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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-0180
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 8 3239-3245
Copyright © 2007 by The Endocrine Society

Ghrelin Affects the Release of Luteolytic and Luteotropic Factors in Human Luteal Cells

Anna Tropea, Federica Tiberi, Francesca Minici, Mariateresa Orlando, Maria Francesca Gangale, Federica Romani, Fiorella Miceli, Stefania Catino, Salvatore Mancuso, Maurizio Sanguinetti, Antonio Lanzone and Rosanna Apa

Cattedra di Fisiopatologia della Riproduzione Umana (A.T., F.Min., M.O., M.F.G., F.R., F.Mic., S.M., R.A.), Istituto Scientifico Internazionale "Paolo VI" (F.T.), and Instituto di Microbiologia (M.S.), Università Cattolica del Sacro Cuore, 00168 Roma, Italy; and Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) "Associazione Oasi Maria SS.-Onlus" (S.C., A.L.), 94018 Troina (EN), Italy

Address all correspondence and requests for reprints to: Rosanna Apa, M.D., Cattedra di Fisiopatologia della Riproduzione Umana, Università Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 Roma, Italy. E-mail: krimisa{at}libero.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: Ghrelin, well-known modulator of food intake and energy balance, is a rather ubiquitous peptide involved in several endocrine and nonendocrine actions. A possible as-yet-unknown role for ghrelin in modulating luteal function has been suggested because both ghrelin and its receptor (GRLN-R) have been immunohistochemically detected in human corpus luteum.

Objective: We first investigated GRLN-R mRNA expression in midluteal phase human luteal cells. Ghrelin effect on basal and human chorionic gonadotropin (hCG)-stimulated progesterone (P) release was then analyzed. Finally, we investigated whether ghrelin could affect luteal release of vascular endothelial growth factor (VEGF), prostaglandin (PG) E2, both luteotropic factors, and PGF2{alpha}, luteolytic modulator. Ghrelin effect on both basal and hypoxia-stimulated VEGF luteal expression was analyzed.

Methods: Human luteal cells were incubated for 24 h with ghrelin (10–13 to 10–7 M) or hCG (100 ng/ml) or CoCl2 (10 µM), chemical hypoxia, or with hCG or CoCl2 in combination with ghrelin. Both GRLN-R mRNA and VEGF mRNA were evaluated by real-time RT-PCR. PGs and P release was assayed by RIA, whereas VEGF release by ELISA.

Results: GRLN-R mRNA expression was demonstrated in human luteal cells. Both basal and hCG-stimulated P release was significantly decreased by ghrelin, which was able to reduce PGE2 and increase PGF2{alpha} luteal release. Both basal and hypoxia-stimulated VEGF release was significantly decreased by ghrelin, which did not affect VEGF mRNA luteal expression.

Conclusions: The present in vitro study provides the first evidence of a direct inhibitory influence of ghrelin on human luteal function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GHRELIN, THE ENDOGENOUS ligand of the GH secretagogue receptor type 1a, is a 28-amino-acid peptide with a peculiar n-octanoyl esterification at serine 3 residue (1).

The octanoylation is required for ghrelin’s ability to bind and activate GH secretagogue receptor type 1a (1, 2), recently designated as the ghrelin receptor (GRLN-R) (3), that mediates neuroendocrine effects of this peptide (2, 4).

The abundant distribution of GRLN-R in the pituitary gland and hypothalamus may account for the important central effects of ghrelin (5). In addition to its potent GH-releasing activity (6), this peptide is involved in the modulation of lactotropic, corticotropic, and gonadotropic axes (7, 8, 9). Interestingly, ghrelin also plays a pivotal role in energy homeostasis (10), being to date the only known peripheral orexigenic hormone. Primarily produced by the stomach in response to hunger and starvation, ghrelin serves as a humoral signal informing brain centers about acute or chronic changes in peripheral energy balance (11). Apart from the stomach, which is certainly the major source of circulating levels of ghrelin (1), many peripheral tissues express both this peptide and its receptor (12). This ubiquitous distribution suggests that, in addition to endocrine actions of gastric-derived peptide, locally produced ghrelin might exert autocrine/paracrine effects in different tissues (13). This should be the case in various reproductive organs, such as endometrium, placenta, testis, and ovary (14). In particular, in the female gonad, ghrelin immunoreactivity was predominantly demonstrated in the luteal compartment (15). Indeed Gaytan et al. (15) detected both ghrelin and GRLN-R immunostaining in luteal cells from young and mature human corpora lutea, suggesting for this peptide a possible as-yet-unknown role in modulating luteal function.

To define this aspect better, in the present study, we investigated the potential regulatory role of ghrelin in steroidogenic cells from fully functional human corpora lutea. First of all, GRLN-R expression was evaluated in midluteal phase human luteal cells. The possible influence of ghrelin on luteal steroidogenesis was then investigated. In particular, we evaluated whether ghrelin could affect basal and/or gonadotropin stimulated luteal progesterone (P) production. Moreover the possible role of ghrelin in modulating the balance between intraovarian luteotropic and luteolytic regulators was investigated. To this aim we analyzed ghrelin effect on luteal release of vascular endothelial growth factor (VEGF), prostaglandin (PG) E2, both local luteotropic factors (16, 17), and PGF2{alpha}, a luteolytic modulator (17). Because in mature corpus luteum VEGF production is mainly stimulated by local hypoxia (18), the ability of ghrelin to influence hypoxia-induced VEGF luteal production was also evaluated. Finally, ghrelin effect in modulating both basal and hypoxia-stimulated VEGF luteal expression was analyzed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals

Human acylated ghrelin was obtained from Clinalfa Inalco SPA (Milano, Italy). Collagenase type IV, antibiotics, and HEPES were purchased from Sigma Chemical Co. (St. Louis, MO). Chorionic gonadotropin, human urine standard grade, was purchased from Calbiochem Inalco (Milano, Italy), nutrient mixture F-12 (Ham) from Flow Laboratories (Milano, Italy), and fetal bovine serum from Euro Clone (Milano, Italy). TRIzol, RT-PCR kit, platinum quantitative RT-PCR ThermoScript reaction mix, and ThermoScript Plus/Platinum Taq mix were purchased from Invitrogen Inc. (Milano, Italy). Primers and probes for real-time RT-PCR were obtained from Sigma-Proligo (Sigma-Aldrich S.r.l., Milano, Italy). RIA progesterone kit was obtained from Radim (Roma, Italy). [3H]PGE2 and [3H]PGF2{alpha} were obtained from NEN Life Science Products (Milano, Italy). Human VEGF ELISA kit was obtained from R&D systems (Minneapolis, MN).

Cell cultures

Corpora lutea (CL) were obtained at the time of hysterectomy or myomectomy performed for nonendocrine gynecological disease (leiomyomatosis) in the midluteal phase of the menstrual cycle (d 5–6 from ovulation). A total of 38 patients (aged 28–41 yr) were included in the present study. All had a history of regular menstrual cycles. The present protocol was approved by the Institutional review Board of Università Cattolica del Sacro Cuore, Roma; all patients provided written informed consent.

The age of CL was determined as follows. All patients were monitored until ovulation by daily measurement of basal body temperature and ultrasound examination of follicular growth. Once the maximal follicular diameter reached 18 mm, daily determination of plasma P values was made. The time of ovulation (d 0) was detected by the biphasic pattern of basal body temperature, the typical ultrasound disappearance of the dominant follicle or the ultrasound detection of CL, and the rise in plasma P concentrations. At the time of surgery, immediately before anesthesia, plasma samples were collected to determine plasma P concentrations.

The removed luteal tissue was immediately freed from blood vessels and ovarian stroma under a dissecting microscope, dissected, and minced. Human CL cultures were performed as previously described (19). The luteal cell identity was confirmed by their positive staining for lipids with oil red O14 (20). Isolated luteal cells were plated on 48-well dishes (250,000 cells/ml) for RIA and ELISA or on six-well dishes (250,000 cells/ml) for real-time RT-PCR and cultured for 24 h in 5% CO2-95% air at 37 C.

At the end of the isolation procedure and 24 h after the treatments mentioned in the next paragraphs, cells were counted in a hemocytometer and viability was determined by the trypan blue exclusion test. No treatment was able to affect either cells count or viability.

Preliminary experiments were performed to exclude that any solvent for dissolving the tested substances could affect luteal cell function and/or viability.

VEGF assay

Luteal cells were incubated for 24 h with serum-free medium alone [control (C)] or with different doses of ghrelin (10–13 to 10–7 M). Moreover, cells were incubated for 24 h with CoCl2 (10 µM) (18) alone or in presence of ghrelin (10–12 to 10–7 M). Three different wells were used for each experimental condition. Experiments were repeated with cells from 12 independent CL. Culture media were separately collected and assayed for VEGF detection. To this aim, commercial culture media-validated VEGF ELISA kits were carried out according to the instructions provided by the manufacturer.

The intra- and interassay coefficients of variation were 3.5 and 6.7%, respectively. The ELISA sensitivity was 5.0 pg/ml.

P assay

To measure P secreted in culture medium, cells were incubated for 24 h with serum-free medium alone (C) or with human chorionic gonadotropin (hCG) (100 ng/ml) (21) or ghrelin (10–13 to 10–7 M). Moreover, cells were incubated for 24 h with hCG (100 ng/ml) in combination with ghrelin (10–13 to 10–7 M). For each experimental condition, three different wells were performed. Experiments were repeated with cells from 11 independent CL. Culture media were separately collected and assayed for P detection. To this aim, a commercial P RIA kit was used according to the manufacturer’s instructions.

The intra- and interassay coefficients of variation were 4 and 10%, respectively. The RIA sensitivity was 5 pg/tube.

PGs assay

Luteal cells were incubated with serum-free medium alone (C) or with different doses of ghrelin (10–12 to 10–7 M). For each experimental condition, three different wells were performed. Experiments were repeated with cells from eight independent CL. Culture media were separately collected and assayed for PGs detection as previously described (22). The RIAs for PGE2 and PGF2{alpha} used in this study were first characterized for measurement of prostanoids in human urine (23) and later used successfully to measure PGs produced and released by several cell types in vitro (24). The detection limit of the assay was 2 pg/tube in all cases. The inter- and intraassay variability coefficients were 2.7 and 2.9% for PGE2, 3.2 and 2.8% for PGF2{alpha}.

Total RNA extraction and quantification

To evaluate GRLN-R mRNA expression, luteal cells were incubated for 24 h with serum-free medium alone (C). As positive control for the mRNA expression of GRLN-R (25) human umbilical vein endothelial cells (HUVECs) were isolated as previously described (22).

To evaluate VEGF mRNA expression, luteal cells were incubated for 24 h with serum-free medium alone (C) or with CoCl2 (10 µM) or with ghrelin (10–10 or 10–7 M). Moreover, cells were incubated for 24 h with CoCl2 (10 µM) (18) in combination with ghrelin (10–10 or 10–7 M).

Luteal cells and HUVECs were treated for total RNA extraction. To this end, the standard TRIzol extraction method was used, according to the instructions provided by the manufacturer. The purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis. Classical and real-time RT-PCR analysis were performed as described in the following paragraph.

RT-PCR

GRLN-R mRNA expression was evaluated by RT-PCR in a thermal cycler (I-Cycler; Bio-Rad Laboratories, Milan, Italy) according to the instruction provided by the manufacturer as previously described (22). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen as housekeeping gene. The PCR conditions were as follows: 95 C for 5 min; 35 cycles for GRLN-R and 28 cycles for GAPDH consisting of 95 C for 45 sec, 58 C for 45 sec, 72 C for 45 sec; and 72 C for 7 min. Starting experiments were performed to ensure being in the linear range of the PCR amplification curve. As negative control, one PCR was performed without prior reverse transcription (RT) of the mRNA. The identity of each PCR product (115 bp for GAPDH and 93 bp for GRLN-R) was confirmed by sequence analysis. Primers used to amplify GRLN-R and GAPDH are shown in Table 1Go. PCR products were separated on a 3.0% agarose gel containing ethidium bromide.


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  		TABLE 1. Primers and fluorescent probes used in RT-PCR

 
Real-time RT-PCR

Real-time RT-PCR was performed to evaluate GRLN-R and VEGF mRNA expression. To analyze VEGF mRNA modulation in human luteal cells, attention has been focused on diffusible VEGF 121 and VEGF 165 isoform mRNA expression (18).

The mRNA expression of GRLN-R and the VEGF 121 and 165 isoforms was evaluated by using the i-Cycler iQ system (Bio-Rad Laboratories, Hercules, CA). For the target genes and the endogenous housekeeping gene, encoding for GAPDH, a primer pair and Taqman probe, which hybridizes to the region between primers, were designed using Beacon Designer 2 v. 3.00 software (Premier Biosoft International, Palo Alto, CA) and synthesized by Sigma Proligo (Table 1Go).

Real-time RT-PCR for GRLN-R and the VEGF 121 and 165 isoforms mRNA expression semiquantification was performed in a 25 µl volume containing the following reagents: 12.5 µl of the Platinum Quantitative RT-PCR ThermoScript reaction mix, 1.5 U of ThermoScript Plus/Platinum Taq mix, each primer pair and Taqman probe at a concentration of 0.5 µM, 5 µl of total RNA sample, and distilled water up to final volume. Samples were subjected to an initial step at 52 C for 45 min for RT; 94 C for 5 min to inactivate the ThermoScript Plus reverse transcriptase and to activate the Platinum Taq polymerase; and 50 cycles, each consisting of 15 sec at 94 C and 1 min at 58 C. Fluorescent data were collected during the 58 C annealing/extension step and analyzed with the iCycler iQ software. Each reaction was run in triplicate. Mean threshold cycle (Ct) was determined for each transcript and was plotted vs. RNA concentration input to calculate the slope. Amplification efficiency for all genes was then determined (26, 27).

For relative quantification of the target genes, each set of primer pairs and Taqman probe were used in combination with that of GAPDH gene in separate reactions. The relative mRNA expression levels of the target genes in each sample were calculated using the comparative cycle time (Ct) method (28). Briefly, the target PCR Ct value (i.e. the cycle number at which emitted fluorescence exceeds 10 times the SD of baseline emissions as measured from cycles 3 to 15) is normalized to the GAPDH PCR Ct value by subtracting the GAPDH Ct value from the target PCR Ct value, which gives the {Delta}Ct value. From this {Delta}Ct value, the relative mRNA expression level to GAPDH for each target PCR can be calculated using the following equation: relative mRNA expression = 2 – (Ct target-Ct GAPDH).

Data analysis

Statistical analysis was performed using ANOVA followed by the Tukey-Kramer test for comparisons of multiple groups or paired Student’s test for comparison of data derived from two groups. Values with P < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Detection of GRLN-R mRNA in human luteal cells

In isolated midluteal phase human luteal cells, GRLN-R mRNA expression was first demonstrated by RT-PCR. As expected, GRLN-R mRNA expression was observed in HUVECs (25). No signal was detected when PCR was carried out without prior RT of the mRNA, thereby ruling out the possibility that genomic DNA has been amplified (Fig. 1Go). Moreover, GRLN-R mRNA expression was confirmed by real-time RT-PCR.


Figure 1
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  		FIG. 1. Detection of GRLN-R mRNA in human luteal cells. Total RNA from human luteal cells and HUVECs was subjected to RT-PCR using a specific set of primers for GRLN-R and GAPDH amplifications, as described in Materials and Methods. The figure shows the ethidium bromide-stained 3% agarose gel with 93- and 115-bp PCR products, which corresponds to GRLN-R (upper panel) and GAPDH (lower panel), respectively. Far left lane, DNA molecular mass standards (MW). Lane 1, Human luteal cells. Lane 2, HUVECs. Lane 3, Negative control, human luteal cell PCR mixture without prior RT.

 
In luteal cells as well as HUVECs, the relative GRLN-R mRNA expression levels in each sample was calculated using the comparative Ct method (28) as described in Materials and Methods. The {Delta}Ct value for GRLN-R mRNA expression relative to the GAPDH gene was 7.7 ± 1.8 in luteal cells (n = 7) and 13.7 ± 0.5 in HUVECs (n = 6). Therefore, in luteal cells GRLN-R mRNA expression was found to be 63.05 ± 3.76-fold higher than in HUVECs (P < 0.001, data not shown).

Effect of ghrelin on basal and hCG-stimulated P release by human luteal cells

Human luteal cells were cultured for 24 h with medium alone (C) or in presence of increasing concentrations of ghrelin (10–13 to 10–7 M). All tested doses of ghrelin were able to significantly decrease basal P luteal release except for the lowest used concentration (10–13 M); the effect was maximal at 10–11 M, and higher doses had no further effect (Fig. 2AGo).


Figure 2
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  		FIG. 2. A, Effect of ghrelin on basal P release by human luteal cells. Luteal cells were cultured for 24 h in medium alone (C) or with ghrelin (10–13 to 10–7 M). Each value represents the mean ± SEM of 11 independent experiments, each done in triplicate. Results are expressed as percentage of control set equal to 100. ***, P < 0.001 vs. C values. Groups with different superscript letters are statistically different (P < 0.05). B, Effect of ghrelin on hCG-stimulated P release by human luteal cells. Luteal cells were cultured for 24 h in medium alone (C) or with hCG (100 ng/ml) in combination or not with ghrelin (10–13 to 10–7 M). Each value represents the mean ± SEM of 11 independent experiments, each done in triplicate. Results are expressed as percentage of control set equal to 100. °°°, P < 0.001 vs. C values; ***, P < 0.001, **, P < 0.01 vs. hCG values. Groups with different superscript letters are statistically different (P < 0.05).

 
To evaluate whether hCG-stimulated P release could be influenced by ghrelin, cells were incubated for 24 h with medium alone (C) or with hCG (100 ng/ml) in combination or not with different concentrations of ghrelin (10–13 to 10–7 M). As expected, P release was significantly increased by hCG (21). We observed that ghrelin from 10–12 to 10–7 M was able to significantly decrease hCG-induced P release. Ghrelin 10–7 M exerted the strongest effect; tested doses from 10–12 to 10–8 M exerted similar effects, significantly lower than that exerted by ghrelin 10–7 M. Ghrelin 10–13 M was not able to affect hCG-induced P release (Fig. 2BGo).

The mean concentration of P in the control was 14.5 ± 1.1 ng/ml.

Effect of ghrelin on PGs release in human luteal cells

We investigated whether ghrelin could affect PGs luteal release. Human luteal cells were incubated for 24 h in medium alone (C) or in presence of increasing concentrations of ghrelin (10–12 to 10–7 M). Prostanoid release was estimated by measuring the amounts of PGF2{alpha} and PGE2 into the conditioned medium.

Figure 3AGo shows that PGE2 release was significantly reduced by all tested doses of ghrelin, except for the lowest used concentration (10–12 M). The effect was maximal for ghrelin 10–10 M, higher doses not being able to exert any further effect.


Figure 3
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  		FIG. 3. A, Effect of ghrelin on PGE2 release in human luteal cells. Luteal cells were cultured for 24 h in medium alone (C) or with ghrelin (10–12 to 10–7 M). Each value represents the mean ± SEM of eight independent experiments, each done in triplicate. Results are expressed as percentage of control set equal to 100. **, P < 0.01, *, P < 0.05 vs. C values. Groups with different superscript letters are statistically different (P < 0.05). B, Effects of ghrelin on PGF2{alpha} release by human luteal cells. Luteal cells were cultured for 24 h in medium alone (C) or with ghrelin (10–12 to 10–7 M). Each value represents the mean ± SEM of eight independent experiments, each done in triplicate. Results are expressed as percentage of control set equal to 100. ***, P < 0.001, **, P < 0.01 vs. C values. Groups with different superscript letters are statistically different (P < 0.05).

 
As shown in Fig. 3BGo, ghrelin from 10–11 to 10–7 M was able to significantly enhance PGF2{alpha} release in a dose-dependent manner. No effect was observed when luteal cells were incubated with ghrelin 10–12 M.

The mean concentration of PGE2 and PGF2{alpha} in the control was 51.5 ± 4.1 and 30.1 ± 3.3 pg/ml, respectively.

Effect of ghrelin on basal and CoCl2-induced VEGF release in human luteal cells

To evaluate whether luteal VEGF release could be influenced by ghrelin, human luteal cells were incubated for 24 h with medium alone (C) or with ghrelin (10–13 to 10–7 M). As shown in Fig. 4AGo, all tested doses of ghrelin were able to significantly decrease VEGF release, except for the lowest used concentration (10–13 M); ghrelin 10–11 M exerted the strongest effect and higher doses had no further effect.


Figure 4
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  		FIG. 4. A, Effect of ghrelin on basal VEGF release in human luteal cells. Luteal cells were cultured for 24 h in medium alone (C) or with ghrelin (10–13 to 10–7 M). Each value represents the mean ± SEM of 12 independent experiments, each done in triplicate. Results are expressed as percentage of control set equal to 100. ***, P < 0.001 vs. C values. Groups with different superscript letters are statistically different (P < 0.05). B, Effect of ghrelin on CoCl2-induced VEGF release in human luteal cells. Luteal cells were cultured for 24 h in medium alone (C) or CoCl2 (10 µM) in combination or not with ghrelin (10–12 to 10–7 M). Each value represents the mean ± SEM of 12 independent experiments, each done in triplicate. Results are expressed as percentage of control set equal to 100. °°°, P < 0.001 vs. C values; ***, P < 0.001, **, P < 0.01 vs. hCG values. Groups with different superscript letters are statistically different (P < 0.05).

 
Moreover, we investigated whether CoCl2-stimulated VEGF release could be influenced by ghrelin. To this aim, human luteal cells were incubated for 24 h with medium alone (C) or with CoCl2 (10 µM) in combination or not with ghrelin (10–12 to 10–7 M). As expected, CoCl2 significantly enhanced VEGF release (18). We demonstrated that ghrelin was able to significantly decrease CoCl2-induced VEGF release at all tested doses, except for the lowest used concentration (10–12 M). The effect was maximal at ghrelin 10–9 M, higher doses not being able to exert any further effect; ghrelin 10–11 to 10–12 M exerted similar effects, significantly lower than that exerted by ghrelin 10–9 M (Fig. 4BGo).

The mean concentration of VEGF in the control was 0.25 ± 0.02 pg/ml.

Effect of ghrelin on VEGF mRNA expression in human luteal cells

The mRNA expression of VEGF 121 and 165 isoforms was quantified by real-time RT-PCR and normalized with the internal control, the GAPDH gene. Ghrelin (10–10 or 10–7 M) failed to significantly affect VEGF 121 and 165 mRNA expression, either basal or CoCl2 induced (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the past years, Gaytan et al. (15) detected both components (ligand and receptor) of ghrelin system in human ovary, suggesting a potential regulatory action in the control of ovarian function. In particular immunohistochemistry revealed ghrelin and GRLN-R in luteal cells from young and mature human corpora lutea.

To our knowledge, our data are the first to demonstrate GRLN-R mRNA expression in purified mid-luteal phase human luteal cells by using classical and real-time RT-PCR.

More relevant for the purpose of this study, our in vitro results provide the first evidence of a direct inhibitory influence of ghrelin on human luteal function. A role of ghrelin in the control of ovarian physiology has been previously postulated in nonmammalian ovary. Actually very recently ghrelin has been suggested to modulate specific ovarian functions, such as hormone release, by using chicken ovarian granulosa cells (29). In line with these observations, in the current study, the ability of ghrelin to significantly decrease both basal and hCG-stimulated release of P has been demonstrated in midluteal phase human luteal cells. In the male gonad, a direct inhibitory effect of ghrelin has been recently suggested as well. Actually in rat testis, the in vitro steroidogenesis has been reported to be negatively influenced by ghrelin (30). Furthermore, very recently ghrelin expression in human Leydig cells has been inversely correlated with serum testosterone levels (31). Our results about ghrelin effect on luteal P production are consistent with the hypothesis that ghrelin might participate in the autolimitation of gonadal steroidogenesis, as proposed for rat testis by Barreiro et al. (32).

Besides inhibiting P secretion, in human luteal cells, ghrelin was also able to affect the release of important intraovarian regulators, such as PGs (17, 33). Indeed we demonstrated that ghrelin decreases luteotropic PGE2 release and increases luteolytic PGF2{alpha} release. The consequent imbalance between luteotropic and luteolytic factors could be a mechanism by which ghrelin might negatively influence luteal function.

Based on this assumption, it is not surprising that in our in vitro system ghrelin was able to significantly decrease both basal and hypoxia-stimulated VEGF release (18). Indeed, VEGF plays a pivotal luteotropic role, being essential for both luteal development and function (34). Interestingly, in our in vitro system, ghrelin was not able to affect either basal or hypoxia-induced VEGF mRNA expression (18). Because for VEGF synthesis several levels of regulation have been reported (35), our results suggest that on luteal VEGF production the influence of ghrelin could occur at a posttranscriptional level. The exact mechanism of such a posttranscriptional regulation needs further investigation.

Notably, in our in vitro study, the effects of ghrelin on most of the parameters under analysis showed similar responses over a wide range of doses. The ability of ghrelin to directly modulate its own sensitivity could at least in part explain our in vitro results, luteal cell number and viability not being affected by ghrelin treatment. Actually a ghrelin-dependent modulation of GRLN-R mRNA expression has been recently reported in some tissues (36, 37). In particular, in rat pituitary GRLN-R mRNA expression was apparently up-regulated by low levels and down-regulated by high levels of its ligand (36). If operative in human luteal cells, this model could also explain the ability of picomolar doses of ghrelin to evoke relevant secretory responses in our in vitro system. Nevertheless, in human luteal cells, the fine modulation of ghrelin system remains to be characterized, by either homologous (i.e. ghrelin) or heterologous (i.e. hCG, CoCl2-hypoxia) signals, together with postreceptorial signaling cascades after ghrelin-GRLN-R interaction. To elucidate these mechanisms, in our laboratory further experimental work is in progress.

Overall, in the current study, a direct inhibitory effect for ghrelin on luteal function has been demonstrated. Because in mature corpus luteum a complete ghrelin system was immunohistochemically demonstrated (15), extrapolating our in vitro results, it is tempting to propose that ghrelin may be involved in the autocrine/paracrine regulation of human luteal function. Moreover, the detection of GRLN-R, by both immunohistochemistry (15) and real-time RT-PCR (present results), in human luteal cells allows us to hypothesize that not only locally produced but also circulating ghrelin may directly affect luteal function. Interestingly, systemic ghrelin levels are chronically increased in malnutritional states (38), when the fertility potential is strongly decreased to avoid the energetic drain imposed by pregnancy (14, 39). In this adaptive response, a possible involvement for ghrelin has been recently suggested. Indeed, enhanced ghrelin levels seem to be able to exert a negative extragonadal influence on the reproductive axis (9) and inhibit development of preimplantation embryos (14, 39). In this assumption, our demonstration of a negative influence of ghrelin on luteal function could further strengthen the role recently proposed for ghrelin as a regulatory signal in the integrated control of energy balance and reproduction (40).


   Acknowledgments
 
The authors thank Dr. Maurizio Guido and Dr. Giovanna Salerno for their help in corpora lutea collection.


   Footnotes
 
This work was supported by grants from the Ministero della Salute (current research, 2006, "La prevenzione dell’handicap mentale: ruolo dei fattori paracrini ovarici ed endometriali nelle prime fasi di gestazione").

Author Disclosure Summary: The authors have nothing to disclose.

First Published Online May 29, 2007

Abbreviations: C, Control; CL, corpora lutea; Ct, threshold cycle; Ct, cycle time; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRLN-R, ghrelin receptor; hCG, human chorionic gonadotropin; HUVEC, human umbilical vein endothelial cell; P, progesterone; PG, prostaglandin; RT, reverse transcription; VEGF, vascular endothelial growth factor.

Received January 24, 2007.

Accepted May 23, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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