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Published-Ahead-of-Print November 1, 2006, DOI:10.2164/jandrol.106.000810
Journal of Andrology, Vol. 28, No. 2, March/April 2007
Copyright © American Society of Andrology
DOI: 10.2164/jandrol.106.000810

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Ghrelin Expression in Human Testis and Serum Testosterone Level

TOMOMOTO ISHIKAWA, HITOSHI FUJIOKA, TAKESHI ISHIMURA, ATSUSHI TAKENAKA AND MASATO FUJISAWA

From the Division of Urology, Kobe University Graduate School of Medicine, Kobe, Japan.

Correspondence to: Dr Tomomoto Ishikawa, Division of Urology, Department of Organs Therapeutics, Faculty of Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, Chuo-Ku, Kobe, Japan 650-0017 (e-mail: iskwtmmt{at}med.kobe-u.ac.jp).
Received for publication June 2, 2006; accepted for publication October 30, 2006.

   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Ghrelin, which is the endogenous ligand for the growth hormone (GH) secretagogue receptor (GHS-R), has been primarily linked to the central neuroendocrine regulation of GH secretion and food intake, although additional peripheral actions of ghrelin have also been reported. Recent research has suggested that ghrelin also affects testosterone (T) secretion in vitro. To investigate the role of ghrelin in human testicular function, we examined the expression of ghrelin in testicular tissues by immunohistochemistry. Testicular tissue samples were collected from the testes of 5 fertile volunteers, 8 patients with obstructive azoospermia, and 36 oligospermic patients with varicocele testis. In the testicular tissues, ghrelin was stained using the antighrelin polyclonal antibody, and the Johnsen score was calculated. The concentrations of serum follicle-stimulating hormone (FSH), lutenizing hormone (LH), and T were determined by chemiluminescence assays. Immunostaining of ghrelin was detected in the interstitium and in Leydig cells. Ghrelin expression by Leydig cells was inversely correlated with the serum T concentration (r = –.50; P < .001), but was not directly related to spermatogenesis. We conclude that steroidogenic dysfunction is associated with increased ghrelin expression in human testes.

     Key words: Leydig


Ghrelin has recently been identified as the endogenous ligand for the growth hormone (GH) secretagogue receptor (GHS-R) (Kojima et al, 1999; Kojima et al, 2002). Ghrelin is a 28-amino acid peptide with an essential n-octanoyl modification at Ser3. This peptide is primarily expressed in the stomach and hypothalamus (Kojima et al, 1999; Kojima et al, 2002). As expected for an endogenous ligand of GHS-R, ghrelin elicits GH secretion in vivo and from anterior pituitary cells in culture (Kojima et al, 1999; Takaya et al, 2000; Wren et al, 2000). In addition to its role in the control of GH release, ghrelin, probably derived from a stomach source and acting on hypothalamic centers, has recently been shown to induce a number of biological responses at the central neuroendocrine level, including stimulation of food intake and adiposity (Kamegai et al, 2000; Takaya et al, 2000; Wren et al, 2000). The biological effects of ghrelin identified to date are executed at the central levels of the hypothalamus and pituitary. In addition to these central biological effects, there is growing evidence for peripheral actions of ghrelin. In this regard, the novel expression of ghrelin in noncentral tissues, such as the ovary and kidney, is of interest (Tschop et al, 2000; Gaytan et al, 2003). Moreover, it has been shown that ghrelin and its functional receptor are expressed in rat and human testes (Barreiro et al, 2002; Tena-Sempereet al, 2002; Gaytan et al, 2004). Testicular function is critically dependent upon the interaction of a plethora of endocrine, paracrine, and autocrine regulatory signals (Skinner MK, 1991; Saez JM, 1994). It has become evident that different factors with key roles in growth axis (eg, GHRH and IGF-I) and body-weight homeostasis (eg, leptin) are involved in the regulation of testicular function (Ciampani et al, 1992; Baker et al, 1996; Tena-Sempere et al, 1999). In the human testis, the expression of ghrelin by Leydig cells is apparently linked to the degree of cell differentiation (Gaytan et al, 2004). Ghrelin has been observed in interstitial rat Leydig cells, and ghrelin induces the inhibition of human CG- and cAMP-stimulated testosterone secretion in vitro (Tena-Sempere et al, 2002). Thus, ghrelin may also participate in the regulation of testicular function. In order to investigate the function of ghrelin in the testes, we evaluated the relationships between the expression of ghrelin in human testes and the levels of serum FSH, LH, and T, as well as its effects on spermatogenesis.


   Materials and Methods
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Results
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Patients

Tissue specimens were obtained from the testes of 8 patients with obstructive azoospermia (mean ± SD age, 31.2 ± 4.2 years; range, 23–41 years), and 36 patients with varicocele (mean ± SD age, 34.8 ± 5.3 years; range, 25–47 years). Tissue specimens were also collected from five healthy fertile men (mean ± SD age, 38.7 ± 5.6 years; range, 32–46 years) as controls. The serum FSH, LH, and T concentrations were determined by chemiluminescence assays. Each blood sample was drawn between 0900 and 1000 hours. All the patients in the varicocele group had clinical varicocele and showed oligozoospermia. The patients in the control group were healthy volunteers who had at least one child and showed normozoospermia. Subject participation was voluntary, as indicated by free and informed consent. Our research at Kobe University is guided by the ethical principles regarding all research that involves humans as subjects. Testicular biopsies were performed after obtaining informed consent.Go


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  		Clinical data for patient groups*
 

Immunohistochemistry

The specimens were divided into two parts. One part was fixed in 10% formalin and the other part was fixed in Bouin solution for 12 hours at room temperature. The tissues were prepared in an automatic tissue processor using ascending ethanol concentrations, xylene, and paraffin wax. Serial paraffin sections (4-µm thick) were mounted on glass slides for staining. The formalin-fixed sections were deparaffinized in xylene through ethanol to phosphate-buffered saline (PBS; pH 7.2). To block endogenous peroxidase activity, 0.3% hydrogen peroxide in methanol was applied for 20 minutes. All of the slides were then incubated in normal goat serum (Vector Laboratories, Burlingame, Calif) for 40 minutes to block nonspecific binding. Primary antibodies were added to the slides and incubated for 60 minutes in a moist chamber at room temperature. Biotinylated anti-rabbit immunoglobulin G (IgG) was applied and incubated for 30 minutes in a moist chamber at room temperature. The development agent used was diaminobenzidine tetrahydrochloride. The tissue was counterstained with hematoxylin. The primary antibody was a rabbit antighrelin polyclonal (Santa Cruz Biotechnology, Santa Cruz, Calif), which was diluted 1:200 in PBS). All of the sides were covered with a coverslip after mounting in buffered glycerin. For the negative control, normal rabbit IgG (Upstate, Lake Placid, NY) was used as the primary antibody.

Slides in which there were at least 20 seminiferous tubular sections were examined under an Olympus light microscope that was equipped with a 40x objective. The number of ghrelin-positive Leydig cells and the total number of Leydig cells were counted. The number of seminiferous tubules was also counted. The average number of ghrelin-positive Leydig cells, total number of Leydig cells per seminiferous tubular section, and the ratio of ghrelin-positive Leydig cells to total Leydig cells were calculated for each case. Leydig cells were identified on the basis of the beta-HSD marker. More than 20 seminiferous tubular sections per testis were calculated.

Hematoxylin-eosin staining was performed on the specimens that were fixed in Bouin solution. The stained slides were observed under a light microscope that was equipped with a 20x objective. More than 20 seminiferous tubular sections per testis were assigned a Johnsen score that ranged from 1 to 10, as described previously (Johnsen, 1970). To calculate the Johnsen score, the sum of all the scores was divided by the total number of seminiferous tubular sections. More than 20 seminiferous tubular sections per testis were analyzed.

Statistics

Statistical analysis was performed using the non-parametric Mann-Whitney U-test to reveal differences between the control and patient groups. Correlations were tested by the Pearson correlation coefficient. P less than .05 was considered to be statistically significant.


   Results
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 Results
Discussion
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Clinical Examination

The average sperm concentration in the normozoospermia group was significantly higher than those in the varicocele (P < .01) and obstructive azoospermia (P < .001) groups. No significant differences in the average concentrations of FSH, LH, and T were found between the groups. The mean Johnsen score for the normozoospermia group was 9.0, which was within the 95% normal limit described by Johnsen (1970). The Johnsen score for the varicocele group was significantly lower than that for the normozoospermia group (P < .05) (Table).

Immunohistochemistry

The patterns of cellular expression of ghrelin protein were assessed by immunohistochemistry in the human testes of each group. Ghrelin-immunostained cells were detected in the interstitum, Leydig cells, seminiferous tubules, and Sertoli cells in all the groups. In the present study, we investigated ghrelin function in steroidogenic Leydig cells. The ratio of the number of ghrelin-immunostained Leydig cells to the total number of Leydig cell was high in subjects with low serum concentrations of T (Figure 1A). On the other hand, the ratio of ghrelin-immunostained Leydig cells was low in subjects with high serum concentrations of T.


Figure 1
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  		Figure 1. Testicular tissue from a patient with varicocele (Serum T concentration is 3.4 ng/ml) was immunostained with the antighrelin antibody (A). Ghrelin-immunostained cells (arrows) were detected in the interstitum, Leydig cells, and in the seminiferous tubule, spermatocytes. (B) For the negative control, normal rabbit IgG was used as the primary antibody.

 

Ghrelin Levels and Clinical Parameters

The ratios of ghrelin-immunostained Leydig cells in all the groups were significantly negatively correlated with the concentrations of T (r = –.50, P < .001; Figure 2), but were not significantly correlated with sperm concentrations (r = .19, P = .20), Johnsen scores (r = .18, P = .19) or the concentrations of LH and FSH (data not shown).


Figure 2
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  		Figure 2. The ratio of ghrelin-immunostained Leydig cell plotted against the serum T concentration, sperm concentration, Johnsen score in all groups.

 
The average ratio of ghrelin-immunostained Leydig cells was 42 ± 11% in all the groups. The ratio of ghrelin-immunostained Leydig cells was 38 ± 5.4%, 43 ± 10%, and 40 ± 9.1%, in the normal, varicocele, and obstructive azoospermia groups, respectively. No significant differences were found between the normal, varicocele, and obstructive azoospermia groups.


   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The biological effects of ghrelin have been restricted to its abilities to induce GH release acting through pituitary and hypothalamic GHS-Rs (Kojima, 1999; Wren, 2000) and to stimulate food intake through modulation of hypothalamic neuropeptide Y (NPY) and agouti-related protein expression (Wren, 2000; Gaytan, 2003). More recently, information has emerged on the biological actions of ghrelin on noncentral endocrine organs, such as the female and male gonads.

Ghrelin expression has been recently demonstrated in rat and human ovaries (Caminos, 2003; Gaytan, 2003). In the cyclic rat ovary, ghrelin immunoreactivity is mainly located in steroidogenic luteal cells. The role of systemic ghrelin in the control of ovarian function remains to be elucidated. Dynamic changes in the profile of rat ovarian expression of ghrelin during the estrus cycle and pregnancy are highly suggestive of a finely tuned regulatory network, in which endocrine (eg, gonadotropins) and locally produced factors (eg, ovarian steroids) are likely to participate (Caminos, 2003). Ghrelin and its functional type 1a receptor (GHS-R1a) are expressed in the cyclic human ovary (Gaytan, 2003).

In the mouse testis, a specific ghrelin gene-deprived transcript has been identified (Tanaka, 2001). The patterns of ghrelin and GHS-R1a expression in the testis are species-specific. In the rat testis, ghrelin immunoreactivity has been localized with high selectively in interstitial Leydig cells (Tena-Sempere, 2002), and GHS-R1a has been localized in Sertoli and Leydig cells (Barreiro, 2003). In human testis, ghrelin immunoreactivity has been localized in Leydig cells and Sertoli cells. On the other hand, GHS-R1a has been located in germ cells, mainly in pachytene spermatocytes, as well as in Leydig cells and Sertoli cells (Gaytan, 2004). Our data demonstrate ghrelin immunoreactivity in Leydig cells and Sertoli cells of the human testis.

In vitro, ghrelin significantly inhibits in a dose-dependent manner both hCG- and cAMP-stimulated T release (Tena-Sempere, 2002). The mechanisms and cell types involved in this inhibitory response are presently under investigation. The fact that ghrelin decreases to similar extents hCG- and cAMP-induced T secretion indicates that this inhibitory action takes place in a step beyond cAMP formation. This inhibitory effect of ghrelin on T secretion has been associated with significant decreases in the hCG-stimulated expression levels of the mRNAs for several key factors in the steroidogenic pathway, which include StAR, P450scc, 3ß-HSD, and testis-specific 17ß-HSD type III (Tena-Sempere, 2002).

On the other hand, acute administration of hCG to intact rats results in a transient increase in the testicular ghrelin mRNA levels (Barreiro, 2002). A tempting explanation for this discrepancy is that ghrelin may operate as a local regulator in the fine tuning of the steroidogenic actions of LH (Barreiro, 2002). In the present study, the serum LH level was not correlated with the expression of ghrelin on Leydig cells of the human testis. Since the pattern of ghrelin expression differs between rats and humans, the LH effect on ghrelin may differ between rats and humans.

There have been no reports concerning the relationship between the expression of ghrelin in human testis and the serum T level. In the present study, ghrelin expression in Leydig cells was inversely correlated with the serum T levels in patients with normozoospermia, obstructive azoospermia or varicocele. The biological actions of ghrelin are implemented via interaction with its receptor, GHS-R1a. Thus, in Leydig cells, the interaction of overexpressed ghrelin with GHS-R1a may lead to the inhibition of T production in the human testis. The mechanisms by which ghrelin inhibits testosterone are under investigation in our laboratory. As mentioned above, the decreased expression of StAR, P450scc, 3ß-HSD, and 17ß-HSD type III mRNAs in the steroidogenic pathway may contribute to ghrelin-induced inhibition of T production in the human testis. It is possible that a positive interaction occurs between the activated pathway of ghrelin and the hCG-adenylate cyclase pathway stimulated concomitantly by ghrelin and hCG.

The spermatogenic function of ghrelin is poorly characterized. In the present study, ghrelin expression in Leydig cells was not correlated with the sperm concentration or Johnsen score of patients with normozoospermia, obstructive azoospermia or varicocele. Regrettably, we did not perform analyses for abnormal spermatogenesis (in subjects with much lower Johnsen scores [ie, with Sertoli cell only syndrome]), and maturation arrest in the present study. Ghrelin may operate as a local regulator in the fine-tuning of spermatogenic function.

In conclusion, our immunohistochemical analyses show that the expression of ghrelin by Leydig cells is inversely correlated with the serum T concentration, and we propose that ghrelin has an indirect effect on spermatogenesis.


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