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Am J Physiol Endocrinol Metab 291: E1281-E1289, 2006. First published July 18, 2006; doi:10.1152/ajpendo.00130.2006
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Effects of galanin-like peptide on luteinizing hormone secretion in the rat: sexually dimorphic responses and enhanced sensitivity at male puberty

J. M. Castellano,1 V. M. Navarro,1 R. Fernández-Fernández,1 J. Roa,1 E. Vigo,1 R. Pineda,1 R. A. Steiner,2 E. Aguilar,1 L. Pinilla,1 and M. Tena-Sempere1

1Department of Cell Biology, Physiology, and Immunology, University of Córdoba, Córdoba, Spain; and 2Departments of Physiology and Biophysics and Obstetrics and Gynecology, University of Washington, Seattle, Washington

Submitted 1 January 2006 ; accepted in final form 14 July 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reproductive function is exquisitely sensitive to adequacy of nutrition and fuel reserves, through mechanisms that are yet to be completely elucidated. Galanin-like peptide (GALP) has recently emerged as another neuropeptide link that couples reproduction and metabolism. However, although the effects of GALP on luteinizing hormone (LH) secretion have been studied, no systematic investigation on how these responses might differ along sexual maturation and between sexes has been reported. Moreover, the influence of metabolic status and potential interplay with other relevant neurotransmitters controlling LH secretion remain ill defined. These facets of GALP physiology were addressed herein. Intracerebral injection of GALP to male rats induced a dose-dependent increase in serum LH levels, the magnitude of which was significantly greater in pubertal than in adult males. In contrast, negligible LH responses to GALP were detected in pubertal or adult female rats at diestrus. Neonatal androgen treatment to females failed to "masculinize" the pattern of LH response to GALP. In addition, metabolic stress by short-term fasting did not prevent but rather amplified LH responses to GALP in pubertal males, whereas these responses were abrogated by pharmacological inhibition of nitric oxide synthesis. We conclude that the ability of GALP to evoke LH secretion is sexually differentiated, with maximal responses at male puberty, a phenomenon which was not reverted by manipulation of sex steroid milieu during the critical neonatal period and was sensitive to metabolic stress. This state of LH hyperresponsiveness may prove relevant for the mechanisms relaying metabolic status to the reproductive axis in male puberty.

gonadotropin-releasing hormone; kisspeptin; hypothalamus

GALANIN-LIKE PEPTIDE (GALP) was originally isolated from the porcine hypothalamus on the basis of its ability to activate galanin receptors (GalRs) in cell-based assays (26). The mature form of GALP is a 60-amino acid neuropeptide cleaved from a larger precursor encoded by a gene distinct from that of galanin, whose residues 9–21 show complete sequence homology with the 13-amino acid NH2-terminal region of galanin (26). Among the three forms of GalRs cloned so far (GalR1–R3), GALP binds GalR2 with higher affinity than galanin, whereas it has relatively low-affinity binding to GalR1 (10, 26). However, there is indirect evidence to suggest that GALP might have its own unique receptor(s) (9, 20, 22). Like galanin, GALP is highly expressed in the arcuate nucleus (Arc) of the hypothalamus (10, 12, 19, 33), a center that plays a critical role in the neuroendocrine regulation of reproduction, metabolism, and body weight (10). Indeed, GALP neurons have been proven targets for regulation by leptin and insulin, and hypothalamic expression of GALP is modulated by the nutritional status (8, 10, 13, 18). Furthermore, intracerebral injections of GALP evoked consistent luteinizing hormone (LH) secretory responses in a variety of species, including the rat, mouse, and macaque (4, 10, 14, 16, 22). Thus GALP neurons would seem poised to play a key role in the integration of metabolism and reproduction (10).

Neurons in the basal forebrain that produce gonadotropin-releasing hormone (GnRH) are the final common pathway through which brain regulates gonadotropin secretion (6, 34). However, in as much as gonadotropin secretion itself, the functionality of the GnRH system dramatically varies along postnatal maturation, with full awakening at puberty (6, 27, 30, 34). Moreover, in the adult rodent, the control of GnRH secretion is sexually differentiated, a phenomenon that relies on the organizing effects of the hormonal milieu during the neonatal critical period (21). Thus the presence of testosterone during this period in the male, and its absence in the female, permanently imprints the functional organization of the neuronal circuitry in the forebrain to allow the female to produce preovulatory GnRH and LH surges and to abolish this capacity in the male (7, 21). The neuroendocrine signals ultimately responsible for such developmental and sexually differentiated patterns of GnRH/LH secretion remain to be fully elucidated.

Despite the proposed role of GALP as a central regulator of the gonadotropin axis, and molecular conduit for peripheral metabolic signals controlling this system, analysis of the reproductive effects of GALP has been so far mostly restricted to adult male animals. Indeed, LH responses to GALP had not been studied in pubertal animals, even though puberty onset is highly sensitive to metabolic hormones and energy fuels (28). Moreover, although the reproductive effects of GALP (in terms of sex behavior and LH secretion) in the female mouse have been very recently documented (14), a systematic comparison on whether LH responses to GALP might be sexually dimorphic had not been previously reported. To cover these facets of GALP physiology, we present herein a comprehensive analysis of the effects of central administration of this neuropeptide on LH secretion in the rat. Considering the proven ability of GALP to evoke a significant (albeit modest) 1.5- to 2.5-fold increase in serum LH levels in the adult male rat (18, 22), three major objectives were set for this work. First, we evaluated the LH-releasing effect of GALP in pubertal animals. Second, we investigated whether the effects of GALP on LH secretion are sexually differentiated. Finally, we analyzed the impact of metabolic stress (by short-term fasting) on GALP-mediated LH secretion, as well as the potential interaction of GALP with other relevant neuroendocrine regulators in the control of LH release.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and drugs. Wistar rats were bred in the vivarium of the University of Córdoba and were used for all experiments. The day the litters were born was considered as day 1 of age. The animals were maintained under controlled conditions of light (14 h of light, from 07:00) and temperature (22°C), and pups were weaned at 21 days of age and divided in groups of four rats per cage, with all animals having free access to pelleted food and tap water, unless otherwise stated. Experimental procedures were approved by Córdoba University Ethics Committee for animal experimentation and were conducted in accordance with the European Union normative for care and use of experimental animals. The animals were humanely killed by decapitation at the end of the experiments. GALP-(1–60) and kisspeptin-(110–119)-NH2 (termed hereafter kisspeptin-10) were obtained from Phoenix Pharmaceuticals (Belmont, CA). Testosterone propionate (TP) was purchased from Sigma Chemical (St. Louis, MO). The antagonist of ionotropic N-methyl-D-aspartate (NMDA) receptors, MK-801, and the antagonist of kainate (KA) and 2-amino-3-hydroxy-5-methyl-4-isoxazol propionic acid (AMPA) receptors, NBQX, were purchased from Research Biochemicals International (RBI, Natick, MA). The inhibitor of nitric oxide (NO) synthases, N{omega}-monomethyl-L-arginine (L-NAME), was obtained from Sigma.

Experimental designs. In the first series of experiments (experiments 1–4), the effects (dose-dependency and time-course responses) of GALP on LH secretion were characterized in pubertal rats and compared with those in adult animals. In experiment 1, the ability of GALP to elicit LH secretion was analyzed in the male, with comparison being made between pubertal and adult male rats over a limited range of doses. Based on previous reports on the timing of sex development in the rat, 35 (pubertal)- and 75 (adult)-day-old males were used (27). Groups of animals (n = 10–12/group) were centrally injected (in the lateral cerebral ventricle; icv) with increasing doses of GALP, and trunk blood samples were collected upon decapitation of the animals at 15 min after GALP injection. Central (icv) administration of the GALP was conducted as previously described (23, 24). Briefly, to allow delivery of the peptide in the lateral cerebral ventricle, the cannulas were 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. Three doses of GALP (3, 1, and 0.3 nmol in 10 µl) were initially tested. These doses were selected on the basis of previous reports on the minimum effective doses of GALP needed to evoke a consistent LH response in adult male rats (18, 22). Animals injected with vehicle (physiological saline) served as controls.

Because results from experiment 1 demonstrated an age-dependent difference in the pattern of response to GALP, we conducted a comparative analysis of the time course of LH responses to an effective dose of GALP in pubertal and adult animals in experiment 2. To this end, 1 nmol GALP was injected intracerebroventricularly to pubertal (35-day-old) and adult (75-day-old) male rats (n = 10–12/group), and blood samples (300 µl) were obtained by jugular venipuncture, under light ether anesthesia, before (time 0) and at 15, 30, and 60 min after the central delivery of the peptide. This procedure was selected since it allows repeated blood determinations without previous surgical manipulation of the animals. Importantly, on the basis of our experience testing the LH-releasing effects of different neuropeptides, we consider that this approach is minimally invasive and only marginally stressful, without inducing major changes in basal LH levels in the experimental animals (24, 25).

In addition, in experiment 3, the dose dependency and time course of LH responses to GALP were analyzed in pubertal and adult female rats. On the basis of previous reports on the timing of sexual maturation in the rat (27), 30 (pubertal)- and 75 (adult)-day-old females were used. For the latter, virgin cyclic female rats at diestrus day 1 were used; only rats showing at least two consecutive 4-day estrous cycles in vaginal cytology were selected. For the dose-response analyses, an experimental protocol similar to that of experiment 1 was used: groups of animals (n = 10–12/group) were injected intracerebroventricularly with a range of doses of GALP (3, 1, and 0.3 nmol in 10 µl), and trunk blood samples were taken upon decapitation of the animals at 15 min after injection of the peptide. Animals injected with vehicle served as controls. Likewise, for time course analyses, groups of pubertal and adult female rats at diestrus day 1 (10–12 rats/group) were injected intracerebroventricularly with 1 nmol GALP, and blood samples (300 µl) were obtained by jugular venipuncture before (time 0) and at 15, 30, and 60 min after central injection of the peptide, as described in experiment 2.

Evaluation of results from experiments 1–3 demonstrated significant differences in the pattern of LH responses to GALP in pubertal male and female rats. To further characterize this phenomenon, in experiment 4, extended dose-response analyses were conducted in pubertal animals. Thus groups of pubertal male (35-day-old) and female (30-day-old) rats (n = 10–12/group) were centrally injected (icv) with GALP over a wider range of doses (3 nmol, 1 nmol, 500 pmol, 100 pmol, 10 pmol, and 1 pmol in 10 µl), following an experimental set up similar to that described in experiment 1. Samples of trunk blood were collected upon decapitation of the animals at 15 min after GALP injection. Animals injected with vehicle served as controls.

Results from experiment 4 demonstrated a striking sexual dimorphism in LH responses to GALP at puberty, with LH responses being much greater in males than females. To ascertain whether this phenomenon might be linked to differences in sexual differentiation of the brain, the LH-releasing effects of GALP were explored after experimental manipulation of the sex steroid milieu during the critical period of neonatal maturation (see Ref. 35). Thus, in experiment 5, the effects of central injection of GALP on LH secretion were assessed in pubertal female rats after neonatal exposure to high doses of androgen. Thus 1-day-old female rats were injected (sc) with a single dose of TP (1.25 mg/rat; dissolved in 100 µl of olive oil), a regimen that has been reported to induce complete androgenization of the female rat (29). Oil-injected females served as controls. On day 30 postpartum, the animals (n = 10–12/group) were subjected to an injection (icv) of the vehicle alone or GALP over a range of doses (10 pmol, 100 pmol, 1 nmol, and 3 nmol in 10 µl). Trunk blood samples were collected upon decapitation of the animals at 15 min after GALP injection. Similar tests were also conducted in control pubertal males for reference purposes.

Additional experiments were conducted in pubertal males (which showed maximal LH responses to GALP) to evaluate the influence of the metabolic status on LH responses to GALP. Thus, in experiment 6, the ability of GALP to elicit LH secretion was evaluated in conditions of negative energy balance. Because moderate sensitization to the LH-releasing effects of GALP had been previously reported in adult models of defective leptin signaling (18), we anticipated that a similar phenomenon might take place in underfed male rats at puberty. As protocol for food deprivation, pubertal male rats were subjected to a 60-h fast (with free access to water) before testing; age-paired males fed ad libitum served as controls. To screen for potential sensitization events, groups of animals (n = 10–12 rats/group) were injected intracerebroventricularly with low doses of GALP or vehicle, and blood samples (300 µl) were obtained by jugular venipuncture before (time 0) and at 15, 30, and 60 min after injection, as described in experiment 2. Based on data from experiment 4, the doses of 10 and 100 pmol were selected for analysis of the LH responses to GALP in food-deprived animals.

Finally, mechanistic studies were performed in pubertal males to assess the relative potency of GALP and kisspeptin, another potent LH secretagogue (2, 23, 24), and to evaluate the potential interplay between GALP and other relevant neurotransmitters in the control of LH secretion, such as excitatory amino acids (EAA) and NO. In experiment 7, the effects of GALP and kisspeptin on LH secretion were comparatively evaluated. To this end, central injections of kisspeptin-10 or GALP were applied over a wide range of doses (3 nmol, 1 nmol, 500 pmol, 100 pmol, 10 pmol, and 1 pmol). Groups of animals (n = 10–12 rats/group) were injected intracerebroventricularly with GALP, kisspeptin, or vehicle, and samples of trunk blood were collected at 15 min after injection. In experiment 8, the LH-releasing effect of central (icv) administration of GALP was monitored after blockade of NMDA and KA/AMPA receptors (the major ionotropic EAA receptors). Groups of pubertal male rats (n = 10–12 rats/group) were intraperitoneally treated with either the NMDA receptor antagonist MK-801 (1 mg/kg) or the KA/AMPA receptor antagonist NBQX (0.5 mg/kg); the doses and regimen of administration were in agreement with previous studies (23, 24). After injection (45 min), 0.5 nmol GALP was delivered intracerebroventricularly, and trunk blood samples were taken upon decapitation of the animals 15 min later. Similarly, NO dependency for the effects of GALP on LH secretion was assessed in experiment 9. Groups of pubertal male rats (n = 10–12 rats/group) were injected intraperitoneally with the blocker of NO synthase, L-NAME (40 mg/kg), as previously described (23, 24). After injection (45 min), 0.5 nmol GALP was injected intracerebroventricularly, and trunk blood samples were taken upon decapitation of the animals 15 min later.

Hormone measurements by specific RIA. Serum LH levels were determined in a volume of 50 µl using a double-antibody method and RIA kits kindly 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 the reference preparation LH-RP-3 as standard. Intra- and interassay coefficients of variation were <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. Serum LH determinations were conducted in duplicate, with a minimal total number of 10 samples/group. Data are shown as means ± SE of absolute hormonal values. In addition, in experiments 2 and 6, integrated LH secretory responses to GALP, defined as the net increase in serum LH concentrations over the prevailing basal levels during a 60-min period after GALP administration, were calculated as area under the curve (expressed as ng·ml–1·60 min–1), using the trapezoidal rule. Results were analyzed for statistically significant differences by means of Student’s t-test or ANOVA followed by the Student-Newman-Keul’s multiple-range test (SigmaStat 2.0; Jandel, San Rafael, CA). Alternatively, two-way repeated-measures ANOVA was applied in studies involving subsequent LH determinations after GALP injection in the presence of additional covariates, such as age (experiment 2) or feeding state (experiment 6). P ≤ 0.05 was considered significant. Finally, in experiments 4 and 7, the mean effective dose (ED50), defined as the dose able to induce 50% of the maximal response, was determined by nonlinear regression (SigmaStat 2.0).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of GALP on LH secretion in pubertal rats: age- and sex-dependent changes. Central injection (icv) of GALP, at the doses of 3, 1, and 0.3 nmol, was initially conducted in pubertal (35-day-old) male rats, and the LH responses were compared with those adult (75-day-old) males. In pubertal male rats, all doses of GALP evoked significant, dose-dependent, LH secretory responses at 15 min after injection. In contrast, in adult males, only the highest doses of GALP (3 and 1 nmol) significantly increased LH concentrations (Fig. 1). In terms of relative responses, LH secretory bursts at 15 min after GALP injection were much greater in pubertal males than in adult animals. Thus GALP evoked ~4.0-fold (0.3 nmol) and ~8.0-fold (1 and 3 nmol) increases in LH levels over basal values in pubertal males. In contrast, moderate responses (~2.0- to 2.5-fold increases) were observed in adult male rats after intracerebroventricular injection of 1 and 3 nmol GALP (Fig. 1).


Figure 1
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  		Fig. 1. Characterization of the luteinizing hormone (LH)-releasing effects of centrally administered galanin-like peptide (GALP) in pubertal (35-day-old) and adult (75-day-old) male rats. Absolute LH levels at 15 min after icv injection of increasing doses of GALP (0.3, 1, and 3 nmol) are presented. Vehicle-injected (0) males served as controls. In addition to absolute levels, relative responses to GALP (expressed as degree of increase over corresponding mean control values) are shown in the insets. Hormonal values are means ± SE of at least 10 independent determinations/group. **P < 0.01 vs. vehicle-injected controls. ¶P < 0.01 vs. animals injected with 0.3 nmol GALP.

 
In addition, the time course of LH responses to GALP was comparatively evaluated in pubertal and adult males. At both age points, 1 nmol GALP evoked significant LH bursts that peaked at 15 min and gradually declined thereafter, with LH levels being similar to preinjection values after 60 min at both age points. However, age-dependent differences were noticed in the magnitude of these responses. Thus, despite lower basal concentrations in 35-day-old males, absolute LH levels at 15 min after GALP injection were significantly higher in pubertal animals. Likewise, net LH responses to GALP (calculated as the net increase in LH secretory mass over the corresponding basal levels during the 60-min period) were significantly greater in pubertal male rats (Fig. 2).


Figure 2
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  		Fig. 2. Time course for the LH-releasing effect of an effective dose of GALP in pubertal and adult male rats. Experimental animals were injected icv with 1 nmol GALP, and blood samples for LH determination were obtained before (0 min) and at 15, 30, and 60 min after GALP administration. In addition to absolute LH levels (left), net integrated LH responses to GALP [calculated as the areas under the curve (AUC) over the corresponding basal LH values] are shown for pubertal and adult males on right. Hormonal values are the means ± SE of at least 10 independent determinations/group. P < 0.01 vs. control values at 0 min (**) and vs. corresponding values in the adult group (¶). For AUC data, groups with different superscript letters are significantly different.

 
Similar analyses were conducted in pubertal females and adult cyclic rats at diestrus day 1. Central injection of GALP, at the doses of 3, 1, and 0.3 nmol, did not elicit LH secretion in adult cyclic females at 15 min after administration, neither did it acutely stimulate LH release in pubertal female animals, except for a modest but significant 1.75-fold increase after injection of 3 nmol GALP in pubertal females (Fig. 3). Time course analysis (over a 60-min period) of the LH responses to central injection of 1 nmol GALP did not demonstrate any significant change in serum LH levels in pubertal or diestrous female rats at any time point (15, 30, and 60 min) studied (data not shown).


Figure 3
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  		Fig. 3. Characterization of the LH-releasing activity of centrally administered GALP in pubertal (30-day-old) and adult (75-day-old) female rats at diestrus day 1 (D1). Absolute LH levels at 15 min after icv injection of increasing doses of GALP (0.3, 1, and 3 nmol) are presented. Vehicle-injected (0) females served as controls. In addition to absolute levels, relative responses to GALP (expressed as degree of increase over corresponding mean control values) are shown in insets. Hormonal values are the means ± SE of at least 10 determinations/group. *P < 0.05 vs. vehicle-injected controls.

 
Extended dose-response curves for the LH-releasing effects of GALP, at 15 min after injection of the peptide, were generated in pubertal male and female rats over a wider range of doses: 3 and 1 nmol and 500, 100, 10, and 1 pmol. In keeping with our initial observations, pubertal males were shown to be much more sensitive to GALP than females. Thus, in male rats, a significant dose-dependent elevation of serum LH levels was demonstrated from 10 pmol GALP onward, with maximal (~8.0-fold increase over controls) LH responses to 1 and 3 nmol GALP and an ED50 of ~300 pmol GALP. In contrast, in pubertal females, none of the doses of GALP tested induced significant changes in LH secretion, except for 3 nmol GALP, which evoked a modest (<2.0-fold) increase in serum LH levels (Fig. 4).


Figure 4
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  		Fig. 4. Extended dose-response analysis of the effects of central administration of GALP upon serum LH levels in pubertal male and female rats. A range of doses of GALP (3 nmol, 1 nmol, 500 pmol, 100 pmol, 10 pmol, and 1 pmol) were tested, and serum LH concentrations were determined 15 min after administration. Vehicle-injected (0) animals served as controls. Values are means ± SE of at least 10 determinations/group. Calculation of the mean effective dose (ED50), defined as the dose of GALP peptide able to induce 50% of the maximal response, was determined by nonlinear regression in male rats. *P < 0.05 and **P < 0.01 vs. corresponding vehicle-injected controls.

 
Effects of GALP on LH secretion at puberty after neonatal manipulation of sex steroid milieu. To explore the mechanisms underlying the above sexual dimorphism, LH responses to GALP (at doses of 3 nmol, 1 nmol, 100 pmol, and 10 pmol) were evaluated in neonatally androgenized female rats at the expected time of normal puberty. Similar analyses were conducted in males and females neonatally injected with vehicle. In line with our initial findings (Fig. 4), the pattern of LH response to increasing doses of GALP was strikingly sexually dimorphic in control animals, with unambiguous LH bursts mostly in males. Neonatal androgenization resulted in a modest but significant decrease in basal LH levels at the expected peripubertal period and a significant lowering of ovarian weight (data not shown), in keeping with previous studies (29). Yet, neonatal exposure to high doses of androgen did not alter the female-like pattern of the LH response to increasing doses of GALP, since significant increases in LH levels were observed only after central injection of maximal doses of 3 nmol GALP (Fig. 5).


Figure 5
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  		Fig. 5. Pattern of LH response to increasing doses of GALP in neonatally androgenized (Neo-TP) female rats at the expected time of puberty. For reference purposes, similar analyses are also presented for control male and female rats (neonatally injected with vehicle). A range of doses of GALP (10 pmol, 100 pmol, 1.0 nmol, and 3.0 nmol) were tested by icv injection, and serum LH concentrations were determined after 15 min. Vehicle-injected animals (0) served as controls. Values are means ± SE of at least 10 independent determinations/group. *P < 0.05 and **P < 0.01 vs. control values at 0 nmol. ¶P < 0.01 vs. corresponding values in control females neonatally injected with vehicle.

 
Effects of GALP on LH secretion after metabolic stress by food deprivation. The effects of low doses of GALP on LH secretion were also explored in pubertal males after metabolic stress by means of 60-h fasting. In control males fed ad libitum, the doses of 10 and 100 pmol GALP evoked a significant, albeit modest, increase in serum LH levels of 1.5- to 2.5-fold over basal concentrations, respectively (Fig. 6). Food deprivation for 60 h induced a significant reduction in body weight (95.0 ± 2.5 g in fasting vs. 118.5 ± 3.0 g in controls) and a decrease in basal (0-min) LH levels, in keeping with previous studies (2). However, despite suppression of basal levels, LH responses to GALP were not only preserved but rather augmented after food restriction, with ~12.0-fold elevation in mean LH concentrations (over basal levels) at 15 min after injection of 10 and 100 pmol GALP. Moreover, net LH responses to GALP, calculated as the net increase in LH secretory mass over the corresponding basal levels during the 60-min period following injection of the peptide, were significantly greater in fasted males than in fed controls (Fig. 6).


Figure 6
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  		Fig. 6. Analysis of the LH-releasing effects of low doses of GALP in pubertal male rats, which were either fed ad libitum or fasted for 60 h. Experimental animals were injected icv with doses of 0.01 or 0.1 nmol of GALP, and blood samples for LH determination were obtained before (0 min) and at 15, 30, and 60 min after GALP administration. In addition to absolute LH responses (left), net integrated LH responses to GALP (calculated as the AUC over the corresponding basal LH values) are shown for fed and fasted animals on right. Hormonal values are means ± SE of at least 10 determinations/group. P < 0.01 vs. control values at 0 min (**) and vs. corresponding values in the group fed ad libitum (¶). For AUC data, groups with different superscript letters are significantly different.

 
Comparative analysis of the effects of GALP and kisspeptin on LH secretion at male puberty. The relative potency and sensitivity of GALP were compared with those of kisspeptin, a very potent elicitor of GnRH/LH function (3, 23, 24). Analysis of the dose dependency of LH responses to GALP demonstrated a minimal effective dose of 10 pmol, a predicted ED50 of ~300 pmol, and maximal responses from 1 nmol GALP onward. In contrast, all doses of kisspeptin-10 tested (from 1 pmol onward) significantly elicited LH release, with a predicted ED50 of ~2 pmol. Yet, maximal responses to kisspeptin-10 (both in terms of absolute hormonal levels and relative degree of increase over control) were similar to those evoked by the highest doses of GALP in pubertal males (Fig. 7).


Figure 7
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  		Fig. 7. Comparative dose-response curves for the effects of central administration of GALP or kisspeptin-10 upon serum LH levels in pubertal male rats. A range of doses of GALP and kisspeptin-10 (3 nmol, 1 nmol, 500 pmol, 100 pmol, 10 pmol, and 1 pmol) were tested by icv injection, and serum LH concentrations were determined after 15 min. Vehicle-injected (0) animals served as controls. Values are means ± SE of at least 10 determinations/group. Calculation of ED50 values for the LH-releasing effects of GALP and kisspeptin were determined by nonlinear regression.

 
Interaction between GALP, EAAs, and NO in the control of LH secretion in pubertal males. Finally, the effects of an effective dose of GALP (0.5 nmol) on LH secretion were assessed after antagonization of ionotropic glutamate receptors, of the NMDA and non-NMDA type, and after inhibition of endogenous NO synthase. Administration of the specific antagonist of NMDA receptors, MK-801, induced a significant decrease in serum LH levels that was not detected after injection of the blocker of KA/AMPA receptors, NBQX. In this setting, neither pretreatment with MK-801 nor administration of NBQX significantly altered the acute LH-releasing effect of GALP (Fig. 8A). In contrast, LH secretory responses to central administration of 0.5 nmol GALP were blunted after pretreatment with the antagonist of NO synthase, L-NAME (Fig. 8B).


Figure 8
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  		Fig. 8. Analysis of potential interactions between GALP, excitatory amino acid (EAA), and nitric oxide (NO) pathways in the central control of LH secretion in pubertal male rats. A: blockade of endogenous EAA pathways did not abolish the potent LH-releasing effect of GALP. Pubertal male rats were treated with the selective antagonists of N-methyl-D-aspartate (NMDA) or kainate (KA)/2-amino-3-hydroxy-5-methyl-4-isoxazol propionic acid (AMPA) receptors (MK-801 and NBQX, respectively) and were subsequently injected icv with 0.5 nmol GALP or vehicle. B: blockade of endogenous NO tone blunted the ability of GALP to stimulate LH secretion. Pubertal male rats were treated with the inhibitor of NO synthase N{omega}-monomethyl-L-arginine (L-NAME) and were subsequently injected icv with 0.5 nmol GALP or vehicle. Hormonal values are means ± SE of at least 10 determinations. P < 0.01 vs. corresponding vehicle-injected controls (**) and vs. corresponding control values in A (¶).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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In recent years, GALP has emerged as a putative neuroendocrine integrator linking metabolism and fertility (10). This contention was based on the region-specific (mostly in the Arc) and metabolically regulated pattern of brain expression of GALP, as well as on its proven ability to modulate food intake and the gonadotropic axis (10). However, although the capacity of GALP to acutely elicit LH secretion in a number of species (from rodents to macaques) is now undisputed, gonadotropic responses to GALP had been evaluated mostly in adult male animals, whereas its potential function in the control of LH secretion at puberty and in the cyclic female has remained largely neglected. Our present experimental work, aimed at targeting these specific aspects of GALP physiology using the rat as an animal model, provides the first evidence that LH responses to GALP are strikingly sexually dimorphic (prominent in males but virtually negligible in females, at least at the functional states tested) and dramatically enhanced at male puberty.

Our hormonal tests unraveled that male rats at puberty are enormously responsive to the LH-releasing effects of central administration of GALP, with maximal LH responses of ~8.0-fold increase over corresponding controls at the high dose range. Such a high responsiveness to GALP at male puberty was further confirmed in a large number of independent experiments (see Figs. 1, 2, and 4–8) testing different doses of the peptide in diverse experimental conditions. In contrast, in adult rats, high doses (1–3 nmol) of GALP elicited only a moderate, albeit significant, 2.0- to 2.5-fold increase in serum LH levels. Such a magnitude in LH responses to GALP in adulthood is strikingly similar to that originally reported in the rat (22). Yet, subtle variations in the time course of LH responses to GALP were noticed between previous and present data, which might be because of differences in GALP doses and the methodological procedures used for blood sampling. In any event, age-dependent changes in LH responsiveness to GALP in the male rat appear to represent a genuine phenomenon. Indeed, detailed dose-response analyses allowed us to evaluate the sensitivity of the LH axis to the effects of GALP in pubertal male rats. Such in vivo analysis demonstrated a minimal effective dose of 10 pmol and a predicted ED50 of ~300 pmol GALP. Although extended dose-response analyses were not conducted in adult males, it is plausible to propose, on the basis of threshold effective doses detected at both age points, that pubertal males are ~100-fold more sensitive to GALP than adult male rats. This magnitude, together with the fact that GALP was injected centrally, makes it highly improbable that the differences in LH responses to GALP might be solely due to a higher dilution of similar doses of GALP in adult animals (whose body weights were only twofold higher than in pubertal males). The mechanisms for such developmental switch remain to be fully addressed, but, considering that GALP has been previously proven to stimulate GnRH release by hypothalamic explants from adult male rats (32), changes in GnRH responsiveness to GALP along postnatal maturation might contribute to such a phenomenon. Alternatively, age-dependent changes in pituitary responsiveness to GnRH might also be involved; yet, GnRH receptor content at the pituitary does not seem to dramatically vary during pubertal maturation in the male rat (5). Both possibilities are presently under evaluation at our laboratory.

Another striking observation of our studies is that, although GALP consistently elicited LH secretion in pubertal and adult males, LH responses to GALP were virtually negligible in the female, except for a moderate increase in LH levels after central injection of 3 nmol GALP in pubertal rats. Of note, however, the LH-releasing effects of GALP were only tested in pubertal and adult diestrous female rats; the latter was taken as a representative stage of low basal secretion of gonadotropins, which is conventionally used for the testing of the effect of different neuropeptides on LH secretion (25). Likewise, we cannot rule out the possibility that doses >3 nmol GALP might elicit significant LH responses; yet, these were not tested to avoid potential unspecific effects of GALP (e.g., a dose of 5 nmol implies the intracerebral injection of 32.5 µg of GALP). In any event, our present findings are suggestive of a previously unsuspected, major sexual dimorphism for the role of GALP in the control of the gonadotropic axis along the life span and point out a more prominent (if not exclusive) function of this neuropeptide in the integrated control of metabolism and reproduction in the male, at least in the rat. Worth noting is that differences in sexual behavior and locomotor activity elicited by GALP have been reported between the rat and mouse (14). Moreover, ovariectomized female mice, primed with estradiol and progesterone, have been very recently shown to respond to central infusion of GALP, for doses as low as 0.5 nmol (14). Although obvious differences exist in the experimental settings used (see Ref. 14 and the present study), the latter might suggest that the ability of GALP to evoke LH secretion in the female is also species specific. Alternatively, the fact that estrogen-primed female mice respond to GALP might reflect a crucial activational role of gonadal steroids in this phenomenon, a possibility that warrants further investigation.

Sexual differentiation of the LH responses to GALP in the rat does not appear to reflect the organizing effects of the neonatal hormonal milieu. We have shown here that treatment of neonatal female rats with androgen, in a regimen known to induce complete androgenization of the female (29), failed to significantly modify the pattern of GALP-induced LH secretion. Thus, as was the case for cyclic rats, androgenized females only marginally responded to the maximal dose of GALP tested. Nonetheless, effective androgenization was evidenced by lower basal LH levels and significantly reduced ovarian weights in these animals. These observations would argue against a major role of androgen during the critical neonatal period of sex differentiation of the hypothalamus in the programming of the differential patterns of LH response to GALP at puberty between males and females. Such a sexual dimorphism might rely on the differential activational effects of circulating sex steroids in male and female rats. Alternatively, it remains possible that the sexually dimorphic patterns of LH response to GALP may become defined prenatally, since some facets of sexual differentiation of the rat brain are known to be sensitive to androgen exposure before birth (11).

Besides analyses on the sexually dimorphic and age-dependent responses to GALP, our study provides novel information on the influence of the metabolic state on GALP-mediated LH secretion. Indeed, the gonadotropic effects of GALP in conditions of metabolic stress (such as fasting) had not been previously evaluated. In spite of the fact that basal LH levels were significantly decreased by fasting, LH responsiveness to GALP was not blunted but rather enhanced in pubertal male rats after food deprivation, since doses as low as 10–100 pmol GALP were able to induce a very robust ~12.0-fold increase in serum LH concentrations over corresponding basal levels. Interestingly, a moderate sensitization to the effects of GALP on LH secretion had been previously reported in adult male Zucker rats. This was attributed to defective leptin signaling in this model, which in turn might bring about decreased GALP expression and increased sensitivity to exogenous GALP (18). It has to be noted, however, that, in the adult Zucker rat, such a sensitization was of much lower magnitude (1.5- to 2.0-fold vs. 12.0-fold increase at puberty) and was evoked only by very high doses of the peptide (1 nmol vs. 10 pmol in this study). Overall, it is tempting to propose that the enhancement in LH responsiveness to exogenous GALP observed in fasted males is caused by a primary decrease in endogenous GALP after food deprivation, thus suggesting that, as it is the case in the adult, hypothalamic expression of GALP at puberty is regulated by the nutritional status and metabolic signals. Yet, it is acknowledged that at least part of the observed sensitization to GALP might derive from changes in pituitary sensitivity to GnRH after food deprivation. Nonetheless, it is remarkable that very low doses of GALP were able to fully override the restrain of the gonadotropic axis observed in pubertal males under fasting conditions.

Considering the prominent LH-releasing effects of GALP in pubertal male rats, we found it relevant to conduct a comparative analysis of the secretagogue actions of GALP and kisspeptin on LH secretion at puberty. In this sense, the KiSS-1 system has recently arisen as an essential gatekeeper for the activation of the GnRH/LH axis at puberty, as well as for its function later in life (3, 31). Moreover, compelling evidence has now firmly demonstrated that kisspeptin is likely the most potent elicitor of GnRH/gonadotropin secretion known so far (2, 23, 24, 31). Dose-response curves for GALP and kisspeptin-10 revealed that, although maximal LH responses to GALP and kisspeptin-10 were similar in pubertal males, the sensitivity to GALP was at least ~100-fold lower than for KiSS-1 at the low dose range. Although potentially illustrative on the relative importance of these two signals in the control of LH secretion in pubertal male rats, this observation per se does not provide solid proof for the relative position of GALP and kisspeptin in the hierarchy of regulatory signals that control the GnRH/LH axis. Furthermore, the potential interactions between GALP and the KiSS-1 system in the central control of the gonadotropic axis are yet to be unraveled. However, experimental analysis of this phenomenon is hampered by the fact that no reliable antagonists of the kisspeptin receptor are currently available. Likewise, pharmacological antagonism of the receptor(s) responsible for the gonadotropic effects of GALP is not yet possible, since those are not apparently conducted through the classical GalR1 or GalR2 (17).

Finally, central interactions of GALP and other relevant neurotransmitters previously implicated in the neuroendocrine modulation of LH release, such as EAAs (glutamate) and NO (1, 15), were explored in vivo. In this sense, a pivotal role of hypothalamic glutamatergic pathways in the pubertal activation of the GnRH/LH system has been demonstrated (1, 27). However, our results from models of pharmacological blockade of ionotropic EAA receptors, of the NMDA and non-NMDA type, demonstrated that the integrity of EAA neurotransmission is not apparently needed for the expression of the potent LH-releasing effects of GALP in pubertal male rats. Of note, the possibility that such a lack of effect might be due to a defective blockade of the endogenous EAA pathways in our experimental settings is ruled out by the observation that growth hormone responses to GALP in the very same animals were totally or severely blunted (our unpublished data). Taken together, these observations suggest that GALP neurons are located in a circuitry independent of (or eventually distal to) glutamate within the central network governing GnRH release. In contrast, inhibition of NO synthase by pretreatment with L-NAME totally abrogated LH responses to GALP. Interestingly, NO has been previously suggested as a key synchronizing factor for the disperse population of GnRH neurons (15), and our current data strongly suggest that the integrity of endogenous NO tone is required for full expression of the LH-releasing effects of GALP.

In summary, we report herein an integral analysis of the effects of GALP on LH secretion in the rat. Our current data unravel some previously neglected aspects of GALP physiology and demonstrate that, at least in the rat, the effects of this neuropeptide on LH secretion are sexually dimorphic and age dependent, with an extraordinarily potent stimulatory action in males, but not apparently in females, at puberty. Such state of LH hyperresponsiveness to GALP may prove relevant for the functional link between the metabolic status and activation of the reproductive axis at male puberty.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Grants BFI 2000-0419-CO3-03, BFI 2002-00176, and BFI 2005-07446 from Ministerio de Educación y Ciencia (Spain), funds from Instituto de Salud Carlos III (Red de Centros RCMN C03/08 and Project PI042082; Ministerio de Sanidad, Spain), and European Union research contract EDEN QLK4-CT-2002-00603.


   ACKNOWLEDGMENTS
 
RIA kits for hormone determinations were kindly supplied by Dr. A. F. Parlow, National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Peptide Program (Torrance, CA).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Tena-Sempere, Physiology Section, Dept. of Cell Biology, Physiology, and Immunology, Faculty of Medicine, Univ. of Córdoba, Avda. Menéndez Pidal, 14004 Córdoba, Spain (e-mail: fi1tesem{at}uco.es)

J. M. Castellano and V. M. Navarro contributed equally to this work.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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