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Departments of Cell Biology, Physiology, and Immunology (F.G., M.L.B., L.P., E.A., M.T.-S.), and Pathology (C.M.), University of Cordoba, 14004 Cordoba, Spain; Departments of Physiology (J.E.C., C.D.) and Medicine (F.F.C.), University of Santiago de Compostela, 15705 Santiago de Compostela, Spain; Center for Molecular Biotechnology, Queensland University of Technology (L.K.C., A.C.H.), Brisbane, Queensland, Australia; Department of Cell Biology and Genetics, University of Alcala (R.P.), 28871 Madrid, Spain; and Department of Morphology, University Autonoma (M.N.), 28029 Madrid, Spain
Address all correspondence and requests for reprints to: Dr. Manuel Tena-Sempere, Physiology Section, Department of Cell Biology, Physiology, and Immunology, Faculty of Medicine, University of Cordoba, Avda. Menéndez Pidal s/n, 14004 Cordoba, Spain. E-mail: fi1tesem{at}uco.es.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The biological actions of ghrelin are mostly conducted through interaction with its specific cell surface receptor, namely the GHS-R. The cognate ghrelin receptor belongs to the large family of G protein-coupled, seven-transmembrane domain receptors (13, 14). This receptor is highly expressed at central neuroendocrine tissues such as the pituitary and hypothalamus (15). Two GHS-R subtypes, generated by alternative splicing of a single gene, have been described: the full-length type 1a receptor and the truncated GHS-R type 1b (13, 14). The GHS-R1a is the functionally active, signal-transducing form of the receptor. In contrast, the GHS-R1b lacks transmembrane domains 6 and 7, and it is apparently devoid of high affinity ligand binding and signal transduction capacity (13). Thus, its functional role, if any, remains unclear. In addition, evidence for GHS-R-independent biological actions of ghrelin as well as of synthetic GHSs has been presented recently (16, 17).
Despite the fact that most of the biological actions of ghrelin are carried out centrally, additional peripheral actions of ghrelin have recently emerged. Among those, a role for this molecule in the direct control of rat testicular function has been suggested. Thus, we have previously reported the expression of the ghrelin gene and protein in interstitial rat Leydig cells under the control of pituitary LH (18, 19). Similarly, expression of the cognate ghrelin receptor in rat testis has been demonstrated (18, 20), and evidence of the ability of ghrelin to modulate stimulated testosterone secretion in vitro has been presented (18). To our knowledge, however, detailed evaluation of the pattern of expression of ghrelin and its functional receptor in testes from nonrodent species, including humans, has not been conducted, although detection of ghrelin mRNA in human testicular tissue has been briefly reported recently (21). In this context, in the present study we evaluated the expression and pattern of cellular distribution of ghrelin and its functional receptor, i.e. the GHS-R1a, in adult human testis. In addition, considering its proposed effects on the proliferative rate of different tumor cell lines (12, 22), ghrelin and GHS-R1a immunoreactivity was also evaluated in different human testicular tumors and dysgenetic testis tissue.
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Materials and Methods |
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Tissue sections from adult human testes, testicular tumors, and dysgenetic testis tissue were obtained from the archives of Department of Pathology, University of Cordoba (Cordoba, Spain), and Department of Morphology, University Autonoma (Madrid, Spain), upon approval of the respective local ethical committees. In detail, from a larger series, sections of five normal testicular specimens were obtained from adult patients undergoing therapeutic orchidectomy unrelated to testicular pathology. In these samples, normal spermatogenesis and homogenous histological appearance were used as indexes to define tissue lacking any pathological condition, following the criteria described previously (23). In addition, representative tissue sections of several testicular disorders were obtained from archival samples. These were previously diagnosed specimens that included germ cell aplasia [Sertoli cell-only syndrome (SCO); two specimens], stromal cell-derived tumors (Leydig cell tumors; three specimens), and germ cell-derived tumors (seminoma and embryonal carcinoma; three and two specimens, respectively). A compilation of normal and pathological testicular samples used in the present study is included in Table 1
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Total RNA was isolated from adult human testes (BD Biosciences, Palo Alto, CA) using the single-step, acid guanidinium thiocyanate-phenol-chloroform extraction method (24). Human testes were obtained from apparently healthy individuals without history of testicular pathology and hormonal treatment, who had suffered sudden death. As positive controls, total RNA samples were obtained from human stomach and brain tissues (BD Biosciences). The expression of ghrelin and type 1a GHS-R mRNAs was assessed by RT-PCR using the primer pairs indicated in Table 2. These sets of primers were synthesized according to the published cDNA sequences of human ghrelin and GHS-R (1, 13). Amplification of human ghrelin cDNA was conducted using a previously reported primer pair (22), and the expression of the mRNA encoding GHS-R1a was assessed in human samples using a type 1a-specific primer pair spanning the single intron of the GHS-R gene, thus allowing amplification of a 205-bp fragment of GHS-R cDNA unique to the 1a form (13). In addition, as an internal control, amplification of a 285-bp fragment of hypoxanthine guanine phosphoribosyl transferase (HPRT) mRNA or a 232-bp fragment of ß-actin mRNA was carried out in parallel in each sample using the primer pair and conditions indicated in Table 2.
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PCR-generated DNA fragments were resolved in Tris-borate-buffered 1.5% agarose gels and were visualized by ethidium bromide staining. The 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). In all assays, reactions without RT were included that yielded negative amplification, thus ruling out the possibility of spurious amplification of the signals.
Immunohistochemistry and polyclonal antighrelin and anti-GHS-R1a antibodies
For analysis of ghrelin peptide expression, a rabbit antighrelin polyclonal antiserum, provided by Drs. Kojima and Kangawa (National Cardiovascular Center Research Institute, Osaka, Japan), was used as primary antibody. This antibody was generated as described in detail previously (25), using [Cys0]rat ghrelin (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) as antigen. This antiserum is able to recognize both rat and human ghrelin (26), and it is devoid of significant cross-reactivity with other peptides, as reported previously (26). In addition, immunohistochemical labeling of GHS-R1a protein was conducted using a rabbit polyclonal antibody generated against a synthetic peptide corresponding to the C-terminal fragment (RAWTESSINTC) of the human GHS-R1a protein conjugated to diphtheria toxin (Mimotopes, Melbourne, Australia), as described in detail previously (22). Western analyses using this antibody demonstrated a single specific band of approximately the predicted size (45 kDa) for the GHS-R1a in the ALVA-41 and DU145 prostate cancer cell lines (Chopin, L. K., and A. C. Herington, unpublished observation), which have been proven to express the GHS-R1a mRNA isoform and GHS-R1a protein (22).
Immunohistochemistry was performed on routinely neutral-buffered, formaldehyde-fixed, paraffin-embedded tissues. In detail, testicular sections (5 µm thick) were placed on poly-L-lysine-coated slides, and after dewaxing in xylene and rehydration in ethanol, the samples were incubated in 2% hydrogen peroxide in methanol for 30 min to quench endogenous peroxidase, followed by washing in PBS. In addition, sections for GHS-R1a immunolabeling were immersed in 10 mM citrate buffer and submitted to antigen retrieval in a microwave oven (twice, 5 min each time, 700 watts). According to routine immunohistochemical procedure, sections were allowed to cool at room temperature, washed in PBS, blocked with normal serum, and incubated overnight with the primary antibody, antighrelin (diluted 1:600) or anti-GHS-R1a (diluted 1:10). The sections were then processed according to the avidin-biotin-peroxidase complex technique following previously described methods (19, 27). Negative controls were run routinely in parallel by replacing the primary antibody with preimmune serum or PBS. In addition, positive controls for ghrelin and GHS-R1a immunostaining were assayed. These included reactions in rat testicular samples and human ovary and pituitary sections, conducted using antighrelin and anti-GHS-R1a primary antibodies, respectively. These yielded strong immunoreactivity, in line with our previous findings (18, 19, 20, 28). As an additional control for the specificity of GHS-R1a antibody, immunohistochemical reactions were carried out in human pituitary and testicular tissues after preabsorption of the antiserum overnight at 4 C with 1 mg/ml of the synthetic peptide (RAWTESSINTC) against which it was raised. In keeping with our previous results (28), this procedure completely abolished immunolabeling of pituitary and testicular sections (data not shown).
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Results |
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Expression of the ghrelin gene in normal adult human testis was demonstrated by means of primer-specific RT-PCR (Fig. 1). Thus, RT-PCR reactions from human testicular RNA, using a primer pair designed to amplify a 264-bp region of preproghrelin mRNA transcript, resulted in the generation of a single amplicon of expected size; its identity was confirmed by direct sequencing. In our assays, the possibility of spurious amplification of the signal was ruled out by the lack of amplification in reactions without RT. For comparative purposes, RT-PCR amplification of ghrelin mRNA was also conducted in RNA samples from human stomach, the major source of systemic ghrelin (1). These reactions yielded strong amplification signals.
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, A and B). The pattern of cellular distribution of ghrelin immunoreactivity was highly conserved within the control group, with almost negligible variation among samples. Strong ghrelin immunostaining was detected in steroidogenic Leydig cells within the testicular interstitium. In addition, ghrelin immunoreactivity was observed, albeit at lower intensity, in seminiferous tubules, where somatic Sertoli cells showed a specific ghrelin signal. In contrast, no staining was detected in germ cells at any stage of spermatogenesis. Similarly, interstitial cell types other than Leydig cells failed to show specific ghrelin immunoreactivity. In positive cells, the cytoplasm was uniformly stained, whereas cell nuclei remained negative. As a control for specificity of detection, omission of primary antighrelin antibody and its substitution by either PBS or preimmune serum resulted in negative staining of Leydig and Sertoli cells (data not shown).
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The pattern of cellular expression of ghrelin protein was also assessed in representative tissue samples of different testicular disorders. Samples of germ cell aplasia (SCO), stromal-cell derived tumors (Leydig cell tumors), and germ cell-derived tumors (seminoma and embryonal carcinoma) were analyzed. SCO testes were considered as those where germ cells were absent from the seminiferous tubules. Among these, hypoplastic tubules were defined as those bearing dysgenetic Sertoli cells, following previously described criteria (30). As was the case in normal testis tissue, strong ghrelin immunolabeling was detected in discernible Leydig cells within the interstitial space of SCO testes. In addition, ghrelin immunoreactivity was observed at lower intensity in dysgenetic Sertoli cells of hypoplastic tubules (Fig. 2C
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Ghrelin immunohistochemistry was also conducted in representative Leydig cell tumors with distinct morphological patterns of cell differentiation. Poorly differentiated Leydig tumor cells, characterized by small cell volume and scarce cytoplasm, failed to show discernible ghrelin immunoreactivity, whereas interstitial Leydig cells in the adjacent normal tissue presented a strong ghrelin signal (Fig. 2, D and E). In contrast, Leydig cell tumors with a higher degree of cell differentiation, defined by a large vacuolated cytoplasm, exhibited specific ghrelin immunostaining, albeit at lower intensity than normal Leydig cells (Fig. 2, F and G). Finally, analysis of ghrelin peptide expression was also carried out in germ cell-derived tumors. Our analysis failed to demonstrate detectable ghrelin signals in sections from testicular seminoma and embryonal carcinoma (Fig. 2, H and I) despite clear-cut ghrelin immunoreactivity detected in remnants of normal testicular tissue in these samples.
A comprehensive compilation of ghrelin expression data in normal and pathological human testis samples is included in Table 1.
GHS-R expression in normal human testis
In addition to the ligand, expression of the mRNA encoding the functional form of the cognate ghrelin receptor, the type 1a GHS-R, was assessed in normal adult human testis by primer-specific RT-PCR. Our molecular analysis demonstrated that, as was the case for ghrelin, GHS-R1a mRNA is expressed in adult human testis (Fig. 3). Thus, RT-PCR reactions from human testicular RNA, using a type 1a-specific primer pair spanning the single intron of GHS-R gene and designed to amplify a 205-bp area unique to the 1a form (13), generated a single amplicon of expected size; its identity was confirmed by direct sequencing. The possibility of spurious amplification of the signal was excluded by the lack of amplification in reactions without RT. As a positive control, RT-PCR amplification of GHS-R1a mRNA was also conducted in human brain samples.
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, A–C). Also, as reported for the ligand, the pattern of cellular location of GHS-R1a immunoreactivity was highly conserved within the control group, with almost negligible variation among samples. However, compared with that of ghrelin, GHS-R1a protein showed a somewhat wider pattern of cellular distribution within the human testis. Thus, clear-cut GHS-R1a immunostaining was detected in steroidogenic Leydig cells within the testicular interstitium, whereas other interstitial cell types were negative. In addition, GHS-R1a immunoreactivity was detected in the seminiferous tubules, where somatic Sertoli cells showed specific GHS-R1a signal. In addition, GHS-R1a immunolabeling was observed in germ cells, mainly in pachytene spermatocytes that showed strong GHS-R1a-specific staining (Fig. 4C). As a control for specificity of detection, substitution of the primary anti-GHS-R1a antibody with preimmune serum or its immunoabsorption with the synthetic peptide (RAWTESSINTC) against which it was raised resulted in negative staining of testicular tissue (data not shown).
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In addition to ghrelin, the pattern of cellular distribution of GHS-R1a protein was evaluated in different testicular disorders, namely germ cell aplasia (SCO), Leydig cell tumors, and germ cell-derived tumors (seminoma and embryonal carcinoma). As was the case in normal testis tissue, intense GHS-R1a immunostaining was detected in discernible Leydig cells within the interstitial space of SCO testes. However, in contrast to normal tubules (see Fig. 4, A and B), GHS-R1a immunoreactivity was observed at negligible levels in dysgenetic Sertoli cells of hypoplastic tubules despite clear GHS-R1a signals in adjacent interstitial Leydig cells (Fig. 4D). In addition, strong GHS-R1a immunolabeling was detected in Leydig cell tumors regardless of their morphological patterns of cell differentiation. Thus, both poorly differentiated Leydig tumor cells, characterized by small cell volume and scarce cytoplasm (Fig. 4E), and highly differentiated Leydig cell tumors (Fig. 4, F and G) showed specific GHS-R1a signals. Finally, analysis of GHS-R1a peptide distribution was conducted in germ cell-derived tumors. Despite the lack of ghrelin expression in these tumors, our assays demonstrated detectable GHS-R1a immunoreactivity, albeit at low intensity, in sections from testicular seminoma and embryonal carcinoma (Fig. 4, H and I).
A comprehensive compilation of GHS-R1a expression data in normal and pathological human testis samples is included in Table 1.
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Discussion |
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Using real-time RT-PCR, the expression of ghrelin gene in human testis has been postulated very recently (21). Our present data further substantiate this initial observation and demonstrate for the first time the actual presence of ghrelin peptide in the human male gonad. Immunolocalization of ghrelin within human testis tissue indicated that this protein is strongly expressed in interstitial mature Leydig cells. Notably, our previous molecular and immunohistochemical analyses in the rat indicated that testicular expression of ghrelin is restricted to mature Leydig cells (18, 19). Moreover, in the human ovary, intense ghrelin immunoreactivity has been demonstrated in hilus interstitial cells (28), i.e. a cell type that shows distinctive morphological characteristics, such as the presence of crystals of Reinke, identical to those of differentiated testicular Leydig cells (32). Thus, the expression of ghrelin is apparently a characteristic feature of both rodent and human Leydig cells that may serve as a useful marker of Leydig cell differentiation in mammalian testis, as previously proposed for other signals, such as relaxin-like factor (33). Interestingly, Leydig cell-specific expression of ghrelin in rat testis is under regulation of pituitary LH (19), and a role for ghrelin as a direct modulator of LH-driven testicular testosterone secretion has been reported (18). Furthermore, ghrelin expression in human ovary has been demonstrated in steroidogenically active luteal and hilus cells (28). In this context, elucidation of the potential direct role of ghrelin in the regulation of androgen secretion in the human testis merits further investigation.
Despite clear similarities in the pattern of expression of ghrelin in rat and human testicular Leydig cells, a specific feature of ghrelin expression in the human testis is the presence of this peptide in Sertoli cells. In fact, ghrelin protein in rat testis was solely detected in interstitial Leydig cells, and seminiferous tubules did not show any significant ghrelin immunoreactivity (18, 19). The basis for such a species divergence remains obscure. In this sense, the promoter of the human ghrelin gene has been recently cloned, and it was shown to be activated by cAMP (34). Notably, cAMP is the major intracellular second messenger in Sertoli cells for a plethora of paracrine and endocrine regulators, including pituitary FSH (35). This phenomenon might also be the basis for ghrelin expression in interstitial Leydig cells that use cAMP as intracellular messenger for its major endocrine regulator, namely pituitary LH (35). In any case, given the presence of the cognate ghrelin receptor in different tubular cell types reported herein, it is possible that local expression of ghrelin, in both Leydig and Sertoli cells may play a role in the paracrine control of seminiferous tubule function. In this context, it is noticeable that ghrelin apparently regulates expression of the stem cell factor (SCF) gene in seminiferous tubules of the rat testis (our unpublished observation). SCF has been pointed out as the major paracrine stimulator of germ cell development, acting as a survival factor for spermatogonia, spermatocytes, and spermatids in the adult rat seminiferous epithelium (36, 37). Whether ghrelin is able to modulate SCF gene expression, or it conducts additional biological functions in human seminiferous tubules remains to be evaluated.
In addition to the ligand, our immunohistochemical analyses of the presence and cellular location of GHS-R1a protein within the adult human testis demonstrated a somewhat wider pattern of distribution than that of ghrelin, with detectable specific signals in germ cells, mainly in pachytene spermatocytes, as well as in somatic Sertoli and Leydig cells. Partially conflicting results have been reported to date on the actual expression of the functional GHS-R1a in rodent and human gonads. In this sense, our recent analyses demonstrated expression of the type 1a GHS-R subtype at the mRNA and peptide levels in rat testis during the adult period (20). In keeping with these data, high levels of GHS/ghrelin-binding sites were demonstrated in human testis (38). Similarly, ghrelin-binding sites were identified in the human ovary (38), thus suggesting the presence of the functional receptor, which was recently confirmed by our immunohistochemical analyses (28). In contrast, however, in a recent report systematic screening of GHS-R1a mRNA expression in a wide array of human tissues, using real-time RT-PCR, failed to detect the GHS-R1a transcript in the human testis (21). Conversely, positive amplification of the mRNA encoding the truncated GHS-R1b form was observed (21). These observations are apparently in conflict with our present RT-PCR and immunohistochemical data. The reasons for such a discrepancy remain unclear. It has to be noted, however, that changes in the balance of expression between 1a and 1b forms of GHS-R may take place in the human testis during development and under certain endocrine conditions. This is certainly the case for the rat testis, where changes in the pattern of alternative splicing of GHS-R gene are observed throughout postnatal development; strong expression of GHS-R1a mRNA is detected from puberty onward, whereas in earlier stages of testicular development the predominant receptor form is probably the truncated GHS-R1b type (20). Thus, detailed description of the endocrine and developmental background of the assayed testis samples may help to explain the conflicting results indicated above (21). Moreover, pending on the availability of a specific antibody, it will be of interest to analyze the cellular expression of GHS-R1b subtype in human testis. Overall, data from previous binding studies (38) and our present immunohistochemical analyses indicate that functional GHS-Rs are expressed in the adult human testis. Furthermore, comparison of the distribution of GHS-R1a protein in rat and human species (Ref. 20 and the present results) is suggestive of a highly conserved pattern of cellular expression of the functional ghrelin receptor in mammalian testis.
Besides analyses in normal testis tissue, immunolocalization of ghrelin, and GHS-R1a proteins were conducted in representative sections of several types of testicular tumors and dysgenetic syndrome. It is worthy noting that the expression of ghrelin has been previously demonstrated in several endocrine tumors, such as pituitary-, stomach-, and pancreas-derived tumors, i.e. tissues that normally express ghrelin (39, 40, 41). In keeping with these observations, our immunohistochemical analyses demonstrated that ghrelin expression is conserved in Leydig cell tumors and dysgenetic Sertoli cells. However, the expression of ghrelin in Leydig tumor cells is apparently linked to the degree of cell differentiation. Thus, highly differentiated Leydig cell tumors showed specific ghrelin immunostaining, albeit at lower intensity than normal Leydig cells. In contrast, poorly differentiated Leydig tumor cells, characterized by small cell volume and scarce cytoplasm, failed to show any discernible ghrelin immunoreactivity. The mechanisms for such a phenomenon have not been directly addressed in our study. However, on the basis of the present findings and our previous data on the expression of ghrelin in fully differentiated rat Leydig cells (18, 19), it is tempting to propose that ghrelin may operate as a marker of Leydig cell differentiation, both during normal development and in tumor transformation. On the latter, loss of ghrelin expression might be an index of cell dedifferentiation. Moreover, ghrelin and its synthetic counterparts (GHSs) have been shown to carry out antiproliferative actions in different tumor cell lines (12, 17). Notably, rat and human mature Leydig cells, which do express ghrelin and its functional receptor, are devoid of significant proliferative activity (42). In contrast, proliferative rat Leydig cell progenitors (19) and poorly differentiated human Leydig tumor cells do not show ghrelin immunoreactivity. On this basis, we are presently investigating whether ghrelin may function as an autocrine regulator of Leydig cell proliferation in both normal and tumor conditions.
In addition to ghrelin, analysis of the expression of GHS-R1a peptide in representative testicular tumors was conducted. In this sense, the expression of GHS/ghrelin-binding sites and/or the GHS-R gene has been previously demonstrated in a wide variety of tumors, including pituitary adenomas and other neuroendocrine tumors (43), neoplastic thyroid tissue (44), human breast carcinoma (45), prostate cancer cell lines (22), and pancreatic islet cell tumors (40). Our present results indicate that GHS-R1a is expressed in Leydig cell tumors regardless of their degree of cellular differentiation as well as in germ cell-derived seminoma and embryonal carcinoma. Given the proposed regulatory effect of ghrelin and GHSs on cell proliferation in different tumor cell lines (12, 17, 46), our present findings provide the basis for a direct action of these compounds on different testicular tumors. It has to be noted, however, that both antiproliferative and proliferative effects of ghrelin and synthetic GHSs have been reported (for a review, see Ref. 46), and that at least some of the antiproliferative actions of GHSs are apparently conducted through a GHS-R1a-independent pathway (17).
In conclusion, our immunohistochemical analyses provide compelling evidence for the presence of ghrelin and its cognate functional receptor, namely the type 1a GHS-R, in the adult human testis. Indeed, the simultaneous expression of both components (ligand and receptor) of this signaling system in different testicular compartments is compatible with a potential action of locally produced ghrelin in the auto-/paracrine regulation of human testis function. Additionally, the wide pattern of GHS-R1a expression in human testis makes it possible that circulating ghrelin may operate upon specific testicular cell targets, as previously proposed for other peripheral signals with key actions in the regulation of body weight and energy expenditure, such as the adipocyte-derived plasma hormone, leptin (47). Moreover, the expression of ghrelin and GHS-R1a was also demonstrated in different testicular tumors. The fact that the pattern of cellular distribution of ghrelin and its receptor is roughly similar in rat and human testes is highly suggestive of a conserved role of this newly discovered molecule in the regulation of mammalian testicular function. The relevance of this novel testicular regulatory network in physiological and pathophysiological conditions merits further investigation.
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Acknowledgments |
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Footnotes |
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Abbreviations: AMV-RT, Avian myeloblastosis virus reverse transcriptase; GHS-R, GH secretagogue receptor; HPRT, hypoxanthine guanine phosphoribosyl transferase; SCF, stem cell factor; SCO, Sertoli cell-only syndrome.
Received August 6, 2003.
Accepted October 14, 2003.
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