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Regular Article |
a Instituto de Neurobiología, Buenos Aires 1414, Argentina
ABSTRACT
The purpose of this study was to investigate whether melanin-concentrating hormone (MCH) acts directly on the median eminence and on the anterior pituitary of female rats regulating LHRH and gonadotropin release. In addition, immunohistochemistry was used to examine the density and distribution of MCH-immunoreactive fibers in the median eminence of proestrous rats. MCH-immunoreactive fibers were found in both the internal and external layers of the median eminence and in close association with hypophysial portal vessels. In the first series of in vitro experiments, median eminences and anterior pituitaries were incubated in Krebs-Ringer bicarbonate buffer containing two MCH concentrations (10-10 and 10-8 M). The lowest MCH concentration (10-10 M) increased (P < 0.01) LHRH release only from proestrous median eminences. Anterior pituitaries incubated with both MCH concentrations also showed that 10-10 M MCH increased gonadotropin release only from proestrous pituitaries. In the second series of experiments, median eminences and pituitaries from proestrous rats were incubated with graded concentrations of MCH. MCH (10-10 and 10-9 M) increased (P < 0.01) LHRH release from the median eminence, and only 10-10 M MCH increased (P < 0.01) LH and FSH release from the anterior pituitary. The effect of MCH on the stimulation of both gonadotropins from proestrous pituitaries was similar to the effect produced by LHRH. Simultaneous incubation of pituitaries with MCH and LHRH did not modify LH but increased the FSH release induced by LHRH. The present results suggest that MCH could be involved in the regulation of preovulatory gonadotropin secretion.
anterior pituitary, FSH, GnRH, hypothalamus, LH, ovulatory cycle
INTRODUCTION
Melanin-concentrating hormone (MCH) is a cyclic 19-amino-acid peptide originally isolated from the teleost pituitary [1]. MCH-immunoreactive neurons also have been described in the salmon brain [2]. In mammals [3, 4], MCH-containing perikarya have been primarily located in the zona incerta and the lateral hypothalamus, projecting throughout the central nervous system with abundant terminals in the preoptic area and the hypothalamus [4–6]. In studying the male rat median eminence, Bittencourt et al. [4] reported that MCH-immunoreactive fibers diplay a moderate density in the internal layer and a few fibers in the external layer of the median eminence.
MCH was first described as regulating skin pigmentation in teleost fish by inducting melanin aggregation within the melanocytes [1]. In mammals, MCH has been shown to be involved in the control of such complex behavioral processes as drinking [7, 8], eating [9], grooming, aggression, exploration, and anxiety [10, 11].
MCH has also been shown to be involved in the reproductive function. Injections of MCH into the medial preoptic area (MPOA) and the ventromedial nucleus stimulate female sexual receptivity [10, 12]. Gonzalez et al. [13] observed that the injection of MCH in the MPOA or in the median eminence increases LH release in steroid-primed ovariectomized rats. They also showed that the infusion of MCH antiserum into the MPOA blocks the progesterone-induced LH discharge in estrogen-primed ovariectomized rats. Additionally, MCH expression in the hypothalamus appears to be estrogen dependent [14]. These results suggest that MCH could play a role in the control of preovulatory gonadotropin release.
The present experiments were designed to examine a direct effect of MCH on LHRH release from the median eminence and on gonadotropin release from the anterior pituitary. Tissue samples were obtained from female rats, and LHRH and gonadotropins were measured in the incubation medium. The distribution and density of MCH fibers were also examined in female rats at proestrus by means of immunohistochemistry.
MATERIALS AND METHODS
Animals
Adult female Sprague-Dawley rats (240–280 g body weight) were used in these experiments. Animals were maintained under standard laboratory conditions, with tap water and regular rat chow ad libitum and controlled lighting conditions (lights-on from 0600 to 2000 h). Daily vaginal smears were taken before 1200 h, and only those rats showing at least two consecutive 4-day cycles were chosen.
Immunohistochemistry
Female rats at proestrus were anesthetized with tribromoethanol (25 mg/100 g body weight, i.p.) before noon (1100 h) and perfused through the left cardiac ventricle with 50 ml of 0.9% NaCl at 37°C, followed by 300–400 ml of an ice-cold mixture of formalin and picric acid (4% paraformaldehyde and 0.3% picric acid) in PBS (0.14 M NaCl, 4 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) + 2.7 mM KCl. Brains were removed, and hypothalamic blocks were dissected out and postfixed for 60 min by immersion in the same fixative at 4°C. After several washes for at least 24 h in PBS containing 20% sucrose and 0.01% sodium azide, the tissue blocks were then sectioned in the coronal plane with a cryostat at 15–20 µm. Each hypothalamic block was cut rostrocaudally from the preoptic area to the mammillary bodies, and consecutive sections were rinsed in the same buffer containing 10% sucrose. Tissue sections were delipidized and treated with 0.05% hydrogen peroxide to block endogenous peroxidase. Sections were then incubated overnight with MCH antiserum raised in rabbit (1:10 000; Phoenix Pharmaceuticals, Mountain View, CA). After several washes in PBS, the tissues were incubated with biotinylated goat anti-rabbit IgG (Vector Elite kit; Vector Laboratories, Burlingame, CA) for 30 min at room temperature followed by several washes in the same buffer. The avidin-biotin-immunoperoxidase procedure (Vector Elite Kit; Vector Laboratories, Burlingame, CA) was then used following the manufacturer's instructions. Peroxidase activity was revealed with glucose oxidase, using diaminobenzidine (Vector Laboratories) as the chromagen and nickel salts for enhancement of the reaction product [15]. To confirm the specificity of the primary antiserum, two different control procedures were used: 1) MCH antiserum preincubated with an excess of MCH (10 µg/ml; Peninsula Laboratories, Belmont, CA) and 2) omission of the primary antiserum. In addition, sections from each rat were stained with Nissl for histological reference. Immunostained sections were observed through a Nikon Microphot FXA microscope.
Median Eminence Incubations
The rats were killed by decapitation between 1200 and 1300 h, and brains and pituitaries were rapidly removed. The median eminence was removed from the rest of the brain with fine scissors under a dissecting microscope according to anatomical boundaries described elsewhere [16]. This preparation, free from surrounding structures, contained the entire median eminence. Each median eminence was placed for 30 min in 500 µl of ice-cold Krebs-Ringer bicarbonate buffer (KRB). The composition of the KRB used in these experiments was 124 mM NaCl, 5 mM KCl, 0.75 mM CaCl2, 1.3 mM MgSO4, 26 mM NaHCO3, 1.2 mM KH2PO4, and 10 mM glucose (pH 7.4). The medium contained 1 mg/ml ascorbic acid. After 30 min at 4°C, the medium was aspirated, discarded, and replaced with 500 µl of fresh KRB. The median eminence was then preincubated for 30 min using a Dubnoff shaking incubator under a constant atmosphere of 95% O2/5% CO2 at 37°C. Following this preincubation, the medium was also discarded and replaced with fresh medium or medium containing MCH. After incubation for 30 min, the medium was collected and replaced with an equal volume of fresh medium or medium containing the same concentration of MCH. The incubation was continued for an additional 30 min. At that time, the medium was aspirated and collected. At the end of the incubation, all the collected samples were boiled for 30 min in a water bath and stored frozen at -70°C until LHRH determination by RIA (no more than 15 days).
Hemipituitary Incubations
The anterior pituitary was dissected in situ (avoiding contamination from pars intermedia), removed, bissected longitudinally, and placed in 500 µl ice-cold KRB. After 30 min, the medium was discarded and each hemipituitary gland was preincubated for 60 min at 37°C with 500 µl of fresh medium in a Dubnoff shaking incubator under the same conditions described for the median eminence. Following this preincubation, medium was removed and discarded, and fresh medium alone or containing the test substances was added. The hemipituitaries were randomly distributed among the experimental groups. Incubation was then carried out for 3 h. At the end of this incubation period, medium was collected and frozen at -20°C until LH and FSH were assayed.
Rat MCH (Peninsula Laboratories Europe, Merseyside, UK) was dissolved in distilled water (10-4 M), aliquoted, and frozen at -70°C. Further dilutions were made with KRB on the day of the experiment. LHRH (Peninsula Laboratories, Belmont, CA) was dissolved in 0.1 N acetic acid (10-4 M) and further diluted with KRB to 4 x 10-9 M.
Experimental Protocols for Incubations
Preliminary experiments were performed in male rats to find out the minimal number of median eminences to be used for obtaining a substantial LHRH release in response to a given stimulus. Under the experimental conditions described above, 28 mM KCl induced a significant increase of LHRH release from a single median eminence (30.65 ± 4.29 to 75.66 ± 4.4 pg/median eminence; P <0.002).
The first series of experiments was performed in female rats to examine whether MCH exerted any direct effect on LHRH and on gonadotropin release and to analyze whether the responses depended on the stage of the estrous cycle. Median eminences and anterior pituitaries from female rats were incubated with two MCH concentrations: 10-10 M and 10-8 M.
The second series of experiments was performed to find out the minimal effective MCH concentration for median eminence LHRH and anterior pituitary gonadotropin release. Based on the results obtained in the first series of experiments, tissue samples were obtained from proestrous rats only. Median eminences and anterior pituitaries were incubated with graded concentrations of MCH (10-11, 10-10, 10-9, and 10-8 M). In the third experiment, anterior pituitaries were incubated with MCH (10-10 M), LHRH (4 x 10-9 M) or LHRH (4 x 10-9 M) plus MCH (10-10 M).
Hormone Assays
Serum LH and FSH levels were measured by RIA using kits provided by the NIDKK National Pituitary Agency (Baltimore, MD). Results were expressed as nanograms per milliliter of LH or FSH released to the medium from each hemipituitary in terms of rat LH RP-3 and FSH RP-2 standards provided with the kits.
LHRH levels in the medium obtained from median eminence incubations were measured by RIA, using LHRH (Peninsula Laboratories, Belmont, CA) for standards and radioiodination. LHRH antiserum was obtained from Ayala Barnea's laboratory (University of Texas Southwestern Medical Center, Dallas, TX). The sensitivity of the assay was 0.80 pg/tube, and the standard curve was linear between 0.80 and 25 pg. Results were expressed as picograms of LHRH released to the medium from a single median eminence.
Data Analysis
Results were expressed as means ± SEM. Data for the two main experiments were analyzed by the Kruskal-Wallis test. Significant results were followed by Dunn's multiple pairwise comparisons. The third experiment was tested via a single-variable four-level analysis of variance followed by the Newman-Keuls multiple comparisons procedure. The experiment was then submitted to a test for the validity of the simple linear combination of stimulus concentrations [17]: [MCH] + [LHRH] = [MCH + LHRH].
RESULTS
Immunohistochemical Observations
Figure 1 shows the MCH-immunoreactive nerve fiber distribution in the median eminence of the proestrous rat. As previously described for the male rat [4], MCH fibers are present in the internal layer of the median eminence. In the external layer, MCH-containing fibers and gross deposits of MCH-immunoreactive material are present around the portal blood vessels.
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Estrous Cycle
LHRH release Because preliminary experiments showed that spontaneous LHRH release was similar in the different stages of the estrous cycle (proestrus: 16.59 ± 2.61, estrus: 14.96 ± 2.42, diestrus: 17.98 ± 2.15 pg/median eminence, n = 9 to 12 from each stage), in subsequent experiments the control group was composed of animals at any stage of the estrous cycle (Fig. 2). The addition of 10-8 M MCH to the incubation medium did not modify LHRH release in any stage of the cycle, neither in the first 30 min of incubation nor in the second incubation period (Fig. 2A). The addition of 10-10 M MCH increased LHRH release (P < 0.01) in only the second period of incubation of the proestrous median eminence (Fig. 2B).
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Gonadotropin release Following a preincubation of 1 h in KRB, the pituitaries were incubated for 3 h in the presence of two different MCH concentrations (10-8 M and 10-10 M). Basal release of LH was similar in all stages of the estrous cycle. Only the lowest concentration (10-10 M) of MCH induced a significant (P < 0.01) stimulation of LH release from proestrous pituitaries (Fig. 3, upper and lower panels).
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Figure 4 shows the effect of two different concentrations of MCH on FSH release. As previously described for LH, basal release of FSH in the incubation medium did not change with the stage of the estrous cycle. The addition of 10-8 M MCH to the incubation medium did not affect FSH release (Fig. 4, upper panel). However, when the anterior pituitary was incubated with 10-10 M MCH, FSH release increased (P < 0.01) from only the pituitary in proestrus (Fig. 4, lower panel).
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From these experiments, proestrous rats were selected for further studies on the effectiveness of graded MCH concentrations on LHRH and gonadotropin release.
Proestrous Rats
LHRH release To explore a dose-related increment in LHRH release with MCH, median eminences from proestrous rats were incubated in either the absence or presence of graded concentrations of MCH. There was no effect of any MCH concentration on LHRH release in the first 30 min of incubation (Fig. 5, left panel). However, 10-10 M and 10-9 M MCH increased (P < 0.01) LHRH release in the second 30 min of incubation (Fig. 5, right panel).
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Gonadotropin release When anterior pituitaries from proestrous rats were incubated with MCH, only MCH 10-10 M affected release of both gonadotropins (Fig. 6). Basal release of LH from proestrous rats was 104.05 ± 8.37 ng/ml; 10-10 M MCH induced a significant (P < 0.01) stimulation of LH release (Fig. 6, upper panel). Basal FSH release from proestrous rats was 11.25 ± 0.89 ng/ml; only 10-10 M MCH enhanced (P < 0.001) FSH release (Fig. 6, lower panel).
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Figure 7 shows the results of pituitaries incubated with MCH (10-10 M), LHRH (4 x 10-9 M), or MCH (10-10 M) plus LHRH (4 x 10-9 M). MCH effect was similar to the effect of LHRH on stimulation of LH release. The stimulation of FSH release by MCH was smaller than the stimulation produced by LHRH (P < 0.01). When both MCH and LHRH were tested together, no significant extra LH release was observed beyond simple addition of individual stimuli. Results from the coincubation of MCH and LHRH are also consistent with the hypothesis of addition of both stimuli to release FSH (Fig. 7, lower panel).
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DISCUSSION
LHRH is the primary regulator of preovulatory gonadotropin release. Several neuronal systems acting directly or indirectly at different levels of the hypophysiotrophic LHRH neurons modify LHRH release. A consistent body of evidence supports the participation of neuropeptides [18–20], classical neurotransmitters [21–23], excitatory and inhibitory amino acids [24], local growth factors [25], nitric oxide [26], and probably leptin [27] in regulating LHRH release at the mediobasal hypothalamus level.
The present study shows MCH-immunoreactive fibers in the internal layer of the median eminence in female rats in agreement with the results published for males [4]. MCH-immunoreactive fibers are also present in the external layer of the median eminence of proestrous rats, in contrast to the few fibers described in male rats [4]. At this level, MCH-immunoreactive fibers are preferentially located at the lateral edges and around the blood portal vessels. A similar distribution of MCH fibers has also been described in the external layer of the monkey median eminence [6].
The presence of MCH immunoreactive fibers in the external layer of the median eminence suggests the existence of interactions of MCH with nerve terminals of hypophysotropic neurons at the level of the median eminence. In the present study, MCH stimulated LHRH release, acting directly on the median eminence, and this effect was only observed when the median eminences were obtained from proestrous rats.
When proestrous median eminences were incubated with a range of MCH concentrations, LHRH release was significantly stimulated by MCH at concentrations as low as 10-10 M and 10-9 M, whereas smaller (10-11 M) or larger (10-8 M) concentrations were ineffective. A similar bell-shaped curve related to the dose has been observed for the effect of leptin [27] and atrial natriuretic peptide (Antunes-Rodriguez, personal communication) on LHRH release. This shape has also been reported in dose-response curves to other peptides, as reviewed by Kastin et al. [28].
The LHRH response to MCH was observed in the second 30-min period of incubation. This latency of at least 30 min has been observed with LHRH [27] and corticotropin-releasing factor [29]. In both cases, the delayed responses to the peptides were mediated by nitric oxide release. Therefore, the delayed response of LHRH to MCH may also be mediated by other neurotransmitters.
The LHRH-releasing effect of MCH is estrous cycle stage dependent; it takes place only in proestrous rats. This effect may be due to the high levels of circulating estrogens, characteristic of this stage of the cycle [30].
Saito et al. [31] and Chambers et al. [32] have recently shown that MCH is the cognate ligand for the orphan G-protein-coupled receptor SLC-1. SLC-1 mRNA has been detected in the brain and in the pituitary [31, 32]. Within the brain, in situ hybridization studies have revealed SLC-1 mRNA in various nuclei of the hypothalamus [31, 32]. Although MCH receptors in the median eminence have not been described yet, our results suggest that the receptive elements for the stimulatory action of MCH reside on the median eminence LHRH terminals.
The results of the present study lead us to propose a stimulatory role for MCH in LHRH release. This hypothesis is supported by previous findings: 1) MCH injection into the MPOA or the median eminence in estrogen-primed ovariectomized rats stimulated LH release [13], 2) MCH antiserum administration in the MPOA inhibited the LH surge elicited by progesterone in estrogen-primed ovariectomized rats [13], and 3) MCH expression in the hypothalamus appears to be estrogen dependent [14].
The zona incerta, a subthalamic structure, is one of the areas where the majority of MCH perikarya are located [4]. This area has also been implicated in the regulation of the preovulatory LH discharge and ovulation [33, 34]. MacKenzie et al. [33] found that electrolytic lesions of the zona incerta induced constant diestrus. In agreement with this finding, bilateral electrolytic lesions of the medial zona incerta made on the morning of proestrus blocked the preovulatory LH surge [34]. Pharmacological evidence suggested that dopamine-containing neurons of the zona incerta may be involved [33, 35]. Moreover, tyrosine hydroxylase-containing neurons of the zona incerta have estrogen receptors [36]. Shughrue et al. [37] also described the presence of ß-estrogen receptors in the zona incerta neurons, suggesting a feedback modulation of MCH neurons by gonadal steroids. Together, these results suggest that MCH neurons of the zona incerta can be involved in the preovulatory LHRH release.
The present immunohistochemical study of proestrous rats also show very dense immunoreactive material around the hypophysial portal vessels, suggesting the release of MCH into the blood to reach the anterior pituitary. The present results show that MCH acts directly on the anterior pituitary to affect gonadotropin release. When anterior pituitaries from different stages of the estrous cycle were incubated with MCH, only proestrous pituitaries released more LH and FSH in response to the peptide; here again, under certain steroid conditions (stage of the estrous cycle), the gonadotropes responded to MCH by increasing the release of LH and FSH.
The incubation of proestrous pituitaries with a range of concentrations of MCH showed that 10-10 M MCH was the effective concentration to stimulate both gonadotropins. As for the LHRH response to MCH, small doses of peptides were more effective than larger doses in obtaining determined responses [28].
In addition to the LHRH-releasing effect of MCH at the median eminence level, MCH also stimulates LH and FSH secretion by acting on the pituitary. Other neuropeptides [18, 19, 27] have shown to elicit both LHRH and gonadotropin release.
The gonadotropin-releasing effects of MCH suggest that the anterior pituitary could be also a site of action of MCH. MCH receptor mRNA has been detected in the anterior pituitary [31]. According to the present results, it is likely that MCH receptors are located on gonadotropes. Because a variety of paracrine interactions occur in the pituitary, the action of MCH could be exerted on other pituitary cell type, which then via paracrine control stimulate LH and FSH secretion.
Pituitary incubations with MCH and LHRH have shown that MCH is almost as effective as LHRH for stimulating LH and FSH release. Exposure of pituitaries to MCH and LHRH together did not further increase LH but did increase FSH release, induced by LHRH. However, the small sample size in this experiment precludes a definitive answer to the question of additivity of individual stimuli.
It is not clear how MCH fits into the established dogma of the hypothalamic pituitary signaling; however, MCH released from the median eminence may act directly on LHRH nerve terminals at that level or may act indirectly through other nerve endings containing different neurotransmitters [38]. Our results showing MCH-immunoreactive material around the portal vessels and the in vitro MCH stimulation of gonadotropin release support the idea of a direct action of MCH on the pituitary. MCH effects were only seen in proestrous samples, i.e., tissues exposed to the endogenous endocrine conditions under which preovulatory gonadotropin surges take place.
The presence of MCH fibers in the external layer of the median eminence and in vitro experiments showing that MCH stimulates both LHRH release from the median eminence and gonadotropin release from the pituitary of proestrous rats lead us to suggest a role for MCH as a neuromodulator participating in the control of preovulatory gonadotropin release.
ACKNOWLEDGMENTS
The authors thank Dr. Armando Garsd for his help with the statistical analyses, Jorge Gaston, Marcela Huerta, Med Vet Marcela Marquez, and Med Vet Silvina Heisecke for their assistance, Dr. Parlow (NIDKK Program) for providing the RIA materials, and Miriam Golía for her assistance with the English version of the paper.
FOOTNOTES
First decision: 17 August 2000.
1 These studies were supported by Fundación Instituto de Neurobiología, Agencia de Promoción Científica y Tecnológica (PICT 97 05-00101-02022), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Fundación Barceló, Buenos Aires, Argentina. 
2 Correspondence: Sara R. Chiocchio, Instituto de Neurobiología, Serrano 669, Buenos Aires 1414, Argentina. FAX: 54 11 4854 5602;idneu{at}neurob.cyt.edu.ar
Accepted: December 21, 2000.
Received: July 20, 2000.
REFERENCES
-adrenergic mechanism. Endocrinology 1990; 127:2431–2436[Abstract] with the secretion of neuroactive substances that stimulate the release of luteinizing hormone-releasing hormone. Endocrinology 1997; 138:19–25 and-ß mRNA in the rat central nervous system. J Comp Neurol 1997; 388:507–525[CrossRef][Medline]This article has been cited by other articles:
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  P. Pissios, R. L. Bradley, and E. Maratos-Flier Expanding the Scales: The Multiple Roles of MCH in Regulating Energy Balance and Other Biological Functions Endocr. Rev., October 1, 2006; 27(6): 606 - 620. [Abstract] [Full Text] [PDF]
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 |
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 G. Segal-Lieberman, H. Rubinfeld, M. Glick, N. Kronfeld-Schor, and I. Shimon Melanin-concentrating hormone stimulates human growth hormone secretion: a novel effect of MCH on the hypothalamic-pituitary axis Am J Physiol Endocrinol Metab, May 1, 2006; 290(5): E982 - E988. [Abstract] [Full Text] [PDF]
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 I. J. Clarke, A. Rao, Y. Chilliard, C. Delavaud, and G. A. Lincoln Photoperiod effects on gene expression for hypothalamic appetite-regulating peptides and food intake in the ram Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2003; 284(1): R101 - R115. [Abstract] [Full Text] [PDF]
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