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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-1147
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 11 4562-4570
Copyright © 2006 by The Endocrine Society

Immunolocalization of Gonadotropin-Releasing Hormone (GnRH)-I, GnRH-II, and Type I GnRH Receptor during Follicular Development in the Human Ovary

Jung-Hye Choi, C. Blake Gilks, Nelly Auersperg and Peter C. K. Leung

Department of Obstetrics and Gynecology (J.-H.C., N.A., P.C.K.L.), Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada V6H 3V; and Department of Pathology (C.B.G.), Vancouver General Hospital and University of British Columbia, Vancouver, British Columbia, Canada V5Z 1M9

Address all correspondence and requests for reprints to: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynecology, University of British Columbia, 2H-30, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. E-mail: peleung{at}interchange.ubc.ca.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: GnRH and its receptor have been detected at the mRNA level in different ovarian cell types, implicating an autocrine role of the GnRH system in the human ovary. However, the expression at the protein level of GnRH and its receptor in specific cell types during follicular development has not been documented in humans.

Objective: We evaluated the immunohistochemical expression of GnRH-I (the classical form of mammalian GnRH), GnRH-II (the novel isoform), and the type I GnRH receptor (GnRHR) that is known to bind both forms of GnRH, in ovaries of premenopausal women.

Main outcome measures: Immunohistochemistry, immunofluorescence, immunoblot assay, and real-time RT-PCR were performed.

Results: GnRH-I, GnRH-II, and GnRHR were not immunostained in the follicles from the primordial to the early antral stage. In preovulatory follicles, both forms of GnRH and their common receptor were localized predominantly to the granulosa cell layer, whereas the theca interna layer was weakly positive. In the corpus luteum, significant levels of GnRH-I, GnRH-II, as well as GnRHR were observed in granulosa luteal cells, but not in theca luteal cells. Both GnRH isoforms and the type I GnRHR were localized also to the ovarian surface epithelium from which over 85% of ovarian cancers are thought to be derived.

Conclusion: The expression of GnRH-I, GnRH-II, and GnRHR protein in the human ovary is temporally and spatially specific and further supports the physiological role of an autocrine regulatory system involving GnRH-I, GnRH-II, and GnRHR in follicular development and corpus luteal function.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
OVARIAN FOLLICULOGENESIS, OVULATION, and luteinization are complex and dynamic processes requiring locally produced hormones and growth factors that act by autocrine or paracrine mechanisms as well as endocrine factors such as the pituitary gonadotropins (1). Our current knowledge of the factors that control folliculogenesis and corpus luteal function, especially in the human ovary, is still limited.

The hypothalamic decapeptide GnRH is a key neuroendocrine regulator in the mammalian reproductive system. It is released in a pulsatile manner from hypothalamic GnRH neurons and regulates the biosynthesis and secretion of gonadotropins from pituitary gonadotropes. To date, 12 isoforms of GnRH have been identified in vertebrates. In addition to the classical form of mammalian GnRH (GnRH-I), a second form of GnRH (GnRH-II) that is identical to chicken GnRH-II has recently been found in the brains of primates, including the human (2). Besides the hypothalamus and pituitary gland, GnRH-I, GnRH-II, and their mutual receptor [i.e. type I GnRH receptor (GnRHR)] have also been shown to be expressed in extrapituitary tissues including the ovary (3, 4).

In the human ovary, we and others have found that GnRH-I, GnRH-II, and GnRHR are expressed, at the mRNA level, in human granulosa luteal cells (hGLC), immortalized ovarian granulosa-luteal (SVOG) cell lines, normal ovarian surface epithelium (OSE) cells, immortalized OSE (IOSE) cells, as well as in ovarian cancer cell lines (4, 5, 6, 7). Furthermore, we have demonstrated the presence of GnRHR protein in cultured primary and immortalized hGLC using Western blot analysis (8). Other investigators have reported specific binding sites for GnRH-I in homogenates of human corpus luteum throughout the luteal phase of the menstrual cycle and early pregnancy (7). Increasing evidence has indicated that both isoforms of GnRH are biologically active in the human ovary. For instance, it has been reported that, similar to rat granulosa cells, GnRH-I and GnRH-II are capable of altering the basal and gonadotropin-stimulated progesterone secretion in hGLC (4, 5, 9, 10). Hoechst 33258 staining showed increased apoptotic bodies in hGLC after GnRH-I treatment (11). In human OSE and ovarian cancer cells, anti-proliferative and apoptosis-inducing effects of GnRH-I and -II have been demonstrated (12, 13, 14). Furthermore, there is evidence that the expression of GnRH-I, GnRH-II, and GnRHR in OSE cells and hGLC is regulated, sometimes differentially, by other reproductive hormones such as ovarian steroids and gonadotropins (4, 5, 6, 15, 16). Thus, these observations in the human ovary have strongly implicated an autocrine regulatory role of GnRH-I and GnRH-II in the development and function of follicles and corpora lutea in the human ovary. The present study was designed to investigate the spatiotemporal expression of GnRH-I, GnRH-II, and GnRHR, at the protein level, during ovarian follicle development in the human. The results will provide direct evidence of GnRH-I, GnRH-II, and GnRHR protein expression in distinct cell populations of the human ovary and support the physiological role of the autocrine GnRH regulatory system in the human ovary.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Tissues and cells

The human pituitary tissue was purchased from Spring Bioscience (Fremont, CA). Paraffin sections were obtained from 11 grossly and histologically normal ovaries of premenopausal women. The mean age of patients was 43.4 ± 4.2 yr (range, 38–48 yr). Diagnoses for ovary were normal (leiomyoma) for one patient, endometriosis for five patients, endometriotic cyst for one patient, cystic corpus luteum for three patients, and unknown reason for two patients. Premenopausal status was confirmed by the presence of follicles or corpora lutea. Carcinoma was excluded in all the ovarian samples used in this study by histopathological assessment. After follicular aspirates were collected during oocyte retrieval from women undergoing in vitro fertilization, hGLC were prepared as previously described (5) and cultured in medium 199:MCDB 105 (1:1; Sigma-Aldrich Corp., St. Louis, MO) containing 10% fetal bovine serum (FBS; Hyclone Laboratories Ltd., Logan, UT), 100 U/ml penicillin G, and 100 µg/ml streptomycin (Life Technologies, Inc., Rockville, MD) in a humidified atmosphere of 5% CO2-95% air at 37 C for 4 d before usage. Fragments of OSE were scraped from the ovarian surface during laparoscopic procedures for nonmalignant gynecological conditions in premenopausal women as previously described (17). Informed patient consent was obtained in accordance with institutional regulations. Primary cultures were maintained in the above-mentioned culture conditions. The use of human tissues and primary cells was approved by committee for ethical review of research involving human subjects of the University of British Columbia. Nontumorigenic immortalized OSE (IOSE) and granulosa luteal cells (SVOG) (18, 19), which were previously immortalized with SV40 large T antigen, a human neuronal medulloblastoma cell line TE671 and muscle-derived rhabdomyosarcoma cell RMS13 were maintained in 199/105/10%.

Antibodies

The antibodies for GnRH-I and GnRH-II were kindly provided by Dr. V. A. Ferro (University of Strathclyde, Glasgow, UK), and the specificity of these antibodies has been described in previous papers (20, 21). Antibody for GnRHR was purchased Lab Vision Corp. (Fremont, CA), and the specificity was tested in human ovarian and pituitary tissues.

Immunoblot assay

The cells were seeded at a density of 2 x 105 cells in 35-mm culture dishes and cultured in a humidified atmosphere of 5% CO2-95% air at 37 C. The cells were washed twice with ice-cold PBS and lysed in ice-cold RIPA buffer [150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris (pH, 7.5), and 1 mM PMSF, 10 g/ml leupeptin, 100 g/ml aprotinin]. The extracts were placed on ice for 15 min and centrifuged to remove cellular debris. Protein amount of supernatants was determined using a Bradford assay (Bio-Rad Laboratories). Thirty micrograms of total protein were run on 10% SDS-PAGE gels and electrotransferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Quebec, Canada). The membrane was immunoblotted using specific primary antibodies at 4 C overnight. After washing, the signals were detected with horseradish peroxidase-conjugated secondary antibody for 1 h, and visualized using the ECL system (Amersham Pharmacia Biotech).

Immunofluorescence

IOSE-80 and SVOG-4M cells were fixed in cold MeOH at –20 C and permeabilized in cold 1:1 MeOH:acetone for 5 min. The cells were blocked with protein blocking solution (Dako, Mississauga, Ontario, Canada) for 30 min at room temperature and incubated for 1 h with anti-GnRH-I (1:800), GnRH-II (1:600), or GnRHR (1:200) followed by Texas Red-labeled goat-antimouse (1:1200) and/or fluorescein isothiocyanate (FITC)-labeled goat-antirat (1:400) secondary antibody (Molecular Probes, Eugene, OR). The nuclei of cells were counterstained with Hoechst 33342. Controls lacked the first antibody.

Immunohistochemistry

The tissue sections were deparaffinized, rehydrated, treated with 30% H2O2 for 30 min, and then submitted to antigen retrieval by microwaving in 10 mM citric acid solution for 7 min. Slides were blocked in 1% BSA and 0.1% Tween 20 in PBS for 30 min followed by incubation with primary antibody anti-GnRH-I (1:200), GnRH-II (1:400), or GnRHR (1:100) overnight at 4 C. The sections were then incubated with Biotin-labeled universal secondary antibody (Dako) and Streptoavidin solution (Dako), respectively, for 20 min at room temperature, developed in diaminobenzine (Dako), and counterstained with Mayers hematoxylin. For peptide neutralization, the primary antibody was combined with GnRHR blocking peptide (Lab Vision) or GnRH-I/-II peptide (Peninsula Laboratories, Inc., Belmont, CA) and then incubated overnight at 4 C. The following morning, the immunohistochemistry protocol was followed under the same conditions described above.

Real-time RT-PCR

At the end of the treatment period, medium was removed from the culture dish and RNA was extracted using TRIzol (Invitrogen Canada, Burlington, Ontario, Canada). The RNA concentration was measured based on the absorbance at 260 nm, and its integrity was confirmed by agarose-formaldehyde gel electrophoresis. Total RNA (2.5 µg) was reverse transcribed into first-strand cDNA (Amersham Pharmacia Biotech) following the manufacturer’s procedure. The primers used for SYBR Green real-time RT-PCR were designed using the Primer Express Software v2.0 (PerkinElmer Applied Biosystems, Foster City, CA) and tested previously. The primers for GnRH-I are: sense, 5'-GCCTTAGAATGAAGCCAATTCAA-3'; and antisense, 5'-TCCACGCACGAAGTCAGTAGA-3'. The primers for GnRH-II are: sense, 5'-TCTGTTCCCCTCCAACTTTCTTC-3' and antisense, 5'-AGGTCCATCCATCTTTCCTTCA-3'. The primers for GnRHR are: sense, 5'-ACCGCTCCCTGGCTATCAC-3'; and 5'-ACTGTTCCGACTTTGCTGTTGCT-3'. Real-time PCR was performed using the ABI prism 7000 Sequence Detection System (PerkinElmer Applied Biosystems) equipped with a 96-well optical reaction plate. The reactions were set up with 12.5 µl SYBR Green PCR Master Mix (PerkinElmer Applied Biosystems). All real-time experiments were run in triplicate, and a mean value was used for the determination of mRNA levels. Negative controls, containing water instead of sample cDNA, were used in each real-time plate. The amount of transcript in each sample was calculated by interpolation using the following formula: (threshold cycle – y intercept)/S. The steady-state concentrations of mRNA for GnRH-I, GnRH-II, and GnRHR in each cell line were normalized to the amount of GAPDH mRNA. To compare the relative amount of GnRH-I, GnRH-II, and GnRHR mRNA in different samples, TE761 cells were designated as the calibrator and the normalized amount of target gene was divided by the averaged calibrator value.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Immunolocalization of type I GnRHR

First, we evaluated the specificity of the GnRHR antibody in the human pituitary tissue. Prominent immunoreactivity for GnRHR was observed in about 30% of pituitary tissue, which is consistent with data reported by La Rosa et al. (22) stating that GnRHR is expressed not only in gonadotropes but also in somatotropes and thyrotropes of the anterior pituitary (Fig. 1Go). Additionally, Western blot assay was performed in TE761 cells, primary culture of hGLC, immortalized ovarian granulosa-luteal cell lines (SVOG-4O and SVOG-4M), and RMS13 cells. Muscle cells were known as GnRHR negative by RT-PCR and Western blot analysis (5, 23). Human neuronal medulloblastoma cell line TE671 and primary/immortalized granulosa luteal cells have been recently demonstrated to express GnRHR (8, 24). As shown in Fig. 1BGo, we found a single band of around 60 kDa in all tested cells but muscle-derived RMS13.


Figure 1
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  		FIG. 1. Validation of antibody to human GnRHR. A, Paraffin-embedded sections of human pituitary tissue were immunostained with anti-GnRHR. B, The protein expression of GnRHR was determined by Western blot analysis. RMS13, Human rhabdomyosarcoma; TE671, human neuronal medulloblastoma; 4O, immortalized granulosa (SVOG-4O); 4M, immortalized granulosa cell line (SVOG-4M).

 
The localization of GnRHR in the cellular subpopulations of the developing ovarian follicles and corpus luteum was elucidated in paraffin sections of 11 premenopausal ovaries. In primordial to early antral follicles, we failed to detect any significant immunoreactivity for GnRHR when it was compared with surrounding stromal cells (Fig. 2Go, A–D). In preovulatory follicles (Fig. 2Go, E and G), GnRHR was expressed predominantly in the granulosa cells and, to a lesser degree, in the theca interna cells. The specificity of the antibody was confirmed by blocking of the antibody with GnRHR blocking peptide (Fig. 2FGo).


Figure 2
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  		FIG. 2. Immunohistochemical staining for GnRHR in human ovaries. A, Primodial or primary follicles; B, secondary or preantral follicle; C and D, multilayered early antral follicles; E and G, preovulatory follicles; H, OSE; I, epithelial inclusion cyst; J, K, and L, corpus luteum; M, corpus albican. F, Section immunostained using antibody reabsorbed with GnRHR blocking peptide was used as a negative control. Panel L is a close up of K. GC, Granulosa cells; TI, theca interna; TE, theca externa. Arrowheads in L show theca luteal cells.

 
In the human and primate corpus luteum, granulosa and theca luteal cells, with their characteristic morphology, remain clustered in distinct areas, whereas other species have relatively homogenous mixtures of those two cell populations. Here, we examined three different types of corpus lutea, including regressed corpus albicans. Strong immunostaining for GnRHR was observed in both developing corpora lutea of Fig. 2Go, J and K. Figure 2LGo shows typical morphology of a mature corpus luteum (25, 26) that has granulosa luteal cells with abundant eosinophilic cytoplasm and a polyhedral to spherical shape, and smaller theca luteal cells are clearly distinguishable by their position and morphology. In contrast, corpus luteum in panel J, where granulosa cells are loosely and radially organized and morphologically indistinguishable from theca luteal cells, seem to be earlier developing corpus luteum (25). Granulosa luteal cells exhibiting a typical steroidogenic morphology, but not theca luteal cells, expressed the GnRHR protein (Fig. 2Go, J–L). No significant GnRHR staining was detected in the corpus albicans (Fig. 2MGo), the last stage of corpus luteum development.

In addition to follicles and corpora lutea, the OSE, which is a simple squamous-to-cuboidal mesothelium covering the ovary (Fig. 2HGo), was strongly positive in GnRHR immunostaining compared with tunica albuginea and stroma, whereas epithelial inclusion cysts were negative (Fig. 2IGo). The stroma in the cortex (Fig. 2HGo) was more immunoreactive than in the medulla.

Immunolocalization of GnRH-I and GnRH-II

To further examine the notion that both GnRH-I and GnRH-II are produced within the human ovary to exert their action in an autocrine/paracrine manner, immunohistochemistry using specific GnRH-I or GnRH-II antibodies (20, 21) was performed. Immunostaining for both isoforms of the hormone was absent in early stages of follicular development and was first detected in the granulosa compartment of follicles at the preovulatory stage (Figs. 3Go and 4Go, E and G). Luteinized granulosa cells were immunostained for GnRH-I and GnRH-II only in the mature corpus lutea containing distinct granulosa and theca layers (Figs. 3Go and 4KGo), but not in the developing corpus lutea and corpus albicans (Figs. 3Go and 4Go, J and M). GnRH-I and GnRH-II immunostaining was weakly positive in theca interna cells of preovulatory follicles, but not in those of a mature corpus luteum (Figs. 3Go and 4Go, E, G, and L). In the OSE, but not in epithelial inclusion cysts, both GnRH-I and GnRH-II were found to be expressed (Figs. 3Go and 4Go, H and I).


Figure 3
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  		FIG. 3. Immunohistochemical staining for GnRH-I in human ovaries. A, Primodial or primary follicles; B, secondary or preantral follicle; C and D, multilayered early antral follicles; E and G, preovulatory follicles; H, OSE; I, epithelial inclusion cyst; J, K, and L, corpus luteum; M, corpus albicans. F, Section immunostained using antibody reabsorbed with GnRH-I peptide was used as a negative control. Panel L is a close up of K. GC, Granulosa cells; TI, theca interna; TE, theca externa. Arrowheads in L show theca luteal cells.

 

Figure 4
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  		FIG. 4. Immunohistochemical staining for GnRH-II in human ovaries. A, Primodial or primary follicles; B, secondary or preantral follicle; C and D, multilayered early antral follicles; E and G, preovulatory follicles; H, OSE; I, epithelial inclusion cyst; J, K, and L, corpus luteum; M, corpus albican. F-1, Section immunostained using antibody reabsorbed with GnRH-II peptide was used as a negative control. F-2, Antibody preabsorbed with GnRH-I peptide still recognize GnRH-II. Panel L is a close up of K. GC, granulosa cells; TI, theca interna; TE, theca externa. Arrowheads in L show theca luteal cells.

 
No notable difference between the expression patterns of the two isoforms of GnRH was observed in the various ovarian compartments. The high sequence homology between the two GnRH peptides may raise the question of cross-reactivity between GnRH-I and GnRH-II antibodies. Thus, we confirmed the specificity of each antibody using a peptide neutralization assay. GnRH-I antibodies were significantly competed by preabsorption with the native peptide GnRH-I, resulting in elimination of the immunoreactivity (Fig. 3FGo). Although preincubation with GnRH-II peptide resulted in a slight GnRH-II staining in the granulosa cell compartment of preovulatory follicle (Fig. 4Go, F-1), GnRH-I peptide did not significantly interfere with GnRH-II staining, suggesting no cross-reactivity of GnRH-II antibody with GnRH-I. This result is correlated with a previous assessment of antibody cross-reactivity using GnRH-BSA-coated ELISA plates (20). Indeed, the two antibodies were used to demonstrate unique immunolocalization patterns in the rhesus monkey hypothalamus (20) and human placenta and decidua (21). Thus, the coexpression of the two hormones in the human ovary is not likely to be explained by any antibody cross-reactivity.

Subcellular localization of the GnRH system in ovarian cell lines

The presence of the GnRH/GnRHR system at the transcription level in OSE and granulosa cells has been established previously (4, 5, 6, 27). Here, we evaluated the subcellular localization of the GnRH-I, GnRH-II, and GnRHR proteins in immortalized OSE and granulosa cells using immunofluorescence staining. Immortalized OSE (IOSE-80) cells and immortalized granulosa luteal cells (SVOG-4M) were labeled with anti-GnRH-I, GnRH-II, or anti-GnRHR primary antibodies and FITC- or Texas Red-conjugated secondary antibody, respectively. DNA of the cells was counterstained with Hoechst 33342 DNA dye (blue). As shown in Fig. 5Go, GnRH-I and GnRH-II staining was found predominantly in the nuclear compartment in SOVG-4M cells, and in both nuclear and cytoplasmic compartments in the IOSE-80 cells. GnRHR was mainly perinuclear in SVOG-4M cells and IOSE-80 cells.


Figure 5
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  		FIG. 5. Immunofluorescence staining by anti-GnRH-I, GnRH-II, and anti-GnRHR antibodies in cultured immortalized granulosa cells (SVOG-4M) and OSE cells (IOSE-80). B and D, GnRHR; J and L, GnRH-I; N and P, GnRH-II; F and H, no-first antibody followed by Texas-red-conjugated secondary antibody; R and T, no-first antibody followed by FITC-conjugated secondary antibody. Cells were double-stained with Hoechst 3324 (blue). A, C, E, G, I, K, M, O, Q, and S represent the same field as B, D, F, H, J, L, N, P, R, and T.

 
Expression of the GnRH system in primary and immortalized ovarian cell lines

To compare the expression levels of GnRH-I, GnRH-II, and GnRHR among primary or immortalized ovarian cells and TE671 cells that have been demonstrated to express both forms of GnRHs and GnRHR (24), quantitative real-time RT-PCR was performed. We found that GnRH-I, GnRH-II, and GnRHR mRNA levels were present in all the ovarian cells examined, albeit at lower levels compared with those observed in TE671 (Fig. 6Go). Primary hGLC and OSE cells expressed similar levels of GnRH-I and GnRH-II mRNA. GnRHR levels are comparable in all lines except TE671 and IOSE-29. It is notable that immortalized cell lines (SVOG and IOSE), which were constructed by transfection of SV-40 large T antigen, expressed higher levels of GnRH-I and GnRH-II, but not of GnRHR, compared with those of the primary cells. These data suggested that immortalized granulosa and OSE cells lines as well as the primary cells, where the GnRH system appears to work in autocrine manner, may provide a suitable experimental model for further functional studies of GnRHs in the human ovary.


Figure 6
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  		FIG. 6. Expression of GnRH-I, GnRH-II, and GnRHR mRNA in primary cells and immortalized ovarian cell lines. First-strand cDNA from TE671, primary GLC, primary OSE, SVOG-4O, SVOG-4M, IOSE-120, and IOSE-80 cells were amplified by real-time RT-PCR using two sets of PCR primers shown in Materials and Methods. The expression levels of GnRH-I, GnRH-II, and GnRHR mRNA were normalized against GAPDH mRNA level. Data are derived from three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
Whereas previous reports have shown that GnRH-I, GnRH-II, and GnRHR are expressed at the mRNA level in primary cultures of hGLC and OSE cells (4, 5, 6), the present study demonstrated, for the first time, the expression of the GnRH/GnRHR system at the protein level, as well as their temporal-spatial distribution, during human ovarian follicle development. Both forms of GnRH and their common receptor were found to be coexpressed in preovulatory follicles and mature corpus luteum. In view of the observations that the type I GnRHR may mediate the biological action of both GnRH-I and GnRH-II, but the circulating level of hypothalamic GnRH is neither sufficient nor stable enough to activate the ovarian GnRHR, coexpression or proximal expression of those two GnRH isoforms and GnRHR in the various ovarian cell populations will argue strongly in favor of their potential role as autocrine/paracrine regulators in the human ovary. It is noteworthy that that peripheral GnRH administration restores the ovulatory cycle in women and monkeys with hypothalamic amenorrhea (28).

It is noteworthy that the immunostaining for GnRHR as well as its ligands was prominently localized to the cytoplasm and nuclei in positive cells of the pituitary and ovary. Despite the GnRHR being a G protein-coupled transmembrane receptor, its cytoplasmic expression is believed to represent neosynthesized or internalized receptors. In various cells, including ovarian granulosa cells and pituitary gonadotropes, binding of GnRHs results in activation of GnRHR followed by cluster formation, microaggregation, and internalization of the receptor into cytoplasm or nuclei (29, 30, 31). Whether or not the different pattern of subcellular localization of GnRHR in IOSE-80 and SVOG-4M reflects functional or signaling differences of GnRH remains to be determined.

Previous animal studies have demonstrated the expression of GnRH-I and GnRHR in the granulosa or luteal compartments of follicles and corpora lutea (5, 7, 32, 33). In rat ovary, GnRH-I and GnRHR mRNA was localized to the granulosa cells of most developing follicles from primary to preovulatory follicle (32, 33). Hybridization signal for GnRHR was not detected in oocyte and theca cell compartment (32, 33), whereas GnRH-I mRNA was found to be expressed in theca interna cells. There are some minor differences between the findings in the rat compared with the human in the present study. For example, we failed to detect significant immunostaining for GnRH-I, GnRH-II, and GnRHR in most early follicles. Also, moderate, but readily detectable, immunostaining for GnRHR was observed in theca interna cells of human preovulatory follicles. These differences may be attributable to the sensitivity of the immunohistochemistry techniques employed in this and the other study, a difference between mRNA and protein expression patterns, or species specificity.

Presence of the GnRH/GnRHR system in the human corpus luteum has been examined by different methodologies such as RT-PCR in hGLC (5, 10) and by RIA in homogenates of human corpus luteum (7). In the present study, we demonstrated the specific expression of GnRH/GnRHR protein in the granulosa luteal layer but not in the theca luteal layer. Moreover, based on the morphological difference between early developing and mature corpus luteum (25, 26), we assumed that early corpus luteum may not produce its own GnRH, such that the GnRHR in the early corpus luteum may be activated in a paracrine manner by GnRH-I and GnRH-II from the other ovarian compartments.

This stage-specific change in GnRH/GnRHR expression in human ovarian folliculogenesis suggested the possible interaction between GnRH and gonadotropin system. Previously, we have demonstrated that treatment with FSH or hCG increased GnRH-II mRNA levels but decreased GnRH-I mRNA levels (4). However, the mechanism by which gonadotropins differentially regulate two types of GnRH and the relevance of differential regulation in ovarian function remain to be elucidated. Moreover, a significant down-regulation of FSH receptor and LH receptor was observed in cells treated with GnRH-I and GnRH-II, suggesting that GnRH-I and GnRH-II may exert their antigonadotropic effect by down-regulating gonadotropin receptors (4).

In the human ovary, both isoforms of GnRH appear to be involved in a number of biological functions such as steroidogenesis and apoptosis. For example, previous findings from our laboratory have demonstrated that both GnRH-I and GnRH-II inhibited gonadotropin-stimulated progesterone secretion (4, 5, 10). The direct action of GnRH-I on progesterone production is mediated by ERK1/2 in hGLC (10). In hGLC, Zhao et al. (11) found that treatment with GnRH-I increased apoptotic DNA fragmentation. In addition to regulation of steroidogenesis and apoptosis, GnRH-I has been shown to play a role in ovulation, luteinization, and luteolysis in the rat ovary. For example, treatment with GnRH-I stimulated tissue-type plasminogen activator secretion (34) and prostaglandin H synthesis induction (35), which are implicated in gonadotropin-induced ovulation, in cultured rat granulosa cells and/or cumulus-oocyte complexes. Progesterone receptor, a critical factor of luteinization, was regulated by GnRH in rat granulosa cells (36). Moreover, GnRH-I induces activity of matrix metalloproteinase-2 and membrane-type matrix metalloproteinase, which degrade collagens type IV and type I/III, and this results in involution of developed corpus luteum as shown by their markedly smaller size (37). The present study has shown that GnRH-I, GnRH-II, and GnRHR are prominently immunolocalized to late preovulatory follicles and corpus luteum in the human ovary, implicating a role for GnRH in ovulation, luteinization, and luteolysis in the human ovary.

Over 85% of ovarian cancers are considered to arise from the OSE, a simple squamous-to-cuboidal mesothelium covering the ovary. Epithelial inclusion cysts, possibly resulting from invagination of OSE into the stroma after ovulation, are hypothesized to be precursor lesions for some epithelial ovarian cancers (38). Native GnRH-I and its synthetic analogs inhibit the growth of numerous GnRHR-bearing ovarian cancer cell lines in vitro. The growth inhibitory effects of GnRH analogs have also been observed in normal OSE cells (12, 13). More recently, it has been established that GnRH-II can induce growth inhibition in IOSE cells and ovarian cancer cells (13, 14). Several reports have shown that GnRH analogs can induce apoptosis or regulate drug-induced apoptosis in ovarian cancers (39, 40). The result of the present study, that both forms of GnRH and their common receptor were readily detectable in surface OSE but not in epithelial inclusion cysts, raises the intriguing possibility that loss of the growth-inhibitory GnRH/GnRHR system in the epithelial cells, after entrapping them within the stroma, contributes to the trigger of tumorigenesis. However, of the 11 ovaries examined, only two had intact surface OSE, such that the conclusion about the significance should be drawn by further study with more cases.


   Acknowledgments
 
We are grateful to Dr. Takayo Ota at Child and Family Research Institute, University of British Columbia, for technical assistance.


   Footnotes
 
This work was supported by the Canadian Institutes of Health Research. P.C.K.L. is the recipient of a Distinguished Scholar Award from the Michael Smith Foundation for Health Research. J.H.C. is the recipient of a Graduate Studentship Award from Interdisciplinary Women’s Reproductive Health Research Training Program, University of British Columbia.

Disclosure statement: The authors have nothing to disclose.

First Published Online September 5, 2006

Abbreviations: FITC, Fluorescein isothiocyanate; GnRHR, GnRH receptor; hGLC, human granulosa luteal cell; IOSE, immortalized OSE; OSE, ovarian surface epithelium.

Received May 26, 2006.

Accepted August 25, 2006.


   References
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 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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