Effects of Chronic Hyperghrelinemia on Puberty Onset and Pregnancy Outcome in the Rat
http://www.100md.com
内分泌学杂志 2005年第7期
Department of Cell Biology, Physiology and Immunology, University of Cordoba (R.F.F., V.M.N., M.L.B., E.M.V., E.A., L.P., M.T.-S.), 14004 Cordoba, Spain; Research Institute of Animal Production (A.V.S.), 94992 Nitra, Slovakia; and Departments of Physiology (S.T., C.D.) and Medicine (F.F.C.), University of Santiago de Compostela, 15705 Santiago de Compostela, Spain
Address all correspondence and requests for reprints to: Dr. Manuel Tena-Sempere, Physiology Section, Department of Cell Biology, Physiology, and Immunology, Faculty of Medicine, University of Cordoba, Avenida Menéndez Pidal s/n, 14004 Cordoba, Spain. E-mail: fi1tesem@uco.es.
Abstract
Ghrelin, the endogenous ligand of the GH secretagogue receptor, has been recently involved in a wide array of biological functions, including signaling of energy insufficiency and energy homeostasis. On the basis of the proven reproductive effects of other regulators of energy balance, such as the adipocyte-derived hormone leptin, we hypothesized that systemic ghrelin may participate in the control of key aspects of reproductive function. To test this hypothesis, the effects of daily treatment with ghrelin were assessed in rats, pair-fed with control animals, in two relevant reproductive states, puberty and gestation, which are highly dependent on proper energy stores. Daily sc injection of ghrelin (0.5 nmol/12 h; between postnatal d 33 and 43) significantly decreased serum LH and testosterone levels and partially prevented balanopreputial separation (as an external index of puberty onset) in pubertal male rats. On the contrary, chronic administration of ghrelin to prepubertal females, between postnatal d 23 and 33, failed to induce major changes in serum levels of gonadotropins and estradiol, nor did it modify the timing of puberty, as estimated by the ages at vaginal opening and first estrus. Moreover, females treated with ghrelin at puberty subsequently displayed normal estrous cyclicity and were fertile. Conversely, ghrelin administration (0.5 nmol/12 h) during the first half of pregnancy (d 1–11) resulted in a significant decrease in pregnancy outcome, as estimated by the number of pups born per litter, without changes in the number of successful pregnancies at term or gestational length. Overall, our data indicate that persistently elevated ghrelin levels, as a putative signal for energy insufficiency, may operate as a negative modifier of key reproductive states, such as pregnancy and (male) puberty onset.
Introduction
GHRELIN, THE RECENTLY identified endogenous ligand of the GH secretagogue receptor, is a 28-amino acid peptide with an essential n-octanoylation at serine 3 residue (1, 2). Ghrelin was originally isolated from the stomach, which is by far the major source of systemic ghrelin, accounting for at least two thirds of its plasma levels (1, 2, 3, 4). Nonetheless, ghrelin has also been detected in a large number of tissues and cell types, including small intestine, pancreas, lymphocytes, placenta, kidney, lung, pituitary, brain, and gonads (3, 4, 5, 6). Apart from its proven ability to elicit GH secretion, a major interest in this peptide derived from the fact that it is involved in the regulation of energy balance by inducing food intake and reducing fat utilization (3, 7). These activities are probably mediated by a complex cellular network at the central nervous system, which is under the control of additional peripheral hormones, such as the adipocyte-derived hormone leptin (3). In this context, ghrelin analogs, with agonistic or antagonistic activity, have appeared as a suitable approach for therapeutic intervention in a variety of disease states linked to alterations in body weight homeostasis. Moreover, on the basis of its diverse biological actions, the (chronic) use of ghrelin has been recently postulated as a potential therapeutic strategy in different pathological conditions, such as cancer cachexia (anabolic agent) (8), severe chronic heart failure (improvement of left ventricular function) (9), and diverse proinflammatory states (inhibitor of cytokine production) (10). In this scenario, complete characterization of the biological effects of repeated administration of ghrelin has become mandatory.
Compelling evidence has demonstrated the close connection between the systems governing energy homeostasis and reproductive function (11). Although the signals and molecular mechanisms responsible for the concerted control of energy stores and reproduction remain to be fully elucidated, leptin has been recently proven to be a key neuroendocrine integrator in such biological function (11, 12). Thus, threshold leptin levels, as peripheral signal for sufficient energy stores, are absolutely essential for normal pubertal development and fertility, whereas disturbance of reproductive function is observed in human and animal models of leptin insufficiency (11, 12, 13). The mechanisms by which leptin regulates fertility probably involve actions at different levels of the gonadotropic axis, the hypothalamus being the primary target for the reproductive actions of leptin (11, 12). Conceivably, other neuroendocrine integrators with key roles in body weight homeostasis would interact with leptin in the integrated control of energy balance and reproduction.
Despite the proposed role of ghrelin in body weight homeostasis as peripheral signal for energy insufficiency (14), its contribution to the control of reproductive functions has received limited attention to date. Nonetheless, fragmentary data strongly suggest the potential involvement of ghrelin in regulation of the reproductive axis. Thus, expression of ghrelin has been demonstrated in human and rodent placenta, and ghrelin has been reported to inhibit early embryo development in vitro (6, 15). In addition, ghrelin was shown to suppress LH secretion in vivo and to decrease LH responsiveness to LHRH in vitro (16, 17). Moreover, expression of ghrelin and its cognate receptor has been demonstrated in rat and human gonads (6), ghrelin was reported to inhibit stimulated testicular testosterone (T) secretion (18), and acquisition of ghrelin expression by Leydig cells has been suggested to play an important role in the control of proliferation of this cell type (19). However, the functional analyses conducted to date have been mostly restricted to acute administration of the peptide and assessment of hormonal responses, whereas the developmental or reproductive effects of continuous administration of ghrelin have been scarcely studied (20). In the present work we aimed at evaluating the effects of chronic administration of ghrelin on two relevant reproductive states, namely, puberty and gestation, which have been proven to be extraordinarily sensitive to appropriate energy stores (14). To this end, daily sc administration of ghrelin was conducted in (pre)pubertal male and female rats as well as in pregnant rats during the first half of gestation. In all settings, ghrelin-treated rats were pair-fed with control animals to minimize the potential bias of differences in body weight on key reproductive parameters, such as conventional indexes of puberty onset (21).
Materials and Methods
Experimental design
Wistar rats bred in the vivarium of University of Cordoba were used. The day the litters were born was considered d 1 of age. The animals were maintained under constant conditions of light (14 h of light, from 0700 h) and temperature (22 C) and were weaned at 21 d of age in groups of five rats per cage with free access to pelleted food and tap water until initiation of the experimental procedures, when the animals were individually caged, and the access to chow was paired between control and treated animals (see below). Experimental procedures were approved by the Cordoba University ethical committee for animal experimentation and were conducted in accordance with the European Union normative for care and use of experimental animals. Rat ghrelin, with n-octanoyl modification at Ser3, was obtained from Bachem AG (Bubendorf, Switzerland).
To monitor the effects of ghrelin on puberty onset and pregnancy outcome, a general protocol of sc injection of ghrelin (0.5 nmol/100 μl saline), twice a day (at 0900 and 1900 h), was used, and different end points were recorded depending on the experimental setting. In all experiments, special efforts were made to minimize the potential bias of major differences in body weight on the different reproductive end points under analysis. Thus, ghrelin-treated rats were pair-fed with control animals. To this end, daily food intake was recorded in reference control animals (pubertal male and female rats as well as pregnant animals), and an equal fixed amount of standard chow was daily provided to control and ghrelin-treated animals. Likewise, to prevent the impact of differences in grouping of the animals on sensitive reproductive parameters, such as puberty onset, control and ghrelin-treated animals were individually caged at the beginning of the experimental procedures, in keeping with our previous studies (22).
In experiment 1, pubertal male rats (n = 12/group) were sc injected with ghrelin (0.5 nmol/12 h) or vehicle (physiological saline) between postnatal d 33 and 43. This period was selected on the basis of previous studies of the normal timing of puberty in the male rat (23) and local data on the occurrence of balanopreputial separation (BPS) in our animal stock. In all experimental animals, body weights were monitored daily, and the occurrence of BPS, defined as complete separation of prepuce from gland penis (balano), was recorded. The animals were sc injected under conscious conditions after careful handling to avoid any stressful influence and were humanely killed by decapitation on d 43, 1 h after the last injection of ghrelin, at which time trunk blood was collected. Additional blood samples were obtained by jugular venipuncture under light ether anesthesia on d 33 and 41, 1 h after sc injection of ghrelin or vehicle.
In addition, in experiment 2, daily sc administration of ghrelin (0.5 nmol/12 h) or vehicle was performed in pubertal female rats (n = 12/group) between postnatal d 23 and 33. This period was selected on the basis of previous references on the normal timing of puberty in the female rat (23), and local data on the occurrence of vaginal opening (VO) in our animal stock. In all experimental animals, body weights were daily monitored, and the ages of occurrence of VO, defined by the complete canalization of the vagina, and of first estrus were recorded. The animals were sc injected under conscious conditions after careful handling to avoid any stressful influence. Blood samples were obtained by jugular venipuncture under light ether anesthesia on d 25, 28, and 31, 1 h after sc injection of ghrelin or vehicle. Additional blood sampling was carried out on the day of VO. Thereafter, estrous cyclicity and fertility were monitored in ghrelin- and vehicle-treated rats by daily vaginal cytology, and mating with adult male rats of proven fertility and recording of percentage of successful pregnancies, respectively.
Finally, in experiment 3, pregnant females (n = 12/group) were sc injected with ghrelin (0.5 nmol/12 h) or vehicle (physiological saline), between d 1 and 11 of pregnancy. This period was specifically selected to target early events in gestation, in line with a previous study of the effects of ghrelin on development of mouse preimplantation embryos in vitro (15). For timing of pregnancy, on the afternoon of proestrus cycling female rats were placed in individual cages with adult males of proven fertility. In the morning of the following day, vaginal smears were collected, and the presence of spermatozoa was checked; animals showing spermatozoa at vaginal smears were considered at d 1 of pregnancy and were included for daily injection of ghrelin or vehicle. Pregnant rats were sc injected under conscious conditions after careful handling to avoid any stressful influence. In all experimental animals, body weights were monitored daily throughout gestation. Pregnancy outcome was recorded by the percentage of successful pregnancies at term and the number of pups per litter at birth. In addition, sex ratio was calculated, and body weight of litters was monitored up to weaning (postnatal d 21).
Hormone measurement by specific RIAs
Serum levels of LH and FSH were measured in a volume of 25–50 μl using a double-antibody method and RIA kits supplied by the National Institutes of Health (Dr. A. F. Parlow, National Institute of Diabetes and Digestive and Kidney Diseases, National Hormone and Peptide Program, Torrance, CA). Rat LH-I-9 and FSH-I-9 were labeled with 125I by the chloramine-T method, and hormone concentrations were expressed using the reference preparations LH-RP-3 and FSH-RP-2 as standards. Intra- and interassay coefficients of variation were 8% and 10%, respectively, for LH and 6% and 9%, respectively, for FSH. The sensitivity of the assays was 5 pg/tube for LH and 20 pg/tube for FSH. In addition, in selected experimental groups, serum ghrelin and GH levels were assayed. For ghrelin measurements, a commercially available RIA kit from Phoenix Pharmaceuticals (Belmont, CA), with an assay sensitivity of 1 pg/tube, was used for determination of total ghrelin levels. Serum GH levels were measured by a double-antibody RIA, using kits provided by the National Institutes of Health. Rat GH-I-7 was labeled as described above, and hormone concentrations were expressed using reference preparation GH-RP-2. Intra- and interassay coefficients of variation were 6% and 9%, respectively, and the sensitivity of the assay was 5 pg/tube. Finally, where indicated, serum T and estradiol levels were determined using commercial kits from MP Biomedicals (Costa Mesa, CA), following the instructions of the manufacturer. The sensitivities of the assay were 0.1 and 0.5 ng/tube for T and estradiol, respectively. The intraassay coefficients of variation were less than 5%. The accuracy of hormone determinations was confirmed by assessment of rat serum samples of known hormone concentrations used as external controls.
Presentation of data and statistics
Serum LH, FSH, GH, ghrelin, T, and estradiol determinations were conducted in duplicate, with a minimum total number of 10 samples/determinations/group. Hormonal data are presented as the mean ± SEM. Results were analyzed for statistically significant differences using Student’s t test or ANOVA, followed by Student-Newman-Keuls multiple range test (SigmaStat 2.0, Jandel Corp., San Rafael, CA). 2 evaluation was used for statistical comparison of data for percentages of BPS and successful pregnancy. P 0.05 was considered significant.
Results
Effects of chronic administration of ghrelin on puberty onset in the male rat
The effects of ghrelin on male puberty onset were assessed by daily sc injection of the peptide (at 0.5 nmol/12 h) between postnatal d 33 and 43 and recording of body weight, hormonal responses, and BPS occurrence. Cumulative body weight gain (data not shown) and total body weights were not significantly different between controls and pair-fed ghrelin-injected males (Fig. 1A). Serum LH and T levels in control animals significantly increased throughout the study period (between d 33 and 43), in line with previous findings (24), whereas serum FSH levels remained unaltered. The age of occurrence of BPS in controls rats was 42.2 ± 0.4 d, and 100% of animals (12 of 12) presented complete preputial separation on d 43. In pair-fed animals, chronic treatment with ghrelin did not modify serum FSH levels. In contrast, sc injection of ghrelin partially prevented the age-dependent increase in serum LH levels: a moderate decrease, which was shortly below the limit of statistical significance, was detected on d 41, whereas a significant inhibition was observed on d 43 in ghrelin-treated animals. In addition, daily injection of ghrelin significantly lowered serum T levels during the study period and partially blunted the age-dependent rise in T concentrations (Fig. 1, B–D). In keeping with these findings, chronic administration of ghrelin partially prevented the normal occurrence of BPS; complete preputial separation was detected in only 58.5% (seven of 12) of the ghrelin-treated animals on d 43 (Fig. 1E). As a control for the validity of the experimental model and the dose of ghrelin used, at this age point, exogenous administration of 0.5 nmol ghrelin was able to induce a significant 2.4-fold increase in serum levels of total ghrelin 1 h after injection (Fig. 1F). Such a response was associated with a moderate, but significant, elevation in basal serum GH levels (1.95 ± 0.217 vs. 0.997 ± 0.216 ng/ml in vehicle-injected animals), in keeping with previous findings (25).
FIG. 1. Compilation of the effects of chronic administration of ghrelin to pubertal male rats. Ghrelin was injected at a dose of 0.5 nmol/12 h between postnatal d 33 and 43. Body weights of the experimental animals are presented in A. Serum LH, FSH, and T levels in vehicle- and ghrelin-treated animals are shown in B–D. Samples were obtained on d 33, 41, and 43, 1 h after the last injection of the peptide. In addition, the cumulative percentage of males with complete BPS on d 43 is presented in E, whereas serum ghrelin levels on d 43 (1 h after the last injection of the peptide) are shown in F. The experimental groups were composed of 12 animals. Where applicable, values are given as the mean ± SEM. For vehicle-injected animals, groups with different superscript letters are statistically different (P < 0.05). **, P < 0.01 vs. corresponding vehicle-injected animals (by ANOVA, followed by Student-Newman-Keuls multiple range test).
Effects of chronic administration of ghrelin on puberty onset in the female rat
The effects of ghrelin on female puberty onset were assessed by daily sc injection of the peptide (at 0.5 nmol/12 h) between postnatal d 23 and 33, and recording of body weight, hormonal responses, and occurrence of VO and first estrus. In addition, estrous cyclicity and fertility were subsequently monitored in adult females treated with ghrelin at puberty. Cumulative body weight gain (data not shown) and total body weights were not significantly different between controls and pair-fed, ghrelin-injected females (Fig. 2A). Serum LH and estradiol levels in control animals significantly increased during the study period (between d 23 and 33), as previously described (23), whereas serum FSH levels remained unaltered. The age of occurrence of VO in controls rats was 34.4 ± 0.6 d, and first estrus was observed at a mean age of 35.6 ± 1.0 d. In pair-fed animals, chronic treatment with ghrelin did not change serum FSH levels. Likewise, sc injection of ghrelin failed to substantially modify the age-dependent increase in serum LH levels, except for a decrease in LH concentrations at the age of VO, nor did it alter serum estradiol concentrations during the study period (Fig. 2, B–D). In line with these observations, chronic administration of ghrelin did not change the age of occurrence of VO and first estrus (Fig. 2E). Similarly, female rats chronically treated with ghrelin during puberty subsequently displayed normal estrous cyclicity and were fertile: 92% (11 of 12) of ghrelin-treated females become pregnant upon mating with males of proven fertility, a percentage similar to that in vehicle-treated animals. Nonetheless, evidence for the validity of the experimental model and the dose of ghrelin used comes from the observation that on postnatal d 31, exogenous administration of 0.5 nmol ghrelin was able to induce a significant 2.6-fold increase in serum levels of total ghrelin 1 h after injection (Fig. 2F). As was the case in males, such a response was associated with a moderate, but significant, elevation of basal serum GH levels (1.332 ± 0.116 vs. 0.726 ± 0.043 ng/ml in vehicle-injected animals).
FIG. 2. Compilation of the effects of chronic administration of ghrelin to pubertal female rats. Ghrelin was injected at a dose of 0.5 nmol/12 h between postnatal d 23 and 33. Body weights of the experimental animals are presented in A. Serum LH, FSH, and estradiol (E2) levels in vehicle- and ghrelin-treated animals are shown in B–D. Samples were obtained on d 25, 28, and 31 as well as on the day of VO, 1 h after the last injection of the peptide. In addition, the ages of occurrence of VO and first estrus are shown in E, whereas serum ghrelin levels on d 31 (1 h after the last injection of peptide) are presented in F. The experimental groups were composed of 12 animals. Values are given as the mean ± SEM. For vehicle-injected animals, groups with different superscript letters are statistically different (P < 0.05). **, P < 0.01 vs. corresponding vehicle-injected animals (by ANOVA, followed by Student-Newman-Keuls multiple range test).
Effects of chronic administration of ghrelin on pregnancy outcome in the rat
Finally, the effects of ghrelin on pregnancy onset were assessed by daily sc injection of the peptide (at 0.5 nmol/12 h) to pregnant dams between d 1 and 11 of gestation. Cumulative body weight gain (data not shown) and total body weights were not significantly different between controls and pair-fed, ghrelin-injected pregnant females (Fig. 3A). Likewise, the percentage of successful pregnancies at term and gestational length were similar between the experimental groups (data not shown). On the contrary, the number of pups born per litter was significantly lower in ghrelin-treated dams, with a male/female ratio of 1.33 vs. 1.25 in vehicle-injected animals (Fig. 3B). After adjustment of litter size (10 pups/mother), daily recording of body weight demonstrated that pups from mothers treated with ghrelin during the first half of pregnancy showed higher body weight values than those of vehicle-treated dams from birth to weaning (Fig. 3C).
FIG. 3. Compilation of the effects of chronic administration of ghrelin to pregnant rats during the first half of gestation. Ghrelin was injected at a dose of 0.5 nmol/12 h between d 1 and 11 of pregnancy. Body weights of pregnant animals are presented in the upper panel (A). The number of pups born per litter is shown in the middle panel (B). In the lower panel (C), body weights of pups from vehicle- and ghrelin-treated dams, from birth to weaning, are presented. Groups of pregnant rats were composed of 12 animals. Values are given as the mean ± SEM. Analysis of number of pups per litter: **, P < 0.01 vs. corresponding litters from vehicle-treated dams; analysis of BW of pups: *, P < 0.05 vs. corresponding litters from vehicle-injected dams (by Student’s t test).
Discussion
Expression and/or actions of ghrelin have been recently demonstrated at different levels of the hypothalamic-pituitary-gonadal axis (6, 17, 18). Yet the actual role of ghrelin in the integrated control of energy homeostasis and reproduction remains largely unexplored. Moreover, despite extensive efforts in characterization of the biological roles of ghrelin, only a limited number of studies analyzing the effects of continuous, rather than acute, administration of the peptide have been reported to date, and these have mostly focused on analysis of its metabolic and orexigenic actions (7, 25, 26). In the present study we evaluated the effects of elevated ghrelin levels on two relevant reproductive states, puberty and gestation, by recording suitable hormonal and phenotypic biomarkers. Considering the expected orexigenic and adipogenic effects of ghrelin treatment (7, 26), special attention was paid to avoid the potential bias of changes in body weight between the experimental groups. Thus, ghrelin-treated rats were pair-fed with control animals, a protocol that resulted in similar body weights among treatments, in keeping with previous studies (27). In addition, the dose of ghrelin selected (0.5 nmol/12 h) was sufficient to produce endocrine responses (28, 29), but is within the limit of induction of overt hyperphagia in rats (26). Indeed, a significant elevation (2.4- to 2.6-fold increase) of total ghrelin levels was demonstrated after systemic injection of 0.5 nmol of the peptide in our experimental groups, whose magnitude was in the range of that induced by fasting (26). Such elevation was associated with a modest, but significant, increase in serum GH levels, which might be regarded as sentinel for the endocrine activity of exogenously administered ghrelin. Overall, it was assumed that the reproductive effects observed after its repeated administration were primarily due to elevated ghrelin levels and were not secondary to changes in body weight induced by the orexigenic hormone.
Under the above experimental conditions, daily sc administration of ghrelin to prepubertal males induced a significant decrease in serum T levels during the study period, which was associated with normal to decreased serum LH levels. In good agreement, normal occurrence of preputial separation (as an external index of puberty) (30) was prevented on d 43 in more than 40% of treated males. The mechanisms for such an effect might involve inhibition of LH secretion at the hypothalamic-pituitary unit and/or direct inhibitory effects on testicular T secretion. Indeed, suppression of serum LH levels has been reported in prepubertal male rats after central injection of ghrelin (17). Likewise, ghrelin has been proven to be an inhibitory signal for stimulated testicular T secretion in vitro (18). Although the two mechanisms are not mutually exclusive, the fact that T levels were significantly lower in ghrelin-treated animals at all ages tested strongly suggests a major contribution of direct inhibitory effects of ghrelin on the prepubertal testis. These might include disturbance of adult-type Leydig cell development, because ghrelin has been recently proven to inhibit the proliferative activity of immature Leydig cells in the pubertal testis (19). Nevertheless, the net end point for the above hormonal responses is the partial disruption of the normal timing of puberty (as estimated by age at BPS). Together, these data strongly suggest that elevated ghrelin levels might be detrimental for normal puberty onset in the male. Considering that circulating ghrelin levels are negatively correlated to body mass index, and the proposed role of ghrelin as a signal for energy insufficiency (14), it is tempting to propose that ghrelin per se might operate as a negative modifier of male puberty, and it might contribute to suppression of the male reproductive axis in situations of negative energy balance, such as starvation (31).
In contrast with data obtained in males, chronic treatment of prepubertal females with ghrelin failed to induce major hormonal changes in LH, FSH, and estradiol secretion (except for a decrease in serum LH levels at the age of VO), nor did it alter the age of occurrence of VO and first estrus, taken as putative indices of puberty onset in the female (23). Moreover, females treated with ghrelin at puberty subsequently displayed normal estrous cyclicity and were fertile. Interestingly, despite the proposed role of ghrelin as a signal for energy insufficiency (14), chronic ghrelin treatment from birth to puberty has been previously reported to advance the age of VO in immature females (20). Such a discrepancy might be related to the fact that in the previous study, doses of 1.0–1.5 nmol ghrelin were daily injected between postnatal d 1 to the day of VO, and the animals were not pair-fed with controls (20). Because we aimed at targeting the specific effects of ghrelin on female puberty, for the present study we selected a protocol of daily treatment from d 23 (beginning of the prepubertal period) to d 33 (around VO). Overall, although we cannot exclude the possibility that higher doses of ghrelin may bring about deleterious effects similar to those observed in males, our data clearly show that male puberty is more sensitive than female puberty to the potential adverse effects of ghrelin. Similarly, prepubertal females were recently proven less sensitive than males to the inhibitory effects of centrally administered ghrelin on serum LH levels in models of acute administration of the peptide (17). It is noticeable, however, that female puberty in mammals is dramatically sensitive to insufficient energy stores, a phenomenon that is mainly signaled by the adipocyte hormone leptin (11, 13). In contrast, male puberty is less responsive to low leptin levels (11, 12), but is apparently more sensitive to high ghrelin levels (our present results). Taken together, our current data are strongly suggestive of a differential contribution of ghrelin and leptin in signaling the energy status to the reproductive axis at puberty between male and female rats.
In addition to its effects at puberty, the impact of chronic ghrelin treatment during the first half of gestation was monitored. A protocol of administration between d 1 to 11 of pregnancy was selected to specifically target, using an in vivo model, the actions of ghrelin during early stages of gestation. In our setting, daily administration of ghrelin did not alter the percentage of successful pregnancies at term or the gestational length, but it significantly decreased pregnancy outcome, as estimated by the number of pups born per litter. Although the precise mechanisms for such an effect were not directly addressed, a tempting possibility is that elevated ghrelin levels might be deleterious for early embryonic developmental events. In this sense, ghrelin has been recently reported to inhibit the development of mouse preimplantation embryos in vitro (15). In this context, the reduction in litter size after chronic ghrelin treatment reported herein reinforces the hypothesis that ghrelin may operate as a signal for energy insufficiency during early stages of gestation, acting as an inhibitory factor in early embryo development to avoid the excessive metabolic drain linked to pregnancy and lactation in situations of malnutrition (15). An interesting observation, however, is that pups from dams treated with ghrelin during the first half of pregnancy showed persistently higher body weight than their corresponding controls from birth to weaning, despite normalization of litter size. This finding is in keeping with a previous report that ghrelin administration in late pregnancy similarly induced an increase in body weight at birth (20). The functional relevance of such a phenomenon awaits further investigation.
Notably, we focused our study on analysis of the reproductive effects of repeated administration of octanoylated (acylated) ghrelin, classically regarded as the active form of the molecule (1, 2, 3, 4). However, despite the original contention that acylation at serine 3 is absolutely essential for ghrelin to conduct its biological effects (1, 2), a wealth of evidence has very recently emerged pointing out that unacylated ghrelin, whose concentration in plasma and half-life largely exceed those of mature ghrelin, is not merely an inert form of the molecule, but is provided with some specific biological actions (3, 4). Moreover, it has been recently reported that the injection of acylated ghrelin elicits a secretory burst of unacylated ghrelin, which might modulate its own biological effects in specific systems (32). Overall, these observations warrant additional studies involving comparative analyses of the reproductive effects of repeated administration of unacylated ghrelin, alone or in combination with the acylated peptide.
Energy homeostasis is maintained by the complex interplay of orexigenic and anorexigenic signals, a network where reciprocal roles of leptin and ghrelin, as signals for energy abundance and insufficiency, respectively, have been proposed (14). On the basis of present and previous data, it is tempting to hypothesize that a similar interplay might operate for the neuroendocrine integration of energy balance and reproductive function. Yet the mechanisms for such a phenomenon (including the possibility that the reproductive effects of ghrelin might be at least partially mediated by changes in leptin levels and/or signaling) remain to be elucidated. Nonetheless, the reproductive relevance of leptin and ghrelin is probably different, as evidenced by genetic models of leptin and ghrelin insufficiency. Thus, in contrast to leptin-deficient animals, mice bearing null mutations of ghrelin and its receptor do not show major reproductive defects (33, 34). An interesting possibility, however, is that although the lack of leptin (as signal for energy abundance) is clearly detrimental for fertility, the absence of ghrelin may not be as deleterious for reproductive function as its overexpression (which is observed in situations of energy deficit). Our current data are in keeping with such a hypothesis and strongly suggest that persistently elevated ghrelin levels, as a putative signal for energy insufficiency, may operate as a negative modifier of key reproductive states, such as pregnancy and (male) puberty onset. Overall, it is proposed that ghrelin may cooperate with other regulatory signals in the integrated control of energy balance and reproduction.
Acknowledgments
RIA kits for hormone determinations were kindly supplied by Dr. A. F. Parlow, National Institute of Diabetes and Digestive and Kidney Disease, National Hormone and Peptide Program (Torrance, CA).
References
Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K 1999 Ghrelin is a growth-hormone acylated peptide from stomach. Nature 402:656–666
Kojima M, Hosoda H, Matsuo H, Kangawa K 2001 Ghrelin: discovery of the natural endogenous ligand for the growth hormone secretagogue receptor. Trends Endocrinol Metab 12:118–122
van der Lely AJ, Tschop M, Heiman ML, Ghigo E 2004 Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocr Rev 25:426–457
Korbonits M, Goldstone AP, Gueorguiev M, Grossman AB 2004 Ghrelin: a hormone with multiple functions. Front Neuroendocrinol 25:27–68
Gualillo O, Lago F, Gomez-Teino J, Casanueva FF, Dieguez C 2003 Ghrelin, a widespread hormone: insights into molecular and cellular regulation of its expression and mechanism of action. FEBS Lett 552:105–109
Barreiro ML, Tena-Sempere M 2004 Ghrelin and reproduction: a novel signal linking energy status and fertility? Mol Cell Endocrinol 226:1–9
Tschop M, Smiley DL, Heiman ML 2000 Ghrelin induces adiposity in rodents. Nature 407:908–913
Hanada T, Toshinai K, Kajimura N, Nara-Ashizawa N, Tsukada T, Hayashi Y, Osuye K, Kangawa K, Matsukura S, Nakazato M 2003 Anti-cachectic effect of ghrelin in nude mice bearing human melanoma cells. Biochem Biophys Res Commun 301:275–279
Nagaya N, Kangawa K 2003 Ghrelin, a novel growth hormone-releasing peptide, in the treatment of chronic heart failure. Regul Pept 114:71–77
Dixit VD, Schaffer EM, Pyle RS, Collins GD, Sakthivel SK, Palaniappan R, Lillard Jr JW, Taub DD 2004 Ghrelin inhibits leptin- and activation-induced proinflammatory cytokine expression by human monocytes and T cells. J Clin Invest 114:57–66
Casanueva FF, Dieguez C 1999 Neuroendocrine regulation and actions of leptin. Front Neuroendocrinol 20:317–363
Tena-Sempere M, Barreiro ML 2002 Leptin in male reproduction: the testis paradigm. Mol Cell Endocrinol 188:9–13
Ahima RS, Saper CB, Flier JS, Elmquist JK 2000 Leptin regulation of neuroendocrine systems. Front Neuroendocrinol 21:263–307
Zigman JM, Elmquist JK 2004 From anorexia to obesity: the yin and yang of body weight control. Endocrinology 144:3749–3756
Kawamura K, Sato N, Fukuda J, Kodama H, Kumegai J, Tanikawa H, Nakamura A, Honda Y, Sato T, Tanaka T 2003 Ghrelin inhibits the development of mouse preimplantation embryos in vitro. Endocrinology 144:2623–2633
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
Fernandez-Fernandez R, Tena-Sempere M, Aguilar E, Pinilla L 2004 Ghrelin effects on gonadotropin secretion in male and female rats. Neurosci Lett 362:103–107
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
Barreiro ML, Gaytan F, Castellano JM, Suominen JS, Roa J, Gaytan M, Aguilar E, Dieguez C, Toppari J, Tena-Sempere M 2004 Ghrelin inhibits the proliferative activity of immature Leydig cells in vivo and regulates stem cell factor messenger ribonucleic acid expression in rat testis. Endocrinology 145:4825–4834
Hayashida T, Nakahara K, Mondal MS, Date Y, Nakazato M, Kojima M, Kangawa K, Murakami N 2002 Ghrelin in neonatal rats: distribution in stomach and its possible role. J Endocrinol 173:239–245
Frisch RE 1980 Pubertal adipose tissue: is it necessary for normal sexual maturation? Evidence from the rat and human female. Fed Proc 39:2395–2400
Navarro VM, Fernandez-Fernandez R, Castellano JM, Roa J, Mayen A, Barreiro ML, Gaytan F, Aguilar E, Pinilla L, Dieguez C, Tena-Sempere M 2004 Advanced vaginal opening and precocious activation of the reproductive axis by KiSS-1 peptide, the endogenous ligand of GPR54. J Physiol 561:379–386
Ojeda SR, Urbanski HF 1994 Puberty in the rat. In: Knobil E, Neill JD, eds. The physiology of reproduction. New York: Raven Press; 363–410
Tena-Sempere M, Navarro J, Pinilla L, Gonzalez LC, Huhtaniemi I, Aguilar E 2000 Neonatal exposure to estrogen differentially alters estrogen receptor and ? mRNA expression in rat testis during postnatal development. J Endocrinol 165:345–357
Nakahara K, Hayashida T, Nakazato M, Kojima M, Hosoda H, Kangawa K, Murakami N 2003 Effects of chronic treatments with ghrelin on milk secretion in lactating rats. Biochem Biophys Res Commun 303:751–755
Wren AM, Small CJ, Abbott CR, Dhillo WS, Seal LJ, Cohen MA, Batterham RL, Taheri S, Stanley SA, Ghatei MA, Bloom SR 2001 Ghrelin causes hyperphagia and obesity in rats. Diabetes 50:2540–2547
Wren AM, Small CJ, Thomas EL, Abbott CR, Ghatei MA, Bell JD, Bloom SR, Continuous subcutaneous administration of ghrelin promotes adiposity, independent of hyperphagia or body weight gain. Proc 12th International Congress of Endocrinology, Lisbon, Portugal 2004, p 121 (Abstract OR100)
Hataya Y, Akamizu T, Takaya K, Kanamoto N, Ariyasu H, Saijo M, Moriyama K, Shimatsu A, Kojima M, Kangawa K, Nakao K 2001 A low dose of ghrelin stimulates growth hormone (GH) release synergistically with GH-releasing hormone in humans. J Clin Endocrinol Metab 86:4552–4555
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
Korenbrot CC, Huhtaniemi IT, Weiner RI 1977 Preputial separation as an external sign of pubertal development in the male rat. Biol Reprod 17:298–303
Bergendahl M, Perheentupa A, Huhtaniemi I 1991 Starvation-induced suppression of pituitary-testicular function in rats is reversed by pulsatile gonadotropin-releasing hormone substitution. Biol Reprod 44:413–419
Gauna C, Meyler FM, Janssen JA, Delhanty PJ, Abribat T, van Koetsveld P, Hofland LJ, Broglio F, Ghigo E, van der Lely AJ 2004 Administration of acylated ghrelin reduces insulin sensitivity, whereas the combination of acylated plus unacylated ghrelin strongly improves insulin sensitivity. J Clin Endocrinol Metab 89:5035–5042
Sun Y, Ahmed S, Smith RG 2003 Deletion of ghrelin impairs neither growth nor appetite. Mol Cell Biol 23:7973–7981
Sun Y, Wang P, Zheng H, Smith RG 2004 Ghrelin stimulation of growth hormone release and appetite is mediated through the growth hormone secretagogue receptor. Proc Natl Acad Sci USA 101:4679–4684(R. Fernández-Fernández1, )
Address all correspondence and requests for reprints to: Dr. Manuel Tena-Sempere, Physiology Section, Department of Cell Biology, Physiology, and Immunology, Faculty of Medicine, University of Cordoba, Avenida Menéndez Pidal s/n, 14004 Cordoba, Spain. E-mail: fi1tesem@uco.es.
Abstract
Ghrelin, the endogenous ligand of the GH secretagogue receptor, has been recently involved in a wide array of biological functions, including signaling of energy insufficiency and energy homeostasis. On the basis of the proven reproductive effects of other regulators of energy balance, such as the adipocyte-derived hormone leptin, we hypothesized that systemic ghrelin may participate in the control of key aspects of reproductive function. To test this hypothesis, the effects of daily treatment with ghrelin were assessed in rats, pair-fed with control animals, in two relevant reproductive states, puberty and gestation, which are highly dependent on proper energy stores. Daily sc injection of ghrelin (0.5 nmol/12 h; between postnatal d 33 and 43) significantly decreased serum LH and testosterone levels and partially prevented balanopreputial separation (as an external index of puberty onset) in pubertal male rats. On the contrary, chronic administration of ghrelin to prepubertal females, between postnatal d 23 and 33, failed to induce major changes in serum levels of gonadotropins and estradiol, nor did it modify the timing of puberty, as estimated by the ages at vaginal opening and first estrus. Moreover, females treated with ghrelin at puberty subsequently displayed normal estrous cyclicity and were fertile. Conversely, ghrelin administration (0.5 nmol/12 h) during the first half of pregnancy (d 1–11) resulted in a significant decrease in pregnancy outcome, as estimated by the number of pups born per litter, without changes in the number of successful pregnancies at term or gestational length. Overall, our data indicate that persistently elevated ghrelin levels, as a putative signal for energy insufficiency, may operate as a negative modifier of key reproductive states, such as pregnancy and (male) puberty onset.
Introduction
GHRELIN, THE RECENTLY identified endogenous ligand of the GH secretagogue receptor, is a 28-amino acid peptide with an essential n-octanoylation at serine 3 residue (1, 2). Ghrelin was originally isolated from the stomach, which is by far the major source of systemic ghrelin, accounting for at least two thirds of its plasma levels (1, 2, 3, 4). Nonetheless, ghrelin has also been detected in a large number of tissues and cell types, including small intestine, pancreas, lymphocytes, placenta, kidney, lung, pituitary, brain, and gonads (3, 4, 5, 6). Apart from its proven ability to elicit GH secretion, a major interest in this peptide derived from the fact that it is involved in the regulation of energy balance by inducing food intake and reducing fat utilization (3, 7). These activities are probably mediated by a complex cellular network at the central nervous system, which is under the control of additional peripheral hormones, such as the adipocyte-derived hormone leptin (3). In this context, ghrelin analogs, with agonistic or antagonistic activity, have appeared as a suitable approach for therapeutic intervention in a variety of disease states linked to alterations in body weight homeostasis. Moreover, on the basis of its diverse biological actions, the (chronic) use of ghrelin has been recently postulated as a potential therapeutic strategy in different pathological conditions, such as cancer cachexia (anabolic agent) (8), severe chronic heart failure (improvement of left ventricular function) (9), and diverse proinflammatory states (inhibitor of cytokine production) (10). In this scenario, complete characterization of the biological effects of repeated administration of ghrelin has become mandatory.
Compelling evidence has demonstrated the close connection between the systems governing energy homeostasis and reproductive function (11). Although the signals and molecular mechanisms responsible for the concerted control of energy stores and reproduction remain to be fully elucidated, leptin has been recently proven to be a key neuroendocrine integrator in such biological function (11, 12). Thus, threshold leptin levels, as peripheral signal for sufficient energy stores, are absolutely essential for normal pubertal development and fertility, whereas disturbance of reproductive function is observed in human and animal models of leptin insufficiency (11, 12, 13). The mechanisms by which leptin regulates fertility probably involve actions at different levels of the gonadotropic axis, the hypothalamus being the primary target for the reproductive actions of leptin (11, 12). Conceivably, other neuroendocrine integrators with key roles in body weight homeostasis would interact with leptin in the integrated control of energy balance and reproduction.
Despite the proposed role of ghrelin in body weight homeostasis as peripheral signal for energy insufficiency (14), its contribution to the control of reproductive functions has received limited attention to date. Nonetheless, fragmentary data strongly suggest the potential involvement of ghrelin in regulation of the reproductive axis. Thus, expression of ghrelin has been demonstrated in human and rodent placenta, and ghrelin has been reported to inhibit early embryo development in vitro (6, 15). In addition, ghrelin was shown to suppress LH secretion in vivo and to decrease LH responsiveness to LHRH in vitro (16, 17). Moreover, expression of ghrelin and its cognate receptor has been demonstrated in rat and human gonads (6), ghrelin was reported to inhibit stimulated testicular testosterone (T) secretion (18), and acquisition of ghrelin expression by Leydig cells has been suggested to play an important role in the control of proliferation of this cell type (19). However, the functional analyses conducted to date have been mostly restricted to acute administration of the peptide and assessment of hormonal responses, whereas the developmental or reproductive effects of continuous administration of ghrelin have been scarcely studied (20). In the present work we aimed at evaluating the effects of chronic administration of ghrelin on two relevant reproductive states, namely, puberty and gestation, which have been proven to be extraordinarily sensitive to appropriate energy stores (14). To this end, daily sc administration of ghrelin was conducted in (pre)pubertal male and female rats as well as in pregnant rats during the first half of gestation. In all settings, ghrelin-treated rats were pair-fed with control animals to minimize the potential bias of differences in body weight on key reproductive parameters, such as conventional indexes of puberty onset (21).
Materials and Methods
Experimental design
Wistar rats bred in the vivarium of University of Cordoba were used. The day the litters were born was considered d 1 of age. The animals were maintained under constant conditions of light (14 h of light, from 0700 h) and temperature (22 C) and were weaned at 21 d of age in groups of five rats per cage with free access to pelleted food and tap water until initiation of the experimental procedures, when the animals were individually caged, and the access to chow was paired between control and treated animals (see below). Experimental procedures were approved by the Cordoba University ethical committee for animal experimentation and were conducted in accordance with the European Union normative for care and use of experimental animals. Rat ghrelin, with n-octanoyl modification at Ser3, was obtained from Bachem AG (Bubendorf, Switzerland).
To monitor the effects of ghrelin on puberty onset and pregnancy outcome, a general protocol of sc injection of ghrelin (0.5 nmol/100 μl saline), twice a day (at 0900 and 1900 h), was used, and different end points were recorded depending on the experimental setting. In all experiments, special efforts were made to minimize the potential bias of major differences in body weight on the different reproductive end points under analysis. Thus, ghrelin-treated rats were pair-fed with control animals. To this end, daily food intake was recorded in reference control animals (pubertal male and female rats as well as pregnant animals), and an equal fixed amount of standard chow was daily provided to control and ghrelin-treated animals. Likewise, to prevent the impact of differences in grouping of the animals on sensitive reproductive parameters, such as puberty onset, control and ghrelin-treated animals were individually caged at the beginning of the experimental procedures, in keeping with our previous studies (22).
In experiment 1, pubertal male rats (n = 12/group) were sc injected with ghrelin (0.5 nmol/12 h) or vehicle (physiological saline) between postnatal d 33 and 43. This period was selected on the basis of previous studies of the normal timing of puberty in the male rat (23) and local data on the occurrence of balanopreputial separation (BPS) in our animal stock. In all experimental animals, body weights were monitored daily, and the occurrence of BPS, defined as complete separation of prepuce from gland penis (balano), was recorded. The animals were sc injected under conscious conditions after careful handling to avoid any stressful influence and were humanely killed by decapitation on d 43, 1 h after the last injection of ghrelin, at which time trunk blood was collected. Additional blood samples were obtained by jugular venipuncture under light ether anesthesia on d 33 and 41, 1 h after sc injection of ghrelin or vehicle.
In addition, in experiment 2, daily sc administration of ghrelin (0.5 nmol/12 h) or vehicle was performed in pubertal female rats (n = 12/group) between postnatal d 23 and 33. This period was selected on the basis of previous references on the normal timing of puberty in the female rat (23), and local data on the occurrence of vaginal opening (VO) in our animal stock. In all experimental animals, body weights were daily monitored, and the ages of occurrence of VO, defined by the complete canalization of the vagina, and of first estrus were recorded. The animals were sc injected under conscious conditions after careful handling to avoid any stressful influence. Blood samples were obtained by jugular venipuncture under light ether anesthesia on d 25, 28, and 31, 1 h after sc injection of ghrelin or vehicle. Additional blood sampling was carried out on the day of VO. Thereafter, estrous cyclicity and fertility were monitored in ghrelin- and vehicle-treated rats by daily vaginal cytology, and mating with adult male rats of proven fertility and recording of percentage of successful pregnancies, respectively.
Finally, in experiment 3, pregnant females (n = 12/group) were sc injected with ghrelin (0.5 nmol/12 h) or vehicle (physiological saline), between d 1 and 11 of pregnancy. This period was specifically selected to target early events in gestation, in line with a previous study of the effects of ghrelin on development of mouse preimplantation embryos in vitro (15). For timing of pregnancy, on the afternoon of proestrus cycling female rats were placed in individual cages with adult males of proven fertility. In the morning of the following day, vaginal smears were collected, and the presence of spermatozoa was checked; animals showing spermatozoa at vaginal smears were considered at d 1 of pregnancy and were included for daily injection of ghrelin or vehicle. Pregnant rats were sc injected under conscious conditions after careful handling to avoid any stressful influence. In all experimental animals, body weights were monitored daily throughout gestation. Pregnancy outcome was recorded by the percentage of successful pregnancies at term and the number of pups per litter at birth. In addition, sex ratio was calculated, and body weight of litters was monitored up to weaning (postnatal d 21).
Hormone measurement by specific RIAs
Serum levels of LH and FSH were measured in a volume of 25–50 μl using a double-antibody method and RIA kits supplied by the National Institutes of Health (Dr. A. F. Parlow, National Institute of Diabetes and Digestive and Kidney Diseases, National Hormone and Peptide Program, Torrance, CA). Rat LH-I-9 and FSH-I-9 were labeled with 125I by the chloramine-T method, and hormone concentrations were expressed using the reference preparations LH-RP-3 and FSH-RP-2 as standards. Intra- and interassay coefficients of variation were 8% and 10%, respectively, for LH and 6% and 9%, respectively, for FSH. The sensitivity of the assays was 5 pg/tube for LH and 20 pg/tube for FSH. In addition, in selected experimental groups, serum ghrelin and GH levels were assayed. For ghrelin measurements, a commercially available RIA kit from Phoenix Pharmaceuticals (Belmont, CA), with an assay sensitivity of 1 pg/tube, was used for determination of total ghrelin levels. Serum GH levels were measured by a double-antibody RIA, using kits provided by the National Institutes of Health. Rat GH-I-7 was labeled as described above, and hormone concentrations were expressed using reference preparation GH-RP-2. Intra- and interassay coefficients of variation were 6% and 9%, respectively, and the sensitivity of the assay was 5 pg/tube. Finally, where indicated, serum T and estradiol levels were determined using commercial kits from MP Biomedicals (Costa Mesa, CA), following the instructions of the manufacturer. The sensitivities of the assay were 0.1 and 0.5 ng/tube for T and estradiol, respectively. The intraassay coefficients of variation were less than 5%. The accuracy of hormone determinations was confirmed by assessment of rat serum samples of known hormone concentrations used as external controls.
Presentation of data and statistics
Serum LH, FSH, GH, ghrelin, T, and estradiol determinations were conducted in duplicate, with a minimum total number of 10 samples/determinations/group. Hormonal data are presented as the mean ± SEM. Results were analyzed for statistically significant differences using Student’s t test or ANOVA, followed by Student-Newman-Keuls multiple range test (SigmaStat 2.0, Jandel Corp., San Rafael, CA). 2 evaluation was used for statistical comparison of data for percentages of BPS and successful pregnancy. P 0.05 was considered significant.
Results
Effects of chronic administration of ghrelin on puberty onset in the male rat
The effects of ghrelin on male puberty onset were assessed by daily sc injection of the peptide (at 0.5 nmol/12 h) between postnatal d 33 and 43 and recording of body weight, hormonal responses, and BPS occurrence. Cumulative body weight gain (data not shown) and total body weights were not significantly different between controls and pair-fed ghrelin-injected males (Fig. 1A). Serum LH and T levels in control animals significantly increased throughout the study period (between d 33 and 43), in line with previous findings (24), whereas serum FSH levels remained unaltered. The age of occurrence of BPS in controls rats was 42.2 ± 0.4 d, and 100% of animals (12 of 12) presented complete preputial separation on d 43. In pair-fed animals, chronic treatment with ghrelin did not modify serum FSH levels. In contrast, sc injection of ghrelin partially prevented the age-dependent increase in serum LH levels: a moderate decrease, which was shortly below the limit of statistical significance, was detected on d 41, whereas a significant inhibition was observed on d 43 in ghrelin-treated animals. In addition, daily injection of ghrelin significantly lowered serum T levels during the study period and partially blunted the age-dependent rise in T concentrations (Fig. 1, B–D). In keeping with these findings, chronic administration of ghrelin partially prevented the normal occurrence of BPS; complete preputial separation was detected in only 58.5% (seven of 12) of the ghrelin-treated animals on d 43 (Fig. 1E). As a control for the validity of the experimental model and the dose of ghrelin used, at this age point, exogenous administration of 0.5 nmol ghrelin was able to induce a significant 2.4-fold increase in serum levels of total ghrelin 1 h after injection (Fig. 1F). Such a response was associated with a moderate, but significant, elevation in basal serum GH levels (1.95 ± 0.217 vs. 0.997 ± 0.216 ng/ml in vehicle-injected animals), in keeping with previous findings (25).
FIG. 1. Compilation of the effects of chronic administration of ghrelin to pubertal male rats. Ghrelin was injected at a dose of 0.5 nmol/12 h between postnatal d 33 and 43. Body weights of the experimental animals are presented in A. Serum LH, FSH, and T levels in vehicle- and ghrelin-treated animals are shown in B–D. Samples were obtained on d 33, 41, and 43, 1 h after the last injection of the peptide. In addition, the cumulative percentage of males with complete BPS on d 43 is presented in E, whereas serum ghrelin levels on d 43 (1 h after the last injection of the peptide) are shown in F. The experimental groups were composed of 12 animals. Where applicable, values are given as the mean ± SEM. For vehicle-injected animals, groups with different superscript letters are statistically different (P < 0.05). **, P < 0.01 vs. corresponding vehicle-injected animals (by ANOVA, followed by Student-Newman-Keuls multiple range test).
Effects of chronic administration of ghrelin on puberty onset in the female rat
The effects of ghrelin on female puberty onset were assessed by daily sc injection of the peptide (at 0.5 nmol/12 h) between postnatal d 23 and 33, and recording of body weight, hormonal responses, and occurrence of VO and first estrus. In addition, estrous cyclicity and fertility were subsequently monitored in adult females treated with ghrelin at puberty. Cumulative body weight gain (data not shown) and total body weights were not significantly different between controls and pair-fed, ghrelin-injected females (Fig. 2A). Serum LH and estradiol levels in control animals significantly increased during the study period (between d 23 and 33), as previously described (23), whereas serum FSH levels remained unaltered. The age of occurrence of VO in controls rats was 34.4 ± 0.6 d, and first estrus was observed at a mean age of 35.6 ± 1.0 d. In pair-fed animals, chronic treatment with ghrelin did not change serum FSH levels. Likewise, sc injection of ghrelin failed to substantially modify the age-dependent increase in serum LH levels, except for a decrease in LH concentrations at the age of VO, nor did it alter serum estradiol concentrations during the study period (Fig. 2, B–D). In line with these observations, chronic administration of ghrelin did not change the age of occurrence of VO and first estrus (Fig. 2E). Similarly, female rats chronically treated with ghrelin during puberty subsequently displayed normal estrous cyclicity and were fertile: 92% (11 of 12) of ghrelin-treated females become pregnant upon mating with males of proven fertility, a percentage similar to that in vehicle-treated animals. Nonetheless, evidence for the validity of the experimental model and the dose of ghrelin used comes from the observation that on postnatal d 31, exogenous administration of 0.5 nmol ghrelin was able to induce a significant 2.6-fold increase in serum levels of total ghrelin 1 h after injection (Fig. 2F). As was the case in males, such a response was associated with a moderate, but significant, elevation of basal serum GH levels (1.332 ± 0.116 vs. 0.726 ± 0.043 ng/ml in vehicle-injected animals).
FIG. 2. Compilation of the effects of chronic administration of ghrelin to pubertal female rats. Ghrelin was injected at a dose of 0.5 nmol/12 h between postnatal d 23 and 33. Body weights of the experimental animals are presented in A. Serum LH, FSH, and estradiol (E2) levels in vehicle- and ghrelin-treated animals are shown in B–D. Samples were obtained on d 25, 28, and 31 as well as on the day of VO, 1 h after the last injection of the peptide. In addition, the ages of occurrence of VO and first estrus are shown in E, whereas serum ghrelin levels on d 31 (1 h after the last injection of peptide) are presented in F. The experimental groups were composed of 12 animals. Values are given as the mean ± SEM. For vehicle-injected animals, groups with different superscript letters are statistically different (P < 0.05). **, P < 0.01 vs. corresponding vehicle-injected animals (by ANOVA, followed by Student-Newman-Keuls multiple range test).
Effects of chronic administration of ghrelin on pregnancy outcome in the rat
Finally, the effects of ghrelin on pregnancy onset were assessed by daily sc injection of the peptide (at 0.5 nmol/12 h) to pregnant dams between d 1 and 11 of gestation. Cumulative body weight gain (data not shown) and total body weights were not significantly different between controls and pair-fed, ghrelin-injected pregnant females (Fig. 3A). Likewise, the percentage of successful pregnancies at term and gestational length were similar between the experimental groups (data not shown). On the contrary, the number of pups born per litter was significantly lower in ghrelin-treated dams, with a male/female ratio of 1.33 vs. 1.25 in vehicle-injected animals (Fig. 3B). After adjustment of litter size (10 pups/mother), daily recording of body weight demonstrated that pups from mothers treated with ghrelin during the first half of pregnancy showed higher body weight values than those of vehicle-treated dams from birth to weaning (Fig. 3C).
FIG. 3. Compilation of the effects of chronic administration of ghrelin to pregnant rats during the first half of gestation. Ghrelin was injected at a dose of 0.5 nmol/12 h between d 1 and 11 of pregnancy. Body weights of pregnant animals are presented in the upper panel (A). The number of pups born per litter is shown in the middle panel (B). In the lower panel (C), body weights of pups from vehicle- and ghrelin-treated dams, from birth to weaning, are presented. Groups of pregnant rats were composed of 12 animals. Values are given as the mean ± SEM. Analysis of number of pups per litter: **, P < 0.01 vs. corresponding litters from vehicle-treated dams; analysis of BW of pups: *, P < 0.05 vs. corresponding litters from vehicle-injected dams (by Student’s t test).
Discussion
Expression and/or actions of ghrelin have been recently demonstrated at different levels of the hypothalamic-pituitary-gonadal axis (6, 17, 18). Yet the actual role of ghrelin in the integrated control of energy homeostasis and reproduction remains largely unexplored. Moreover, despite extensive efforts in characterization of the biological roles of ghrelin, only a limited number of studies analyzing the effects of continuous, rather than acute, administration of the peptide have been reported to date, and these have mostly focused on analysis of its metabolic and orexigenic actions (7, 25, 26). In the present study we evaluated the effects of elevated ghrelin levels on two relevant reproductive states, puberty and gestation, by recording suitable hormonal and phenotypic biomarkers. Considering the expected orexigenic and adipogenic effects of ghrelin treatment (7, 26), special attention was paid to avoid the potential bias of changes in body weight between the experimental groups. Thus, ghrelin-treated rats were pair-fed with control animals, a protocol that resulted in similar body weights among treatments, in keeping with previous studies (27). In addition, the dose of ghrelin selected (0.5 nmol/12 h) was sufficient to produce endocrine responses (28, 29), but is within the limit of induction of overt hyperphagia in rats (26). Indeed, a significant elevation (2.4- to 2.6-fold increase) of total ghrelin levels was demonstrated after systemic injection of 0.5 nmol of the peptide in our experimental groups, whose magnitude was in the range of that induced by fasting (26). Such elevation was associated with a modest, but significant, increase in serum GH levels, which might be regarded as sentinel for the endocrine activity of exogenously administered ghrelin. Overall, it was assumed that the reproductive effects observed after its repeated administration were primarily due to elevated ghrelin levels and were not secondary to changes in body weight induced by the orexigenic hormone.
Under the above experimental conditions, daily sc administration of ghrelin to prepubertal males induced a significant decrease in serum T levels during the study period, which was associated with normal to decreased serum LH levels. In good agreement, normal occurrence of preputial separation (as an external index of puberty) (30) was prevented on d 43 in more than 40% of treated males. The mechanisms for such an effect might involve inhibition of LH secretion at the hypothalamic-pituitary unit and/or direct inhibitory effects on testicular T secretion. Indeed, suppression of serum LH levels has been reported in prepubertal male rats after central injection of ghrelin (17). Likewise, ghrelin has been proven to be an inhibitory signal for stimulated testicular T secretion in vitro (18). Although the two mechanisms are not mutually exclusive, the fact that T levels were significantly lower in ghrelin-treated animals at all ages tested strongly suggests a major contribution of direct inhibitory effects of ghrelin on the prepubertal testis. These might include disturbance of adult-type Leydig cell development, because ghrelin has been recently proven to inhibit the proliferative activity of immature Leydig cells in the pubertal testis (19). Nevertheless, the net end point for the above hormonal responses is the partial disruption of the normal timing of puberty (as estimated by age at BPS). Together, these data strongly suggest that elevated ghrelin levels might be detrimental for normal puberty onset in the male. Considering that circulating ghrelin levels are negatively correlated to body mass index, and the proposed role of ghrelin as a signal for energy insufficiency (14), it is tempting to propose that ghrelin per se might operate as a negative modifier of male puberty, and it might contribute to suppression of the male reproductive axis in situations of negative energy balance, such as starvation (31).
In contrast with data obtained in males, chronic treatment of prepubertal females with ghrelin failed to induce major hormonal changes in LH, FSH, and estradiol secretion (except for a decrease in serum LH levels at the age of VO), nor did it alter the age of occurrence of VO and first estrus, taken as putative indices of puberty onset in the female (23). Moreover, females treated with ghrelin at puberty subsequently displayed normal estrous cyclicity and were fertile. Interestingly, despite the proposed role of ghrelin as a signal for energy insufficiency (14), chronic ghrelin treatment from birth to puberty has been previously reported to advance the age of VO in immature females (20). Such a discrepancy might be related to the fact that in the previous study, doses of 1.0–1.5 nmol ghrelin were daily injected between postnatal d 1 to the day of VO, and the animals were not pair-fed with controls (20). Because we aimed at targeting the specific effects of ghrelin on female puberty, for the present study we selected a protocol of daily treatment from d 23 (beginning of the prepubertal period) to d 33 (around VO). Overall, although we cannot exclude the possibility that higher doses of ghrelin may bring about deleterious effects similar to those observed in males, our data clearly show that male puberty is more sensitive than female puberty to the potential adverse effects of ghrelin. Similarly, prepubertal females were recently proven less sensitive than males to the inhibitory effects of centrally administered ghrelin on serum LH levels in models of acute administration of the peptide (17). It is noticeable, however, that female puberty in mammals is dramatically sensitive to insufficient energy stores, a phenomenon that is mainly signaled by the adipocyte hormone leptin (11, 13). In contrast, male puberty is less responsive to low leptin levels (11, 12), but is apparently more sensitive to high ghrelin levels (our present results). Taken together, our current data are strongly suggestive of a differential contribution of ghrelin and leptin in signaling the energy status to the reproductive axis at puberty between male and female rats.
In addition to its effects at puberty, the impact of chronic ghrelin treatment during the first half of gestation was monitored. A protocol of administration between d 1 to 11 of pregnancy was selected to specifically target, using an in vivo model, the actions of ghrelin during early stages of gestation. In our setting, daily administration of ghrelin did not alter the percentage of successful pregnancies at term or the gestational length, but it significantly decreased pregnancy outcome, as estimated by the number of pups born per litter. Although the precise mechanisms for such an effect were not directly addressed, a tempting possibility is that elevated ghrelin levels might be deleterious for early embryonic developmental events. In this sense, ghrelin has been recently reported to inhibit the development of mouse preimplantation embryos in vitro (15). In this context, the reduction in litter size after chronic ghrelin treatment reported herein reinforces the hypothesis that ghrelin may operate as a signal for energy insufficiency during early stages of gestation, acting as an inhibitory factor in early embryo development to avoid the excessive metabolic drain linked to pregnancy and lactation in situations of malnutrition (15). An interesting observation, however, is that pups from dams treated with ghrelin during the first half of pregnancy showed persistently higher body weight than their corresponding controls from birth to weaning, despite normalization of litter size. This finding is in keeping with a previous report that ghrelin administration in late pregnancy similarly induced an increase in body weight at birth (20). The functional relevance of such a phenomenon awaits further investigation.
Notably, we focused our study on analysis of the reproductive effects of repeated administration of octanoylated (acylated) ghrelin, classically regarded as the active form of the molecule (1, 2, 3, 4). However, despite the original contention that acylation at serine 3 is absolutely essential for ghrelin to conduct its biological effects (1, 2), a wealth of evidence has very recently emerged pointing out that unacylated ghrelin, whose concentration in plasma and half-life largely exceed those of mature ghrelin, is not merely an inert form of the molecule, but is provided with some specific biological actions (3, 4). Moreover, it has been recently reported that the injection of acylated ghrelin elicits a secretory burst of unacylated ghrelin, which might modulate its own biological effects in specific systems (32). Overall, these observations warrant additional studies involving comparative analyses of the reproductive effects of repeated administration of unacylated ghrelin, alone or in combination with the acylated peptide.
Energy homeostasis is maintained by the complex interplay of orexigenic and anorexigenic signals, a network where reciprocal roles of leptin and ghrelin, as signals for energy abundance and insufficiency, respectively, have been proposed (14). On the basis of present and previous data, it is tempting to hypothesize that a similar interplay might operate for the neuroendocrine integration of energy balance and reproductive function. Yet the mechanisms for such a phenomenon (including the possibility that the reproductive effects of ghrelin might be at least partially mediated by changes in leptin levels and/or signaling) remain to be elucidated. Nonetheless, the reproductive relevance of leptin and ghrelin is probably different, as evidenced by genetic models of leptin and ghrelin insufficiency. Thus, in contrast to leptin-deficient animals, mice bearing null mutations of ghrelin and its receptor do not show major reproductive defects (33, 34). An interesting possibility, however, is that although the lack of leptin (as signal for energy abundance) is clearly detrimental for fertility, the absence of ghrelin may not be as deleterious for reproductive function as its overexpression (which is observed in situations of energy deficit). Our current data are in keeping with such a hypothesis and strongly suggest that persistently elevated ghrelin levels, as a putative signal for energy insufficiency, may operate as a negative modifier of key reproductive states, such as pregnancy and (male) puberty onset. Overall, it is proposed that ghrelin may cooperate with other regulatory signals in the integrated control of energy balance and reproduction.
Acknowledgments
RIA kits for hormone determinations were kindly supplied by Dr. A. F. Parlow, National Institute of Diabetes and Digestive and Kidney Disease, National Hormone and Peptide Program (Torrance, CA).
References
Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K 1999 Ghrelin is a growth-hormone acylated peptide from stomach. Nature 402:656–666
Kojima M, Hosoda H, Matsuo H, Kangawa K 2001 Ghrelin: discovery of the natural endogenous ligand for the growth hormone secretagogue receptor. Trends Endocrinol Metab 12:118–122
van der Lely AJ, Tschop M, Heiman ML, Ghigo E 2004 Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocr Rev 25:426–457
Korbonits M, Goldstone AP, Gueorguiev M, Grossman AB 2004 Ghrelin: a hormone with multiple functions. Front Neuroendocrinol 25:27–68
Gualillo O, Lago F, Gomez-Teino J, Casanueva FF, Dieguez C 2003 Ghrelin, a widespread hormone: insights into molecular and cellular regulation of its expression and mechanism of action. FEBS Lett 552:105–109
Barreiro ML, Tena-Sempere M 2004 Ghrelin and reproduction: a novel signal linking energy status and fertility? Mol Cell Endocrinol 226:1–9
Tschop M, Smiley DL, Heiman ML 2000 Ghrelin induces adiposity in rodents. Nature 407:908–913
Hanada T, Toshinai K, Kajimura N, Nara-Ashizawa N, Tsukada T, Hayashi Y, Osuye K, Kangawa K, Matsukura S, Nakazato M 2003 Anti-cachectic effect of ghrelin in nude mice bearing human melanoma cells. Biochem Biophys Res Commun 301:275–279
Nagaya N, Kangawa K 2003 Ghrelin, a novel growth hormone-releasing peptide, in the treatment of chronic heart failure. Regul Pept 114:71–77
Dixit VD, Schaffer EM, Pyle RS, Collins GD, Sakthivel SK, Palaniappan R, Lillard Jr JW, Taub DD 2004 Ghrelin inhibits leptin- and activation-induced proinflammatory cytokine expression by human monocytes and T cells. J Clin Invest 114:57–66
Casanueva FF, Dieguez C 1999 Neuroendocrine regulation and actions of leptin. Front Neuroendocrinol 20:317–363
Tena-Sempere M, Barreiro ML 2002 Leptin in male reproduction: the testis paradigm. Mol Cell Endocrinol 188:9–13
Ahima RS, Saper CB, Flier JS, Elmquist JK 2000 Leptin regulation of neuroendocrine systems. Front Neuroendocrinol 21:263–307
Zigman JM, Elmquist JK 2004 From anorexia to obesity: the yin and yang of body weight control. Endocrinology 144:3749–3756
Kawamura K, Sato N, Fukuda J, Kodama H, Kumegai J, Tanikawa H, Nakamura A, Honda Y, Sato T, Tanaka T 2003 Ghrelin inhibits the development of mouse preimplantation embryos in vitro. Endocrinology 144:2623–2633
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
Fernandez-Fernandez R, Tena-Sempere M, Aguilar E, Pinilla L 2004 Ghrelin effects on gonadotropin secretion in male and female rats. Neurosci Lett 362:103–107
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
Barreiro ML, Gaytan F, Castellano JM, Suominen JS, Roa J, Gaytan M, Aguilar E, Dieguez C, Toppari J, Tena-Sempere M 2004 Ghrelin inhibits the proliferative activity of immature Leydig cells in vivo and regulates stem cell factor messenger ribonucleic acid expression in rat testis. Endocrinology 145:4825–4834
Hayashida T, Nakahara K, Mondal MS, Date Y, Nakazato M, Kojima M, Kangawa K, Murakami N 2002 Ghrelin in neonatal rats: distribution in stomach and its possible role. J Endocrinol 173:239–245
Frisch RE 1980 Pubertal adipose tissue: is it necessary for normal sexual maturation? Evidence from the rat and human female. Fed Proc 39:2395–2400
Navarro VM, Fernandez-Fernandez R, Castellano JM, Roa J, Mayen A, Barreiro ML, Gaytan F, Aguilar E, Pinilla L, Dieguez C, Tena-Sempere M 2004 Advanced vaginal opening and precocious activation of the reproductive axis by KiSS-1 peptide, the endogenous ligand of GPR54. J Physiol 561:379–386
Ojeda SR, Urbanski HF 1994 Puberty in the rat. In: Knobil E, Neill JD, eds. The physiology of reproduction. New York: Raven Press; 363–410
Tena-Sempere M, Navarro J, Pinilla L, Gonzalez LC, Huhtaniemi I, Aguilar E 2000 Neonatal exposure to estrogen differentially alters estrogen receptor and ? mRNA expression in rat testis during postnatal development. J Endocrinol 165:345–357
Nakahara K, Hayashida T, Nakazato M, Kojima M, Hosoda H, Kangawa K, Murakami N 2003 Effects of chronic treatments with ghrelin on milk secretion in lactating rats. Biochem Biophys Res Commun 303:751–755
Wren AM, Small CJ, Abbott CR, Dhillo WS, Seal LJ, Cohen MA, Batterham RL, Taheri S, Stanley SA, Ghatei MA, Bloom SR 2001 Ghrelin causes hyperphagia and obesity in rats. Diabetes 50:2540–2547
Wren AM, Small CJ, Thomas EL, Abbott CR, Ghatei MA, Bell JD, Bloom SR, Continuous subcutaneous administration of ghrelin promotes adiposity, independent of hyperphagia or body weight gain. Proc 12th International Congress of Endocrinology, Lisbon, Portugal 2004, p 121 (Abstract OR100)
Hataya Y, Akamizu T, Takaya K, Kanamoto N, Ariyasu H, Saijo M, Moriyama K, Shimatsu A, Kojima M, Kangawa K, Nakao K 2001 A low dose of ghrelin stimulates growth hormone (GH) release synergistically with GH-releasing hormone in humans. J Clin Endocrinol Metab 86:4552–4555
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
Korenbrot CC, Huhtaniemi IT, Weiner RI 1977 Preputial separation as an external sign of pubertal development in the male rat. Biol Reprod 17:298–303
Bergendahl M, Perheentupa A, Huhtaniemi I 1991 Starvation-induced suppression of pituitary-testicular function in rats is reversed by pulsatile gonadotropin-releasing hormone substitution. Biol Reprod 44:413–419
Gauna C, Meyler FM, Janssen JA, Delhanty PJ, Abribat T, van Koetsveld P, Hofland LJ, Broglio F, Ghigo E, van der Lely AJ 2004 Administration of acylated ghrelin reduces insulin sensitivity, whereas the combination of acylated plus unacylated ghrelin strongly improves insulin sensitivity. J Clin Endocrinol Metab 89:5035–5042
Sun Y, Ahmed S, Smith RG 2003 Deletion of ghrelin impairs neither growth nor appetite. Mol Cell Biol 23:7973–7981
Sun Y, Wang P, Zheng H, Smith RG 2004 Ghrelin stimulation of growth hormone release and appetite is mediated through the growth hormone secretagogue receptor. Proc Natl Acad Sci USA 101:4679–4684(R. Fernández-Fernández1, )