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Departments of Physiology and Biophysics (J.T.S., M.J.C., R.A.S.) and Obstetrics and Gynecology (D.K.C., R.A.S.), University of Washington, Seattle Washington 98195-7290; and Department of Biochemistry and Molecular Genetics (E.F.R.), University of Virginia, Charlottesville, Virginia 22908
Address all correspondence and requests for reprints to: Robert A. Steiner, Department of Physiology and Biophysics, Health Sciences Building, G-424, School of Medicine, University of Washington, Box 357290, Seattle, Washington 98195-7290. E-mail: steiner{at}u.washington.edu.
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Abstract |
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or ERß, we evaluated the effects of E2 in OVX mice that lacked functional ER or ERß. In OVX mice that lacked functional ER, KiSS-1 mRNA did not respond to E2 in either the Arc or AVPV, suggesting that ER is essential for mediating the inhibitory and stimulatory effects of E2. In contrast, KiSS-1 mRNA in OVX mice that lacked functional ERß responded to E2 exactly as wild-type animals. Double-label in situ hybridization revealed that virtually all KiSS-1-expressing neurons in the Arc and AVPV coexpress ER, suggesting that the effects of E2 are mediated directly through KiSS-1 neurons. We conclude that KiSS-1 neurons in the Arc, which are inhibited by E2, may play a role in the negative feedback regulation of GnRH secretion, whereas KiSS-1 neurons in the AVPV, which are stimulated by E2, may participate in the positive feedback regulation of GnRH secretion. |
Introduction |
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) (3), it is generally thought that other steroid-sensitive neurons mediate the predominant physiological effects of sex steroids on GnRH secretion (4, 5, 6). Steroid receptors are expressed throughout the forebrain, particularly within the arcuate nucleus (Arc) and the anteroventral periventricular nucleus (AVPV) (7, 8, 9), and these areas are known to send projections to the preoptic area in close proximity to GnRH neurons (6, 10, 11, 12). Nevertheless, the phenotypic identities of steroid-sensitive neurons in the Arc and AVPV that couple directly to GnRH neurons have yet to be revealed. The Kiss1 gene codes for neuropeptides called kisspeptins, which bind to a G protein-coupled receptor, GPR54 (13, 14, 15). Kisspeptins and GPR54 have recently been implicated in the neuroendocrine regulation of GnRH secretion, based on several lines of evidence. First, mutations and targeted deletions in GPR54 in humans and mice result in sexual infantilism and hypogonadotropic hypogonadism, caused by GnRH deficiency (16, 17, 18). Second, KiSS-1 mRNA is expressed in the forebrain of the rat, mouse, and monkey (19, 20, 21), and administration of kisspeptin evokes GnRH-dependent gonadotropin secretion in these species (19, 21, 22, 23). Finally, sex steroids inhibit hypothalamic expression of KiSS-1 mRNA in the rat and monkey (20, 21, 24). Thus, it would appear that KiSS-1 neurons provide a link between the physiological action of sex steroids and the regulation of GnRH secretion; however, these initial studies of KiSS-1 regulation lack precise anatomical resolution. This limitation is compounded by the fact that KiSS-1 is expressed discretely in several hypothalamic nuclei, including the Arc and AVPV, which are each thought to play separate (and unique) roles in the regulation of GnRH and gonadotropin secretion (25, 26).
The objectives of our studies were 2-fold: to dissect the anatomical regulation of KiSS-1 mRNA expression by E2 in the hypothalamus and identify the molecular pathway through which E2 exerts its action. To study the regulation of Kiss1 gene expression as a function of area, we examined the effects of E2 on KiSS-1 mRNA expression in individual nuclei of the female mouse. To determine whether the effects of E2 were mediated by either ER or ERß, we evaluated the effects of E2 on KiSS-1 expression in mice bearing genetically targeted deletions of these receptors (ERKO and ERßKO, respectively). Finally, to determine whether E2 acts directly on KiSS-1 neurons, we performed double-label in situ hybridization for KiSS-1 mRNA coupled with ER or ERß mRNA.
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Materials and Methods |
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null mice were produced by breeding pairs, each carrying a single copy of the disrupted ER gene, as previously described (27). Female ERß null mice were purchased from Taconic (Germantown, NY). Animals were individually housed, maintained on a 12-h light, 12-h dark cycle (lights on at 0600 h) and had access to standard rodent chow and water ad libitum. All procedures were approved by the Animal Care Committee of the School of Medicine of the University of Washington as well as the Animal Use and Care Committee at the University of Virginia, in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.
Ovariectomy (OVX) and steroid replacement
Gonads were removed from adult mice under isoflurane inhalation anesthesia (Abbott Laboratory, North Chicago, IL) delivered by a vaporizer (Veterinary Anesthesia Systems, Bend, OR). Vasculature to the ovary was sutured and wound clips were used to close the incision. Immediately after OVX, E2-filled capsules were implanted sc via a small incision at the base of the neck; wound clips were used to close the incision.
For E2 implants, SILASTIC brand tubing (inner diameter = 1.47 mm; outer diameter = 1.95 mm; Dow Corning, Midland, MI) was cut to 9 mm; one end sealed with silicone cement and allowed to cure overnight. The dose of crystalline E2 (Sigma, St. Louis, MO) was based on a previous study (28) and involved packing silastic tubing with 4 mm of an E2/cholesterol mix (1:4). The day before surgery, implants were washed with 2 x 10 min changes of 100% ethanol and then placed in physiological saline overnight. All untreated animals received empty (sham) capsules.
Experimental design
Experiment 1.
The purpose of this experiment was to examine the effects of OVX and E2 replacement on hypothalamic KiSS-1 mRNA. Female mice were divided into three groups (n = 6–8 per group): ovary intact (diestrus), castrated, and castrated plus E2 replacement. For intact mice, vaginal cytology was examined for two consecutive cycles. At approximately 0900 h either on diestrus or 7 d after castration, mice were weighed, anesthetized with isoflurane, and killed by decapitation. Trunk blood was collected for E2 and LH RIA. Brains were removed for KiSS-1 mRNA in situ hybridization, frozen on dry ice, and then stored at –80 C until sectioned. Five sets of 20-µm sections in the coronal plane were cut on a cryostat (from the diagonal band of Broca to the mammillary bodies), thaw mounted onto SuperFrost Plus slides (VWR Scientific, West Chester, PA), and stored at –80 C. A single set was used for in situ hybridization (adjacent sections 100 µm apart).
Experiment 2.
The purpose of this experiment was to determine whether E2 can regulate expression of KiSS-1 mRNA in ERKO female mice. Seven female ERKO mice and seven wild-type (WT) littermates were OVX and four from each group received E2 replacement. Tissue collection and preparation for KiSS-1 mRNA in situ hybridization was carried out as described in experiment 1.
Experiment 3.
The purpose of this experiment was to determine whether E2 can regulate expression of KiSS-1 mRNA in ERßKO female mice. Twelve female ERßKO mice and 12 WT littermates were OVX and half from each group received E2 replacement. Tissue collection and preparation for KiSS-1 mRNA in situ hybridization occurred as described in experiment 1.
Experiment 4.
The purpose of this experiment was to determine whether KiSS-1 neurons in the female forebrain coexpress ER and ERß. We performed double-label in situ hybridization on a set of coronal sections from brains of ovary-intact and OVX female mice (used in experiment 1, n = 3–4). Intact (diestrus) mice were used to examine coexpression in the AVPV and periventricular nucleus (PeN), whereas OVX mice were used to determine coexpression in the Arc because this treatment allows for adequate visualization of KiSS-1 mRNA in the Arc via digoxigenin (DIG)-labeled riboprobes.
Radioimmunoassays
Serum levels of LH and E2 were measured at Northwestern University (Evanston, IL). Reagents for the LH assay were from the NIH, the antiserum used was anti-r-LH-S11, and the standard was rLH-RP3. The assay sensitivity was 0.2 ng/ml, and the intraassay coefficient of variation was 4%. E2 was measured with a double-antibody kit (Diagnostics Production Corp., Los Angeles, CA). The assay sensitivity was 2.0 pg/ml, and the intraassay coefficient of variation was 6%.
Radiolabeled KiSS-1 cRNA riboprobes
Antisense and sense mouse KiSS-1 riboprobes were generated as previously described (19). The KiSS-1-specific sequence spanned bases 76–486 of the mouse cDNA sequence (GenBank accession no. AF472576). We previously demonstrated that there is no signal with excess unlabeled antisense probe or radiolabeled sense probe (19).
In situ hybridization
Radioactive in situ hybridization was performed as described (29). Radiolabeled (33P), antisense KiSS-1 riboprobe was denatured, diluted in hybridization buffer at a concentration of 0.03 pmol/ml along with tRNA (2 mg/ml), and applied to slides (100 µl/slide). After hybridization, slides were treated with RNase (32 µg/ml), washed, and dehydrated as previously reported (29). Slides were then dipped in NTB-3 liquid emulsion (Eastman Kodak Co., Rochester, NY) and stored at 4 C. Slides were developed 3 d later.
KiSS-1 mRNA quantification and analysis
All slides were assigned a random three-letter code, alphabetized, and read unilaterally under dark-field illumination with custom software designed to count the total number of cells and the number of silver grains (corresponding to radiolabeled KiSS-1 mRNA) over each cell (30). Cells were counted as KiSS-1 mRNA positive when the number of silver grains in a cluster exceeded that of background. Data are expressed as the total number of identifiable cells and grains/cell (a semiquantitative index of mRNA content/cell).
Double-label in situ hybridization for KiSS-1 mRNA/ER mRNA and KiSS-1 mRNA/ERß mRNA
The cDNA template for the ER riboprobe was generated by PCR as previously described for GPR54 (20), with primers that were designed to contain promoters for T7 RNA polymerase in the antisense direction and T3 RNA polymerase in the sense direction (antisense: CCAAGCCTTC TAATACGACT CACTATAGGG AGAGGGAGCT CTCAGATCG; sense: CAGAGATGCA ATTAACCCTC ACTAAAGGGA GAACCGCCCA TGATCTATTC TG). The ER-specific sequence spanned bases 1163–1990 of the mouse cDNA sequence (GenBank accession no. NM_007956). Antisense and sense mouse ER probes were transcribed from the cDNA template as previously described for the radiolabeled KiSS-1 cRNA riboprobe (19).
The cDNA template for the ERß ribroprobe was generated by PCR with primers as above for ER (antisense: CCAAGCCTTC TAATACGACT CACTATAGGG AGACATCAGC ACCTCCATCC AGC; sense: CAGAGATGCA ATTAACCCTC ACTAAAGGGA GAACTCTCCT TTAGCCACCC ACTGC). The ERß-specific sequence spanned bases 662-1424 of the mouse cDNA sequence (GenBank accession no. NM_207707). Antisense and sense mouse ERß probes were transcribed from the cDNA template as above for ER.
The cDNA template for the KiSS-1 riboprobe was prepared as above for single label in situ hybridization. DIG-labeled antisense cRNA was synthesized with T7 RNA polymerase and DIG labeling mix (Roche, Indianapolis, IN) according to the manufacturer’s protocol.
Slides were processed for in situ hybridization as above with modifications. Radiolabeled antisense ER (0.03 pmol/ml) or ERß (0.05 pmol/ml) and DIG-label KiSS-1 riboprobes (concentration determined empirically) were denatured, dissolved in the same hybridization buffer along with tRNA (1.9 mg/ml), and applied to slides. Slides were hybridized, treated with RNase, and washed as above. KiSS-1 mRNA-positive cells were visualized using anti-DIG fragments conjugated to alkaline phosphatase (diluted 1:300; Roche) and Vector Red substrate (SK-5100; Vector Laboratories, Burlingame, CA) under the manufacturer’s directions. Slides were dipped in 70% ethanol, air dried, and then dipped in NTB-3 liquid emulsion (Eastman Kodak). Slides were developed 7–18 d later.
KiSS-1 mRNA-containing cells were identified under fluorescent illumination, and custom-designed software was used to count the silver grains (corresponding to radiolabeled ER or ERß mRNA) over each cell (30). Signal to background ratios (SBRs) for individual cells were calculated; a cell was considered to be double labeled if it had a SBR of 3 or more. For each animal, the amount of double labeling was calculated as a percentage of the total number of KiSS-1 mRNA-expressing cells and then averaged across animals to produce a mean ± SEM.
Statistical analysis
All data are expressed as mean ± SEM for each group. Variation in KiSS-1 mRNA expression among treatment groups was assessed by one-way ANOVA. For experiments 3 and 4, variation among genotype and treatment groups was assessed by two-way ANOVA. Where the F test for the ANOVA reached statistical significance (P < 0.05), differences among means were assessed by least significant difference tests. All analyses were performed with Statview 5.0.1 for Macintosh (SAS Institute, Cary, NC).
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), and data from these areas and the PeN are presented in the descriptions that follow.
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). In the AVPV, the results were the opposite of those found in the Arc. OVX decreased the number of identifiable KiSS-1 mRNA-containing cells by 58% (P < 0.01), and E2 treatment restored this number to that of intact mice (P < 0.01; OVX vs. OVX + E2; Fig. 2). A similar effect was seen for mRNA content/cell (P < 0.001; intact vs. OVX; P < 0.0001; OVX vs. OVX + E2; Fig. 2). Results in the PeN were similar to those in the AVPV; however, in the PeN cell number differed significantly only between OVX and OVX + E2 (P < 0.01). KiSS-1 mRNA content/ cell in the PeN decreased with OVX (52%, P < 0.01) and was restored by E2 treatment (Fig 2).
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KO miceKO mice in any anatomical area (Fig. 3 and Table 1). The effects of E2 in the WT animals confirmed those found in experiment 1. In the Arc of WT animals, E2 treatment reduced the number of identifiable KiSS-1 neurons and the per-cell content of KiSS-1 mRNA by 56 and 55%, respectively (compared with OVX/sham-treated controls; P < 0.01 for both). In the AVPV of WT mice, compared with OVX controls, E2 treatment significantly increased the number of identifiable KiSS-1 cells by 2.4-fold (P < 0.01) and increased the KiSS-1 mRNA grains per cell by 1.6-fold, although that increase was not statistically significant. A similar effect of E2 was seen in the PeN of WT mice. Here, E2 treatment significantly increased KiSS-1 cell number by 2.2-fold (P < 0.01) and also led to a 1.6-fold increase in KiSS-1 mRNA grains/cell that was not statistically significant.
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). This was confirmed by two-way ANOVA, which detected a significant effect of treatment (P < 0.001), but not a significant effect of genotype nor a significant interaction between treatment and genotype for both the number of cells and grains per cell in each area.
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and ERß mRNAs mRNA were observed in areas in which they have previously been reported, including the preoptic area, AVPV, PeN, Arc, ventromedial hypothalamus, medial amygdala, and BnST. The vast majority of identifiable KiSS-1 mRNA-positive neurons in the Arc, AVPV, and PeN had clusters of silver grains (representing ER mRNA) overlying them (Fig. 4A). Quantitative analysis, with a criterion for double labeling of signal 3 times over background, indicated that 99.8 ± 0.1% of all KiSS-1 mRNA-expressing cells in the Arc, 98.7 ± 0.8% of all KiSS-1 mRNA-expressing cells in the AVPV, and 97.9 ± 1.8% of all KiSS-1 mRNA-expressing cells in the PeN also expressed ER mRNA.
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). Quantitative analysis, with a criterion for double labeling of signal 3 times over background, indicated that that 25.0 ± 2.6% of all KiSS-1 mRNA-expressing cells in the Arc, 30.5 ± 5.1% of all KiSS-1 mRNA-expressing cells in the AVPV, and 43.3 ± 10.8% of all KiSS-1 mRNA-expressing cells in the PeN also expressed ERß mRNA.
Body weight and serum hormone concentrations
Body weight gain during the 7-d treatment period was similar among intact and OVX groups in all experiments, whereas OVX + E2 treated mice gained slightly more weight (P < 0.05, data not shown). Serum E2 levels were, as expected, undetectable in OVX mice, within the physiological range in diestrus mice (average 8 ± 3 pg/ml) and elevated in OVX mice treated with E2 (average 188 ± 16 pg/ml). Serum levels of E2 did not appear to differ between WT and ERKO or ERßKO mice (data not shown). Serum LH levels were measured for experiment 1 only and as expected were minimal in intact mice (0.5 ± 0.2 ng/ml), elevated with OVX (3.7 ± 0.5 ng/ml), and undetectable in OVX mice treated with E2.
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because KiSS-1 expression is unresponsive to E2 treatment in mice with genetically targeted deletions of this ER receptor isoform. Finally, the effect of E2 on KiSS-1 mRNA is likely to be direct on KiSS-1 neurons because virtually all KiSS-1 mRNA-expressing cells in the forebrain also coexpress ER mRNA.
Steroid-sensitive projections from the Arc to the medial preoptic area have long been implicated for a role in the negative feedback control of GnRH secretion by E2 (7, 8, 9, 10, 11, 12, 31). The identity of the cells that mediate this signal is unknown, but KiSS-1 neurons fulfill most of the major criteria. First, GnRH neurons are direct targets for kisspeptin because most GnRH neurons express the kisspeptin receptor (20, 32). Second, kisspeptin activates GnRH neurons (inducing Fos expression) and stimulates GnRH-dependent gonadotropin secretion (19, 20, 22, 23). Third, the present results demonstrate that all KiSS-1 neurons express ER, which is thought to mediate the predominant effects of sex steroids on GnRH secretion (4, 33). Finally, low circulating levels of E2 (e.g. OVX) stimulate KiSS-1 mRNA expression in the Arc, whereas high levels of E2 inhibit the expression of KiSS-1 in the Arc. Together these observations suggest that afferents from KiSS-1 neurons in the Arc provide tonic stimulatory input to GnRH neurons, which increases in the absence and decreases in the presence of E2 and that E2 acts directly on KiSS-1 neurons to regulate the expression of the KiSS-1 mRNA through ER.
In the AVPV and PeN, E2 induces the expression of KiSS-1 mRNA, which is precisely the opposite effect of E2 in the Arc. The AVPV is a sexually dimorphic nucleus and is recognized to play an important role in the preovulatory surge of GnRH and LH (26, 34). KiSS-1 mRNA expression in the AVPV is sexually differentiated, with the female AVPV harboring far more KiSS-1-positive cells than the male (35). Furthermore, the expression of KiSS-1 mRNA in the AVPV is elevated during the afternoon of proestrus in the female rat, during the time of the LH surge (Smith, J. T., S. M. Popa, D. K Clifton, and R. A. Steiner, unpublished observations). If KiSS-1 neurons in the AVPV are necessary for the generation of the GnRH/LH surge, it would appear that increased KiSS-1 expression alone is insufficient for its generation because elevated levels of KiSS-1 mRNA in the AVPV were not accompanied by high levels of circulating LH (as shown in experiment 1). This is not unexpected because the generation of the GnRH/LH surge in the rodent is dependent on not only high levels of E2 (which induces KiSS-1 in the AVPV) but also a circadian signal that restricts the LH surge to the late afternoon (36, 37). Thus, if KiSS-1 neurons in the AVPV are involved in generating the LH surge, it seems likely that they are coupled to circadian circuits that originate in the suprachiasmatic nucleus (SCN) (38, 39, 40). The SCN sends projections to estrogen-responsive neurons in the AVPV (41, 42, 43), and the AVPV in turn sends projections to GnRH neurons (11, 34, 44). Thus, it is plausible that estrogen-sensitive KiSS-1 neurons in the AVPV are the conduit between the circadian oscillator in the SCN and GnRH neurons in the medial preoptic area.
The molecular mechanisms by which E2 produces reverse effects on KiSS-1 mRNA expression in the Arc and AVPV are unknown. Whereas E2 apparently acts through the same receptor (ER) to produce opposite results in the Arc and AVPV/PeN, it seems likely that something intrinsic to the two cell types causes the opposing regulatory effects of E2. We initially postulated that the opposing effect of E2 in these two regions might be attributed to differential expression of ER isoforms (ER and ERß) between these regions of the brain. These two ER isoforms have been shown to produce opposing molecular actions (45, 46). ER and ERß are colocalized in many areas of the forebrain and can form heterodimers (47). In the presence of ER/ERß heterodimers, ERß appears to be the dominant regulator and can oppose ER-mediated gene transcription (45). In the AVPV, there appears to be a greater ratio of ERß to ER expression than that in the Arc (8). However, our data show that in the absence of ER, both negative (in the Arc) and positive (in the AVPV) regulation of KiSS-1 mRNA by E2 is completely voided, whereas in the absence of ERß, E2 fully retains its capacity to regulate KiSS-1. Because all KiSS-1 cells in the Arc, AVPV, and PeN express ER and only a minority coexpress ERß, it would appear that the ERß isoform does not play a significant role in mediating the effects of E2 on KiSS-1 mRNA expression. Thus, it is probable that regional differences in the regulation of KiSS-1 expression are the result of E2 acting through ER alone, which may recruit coactivators of transcription in KiSS-1 neurons of the AVPV and corepressors of transcription in the Arc (48).
Our results demonstrate that the expression of ERß is not essential for the regulation of KiSS-1 mRNA by E2. In the Arc, the decline in KiSS-1 mRNA in response to E2 appeared greater in the ERßKO than in WT mice. It is tempting to speculate that under normal circumstances the formation of ERß/ER heterodimers in KiSS-1 neurons in the Arc attenuates E2-dependant transcriptional regulation of the Kiss1 gene (45). Thus, in the absence of ERß, the inhibitory effect of E2 on transcription of KiSS-1 is enhanced.
OVX mice lacking ER appeared to express more KiSS-1 mRNA in the Arc and less in the AVPV, compared with OVX WT controls. One explanation for this phenomenon may be that ER retains a small level of intrinsic activity in WT OVX animals. This could reflect low-level binding of estrogenic ligands that remain despite OVX. Alternatively, ER may be able to activate/repress KiSS-1 expression, even when unoccupied. However, a more likely possibility is that the congenital absence of ER alters the development of KiSS-1-expressing neurons, leading to altered basal KiSS-1 expression in the adult. This argument seems tenable, considering that perinatal exposure to E2 has long been known to direct the formation of sexually dimorphic circuits within the brain, particularly within the AVPV, and at least part of this effect is thought to be mediated by ER (12). Whether KiSS-1 neurons are organized in a similar fashion and whether their development is altered in the absence of ER is yet to be determined.
In summary, we have shown that E2 differentially regulates the expression of KiSS-1 mRNA in distinct forebrain nuclei and that these effects are mediated by ER. We have also shown that the effect of E2 on KiSS-1 mRNA appears to be direct on these cells because nearly all KiSS-1 neurons (in all hypothalamic regions) coexpress ER mRNA. We conclude that KiSS-1 neurons in the Arc are likely to be involved in the E2-mediated negative feedback control of gonadotropin secretion, whereas KiSS-1 neurons in the AVPV and PeN are involved in E2-mediated positive feedback control of gonadotropin secretion.
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Acknowledgments |
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Footnotes |
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First Published Online May 26, 2005
Abbreviations: Arc, Arcuate nucleus; AVPV, anteroventral periventricular nucleus; BnST, bed nucleus of the stria terminalis; DIG, digoxigenin; E2, estradiol; ER, estrogen receptor; ERKO, mice that lack functional ER; ERßKO, mice that lack functional ERß; GPR, G protein-coupled receptor; OVX, ovariectomized; PeN, periventricular nucleus; SBR, signal to background ratio; SCN, suprachiasmatic nucleus; WT, wild type.
Received April 25, 2005.
Accepted for publication May 17, 2005.
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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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 E. Gianetti and S. Seminara Kisspeptin and KISS1R: a critical pathway in the reproductive system Reproduction, September 1, 2008; 136(3): 295 - 301. [Abstract] [Full Text] [PDF]
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I. Dumalska, M. Wu, E. Morozova, R. Liu, A. van den Pol, and M. Alreja Excitatory Effects of the Puberty-Initiating Peptide Kisspeptin and Group I Metabotropic Glutamate Receptor Agonists Differentiate Two Distinct Subpopulations of Gonadotropin-Releasing Hormone Neurons J. Neurosci., August 6, 2008; 28(32): 8003 - 8013. [Abstract] [Full Text] [PDF]
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K. L. Keen, F. H. Wegner, S. R. Bloom, M. A. Ghatei, and E. Terasawa An Increase in Kisspeptin-54 Release Occurs with the Pubertal Increase in Luteinizing Hormone-Releasing Hormone-1 Release in the Stalk-Median Eminence of Female Rhesus Monkeys in Vivo Endocrinology, August 1, 2008; 149(8): 4151 - 4157. [Abstract] [Full Text] [PDF]
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S. T. Page, J. K. Amory, and W. J. Bremner Advances in Male Contraception Endocr. Rev., June 1, 2008; 29(4): 465 - 493. [Abstract] [Full Text] [PDF]
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J. Roa, E. Vigo, D. Garcia-Galiano, J. M. Castellano, V. M. Navarro, R. Pineda, C. Dieguez, E. Aguilar, L. Pinilla, and M. Tena-Sempere Desensitization of gonadotropin responses to kisspeptin in the female rat: analyses of LH and FSH secretion at different developmental and metabolic states Am J Physiol Endocrinol Metab, June 1, 2008; 294(6): E1088 - E1096. [Abstract] [Full Text] [PDF]
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D. Gonzalez-Martinez, C. De Mees, Q. Douhard, C. Szpirer, and J. Bakker Absence of Gonadotropin-Releasing Hormone 1 and Kiss1 Activation in {alpha}-Fetoprotein Knockout Mice: Prenatal Estrogens Defeminize the Potential to Show Preovulatory Luteinizing Hormone Surges Endocrinology, May 1, 2008; 149(5): 2333 - 2340. [Abstract] [Full Text] [PDF]
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S. Kanda, Y. Akazome, T. Matsunaga, N. Yamamoto, S. Yamada, H. Tsukamura, K.-i. Maeda, and Y. Oka Identification of KiSS-1 Product Kisspeptin and Steroid-Sensitive Sexually Dimorphic Kisspeptin Neurons in Medaka (Oryzias latipes) Endocrinology, May 1, 2008; 149(5): 2467 - 2476. [Abstract] [Full Text] [PDF]
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J. W. Hill, J. K. Elmquist, and C. F. Elias Hypothalamic pathways linking energy balance and reproduction Am J Physiol Endocrinol Metab, May 1, 2008; 294(5): E827 - E832. [Abstract] [Full Text] [PDF]
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J. Pielecka-Fortuna, Z. Chu, and S. M. Moenter Kisspeptin Acts Directly and Indirectly to Increase Gonadotropin-Releasing Hormone Neuron Activity and Its Effects Are Modulated by Estradiol Endocrinology, April 1, 2008; 149(4): 1979 - 1986. [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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C. T. Phillips and R. D. Palmiter Role of Agouti-Related Protein-Expressing Neurons in Lactation Endocrinology, February 1, 2008; 149(2): 544 - 550. [Abstract] [Full Text] [PDF]
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 A. L Filby, R. v. Aerle, J. Duitman, and C. R Tyler The Kisspeptin/Gonadotropin-Releasing Hormone Pathway and Molecular Signaling of Puberty in Fish Biol Reprod, February 1, 2008; 78(2): 278 - 289. [Abstract] [Full Text] [PDF]
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R. L. Goodman, M. N. Lehman, J. T. Smith, L. M. Coolen, C. V. R. de Oliveira, M. R. Jafarzadehshirazi, A. Pereira, J. Iqbal, A. Caraty, P. Ciofi, et al. Kisspeptin Neurons in the Arcuate Nucleus of the Ewe Express Both Dynorphin A and Neurokinin B Endocrinology, December 1, 2007; 148(12): 5752 - 5760. [Abstract] [Full Text] [PDF]
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H. M. Dungan, M. L. Gottsch, H. Zeng, A. Gragerov, J. E. Bergmann, D. K. Vassilatis, D. K. Clifton, and R. A. Steiner The Role of Kisspeptin GPR54 Signaling in the Tonic Regulation and Surge Release of Gonadotropin-Releasing Hormone/Luteinizing Hormone J. Neurosci., October 31, 2007; 27(44): 12088 - 12095. [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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R. Lapatto, J. C. Pallais, D. Zhang, Y.-M. Chan, A. Mahan, F. Cerrato, W. W. Le, G. E. Hoffman, and S. B. Seminara Kiss1 / Mice Exhibit More Variable Hypogonadism than Gpr54 / Mice Endocrinology, October 1, 2007; 148(10): 4927 - 4936. [Abstract] [Full Text] [PDF]
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D. Li, D. Mitchell, J. Luo, Z. Yi, S.-G. Cho, J. Guo, X. Li, G. Ning, X. Wu, and M. Liu Estrogen Regulates KiSS1 Gene Expression through Estrogen Receptor {alpha} and SP Protein Complexes Endocrinology, October 1, 2007; 148(10): 4821 - 4828. [Abstract] [Full Text] [PDF]
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 D. Marot, I. Bieche, C. Aumas, S. Esselin, C. Bouquet, S. Vacher, G. Lazennec, M. Perricaudet, F. Kuttenn, R. Lidereau, et al. High tumoral levels of Kiss1 and G-protein-coupled receptor 54 expression are correlated with poor prognosis of estrogen receptor-positive breast tumors Endocr. Relat. Cancer, September 1, 2007; 14(3): 691 - 702. [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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 X. d'Anglemont de Tassigny, L. A. Fagg, J. P. C. Dixon, K. Day, H. G. Leitch, A. G. Hendrick, D. Zahn, I. Franceschini, A. Caraty, M. B. L. Carlton, et al. Hypogonadotropic hypogonadism in mice lacking a functional Kiss1 gene PNAS, June 19, 2007; 104(25): 10714 - 10719. [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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A. S. Kauffman, M. L. Gottsch, J. Roa, A. C. Byquist, A. Crown, D. K. Clifton, G. E. Hoffman, R. A. Steiner, and M. Tena-Sempere Sexual Differentiation of Kiss1 Gene Expression in the Brain of the Rat Endocrinology, April 1, 2007; 148(4): 1774 - 1783. [Abstract] [Full Text] [PDF]
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J. T. Smith, C. M. Clay, A. Caraty, and I. J. Clarke KiSS-1 Messenger Ribonucleic Acid Expression in the Hypothalamus of the Ewe Is Regulated by Sex Steroids and Season Endocrinology, March 1, 2007; 148(3): 1150 - 1157. [Abstract] [Full Text] [PDF]
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C. Mastronardi, G. G. Smiley, J. Raber, T. Kusakabe, A. Kawaguchi, V. Matagne, A. Dietzel, S. Heger, A. E. Mungenast, R. Cabrera, et al. Deletion of the Ttf1 Gene in Differentiated Neurons Disrupts Female Reproduction without Impairing Basal Ganglia Function J. Neurosci., December 20, 2006; 26(51): 13167 - 13179. [Abstract] [Full Text] [PDF]
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J. Clarkson and A. E. Herbison Postnatal Development of Kisspeptin Neurons in Mouse Hypothalamus; Sexual Dimorphism and Projections to Gonadotropin-Releasing Hormone Neurons Endocrinology, December 1, 2006; 147(12): 5817 - 5825. [Abstract] [Full Text] [PDF]
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W. S. Dhillo, P. Savage, K. G. Murphy, O. B. Chaudhri, M. Patterson, G. M. Nijher, V. M. Foggo, G. S. Dancey, H. Mitchell, M. J. Seckl, et al. Plasma kisspeptin is raised in patients with gestational trophoblastic neoplasia and falls during treatment Am J Physiol Endocrinol Metab, November 1, 2006; 291(5): E878 - E884. [Abstract] [Full Text] [PDF]
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 J. M. Castellano, V. M. Navarro, R. Fernandez-Fernandez, J. Roa, E. Vigo, R. Pineda, C. Dieguez, E. Aguilar, L. Pinilla, and M. Tena-Sempere Expression of Hypothalamic KiSS-1 System and Rescue of Defective Gonadotropic Responses by Kisspeptin in Streptozotocin-Induced Diabetic Male Rats Diabetes, September 1, 2006; 55(9): 2602 - 2610. [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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J. Roa, E. Vigo, J. M. Castellano, V. M. Navarro, R. Fernandez-Fernandez, F. F. Casanueva, C. Dieguez, E. Aguilar, L. Pinilla, and M. Tena-Sempere Hypothalamic Expression of KiSS-1 System and Gonadotropin-Releasing Effects of Kisspeptin in Different Reproductive States of the Female Rat Endocrinology, June 1, 2006; 147(6): 2864 - 2878. [Abstract] [Full Text] [PDF]
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J. T Smith, D. K Clifton, and R. A Steiner Regulation of the neuroendocrine reproductive axis by kisspeptin-GPR54 signaling. Reproduction, April 1, 2006; 131(4): 623 - 630. [Abstract] [Full Text] [PDF]
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H. M. Dungan, D. K Clifton, and R. A. Steiner Minireview: Kisspeptin Neurons as Central Processors in the Regulation of Gonadotropin-Releasing Hormone Secretion Endocrinology, March 1, 2006; 147(3): 1154 - 1158. [Abstract] [Full Text] [PDF]
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S. Pompolo, A. Pereira, K. M. Estrada, and I. J. Clarke Colocalization of Kisspeptin and Gonadotropin-Releasing Hormone in the Ovine Brain Endocrinology, February 1, 2006; 147(2): 804 - 810. [Abstract] [Full Text] [PDF]
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S.-K. Han, M. L. Gottsch, K. J. Lee, S. M. Popa, J. T. Smith, S. K. Jakawich, D. K. Clifton, R. A. Steiner, and A. E. Herbison Activation of Gonadotropin-Releasing Hormone Neurons by Kisspeptin as a Neuroendocrine Switch for the Onset of Puberty J. Neurosci., December 7, 2005; 25(49): 11349 - 11356. [Abstract] [Full Text] [PDF]
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W. S. Dhillo, O. B. Chaudhri, M. Patterson, E. L. Thompson, K. G. Murphy, M. K. Badman, B. M. McGowan, V. Amber, S. Patel, M. A. Ghatei, et al. Kisspeptin-54 Stimulates the Hypothalamic-Pituitary Gonadal Axis in Human Males J. Clin. Endocrinol. Metab., December 1, 2005; 90(12): 6609 - 6615. [Abstract] [Full Text] [PDF]
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M. Tena-Sempere Hypothalamic KiSS-1: The Missing Link in Gonadotropin Feedback Control? Endocrinology, September 1, 2005; 146(9): 3683 - 3685. [Full Text] [PDF]
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