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Expression of Human Prepro-Orexin and Signaling Characteristics of Orexin Receptors in the Male Reproductive System

Biomedical Research Institute, Department of Biological Sciences, University of Warwick, Coventry, United Kingdom CV4 7AL

Address all correspondence and requests for reprints to: Dr. Harpal S. Randeva, Department of Biological Sciences, University of Warwick, Gibbet Hill Road, Coventry, United Kingdom CV4 7AL. E-mail: hrandeva{at}bio.warwick.ac.uk.

	Abstract

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Orexin-A (OR-A) and OR-B are derived from a common 130-amino acid precursor peptide, prepro-OR, by proteolytic cleavage. Orexins orchestrate their actions by binding and activating two types of G protein-coupled receptors, OR-1 receptor (OX1R) and OR-2 receptor (OX2R). Besides playing a role in the regulation of feeding and energy homeostasis in rats, ORs appear to increase sexual arousal as well as copulatory performance in rats. Furthermore, OR-A and -B immunoreactivity has been detected in rat testis. In view of these findings we investigated the expression of OR receptors and their signaling characteristics in the human male reproductive system. Using RT-PCR analysis, we were able to demonstrate that both OX1R and OX2R are expressed in the testis, epididymis, penis, and seminal vesicle, whereas prepro-OR was expressed only in the epididymis and penis. Protein expression of both OR receptors in human testis was confirmed by Western blotting analysis, with apparent molecular masses of 50 kDa for OX1R and 40 kDa for OX2R. Immunofluorescent studies revealed staining for both OX1R and OX2R in Leydig cells, myoid cells of the seminiferous tubules, and Sertoli cells. To test the ability of ORs to activate testicular phospholipase C, we determined the effects of OR-A and OR-B on inositol trisphosphate production. When membranes from testicular biopsies were incubated with OR-A or OR-B, there was a rapid inositol trisphosphate turnover. This effect appeared to be dose dependent. These data provide a novel insight into the expression and signaling characteristics of OR receptors in the human male reproductive system and potentially further implicate these peptides, acting in an autocrine/paracrine manner, in the regulation of arousal mechanisms in humans.

	Introduction

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OREXIN A (OR-A) and OR-B were cloned from rat hypothalamus in 1998 (1, 2). They are derived from a common 130-amino acid precursor peptide, prepro-OR, by proteolytic cleavage. Orexin-A, a 33-amino acid peptide, and OR-B, a 28-amino acid peptide, share 46% homology. Besides playing a role in the regulation of feeding and energy homeostasis (3, 4), intracerebroventricular administration of OR-A or -B has been reported to exert divergent actions, including modulation of physiological behaviors (5), cardiovascular regulation (6), and regulation of sleep-wake cycle (7), with the recent demonstration of reduced cerebrospinal fluid concentrations of ORs in patients with narcolepsy (8). The distribution of OR-immunoreactive neurons in the central nervous system suggests potentially important roles for these peptides in the regulation of the hypothalamo-pituitary-gonadal axis and sexual behavior (9, 10). In a more recent study, OR-A application at the medial preoptic area, which is important for the regulation of male sexual behavior in rats, increased sexual arousal as well as copulatory performance (11). To date, it has been presumed that the excitatory effects of ORs are mediated via the central nervous system; however, the detection of OR-A and OR-B immunoreactivity in rat testis may implicate these peptides at a local (gonad) level (12, 13), with paracrine/autocrine effects.

Orexins orchestrate their actions by binding and activating two types of G protein-coupled receptors, OR-1 receptor (OX1R) and OR-2 receptor (OX2R), which display 64% homology in their amino acid level (1). The rat and human receptors display 94% and 95% homology for OX1R and OX2R, respectively, suggesting that they are highly conserved between mammalian species (1). OX1R preferentially binds OR-A, whereas OX2R binds both OR-A and -B, apparently with similar affinity. Orexin receptors were originally shown to be present only in the hypothalamus (1), but now their presence has been noted in peripheral tissues, including the myenteric plexus of the enteric nervous system and the endocrine cells of the gut (14) and adrenal gland (15, 16), implicating ORs in an increasing number of physiological responses in peripheral tissues.

Studies in rats revealed that OX1R, but not OX2R, are expressed in the testis (17), although no data exist in the literature regarding the presence of OR receptors in the human reproductive system. Therefore, our aim was to elucidate the expression of prepro-OR and OR receptors in the human male reproductive system and investigate their signaling characteristics at the testicular level.

To this end, the expression of prepro-OR and OR receptors was investigated by RT-PCR using cDNA from human testis, epididymis, penis, and seminal vesicles. Localization of OR receptor protein was studied using Western blotting and immunofluorescent analysis in sections prepared from human testis. Lastly, functionality of the testicular OR receptors was assessed by performing a radioligand binding assay and measuring their responses to OR-A and OR-B in terms of inositol triphosphate turnover.

	Materials and Methods

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Chemicals

Human OR-A, OR-B, [¹²⁵I]OR-A, and [¹²⁵I]OR-B were purchased from Phoenix Pharmaceuticals (Belmont, CA). All PCRs were carried out using a Taq DNA polymerase kit from Life Technologies, Inc. (Paisley, UK). All primary antibodies were raised in rabbits and were polyclonal. OX1R and OX2R antibodies and their blocking peptides were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), whereas antibodies for OR-A and OR-B were purchased from Phoenix Pharmaceuticals. All electrophoretic reagents were of the highest grade available and were obtained from Bio-Rad Laboratories (Richmond, CA). The inositol trisphosphate (IP₃) RIA kit was purchased from Amersham Life Sciences (Little Chalfont, UK).

Subjects

To detect the expression of prepro-OR and OR receptors, we used a male reproductive cDNA panel (AMS Biotechnology, Abingdon, UK). This male cDNA panel was constructed using RNA from normal pooled samples without any genetic disorders or cancer. The age range of donors for testis, epididymis, and penis was 21–24 yr, and the age of donors for the seminal vesicles was 27 yr. For the immunofluorescent studies we used fresh paraffin-embedded testis, 5 µm in thickness, obtained from a normal 23-yr-old male (AMS Biotechnology) and archival testicular tissue (n = 8) obtained from postpubertal patients with unilateral cryptorchidism, who were counseled to undergo surgical intervention.

For protein and signaling studies, testicular tissue was also obtained from men (n = 6) undergoing surgical correction of vasectomy. The protocol was approved by the local ethics committee. Tissues for extraction of protein for Western blot and signaling studies were chopped into 0.5-cm fragments, snap-frozen in liquid nitrogen, and stored at –70 C before use.

PCR

All PCRs were carried out using Taq DNA polymerase (Life Technologies, Inc.) with 200 ng cDNA for each amplification, as previously described (18). Briefly, cDNAs were amplified at 94 C (45 sec), 58 C (45 sec), and 72 C (1 min) for a total of 35 cycles, with a final extension step at 72 C for 10 min. The sets of primers used for RT-PCR amplification were: for OX1R: sense, 5'-CCTGTGCCTCCAGACTAT GA-3'; and antisense, 5'-ACACTGCTGCATTCCATGAC-3'; for OX2R: sense, 5'-TAGTTCCTCAGCTGCCTA TC-3'; and antisense, 5'-CGTCCTCATGTGGTGGTTCT-3'; and for prepro-OR: sense, 5'-CAATTGACAGCCTCAAGGTTC-3'; and antisense, 5'-CAGAGGCCTGGGCCAGG AC AG-3'. Ten microliters of the reaction mixture were electrophoresed on a 1.8% agarose gel and visualized by ethidium bromide using a 1-kb DNA ladder (Life Technologies, Inc.) to estimate the band sizes. As a negative control for all reactions, distilled water was used in place of cDNA. The resultant PCR products were sequenced in an automated DNA sequencer, and the sequence data were analyzed using Blast Nucleic Acid Database Searches from the National Center for Biotechnology Information.

Preparation of testicular membranes

For the detection of OX1R and OX2R, testicular tissues (n = 4) were weighed and homogenized in 6 ml Dulbecco’s PBS containing 10 mM MgCl ₂, 2 mM EGTA, 0.15% (wt/vol) BSA, 0.15 mM bacitracin, and 1 mM phenylmethylsulfonylfluoride (PMSF), pH 7.2, at 22 C for 40 sec. The homogenate was centrifuged at 3,000 rpm for 30 min at 4 C, and the supernatant was centrifuged (19,000 rpm) for an additional 60 min at 4 C. The resultant pellet was washed, resuspended in extraction buffer, and spun at 19,000 rpm for 60 min at 4 C. The final pellet was resuspended in extraction buffer using homogenizer for 20 sec, and protein aliquots of 100 µg were stored at –70 C until further use.

Total lysate was prepared for Western blotting studies for OR peptides. Testicular tissues (n = 4) were thawed and lysed in cold modified radioimmunoprecipitation buffer containing 1x PBS, 1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 10 mg/ml PMSF. The tissue was further homogenized, followed by the addition of 10 mg/ml PMSF/g tissue, and incubated on ice for 30 min. The homogenate was then transferred to microcentrifuge tubes and centrifuged at 13,000 rpm for 10 min at 4 C. The resultant supernatant fluid contained the total lysate.

Binding studies

Testicular membrane suspensions (100 µg) were added to polypropylene tubes with 50 µl [¹²⁵I]OR-A/-B (50,000 cpm) and 50 µl extraction buffer or unlabeled OR-A and OR-B (diluted in buffer). The unrelated peptide urocortin (UCN) was used as a control. The tubes were incubated at 22 C for 2 h, after which the reaction was terminated by the addition of 1 ml ice-cold polyethylene glycol (20%, wt/vol). The tubes were spun at 3,000 rpm for 30 min at 4 C. The supernatant was discarded, and the membrane-bound radioactivity present in the pellet was measured in a -counter.

Western blotting

Human testicular membranes (100 µg) were centrifuged at 13,000 rpm for 15 min at 4 C. The supernatant was then discarded, and the resultant pellets were solubilized with Laemmli buffer (5 M urea, 0.17 M SDS, 0.4 M dithiothreitol, and 50 mM Tris-HCl, pH 8.0), mixed, placed in a boiling water bath for 5 min, and allowed to cool at room temperature.

Samples were separated on an SDS-12% polyacrylamide gel, and proteins were electrophoretically transferred to a nitrocellulose filter at 250 mA for 16–18 h in a transfer buffer containing 20 mM Tris, 150 mM glycine, and 20% methanol. The filter was then blocked in PBS containing 0.1% Tween 20 and 5% (wt/vol) dried milk powder for 2 h at room temperature. After three washes (20 min each) with PBS-0.1% Tween, the nitrocellulose filters were incubated with primary antibody for OX1R and OX2R (Santa Cruz Biotechnology, Inc.). The primary antiserum was used at a 1:1000 dilution in PBS-0.1% Tween, and 5% BSA and blocked overnight at 4 C. The filters were washed thoroughly for 30 min with PBS-0.1% Tween before incubation with the secondary antirabbit horseradish peroxidase-conjugated Ig (1:2000) for 1 h at room temperature and further washing for 30 min with PBS-0.1% Tween and 5% dried milk powder. Antibody complexes were visualized using chemiluminescence, as previously described (16). To ensure specificity, we also performed preabsorption of both OX1R and OX2R Abs with their blocking peptides before Western blotting.

For the detection of OR-A and OR-B, similar procedures were followed, using specific noncross-reactive antibodies (Phoenix Pharmaceuticals) and total testicular tissue lysate.

Immunofluorescence

Paraffin-embedded sections of testicular tissue were dewaxed and dehydrated. The primary goat polyclonal OX1R and OX2R antibodies (Santa Cruz Biotechnology, Inc.) were used at a 1:100 dilution. All dilutions were made in 3% BSA in PBS. Specimens were incubated with primary antibody for 60 min, then washed three times with PBS (5 min each time) before incubation with antigoat IgG-fluorescein isothiocyanate-conjugated secondary antibody (Santa Cruz Biotechnology, Inc.) for 45 min. The slides were then thoroughly rinsed with PBS, and the cell nuclei were visualized by applying the DNA-specific dye 4,6-diamido-2-phenylindole at a final concentration of 1 µg/ml.

IP₃ assay

For the IP₃ assay, testicular membrane suspensions were incubated with increasing concentrations of OR-A and OR-B, followed by the addition of 200 µl IP ₃ generation buffer containing 25 mM Tris-acetate buffer (pH 7.2), 5 mM magnesium acetate, 1 mM dithiothreitol, 0.5 mM ATP, 0.1 mM CaCl₂, 0.1 mg/ml BSA, and 10 µM GTP. Membranes were incubated for 3 min at 37 C, and the reaction was terminated by the addition of 1 M ice-cold trichloroacetic acid, followed by extraction of inositol phosphates and neutralization. IP₃ levels were estimated by RIA based on the displacement of [³H]IP₃ from a specific bovine adrenocortical IP₃ binding protein. The interassay coefficient of variation was 9.7%.

Statistical analysis

For the IP₃ study, data are shown as the mean ± SEM of each measurement (n = 4). In each case, results were evaluated between groups using two-tailed t test, with significance determined at the level of P < 0.05.

	Results

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RT-PCR of OR receptor mRNA

The expression of prepro-OR and OR receptors in the human male reproductive system was analyzed by RT-PCR using a cDNA panel. Both OX1R and OX2R appear to be expressed in human testis, epididymis, penis, and seminal vesicle, as a 204-bp PCR product for the OX1R and a 261-bp PCR product for the OX2R. Interestingly, prepro-OR appeared to have a more confined expression only in epididymis and penis as a 519-bp PCR fragment, whereas testis and seminal vesicle were void of prepro-OR mRNA (Fig. 1A).

View larger version (49K): [in this window] [in a new window]   FIG. 1. A, RT-PCR analysis of OX1R, OX2R, and prepro-OR in human male reproductive tissues. Lane M, DNA ladder marker; lane 1, testis cDNA; lane 2, epididymis cDNA; lane 3, penis cDNA; lane 4, seminal vesicles cDNA; lane 5, negative control (no cDNA input). B, Western blot analysis of protein extracts from rat brain (lane 1) and human testis (lane 2) demonstrate that the antibody against OX1R recognized a band with an apparent molecular mass of 50 kDa. Similarly, when the specific OX2R antibody was used, it recognized a single band with an apparent molecular mass of 40 kDa. Both bands appeared to be specific for OR receptors, as when the antibodies were preabsorbed with their respective blocking peptides (lanes 3 and 4 for rat brain and human testis, respectively), there was no or very little immunodetection.

To determine the apparent molecular mass of the membrane-bound OX1R in human testicular tissues, SDS-PAGE of membrane proteins was performed, using a goat polyclonal antibody raised against the C terminus of the human OX1R. A protein with an apparent molecular mass of approximately 50 kDa was detected in preparations from testicular tissue (Fig. 1B).

Protein expression of OX2R in human testicular preparations was confirmed by immunoblotting using a specific goat polyclonal antibody raised against a peptide mapping at the N terminus of the OX2R of human origin. The detected protein has a molecular mass of approximately 40 kDa (Fig. 1B). The specificity of the response was confirmed by preincubation of OX1R and OX2R antibodies with their blocking peptides (Fig. 1B). There was no expression of OR-A in testicular lysates, although its presence was detected in the rat brain as a 4-kDa band, which was used as a positive control. Similarly, no apparent expression of OR-B was evident (data not shown).

Immunofluorescence analysis

To investigate further the cellular distribution of OR receptors in human testis, immunofluorescence was carried out using OX1R and OX2R antibodies. Positive staining was evident for OX1R in Leydig cells, testicular peritubular myoid cells (TPMCs), and some Sertoli cells (Fig. 2). This immunostaining appeared to be consistent when different paraffin-embedded tissues were tested (n = 8). Similar immunostaining was evident for OX2R (data not shown).

View larger version (52K): [in this window] [in a new window]   FIG. 2. Immunofluorescence analysis for OX1R of serial sections of human testis (n = 3). Immunoreactive staining for the OX1R is distributed exclusively in the interstitial space and on the outer surface of the seminiferous tubules of the human testis (A). When the antibody was preabsorbed, there was no apparent staining (B). Original magnification, x400 (A and B). Higher magnification (C) showed that there was staining for OX1R in Leydig cells and less intense staining in TPMCs and Sertoli cells. D, The magnified immunofluorescent image reveals a detailed granulous staining for OR receptor around the Leydig cells. E, Negative control. Original magnifications, x1000 (C–E).

The presence of functional OR receptors in testicular tissue was confirmed by binding displacement studies. Human OR-A and -B were able to displace their respective radiolabeled ligands from their binding sites in a concentration-dependent manner in human testicular membranes (Fig. 3A). When analyzed, there were no significant differences in binding of both peptides in human testis. The specificity of the receptors was assessed by coincubating [¹²⁵I]OR-A/OR-B with the unrelated peptide UCN (at concentrations up to 100 nM), which was unable to displace either [¹²⁵I]OR-A or [¹²⁵I]OR-B from their binding sites (Fig. 3A).

View larger version (13K): [in this window] [in a new window]   FIG. 3. A, Displacement curves for binding of [125I]OR-A and [125I]OR-B to human testicular membranes. The unrelated peptide UCN was unable to displace either [125I]OR-A or [125I]OR-B. Each point is the mean of four estimations. B, Orexins induced IP3 accumulation from human testicular membranes. Membranes (100 µg) were incubated with OR-A and OR-B (10–6–10–9 M) for 5 min at room temperature, followed by the addition of 200 µl IP3 generation buffer and further incubation for 3 min at 37 C. This was followed by extraction of inositol phosphates and neutralization. IP3 levels were determined by competitive binding assay. Results are expressed as the mean ± SEM of four estimations from three independent experiments. *, P < 0.05 compared with basal; +, P < 0.05 for OR-B compared with OR-A. B/Bo, Bound/free ratio.

To test whether ORs are able to activate testicular PLC in human testes, we determined the effects of OR-A and OR-B on IP3 production. When membranes from testicular tissue were incubated with various concentrations of ORs (10^–6–10^–9 M), there was rapid IP₃ turnover produced by both OR-A and OR-B. This effect has a threshold of 1 nM for both ORs and appeared to be dose specific. Interestingly, the maximum response at 100 nM appeared to be different, with OR-B exerting a more potent effect (270 ± 20% of basal) than OR-A (240 ± 20% of basal; Fig. 3B). This might be due to the different affinities of the peptides for the OR receptors and suggests that OX2R might be the predominant receptor in our preparations. Collectively, our data suggest that the testicular OR receptors can couple to G_q-subunits and subsequently activate the PLC/IP₃ pathway.

	Discussion

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The novel data presented in this study demonstrate that OX1R and OX2R are expressed in the human male reproductive tract. The current study localized for the first time OX1R and OX2R proteins to Leydig cells and TPMCs and Sertoli cells. In Leydig cells there was strong membrane and cytoplasmic granulous staining for both receptors. The expression of OR receptors in these cells is consistent with the role of testosterone (T) in the expression of peripheral OR receptors. In a recent study it has been shown that rat adrenal OX2R is regulated by T at the mRNA level (19). In gonadectomized rats there was a significant down-regulation of adrenal OX2R, an effect that was reversed upon T replacement (19). Moreover, the effects of ORs on steroidogenesis are well documented. For example, ORs stimulate corticosterone and cortisol production from dispersed rat and human adrenocortical cells through activation of the adenylate cyclase-dependent signaling cascade (18). It is possible, therefore, that ORs, acting via their receptors in Leydig cells, might influence T production, which, in turn, can regulate receptor expression in peripheral tissues, thus creating a positive feedback loop. Further research is required to assess the precise effects ORs exert on Leydig cells. However, caution should be exercised in interpretation of any future data, given that many G protein-coupled receptors appear to act in a tissue- and species-specific manner in these cells. In rat Leydig cells, CRH did not stimulate cAMP production and did not induce T production (20), in contrast with mouse Leydig cells, where CRH agonists stimulate T production through cAMP (21).

Orexin receptors have also been localized in the TPMCs, which are contractile cells and express all of the cytoskeletal markers of true smooth muscle cells (22). Seminiferous tubule contraction is an important step in the regulation of spermatogenesis and testicular sperm output. Several hormones, such as endothelin-1, angiotensin II, and oxytocin, acting via G protein-coupled receptors, appear to modulate TPMC function in an endocrine/paracrine/autocrine manner (22, 23, 24). In the present study, using testicular membrane preparations, ORs were able to activate the PLC pathway by inducing IP₃ production, with OR-B being more potent. This is in agreement with previous studies from our laboratory in which we showed that OR receptors can couple to G_q and activate the PLC/PKC/IP₃ system (16). In a recent study, angiotensin II induced contractions acting on rat TPMCs via a protein kinase C-dependent mechanism (22). Given their signaling similarities, it is attractive to speculate that ORs, too, can modulate the contractile state of TPMCs via activation of PLC/IP₃ and, as a result, provide structural integrity to seminiferous tubules. Contrary to the immunodetection of OR-A in rat testis (12, 13), there was no expression in the human samples examined by us. This is in agreement with a recent study in which OR-A in undetectable in human testis (25). Interestingly, there was some scattered expression of OR receptors in Sertoli cells. The protein expression in these cells suggests that they are implicated in signals that regulate the rate of new sperm cell production. However, this observation should be interpreted with caution, given the low expression of OR receptors compared with that in interstitial cells. Future studies should determine whether ORs are involved in spermatogenic-specific events.

Expression of OR receptors was also evident in human penis and seminal vesicles, potentially implicating ORs in the control of penile function. One of the key molecules with fundamental significance in the maintenance of penile erection is nitric oxide (NO) (26). NO is produced after catalytic oxidation of L-arginine by the enzyme NO synthase (NOS). NOS occurs in three distinct isoforms: two constitutive, Ca²⁺-dependent, isoforms, endothelial NOS and neuronal NOS (nNOS), and the Ca²⁺-independent inducible NOS (27). Recent studies revealed coexpression of OR and nNOS activities in rat hypothalamus (28). The Ca²⁺-dependent nNOS is also expressed in the human penis (29). Moreover, exogenous OR-A induced relaxation in the mouse small intestine, which was completely inhibited by the NO synthesis inhibitor, N^G-nitro-L-arginine (30). Given that ORs are expressed in the penis, it is possible that they can influence erectile function by inducing NO via activation of a Ca²⁺-dependent NOS isoform, as they have been shown to induce Ca²⁺responses in various cell types.

In conclusion, this study provides support for the view that OR receptors may act as regulators of reproductive function in the human male reproductive tract. The expression of functional OR receptors in human Leydig cells potentially implicates ORs in steroidogenesis, whereas the expression of these receptors in TPMCs and the activation of the PLC/IP₃ cascade promote further overall testicular function. In addition, the expression of OR receptors in Sertoli cells, epididymis, seminal vesicle, and penis may account for differential effects in each tissue, in agreement with the pleiotropic effects of ORs acting in numerous peripheral tissues and activating multiple signaling pathways. Future studies should focus on elucidating further the potential effects of ORs on steroidogenesis and their roles in the control of the arousal system.

	Footnotes

 
This work was supported by the General Charities of City of Coventry.

E.K. and J.C. should be considered first coauthors by virtue of their equal contributions to the study.

Abbreviations: IP₃, Inositol trisphosphate; OR, orexin; OX1R, orexin-1 receptor; OX2R, orexin-2 receptor; nNOS, neuronal NO synthase; NO, nitric oxide; NOS, NO synthase; PLC, phospholipase C; PMSF, phenylmethylsulfonylfluoride; SDS, sodium dodecyl sulfate; T, testosterone; TPMC, testicular peritubular myoid cell; UCN, urocortin.

Received October 10, 2003.

Accepted January 13, 2004.

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