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Endocrinology Vol. 148, No. 3 1150-1157
Copyright © 2007 by The Endocrine Society

KiSS-1 Messenger Ribonucleic Acid Expression in the Hypothalamus of the Ewe Is Regulated by Sex Steroids and Season

Jeremy T. Smith, Colin M. Clay, Alain Caraty and Iain J. Clarke

Department of Physiology (J.T.S., I.J.C.), Monash University, Victoria 3880, Australia; Department of Biomedical Sciences (C.M.C.), Colorado State University, Fort Collins, Colorado 80523; and Unité Mixte de Recherche 6175 (A.C.), Institut National de la Recherche Agronomique/Centre National de la Recherche Scientifique, Université de Tours, Haras Nationaux, Institut Fédératif de Recherche 135, Nouzilly, France

Address all correspondence and requests for reprints to: Prof. Iain Clarke, Department of Physiology, P.O. Box 13F, Monash University, Victoria 3800, Australia. E-mail: iain.clarke{at}med.monash.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The KiSS-1 gene encodes a family of peptides called kisspeptins, which are endogenous ligands for the G protein-coupled receptor GPR54. Kisspeptin function appears to be critical for GnRH secretion and the initiation of puberty. To test the hypothesis that steroid hormones regulate KiSS-1 mRNA expression in the ewe, we examined the brains of ovary-intact (luteal phase) and ovariectomized (OVX) ewes, as well as OVX ewes that received estradiol (E) or progesterone (P) replacement. KiSS-1 mRNA-expressing cells were predominantly located in the arcuate nucleus (ARC). Here, expression was increased after OVX but returned to the level of gonad-intact animals with E treatment. Treatment with P partially restored KiSS-1 expression toward gonad-intact levels. Double-label immunohistochemistry revealed that approximately 86% of kisspeptin-immunoreactive cells in the ARC are also P-receptor positive. Finally, we tested the hypothesis that KiSS-1 mRNA is lower during anestrus, due to a non-steroid-dependent seasonal effect. In OVX ewes, expression in the ARC was lower at the time of year corresponding to anestrus. We conclude that KiSS-1 expression in the ARC of the ewe brain is negatively regulated by chronic levels of E and P, suggesting that both steroids may exert negative feedback control on GnRH secretion through altered kisspeptin signaling. Furthermore, a seasonal alteration in KiSS-1 expression in the ARC of OVX ewes strongly suggests that kisspeptin is fundamentally involved in the control of seasonal changes in reproductive function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
KISSPEPTINS ARE A family of neuropeptides derived from the translation product of the KiSS-1 gene (1, 2, 3, 4). Posttranslational processing of a 145-amino-acid precursor peptide results in the formation of smaller C-terminal peptides (kisspeptin-54, -14, -13, and -10), which activate the G protein-coupled receptor GPR54 with equal efficacy (1, 2, 3, 4). In humans and mice, inactivating mutations in GPR54 result in the failure to initiate puberty and subsequent hypogonadotropic hypogonadism (5, 6, 7). Practically all GnRH neurons express GPR54 (8, 9, 10), and central and peripheral administration of kisspeptin stimulates GnRH and gonadotropin secretion in various species (11, 12, 13, 14). Kisspeptin-producing neurons (or KiSS-1 neurons) have been localized to various regions of the forebrain in rodents, primates, and sheep (11, 14, 15, 16, 17). In sheep, most KiSS-1 neurons are found in the arcuate nucleus (ARC), with a smaller population present in the preoptic area (POA) (15, 16, 18).

In the female, the pulsatile release of GnRH is tightly regulated by the negative and positive feedback of gonadal steroids (19, 20, 21). Although GnRH neurons express estrogen receptor (ER)ß (22), they do not express ER{alpha} (23, 24) or progesterone receptor (PR) (25), and so other steroid-sensitive neurons must mediate the feedback effects of sex steroids on GnRH secretion. In female rodents, ovarian steroids inhibit KiSS-1 mRNA expression in the ARC but stimulate expression in the anteroventral periventricular nucleus (AVPV) (26, 27). These observations have led to the notion that kisspeptin-producing neurons in the former mediate the negative-feedback effects of estrogen (E), whereas the positive-feedback effects of steroids on GnRH cells are mediated by kisspeptin-producing neurons in the latter (26, 27). This is consistent with our understanding that the AVPV is the surge center in the rodent (28, 29), but this is not the case in the sheep. Indeed, implantation of E into the mediobasal hypothalamus elicits GnRH/LH surges in the ewe (30). Consistent with the notion that the GnRH/LH surge center is within the basal hypothalamus and that kisspeptin may relay the positive-feedback effect of E to GnRH cells, we recently reported that KiSS-1 mRNA is up-regulated in the ARC, just before and during the GnRH/LH surge in the ewe (15). Unlike the female rat, the ewe does not show up-regulation of KiSS-1 in the POA of the brain at the time of the cyclic surge. Thus, it appears that both negative- and positive-feedback effects of E and progesterone (P) on GnRH secretion in the sheep may be mediated by KiSS-1 neurons in the ARC. Whether E or P is able to inhibit KiSS-1 expression in the ARC is unknown, although we have shown that kisspeptin immunoreactivity is increased by ovariectomy (18).

In sheep, reproductive activity is seasonal, being activated by short-day photoperiod and inhibited by long days. During the nonbreeding (anestrous) season, GnRH secretion is reduced due to steroid-independent (31, 32, 33) and steroid-dependent (34, 35) effects of photoperiod. We hypothesize that the mechanism underlying the regulation of GnRH secretion and, in turn, seasonal changes in reproductive function is changing KiSS-1 mRNA expression.

The primary objectives of the present study were 3-fold. The first was to determine the regulation of KiSS-1 mRNA by E or P. Accordingly, we used single-label in situ hybridization to compare expression of KiSS-1 mRNA in groups of ovary-intact (luteal phase) and ovariectomized (OVX) ewes as well as OVX ewes receiving chronic E or P replacement. The second objective was to test whether P is likely to act directly on kisspeptin neurons, using double-label immunohistochemistry for kisspeptin and PR. Our final objective was to test the hypothesis that KiSS-1 mRNA is regulated across the breeding and nonbreeding seasons of the ewe in a steroid-independent manner. Accordingly, we examined expression of KiSS-1 in the brains of OVX ewes killed in February (early breeding season), May (late breeding season), October (mid nonbreeding season), and December (late nonbreeding season).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Corriedale ewes of similar age (5–6 yr) and weight were maintained under natural conditions at the Monash University Sheep Facility, Werribee, Victoria. All experiments were carried out according to the guidelines established by the Australian Prevention of Cruelty to Animals Act 1986 and was approved by the Monash University Animal Ethics Committee. Brains of ovary-intact animals were collected during the luteal phase of the estrous cycle. The estrous cycles of ovary-intact animals were synchronized by im injection of 125 µg of the synthetic luteolysin Cloprostenol (Estrumate; Pitman-Moore, Sydney, Australia), and brains were collected on d 10 of the ensuing estrous cycle (luteal phase). Verification of the stage of cycle for these animals has been previously published (36). Briefly, ovaries contained at least one active corpus luteum, plasma LH concentrations were less than 1.0 ng/ml, and plasma P concentrations were elevated (3.2 ± 0.5 ng/ml).

Ovariectomy and steroid replacement
Ovariectomy was performed as previously described (37). Chronic E treatment was achieved with a 3-cm sc implant [SILASTIC brand tubing (Dow Corning Corp., Midland, MI) with an inner diameter of 3.35 mm and outer diameter of 4.65 mm packed with crystalline estradiol-17ß (Sigma Chemical Co., St. Louis, MO). Implants were inserted into the axillary region 2 wk before tissue collection. These implants were designed to produce circulated E levels of approximately 3–5 pg/ml (38), similar to that observed in the normal luteal phase. P treatment was via intravaginal controlled internal drug release devices containing 0.3 g P (Riverina; Artificial Breeders Ltd., Albury, Australia). Controlled internal drug release devices were inserted 2 wk before tissue collection, and plasma levels of P in these animals, which were previously described (39), were 2.0 ± 0.3 ng/ml.

Experimental design
Experiment 1.
The purpose of this experiment was to determine the effects of ovariectomy and E or P replacement on hypothalamic KiSS-1 mRNA. Ewes were divided into the following four groups: ovary intact (luteal, n = 6), OVX (n = 5), OVX plus E replacement (n = 4), and OVX plus P replacement (n = 4). The animals were treated during the normal breeding season, and ovariectomy took place 5–7 wk before tissue collection. Hypothalami were collected for KiSS-1 mRNA in situ hybridization as follows. The ewes were killed by an iv overdose of sodium pentobarbital (Lethabarb; Virbac, Peakhurst, Australia), and the heads were perfused with 2 liters of heparinized saline (12.5 U/ml) followed by 2 liters of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and 1 liter of the same fixative containing 20% sucrose. The brains were removed and the hypothalamus dissected out and postfixed in fixative containing 30% sucrose for 7 d and then frozen in powdered dry ice. Coronal sections (20 µm) were cut on a cryostat (from the diagonal band of Broca to the mammillary bodies) and placed into cryoprotectant (30% ethylene glycol, 20% glycerol in sodium phosphate buffer) with 2% paraformaldehyde and stored at –20 C until used for in situ hybridization.

Experiment 2.
The purpose of this experiment was to determine whether PR is present in KiSS-1 neurons in the ARC of the ewe brain. Brains were collected for double-label immunohistochemistry for kisspeptin and PR from three ovary-intact animals (luteal phase) as described above with the following modifications. These brains were perfused with 2 liters of heparinized saline followed by 2 liters of fixative, containing 4% paraformaldehyde plus 15% picric acid in 0.1 M phosphate buffer (pH 7.4) and 1 liter of the same fixative containing 20% sucrose. The hypothalamus was dissected out and then placed in a solution of 0.1 M phosphate buffer containing 30% sucrose. Coronal sections (40 µm) were placed in cryoprotectant without paraformaldehyde and stored at –20 C until used for immunohistochemistry.

Experiment 3.
The purpose of this experiment was to determine whether KiSS-1 mRNA expression in the hypothalamus changes in a steroid-independent manner between the normal breeding and nonbreeding seasons. Sheep are short-day breeders, and for this breed in this location, the normal breeding season (when ewes exhibit regular estrous cycles) is between February and June, with transitional periods (with irregular cycles or silent estrus) in January and July (Clarke, I. J., and B. D. Doughton, unpublished data). In this experiment, animals were run on pasture, so that they were subject to the normal change in photoperiod as well as environmental fluctuations in temperature. Groups of OVX ewes were sampled in February (early breeding season, n = 4), May (mid-breeding season, n = 4), October (mid nonbreeding season, n = 6), and December (late nonbreeding season, n = 5). OVX ewes were used so that we could discern the effect of season in the absence of fluctuating levels of gonadal steroids, because the feedback effects of E and P change with season (34), and previous experiments showed an effect of these steroids on KiSS-1 mRNA (17, 26). Collection of the brains and processing of the hypothalami were as in experiment 1, and in situ hybridization was performed to quantify KiSS-1 expression.

Radiolabeled KiSS-1 cRNA riboprobe
A 375-base sequence of the ovine KiSS-1 gene (GenBank accession no. DQ059506) was inserted into a pGemT-easy plasmid. The antisense ovine KiSS-1 probe was transcribed from linearized plasmid containing the ovine KiSS-1 insert with SP6 polymerase (Promega Corp., Madison, WI) and [35S]UTP (GE Healthcare Life Sciences, Boston, MA) under a standard transcription protocol (40). The riboprobe was separated from unincorporated nucleotides on a Sephadex G-25 column.

In situ hybridization
In situ hybridization was performed as described for KiSS-1 expression in ewes during the estrous cycle (15). Three sections representing the rostral, medial, and caudal regions of the ARC and three sections through the POA were chosen from each ewe, mounted on SuperFrost slides, and prepared for in situ hybridization. Radiolabeled (35S) antisense KiSS-1 riboprobe was denatured, diluted in hybridization buffer at a concentration of 5 x 106 cpm/ml along with tRNA, and applied to slides (120 µl/slide). After hybridization (53 C for 16 h), slides were treated with RNase A, washed in decreasing concentrations of saline-sodium citrate, and dehydrated. Slides were then dipped in Ilford K5 photographic emulsion (Ilford Imaging, Melbourne, Australia), stored in the dark at 4 C, and developed 7–10 d later. No signal was observed after the application of radiolabeled sense probe (data not shown).

KiSS-1 quantification and analysis
Image analysis was carried out using randomly coded slides under dark-field illumination with custom-designed software designed to count the total number of cells and the number of silver grains per cell (41). Cells were counted when silver grain density was more than five times background. Data are expressed as the mean number of identifiable KiSS-1 cells and the mean number of silver grains per cell (a semiquantitative index of mRNA expression per cell).

Double-label immunohistochemistry for kisspeptin and PR
Three sections representing the rostral, medial, and caudal regions of the ARC were chosen from each ewe and mounted on SuperFrost slides. Antigen retrieval was performed using 1 M citrate buffer (pH 6) in a microwave oven at 1000 W (2 x 5 min). After cooling (20 min), the sections were washed in Tris-buffered saline (TBS), and a blocking solution containing 10% normal goat serum and 0.3% Triton X-100 in TBS was applied. Sections were then incubated for 72 h at 4 C with a cocktail containing a rabbit polyclonal antibody against mouse kisspeptin-10 (no. 566 used at a dilution of 1:2000) (16) and a mouse monoclonal antibody against the PR (Ab-8 used at a dilution of 1:100; Neomarkers, Union City, CA). Both antibodies have been used successfully and their specificity tested previously (16, 25). Slides were then washed in TBS and sections incubated with a mixture of goat antirabbit Alexa 448 and goat antimouse Alexa 546 for 2 h at room temperature (both 1:400; Molecular Probes Inc., Eugene, OR). Slides were again washed in TBS, and sections were stained with 0.3% Sudan Black B to minimize autofluorescence. After rinses in TBS and then phosphate buffer, coverslips were applied using antifade mounting solution (Dako, Carpinteria, CA). The slides were stored in the dark. Kisspeptin and PR-immunoreactive (ir) cells were identified under fluorescent illumination, with a single observer counting the total number of cells stained. For each ewe, the degree of double labeling was calculated as a percentage of the total number of kisspeptin-ir cells and then averaged across animals to produce a mean (± SEM).

Statistical analysis
All data are expressed as the means (± SEM), and statistical analysis was by one-way ANOVA. Two-way ANOVA was initially employed to determine the treatment effects in the rostral, medial, and caudal divisions of the ARC. The level of P < 0.05 was considered significant and the least significant differences test was used as a post hoc test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distribution of KiSS-1 mRNA in the hypothalamus of the ewe
Cells expressing KiSS-1 mRNA were readily identifiable in the ARC (Fig. 1Go), and a smaller population was also seen in the POA. No KiSS-1-expressing cells were found in any other neighboring regions of the hypothalamus.


Figure 1
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  		FIG. 1. Dark-field photomicrographs showing representative sections of the middle ARC and KiSS-1 mRNA-expressing cells (as shown by the presence of silver grain clusters) from gonad-intact, OVX, OVX plus E, and OVX plus P ewes. 3V, Third ventricle. Scale bars, 200 µm.

 
Experiment 1: effect of ovariectomy and E or P replacement on KiSS-1 expression
In the ARC, ovariectomy increased the number of cells expressing KiSS-1 mRNA by over 3-fold (P < 0.001) and increased the level of expression per cell by 40% (P < 0.05) compared with intact (luteal) controls (Figs. 1Go and 2Go). Treatment with E negated the effect of ovariectomy in terms of cell number (P < 0.001 compared with OVX) and expression per cell (P < 0.05 compared with OVX). Treatment of OVX ewes with P reduced the number of KiSS-1 mRNA-expressing cells by 37% (P < 0.01 compared with OVX) but not to the same extent as E treatment. Hence, the number of KiSS-1-expressing cells in the ARC of OVX plus P animals was 2-fold higher than the level in the ARC of luteal-phase animals (P < 0.05). P treatment also diminished the effect of ovariectomy on KiSS-1 mRNA per cell (P < 0.05 compared with OVX). The change in the level of expression with E or P treatment was uniform across the entire ARC, with no difference between rostral, medial, and caudal divisions. There was no difference between groups in the number of KiSS-1 mRNA-expressing cells or the level of expression of KiSS-1 in the POA (Fig. 2Go), although the effect of E appeared to reduce the number of cells (but not expression per cell). It should further be noted that the number of cells in the POA of OVX animals was one tenth that of the ARC and the direction of change in the data for the POA was not the same as that seen in the ARC.


Figure 2
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  		FIG. 2. Effect of ovariectomy and sex-steroid replacement on KiSS-1 expression in the ARC and the POA of the brain of the ewe. The number of KiSS-1 mRNA-expressing cells and the number of silver grains per KiSS-1 cell (reflecting expression per cell) is represented for animals in the luteal phase of the estrous cycle (intact), OVX, and in OVX animals given either E or P replacement. Values are means (± SEM). *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with intact.

 
Experiment 2: kisspeptin/PR coexpression in the ARC
Cells expressing PR were found throughout the ARC, and the majority of kisspeptin-ir cells colocalized with PR (Fig. 3Go). Quantitative analysis indicated that 86 ± 3% of all kisspeptin-ir cells also immunostained for PR.


Figure 3
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  		FIG. 3. Representative photomicrograph showing costaining of cells for kisspeptin and PR in the ARC of the ewe brain. Kisspeptin-ir cells are green and PR-ir cells are red. Arrows indicate coexpressing cells. The open arrowhead indicates a kisspeptin-positive PR-negative cell. Scale bar, 50 µm.

 
Experiment 3: seasonal expression of KiSS-1 mRNA expression
In the ARC, the number of cells expressing KiSS-1 mRNA was over 2-fold higher in February than in December (P < 0.001; Figs. 4Go and 5Go). The number KiSS-1-expressing cells fell between February and May, although this did not reach statistical significance. Expression was 44% lower in October than in February (P < 0.01). The level of KiSS-1 expression per cell did not change across the year (Fig. 5Go). There was no effect of season on KiSS-1 expression in the POA, even though the trend was similar to that seen in the ARC (Fig. 5Go).


Figure 4
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  		FIG. 4. Dark-field photomicrographs showing KiSS-1 mRNA-expressing cells (as shown by the presence of silver grain clusters) in representative sections of the middle ARC of OVX ewes at four times of the year. 3V, Third ventricle. Scale bars, 200 µm.

 

Figure 5
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  		FIG. 5. The number of KiSS-1 mRNA-expressing cells and the number of silver grains per cell (reflecting expression per cell) in the ARC and POA of OVX ewes at four times of the year. Black bars represent the breeding season. Values are the means (± SEM). **, P < 0.01; ***, P < 0.001 compared with February.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that kisspeptin neurons in the ARC of the ewe brain are regulated by both E and P and are targets for P and that these kisspeptin neurons also display steroid-independent seasonal regulation. These data are consistent with previous reports in the mouse and rat, in which KiSS-1 mRNA in the ARC was found to be regulated by gonadal steroids (9, 17, 26, 27). In the ewe, KiSS-1 expression in the ARC was up-regulated by ovariectomy and corrected with E replacement, but physiological replacement with P only partially countered the effect of ovariectomy. Furthermore, the actions of these sex steroids on KiSS-1 appears to be direct, because the presence of ER{alpha} in kisspeptin-ir neurons of the ARC of the ewe has been established previously (16), and we show that most KiSS-1 neurons express PR. Thus, KiSS-1 neurons in the ARC of the ewe are plausible candidates as mediators of the negative-feedback effects of gonadal steroids on GnRH neurons.

Steroid-responsive cells of the ARC of the ewe brain that project to the medial POA have been implicated in the feedback regulation of GnRH cells (42, 43). The mediobasal hypothalamic region of the ewe brain is recognized as the site at which E acts to evoke a positive-feedback effect on GnRH/LH secretion (30, 44), so the location of KiSS-1-expressing cells in the ARC is highly relevant in this regard. Consistent with a major role of kisspeptin in the regulation of the reproductive cycle, we recently reported that KiSS-1 expression in the ARC of the ewe is up-regulated immediately before, and during, the preovulatory GnRH/LH surge, suggesting involvement in the positive-feedback event (15). The present observations suggest that kisspeptin also participates in the relay of negative-feedback regulation of GnRH cells. Interestingly, it appears that only the KiSS-1 neurons located at the caudal portion of the ARC are activated just before the GnRH/LH surge, although a population in the rostral ARC seems to be recruited during the surge (15). The data of the present study indicate that KiSS-1 neurons across the entire ARC are responsive to ovariectomy and steroidal manipulations. This leads us to the enticing proposition that distinct regions of KiSS-1 neurons in the ARC are involved in the negative and positive regulation of GnRH. It remains to be determined whether the kisspeptin-producing cells of the ARC of the ewe brain provide direct input to GnRH cells, which is a focus of attention in our laboratory at present. Such studies may identify the subpopulations that are responsible for the different modes of feedback.

In rodents, KiSS-1 expression in the AVPV appears to be critical in the production of the preovulatory LH surge (27), but this region of forebrain is void of kisspeptin-producing cells in the ewe (15, 16). Nevertheless, KiSS-1 mRNA-expressing and kisspeptin-ir cells are found in the POA of the ewe (15, 16). Using an antibody from Phoenix Pharmaceuticals Inc. (Belmont, CA), a high degree of colocalization of kisspeptin and GnRH was found in the cells of the POA (18), although a different antibody (Caraty no. 566) did not show the same (16). The possibility of a lack of specificity of the Phoenix antibody, at least in our hands, is under active investigation. In the present study, we used the Caraty antibody. The in situ hybridization data of the present study complement earlier immunohistochemical data, confirming that kisspeptin is produced in cells found in the POA of the ewe brain. Our data show that KiSS-1 mRNA expression in the POA is not regulated by either E or P, because differences between groups did not reach statistical significance. Despite this, 50% of kisspeptin-ir cells in the POA express ER{alpha} (16).

Many studies have shown that cells within the mediobasal hypothalamus of the ewe are substantially involved in the negative and positive feedback effects of E and P on GnRH secretion (42, 43, 45, 46, 47). Although subsets of a wide range of cell types express ER and/or PR, the proportion of these cells that possess steroid receptors is generally low, with the exception of glutamatergic and dynorphin neurons (47, 48). Thus, it is of major significance that virtually all kisspeptin-producing cells in the ARC express ER{alpha}, and we have now shown that a very high percentage also express PR. Other studies indicate that there is a high level of coexpression of ER{alpha} and PR in the hypothalamus of the ewe (49), so the result is not surprising. It is possible that the glutamate/ER{alpha} cells in the ARC of the ewe brain are the same as those that produce kisspeptin. Irrespective of what other transmitters/neuropeptides are found in the kisspeptin-producing neurons, the possession of the relevant steroid receptors and the demonstration of regulation of KiSS-1 expression by E and P suggests that these cells could be integrally involved in feedback regulation of the reproductive system.

In our study, we demonstrate that P treatment was unable to fully suppress KiSS-1 mRNA expression in the ARC of OVX ewes. This result is surprising given that P has been shown to exert a strong negative feedback effect on LH secretion (50, 51) by action at the level of the mediobasal hypothalamus (52). It is possible that the lesser effect of P on KiSS-1 expression in OVX ewes may be due to reduced PR expression in the absence of E (53). In OVX ewes, the suppressive effects of P on LH secretion are eventually lost in the continued absence of E (51). Moreover, E treatment is known to increase the number of PR-expressing neurons in the rat brain (53). The mediobasal hypothalamus is the major site of P negative feedback in the ewe (52), and we have shown that kisspeptin cells located here express a level of PR greater than that seen in cells that produce dynorphin (54), ß-endorphin (54), tyrosine hydroxylase (42), and neuropeptide Y (42). Furthermore, these aforementioned cells are known to project from the ARC to GnRH-rich regions of the ovine POA (42, 54). We show that KiSS-1 expression at the ARC is regulated by P, leading us to conclude that kisspeptin cells are most likely involved in the transmission of P feedback to GnRH cells, whether this is through direct or indirect input to the latter. P does not exert such negative feedback when implanted into the POA (52), so we did not examine kisspeptin/PR coexpression at this level. It will be instructive to ascertain whether the kisspeptin cells of the ARC project directly to GnRH cells in the ovine brain, and appropriate neural tracing studies are in progress.

Sheep are seasonally breeding mammals, with an annual cycle of reproductive function that is controlled by photoperiod. This is reflected in alterations in GnRH and LH secretion that are regulated by steroid-dependent and steroid-independent mechanisms (31, 32, 33, 34, 35). In the present study, we investigated the steroid-independent mechanism by examining the expression of KiSS-1 in OVX ewes at different times of the year, which represented the breeding and the nonbreeding seasons. We showed a marked elevation of KiSS-1 mRNA in the ARC between the end of the anestrous season (December) and the onset of the breeding season (February). This change in KiSS-1 expression may act as a neuroendocrine switch allowing the onset of the breeding season, in a similar fashion to that recently proposed for the onset of puberty in the mouse (8). Expression of KiSS-1 in cells localized in the ARC of the subhuman primate also increases across puberty onset (14). If, as has been suggested (55, 56), the transition from the anestrous season to the breeding season represents a similar mechanism, this could provide a model relevant to puberty. We do not know whether the seasonal shift in the sensitivity of the reproductive neuroendocrine axis to E negative feedback also involves a further alteration in KiSS-1 expression, but appropriate studies are in progress.

The frequency of pulsatile episodes of LH secretion (reflecting GnRH secretion) is reduced during anestrus in OVX ewes (31, 32, 33), and this may be a direct function of reduced kisspeptin action. To test this hypothesis, it will be necessary to measure GnRH/LH secretion in response to kisspeptin infusion at different stages of the circannual breeding cycle. One might hypothesize that there is a seasonal alteration in responsiveness to kisspeptin in terms of GnRH/LH secretion. Because there is no morphological difference in GnRH neurons between the breeding and nonbreeding season (57), it appears that afferents regulating GnRH release would be of critical importance to this phenomenon. Indeed, synaptic inputs on to ovine GnRH cells undergo marked seasonal rearrangements that are independent of gonadal steroid hormones and may reflect the intrinsic seasonality of the brain (58, 59). It is possible that there is some seasonal alteration in the degree to which kisspeptin provides input to GnRH cells.

Similar results to ours have been seen in the hamster (60), which is a long-day breeder. Accordingly, in this species, limited data indicate that KiSS-1 expression is higher under long-day photoperiod than under short days. The question remains as to what the regulatory stimulus might be for the change in KiSS-1 expression. One possibility is that melatonin signaling alters function of the KiSS-1-expressing cells, because melatonin receptors are found in the basal hypothalamus (61) and melatonin acts at this level in the ovine brain (61, 62, 63). Alternatively, it may be that the seasonal alteration in KiSS-1 expression is related to a change in nutrition (64). Certainly there is a link between nutrition and kisspeptin, because mice lacking leptin (ob/ob mice) have down-regulated KiSS-1 expression (65) and other work in rodent models shows that expression is regulated by nutritional manipulation (66, 67).

In summary, we show that KiSS-1 neurons in the ARC of the brain of the ewe are regulated by chronic E or P treatment, suggesting a that these cells participate in the negative-feedback control of GnRH secretion. This effect is most probably due to direct steroid action on kisspeptin-producing cells, because they possess the relevant receptors. In contrast, the KiSS-1-expressing cells of the POA are not significantly affected by gonadectomy or E or P replacement. We also report that KiSS-1 mRNA expression is up-regulated between the end of the anestrous season and the beginning of the onset of the breeding season, indicating that kisspeptin may be involved in the activation of central mechanisms controlling seasonal changes in reproductive function.


   Acknowledgments
 
We thank Ms. A. Rao, Mr. B. Doughton, Ms. L. Morrish, Dr. C. J. Scott, and Dr. K. M. Estrada for technical assistance.


   Footnotes
 
J.T.S. is supported by a Peter Doherty Fellowship and The National Health and Medical Research Council, Australia.

Disclosure Summary: All authors have nothing to declare.

First Published Online December 21, 2006

Abbreviations: ARC, Arcuate nucleus; AVPV, anteroventral periventricular nucleus; E, estrogen; ER, estrogen receptor; ir, immunoreactive; P, progesterone, POA, preoptic area; PR, progesterone receptor; TBS, Tris-buffered saline.

Received October 26, 2006.

Accepted for publication December 11, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. 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[Abstract/Free Full Text]
  2. 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 CG, 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[Abstract/Free Full Text]
  3. 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, Kitada 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[CrossRef][Medline]
  4. Stafford LJ, Xia C, Ma W, Cai Y, Liu M 2002 Identification and characterization of mouse metastasis-suppressor KiSS1 and its G-protein-coupled receptor. Cancer Res 62:5399–5404[Abstract/Free Full Text]
  5. 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[Abstract/Free Full Text]
  6. Funes S, Hedrick JA, Vassileva G, Markowitz L, Abbondanzo S, Golovko A, Yang S, Monsma FJ, Gustafson EL 2003 The KiSS-1 receptor GPR54 is essential for the development of the murine reproductive system. Biochem Biophys Res Commun 312:1357–1363[CrossRef][Medline]
  7. Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno Jr 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 Jr WF, Aparicio SA, Colledge WH 2003 The GPR54 gene as a regulator of puberty. N Engl J Med 349:1614–1627[Abstract/Free Full Text]
  8. Han SK, Gottsch ML, Lee KJ, Popa SM, Smith JT, Jakawich SK, Clifton DK, Steiner RA, Herbison AE 2005 Activation of gonadotropin-releasing hormone neurons by kisspeptin as a neuroendocrine switch for the onset of puberty. J Neurosci 25:11349–11356[Abstract/Free Full Text]
  9. 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[CrossRef][Medline]
  10. Parhar IS, Ogawa S, Sakuma Y 2004 Laser-captured single digoxigenin-labeled neurons of gonadotropin-releasing hormone types reveal a novel G protein-coupled receptor (Gpr54) during maturation in cichlid fish. Endocrinology 145:3613–3618[Abstract/Free Full Text]
  11. 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[Abstract/Free Full Text]
  12. 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[CrossRef][Medline]
  13. 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 release via G protein-coupled receptor 54. Proc Natl Acad Sci USA 102:1761–1766[Abstract/Free Full Text]
  14. 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[Abstract/Free Full Text]
  15. Estrada KM, Clay CM, Pompolo S, Smith JT, Clarke IJ 2006 Elevated KiSS-1 expression in the arcuate nucleus prior to the cyclic preovulatory gonadotrophin-releasing hormone/lutenising hormone surge in the ewe suggests a stimulatory role for kisspeptin in oestrogen-positive feedback. J Neuroendocrinol 18:806–809[CrossRef][Medline]
  16. Franceschini I, Lomet D, Cateau M, Delsol G, Tillet Y, Caraty A 2006 Kisspeptin immunoreactive cells of the ovine preoptic area and arcuate nucleus co-express estrogen receptor {alpha}. Neurosci Lett 401:225–230[CrossRef][Medline]
  17. Smith JT, Dungan HM, Stoll EA, Gottsch ML, Braun RE, Eacker SM, Clifton DK, Steiner RA 2005 Differential regulation of KiSS-1 mRNA expression by sex steroids in the brain of the male mouse. Endocrinology 146:2976–2984[Abstract/Free Full Text]
  18. Pompolo S, Pereira A, Estrada KM, Clarke IJ 2006 Colocalization of kisspeptin and gonadotropin-releasing hormone in the ovine brain. Endocrinology 147:804–810[Abstract/Free Full Text]
  19. Caligaris L, Astrada JJ, Taleisnik S 1971 Release of luteinizing hormone induced by estrogen injection into ovariectomized rats. Endocrinology 88:810–815[Medline]
  20. Clarke IJ 1996 The Hypothalamo-pituitary axis. In: Hillier SG, Kitchener HC, Neilsen JP, eds. Scientific essentials of reproductive medicine. London: WB Saunders; 120–132
  21. Moore CR, Price D 1932 Gonadal hormone functions and the reciprocal influence between gonads and hypophysis, with its bearing on sex hormone antagonism. Am J Anat 50:13–71[CrossRef]
  22. Hrabovszky E, Steinhauser A, Barabas K, Shughrue PJ, Petersen SL, Merchenthaler I, Liposits Z 2001 Estrogen receptor-ß immunoreactivity in luteinizing hormone-releasing hormone neurons of the rat brain. Endocrinology 142:3261–3264[Abstract/Free Full Text]
  23. Herbison AE, Theodosis DT 1992 Localization of oestrogen receptors in preoptic neurons containing neurotensin but not tyrosine hydroxylase, cholecystokinin or luteinizing hormone-releasing hormone in the male and female rat. Neuroscience 50:283–298[CrossRef][Medline]
  24. Shivers BD, Harlan RE, Morrell JI, Pfaff DW 1983 Absence of oestradiol concentration in cell nuclei of LHRH-immunoreactive neurones. Nature 304:345–347[CrossRef][Medline]
  25. Skinner DC, Caraty A, Allingham R 2001 Unmasking the progesterone receptor in the preoptic area and hypothalamus of the ewe: no colocalization with gonadotropin-releasing neurons. Endocrinology 142:573–579[Abstract/Free Full Text]
  26. Smith JT, Cunningham MJ, Rissman EF, Clifton DK, Steiner RA 2005 Regulation of Kiss1 gene expression in the brain of the female mouse. Endocrinology 146:3686–3692[Abstract/Free Full Text]
  27. Smith JT, Popa SM, Clifton DK, Hoffman GE, Steiner RA 2006 Kiss1 neurons in the forebrain as central processors for generating the preovulatory luteinizing hormone surge. J Neurosci 26:6687–6694[Abstract/Free Full Text]
  28. Gu GB, Simerly RB 1997 Projections of the sexually dimorphic anteroventral periventricular nucleus in the female rat. J Comp Neurol 384:142–164[CrossRef][Medline]
  29. Simerly RB 1998 Organization and regulation of sexually dimorphic neuroendocrine pathways. Behav Brain Res 92:195–203[CrossRef][Medline]
  30. Caraty A, Fabre-Nys C, Delaleu B, Locatelli A, Bruneau G, Karsch FJ, Herbison A 1998 Evidence that the mediobasal hypothalamus is the primary site of action of estradiol in inducing the preovulatory gonadotropin releasing hormone surge in the ewe. Endocrinology 139:1752–1760[Abstract/Free Full Text]
  31. Barker-Gibb ML, Clarke IJ 2000 Effect of season on neuropeptide Y and galanin within the hypothalamus of the ewe in relation to plasma luteinizing hormone concentrations and the breeding season: an immunohistochemical analysis. J Neuroendocrinol 12:618–626[CrossRef][Medline]
  32. Barrell GK, Moenter SM, Caraty A, Karsch FJ 1992 Seasonal changes of gonadotropin-releasing hormone secretion in the ewe. Biol Reprod 46:1130–1135[Abstract]
  33. Robinson JE, Radford HM, Karsch FJ 1985 Seasonal changes in pulsatile luteinizing hormone (LH) secretion in the ewe: relationship of frequency of LH pulses to day length and response to estradiol negative feedback. Biol Reprod 33:324–334[Abstract]
  34. Karsch FJ, Dahl GE, Evans NP, Manning JM, Mayfield KP, Moenter SM, Foster DL 1993 Seasonal changes in gonadotropin-releasing hormone secretion in the ewe: alteration in response to the negative feedback action of estradiol. Biol Reprod 49:1377–1383[Abstract]
  35. Legan SJ, Karsch FJ, Foster DL 1977 The endocrine control of seasonal reproductive function in the ewe: a marked change in response to the negative feedback action of estradiol on luteinizing hormone secretion. Endocrinology 101:818–824[Medline]
  36. Scott CJ, Pereira AM, Tilbrook AJ, Rawson JA, Clarke IJ 2001 Changes in preoptic and hypothalamic levels of progesterone receptor mRNA across the oestrous cycle of the ewe. J Neuroendocrinol 13:401–406[CrossRef][Medline]
  37. Barker-Gibb ML, Scott CJ, Boublik JH, Clarke IJ 1995 The role of neuropeptide Y (NPY) in the control of LH secretion in the ewe with respect to season, NPY receptor subtype and the site of action in the hypothalamus. J Endocrinol 147:565–579[Abstract/Free Full Text]
  38. Karsch FJ, Foster DL 1975 Sexual differentiation of the mechanism controlling the preovulatory discharge of luteinizing hormone in sheep. Endocrinology 97:373–379[Abstract]
  39. Estrada KM, Pompolo S, Morris MJ, Tilbrook AJ, Clarke IJ 2003 Neuropeptide Y (NPY) delays the oestrogen-induced luteinizing hormone (LH) surge in the ovariectomized ewe: further evidence that NPY has a predominant negative effect on LH secretion in the ewe. J Neuroendocrinol 15:1011–1020[CrossRef][Medline]
  40. Sambrook J, Fritsch EF, Maniatis T 1989 Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press
  41. Chowen JA, Steiner RA, Clifton DK 1991 Semiquantitative analysis of cellular somatostatin mRNA levels by in situ hybridization histochemistry. Methods Neurosci 5:137–158
  42. Dufourny L, Caraty A, Clarke IJ, Robinson JE, Skinner DC 2005 Progesterone-receptive dopaminergic and neuropeptide Y neurons project from the arcuate nucleus to gonadotropin-releasing hormone-rich regions of the ovine preoptic area. Neuroendocrinology 82:21–31[CrossRef][Medline]
  43. Pompolo S, Rawson JA, Clarke IJ 2001 Projections from the arcuate/ventromedial region of the hypothalamus to the preoptic area and bed nucleus of stria terminalis in the brain of the ewe: lack of direct input to gonadotropin-releasing hormone neurons. Brain Res 904:1–12[CrossRef][Medline]
  44. Blache D, Fabre-Nys CJ, Venier G 1991 Ventromedial hypothalamus as a target for oestradiol action on proceptivity, receptivity and luteinizing hormone surge of the ewe. Brain Res 546:241–249[CrossRef][Medline]
  45. Blache D, Batailler M, Fabre-Nys C 1994 Oestrogen receptors in the preoptico-hypothalamic continuum: immunohistochemical study of the distribution and cell density during induced oestrous cycle in ovariectomized ewe. J Neuroendocrinol 6:329–339[CrossRef][Medline]
  46. Clarke IJ, Pompolo S, Scott CJ, Rawson JA, Caddy D, Jakubowska AE, Pereira AM 2001 Cells of the arcuate nucleus and ventromedial nucleus of the ovariectomized ewe that respond to oestrogen: a study using Fos immunohistochemistry. J Neuroendocrinol 13:934–941[CrossRef][Medline]
  47. Pompolo S, Pereira A, Scott CJ, Fujiyma F, Clarke IJ 2003 Evidence for estrogenic regulation of gonadotropin-releasing hormone neurons by glutamatergic neurons in the ewe brain: an immunohistochemical study using an antibody against vesicular glutamate transporter-2. J Comp Neurol 465:136–144[CrossRef][Medline]
  48. Foradori CD, Coolen LM, Fitzgerald ME, Skinner DC, Goodman RL, Lehman MN 2002 Colocalization of progesterone receptors in parvicellular dynorphin neurons of the ovine preoptic area and hypothalamus. Endocrinology 143:4366–4374[Abstract/Free Full Text]
  49. Dufourny L, Skinner DC 2002 Progesterone receptor, estrogen receptor {alpha}, and the type II glucocorticoid receptor are coexpressed in the same neurons of the ovine preoptic area and arcuate nucleus: a triple immunolabeling study. Biol Reprod 67:1605–1612[Abstract/Free Full Text]
  50. Goodman RL, Karsch FJ 1980 Pulsatile secretion of luteinizing hormone: differential suppression by ovarian steroids. Endocrinology 107:1286–1290[Abstract]
  51. Skinner DC, Evans NP, Delaleu B, Goodman RL, Bouchard P, Caraty A 1998 The negative feedback actions of progesterone on gonadotropin-releasing hormone secretion are transduced by the classical progesterone receptor. Proc Natl Acad Sci USA 95:10978–10983[Abstract/Free Full Text]
  52. Blache D, Fabre-Nys C, Venier G 1996 Inhibition of sexual behaviour and the luteinizing hormone surge by intracerebral progesterone implants in the female sheep. Brain Res 741:117–122[CrossRef][Medline]
  53. Shughrue PJ, Lane MV, Merchenthaler I 1997 Regulation of progesterone receptor messenger ribonucleic acid in the rat medial preoptic nucleus by estrogenic and antiestrogenic compounds: an in situ hybridization study. Endocrinology 138:5476–5484[Abstract/Free Full Text]
  54. Dufourny L, Caraty A, Clarke IJ, Robinson JE, Skinner DC 2005 Progesterone-receptive ß-endorphin and dynorphin B neurons in the arcuate nucleus project to regions of high gonadotropin-releasing hormone neuron density in the ovine preoptic area. Neuroendocrinology 81:139–149[CrossRef][Medline]
  55. Ebling FJ, Foster DL 1988 Photoperiod requirements for puberty differ from those for the onset of the adult breeding season in female sheep. J Reprod Fertil 84:283–293[Abstract/Free Full Text]
  56. Foster DL, Ebling FJ, Claypool LE, Woodfill CJ 1988 Cessation of long day melatonin rhythms time puberty in a short day breeder. Endocrinology 123:1636–1641[Abstract]
  57. Lehman MN, Robinson JE, Karsch FJ, Silverman AJ 1986 Immunocytochemical localization of luteinizing hormone-releasing hormone (LHRH) pathways in the sheep brain during anestrus and the mid-luteal phase of the estrous cycle. J Comp Neurol 244:19–35[CrossRef][Medline]
  58. Lehman MN, Coolen LM, Goodman RL, Viguie C, Billings HJ, Karsch FJ 2002 Seasonal plasticity in the brain: the use of large animal models for neuroanatomical research. Reprod Suppl 59:149–165[Medline]
  59. Pompolo S, Pereira A, Kaneko T, Clarke IJ 2003 Seasonal changes in the inputs to gonadotropin-releasing hormone neurones in the ewe brain: an assessment by conventional fluorescence and confocal microscopy. J Neuroendocrinol 15:538–545[Medline]
  60. Revel FG, Saboureau M, Masson-Pevet M, Pevet P, Mikkelsen JD, Simonneaux V 2006 Kisspeptin mediates the photoperiodic control of reproduction in hamsters. Curr Biol 16:1730–1735[CrossRef][Medline]
  61. Malpaux B, Daveau A, Maurice-Mandon F, Duarte G, Chemineau P 1998 Evidence that melatonin acts in the premammillary hypothalamic area to control reproduction in the ewe: presence of binding sites and stimulation of luteinizing hormone secretion by in situ microimplant delivery. Endocrinology 139:1508–1516[Abstract/Free Full Text]
  62. Lincoln GA 1994 Effects of placing micro-implants of melatonin in the pars tuberalis, pars distalis and the lateral septum of the forebrain on the secretion of FSH and prolactin, and testicular size in rams. J Endocrinol 142:267–276[Abstract/Free Full Text]
  63. Lincoln GA, Maeda K 1992 Effects of placing micro-implants of melatonin in the mediobasal hypothalamus and preoptic area on the secretion of prolactin and ß-endorphin in rams. J Endocrinol 134:437–448[Abstract/Free Full Text]
  64. Clarke IJ, Scott CJ, Rao A, Pompolo S, Barker-Gibb ML 2000 Seasonal changes in the expression of neuropeptide Y and pro-opiomelanocortin mRNA in the arcuate nucleus of the ovariectomized ewe: relationship to the seasonal appetite and breeding cycles. J Neuroendocrinol 12:1105–1111[CrossRef][Medline]
  65. Smith JT, Acohido BV, Clifton DK, Steiner RA 2006 KiSS-1 neurones are direct targets for leptin in the ob/ob mouse. J Neuroendocrinol 18:298–303[CrossRef][Medline]
  66. Castellano JM, Navarro VM, Fernandez-Fernandez R, Nogueiras R, Tovar S, Roa J, Vazquez MJ, Vigo E, Casanueva FF, Aguilar E, Pinilla L, Dieguez C, Tena-Sempere M 2005 Changes in hypothalamic KiSS-1 system and restoration of pubertal activation of the reproductive axis by kisspeptin in undernutrition. Endocrinology 146:3917–3925[Abstract/Free Full Text]
  67. Castellano JM, Navarro VM, Fernandez-Fernandez R, Roa J, Vigo E, Pineda R, Dieguez C, Aguilar E, Pinilla L, Tena-Sempere M 2006 Expression of hypothalamic KiSS-1 system and rescue of defective gonadotropic responses by kisspeptin in streptozotocin-induced diabetic male rats. Diabetes 55:2602–2610[Abstract/Free Full Text]



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