help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Caminos, J. E.
Right arrow Articles by Diéguez, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Caminos, J. E.
Right arrow Articles by Diéguez, C.
Endocrinology Vol. 144, No. 4 1594-1602
Copyright © 2003 by The Endocrine Society

ARTICLE

Expression of Ghrelin in the Cyclic and Pregnant Rat Ovary

J. E. Caminos1, M. Tena-Sempere1, F. Gaytán, J. E. Sanchez-Criado, M. L. Barreiro, R. Nogueiras, F. F. Casanueva, E. Aguilar and C. Diéguez

Departments of Physiology (J.E.C., R.N., C.D.) and Medicine, Molecular Endocrinology Section (F.F.C.), University of Santiago de Compostela School of Medicine, 15705 Santiago de Compostela, Spain; and Department of Cell Biology, Physiology, and Immunology (M.T.-S., F.G., J.E.S.-C., M.L.B., E.A.), University of Córdoba, 14004 Córdoba, Spain

Address all correspondence and requests for reprints to: Prof. Carlos Diéguez, Department of Physiology, University of Santiago de Compostela School of Medicine, C/S. Francisco 1, 15705 Santiago de Compostela, Spain. E-mail: fscadigo{at}usc.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ghrelin, a 28-amino acid acylated peptide, has been recently identified as the endogenous ligand for the GH secretagogue receptor. Previous studies demonstrated that ghrelin, acting centrally, strongly stimulates GH release and food intake. In this study we provide novel evidence for the expression of ghrelin in the cyclic and pregnant rat ovary. Persistent expression of ghrelin gene was demonstrated in rat ovary throughout the estrous cycle, although its relative mRNA levels varied depending on the stage of the cycle, with the lowest levels in proestrus and peak expression values on diestrous d 1, i.e. during the luteal phase of the cycle. Ghrelin immunoreactivity was predominantly located in the luteal compartment of the ovary; with intense immunostaining being detected in steroidogenic cells from corpus luteum of the current cycle as well as in all generations of regressing corpora lutea. Indeed, predominant expression of ghrelin in the corpus luteum was confirmed using a pseudopregnant rat model, where maximum ghrelin mRNA levels were detected in dissected luteal tissue. To note, the cyclicity in the profile of ovarian expression of ghrelin appeared to be tissue specific, as it was not detected in the stomach, nor was it observed in terms of circulating ghrelin levels. In addition, cyclic expression of ovarian ghrelin mRNA was disrupted by blockade of the preovulatory gonadotropin surge and ovulation by means of administration of a potent GnRH antagonist. Finally, ghrelin mRNA expression was persistently detected in rat ovary throughout pregnancy, with higher levels in early pregnancy and lower expression during the later part of gestation. In conclusion, our data provide novel evidence for the expression of ghrelin in the cyclic and pregnant rat ovary. Dynamic changes in the profile of ghrelin expression were detected during the estrous cycle and throughout pregnancy, thus suggesting a precise regulation of ovarian expression of ghrelin. Overall, our present findings may represent an additional link between body weight homeostasis and female reproductive function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GH SECRETAGOGUES (GHSs) are artificial compounds that induce GH release in all species tested to date. Until 1999, these molecules were assumed to mimic an unknown endogenous factor with the ability to activate the GHS-receptor (GHS-R) (1). The earlier cloning of GHS-R suggested that an endogenous ligand for this receptor might exist (2). Indeed, after intensive research by different groups, isolation of an endogenous ligand for the GHS-R, named ghrelin, was recently reported (3). The purified ligand was found to be a peptide of 28 amino acids, where the serine 3 residue was n-octanoylated. More recently, a second endogenous ligand for the GHS-R named des-Gln14-ghrelin, whose biological activity and sequence were identical to ghrelin, except for one glutamine in position 14, has been purified and characterized (4). These peptides have been shown to exert a very potent and specific GH-releasing activity in vitro and in vivo as well as to induce Pit-gene transcription (3, 5, 6, 7, 8, 9). Taking into account that ghrelin is secreted prevalently for the stomach and is present in normal subjects at considerable plasma concentrations, it has been postulated that stomach-derived circulating ghrelin stimulates GH synthesis and secretion by the somatotrophs (3). Moreover, ghrelin has emerged as a regulatory signal involved in energy homeostasis (10), reproduction (11), and gastrointestinal (12, 13) and cardiovascular (14, 15, 16) function, among others.

In this context, recent data have led to the recognition that ghrelin plays an important role in energy homeostasis. Thus, ghrelin administration induces a positive energy balance in rodents by decreasing fat utilization without significantly changing energy expenditure or locomotor activity (9, 17). Such an effect appears to be exerted at the central levels, and chronic ghrelin administration is associated with metabolic changes that lead to an efficient metabolic state, resulting in increased body weight and fat mass (9, 17). In good agreement, an inverse relationship between plasma ghrelin levels and body mass index has been reported in humans (18).

Data gleaned in recent years have shown the existence of an important interrelationship between gonadal function and energy homeostasis (19). Gonadal steroids affect energy balance and adiposity in a variety of mammalian species, via multiple redundant mechanisms (20). These include regulation of food intake at the central level, modulation of enzyme activity at the tissue level, and regulation of energy expenditure (21). Similarly, alterations in nutritional status, particularly fat mass, markedly influence the hypothalamus-pituitary-gonadal axis (20, 22). Noteworthy, some of the effects exerted by adipose tissue on gonadal function appear to be mediated by leptin, a relevant signal in body weight homeostasis and neuroendocrine function (10, 23), features that are shared by ghrelin. Interestingly, a wide range of endocrine and nonendocrine tissues, including the gonads, possess GHS-binding sites, and many of these tissues also have significant levels of ghrelin mRNA (24). Moreover, we have recently demonstrated the expression and functional role of ghrelin in rat testis, thus providing evidence for an unexpected reproductive facet of this newly discovered hormone (25).

As yet, expression of the ghrelin gene in the rat ovary remains unexplored. The aim of this paper was to characterize in detail the pattern of expression of ghrelin in the rat ovary, with special attention to the cellular distribution of ghrelin peptide within the ovarian tissue as well as the influence of estrous cyclicity, hormonal (gonadotropin) background, and pregnancy on ovarian ghrelin mRNA expression levels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Groups of virgin and pregnant female rats of the Sprague Dawley strain were housed in plastic cages under controlled conditions of lights (lights on, 0700–1900 h) and temperature (20–23 C), with free access to tap water an food available ad libitum. Vaginal smears were examined daily, and only rats exhibiting at least three consecutive 4-d estrous cycles were included in the study. After completion of the experiments, rats were anesthetized (ketamine hydrochloride, 80 mg/kg, im; xylazine, 10 mg/kg, im) and killed by decapitation, and trunk blood samples were collected in tubes containing EDTA·2Na (1 mg/ml blood) and aprotinin (500 U/ml blood; Sigma-Aldrich, St. Louis, MO). Plasma was separated by centrifugation, and samples were kept at -20 C until ghrelin analysis (26). The gastric fundus and ovaries were dissected, frozen in liquid nitrogen, and stored at -80 C. All experimental procedures were approved by the animal care committee on research from the University of Santiago de Compostela, Spain.

Experimental protocols
Experiment 1.
The pattern of expression of ghrelin mRNA was examined in ovarian and gastric tissue from adult cycling rats. Plasma ghrelin levels were also assessed. Animals (60 d old) were monitored for reproductive cyclicity by daily examination of vaginal cytology. Once an animal exhibited at least three subsequent 4-d estrous cycles, plasma, stomach, and ovaries were collected at 1700 h on the days of proestrus and estrus, diestrous d 1, and diestrous d 2. Collection and processing of plasma and tissue samples were conducted as described above. In addition, ovarian samples were taken at different stages of the estrous cycle for immunohistochemical analysis of ghrelin peptide expression.

Experiment 2.
Ovarian expression of ghrelin mRNA was investigated after blockade of preovulatory gonadotropin surge and ovulation by means of administration of a potent GnRH antagonist (Organon 30276, Organon, Oss, The Netherlands). Cycling females received a single ip injection of the antagonist (5 mg/kg) on the day of proestrus (1100 h), as this regimen is known to prevent the preovulatory rise in serum LH and FSH levels and ovulation in the rat (27). Vehicle-injected rats served as controls. The rats were killed on the afternoon of estrus, diestrous d 1, and diestrous d 2. Ovaries were removed and frozen at -80 C until analysis.

Experiment 3.
Previous experiments indicated predominant expression of ghrelin in the corpus luteum within the rat ovary. To further confirm this contention, an adult pseudopregnant rat model was used to discriminate the expression of ghrelin mRNA between luteal tissue (LT) and the remainder of ovary (NLT). During mating in rats, the male provides vaginocervical stimulation to the female via intromissions. Vaginocervical stimulation provided manually mimics many aspects of mating, including facilitation of lordosis, induction of sexual receptivity, abbreviation of the period of sexual receptivity, and induction of twice daily PRL surges, which result in pseudopregnancy (28). Day 1 of pseudopregnancy was defined as the day when a vaginal plug was recorded. Animals were killed by decapitation at 1700 h on d 7 of pseudopregnancy, and ovaries were collected. Ovarian tissue was separated by dissection into two compartments: LT of pseudopregnancy and NLT. Whole ovaries at the proestrous stage of normal cycling rats were used for comparison. Upon dissection, LT and NLT were pooled, snap-frozen, and stored at -80 C until use for RNA analysis.

Experiment 4.
Ovarian ghrelin mRNA expression was studied throughout gestation in Sprague Dawley rats (n = 6). The first day of pregnancy was documented by the presence of a vaginal plug with sperm after mating. Whole ovary was dissected from pregnant rats killed on d 5, 10, 15, and 21 of gestation. Tissues were immediately snap-frozen and stored at 80 C until RNA isolation. Ovarian samples from virgin cycling rats (75 d old) were obtained on the afternoon of proestrus and used as controls.

Gastric RNA isolation and Northern blot analysis
Total gastric fundus RNA from each stage of the estrous cycle was extracted by the acid guanidinium thiocyanate-phenol-chloroform method (29). Twenty micrograms of total gastric RNA were denatured with formaldehyde, electrophoresed in 1.5% agarose gel, and blotted onto a Hybond-N+ membrane (Amersham Pharmacia Biotech, Arlington Heights, IL). The membranes were hybridized with a 32P-labeled cDNA probe for ghrelin mRNA, as previously described (26, 30). The total integrated densities of hybridization signal were normalized using an image analyzer (Gel 2000, Bio-Rad Laboratories, Inc., Richmond, CA) and normalized for 18S RNA signal intensity.

RNA preparation and RT-PCR
Ovarian expression of the mRNA encoding ghrelin was assessed by semiquantitative RT-PCR. Total RNA was isolated from ovary samples from the different experimental settings, as previously described (25). For amplification of the different signals, the primer pairs indicated in Table 1Go were used. These sets of primers were synthesized according to the published cDNA sequences of rat ghrelin (3), based on previous references (25). In addition, to provide an appropriate internal control, parallel amplification of a 603-bp fragment of ß-actin mRNA was carried out in each sample using the primer pairs and conditions indicated in Table 1Go, as described in detail previously (30).


View this table:
[in this window]
[in a new window]
  		Table 1. Targets and primer pairs

 
For amplification of the targets, RT and PCR were run in two separate steps. Briefly, equal amounts of total ovary RNA (2 µg) were used as a template to generate cDNA by RT with random hexamer priming (25, 26). For semiquantitative PCR, 3 µl of the RT products were amplified in separate reactions using primers for rat ghrelin cDNA and ß-actin. PCR consisted of a variable number of cycles of amplification defined by denaturation at 98 C for 15 sec, annealing at 60 C for 1 min, and extension at 72 C for 1 min. A final extension cycle of 72 C for 10 min was included. In addition, different numbers of cycles were tested to optimize amplification in the exponential phase of PCR (data not show), and based on current data and previous references (25), the number of PCR cycles indicated for each target in Table 1Go was chosen for further analysis. Amplification of ghrelin signal from rat stomach was conducted as a positive control, using 26 PCR cycles to achieve amplification in the exponential phase. PCR products were gel-electrophoresed, transferred to nylon membrane, and subsequently incubated with a radiolabeled cDNA probe that hybridized to ghrelin. Hybridization signal was visualized by an image analyzer (Gel 2000, Bio-Rad Laboratories, Inc.), and band intensity was evaluated by image analysis software. In all samples, specific ghrelin hybridization signals were corrected by ß-actin as previously described (25, 30).

Ghrelin immunohistochemistry
Ovarian samples from adult (60-d-old) cycling rats were fixed in 4% paraformaldehyde in Sorensen buffer (pH 7.3) for 24 h and processed for paraffin embedding. Four-micrometer-thick sections were cut, placed on poly-L-lysine-coated slides, and used for immunohistochemistry. Ghrelin protein was detected using a rabbit antighrelin polyclonal antibody and avidin-biotin-peroxidase complex method, as described in detail previously (25).

RIA for rat ghrelin
Plasma ghrelin levels were assessed throughout the estrous cycle. Ghrelin levels were determined by means of a double antibody RIA using reagents kit and methods provided by Phoenix Pharmaceuticals, Inc. (Belmont, CA). Trunk blood samples were obtained by decapitation and were collected in tubes containing EDTA·2Na (1 mg/ml blood) and aprotinin (500 U/ml blood; Sigma-Aldrich). Samples were immediately centrifuged and then subjected to RIA, as previously described (26). The limit of assay sensitivity was 2 pg/ml; the intra- and interassay coefficients of variation were 5% and 13%, respectively.

Statistical analysis
All values were expressed as the mean ± SEM (n = 6/group). Statistical significance was determined by ANOVA in conjunction with a post hoc multiple comparison test. P < 0.05 compared with the appropriate control was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of ghrelin gene and protein in the cyclic rat ovary
Initial RT-PCR and Southern blot analyses revealed that ghrelin mRNA transcripts are expressed in random cycling rat ovary. As anticipated, similar signals were amplified from the stomach and used as a positive control. In detail, RT-PCR assays using ghrelin-specific primers resulted in the generation of amplicons of the expected size of 254 bp. Relative expression levels of ghrelin signal in rat stomach were much higher than those in random cyclic ovaries. Thus, exponential amplification of the target was conducted at 26 and 36 cycles for stomach and ovarian samples, respectively. In each sample, parallel amplification using a specific primer set of rat ß-actin served as an internal control. No products were detected from the negative controls (Fig. 1AGo).



View larger version (41K):
[in this window]
[in a new window]
  		Figure 1. A, Upper panels, Assessment of the expression of ghrelin mRNA in the random cyclic rat ovary. A representative RT-PCR assay of the expression of ghrelin mRNA in two independent ovarian samples is represented. Cloned ghrelin cDNA (lane 1), 100-bp molecular weight marker (lane 2), stomach (lane 3), ovary (lanes 7 and 9), ß-actin (lanes 4, 8, and 10), and RT-PCR controls (lanes 5, 6, 11, and 12) are shown. The specificity of the amplicons is demonstrated by Southern hybridization using a specific cDNA probe. B, Lower panels, Analysis of ghrelin mRNA expression in rat ovaries at different stages of the estrous cycle is presented. A representative RT-PCR and Southern blot of ghrelin in the experimental groups is shown. The integrity and loading of RNA were confirmed by amplification of ß-actin. The bottom panel is a summary of the ratio profiles of ghrelin mRNA/ß-actin. The data are represented as the mean ± SEM (n = 6/group). Histograms with different superscript letters are statistically different (P < 0.05). pro, Proestrus; di1, diestrous d 1; di2, diestrous d 2.

 
In a next step, ovarian ghrelin mRNA expression was evaluated throughout the estrous cycle by means of semiquantitative RT-PCR. A linear relationship between PCR products and amplification cycles was obtained in all assays (data no shown). Our analysis revealed that ghrelin mRNA is present in rat ovary at all stages of the estrous cycle, although its relative levels of expression varied depending on the cycle phase. Thus, ghrelin mRNA expression was lower during proestrus and increased significantly until diestrous d 1, when mRNA levels appeared to peak, declining thereafter during diestrous d 2 (Fig. 1BGo). In contrast, Northern blot analyses from rat stomach RNA showed that ghrelin mRNA levels remain at relatively constant values throughout the estrous cycle (Fig. 2AGo). In good agreement, plasma ghrelin levels did not significantly differ from one another (P > 0.05) throughout the estrous cycle in adult cycling rats (Fig. 2BGo).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 2. A, Northern blot analysis of ghrelin mRNA expression in rat stomach collected at the different stages of the estrous cycle. The levels of ghrelin transcript were normalized to the levels of 18S RNA and expressed in arbitrary units. B, Concentrations of plasma ghrelin during the different stages of the estrous cycle. Plasma ghrelin levels were measured by RIA. The data are represented as the mean ± SEM (n = 6/group). pro, Proestrus; di1, diestrous d 1; di2, diestrous d 2.

 
In addition, the distribution of ghrelin protein within ovarian tissue was examined by immunohistochemistry in adult cycling rats. Growing follicles and at least four generations of corpora lutea (CL) were present at any stage of the cycle. Growing and preovulatory follicles showed almost negligible immunostaining, which was impossible to differentiate from the background and thus was considered negative (Fig. 3AGo). In contrast, a weak ghrelin signal was detected in the newly formed CL (on the morning of estrus; data not shown), whereas strong ghrelin immunoreactivity was observed in CL of the current cycle, from diestrous d 1 to early proestrus (Fig. 3Go, B and C, and Fig. 4Go, A and B) as well as in all generations of regressing CL (Figs. 4CGo and 5AGo). Noteworthy, ghrelin immunostaining was located in the cytoplasm of steroidogenic luteal cells from both current cycle and regressing CL, whereas stromal luteal cells as well as apoptotic cells in regressing CL were negative (Fig. 4Go, B and C, and Fig. 5AGo). Ghrelin immunostaining was also detected in cells from the interstitial gland, although they showed weaker immunostaining than CL cells (Fig. 5BGo).



View larger version (72K):
[in this window]
[in a new window]
  		Figure 3. Sections of ovaries from adult cycling rats at proestrus (A), diestrous d 1 (B), and diestrous d 2 (C), immunostained with a rabbit polyclonal antighrelin antibody and counterstained with hematoxylin. Strong ghrelin signal was selectively detected in CL, whereas weak-to-negligible ghrelin immunostaining was observed in interstitial tissue and follicles. Scale bar, 100 µm.

 


View larger version (92K):
[in this window]
[in a new window]
  		Figure 4. Higher magnification of sections of CL, immunostained with a polyclonal antighrelin antibody. Steroidogenic cells in the CL of the current cycle, on diestrous d 1 (A), and in early proestrus (B) as well as of the previous cycle at estrus (C) show intense ghrelin immunoreactivity. Apoptotic cells in regressing CL at estrus are indicated by arrows. Scale bar, 25 µm.

 


View larger version (144K):
[in this window]
[in a new window]
  		Figure 5. Section of two-cycle regressing CL (A) and interstitial gland (B) at high magnification, immunostained with antighrelin antibody. Clear-cut ghrelin signal is detected in nonapoptotic steroidogenic cells from regressing CL. Similarly, weak immunostaining is detected in cells from interstitial gland. Scale bar, 25 µm.

 
Ovarian ghrelin mRNA expression after GnRH antagonist treatment
Administration of a potent GnRH antagonist (Organon 30276) on the day of proestrus (1100 h) in a regimen known to abolish the preovulatory gonadotropin surge and ovulation (27) significantly altered the pattern of ghrelin mRNA expression in the cyclic ovary. In detail, a clear-cut decrease in relative ghrelin mRNA levels (P < 0.01) was detected throughout luteal stages (i.e. diestrous d 1 and 2) of the cycle with respect to those in vehicle-treated rats (Fig. 6Go).



View larger version (14K):
[in this window]
[in a new window]
  		Figure 6. Effect of acute GnRH antagonist (Organon 30276) treatment on ovarian ghrelin mRNA expression. Normal cycling rats were treated in proestrus (1100 h) with a single ip injection of GnRH antagonist (5 mg/kg) or vehicle. The rats were killed on the afternoon of estrus, diestrous d 1 (di1), and diestrous d 2 (di2). The ghrelin mRNA/ß-actin ratio was measured in total RNA by RT-PCR/Southern blot analysis. The data are presented as the mean ± SEM (n = 6/group). **, P < 0.01 vs. corresponding stage in vehicle-treated animals. pro, Proestrus; di1, diestrous d 1; di2, diestrous d 2; Ant, GnRH antagonist.

 
Ghrelin mRNA expression in LT and NLT compartments of the pseudopregnant rat ovary
To obtain a more detailed localization of ghrelin mRNA expression in the ovary, CL were dissected out from ovaries of pseudopregnant rats. Thus, from the pseudopregnant rat ovary, two compartments were assessed for ghrelin mRNA expression: LT and NLT. In keeping with our previous results pointing to a predominant expression of ghrelin in the luteal tissue (see Figs. 3–5GoGoGo), significantly higher levels of ghrelin mRNA were detected in the LT vs. the NLT compartment (P < 0.01). No differences in ghrelin expression could be seen between NLT and virgin rat ovary in the proestrous stage (Fig. 7Go).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 7. Comparison of ghrelin mRNA expression in LT and NLT compartments of pseudopregnant rat ovary. Upper panel, Representative RT-PCR and Southern blot of ghrelin mRNA from LT, NLT, and total RNA from intact cyclic ovary on proestrus. Lower panel, Ghrelin mRNA levels were standardized by ß-actin mRNA levels, and the results were expressed as arbitrary units. The data are presented as the mean ± SEM (n = 6/group). Histograms with different superscript letters are statistically different (P < 0.05).

 
Ghrelin mRNA expression in rat ovary during pregnancy
The profile of ghrelin mRNA expression in rat ovary was evaluated throughout gestation (Fig. 8Go). Ghrelin mRNA was detected in rat ovary at all gestational stages examined. In detail, the expression of ghrelin transcript was highest at the earliest date examined (d 5), and decreased gradually over the course of pregnancy. Ghrelin mRNA levels were significantly lower on d 21 of pregnancy compared with those on d 5 (P < 0.01), d 10 (P < 0.01), and d 15 (P < 0.01) of gestation. In contrast, ghrelin mRNA levels on d 21 of pregnancy were similar to those in virgin rat ovaries at the proestrous stage.



View larger version (27K):
[in this window]
[in a new window]
  		Figure 8. Changes in the expression of ghrelin mRNA in the rat ovary throughout gestation. Upper panel, Representative RT-PCR and Southern blot analysis of ghrelin mRNA expression in pregnant rat ovaries at different stages of gestation. Lower panel, Ghrelin mRNA levels were normalized to levels of ß-actin mRNA. Ghrelin mRNA expression in cyclic ovaries at the proestrous phase (pro) served as a control for comparison. The data are presented as the mean ± SEM (n = 6/group). Histograms with different superscript letters are statistically different (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of this study provide the first demonstration of the expression of ghrelin (at the mRNA and peptide levels) in the adult rat ovary. In our study identification of the expression of ghrelin was accomplished by molecular (RT-PCR and Southern hybridization) and immunological approaches, thus allowing detailed characterization of the pattern of cellular distribution of this novel signal within the ovary as well as evaluation of the influence of estrous cyclicity, hormonal (gonadotropin) background, and pregnancy on ovarian ghrelin mRNA expression levels. In this sense, although the presence of ghrelin mRNA in human ovary has been preliminarily reported very recently (31), no detailed characterization of the pattern of expression of ghrelin in the female gonad had been yet attempted.

To ascertain the physiological regulation of the ghrelin gene expression in rat ovary, assessment of relative mRNA levels of this message was conducted through the different stages of the estrous cycle. Interestingly, despite persistent expression of the signal at all stages tested, ghrelin mRNA levels significantly varied depending on the phase of the cycle, with the lowest expression levels in proestrus and maximum values in the diestrous d 1 phase. Such a cyclic profile of expression, with peak levels in the luteal stages, is highly suggestive of predominant expression of ghrelin in the CL of the current cycle. Thus, ghrelin mRNA levels reached its highest values when CL entered into its functional phase and remained lower during CL formation and regression. This contention was further substantiated by our immunohistochemical analyses, which showed intense and specific ghrelin immunoreactivity in the cytoplasm of steroidogenic luteal cells from current cycle CL. In addition, nonapoptotic cells in regressing CL from previous cycles and, to a lesser extent, cells from the interstitial gland showed detectable ghrelin immunostaining. The profile of ghrelin expression in the CL was roughly coincident with its peak in functional activity and paralleled the pattern of progesterone secretion (32), which is suggestive of a potential functional role of ghrelin in the regulation of luteal development and/or function in the rat. Nevertheless, whether ghrelin is actually involved in steroidogenesis, angiogenesis, tissue remodeling, and growth of the CL remains to be established. Overall, our current data provide evidence for the first time that during the estrous cycle the expression of the gene encoding ghrelin undergoes dynamic changes that result in predominant expression of the peptide in the CL. Interestingly, our results also indicate that such a cyclic profile of ovarian ghrelin gene expression is tissue specific, as we failed to find any meaningful modification in ghrelin mRNA expression levels in the stomach. In good agreement, considering that the stomach accounts for more than 65% of circulating ghrelin (10), we did not observe any variation in plasma ghrelin levels during the different stages of the estrous cycle.

In keeping with our functional and morphological data pointing to a predominant expression of ghrelin in the CL within the cyclic ovary, blockade of the preovulatory surge of gonadotropins and subsequent ovulation by means of administration of a potent GnRH antagonist significantly disturbed the cyclic profile of ovarian ghrelin mRNA expression. Indeed, ghrelin mRNA levels in ovaries from rats treated with a single dose of GnRH antagonist persistently remained at values similar to those in the proestrous stage and were significantly lower than those in paired diestrous d 1 and diestrous d 2 cyclic ovaries. It is likely that prevention of ovulation, which, in turn, blocks formation of the new CL, accounted for the decrease in ghrelin mRNA expression in ovaries from GnRH antagonist-treated rats. Additionally, the reduction of circulating LH levels after GnRH antagonist treatment may be primarily involved in the decrease in ovarian ghrelin mRNA levels. In this sense, we have recently provided evidence for a direct stimulatory role of LH/CG in the control of testicular ghrelin gene expression in interstitial Leydig cells, i.e. the major steroidogenic cell type of the testis (33). Whether an analogous phenomenon also operates in the ovary awaits further investigation. Interestingly, it has been shown that central ghrelin administration inhibits LH pulsatility in female rats (11). Those and the present data strongly suggest the existence of a relationship between ghrelin and the reproductive axis (11). Taking into account that the reproductive axis is highly dependent on nutritional status (19), ghrelin, acting at central and peripheral levels, could be one of the signaling mechanisms linking the nutritional status and the hypothalamus-pituitary-ovary axis.

In addition to the cyclic ovary, ghrelin mRNA expression was assessed in the pseudopregnant and pregnant rat ovary. In the pseudopregnant rat model, manually provided vaginocervical stimulation to the female mimics many aspects of mating, including facilitation of lordosis, induction of sexual receptivity, abbreviation of the period of sexual receptivity, and induction of twice daily PRL surges, which results in pseudopregnancy (28). This, in turn, induces maintenance and enlargement of the CL, thus making it feasible to dissect the ovarian LT from the NLT. In this setting, and in keeping with our observations in the cyclic ovary, ghrelin mRNA expression levels were much higher in the LT compartment than in the NLT. It has to be stressed, however, that the NLT fraction is likely to contain minute amounts of extremely small, regressing CL that are impossible to dissect out of the remainder of ovary, which might contribute to ghrelin mRNA expression in this compartment. Nevertheless, NLT showed ghrelin mRNA levels similar to those of the cyclic ovary at the proestrous stage.

In addition, ovarian ghrelin gene expression was monitored throughout pregnancy. In our analysis, expression levels of the messenger encoding ghrelin were higher in early pregnancy, and a significant decrease was detected during the later part of gestation. In the rat, extension of the luteal function, which, in turn, acts to maintain pregnancy during the first week of gestation, is modulated by LH and PRL (34). However, gestation is maintained during the latter half of pregnancy by placental production of rat lactogen and androgens and by estrogen and progesterone produced by CL (35). Most studies on the biochemistry and structure of the CL suggest that during late pregnancy the CL of the rat is at the very early stages of structural regression, with no changes at the morphological level, but with changes at the molecular level (functional regression). Overall, our data on the profile of expression of ghrelin mRNA in the cyclic and pregnant rat ovary strongly suggest that ghrelin is maximally expressed in functional CL from both current cycle and gestation, with declining expression levels along functional regression of CL. Nevertheless, detailed analyses of the pattern of ghrelin immunoreactivity in rat ovary throughout pregnancy are needed to fully substantiate this hypothesis. In addition, recent reports showed a sharp peak of ghrelin mRNA expression on d 16 of gestation in the labyrinth trophoblast of rat placenta (30). On the basis of our current and previous data, we postulate that LH (and PRL) regulate the expression of ghrelin in the ovary during early pregnancy, whereas placental lactogen could play a pivotal role in the regulation of placental ghrelin mRNA expression during the latter half of pregnancy.

In summary, we have demonstrated that the novel peptide, ghrelin, is expressed in the rat ovary and that the functional CL is the major site for ghrelin expression within ovarian tissue. Dynamic changes in the profile of ovarian expression of ghrelin during the estrous cycle and pregnancy are highly suggestive of a finely tuned regulatory network, where endocrine (e.g. gonadotropins) and locally produced factors (e.g. ovarian steroids) are likely to participate. In turn, ghrelin may operate as an autocrine/paracrine regulator of ovarian physiology. Overall, our present findings open up the possibility that ghrelin may represent an additional link between body weight homeostasis and reproductive function.


   Footnotes
 
This work was supported by grants from the DGICYT and the Xunta de Galicia.

1 J.E.C. and M.T.-S. contributed equally to this work. Back

Abbreviations: CL, Corpora lutea; GHS, GH secretagogue; GHS-R, GH secretagogue receptor; LT, corpora lutea tissue; NLT, nonluteal tissue (remaining ovary).

Received October 11, 2002.

Accepted for publication December 31, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Casanueva FF, Dieguez C 1999 Growth hormone secretagogues: physiological role and clinical utility. Trends Endocrinol Metab 10:30–38[CrossRef][Medline]
  2. Smith RG, Van der Ploeg LH, Howard AD, Feighner SD, Cheng K, Hickey GJ, Wyvratt Jr MJ, Fisher MH, Nargund RP, Patchett AA 1997 Peptidomimetic regulation of growth hormone secretion. Endocr Rev 18:621–645[Abstract/Free Full Text]
  3. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K 1999 Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402:656–660[CrossRef][Medline]
  4. Hosoda H, Kojima M, Matsuo H, Kangawa K 2000 Purification and characterization of rat des-Gln14-ghrelin, a second endogenous ligand for the growth hormone secretagogue receptor. J Biol Chem 275:21995–22000[Abstract/Free Full Text]
  5. Seoane LM, Tovar S, Baldelli R, Arvat E, Ghigo E, Casanueva FF, Dieguez C 2000 Ghrelin elicits a marked stimulatory effect on GH secretion in freely-moving rats. Eur J Endocrinol 143:R7–R9
  6. Garcia A, Alvarez CV, Smith RG, Dieguez C 2001 Regulation of Pit-1 expression by ghrelin and GHRP-6 through the GH secretagogue receptor. Mol Endocrinol 15:1484–1495[Abstract/Free Full Text]
  7. Tamura H, Kamegai J, Shimizu T, Ishii S, Sugihara H, Oikawa S 2002 Ghrelin stimulates GH but not food intake in arcuate nucleus ablated rats. Endocrinology 143:3268–3275[Abstract/Free Full Text]
  8. Yamazaki M, Nakamura K, Kobayashi H, Matsubara M, Hayashi Y, Kangawa K, Sakai T 2002 Regulational effect of ghrelin on growth hormone secretion from perifused rat anterior pituitary cells. J Neuroendocrinol 14:156–162[CrossRef][Medline]
  9. Wren AM, Small CJ, Ward HL, Murphy KG, Dakin CL, Taheri S, Kennedy AR, Roberts GH, Morgan DG, Ghatei MA, Bloom SR 2000 The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion. Endocrinology 141:4325–4328[Abstract/Free Full Text]
  10. Horvath TL, Diano S, Sotonyi P, Heiman M, Tschop M 2001 Minireview: ghrelin and the regulation of energy balance—a hypothalamic perspective. Endocrinology 142:4163–4169[Abstract/Free Full Text]
  11. Furuta M, Funabashi T, Kimura F 2001 Intracerebroventricular administration of ghrelin rapidly suppresses pulsatile luteinizing hormone secretion in ovariectomized rats. Biochem Biophys Res Commun 288:780–785[CrossRef][Medline]
  12. Masuda Y, Tanaka T, Inomata N, Ohnuma N, Tanaka S, Itoh Z, Hosoda H, Kojima M, Kangawa K 2000 Ghrelin stimulates gastric acid secretion and motility in rats. Biochem Biophys Res Commun 276:905–908[CrossRef][Medline]
  13. Date Y, Nakazato M, Murakami N, Kojima M, Kangawa K, Matsukura S 2001 Ghrelin acts in the central nervous system to stimulate gastric acid secretion. Biochem Biophys Res Commun 280:904–907[CrossRef][Medline]
  14. Nagaya N, Kojima M, Uematsu M, Yamagishi M, Hosoda H, Oya H, Hayashi Y, Kangawa K 2001 Hemodynamic and hormonal effects of human ghrelin in healthy volunteers. Am J Physiol 280:R1483–R1487
  15. Nagaya N, Uematsu M, Kojima M, Date Y, Nakazato M, Okumura H, Hosoda H, Shimizu W, Yamagishi M, Oya H, Koh H, Yutani C, Kangawa K 2001 Elevated circulating level of ghrelin in cachexia associated with chronic heart failure: relationships between ghrelin and anabolic/catabolic factors. Circulation 104:2034–2038[Abstract/Free Full Text]
  16. Nagaya N, Uematsu M, Kojima M, Ikeda Y, Yoshihara F, Shimizu W, Hosoda H, Hirota Y, Ishida H, Mori H, Kangawa K 2001 Chronic administration of ghrelin improves left ventricular dysfunction and attenuates development of cardiac cachexia in rats with heart failure. Circulation 104:1430–1435[Abstract/Free Full Text]
  17. Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, Matsukura S 2001 A role for ghrelin in the central regulation of feeding. Nature 409:194–198[CrossRef][Medline]
  18. Tschop M, Weyer C, Tataranni PA, Devanarayan V, Ravussin E, Heiman ML 2001 Circulating ghrelin levels are decreased in human obesity. Diabetes 50:707–709[Abstract/Free Full Text]
  19. Mystkowski P, Schwartz MW 2000 Gonadal steroids and energy homeostasis in the leptin era. Nutrition 16:937–946[CrossRef][Medline]
  20. Casanueva FF, Dieguez C 1999 Neuroendocrine regulation and actions of leptin. Front Neuroendocrinol 20:317–363[CrossRef][Medline]
  21. Jones ME, Thorburn AW, Britt KL, Hewitt KN, Wreford NG, Proietto J, Oz OK, Leury BJ, Robertson KM, Yao S, Simpson ER 2000 Aromatase-deficient (ArKO) mice have a phenotype of increased adiposity. Proc Natl Acad Sci USA 97:12735–12740[Abstract/Free Full Text]
  22. Tena-Sempere M, Barreiro ML 2002 Leptin in male reproduction: the testis paradigm. Mol Cell Endocrinol 188:9–13[CrossRef][Medline]
  23. Altman J 2002 Weight in the balance. Neuroendocrinology 76:131–136[CrossRef][Medline]
  24. Muccioli G, Tschop M, Papotti M, Deghenghi R, Heiman M, Ghigo E 2002 Neuroendocrine and peripheral activities of ghrelin: implications in metabolism and obesity. Eur J Pharmacol 440:235–254[CrossRef][Medline]
  25. Tena-Sempere M, Barreiro ML, Gonzalez LC, Gaytan F, Zhang FP, Caminos JE, Pinilla L, Casanueva FF, Dieguez C, Aguilar E 2002 Novel expression and functional role of ghrelin in rat testis. Endocrinology 143:717–725[Abstract/Free Full Text]
  26. Gualillo O, Caminos JE, Nogueiras R, Seoane LM, Arvat E, Ghigo E, Casanueva FF, Dieguez C 2002 Effect of food restriction on ghrelin in normal-cycling female rats and in pregnancy. Obes Res 10:682–687[Medline]
  27. Nekola MV, Coy DH 1985 Direct and indirect inhibition of ovulation in rats by an antagonist of luteinizing hormone-releasing hormone. Endocrinology 116:756–760[Abstract]
  28. Komar CM, Curry Jr TE 2002 Localization and expression of messenger RNAs for the peroxisome proliferator-activated receptors in ovarian tissue from naturally cycling and pseudopregnant rats. Biol Reprod 66:1531–1539[Abstract/Free Full Text]
  29. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  30. Gualillo O, Caminos J, Blanco M, Garcia-Caballero T, Kojima M, Kangawa K, Dieguez C, Casanueva F 2001 Ghrelin, a novel placental-derived hormone. Endocrinology 142:788–794[Abstract/Free Full Text]
  31. Gnanapavan S, Kola B, Bustin SA, Morris DG, McGee P, Fairclough P, Bhattacharya S, Carpenter R, Grossman AB, Korbonits M 2002 The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans. J Clin Endocrinol Metab 87:2988–2991[Abstract/Free Full Text]
  32. Duggal PS, Weitsman SR, Magoffin DA, Norman RJ 2002 Expression of the long (OB-RB) and short (OB-RA) forms of the leptin receptor throughout the oestrous cycle in the mature rat ovary. Reproduction 123:899–905[Abstract]
  33. Barreiro ML, Gaytan F, Caminos JE, Pinilla L, Casanueva FF, Aguilar E, Dieguez C, Tena-Sempere M 2002 Cellular location and hormonal regulation of ghrelin expression in rat testis. Biol Reprod 67:1768–1776[Abstract/Free Full Text]
  34. Freeman ME, Kanyicska B, Lerant A, Nagy G 2000 Prolactin: structure, function, and regulation of secretion. Physiol Rev 80:1523–1631[Abstract/Free Full Text]
  35. Ben-Zimra M, Koler M, Melamed-Book N, Arensburg J, Payne AH, Orly J 2002 Uterine and placental expression of steroidogenic genes during rodent pregnancy. Mol Cell Endocrinol 187:223–231[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Hum ReprodHome page
C. I. Messini, K. Dafopoulos, N. Chalvatzas, P. Georgoulias, and I. E. Messinis
Effect of ghrelin on gonadotrophin secretion in women during the menstrual cycle
Hum. Reprod., December 18, 2008; (2008) den458v1.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
I. Viani, A. Vottero, F. Tassi, G. Cremonini, C. Sartori, S. Bernasconi, B. Ferrari, and L. Ghizzoni
Ghrelin Inhibits Steroid Biosynthesis by Cultured Granulosa-Lutein Cells
J. Clin. Endocrinol. Metab., April 1, 2008; 93(4): 1476 - 1481.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
C. Vidal, J. Roa, L. Pinilla, A. Pellicer, and M. Tena-Sempere
Maternal serum ghrelin levels in early IVF pregnancies: lack of prognostic value for viable pregnancy and altered post-prandial responses
Hum. Reprod., April 1, 2008; 23(4): 958 - 963.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
L. J. Moran, M. Noakes, P. M. Clifton, and R. J. Norman
The Use of Anti-Mullerian Hormone in Predicting Menstrual Response after Weight Loss in Overweight Women with Polycystic Ovary Syndrome
J. Clin. Endocrinol. Metab., October 1, 2007; 92(10): 3796 - 3802.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
G. M. Raso, G. Bianco, A. Iacono, E. Esposito, G. Autore, M. C. Ferrante, A. Calignano, and R. Meli
Maternal adaptations to pregnancy in spontaneously hypertensive rats: leptin and ghrelin evaluation
J. Endocrinol., September 1, 2007; 194(3): 611 - 619.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
M. Kluge, P. Schussler, M. Uhr, A. Yassouridis, and A. Steiger
Ghrelin Suppresses Secretion of Luteinizing Hormone in Humans
J. Clin. Endocrinol. Metab., August 1, 2007; 92(8): 3202 - 3205.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
M. C Garcia, M. Lopez, C. V Alvarez, F. Casanueva, M. Tena-Sempere, and C. Dieguez
Role of ghrelin in reproduction
Reproduction, March 1, 2007; 133(3): 531 - 540.
[Abstract] [Full Text] [PDF]

Home page
 J AndrolHome page
T. Ishikawa, H. Fujioka, T. Ishimura, A. Takenaka, and M. Fujisawa
Ghrelin Expression in Human Testis and Serum Testosterone Level
J Androl, March 1, 2007; 28(2): 320 - 324.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. M. Castellano, M. Gaytan, J. Roa, E. Vigo, V. M. Navarro, C. Bellido, C. Dieguez, E. Aguilar, J. E. Sanchez-Criado, A. Pellicer, et al.
Expression of KiSS-1 in Rat Ovary: Putative Local Regulator of Ovulation?
Endocrinology, October 1, 2006; 147(10): 4852 - 4862.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
K. Nakahara, M. Nakagawa, Y. Baba, M. Sato, K. Toshinai, Y. Date, M. Nakazato, M. Kojima, M. Miyazato, H. Kaiya, et al.
Maternal Ghrelin Plays an Important Role in Rat Fetal Development during Pregnancy
Endocrinology, March 1, 2006; 147(3): 1333 - 1342.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
K. J. Pulman, W. M. Fry, G. T. Cottrell, and A. V. Ferguson
The Subfornical Organ: A Central Target for Circulating Feeding Signals
J. Neurosci., February 15, 2006; 26(7): 2022 - 2030.
[Abstract] [Full Text] [PDF]

Home page
 J ANIM SCIHome page
B. W. Hess, S. L. Lake, E. J. Scholljegerdes, T. R. Weston, V. Nayigihugu, J. D. C. Molle, and G. E. Moss
Nutritional controls of beef cow reproduction
J Anim Sci, June 1, 2005; 83(13_suppl): E90 - 106.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
P. L. Jeffery, R. P. Duncan, A. H. Yeh, R. A. Jaskolski, D. S. Hammond, A. C. Herington, and L. K. Chopin
Expression of the Ghrelin Axis in the Mouse: An Exon 4-Deleted Mouse Proghrelin Variant Encodes a Novel C Terminal Peptide
Endocrinology, January 1, 2005; 146(1): 432 - 440.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
G. Rindi, A. Torsello, V. Locatelli, and E. Solcia
Ghrelin Expression and Actions: A Novel Peptide for an Old Cell Type of the Diffuse Endocrine System
Experimental Biology and Medicine, November 1, 2004; 229(10): 1007 - 1016.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
A. J. van der Lely, M. Tschop, M. L. Heiman, and E. Ghigo
Biological, Physiological, Pathophysiological, and Pharmacological Aspects of Ghrelin
Endocr. Rev., June 1, 2004; 25(3): 426 - 457.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
M. J Iglesias, R. Pineiro, M. Blanco, R. Gallego, C. Dieguez, O. Gualillo, J. R Gonzalez-Juanatey, and F. Lago
Growth hormone releasing peptide (ghrelin) is synthesized and secreted by cardiomyocytes
Cardiovasc Res, June 1, 2004; 62(3): 481 - 488.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
F. Gaytan, M. L. Barreiro, J. E. Caminos, L. K. Chopin, A. C. Herington, C. Morales, L. Pinilla, R. Paniagua, M. Nistal, F. F. Casanueva, et al.
Expression of Ghrelin and Its Functional Receptor, the Type 1a Growth Hormone Secretagogue Receptor, in Normal Human Testis and Testicular Tumors
J. Clin. Endocrinol. Metab., January 1, 2004; 89(1): 400 - 409.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
J. E. Caminos, R. Nogueiras, M. Blanco, L. M. Seoane, S. Bravo, C. V. Alvarez, T. Garcia-Caballero, F. F. Casanueva, and C. Dieguez
Cellular Distribution and Regulation of Ghrelin Messenger Ribonucleic Acid in the Rat Pituitary Gland
Endocrinology, November 1, 2003; 144(11): 5089 - 5097.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
U. Pagotto, A. Gambineri, C. Pelusi, S. Genghini, M. Cacciari, B. Otto, T. Castaneda, M. Tschop, and R. Pasquali
Testosterone Replacement Therapy Restores Normal Ghrelin in Hypogonadal Men
J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4139 - 4143.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article