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Expression of Growth Hormone Secretagogue Receptor Type 1a, the Functional Ghrelin Receptor, in Human Ovarian Surface Epithelium, Mullerian Duct Derivatives, and Ovarian Tumors

Departments of Cell Biology, Physiology, and Immunology (F.G., M.L.B., E.A., M.T.-S.) and Pathology (C.M.), University of Córdoba, 14004 Córdoba, Spain; Departments of Physiology (C.D.) and Medicine (F.F.C.), University of Santiago de Compostela, 15705 Santiago de Compostela, Spain; and School of Life Sciences (P.J., L.K.C., A.C.H.), Cluster for Molecular Biotechnology, Queensland University of Technology, 4001 Brisbane, Queensland, Australia

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

	Abstract

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Ghrelin, the endogenous ligand of the GH secretagogue receptor (GHS-R), is a newly identified, ubiquitously expressed molecule that has been involved in a wide array of endocrine and nonendocrine functions, including cell proliferation. In this context, our group recently reported the expression of ghrelin and its functional receptor, the GHS-R type 1a, in the human ovary and testis as well as several testicular tumors. Ovarian malignancies, however, remain unexplored. Notably, a vast majority of ovarian tumors derive from the surface epithelium, which originates from the celomic epithelium. Considering the proven expression of ghrelin in the human ovary, and its reported effects in the proliferative activity of different cancer cell lines, we aimed at evaluating whether the ovarian surface epithelium as well as related reproductive structures and tumors are potential targets of ghrelin. To this end, expression of GHS-R1a was analyzed by immunohistochemistry in a panel of normal, metaplastic, and neoplastic tissues. Uniform GHS-R1a immunostaining was detected throughout the ovarian surface epithelium. Likewise, ciliated cells within the fallopian tube epithelium showed strong GHS-R1a expression. In contrast, other celomic derivatives, such as endometrium and endocervix, were negative for GHS-R1a immunoreactivity. In keeping with data from normal tissues, inclusion cysts from the surface epithelium expressed GHS-R1a. Similarly, benign serous tumors resembling fallopian tube epithelium were also positive, whereas serous cystadenocarcinomas showed GHS-R1a expression only in highly differentiated specimens. In contrast, other neoplasms, such as mucinous cystadenomas and cystadenocarcinomas, endometrioid tumors, clear cell carcinomas, and Brenner tumors, did not express GHS-R1a. In conclusion, our results demonstrate that the ovarian surface epithelium and related tumors are potential targets for systemic or locally produced ghrelin because they express the functional type 1a GHS-R. Considering the relevant role of the ovarian surface epithelium in key physiological events (such as ovulation) and neoplastic transformation of the ovary, the potential actions of ghrelin in those phenomena merit further investigation.

	Introduction

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GHRELIN HAS BEEN recently identified as the endogenous ligand for the GH-secretagogue receptor (GHS-R) (1, 2). Although this molecule was originally proven to elicit GH secretion in different species, including humans (1, 2, 3, 4, 5) and induce food intake in humans and rodents (3, 4, 5, 6, 7), compelling evidence has now demonstrated that ghrelin carries out an array of additional endocrine and nonendocrine biological actions, which include neuroendocrine control of lactotropic, corticotropic, and gonadotropic axes; metabolic and cardiovascular effects; and regulation of cell proliferation in different tissues and tumors (3, 4, 8, 9, 10, 11). The cognate ghrelin receptor, the GHS-R, belongs to the large family of G protein-coupled, seven transmembrane domain receptors (12, 13). Two GHS-R subtypes, generated by alternative splicing of a single gene, have been described so far: the full-length type 1a receptor and the truncated GHS-R type 1b (12, 13). The GHS-R1a is the functionally active, signal transducing form of the receptor. In contrast, the GHS-R1b lacks the transmembrane domains 6 and 7, and it is apparently devoid of high-affinity ligand binding and signal transduction capacity (12). Thus, its functional role, if any, remains unclear.

The mammalian ovary is a complex endocrine organ responsible for oocyte release at ovulation and hormonogenesis (14). At adulthood the cellular structures supporting those functions are arranged into several tissue compartments including ovarian follicles at different stages of development, corpora lutea, ovarian stroma, and hilus (14, 15). Notably, ovarian parenchyma is encased by a layer of cells resting on a basement membrane, which forms the ovarian surface epithelium (OSE). The OSE varies morphologically from cuboidal to columnar, and it is embryologically derived from the celomic epithelium covering the gonadal ridge (16). In addition, the celomic epithelium in the vicinity of the gonadal ridges invaginates at early stages of development to give rise also to the Mullerian ducts, from which the epithelia of the fallopian tubes, endometrium, and cervix are derived (17).

Despite the classical contention that the OSE is devoid of a major functional role in ovarian physiology, emerging evidence indicates that, as an obligate component of the ovulatory process, it is essential during follicle rupture at the ovarian surface and in postovulatory wound repair (16, 18). Aside from its role in ovulation, considerable attention has been recently focused in the OSE because it became apparent that almost 90% of ovarian cancers, the most lethal among gynecological malignancies, arise from the surface epithelium or its derivatives (i.e. epithelial inclusion cysts) (16, 18, 19). Although the etiology of ovarian cancer remains unsolved, an intriguing aspect of the OSE is its tendency to differentiate into the more complex Mullerian duct-derived epithelia, acquiring the architectural and functional characteristics of Mullerian duct derivatives (i.e. Mullerian metaplasia) (16, 20). This likely reflects the close embryological relationship between the OSE and Mullerian duct derivatives and suggests that ovarian epithelial cells are less committed than other celomic-derived epithelia and have the potential to undergo metaplastic changes, as well as epithelial-mesenchymal transitions, in the course of their neoplastic progression (21).

Recently we provided evidence for the expression of ghrelin and its functional receptor in the human ovary. In detail, the presence of ghrelin was demonstrated in ovarian hilus interstitial cells as well as young and mature corpora lutea, whereas ghrelin was not detected in ovarian follicles at any developmental stage (22). In turn, expression of GHS-R1a protein in the human ovary showed a wider pattern of tissue distribution, with detectable expression in oocytes; somatic follicular cells; luteal cells from young, mature, old, and regressing corpora lutea; and, to a lesser extent, interstitial hilus cells (22). To date, the functional role of the ghrelin system in the ovary remains unexplored. Notably, the biological actions of ghrelin have been addressed in the male gonad, in which it is involved in the regulation of steroidogenesis and, probably, proliferation of interstitial Leydig cells (23, 24).

Among other peripheral actions, a role for ghrelin in the autocrine/paracrine control of cell proliferation and cancer has been recently proposed. However, conflicting results have thus far been reported (11). Thus, proliferative actions of ghrelin (or its synthetic analogs, the GHSs) have been observed in prostate (PC-3) and thyroid (ARO) cancer cell lines as well as the cardiomyocyte H9c2 cell line and cultured adrenocortical cells (11, 25, 26, 27). However, antiproliferative effects of ghrelin/GHSs have also been reported in several thyroid (NPA, WRO), lung (CALU-1), and breast (MCF7, T47D, MDA-MB231) cancer cell lines (25, 28, 29) as well as immature Leydig cells of the testis (24). Although the basis for such a discrepancy remains obscure, it is likely that differences in cell types and expression of GHS-Rs may contribute to this phenomenon.

On the basis of the proven expression of ghrelin in the cyclic human ovary (24) and the reported effects of this molecule on the proliferative activity of different normal and neoplastic cell types (11, 24, 25, 26, 27, 28, 29), we aimed at evaluating whether the OSE and related genital structures and tumors are potential targets for the actions of ghrelin. To this end, expression of GHS-R1a, the functional ghrelin receptor, was analyzed by immunohistochemistry in a panel of normal, metaplastic, and neoplastic tissues, including the surface epithelium of the ovary, Mullerian duct derivatives (such as fallopian tube and endometrial and endocervix epithelia), epithelial inclusion cysts, and different benign and malignant ovarian tumors.

	Materials and Methods

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Tissue samples

Sections from normal human cyclic ovaries and Mullerian duct derivatives were obtained from the files of the Department of Pathology of the University of Cordoba, on approval of the University of Cordoba Bioethics Committee. In detail, tissue sections from hysterectomized and bilaterally salpingo-oophorectomized women were used. Retrospective review of their medical records confirmed that patients did not show endocrine ovarian pathology, and they were not undergoing hormonal treatment. Concerning the ovarian tissue, normal cyclicity was confirmed by the presence of a corpus luteum of the current cycle (during the luteal phase) and a regressing corpora lutea of the preceding cycle (during the follicular phase). The day of the cycle was assigned by considering the menstrual history and dating of the endometrium (30) and corpus luteum (31). The standard cycle was considered to be 28 d and was divided into follicular (from d 1 to d 14) and luteal (from d 15 to d 28) phases. At least five ovaries per phase were studied. Similarly, at least five specimens of fallopian tube and endometrial and endocervix tissue were analyzed. A summary of the characteristics of the normal tissues processed for immunohistochemical detection of GHS-R1a is shown in Table 1.

Polyclonal anti-GHS-R1a antibody

For analysis of GHS-R1a protein expression, immunohistochemical labeling was conducted using a rabbit polyclonal antibody generated against a synthetic peptide corresponding to the C-terminal fragment (RAWTESSINTC) of the human GHS-R1a protein conjugated to diphtheria toxin (Mimotopes, Melbourne, Australia), as described in detail previously (32). Western analyses using this antibody demonstrated a single specific band of approximately the predicted size (45 kDa) for the GHS-R1a in the ALVA-41 and DU145 prostate cancer cell lines (Chopin, L. K., and A. C. Herington, personal observation), which have been proven to express the GHS-R1a mRNA isoform and GHS-R1a protein (32). This antiserum has been previously used by our group for the immunohistochemical detection of GHS-R1a protein in human male and female gonads (22, 33).

Immunohistochemistry

Immunohistochemistry was performed on routinely neutral-buffered formaldehyde-fixed, paraffin-embedded tissues. In detail, ovarian sections (5 µm thick) were placed on poly-L-lysine-coated slides and, after dewaxing in xylene and rehydration in ethanol, the samples were incubated in 2% hydrogen peroxide in methanol for 30 min to quench endogenous peroxidase followed by washing in PBS. In addition, sections were immersed in 10 mM citrate buffer and submitted to antigen retrieval in a microwave oven (2 x 5 min at 700 W). Sections were allowed to cool at room temperature, washed in PBS, blocked with normal serum, and incubated overnight with the primary anti-GHS-R1a antibody (diluted 1:10). The sections were then processed according to the avidin-biotin-peroxidase complex technique following previously described methods (34). Negative controls were run routinely in parallel by replacing the primary antibody by preimmune serum or PBS. In addition, positive controls for GHS-R1a immunostaining were assayed. These included reactions in human ovarian (parenchyma), testicular, and pituitary sections, conducted using anti-GHS-R1a primary antibody, which yielded strong immunoreactivity in line with our previous data (22, 33). As an additional control for the specificity of GHS-R1a antibody, immunohistochemical reactions were carried out in human pituitary and testicular tissues after preabsorption of the antiserum overnight at 4 C with 1 mg/ml of the synthetic peptide (RAWTESSINTC) against which it was raised. In keeping with our previous results (22, 33), this procedure completely abolished immunolabeling of pituitary and testicular sections (data not shown). For presentation of data (Table 1), intensity of specific GHS-R1a immunostaining in the OSE, related tissue samples and tumor specimens were estimated on a semiquantitative basis, using a 0–3 scale: +++, strongly positive; ++, clearly positive; +, weakly positive; and –, negative, in agreement with previous reports (33).

	Results

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GHS-R1a expression in OSE and Mullerian duct derivatives

Immunohistochemical analyses, using a previously characterized specific anti-GHS-R1a polyclonal antibody (22, 32, 33), demonstrated the presence of GHS-R1a protein in the normal human surface epithelium of the ovary, in which uniform GHS-R1a immunostaining was found in areas showing either low cuboidal or tall columnar cells (Fig. 1A). Evenly distributed GHS-R1a immunoreactivity was detected in the OSE regardless of the phase (follicular or luteal) of the cycle. Concerning Mullerian duct derivatives, strong GHS-R1a immunostaining was detected in the epithelium of the fallopian tubes, although the signal was restricted to ciliated cells, whereas the protein was absent in secretory cells (Fig. 1, B and C). Cell-specific immunoreactivity in the fallopian tube epithelium was independent of the phase of the cycle (Fig. 1, B and C). On the contrary, no GHS-R1a immunostaining was present in the endometrium (in either the proliferative or secretory phase of the cycle) or endocervix (Fig. 1, D and E). A comprehensive compilation of GHS-R1a expression data in normal human tissue samples under analysis is included in Table 1.

View larger version (136K): [in this window] [in a new window]   FIG. 1. Analysis of immunolocalization of GHS-R1a protein in normal human OSE (A, inset) and several Mullerian duct derivatives, including fallopian tube epithelium (B and C) and endometrium and endocervix (D and E). Clear-cut GHS-R1a immunostaining was detected in the OSE, with a continuous pattern regardless of the presence of low cuboidal or tall columnar cells (A). Similarly, strong GHS-R1a immunoreactivity was observed in the fallopian tube epithelium, although peptide signal was selectively restricted to ciliated cells (secretory cells lacked specific GHS-R1a immunoreactivity) regardless of the follicular (B) or luteal (C) phase of the cycle. In contrast, neither endometrial (D) nor endocervix (E) tissue sections showed specific GHS-R1a immunostaining in any of the specimens under analysis. Scale bars are shown in the panels.

In addition to normal tissues, the pattern of cellular distribution of GHS-R1a protein was evaluated in different metaplastic and neoplastic ovarian samples. In detail, a number of epithelial inclusion cysts and ovarian neoplasms were analyzed. For the latter, specimens corresponding to the most frequent ovarian surface-derived tumors included serous benign cystadenoma, mucinous benign cystadenoma, mixed benign cystadenoma, serous cystadenocarcinoma, mucinous cystadenocarcinoma, endometrioid carcinoma, Brenner tumor, and clear cell carcinoma. Epithelial inclusion cysts showed strong GHS-R1a immunoreactivity (Fig. 2, A and B). Notably, cysts without obvious metaplasia exhibited an evenly distributed pattern of GHS-R1a immunostaining (Fig. 2A), whereas in those cysts showing clear ductal metaplasia, i.e. cells in which morphological features of secretory and ciliated cells were observed, GHS-R1a immunoreactivity presented a discontinuous pattern, the signal being strongly located only in ciliated cells (Fig. 2B). Serous ovarian tumors were characterized by cell types resembling those of the fallopian tube epithelium. In benign serous tumors (serous cystadenoma), the thin-walled cysts were lined by ciliated epithelial cells and, less frequently, nonciliated secretory cells, with strong GHS-R1a immunostaining being observed mainly in ciliated cells (Fig. 2C). In contrast, in benign mucinous tumors (mucinous cystadenoma), characterized by the presence of glands and cysts lined by mucin-filled columnar cells resembling those of the endocervix, immunostaining was totally absent (Fig. 2D). Accordingly, mixed benign tumors, composed of an admixture of serous and mucinous components, showed positive GHS-R1a immunoreactivity in the serous areas and negative in mucinous ones (Fig. 2, E and F). In malignant serous tumors (serous cystadenocarcinoma), the presence of immunostaining was dependent on the degree of differentiation. Well-differentiated (low-grade) serous cystadenocarcinomas, showing papillary structures, were weakly positive (Fig. 2G), whereas immunostaining was negative in undifferentiated (high-grade) serous carcinomas, showing confluent cellular growth and solid sheets of cells (data not shown). Otherwise, benign Brenner tumors (composed of nests of epithelial cells resembling urothelium), malignant mucinous tumors (mucinous cystadenocarcinoma), endometrioid tumors (closely resembling endometrial tumors), and clear cell carcinomas (showing polyhedral cells with clear cytoplasm) were negative (data not shown). A comprehensive compilation of GHS-R1a expression data in pathological human samples under analysis is included in Table 1.

View larger version (128K): [in this window] [in a new window]   FIG. 2. Analysis of immunolocalization of GHS-R1a protein in pathological samples from human ovaries. In detail, sections from epithelial inclusion cysts (A and B), serous cystadenoma (C), mucinous cystadenoma (D), mixed cystadenoma (E and F), and serous cystadenocarcinoma (G) are shown. Epithelial inclusion cysts showed strong GHS-R1a immunostaining, with a continuous pattern in areas without obvious tubal metaplasia (A; arrows). In contrast, inclusion cysts displaying clear tubal metaplasia exhibited a discontinuous pattern with strongly immunostained ciliated cells (B; arrows and inset). Similarly, benign serous cystadenomas showed a fallopian tube-like immunostaining, with strongly GHS-R1a positive ciliated cells (C; arrows and inset). In contrast, mucinous cystadenomas were negative for GHS-R1a immunolabeling (D). In mixed cystadenomas (E and F), serous areas were positive for GHS-R1a signal, whereas mucinous areas did not show GHS-R1a immunoreactivity. Finally, positive GHS-R1a immunostaining was detected in low-grade cystadenocarcinomas (G), whereas undifferentiated (high-grade) serous carcinomas were negative for GHS-R1a signals. Scale bars are indicated in the panels.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Concerning normal specimens, GHS-R1a protein was detected in some of the structures derived from the celomic epithelium (OSE and fallopian tube) but not in others (endometrium and endocervix). The physiological significance of such a tissue-specific pattern of expression remains to be elucidated. Notably, both the OSE and fallopian tube epithelium play important roles in key reproductive events such as ovulation and postovulatory repair (ovarian epithelium) and conditioning of the microenvironment for fertilization and early embryo development (fallopian epithelium) (16, 18). Despite its morphological simplicity, recent studies pointed out the complexity of the OSE, whose pattern of cellular growth and function is under the precise control of a plethora of different regulatory signals, including systemic hormones and locally produced factors (16, 18). In this sense, besides the well-proven regulatory roles of gonadotropins and sex steroids (16, 18, 35, 36, 37, 38), the contribution of an array of autocrine/paracrine growth factors in the control of the OSE has been recently demonstrated. Considering our previous data on the expression of ghrelin in human ovarian parenchyma (22), it is tempting to propose that the ghrelin/GHS-R1a system might be a novel member of the regulatory network involved in the local control of the OSE in normal conditions. Noteworthy, although detailed expression analyses of ghrelin itself are yet to be completed, our preliminary assays evidence that ghrelin immunoreactivity is also detectable in the normal human OSE (our unpublished observation).

Previous data from our group demonstrated that ghrelin is involved in the control of steroidogenesis and cell proliferation in rat testis (23, 24). However, direct assessment of the potential direct ovarian actions of ghrelin has not yet been conducted. Our present data provide the basis for the analysis of ghrelin actions on the OSE and related structures. In this sense, the OSE is not considered as a classical steroidogenic tissue. Yet it possesses some of the phenotypic characteristics of steroid-producing tissues, such as expression of steroidogenic factor 1, some steroidogenic enzymes, and gonadotropin receptors (38, 39, 40). Ghrelin, probably acting through GHS-R1a, has been previously reported to inhibit the expression of several key factors in the steroidogenic route in the testis (23, 41). Whether similar effects are conducted on the ovarian surface epithelial cells remains to be evaluated. In addition, ghrelin has been recently involved in the control of testicular cell proliferation because it suppressed the proliferative rate of immature testicular Leydig cells and was able to inhibit stem cell factor (SCF) gene expression in the adult testis (24). Because SCF has been identified as a relevant growth factor in the local control of the surface epithelium of the ovary (42), our current data on the expression of GHS-R1a in this system open up the possibility that ghrelin might participate in regulation of the OSE through modulation of SCF expression. This hypothesis is yet to be proven.

The precise etiology of ovarian cancer is still a matter of debate (for a review, see Refs.16 and 18). Nevertheless, experimental and epidemiological data support the contention that mitotically active ovarian cells are more susceptible to mutagenic events than quiescent cells. Considering that almost 90% of ovarian tumors originate from the OSE or its derivatives, i.e. epithelial inclusion cysts (16, 18, 19), it becomes evident that complete knowledge of the factors regulating ovarian surface epithelial cell proliferation is essential for a better understanding of the mechanisms underlying the neoplastic transformation of this epithelium. On the basis of our expression data in normal tissues and the reported effects of ghrelin on the proliferation of different tumor cell types (10, 11), a number of metaplastic and neoplastic ovarian samples were assayed for GHS-R1a immunoreactivity. Assumedly, larger series of specimens for each pathological condition might be required to unequivocally establish epidemiological trends. Nonetheless, our current analysis in representative metaplastic lesions and tumors of the ovary demonstrated that the pattern of expression and cellular distribution of the functional ghrelin receptor closely correlates with that observed in normal tissues and is partially defined by the degree of differentiation of the neoplasm. Thus, inclusion cysts from the surface epithelium and benign serous tumors resembling fallopian tube epithelium expressed GHS-R1a, whereas serous cystadenocarcinomas showed GHS-R1a expression, albeit at lower levels, only in highly differentiated specimens. In contrast, other tumors, such as mucinous cystadenomas and cystadenocarcinomas, endometrioid tumors, clear cell carcinomas, and Brenner tumors, did not express GHS-R1a. Overall, such a pattern of tissue- and cell-specific expression of GHS-R1a in normal, metaplastic, and neoplastic specimens reinforces the contention that ghrelin receptors are selectively present in some of the structures derived from the celomic epithelium (OSE, fallopian tube, and similar neoplasms) but not in others (endometrium, endocervix, and like tumors). Noteworthy, malignant tumors derived from the OSE showed null, or at the best low levels of, GHS-R1a immunoreactivity, a phenomenon whose relevance merits further investigation.

Expression of GHS/ghrelin binding sites and/or the GHS-R gene has been previously demonstrated in a wide variety of tumors, including pituitary adenomas and other neuroendocrine tumors, neoplastic thyroid tissue, human breast carcinoma, prostate cancer cell lines, pancreatic islet cell tumors, and different testicular tumors (25, 28, 32, 33, 43, 44, 45). Present data enlarge those previous observations and evidence that GHS-R1a immunoreactivity is detectable in relevant ovarian tumors. Given the proposed regulatory effect of ghrelin and GHSs on cell proliferation in different tumor cell lines (10, 11), our present findings provide the basis for direct action of these compounds on different ovarian tumors and metaplastic lesions, e.g. epithelial inclusion cysts, serous cystadenomas and well-differentiated cystadenocarcinomas, as well as the potential role of ghrelin signaling in the control of proliferation of ovarian tumor cells. It has to be noted, however, that, depending on the tumor type, antiproliferative and proliferative effects of ghrelin and synthetic GHSs have been reported (11) and that at least some of the antiproliferative actions of GHSs are apparently conducted through a GHS-R1a-independent pathway (29).

In conclusion, our immunohistochemical analyses provide compelling evidence for the presence of the putative functional receptor of ghrelin, the type 1a GHS-R, in the surface epithelium of the adult cyclic ovary as well as some of the Mullerian duct derivatives (fallopian tube epithelium) and several metaplastic and neoplastic lesions from the ovarian epithelium. Considering the relevant role of the OSE in key physiological events (such as ovulation) and neoplastic transformation of the ovary, the potential actions of ghrelin in those phenomena merit further investigation.

	Acknowledgments

 
Helpful discussions with L. Pinilla during preparation of the manuscript are cordially appreciated. The authors are indebted to P. Cano for her excellent technical support in conducting immunohistochemical analyses. The skillful assistance of E. Tarradas in preparation of photomicrographs is acknowledged.

	Footnotes

 
This work was supported by Grants BFI2000-0419-CO3-03 and BFI2002-00176 from DGESIC (Ministerio de Ciencia y Tecnología, Spain), European Union Research Contract EDEN QLK4-CT-2002-00603, and the National Health and Medical Research Council of Australia.

First Published Online December 7, 2004

Abbreviations: GHS-R, GH secretagogue receptor; OSE, ovarian surface epithelium; SCF, stem cell factor.

Received August 3, 2004.

Accepted November 23, 2004.

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