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Department of Cell Biology, Physiology and Immunology (J.R., J.M.C., V.M.N., R.F.-F., E.A., L.P., M.T.-S.), University of Córdoba, 14004 Córdoba, Spain; and Departments of Physiology (C.D.) and Medicine (F.F.C.), University of Santiago de Compostela, 15705 Santiago de Compostela, Spain
Address all correspondence and requests for reprints to: Manuel Tena-Sempere, Physiology Section, Department of Cell Biology, Physiology and Immunology, Faculty of Medicine, University of Córdoba, Avenida Menéndez Pidal s/n, 14004 Córdoba, Spain. E-mail: fi1tesem{at}uco.es.
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In this scenario, a major breakthrough in our understanding of the systems controlling puberty onset and reproductive function in mammals came in late 2003 by the identification of the causative link between inactivating mutations in the gene encoding the G protein-coupled receptor 54 (GPR54) and forms of hypogonadotropic hypogonadism in humans and mice (6, 7). These seminal observations drew immediate attention onto the reproductive roles of the ligands of GPR54, a family of structurally related peptides, encoded by the metastasis-suppressor KiSS-1 gene, globally termed kisspeptins (8, 9, 10). Indeed, in the last year, a number of studies on the reproductive facet of the KiSS-1 system have demonstrated that hypothalamic expression of KiSS-1 gene, and to a lower extent of GPR54, is developmentally (maximum at puberty) and hormonally (by sex steroids) regulated (11, 12, 13, 14, 15). In addition, pharmacological tests have now proven the extraordinary potency of kisspeptins in inducing gonadotropin release in a number of species, such as the rat, mouse, sheep, monkey, and, very recently, the human (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23). Moreover, functional studies have evidenced a relevant role of KiSS-1 signaling in the timing of puberty onset in rodent and primate species (13, 18). In terms of mechanism of action, it is globally accepted that the effects of kisspeptins upon the gonadotropic axis primarily stem from direct stimulatory actions upon the hypothalamic GnRH system, as activation of GnRH neurons and GnRH release by kisspeptins have been reported (12, 19, 22, 24). Yet, the possibility of additional sites of action of kisspeptins cannot be ruled out (18). Altogether, this body of evidence strongly supports the contention that the KiSS-1 system is a major gatekeeper of reproductive function in mammals (25).
Despite considerable progress in the field, most of the physiological and pharmacological studies on the reproductive roles of the KiSS-1 system have been conducted in pubertal animals or adult male rodents (11, 12, 13, 14, 16, 17, 18, 19, 20, 21). Indeed, although the functionality of the gonadotropic axis in the cyclic adult female undergoes striking changes, both along the cycle and across the reproductive life span, limited attention has been paid so far to the characterization of the role of KiSS-1 system at this period and, to our knowledge, testing of the gonadotropin-releasing effects of kisspeptin at different functional states of the female reproductive axis had not been reported to date. To cover this relevant aspect of KiSS-1 physiology, the present experimental work was undertaken to evaluate LH and FSH secretory responses to kisspeptin-10 (maximal responsiveness and/or sensitivity to low doses) in different physiological (different stages of the estrous cycle, mid- and late pregnancy, and early lactation) and experimental [ovariectomy (OVX) with or without sex steroid replacement] settings in the adult female rat. In addition, when relevant, pharmacological tests were complemented with expression analyses of KiSS-1 and GPR54 genes at the hypothalamus is some of the above experimental models.
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Experimental designs
In experiment 1, the gonadotropin-releasing effects of kisspeptin were tested in adult female rats in vivo, at different stages of the estrous cycle. To this end, adult virgin female rats, weighing 210 ± 15 g, were monitored for estrous cyclicity by daily vaginal cytology. Only rats with at least two consecutive regular 4-d estrous cycles were used in the subsequent pharmacological studies. Groups of cyclic females (n = 10–12 per phase), at estrus, metaestrus or diestrus-1 (D1), diestrus-2 (D2), and proestrus, were selected for functional testing. All tests were conducted between 0900 and 1000 h (i.e. in the morning of the corresponding stage of the cycle), except for proestrus, when tests were conducted at 1800, coinciding with the preovulatory surges of LH and FSH (3). A protocol of intracerebroventricular (i.c.v.) administration of 1 nmol kisspeptin-10 was carried out, as described elsewhere (11, 20, 21, 24). To allow delivery of kisspeptin into the lateral cerebral ventricle, the animals were implanted with i.c.v. cannulae lowered to a depth of 3 mm beneath the surface of the skull; the insert point was 1 mm posterior and 1.2 mm lateral to bregma. The 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 and FSH secretion in pubertal rats and adult males (11, 20, 21). Blood samples were obtained by jugular veni-puncture before (0 min) and at 15 and 60 min after kisspeptin injection. Animals injected with vehicle (NaCl 0.9%) served as controls.
In experiment 2, the gonadotropin responses to kisspeptin were studied in gonadectomized females, with or without sex steroid replacement. Regularly cycling adult virgin female rats were subjected to bilateral OVX, under ether anesthesia, at random stages of the estrous cycle. At the time of surgery, groups of animals (n = 10–12) were implanted with SILASTIC brand silicon tubing (Dow Corning, Midland, MI) elastomers (20 mm length; inner diameter, 0.062 cm; exterior diameter, 0.125 cm) containing estradiol or progesterone. Selection of dosage and capsule length was based on previous physiological studies in the OVX female rat (26, 27). Additional experimental groups (n = 10–12) were implanted with empty capsules (OVX without replacement), or capsules containing estradiol and progesterone. Functional testing of in vivo gonadotropic responses to 1 nmol kisspeptin-10 was conducted at d 7 after OVX, after a procedure similar to that of experiment 1. In addition, expression analyses of KiSS-1 and GPR54 mRNAs at the hypothalamus were conducted in this experimental setting. To this end, additional groups of OVX animals (n = 5), either bearing empty capsules or elastomers containing estradiol, progesterone, or estradiol and progesterone, were generated as described above, and hypothalamic samples were excised at d 7 after OVX (with or without replacement), and stored at –80 C until use for RNA analysis.
In an additional set of experiments, LH and FSH secretory responses to kisspeptin, as well as expression of KiSS-1 and GPR54 genes at the hypothalamus, were studied in two relevant reproductive states of the adult female, namely pregnancy and lactation, when striking changes in the functionality of the gonadotropic axis take place (4, 5). Studies on KiSS-1 function at pregnancy were carried out in experiment 3. In detail, functional tests and expression analyses were implemented at two distinct time-points pregnancy: d 13.5 (mid-pregnancy) and d 20.5 (late pregnancy). For comparative purposes, a group of cyclic females at D1 was also included in the analysis. For timing of pregnancy, cycling female rats were placed, at the afternoon of proestrus, in individual cages with adult males of proven fertility. In the morning of the following day, vaginal smears were collected and the presence of spermatozoa was checked; those showing spermatozoa at vaginal smears were considered at d 0.5 of pregnancy and included for pharmacological tests or tissue sampling. For the former, groups of pregnant dams (n = 10–12) were implanted with i.c.v. cannulae and a protocol of central injection of kisspeptin-10 and blood sampling, similar to that described for experiment 1, was carried out. Additional groups of dams (n = 5), at d 13.5 and 20.5 of pregnancy, were taken for sampling of hypothalamic tissue. Dissection and processing of hypothalamic samples for RNA analysis were conducted as described in experiment 2.
In experiment 4, the functionality of KiSS-1 system was studied by means of pharmacological testing and expression analyses in female rats at early stages of lactation. The day of delivery was considered d 1 of lactation. At this time, litter size was adjusted to 10 pups, and lactating rats were allowed to suckle their pups undisturbed. Groups of lactating dams (n = 10–12) were implanted with i.c.v. cannulae and, on d 5 of lactation, were subjected to a protocol of central administration of 1 nmol kisspeptin-10 and blood sampling as described in previous experiments. For comparative purposes, a group of cyclic females at D1 was also included in the in vivo tests. Additional groups of lactating dams (n = 5), at d 5 of lactation, were taken for sampling of hypothalamic tissue. Dissection and processing of hypothalamic samples for RNA analysis were conducted as described in experiments 2 and 3.
In previous experiments, gonadotropin responses to a high dose (1 nmol/rat) of kisspeptin were explored in different functional states of the female reproductive axis. Although this procedure allowed us testing of maximal responsiveness, it did not provide information concerning changes in gonadotropin sensitivity to kisspeptin stimulation. To cover the latter, in experiment 5, the effects of low doses of kisspeptin-10 upon LH and FSH secretion were assayed in representative physiological states of the adult female rat. To minimize the number of animals used, and considering results from experiments 1–4, groups of cyclic females (n = 10–12) at D1 and estrus were selected for functional testing. In addition, groups of pregnant females (at d 13.5 of pregnancy; mid-gestation) and lactating dams (at d 5 of lactation) were also tested (n = 8–11). General procedures for animal selection and central (i.c.v.) injection of kisspeptin were as described in experiments 1, 3, and 4. Based in our previous dose-response analyses in the male rat (23, 24), two doses of kisspeptin-10 (0.1 and 10 pmol/rat) were selected for assessment of gonadotropin responses at the low dose range. Blood samples were obtained by jugular venipuncture 15 and 60 min after kisspeptin injection. Animals injected with vehicle served as controls.
RNA analysis by semiquantitative (semi-Q) RT-PCR
Total RNA was isolated from hypothalamic samples using the single-step, acid guanidinium thiocyanate-phenol-chloroform extraction method. Hypothalamic expression of KiSS-1 and GPR54 mRNAs was assessed by RT-PCR, optimized for semi-Q detection, using previously defined primer pairs and conditions (11, 24). 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. 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 (11), 32 and 24 PCR cycles were chosen for semi-Q 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). General procedures for real-time RT-PCR were as previously described (11, 24). The synthesized cDNAs were further amplified (1/10th) 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 MgCl2, and 2.5 U iTaq DNA polymerase (Bio-Rad Laboratories), 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 and 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 (28). 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. 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 National Institutes of Health (Dr. A. F. Parlow, National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Peptide Program, Torrance, CA). Rat LH-I-9 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 experimental groups (experiment 2), serum estradiol and progesterone levels were measured using commercial kits purchased from MP Biomedicals (Costa Mesa, CA), following the instructions of the manufacturer. The sensitivity of the assay was 0.5 and 20 pg/tube for estradiol and progesterone, respectively; all samples were measured in the same assay.
Presentation of data and statistics
Hormonal determinations were conducted in duplicate, with a minimal total number of 10 samples per group. When appropriate, besides individual time-point determinations, integrated LH and FSH secretory responses were calculated as the area under the curve (AUC), calculated following the trapezoidal rule, over the 60-min period after administration of kisspeptin. Semi-Q RT-PCR analyses were carried out in duplicate from five 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 single or repeated ANOVA followed by Student-Newman-Keuls multiple range test (SigmaStat 2.0; Jandel Corp., San Rafael, CA). P 
0.05 was considered significant.
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9.0-fold increase in terms of integrated LH secretion vs. corresponding controls at D1; P < 0.01). Such an increase was completely blunted by simultaneous replacement with estradiol (terminal serum estradiol concentrations: 206.5 ± 41.9 vs. 21.9 ± 3.6 pg/ml in OVX animals), whereas substitutive administration of progesterone only marginally reduced basal LH levels in OVX females. Yet, effective release of progesterone was confirmed by specific RIA, with terminal serum concentrations grossly similar to those of females at diestrus (data not shown). Hormonal replacement by a combination of estradiol plus progesterone was maximally effective in suppressing stimulated LH levels in OVX animals, with a reduction which was significantly higher than that induced by estradiol alone. In this experimental scenario, central administration of kisspeptin-10 elicited significant LH secretory responses in all the settings tested. However, the magnitude of such responses, in terms of absolute levels and relative increases over corresponding control values, strikingly varied among the experimental groups. In OVX animals, despite highly stimulated basal levels, kisspeptin was able to evoke a further increase in LH concentrations of approximately 2.0-fold over vehicle-injected controls. Likewise, in OVX females replaced with progesterone alone, basal levels were highly elevated but kisspeptin elicited a similar 2.0-fold increase in serum LH concentrations. Administration of estradiol to OVX rats not only resulted in a drastic suppression of basal LH levels but also induced a significant attenuation of absolute LH secretory responses of kisspeptin (P < 0.01 vs. OVX animals). Yet, due to suppression of basal concentrations, relative responses over corresponding controls were of approximately 3.0-fold increase. Strikingly, despite maximal suppression of basal LH levels, combined replacement with estradiol and progesterone significantly augmented the magnitude of LH secretory responses to kisspeptin, with an approximately 22.0-fold increase in terms of integrated LH responses vs. corresponding controls (Fig. 3). Of note, when comparing LH responses to kisspeptin between OVX + E and OVX + E/P groups, it became apparent that the magnitude of such responses was 3.5 times higher in terms of absolute LH levels, and 7.0 times higher in terms of relative responses, in OVX rats supplemented with estradiol and progesterone than in OVX animals replaced with estradiol alone.
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10.0-fold increase in terms of integrated FSH secretion vs. corresponding controls at D1; P < 0.01). Such an increase was considerably attenuated by simultaneous replacement with estradiol. In contrast, substitutive administration of progesterone failed to reduce the stimulated FSH levels in OVX females, neither did it enhance the magnitude of estradiol-induced FSH suppression when coadministered with the estrogen. In contrast to LH responses, central injection of kisspeptin-10 only marginally elicited FSH secretion in OVX rats (1.2-fold increase over controls). Such a modest 1.2-fold increase in serum FSH levels was also observed after kisspeptin injection to OVX rats treated with estradiol; yet, absolute FSH levels after kisspeptin administration in this group were approximately one fourth of those observed in OVX animals. In contrast, kisspeptin-10 failed to further increase serum FSH levels in OVX rats replaced with progesterone. Finally, combined replacement of OVX rats with estradiol and progesterone allowed a marginal enhancement of FSH secretory responses to kisspeptin, with a relative increase of approximately 1.8-fold over corresponding controls; i.e. the magnitude of kisspeptin responses was approximately 50% higher, both in terms of absolute FSH levels and relative increase, in OVX rats supplemented with estradiol and progesterone than in OVX animals replaced with estradiol alone (Fig. 4).
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, 6, and 7), and was aimed at limiting the number of experimental animals used. In keeping with our previous experiments, in vehicle-injected animals, basal LH concentrations appeared similar in cyclic female at estrus and diestrus, but were dramatically suppressed (P < 0.01) in pregnant and lactating females. In contrast, serum FSH levels were elevated at the morning of estrus and mildly decreased in lactation. Central administration of a dose as low as 0.1 pmol kisspeptin-10 induced significant LH responses in D1 and pregnant females, whereas i.c.v. injection of 10 pmol kisspeptin was able to elicit unambiguous LH secretory bursts in all the models tested, except for lactation. Moreover, although responses to 0.1 pmol were strikingly short-lived (only at diestrus these responses were detected at 60 min after injection), elevation of LH concentrations after central injection of 10 pmol kisspeptin persisted after 60 min in diestrus, estrus, and mid-gestation. Conversely, FSH secretion remained unaltered after central injection of low doses of kisspeptin-10 at all the functional states tested, except for pregnant rats, where a rise in serum FSH concentrations was detected 15 and 60 min after i.c.v. administration of 10 pmol kisspeptin (Fig. 9).
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One of the most remarkable observations of our present study was that, despite striking differences in preexisting serum LH levels, kisspeptin efficiently elicited LH secretory responses in the wide variety of physiological and experimental conditions tested. For instance, along the estrous cycle, despite injection at different times of the day, high doses of kisspeptin evoked clear-cut LH bursts at the morning of estrus and diestrus (low prevailing LH levels), as well as in the afternoon of proestrus (high prevailing LH levels). Interestingly, however, maximal responses to kisspeptin appeared to occur at estrus, when absolute and relative responses to kisspeptin were significantly higher than those at diestrus, despite the fact that the preovulatory LH surge had taken place less than 12 h before. Apparently, at early estrus, intracerebral injection of kisspeptin is still able to evoke a massive LH burst, reminiscent of the preovulatory secretory peak. Of note, recent data demonstrated that immunoneutralization of endogenous central metastin (kisspeptin-54) totally prevented the LH surge at proestrus (30). Taken together, these two sets of data jointly point to the major contribution of a proper balance between activation and inactivation of the hypothalamic KiSS-1 system in the precise generation of a key neuroendocrine event in the ovulatory process: the preovulatory rise in serum LH levels at the afternoon of proestrus, and its cessation thereafter. Further expression analyses, involving characterization of hypothalamic, nucleus-specific changes in KiSS-1 and GPR54 expression at the proestrus-to-estrus transition, will help to better define such a phenomenon.
From a mechanistic standpoint, the reported changes in the magnitude of the gonadotropin-releasing effects of high doses of kisspeptin (maximal responsiveness) along the estrous cycle may stem from changes in the efficiency of kisspeptin to elicit the hypothalamic release of GnRH and/or changes in pituitary sensitivity to the releasing effects of GnRH. Although the former possibility remains plausible, it is worthy noting that changes in pituitary responsiveness to GnRH in terms of LH release have been reported across the estrous cycle, with maximal responses at proestrus and minimum releasing effects at diestrus (31). Moreover, it is well established that a dramatic increase in pituitary sensitivity to GnRH is brought about at proestrus by a self-priming effect of GnRH (Ref.32 and references therein), which is strictly an estrogen-dependent phenomenon that can be significantly augmented by progesterone (33, 34). Overall, those observations might explain (at least partially) the window of maximal responsiveness to high doses of kisspeptin in cyclic females, which is detected in the afternoon of proestrus—despite very high prevailing LH levels—and is extended until the morning of estrus. Likewise, those data are also compatible with our present findings on the maximal LH-releasing capacity of kisspeptin in OVX rats supplemented with both estradiol and progesterone. In this sense, it is noticeable that by addition of progesterone, the magnitude of kisspeptin responses was augmented by 3.5 times in terms of absolute LH levels, and by 7.0 times in terms of relative responses, vs. those of OVX rats replaced with estradiol alone. This observation clearly evidences the need for proper preexposure to estradiol and progesterone to achieve maximal LH releasing responses to kisspeptin in the reproductive female.
Interestingly, although maximal responses to high doses of kisspeptin appeared the highest at estrus, the sensitivity to low doses of kisspeptin in terms of LH release was not enhanced at this stage of the cycle. On the contrary, doses as low as 0.1 pmol kisspeptin-10 efficiently elicited LH secretion at diestrus but not in estrous females, where significant LH bursts were only observed after central injection of 10 pmol. These observations suggest that the sensitivity and maximal responsiveness to kisspeptin are distinctly regulated along the estrous cycle. Moreover, different mechanisms (e.g. GnRH responsiveness to kisspeptin, pituitary sensitivity to GnRH) might contribute to such a disparate regulation. Of note, a similar dichotomy seems to be operative also during pubertal maturation as, despite decreased sensitivity to low doses of kisspeptin is detected in juvenile mice, these are able to generate much higher net LH responses to high doses of kisspeptin than adult animals (35).
In contrast to hormonal data, our expression analyses in OVX models evidenced that progesterone does not seem to play a major role in the regulation of hypothalamic KiSS-1 gene expression, which appeared to be solely inhibited by estrogen. In the mouse, a prominent population of KiSS-1 neurons at the arcuate nucleus has been proposed as an essential element in relaying the inhibitory feedback actions of estrogen upon the GnRH/LH system (14, 15). Assuming that most of the KiSS-1 signal in our RT-PCR assays (using whole hypothalamic fragments) comes from the arcuate nucleus (as KiSS-1 mRNA was negatively regulated by estrogen), it is tempting to propose that, at this site, progesterone is devoid of major regulatory actions. This observation is in line with the marginal feedback suppression of serum LH levels in OVX rats supplemented with progesterone alone (see Fig. 3). In addition, progesterone also failed to modify hypothalamic mRNA levels of GPR54. Altogether, our present and previous data (see Ref.11) jointly point out that sex steroid regulation of hypothalamic KiSS-1 system in the female rat is mostly conducted by estrogen through modulation of KiSS-1 mRNA levels.
In addition to responses along the estrous cycle, the ability of high doses of kisspeptin-10 to elicit LH secretion was also tested at two relevant reproductive states of the adult female, namely pregnancy and lactation, when, among others, important changes in the functionality of the gonadotropic axis do take place (4, 5). At pregnancy, a tendency toward a decrease has been documented for serum LH levels, which become the lowest at mid-pregnancy and tend to recover by the end of gestation (4, 36). Considering the very potent LH releasing ability of systemically delivered KiSS-1 peptides in different species (16, 20, 21, 22, 23), this phenomenon seems to be at odds with the reported dramatic increase in serum metastin (kisspeptin-54) concentrations in human pregnancy (37). Assuming that a similar increase in circulating kisspeptin levels does take place in rat pregnancy, we speculated that central desensitization to the releasing effects of kisspeptin-10 might occur during gestation, thus contributing to the reported profiles of low circulating LH. Surprisingly, however, despite the suppression (to a variable degree) of basal levels, LH responses to a high (1 nmol) dose of kisspeptin-10 were not only preserved during gestation, but equaled in terms of absolute levels, and even exceeded in terms of relative responses, those observed at estrus in the cyclic female rat (i.e. the stage of maximal responsiveness to kisspeptin along the cycle). Moreover, significant LH responses were observed after central injection of doses as low as 0.1 pmol, suggesting a preserved (if not enhanced) sensitivity to kisspeptin during gestation. In good agreement, relative levels of GPR54 mRNA at the hypothalamus remained invariant at mid- and late pregnancy; a phenomenon which would argue against a potential homologous down-regulation of this receptor by exposure to persistently elevated kisspeptin levels. The state of (enhanced) responsiveness to kisspeptin at pregnancy may derive from the combined exposure to high levels of progesterone and estradiol during gestation, as suggested by data from our OVX experiments. However, expression analyses in OVX animals supplemented with estradiol plus progesterone strongly suggest that this endocrine background cannot explain the reported changes in hypothalamic KiSS-1 mRNA levels, which appeared to increase along pregnancy; a phenomenon that might parallel the reported increase in circulating metastin levels in humans, and whose functional relevance remains to be determined. Overall, the contribution of regulatory factors, other than estrogen and progesterone, to the observed increase in hypothalamic KiSS-1 gene expression during pregnancy is strongly suggested. Nonetheless, additional mechanistic analyses are needed to reconcile the observed decrease in basal LH levels and the (predicted) elevation in serum kisspeptin during pregnancy, in face of the paradoxically preserved LH responsiveness and sensitivity to central activation of GPR54.
Besides pregnancy, another reproductive condition targeted in our study was lactation. Interestingly, lactation is defined by a well known state of suppression of GnRH and LH secretion, which is centrally driven and directly related with the suckling stimulus (5). However, the ultimate mechanism(s) whereby suckling information is relied onto the hypothalamic GnRH system remains to be fully unraveled. Of particular note, despite the presumed suppression of GnRH neuronal activity during lactation, GnRH mRNA levels are conserved at this state, thus suggesting the crucial role of regulatory elements up-stream the GnRH neuron (5). Given the proposed role of central KiSS-1 system as major gatekeeper of the GnRH/LH axis (25), we explored whether substantial changes in terms of kisspeptin responsiveness and sensitivity, as well as in hypothalamic KiSS-1 gene expression, take place at early lactation. Our results evidenced that, despite dramatic suppression of circulating LH to virtually negligible levels, central injection of a maximal dose of kisspeptin-10 was able to elicit significant LH secretory bursts, which were only two thirds of those seen in cycling females at D1. Of note, however, sensitivity to kisspeptin appeared to be dramatically reduced during lactation, as no LH responses were detected after i.c.v. injection of 0.1 and 10 pmol doses, which were fully effective in diestrous females. It is noticeable that other well-known elicitors of the gonadotropic axis, such as the excitatory amino acids, failed to activate the GnRH/LH system in lactating rats (8, 38). Overall, it is tempting to propose that the suppressed function of GnRH/LH axis at lactation is not primarily due to a defective capacity to respond to maximal kisspeptin stimulation but, instead, might derive from a decrease in the sensitivity to kisspeptin and/or suppression of its central tone. Interestingly, the hypothalamic KiSS-1 system has been proposed as an essential down-stream integrator relaying a variety of regulatory inputs (such as sex steroids and energy status) onto GnRH neurons (11, 14, 15, 24, 28). This feature makes it feasible that the inhibition of the gonadotropic axis during lactation might be ultimately conveyed by the suppression of KiSS-1 secretion at central levels. In our experiments, transition from pregnancy to lactation was associated to a clear-cut decrease in hypothalamic KiSS-1 mRNA levels. However, at early lactation, neither KiSS-1 nor GPR54 mRNA levels appeared to be lower than in reference cyclic females at diestrus. This observation casts doubts on the potential contribution of decreased hypothalamic expression of KiSS-1 to the observed suppression of the gonadotropic axis during lactation. Yet, it is to be noted that the lack of anatomical resolution of our RT-PCR assays may hamper identification of subtle, nucleus-specific changes in gene expression. Likewise, our current results cannot rule out that changes in hypothalamic KiSS-1 system at lactation may take place at a posttranscriptional level (e.g. secretion).
Finally, consistent with the fact that FSH appears to be more constitutively secreted than LH (39), changes in FSH responses to kisspeptin were less striking than those observed for LH at the different experimental scenarios tested. Indeed, a roughly conserved 2.0-fold increase in serum FSH concentrations was detected after central administration of a high dose (1 nmol) of kisspeptin-10 in cyclic female rats at diestrus, in OVX rats supplemented with estradiol plus progesterone, and in pregnant and lactating females. In contrast, marginal to negligible responses were observed in cyclic females at proestrus or estrus, as well as in OVX (even after individual replacement with estradiol or progesterone alone), despite maximal kisspeptin stimulation. In addition, significant FSH responses to low (10 pmol) doses of kisspeptin were only detected in female rats at mid-gestation, thus confirming the state of enhanced sensitivity to kisspeptin during pregnancy. Overall, these data provide further evidence for dissociated LH and FSH responses to kisspeptin at different states of the gonadotropic axis in the reproductive female, in keeping with our previous data from pubertal male rats (21). The functional relevance of these observations is yet to be determined.
In summary, in this study, we provide an integral analysis of the gonadotropin-releasing effects of kisspeptin in relevant functional states of the reproductive female rat, including the phases of the estrous cycle, pregnancy, and lactation, as well as after manipulation of key feedback signals, by means of gonadectomy with or without sex steroid replacement. Strikingly, kisspeptin-10 was able to induce robust LH (and to a lower extent FSH) responses in all the physiological settings and experimental conditions tested, which further substantiates the key role of KiSS-1-derived peptides as major elicitors of the GnRH/gonadotropin axis. Yet, differences in terms of maximal responsiveness and sensitivity to kisspeptin were evidenced across the experimental models. These, together with the observed changes in hypothalamic expression of KiSS-1 system, and the reported divergences between LH and FSH responses, will be of help to enlarge our current knowledge on the reproductive physiology of KiSS-1 system in the female, and may prove useful to define the potential therapeutic use of kisspeptin analogs in the pharmacological manipulation of the female gonadotropic axis.
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Acknowledgments |
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Footnotes |
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The authors (J.R., E.V., J.M.C., V.M.N., R.F.-F., F.F.C., C.D., E.A., L.P., and M.T.-S.) have nothing to declare.
First Published Online March 9, 2006
Abbreviations: AUC, Area under the curve; CT, cycle threshold; D1, diestrus-1; D2, diestrus-2; GPR54, G protein-coupled receptor 54; i.c.v., intracerebroventricular; OVX, ovariectomy; semi-Q, semiquantitative.
Received November 17, 2005.
Accepted for publication February 28, 2006.
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 G. Neal-Perry, D. Lebesgue, M. Lederman, J. Shu, G. D. Zeevalk, and A. M. Etgen The Excitatory Peptide Kisspeptin Restores the Luteinizing Hormone Surge and Modulates Amino Acid Neurotransmission in the Medial Preoptic Area of Middle-Aged Rats Endocrinology, August 1, 2009; 150(8): 3699 - 3708. [Abstract] [Full Text] [PDF]
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 R. M Reynolds, J. J Logie, A. K Roseweir, A. J McKnight, and R. P Millar A role for kisspeptins in pregnancy: facts and speculations Reproduction, July 1, 2009; 138(1): 1 - 7. [Abstract] [Full Text] [PDF]
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L. Pinilla, J. M. Castellano, M. Romero, M. Tena-Sempere, F. Gaytan, and E. Aguilar Delayed Puberty in Spontaneously Hypertensive Rats Involves a Primary Ovarian Failure Independent of the Hypothalamic KiSS-1/GPR54/GnRH System Endocrinology, June 1, 2009; 150(6): 2889 - 2897. [Abstract] [Full Text] [PDF]
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V. M. Navarro, M. A. Sanchez-Garrido, J. M. Castellano, J. Roa, D. Garcia-Galiano, R. Pineda, E. Aguilar, L. Pinilla, and M. Tena-Sempere Persistent Impairment of Hypothalamic KiSS-1 System after Exposures to Estrogenic Compounds at Critical Periods of Brain Sex Differentiation Endocrinology, May 1, 2009; 150(5): 2359 - 2367. [Abstract] [Full Text] [PDF]
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J. K. Hiney, V. K. Srivastava, M. D. Pine, and W. L. Dees Insulin-Like Growth Factor-I Activates KiSS-1 Gene Expression in the Brain of the Prepubertal Female Rat Endocrinology, January 1, 2009; 150(1): 376 - 384. [Abstract] [Full Text] [PDF]
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 A. S. Mechaly, J. Vinas, and F. Piferrer Identification of Two Isoforms of the Kisspeptin-1 Receptor (kiss1r) Generated by Alternative Splicing in a Modern Teleost, the Senegalese Sole (Solea senegalensis) Biol Reprod, January 1, 2009; 80(1): 60 - 69. [Abstract] [Full Text] [PDF]
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J. Roa, E. Vigo, J. M. Castellano, F. Gaytan, D. Garcia-Galiano, V. M. Navarro, E. Aguilar, F. A. Dijcks, A. G. H. Ederveen, L. Pinilla, et al. Follicle-Stimulating Hormone Responses to Kisspeptin in the Female Rat at the Preovulatory Period: Modulation by Estrogen and Progesterone Receptors Endocrinology, November 1, 2008; 149(11): 5783 - 5790. [Abstract] [Full Text] [PDF]
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 M. Torricelli, L. Galleri, C. Voltolini, G. Biliotti, P. Florio, M. De Bonis, and F. Petraglia Changes of Placental Kiss-1 mRNA Expression and Maternal/Cord Kisspeptin Levels at Preterm Delivery Reproductive Sciences, October 1, 2008; 15(8): 779 - 784. [Abstract] [PDF]
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 C. Zhang, T. A. Roepke, M. J. Kelly, and O. K. Ronnekleiv Kisspeptin Depolarizes Gonadotropin-Releasing Hormone Neurons through Activation of TRPC-Like Cationic Channels J. Neurosci., April 23, 2008; 28(17): 4423 - 4434. [Abstract] [Full Text] [PDF]
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J. Roa, E. Vigo, J. M. Castellano, F. Gaytan, V. M. Navarro, E. Aguilar, F. A. Dijcks, A. G. H. Ederveen, L. Pinilla, P. I. van Noort, et al. Opposite Roles of Estrogen Receptor (ER)-{alpha} and ER{beta} in the Modulation of Luteinizing Hormone Responses to Kisspeptin in the Female Rat: Implications for the Generation of the Preovulatory Surge Endocrinology, April 1, 2008; 149(4): 1627 - 1637. [Abstract] [Full Text] [PDF]
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 E. Vigo, J. Roa, R. Pineda, J. M. Castellano, V. M. Navarro, E. Aguilar, L. Pinilla, and M. Tena-Sempere Novel role of the anorexigenic peptide neuromedin U in the control of LH secretion and its regulation by gonadal hormones and photoperiod Am J Physiol Endocrinol Metab, November 1, 2007; 293(5): E1265 - E1273. [Abstract] [Full Text] [PDF]
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 W. S. Dhillo, O. B. Chaudhri, E. L. Thompson, K. G. Murphy, M. Patterson, R. Ramachandran, G. K. Nijher, V. Amber, A. Kokkinos, M. Donaldson, et al. Kisspeptin-54 Stimulates Gonadotropin Release Most Potently during the Preovulatory Phase of the Menstrual Cycle in Women J. Clin. Endocrinol. Metab., October 1, 2007; 92(10): 3958 - 3966. [Abstract] [Full Text] [PDF]
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A. M. Rometo, S. J. Krajewski, M. Lou Voytko, and N. E. Rance Hypertrophy and Increased Kisspeptin Gene Expression in the Hypothalamic Infundibular Nucleus of Postmenopausal Women and Ovariectomized Monkeys J. Clin. Endocrinol. Metab., July 1, 2007; 92(7): 2744 - 2750. [Abstract] [Full Text] [PDF]
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S. Yamada, Y. Uenoyama, M. Kinoshita, K. Iwata, K. Takase, H. Matsui, S. Adachi, K. Inoue, K.-I. Maeda, and H. Tsukamura Inhibition of Metastin (Kisspeptin-54)-GPR54 Signaling in the Arcuate Nucleus-Median Eminence Region during Lactation in Rats Endocrinology, May 1, 2007; 148(5): 2226 - 2232. [Abstract] [Full Text] [PDF]
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E. Vigo, J. Roa, M. Lopez, J. M. Castellano, R. Fernandez-Fernandez, V. M. Navarro, R. Pineda, E. Aguilar, C. Dieguez, L. Pinilla, et al. Neuromedin S as Novel Putative Regulator of Luteinizing Hormone Secretion Endocrinology, February 1, 2007; 148(2): 813 - 823. [Abstract] [Full Text] [PDF]
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 M. Tena-Sempere GPR54 and kisspeptin in reproduction Hum. Reprod. Update, September 1, 2006; 12(5): 631 - 639. [Abstract] [Full Text] [PDF]
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J. T. Smith, S. M. Popa, D. K. Clifton, G. E. Hoffman, and R. A. Steiner Kiss1 neurons in the forebrain as central processors for generating the preovulatory luteinizing hormone surge. J. Neurosci., June 21, 2006; 26(25): 6687 - 6694. [Abstract] [Full Text] [PDF]
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