Ghrelin, the endogenous ligand for the 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. In this context, the expression of ghrelin and its cognate receptor has been recently demonstrated in rat testis, suggesting a role for this molecule in the direct control of male gonadal function. However, whether this signaling system is present in human testis remains largely unexplored. In this study we report the expression and cellular location of ghrelin and its functional receptor, the type 1a GHS-R, in adult human testis. In addition, evaluation of ghrelin and GHS-R1a immunoreactivity in testicular tumors and dysgenetic tissue is presented. The expression of the mRNAs encoding ghrelin and GHS-R1a was demonstrated in human testis specimens by RT-PCR, followed by direct sequencing. In normal testis, ghrelin immunostaining was demonstrated in interstitial Leydig cells and, at lower intensity, in Sertoli cells within the seminiferous tubules. In contrast, ghrelin was not detected in germ cells at any stage of spermatogenesis. The cognate ghrelin receptor showed a wider pattern of cellular distribution, with detectable GHS-R1a protein in germ cells, mainly in pachytene spermatocytes, as well as in somatic Sertoli and Leydig cells. Ghrelin immunoreactivity was absent in poorly differentiated Leydig cell tumor, which retained the expression of GHS-R1a peptide. In contrast, highly differentiated Leydig cell tumors expressed both the ligand and the receptor. The expression of ghrelin and GHS-R1a was also detected in dysgenetic Sertoli cell-only seminiferous tubules, whereas germ cell tumors (seminoma and embryonal carcinoma) were negative for ghrelin and were weakly positive for GHS-R1a. In conclusion, our results demonstrate that ghrelin and the type 1a GHS-R are expressed in adult human testis and testicular tumors. Overall, the expression of ghrelin and its functional receptor in human and rat testis, with roughly similar patterns of cellular distribution, is highly suggestive of a conserved role for this newly discovered molecule in the regulation of mammalian testicular function.

Taken together, our results suggest that, at least in rodents, adiponectin may be involved in the regulation of several placental functions.

Objective: To assess whether some of the alterations in energy homeostasis present in thyroid function disorders and GH deficiency could be mediated by ghrelin. Design: To assess the influence of thyroid status on ghrelin, adult male Sprague -Dawley rats were treated with vehicle (euthyroid), amino-triazole (hypothyroid) or l-thyroxine (hyperthyroid). The influence of GH on ghrelin was assessed in wild-type (control) and GH-deficient (dwarf) Lewis rats. Evaluation of gastric ghrelin mRNA expression in the stomach was carried out by Northern blot. Circulating levels of ghrelin were measured by radioimmunoassay. Results: Hypothyroidism resulted in an increase in gastric ghrelin mRNA levels (euthyroid: 1003 :2% vs hypothyroid: 127:3^6:5%; P , 0:01), being decreased in hyperthyroid rats (70^5:4%; P , 0:01). In keeping with these results, circulating plasma ghrelin levels were increased in hypothyroid (euthyroid: 124^11 pg=ml vs hypothyroid: 262^39 pg=ml; P , 0:01) and decreased in hyperthyroid rats (75^6 pg=ml; P , 0:01). Using an experimental model of GH deficiency, namely the dwarf rat, we found a decrease in gastric ghrelin mRNA levels (controls: 100^6% vs dwarf: 66^5:5%; P , 0:01) and circulating plasma ghrelin levels (controls: 124^12 pg=ml vs dwarf: 81^7 pg=ml; P , 0:01). Conclusion: This study provides the first evidence that ghrelin gene expression is influenced by thyroid hormones and GH status and provides further evidence that ghrelin may play an important role in the alteration of energy homeostasis and body weight present in these pathophysiological states.

Ghrelin, a GH-releasing acylated peptide, has been recently identified from the rat stomach. The purified peptide consists of 28 amino acids in which the serine 3 residue is n-octanoylated. Here we show that ghrelin messenger RNA and ghrelin peptide are present in the human as well as in rat placentae. In human placenta, ghrelin was detected by PCR at both first trimester and after delivery. While ghrelin was not detected by immunohistochemistry in human placenta at term, it was easily identified by immunohistochemistry at first trimester being mainly expressed in cytotrophoblast cells and scarcely in syncytiotrophoblast ones. Ghrelin was also identified in a human choriocarcinoma cell line, the BeWo cells. Ghrelin was found, by immunohistochemistry, in the cytoplasm of labyrinth trophoblast of rat placenta, whereas other placental cell types seems to be negative for ghrelin immunostaining. Moreover, placental ghrelin messenger RNA, in pregnant rats, showed a characteristic profile of expression being practically undetectable during early pregnancy, with a sharp peak of expression at day 16 and decreasing in the latest stages of gestation. In conclusion, ghrelin has been detected in human and rat placenta showing a pregnancy-related time course of expression. Whether placenta-derived ghrelin is involved in the modulation of GH release, or placental cell growth and differentiation remains to be established.

Objective: The recently isolated endogenous GH secretagogue, named ghrelin, is a gastric peptide of 28 amino acids with an n-octanoylation in the serine 3 that confers the biological activity to this factor. Ghrelin has been shown to directly stimulate GH release in vivo and in vitro and to be involved in the regulation of gastric acid secretion and motility. In the present work we have studied gender and gonadal dependency of ghrelin mRNA expression in rat stomach. Design and Methods: We analysed ghrelin mRNA expression in rat stomach by Northern blot analysis. We also examined the effect of gonadal steroid deprivation on ghrelin mRNA expression. Results and Conclusions:The results obtained showed clearly that ghrelin gastric mRNA expression increased with age in young rats (up to 90 days old) but exhibited no significant sex difference at each age tested. Ghrelin mRNA levels were lowest at postnatal day 9, reaching a stable level of expression at day 40 in both female and male rats, although the increase in female rats appears much more gradual than that in males. Moreover, neither ovariectomy nor orchidectomy significantly modified ghrelin mRNA gastric levels in adult rats. In conclusion, these data indicate that ghrelin mRNA expression is associated with age and that a progressive increase is present from the perinatal period up to a stable level after puberty. Gonadal hormones did not alter ghrelin mRNA levels. Taken together, these data showed that ghrelin mRNA levels in young rats are age but not gender dependent, and are not influenced by gonadal steroids.