Pattern of Orexin Expression and Direct Biological Actions of Orexin-A in Rat Testis
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《内分泌学杂志》
Department of Cell Biology, Physiology and Immunology (M.L.B., R.P., F.G., J.M.C., L.P., E.A., M.T.-S.), University of Cordoba, 14004 Cordoba, Spain
Department of Histology and Pathology (M.A., M.A.B.), University of Navarra, 31080 Pamplona, Spain
Department of Physiology (C.D.), University of Santiago de Compostela, 15705 Santiago de Compostela, Spain
Departments of Physiology and Pediatrics (H.H., M.N., J.T.), University of Turku, 20520 Turku, Finland
Abstract
Orexins, hypothalamic neuropeptides initially involved in the control of food intake and sleep-wake cycle, have recently emerged as pleiotropic regulators of different biological systems, including the reproductive axis. Besides central actions, peripheral expression and functions of orexins have been reported, and prepro-orexin and orexin type-1 receptor mRNAs have been detected in the testis. However, the pattern of expression and biological actions of orexin in the male gonad remain mostly unexplored. In this study, we report analyses on testicular prepro-orexin mRNA expression and orexin-A immunoreactivity in different experimental settings, and on direct effects of orexin-A on seminiferous tubule functions. Expression of prepro-orexin mRNA was demonstrated in the rat testis at different stages of postnatal development, with negligible levels at early juvenile period and maximum values in adulthood. Likewise, orexin-A immunoreactivity was demonstrated along postnatal maturation, with strong peptide signal in Leydig cells and spermatocytes at specific stages of meiosis. Testicular expression of prepro-orexin mRNA appeared hormonally regulated; its levels decreased after hypophysectomy and increased after gonadotropin replacement and ghrelin stimulation. Finally, orexin-A suppressed the expression of key Sertoli cell genes, such as Müllerian-inhibiting substance and stem cell factor, and inhibited DNA synthesis in specific stages of the seminiferous epithelium. In conclusion, we provide evidence for the regulated expression of orexin in the rat testis and its potential involvement in the control of seminiferous tubule functions. Together with our recent results on the expression of orexin type-1 receptor in the rat testis, our data further document a novel testicular site of action of orexins in the control of male reproductive axis.
Introduction
THE MAMMALIAN TESTIS is a multifaceted endocrine organ responsible for production of spermatozoa as well as secretion of steroid and peptide hormones (1). These biological functions are based on a complex cellular network composed of different cell types, which undergo dramatic changes throughout development and are arranged in two major tissue compartments, the interstitium and the seminiferous tubules (1). The pituitary gonadotropins, LH and FSH, are the pivotal endocrine regulators of testicular functions; LH is the major elicitor of androgen secretion, and spermatogenesis is tightly controlled by FSH and testosterone (T) (2). In addition, during the last decades, we have become aware of an ever-growing number of signals, of peripheral or local origin, that are also involved in the endocrine and auto-/paracrine control of testicular functions (3, 4). In this context, compelling evidence has been recently presented for the testicular expression and/or direct biological actions of a number of molecules with key roles in energy homeostasis, such as the adipocyte-derived hormones leptin and resistin, and the gut-secreted peptide ghrelin (5, 6, 7, 8). Conceivably, this phenomenon might contribute to the mechanisms by which energy balance and reproductive function are jointly regulated, and clearly illustrates the functional diversity of an array of newly cloned molecules whose biological functions largely exceed those linked to food intake control and metabolism.
Orexins, also termed hypocretins, are hypothalamic neuropeptides, primarily expressed in the lateral hypothalamic area, with a key role in the control of feeding behavior (9, 10). Two different orexin molecules have been identified so far, orexin-A and orexin-B, which derive from the proteolysis of a common 130-amino-acid precursor named prepro-OX. 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), with partially different binding properties: OX1R is highly selective for orexin-A, whereas OX2R is a nonselective receptor that binds both orexin-A and orexin-B (10). Besides their proven role in food intake, orexins have also been involved in the central control of a wide array of biological functions such as regulation of the sleep-wake cycle and arousal (11), stress reactions (12), arterial blood pressure and heart rate (13), as well as several neuroendocrine systems, including the gonadotropic axis (14, 15, 16). On the latter, hypothalamic GnRH-neurons have been shown to express OX1R and to receive direct contacts from orexin fibers (17, 18), and orexin-A was proven to stimulate GnRH release from rat hypothalamic explants (16). Interestingly, the net effects of orexins on LH secretion seem to be dependent on the prevailing sex steroid background, because central injection of orexin-A elicited LH release in steroid-primed ovariectomized rats, but it decreased LH secretion in the absence of ovarian steroids (14, 19, 20). Potential actions of orexins at other sites of the hypothalamo-pituitary-gonadal axis remain largely unexplored, although orexin-A has been reported to inhibit GnRH-induced LH release by dispersed pituitary cells (16).
In the context of the reproductive actions of orexins, it is noticeable that significant amounts of prepro-orexin mRNA, as well as of radioimmunoassayable orexin-A, have been detected in the rat testis; a tissue that, after the brain, holds the highest orexin expression levels reported so far (21, 22). In addition, we have recently provided evidence for the developmental and hormonally regulated expression of OX1R gene in rat testis, and for direct actions of orexin-A on testicular T secretion (23). Moreover, expression of OX1R has been also demonstrated in human and sheep testis (24, 25). Taken together, the available data strongly suggest that the male gonad is a potential site for expression and/or biological actions of orexins. Yet, the pattern of orexin expression in the testis and its eventual involvement in the direct control of testicular functions other than T secretion remain so far unexplored. To cover these issues, we undertook the detailed characterization of testicular expression of prepro-orexin mRNA, as well as orexin-A immunoreactivity, in different developmental stages and experimental settings. In addition, based on our previous findings on the prominent expression of OX1R gene at different stages of the seminiferous epithelial cycle, we assayed the effects of orexin-A upon the expression levels of relevant Sertoli cell genes and the rate of DNA synthesis in staged seminiferous tubule preparations.
Materials and Methods
Animals and drugs
Wistar male rats bred in the vivarium of the University of Cordoba were used, unless otherwise stated. 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 h) 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 Cordoba University Ethical Committee for animal experimentation and were conducted in accordance with the European Union normative for care and use of experimental animals. The animals were killed by decapitation at the end of all experimental procedures. In experiments involving mRNA analysis, testes were immediately removed upon decapitation, frozen in liquid nitrogen, and stored at –80 C until processing. Rat orexin-A and ghrelin (octanoylated at Ser 3) were 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). Ethylene dimethane sulfonate (EDS) was synthesized in our laboratory as described in detail elsewhere (26), and dissolved in dimethylsulfoxide-water (1:3, vol/vol).
Experimental designs
In the first set of experiments, expression of prepro-orexin gene was explored in rat testis at different stages of postnatal development and after selective elimination of mature Leydig cells. To this end, in experiment 1, testicular samples were obtained from 5 (n = 10), 15 (n = 8), 30 (n = 5), 45 (n = 5), and 75-d-old rats (n = 5 per group), corresponding to the neonatal-infantile (5 d), juvenile (15 d), pubertal (30 d), early adult (45 d), and adult (75 d) stages of postnatal maturation. In addition, in experiment 2, testicular expression of prepro-orexin mRNA was studied at different time-points after systemic administration of a single dose of the cytotoxic drug EDS (75 mg/kg, ip). This in vivo model (where mature Leydig cell are selectively and rapidly eliminated from testis interstitium) provides an optimal experimental background in which to test Leydig cell-specific expression and/or regulation of testicular factors (for example, see Ref.6). Thus, testicular samples were obtained from adult 75-d-old rats (n = 5 per group), before (0) and 3, 5, 15, 20, and 40 d after EDS administration. Selective elimination of mature Leydig cells in EDS-treated rats was confirmed by determination of T levels in trunk blood samples and histological examination of testicular tissue, as described in detail elsewhere (23). Finally, in addition to RNA analyses, the pattern of expression and cellular location of orexin-A peptide in rat testis was evaluated throughout postnatal development in experiment 3. To this end, samples of testicular tissue were obtained from 5-, 15-, 20-, 30-, 45-, and 75-d-old rats (n = 5 per group) and subjected to immunohistochemical analysis for detection of orexin-A peptide using a polyclonal antibody (see below).
In the second series of experiments, the pattern of hormonal regulation of prepro-orexin mRNA expression in rat testis was explored. Thus, in experiment 4, the role of pituitary gonadotropins was monitored in vivo by analysis of prepro-orexin mRNA levels 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 (as superagonist of LH; 10 IU/rat per 24 h) or recombinant FSH (7.5 IU/rat per 24 h) for 7 d before sampling. In addition, the ability of gonadotropins to acutely regulate testicular prepro-orexin mRNA levels was assessed in experiment 5. To this end, relative prepro-orexin mRNA was assayed in testes from adult (75 d old) intact rats injected at 1000 h with hCG (25 IU/rat) or recombinant FSH (12.5 IU/rat) and sampled at 2, 4, 8, and 24 h after injection. Vehicle-treated rats served as controls. Finally, in experiment 6, the effects of ghrelin upon testicular expression of prepro-orexin gene were studied. In this sense, ghrelin has been reported as putative regulator of several testicular functions (6, 7, 27), and hypothalamic orexin neurons appear to be modulated by ghrelin (28, 29). Single intratesticular (i.t.) injection of ghrelin (15 μg in 50 μl) was applied to intact adult rats. Of note, each animal received unilateral i.t. injections of ghrelin (right testis), and the contralateral (left) testis was injected with 50 μl of vehicle for control. Groups of animals (n = 5) were sequentially killed at 3, 6, 12, and 24 h after injection, and testis samples were obtained for RNA analysis as indicated above.
Finally, in the third set of experiments, direct biological actions of orexin-A upon rat testis were explored. Based on our previous findings on the prominent expression of OX1R gene at different stages of the seminiferous epithelial cycle (23), we focused our analyses on the potential effects of orexin-A upon the expression levels of relevant Sertoli cell genes and the rate of DNA synthesis in the seminiferous epithelium. Thus, in experiment 7, a panel of Sertoli cell genes were assayed after exposure to orexin-A using static incubations of adult rat tissue, as described in detail elsewhere (23). As experimental setting, slices of testicular tissue were incubated for 180 min in the presence of increasing doses (10–10, 10–8, 10–6 M) of rat orexin-A (n = 8–10 samples per group), as described in detail elsewhere (23), and processed for RNA analysis at the end of the incubation period. In addition, in experiment 8, direct actions of orexin-A upon the rate of DNA synthesis of the seminiferous epithelium was explored in vitro, using static incubations of staged fragments of seminiferous tubules and [3H]thymidine labeling, as described in detail below.
RNA analysis by semiquantitative RT-PCR
Poly(A) RNA was isolated from testicular samples using the PolyATrack mRNA Isolation System, following the instructions of the manufacturer (Promega, Madison, WI). Testicular expression of prepro-orexin mRNA was assessed by RT-PCR, optimized for semiquantitative detection, using the following specific primer pairs: OX-forward (5'-CGGATTGCCTCTCCCTGAGC-3') and OX-reverse (5'-CTAAAGCGGTGGCGGTTGC-3'), flanking 397 bp of the coding area of rat prepro-orexin cDNA (GenBank accession no. NM 013179). These sets of primers were selected on the basis of previous references (30). In addition, in selected experimental settings (see experiment 2), the expression levels of LH receptor mRNA were assessed by semiquantitative RT-PCR, using the following primer pairs: LH receptor-forward (5'-ACTGCTGCGCCTTCAGGAAT-3') and LH receptor-reverse (5'-ATAGCCCATAATATCTTCACA-3'), flanking 245 bp of the coding area of rat LH receptor cDNA (GenBank accession no. NM 012978). Likewise, the expression levels of several Sertoli cell genes, such as inhibin- subunit, inhibin-B subunit, stem cell factor (SCF), and Müllerian inhibiting substance (MIS), were assayed in experiment 7 using specific primer pairs, described in detail elsewhere (27, 31). As internal control for reverse transcriptase (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 (6, 23).
For amplification of the targets, 0.5 μg of poly(A) 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 the specific signals and L19 ribosomal protein transcript was carried out in separate reactions with a different number of cycles, but using similar amounts of the corresponding cDNA templates, generated in single RT reactions, as previously described (6, 23). PCR 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 min. A final extension cycle of 72 C for 15 min was included. Annealing temperature was adjusted for each target: 62 C for prepro-orexin, 60 C for SCF, 58 C for MIS, 57 C for LH receptor, and 56 C for inhibin subunit and RP-L19 transcripts. For the different targets, different numbers of cycles were tested to optimize amplification in the exponential phase of PCR. This procedure was conducted in detail for prepro-orexin transcript, using RNA from adult rat testis. Analysis of intensity of PCR signals as function of the number of amplification cycles revealed a strong linear relationship between cycles 28 and 40, with a correlation coefficient r2 = 0.982 (Fig. 1). Moreover, a strong exponential (log-linear) relationship between signal intensity and cycle number was observed for less than 35 cycles. Thus, considering the diversity of experimental samples to be tested (with different relative amounts of the target), 34 PCR cycles were selected for semiquantitative analyses of prepro-orexin mRNA levels. In addition, previous optimization assays conducted at our laboratory demonstrated amplification in the linear range between 28 and 38 cycles in the case of SCF (r2 0.97), cycles 26–36 in the case of MIS and inhibin subunits (r2 0.97), and cycles 19–29 in the case of RP-L19 (r2 = 0.992) (see Ref.27). On this basis, 35 (LH receptor), 34 (SCF), 32 (inhibin- and -B subunits), 30 (MIS), and 23 (RP-L19) PCR cycles were chosen for further analyses of these targets.
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). 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.
Orexin-A immunohistochemistry
Detection of orexin-A protein was carried out in rat testicular sections at different stages of postnatal development by means of immunohistochemistry using a polyclonal antiserum specific for orexin-A (OXA11A; Alpha Diagnostic, San Antonio, TX). Testis specimens from 5-, 15-, 20-, 30-, 45-, and 75-d-old rats were obtained, fixed for at least 24 h in Bouin fluid, and routinely processed for paraffin embedding. For immunohistochemical analyses, serial 5-μm-thick sections were generated and mounted on Vectabond-coated slides (Vector Laboratories, Burlingame, CA). The sections were dewaxed in xylene, rehydrated in ethanol, and incubated in 3% hydrogen peroxide to quench endogenous peroxidase activity. In addition, the sections were immersed in 10 mM citrate buffer and submitted to antigen retrieval in a microwave oven for 15 min at 1150 W followed by 15 min at 900 W. Immunohistochemical staining for orexin-A was performed using EnVision+System-HRP, consisting of a secondary antibody (goat antirabbit Ig) coupled to a peroxidase-labeled dextran polymer (Dako, Carpinteria, CA) as described in detail elsewhere (32). To prevent nonspecific binding, the sections were treated with goat normal serum, 1:20 in Tris-buffered saline, prior overnight incubation at 4 C in the presence of the primary antiserum (at 1:200 dilution). Thereafter, the slides were incubated for 30 min in EnVision-HRP, and the peroxidase activity was revealed by incubation in the presence of 0.03% 3,3'-diaminobenzidine (DAB). Immunohistochemical controls included omission of the primary anti-orexin antibody (negative control) and the use of sections of rat intestine as positive control. As additional control for the specificity of the primary antibody, immunohistochemical reactions were carried out in slices of rat testicular tissue after preabsorption of the antiserum overnight at 4 C with 10 nmol/ml of the synthetic antigen (OXA11P; Alpha Diagnostic). In keeping with previous results in mouse gut (32), this procedure completely abolished orexin immunolabeling in testis sections (Fig. 1).
Microdissection of seminiferous tubule fragments and assessment of DNA synthesis
Microdissection of seminiferous tubule segments of testes from Sprague Dawley rats was carried out as described in detail elsewhere (33). Briefly, testes from 75-d-old rats were decapsulated, and 2 mm seminiferous tubule segments were isolated under a trans-illuminating stereomicroscope; specific stages of the seminiferous epithelial cycle were identified as described previously (34). Stages I, V, VIIa, VIII–XI, and XII of the cycle were selected, as they contain cells at the representative phases of mitotic and meiotic DNA synthesis (33, 34). Microdissection of tubule segments was performed in DMEM-Ham’s F-12 medium (1:1; Life Technologies, Paisley, UK) supplemented with 1.25 g/liter sodium bicarbonate, 10 mg/liter gentamicin sulfate, and 1 g/liter BSA. For in vitro analysis of orexin-A effects upon DNA synthesis, staged seminiferous tubule preparations were challenged for 24 and 48 h with increasing concentrations of orexin-A (10–10–10–6 M). Tubule fragments were pulse labeled during the last 4 h of culture by addition of 20 kBq [methyl-3H]thymidine (Amersham, Aylesbury, UK), as described in detail elsewhere (33). The cultures were harvested on filter disks (Whatman, Clifton, NJ) with a continuous flow of distilled water for 1 min. A scintillation wax (MeltiLex A 1450-441; Wallac, Turku, Finland) was melted on the filters, and the radioactivity was measured in a liquid scintillation counter (Wallac). Four separate experiments were performed, each with three replicate samples.
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 5-d-old rat testes, which were pooled (n = 2–4) before RNA isolation. Data are presented as mean ± SEM. Results were analyzed for statistically significant differences using ANOVA, followed by Student-Newman-Keuls multiple range test. P 0.05 was considered significant.
Results
Prepro-orexin mRNA in rat testis along postnatal development and after Leydig cell elimination
Expression of the gene encoding prepro-orexin was first evaluated in the rat testis at different stages of postnatal maturation by means of semiquantitative RT-PCR assays. In detail, representative stages of development, corresponding to neonatal (5 d old), juvenile (15 d old), pubertal (30 d old), early adult (45 d old), and adult (75 d old) periods, were explored. Although persistent expression of prepro-orexin mRNA was observed at the different phases of postnatal maturation, our analysis evidenced striking differences in the relative levels of this messenger. Thus, low expression of prepro-orexin mRNA was detected in the neonatal testis, and virtually negligible levels were observed at the early juvenile phase. In contrast, prepro-orexin mRNA levels progressively increased from puberty onwards, and reached maximum values at adulthood (Fig. 2).
In addition, semiquantitative RT-PCR assays of prepro-orexin mRNA were conducted in testis samples at different time-points after selective Leydig cell elimination in vivo by administration of the cytotoxic compound EDS. Complete Leydig cell elimination was confirmed by histological examination of testicular sections and measurement of serum T concentrations in EDS-treated groups, which dropped to nearly undetectable values at d 3 and 5 after EDS (data not shown). In this setting, persistent expression of prepro-orexin mRNA was demonstrated throughout a 40-d period after EDS administration, with a significant increase in prepro-orexin mRNA levels observed at d 3 and 5 post-EDS, which was followed by a decrease in its relative levels to control values thereafter, except for a transient, minor elevation at d 20 (Fig. 3). In contrast, LH receptor mRNA become undetectable in rat testis at d 3, 5, and 15 after EDS, and gradually reappeared from this time-point onwards, co- inciding with the process of Leydig cell repopulation (34). To be noted, within the testis, LH receptors are selectively expressed in Leydig cells at advanced stages of maturation (2). Thus, expression of this messenger was used as molecular marker for the lack of mature Leydig cells at early periods after EDS administration.
Pattern of cellular expression of orexin-A in rat testis along postnatal development
The pattern of orexin-A immunoreactivity was evaluated in testis tissue sections from representative stages of postnatal development, including neonatal (5 d old), juvenile (15 and 20 d old), pubertal (30 d old), early adult (45 d old), and adult (75 d old) periods. Our analyses evidenced the presence of specific orexin-A immunostaining in both the interstitial compartment and the seminiferous tubules of the rat testis. Concerning testis interstitium, orexin-A immunoreactivity was selectively detected in Leydig cells of the fetal- and adult-type origin, mostly at advanced stages of differentiation (Fig. 4). In detail, strong orexin-A signal was observed in fetal-type Leydig cells in testes from 5-d-old rats (Fig. 4A). Likewise, intense orexin-A immunostaining was detected in adult-type Leydig cells in testis samples from puberty onwards, with an apparent increase in the intensity of the signal in adulthood (Fig. 4, D–G). In contrast, negligible orexin-A immunoreactivity was observed in undifferentiated Leydig cell progenitors, present in the interstitial space of testes from juvenile (15 and 20 d old) rats (Fig. 4, B and C). Of note, in all positive cells, orexin-A immunoreactivity showed specific cytoplasmic location. Other interstitial cell types, such as macrophages, were negative for orexin-A at all age-points studied.
Orexin-A immunoreactivity was also detected in the seminiferous epithelium, with prominent signals in spermatocytes at defined stages of meiosis. Indeed, ontogenetic analyses revealed that orexin-A immunostaining was virtually negligible in the tubular compartment before postnatal d 20, whereas its intensity apparently increased thereafter (Fig. 4). Detailed immunohistochemical analyses in adult testis sections demonstrated a stage-specific pattern of orexin-A peptide expression, with specific signals being detected in preleptotene (stages VII–VIII), leptotene (stages IX–XI), zygotene (stages XII–XIII), and pachytene (stages XIV–IV) spermatocytes (Fig. 5). To be noted, weak to negligible orexin-A immunoreactivity was observed in pachytene spermatocytes at tubules between stages IV and XII. Likewise, other cell types within the seminiferous epithelium were negative for orexin-A immunoreactivity. In all positive tubular cells, orexin-A immunoreactivity showed specific cytoplasmic location. A schematic presentation of the pattern of orexin-A immunoreactivity within the seminiferous epithelium, at specific cell types and stages of the cycle, is depicted in Fig. 5.
Hormonal regulation of testicular expression of prepro-orexin mRNA
Hormonal regulation of testicular expression of prepro-orexin mRNA by pituitary gonadotropins was first evaluated using the HPX rat. Gonadotropins were explored as putative modulators of prepro-orexin gene expression given their essential role in testicular development and function (2). Long-term (4-wk) HPX resulted in a clear-cut decrease of serum T levels and the regression of all testicular compartments, with atrophic Leydig cells in the interstitial space and regressing seminiferous epithelium (data not shown). Semiquantitative RT-PCR analyses demonstrated that 4-wk HPX induced a significant decrease in prepro-orexin mRNA expression levels; a decrease that was partially counteracted by administration of hCG (as superagonist of LH; 10 IU/rat/24 h) and fully prevented by replacement with recombinant FSH (7.5 IU/rat/24 h) (Fig. 6).
In addition, the effects of acute administration of hCG and FSH upon testicular prepro-orexin gene expression were evaluated in intact rats. Injection of a single bolus of hCG (25 IU/rat) or recombinant FSH (12.5 IU/rat) evoked a significant increase in testicular prepro-orexin mRNA levels, with peak values at 2 h after administration. However, the profiles of response to each gonadotropin were partially different, as prepro-orexin mRNA levels returned to control values 8 h after hCG injection, whereas they remained significantly elevated at all time-points studied (2, 4, 8, and 24 h) after FSH administration (Fig. 7).
Finally, the ability of ghrelin to modulate testicular expression of prepro-orexin mRNA was also evaluated. Assessment of the potential effects of ghrelin was based on our previous studies on the role of ghrelin as putative regulator of several testicular functions (6, 7, 27), and the fact that hypothalamic orexin system appears to be modulated by ghrelin (28, 29). A single i.t. injection of ghrelin was applied to intact adult rats, where each animal received a unilateral injection of ghrelin (right testis); the contralateral (left) testis (injected with vehicle) serving as control. Prepro-orexin mRNA levels were assayed at 3, 6, 12, and 24 h after ghrelin injection. Expression levels of prepro-orexin mRNA remained unaltered in control (vehicle-injected) testes throughout the study period, thus they were pooled for further analyses. In this model, i.t. injection of ghrelin (15 μg/testis) failed to alter gene expression at 3 and 6 h, but evoked a significant increase in relative prepro-orexin mRNA levels at 12 and 24 h after ghrelin administration (Fig. 8).
Effects of orexin-A on seminiferous tubule functions in vitro
In addition to present data on the pattern of expression of orexin in rat testis (see Results), previous studies of our group evidenced a prominent expression of OX1R gene in the tubular compartment of rat testis (23). Accordingly, the ability of orexin-A, the cognate ligand of OX1R, to modulate key seminiferous tubule functions was explored using in vitro settings. In a first step, the effects of increasing concentrations of orexin-A (10–10, 10–8, 10–6 M) upon the relative mRNA levels of a panel of Sertoli cell genes were explored using static incubations of testicular tissue. In detail, inhibin- and inhibin-B subunit, SCF, and MIS mRNAs were assayed as Sertoli cell-specific signals within the tubular compartment of the adult rat testis (27, 33, 35). In our in vitro setting, exposure to orexin-A failed to modify the relative expression levels of inhibin- and inhibin-B mRNAs, at any of the doses tested. In contrast, challenge with orexin-A significantly inhibited, in a dose-dependent manner, relative expression levels of MIS and, to a lesser extent, SCF mRNAs. Thus, MIS mRNA levels were significantly decreased after 180 min of incubation in the presence of 10–10, 10–8, and 10–6 M orexin-A; the effect of 10–8 and 10–6 M orexin-A (80% decrease vs. controls) was significantly higher than that of the 10–10 M dose (50% decrease). In the case of SCF gene, only the highest dose tested (10–6 M) evoked a significant approximately 55% decrease in mRNA levels vs. control values (Fig. 9). Of note, two SCF transcripts were detected upon RT-PCR using our assay conditions: the one encoding the soluble form (SCFs) and the other coding for the membrane-bound form of SCF (SCFm), in keeping with previous studies (27). Because both transcripts were equally decreased by 10–6 M orexin-A, the net combined reduction of SCF mRNA levels is presented in Fig. 9.
In addition, the ability of different doses of orexin-A to modify spermatogonial DNA synthesis was evaluated by means of analysis of [3H]thymidine incorporation in 24-h and 48-h cultures of staged seminiferous tubule preparations. Exposure to increasing doses of orexin-A (10–10, 10–8, 10–6 M) was conducted in tubule fragments corresponding to stages I, V, VIIa, VIII–IX, and XII of the epithelial cycle. In basal conditions, the profile of thymidine incorporation throughout the seminiferous epithelial cycle was roughly similar to that reported previously (32). Exposure to different doses of orexin-A for 24 h failed to significantly alter the rate of DNA synthesis of the seminiferous epithelium, at any of the stages and doses tested. In contrast, challenge of tubule preparations with orexin-A for 48 h induced a significant inhibition of thymidine incorporation at defined stage V of the epithelial cycle (Fig. 10). This inhibitory effect was detected, with a similar magnitude, for all doses tested.
Discussion
To our knowledge, this study provides the first comprehensive evaluation of the pattern of expression and hormonal regulation of orexin in rat testis. Fragmentary information had previously suggested that the testis is likely the major site for peripheral expression of prepro-orexin gene in the rat (21); a tissue in which radioimmunoassayable levels of orexin-A have been reported (22). However, not a single study had previously addressed the major characteristics of expression of orexin in the testis. In this study, we report evidence for a developmental, stage-specific, and hormonally regulated pattern of orexin expression in rat testis. Taken together with our recent data on the profile of testicular expression of the gene encoding OX1R, the putative receptor for orexin-A (23), it is tempting to consider that the orexin system represents a novel modulator of the function of mammalian male gonad, as indirectly suggested also by recent studies in human and sheep species (24, 25). To be noted, however, differences might exist on the actual expression of orexin in the testis among different species as, in contrast to the rat (21, 22, 30), recent studies failed to detected measurable prepro-orexin mRNA or orexin-A immunoreactivity in the human testis (24, 36).
Testicular expression of prepro-orexin gene and orexin-A peptide appeared strikingly regulated during development and throughout the seminiferous epithelial cycle. Moreover, a close correlation between mRNA and protein levels was apparent along postnatal maturation. Thus, expression levels of prepro-orexin gene were low (neonatal) to negligible (early juvenile) before initiation of puberty; a developmental time-frame when orexin-A immunoreactivity was only detected in mature fetal-type Leydig cells. The fact that this cell population represents only a minute amount of the whole cell mass of the neonatal/infantile testis likely justify the very modest mRNA levels of prepro-orexin at this period (2). In contrast, expression levels of prepro-orexin mRNA and orexin-A immunoreactivity dramatically increased from puberty onwards. Two major phenomena likely contribute to this profile: the appearance of primary spermatocytes at defined stages of meiosis, and the development of adult-type Leydig cells, whose number increase progressively along pubertal maturation. The fact that orexin-A is expressed in steroidogenically active mature Leydig cells (present results), and it is provided with specific modulatory (stimulatory) effects upon testicular T secretion in vitro, strongly suggests that orexin may represent a novel autocrine regulator of testicular steroidogenic function. Moreover, the fact that orexin receptors appear to be regulated by androgen in other tissues (such as the pituitary; see Ref.37) makes it plausible that a precise interplay between the orexin system and androgen might be operative at different levels of the hypothalamic-pituitary-gonadal axis. Nonetheless, the overall contribution of Leydig cells to the net testicular expression of prepro-orexin is yet to be determined. However, the fact that prepro-orexin mRNA was persistently detected in rat testis after selective Leydig cell elimination suggests that the tubular compartment is likely the major source of orexin expression in the rat testis.
Concerning the pattern of expression of orexin-A in the seminiferous epithelium, it is noticeable that orexin-A immunoreactivity was selectively detected in spermatocytes at defined stages of meiosis. In detail, specific orexin-A signals were observed in preleptotene, leptotene, zygotene, and pachytene (at stages XIV–IV, but not at stages VI–XII) spermatocytes, whereas other cell types of the seminiferous epithelium were negative. These data allow us to predict the tentative stage-specific pattern of expression of orexin-A peptide throughout the seminiferous epithelial cycle, with low values at stages V–VI and increased expression thereafter. The physiological meaning of such a staged pattern of expression awaits further investigation, although it is tempting to point out that stage V (when endogenous expression of orexin-A is apparently the lowest at the seminiferous epithelium) corresponds to the stage of the cycle when prominent inhibitory effects of exogenous orexin-A upon DNA spermatogonial DNA synthesis were evidenced in our in vitro system. Thus, the transient decrease in spermatocyte expression of orexin-A might prevent a decrease in DNA synthesis in spermatogonia B. This would be a novel paracrine feed-back loop between meiotic spermatocytes and premeiotic spermatogonia, likely operating through Sertoli cells. To be noted, expression of OX1R gene in the seminiferous tubules was also proven stage dependent (23). Overall, our data on the expression of both components (ligand and receptor) of orexin system in the seminiferous epithelium strongly suggests the putative role of orexin in the control of seminiferous tubule functions. This contention is further supported by our present data using in vitro systems.
Testicular expression of prepro-orexin gene in the adult testis appeared to be exquisitely regulated. First, the expression levels of this mRNA were significantly up-regulated immediately after withdrawal of mature Leydig cells, which suggests that tubular expression of prepro-orexin gene in the adult testis is tonically repressed by Leydig cell factor(s), whose nature is yet to be identified. A similar phenomenon has been previously reported for inhibin- gene (35). Among other Leydig cells products, the possibility that T might contribute to such a repression appears unlikely because hCG, superagonist of LH and potent elicitor of T release, evoked a modest increase (rather than decrease) in prepro-orexin mRNA levels. In fact, both gonadotropins were demonstrated as putative stimulators of testicular expression of prepro-orexin. However, FSH appeared to play a more prominent role as elicitor of prepro-orexin gene expression in the rat testis, as evidenced by replacement protocols in HPX rats and after acute administration. This contention is in good agreement with the fact that the seminiferous epithelium is likely the major source of orexin expression in adult rat testis and that FSH, acting through specific receptors located solely in Sertoli cells, is one of the major tropic factors for the tubular compartment of the testis (2). Thus, it is likely that the observed rise in prepro-orexin gene expression may represent a genuine increase in spermatocyte expression of this messenger after FSH stimulation. Nonetheless, LH/CG likely contributes also to the regulation of orexin expression in the rat testis, as LH receptors are located in Leydig cells, which express also orexin-A, and prepro-orexin mRNA levels were moderately increased after hCG stimulation.
In terms of hormonal control, another putative regulator of testicular expression of orexin is the newly cloned orexigenic peptide ghrelin. Notably, the hypothalamic orexin system appears to be modulated by ghrelin (28, 29). In addition, in recent years, we have presented compelling evidence for the expression and functional role of ghrelin in the mammalian testis (6, 7, 27, 38). Our observations indicate that an i.t. injection of ghrelin is able to evoke a delayed increase in testicular prepro-orexin mRNA levels. The contribution of such a phenomenon to the documented effects of ghrelin upon testicular functions (such as T secretion and Leydig cell proliferation; see Refs.6 and 27) remains to be elucidated. Nonetheless, considering that expression of both ghrelin and its functional receptor, the GH secretagogue receptor type 1a, has been reported in the testis, it is plausible that modulation of testicular expression of orexin might be conducted by systemic and/or locally produced ghrelin. From a general standpoint, it is intriguing to note that, although serving different functions, a similar cross-talk between ghrelin and orexin systems appears to be operative both in the brain and testis.
Finally, our current data provide novel evidence for a functional role of orexin-A in the control of key seminiferous tubule functions. In this sense, previous data from our group substantiated a potential role of orexin-A in the regulation of T secretion in rat testis (23), and solid evidence for expression of functional orexin receptors in the human testis was recently reported by Karteris and coworkers (24). In this context, using in vitro settings, orexin-A was proven to regulate the expression of relevant Sertoli cell genes, such as MIS and SCF, and to inhibit spermatogonial DNA synthesis in a stage-specific manner. These findings are in line with our previous data on the prominent expression of OX1R gene in the tubular compartment of the testis (23), and pave the way for further analyses of the functional relevance of such regulated phenomena in testicular physiology. Moreover, our present results on orexin-A effects on spermatogonial DNA synthesis warrant additional studies on its actions upon germ cell-specific gene expression. Concerning expression analyses of Sertoli cell genes, several targets, including inhibin- and inhibin-B subunits, as well as SCF and MIS, were selected on the basis of their proven key roles in testis development and/or function. Thus, in addition to their endocrine actions, inhibins and activins have been involved in the local control of testicular T secretion (39); inhibin B (heterodimer of inhibin- and -B subunits) being the major inhibin form in the male (40). On the other hand, SCF has been pointed out as the major paracrine stimulator of germ cell development, acting as survival factor for spermatogonia in the adult rat seminiferous epithelium (33, 41), whereas MIS, besides its key developmental actions, has been involved in a variety of regulatory functions in the adult testis including the control adult-type Leydig cell proliferation and the inhibition of T secretion (42, 43). Overall, it is tempting to hypothesize that the observed changes in gene expression after exposure to orexin-A might have mechanistic implications, as orexin-A-induced decrease in MIS gene expression may contribute to its reported stimulatory effect upon T secretion (23), whereas the ability of orexin-A to lower SCF mRNA might have a role in its inhibitory effect upon spermatogonial DNA synthesis.
To be noted, there is no reported evidence for major reproductive defects in orexin null models. This would argue against an essential role of orexin in the control of testicular function. However, it has to be stressed that despite the proven role or orexin (as appetite promoting factor) in the control of food intake (10), orexin knockout mice are devoid also of a major metabolic phenotype, as they show a rather modest trend to reduction in food intake and mild tendency toward obesity (44, 45). This paradoxical finding, which has been observed also for other well-known orexigenic factors, has been interpreted as evidence for partial redundancy in the networks positively controlling food intake. In addition, the possibility that potential compensatory mechanisms might be activated in face of persistent absence of orexin signaling throughout development cannot be excluded. Similar possibilities may apply also for testicular orexin. However, the lack of specific testicular studies in orexin null models makes such possibility merely speculative. Overall, although orexin appears to be dispensable for gross testicular development and function, the lack of testicular phenotype in orexin-deficient animals cannot rule out a potential role of locally produced orexin in the fine tuning of testis function, as suggested by our present data.
In summary, our present data document for the first time the pattern of orexin expression in rat testis that takes place in a cell-, developmental-, and stage-specific manner, under the control of an array of hormones and regulators, which include Leydig cell factor(s), pituitary gonadotropins and ghrelin. In addition, our results substantiate a novel role of orexin-A as putative regulator of key seminiferous tubule functions, such as Sertoli cell gene expression and spermatogonial DNA synthesis. Although some aspects of orexin system in the testis (such as the potential expression of orexin-B) are yet to be covered, the current study provides evidence for a novel testicular site of action of orexins in the control of male reproductive axis. In the context of our recent data on the testicular expression and/or direct biological actions of a number of molecules with key roles in energy homeostasis, such as leptin, resistin, and ghrelin (5, 6, 7, 8), the contribution of such phenomenon to the joint control of energy balance and reproduction merits further investigation.
Acknowledgments
We are indebted to V. M. Navarro, R. Fernandez-Fernandez, and J. Roa 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 Direccion General de Educacion Superior e Investigacion Científica (Ministerio de Ciencia y Tecnología, Spain), funds from Instituto de Salud Carlos III (Red de Centros RCMN C03/08 and Project PI042082; Ministerio de Sanidad, Spain), and the Academy of Finland and Turku University Central Hospital.
First Published Online September 1, 2005
1 M.L.B. and R.P. contributed equally to this study and should be conisdered joint first authors.
Abbreviations: EDS, Ethylene dimethane sulfonate; hCG, human chorionic gonadotropin; HPX, hypophysectomized; i.t., intratesticular; MIS, Müllerian inhibiting substance; RT, reverse transcriptase; SCF, stem cell factor; T, testosterone.
Accepted for publication August 18, 2005.
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Department of Histology and Pathology (M.A., M.A.B.), University of Navarra, 31080 Pamplona, Spain
Department of Physiology (C.D.), University of Santiago de Compostela, 15705 Santiago de Compostela, Spain
Departments of Physiology and Pediatrics (H.H., M.N., J.T.), University of Turku, 20520 Turku, Finland
Abstract
Orexins, hypothalamic neuropeptides initially involved in the control of food intake and sleep-wake cycle, have recently emerged as pleiotropic regulators of different biological systems, including the reproductive axis. Besides central actions, peripheral expression and functions of orexins have been reported, and prepro-orexin and orexin type-1 receptor mRNAs have been detected in the testis. However, the pattern of expression and biological actions of orexin in the male gonad remain mostly unexplored. In this study, we report analyses on testicular prepro-orexin mRNA expression and orexin-A immunoreactivity in different experimental settings, and on direct effects of orexin-A on seminiferous tubule functions. Expression of prepro-orexin mRNA was demonstrated in the rat testis at different stages of postnatal development, with negligible levels at early juvenile period and maximum values in adulthood. Likewise, orexin-A immunoreactivity was demonstrated along postnatal maturation, with strong peptide signal in Leydig cells and spermatocytes at specific stages of meiosis. Testicular expression of prepro-orexin mRNA appeared hormonally regulated; its levels decreased after hypophysectomy and increased after gonadotropin replacement and ghrelin stimulation. Finally, orexin-A suppressed the expression of key Sertoli cell genes, such as Müllerian-inhibiting substance and stem cell factor, and inhibited DNA synthesis in specific stages of the seminiferous epithelium. In conclusion, we provide evidence for the regulated expression of orexin in the rat testis and its potential involvement in the control of seminiferous tubule functions. Together with our recent results on the expression of orexin type-1 receptor in the rat testis, our data further document a novel testicular site of action of orexins in the control of male reproductive axis.
Introduction
THE MAMMALIAN TESTIS is a multifaceted endocrine organ responsible for production of spermatozoa as well as secretion of steroid and peptide hormones (1). These biological functions are based on a complex cellular network composed of different cell types, which undergo dramatic changes throughout development and are arranged in two major tissue compartments, the interstitium and the seminiferous tubules (1). The pituitary gonadotropins, LH and FSH, are the pivotal endocrine regulators of testicular functions; LH is the major elicitor of androgen secretion, and spermatogenesis is tightly controlled by FSH and testosterone (T) (2). In addition, during the last decades, we have become aware of an ever-growing number of signals, of peripheral or local origin, that are also involved in the endocrine and auto-/paracrine control of testicular functions (3, 4). In this context, compelling evidence has been recently presented for the testicular expression and/or direct biological actions of a number of molecules with key roles in energy homeostasis, such as the adipocyte-derived hormones leptin and resistin, and the gut-secreted peptide ghrelin (5, 6, 7, 8). Conceivably, this phenomenon might contribute to the mechanisms by which energy balance and reproductive function are jointly regulated, and clearly illustrates the functional diversity of an array of newly cloned molecules whose biological functions largely exceed those linked to food intake control and metabolism.
Orexins, also termed hypocretins, are hypothalamic neuropeptides, primarily expressed in the lateral hypothalamic area, with a key role in the control of feeding behavior (9, 10). Two different orexin molecules have been identified so far, orexin-A and orexin-B, which derive from the proteolysis of a common 130-amino-acid precursor named prepro-OX. 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), with partially different binding properties: OX1R is highly selective for orexin-A, whereas OX2R is a nonselective receptor that binds both orexin-A and orexin-B (10). Besides their proven role in food intake, orexins have also been involved in the central control of a wide array of biological functions such as regulation of the sleep-wake cycle and arousal (11), stress reactions (12), arterial blood pressure and heart rate (13), as well as several neuroendocrine systems, including the gonadotropic axis (14, 15, 16). On the latter, hypothalamic GnRH-neurons have been shown to express OX1R and to receive direct contacts from orexin fibers (17, 18), and orexin-A was proven to stimulate GnRH release from rat hypothalamic explants (16). Interestingly, the net effects of orexins on LH secretion seem to be dependent on the prevailing sex steroid background, because central injection of orexin-A elicited LH release in steroid-primed ovariectomized rats, but it decreased LH secretion in the absence of ovarian steroids (14, 19, 20). Potential actions of orexins at other sites of the hypothalamo-pituitary-gonadal axis remain largely unexplored, although orexin-A has been reported to inhibit GnRH-induced LH release by dispersed pituitary cells (16).
In the context of the reproductive actions of orexins, it is noticeable that significant amounts of prepro-orexin mRNA, as well as of radioimmunoassayable orexin-A, have been detected in the rat testis; a tissue that, after the brain, holds the highest orexin expression levels reported so far (21, 22). In addition, we have recently provided evidence for the developmental and hormonally regulated expression of OX1R gene in rat testis, and for direct actions of orexin-A on testicular T secretion (23). Moreover, expression of OX1R has been also demonstrated in human and sheep testis (24, 25). Taken together, the available data strongly suggest that the male gonad is a potential site for expression and/or biological actions of orexins. Yet, the pattern of orexin expression in the testis and its eventual involvement in the direct control of testicular functions other than T secretion remain so far unexplored. To cover these issues, we undertook the detailed characterization of testicular expression of prepro-orexin mRNA, as well as orexin-A immunoreactivity, in different developmental stages and experimental settings. In addition, based on our previous findings on the prominent expression of OX1R gene at different stages of the seminiferous epithelial cycle, we assayed the effects of orexin-A upon the expression levels of relevant Sertoli cell genes and the rate of DNA synthesis in staged seminiferous tubule preparations.
Materials and Methods
Animals and drugs
Wistar male rats bred in the vivarium of the University of Cordoba were used, unless otherwise stated. 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 h) 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 Cordoba University Ethical Committee for animal experimentation and were conducted in accordance with the European Union normative for care and use of experimental animals. The animals were killed by decapitation at the end of all experimental procedures. In experiments involving mRNA analysis, testes were immediately removed upon decapitation, frozen in liquid nitrogen, and stored at –80 C until processing. Rat orexin-A and ghrelin (octanoylated at Ser 3) were 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). Ethylene dimethane sulfonate (EDS) was synthesized in our laboratory as described in detail elsewhere (26), and dissolved in dimethylsulfoxide-water (1:3, vol/vol).
Experimental designs
In the first set of experiments, expression of prepro-orexin gene was explored in rat testis at different stages of postnatal development and after selective elimination of mature Leydig cells. To this end, in experiment 1, testicular samples were obtained from 5 (n = 10), 15 (n = 8), 30 (n = 5), 45 (n = 5), and 75-d-old rats (n = 5 per group), corresponding to the neonatal-infantile (5 d), juvenile (15 d), pubertal (30 d), early adult (45 d), and adult (75 d) stages of postnatal maturation. In addition, in experiment 2, testicular expression of prepro-orexin mRNA was studied at different time-points after systemic administration of a single dose of the cytotoxic drug EDS (75 mg/kg, ip). This in vivo model (where mature Leydig cell are selectively and rapidly eliminated from testis interstitium) provides an optimal experimental background in which to test Leydig cell-specific expression and/or regulation of testicular factors (for example, see Ref.6). Thus, testicular samples were obtained from adult 75-d-old rats (n = 5 per group), before (0) and 3, 5, 15, 20, and 40 d after EDS administration. Selective elimination of mature Leydig cells in EDS-treated rats was confirmed by determination of T levels in trunk blood samples and histological examination of testicular tissue, as described in detail elsewhere (23). Finally, in addition to RNA analyses, the pattern of expression and cellular location of orexin-A peptide in rat testis was evaluated throughout postnatal development in experiment 3. To this end, samples of testicular tissue were obtained from 5-, 15-, 20-, 30-, 45-, and 75-d-old rats (n = 5 per group) and subjected to immunohistochemical analysis for detection of orexin-A peptide using a polyclonal antibody (see below).
In the second series of experiments, the pattern of hormonal regulation of prepro-orexin mRNA expression in rat testis was explored. Thus, in experiment 4, the role of pituitary gonadotropins was monitored in vivo by analysis of prepro-orexin mRNA levels 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 (as superagonist of LH; 10 IU/rat per 24 h) or recombinant FSH (7.5 IU/rat per 24 h) for 7 d before sampling. In addition, the ability of gonadotropins to acutely regulate testicular prepro-orexin mRNA levels was assessed in experiment 5. To this end, relative prepro-orexin mRNA was assayed in testes from adult (75 d old) intact rats injected at 1000 h with hCG (25 IU/rat) or recombinant FSH (12.5 IU/rat) and sampled at 2, 4, 8, and 24 h after injection. Vehicle-treated rats served as controls. Finally, in experiment 6, the effects of ghrelin upon testicular expression of prepro-orexin gene were studied. In this sense, ghrelin has been reported as putative regulator of several testicular functions (6, 7, 27), and hypothalamic orexin neurons appear to be modulated by ghrelin (28, 29). Single intratesticular (i.t.) injection of ghrelin (15 μg in 50 μl) was applied to intact adult rats. Of note, each animal received unilateral i.t. injections of ghrelin (right testis), and the contralateral (left) testis was injected with 50 μl of vehicle for control. Groups of animals (n = 5) were sequentially killed at 3, 6, 12, and 24 h after injection, and testis samples were obtained for RNA analysis as indicated above.
Finally, in the third set of experiments, direct biological actions of orexin-A upon rat testis were explored. Based on our previous findings on the prominent expression of OX1R gene at different stages of the seminiferous epithelial cycle (23), we focused our analyses on the potential effects of orexin-A upon the expression levels of relevant Sertoli cell genes and the rate of DNA synthesis in the seminiferous epithelium. Thus, in experiment 7, a panel of Sertoli cell genes were assayed after exposure to orexin-A using static incubations of adult rat tissue, as described in detail elsewhere (23). As experimental setting, slices of testicular tissue were incubated for 180 min in the presence of increasing doses (10–10, 10–8, 10–6 M) of rat orexin-A (n = 8–10 samples per group), as described in detail elsewhere (23), and processed for RNA analysis at the end of the incubation period. In addition, in experiment 8, direct actions of orexin-A upon the rate of DNA synthesis of the seminiferous epithelium was explored in vitro, using static incubations of staged fragments of seminiferous tubules and [3H]thymidine labeling, as described in detail below.
RNA analysis by semiquantitative RT-PCR
Poly(A) RNA was isolated from testicular samples using the PolyATrack mRNA Isolation System, following the instructions of the manufacturer (Promega, Madison, WI). Testicular expression of prepro-orexin mRNA was assessed by RT-PCR, optimized for semiquantitative detection, using the following specific primer pairs: OX-forward (5'-CGGATTGCCTCTCCCTGAGC-3') and OX-reverse (5'-CTAAAGCGGTGGCGGTTGC-3'), flanking 397 bp of the coding area of rat prepro-orexin cDNA (GenBank accession no. NM 013179). These sets of primers were selected on the basis of previous references (30). In addition, in selected experimental settings (see experiment 2), the expression levels of LH receptor mRNA were assessed by semiquantitative RT-PCR, using the following primer pairs: LH receptor-forward (5'-ACTGCTGCGCCTTCAGGAAT-3') and LH receptor-reverse (5'-ATAGCCCATAATATCTTCACA-3'), flanking 245 bp of the coding area of rat LH receptor cDNA (GenBank accession no. NM 012978). Likewise, the expression levels of several Sertoli cell genes, such as inhibin- subunit, inhibin-B subunit, stem cell factor (SCF), and Müllerian inhibiting substance (MIS), were assayed in experiment 7 using specific primer pairs, described in detail elsewhere (27, 31). As internal control for reverse transcriptase (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 (6, 23).
For amplification of the targets, 0.5 μg of poly(A) 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 the specific signals and L19 ribosomal protein transcript was carried out in separate reactions with a different number of cycles, but using similar amounts of the corresponding cDNA templates, generated in single RT reactions, as previously described (6, 23). PCR 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 min. A final extension cycle of 72 C for 15 min was included. Annealing temperature was adjusted for each target: 62 C for prepro-orexin, 60 C for SCF, 58 C for MIS, 57 C for LH receptor, and 56 C for inhibin subunit and RP-L19 transcripts. For the different targets, different numbers of cycles were tested to optimize amplification in the exponential phase of PCR. This procedure was conducted in detail for prepro-orexin transcript, using RNA from adult rat testis. Analysis of intensity of PCR signals as function of the number of amplification cycles revealed a strong linear relationship between cycles 28 and 40, with a correlation coefficient r2 = 0.982 (Fig. 1). Moreover, a strong exponential (log-linear) relationship between signal intensity and cycle number was observed for less than 35 cycles. Thus, considering the diversity of experimental samples to be tested (with different relative amounts of the target), 34 PCR cycles were selected for semiquantitative analyses of prepro-orexin mRNA levels. In addition, previous optimization assays conducted at our laboratory demonstrated amplification in the linear range between 28 and 38 cycles in the case of SCF (r2 0.97), cycles 26–36 in the case of MIS and inhibin subunits (r2 0.97), and cycles 19–29 in the case of RP-L19 (r2 = 0.992) (see Ref.27). On this basis, 35 (LH receptor), 34 (SCF), 32 (inhibin- and -B subunits), 30 (MIS), and 23 (RP-L19) PCR cycles were chosen for further analyses of these targets.
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). 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.
Orexin-A immunohistochemistry
Detection of orexin-A protein was carried out in rat testicular sections at different stages of postnatal development by means of immunohistochemistry using a polyclonal antiserum specific for orexin-A (OXA11A; Alpha Diagnostic, San Antonio, TX). Testis specimens from 5-, 15-, 20-, 30-, 45-, and 75-d-old rats were obtained, fixed for at least 24 h in Bouin fluid, and routinely processed for paraffin embedding. For immunohistochemical analyses, serial 5-μm-thick sections were generated and mounted on Vectabond-coated slides (Vector Laboratories, Burlingame, CA). The sections were dewaxed in xylene, rehydrated in ethanol, and incubated in 3% hydrogen peroxide to quench endogenous peroxidase activity. In addition, the sections were immersed in 10 mM citrate buffer and submitted to antigen retrieval in a microwave oven for 15 min at 1150 W followed by 15 min at 900 W. Immunohistochemical staining for orexin-A was performed using EnVision+System-HRP, consisting of a secondary antibody (goat antirabbit Ig) coupled to a peroxidase-labeled dextran polymer (Dako, Carpinteria, CA) as described in detail elsewhere (32). To prevent nonspecific binding, the sections were treated with goat normal serum, 1:20 in Tris-buffered saline, prior overnight incubation at 4 C in the presence of the primary antiserum (at 1:200 dilution). Thereafter, the slides were incubated for 30 min in EnVision-HRP, and the peroxidase activity was revealed by incubation in the presence of 0.03% 3,3'-diaminobenzidine (DAB). Immunohistochemical controls included omission of the primary anti-orexin antibody (negative control) and the use of sections of rat intestine as positive control. As additional control for the specificity of the primary antibody, immunohistochemical reactions were carried out in slices of rat testicular tissue after preabsorption of the antiserum overnight at 4 C with 10 nmol/ml of the synthetic antigen (OXA11P; Alpha Diagnostic). In keeping with previous results in mouse gut (32), this procedure completely abolished orexin immunolabeling in testis sections (Fig. 1).
Microdissection of seminiferous tubule fragments and assessment of DNA synthesis
Microdissection of seminiferous tubule segments of testes from Sprague Dawley rats was carried out as described in detail elsewhere (33). Briefly, testes from 75-d-old rats were decapsulated, and 2 mm seminiferous tubule segments were isolated under a trans-illuminating stereomicroscope; specific stages of the seminiferous epithelial cycle were identified as described previously (34). Stages I, V, VIIa, VIII–XI, and XII of the cycle were selected, as they contain cells at the representative phases of mitotic and meiotic DNA synthesis (33, 34). Microdissection of tubule segments was performed in DMEM-Ham’s F-12 medium (1:1; Life Technologies, Paisley, UK) supplemented with 1.25 g/liter sodium bicarbonate, 10 mg/liter gentamicin sulfate, and 1 g/liter BSA. For in vitro analysis of orexin-A effects upon DNA synthesis, staged seminiferous tubule preparations were challenged for 24 and 48 h with increasing concentrations of orexin-A (10–10–10–6 M). Tubule fragments were pulse labeled during the last 4 h of culture by addition of 20 kBq [methyl-3H]thymidine (Amersham, Aylesbury, UK), as described in detail elsewhere (33). The cultures were harvested on filter disks (Whatman, Clifton, NJ) with a continuous flow of distilled water for 1 min. A scintillation wax (MeltiLex A 1450-441; Wallac, Turku, Finland) was melted on the filters, and the radioactivity was measured in a liquid scintillation counter (Wallac). Four separate experiments were performed, each with three replicate samples.
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 5-d-old rat testes, which were pooled (n = 2–4) before RNA isolation. Data are presented as mean ± SEM. Results were analyzed for statistically significant differences using ANOVA, followed by Student-Newman-Keuls multiple range test. P 0.05 was considered significant.
Results
Prepro-orexin mRNA in rat testis along postnatal development and after Leydig cell elimination
Expression of the gene encoding prepro-orexin was first evaluated in the rat testis at different stages of postnatal maturation by means of semiquantitative RT-PCR assays. In detail, representative stages of development, corresponding to neonatal (5 d old), juvenile (15 d old), pubertal (30 d old), early adult (45 d old), and adult (75 d old) periods, were explored. Although persistent expression of prepro-orexin mRNA was observed at the different phases of postnatal maturation, our analysis evidenced striking differences in the relative levels of this messenger. Thus, low expression of prepro-orexin mRNA was detected in the neonatal testis, and virtually negligible levels were observed at the early juvenile phase. In contrast, prepro-orexin mRNA levels progressively increased from puberty onwards, and reached maximum values at adulthood (Fig. 2).
In addition, semiquantitative RT-PCR assays of prepro-orexin mRNA were conducted in testis samples at different time-points after selective Leydig cell elimination in vivo by administration of the cytotoxic compound EDS. Complete Leydig cell elimination was confirmed by histological examination of testicular sections and measurement of serum T concentrations in EDS-treated groups, which dropped to nearly undetectable values at d 3 and 5 after EDS (data not shown). In this setting, persistent expression of prepro-orexin mRNA was demonstrated throughout a 40-d period after EDS administration, with a significant increase in prepro-orexin mRNA levels observed at d 3 and 5 post-EDS, which was followed by a decrease in its relative levels to control values thereafter, except for a transient, minor elevation at d 20 (Fig. 3). In contrast, LH receptor mRNA become undetectable in rat testis at d 3, 5, and 15 after EDS, and gradually reappeared from this time-point onwards, co- inciding with the process of Leydig cell repopulation (34). To be noted, within the testis, LH receptors are selectively expressed in Leydig cells at advanced stages of maturation (2). Thus, expression of this messenger was used as molecular marker for the lack of mature Leydig cells at early periods after EDS administration.
Pattern of cellular expression of orexin-A in rat testis along postnatal development
The pattern of orexin-A immunoreactivity was evaluated in testis tissue sections from representative stages of postnatal development, including neonatal (5 d old), juvenile (15 and 20 d old), pubertal (30 d old), early adult (45 d old), and adult (75 d old) periods. Our analyses evidenced the presence of specific orexin-A immunostaining in both the interstitial compartment and the seminiferous tubules of the rat testis. Concerning testis interstitium, orexin-A immunoreactivity was selectively detected in Leydig cells of the fetal- and adult-type origin, mostly at advanced stages of differentiation (Fig. 4). In detail, strong orexin-A signal was observed in fetal-type Leydig cells in testes from 5-d-old rats (Fig. 4A). Likewise, intense orexin-A immunostaining was detected in adult-type Leydig cells in testis samples from puberty onwards, with an apparent increase in the intensity of the signal in adulthood (Fig. 4, D–G). In contrast, negligible orexin-A immunoreactivity was observed in undifferentiated Leydig cell progenitors, present in the interstitial space of testes from juvenile (15 and 20 d old) rats (Fig. 4, B and C). Of note, in all positive cells, orexin-A immunoreactivity showed specific cytoplasmic location. Other interstitial cell types, such as macrophages, were negative for orexin-A at all age-points studied.
Orexin-A immunoreactivity was also detected in the seminiferous epithelium, with prominent signals in spermatocytes at defined stages of meiosis. Indeed, ontogenetic analyses revealed that orexin-A immunostaining was virtually negligible in the tubular compartment before postnatal d 20, whereas its intensity apparently increased thereafter (Fig. 4). Detailed immunohistochemical analyses in adult testis sections demonstrated a stage-specific pattern of orexin-A peptide expression, with specific signals being detected in preleptotene (stages VII–VIII), leptotene (stages IX–XI), zygotene (stages XII–XIII), and pachytene (stages XIV–IV) spermatocytes (Fig. 5). To be noted, weak to negligible orexin-A immunoreactivity was observed in pachytene spermatocytes at tubules between stages IV and XII. Likewise, other cell types within the seminiferous epithelium were negative for orexin-A immunoreactivity. In all positive tubular cells, orexin-A immunoreactivity showed specific cytoplasmic location. A schematic presentation of the pattern of orexin-A immunoreactivity within the seminiferous epithelium, at specific cell types and stages of the cycle, is depicted in Fig. 5.
Hormonal regulation of testicular expression of prepro-orexin mRNA
Hormonal regulation of testicular expression of prepro-orexin mRNA by pituitary gonadotropins was first evaluated using the HPX rat. Gonadotropins were explored as putative modulators of prepro-orexin gene expression given their essential role in testicular development and function (2). Long-term (4-wk) HPX resulted in a clear-cut decrease of serum T levels and the regression of all testicular compartments, with atrophic Leydig cells in the interstitial space and regressing seminiferous epithelium (data not shown). Semiquantitative RT-PCR analyses demonstrated that 4-wk HPX induced a significant decrease in prepro-orexin mRNA expression levels; a decrease that was partially counteracted by administration of hCG (as superagonist of LH; 10 IU/rat/24 h) and fully prevented by replacement with recombinant FSH (7.5 IU/rat/24 h) (Fig. 6).
In addition, the effects of acute administration of hCG and FSH upon testicular prepro-orexin gene expression were evaluated in intact rats. Injection of a single bolus of hCG (25 IU/rat) or recombinant FSH (12.5 IU/rat) evoked a significant increase in testicular prepro-orexin mRNA levels, with peak values at 2 h after administration. However, the profiles of response to each gonadotropin were partially different, as prepro-orexin mRNA levels returned to control values 8 h after hCG injection, whereas they remained significantly elevated at all time-points studied (2, 4, 8, and 24 h) after FSH administration (Fig. 7).
Finally, the ability of ghrelin to modulate testicular expression of prepro-orexin mRNA was also evaluated. Assessment of the potential effects of ghrelin was based on our previous studies on the role of ghrelin as putative regulator of several testicular functions (6, 7, 27), and the fact that hypothalamic orexin system appears to be modulated by ghrelin (28, 29). A single i.t. injection of ghrelin was applied to intact adult rats, where each animal received a unilateral injection of ghrelin (right testis); the contralateral (left) testis (injected with vehicle) serving as control. Prepro-orexin mRNA levels were assayed at 3, 6, 12, and 24 h after ghrelin injection. Expression levels of prepro-orexin mRNA remained unaltered in control (vehicle-injected) testes throughout the study period, thus they were pooled for further analyses. In this model, i.t. injection of ghrelin (15 μg/testis) failed to alter gene expression at 3 and 6 h, but evoked a significant increase in relative prepro-orexin mRNA levels at 12 and 24 h after ghrelin administration (Fig. 8).
Effects of orexin-A on seminiferous tubule functions in vitro
In addition to present data on the pattern of expression of orexin in rat testis (see Results), previous studies of our group evidenced a prominent expression of OX1R gene in the tubular compartment of rat testis (23). Accordingly, the ability of orexin-A, the cognate ligand of OX1R, to modulate key seminiferous tubule functions was explored using in vitro settings. In a first step, the effects of increasing concentrations of orexin-A (10–10, 10–8, 10–6 M) upon the relative mRNA levels of a panel of Sertoli cell genes were explored using static incubations of testicular tissue. In detail, inhibin- and inhibin-B subunit, SCF, and MIS mRNAs were assayed as Sertoli cell-specific signals within the tubular compartment of the adult rat testis (27, 33, 35). In our in vitro setting, exposure to orexin-A failed to modify the relative expression levels of inhibin- and inhibin-B mRNAs, at any of the doses tested. In contrast, challenge with orexin-A significantly inhibited, in a dose-dependent manner, relative expression levels of MIS and, to a lesser extent, SCF mRNAs. Thus, MIS mRNA levels were significantly decreased after 180 min of incubation in the presence of 10–10, 10–8, and 10–6 M orexin-A; the effect of 10–8 and 10–6 M orexin-A (80% decrease vs. controls) was significantly higher than that of the 10–10 M dose (50% decrease). In the case of SCF gene, only the highest dose tested (10–6 M) evoked a significant approximately 55% decrease in mRNA levels vs. control values (Fig. 9). Of note, two SCF transcripts were detected upon RT-PCR using our assay conditions: the one encoding the soluble form (SCFs) and the other coding for the membrane-bound form of SCF (SCFm), in keeping with previous studies (27). Because both transcripts were equally decreased by 10–6 M orexin-A, the net combined reduction of SCF mRNA levels is presented in Fig. 9.
In addition, the ability of different doses of orexin-A to modify spermatogonial DNA synthesis was evaluated by means of analysis of [3H]thymidine incorporation in 24-h and 48-h cultures of staged seminiferous tubule preparations. Exposure to increasing doses of orexin-A (10–10, 10–8, 10–6 M) was conducted in tubule fragments corresponding to stages I, V, VIIa, VIII–IX, and XII of the epithelial cycle. In basal conditions, the profile of thymidine incorporation throughout the seminiferous epithelial cycle was roughly similar to that reported previously (32). Exposure to different doses of orexin-A for 24 h failed to significantly alter the rate of DNA synthesis of the seminiferous epithelium, at any of the stages and doses tested. In contrast, challenge of tubule preparations with orexin-A for 48 h induced a significant inhibition of thymidine incorporation at defined stage V of the epithelial cycle (Fig. 10). This inhibitory effect was detected, with a similar magnitude, for all doses tested.
Discussion
To our knowledge, this study provides the first comprehensive evaluation of the pattern of expression and hormonal regulation of orexin in rat testis. Fragmentary information had previously suggested that the testis is likely the major site for peripheral expression of prepro-orexin gene in the rat (21); a tissue in which radioimmunoassayable levels of orexin-A have been reported (22). However, not a single study had previously addressed the major characteristics of expression of orexin in the testis. In this study, we report evidence for a developmental, stage-specific, and hormonally regulated pattern of orexin expression in rat testis. Taken together with our recent data on the profile of testicular expression of the gene encoding OX1R, the putative receptor for orexin-A (23), it is tempting to consider that the orexin system represents a novel modulator of the function of mammalian male gonad, as indirectly suggested also by recent studies in human and sheep species (24, 25). To be noted, however, differences might exist on the actual expression of orexin in the testis among different species as, in contrast to the rat (21, 22, 30), recent studies failed to detected measurable prepro-orexin mRNA or orexin-A immunoreactivity in the human testis (24, 36).
Testicular expression of prepro-orexin gene and orexin-A peptide appeared strikingly regulated during development and throughout the seminiferous epithelial cycle. Moreover, a close correlation between mRNA and protein levels was apparent along postnatal maturation. Thus, expression levels of prepro-orexin gene were low (neonatal) to negligible (early juvenile) before initiation of puberty; a developmental time-frame when orexin-A immunoreactivity was only detected in mature fetal-type Leydig cells. The fact that this cell population represents only a minute amount of the whole cell mass of the neonatal/infantile testis likely justify the very modest mRNA levels of prepro-orexin at this period (2). In contrast, expression levels of prepro-orexin mRNA and orexin-A immunoreactivity dramatically increased from puberty onwards. Two major phenomena likely contribute to this profile: the appearance of primary spermatocytes at defined stages of meiosis, and the development of adult-type Leydig cells, whose number increase progressively along pubertal maturation. The fact that orexin-A is expressed in steroidogenically active mature Leydig cells (present results), and it is provided with specific modulatory (stimulatory) effects upon testicular T secretion in vitro, strongly suggests that orexin may represent a novel autocrine regulator of testicular steroidogenic function. Moreover, the fact that orexin receptors appear to be regulated by androgen in other tissues (such as the pituitary; see Ref.37) makes it plausible that a precise interplay between the orexin system and androgen might be operative at different levels of the hypothalamic-pituitary-gonadal axis. Nonetheless, the overall contribution of Leydig cells to the net testicular expression of prepro-orexin is yet to be determined. However, the fact that prepro-orexin mRNA was persistently detected in rat testis after selective Leydig cell elimination suggests that the tubular compartment is likely the major source of orexin expression in the rat testis.
Concerning the pattern of expression of orexin-A in the seminiferous epithelium, it is noticeable that orexin-A immunoreactivity was selectively detected in spermatocytes at defined stages of meiosis. In detail, specific orexin-A signals were observed in preleptotene, leptotene, zygotene, and pachytene (at stages XIV–IV, but not at stages VI–XII) spermatocytes, whereas other cell types of the seminiferous epithelium were negative. These data allow us to predict the tentative stage-specific pattern of expression of orexin-A peptide throughout the seminiferous epithelial cycle, with low values at stages V–VI and increased expression thereafter. The physiological meaning of such a staged pattern of expression awaits further investigation, although it is tempting to point out that stage V (when endogenous expression of orexin-A is apparently the lowest at the seminiferous epithelium) corresponds to the stage of the cycle when prominent inhibitory effects of exogenous orexin-A upon DNA spermatogonial DNA synthesis were evidenced in our in vitro system. Thus, the transient decrease in spermatocyte expression of orexin-A might prevent a decrease in DNA synthesis in spermatogonia B. This would be a novel paracrine feed-back loop between meiotic spermatocytes and premeiotic spermatogonia, likely operating through Sertoli cells. To be noted, expression of OX1R gene in the seminiferous tubules was also proven stage dependent (23). Overall, our data on the expression of both components (ligand and receptor) of orexin system in the seminiferous epithelium strongly suggests the putative role of orexin in the control of seminiferous tubule functions. This contention is further supported by our present data using in vitro systems.
Testicular expression of prepro-orexin gene in the adult testis appeared to be exquisitely regulated. First, the expression levels of this mRNA were significantly up-regulated immediately after withdrawal of mature Leydig cells, which suggests that tubular expression of prepro-orexin gene in the adult testis is tonically repressed by Leydig cell factor(s), whose nature is yet to be identified. A similar phenomenon has been previously reported for inhibin- gene (35). Among other Leydig cells products, the possibility that T might contribute to such a repression appears unlikely because hCG, superagonist of LH and potent elicitor of T release, evoked a modest increase (rather than decrease) in prepro-orexin mRNA levels. In fact, both gonadotropins were demonstrated as putative stimulators of testicular expression of prepro-orexin. However, FSH appeared to play a more prominent role as elicitor of prepro-orexin gene expression in the rat testis, as evidenced by replacement protocols in HPX rats and after acute administration. This contention is in good agreement with the fact that the seminiferous epithelium is likely the major source of orexin expression in adult rat testis and that FSH, acting through specific receptors located solely in Sertoli cells, is one of the major tropic factors for the tubular compartment of the testis (2). Thus, it is likely that the observed rise in prepro-orexin gene expression may represent a genuine increase in spermatocyte expression of this messenger after FSH stimulation. Nonetheless, LH/CG likely contributes also to the regulation of orexin expression in the rat testis, as LH receptors are located in Leydig cells, which express also orexin-A, and prepro-orexin mRNA levels were moderately increased after hCG stimulation.
In terms of hormonal control, another putative regulator of testicular expression of orexin is the newly cloned orexigenic peptide ghrelin. Notably, the hypothalamic orexin system appears to be modulated by ghrelin (28, 29). In addition, in recent years, we have presented compelling evidence for the expression and functional role of ghrelin in the mammalian testis (6, 7, 27, 38). Our observations indicate that an i.t. injection of ghrelin is able to evoke a delayed increase in testicular prepro-orexin mRNA levels. The contribution of such a phenomenon to the documented effects of ghrelin upon testicular functions (such as T secretion and Leydig cell proliferation; see Refs.6 and 27) remains to be elucidated. Nonetheless, considering that expression of both ghrelin and its functional receptor, the GH secretagogue receptor type 1a, has been reported in the testis, it is plausible that modulation of testicular expression of orexin might be conducted by systemic and/or locally produced ghrelin. From a general standpoint, it is intriguing to note that, although serving different functions, a similar cross-talk between ghrelin and orexin systems appears to be operative both in the brain and testis.
Finally, our current data provide novel evidence for a functional role of orexin-A in the control of key seminiferous tubule functions. In this sense, previous data from our group substantiated a potential role of orexin-A in the regulation of T secretion in rat testis (23), and solid evidence for expression of functional orexin receptors in the human testis was recently reported by Karteris and coworkers (24). In this context, using in vitro settings, orexin-A was proven to regulate the expression of relevant Sertoli cell genes, such as MIS and SCF, and to inhibit spermatogonial DNA synthesis in a stage-specific manner. These findings are in line with our previous data on the prominent expression of OX1R gene in the tubular compartment of the testis (23), and pave the way for further analyses of the functional relevance of such regulated phenomena in testicular physiology. Moreover, our present results on orexin-A effects on spermatogonial DNA synthesis warrant additional studies on its actions upon germ cell-specific gene expression. Concerning expression analyses of Sertoli cell genes, several targets, including inhibin- and inhibin-B subunits, as well as SCF and MIS, were selected on the basis of their proven key roles in testis development and/or function. Thus, in addition to their endocrine actions, inhibins and activins have been involved in the local control of testicular T secretion (39); inhibin B (heterodimer of inhibin- and -B subunits) being the major inhibin form in the male (40). On the other hand, SCF has been pointed out as the major paracrine stimulator of germ cell development, acting as survival factor for spermatogonia in the adult rat seminiferous epithelium (33, 41), whereas MIS, besides its key developmental actions, has been involved in a variety of regulatory functions in the adult testis including the control adult-type Leydig cell proliferation and the inhibition of T secretion (42, 43). Overall, it is tempting to hypothesize that the observed changes in gene expression after exposure to orexin-A might have mechanistic implications, as orexin-A-induced decrease in MIS gene expression may contribute to its reported stimulatory effect upon T secretion (23), whereas the ability of orexin-A to lower SCF mRNA might have a role in its inhibitory effect upon spermatogonial DNA synthesis.
To be noted, there is no reported evidence for major reproductive defects in orexin null models. This would argue against an essential role of orexin in the control of testicular function. However, it has to be stressed that despite the proven role or orexin (as appetite promoting factor) in the control of food intake (10), orexin knockout mice are devoid also of a major metabolic phenotype, as they show a rather modest trend to reduction in food intake and mild tendency toward obesity (44, 45). This paradoxical finding, which has been observed also for other well-known orexigenic factors, has been interpreted as evidence for partial redundancy in the networks positively controlling food intake. In addition, the possibility that potential compensatory mechanisms might be activated in face of persistent absence of orexin signaling throughout development cannot be excluded. Similar possibilities may apply also for testicular orexin. However, the lack of specific testicular studies in orexin null models makes such possibility merely speculative. Overall, although orexin appears to be dispensable for gross testicular development and function, the lack of testicular phenotype in orexin-deficient animals cannot rule out a potential role of locally produced orexin in the fine tuning of testis function, as suggested by our present data.
In summary, our present data document for the first time the pattern of orexin expression in rat testis that takes place in a cell-, developmental-, and stage-specific manner, under the control of an array of hormones and regulators, which include Leydig cell factor(s), pituitary gonadotropins and ghrelin. In addition, our results substantiate a novel role of orexin-A as putative regulator of key seminiferous tubule functions, such as Sertoli cell gene expression and spermatogonial DNA synthesis. Although some aspects of orexin system in the testis (such as the potential expression of orexin-B) are yet to be covered, the current study provides evidence for a novel testicular site of action of orexins in the control of male reproductive axis. In the context of our recent data on the testicular expression and/or direct biological actions of a number of molecules with key roles in energy homeostasis, such as leptin, resistin, and ghrelin (5, 6, 7, 8), the contribution of such phenomenon to the joint control of energy balance and reproduction merits further investigation.
Acknowledgments
We are indebted to V. M. Navarro, R. Fernandez-Fernandez, and J. Roa 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 Direccion General de Educacion Superior e Investigacion Científica (Ministerio de Ciencia y Tecnología, Spain), funds from Instituto de Salud Carlos III (Red de Centros RCMN C03/08 and Project PI042082; Ministerio de Sanidad, Spain), and the Academy of Finland and Turku University Central Hospital.
First Published Online September 1, 2005
1 M.L.B. and R.P. contributed equally to this study and should be conisdered joint first authors.
Abbreviations: EDS, Ethylene dimethane sulfonate; hCG, human chorionic gonadotropin; HPX, hypophysectomized; i.t., intratesticular; MIS, Müllerian inhibiting substance; RT, reverse transcriptase; SCF, stem cell factor; T, testosterone.
Accepted for publication August 18, 2005.
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