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Endocrinology, doi:10.1210/en.2007-1582
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Endocrinology Vol. 149, No. 7 3390-3402
Copyright © 2008 by The Endocrine Society

Novel Expression and Direct Effects of Adiponectin in the Rat Testis

J. E. Caminos, R. Nogueiras, F. Gaytán, R. Pineda, C. R. González, M. L. Barreiro, J. P. Castaño, M. M. Malagón, L. Pinilla, J. Toppari, C. Diéguez and M. Tena-Sempere

Department of Physiology (J.E.C., R.N., C.R.G., C.D.), University of Santiago de Compostela, 15705 Santiago de Compostela, Spain; Department of Physiology and Genetic Institute (J.E.C.), Faculty of Medicine, National University of Colombia, Bogotá, Colombia; Departments of Physiology and Pediatrics (J.T.), University of Turku, 20520 Turku, Finland; Department of Cell Biology, Physiology and Immunology (F.G., R.P., M.L.B., J.P.C., M.M.M., L.P., M.T.-S.), University of Córdoba, and CIBER (CB06/03) Fisiopatología de la Obesidad y Nutrición (J.P.C., M.M.M., L.P., C.D., M.T.-S.), Instituto de Salud Carlos III, 14004 Córdoba, Spain

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adiponectin is an adipocyte hormone, with relevant roles in lipid metabolism and glucose homeostasis, recently involved in the control of different endocrine organs, such as the placenta, pituitary and, likely, the ovary. However, whether as described previously for other adipokines, such as leptin and resistin, adiponectin is expressed and/or conducts biological actions in the male gonad remains unexplored. In this study, we provide compelling evidence for the expression, putative hormonal regulation, and direct effects of adiponectin in the rat testis. Testicular expression of adiponectin was demonstrated along postnatal development, with a distinctive pattern of RNA transcripts and discernible protein levels that appeared mostly located at interstitial Leydig cells. Testicular levels of adiponectin mRNA were marginally regulated by pituitary gonadotropins but overtly modulated by metabolic signals, such as glucocorticoids, thyroxine, and peroxisome proliferator-activated receptor-{gamma}, whose effects were partially different from those on circulating levels of adiponectin. In addition, expression of the genes encoding adiponectin receptor (AdipoR)-1 and AdipoR2 was detected in the rat testis, with developmental changes and gonadotropin regulation for AdipoR2 mRNA, and prominent levels of AdipoR1 in seminiferous tubules. Moreover, recombinant adiponectin significantly inhibited basal and human choriogonadotropin-stimulated testosterone secretion ex vivo, whereas it failed to change relative levels of several Sertoli cell-expressed mRNAs, such as stem cell factor and anti-Müllerian hormone. In summary, our data are the first to document the expression, regulation and functional role of adiponectin in the rat testis. Taken together with its recently reported expression in the ovary and its effects on LH secretion and ovarian steroidogenesis, these results further substantiate a multifaceted role of adiponectin in the control of the reproductive axis, which might operate as endocrine integrator linking metabolism and gonadal function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ADIPONECTIN, ALSO TERMED Acrp30 (adipocyte complement related protein), apMI (adipose most abundant gene transcript I), GBP28 (gelatin binding protein 28), or AdipoQ, was identified in 1995–1996 as an adipocyte-specific secreted factor with structural features of the collagen superfamily (1, 2, 3, 4). Its monomeric form is a 30-kDa protein that oligomerize to form trimers, which can further bind to generate 180-kDa hexamers or even high-molecular-weight polymers (>400 kDa), the latter being the most abundant circulating form of adiponectin in humans (5). Such a multimerization process has been documented as essential for the biological actions of adiponectin (6, 7).

Although adiponectin was identified as an adipose-secreted factor more than a decade ago, approximately coincident with the cloning of leptin (5), its full physiological relevance has emerged only in recent years, when adiponectin has been recognized as an antidiabetic adipokine, with potent insulin-sensitizing functions and thus promising therapeutic properties (5, 8, 9, 10). Of note, circulating levels of adiponectin are inversely correlated with the degree of adiposity and are overtly reduced in obesity and type 2 diabetes (4, 11, 12), whereas adiponectin administration has been shown to ameliorate insulin resistance and to induce glucose lowering in animal models of obesity (9, 13, 14). As a whole, these observations strongly suggest that decreased adiponectin levels are likely to operate as causative link between excess of adiposity and related comorbidities (15). As further proof for the relevant regulatory role of adiponectin in lipid and glucose homeostasis, its adipose expression appears tightly regulated by a wide array of metabolic cues and hormones, with either stimulatory [peroxisome proliferator-activated receptor (PPAR)-{gamma} ligands] or inhibitory (insulin, glucocorticoids, IL-6, TNF{alpha}, GH) effects (5, 16, 17, 18).

The biological actions of adiponectin are mediated by two distinct but structurally related receptors, AdipoR1 and AdipoR2. These are seven-transmembrane spanning receptors but with differential features from the superfamily of G protein-coupled receptors, such as their inverted orientation and their predominant signaling via AMP kinase (19). Initial reports demonstrated that AdipoR1 is abundantly expressed in skeletal muscle, whereas AdipoR2 is predominantly located in the liver (19). The physiological importance of these receptors in terms of metabolic control is now firmly established. AdipoR1–/– mice showed increased adiposity associated with decreased glucose tolerance, spontaneous locomotor activity, and energy expenditure. In contrast, AdipoR2–/– mice were lean and resistant to high-fat diet-induced obesity and showed improved glucose tolerance and reduced plasma cholesterol levels (20). Whether additional receptors can mediate these or other biological effects of adiponectin is still a matter of debate.

Besides its dominant metabolic roles, solid evidence points out that adiponectin is a rather pleiotropic regulator of a large set of biological functions, ranging from endothelial responses and pituitary hormone secretion to cell proliferation and tumor progression (5, 21, 22, 23). This contention is further supported by the recently recognized, widespread pattern of expression of AdipoR1 and AdipoR2, which include not only muscle, liver, and adipocytes but also hypothalamus, pituitary, pancreatic β-cells, endothelial cells, and placenta (19, 24, 25, 26, 27). Likewise, adiponectin expression has been recently proven more scattered than originally recognized, with detectable adiponectin mRNA levels in skeletal muscle, heart, placenta, pituitary, and osteoblasts (22, 24, 28, 29, 30).

It is well known that reproductive capacity is metabolically gated, and an ever growing number of neuropeptides and hormone signals, primarily involved in the control of energy balance and metabolism, have been recently proven as putative regulators of puberty maturation, gonadotropin function, and/or fertility (31). Among those, the prominent role of the adipocyte-derived hormone, leptin, in the control of reproduction has been well characterized over the last decade (32, 33, 34). In contrast, the physiological role, if any, of other adipose-born signals in the modulation of reproductive function remains ill defined. Notwithstanding, given its functional profile, the putative reproductive functions of adiponectin have begun to be explored recently. These analyses were initially focused in the eventual implication of adiponectin in female reproductive disorders linked to obesity and insulin resistance, such as polycystic ovarian syndrome. Indeed, a decrease in circulating adiponectin levels has been reported in polycystic ovarian syndrome patients (35, 36, 37). Further evidence for a physiological link between adiponectin and reproductive function came form the observation that adiponectin concentrations are invariantly higher in females than in males (38) and that androgens inhibit adiponectin secretion (39, 40). Very recently expression of adiponectin and its receptors has been documented in the rat ovary, in which adiponectin has been demonstrated to modestly stimulate progesterone and estradiol secretion in response to IGF-I (41). Moreover, the presence of adiponectin in rat oviduct has been recently documented (42). Whether adiponectin is expressed and/or able to conduct direct actions in the testis and/or male reproductive tract remains unexplored to date.

Worth noting, a number of hormonal signals with key roles in energy homeostasis and metabolism are expressed and/or conduct biological actions directly at the testicular level. These have been reported in a diversity of species (from rodents to humans) and include not only leptin (for a recent review see Ref. 33) but also ghrelin, the gut-derived orexigenic signal, and resistin (33, 43, 44). On the above basis, the present experimental work was undertaken to evaluate the potential expression of adiponectin and its cognate receptors, AdipoR1 and AdipoR2, in the rat testis. Our initial identification of the testicular expression of adiponectin prompted us to evaluate in detail its hormonal and metabolic regulation as well as the eventual direct testicular actions of this adipokine.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and drugs
Sprague Dawley male rats bred in the vivarium of our institution were used. The animals were housed under constant conditions of light (14 h of light; from 0700 h) and temperature (22 C). Experimental procedures were approved by local ethical committees and conducted in accordance with the European Union normative for care and use of experimental animals. In all experiments, the animals were killed by decapitation and testes were immediately removed, decapsulated (free of surrounding epididymal fat), frozen in liquid nitrogen, and stored at –80 C until processing. Recombinant mouse gAdiponectin/Acrp30 was purchased from R&D Systems Inc. (Minneapolis, MN). This recombinant protein is a noncovalent homotrimer generated by expressing a cDNA containing the globular domain of mouse adiponectin in the mouse myeloma cell line, BS0. Highly purified human choriogonadotropin (hCG) and human recombinant FSH were obtained from Serono (Madrid, Spain). The selective agonist of peroxisome proliferator-activated receptor (PPAR)-{gamma}, rosiglitazone maleate, was obtained from Calbiochem (La Jolla, CA). Human leptin was supplied by Eli Lilly (Indianapolis, IN). L-Thyroxine (sodium salt pentahydrate) and 3-amino-1,2,4-triazole (AMT) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO).

Experimental designs
Our first set of experiments was focused on the analysis of adiponectin expression in the rat testis in a number of physiological and experimental settings. First, the pattern of expression of adiponectin, at the mRNA and peptide levels, was explored in testes from adult rats. Testicular samples were obtained from 90-d-old animals (n = 10) and processed for RNA (RT-PCR and Northern blot) or protein (Western blot) isolation, as described in the following sections. Additional testicular samples (n = 4) from adult animals were subjected to immunohistochemical analysis using antiadiponectin polyclonal antibodies (see below).

Second, testicular expression of adiponectin mRNA was assayed at different stages of postnatal development. Samples of testis tissue were obtained from 5-, 15-, 30-, 60- and 90-d-old rats (n = 5–10/group), corresponding to the neonatal-infantile (5 d), late infantile (15 d), prepubertal (30 d), early adult (60 d), and adult (90 d) stages of postnatal maturation.

Third, the ability of pituitary gonadotropins to modulate testicular expression of adiponectin gene was evaluated. To this end, adiponectin mRNA levels were measured in control and long-term hypophysectomized (HPX) rats, i.e. 4 wk after pituitary removal via the parapharyngeal route, with or without gonadotropin replacement: hCG (10 IU/ rat per 24 h) or recombinant FSH (7.5 IU/rat per 24 h) for 7 d before sampling. In addition to mRNA analyses, plasma levels of adiponectin were assayed in these groups.

Finally, we explored the potential regulation of testicular expression of adiponectin mRNA by a number of metabolic hormones and cues in adult rats. These experiments included: 1) analysis of the effects of food deprivation for 48 and 72 h, with or without coadministration of leptin, as described in detail previously (44); 2) analysis of the effects of glucocorticoid hormones by means of administration of dexamethasone (40 µg per rat per day) for 1, 3, and 7 d, as reported previously (45); and 3) analysis of the effects of thyroid hormones by means of induction of states of hypo- or hyperthyroidism; the former was experimentally induced by administration of 0.1% amino-triazole in drinking water for 3 wk, whereas the latter was caused by repeated sc administration of 100 µg/d L-thyroxine, in keeping with previous references (45, 46). Besides mRNA analyses, in some of the above experimental groups, plasma levels of adiponectin were assayed by specific RIA. In addition, considering the previously reported effects of PPAR{gamma} ligands on adipose expression of adiponectin gene (16), the ability of the selective agonist of PPAR{gamma}, rosiglitazone, to modulate testicular adiponectin mRNA expression was assessed in vitro. Slices of testicular tissue were obtained from adult rats and incubated for 180 min in the presence of increasing concentrations (10–10 to 10–4 M) of rosiglitazone, as described in detail elsewhere (44). At the end of the incubation period, testis samples were processed for RNA analysis.

Our second set of experiments aimed to evaluate the expression of the genes encoding the putative adiponectin receptors, AdipoR1 and AdipoR2, in some of the experimental conditions tested above. Expression analyses were conducted in testicular samples from rats along postnatal development, after HPX, with or without gonadotropin supplementation, and after food deprivation. In addition, expression of AdipoR1 and AdipoR2 mRNAs (and the ligand) was assessed in microdissected seminiferous tubule fragments, isolated following previously described procedures (47). Specific stages of the seminiferous epithelial cycle were identified and pooled in four major groups corresponding to stages II–VI, stages VII–VIII, stages IX–XII, and stages XIII–I of the cycle. After exhaustive washing, tubular tissue was processed for RNA analysis as described below. In addition, staged tubule preparations (20 5 mm segments per well) were cultured in the presence of FSH (10 ng/ml) for 24 h. Samples incubated in the presence of medium alone served as controls.

Finally, in our third set of experiments, the direct biological effects of recombinant adiponectin on basal and stimulated testosterone (T) secretion were assessed in vitro using static incubations of adult rat tissue, as described elsewhere (48, 49). Tissue samples were incubated in the presence of increasing doses of adiponectin (0.01, 0.1, and 1 µg/ml), under basal or stimulated conditions (coincubation with 10 IU/ml hCG). In addition to secretory T responses, the effects of adiponectin on the mRNA levels of several Sertoli cell-expressed genes, such as stem cell factor (SCF), anti-Müllerian hormone (AMH), inhibin-{alpha}, and inhibin-βB, were evaluated in the same tissue samples following previously published protocols (50, 51).

RNA analysis by final-time RT-PCR
Total RNA was isolated from testicular samples (or seminiferous tubule fragments) by the single-step, acid guanidinium thiocyanate-phenol-chloroform extraction method. Testicular expression of adiponectin, AdipoR1, and AdipoR2 mRNAs was assessed by RT-PCR, using specific primer pairs and conditions as described in detail elsewhere (24). In addition, in selected experimental designs, semiquantitative RT-PCR analysis of SCF, AMH, inhibin-{alpha}, and inhibin-βB mRNAs was conducted, using previously reported primer pairs and conditions (50, 51). As internal standard, hypoxanthine phosphor-ribosyltransferase or ribosomal protein RP-L19 transcripts were amplified in parallel, as previously described (24, 50).

For amplification of the targets, total RNA (2 µg) was reverse transcribed using random primers and SuperScript II reverse transcriptase (Invitrogen Life Technologies, Inc., Carlsbad, CA). For PCR amplification of adiponectin, AdipoR1, and AdipoR2 transcripts, the following conditions were used: denaturation at 95 C for 1 min, annealing at 60 C for 30 sec, and extension at 72 C for 1 min for 34 cycles (24). PCR amplification of SCF, AMH, and inhibin transcripts was conducted as previously reported, using conditions optimized for amplification in the exponential phase for each transcript (50).

PCR-generated DNA fragments were resolved in Tris-borate buffered 1.5% agarose gels and visualized by ethidium bromide staining; identity of the amplicons was routinely conducted by double-strand sequencing. In all assays, liquid controls and reactions without reverse transcription resulted in negative amplification. When relevant, quantitative evaluation of RT-PCR signals was carried out by densitometric scanning using an image analysis system (Gel-2000 documentation system; Bio-Rad Laboratories, Inc., Richmond, CA), and values of the specific targets were normalized to those of internal controls to express arbitrary units of relative expression. In the case of adiponectin, specificity of PCR products was further confirmed by Southern blot using a 32P cDNA specific probe for the rat cDNA, as described in detail elsewhere (24).

Analysis of adiponectin RNA by Northern hybridization
The pattern of expression of adiponectin mRNA was evaluated in the rat testis by means of Northern hybridization. Total RNA was isolated from adult rat testicular samples as described above. Using denaturing agarose gels, samples of 25 µg of total RNA per lane were electrophoretically resolved and transferred to nitrocellulose membranes following standard procedures (24). The filters were subsequently hybridized using a radiolabeled cDNA probe, generated using a 658-bp fragment of rat adiponectin cDNA (24). Hybridization signals were digitalized and processed by densitometric scanning using an image analysis system (Gel-2000 documentation system; Bio-Rad Laboratories).

Real-time semiquantitative RT-PCR
Real-time RT-PCR analyses for quantification of changes in relative expression levels of adiponectin, AdipoR1, and AdipoR2 mRNAs were conducted in testicular samples using a fluorescent thermal cycler (LightCycler; Roche, Mannheim, Germany). Reverse transcription was conducted as described above, using 2 µg of total RNA per sample. The synthesized cDNAs were further amplified by PCR using SYBR green I as fluorescent dye and 1x LightCycler DNA master mix (Roche) containing 300 nmol/liter of forward and reverse primers, in a final volume of 20 µl. All reactions were carried out using the following cycling parameters: initial denaturation at 96 C for 1 min, followed by 40 cycles of 96 C for 15 sec, 60 C for 15 sec, and 72 C for 15 sec. After PCR, cycle threshold values were obtained for each sample using the software provided by the manufacturer. Relative quantification of PCR products was carried out by means of the comparative threshold cycle method after normalization of specific signals to those of the standard control (hypoxanthine phosphor-ribosyltransferase), as described previously (24). Product purity was confirmed by dissociation curves and random agarose gel electrophoresis. No-template controls were included in all assays, yielding no amplification.

Western blot analysis of adiponectin
Western blot analyses of rat adiponectin was conducted in homogenates of adult testicular tissue, as described in detail previously (24). Portions of adult rat testes were homogenized at 4 C in ice-cold lysis buffer and centrifuged at 11,000 x g for 30 min. The supernatant protein concentration was determined (protein assay kit; Bio-Rad Laboratories), and 100-µg protein samples were resolved in 10% SDS-PAGE gels and transferred to nitrocellulose membranes following standard procedures. Protein loading and membrane transfer were inspected under Ponceau S dye staining (ICN Biomedicals Inc., Aurora, OH). The membranes were sequentially incubated with a rabbit antiadiponectin primary antibody (ACRP 301-A; working dilution 1:100; {alpha}Diagnostic International, San Antonio, TX) and secondary antirabbit antiserum, as previously described (24). As protein control, the filters were washed and sequentially incubated with monoclonal {alpha}-tubulin antibody (dilution 1:1000; Sigma) and secondary antimouse antiserum. Detection was carried out using a chemiluminescent system (Tropix, Bedford, MA).

Adiponectin immunohistochemistry
Immunohistochemical detection of adiponectin peptide was carried out in 4% paraformaldehyde fixed sections of testes from adult rats. Two different primary antibodies raised against adiponectin were routinely used: rabbit anti-[Cys0]-adiponectin primary antibody (Phoenix Pharmaceuticals, Belmont, CA) and rabbit Acrp30 (N-20)-R (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). For immunolabeling, testicular sections (5 µm thick) were submitted to antigen retrieval in a microwave oven and incubated overnight with the primary antibody (diluted 1:200). The sections were then processed according to the avidin-biotin-peroxidase complex technique, as described elsewhere (52). Adiponectin immunoreactivity was identified as brown cytoplasmic staining in testicular sections counterstained with hematoxylin. Similar patterns of immunoreactivity were detected in testis specimens with the two primary antisera used. Testicular cell types were identified based on morphological criteria, in keeping with previous references (44, 53). Negative controls were run routinely in parallel by replacing the primary antibody by preimmune serum. In addition, as control for antibody specificity, immunohistochemical reactions were carried out after preabsorption of the antiserum overnight at 4 C with recombinant adiponectin.

T measurements by specific RIA
T levels in static incubation media were measured using a commercial kit from MP Biomedicals (Costa Mesa, CA). All medium samples were measured in the same assay. The sensitivity of the assay was 1 ng/ml and intraassay coefficient of variation was 4.5%.

Adiponectin measurements by specific RIA
Plasma levels of adiponectin were assayed in selected experimental groups using a commercial double-antibody RIA kit from Linco Research (St. Charles, MO), as previously described (24). All samples were assayed in duplicate in the same assay. The limit of the assay sensitivity was 1 ng/ml and interassay coefficient of variation was 6.6%.

Presentation of data and statistics
Semiquantitative, real-time RT-PCR analyses were carried out, at least in quadruplicate, using independent RNA samples. For presentation, in each experimental design, the expression levels in control/reference groups were assigned to a value of 100, and the others were normalized accordingly. Tissue incubations were conducted in duplicate, with a total number of 10–12 samples/determinations per group. Quantitative data are presented as mean ± SEM. Results were analyzed for statistically significant differences using ANOVA, followed by Tukey’s test. P ≤ 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Adiponectin expression in rat testis: developmental, hormonal and metabolic regulation
Testicular expression of adiponectin, at the mRNA and protein levels, was first explored in samples from adult rats using a combination of RT-PCR/Southern blot, Northern hybridization, Western blot, and immunohistochemistry. Initial RT-PCR analyses revealed that adiponectin gene is expressed in adult testis tissue, with amplicons similar in size to those obtained from white adipose tissue (WAT), whose identity was confirmed by Southern blotting and direct sequencing (Fig. 1AGo). Northern hybridization of adiponectin mRNA in adult rat testis and WAT, using a specific cDNA probe and stringent conditions, revealed that, whereas expression of adiponectin gene in fat tissue results in the expected three major transcripts of 2.5, 1.8, and 1.2 kb (54), adiponectin mRNA exists in the rat testis as two major species of 1.2-kb and less than 1.0-kb size (Fig. 1BGo).


Figure 1
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  		FIG. 1. Analysis of adiponectin expression in adult rat testis. A, Assessment of adiponectin mRNA expression in random testis specimens by RT-PCR and Southern blot. MW, Molecular weight; RT, reverse transcription. B, Representative Northern hybridization of total testicular RNA using a rat adiponectin cDNA probe. C, Western blot analysis of rat adiponectin expression in protein extracts from adult testicular tissue; immunolabeling of tubulin is shown as loading control. In all assays, assessment of adiponectin expression, at mRNA or protein level, in WAT is presented as positive control.

 
Besides mRNA analysis, assessment of adiponectin protein in rat testicular tissue was conducted by means of Western blot and immunohistochemistry. The former detected a major peptide band of 30 kDa in testicular homogenates, similar in size to that observed in WAT (Fig. 1CGo). For both testicular and WAT samples, additional bands of higher molecular mass (>50 kDa) were also detected, whereas omission of primary antibody abolished immunolabeling (data not shown). In addition, the presence of adiponectin immunoreactivity was demonstrated in adult rat testicular sections, with a predominant location in Leydig cells, identified at high magnification as cellular clusters in the interstitial space. In contrast, weak to negligible adiponectin immunolabeling was observed in the seminiferous tubules (Sertoli and germ cells) (Fig. 2Go, A and B). Specificity of adiponectin immunostaining was confirmed by omission and preabsorption of the primary antibody, procedures that completely blocked labeling of testis sections (Fig. 2CGo).


Figure 2
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  		FIG. 2. Immunolocalization of adiponectin protein in adult rat testis sections. Representative sections, at two different magnifications, are presented in A and B, in which prominent adiponectin immunoreactivity is detected at the cytoplasm of interstitial Leydig cells. In contrast, in Sertoli and germ cells within the seminiferous tubules, weak to negligible immunostaining for adiponectin was observed, which was difficult to discriminate form the background. Macrophages were negative for adiponectin immunoreactivity. C, A representative reaction after preadsorption of the primary antibody, showing absence of labeling. Scale bars, 100 µm (A); 25 µm (B and C). ST, Seminiferous tubules.

 
Subsequent analyses were directed toward the characterization of developmental, hormonal, and metabolic regulation of adiponectin gene expression in the rat testis. Assessment of mRNA levels of adiponectin throughout postnatal development, from the neonatal period to adulthood, demonstrated the persistent expression of the transcript in the rat testis. Yet its relative levels significantly varied along the age: moderate levels were observed before puberty (≤30 d old rats) that increased thereafter, with peak expression in early adult (60 d old) samples (Fig. 3AGo). In addition, the influence on testicular adiponectin mRNA expression of the major hormonal regulators of the testis, namely pituitary gonadotropins, was assessed using HPX rats, with or without gonadotropin replacement. Long-term (4 wk) HPX failed to induce major changes in the relative levels of adiponectin mRNA, which were not further affected by replacement with hCG (10 IU/rat per 24 h for 7 d; hCG as a superagonist of LH); or FSH (7.5 IU/rat per 24 h for 7 d) (Fig. 3BGo). Of notice, however, long-term HPX induced dramatic changes in testis weight (HPX: 309.0 ± 17.5 mg/testis; HPX+hCG: 585.0 ± 27.0 mg/testis; HPX+FSH: 447.0 ± 15.0 mg/testis; controls: 995.0 ± 37.0 mg/testis). Thus, in addition to relative levels of the transcript (i.e. relative mRNA abundance in 2 µg of total RNA), total expression of adiponectin mRNA per testis was also calculated after correction of relative values by testis weight (arbitrary units per milligram testis weight). In contrast to relative levels, 4 wk HPX induced a significant decrease in absolute expression of adiponectin mRNA per testis; a response that was partially prevented by hCG treatment. Conversely, FSH supplementation to HPX rats evoked only a marginal increase in total adiponectin mRNA levels (Fig. 3CGo). Contrary to its testicular effects, 4 wk HPX caused a significant rise in circulating levels of adiponectin that was not prevented by protocols of hCG or FSH replacement (Fig. 4AGo).


Figure 3
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  		FIG. 3. Testicular expression of adiponectin mRNA along postnatal development and its modulation by gonadotropins. A, Relative expression levels of adiponectin mRNA in testicular samples from 5-, 15-, 30-, 60-, and 90-d-old rats. B, Relative expression levels of adiponectin mRNA in testes from HPX rats, with or without replacement with hCG (10 IU/rat per 24 h for 7 d) or recombinant FSH (7.5 IU/rat per 24 h for 7 d). In addition, total levels of expression of adiponectin mRNA per testis are presented (C); these were obtained after correction of relative values, i.e. relative mRNA abundance in 2 µg total RNA, expressed in arbitrary units (AU) for testicular weight (TW). Semiquantitative values are the mean ± SEM of at least five independent determinations. Groups with different superscript letters are statistically different (P < 0.01; ANOVA followed by Tukey’s test).

 

Figure 4
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  		FIG. 4. Plasma adiponectin levels in adult male rats under different experimental conditions. A, Adiponectin concentrations in HPX rats, treated or not with hCG or FSH. B, The effects of 48 h fasting on plasma adiponectin levels. C, The effects of dexamethasone treatment (DEX). D, Plasma adiponectin concentrations are presented from hypothyroid (AMT) and hyperthyroid (T4) male rats. For each graph, groups with different superscript letters are statistically different (P < 0.01; ANOVA followed by Tukey’s test).

 
Metabolic regulation of testicular expression of adiponectin mRNA was explored in a number of experimental settings. Food deprivation for 48 or 72 h did not induce detectable changes in the relative mRNA levels of adiponectin (Fig. 5AGo), even after central supplementation of fasted rats with leptin (data not shown). Notably, 48 h fasting evoked the expected rise in plasma levels of adiponectin (Fig. 4BGo). Chronic treatment with dexamethasone in vivo decreased (Fig. 5BGo), whereas repeated administration of L-thyroxine increased (Fig. 5CGo), testicular expression of adiponectin mRNA; hypothyroidism was without any effect. However, the circulating levels of adiponectin increased in hypothyroid rats, remained unaltered after L-thyroxine administration and were decreased by dexamethasone (Fig. 4Go, C and D). Finally, in vitro exposure to increasing doses (10–10 to 10–4 M) of the agonist of PPAR{gamma}, rosiglitazone, evoked a significant reduction in the testicular levels of adiponectin mRNA, which was statistically significant at all doses tested (Fig. 6Go).


Figure 5
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  		FIG. 5. Regulation of adiponectin gene expression by metabolic hormones. A, Testicular levels of adiponectin mRNA in testes from adult rats subjected to complete food deprivation (fast) for 48 and 72 h. B, Relative mRNA levels of adiponectin are shown from testes of adult rats injected with dexamethasone (DEX), for 1, 3, and 7 d. Finally, adiponectin mRNA levels in testes of adult rats treated with AMT (to induce hypothyroidism) or L-thyroxine (T4; to induce hyperthyroidism) are presented (C). Semiquantitative values are the mean ± SEM of at least five independent determinations. Groups with different superscript letters are statistically different (P < 0.01; ANOVA followed by Tukey’s test).

 

Figure 6
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  		FIG. 6. Regulation of adiponectin mRNA expression in rat testis by the selective ligand of PPAR{gamma}, rosiglitazone. Relative levels of adiponectin mRNA were assayed by RT-PCR in slices of testicular tissue after in vitro exposure for 180 min to rosiglitazone maleate (TZD) at 10–10 to 10–4 M doses. Semiquantitative values are the mean ± SEM of at least five independent determinations. Groups with different superscript letters are statistically different (P < 0.01; ANOVA followed by Tukey’s test).

 
Expression of AdipoR1 and AdipoR2 genes in rat testis and direct testicular effects of adiponectin
Expression of AdipoR1 and AdipoR2 mRNAs was initially demonstrated in adult rat testes by means of RT-PCR, with amplicons similar in size to those obtained from skeletal muscle (AdipoR1) and liver (AdipoR2), and whose identity was confirmed by direct sequencing (Fig. 7AGo). Considering the proven expression of AdipoR1 mRNA in blood cells (24) and given the difficulty to discriminate whether amplification of this transcript in homogenates from whole testicular tissue might be due to blood contamination, RT-PCR analyses were also performed in homogenates of seminiferous tubule fragments, isolated under the stereomicroscope and thoroughly washed prior RNA isolation (i.e. to eliminate the presence of blood cells). These assays showed that AdipoR1 mRNA, but not AdipoR2, is expressed in the seminiferous epithelium (Fig. 7BGo), thus suggesting a compartmentalized distribution of AdipoRs within testicular tissue: AdipoR2 is not expressed in the seminifeorus epithelium (but rather in the interstitium), whereas AdipoR1 is expressed in the tubular compartment. On this basis, and considering the methodological limitations indicated above, samples of whole testicular tissue were used to specifically evaluate the developmental and hormonal regulation of AdipoR2 gene expression, whereas analyses on AdipoR1 mRNA were selectively conducted in staged seminiferous tubule fragments.


Figure 7
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  		FIG. 7. Analysis of AdipoR1 and AdipoR2 mRNA expression in adult rat testis by means of final-time RT-PCR. For each target, amplicons similar in size to those obtained in skeletal muscle (M; AdipoR1) and liver (L; AdipoR2) were obtained in random testis samples (T1, T2), whose identity was confirmed by direct sequencing. In addition to amplification in whole-testis homogenates, RT-PCR analyses were applied to pooled seminiferous tubule segments (ST1, ST2), isolated from fresh living preparations as described in Materials and Methods and devoid of blood cell contamination.

 
As was the case for the ligand, persistent expression of AdipoR2 mRNA was detected in the rat testis along postnatal development. However, in contrast to adiponectin, relative levels of this transcript were high at the neonatal period, declined thereafter during the infantile and prepubertal stages, and partially increased in adult animals (Fig. 8AGo). In addition, testicular expression of AdipoR2 appeared sensitive to gonadotropin regulation because its relative mRNA levels were significantly increased by hCG treatment in long-term HPX rats (Fig. 8BGo). Moreover, when total expression of AdipoR2 mRNA per testis was calculated (see above), it was noticed that 4 wk HPX induced a significant decrease in absolute expression of AdipoR2 mRNA per testis, whereas hCG, and to a lower extent FSH, treatment induced an increase in total AdipoR2 mRNA content (Fig. 8CGo).


Figure 8
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  		FIG. 8. Testicular expression of AdipoR2 mRNA throughout postnatal development and its modulation of by gonadotropins. A, Relative expression levels of AdipoR2 mRNA in testicular samples from 5-, 15-, 30-, 60-, and 90-d-old rats. B, Relative expression levels of AdipoR2 mRNA in testes from HPX rats, with or without replacement with hCG or recombinant FSH. In addition, total levels of expression of AdipoR2 mRNA per testis are presented (C); these were obtained after correction of relative values [i.e. relative mRNA abundance in 2 µg of total RNA, expressed in arbitrary units (AU)] for testicular weight (TW). Semiquantitative values are the mean ± SEM of at least five independent determinations. Groups with different superscript letters are statistically different (P < 0.01; ANOVA followed by Tukey’s test).

 
Expression of AdipoR1 mRNA in the seminiferous epithelium was confirmed by real-time RT-PCR analyses using staged tubule fragments devoid of blood cell contamination. These analyses revealed that AdipoR1 mRNA levels are the highest at stages II–VI, significantly decrease during stages VII–VIII and IX–XII, and increase again at stages XIII–I of the epithelial cycle. Exposure to FSH in vitro for 24 h (10 ng/ml) failed to change the expression levels of AdipoR1 at any of the stages tested (Fig. 9Go). Of note, adiponectin mRNA, albeit at low levels, was also detected in seminiferous epithelial preparations, yet its relative levels remained invariant regardless of the stage of the cycle and FSH treatment (data not shown).


Figure 9
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  		FIG. 9. Profile of expression of AdipoR1 in seminiferous tubule segments at different stages of the epithelial cycle. Relative levels of AdipoR1 mRNA were assayed by real-time RT-PCR in tubule preparations isolated at stages II–VI, VII–VIII, IX–XII, and XIII–I of the cycle, either in control conditions or after 24 h culture in the presence of FSH (10 ng/ml). Semiquantitative values are the mean ± SEM of at least five independent determinations. For presentation of data, the level of expression of AdipoR1 mRNA in stages II–VI at control conditions was taken as 100%, and the other values were normalized accordingly. Groups with different superscript letters are statistically different (P < 0.01; ANOVA followed by Tukey’s test). Co, Control; Co-0h, freshly isolated; Co-24h, incubated with medium alone.

 
Finally, demonstration of AdipoR1 and AdipoR2 gene expression in the rat testis prompted us to explore the potential effects of adiponectin directly at the testicular level. On the basis of previous data on the direct actions of relevant metabolic signals in rat testis (44, 48, 49, 50), two phenomena were selectively evaluated using static incubations of testicular tissue. First, T responses to increasing doses of adiponectin (0.01, 0.1, 1 µg/ml) were assayed, at 90 and 180 min incubation, in basal and hCG-stimulated conditions. Recombinant adiponectin, in a dose-dependent manner, was able to inhibit basal T secretion by incubated testicular tissue, with a significant lowering of T levels being detected after challenge with 0.01 and 0.1 µg/ml doses of adiponectin at both time points tested. Likewise, hCG-stimulated testosterone secretion was significantly suppressed by coincubation with adiponectin, at all doses studied (Fig. 10Go). Second, the potential actions of adiponectin on key seminiferous tubule functions were explored in vitro by monitoring its effects on a panel of Sertoli cell-expressed genes. In detail, the effects of increasing concentrations of adiponectin (0.01, 0.1, 1 µg/ml) upon the relative mRNA levels of inhibin-{alpha} and inhibin-βB subunits, SCF and AMH, because Sertoli cell-specific signals within the tubular compartment of the adult rat testis (51), were explored. In our setting, exposure to adiponectin, at the different doses tested, failed to modify the relative expression levels of any of the targets under analysis (data not shown).


Figure 10
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  		FIG. 10. Regulation of testicular T secretion by adiponectin ex vivo. Slices of testicular tissue were incubated with increasing doses of adiponectin (0.01, 0.1, and 1 µg/ml), and the concentration of T in the media was monitored after 90 and 180 min incubation. Because similar results were obtained at both time points, only results from the latter are shown. Values are normalized per gram of incubated tissue. Data are expressed as mean ± SEM (n = 10–12 samples/group). In addition, individual T values are plotted for each group. **, P < 0.01 vs. corresponding controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Among the diversity of target tissues and cellular functions (55), expression and/or direct actions of different adipokines have been reported in the gonads (41, 44). To our knowledge, our study is the first to provide a detailed characterization of the pattern of expression and hormonal regulation of adiponectin in the testis and document the presence of functional receptors and direct biological effects of this adipokine in the male gonad. Taken together with very recent reports on the presence of adiponectin and its cognate receptors in the rat ovary (41), our data unveil the putative role of adiponectin as novel regulator of gonadal function in rodents.

The expression of adiponectin in the rat testis was documented by a combination of analytic approaches at the mRNA and protein levels. Intriguingly, a distinctive pattern of mRNA transcripts, partially different to that observed in the WAT, was detected, with two prominent species of 1.2- and less than 1.0 kb (see Fig. 1Go). Although the sequence identity of those transcripts was not directly determined, the combined evaluation of Northern and RT-PCR analyses strongly suggests that differences in size between adipose and testicular transcripts are likely to reside in the 3'-untranslated region. Of note, a similar phenomenon has been described for other sites of extraadipose expression of adiponectin, such as the placenta (24). Nonetheless, the fact that a similar protein band, of the expected 30-kDa size, was obtained in homogenates from fat and testicular tissues evidence that the 1.2-kDa mRNA transcript is likely to encode functional adiponectin monomers, in keeping with previous data in the mouse showing that the 1.2-kb species contains the complete open reading frame of adipoinectin (54). In good agreement, discernible adiponectin immunoreactivity was observed in testicular sections from adult rats, with prominent signals being detected in interstitial Leydig cells. In contrast, faint to negligible immunostaining was observed in the seminiferous tubules, regardless of the stage of the epithelial cycle. This observation strongly suggests that the major source of testicular expression of adiponectin is located at the interstitium, likely in steroidogenic Leydig cells, in which this adipokine might play a functional role in the local (autocrine) control of testosterone secretion (see below). Yet weak expression of adiponectin at the seminiferous epithelium is likely to occur because its mRNA was detected, albeit at low levels, in tubular fragments throughout the epithelial cycle.

Testicular expression of adiponectin gene appears to be under the regulation of developmental cues and hormonal factors. On the former, moderate mRNA levels of adiponectin were detected in the rat testis before puberty, which significantly increased after pubertal maturation. The basis for such observation is yet to be defined but may involve the expansion of the adult-type Leydig cell population that takes place along the puberty transition (56). In addition, the contribution of pituitary gonadotropins, whose circulating levels increase at puberty, to this phenomenon cannot be excluded. Yet it is noticeable that relative levels of adiponectin mRNA were not overtly increased by treatment of HPX rats with either hCG (as superagonist of LH) or FSH. Nonetheless, the total content of adiponectin mRNA per testis was significantly decreased after HPX (a procedure that markedly reduced testis weight) and partially rescued by gonadotropin replacement, especially by hCG treatment. This is likely to reflect the trophic effect of hCG administration on the testis (57), and it is fully compatible with the preferential expression of adiponectin in Leydig cells within the testis because these cells represent the primary site of testicular expression of LH/CG receptors (58). Of note, circulating levels of adiponectin increased after HPX, which might be due (at least partially) to reduced secretion of androgen, which is a putative inhibitor of adiponectin expression in the adipose (39). Yet our protocols of gonadotropin replacement were unable to normalize adiponectin plasma concentrations, suggesting the involvement of other pituitary hormones in this phenomenon.

Contrary to the modest regulatory effects of gonadotropins, testicular expression of adiponectin gene was clearly modulated by a number of metabolic hormones and factors. Among those, administration of the synthetic glucocorticoid, dexamethasone, significantly decreased, whereas repeated injection of thyroxine markedly increased, adiponectin mRNA levels in the testis. In addition, in vitro exposure to the ligand of the transcription factor PPAR{gamma}, rosiglitazone, significantly suppressed testicular expression of adiponectin mRNA; an effect detected for doses as low as 100 pM, the lowest dose tested in our experiments. In contrast, food deprivation, for up to 72 h, failed to induce overt changes in adiponectin mRNA levels in the testis, which were not affected by leptin administration either. Of note, whereas some of the above regulators (e.g. glucocorticoids) have been reported to induce similar effects in terms of adipose expression of adiponectin gene (5), significant differences become apparent when comparing the actions of proven modulators of adiponectin expression in fat tissue, such as ligands of PPAR{gamma} and fasting, which increase adiponectin expression in the adipose (5), but either decrease (PPAR{gamma}) or do not affect (fasting) it in the testis. Likewise, measurement of circulating adiponectin levels in our models suggested dissimilar effects of thyroid hormones on testicular and adipose expression of adiponectin because T4 increased adiponectin mRNA levels in the testis, whereas its plasma levels were raised in conditions of hypothyroidism. Overall, these comparative analyses evidence that, whereas testicular and adipose expression of adiponectin gene may share common regulatory signals, the effects of some of those regulators appear to be tissue specific. The relevance of such metabolic modulation of adiponectin expression in the rat testis merits further investigation.

In addition to the ligand, our study is also the first to document the presence of functional adiponectin receptors in the rat testis. Thus, RT-PCR analyses demonstrated the testicular expression of both AdipoR1 and AdipoR2 mRNAs, with a rather compartmentalized pattern of distribution: AdipoR1 mRNA was clearly expressed in the seminiferous epithelium, whereas expression of AdipoR2 mRNA was not detected in the seminiferous epithelium, but likely confined to the interstitium. The latter was under the regulation of developmental cues and gonadotropins because AdipoR2 mRNA levels increased at puberty transition and were significantly enhanced by the superagonist of LH, hCG, a phenomenon that might be causative for the observed pubertal rise of AdipoR2 mRNA in the rat testis. Admittedly, the interstitial expression of AdipoR1 gene cannot be excluded because this could not be reliably assessed in our analyses given the presence of AdipoR1 transcripts in blood cells (24), which are likely to contaminate whole-testis homogenates. Nonetheless, clear-cut AdipoR1 transcripts were detected in isolated tubule fragments, at the different stages of the seminiferous epithelial cycle, with varying relative levels across the cycle: peak values at stages II–VI and nadir levels at stages VII–XII. This profile is suggestive of a fine tuning of adiponectin signaling in the seminiferous epithelium. Yet it is noticeable that FSH, as a key endocrine regulator of spermatogenesis (58), is not apparently posed with major regulatory effects on the tubular expression of AdipoR1 gene.

Further proof for the presence of functional adiponectin receptors in rat testis came from our in vitro analyses, testing the effects of increasing doses of the adipokine on several indices of testis function. As most salient finding, such analyses revealed that adiponectin is able to significantly inhibit basal and hCG-stimulated testosterone secretion by testicular explants. The relevance of this observation is 2-fold: 1) it further documents the ability of a number of metabolic signals, such as leptin, ghrelin, orexin-A, and resistin, to modulate testicular steroidogenesis (33, 44, 49, 59), a phenomenon that is likely to contribute to the functional coupling of metabolic status and reproduction (33); and 2) it demonstrates for the first time the ability of adiponectin to directly regulate male gonadal function. The physiological relevance and mechanisms of the latter are yet to be fully defined. Of note, our preliminary expression analyses in testicular explants did not identify any detectable effect of adiponectin on the relative mRNA levels of key factors of the steroidogenic route, such as steroidogenic acute regulatory protein, P450 side-chain cleavage enzyme, 3β- hydroxysteroid dehydrogenase, and 17β-hydroxysteroid dehydrogenase type III (our unpublished data), some of which are targets for the testicular effects of leptin (60). Notwithstanding, the inhibitory effects of adiponectin on basal and stimulated testosterone secretion reported here might contribute to the well-known suppression of testicular function in conditions of persistent negative energy balance (31), when systemic adiponectin levels are known to increase (5). Of note, a decrease in serum total levels of adiponectin has been reported in human males through puberty (61), a phenomenon that may play a permissive role in the pubertal increase of androgen secretion, provided that similar inhibitory effects take place in humans. Interestingly, our recent data demonstrate that adiponectin is also able to suppress basal and GnRH-stimulated LH secretion at the pituitary level (22). Thus, adiponectin could be considered an integral negative modifier of reproductive function in conditions of low adiposity linked to hyperadiponectinemia (5) via its combined actions at central (pituitary) and peripheral (testis) levels. In contrast, adiponectin is not likely to contribute to the hypoandrogenism frequently observed states of obesity and insulin resistance (62, 63) because adiponectin levels are reported to decrease in those conditions (4, 11, 12).

As reported herein for adiponectin, the expression and direct effects of resistin, another adipokine putatively involved in the regulation of glucose homeostasis and insulin sensitivity (5), have been previously documented by our group in the rat testis (44). However, the regulation and testicular actions of resistin and adiponectin appears to be markedly different because testicular expression of resistin was overtly modulated by gonadotropins and fasting in vivo, and it significantly enhanced basal and stimulated testosterone secretion in vitro (44). Of note, antagonistic actions have been reported for resistin and adiponectin in the modulation of insulin sensitivity, and their circulating levels are known to inversely change in different metabolic conditions (5). Likewise, the effects of resistin and adiponectin in terms of control of testosterone secretion appear to be opposite. Moreover, whereas a reciprocal stimulatory loop between androgen and resistin has been described [because androgens enhance adipose expression of resistin, whereas the latter increases testosterone secretion (44)], the opposite seems to be the case for adiponectin because androgens are known to decrease adiponectin expression (39), whereas adiponectin is able to suppress basal and stimulated testosterone secretion (present results). The relevance of the potential cross talk between resistin and adiponectin, not only in metabolic control but also in the regulation of testis function, is yet to be defined.

In summary, we provide herein novel evidence for the expression and potential functional role of adiponectin in the rat testis. Admittedly, some key aspects of adiponectin expression and action in the male gonad remain to be fully disclosed, including, among others: 1) its ontogenetic profile; 2) the relative importance of local vs. systemic adiponectin; 3) the mechanisms for its effects on testosterone secretion; and 4) the possibility of additional regulatory signals and functional roles (e.g. at the seminiferous epithelium). Yet our present observations are the first to disclose the potential role of adiponectin in the direct control of testicular function. From a more general perspective, our data, together with recent findings on the expression and direct actions of adiponectin at the pituitary and ovary (22, 41), substantiate the putative role of this adipokine as a metabolic modulator of the reproductive axis; a function whose physiological relevance (e.g. in coupling of energy reserves, metabolism, and fertility) and eventual physiopathological implications (e.g. in conditions of disturbed adiposity, insulin resistance, and gonadal dysfunction) merit further investigation.


   Acknowledgments
 
The authors are indebted to Dr. J. S. Suominen for help in microdissection and cultures of seminiferous tubule segments.


   Footnotes
 
This work was supported by grants from Instituto de Salud Carlos III (Red de Centros RCMN C03/08 and CIBER Fisiopatología de la Obesidad y Nutrición); Ministerio de Sanidad, Spain; the Academy of Finland; and the Sigrid Jusélius Foundation. CIBER is an initiative of Instituto de Salud Carlos III.

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 10, 2008

Abbreviations: AdipoR, Adiponectin receptor; AMH, anti-Müllerian hormone; AMT, 3-amino-1,2,4-triazole; hCG, human choriogonadotropin; HPX, hypophysectomized; PPAR, peroxisome proliferator-activated receptor; SCF, stem cell factor; T, testosterone; WAT, white adipose tissue.

Received November 19, 2007.

Accepted for publication April 2, 2008.


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 Introduction
 Materials and Methods
 Results
 Discussion
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