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REPRODUCTION-DEVELOPMENT |
Departments of Physiology (M.T.-S., M.L.B., L.C.G., L.P., E.G.) and Cell Biology (F.G.), University of Córdoba, 14004 Córdoba, Spain; Department of Physiology (F.-P.Z.), University of Helsinki, FIN-00014 Helsinki, Finland; and Departments of Physiology (J.E.C., C.D.) and Medicine (F.F.C.), University of Santiago de Compostela, 15705 Santiago de Compostela, Spain
Address all correspondence and requests for reprints to: Dr. M. Tena-Sempere, Department of Physiology, Faculty of Medicine, University of Córdoba, Avda. Menéndez Pidal s/n, 14004 Córdoba, Spain. E-mail: fi1tesem{at}lucano.uco.es
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Introduction |
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Notably, the biological effects of ghrelin known to date are carried out at central levels, i.e. the hypothalamus and/or pituitary. However, additional as yet unknown peripheral actions of ghrelin cannot be ruled out. In this sense, it was shown recently that a wide range of endocrine and nonendocrine tissues possess GHS binding sites in humans (10). Moreover, novel expression of ghrelin in noncentral tissues, such as placenta and kidney, has been reported very recently (11, 12). Nevertheless, the functional roles, if any, of ghrelin in such peripheral systems remain unexplored.
The testis is a complex endocrine organ in which different cell types interplay in the fine tuning of the reproductive function under the control of a plethora of endocrine, paracrine, and autocrine regulatory signals (13). In recent years, it has become evident that different factors with key roles in the growth axis (e.g. GHRH and IGF-I) and body weight homeostasis (e.g. leptin) are potentially involved in the regulation of testicular function (14, 15, 16, 17). The identification of ghrelin as a novel endogenous factor implicated in growth and body weight regulation (1, 2, 7, 8, 9) prompted us to evaluate whether this signal and its functional receptor are expressed in rat testis. Our current data are suggestive of a possible involvement of ghrelin signaling in the direct control of gonadal function in the male rat, underscoring an unexpected reproductive facet of this newly discovered molecule.
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Materials and Methods |
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Experimental designs
Assessment of ghrelin expression in rat testis was carried out using different experimental approaches. First, testicular expression of ghrelin gene was evaluated by means of RT-PCR at different age points throughout postnatal development. In detail, based on previous reports on the timing of postnatal sex development in the rat (18), testis samples from 15-, 30-, 45-, 60-, and 90-d-old rats were assayed for expression of ghrelin mRNA. Secondly, expression and cellular location of ghrelin peptide within testicular tissue from adult rat specimens was studied by immunohistochemistry (see Ghrelin immunohistochemistry). In addition, because the latter suggested location of ghrelin protein in Leydig cells within rat testis, testicular ghrelin mRNA expression was analyzed at different time points after selective Leydig cell elimination by systemic administration of the cytotoxic drug ethylene dimethane sulfonate (EDS). In this model, mature Leydig cells are completely and selectively eliminated from the testicular interstitium within 24–48 h after administration of the toxicant in vivo, a phenomenon that is followed by reappearance of a newly formed population of Leydig cells in approximately 3–4 wk (Ref. 19 and references therein). Thus, this setting provides an optimal experimental background in which to test Leydig cell-specific expression of testis-derived factors (as an example, see Ref. 20).
In a second set of experiments, evaluation of testicular expression of the cognate receptor for ghrelin, i.e. GHS-R, was undertaken. Using an experimental approach similar to that used for analysis of the ligand, assessment of GHS-R mRNA expression was conducted in testis samples by RT-PCR at different representative age points of postnatal development: 15, 30, 45, and 75 d of age. In addition, testicular GHS-R mRNA expression was analyzed in a model of selective Leydig cell destruction. Thus, relative GHS-R mRNA levels were assayed in adult rat testis before 0, 5, 15, 20, 30, and 40 d after systemic administration of EDS.
In a third group of experiments, the potential functional role of ghrelin signaling in the control of testicular function was explored. To this end, assessment of the effect of ghrelin upon basal and stimulated T secretion in vitro was carried out using static incubations of adult rat testicular tissue, as described below. In addition to secretory responses, the effects of ghrelin on the mRNA expression levels of several key factors in the steroidogenic route were explored in this setting. In detail, four targets were evaluated: steroidogenic acute regulatory (StAR) protein, cytochrome P450 side-chain cleavage enzyme (P450scc), 3ß- hydroxy steroid dehydrogenase (HSD), and testis-specific 17ß-HSD type III. They were selected given their crucial role as hormonally regulated and/or pivotal steps in T biosynthesis in rat testis (21, 22, 23, 24).
RNA analysis by semiquantitative RT-PCR
Testicular expression of the mRNAs encoding ghrelin and its cognate GHS-R was assessed by semiquantitative RT-PCR. Similarly, this approach was used for analysis of the relative expression levels of the messages encoding StAR protein and enzymes P450scc, 3ß-HSD, and 17ß-HSD type III in incubated testicular tissue. Total RNA was isolated from testis samples from different experimental settings using the single-step acid guanidinium thiocyanate-phenol-chloroform extraction method (25). For amplification of the different signals, the primer pairs indicated in Table 1
were used. These sets of primers were synthesized according to the published cDNA sequences of rat ghrelin (1) and GHS-R ( 5) and the factors of the steroidogenic pathway under analysis (24, 26, 27, 28), and whenever possible, they were selected based on previous references (11, 29). In addition, to provide an appropriate internal control, parallel amplification of a 290-bp fragment of L19 ribosomal protein mRNA was carried out in each sample using the primer pairs and conditions indicated in Table 1, as described in detail elsewhere (11, 17).
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). PCR consisted in 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 1.5 min, annealing for 1.5 min, and extension at 72 C for 3 min. A final extension cycle of 72 C for 15 min was included. Annealing temperature was adjusted for each target: 55 C for ghrelin, 60 C for GHS-R and StAR protein, 61 C for P450scc, 64 C for 3ß-HSD, and 59 C for 17ß-HSD type III. In addition, different numbers of cycles were tested to optimize amplification in the exponential phase of PCR (data not shown), and based on current data and previous references (11, 17), the PCR cycles indicated for each target in Table 1 were chosen for further analysis. 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 (NewBiotechnic Ltd., Sevilla, Spain) or by Southern hybridization using radiolabeled nested oligonucleotide primers, as described elsewhere (11). In all assays, liquid controls and reactions without reverse transcriptase were included, yielding negative amplification. When relevant, quantitative evaluation of RT-PCR signals was carried out by densitometric scanning using an image analysis system (1-D Manager; TDI Ltd., Madrid, Spain), with the values for the specific targets being normalized to those of internal controls.
Ghrelin immunohistochemistry
Detection of ghrelin protein was carried out in 4% paraformaldehyde-fixed sections of adult (75-d-old) rat testis using a rabbit antighrelin polyclonal antibody and the avidin-biotin-peroxidase complex method, as described in detail previously (11).
Tissue incubation and T measurements
The general procedure for static incubations of testicular tissue has been described in detail elsewhere (16, 17). In this setting, testis samples were incubated in fresh medium or medium containing increasing doses of ghrelin (10-9 to 10-7 M) alone (basal) or supplemented with human hCG (10 IU/ml; stimulated). Moreover, the ability of ghrelin to modulate cAMP-stimulated T secretion was tested in additional samples incubated with Bu2-cAMP (10-4 M) alone or in combination with 10-7 M ghrelin. T was measured from diethyl ether extracts of incubation media, at 90 and 180 min, as described elsewhere (16). The levels of T in the media were expressed as normalized values per gram of incubated tissue. At the end of the incubation period, samples of testicular tissue from the different experimental groups were frozen in liquid nitrogen and stored at -70 C until used for RNA analysis (see RNA analysis by semiquantitative RT-PCR).
Presentation of data and statistics
RT-PCR analyses were carried out in triplicate using independent RNA samples. Tissue incubations were carried out in duplicate, with a total number of 12 determinations per group. When relevant, data are presented as mean ± SEM. Quantitative results were analyzed for statistically significant differences using ANOVA, followed by Tukey’s test. Values of P < 0.05 were considered significant.
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Results |
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). In detail, four representative stages of development were explored: infantile (15-d-old), prepubertal (30-d-old), pubertal-early adult (45-d-old), and adult (60- and 90-d-old). Quantitative analysis of the RT-PCR signals revealed, however, that the expression levels of ghrelin message in rat testis changed along the study period, with the highest values being detected during the adult period. In addition, clear-cut ghrelin immunostaining was observed in the testicular interstitium of adult rat specimens using a rabbit antighrelin polyclonal antibody. In contrast, negligible staining in the seminiferous tubules was impossible to differentiate from background and was considered negative. In the interstitial areas, ghrelin peptide was strongly located in mature Leydig cells (Fig. 2). In good agreement, testicular expression of ghrelin mRNA became undetectable 5 and 15 d after administration of the Leydig cell toxicant EDS. In this time frame, testicular interstitium is completely devoid of mature Leydig cells (Ref. 19 and references therein). Along with Leydig cell repopulation, reappearance of ghrelin mRNA was detected in rat testis 30 and 40 d after EDS administration (Fig. 3).
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). Quantitative evaluation of the intensity of amplicons indicated maximum expression levels of GHS-R mRNA in adult samples, in keeping with above observations on the pattern of testicular ghrelin expression. However, unlike the ligand, minimum expression levels of GHS-R mRNA were detected in pubertal 45-d-old testis samples.
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). In detail, 5 and 15 d after EDS administration, a period when no mature Leydig cells are detected in the testicular tissue (19), positive amplification of GHS-R signal was clearly obtained, thus suggesting an alternative cell source for testicular expression of GHS-R gene.
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). In good agreement, 10-4 M cAMP-stimulated T secretion in vitro was significantly inhibited by coexposure to an effective 10-7 M dose of ghrelin (Fig. 7).
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Discussion |
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Identification of testicular expression of ghrelin was accomplished by molecular (RT-PCR and Southern hybridization) and immunological approaches. Our analyses demonstrated that ghrelin gene is expressed in rat testis throughout postnatal development, although the relative mRNA levels changed sharply along the study period: the lowest expression values were detected in infantile-prepubertal testicular samples, whereas the highest levels were observed during the adult (60- and 90-d-old) period. By means of immunohistochemistry, ghrelin protein within the testis structure was located with high selectivity in interstitial Leydig cells, i.e. the steroidogenic cell-type of the testis. In good agreement, ghrelin mRNA expression became undetectable in rat testis after selective withdrawal of adult-type Leydig cells by administration of the cytotoxic compound EDS. Conversely, repopulation of this cell type was associated to recovery of testicular ghrelin mRNA signal. Moreover, our preliminary immunohistochemical analysis demonstrated absence of ghrelin protein in testis tissue at early stages (i.e. 5 and 15 d) after EDS administration (data not shown). Overall, our present results strongly indicate that Leydig cells are the primary source of ghrelin expression in rat testis.
In addition to the cognate ligand, our current data document the expression of functional receptors for ghrelin in rat testis. In this sense, RT-PCR analysis was performed to evaluate whether the message encoding the previously cloned GHS-R (4, 5) is expressed in rat testis. Our assays demonstrated positive amplification of GHS-R signal in testicular samples at different stages of postnatal development. As was the case for the ligand, expression levels of GHS-R message changed throughout the period under analysis, with the highest expression levels being detected in adult tissue. In this sense, although subtle differences in the pattern of temporal expression of the messages encoding the GHS-R and ghrelin itself can be noted, it is apparent from our analyses that both genes are maximally expressed in rat testis at the adult age. Worthy to note, expression of GHS-R mRNA in adult testes was persistently detected after selective Leydig cell destruction by EDS, thus suggesting that, unlike the cognate ligand, the major cellular source of testicular GHS-R signal is not Leydig cells. However, expression of GHS-R in this cell type cannot be ruled out on the basis of our current data.
Further evidence on the expression of functional ghrelin receptors in rat testis is provided by our studies using incubated testicular tissue. In this setting, basal T secretion remained unaffected after exposure to increasing concentrations of ghrelin. However, ghrelin, in a dose-dependent manner, was able to significantly inhibit both hCG- and cAMP-stimulated T release in vitro. The mechanisms and cell types involved in such an inhibitory response are presently under investigation. The fact that ghrelin equally decreased hCG- and cAMP-induced T secretion indicates that this inhibitory action must take place in a step beyond cAMP formation. Concerning cell types involved, our results in vitro are compatible either with a direct inhibitory effect of ghrelin upon the steroidogenic Leydig cells, or indirect actions mediated through other testicular cell type(s). In favor of the latter, invariant levels of GHS-R mRNA were detected after selective Leydig cell destruction (see Fig. 5). Moreover, our preliminary functional analyses, including assessment of expression and hormonal regulation of GHS-R gene in testicular cell lines and tissue, as well as evaluation of the ability of ghrelin to modulate gene expression of several non-Leydig cell products, strongly suggest that Leydig cells are not the primary testicular target of ghrelin (Tena- Sempere, M., and M. L. Barreiro, manuscript in preparation). Direct assessment of the cellular location of GHS-R and biological actions of ghrelin in purified testicular cell preparations (e.g. Sertoli and Leydig cells) will help to identify the targets of this molecule within the rat testis.
The inhibitory effect of ghrelin upon T secretion was associated with a significant decrease in hCG-stimulated expression levels of the mRNAs encoding several key factors in the steroidogenic route: StAR and enzymes P450scc, 3ß-HSD, and testis-specific 17ß-HSD type III. It must be stressed, however, that causative relationship between these phenomena is yet to be proven. Nevertheless, our data showing that ghrelin was able to consistently inhibit stimulated T secretion, both after coincubation with hCG or Bu2-cAMP, and to decrease mRNA expression levels of several key steroidogenic factors strongly suggest that functional ghrelin receptors are expressed in rat testis and that ghrelin signaling negatively regulates testicular steroidogenic function. In our laboratory, we are currently assessing the effects of blockade of endogenous ghrelin upon testicular T secretion in vivo to evaluate the physiological relevance of such a phenomenon.
Interestingly, the pattern of response to ghrelin in terms of StAR and P450scc mRNA expression closely mirrored that observed in terms of T release: a lack of inhibitory effect of 10-9 M ghrelin was followed by significant decreases after challenge with 10-8 to 10-7 M ghrelin. In this sense, cholesterol translocation to the inner mitochondrial membrane (StAR-mediated event) and its subsequent conversion to pregnenolone (P450scc-mediated event) are the first and rate-limiting steps in steroid biosynthesis (21, 22). Moreover, it is well documented that regulation of steroidogenesis by various hormonal signals is tightly correlated with concomitant changes in StAR and P450scc gene expression in different experimental settings (21, 22, 30, 31). However, the possibility that ghrelin-induced decrease in StAR and P450scc expression levels may directly contribute to the inhibition of stimulated T secretion after exposure to ghrelin in vitro must be substantiated by additional experimental work, including analysis of protein expression and/or activity of the above steroidogenic factors. From a general standpoint, the facts that the three major steroidogenic tissues, namely adrenal, testis, and ovary, possess high amounts of GHS binding sites in humans (10) and that, besides the testis, GHS-R gene is expressed in rat adrenal and ovary (our unpublished observation) make it worthy to evaluate the potential effects and mechanism(s) of action of ghrelin upon the steroidogenic function in different systems.
Notably, a similar direct inhibitory action on testicular T secretion was recently documented for leptin, the adipocyte-derived plasma hormone (16, 32). Both leptin and ghrelin appear as regulatory signals in growth and body weight homeostasis (1, 2, 7, 8, 33). Moreover, the involvement of leptin in the control of the reproductive axis has been well established (33). In this context, our current data on the expression of ghrelin and its functional receptor in rat testis open up the possibility that ghrelin may represent an additional regulatory signal linking growth, food intake, and reproductive function.
The testis is a complex endocrine organ in which different cell types cooperate to ensure adequate male fertility. Besides pituitary gonadotropins, an ever-growing group of extragonadal and intragonadal hormones and growth factors have been implicated in recent years in the control of testicular function (13). Overall, the results presented herein strongly suggest that ghrelin participates in such a regulatory network, thus providing evidence for an unexpected reproductive facet of this newly discovered molecule.
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Acknowledgments |
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Footnotes |
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Abbreviations: EDS, Ethylene dimethane sulfonate; GHS, GH secretagogue; GHS-R, GH-secretagogue receptor; hCG, human CG; HSD, hydroxy steroid dehydrogenase; P450scc, cytochrome P450 cholesterol side-chain cleavage; StAR, steroid acute regulatory.
Received July 24, 2001.
Accepted for publication October 16, 2001.
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5-4 isomerase cDNAs and differential tissue-specific expression of the corresponding mRNAs in steroidogenic and peripheral tissues. J Biol Chem 266:583–593This article has been cited by other articles:
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