved by the Córdoba University Ethical Committee for animal experimentation and were conducted in accordance with the European Union normative for care and use of experimental animals. Special attention was taken to minimize the number of animals per experimental group. In all experiments, the animals were killed by decapitation, and for those experiments involving mRNA analysis (experiments 1–6), testes were immediately removed, frozen in liquid nitrogen, and stored at –80 C until processing. In addition, in specific settings, trunk blood was collected, and samples of testicular tissue were taken and processed for histological evaluation (experiments 2 and 4). Rat orexin-A was obtained from Bachem AG (Bubendorf, Switzerland). Highly purified human chorionic gonadotropin (hCG; Profasi) and human recombinant FSH (Gonal-F) were purchased from Serono (Madrid, Spain). Estradiol benzoate was obtained from Sigma (St. Louis, MO), and the GnRH antagonist Org 30276 (Ac-D-pClPhe-D-pClPhe-D-Trp-Ser-Tyr-D-Arg-Leu-Arg-Pro-D-Ala-NH2CH3COOH) was generously supplied by Organon (Oss, Netherlands). Ethylene dimethane sulfonate (EDS) was synthesized in our laboratory and dissolved in dimethylsulfoxide-water (1:3, vol/vol).

Experimental designs
In experiment 1, analysis of testicular expression of OX1R and OX2R mRNAs 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-old rats (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 studied after selective Leydig cell elimination by systemic administration of the cytotoxic drug EDS (single dose of 75 mg/kg, ip). This in vivo model provides an optimal experimental background in which to test Leydig cell-specific expression and/or regulation of testicular factors (for example, see Refs. 27 and 28). Testicular samples were obtained from adult 75-d-old rats (n = 5 per group) before (0) and 5, 15, 20, 30, and 40 d after EDS administration and assayed for OX1R mRNA expression. In addition, to monitor elimination of Leydig cells in EDS-treated rats, trunk blood samples were collected upon decapitation of the animals, and testis specimens were taken and processed for histological examination, as described in detail elsewhere (29).

In experiment 3, expression of OX1R mRNA was assessed in seminiferous tubule preparations at different stages of the epithelial cycle. Microdissection of seminiferous tubule segments was carried out as described in detail elsewhere (30). Briefly, testes from adult rats were decapsulated, and 5-mm seminiferous tubule segments were isolated under a transilluminating stereomicroscope. Specific stages of the seminiferous epithelial cycle were identified and pooled in four major groups corresponding to stages II–VI, stages VII–VIII, stages IX–XII, and stages XIII–I of the cycle. After exhaustive washing, tubular tissue was processed for RNA analysis as described in RNA analysis by semiquantitative RT-PCR. Quadruplicate pools per stage of the seminiferous epithelium were processed.

In experiment 4, the ability of pituitary gonadotropins to regulate testicular expression of OX1R gene was monitored. To this end, testicular OX1R mRNA levels were analyzed in groups (n = 5) of adult (75-d-old) control and long-term hypophysectomized (HPX) rats (i.e. 4 wk after pituitary removal) with or without gonadotropin replacement: hCG (10 IU/rat·24 h) or recombinant FSH (7.5 IU/rat·24 h) for 7 d before sampling. In addition, in experiment 5, the regulation of testicular OX1R mRNA levels by its cognate ligand was assayed in vitro. As experimental setting, slices of testicular tissue were prepared from 75-d-old rats and incubated 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 detail elsewhere (28).

In experiment 6, the effects of neonatal exposure to estrogen on the expression levels of OX1R mRNA in rat testis were evaluated. In this setting, 1-d-old male rats were injected sc with a single dose of estradiol benzoate (500 µg/rat; dissolved in 100 µl of olive oil), a regimen that has been reported to induce complete estrogenization in the male rat, without major systemic toxicity (31, 32). Groups of males (n = 5) were killed on d 15, 30, and 75 of age. Vehicle (oil)-injected animals served as controls. In this experiment, the potential mechanism(s) for the effects of neonatal estrogen exposure on testicular OX1R gene expression was also assessed. In this sense, because the effect of neonatal estrogen on the developing testis may be direct or mediated by postnatal suppression of gonadotropins (31), the level of OX1R mRNA was determined also in testes of rats (n = 5) treated neonatally with a potent GnRH antagonist, as described previously (33). Groups of male rats were injected sc with GnRH antagonist (5 mg/kg body weight) on d 1, 4, 7, 10, 13, and 15 postpartum, and tissue sampling was conducted on d 15 (4 h after the last injection), 30, and 75.

Finally, in experiment 7, the effects of orexin-A on basal and stimulated testosterone (T) secretion in vitro were assessed using static incubations of adult rat tissue, as described in detail elsewhere (28, 34). Tissue samples (n = 10–12 per group) were incubated in the presence of increasing doses of orexin-A (10–10–10–6 M), under basal or stimulated (co-incubation with 10 IU/ml hCG) conditions, and T concentrations in the incubation media were determined at 90 and 180 min. In addition, in experiment 8, the effects of orexin-A on T secretion were evaluated in vivo. To this end, bilateral intratesticular injection of orexin-A (10–6 M; 50 µl/testis) or vehicle was performed (n = 12 rats per group), and systemic blood samples were obtained by jugular venipuncture before (0) and at 30, 60, 120, 180, and 240 min after orexin-A administration.

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 by RT-PCR, optimized for semiquantitative detection, using the following specific primer pairs: OX1 forward (5'-TGGGCTGTGTCGCTGGCTG-3') and OX1 reverse (5'-GTTGGGGCTCTGTACACAGG-3'), flanking a 328-bp coding area of rat OX1R cDNA (GenBank accession no. AF041244); OX2 forward (5'-TTGGGGTTCATCGTCGTC AAG-3') and OX2 reverse (5'-AGCCAGGTGGACAGGAGTGA-3'), flanking a 226-bp coding area of rat OX2R cDNA (GenBank accession no. AF041246). These sets 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 carried out in parallel in each sample, using the primer pair: L19 forward (5'-GAAATCGCCAATGCCAACTC-3') and L19 reverse (5'-ACCTTCAGGTACAGGCTGTG-3'), as described in detail elsewhere (28, 33). Given its constitutive expression and rather constant mRNA levels along postnatal development and under different experimental conditions, the RP-L19 gene has been widely used by our group as internal control for RT-PCR assessment of testicular gene expression (for examples, see Refs.28 ,33).

For amplification of the targets, 4 µg of total RNA was used to perform RT-PCR in two consecutive separate steps. In addition, to enable appropriate amplification in the exponential phase for each target, PCR amplification of specific signals (OX1R and OX2R) and L19 ribosomal protein transcript was carried out in separate reactions with different numbers of cycles but using similar amounts of the corresponding cDNA templates, generated in single RT reactions, as previously described (28, 33). PCRs consisted of a first denaturing cycle at 97 C for 5 min, followed by a variable number of cycles of amplification defined by denaturation at 96 C for 30 sec, annealing for 30 sec, and extension at 72 C for 1:30 min. A final extension cycle of 72 C for 15 min was included. Annealing temperature was adjusted for each target: 66 C for OX1R, 63 C for OX2R, and 56 C for RP-L19 transcripts. For OX1R and RP-L19, different numbers of cycles were tested to optimize amplification in the exponential phase of PCR. In detail, analysis of intensity of PCR signals as function of the number of amplification cycles revealed a 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 this basis, 33 and 23 PCR cycles were chosen for further analysis of the OX1R and L19 species, respectively.

PCR-generated DNA fragments were resolved in Tris-borate-buffered 1.5% agarose gels and visualized by ethidium bromide staining. Specificity of PCR products was confirmed by direct sequencing using a 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 densitometric scanning using an image analysis system (1-D Manager; TDI Ltd., Madrid, Spain), and values of the specific targets were normalized to those of internal controls to express arbitrary units of relative expression. In all assays, liquid controls and reactions without RT resulted in 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 using the iCycler iQ Real-Time PCR detection system (Bio-Rad Laboratories, Hercules, CA). In detail, OX1R mRNA levels were assayed in representative testicular samples of different stages of postnatal development (15-, 45-, and 75-d-old rats) and after EDS treatment (at 0 and 5 and 40 d after EDS) and from HXP rats with or without gonadotropin replacement. RT of total RNA was conducted as described earlier. The synthesized cDNAs were further amplified (1/10th) in triplicate by PCR using SYBR green I (Bio-Rad) as fluorescent dye and 1 x iQ Supermix containing 50 mM KCl, 20 mM Tris-HCl, 0.2 mM deoxynucleotide triphosphates, 3 mM MgCl2, and 2.5 U iTaq DNA polymerase (Bio-Rad), in a final volume of 25 µl. The PCR cycling conditions were as follows: initial denaturation and enzyme activation at 95 C for 5 min, followed by 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. Product purity was confirmed by dissociation curves and random agarose gel electrophoresis. No-template controls were included in all assays, yielding no consistent amplification. Calculation of relative expression levels of OX1R mRNA was conducted based on the cycle threshold (CT) method (38). The CT for each sample was calculated using the iCycler iQ Real-Time PCR detection system software with an automatic fluorescence threshold setting. Accordingly, fold expression of OX1R mRNA over reference values was calculated by the equation 2–{Delta}{Delta}CT, where {Delta}CT is determined by subtracting the corresponding RP-L19 CT value (internal control) from the specific CT of the target (OX1R), and {Delta}{Delta}CT is obtained by subtracting the {Delta}CT of each experimental sample from that of the reference sample (taken as reference value 100). For the experimental groups assayed, reference samples were arbitrarily taken from 15-d-old testis (postnatal development), control testis before EDS administration (EDS model), and control adult testis (HPX model). To note, no significant differences in CT values were observed for RP-L19 between the treatment groups.

T measurement by specific RIA
T levels in static incubation media and serum samples were measured using a commercial kit from ICN Biomedicals (Costa Mesa, CA), following the instructions of the manufacturer. All medium samples were measured 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 hormone determination was confirmed by assessment of rat serum samples of known T concentrations used as external controls.

Presentation of data and statistics
Semiquantitative RT-PCR analyses were carried out in duplicate from at least four independent RNA samples of each experimental group. For generation 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 in triplicate using independent RNA samples. Tissue incubations for T measurement were conducted in duplicate, with a total number of 10–12 samples/determinations per group. In addition, integrated T secretory responses in vivo were expressed as the area under the curve, calculated following the trapezoidal rule, over a 240-min period. Data are presented as mean ± SEM. Results were analyzed for statistically significant differences using ANOVA, followed by the Student-Newman-Keuls multiple range test. P <= 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Testicular expression of OX1R mRNA along postnatal development, after selective Leydig cell elimination, and in staged seminiferous tubule preparations
Expression of the genes encoding OX1R and OX2R was evaluated in the rat testis at different stages of postnatal maturation. Our semiquantitative RT-PCR assays demonstrated persistent expression of OX1R mRNA throughout postnatal development (Fig. 1Go). In detail, representative stages of development, corresponding to neonatal (1 and 5 d old), 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-PCR signals demonstrated rather constant relative expression levels of OX1R mRNA along the study period, with peak values in neonatal and pubertal to early adult samples. Such an expression profile was confirmed by real-time RT-PCR analysis of representative testicular samples that showed maximum expression levels in 45-d-old rat testis (Table 1Go). Contrary to OX1R transcript, OX2R mRNA was not detectable in testicular samples at any developmental stage assayed, from the neonatal period to adulthood. However, using similar RT-PCR conditions, OX2R mRNA was detected in adult hypothalamic samples, used as positive controls (data not shown). Consequently, further studies were focused in the analysis of testicular expression of OX1R transcript in different experimental settings.



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  		FIG. 1. Developmental profile of expression of OX1R gene in rat testis throughout postnatal maturation. In the upper panel, a representative RT-PCR assay is presented of expression levels of OX1R mRNA in testicular samples from 1-, 5-, 10-, 15-, 20-, 30-, 45-, and 75-d-old rats. Parallel amplification of L19 ribosomal protein mRNA served as internal control. In the lower panel, semiquantitative values are the mean ± SEM of at least four independent determinations. For presentation of data, the level of expression of OX1R mRNA on d 1 postpartum was taken as 100%, and the other values were normalized accordingly, thus allowing semiquantitative comparison between the different age points. Groups with different lowercase letters are statistically different (P < 0.05; ANOVA followed by Student-Newman-Keuls multiple range test).

 

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  		TABLE 1. Compilation of real-time RT-PCR data from triplicate analyses of testicular OX1R mRNA levels in representative experimental groups

 
In the next step, semiquantitative RT-PCR assays of OX1R mRNA expression were conducted in testis samples at different time points after selective Leydig cell elimination in vivo by administration of the cytotoxic compound EDS. Efficiency of Leydig cell destruction was monitored by histological examination of testicular tissue sections and measurement of serum T concentrations in EDS-treated groups. In line with previous references (29), serum T levels dropped to nearly undetectable values, and no mature Leydig cells were detected in testicular interstitium 5 and 15 d after EDS (data not shown). In this setting, persistent testicular expression of specific OX1R mRNA was demonstrated throughout a 40-d period after EDS administration (Fig. 2Go). In detail, 5 and 15 d after EDS administration, amplification of OX1R signal was clearly obtained at levels similar to those of control animals. However, although persistent expression of OX1R message was detected, the relative levels of the signal progressively declined from d 20 onwards and remained lower than controls at d 40 after EDS treatment. Such a pattern of expression, with persistent levels of OX1R mRNA 5 d after EDS and decreased levels at d 40, was confirmed by real-time RT-PCR assays (Table 1Go).



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  		FIG. 2. Expression of OX1R mRNA in rat testis after selective withdrawal of mature adult-type Leydig cells. In the upper panel, a representative RT-PCR assay is presented of the expression levels of OX1R transcript in testicular samples from adult rats before (0) and 5, 15, 20, 30, and 40 d after administration of the Leydig cell-specific toxicant EDS. Parallel amplification of L19 ribosomal protein mRNA served as internal control. In the lower panel, semiquantitative values are the mean ± SEM of at least four independent determinations. For presentation of data, the level of expression of OX1R mRNA in control (0) samples (solid bar) was taken as 100%, and the other values were normalized accordingly, thus allowing semiquantitative comparison between the different time points after EDS. Groups with different lowercase letters are statistically different (P < 0.05; ANOVA followed by Student-Newman-Keuls multiple range test).

 
Because these data pointed out that OX1R gene is expressed in the tubular compartment of the testis, analysis of mRNA expression was undertaken in preparations of seminiferous tubule fragments isolated at different stages of the seminiferous epithelial cycle. Tubule segments were separated in four major groups corresponding to stages II–VI, VII–VIII, IX–XII, and XIII–I of the cycle. Positive amplification of OX1R mRNA was obtained in seminiferous tubule preparations at all stages of the cycle. However, the relative levels of OX1R mRNA varied throughout the cycle, with maximum values in stages VII-VIII and minimum expression levels in stages XIII-I (Fig. 3Go).



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  		FIG. 3. Expression profile of OX1R gene in seminiferous tubule segments at different stages of the seminiferous epithelial cycle. In the upper panel, a representative RT-PCR assay is presented of the relative levels of OX1R mRNA in tubule preparations isolated at stages II–VI, VII–VIII, IX–XII, and XIII–I of the cycle, as described in Materials and Methods. Parallel amplification of L19 ribosomal protein mRNA served as internal control. In the lower panel, semiquantitative values are the mean ± SEM of at least four independent determinations. For presentation of data, the level of expression of OX1R mRNA in stages II–VI (solid bar) was taken as 100%, and the other values were normalized accordingly, thus allowing semiquantitative comparison between different stages of the seminiferous epithelial cycle. Groups with different lowercase letters are statistically different (P < 0.05; ANOVA followed by Student-Newman-Keuls multiple range test).

 
Hormonal regulation of testicular expression of OX1R mRNA
In the first step, hormonal regulation of testicular expression of OX1R mRNA by pituitary gonadotropins was assessed using the HPX rat as experimental model. Long-term (4-wk) HPX resulted in a clear-cut decrease of serum T levels and the atrophy of all testicular compartments, with atrophic Leydig cells in the interstitial space and regressing seminiferous epithelium with apparent arrest of spermatogenesis at early meiosis phase within the tubules (data not shown). Semiquantitative RT-PCR analyses demonstrated that 4-wk HPX induced a significant decrease in OX1R mRNA expression levels; a response that was partially prevented by replacement with recombinant FSH (7.5 IU/rat·24 h), but not with hCG (10 IU/rat·24 h), for 7 d before sampling (Fig. 4Go). Similar responses to HPX and gonadotropin replacement were detected for testicular OX1R mRNA levels using real-time RT-PCR assays (Table 1Go).



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  		FIG. 4. Effects of HPX and gonadotropin replacement on testicular expression 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·24 h for 7 d) or FSH (7.5 IU/rat·24 h for 7 d). Parallel amplification of L19 ribosomal protein mRNA served as internal control. In the lower panel, semiquantitative values are the mean ± SEM of at least four independent determinations. For presentation of data, the level of expression of OX1R mRNA in control (Co; solid bar) samples was taken as 100%, and the other values were normalized accordingly, thus allowing semiquantitative comparison between the different experimental groups. Groups with different lowercase letters are statistically different (P < 0.05; ANOVA followed by Student-Newman-Keuls multiple range test).

 
In addition, the ability of the cognate ligand (orexin-A) to modulate testicular levels of OX1R mRNA was evaluated in vitro. Challenge of testicular tissue for 180 min with increasing doses of orexin-A (10–10, 10–8, and 10–6 M) induced a significant decrease in OX1R mRNA levels. Of note, dose responsiveness to orexin-A exhibited a biphasic pattern, with a progressive decline in OX1R mRNA levels between 10–10 and 10–8 M, and a trend to normalization of expression values for doses 10–6 M (Fig. 5Go).



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  		FIG. 5. Homologous regulation of OX1R mRNA expression in rat testis by the cognate ligand, orexin-A. In the upper panel, a representative RT-PCR assay is presented of OX1R mRNA levels in testes after in vitro exposure to increasing concentrations of orexin-A (10–10–10–6 M). Parallel amplification of L19 ribosomal protein mRNA served as internal control. In the lower panel, semiquantitative values are the mean ± SEM of at least four independent determinations. For presentation of data, the level of expression of OX1R mRNA in control (0) samples, incubated in the presence of medium alone, was taken as 100%, and the other values were normalized accordingly, thus allowing semiquantitative comparison between the different doses. Groups with different lowercase letters are statistically different (P < 0.05; ANOVA followed by Student-Newman-Keuls multiple range test).

 
Finally, the effects of neonatal estrogen exposure on the expression levels of OX1R mRNA in rat testis were monitored. Neonatal administration of estradiol benzoate (500 µg/rat, on d 1 postpartum) did not induce significant changes in OX1R mRNA levels in 15-d-old animals, whereas a significant decline to nearly undetectable levels was detected on d 30 and 75 postpartum. In addition, expression levels of OX1R mRNA were assayed in testes of rats treated neonatally with a potent GnRH antagonist. This treatment has been previously reported to induce a significant suppression in serum LH, FSH, and T levels at the end of the 15-d period of treatment, which is similar in magnitude to that induced by neonatal estrogenization in pair-aged animals (39). In contrast to neonatal estrogen exposure, treatment with a potent GnRH antagonist did not induce significant changes in OX1R mRNA levels at any age point studied (Fig. 6).

Effects of orexin-A stimulation on testicular T secretion in vivo 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 responses to different doses of orexin-A (10–10–10–6 M) were assayed at 90 and 180 min after incubation in basal and hCG-stimulated conditions. Orexin-A, in a dose-dependent manner, was able to significantly stimulate basal T secretion by incubated testicular tissue; an increase in T levels in the media was detected after challenge with 10–8 and 10–6 M doses but not with 10–10 M orexin-A (Fig. 7Go). However, the response to 10–8 M orexin-A at 180 min was shortly below the limit of statistical significance (P < 0.06). In contrast to basal conditions, hCG-stimulated T secretion was not significantly modified by co-incubation with orexin-A at any of the doses tested (data not shown).



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

 
In addition, the effects of orexin-A on testicular T secretion were monitored in vivo. Bilateral intratesticular injection of orexin-A (10–6 M; 50 µl per testis) resulted in a significant increase in serum T levels over basal (0 min) values at 30 min after 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. Accordingly, integrated T secretion during the study period (240 min), as estimated by the area under the curve, was significantly increased by intratesticular injection of orexin-A (Fig. 8Go). Of note, a trend to decline in serum T levels was observed for both vehicle- and orexin-A-injected groups at advanced time points of the study.



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  		FIG. 8. Regulation of testicular T secretion by orexin-A in vivo. Bilateral intratesticular injections of orexin-A (10–6 M; 50 µl per testis) were performed. Rats intratesticularly injected with vehicle served as controls. Blood sampling was conducted by jugular venipuncture before (0) and at 30, 60, 120, 180, and 240 min after drug administration. Profiles of T secretion along the study period in the experimental groups are presented. In addition, integrated secretory responses over the study period (240 min) are shown as area under the curve (AUC) in the inset. **, P < 0.01 vs. corresponding controls; a, P < 0.01 vs. basal (0) values (ANOVA followed by Student-Newman-Keuls multiple range test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In our study, assessment of testicular OX1R mRNA expression was conducted by semiquantitative RT-PCR and, in selected representative samples, by real-time RT-PCR. Such analyses demonstrated that OX1R gene is expressed in rat testis throughout postnatal development, with peak values being detected in the neonatal and pubertal to early adult periods. Although direct evaluation of its cellular location within the rat testis was not conducted, persistent testicular expression of OX1R message was also detected after selective elimination of mature Leydig cells. In this model, mature Leydig cells are completely and selectively eliminated from the testicular interstitium within 24–48 h after administration of the toxicant in vivo (40). This suggests that this cell type is not the major source for the testicular expression of OX1R gene. Conversely, our results point out a predominant location of this transcript in the seminiferous tubules within rat testis. It has to be stressed, however, that our experimental approach does not rule out the possibility of low expression of OX1R signal in Leydig cells. Of note, despite persistent expression, a progressive decline in testicular expression levels of OX1R mRNA was observed from d 15 after EDS onwards. 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 unaltered after short-term hCG replacement to HPX rats strongly suggests that the potential role of T in the acute regulation of OX1R expression, if any, might be mostly permissive and likely related with its ability to maintain the spermatogenic epithelium in the adult rat.

Assessment of the tubular expression of OX1R gene was carried out using preparations of seminiferous tubules isolated at different stages of the epithelial cycle. Positive amplification of OX1R was obtained in tubule preparations at all stages of the spermatogenic cycle, but relative levels of expression of OX1R transcript varied throughout the cycle, with maximum values in stages VII–VIII and minimum expression levels in stages XIII–I. Of note, stages VII–VIII are the most androgen-dependent phases of spermatogenesis (41, 42), and, as reported herein for OX1R mRNA, androgen receptor expression is the highest at stages VII–VIII (43). This observation is in keeping with the potential involvement of T in the regulation of testicular OX1R mRNA levels. Overall, the staged profile of expression of OX1R gene in rat seminiferous epithelium is highly suggestive of direct actions of orexin-A (on Sertoli and/or germ cells) in the control of spermatogenesis. This possibility is presently under evaluation in our laboratory.

Among others, a candidate for the hormonal control of OX1R mRNA in rat testis is pituitary FSH because treatment of HPX rats with FSH, but not with hCG (as super-agonist of LH), was able to restore testicular OX1R mRNA levels. Of note, FSH receptors are solely expressed in Sertoli cells within the tubular compartment of the testis (44). Thus, it is likely that OX1R gene is expressed in Sertoli cells, and the above stimulatory effect may be a direct action of FSH on Sertoli cells. This contention is indirectly supported also by our observation of high expression levels of OX1R mRNA in the neonatal testis, when Sertoli cells actively proliferate (44). Alternatively, an indirect action of FSH on other cell types within the testis (either at the interstitium or the tubular compartment) cannot be ruled out. An additional signal regulating testicular expression levels of OX1R mRNA is likely the cognate ligand, orexin-A. Thus, orexin may participate in the autolimitation of its biological effects at the testis through ligand-induced down-regulation of OX1R gene expression. Notably, homologous regulation has also been demonstrated by our group for the testicular expression of leptin and ghrelin receptors (33, 45). From a functional standpoint, the hormonally regulated pattern of expression of OX1R gene reported herein strongly suggests a finely tuned, direct role of orexins in the control of testicular function. This contention is supported by our data on the effects of orexin-A in the control of T secretion in vivo and in vitro.

In addition to acute regulation by FSH and orexin-A, testicular expression of OX1R mRNA appears to be sensitive also to the organizing effects of neonatal estrogen. A physiological role for estrogen in the regulation of testicular development and function has recently emerged (46). Indeed, key events in testicular maturation, such as Sertoli cell development (47), Leydig cell differentiation and function (32), and expression of estrogen receptor {alpha} and ß genes (39), are likely imprinted by neonatal estrogen. Our current data point out that the neonatal endocrine (estrogen) milieu can imprint also the pattern of expression of OX1R gene in rat testis. In fact, relative OX1R mRNA levels were persistently suppressed by neonatal estrogenization up to adulthood. Such an effect cannot be attributed to estrogen-induced suppression of serum gonadotropin levels during the neonatal period because it was not mimicked by postnatal administration of a potent GnRH antagonist. This suggests a direct action of estrogen, during early developmental stages, in the control of testicular OX1R gene expression. It is worthy of note that neonatal estrogen exposure induces a strikingly opposite effect upon testicular expression levels of leptin receptors (33). The contribution of persistently decreased OX1R expression to the plethora of functional defects in rat testis after neonatal exposure to supraphysiological doses of estrogen remains to be elucidated.

Further evidence for the presence of functional OX1R in the rat testis and for a role of orexin-A in the control of testicular function is provided by its ability to moderately but significantly elicit basal T secretion both in vitro and in vivo. The physiological relevance of this phenomenon, as well as the site of action of orexin-A for the direct control of testicular steroidogenesis, has yet to be established. On the latter, our results are suggestive of a primary action of orexin-A on the seminiferous tubules (likely on Sertoli cells) to modulate testicular T secretion, although direct effects of this molecule on steroidogenic Leydig cells cannot be completely ruled out on the basis of our present data. Of note, evidence for a stimulatory role of orexin-A in the control of adrenal steroidogenesis has been presented in rats, pigs, and humans (20, 21, 48). Thus, orexin may operate as a common secretagogue in different steroidogenic tissues (such as the adrenal and testis). Under physiological conditions, the source of orexin-A for the stimulation of testicular T secretion may be either systemic, because orexin has been detected in plasma (23), or locally produced, because significant levels of expression of prepro-orexin gene have been reported in the rat testis (22). In contrast to the stimulatory effect of orexin, we have recently demonstrated that leptin and ghrelin, other relevant signals in the control of energy homeostasis, are direct inhibitory factors in the regulation of testicular steroidogenic function (28, 49). Interactions between leptin, ghrelin, and orexin systems in the control of food intake have been demonstrated at central levels (3, 50). Whether an analogous crosstalk between stimulatory (orexin) and inhibitory (leptin and ghrelin) signals of steroidogenesis operates within the rat testis merits further investigation.

Over the last years, compelling evidence has demonstrated a close link between energy status and reproductive function (51, 52). The integrated control of these pivotal physiological systems is probably conducted by a plethora of signals acting at different levels of the neuroendocrine axes governing food intake, energy homeostasis, metabolism, and fertility. Among others, the adipocyte-derived hormone, leptin, and the gut-derived peptide, ghrelin, have been identified as putative neuroendocrine integrators, mainly through actions at central hypothalamic levels (51, 52). However, expression of their cognate receptors and direct actions of leptin and ghrelin in the rat testis have also been reported (28, 32, 33). In this context, our present data demonstrate that the gene encoding the putative receptor for orexin-A, OX1R, but not the OX2R gene, is expressed in rat testis along postnatal development, under the regulation of an array of hormones and 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-A in the direct control of testicular steroidogenic function in rats. Overall, our data point out a novel site of action of orexin in the control of male reproductive axis, thus suggesting a complex, multifaceted role of this molecule in the integrated control of energy homeostasis and reproductive function.



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  		FIG. 6. Regulation of OX1R gene expression in rat testis by neonatal exposure to estrogen. In the upper panel, a representative RT-PCR assay is presented of OX1R mRNA levels in testes from controls (C), neonatally estrogenized (EB) rats, and rats treated neonatally with a potent GnRH antagonist (A). The following three age points were analyzed: d 15, d 30, and d 75 postpartum. Parallel amplification of L19 ribosomal protein mRNA served as internal control. In the lower panel, semiquantitative values are the mean ± SEM of at least four independent determinations. For presentation of data, the level of expression of OX1R mRNA in control samples was taken as 100%, and the other values were normalized accordingly, thus allowing semiquantitative comparison between the different experimental groups. Groups with different lowercase letters are statistically different (P < 0.05; ANOVA followed by Student-Newman-Keuls multiple range test).

 

   Acknowledgments
 
We are indebted to R. Fernandez-Fernandez and A. Mayen for outstanding assistance during conduction of some of the experimental studies.


   Footnotes
 
This work was supported by Grants BFI 2000-0419-CO3-03 and BFI 2002-00176 from DGESIC (Ministerio de Ciencia y Tecnología, Spain), the Academy of Finland, and Turku University Central Hospital.

Abbreviations: CT, Cycle threshold; EDS, ethylene dimethane sulfonate; hCG, human chorionic gonadotropin; HPX, hypophysectomized; OX1R, orexin receptor 1; OX2R, orexin receptor 2; RT, reverse transcription; T, testosterone.

Received October 20, 2003.

Accepted for publication January 26, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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