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Ghrelin effects on gonadotropin secretion in male and female rats
Neuroscience Letters 362 (2004) 103–107 www.elsevier.com/locate/neulet
Ghrelin effects on gonadotropin secretion in male and female rats Rafael Ferna´ndez-Ferna´ndez, Manuel Tena-Sempere, Enrique Aguilar, Leonor Pinilla* Department of Physiology, Faculty of Medicine, University of Co´rdoba, 14004 Cordoba, Spain Received 10 February 2004; received in revised form 29 February 2004; accepted 3 March 2004
Abstract Ghrelin is a 28-amino acid peptide primarily involved in the control of food intake and growth hormone secretion. The present experiments were carried out to analyze the potential involvement of ghrelin in the control of gonadotropin secretion. Prepubertal intact and gonadectomized female and male rats, cyclic rats in diestrus, lactating rats and aged female rats were i.c.v. injected with ghrelin (3 nmol/rat) and blood samples were obtained by decapitation 15 min later. In addition, we analyzed the effects of ghrelin on in vitro basal and luteinizing hormone-releasing hormone (LHRH)-stimulated gonadotropin secretion. Our present results indicate that ghrelin inhibited luteinizing hormone (LH) secretion in vivo in prepubertal males as well as gonadectomized males and females, whereas follicle-stimulating hormone (FSH) remained unaffected. In vitro, ghrelin stimulated the secretion of both gonadotropins, and differentially modulated the response to LHRH; the LH response was inhibited, while the FSH response was enhanced. Overall, our current data open up the possibility that ghrelin may be involved in the control of LH secretion, and in the dissociation of both gonadotropins that takes place in many physiological, pathological and experimental situations. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Ghrelin; Follicle-stimulating hormone; Luteinizing hormone; Luteinizing hormone-releasing hormone
Ghrelin is a 28-amino acid peptide identified as the endogenous ligand for growth hormone secretagogue (GHS) receptor [16,17]. Ghrelin is primarily expressed in stomach and hypothalamus [16,17], and stimulates GH secretion in humans and rats [1 – 3,9,11,16,17,23,29,31,38]. In rats, a limited number of studies have been carried out to analyze the effects of ghrelin on the pituitary secretion of hormones other than GH. The data obtained so far indicate that in prepubertal male rats intracerebroventricular (i.c.v.) administration of ghrelin inhibits luteinizing hormone (LH) and prolactin secretion [24], whereas in estradiol-treated ovariectomized rats, i.c.v. administration of ghrelin suppresses pulsatile LH secretion [7]. In addition, intravenous administration of ghrelin did not change plasma concentrations of prolactin, ACTH and leptin [33]. The regulation of gonadotropin secretion is carried out through a complex interaction between hypothalamic luteinizing hormone-releasing hormone (LHRH), other hypothalamic peptides [6,20], locally-produced pituitary signals such as activin, inhibin and follistatin [15,19], and gonadal-derived steroids and peptides. Preliminary data * Corresponding author. Tel.: þ 34-957-218280; fax: þ 34-957-218288. E-mail address: [email protected] (L. Pinilla).
from our laboratory indicated that ghrelin inhibits LH secretion in prepubertal male rats [24]. The present experiments were carried out in vivo and in vitro in order to analyze the potential effects of ghrelin on basal and LHRH-stimulated gonadotropin secretion. In Experiment 1, male and female rats (23 days old) were gonadectomized or sham-gonadectomized under light ether anesthesia. The animals were i.c.v. injected 7 days later with 3 nmol/rat of ghrelin dissolved in 10 ml of vehicle or vehicle alone (physiological saline; NaCl 0.9%) and decapitated 15 min later. Blood samples were collected. The procedure for i.c.v. injections was as described previously [25]. In Experiment 2, cyclic females in diestrus, lactating rats and aged (24 months) female animals were i.c.v. injected with ghrelin (3 nmol/rat dissolved in 10 ml of vehicle) or vehicle and decapitated 15 min later. Blood samples were collected. In order to detect a potential primary action of ghrelin at the pituitary level, in Experiment 3, anterior pituitaries were obtained from 30-day-old male rats and placed in glass scintillation vials (one per vial) in a Dubnoff shaker at 37 8C under an atmosphere of 96% O2/5% CO2. Each vial contained 1 ml Dulbecco’s Minimal Essential Medium (DMEM). After preincubation for 60 min, the medium was replaced by fresh medium containing ghrelin (1028 and
0304-3940/03/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.03.003
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1026 M). Medium samples were obtained at 60, 120 and 180 min of the incubation period. In addition, the combined effects of LHRH (1029 and 1027 M) and ghrelin (1026 M) were also analyzed. Each group consisted of eight to 12 pituitary samples. In all experiments, special precautions were taken to avoid any stressing influence (all the animals were handled daily for a week before the experiment and killed by the same person). Experiments were carried out between 11:00 and 12:00 h. Experimental procedures were approved by the Co´rdoba University Ethical Committee for animal experimentation and were conducted in accordance with the European Union normative for care and use of experimental animals. After centrifugation (1600 £ g at 4 8C for 20 min), serum was collected, frozen and stored at 2 20 8C until use. The concentrations of gonadotropins were measured in 5 – 50 ml by a double-antibody method using a radioimmunoassay kit supplied by NIDDK (Bethesda, MD). Rat LH-I-10 and follicle-stimulating hormone (FSH)-I-7 were labeled with I125 by the chloramine T method and hormone concentrations were expressed using a reference preparation LH-RP3 and FSH-RP2 as standard. Intra- and interassay variations were 8 and 10% for LH and 6 and 9% for FSH. The sensitivities of the assay were 75 pg/ml for LH and 0.4 ng/ml for FSH. All samples of each experiment were measured in the same assay. Values are expressed as means ^ SEM. Differences between groups were analyzed using one- or twoway ANOVA followed by Tukey’s test. Central (i.c.v.) administration of ghrelin (3 nmol/rat) significantly decreased serum LH levels in intact and orchidectomized prepubertal males, in ovariectomized prepubertal females and in aged (24-month-old) female rats, whereas no significant effect on LH concentrations was detected in lactating females (Table 1). Due to technical reasons, the effects of central injection of ghrelin on serum LH levels were not determined in cyclic female rats in the diestrus phase. In none of the groups studied did serum FSH concentrations change significantly after ghrelin treatment, although a trend to increase, close to the limits of the
statistical significance, was observed in prepubertal ovariectomized and aged females (Table 1). Gonadotropin concentrations in the incubation media increased significantly along the incubation period (Figs. 1 and 2). Ghrelin stimulated LH secretion at 60, 120 and 180 min of the incubation period, while the concentrations of FSH were increased only at 60 and 120 min after exposure to ghrelin (Fig. 1). LHRH at the doses of 1029 and 1027 M stimulated gonadotropin release, and the magnitude of this effect was dose-dependent (Fig. 2). Under these stimulated conditions, ghrelin initially potentiated (at 60 min) the effect of LHRH (1029 M) on LH and FSH secretion (Fig. 2), but a clear dissociation at 120 and 180 min was observed, since ghrelin inhibited the LH response to LHRH while it enhanced that of FSH (Fig. 2). The presence of ghrelin receptors in the hypothalamus and pituitary [10,12,18] opens the possibility that ghrelin participates in the control of pituitary secretion and, in addition to its effects on GH, ghrelin actions on prolactin and LH release have been reported [3,7,9,24]. Our results demonstrate that in prepubertal rats ghrelin is involved in the control of gonadotropin secretion, since ghrelin (a) decreased both basal and post-orchidectomy LH secretion in vivo without affecting the release of FSH, (b) stimulated the secretion of both gonadotropins in vitro, and (c) differentially affected the response of both gonadotropins to LHRH, decreasing the LH response and increasing the FSH response. The inhibitory effect of ghrelin on LH secretion observed in vivo can be explained by the decrease of LH response to LHRH detected in vitro. It should be noted that the suppressive effect of ghrelin was more potent after gonadectomy, when LHRH release is increased, and after stimulation of LHRH release by NMDA (data not shown). Intriguingly, no inhibitory effect of ghrelin upon serum LH levels was detected in lactating female rats, a phenomenon whose physiological relevance is presently under investigation in our laboratory. In contrast with data obtained for LH, FSH secretion in vivo was apparently independent of ghrelin action, since central administration of the peptide was ineffective in prepubertal male and female rats, as well as in cyclic, lactating and aged female rats. Such a lack of
Table 1 Effects of central (i.c.v.) administration of ghrelin (3 nmol/rat) on serum LH and FSH concentrations in different experimental models Group and treatment
Females, intact prepubertal Females, OVX prepubertal Males, intact prepubertal Males, ORX prepubertal Females, diestrus Females, lactating Females, aging (24 months)
LH (ng/ml)
FSH (ng/ml)
Vehicle
Ghrelin
Vehicle
Ghrelin
0.37 ^ 0.07 8.21 ^ 0.20 0.40 ^ 0.05 4.74 ^ 0.40 ND 0.21 ^ 0.08 0.70 ^ 0.08
0.21 ^ 0.03 4.71 ^ 0.30** 0.17 ^ 0.05** 2.12 ^ 0.20** ND 0.22 ^ 0.06 0.33 ^ 0.06**
6.97 ^ 0.70 28.90 ^ 1.10 5.40 ^ 0.30 34.53 ^ 2.10 2.79 ^ 0.20 1.63 ^ 0.30 2.87 ^ 0.30
9.78 ^ 0.90 32.21 ^ 0.20 4.53 ^ 0.30 33.05 ^ 2.50 2.70 ^ 0.30 1.89 ^ 0.20 4.50 ^ 0.90
Values are expressed as means ^ SEM (n ¼ 8 – 12 animals/group). OVX, ovariectomized; ORX, orchidectomized; ND, not determined. **P # 0:01 vs. corresponding vehicle-injected group (ANOVA followed by Tukey’s test).
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Fig. 1. LH and FSH secretion by pituitaries from 23-day-old male rats 60, 120 and 180 min after incubation in the presence of medium (DMEM) or two different concentrations of ghrelin (1028 and 1026 M). Each experimental group included ten pituitary samples. Values are given as mean ^ SEM. **P # 0:01 vs. DMEM (ANOVA followed by Tukey’s test).
effect of ghrelin was also observed after the increase of serum FSH concentrations by gonadectomy. These results
Fig. 2. LH and FSH secretion by pituitaries of 23-day-old male rats 60, 120 and 180 min after incubation in the presence of medium (DMEM) or two different concentrations of LHRH (1029 and 1027 M) with or without ghrelin (1026 M). Each experimental group included ten pituitary samples. Values are given as mean ^ SEM. **P # 0:01 vs. DMEM; aP # 0:01 vs. corresponding LHRH-treated groups (ANOVA followed by Tukey’s test).
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are in line with data obtained in humans, where ghrelin was also unable to control FSH secretion [31]. In clear-cut contrast with its effects in vivo, ghrelin stimulated LH and FSH secretion in vitro. The mechanism involved in this effect remains unknown. Since the presence of the cognate ghrelin receptor, the GHS-R, in the gonadotropes has not been proven, it is possible to postulate that ghrelin can modify pituitary signals involved in the paracrine control of gonadotropin secretion. A possible candidate to mediate the ghrelin effect on gonadotropin secretion is nitric oxide. Nitric oxide directly stimulates LH and FSH secretion throughout a calciumdependent, cGMP-independent mechanism [26], and it mediates the effects of ghrelin on GH secretion [23], vascular relaxation [30], and food intake [8]. In addition, hypothalamic NOS is increased by ghrelin [8]. Additionally, the stimulatory effect of ghrelin upon gonadotropin secretion directly at the pituitary level could be explained through changes in expression of locally-produced activins, inhibins and follistatin. These peptides have been involved, for example, in the facilitatory action of progesterone and corticosterone for the generation of the secondary FSH surge in cyclic adult rats [32]. Finally, the possibility exists that ghrelin may increase gonadotropin secretion by acting through an, as yet unknown, alternative receptor distinct to the GHS-R, since the existence of different GHS-R subtypes has been previously proposed [4,5,21]. Our data indicate that ghrelin dissociated gonadotropin secretion. Such a dissociation in the response of both gonadotropins in vivo after i.c.v. administration of ghrelin (see Table 1) can be explained through its differential effects upon LH and FSH responsiveness to LHRH observed in vitro (Fig. 2). Secretion of FSH and LH is dissociated in many physiological and experimental conditions in rats [20, 27,28]. With the discovery of a single gonadotropinreleasing hormone, different mechanisms, such as differences in the half-life of both gonadotropins, different modulation by steroids and inhibins, and different sensitivity to LHRH, have been implicated in the non-parallelism between FSH and LH levels observed in different conditions. However, another possibility is that the response of both gonadotropins to LHRH stimulation could be modulated in a different manner by peptides acting upon anterior pituitary cells. The potentiation of FSH response to LHRH by ghrelin and its opposite action on LH suggests a potential role of ghrelin in the dissociation of both gonadotropins. The mechanism whereby ghrelin dissociates the effects of LHRH on gonadotropin secretion is unknown. It might be possible that ghrelin modulates the intracellular actions of LHRH, a finding previously described for peptides involved in the control of LHRH action, such as NPY [6,22], galanin [22] or endothelins [13]. The divergent effect of a regulatory signal on the LHRH-stimulated secretion of both gonadotropins has been previously described for other factors, such
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as NPY or progesterone, which had a stimulatory effect on LHRH-induced LH secretion and a suppressive effect on LHRH-stimulated FSH secretion [14]. Assuming that only one type of gonadotrope exists in the rat pituitary, the explanation for this dissociation is difficult, but subcellular processes might be involved in this phenomenon. In male rat gonadotropes, two distinct subsets of secretory granules have been described (small dense granules rich in LH and large lucent granules rich in FSH) [34]. These granules contain chromogranin A and secretogranin II [36,37], but they show a specific localization depending on the granule subtype [35]. It is possible that ghrelin differentially modulates the intracellular signals activated by LHRH, thus triggering the release of the different granules. In conclusion, the present experiments show that in the rat ghrelin (a) directly stimulates gonadotropin secretion in vitro, (b) differentially modulates the effects of LHRH on both gonadotropins in vitro (reduced LH response and enhanced FSH response), and (c) decreases basal and postorchidectomy LH secretion in vivo, probably by decreasing the LH response to LHRH at the pituitary level. Overall, these data reinforce the concept that ghrelin participates in the control of pituitary hormones other that GH, and suggest the potential involvement of ghrelin in the dissociation of LH and FSH secretion observed in several physiological and experimental conditions.
Acknowledgements This work was supported by Grant BFI 2002-00176 from DGESIC (Ministerio de Ciencia y Tecnologı´a, Spain).
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Ghrelin effects on gonadotropin secretion in male and female rats Rafael Ferna´ndez-Ferna´ndez, Manuel Tena-Sempere, Enrique Aguilar, Leonor Pinilla* Department of Physiology, Faculty of Medicine, University of Co´rdoba, 14004 Cordoba, Spain Received 10 February 2004; received in revised form 29 February 2004; accepted 3 March 2004
Abstract Ghrelin is a 28-amino acid peptide primarily involved in the control of food intake and growth hormone secretion. The present experiments were carried out to analyze the potential involvement of ghrelin in the control of gonadotropin secretion. Prepubertal intact and gonadectomized female and male rats, cyclic rats in diestrus, lactating rats and aged female rats were i.c.v. injected with ghrelin (3 nmol/rat) and blood samples were obtained by decapitation 15 min later. In addition, we analyzed the effects of ghrelin on in vitro basal and luteinizing hormone-releasing hormone (LHRH)-stimulated gonadotropin secretion. Our present results indicate that ghrelin inhibited luteinizing hormone (LH) secretion in vivo in prepubertal males as well as gonadectomized males and females, whereas follicle-stimulating hormone (FSH) remained unaffected. In vitro, ghrelin stimulated the secretion of both gonadotropins, and differentially modulated the response to LHRH; the LH response was inhibited, while the FSH response was enhanced. Overall, our current data open up the possibility that ghrelin may be involved in the control of LH secretion, and in the dissociation of both gonadotropins that takes place in many physiological, pathological and experimental situations. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Ghrelin; Follicle-stimulating hormone; Luteinizing hormone; Luteinizing hormone-releasing hormone
Ghrelin is a 28-amino acid peptide identified as the endogenous ligand for growth hormone secretagogue (GHS) receptor [16,17]. Ghrelin is primarily expressed in stomach and hypothalamus [16,17], and stimulates GH secretion in humans and rats [1 – 3,9,11,16,17,23,29,31,38]. In rats, a limited number of studies have been carried out to analyze the effects of ghrelin on the pituitary secretion of hormones other than GH. The data obtained so far indicate that in prepubertal male rats intracerebroventricular (i.c.v.) administration of ghrelin inhibits luteinizing hormone (LH) and prolactin secretion [24], whereas in estradiol-treated ovariectomized rats, i.c.v. administration of ghrelin suppresses pulsatile LH secretion [7]. In addition, intravenous administration of ghrelin did not change plasma concentrations of prolactin, ACTH and leptin [33]. The regulation of gonadotropin secretion is carried out through a complex interaction between hypothalamic luteinizing hormone-releasing hormone (LHRH), other hypothalamic peptides [6,20], locally-produced pituitary signals such as activin, inhibin and follistatin [15,19], and gonadal-derived steroids and peptides. Preliminary data * Corresponding author. Tel.: þ 34-957-218280; fax: þ 34-957-218288. E-mail address: [email protected] (L. Pinilla).
from our laboratory indicated that ghrelin inhibits LH secretion in prepubertal male rats [24]. The present experiments were carried out in vivo and in vitro in order to analyze the potential effects of ghrelin on basal and LHRH-stimulated gonadotropin secretion. In Experiment 1, male and female rats (23 days old) were gonadectomized or sham-gonadectomized under light ether anesthesia. The animals were i.c.v. injected 7 days later with 3 nmol/rat of ghrelin dissolved in 10 ml of vehicle or vehicle alone (physiological saline; NaCl 0.9%) and decapitated 15 min later. Blood samples were collected. The procedure for i.c.v. injections was as described previously [25]. In Experiment 2, cyclic females in diestrus, lactating rats and aged (24 months) female animals were i.c.v. injected with ghrelin (3 nmol/rat dissolved in 10 ml of vehicle) or vehicle and decapitated 15 min later. Blood samples were collected. In order to detect a potential primary action of ghrelin at the pituitary level, in Experiment 3, anterior pituitaries were obtained from 30-day-old male rats and placed in glass scintillation vials (one per vial) in a Dubnoff shaker at 37 8C under an atmosphere of 96% O2/5% CO2. Each vial contained 1 ml Dulbecco’s Minimal Essential Medium (DMEM). After preincubation for 60 min, the medium was replaced by fresh medium containing ghrelin (1028 and
0304-3940/03/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.03.003
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1026 M). Medium samples were obtained at 60, 120 and 180 min of the incubation period. In addition, the combined effects of LHRH (1029 and 1027 M) and ghrelin (1026 M) were also analyzed. Each group consisted of eight to 12 pituitary samples. In all experiments, special precautions were taken to avoid any stressing influence (all the animals were handled daily for a week before the experiment and killed by the same person). Experiments were carried out between 11:00 and 12:00 h. Experimental procedures were approved by the Co´rdoba University Ethical Committee for animal experimentation and were conducted in accordance with the European Union normative for care and use of experimental animals. After centrifugation (1600 £ g at 4 8C for 20 min), serum was collected, frozen and stored at 2 20 8C until use. The concentrations of gonadotropins were measured in 5 – 50 ml by a double-antibody method using a radioimmunoassay kit supplied by NIDDK (Bethesda, MD). Rat LH-I-10 and follicle-stimulating hormone (FSH)-I-7 were labeled with I125 by the chloramine T method and hormone concentrations were expressed using a reference preparation LH-RP3 and FSH-RP2 as standard. Intra- and interassay variations were 8 and 10% for LH and 6 and 9% for FSH. The sensitivities of the assay were 75 pg/ml for LH and 0.4 ng/ml for FSH. All samples of each experiment were measured in the same assay. Values are expressed as means ^ SEM. Differences between groups were analyzed using one- or twoway ANOVA followed by Tukey’s test. Central (i.c.v.) administration of ghrelin (3 nmol/rat) significantly decreased serum LH levels in intact and orchidectomized prepubertal males, in ovariectomized prepubertal females and in aged (24-month-old) female rats, whereas no significant effect on LH concentrations was detected in lactating females (Table 1). Due to technical reasons, the effects of central injection of ghrelin on serum LH levels were not determined in cyclic female rats in the diestrus phase. In none of the groups studied did serum FSH concentrations change significantly after ghrelin treatment, although a trend to increase, close to the limits of the
statistical significance, was observed in prepubertal ovariectomized and aged females (Table 1). Gonadotropin concentrations in the incubation media increased significantly along the incubation period (Figs. 1 and 2). Ghrelin stimulated LH secretion at 60, 120 and 180 min of the incubation period, while the concentrations of FSH were increased only at 60 and 120 min after exposure to ghrelin (Fig. 1). LHRH at the doses of 1029 and 1027 M stimulated gonadotropin release, and the magnitude of this effect was dose-dependent (Fig. 2). Under these stimulated conditions, ghrelin initially potentiated (at 60 min) the effect of LHRH (1029 M) on LH and FSH secretion (Fig. 2), but a clear dissociation at 120 and 180 min was observed, since ghrelin inhibited the LH response to LHRH while it enhanced that of FSH (Fig. 2). The presence of ghrelin receptors in the hypothalamus and pituitary [10,12,18] opens the possibility that ghrelin participates in the control of pituitary secretion and, in addition to its effects on GH, ghrelin actions on prolactin and LH release have been reported [3,7,9,24]. Our results demonstrate that in prepubertal rats ghrelin is involved in the control of gonadotropin secretion, since ghrelin (a) decreased both basal and post-orchidectomy LH secretion in vivo without affecting the release of FSH, (b) stimulated the secretion of both gonadotropins in vitro, and (c) differentially affected the response of both gonadotropins to LHRH, decreasing the LH response and increasing the FSH response. The inhibitory effect of ghrelin on LH secretion observed in vivo can be explained by the decrease of LH response to LHRH detected in vitro. It should be noted that the suppressive effect of ghrelin was more potent after gonadectomy, when LHRH release is increased, and after stimulation of LHRH release by NMDA (data not shown). Intriguingly, no inhibitory effect of ghrelin upon serum LH levels was detected in lactating female rats, a phenomenon whose physiological relevance is presently under investigation in our laboratory. In contrast with data obtained for LH, FSH secretion in vivo was apparently independent of ghrelin action, since central administration of the peptide was ineffective in prepubertal male and female rats, as well as in cyclic, lactating and aged female rats. Such a lack of
Table 1 Effects of central (i.c.v.) administration of ghrelin (3 nmol/rat) on serum LH and FSH concentrations in different experimental models Group and treatment
Females, intact prepubertal Females, OVX prepubertal Males, intact prepubertal Males, ORX prepubertal Females, diestrus Females, lactating Females, aging (24 months)
LH (ng/ml)
FSH (ng/ml)
Vehicle
Ghrelin
Vehicle
Ghrelin
0.37 ^ 0.07 8.21 ^ 0.20 0.40 ^ 0.05 4.74 ^ 0.40 ND 0.21 ^ 0.08 0.70 ^ 0.08
0.21 ^ 0.03 4.71 ^ 0.30** 0.17 ^ 0.05** 2.12 ^ 0.20** ND 0.22 ^ 0.06 0.33 ^ 0.06**
6.97 ^ 0.70 28.90 ^ 1.10 5.40 ^ 0.30 34.53 ^ 2.10 2.79 ^ 0.20 1.63 ^ 0.30 2.87 ^ 0.30
9.78 ^ 0.90 32.21 ^ 0.20 4.53 ^ 0.30 33.05 ^ 2.50 2.70 ^ 0.30 1.89 ^ 0.20 4.50 ^ 0.90
Values are expressed as means ^ SEM (n ¼ 8 – 12 animals/group). OVX, ovariectomized; ORX, orchidectomized; ND, not determined. **P # 0:01 vs. corresponding vehicle-injected group (ANOVA followed by Tukey’s test).
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Fig. 1. LH and FSH secretion by pituitaries from 23-day-old male rats 60, 120 and 180 min after incubation in the presence of medium (DMEM) or two different concentrations of ghrelin (1028 and 1026 M). Each experimental group included ten pituitary samples. Values are given as mean ^ SEM. **P # 0:01 vs. DMEM (ANOVA followed by Tukey’s test).
effect of ghrelin was also observed after the increase of serum FSH concentrations by gonadectomy. These results
Fig. 2. LH and FSH secretion by pituitaries of 23-day-old male rats 60, 120 and 180 min after incubation in the presence of medium (DMEM) or two different concentrations of LHRH (1029 and 1027 M) with or without ghrelin (1026 M). Each experimental group included ten pituitary samples. Values are given as mean ^ SEM. **P # 0:01 vs. DMEM; aP # 0:01 vs. corresponding LHRH-treated groups (ANOVA followed by Tukey’s test).
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are in line with data obtained in humans, where ghrelin was also unable to control FSH secretion [31]. In clear-cut contrast with its effects in vivo, ghrelin stimulated LH and FSH secretion in vitro. The mechanism involved in this effect remains unknown. Since the presence of the cognate ghrelin receptor, the GHS-R, in the gonadotropes has not been proven, it is possible to postulate that ghrelin can modify pituitary signals involved in the paracrine control of gonadotropin secretion. A possible candidate to mediate the ghrelin effect on gonadotropin secretion is nitric oxide. Nitric oxide directly stimulates LH and FSH secretion throughout a calciumdependent, cGMP-independent mechanism [26], and it mediates the effects of ghrelin on GH secretion [23], vascular relaxation [30], and food intake [8]. In addition, hypothalamic NOS is increased by ghrelin [8]. Additionally, the stimulatory effect of ghrelin upon gonadotropin secretion directly at the pituitary level could be explained through changes in expression of locally-produced activins, inhibins and follistatin. These peptides have been involved, for example, in the facilitatory action of progesterone and corticosterone for the generation of the secondary FSH surge in cyclic adult rats [32]. Finally, the possibility exists that ghrelin may increase gonadotropin secretion by acting through an, as yet unknown, alternative receptor distinct to the GHS-R, since the existence of different GHS-R subtypes has been previously proposed [4,5,21]. Our data indicate that ghrelin dissociated gonadotropin secretion. Such a dissociation in the response of both gonadotropins in vivo after i.c.v. administration of ghrelin (see Table 1) can be explained through its differential effects upon LH and FSH responsiveness to LHRH observed in vitro (Fig. 2). Secretion of FSH and LH is dissociated in many physiological and experimental conditions in rats [20, 27,28]. With the discovery of a single gonadotropinreleasing hormone, different mechanisms, such as differences in the half-life of both gonadotropins, different modulation by steroids and inhibins, and different sensitivity to LHRH, have been implicated in the non-parallelism between FSH and LH levels observed in different conditions. However, another possibility is that the response of both gonadotropins to LHRH stimulation could be modulated in a different manner by peptides acting upon anterior pituitary cells. The potentiation of FSH response to LHRH by ghrelin and its opposite action on LH suggests a potential role of ghrelin in the dissociation of both gonadotropins. The mechanism whereby ghrelin dissociates the effects of LHRH on gonadotropin secretion is unknown. It might be possible that ghrelin modulates the intracellular actions of LHRH, a finding previously described for peptides involved in the control of LHRH action, such as NPY [6,22], galanin [22] or endothelins [13]. The divergent effect of a regulatory signal on the LHRH-stimulated secretion of both gonadotropins has been previously described for other factors, such
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as NPY or progesterone, which had a stimulatory effect on LHRH-induced LH secretion and a suppressive effect on LHRH-stimulated FSH secretion [14]. Assuming that only one type of gonadotrope exists in the rat pituitary, the explanation for this dissociation is difficult, but subcellular processes might be involved in this phenomenon. In male rat gonadotropes, two distinct subsets of secretory granules have been described (small dense granules rich in LH and large lucent granules rich in FSH) [34]. These granules contain chromogranin A and secretogranin II [36,37], but they show a specific localization depending on the granule subtype [35]. It is possible that ghrelin differentially modulates the intracellular signals activated by LHRH, thus triggering the release of the different granules. In conclusion, the present experiments show that in the rat ghrelin (a) directly stimulates gonadotropin secretion in vitro, (b) differentially modulates the effects of LHRH on both gonadotropins in vitro (reduced LH response and enhanced FSH response), and (c) decreases basal and postorchidectomy LH secretion in vivo, probably by decreasing the LH response to LHRH at the pituitary level. Overall, these data reinforce the concept that ghrelin participates in the control of pituitary hormones other that GH, and suggest the potential involvement of ghrelin in the dissociation of LH and FSH secretion observed in several physiological and experimental conditions.
Acknowledgements This work was supported by Grant BFI 2002-00176 from DGESIC (Ministerio de Ciencia y Tecnologı´a, Spain).
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