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Endocrinology, doi:10.1210/en.2006-0636
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Endocrinology Vol. 148, No. 2 813-823
Copyright © 2007 by The Endocrine Society

Neuromedin S as Novel Putative Regulator of Luteinizing Hormone Secretion

E. Vigo, J. Roa, M. López, J. M. Castellano, R. Fernandez-Fernandez, V. M. Navarro, R. Pineda, E. Aguilar, C. Diéguez, L. Pinilla and M. Tena-Sempere

Department of Cell Biology, Physiology, and Immunology (E.V., J.R., J.M.C., R.F.-F., V.M.N., R.P., E.A., L.P., M.T.-S.), University of Córdoba, 14004 Córdoba, Spain; and Department of Physiology (M.L., C.D.), University of Santiago de Compostela, 15705 Santiago de Compostela, Spain

Address all correspondence and requests for reprints to: M. Tena-Sempere, Physiology Section, Department of Cell Biology, Physiology, and Immunology, Faculty of Medicine, University of Córdoba, Avda. Menéndez Pidal s/n, 14004 Córdoba, Spain. E-mail: fi1tesem{at}uco.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neuromedin S (NMS), a 36 amino acid peptide structurally related to neuromedin U, was recently identified in rat brain as ligand for the G protein-coupled receptor FM4/TGR-1, also termed neuromedin U receptor type-2 (NMU2R). Central expression of NMS appears restricted to the suprachiasmatic nucleus, and NMS has been involved in the regulation of dark-light rhythms and suppression of food intake. Reproduction is known to be tightly regulated by metabolic and photoperiodic cues. Yet the potential contribution of NMS to the control of reproductive axis remains unexplored. We report herein analyses of hypothalamic expression of NMS and NMU2R genes, as well as LH responses to NMS, in different developmental and functional states of the female rat. Expression of NMS and NMU2R genes was detected at the hypothalamus along postnatal development, with significant fluctuations of their relative levels (maximum at prepubertal stage and adulthood). In adult females, hypothalamic expression of NMS (which was confined to suprachiasmatic nucleus) and NMU2R significantly varied during the estrous cycle (maximum at proestrus) and was lowered after ovariectomy and enhanced after progesterone supplementation. Central administration of NMS evoked modest LH secretory responses in pubertal and cyclic females at diestrus, whereas exaggerated LH secretory bursts were elicited by NMS at estrus and after short-term fasting. Conversely, NMS significantly decreased elevated LH concentrations of ovariectomized rats. In summary, we provide herein novel evidence for the ability of NMS to modulate LH secretion in the female rat. Moreover, hypothalamic expression of NMS and NMU2R genes appeared dependent on the functional state of the female reproductive axis. Our data are the first to disclose the potential implication of NMS in the regulation of gonadotropic axis, a function that may contribute to the integration of circadian rhythms, energy balance, and reproduction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NEUROMEDIN S (NMS) was very recently identified in rat brain by means of reverse pharmacology as a novel 36-amino acid peptide with ability to bind the G protein-coupled receptor FM4/TGR-1, also termed neuromedin U receptor type-2 (NMU2R) (1). Structural analyses revealed that NMS shows complete sequence homology with the C-terminal seven amino acid region of NMU, a brain-gut peptide originally isolated for porcine spinal cord (2). Yet NMU and NMS are encoded by different genes (1). In keeping with structural data, functional assays demonstrated that, besides NMU2R, NMS is able to bind to and activate FM-3/GPR66, also known as NMU receptor type-1 (NMU1R) (1). Interestingly, NMU1R and NMU2R show quite different patterns of distribution, with NMU1R being detected in a wide range of peripheral tissues (such as intestine, testis, pancreas, uterus, lung, and kidney) and NMU2R expression being limited to discrete brain areas as the paraventricular and arcuate (ARC) nuclei (3, 4, 5, 6, 7). On the basis of their structural similarities and the usage of common receptor pathways, it was anticipated that NMU and NMS may share their biological actions. Of note, however, expression analyses evidenced that the pattern of distribution of NMS is more restricted than that of NMU, with consistent NMS expression being found only at the suprachiasmatic nucleus (SCN) within the brain and the spleen and testis at the periphery (8). These findings suggested a limited array of functions for NMS selectively at those sites. Whereas the peripheral biological actions of NMS remain unexplored, NMS at the SCN has been implicated in the regulation of dark-light rhythms (1), and central administration of NMS has been shown to induce clear-cut anorexigenic responses (8). These biological actions had been previously demonstrated for NMU (9, 10, 11); yet the biopotency for such central effects was significantly higher for NMS (1, 8).

A wealth of data has now demonstrated that a large number of peripheral regulators of food intake (such as leptin, ghrelin, and peptide YY3–36) are also implicated in the control of the gonadotropic axis, thereby contributing to the joint regulation of energy balance and reproduction (12, 13, 14, 15). Moreover, the function of reproductive axis is also highly sensitive to photoperiodic cues and endogenous rhythms (16, 17). However, the ultimate central effectors relaying metabolic status and rhythm information onto the hypothalamic centers governing the reproductive axis remain to be fully unfolded. In this context, the biological profiles of NMS (as neuropeptide regulator of circadian rhythms and food intake) make it a suitable candidate for putative modulator of the reproductive system. Indeed, NMU has been proven to induce changes in pulsatile LH secretion in the rat via regulation of central CRH expression (18, 19). Yet characterization of the gonadotropic actions of NMU has been solely conducted in the ovariectomized rat. In fact, a counterintuitive effect of NMU as inhibitor of LH secretion (despite it being a satiety factor) has been reported in this model (18, 19). To our knowledge, not a single study on the effects of NMS on the reproductive system has been reported to date.

On the above basis, we found it relevant to explore the potential reproductive facet of NMS. To this end, we evaluated the pattern of hypothalamic expression of the genes encoding NMS and its putative central receptor, NMU2R, at different developmental and functional states (i.e. stages of postnatal maturation, phases of estrous cycle, and ovariectomy, with or without sex steroid replacement) of the female rat. In addition, hormonal tests were conducted to assess the effects of central administration of NMS upon LH secretion in a number of physiological and experimental settings (i.e. pubertal and adult cyclic females, ovariectomized rats, and short-term fasting). Altogether our data are the first to demonstrate the potential implication of NMS in the functional regulation of the reproductive axis in the female rat.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and drugs
Wistar female rats bred in the vivarium of the University of Córdoba were used. The day the litters were born was considered as d 1 of age. Unless otherwise stated, the animals were maintained under regular light-dark cycles (14 h light, from 0700 h) and temperature (22 C) and were weaned at d 21 of age in groups of four rats per cage with free access to pelleted food and tap water. Experimental procedures were approved by the Córdoba University Ethical Committee for animal experimentation and were conducted in accordance with the European Union normative for care and use of experimental animals. In all experiments, the animals were killed by decapitation at the end of the procedures, and in those studies involving mRNA analysis by semiquantitative (semi-Q) RT-PCR, hypothalami were dissected out from the experimental animals as described in detail previously (20), snap frozen in liquid nitrogen, and stored at –80 C until used for RNA analysis. In addition, in selected experimental settings (experiments 2 and 5), brains were removed intact for in situ hybridization (ISH; see below). For experiments involving hormonal tests, rat NMS was obtained from Phoenix Pharmaceuticals Ltd. (Belmont, CA). 17ß-Estradiol and progesterone were purchased from Sigma Chemical Co. (St. Louis, MO).

Experimental designs
In the first set of experiments, expression analyses of NMS and NMU2R genes at the hypothalamus were conducted in different physiological states and experimental models of the female rat. Unless otherwise stated, tissue sampling was conducted in all experiments between 0900 and 1000 h, to exclude the potential interference of diurnal fluctuations in the expression of NMS and NMU2R genes. In experiment 1, hypothalamic levels of NMS and NMU2R mRNAs were assayed by means of semi-Q RT-PCR at different stages of postnatal maturation. Samples were obtained from female rats at 1, 5, 10, 15, 20, 30, and 60 d postpartum (n = 5–10 per group), corresponding to the neonatal (1 and 5 d), infantile (10 d and 15 d), prepuberal (20 d), pubertal (30 d), and early adult stages of postnatal development (21). Based on endocrine profiles and timing of vaginal opening (around d 34 in our stock; see Ref. 22), 30-d-old female rats were taken as representative of the period of pubertal activation, in keeping with our previous references (20, 22). Concerning adulthood, 60-d-old female rats were monitored for estrous cyclicity by daily vaginal cytology, and only rats with at least two consecutive regular 4-d estrous cycles were used in expression analyses. Hypothalamic tissue from cyclic females at diestrus-1 was used in this experiment.

In experiment 2, potential changes in NMS and NMU2R mRNA at the hypothalamus were analyzed by semi-Q RT-PCR along the estrous cycle. Assessment of estrous cyclicity was conducted as described for experiment 1. Groups of cyclic females (n = 6), at estrus, diestrus-1 (d 1), diestrus-2 (d 2), and proestrus, were used. For each phase, tissue sampling was conducted between 0900 and 1000 h. An additional sampling group (n = 6) was set at 1800 h of proestrus, i.e. coinciding with the preovulatory surge of gonadotropins (23). Because initial RT-PCR results evidenced significant changes in mRNA levels of NMS (and its receptor) along the cycle, in experiment 3, additional groups of cyclic females (n = 6) were killed at proestrus (peak levels) or diestrus (nadir levels), and brains were removed for ISH (see below).

In addition, in experiment 4, expression analyses of NMS and NMU2R mRNAs were conducted by semi-Q RT-PCR in ovariectomized (OVX) rats as a means to evaluate their potential modulation by ovarian signals. Cyclic female rats were bilaterally OVX under ether anesthesia at random stages of the estrous cycle, and hypothalamic samples (n = 6 per group) were obtained 1 wk after surgery. Sham-operated females at proestrus served as controls. As initial expression analyses demonstrated significant changes in mRNA levels of NMS (and its receptor) after OVX, the endocrine basis for such a phenomenon was further explored in experiment 5. First, groups of bilaterally OVX females (n = 10–12) were implanted at the time of surgery with SILASTIC brand silicon tubing (Dow Corning, Midland, MI) elastomers (20 mm length; inner diameter, 0.062 cm; exterior diameter, 0.125 cm) containing estradiol. Selection of dosage and capsule length was based on previous physiological studies in the OVX female rat (24). Additional groups (n = 10–12) were implanted with empty capsules. At the end of the experimental procedure (1 wk), one set of brains (n = 5–6/group) were removed intact for ISH, whereas in the remaining samples the hypothalami were excised for semi-Q RT-PCR assays. Because results from this initial experiment indicated that estradiol replacement did not prevent (but rather enhanced) the decrease in NMS mRNA levels after OXV, an extended experimental protocol was implemented, in which groups of bilaterally OVX females (n = 6) were implanted with SILASTIC brand capsules containing estradiol, progesterone or estradiol plus progesterone, as recently described elsewhere (24). One week after surgery, the animals were killed and hypothalamic samples dissected for semi-Q RT-PCR analysis as described above.

Besides expression analyses, in the second set of experiments, hormonal tests were conducted to evaluate the effects of centrally administered NMS upon LH secretion in the female rat. To this end, different physiological and experimental settings were tested. As general procedure, a protocol of central intracerebroventricular (i.c.v.) administration of 1 nmol NMS (in 10 µl of physiological saline) followed by blood sampling via jugular venipuncture under light ether anesthesia, at 15 and 60 min after injection, was implemented. Selection of the dose and time points for blood sampling was based on previous references, testing the neuroendocrine effects of NMS and other centrally delivered neuropeptides (8, 20, 25, 26, 27, 28). In all experiments, hormonal tests were conducted between 0900 and 1000 h to avoid the potential interference of diurnal changes in the endogenous NMS system.

In experiment 6, the effects of intracerebral injection of NMS were tested in peripubertal and adult cyclic female rats. For the latter, groups (n = 10–12) of regularly cycling adult female rats, selected as described for experiment 2, were used. For pubertal animals, 30-d-old females (n = 10–12) were implanted with i.c.v. cannulae lowered to a depth of 3 mm beneath the surface of the skull; the insert point was 1 mm posterior and 1.2 mm lateral to bregma, as described in detail elsewhere (20). For adult animals, cyclic female rats at estrus and d 1 (when low basal LH levels are expected) were used. The procedure for implantation of i.c.v. cannulae was similar to that described for pubertal animals, except that cannulae were lowered to a depth of 4 mm beneath the skull (20). The animals were injected under conscious conditions after careful handling to avoid any stressful influence.

In experiment 7, the effects of NMS on prestimulated LH levels were explored. To this end, young adult female rats (n = 10–12) were submitted to bilateral OVX, as described for experiment 4. Hormonal tests (central NMS administration and serial blood sampling at 15 and 60 min) were conducted 1 wk after OVX.

Finally, in experiment 8, the ability of NMS to modulate LH secretion was explored after metabolic stress by short-term fasting. Adult, regularly cycling female rats were subjected to food deprivation (with free access to tap water) for 48 h, and hormonal tests were applied after this period (between 0900 and 1000 h of corresponding d 1), as described above. Adult cycling rats at d 1, fed ad libitum, were tested in parallel for reference purposes. In experiments 6–8, animals injected with vehicle (physiological saline, 0.9% NaCl) served as controls. In addition, in experiments involving intracerebral administration, the correct positioning of i.c.v. cannulae was routinely inspected before injection (to exclude animals with obvious mislocation or deattachment) and checked postmortem by visualization of their inner insert point (after decapitation), as described elsewhere (22).

RNA analysis by semi-Q RT-PCR
Total RNA was isolated from hypothalamic samples using the single-step, acid guanidinium thiocyanate-phenol-chloroform extraction method. Hypothalamic expression of NMS and NMU2R mRNAs was assessed by RT-PCR, optimized for semi-Q detection, using the primer pairs and conditions indicated in Table 1Go. These sets of primers were generated on the basis of the published sequences of rat NMS and NMU2R genes (GenBank accession no. NM 001012233 and NM 022275) and designed to span over intron sequences. As internal control for reverse transcription (RT) and reaction efficiency, amplification of a 290-bp fragment of L19 ribosomal protein mRNA was carried out in parallel in each sample, using the primer pair and conditions indicated in Table 1Go.


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  		TABLE 1. Oligoprimer pairs used for RT-PCR amplification of NMS, NMU2R, and RP-L19 transcripts in hypothalamic samples

 
For amplification of the targets, RT and PCR were run in two separate steps. Thus, equal amounts of total RNA (2 µg) were used as a template for cDNA synthesis by RT with random hexamer priming (20). For semi-Q PCR, 3 µl of RT products were amplified in separate reactions using specific primers for rat NMS, NMU2R, and L19. PCR consisted of a first denaturing cycle at 97 C for 5 min, followed by a variable number of cycles of amplification, defined by denaturation at 96 C for 45 sec, annealing for 1 min, and extension at 72 C for 1 min. A final extension cycle of 72 C for 10 min was included. Annealing temperature was adjusted for each target and primer pair: 60 C for NMS and NMU2R products and 55 C for RP-L19 transcripts. Different numbers of cycles were tested to optimize amplification in the exponential phase of PCR. On this basis, 30 and 25 cycles were chosen for semi-Q analysis of specific targets (NMS and NMU2R) and RP-L19, respectively. Identity of PCR products was confirmed by direct sequencing (Central Sequencing Service, University of Cordoba, Cordoba, Spain). Quantification of intensity of RT-PCR signals was carried out by densitometric scanning using an image analysis system (1-D Manager; TDI Ltd., Madrid, Spain), and values of the specific targets were normalized to those of internal controls to express arbitrary units of relative expression. In all assays, liquid controls and reactions without RT resulted in negative amplification.

ISH
Coronal hypothalamic sections (16 µm) were cut on a cryostat, and immediately stored at –80 C until hybridization. A specific oligonucleotide probe for rat NMS (5'-CAA ACC ATC AGG AGG ACC AGC TAA AGG TGG AGA AGC TC-3'; 5' position: 130 of NMS cDNA) was used. This probe was 3'-end labeled with 35S-{alpha}dATP using terminal deoxynucleotidyl transferase (Amersham Biosciences, Little Chalfont, UK). General procedures for ISH were as previously described (29, 30). Similar anatomical regions were analyzed using the rat brain atlas of Paxinos and Watson (31). The slides from all experimental groups were exposed to the same autoradiographic film. All sections were scanned and the specific hybridization signal was quantified by densitometry using a digital imaging system (ImageJ 1.33; National Institutes of Health, Bethesda, MD) (29, 30). The OD of the hybridization signal was determined and subsequently corrected by the OD of its adjacent background value. A rectangle, with the same dimensions in each case, was drawn enclosing the hybridization signal over each nucleus and over adjacent brain areas of each section (background). For in situ analysis, groups of n = 5–7 animals were used. From each animal, 16–20 sections were used (four to five slides with four sections per slide). The mean of these 16–20 values was used as the densitometry value for each animal.

Hormone measurements
Serum LH levels were measured in a volume of 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 was labeled with 125I by the chloramine-T method, and the hormone concentrations were expressed using reference preparations LH-RP-3 as standard. Intra- and interassay coefficients of variation were less than 8 and 10%, respectively. The sensitivity of the assay was 5 pg/tube. 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
Hormonal determinations were conducted in duplicate, with a minimal total number of 10 samples per group. When appropriate, besides individual time-point determinations, integrated LH secretory responses were calculated as the area under the curve (AUC), calculated following the trapezoidal rule, over the 60-min period after administration of NMS. Semi-Q RT-PCR analyses were carried out in duplicate from at least five independent RNA samples of each experimental group. RNA and hormonal data are presented as 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). P ≤ 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hypothalamic expression of NMS and NMU2R genes in the female rat: developmental and hormonal regulation
Expression analyses of the genes encoding NMS and its putative central receptor, NMU2R, were first conducted by semi-Q RT-PCR in hypothalamic samples from female rats at different stages of postnatal development, from the neonatal period to adulthood. Expression of NMS mRNA was low to negligible during the neonatal period (<10 d postpartum) and consistently increased thereafter, with high levels during the late-infantile and juvenile stages of postnatal development (d 15 and 20). Around puberty (d 30), a clear-cut decrease in relative mRNA levels of NMS was detected, which was followed by sharp increase in the adult period when peak expression of NMS was observed. The developmental profile of expression of NMU2R mRNA at female rat hypothalamus was grossly similar to that of NMS: moderate levels were detected at birth that increased during prepubertal stage, declined at puberty and reach maximal expression levels in adulthood (Fig. 1AGo).


Figure 1
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  		FIG. 1. Profiles of expression of NMS and NMU2R genes in the hypothalamus of female rats at different stages of postnatal development and across the estrous cycle. In the upper panels (A), representative RT-PCR assays are presented of expression levels of NMS and NMU2R mRNAs in hypothalamic samples from 1-, 5-, 10-, 15-, 20-, 30-, and 60-d-old female rats. On the latter, hypothalamic samples at diestrous-1 (d 1) phase on the ovarian cycle were used. In the lower panels (B), RT-PCR assays are shown of NMS and NMU2R mRNA levels in hypothalamic samples from cyclic female rats at the morning (1000 h) and afternoon (1800 h) of proestrus (P), and the morning (1000 h) of estrus (E), diestrus-1 (d 1) and diestrus-2 (d 2). Parallel amplification of L-19 ribosomal protein mRNA served as internal control. Semiquantitative values of NMS and NMUR2 mRNA levels are the mean ± SEM of at least four to six independent determinations. Groups with different superscript letters are statistically different (P < 0.05; ANOVA followed by Student-Newman-Keuls multiple range test).

 
Expression analyses of NMS and NMU2R transcripts at the hypothalamus were also conducted by semi-Q RT-PCR at different stages of the estrous cycle. Hypothalamic expression of NMS and NMU2R mRNAs was persistently detected at all stages of the cycle, yet their relative levels significantly fluctuated across its phases. For NMS, peak expression was observed at proestrus (1000 and 1800 h), whereas its mRNA levels significantly decreased at estrus and remained at low to moderate values during diestrus (d 1 and 2). Likewise, NMU2R mRNA was low at estrus and diestrus-1, increased at diestrus-2, and peaked at proestrus (P), with a trend to modestly decline between P1000 and P1800, which was not statistically significant (Fig. 1BGo).

Based on its persistent expression and the fluctuation of its levels along the estrous cycle, the distribution and relative changes in hypothalamic NMS mRNA were further explored in cyclic female rats by means of ISH. Brain samples from females at proestrus (1000 h) and diestrus-1 were selectively analyzed. ISH revealed that hypothalamic NMS mRNA is selectively expressed in the SCN of the cyclic female rat, in keeping with previous reports in the male (1). Moreover, these analyses fully confirmed the validity of results from our semi-Q RT-PCR assays, as a significant decrease in NMS mRNA levels at SCN, similar in magnitude to that detected in whole hypothalamic samples, was observed between proestrus and diestrus (Fig. 2Go).


Figure 2
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  		FIG. 2. Representative autoradiographic images of brain coronal sections from cyclic female rats at the morning (1000 h) of proestrus and diestrus-1, incubated with a 35S-labeled antisense oligonucleotide probes against NMS, as described in Materials and Methods. A rectangle encompassing the SNC is presented at higher (x2) magnification. In the lower panel, relative NMS mRNA levels at proestrus and diestrus, as determined by ISH in the experimental samples, are presented. Values are the mean ± SEM of five to seven individuals, with 16–20 determinations each. **, P < 0.01 vs. values at proestrus (Student’s t test).

 
Further proof for the ovarian regulation of hypothalamic NMS and NMU2R mRNAs was provided by expression analysis in OVX animals, in which semi-Q RT-PCR assays showed a persistent decrease in the relative levels of both targets 1 wk after bilateral gonadectomy vs. the expression values in cyclic females at proestrus (1000 h), used as reference controls (Fig. 3Go). The hormonal basis for the ovarian regulation of NMS and NMU2R mRNAs was explored by the use of models of sex steroid replacement in OVX animals. Initial analyses (by combination of semi-Q RT-PCR and ISH) revealed that tonic estradiol replacement for 1 wk did not rescue, but rather further decreased, NMS mRNA levels at the hypothalamus (Fig. 4Go). To further characterize this paradoxical observation, a larger series of hypothalamic samples, from OVX rats treated for 1 wk with estradiol, progesterone or both, were assayed for NMS and NMU2R mRNA levels using semi-Q RT-PCR. Concerning NMS, its mRNA levels were decreased by OVX (vs. reference values at proestrus) and were further inhibited, albeit modestly, by estradiol supplementation, in keeping with our ISH results (see Fig. 4Go). Conversely, progesterone administration to OVX rats significantly enhanced NMS mRNA levels, which reached values similar to those at proestrus. Moreover, progesterone was able to fully prevent the inhibitory effect of estradiol on NMS expression. Similar patterns were detected for NMU2R mRNA in the different experimental groups, except for the fact that estradiol alone did not further inhibit its expression levels in OVX animals (Fig. 5Go).


Figure 3
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  		FIG. 3. Expression of NMS and NMU2R genes in the hypothalamus of female rats 1 wk after bilateral OVX. In the left panels, representative RT-PCR assays are presented of expression levels of NMS and NMU2R mRNAs in hypothalamic samples from cyclic female rats at proestrus (P, 1000 h) and 1-wk OVX rats. Parallel amplification of L-19 ribosomal protein mRNA served as internal control. In the right panels, semiquantitative values of NMS and NMUR2 mRNA levels are the mean ± SEM of at least four to six independent determinations. Groups with different superscript letters are statistically different (P < 0.05; Student’s t test).

 

Figure 4
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  		FIG. 4. Representative autoradiographic images of brain coronal sections from OVX rats implanted or not with SILASTIC elastomers containing estradiol (E2), incubated with a 35S-labeled antisense oligonucleotide probe against NMS, as described in Materials and Methods. A rectangle encompassing the SNC is presented at higher (x2) magnification. In the lower panel, relative NMS mRNA levels in OVX and OVX+E2 groups, as determined by ISH in the experimental samples, are presented. For reference purposes, expression levels of NMS mRNA at proestrus are also shown. Values are the mean ± SEM of five to seven individuals, with 16–20 determinations each. *, P < 0.05 vs. values at proestrus; a, P < 0.05 vs. values in OVX animals (ANOVA followed by Student-Newman-Keuls multiple range test).

 

Figure 5
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  		FIG. 5. Expression of NMS and NMU2R genes in the hypothalamus of female rats 1 wk after bilateral OVX, subjected or not to hormone replacement with estradiol (E), progesterone (P), or E+P. For reference purposes, samples from intact, cyclic female rats at proestrus (P, 1000 h) and diestrus-1 (D1, 10:00 h) are also included. In the left panels, representative RT-PCR assays are presented of expression levels of NMS and NMU2R mRNAs in hypothalamic samples from the experimental groups. Parallel amplification of L-19 ribosomal protein mRNA served as internal control. In the right panels, semiquantitative values of NMS and NMUR2 mRNA levels are the mean ± SEM of at least six independent determinations. Groups with different superscript letters are statistically different (P < 0.05; ANOVA followed by Student-Newman-Keuls multiple range test).

 
Effects of NMS on LH secretion at different functional states of the female rat
In addition to expression analyses, functional tests were conducted to define the potential role of NMS in the regulation of LH secretion in different physiological and experimental settings. Intracerebral administration of 1 nmol NMS to pubertal (30 d old) female rats evoked a transient, significant increase in serum LH levels, which was detected at 15 min, but not at 60 min, after injection. Accordingly, the integrated LH secretion during the 1-h period after NMS administration was increased approximately 70% vs. corresponding controls injected with vehicle (Fig. 6AGo). Likewise, central injection of NMS to cyclic females at d 1 elicited a modest, but significant, increase in circulating LH concentrations that were elevated over corresponding control values at 15 and 60 min after injection, with a net increase of integrated LH secretion of approximately 30% over controls. Of note, intracerebral administration of NMS to cyclic rats at estrus induced exaggerated LH responses, that peaked at 15 min (8-fold increase over basal levels) and remained elevated at 60 min (4-fold increase) after injection. Accordingly, 1 h integrated LH secretion after NMS injection was 7-fold higher than that observed in vehicle-injected controls (Fig. 6BGo).


Figure 6
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  		FIG. 6. Testing of the effects of NMS on LH secretion in peripubertal (upper panel, A) and adult (lower panel, B) female rats. On the latter, two representative stages of the estrous cycle were tested: diestrus-1 (D1) and estrus (E). The experimental animals were injected i.c.v. with a dose of 1 nmol NMS or vehicle (Veh; denoted by arrow), and blood samples for LH determination were obtained before (0 min) and at 15 and 60 min after NMS administration. In addition to time-course profiles, integrated secretory responses to central administration of NMS (calculated as AUC over the 60-min study period) are shown. Hormonal values are the mean ± SEM of at least 10 independent determinations per group. *, P < 0.05; **, P < 0.01 vs. corresponding control values; in adult animals; a, P < 0.05 vs. corresponding values in female rats at estrus (ANOVA followed by Student-Newman-Keuls multiple range test). Note that scales of y-axes in A and B are different.

 
The effects of NMS on circulating LH levels were also explored in conditions of prestimulated gonadotropin secretion by means of 1 wk gonadectomy. Bilateral OVX induced the expected rise in circulating LH levels (~11-fold increase) in young adult female rats. Central injection of NMS to OVX animals evoked a sustained reduction of serum LH levels, which was statistically significant at 15 min after i.c.v. administration. In terms of integrated responses, net LH secretion during the 1-h period after NMS injection was 35% lower than that observed in OVX females i.c.v. injected with vehicle (Fig. 7Go).


Figure 7
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  		FIG. 7. Testing of the effects of NMS on LH secretion in gonadectomized female rats 1 wk after bilateral OVX. The experimental animals were injected i.c.v. with a dose of 1 nmol NMS or vehicle (denoted by arrow), and blood samples for LH determination were obtained before (0 min) and at 15 and 60 min after NMS administration. In addition to time-course profiles, integrated secretory responses to central administration of NMS (calculated as AUC over the 60-min study period) are shown for each group in the right panel. Hormonal values are the mean ± SEM of at least 10 independent determinations per group. **, P < 0.01 vs. corresponding control values (ANOVA followed by Student-Newman-Keuls multiple range test or Student t test for AUC data).

 
Finally, functional tests were also performed in cyclic female rats after metabolic stress by food deprivation for 48 h. Short-term fasting induced the expected decrease in serum LH levels, which were approximately half of those in cyclic d 1 females fed ad libitum, in keeping with previous reports (32). However, despite such a suppression in basal LH levels, intracerebral administration of NMS to fasted rats elicited robust LH responses at 15 min after injection that, due to the significant lowering of prevailing LH levels, represented an approximately 4-fold increase over corresponding preinjection values. In contrast, modest, but significant, LH secretory responses were induced by intracerebral injection of NMS in cyclic d 1 females fed ad libitum. Accordingly, net LH responses to NMS (calculated as the net increase in LH secretory mass over the corresponding basal levels during the 60-min period) were significantly greater (>2-fold) in fasted animals than in the corresponding controls (Fig. 8Go).


Figure 8
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  		FIG. 8. Testing of the effects of NMS on LH secretion in female rats after short-term metabolic stress by 48-h food-restriction (FR). Female controls, fed ad libitum and paired to the same stage of the cycle (diestrus-1), were used as reference controls. The experimental animals were injected i.c.v. with a dose of 1 nmol NMS or vehicle (Veh; denoted by arrow), and blood samples for LH determination were obtained before (0 min) and at 15 and 60 min after NMS administration. In addition to time-course profiles, net secretory responses to NMS (calculated as net increase in AUC over corresponding vehicle-injected levels along the 60-min study period) are shown for each group in the right panel, relative fold increase over corresponding controls being indicated in the insets. Hormonal values are the mean ± SEM of at least 10 independent determinations per group. **, P < 0.01 vs. corresponding control values; a, P < 0.01 vs. corresponding values in control animals fed ad libitum (ANOVA followed by Student-Newman-Keuls multiple range test or Student’s t test for AUC data).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Compelling evidence indicates that the control of reproduction is tightly linked to the status of body fuels and highly sensitive to photoperiodic cues. Yet the neuroendocrine circuitry and signals underlying such a concerted regulation are far from being totally elucidated. Recently NMS has been identified as a novel neuropeptide, preferentially expressed at the SCN within the brain, which is involved in the control of circadian rhythms and also exerts a potent anorexigenic activity (1, 8). On the basis of this biological profile, we tested the hypothesis of whether NMS may participate also in the central regulation of the gonadotropic axis. Our data are the first to demonstrate that the expression of NMS and NMU2R genes at the hypothalamus depends on the functional status of the gonadal axis, with prominent mRNA levels in cyclic rats at proestrus, and significantly decreased expression after ovariectomy. Moreover, central administration of NMS acutely elicited LH secretion in female rats at different physiological and experimental conditions, with exaggerated LH responses in females at estrus and under metabolic stress by food deprivation. Altogether these evidences unravel a previously unsuspected role of NMS as putative modulator of the female gonadotropic axis.

The ability of NMS to influence gonadotropin secretion was not totally unpredicted, as actions of NMU, which operates through the same NMU2R centrally, on LH release had been previously demonstrated in ovariectomized female rats (18, 19). Strikingly, however, the reported effects NMU on LH secretion were solely inhibitory, a finding that was somewhat counterintuitive and opposed to the expected increase of the activity of the gonadotropic axis, which is usually induced by factors signaling satiety, such as leptin (12, 13). On the contrary, our current data demonstrate that the predominant effect of NMS on LH secretion in the normal female rat is actually stimulatory. The validity of such observation is supported by its repeated detection at different developmental and functional states of the female gonadotropic axis, at a dose level (1 nmol i.c.v.), which has been previously demonstrated to induce anorexigenic responses in the rat (8). The neuroendocrine circuitry responsible for such a positive effect of NMS on LH secretion remains to be elucidated. However, considering that NMS is able to modulate neuropeptide expression at the ARC (8), which is a major center for the integrated control of energy balance and reproduction with abundant expression of NMU2R, it is plausible that the central mechanism whereby NMS stimulates LH secretion involves the activation of ARC pathways. Potential candidates for such an intermediary action, such as kisspeptin and galanin-like peptide, which are prominently expressed at the ARC (32, 33), are presently under investigation at our laboratory.

The stimulatory effects of NMS on LH secretion were also detected after suppression of gonadotropin secretion by short-term fasting, with exaggerated responses vs. control females at diestrus. Of note, enhanced LH secretory responses to different stimuli (such as kisspeptin and galanin-like peptide) have been detected in underfed animals (34, 35). In any event, this observation evidences that NMS is able to counteract the inhibitory effect of energy insufficiency on the gonadotropic axis, thus reinforcing the potential role of this neuropeptide in the joint regulation of reproductive function and energy balance. Interestingly, based on the comparison of the effects of leptin on hypothalamic NMU release and food intake in normal and NMU-deficient rodents, it was suggested that leptin might use central NMS as effector for its satiety actions (36). On the basis of our present data, it is tempting to propose that NMS might also contribute to the well-known positive effects of leptin on female gonadotropic axis (12, 13).

Besides stimulatory effects, inhibitory LH responses to NMS were also detected, selectively in gonadectomized females, in line with previous reports on the effects of NMU on LH secretion in OVX rats (18, 19). It is possible that such inhibitory actions of NMS might be mediated via CRH pathways, as proposed previously for NMU (18, 19). Altogether, our findings on the effects of NMS on LH secretion strongly suggest a potential dual modulatory effect of this neuropeptide on the centers governing GnRH/LH release, with inhibitory actions being detected only at situations of elevated LH levels, such as gonadectomy, whose physiological relevance awaits to be elucidated. Alternatively, we cannot exclude the possibility that the inhibitory effects of NMS on LH secretion reported herein might reflect the pharmacological activation of NMU receptors, NMU being the endogenous ligand for such an effect. This possibility, however, seems unlikely because we observed modest, but significant, stimulatory LH responses after central injection of NMU in cyclic female rats (our unpublished data).

The reproductive facet of the NMS system is reinforced by our expression analyses that demonstrated a close correlation between the relative levels of NMS and NMU2R mRNAs at the hypothalamus and the functional status of the gonadal axis in the female rat. Thus, the combination of semi-Q RT-PCR and ISH assays evidenced that expression of NMS gene, which is confined to the SCN at the hypothalamus in the adult female rat, significantly varies along the estrous cycle and after manipulation of the ovarian sex steroid milieu. Likewise, similar fluctuations were detected for NMU2R mRNA using semi-Q RT-PCR. Along the ovarian cycle, the expression levels of NMS and NMU2R were the highest at proestrus (morning and afternoon), and the lowest at estrus and diestrus-1. This pattern is suggestive of the regulation of expression of both genes by ovarian factors; the preovulatory rise of estradiol at early proestrus being a potential modulator. In good agreement, removal of sex steroids by ovariectomy evoked a significant decrease in NMS and NMUR2 mRNA levels at the hypothalamus vs. values at proestrus. Intriguingly, however, tonic replacement with estradiol did not prevent such a decrease but rather further inhibited (albeit modestly) NMS mRNA expression in OVX rats. This observation suggests that estradiol is not responsible for the rise in mRNA levels of NMS and its receptor at proestrus, and their decline after gonadectomy. Alternatively, it remains possible that the effects of tonically elevated estradiol levels upon NMS expression might not be analogous to those evoked by the acute increase in circulating estradiol, as that observed at proestrus. In addition, the mechanism(s) for the observed decrease of NMS and NMU2R mRNAs at diestrus remains to be defined (see below).

In fact, to further characterize the regulation of NMS and NMU2R gene expression at the hypothalamus by ovarian sex steroids, we conducted analyses after hormonal replacement of OVX rats with progesterone, alone or in combination to estradiol. Contrary to estradiol supplementation, progesterone treatment significantly enhanced NMS and NMU2R mRNA levels that reached maximum values of proestrus. This is in apparent contrast to the observed decrease in the expression of both targets at diestrus, and suggests additional regulatory signals at this phase. Moreover, in the presence of progesterone, the inhibitory effect of estradiol on NMS mRNA was not detected. Altogether these observations disclose a major stimulatory role of ovarian progesterone on the expression of NMS and its receptor at the hypothalamus. Considering the potent LH-releasing effects of NMS at estrus, it is plausible that progesterone-induced NMS (and/or NMU2R) expression might contribute to the well-known positive modulatory actions of progesterone on the preovulatory LH surge (37, 38). In fact, activation of progesterone receptors, in the presence of the estrogen surge, is mandatory for maximal secretion of LH at proestrus, in a mechanism that involves, at least partially, the enhanced activation of GnRH neurons (38). Our current findings would allow to propose that NMS might operate as one of the central effectors for such a phenomenon.

In addition to changes along the ovarian cycle, expression of NMS and NMU2R genes steadily increased along postnatal maturation, with maximal levels at the juvenile period and adult stage. This trend, however, was abruptly interrupted at puberty, when a significant drop in the expression levels of NMS and NMU2R genes was detected; a phenomenon whose mechanisms are yet to be determined. Yet, it is possible to speculate that the rise in estrogen levels at the time of puberty, in the absence of progesterone (because first ovulation and corpus luteum formation is delayed several days after vaginal opening), might contribute to the observed decrease in NMS and NMU2R mRNA levels in 30-d-old female rats. Worthy to note, a similar expression profile has been identified by our group for NMS and NMU2R genes in male rats at puberty (our unpublished data), suggesting that reduced hypothalamic expression of both genes is a genuine phenomenon at rat puberty, whose physiological relevance merits further investigation.

Female reproductive function is an extremely timely phenomenon, characterized by successive episodes of ovulation driven by a complex sequence of neuroendocrine events that conform the preovulatory surge of gonadotropins (16, 17). Complete expression of such a surge depends on two major determinants: the rise of estradiol (together with progesterone receptor activation) at the morning-afternoon of proestrus and the delivery of a central, neuronal signal that entrains the surge to endogenous circadian rhythms. Such a neuronal signal is thought to be generated at the SCN, which operates as the master circadian pacemaker (16, 17). However, the nature of such signal(s) has remained partially elusive and different neuropeptides, such as vasopressin and vasoactive intestinal polypeptide, have been involved in this phenomenon (39, 40, 41, 42). Our current data make it tempting to hypothesize that NMS, as signal from the SCN (1), might cooperate in the rhythmic regulation of the centers governing the gonadotropic axis at the preovulatory surge. This contention is supported by our observations that hypothalamic NMS (and NMU2R) gene expression is modulated along the estrous cycle, with peak levels at proestrus and minimal expression at estrus-diestrus, and that LH responsiveness to exogenous NMS is exaggerated at estrus (i.e. after the state of maximal pituitary responsiveness at the afternoon of proestrus) but minimal at diestrus-1. Of note, the state of high responsiveness at estrus took place even in the presence of low expression levels of NMU2R mRNA, suggesting additional regulatory mechanisms. Overall, these observations suggest that the elevated hypothalamic expression of NMS gene at proestrus, and its sharp decrease at the estrus transition, might contribute to the generation of the preovulatory LH surge at the afternoon of proestrus and its cessation thereafter.

The central effects of NMS on gonadotropin secretion reported herein do not exclude additional actions of this neuropeptide at other levels of the gonadotropic axis. In this sense, expression of NMU1R and NMU2R has been very recently described in mouse pituitary, in which the presence of NMU mRNA has been also detected (43). These observations rise the possibility that, in addition to its central actions, NMS might modulate LH secretion acting directly at the pituitary level. This potential mechanism, however, could not explain the stimulatory effects of NMS after its intracerebral administration (see Figs. 6Go and 8Go). Moreover, considering its biological profile, as neuropeptide involved in the central control of key functions such as circadian rhythms and food intake, and our current observations of changes in its hypothalamic expression depending on the gonadal status, it is reasonable to predict that NMS acts predominantly at central levels to regulate gonadotropin secretion. The possibility of additional pituitary effects of NMS, and their relevance in the control of the gonadotropic axis, awaits further investigation.

In conclusion, we provide herein novel evidence for the potential involvement NMS, a newly cloned neuropeptide primarily produced at the SCN, in the regulation of the female gonadotropic axis. Such a contention is based in our observations on the pattern of hypothalamic expression of NMS and NMU2R genes, which appears to be tightly coupled to the developmental and functional state of the reproductive system as well as on the characterization of the ability of centrally delivered NMS to evoke LH responses in a number of physiological and experimental conditions of the female rat, including different states of sexual maturation, stages of the ovarian cycle, and metabolic conditions. It is assumed that some critical aspects of NMS function, such as its relative importance over the related neuropeptide NMU, its potential interaction with essential central regulators of the GnRH/LH axis, such as kisspeptin, and its eventual role and modulation at other levels of the hypothalamic-pituitary-gonadal axis, remain to be elucidated. Nonetheless, our current data are the first to disclose a previously unsuspected reproductive dimension of NMS, which might operate as novel neuroendocrine processor, linking the control of circadian rhythms, energy balance, and reproductive function in the female.


   Footnotes
 
This work was supported by Grants BFI 2002-00176, BFU 2005-01443, and BFU 2005-07446 (Ministerio de Educacion y Ciencia, Spain), funds from Instituto de Salud Carlos III [Red de Centros RCMN C03/08, Project PI042082, and CIBER (Centros de Investigación Biomédica en Red) Physiopathology of Obesity and Nutrition], and European Union Research Contract EDEN QLK4-CT-2002-00603.

Disclosure Statement: The authors (E.V., J.R., M.L., J.M.C., R.F.-F., V.M.N., R.P., E.A., C.D., L.P., and M.T.-S.) have nothing to disclose.

First Published Online November 16, 2006

Abbreviations: ARC, Arcuate nucleus; AUC, area under the curve; i.c.v., intracerebroventricular; ISH, in situ hybridization; NMS, neuromedin S; NMU, neuromedin U; NMUR, neuromedin U receptor; OVX, ovariectomized; RT, reverse transcription; SCN, suprachiasmatic nucleus; semi-Q, semiquantitative.

Received May 11, 2006.

Accepted for publication November 7, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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E. Vigo, J. Roa, R. Pineda, J. M. Castellano, V. M. Navarro, E. Aguilar, L. Pinilla, and M. Tena-Sempere
Novel role of the anorexigenic peptide neuromedin U in the control of LH secretion and its regulation by gonadal hormones and photoperiod
Am J Physiol Endocrinol Metab, November 1, 2007; 293(5): E1265 - E1273.
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