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The present study was designed to determine the distribution of GHS-R1a in the testis in rats, the effects of the intracerebroventricular (i.c.v) administration of ghrelin on testosterone secretion and the expression of androgen receptor (AR) mRNA. GHS-R1a immunoreactivity was demonstrated mainly in Sertoli and Leydig cells, primary spermatocytes, and secondary spermatocytes. Ghrelin, at dosages of 1 and 3 nmol, significantly inhibited testosterone secretion. At dosage of 3 nmol, ghrelin significantly inhibited AR mRNA expression. Overall, our data provide initial evidence that the i.c.v injection of ghrelin plays a physiological role in testosterone secretion and the expression of AR mRNA, further documenting the reproductive role of ghrelin.
Ghrelin, a 28-amino-acid peptide with an essential n-octanoylation at serrine 3, is the natural ligand of the growth hormone (GH) secretagogue receptor (GHS-R), which belongs to a large family of G protein-coupled seven-transmembrane receptors [Kojima et al. 1999]. Ghrelin has been demonstrated to be a pleiotropic regulator involved in a large array of endocrine and non-endocrine functions, including food intake and energy balance [Wren et al. 2000; Horvath et al. 2001]. Moreover, accumulated evidence suggests that ghrelin plays a role in the central regulation of reproduction. For example, intracerebroventricular (i.c.v) administration of 3 nmol of ghrelin evokes a significant inhibition of luteinizing hormone (LH) secretion in cyclic female rats throughout their estrous cycle [Fernandez-Fernandez et al. 2006]. Ghrelin has been shown to inhibit human chorionic gonadotropin (hCG) and cyclic adenosine monophosphate (cAMP)-stimulated testicular testosterone secretion in rats in vitro [Tena-Sempere et al. 2002]. Testosterone (T) is a critical steroid hormone that is essential for spermatogenesis, fertility, and maintenance of the male phenotype, including the outward development of secondary sex characteristics [Wang et al. 2009]. Such actions are mediated by the androgen receptor (AR), a member of the nuclear receptor superfamily. The impacts of deficient AR in the testis include spermatogenesis arrest and abnormal fertility [Wang et al. 2009; Tsai et al. 2006; Xu et al. 2007; Chang et al. 2004]. Increasing evidence suggests that many factors are involved in the control of AR mRNA expression, such as follicle-stimulating hormone (FSH), estrogen (E2), and so on [Sanborn et al. 1991; Pelletier et al. 2004]. It is still unknown whether the i.c.v injection of ghrelin can regulate testosterone secretion and AR mRNA expression. To gain further knowledge about these, a set of experiments was carried out.
Serum LH, FSH, and T concentration changes after i.c.v. injection of ghrelin
Ghrelin, at dosages of 1 and 3 nmol, significantly inhibited LH secretion (P <0.05). Dosage of 0.3 nmol significantly inhibited T secretion. FSH secretion was significantly inhibited by 3nmol ghrelin. (Table 2)
GHS-R1a iimmunohistochemistry in rat testis
The GHS-R1a protein showed a wide pattern of tissue distribution in rat testis, detected mainly in the Sertoli and Leydig cells. Primary spermatocytes and secondary spermatocytes also showed clear-cut signals, whereas spermatogonia and spermatids were not stained (Fig. 1).
Gene expression of AR after i.c.v injection of ghrelin
Results from Fig. 2 are summarized. The AR mRNA level was significantly inhibited at the dose of 3 nmol (P<0.05). No significant changes in AR mRNA expression were observed at doses of 0.3 and 1 nmol (Fig. 2).
Ghrelin has been demonstrated to be highly selectivity in mature Leydig cells of rat testis [Tena-Sempere et al. 2002]. In addition, the expression of the functional ghrelin receptor, GHS-R1a, has been shown in the Sertoli and Leydig cells of the same organ [Gaytan et al. 2004]. Immunohistochemistry results showed that clear-cut GHS-R1a immunostaining was observed in primary and secondary spermatocytes. In contrast, negligible staining was observed in spermatogonia and spermatids, suggesting that GHS-R1a is expressed in a stage-dependent manner in spermatogenesis. From the above results, we strongly believe that ghrelin may be involved in the control of some aspects of testicular function. Testosterone secretion and the expression of AR mrna have been demonstrated to play important roles in spermatogenesis and fertility. Whether or not ghrelin is involved in the modulation of testosterone secretion and the expression of AR mrna, however, remains to be established.
Ghrelin has been proven to inhibit testicular testosterone secretion by dose-dependent actions in vitro [Tena-Sempere et al. 2002]. The present study demonstrates that the i.c.v injection of ghrelin in rats affects their serum testosterone and AR mrna levels. In particular, ghrelin doses of 0.3 nmol significantly inhibit the secretion of testosterone, while a ghrelin dose of 1nmolã€3 nmol does not. This suggests that ghrelin has an inhibitory action on testosterone release in vivo limited to a certain dosage range. The mechanism of the inhibitory effects of ghrelin on testosterone secretion could be theoretically explained by one or more of the following possibilities. First, the inhibitory effect may be related to the proliferation of Leydig cells. Injection of ghrelin significantly decreases the proliferation activity of differentiating immature Leydig cells. Ghrelin also induces a significant decrease in Stem cell factor (SCF) mRNA levels, a key regulator signal of Leydig cell development [Barreiro et al. 2004; Yan et al. 2000]. Second, LH may be involved in the decrease of testosterone. Testicular LH receptors are selectively expressed in Leydig cells. LH stimulates serum testosterone concentrations through its functional receptor. It was observed that 1ã€3 nmol of ghrelin significantly inhibits LH secretion. Ghrelin may inhibit testosterone secretion by decreasing LH release. Finally, Tena-Sempere M et al. found that ghrelin was significantly decreased in hCG-stimulated expression levels of some mRNAs that encode several key factors in the steroidogenic route, such as steroid acute regulatory protein (StAR), P450 cholesterol side-chain cleavage (P450scc), 3ß-hydroxy steroid dehydrogenase (3ß-HSD), and testis-specific 17ß-hydroxy steroid dehydrogenase (17ß-HSD) type III [Tena-Sempere et al. 2002].
Although ghrelin has been reported to regulate testosterone secretion, information regarding the control of gene expression at the mRNA level remains incomplete. Our present results demonstrate that ghrelin doses of 3 nmol could significantly inhibit AR mRNA expression; however, gherlin doses of 0.3 and 1 nmol do not. These observations suggest that ghrelin has an inhibitory action on AR mRNA expression. Germ cell development within the mammalian testis requires testosterone stimulation of somatic Sertoli cells via interaction with intracellular AR. AR mRNA expression levels undergo marked changes after injection of 3 nmol of ghrelin, suggesting that the modulation of AR mRNA expression is an important mechanism for regulating Sertoli cell responsiveness to testosterone [Kim et al. 2000]. Adult rats treated with ethane dimethane sulphonate to eliminate the presence Leydig cells showed very low levels of testosterone in their blood and testis, the AR mRNA levels of these rats were unchanged, indicating that the inhibitory action must take place in a step beyond testosterone secretion, although ghrelin could also significantly inhibit the secretion of testosteronep Blok et al. 1992]. In recent years, FSH have been reported to increase AR mRNA in Sertoli cells [Sanborn et al. 2004]. I.c.v injection of 3nmol ghrelin significantly inhibit the secretion of FSH. Therefore, the inhibition may be related to FSH. To this date, the molecular mechanisms governing the inhibitory actions of ghrelin on AR mRNA expression are not fully understood.
In summary, gonads are complex endocrine organs. Although tests of its effects via acute administration have suggested a possible role for ghrelin in the control of testosterone secretion and expression in AR mRNA, the mechanisms of such actions remain unclear and further research is necessary to identify the precise functional role of ghrelin.
MATERIALS AND METHODS
Animals and Sampling
Groups of adult male Sprague Dawley rats (180-240 g) were kept under controlled conditions of light (12 h light, 12 h darkness, light at 07:00 h) and temperature (22° C), with free access to food and tap water. Each experimental group consisted of eight animals. ghrelin was obtained from Sigma (USA) and dissolved in saline solution immediately before use. The rats were intracerebroventricularly injected with different doses of ghrelin (0.3nmol, 1nmol, 3nmol and saline). All animals were killed by decapitation 15 min after injection with ghrelin. The trunk blood of the animals was collected for other determinations, while their testis was rapidly dissected out and then stored at -80° C until analysis. Other testis samples were taken for the immunohistochemical analysis of GHS-R1a peptide expression.
GHS-R1a Immunohistochemistry in Rat Testis
GHS-R1a immunostaining was conducted in paraffin sections 5 µm thick. After dewaxing and rehydrating the samples, and antigen retrieval in a microwave oven (3-5 min at 700 w), endogenous peroxidase was inhibited by incubation in a 3 % hydrogen peroxide solution for 10 min. After washing in phosphate buffer saline (PBS), sections were blocked with normal goat serum and incubated overnight with anti-GHS-R1a antibody (dilution: 1:40). The sections were then processed according to PV-9000 two-step method detection kit instructions (Golden Bridge, Beijine), treated with polymer helper for 20 min (37° C), washed in PBS (3-5 min), and incubated in polyperoxidase-anti-Rabbit IgG for 30 min (37° C). Antibody binding was visualized by a DAB detection system, with sections counterstained with hematoxylin. In all reactions, negative controls were run in parallel by omitting the primary anti-GHS-R1a antibody (substituted by PBS) to demonstrate the absence of specific immunoreactivity.
LH, FSH, and T determination
LH and FSH concentrations were measured in the serum using a double-antibody method (ELISA kits were purchased from Shanghai Westang Biological Engineering Company). The mean intra- and inter-assay coefficients of variation were 9.7% for LH and 10% for FSH. T concentrations were measured in the serum using commercially available radioimmunoassay kits (purchased from Beijing Furui Biological Engineering Company). The mean intra- and inter-assay coefficients of variation were 9.5% and 10.3% for T, respectively. The assay sensitivity was 0.002 ng/mL for T.
Total RNA Isolation and Reverse Transcription
Total RNA was extracted from testis using Trizol reagent (TransGen, Beijing, China) according to the manufacturer's protocol. The quality of total RNA was assessed by formaldehyde gel electrophoresis. Samples that showed good RNA quality were selected for further reverse transcription. Reverse transcription was performed using the TransScript First-Strand cDNA Synthesis SuperMix (TransGen, Beijing, China) according to the manufacturer's protocol. Approximately 2 μL of total RNA was used for reverse transcription.
Primer information related to AR target genes and β-actin as the housekeeping internal control gene are listed in Table 1. Both gene sequences are available at http://www.ncbi.nlm.nih.gov (GenBank Accession Nos. M20133.1 and AF122902.1, respectively). All primers were designed using Primer 5.0 and allowed the amplification of regions that span introns. Real-time PCR was performed using SYBR Premix Ex TaqTM Kits (TaKaRa, Dalian, China) and a Rotor-Gene 6000 instrument (Corbett Life Science, Australia). Each reaction well was loaded with 2.0 μL (10 pmol/μL) of the forward and reverse primers, 12.5 μL of SYBR Premix Ex TaqTM, 2 μL of cDNA, and 8.5 μL of double-distilled water. The final reaction volume was 25 μL. All samples were run in triplicate. The following PCR conditions were used: preliminary denaturation at 95° C for 30 s, followed by 40 cycles with a temperature profile of 5 s at 95° C, 20 s at 55° C, and 15 s at 72° C. Melting curves were also determined. Quantitative analysis was performed using the relative standard curve method. After analysis, data are shown as the ratio of analyzed gene expression to β-actin.
Results are expressed as mean±SEM. Differences in the serum T, LH, FSH, and AR mRNA relative quantities were carried out using one-way repeated-measures analysis of variance. The level of P < 0.05 was considered statistically significant.