Changes in Hypothalamic KiSS-1 System and Restoration of Pubertal Activation of the Reproductive Axis by Kisspeptin in Undernutrition
http://www.100md.com
《内分泌学杂志》
Department of Cell Biology, Physiology, and Immunology (J.M.C., V.M.N., R.F.-F., J.R., E.V., E.A., L.P., M.T.-S.), University of Córdoba, 14004 Córdoba
Departments of Physiology (R.N., S.T., M.J.V., C.D.), and Medicine (F.F.C.), University of Santiago de Compostela, 15705 Santiago de Compostela, Spain
Abstract
Activation of the gonadotropic axis critically depends on sufficient body energy stores, and conditions of negative energy balance result in lack of puberty onset and reproductive failure. Recently, KiSS-1 gene-derived kisspeptin, signaling through the G protein-coupled receptor 54 (GPR54), has been proven as a pivotal regulator in the control of gonadotropin secretion and puberty. However, the impact of body energy status upon hypothalamic expression and function of this system remains unexplored. In this work, we evaluated the expression of KiSS-1 and GPR54 genes at the hypothalamus as well as the ability of kisspeptin-10 to elicit GnRH and LH secretion in prepubertal rats under short-term fasting. In addition, we monitored the actions of kisspeptin on food intake and the effects of its chronic administration upon puberty onset in undernutrition. Food deprivation induced a concomitant decrease in hypothalamic KiSS-1 and increase in GPR54 mRNA levels in prepubertal rats. In addition, LH responses to kisspeptin in vivo were enhanced, and its GnRH secretagogue action in vitro was sensitized, under fasting conditions. Central kisspeptin administration failed to change food intake patterns in animals fed ad libitum or after a 12-h fast. However, chronic treatment with kisspeptin was able to restore vaginal opening (in 60%) and to elicit gonadotropin and estrogen responses in a model of undernutrition. In summary, our data are the first to show an interaction between energy status and the hypothalamic KiSS-1 system, which may constitute a target for disruption (and eventual therapeutic intervention) of pubertal development in conditions of negative energy balance.
Introduction
PITUITARY GONADOTROPINS LH and FSH are structurally related glycoproteins responsible for the control of gonadal development and function (1, 2). Secretion of both gonadotropins is primarily driven by the pulsatile release of the hypophysiotropic hypothalamic decapeptide GnRH, also termed GnRH-I (1, 2, 3, 4, 5). Full activation of the gonadotropic axis takes place at puberty, which is the culmination of a cascade of developmental events that ultimately leads to enhanced activity of the GnRH pulse generator, increased plasma levels of LH and FSH, and attainment of reproductive capacity (2, 3, 4, 5). Awakening of the reproductive axis at puberty, and its subsequent functioning in adulthood, is critically dependent on sufficient body energy stores (5, 6). Indeed, diverse conditions of persistent negative energy balance, such as undernutrition and extreme physical exercise, are associated with lack of puberty onset and reproductive failure, both in humans and experimental animals (6, 7, 8). Despite recent developments in the field, including characterization of the pivotal role of leptin in this function, the signals and circuitries responsible for the concerted control of energy balance and reproduction are yet to be totally elucidated (9). Likewise, although decreased pulsatile secretion of GnRH has been ultimately invoked (8), the molecular mechanisms underlying disruption of reproductive function in situations of energy insufficiency remain to be fully characterized.
The master position of hypothalamic GnRH in the hierarchy of signals controlling the gonadotropic axis makes it the target of multiple regulators of central and peripheral origin, and a wide array of excitatory and inhibitory circuits governing GnRH secretion have been identified over the last decades (3, 4, 5). In this context, an unsuspected key role for the KiSS-1/G protein-coupled receptor 54 (GPR54) system in the central control of the GnRH-gonadotropin axis has recently arisen on the basis of genetic and pharmacological studies (10). KiSS-1 was originally identified as a metastasis suppressor gene encoding a number of structurally related peptides, generated by the differential proteolytic processing of a common precursor, globally termed kisspeptins (11, 12, 13). The biological actions of kisspeptins are conducted through interaction with the previously orphan G protein-coupled receptor GPR54, also termed AXOR12 or hOT7T175 (11, 12, 13). A number of point mutations and deletions of the GPR54 gene were recently found in patients suffering idiopathic hypogonadotropic hypogonadism (14, 15, 16), a syndrome that was reproduced in mouse models carrying null mutations of the GPR54 gene (15). Thereafter, hypothalamic expression of KiSS-1 and GPR54 genes has been proven as developmentally (maximum at puberty) and hormonally (by sex steroids) regulated (17, 18, 19), and the ability of kisspeptins to potently elicit gonadotropin secretion has been simultaneously reported by different groups, including ours (17, 18, 19, 20, 21, 22, 23, 24, 25, 26). Moreover, direct stimulatory actions of kisspeptin upon hypothalamic GnRH neurons and/or GnRH release have been very recently documented (18, 19, 23), and mechanistic studies have suggested a relevant role of KiSS-1 signaling in puberty onset in rodent and primate species (19, 22). In fact, comparative meta-analyses with published data on the LH-releasing activity of other neuropeptides and neurotransmitters, such as glutamate and galanin-like peptide, evidence that the KiSS-1 system is likely the most potent elicitor of the GnRH-gonadotropin axis known so far (24, 25). Indeed, although evaluation of its potential interplay with other well known modulators of the reproductive system has been initiated only recently (24, 25), the KiSS-1 system has been already proposed as an essential gatekeeper for proper function of the gonadotropic axis (15, 19).
In the above scenario, we hypothesized that the mechanisms whereby conditions of negative energy balance hamper the functioning of reproductive axis may target directly the hypothalamic KiSS-1 system. To test this hypothesis, expression analyses of KiSS-1 and GPR54 genes at the hypothalamus were conducted, and reproductive responses to kisspeptin (in terms of hormone secretion and biomarkers of puberty onset) were monitored at puberty in models of severe undernutrition.
Materials and Methods
Animals and drugs
Wistar 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. The animals were maintained under constant conditions of light (14 h of light, from 0700 h) and temperature (22 C), and were weaned at 21 d of age in groups of five rats per cage with free access to pelleted food and tap water, unless otherwise stated. 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. The animals were humanely killed by decapitation at the end of the experimental settings. Mouse KiSS-1 (110–119)-NH2, the rodent analog of the C-terminal KiSS-1 decapeptide KiSS-1 (112–121)-NH2, was obtained from Phoenix Pharmaceuticals Ltd. (Belmont, CA). This peptide fragment, which has been previously shown to maximally bind and activate GPR54 in transfected CHO cells (12, 13), will be referred hereafter as kisspeptin-10.
Experimental designs
In experiment 1, the effects of conditions of negative energy balance on the expression of the KiSS-1 system at the hypothalamus were monitored in prepubertal animals. This stage of postnatal maturation was selected given the well known dependence of normal pubertal development on sufficient energy stores (3). Groups of male and female rats (30 d old; n = 10–12 per group) were subjected to a period of 72 h of fasting (only access to tap water); age-matched rats fed ad libitum served as controls. At the end of the fasting period, the animals were killed by decapitation, and the hypothalamus was immediately dissected out, as described in detail elsewhere (17), by a horizontal cut of about 2 mm depth with the following limits: 1 mm anteriorly from the optic chiasm (to include the preoptic area), the posterior border of mamillary bodies, and the hypothalamic fissures. Hypothalamic samples were frozen in liquid nitrogen and stored at –80 C until processing for RNA analysis.
In experiment 2, the functionality of the KiSS-1 system, in terms of gonadotropic responses, was tested under conditions of severe energy deficit. To this end, the ability of kisspeptin-10 to centrally elicit LH secretion was assessed in prepubertal male and female rats (30 d old; n = 10–12 per group) previously subjected to food deprivation for 72 h. A protocol of intracerebroventricular (icv) administration of 1 nmol kisspeptin-10 was carried out, as described elsewhere (17, 22, 24, 25). To allow delivery of kisspeptin into the lateral cerebral ventricle, the animals were implanted with icv 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. A dose of 1 nmol kisspeptin in 10 μl per rat was selected on the basis of our recent data on the ability of this dose to potently elicit LH secretion in rats fed ad libitum (17, 24). Trunk blood samples were collected upon decapitation at 15 min after kisspeptin injection. Animals injected with vehicle (NaCl 0.9%) served as controls.
In experiment 3, the central mechanisms for the effects of kisspeptin upon gonadotropin secretion in situations of negative energy balance were explored. To this end, the ability of kisspeptin-10 to elicit GnRH secretion was tested using a static incubation system and hypothalamic fragments from prepubertal female rats (n = 10–12 hypothalamic samples per group) at two different prevailing metabolic states: feeding ad libitum and 72 h of fasting. Upon decapitation of the animals, hypothalamic fragments were excised following the tissue limits indicated in experiment 2 and placed into individual incubation chambers containing 250 μl of phenol red-free DMEM for 30 min of preincubation, using a Dubnoff incubator at 37 C with constant shaking (60 cycles/min), under an atmosphere of 95% O2/5% CO2. After this period, preincubation media were replaced by DMEM alone or medium containing increasing concentrations of kisspeptin-10 (10–12, 10–10, 10–8, and 10–6 M). Incubations were terminated after 30 min, when media were boiled to inactivate endogenous protease activity, and kept at –80 C until assayed for GnRH levels.
In experiment 4, the effects of kisspeptin upon food intake pattern were studied. To this end, groups of male rats (n = 8 per group) were implanted with icv cannulae, and daily intracerebral injection of 1 nmol kisspeptin was conducted at early light phase (0900 h) for 7 consecutive days. As control for the experimental procedure, an additional group of animals (n = 8) were icv injected with 1 nmol ghrelin, a proven orexigenic factor (9). To explore the impact of the prevailing energy status upon the effects of kisspeptin on food intake, the animals were initially allowed to have free access to chow during the first 4 d, and cumulative food intake was monitored at 1.5, 3, 6, 12, and 24 h after each kisspeptin injection. Thereafter, the animals were subjected to 12 h of fasting during the dark phase preceding kisspeptin injection for the last 3 d of treatment, and cumulative food intake was monitored at 1.5, 3, 6, and 12 h after injection.
Finally, in experiment 5, the effects of chronic central administration of kisspeptin-10 on puberty onset in immature female rats under severe undernutrition were monitored. A protocol of 30% restriction in daily food intake vs. age-matched control females (fed ad libitum) was initiated on d 23 postpartum. Daily icv administration of kisspeptin in the lateral cerebral ventricle was conducted between d 30–37 postpartum in food-restricted females (n = 12), as described in detail elsewhere (22). The treatment regimen was set at a dose of 1 nmol KiSS-1 per animal in 10 μl, every 12 h. Pair-aged females (n = 10), at 30% food restriction, injected with vehicle served as controls. The animals were icv injected under conscious conditions after careful handling to avoid any stressful influence, in keeping with our previous references (22). In all experimental animals, body weight and vaginal opening were daily monitored. On the latter, detailed inspection was conducted in each animal to determine the date of complete canalization of the vagina. At the end of treatment (37 d postpartum), the animals were killed by decapitation, 60 min after the last injection of kisspeptin or vehicle, and trunk blood was collected. For determination of the normal date of vaginal opening in animals fed ad libitum, an additional group of females (n = 20), without food restriction and icv injected with vehicle, were maintained on daily inspection of canalization of the vagina up to d 37 postpartum.
RNA analysis by semiquantitative RT-PCR
Total RNA was isolated from hypothalamic samples using the single-step, acid guanidinium thiocyanate-phenol-chloroform extraction method. Hypothalamic expression of KiSS-1 and GPR54 mRNAs was assessed by RT-PCR, optimized for semiquantitative detection, using previously defined primer pairs and conditions (17). As internal control for RT and reaction efficiency, amplification of a 240-bp fragment of S11 ribosomal protein mRNA was carried out in parallel in each sample (17). 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 30 sec, annealing for 30 sec, and extension at 72 C for 1 min. A final extension cycle of 72 C for 15 min was included. Annealing temperature was adjusted for each target and primer pair: 62.5 C for KiSS-1, 63.5 C for GPR54, and 58 C for RP-S11 transcripts. In keeping with previous optimization tests (17), 32 and 24 PCR cycles were chosen for semiquantitative analysis of specific targets (KiSS-1 and GPR54) and RP-S11 internal control, respectively. Specificity of PCR products was confirmed by direct sequencing (Central Sequencing Service, University of Córdoba, Córdoba, 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.
RNA analysis by real-time RT-PCR
To verify changes in gene expression, real-time RT-PCR was performed in the experimental samples using the iCycler iQ Real-Time PCR detection system (Bio-Rad Laboratories, Hercules, CA). In detail, KiSS-1 and GPR54 mRNA levels were assayed in hypothalamic samples from prepubertal male and female rats subjected to 72 h of fasting and their respective controls fed ad libitum. General procedures for real-time RT-PCR were as previously described (17). The synthesized cDNAs were further amplified in triplicate by PCR using SYBR green I as fluorescent dye and 1x iQ Supermix containing 50 mM KCl, 20 mM Tris-HCl, 0.2 mM dNTPs, 3 mM Mg Cl2, and 2.5 U iTaq DNA polymerase (Bio-Rad), in a final volume of 25 μl. The PCR cycling conditions were as follows: initial denaturation and enzyme activation at 95 C for 5 min, followed by 40 cycles of denaturation at 95 C for 15 sec, annealing at 62.5 C (KiSS-1), 63.5 C (GPR54), or 58 C (RP-S11) for 15 sec, and extension at 72 C for 1 min. Calculation of relative expression levels of the target mRNAs was conducted based on the cycle threshold (CT) method (27). The CT for each sample was calculated using the iCycler iQ Real-Time PCR detection system software with an automatic fluorescence threshold (Rn) setting. Accordingly, fold expression of target mRNAs over reference values was calculated by the equation 2–CT, where CT is determined by subtracting the corresponding RP-S11 CT value (internal control) from the specific CT of the target (KiSS-1 or GPR54), and CT is obtained by subtracting the CT of each experimental sample from that of the reference sample (taken as reference value 100). No significant differences in CT values were observed for RP-S11 between the treatment groups.
Hormone measurement by specific RIAs
Serum LH and FSH levels were determined in a volume of 25–50 μl using a double-antibody method and RIA kits kindly supplied by the NIH (Dr. A. F. Parlow, National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Peptide Program, Torrance, CA). Rat LH-I-9 and FSH-I-9 were labeled with 125I by the chloramine-T method, and the hormone concentrations were expressed using the reference preparation LH-RP-3 and FSH-RP2 as standards. Intra- and interassay coefficients of variation were less than 8 and 10% for LH, and 6 and 9% for FSH, respectively. The sensitivity of the assay was 5 pg/tube for LH and 20 pg/tube for FSH. In addition, in selected serum samples (experiment 5), serum estradiol levels were determined using a commercial kit from MP Biomedicals (Costa Mesa, CA), following the instructions of the manufacturer. The sensitivity of the assay was 0.5 pg/tube, and the intraassay coefficient of variation was less than 5%. Finally, GnRH levels in incubation media (experiment 3) were assayed using a commercial RIA kit (Peninsula Laboratories, San Carlos, CA), following the instructions of the manufacturer. The sensitivity of the assay was 1 pg/tube. For each hormone, all the samples were measured in the same assay.
Presentation of data and statistics
Hormonal determinations (LH, FSH, and estradiol in serum and GnRH in incubation medium) were conducted in duplicate, with a minimal total number of 10 samples per group. Semiquantitative RT-PCR analyses were carried out in duplicate from at least four independent RNA samples of each experimental group. Quantitative 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. When appropriate (see experiment 3), the mean effective dose (ED50), defined as the dose of KiSS-1 peptide able to induce 50% of the maximal response, was determined by nonlinear regression (SigmaStat 2.0).
Results
Effects of severe undernutrition upon hypothalamic expression of the KiSS-1 system
To evaluate the effects of situations of negative energy balance on the hypothalamic expression of the KiSS-1 system, relative levels of KiSS-1 and GPR54 mRNAs were assayed by means of final-time and real-time RT-PCR in whole hypothalamic fragments from prepubertal male and female rats after 72 h of fasting. Food deprivation in prepubertal (30 d old) females induced a significant decrease in hypothalamic KiSS-1 mRNA levels that was associated with a moderate but significant increase in GPR54 mRNA expression levels (Fig. 1A). Likewise, a concomitant decrease in KiSS-1 mRNA and increase in GPR54 mRNA levels was observed in hypothalamic samples from prepubertal (30 d old) male rats subjected to 72 h of fasting (Fig. 1B).
Gonadotropic responses to kisspeptin under severe undernutrition
The functionality of the central KiSS-1 system in situations of negative energy balance was explored by assessing the LH- and GnRH-releasing effects of kisspeptin-10 using in vivo and in vitro models, respectively. For the analysis of LH responses to kisspeptin in vivo, prepubertal (30 d old) male and female rats were subjected to food deprivation for 72 h, a procedure that induced a significant reduction in body weight (females, 64.7 ± 1.9 g in fasting vs. 77.5 ± 2.5 g in controls; males, 67.0 ± 2.1 g in fasting vs. 81.7 ± 2.9 g in controls) and serum LH levels (Fig. 2). In age-matched rats fed ad libitum, intracerebral injection of 1 nmol kisspeptin-10 induced robust LH secretory responses, at 15 min after injection, both in females (mean response, 9.7-fold increase over vehicle-injected animals) and males (mean response, 9.0-fold increase over vehicle-injected animals). In fasted animals, the ability of similar doses (1 nmol/rat icv) of kisspeptin-10 to elicit LH secretion in vivo was preserved. Moreover, the effectiveness of kisspeptin in terms of induction of LH secretion was notably enhanced, as estimated by the absolute LH levels reached at 15 min after injection of the peptide (significantly higher in fasted animals than in controls) as well as by the relative fold increase in serum LH concentrations induced by kisspeptin over control levels in fasted animals injected with vehicle (females, 62.5-fold increase; males, 53.5-fold increase) (Fig. 2).
In addition, the ability of increasing doses of kisspeptin-10 (10–12 to 10–6 M) to elicit the release of GnRH in conditions of negative energy balance was assessed in vitro using static incubations of hypothalamic fragments from prepubertal female rats, either fed ad libitum or after 72 h of fasting. Kisspeptin-10 was able to dose-dependently stimulate GnRH release by hypothalamic tissue from fed animals, with a threshold no-observed effect level of 10–10 M, a predicted ED50 of approximately 10–9 M, and a maximal response of 2.6-fold increase over control values (Fig. 3). It is to be noted that such doses are in good agreement with the mean effective and threshold values recently reported by our group for the stimulatory effect of kisspeptin upon LH secretion in vivo (24). Fasting for 72 h induced a significant decrease (P 0.01) in basal release of GnRH in vitro. However, the ability of kisspeptin-10 to dose-dependently elicit GnRH secretion was preserved after food deprivation. Moreover, in this model, the sensitivity to kisspeptin in terms of induction of GnRH secretion was significantly enhanced, as estimated by the lower NOEL (10–12 M) and predicted ED50 value (10–10 M). In addition, maximal relative responses to kisspeptin over corresponding control values (4.0-fold) were increased in fasted animals (Fig. 3).
Effects of kisspeptin upon food intake patterns in rats fed ad libitum and after prefasting
Considering the impact of body energy status upon the expression and function of the KiSS-1 system, the effects of repeated intracerebral injections of kisspeptin-10 upon food intake were explored. To this end, 1 nmol kisspeptin was daily injected into male rats at the early light phase (0900 h), and cumulative food intake was recorded at different time intervals over a 7-d period. The potential effects of kisspeptin were tested against two different prevailing nutritional states: rats fed ad libitum without food restriction (during the first 4 d of the experiment) and rats subjected to a period of 12 h of food deprivation during the dark phase before injection of kisspeptin (for the last 3 d). In these two models, kisspeptin was unable to significantly modify the pattern of food intake vs. vehicle-injected controls. For illustrative purposes, the patterns of cumulative food intake on d 1 and 7 of treatment are shown in Fig. 4. As positive control for the experimental procedure in this setting, icv injection of 1 nmol ghrelin evoked significant orexigenic responses throughout the study period. For example, on d 1 of treatment, intracerebral administration of ghrelin acutely increase food intake (from 0.7 ± 0.4 g in control animals to 1.91 ± 0.45 in ghrelin-treated rats at 1.5 h after injection; P 0.01), in keeping with previous references (9).
Effects of KiSS-1 administration on puberty onset under severe undernutrition
The effects of KiSS-1 upon the activation of the reproductive axis were monitored in immature female rats subjected to a protocol of 30% restriction in daily food intake, initiated on d 23 postpartum. A model of repeated intracerebral injection of kisspeptin, starting at d 30 postpartum, was selected to specifically target central (e.g. hypothalamic) actions of KiSS-1. Yet, this prevented us from extending the treatment protocol beyond 37 d postpartum (i.e. 13 injections of KiSS-1 over 7 d) to avoid misadministration of kisspeptin caused by displacement of the cannulae that takes place (approximately) 7–8 d after their surgical implantation. As an external index of puberty onset, vaginal opening (defined as complete canalization of the vagina) was daily monitored. Such a marker was selected because it has been conventionally considered a reliable external sign of puberty, allowing recapitulation of proper functional activation of all the levels of the reproductive axis (Ref. 22 and references therein). For reference purposes, control reference females fed ad libitum were also daily monitored, a group that showed complete vaginal opening at a mean age of 34.9 ± 0.25 d postpartum and mean body weight of 98.5 ± 2.1 g at 37 d postpartum. Chronic food restriction induced a significant reduction in body weight of approximately 30% from controls (mean body wieght, 68.4 ± 1.0 g; n = 22). This regimen totally prevented normal puberty onset because none of the vehicle-injected females in undernutrition presented vaginal opening by 37 d postpartum (mean body weight, 69.3 ± 0.95 g; n = 10). In addition, significantly suppressed basal LH and estradiol levels were detected in food-restricted animals vs. fed controls. Likewise, serum FSH levels were significantly lower in underfed females (2.48 ± 0.3 ng/ml vs. 5.05 ± 0.21 ng/ml in controls). In contrast, chronic icv administration of kisspeptin-10 (1 nmol/12 h) between d 30 and 37 to females with undernutrition induced complete vaginal opening in seven of 12 treated animals (60%), with a mean body weight of 67.6 ± 1.0 g. As additional indices of rescue of pubertal activation of the reproductive axis, serum LH and estradiol levels were significantly increased in every single food-restricted female rat after repeated icv injection of kisspeptin (Fig. 5). Similarly, FSH levels in kisspeptin-treated females were significantly higher than in underfed animals injected with vehicle (5.2 ± 0.77 ng/ml vs. 2.48 ± 0.3 ng/ml).
Discussion
Activation of the reproductive axis at puberty, and its subsequent functioning in adulthood, critically depends on sufficient energy stores (6, 7, 8). It is assumed that the mechanism whereby situations of energy deficit ultimately impair fertility involves a decrease in the activity of the hypothalamic GnRH pulse generator (8). A conspicuous feature, however, of this phenomenon is that GnRH gene expression at the hypothalamus is not apparently reduced in fasting conditions (28). This suggests that central modulation of the GnRH-gonadotropic axis by the energy status and metabolic factors likely takes place at the level of upstream regulatory signals (involved in the control of GnRH release), whose nature is yet to be fully determined. Among others, a pivotal role for the KiSS-1 system in the central control of the reproductive axis has very recently arisen (10). Indeed, different reports have now provided compelling evidence on the ability of kisspeptins to directly activate GnRH neurons and to elicit GnRH secretion (18, 19, 23) (present results). Yet, despite the proposed function of the KiSS-1 system as a major gatekeeper of GnRH neurons (15, 19), its potential involvement in conveying the impact of (insufficient) body energy stores onto the reproductive axis remains to be determined. To our knowledge, the present study provides the first comprehensive mechanistic overview of the interaction between energy status and the KiSS-1 system in the central control of puberty onset and gonadotropin secretion.
The experimental data presented herein evidence that situations of negative energy balance induce a decrease in the hypothalamic expression of kisspeptin, as suggested by the significant reduction in the relative levels of KiSS-1 mRNA observed in prepubertal male and female rats under short-term fasting. Considering its physiological role in the control of GnRH neurons, it is reasonable to predict that reduced KiSS-1 signaling might operate as a major contributing factor for the reproductive failure (i.e. hypogonadotropic hypogonadism) induced by food deprivation. Additional evidence for alterations in the KiSS-1 system in situations of energy deficit comes from the observation of increased GPR54 mRNA levels at the hypothalamus in fasted animals. A tempting explanation for such a paradoxical finding is that a primary decrease in ligand (KiSS-1) expression might bring about a compensatory increase in the expression of its putative receptor (GPR54), inducing a state of sensitization to the effects of kisspeptin. Indeed, this is apparently the case, as evidenced by our in vivo and in vitro data in short-term fasting. Thus, LH responses to kisspeptin in vivo were preserved, and even enhanced, in food-deprived animals, suggesting that replacement of endogenous KiSS-1 levels is sufficient to fully activate the GnRH-LH axis despite adverse metabolic conditions. Likewise, the sensitivity to kisspeptin in terms of induction of GnRH secretion in vitro was significantly enhanced (as evidenced by lower mean and minimal effective doses), and its maximal responses increased, in fasted animals. On the former, it has to be noted that 10–10 M kisspeptin was able to elicit GnRH release in vitro in underfed animals but not in those fed ad libitum, which support the contention that the sensitivity to kisspeptin is significantly increased in situations of negative energy balance. Interestingly, in our setting, basal release of GnRH was significantly reduced (2.0-fold) by previous short-term fasting. Such a decrease, however, was fully reversed by incubation of hypothalamic fragments from fasted animals in the presence of 10–10 M kisspeptin (see Fig. 3). This suggests that the physiological level of hypothalamic kisspeptin may range between 10 and 100 pM and that its replacement in conditions of negative energy balance is apparently sufficient to restore normal GnRH release.
Assumably, the molecular basis for the observed changes in the expression and function of the KiSS-1 system in fasting conditions is yet to be elucidated, but in principle, different signals with proven actions in the joint control of energy balance and reproduction might be involved. Among others, the adipocyte hormone leptin has been identified as a pivotal peripheral factor for signaling the amount of body energy stores to the hypothalamic centers governing reproduction. However, the ultimate mechanisms whereby leptin conducts this relevant function are still a matter of debate, and direct or indirect effects of leptin upon GnRH neurons have been proposed (8). Interestingly, leptin has been involved in the control of the hypothalamic expression of galanin-like peptide (GALP), another potent elicitor of GnRH-LH secretion, and a sensitization phenomenon, analogous to that reported herein for KiSS-1, has been recently described for the LH-releasing effects of GALP in conditions of leptin insufficiency (29). Indeed, hyperresponsiveness to a number of elicitors of the hypothalamic-pituitary-gonadal axis is apparently detected in conditions of negative energy balance and/or hypoleptinemia, a phenomenon where the potential contribution of altered KiSS-1 functions (as a relevant downstream regulator of the GnRH system) merits further investigation. Nonetheless, whether similar mechanisms underlie the reported sensitization to kisspeptin and GALP is yet to be determined. In fact, our preliminary observations evidence that, in contrast to GALP, KiSS-1 mRNA levels at the hypothalamus are not significantly modified in conditions of leptin deficiency (i.e. ob/ob mouse) (our unpublished data), suggesting that leptin is not an essential regulator of KiSS-1 gene expression at the hypothalamus. Alternatively, leptin modulation of the KiSS-1 system may take place at a posttranscriptional step, and/or additional neuroendocrine integrators might be involved. The latter possibility is presently under investigation at our laboratory.
Expression of KiSS-1 and GPR54 mRNAs, as well as metastin-like immunoreactivity, has been detected in several hypothalamic areas involved in feeding regulation (21, 30, 31), including the arcuate nucleus. This area has been highlighted as a major center for the integrated regulation of food intake, where neuropeptide Y (NPY)/Agouti-related peptide (orexigenic) and proopiomelanocortin/cocaine- and amphetamine-regulated transcript (anorexigenic) neurons reciprocally operate under the regulation of peripheral factors, such as leptin (29 and references therein). Our present results document, however, that central injection of kisspeptin is not able to significantly alter the pattern of food intake, either in rats fed ad libitum or subjected to previous 12 h of fasting. In good agreement, intracerebral administration of kisspeptin, at a dose effective to maximally elicit LH secretion, failed to change hypothalamic expression levels of NPY, Agouti-related peptide, proopiomelanocortin, and cocaine- and amphetamine-regulated transcript mRNAs (our unpublished data). Altogether, our current results demonstrate that, although body energy stores impact the expression and function of the KiSS-1 system at the hypothalamus, kisspeptin is not provided with specific regulatory actions upon feeding. In fact, during conduction of this study, this contention was further supported by an independent study (23). This finding is contrast with other well-known regulators of food intake and the reproductive axis, such as leptin, NPY, orexin, GALP, and likely ghrelin, for which direct, independent actions in the control of feeding and reproductive axis have been reported (9, 29, 32, 33), and strengthens the role of the KiSS-1 system as a selective downstream regulatory signal essential for the proper function of the GnRH-LH axis.
Additional evidence for the pivotal involvement of the KiSS-1 system in the mechanisms whereby energy status controls activation of the reproductive axis at puberty is provided by our experiments involving chronic administration of kisspeptin in conditions of undernutrition. In our setting, a persistent decrease in daily food intake of 30% from controls was able to totally block normal pubertal development, as estimated by lack of vaginal opening and decreased serum gonadotropin levels. In this model, repeated administration of kisspeptin was sufficient to restore vaginal opening in a significant number (60%) of cases as well as to induce gonadotropin and estrogen secretion in every single animal tested. On the basis of present and previous data (18, 19, 23), this activational response is thought to be mediated by central stimulation of the GnRH system. Yet, in keeping with our previous results (24), such an effect did not involve increased hypothalamic expression of GnRH mRNA, nor was it associated with significant changes in the relative levels of GnRH receptor mRNA at the pituitary (our unpublished observations). The reasons for the lack of vaginal opening in a proportion of food-restricted females injected with kisspeptin remain to be solved, but subtle individual variations in the responses to subnutrition and/or kisspeptin administration cannot be excluded. Moreover, it is plausible that occurrence of vaginal opening might have taken place in the remaining kisspeptin-injected animals (which actually had significantly elevated LH and estradiol levels over vehicle-treated underfed females at d 37) if longer treatment protocols could have been implemented.
The relevance of the present observations is stressed by the fact that, although kisspeptin was able to rescue vaginal opening in a large percentage of food-restricted females, the very same treatment protocol (between d 30 and 37 postpartum) dramatically depressed serum LH levels and prevented normal occurrence of canalization of the vagina in more than 50% of normally fed female rats at puberty (manuscript in preparation), i.e. when full activation of the endogenous KiSS-1 system occurs (17). This illustrates the exquisite sensitivity of this system not only to down-regulation (as observed in food deprivation) but also to hyperactivation, where desensitization of the gonadotropic axis is likely taking place. Additional studies, involving repeated administration of kisspeptin at different stages of maturation, and testing of hypothalamic secretion and pituitary sensitivity to GnRH, are presently in progress in our laboratory to cover this relevant issue. Nonetheless, although ultimate occurrence of ovulation as definitive proof of complete puberty was not monitored in our experimental setting, in the context of the reported decrease in KiSS-1 expression in situations of negative energy balance, our present data (involving assessment of different end-points such as pituitary LH and FSH secretion, ovarian-derived estrogen levels, and vaginal opening as biomarker of estrogenic action) strongly suggest that restoration of endogenous KiSS-1 tone is sufficient to rescue (at least partially) defective pubertal activation of the reproductive axis in undernutrition. This is not only relevant from a mechanistic standpoint, but it may pose also interesting therapeutic implications, especially considering the extraordinarily potent gonadotropin-releasing activity of systemically delivered kisspeptins (24, 25).
In summary, we provide herein an integral analysis of the potential interaction of energy balance and the KiSS-1 system in the control of reproductive function, with special attention to the role of this novel signaling system in conveying the impact of (insufficient) body energy stores onto the gonadotropic axis at puberty. Our data demonstrate for the first time that, despite its lack of direct effects on feeding and related neuropeptide gene expression, food deprivation induces significant changes in the expression and function of the central (hypothalamic) KiSS-1 system, which may represent a previously uncharacterized target for disruption (and eventual therapeutic intervention) of pubertal development in conditions of negative energy balance.
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
This work was supported by Grants BFI 2000-0419-CO3-03 and BFI 2002-00176 from DGESIC (Ministerio de Ciencia y Tecnología, Spain), funds from Instituto de Salud Carlos III (Red de Centros RCMN C03/08 and Project PI042082; Ministerio de Sanidad, Spain), and EU research contract EDEN QLK4-CT-2002-00603.
1 J.M.C. and V.M.N. equally contributed to this work and should be considered as joint first authors.
Abbreviations: CT, Cycle threshold; GALP, galanin-like peptide; GPR54, G protein-coupled receptor 54; icv, intracerebroventricular; NPY, neuropeptide Y.
References
Fink G 2000 Neuroendocrine regulation of pituitary function: general principles. In: Conn PM, Freeman ME, eds. Neuroendocrinology in physiology and medicine. Totowa, NJ: Humana Press; 107–134
Tena-Sempere M, Huhtaniemi I 2003 Gonadotropins and gonadotropin receptors. In: Fauser BCJM, ed. Reproductive medicine: molecular, cellular and genetic fundamentals. New York: Parthenon Publishing; 225–244
Ojeda SR, Urbanski HF 1994 Puberty in the rat. In: Knobil E, Neill JD, eds. The physiology of reproduction. New York: Raven Press; 363–410
Grumbach MM 2002 The neuroendocrinology of human puberty revisited. Horm Res 57:2–14
Plant TM, Barker-Gibb ML 2004 Neurobiological mechanisms of puberty in higher primates. Hum Reprod Update 10:67–77
Parent AS, Teilmann G, Juul A, Skakkebaek NE, Toppari J, Bourguignon JP 2003 The timing of normal puberty and the age limits of sexual precocity: variations around the world, secular trends, and changes after migration. Endocr Rev 24:668–693
Frisch RE 1987 Body fat, menarche, fitness and fertility. Hum Reprod 2:521–533
Cunningham MJ, Clifton DK, Steiner RA 1999 Leptin’s actions on the reproductive axis: perspectives and mechanisms. Biol Reprod 60:616–622
Barreiro ML, Tena-Sempere M 2004 Ghrelin and reproduction: a novel signal linking energy status and fertility Mol Cell Endocrinol 226:1–9
Colledge WH 2004 GPR54 and puberty. Trends Endocrinol Metab 15:448–453
Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, Terao Y, Kumano S, Takatsu Y, Masuda Y, Ishibashi Y, Watanabe T, Asada M, Yamada T, Suenaga M, Fujino C, Usuki S, Kurokawa T, Onda H, Nishimura O, Fujino M 2001 Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G protein-coupled receptor. Nature 411:613–617
Muir AI, Chamberlain L, Elshourbagy NA, Michalovich D, Moore DJ, Calamari A, Szekeres PG, Sarau HM, Chambers JK, Murdock P, Steplewski K, Shabon U, Miller JE, Middleton SE, Darker JG, Larminie CGC, Wilson S, Bergsma DJ, Emson P, Faull R, Philpott KL, Harrison DC 2001 AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1. J Biol Chem 276:28969–28975
Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, Le Poul E, Brezillon S, Tyldesley R, Suarez-Huerta N, Vandeput F, Blanpain C, Schiffmann SN, Vassart G, Parmentier M 2001 The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. J Biol Chem 276:34631–34636
de Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E 2003 Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci USA 100:10972–10976
Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno JS, Shagoury JK, Bo-Abbas Y, Kuohung W, Schwinof KM, Hendrick AG, Zahn D, Dixon J, Kaiser UB, Slaugenhaupt SA, Gusella JF, O’Rahilly S, Carlton MB, Crowley WF, Aparicio SA, Colledge WH 2003 The GPR54 gene as a regulator of puberty. N Engl J Med 349:1614–1627
Semple RK, Achermann JC, Ellery J, Farooqi IS, Karet FE, Stanhope RG, O’Rahilly S, Aparicio SA 2005 Two novel missense mutations in G-protein-coupled receptor 54 in a patient with hypogonadotropic hypogonadism. J Clin Endocrinol Metab 90:1849–1855
Navarro VM, Castellano JM, Fernandez-Fernandez R, Barreiro ML, Roa J, Sanchez-Criado JE, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M 2004 Developmental and hormonally regulated messenger ribonucleic acid expression of KiSS-1 and its putative receptor GPR54 in rat hypothalamus and potent LH releasing activity of KiSS-1 peptide. Endocrinology 145:4565–4574
Irwig MS, Fraley GS, Smith JT, Acohido BV, Popa SM, Cunningham MJ, Gottsch ML, Clifton DK, Steiner RA 2004 Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat. Neuroendocrinology 80:264–272
Shahab M, Mastronardi C, Seminara SB, Crowley WF, Ojeda SR, Plant TM 2005 Increased hypothalamic GPR54 signaling: a potential mechanism for initiation of puberty in primates. Proc Natl Acad Sci USA 102:2129–2134
Matsui H, Takatsu Y, Kumano S, Matsumoto H, Ohtaki T 2004 Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat. Biochem Biophys Res Commun 320:383–388
Gottsch ML, Cunningham MJ, Smith JT, Popa SM, Acohido BV, Crowley WF, Seminara S, Clifton DK, Steiner RA 2004 A role for kisspeptins in the regulation of gonadotropin secretion in the mouse. Endocrinology 145:4073–4077
Navarro VM, Fernandez-Fernandez R, Castellano JM, Roa J, Mayen A, Barreiro ML, Gaytan F, Aguilar E, Pinilla L, Dieguez C, Tena-Sempere M 2004 Advanced vaginal opening and precocious activation of the reproductive axis by KiSS-1 peptide, the endogenous ligand of GPR54. J Physiol 561:379–386
Thompson EL, Patterson M, Murphy KG, Smith KL, Dhillo WS, Todd JF, Ghatei MA, Bloom SR 2004 Central and peripheral administration of kisspeptin-10 stimulates the hypothalamo-pituitary-gonadal axis. J Neuroendocrinol 16:850–858
Navarro VM, Castellano JM, Fernandez-Fernandez R, Tovar S, Roa J, Mayen A, Nogueiras R, Vazquez MJ, Barreiro ML, Magni P, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M 2005 Characterization of the potent LH releasing activity of KiSS-1 peptide, the natural ligand of GPR54. Endocrinology 146:156–163
Navarro VM, Castellano JM, Fernandez-Fernandez R, Tovar S, Roa J, Mayen A, Barreiro ML, Casanueva FF, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M 2005 Effects of KiSS-1 peptide, the natural ligand of GPR54, on follicle-stimulating hormone secretion in the rat. Endocrinology 146:1689–1697
Messager S, Chatzidaki EE, Ma D, Hendrick AG, Zahn D, Dixon J, Thresher RR, Malinge I, Lomet D, Carlton MB, Colledge WH, Caraty A, Aparicio SA 2005 Kisspeptin directly stimulates gonadotropin-releasing hormone secretion via G protein-coupled receptor 54. Proc Natl Acad Sci USA 102:1761–1766
Higuchi R, Fockler C, Dollinger G, Watson R 1993 Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 11:1026–1030
Bergendahl M, Wiemann JN, Clifton DK, Huhtaniemi I, Steiner RA 1992 Short-term starvation decreases POMC mRNA but does not alter GnRH mRNA in rat brain. Neuroendocrinology 56:913–920
Kumano S, Matsumoto H, Takatsu Y, Noguchi J, Kitada C, Ohtaki T 2003 Changes in hypothalamic expression levels of galanin-like peptide in rat and mouse models support that it is a leptin-target peptide. Endocrinology 144:2634–2643
Lee DK, Nguyen T, O’Neill GP, Chang R, Liu Y, Howard AD, Coulombe N, Tan CP, Tang-Nguyen AT, George SR, O’Dowd BF 1999 Discovery of a receptor related to the galanin receptors. FEBS Lett 446:103–107
Brailoiu GC, Dub SL, Ohsawa M, Yin D, Yang J, Chang JK, Brailoiu E, Dun NJ 2005 KiSS-1 expression and metastin-like immunoreactivity in the rat brain. J Comp Neurol 481:314–329
Tena-Sempere M, Barreiro ML 2002 Leptin in male reproduction: the testis paradigm. Mol Cell Endocrinol 188:9–13
Russell SH, Small CJ, Kennedy AR, Stanley SA, Seth A, Murphy KG, Taheri S, Ghatei MA, Bloom SR 2001 Orexin A interactions in the hypothalamo-pituitary gonadal axis. Endocrinology 142:5294–5302(J. M. Castellano1, V. M. )
Departments of Physiology (R.N., S.T., M.J.V., C.D.), and Medicine (F.F.C.), University of Santiago de Compostela, 15705 Santiago de Compostela, Spain
Abstract
Activation of the gonadotropic axis critically depends on sufficient body energy stores, and conditions of negative energy balance result in lack of puberty onset and reproductive failure. Recently, KiSS-1 gene-derived kisspeptin, signaling through the G protein-coupled receptor 54 (GPR54), has been proven as a pivotal regulator in the control of gonadotropin secretion and puberty. However, the impact of body energy status upon hypothalamic expression and function of this system remains unexplored. In this work, we evaluated the expression of KiSS-1 and GPR54 genes at the hypothalamus as well as the ability of kisspeptin-10 to elicit GnRH and LH secretion in prepubertal rats under short-term fasting. In addition, we monitored the actions of kisspeptin on food intake and the effects of its chronic administration upon puberty onset in undernutrition. Food deprivation induced a concomitant decrease in hypothalamic KiSS-1 and increase in GPR54 mRNA levels in prepubertal rats. In addition, LH responses to kisspeptin in vivo were enhanced, and its GnRH secretagogue action in vitro was sensitized, under fasting conditions. Central kisspeptin administration failed to change food intake patterns in animals fed ad libitum or after a 12-h fast. However, chronic treatment with kisspeptin was able to restore vaginal opening (in 60%) and to elicit gonadotropin and estrogen responses in a model of undernutrition. In summary, our data are the first to show an interaction between energy status and the hypothalamic KiSS-1 system, which may constitute a target for disruption (and eventual therapeutic intervention) of pubertal development in conditions of negative energy balance.
Introduction
PITUITARY GONADOTROPINS LH and FSH are structurally related glycoproteins responsible for the control of gonadal development and function (1, 2). Secretion of both gonadotropins is primarily driven by the pulsatile release of the hypophysiotropic hypothalamic decapeptide GnRH, also termed GnRH-I (1, 2, 3, 4, 5). Full activation of the gonadotropic axis takes place at puberty, which is the culmination of a cascade of developmental events that ultimately leads to enhanced activity of the GnRH pulse generator, increased plasma levels of LH and FSH, and attainment of reproductive capacity (2, 3, 4, 5). Awakening of the reproductive axis at puberty, and its subsequent functioning in adulthood, is critically dependent on sufficient body energy stores (5, 6). Indeed, diverse conditions of persistent negative energy balance, such as undernutrition and extreme physical exercise, are associated with lack of puberty onset and reproductive failure, both in humans and experimental animals (6, 7, 8). Despite recent developments in the field, including characterization of the pivotal role of leptin in this function, the signals and circuitries responsible for the concerted control of energy balance and reproduction are yet to be totally elucidated (9). Likewise, although decreased pulsatile secretion of GnRH has been ultimately invoked (8), the molecular mechanisms underlying disruption of reproductive function in situations of energy insufficiency remain to be fully characterized.
The master position of hypothalamic GnRH in the hierarchy of signals controlling the gonadotropic axis makes it the target of multiple regulators of central and peripheral origin, and a wide array of excitatory and inhibitory circuits governing GnRH secretion have been identified over the last decades (3, 4, 5). In this context, an unsuspected key role for the KiSS-1/G protein-coupled receptor 54 (GPR54) system in the central control of the GnRH-gonadotropin axis has recently arisen on the basis of genetic and pharmacological studies (10). KiSS-1 was originally identified as a metastasis suppressor gene encoding a number of structurally related peptides, generated by the differential proteolytic processing of a common precursor, globally termed kisspeptins (11, 12, 13). The biological actions of kisspeptins are conducted through interaction with the previously orphan G protein-coupled receptor GPR54, also termed AXOR12 or hOT7T175 (11, 12, 13). A number of point mutations and deletions of the GPR54 gene were recently found in patients suffering idiopathic hypogonadotropic hypogonadism (14, 15, 16), a syndrome that was reproduced in mouse models carrying null mutations of the GPR54 gene (15). Thereafter, hypothalamic expression of KiSS-1 and GPR54 genes has been proven as developmentally (maximum at puberty) and hormonally (by sex steroids) regulated (17, 18, 19), and the ability of kisspeptins to potently elicit gonadotropin secretion has been simultaneously reported by different groups, including ours (17, 18, 19, 20, 21, 22, 23, 24, 25, 26). Moreover, direct stimulatory actions of kisspeptin upon hypothalamic GnRH neurons and/or GnRH release have been very recently documented (18, 19, 23), and mechanistic studies have suggested a relevant role of KiSS-1 signaling in puberty onset in rodent and primate species (19, 22). In fact, comparative meta-analyses with published data on the LH-releasing activity of other neuropeptides and neurotransmitters, such as glutamate and galanin-like peptide, evidence that the KiSS-1 system is likely the most potent elicitor of the GnRH-gonadotropin axis known so far (24, 25). Indeed, although evaluation of its potential interplay with other well known modulators of the reproductive system has been initiated only recently (24, 25), the KiSS-1 system has been already proposed as an essential gatekeeper for proper function of the gonadotropic axis (15, 19).
In the above scenario, we hypothesized that the mechanisms whereby conditions of negative energy balance hamper the functioning of reproductive axis may target directly the hypothalamic KiSS-1 system. To test this hypothesis, expression analyses of KiSS-1 and GPR54 genes at the hypothalamus were conducted, and reproductive responses to kisspeptin (in terms of hormone secretion and biomarkers of puberty onset) were monitored at puberty in models of severe undernutrition.
Materials and Methods
Animals and drugs
Wistar 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. The animals were maintained under constant conditions of light (14 h of light, from 0700 h) and temperature (22 C), and were weaned at 21 d of age in groups of five rats per cage with free access to pelleted food and tap water, unless otherwise stated. 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. The animals were humanely killed by decapitation at the end of the experimental settings. Mouse KiSS-1 (110–119)-NH2, the rodent analog of the C-terminal KiSS-1 decapeptide KiSS-1 (112–121)-NH2, was obtained from Phoenix Pharmaceuticals Ltd. (Belmont, CA). This peptide fragment, which has been previously shown to maximally bind and activate GPR54 in transfected CHO cells (12, 13), will be referred hereafter as kisspeptin-10.
Experimental designs
In experiment 1, the effects of conditions of negative energy balance on the expression of the KiSS-1 system at the hypothalamus were monitored in prepubertal animals. This stage of postnatal maturation was selected given the well known dependence of normal pubertal development on sufficient energy stores (3). Groups of male and female rats (30 d old; n = 10–12 per group) were subjected to a period of 72 h of fasting (only access to tap water); age-matched rats fed ad libitum served as controls. At the end of the fasting period, the animals were killed by decapitation, and the hypothalamus was immediately dissected out, as described in detail elsewhere (17), by a horizontal cut of about 2 mm depth with the following limits: 1 mm anteriorly from the optic chiasm (to include the preoptic area), the posterior border of mamillary bodies, and the hypothalamic fissures. Hypothalamic samples were frozen in liquid nitrogen and stored at –80 C until processing for RNA analysis.
In experiment 2, the functionality of the KiSS-1 system, in terms of gonadotropic responses, was tested under conditions of severe energy deficit. To this end, the ability of kisspeptin-10 to centrally elicit LH secretion was assessed in prepubertal male and female rats (30 d old; n = 10–12 per group) previously subjected to food deprivation for 72 h. A protocol of intracerebroventricular (icv) administration of 1 nmol kisspeptin-10 was carried out, as described elsewhere (17, 22, 24, 25). To allow delivery of kisspeptin into the lateral cerebral ventricle, the animals were implanted with icv 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. A dose of 1 nmol kisspeptin in 10 μl per rat was selected on the basis of our recent data on the ability of this dose to potently elicit LH secretion in rats fed ad libitum (17, 24). Trunk blood samples were collected upon decapitation at 15 min after kisspeptin injection. Animals injected with vehicle (NaCl 0.9%) served as controls.
In experiment 3, the central mechanisms for the effects of kisspeptin upon gonadotropin secretion in situations of negative energy balance were explored. To this end, the ability of kisspeptin-10 to elicit GnRH secretion was tested using a static incubation system and hypothalamic fragments from prepubertal female rats (n = 10–12 hypothalamic samples per group) at two different prevailing metabolic states: feeding ad libitum and 72 h of fasting. Upon decapitation of the animals, hypothalamic fragments were excised following the tissue limits indicated in experiment 2 and placed into individual incubation chambers containing 250 μl of phenol red-free DMEM for 30 min of preincubation, using a Dubnoff incubator at 37 C with constant shaking (60 cycles/min), under an atmosphere of 95% O2/5% CO2. After this period, preincubation media were replaced by DMEM alone or medium containing increasing concentrations of kisspeptin-10 (10–12, 10–10, 10–8, and 10–6 M). Incubations were terminated after 30 min, when media were boiled to inactivate endogenous protease activity, and kept at –80 C until assayed for GnRH levels.
In experiment 4, the effects of kisspeptin upon food intake pattern were studied. To this end, groups of male rats (n = 8 per group) were implanted with icv cannulae, and daily intracerebral injection of 1 nmol kisspeptin was conducted at early light phase (0900 h) for 7 consecutive days. As control for the experimental procedure, an additional group of animals (n = 8) were icv injected with 1 nmol ghrelin, a proven orexigenic factor (9). To explore the impact of the prevailing energy status upon the effects of kisspeptin on food intake, the animals were initially allowed to have free access to chow during the first 4 d, and cumulative food intake was monitored at 1.5, 3, 6, 12, and 24 h after each kisspeptin injection. Thereafter, the animals were subjected to 12 h of fasting during the dark phase preceding kisspeptin injection for the last 3 d of treatment, and cumulative food intake was monitored at 1.5, 3, 6, and 12 h after injection.
Finally, in experiment 5, the effects of chronic central administration of kisspeptin-10 on puberty onset in immature female rats under severe undernutrition were monitored. A protocol of 30% restriction in daily food intake vs. age-matched control females (fed ad libitum) was initiated on d 23 postpartum. Daily icv administration of kisspeptin in the lateral cerebral ventricle was conducted between d 30–37 postpartum in food-restricted females (n = 12), as described in detail elsewhere (22). The treatment regimen was set at a dose of 1 nmol KiSS-1 per animal in 10 μl, every 12 h. Pair-aged females (n = 10), at 30% food restriction, injected with vehicle served as controls. The animals were icv injected under conscious conditions after careful handling to avoid any stressful influence, in keeping with our previous references (22). In all experimental animals, body weight and vaginal opening were daily monitored. On the latter, detailed inspection was conducted in each animal to determine the date of complete canalization of the vagina. At the end of treatment (37 d postpartum), the animals were killed by decapitation, 60 min after the last injection of kisspeptin or vehicle, and trunk blood was collected. For determination of the normal date of vaginal opening in animals fed ad libitum, an additional group of females (n = 20), without food restriction and icv injected with vehicle, were maintained on daily inspection of canalization of the vagina up to d 37 postpartum.
RNA analysis by semiquantitative RT-PCR
Total RNA was isolated from hypothalamic samples using the single-step, acid guanidinium thiocyanate-phenol-chloroform extraction method. Hypothalamic expression of KiSS-1 and GPR54 mRNAs was assessed by RT-PCR, optimized for semiquantitative detection, using previously defined primer pairs and conditions (17). As internal control for RT and reaction efficiency, amplification of a 240-bp fragment of S11 ribosomal protein mRNA was carried out in parallel in each sample (17). 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 30 sec, annealing for 30 sec, and extension at 72 C for 1 min. A final extension cycle of 72 C for 15 min was included. Annealing temperature was adjusted for each target and primer pair: 62.5 C for KiSS-1, 63.5 C for GPR54, and 58 C for RP-S11 transcripts. In keeping with previous optimization tests (17), 32 and 24 PCR cycles were chosen for semiquantitative analysis of specific targets (KiSS-1 and GPR54) and RP-S11 internal control, respectively. Specificity of PCR products was confirmed by direct sequencing (Central Sequencing Service, University of Córdoba, Córdoba, 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.
RNA analysis by real-time RT-PCR
To verify changes in gene expression, real-time RT-PCR was performed in the experimental samples using the iCycler iQ Real-Time PCR detection system (Bio-Rad Laboratories, Hercules, CA). In detail, KiSS-1 and GPR54 mRNA levels were assayed in hypothalamic samples from prepubertal male and female rats subjected to 72 h of fasting and their respective controls fed ad libitum. General procedures for real-time RT-PCR were as previously described (17). The synthesized cDNAs were further amplified in triplicate by PCR using SYBR green I as fluorescent dye and 1x iQ Supermix containing 50 mM KCl, 20 mM Tris-HCl, 0.2 mM dNTPs, 3 mM Mg Cl2, and 2.5 U iTaq DNA polymerase (Bio-Rad), in a final volume of 25 μl. The PCR cycling conditions were as follows: initial denaturation and enzyme activation at 95 C for 5 min, followed by 40 cycles of denaturation at 95 C for 15 sec, annealing at 62.5 C (KiSS-1), 63.5 C (GPR54), or 58 C (RP-S11) for 15 sec, and extension at 72 C for 1 min. Calculation of relative expression levels of the target mRNAs was conducted based on the cycle threshold (CT) method (27). The CT for each sample was calculated using the iCycler iQ Real-Time PCR detection system software with an automatic fluorescence threshold (Rn) setting. Accordingly, fold expression of target mRNAs over reference values was calculated by the equation 2–CT, where CT is determined by subtracting the corresponding RP-S11 CT value (internal control) from the specific CT of the target (KiSS-1 or GPR54), and CT is obtained by subtracting the CT of each experimental sample from that of the reference sample (taken as reference value 100). No significant differences in CT values were observed for RP-S11 between the treatment groups.
Hormone measurement by specific RIAs
Serum LH and FSH levels were determined in a volume of 25–50 μl using a double-antibody method and RIA kits kindly supplied by the NIH (Dr. A. F. Parlow, National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Peptide Program, Torrance, CA). Rat LH-I-9 and FSH-I-9 were labeled with 125I by the chloramine-T method, and the hormone concentrations were expressed using the reference preparation LH-RP-3 and FSH-RP2 as standards. Intra- and interassay coefficients of variation were less than 8 and 10% for LH, and 6 and 9% for FSH, respectively. The sensitivity of the assay was 5 pg/tube for LH and 20 pg/tube for FSH. In addition, in selected serum samples (experiment 5), serum estradiol levels were determined using a commercial kit from MP Biomedicals (Costa Mesa, CA), following the instructions of the manufacturer. The sensitivity of the assay was 0.5 pg/tube, and the intraassay coefficient of variation was less than 5%. Finally, GnRH levels in incubation media (experiment 3) were assayed using a commercial RIA kit (Peninsula Laboratories, San Carlos, CA), following the instructions of the manufacturer. The sensitivity of the assay was 1 pg/tube. For each hormone, all the samples were measured in the same assay.
Presentation of data and statistics
Hormonal determinations (LH, FSH, and estradiol in serum and GnRH in incubation medium) were conducted in duplicate, with a minimal total number of 10 samples per group. Semiquantitative RT-PCR analyses were carried out in duplicate from at least four independent RNA samples of each experimental group. Quantitative 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. When appropriate (see experiment 3), the mean effective dose (ED50), defined as the dose of KiSS-1 peptide able to induce 50% of the maximal response, was determined by nonlinear regression (SigmaStat 2.0).
Results
Effects of severe undernutrition upon hypothalamic expression of the KiSS-1 system
To evaluate the effects of situations of negative energy balance on the hypothalamic expression of the KiSS-1 system, relative levels of KiSS-1 and GPR54 mRNAs were assayed by means of final-time and real-time RT-PCR in whole hypothalamic fragments from prepubertal male and female rats after 72 h of fasting. Food deprivation in prepubertal (30 d old) females induced a significant decrease in hypothalamic KiSS-1 mRNA levels that was associated with a moderate but significant increase in GPR54 mRNA expression levels (Fig. 1A). Likewise, a concomitant decrease in KiSS-1 mRNA and increase in GPR54 mRNA levels was observed in hypothalamic samples from prepubertal (30 d old) male rats subjected to 72 h of fasting (Fig. 1B).
Gonadotropic responses to kisspeptin under severe undernutrition
The functionality of the central KiSS-1 system in situations of negative energy balance was explored by assessing the LH- and GnRH-releasing effects of kisspeptin-10 using in vivo and in vitro models, respectively. For the analysis of LH responses to kisspeptin in vivo, prepubertal (30 d old) male and female rats were subjected to food deprivation for 72 h, a procedure that induced a significant reduction in body weight (females, 64.7 ± 1.9 g in fasting vs. 77.5 ± 2.5 g in controls; males, 67.0 ± 2.1 g in fasting vs. 81.7 ± 2.9 g in controls) and serum LH levels (Fig. 2). In age-matched rats fed ad libitum, intracerebral injection of 1 nmol kisspeptin-10 induced robust LH secretory responses, at 15 min after injection, both in females (mean response, 9.7-fold increase over vehicle-injected animals) and males (mean response, 9.0-fold increase over vehicle-injected animals). In fasted animals, the ability of similar doses (1 nmol/rat icv) of kisspeptin-10 to elicit LH secretion in vivo was preserved. Moreover, the effectiveness of kisspeptin in terms of induction of LH secretion was notably enhanced, as estimated by the absolute LH levels reached at 15 min after injection of the peptide (significantly higher in fasted animals than in controls) as well as by the relative fold increase in serum LH concentrations induced by kisspeptin over control levels in fasted animals injected with vehicle (females, 62.5-fold increase; males, 53.5-fold increase) (Fig. 2).
In addition, the ability of increasing doses of kisspeptin-10 (10–12 to 10–6 M) to elicit the release of GnRH in conditions of negative energy balance was assessed in vitro using static incubations of hypothalamic fragments from prepubertal female rats, either fed ad libitum or after 72 h of fasting. Kisspeptin-10 was able to dose-dependently stimulate GnRH release by hypothalamic tissue from fed animals, with a threshold no-observed effect level of 10–10 M, a predicted ED50 of approximately 10–9 M, and a maximal response of 2.6-fold increase over control values (Fig. 3). It is to be noted that such doses are in good agreement with the mean effective and threshold values recently reported by our group for the stimulatory effect of kisspeptin upon LH secretion in vivo (24). Fasting for 72 h induced a significant decrease (P 0.01) in basal release of GnRH in vitro. However, the ability of kisspeptin-10 to dose-dependently elicit GnRH secretion was preserved after food deprivation. Moreover, in this model, the sensitivity to kisspeptin in terms of induction of GnRH secretion was significantly enhanced, as estimated by the lower NOEL (10–12 M) and predicted ED50 value (10–10 M). In addition, maximal relative responses to kisspeptin over corresponding control values (4.0-fold) were increased in fasted animals (Fig. 3).
Effects of kisspeptin upon food intake patterns in rats fed ad libitum and after prefasting
Considering the impact of body energy status upon the expression and function of the KiSS-1 system, the effects of repeated intracerebral injections of kisspeptin-10 upon food intake were explored. To this end, 1 nmol kisspeptin was daily injected into male rats at the early light phase (0900 h), and cumulative food intake was recorded at different time intervals over a 7-d period. The potential effects of kisspeptin were tested against two different prevailing nutritional states: rats fed ad libitum without food restriction (during the first 4 d of the experiment) and rats subjected to a period of 12 h of food deprivation during the dark phase before injection of kisspeptin (for the last 3 d). In these two models, kisspeptin was unable to significantly modify the pattern of food intake vs. vehicle-injected controls. For illustrative purposes, the patterns of cumulative food intake on d 1 and 7 of treatment are shown in Fig. 4. As positive control for the experimental procedure in this setting, icv injection of 1 nmol ghrelin evoked significant orexigenic responses throughout the study period. For example, on d 1 of treatment, intracerebral administration of ghrelin acutely increase food intake (from 0.7 ± 0.4 g in control animals to 1.91 ± 0.45 in ghrelin-treated rats at 1.5 h after injection; P 0.01), in keeping with previous references (9).
Effects of KiSS-1 administration on puberty onset under severe undernutrition
The effects of KiSS-1 upon the activation of the reproductive axis were monitored in immature female rats subjected to a protocol of 30% restriction in daily food intake, initiated on d 23 postpartum. A model of repeated intracerebral injection of kisspeptin, starting at d 30 postpartum, was selected to specifically target central (e.g. hypothalamic) actions of KiSS-1. Yet, this prevented us from extending the treatment protocol beyond 37 d postpartum (i.e. 13 injections of KiSS-1 over 7 d) to avoid misadministration of kisspeptin caused by displacement of the cannulae that takes place (approximately) 7–8 d after their surgical implantation. As an external index of puberty onset, vaginal opening (defined as complete canalization of the vagina) was daily monitored. Such a marker was selected because it has been conventionally considered a reliable external sign of puberty, allowing recapitulation of proper functional activation of all the levels of the reproductive axis (Ref. 22 and references therein). For reference purposes, control reference females fed ad libitum were also daily monitored, a group that showed complete vaginal opening at a mean age of 34.9 ± 0.25 d postpartum and mean body weight of 98.5 ± 2.1 g at 37 d postpartum. Chronic food restriction induced a significant reduction in body weight of approximately 30% from controls (mean body wieght, 68.4 ± 1.0 g; n = 22). This regimen totally prevented normal puberty onset because none of the vehicle-injected females in undernutrition presented vaginal opening by 37 d postpartum (mean body weight, 69.3 ± 0.95 g; n = 10). In addition, significantly suppressed basal LH and estradiol levels were detected in food-restricted animals vs. fed controls. Likewise, serum FSH levels were significantly lower in underfed females (2.48 ± 0.3 ng/ml vs. 5.05 ± 0.21 ng/ml in controls). In contrast, chronic icv administration of kisspeptin-10 (1 nmol/12 h) between d 30 and 37 to females with undernutrition induced complete vaginal opening in seven of 12 treated animals (60%), with a mean body weight of 67.6 ± 1.0 g. As additional indices of rescue of pubertal activation of the reproductive axis, serum LH and estradiol levels were significantly increased in every single food-restricted female rat after repeated icv injection of kisspeptin (Fig. 5). Similarly, FSH levels in kisspeptin-treated females were significantly higher than in underfed animals injected with vehicle (5.2 ± 0.77 ng/ml vs. 2.48 ± 0.3 ng/ml).
Discussion
Activation of the reproductive axis at puberty, and its subsequent functioning in adulthood, critically depends on sufficient energy stores (6, 7, 8). It is assumed that the mechanism whereby situations of energy deficit ultimately impair fertility involves a decrease in the activity of the hypothalamic GnRH pulse generator (8). A conspicuous feature, however, of this phenomenon is that GnRH gene expression at the hypothalamus is not apparently reduced in fasting conditions (28). This suggests that central modulation of the GnRH-gonadotropic axis by the energy status and metabolic factors likely takes place at the level of upstream regulatory signals (involved in the control of GnRH release), whose nature is yet to be fully determined. Among others, a pivotal role for the KiSS-1 system in the central control of the reproductive axis has very recently arisen (10). Indeed, different reports have now provided compelling evidence on the ability of kisspeptins to directly activate GnRH neurons and to elicit GnRH secretion (18, 19, 23) (present results). Yet, despite the proposed function of the KiSS-1 system as a major gatekeeper of GnRH neurons (15, 19), its potential involvement in conveying the impact of (insufficient) body energy stores onto the reproductive axis remains to be determined. To our knowledge, the present study provides the first comprehensive mechanistic overview of the interaction between energy status and the KiSS-1 system in the central control of puberty onset and gonadotropin secretion.
The experimental data presented herein evidence that situations of negative energy balance induce a decrease in the hypothalamic expression of kisspeptin, as suggested by the significant reduction in the relative levels of KiSS-1 mRNA observed in prepubertal male and female rats under short-term fasting. Considering its physiological role in the control of GnRH neurons, it is reasonable to predict that reduced KiSS-1 signaling might operate as a major contributing factor for the reproductive failure (i.e. hypogonadotropic hypogonadism) induced by food deprivation. Additional evidence for alterations in the KiSS-1 system in situations of energy deficit comes from the observation of increased GPR54 mRNA levels at the hypothalamus in fasted animals. A tempting explanation for such a paradoxical finding is that a primary decrease in ligand (KiSS-1) expression might bring about a compensatory increase in the expression of its putative receptor (GPR54), inducing a state of sensitization to the effects of kisspeptin. Indeed, this is apparently the case, as evidenced by our in vivo and in vitro data in short-term fasting. Thus, LH responses to kisspeptin in vivo were preserved, and even enhanced, in food-deprived animals, suggesting that replacement of endogenous KiSS-1 levels is sufficient to fully activate the GnRH-LH axis despite adverse metabolic conditions. Likewise, the sensitivity to kisspeptin in terms of induction of GnRH secretion in vitro was significantly enhanced (as evidenced by lower mean and minimal effective doses), and its maximal responses increased, in fasted animals. On the former, it has to be noted that 10–10 M kisspeptin was able to elicit GnRH release in vitro in underfed animals but not in those fed ad libitum, which support the contention that the sensitivity to kisspeptin is significantly increased in situations of negative energy balance. Interestingly, in our setting, basal release of GnRH was significantly reduced (2.0-fold) by previous short-term fasting. Such a decrease, however, was fully reversed by incubation of hypothalamic fragments from fasted animals in the presence of 10–10 M kisspeptin (see Fig. 3). This suggests that the physiological level of hypothalamic kisspeptin may range between 10 and 100 pM and that its replacement in conditions of negative energy balance is apparently sufficient to restore normal GnRH release.
Assumably, the molecular basis for the observed changes in the expression and function of the KiSS-1 system in fasting conditions is yet to be elucidated, but in principle, different signals with proven actions in the joint control of energy balance and reproduction might be involved. Among others, the adipocyte hormone leptin has been identified as a pivotal peripheral factor for signaling the amount of body energy stores to the hypothalamic centers governing reproduction. However, the ultimate mechanisms whereby leptin conducts this relevant function are still a matter of debate, and direct or indirect effects of leptin upon GnRH neurons have been proposed (8). Interestingly, leptin has been involved in the control of the hypothalamic expression of galanin-like peptide (GALP), another potent elicitor of GnRH-LH secretion, and a sensitization phenomenon, analogous to that reported herein for KiSS-1, has been recently described for the LH-releasing effects of GALP in conditions of leptin insufficiency (29). Indeed, hyperresponsiveness to a number of elicitors of the hypothalamic-pituitary-gonadal axis is apparently detected in conditions of negative energy balance and/or hypoleptinemia, a phenomenon where the potential contribution of altered KiSS-1 functions (as a relevant downstream regulator of the GnRH system) merits further investigation. Nonetheless, whether similar mechanisms underlie the reported sensitization to kisspeptin and GALP is yet to be determined. In fact, our preliminary observations evidence that, in contrast to GALP, KiSS-1 mRNA levels at the hypothalamus are not significantly modified in conditions of leptin deficiency (i.e. ob/ob mouse) (our unpublished data), suggesting that leptin is not an essential regulator of KiSS-1 gene expression at the hypothalamus. Alternatively, leptin modulation of the KiSS-1 system may take place at a posttranscriptional step, and/or additional neuroendocrine integrators might be involved. The latter possibility is presently under investigation at our laboratory.
Expression of KiSS-1 and GPR54 mRNAs, as well as metastin-like immunoreactivity, has been detected in several hypothalamic areas involved in feeding regulation (21, 30, 31), including the arcuate nucleus. This area has been highlighted as a major center for the integrated regulation of food intake, where neuropeptide Y (NPY)/Agouti-related peptide (orexigenic) and proopiomelanocortin/cocaine- and amphetamine-regulated transcript (anorexigenic) neurons reciprocally operate under the regulation of peripheral factors, such as leptin (29 and references therein). Our present results document, however, that central injection of kisspeptin is not able to significantly alter the pattern of food intake, either in rats fed ad libitum or subjected to previous 12 h of fasting. In good agreement, intracerebral administration of kisspeptin, at a dose effective to maximally elicit LH secretion, failed to change hypothalamic expression levels of NPY, Agouti-related peptide, proopiomelanocortin, and cocaine- and amphetamine-regulated transcript mRNAs (our unpublished data). Altogether, our current results demonstrate that, although body energy stores impact the expression and function of the KiSS-1 system at the hypothalamus, kisspeptin is not provided with specific regulatory actions upon feeding. In fact, during conduction of this study, this contention was further supported by an independent study (23). This finding is contrast with other well-known regulators of food intake and the reproductive axis, such as leptin, NPY, orexin, GALP, and likely ghrelin, for which direct, independent actions in the control of feeding and reproductive axis have been reported (9, 29, 32, 33), and strengthens the role of the KiSS-1 system as a selective downstream regulatory signal essential for the proper function of the GnRH-LH axis.
Additional evidence for the pivotal involvement of the KiSS-1 system in the mechanisms whereby energy status controls activation of the reproductive axis at puberty is provided by our experiments involving chronic administration of kisspeptin in conditions of undernutrition. In our setting, a persistent decrease in daily food intake of 30% from controls was able to totally block normal pubertal development, as estimated by lack of vaginal opening and decreased serum gonadotropin levels. In this model, repeated administration of kisspeptin was sufficient to restore vaginal opening in a significant number (60%) of cases as well as to induce gonadotropin and estrogen secretion in every single animal tested. On the basis of present and previous data (18, 19, 23), this activational response is thought to be mediated by central stimulation of the GnRH system. Yet, in keeping with our previous results (24), such an effect did not involve increased hypothalamic expression of GnRH mRNA, nor was it associated with significant changes in the relative levels of GnRH receptor mRNA at the pituitary (our unpublished observations). The reasons for the lack of vaginal opening in a proportion of food-restricted females injected with kisspeptin remain to be solved, but subtle individual variations in the responses to subnutrition and/or kisspeptin administration cannot be excluded. Moreover, it is plausible that occurrence of vaginal opening might have taken place in the remaining kisspeptin-injected animals (which actually had significantly elevated LH and estradiol levels over vehicle-treated underfed females at d 37) if longer treatment protocols could have been implemented.
The relevance of the present observations is stressed by the fact that, although kisspeptin was able to rescue vaginal opening in a large percentage of food-restricted females, the very same treatment protocol (between d 30 and 37 postpartum) dramatically depressed serum LH levels and prevented normal occurrence of canalization of the vagina in more than 50% of normally fed female rats at puberty (manuscript in preparation), i.e. when full activation of the endogenous KiSS-1 system occurs (17). This illustrates the exquisite sensitivity of this system not only to down-regulation (as observed in food deprivation) but also to hyperactivation, where desensitization of the gonadotropic axis is likely taking place. Additional studies, involving repeated administration of kisspeptin at different stages of maturation, and testing of hypothalamic secretion and pituitary sensitivity to GnRH, are presently in progress in our laboratory to cover this relevant issue. Nonetheless, although ultimate occurrence of ovulation as definitive proof of complete puberty was not monitored in our experimental setting, in the context of the reported decrease in KiSS-1 expression in situations of negative energy balance, our present data (involving assessment of different end-points such as pituitary LH and FSH secretion, ovarian-derived estrogen levels, and vaginal opening as biomarker of estrogenic action) strongly suggest that restoration of endogenous KiSS-1 tone is sufficient to rescue (at least partially) defective pubertal activation of the reproductive axis in undernutrition. This is not only relevant from a mechanistic standpoint, but it may pose also interesting therapeutic implications, especially considering the extraordinarily potent gonadotropin-releasing activity of systemically delivered kisspeptins (24, 25).
In summary, we provide herein an integral analysis of the potential interaction of energy balance and the KiSS-1 system in the control of reproductive function, with special attention to the role of this novel signaling system in conveying the impact of (insufficient) body energy stores onto the gonadotropic axis at puberty. Our data demonstrate for the first time that, despite its lack of direct effects on feeding and related neuropeptide gene expression, food deprivation induces significant changes in the expression and function of the central (hypothalamic) KiSS-1 system, which may represent a previously uncharacterized target for disruption (and eventual therapeutic intervention) of pubertal development in conditions of negative energy balance.
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
This work was supported by Grants BFI 2000-0419-CO3-03 and BFI 2002-00176 from DGESIC (Ministerio de Ciencia y Tecnología, Spain), funds from Instituto de Salud Carlos III (Red de Centros RCMN C03/08 and Project PI042082; Ministerio de Sanidad, Spain), and EU research contract EDEN QLK4-CT-2002-00603.
1 J.M.C. and V.M.N. equally contributed to this work and should be considered as joint first authors.
Abbreviations: CT, Cycle threshold; GALP, galanin-like peptide; GPR54, G protein-coupled receptor 54; icv, intracerebroventricular; NPY, neuropeptide Y.
References
Fink G 2000 Neuroendocrine regulation of pituitary function: general principles. In: Conn PM, Freeman ME, eds. Neuroendocrinology in physiology and medicine. Totowa, NJ: Humana Press; 107–134
Tena-Sempere M, Huhtaniemi I 2003 Gonadotropins and gonadotropin receptors. In: Fauser BCJM, ed. Reproductive medicine: molecular, cellular and genetic fundamentals. New York: Parthenon Publishing; 225–244
Ojeda SR, Urbanski HF 1994 Puberty in the rat. In: Knobil E, Neill JD, eds. The physiology of reproduction. New York: Raven Press; 363–410
Grumbach MM 2002 The neuroendocrinology of human puberty revisited. Horm Res 57:2–14
Plant TM, Barker-Gibb ML 2004 Neurobiological mechanisms of puberty in higher primates. Hum Reprod Update 10:67–77
Parent AS, Teilmann G, Juul A, Skakkebaek NE, Toppari J, Bourguignon JP 2003 The timing of normal puberty and the age limits of sexual precocity: variations around the world, secular trends, and changes after migration. Endocr Rev 24:668–693
Frisch RE 1987 Body fat, menarche, fitness and fertility. Hum Reprod 2:521–533
Cunningham MJ, Clifton DK, Steiner RA 1999 Leptin’s actions on the reproductive axis: perspectives and mechanisms. Biol Reprod 60:616–622
Barreiro ML, Tena-Sempere M 2004 Ghrelin and reproduction: a novel signal linking energy status and fertility Mol Cell Endocrinol 226:1–9
Colledge WH 2004 GPR54 and puberty. Trends Endocrinol Metab 15:448–453
Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, Terao Y, Kumano S, Takatsu Y, Masuda Y, Ishibashi Y, Watanabe T, Asada M, Yamada T, Suenaga M, Fujino C, Usuki S, Kurokawa T, Onda H, Nishimura O, Fujino M 2001 Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G protein-coupled receptor. Nature 411:613–617
Muir AI, Chamberlain L, Elshourbagy NA, Michalovich D, Moore DJ, Calamari A, Szekeres PG, Sarau HM, Chambers JK, Murdock P, Steplewski K, Shabon U, Miller JE, Middleton SE, Darker JG, Larminie CGC, Wilson S, Bergsma DJ, Emson P, Faull R, Philpott KL, Harrison DC 2001 AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1. J Biol Chem 276:28969–28975
Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, Le Poul E, Brezillon S, Tyldesley R, Suarez-Huerta N, Vandeput F, Blanpain C, Schiffmann SN, Vassart G, Parmentier M 2001 The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. J Biol Chem 276:34631–34636
de Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E 2003 Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci USA 100:10972–10976
Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno JS, Shagoury JK, Bo-Abbas Y, Kuohung W, Schwinof KM, Hendrick AG, Zahn D, Dixon J, Kaiser UB, Slaugenhaupt SA, Gusella JF, O’Rahilly S, Carlton MB, Crowley WF, Aparicio SA, Colledge WH 2003 The GPR54 gene as a regulator of puberty. N Engl J Med 349:1614–1627
Semple RK, Achermann JC, Ellery J, Farooqi IS, Karet FE, Stanhope RG, O’Rahilly S, Aparicio SA 2005 Two novel missense mutations in G-protein-coupled receptor 54 in a patient with hypogonadotropic hypogonadism. J Clin Endocrinol Metab 90:1849–1855
Navarro VM, Castellano JM, Fernandez-Fernandez R, Barreiro ML, Roa J, Sanchez-Criado JE, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M 2004 Developmental and hormonally regulated messenger ribonucleic acid expression of KiSS-1 and its putative receptor GPR54 in rat hypothalamus and potent LH releasing activity of KiSS-1 peptide. Endocrinology 145:4565–4574
Irwig MS, Fraley GS, Smith JT, Acohido BV, Popa SM, Cunningham MJ, Gottsch ML, Clifton DK, Steiner RA 2004 Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat. Neuroendocrinology 80:264–272
Shahab M, Mastronardi C, Seminara SB, Crowley WF, Ojeda SR, Plant TM 2005 Increased hypothalamic GPR54 signaling: a potential mechanism for initiation of puberty in primates. Proc Natl Acad Sci USA 102:2129–2134
Matsui H, Takatsu Y, Kumano S, Matsumoto H, Ohtaki T 2004 Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat. Biochem Biophys Res Commun 320:383–388
Gottsch ML, Cunningham MJ, Smith JT, Popa SM, Acohido BV, Crowley WF, Seminara S, Clifton DK, Steiner RA 2004 A role for kisspeptins in the regulation of gonadotropin secretion in the mouse. Endocrinology 145:4073–4077
Navarro VM, Fernandez-Fernandez R, Castellano JM, Roa J, Mayen A, Barreiro ML, Gaytan F, Aguilar E, Pinilla L, Dieguez C, Tena-Sempere M 2004 Advanced vaginal opening and precocious activation of the reproductive axis by KiSS-1 peptide, the endogenous ligand of GPR54. J Physiol 561:379–386
Thompson EL, Patterson M, Murphy KG, Smith KL, Dhillo WS, Todd JF, Ghatei MA, Bloom SR 2004 Central and peripheral administration of kisspeptin-10 stimulates the hypothalamo-pituitary-gonadal axis. J Neuroendocrinol 16:850–858
Navarro VM, Castellano JM, Fernandez-Fernandez R, Tovar S, Roa J, Mayen A, Nogueiras R, Vazquez MJ, Barreiro ML, Magni P, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M 2005 Characterization of the potent LH releasing activity of KiSS-1 peptide, the natural ligand of GPR54. Endocrinology 146:156–163
Navarro VM, Castellano JM, Fernandez-Fernandez R, Tovar S, Roa J, Mayen A, Barreiro ML, Casanueva FF, Aguilar E, Dieguez C, Pinilla L, Tena-Sempere M 2005 Effects of KiSS-1 peptide, the natural ligand of GPR54, on follicle-stimulating hormone secretion in the rat. Endocrinology 146:1689–1697
Messager S, Chatzidaki EE, Ma D, Hendrick AG, Zahn D, Dixon J, Thresher RR, Malinge I, Lomet D, Carlton MB, Colledge WH, Caraty A, Aparicio SA 2005 Kisspeptin directly stimulates gonadotropin-releasing hormone secretion via G protein-coupled receptor 54. Proc Natl Acad Sci USA 102:1761–1766
Higuchi R, Fockler C, Dollinger G, Watson R 1993 Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology 11:1026–1030
Bergendahl M, Wiemann JN, Clifton DK, Huhtaniemi I, Steiner RA 1992 Short-term starvation decreases POMC mRNA but does not alter GnRH mRNA in rat brain. Neuroendocrinology 56:913–920
Kumano S, Matsumoto H, Takatsu Y, Noguchi J, Kitada C, Ohtaki T 2003 Changes in hypothalamic expression levels of galanin-like peptide in rat and mouse models support that it is a leptin-target peptide. Endocrinology 144:2634–2643
Lee DK, Nguyen T, O’Neill GP, Chang R, Liu Y, Howard AD, Coulombe N, Tan CP, Tang-Nguyen AT, George SR, O’Dowd BF 1999 Discovery of a receptor related to the galanin receptors. FEBS Lett 446:103–107
Brailoiu GC, Dub SL, Ohsawa M, Yin D, Yang J, Chang JK, Brailoiu E, Dun NJ 2005 KiSS-1 expression and metastin-like immunoreactivity in the rat brain. J Comp Neurol 481:314–329
Tena-Sempere M, Barreiro ML 2002 Leptin in male reproduction: the testis paradigm. Mol Cell Endocrinol 188:9–13
Russell SH, Small CJ, Kennedy AR, Stanley SA, Seth A, Murphy KG, Taheri S, Ghatei MA, Bloom SR 2001 Orexin A interactions in the hypothalamo-pituitary gonadal axis. Endocrinology 142:5294–5302(J. M. Castellano1, V. M. )