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. 2008 Jul 10;149(11):5770–5782. doi: 10.1210/en.2008-0581

Variation in Kisspeptin and RFamide-Related Peptide (RFRP) Expression and Terminal Connections to Gonadotropin-Releasing Hormone Neurons in the Brain: A Novel Medium for Seasonal Breeding in the Sheep

Jeremy T Smith 1,a, Lique M Coolen 1,a, Lance J Kriegsfeld 1, Ika P Sari 1, Mohammad R Jaafarzadehshirazi 1, Matthew Maltby 1, Katherine Bateman 1, Robert L Goodman 1, Alan J Tilbrook 1, Takayoshi Ubuka 1, George E Bentley 1, Iain J Clarke 1, Michael N Lehman 1
PMCID: PMC2584593  PMID: 18617612

Abstract

Reproductive activity in sheep is seasonal, being activated by short-day photoperiods and inhibited by long days. During the nonbreeding season, GnRH secretion is reduced by both steroid-independent and steroid-dependent (increased response to estradiol negative feedback) effects of photoperiod. Kisspeptin (also known as metastin) and gonadotropin-inhibitory hormone (GnIH, or RFRP) are two RFamide neuropeptides that appear critical in the regulation of the reproductive neuroendocrine axis. We hypothesized that expression of kisspeptin and/or RFRP underlies the seasonal change in GnRH secretion. We examined kisspeptin and RFRP (protein and mRNA) expression in the brains of ovariectomized (OVX) ewes treated with estradiol (OVX+E) during the nonbreeding and breeding seasons. In OVX+E ewes, greater expression of kisspeptin and Kiss1 mRNA in the arcuate nucleus and lesser expression of RFRP (protein) in the dorsomedial nucleus of the hypothalamus were concurrent with the breeding season. There was also a greater number of kisspeptin terminal contacts onto GnRH neurons and less RFRP-GnRH contacts during the breeding season (compared with the nonbreeding season) in OVX+E ewes. Comparison of OVX and OVX+E ewes in the breeding and nonbreeding season revealed a greater effect of steroid replacement on inhibition of kisspeptin protein and Kiss1 mRNA expression during the nonbreeding season. Overall, we propose that the two RFamide peptides, kisspeptin and RFRP, act in concert, with opposing effects, to regulate the activity of GnRH neurons across the seasons, leading to the annual change in fertility and the cyclical seasonal transition from nonbreeding to breeding season.

IN MANY SPECIES, reproductive activity is seasonal, resulting from annual fluctuations in GnRH secretion (1). In mammals, neural signals relaying photoperiodic stimuli are transduced by the pineal gland into a hormonal signal, melatonin, which in turn acts in the hypothalamus to control activity of the hypothalamo-pituitary-gonadal axis (2). In sheep, the long-day melatonin signal acts to inhibit GnRH secretion during the nonbreeding (anestrous) season via both steroid-independent (3,4,5) and steroid-dependent (6,7) pathways. Although the input and output pathways controlling seasonality are thus well defined, the link connecting the two remains elusive. Two recently discovered RFamide peptides (peptides with a common Arg-Phe-NH2 C-terminus), kisspeptin and gonadotropin-inhibiting hormone (GnIH) may well provide this link (8).

Kisspeptins are a family of neuropeptides encoded by the Kiss1 gene, which are the endogenous ligands for the G protein-coupled receptor GPR54 (9,10,11). It is now unequivocal that kisspeptins play a major role in the maintenance of the reproductive axis and may be critical for the onset of puberty (12,13). Kisspeptins stimulate gonadotropin secretion in rodents and sheep (14,15) with an effect that appears to be mediated exclusively by GnRH neurons, which express GPR54 (16,17). In sheep, kisspeptin neurons have been identified in the ovine arcuate nucleus (ARC) and preoptic area (POA) (18,19), and the former appears to be pivotal to the preovulatory LH surge in this species (20). Moreover, evidence suggests that Kiss1 expression in the forebrain may be regulated as a function of seasonal breeding in the hamster (21,22) and sheep (19).

GnIH was first identified in the quail hypothalamo-hypophysial system (23) and named for its ability to inhibit gonadotropin synthesis and release (24). However, GnIH may also decrease GnRH release because GnIH neurons make axosomatic contact with GnRH neurons in birds and mammals (25,26), and GnIH receptors have been identified on avian GnRH neurons (27). Putative and mature mammalian RFamide-related peptides (RFRP) homologs to GnIH (RFRP-1, RFRP-2, RFRP-3) have since been identified in several mammalian species (28) as well as their receptors GPR147/OT7T022 and GPR74 (28,29). Subsequently GnIH/RFRP peptide and mRNA were localized to the dorsomedial nucleus of the hypothalamus (DMH) in rats and hamsters (26,28), and RFRP inhibits LH secretion in hamsters when given either intracerebroventricularly or peripherally (26). Recently RFRP-3 has been localized to the DMH of ewes and inhibits gonadotropin secretion (Clarke, I. J., unpublished observation).

Given the importance of these two RFamide neuropeptide systems in the control of reproduction, we hypothesized that they both play a role in the regulation of seasonal reproduction in the ewe. If this is the case, one would predict that expression of kisspeptin would be increased in the breeding season, whereas RFRP expression would be decreased. Thus, this study had two objectives: first, to determine whether there are seasonal changes in expression of kisspeptin and/or RFRP containing perikarya and/or close contacts on to GnRH neurons; and second, to determine whether these changes were dependent or independent of the ovarian steroid, estradiol.

Materials and Methods

Animals

For experiment 1, adult Blackface ewes were housed first in an open barn, exposed to ambient environment, and then indoors beginning 1–2 d before ovariectomy. Indoor illumination was adjusted to daylight hours. Animals were fed a maintenance ration daily and had free access to water. All procedures involving animals were approved by the West Virginia University Animal Care and Use Committee. For experiment 2, Corriedale ewes of similar age (5–6 yr) and weight were maintained under natural conditions at the Monash University Sheep Facility, Werribee, Victoria, Australia. Ewes were subject to ambient photoperiod as well as environmental fluctuations in temperature. Experiments were carried out according to the National Health and Medical Research Council/Commonwealth Scientific and Industrial Research Organisation/Australian Animal Commission Code of Practice for the Care and Use of Animals for Experimental Purposes and were approved by the Monash University, School of Biomedical Sciences Animal Ethics Committee.

Ovariectomy and steroid replacement

Ovariectomy was performed as described (30). Chronic estradiol-17β (E) treatment in ovariectomized (OVX) ewes was achieved using sc implant (3 cm, inner diameter 3.35 mm, outer diameter 4.65 mm; SILASTIC brand silicon tubing; Dow Corning Corp., Midland, MI) packed with crystalline estradiol-17β (Sigma Chemical Co., St. Louis, MO). Implants were inserted into the axillary region of ewes either at the time of ovariectomy (experiment 1) or 2 wk after ovariectomy (experiment 2) and remained for 2 wk until tissue collection. Implants were designed to produce circulating levels of E in the range of 3–5 pg/ml (31), similar to that observed during the luteal phase of the estrous cycle.

Experiment 1: are there seasonal changes in kisspeptin or RFRP expression in ewes?

This experiment determined whether there are seasonal changes in the number of kisspeptin- and RFRP-immunoreactive cells in the hypothalamus using OVX+E ewes to normalize ovarian steroid concentrations across seasons. The number of close contacts between kisspeptin- or RFRP-containing varicosities and GnRH neurons in the POA, anterior hypothalamic area (AHA) and mediobasal hypothalamus (MBH) was also determined in nonbreeding (n = 5) and breeding seasons (n = 5). Ewes received injections of heparin (two injections of 25,000 U heparin iv 10 min apart) and were euthanized with an overdose of sodium pentobarbital (∼2 g in 7 ml of saline iv). Heads were then perfused bilaterally via the carotid arteries with 6 liters of 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.3), with 0.1% sodium nitrite added as a vasodilator and 10 U heparin per milliliter. After removal of the brains, POA/hypothalami were dissected out, stored overnight in fixative at 4 C, and then cryoprotected in 30% sucrose in 0.1 m phosphate buffer at 4 C. Coronal sections (50 μm) were cut on a freezing microtome and stored in a cryopreservative solution at (30% ethylene glycol, 1% polyvinylpyrrolidone, 30% sucrose in sodium phosphate buffer) at −20 C until processed for immunohistochemistry.

Immunohistochemistry for kisspeptin, RFRP, and GnRH

Kisspeptin and GnRH.

A series of every fourth section was immunostained for kisspeptin and GnRH using a dual immunoperoxidase technique (32). Briefly, kisspeptin was first detected using an avidin-biotin-immunoperoxidase protocol in which nickel-enhanced diaminobenzidine (DAB) was used as the chromogen to produce a blue-black reaction product. GnRH was then visualized with an avidin-biotin-immunoperoxidase protocol procedure using DAB to produce a brown reaction product. To detect kisspeptin, we used a polyclonal rabbit antibody against mouse kisspeptin-10 [1:100,000; 17 h at room temperature (RT); gift from A. Caraty, Université Tours, Nouzilly, France] (18) previously validated for use in sheep tissues (33). For detection of GnRH, we used a rabbit polyclonal antibody (1:50,000; 17 h at RT; LR-5, gift from R. Benoit, Montréal General Hospital, Montréal, Canada). Controls for double labeling included omission of one or both primary antisera, which eliminated all specific staining for the corresponding antigen(s).

For each animal, the total number of kisspeptin-immunoreactive (ir) cells was counted under bright-field illumination in the POA and rostral, middle, and caudal levels of the ARC and expressed as the mean per section (three sections per ewe for each level). The total number of GnRH-ir cells and the number of close associations between kisspeptin-ir varicosities and GnRH-ir neurons were counted in each animal in every fourth section through the POA, AHA and MBH. Close appositions were defined as kisspeptin-ir varicosities in direct contact with GnRH somas or dendrites viewed at the same plane of focus under high (×100) magnification. For each animal, the percentage of GnRH neurons that possessed at least one close association and the mean number of these close associations between kisspeptin-ir varicosities and each GnRH neuron were calculated for each region.

In addition, a subset of alternate sections through the POA and MBH was processed for triple-label immunofluorescent detection of synaptophysin, kisspeptin and GnRH to confirm that close contacts actually represented presynaptic terminals. Briefly, after washing in 0.1 m PBS, sections were incubated in PBS containing 0.4% Triton X-100 and 20% normal goat serum (NGS) for 1 h at RT and then for 17 h at RT with 1:300,000 antikisspeptin antiserum (A. Caraty, Université Tours). The primary antiserum was then labeled (1 h at RT) with biotinylated goat antirabbit serum (1:500 in NGS; Vector Laboratories, Burlingame, CA) and sections sequentially incubated for 1 h in ABC-elite (1:500 in PBS; Vector Laboratories), for 10 min in tyramide amplification solution (1:250 in PBS; NEN Life Science Products Life Sciences, Boston MA), and for 30 min in streptavidin-CY5 (1:100 in PBS; Jackson Laboratories, Bar Harbor, ME) with washing between each incubation. After thorough washing in PBS, sections were incubated with rabbit anti-GnRH antisera (1:1000; LR5; in NGS) and mouse anti-synaptophysin (Sigma; S5768, 1:1000) for 17 h, washed, and incubated in Alexa 555 conjugated to goat antirabbit and Alexa 488 conjugated to goat antimouse (1:100 in NGS; Molecular Probes, Eugene, OR) for 30 min. Finally, sections were washed, mounted on glass slides, dried, and coverslipped with gelvatol.

Putative contacts of kisspeptin fibers on GnRH neurons were examined with a laser-scanning confocal microscope system (LSM510; Zeiss, Thornwood, NY). Alexa 555 and CY5 fluorescence was imaged with a 567- and 680-nm emission filter and a HeNe laser, and Alexa 488- with 505-nm emission filter and Argon laser. Z-stacks of optical sections (1 μm; ×126 magnification) were captured through GnRH-IR neurons. Putative contacts were defined as a direct contact between the terminal and the GnRH-ir soma or proximal dendrite (and confirmed by synaptophysin labeling where appropriate).

RFRP and GnRH.

A series of every fourth section was washed in PBS, incubated in 0.5% H2O2, and incubated in normal goat serum in 0.1% Triton X-100 for 1 h. Sections were then incubated for 48 h at 4 C in antiserum generated against RFRP (PAC 123a; GE Bentley, University of California Berkley, Berkley, CA) diluted at 1:100,000 with 0.1% normal goat serum in 0.1% Triton X-100 as previously described (26) and optimized in ovine tissue (data not shown). After incubation in anti-RP, brains were incubated for 1 h in biotinylated goat antirabbit (1:300; Vector Laboratories), followed by incubation in avidin-biotin-horseradish peroxidase complex (1:200; ABC Elite kit; Vector Laboratories). Brains were then incubated in a biotinylated tyramide solution (0.6%) for 30 min. After biotinylated tyramide treatment, brains were incubated a second time in avidin-biotin-horseradish peroxidase complex (1:200). Cells were then labeled using nickel-enhanced DAB (Vector Laboratories) as the chromogen. After labeling for RFRP, brains were incubated in an antimouse GnRH antibody (1:2000; SMI-41; Sternberger Inc., Baltimore, MD) for 48 h at 4 C. After incubation, brains were incubated for 1 h in biotinylated goat antimouse (1:300; Vector Laboratories), followed by incubation in avidin-biotin-horseradish peroxidase complex (1:400; ABC Elite kit; Vector Laboratories). GnRH was labeled using DAB as the chromogen. Sections were mounted onto glass slides, dehydrated in a graded series of ethanols, cleared in xylenes, and coverslips were applied.

For each animal, the total number of RFRP-ir cells was counted through the rostral-caudal extent of the mediobasal hypothalamus and expressed per section. To assess close appositions between RFRP-ir terminals and GnRH perikarya, every fourth section through the POA, AHA, and MBH was analyzed. GnRH cells were counted, and contacts of RFRP-ir fibers on GnRH somas and dendrites were counted. A contact was scored only if a RFRP-ir varicosity was in direct contact with the GnRH neuron under high (×100) magnification, with both the varicosity and perikarya being in the same plane of focus. The percentage of GnRH neurons in each region that possessed at least one close association was calculated.

To determine whether the RFRP antisera cross-reacts with other RFamide peptides in sheep tissue, we preincubated antiserum (diluted 1:10,000) with the following peptides (1 μg/ml): GnIH (quail), GnRH (Auspep, Parkville, Melbourne, Australia), kisspeptin [murine C-terminal Kiss1 decapeptide (110–119)NH2], human neuropeptide FF (NFF), Chemerin (human 145–157-amide), pyroglutamylated RFamide peptide (QRFP-43), or prolactin-releasing peptide (a gift from Dr. S. Anderson, The University of Queensland, Brisbane, Australia) using a method previously described for kisspeptin antibodies (27). GnIH, kisspeptin, NFF, Chemerin, and QRFP-43 were all purchased from Phoenix Pharmaceuticals, Inc. (Burlingame, CA). The ability of RFRP antiserum to identify cells in the ovine DMH was diminished by preadsorption of the antiserum with GnIH (Fig. 1). Moreover, preadsorption with GnRH, kisspeptin (Fig. 1) or NFF, Chemerin QRFP-43, or prolactin-releasing peptide (data not shown) had no effect on RFRP immunoreactivity.

Figure 1.

Figure 1

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Controls for specificity of the GnIH(RFRP) antisera. Images are of the DMH after antibody preabsorption with GnIH, GnRH, and kisspeptin at a concentration of 1 μg/ml. Control is no preadsorption of antibody. Arrows indicate GnIH(RFRP) positive cells. Scale bar, 50 μm.

Experiment 2: are seasonal changes in kisspeptin and RFRP dependent on estradiol?

To determine the number of Kiss1 mRNA-, kisspeptin immunoreactive-, and RFRP mRNA-expressing cells in the hypothalamus of OVX and OVX+E ewes during the nonbreeding and breeding seasons, ewes were divided into four groups: nonbreeding season OVX, nonbreeding season OVX+E, breeding season OVX, breeding season OVX+E (n = 4/group). Hypothalamic blocks, from the diagonal band of Broca to the mammillary bodies, were collected as follows: ewes were killed by an iv overdose of sodium pentobarbital (Lethabarb; Virbarc, Peakhurst, Australia), and heads were perfused with 2 liters of heparinized saline (12.5 U/ml), 2 liters of 4% paraformaldehyde plus 15% picric acid in 0.1 m phosphate buffer (pH 7.4), and then 1 liter of the same fixative containing 20% sucrose. Brains were subsequently removed and the hypothalamus dissected out and postfixed in fixative containing 30% sucrose overnight at 4 C. Hypothalami were then transferred to a solution of 0.1 m phosphate buffer containing 30% sucrose for 7 d and then frozen in powdered dry ice. Coronal sections (40 μm) were cut on a cryostat (extending the rostral POA to the caudal MBH) and placed into cryoprotectant (30% ethylene glycol, 20% glycerol in sodium phosphate buffer) until used for immunohistochemistry, or cryoprotectant with 2% paraformaldehyde until used for in situ hybridization. All tissue was stored at −20 C.

Immunohistochemistry for kisspeptin

Sections representing the rostral, middle, and caudal regions of the ARC and six continuous sections through the POA (sections 200 μm apart) 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 × 5 min). A blocking solution containing 10% normal goat serum and 0.3% Triton X-100 in 0.1 m Tris-buffered saline (TBS) was applied, and then sections were incubated for 72 h at 4 C with a rabbit polyclonal antibody against mouse kisspeptin-10 (no. 566 used at a dilution of 1:2000) (18). Slides were washed in TBS and sections incubated with goat antirabbit Alexa 448 for 2 h at RT (1:400; Molecular Probes). Slides were again washed in TBS and sections counterstained with 0.3% Sudan Black B to minimize autofluorescence. After rinses in TBS and then 0.1 m phosphate buffer, coverslips were applied using antifade mounting solution (Dako, Capenteria, CA). Kisspeptin-ir cells were identified under fluorescent illumination, with a single observer counting the total number of cells. For each ewe, the number of kisspeptin-ir cells per section in each region was averaged to produce a mean (± sem).

Radiolabeled cRNA riboprobes

Kiss1.

A 375-base cDNA sequence of the ovine Kiss1 gene (GenBank accession no. DQ059506) was cloned as previously described (19).

RFRP.

A 460-base cDNA sequence of the ovine RFRP precursor was cloned using primers based on the known bovine RFRP sequence (GenBank accession no. NM_174168) (28) and inserted into a pGemT-easy plasmid (GenBank accession no. for sheep RFRP precursor mRNA is EU177779).

The antisense ovine Kiss1 and RFRP riboprobes were transcribed from linearized plasmid containing the ovine Kiss1/RFRP insert with SP6 and T7 polymerase, respectively (Promega Corp., Madison, WI) and 35S-uridine 5-triphosphate (GE Healthcare Life Sciences, Boston, MA) under a standard transcription protocol. The riboprobe was separated from unincorporated nucleotides on a Sephadex G-25 column.

In situ hybridization

Kiss1 mRNA.

In situ hybridization was performed as previously described (19,20). Two sections (200 μm apart) representing the rostral, middle, and caudal regions of the ARC (six sections total) and six continuous sections through the POA (sections 200 μm apart) were chosen from each ewe and mounted on SuperFrost slides and prepared for in situ hybridization. Radiolabeled (35S) antisense Kiss1 riboprobe was denatured, diluted in hybridization buffer at a concentration of 5 × 106 cpm/ml along with tRNA, and applied to slides (120 μl/slide). After hybridization (53 C for 16 h), slides were treated with ribonuclease 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 d later. No signal was observed after the application of radiolabeled sense probe (data not shown).

RFRP mRNA.

Three sections through the rostral to caudal extent of the DMH were chosen from each ewe and prepared for in situ hybridization. Radiolabeled (35S) antisense RFRP riboprobe was denatured, diluted in hybridization buffer at a concentration of 5 × 106 cpm/ml along with tRNA and applied to slides (120 μl/slide). Hybridization and postwashes took place as above, and slides were dipped in photographic emulsion and developed 10 d later. No signal was observed after the application of radiolabeled sense probe (data not shown).

Kiss1 and RFRP mRNA quantification and analysis

Image analysis was carried out using randomly coded slides under dark-field illumination with software designed to count the total number of cells and the number of silver grains per cell (ImagePro plus, Media Cybernetics, Inc., Silver Spring, MD). Cells were counted when silver grain density was greater than five times background. Data are expressed as the mean number of identifiable cells per section and the mean number of silver grains per cell (a semiquantitative index of mRNA expression/cell).

Statistical analysis

All data are expressed as the mean (± sem). For experiment 1, statistical analysis was accomplished by one-way ANOVA (nonbreeding vs. breeding season). Variations in the percentage of GnRH neurons with kisspeptin/GnIH contacts was assessed by one-way ANOVAs on arcsine-transformed data. Two-way ANOVA was used to determine the treatment (OVX vs. OVX+E) and season (nonbreeding vs. breeding season) effects in experiment 2. Where significant interactions were reached (P < 0.05), differences among means were assessed by least significant difference test.

Results

Experiment 1: are there seasonal changes in kisspeptin or RFRP expression in ewes?

Kisspeptin.

Kisspeptin-ir cell bodies were localized to the POA and ARC, with the latter containing far more cells in the middle and caudal regions (Fig. 2). The number of kisspeptin-ir cells in the middle and caudal ARC was more than 4-fold higher during the breeding season, compared with the nonbreeding season (middle, P < 0.01; caudal, P < 0.01; Fig. 2). In the POA, the number of cells expressing kisspeptin was similar between nonbreeding and breeding season (Fig. 2). Kisspeptin-ir terminals made contacts with GnRH neurons located in the POA, AHA, and MBH (Fig. 3). The percentage of GnRH neurons with kisspeptin contacts was approximately 1.9-fold higher during the breeding season, compared with nonbreeding (P < 0.05), in the MBH, but was unchanged in the POA and AHA. Given that virtually all GnRH neurons in the MBH had kisspeptin contacts during the breeding season, we further examined the number of kisspeptin contact per GnRH neuron. The number of kisspeptin contacts was more than 2-fold greater during the breeding season in AHA GnRH neurons (P < 0.05) and MBH GnRH neurons (P < 0.05, Fig. 3), compared with the nonbreeding season; but no change in the number of kisspeptin contacts was seen in the POA GnRH neurons. In addition, to confirm that close contacts observed represented synaptic inputs, we examined a total of 49 MBH GnRH neurons (23 breeding season and 26 nonbreeding season neurons) for triple-label immunofluorescent detection of kisspeptin, GnRH and the synaptic vesicle protein, synaptophysin (Fig. 4). Confocal images of these cells revealed that in every instance of a close contact, the kisspeptin-positive varicosity in contact with the GnRH neuron was also immunopositive for synaptophysin, strongly suggesting that these represented bona fide synaptic inputs. Finally, the number of GnRH neurons did not vary between breeding and nonbreeding season.

Figure 2.

Figure 2

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Representative photomicrographs of immunohistochemistry showing kisspeptin-ir cells in the POA (A) and middle ARC (B) of the ewe brain. 3V, Third ventricle. Scale bar, 100 μm. C, Effect of season on kisspeptin expressing perikarya in the POA and ARC of the ewe. The number of kisspeptin-ir cells per section is shown for nonbreeding and breeding season ewes in the POA and rostral, middle, and caudal ARC. Values are means (± sem). *, P < 0.05 nonbreeding vs. breeding season.

Figure 3.

Figure 3

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A, Photomicrographs of GnRH and kisspeptin double-label immunohistochemistry in the MBH of nonbreeding and breeding season ewes showing kisspeptin-ir terminal (black) contacts with GnRH-ir cell bodies (brown). Arrows indicate points of contact. Scale bar, 15 μm. B, The percentage of GnRH neurons in the POA, AHA, and MBH with kisspeptin contacts in nonbreeding and breeding season ewes. C, The number of kisspeptin contacts per GnRH cell. Values are means (± sem). *, P < 0.05 nonbreeding vs. breeding season.

Figure 4.

Figure 4

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Confocal images (1 μm thickness) of GnRH, kisspeptin, and synaptophysin triple-label immunofluorescence in the MBH of a breeding season ewe. Arrow indicates one example of a dual-labeled kisspeptin and synaptophysin-positive terminal in close contact with a GnRH soma. Scale bar, 10 μm.

RFRP.

RFRP-ir cell bodies were present within the paraventricular nucleus (PVN) and DMH, with approximately 40% more cells identifiable during the nonbreeding season, compared with the breeding season (P < 0.05, Fig. 5). RFRP-ir terminal contacts to more rostral GnRH neurons were also significantly greater during the nonbreeding season. Specifically, the percentage of GnRH neurons in the AHA with RFRP contacts was more than double during the nonbreeding relative to the breeding season (P < 0.05), with a similar result for GnRH neurons located in the POA (80% more contacts during the nonbreeding season, P < 0.05) but no difference seen in the MBH (Fig. 5). Again, the number of GnRH neurons did not vary between breeding and nonbreeding season.

Figure 5.

Figure 5

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A, Representative photomicrographs of immunohistochemistry showing GnIH(RFRP)-ir cells in the DMH of the ewe brain. Arrows indicate positive cells. Scale bar, 100 μm. B, The number of GnIH-ir cells per section is shown for nonbreeding and breeding season ewes in the DMH. Values are means (± sem). *, P < 0.05 nonbreeding vs. breeding season. C, Photomicrograph (low and high power) of GnRH and GnIH double-label immunohistochemistry in the POA showing GnIH-ir terminal (black) contacts with a GnRH-ir cell body (brown). Arrows indicate points of contact. Scale bar, 100 μm. D, The percentage of GnRH neurons in the POA, AHA, and MBH with GnIH contacts in nonbreeding and breeding season ewes. Values are means (± sem). *, P < 0.05; **, P < 0.01 nonbreeding vs. breeding season.

Experiment 2: are seasonal changes in kisspeptin and RFRP dependent on estradiol?

Kisspeptin immunoreactivity.

In concordance with experiment 1, kisspeptin-ir cell bodies were located in the POA and ARC (Fig. 6). In the POA, kisspeptin-ir cells differed with steroid treatment (P < 0.05) but not season. Specifically, kisspeptin cell number was more than 4-fold greater after E treatment in OVX ewes at both the nonbreeding and breeding season (P < 0.05 for both, Fig. 7). Again, relatively few kisspeptin cells were observed in the rostral ARC, and there was no significant effect of E treatment or season. In the middle ARC, kisspeptin-ir cell number was 74% lower in OVX+E ewes during the nonbreeding season and 69% lower during the breeding season (compared with OVX, both P < 0.05); the number of kisspeptin cells in the middle ARC of OVX+E ewes was 1.9-fold greater during the breeding season, compared with nonbreeding, but this did not reach statistical significance (P = 0.09). In the caudal ARC, the number of kisspeptin-ir cells differed with steroid treatment and season. During the nonbreeding season, E treatment resulted in a 51% reduction in kisspeptin-ir cell number (P < 0.05), whereas no change was seen during the breeding season. Consequently, kisspeptin-ir cell number was 2.2-fold greater in the caudal ARC during the breeding season in OVX+E ewes, compared with the nonbreeding season (P < 0.05, Fig. 7).

Figure 6.

Figure 6

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Representative photomicrographs showing kisspeptin-ir cells in sections of the POA and caudal ARC from OVX and OVX+E ewes during the nonbreeding and breeding season. 3V, Third ventricle. Scale bar, 100 μm.

Figure 7.

Figure 7

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Effect of ovariectomy and E replacement on the number of kisspeptin-ir cells per section in the POA and ARC (rostral, middle, caudal) of nonbreeding and breeding season ewes. Values are means (± sem). *, P < 0.05 OVX vs. OVX+E. Letters (a, b) designate significant difference for nonbreeding vs. breeding season.

Kiss1 mRNA.

As with the immunohistochemical data, cells expressing Kiss1 mRNA were identifiable in the POA and ARC, with the majority of the cells in the latter present in the middle and caudal regions (Fig. 8). In the POA, the number of Kiss1 mRNA-expressing cells was 3.5-fold greater after E treatment during the nonbreeding season (P < 0.05, Fig. 9), but there was no significant change during the breeding season. The level of Kiss1 mRNA expression per cell in the POA was similar among all groups. In the rostral portion of the ARC, Kiss1 cell numbers were unchanged with E treatment or season, but Kiss1 mRNA expression per cell differed with treatment, season, and there was a significant interaction (P < 0.05). Thus, expression per cell was reduced 51% in E-treated OVX ewes during the nonbreeding season (P < 0.01) but was unchanged during the breeding season (Fig. 9). In the middle and caudal ARC, the number of Kiss1 mRNA-expressing cells was reduced by E treatment during both the nonbreeding and breeding season (all P < 0.01) but similar to the rostral ARC, a significant interaction between season and treatment was observed in Kiss1 mRNA expression per cell. Again, expression per cell was significantly lower in E-treated OVX ewes during the nonbreeding season (middle ARC 50%, P < 0.01; caudal ARC 45%, P < 0.001) but was unchanged with E treatment during the breeding season (Fig. 9). Therefore, Kiss1 mRNA expression per cell was significantly higher in the ARC of OVX+E ewes during the breeding season (rostral ARC, 3.0-fold, P < 0.01; middle ARC, 4.0-fold, P < 0.05; caudal ARC, 2.8-fold, P < 0.01; Fig. 9). In OVX ewes, Kiss1 mRNA expression per cell was slightly, but significantly higher in the ARC during the breeding season, compared with the nonbreeding season (rostral ARC, 30%, P < 0.05; middle ARC, 25%, P < 0.05; caudal ARC, 34%, P < 0.001; Fig. 9).

Figure 8.

Figure 8

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Dark-field photomicrographs showing representative sections of the POA and caudal ARC and Kiss1 mRNA-expressing cells (as shown by the presence of silver grain clusters) from OVX and OVX+E ewes during the nonbreeding and breeding season. 3V, Third ventricle. Scale bar, 200 μm.

Figure 9.

Figure 9

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Effect of ovariectomy and E replacement on Kiss1 expression in the POA and ARC (rostral, middle, caudal) of nonbreeding and breeding season ewes. Left panels depict the number of Kiss1 mRNA-expressing cells per section, and right panels show the number of silver grains per Kiss1 cell (reflecting expression per cell). Values are means (± sem). *, P < 0.05; **, P < 0.01; ***, P < 0.001; OVX vs. OVX+E. Letters (a and b) designate significant difference for nonbreeding vs. breeding season.

RFRP mRNA.

RFRP mRNA-expressing cells were localized to the medial DMH, and there also appeared to be cells located in the medial portion of the PVN (Fig. 10). The number of RFRP mRNA expressing cells and the cellular content of RFRP mRNA did not differ with E treatment or season (Fig. 10).

Figure 10.

Figure 10

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A, Dark-field photomicrograph of the DMH showing GnIH(RFRP) mRNA-expressing cells. 3V, Third ventricle. Scale bar, 200 μm. B, Effect of ovariectomy and E replacement on GnIH mRNA expression in the DMH/PVN of nonbreeding and breeding season ewes. Left panel depicts the number of GnIH mRNA-expressing cells per section, and the right panel shows the number of silver grains per GnIH cell (reflecting expression per cell) is shown. Values are means (± sem).

Discussion

Kisspeptin and RFRP appear well placed to play roles in the annual shift in fertility in the ewe. First, both kisspeptin and RFRP are potent and opposing (kisspeptin, positive; RFRP, negative) regulators of gonadotropin secretion and the reproductive system (14,15,23,26). Second, our data show higher kisspeptin expression (protein and mRNA) in the ARC and lower RFRP expression (protein) in the PVN and DMH in OVX+E ewes concurrent with the breeding season. Finally, we observed inverse seasonal changes in the number of kisspeptin- and RFRP-containing contacts onto GnRH neurons. Specifically, kisspeptin-contacts were higher in the MBH GnRH neurons and RFRP-contacts were lower in the POA GnRH neurons during the breeding season. Given that kisspeptin can stimulate ovulation in seasonally acyclic ewes (34), and the relationship between RFRP and seasonal breeding in avian species (24,25,27), our data support a role for these two RFamides in the control of seasonal breeding.

Seasonal regulation of GnRH secretion is known to be mediated by steroid-independent (4,5) and steroid-dependent effects of photoperiod, with the latter reflecting a seasonal shift in the response to E-negative feedback evident in OVX+E ewes (6,7). The changes in kisspeptin and GnIH expressing perikarya observed in experiment 1 suggest that these neurons could contribute to the seasonal regulation of GnRH and the results of experiment 2 suggest kisspeptin neurons in the caudal (and possibly middle) ARC as potential mediators of seasonal changes in E-negative feedback. Interestingly, we show kisspeptin immunoreactivity was higher during the breeding season in OVX+E-treated ewes in both the middle and caudal ARC in experiment 1 but only the caudal ARC in experiment 2. We feel, however, that the two experiments are qualitatively the same (a trend for a breeding season increase in kisspeptin cells in the middle ARC was apparent in experiment 2), and any difference between the two studies may relate to the differing breeds of sheep used. Moreover, in experiment 2 the feedback effect of E appeared to be lost on kisspeptin immunoreactive cells in the caudal ARC during the breeding season, but this was not the case with the number of Kiss1 mRNA-positive cells. It should be noted the per cell content of Kiss1 mRNA during the breeding season did not change with E treatment. We believe the net effect of Kiss1 mRNA cell number and mRNA content per cell equate to a reduced effect of E during the breeding season in the ARC, consistent with the immunohistochemical data. In contrast, we did not observe seasonal differences in RFRP mRNA expression in experiment. 2. This inconsistency may relate to the nature of our RFRP riboprobe, which spans the 460-nucleotide ovine RFRP precursor. It is conceivable that posttranslational proteolytic cleavage of the RFRP preproprotein (28) may be regulated by season (or E), leading to specific regulation of mature RFRP peptides. Finally, the modest seasonal changes in expression of Kiss1 mRNA per cell in OVX ewes raises the possibility that this ARC population also contributes to the steroid-independent effects of photoperiod, although this was not evident in changes of kisspeptin immunoreactivity.

The reciprocal seasonal changes in kisspeptin and RFRP input to GnRH neurons strengthen the proposed role for these neurons in control of seasonal breeding. Interestingly, these seasonal changes in RFamide input appear to target different sets of GnRH neurons, such that RFRP input may control more rostral GnRH neurons, whereas kisspeptin regulates the posterior subpopulation. Morphological plasticity in neural circuitry, particularly of those that input to GnRH neurons, has been reported previously in the ewe, with a greater number of synaptic inputs to GnRH neurons observed during the breeding than nonbreeding season (35); our observations suggest that kisspeptin afferents could account for some of these changes.

The anatomical origin of RFRP and kisspeptin inputs to GnRH neurons is yet to be determined. RFRP afferents most likely derive from the DMH/PVN because this is the sole site of RFRP perikarya in the ovine hypothalamus (Clarke, I. J., unpublished observation). Moreover, retrograde tracing has shown that neurons from the DMH/PVN, in a similar distribution to RFRP cells, project to the POA in the ewe (36). Similar projections from RFRP neurons have also been reported in birds (25), hamsters, rats, and mice (26). The positive correlation of ARC kisspeptin perikarya and kisspeptin close contacts onto posterior GnRH neurons suggests that that latter arises from ARC kisspeptin neurons. This is supported by recent evidence that nearly all kisspeptin neurons in the ARC, but not those in the POA, coexpress dynorphin and neurokinin B (NKB) (33) and that MBH GnRH neurons receive synaptic input from terminals that contain dynorphin (37) and colocalize NKB (Lehman, M. N., unpublished observation). Conversely, the close contacts on POA GnRH neurons may come from more rostral kisspeptin neurons. This possibility is supported by anterograde tract tracing data in the ewe, indicating that projections from the lateral and medial regions of the POA provide input to POA GnRH cells (38). On the other hand, ARC kisspeptin neurons may also project to POA GnRH cells because the latter, like MBH GnRH neurons, receive inputs that contain both dynorphin and NKB (Lehman, M. N., unpublished observations). Moreover, it is important to note that GnRH neurons possess extensive dendritic spines (39), which are likely to enable further synaptic inputs. Thus, it is a distinct possibility that our data underestimate the number of kisspeptin- and RFRP-containing inputs to GnRH neurons.

In contrast to kisspeptin expression in the ARC, E treatment appeared to stimulate kisspeptin expression in the subpopulation of cell bodies in the POA. It should also be noted that season did not have any effect on POA kisspeptin expression; thus, we propose that these kisspeptin neurons are not involved in seasonal breeding. This regulatory effect of E in the ewe is intriguing and points to similarities to the rodent, in which stimulatory regulation of Kiss1 mRNA to steroid treatment expression in the anteroventral periventricular nucleus (AVPV) has been previously reported (40,41) and raises the possibility that POA kisspeptin neurons in sheep participate in the E-induced preovulatory LH surge, as do AVPV neurons in rodents (42). However, in the ewe E acts in the MBH, not the POA, to induce the LH surge (43,44) so that ovine POA kisspeptin neurons would most likely be activated indirectly by the positive feedback action of E. In experiment 1, no GnRH-ir cell bodies in the POA were seen to colocalize kisspeptin, as has been previously reported (45). In this previous paper, colocalization of kisspeptin and GnRH was observed in the sheep with one antiserum (Phoenix Pharmaceuticals) (45), but this was not the case with another (A. Caraty, Université Tours) (Franceschini, I., and A. Caraty, personal communication), and it was subsequently shown that this was likely due to nonspecificity of the former (33). Thus, it is unlikely that the colocalization of GnRH and kisspeptin in the ewe is accurate.

The question remains as to what the regulatory stimulus is for these seasonal changes in kisspeptin and RFRP expression. Previous work (46) on E-negative feedback has identified estrogen-response elements on neurons in the ovine POA and retrochiasmatic area that stimulate inhibitory A15 dopaminergic neurons in the nonbreeding season but not the breeding season. Thus, one possible regulatory system would be that these A15 dopaminergic neurons inhibit GnRH by stimulating RFRP and inhibiting kisspeptin. The low number and lack of seasonal changes in dopaminergic close contacts on GnRH neurons (47,48) supports this possibility, particularly in light of the strong seasonal changes in RFamide input to GnRH cells observed in this study. Because most ARC kisspeptin neurons contain steroid receptors (18,19), a second possibility is that E acts directly on these neurons to inhibit them and thus GnRH secretion. Finally, given that seasonal breeding in the ewe is regulated by melatonin (1), a third possibility is that melatonin acts directly on kisspeptin and/or RFRP cells to regulate their activity. Melatonin receptors are present in high density in the ovine premammillary region, which contains the caudal portion of the ARC (49), and melatonin binding is seen, to a lesser extent, in the DMH and PVN (2). Moreover, a direct effect of melatonin on RFRP has been demonstrated in the hamster (50) and quail with melatonin receptor expressed in RFRP cells (51).

There is evidence that RFRP contributes to seasonality in birds. Specifically, RFRP peptide levels in song sparrows are highest at the end of the breeding season, when GnRH levels were lowest (25). Moreover, RFRP axons in the POA were reduced during the breeding season in rufous-winged sparrows (52). Our data show that RFRP changes in a similar manner in mammals. Photoperiodic effects on kisspeptin have been observed in the hamster (21,22), but these two reports are difficult to interpret because of conflicting data. One group found that stimulatory photoperiod increased kisspeptin expression in the AVPV (21), whereas the other could not detect Kiss1 mRNA in this area, regardless of photoperiod (22). With regard to expression in the ARC, inhibitory photoperiod was found to suppress (22) and stimulate (21) Kiss1 mRNA.

Overall, these studies show a higher level of kisspeptin and a lower level of RFRP expression in the hypothalamus of the ewe during the breeding season, compared with the nonbreeding season. Moreover, we show that the number of kisspeptin terminal contacts to GnRH neurons is higher during the breeding season and that terminal contacts from RFRP cells to GnRH neurons is higher during the nonbreeding season. The net result of these changes produces an increase in stimulatory (kisspeptin) input to GnRH neurons during the breeding season and an increase in inhibitory (RFRP) input during the nonbreeding season. Hence, we proposed the two neuropeptides act as a balance controlling the activity of GnRH neurons across the seasons, which would potentially mediate the cyclical seasonal transition from nonbreeding to breeding season.

Acknowledgments

We thank Ms. S. Saleh, Mr. B. Doughton, and Ms. L. Morrish for technical assistance.

Footnotes

This work was supported by the National Health and Medical Research Council of Australia and National Institutes of Health Grants HD050470 and HD17864. J.T.S. is supported by a Peter Doherty Fellowship and is a recipient of an Endocrine Society of Australia Postdoctoral Award.

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 10, 2008

Abbreviations: AHA, Anterior hypothalamic area; ARC, arcuate nucleus; AVPV, anteroventral periventricular nucleus; DAB, diaminobenzidine; DMH, dorsomedial nucleus of the hypothalamus; E, estradiol-17β; GnIH, gonadotropin-inhibiting hormone; GPR, G protein-coupled receptor; ir, immunoreactive; MBH, mediobasal hypothalamus; NFF, neuropeptide FF; NGS, normal goat serum; NKB, neurokinin B; OVX, ovariectomized; POA, preoptic area; PVN, paraventricular nucleus; RFRP, RFamide-related peptide; RT, room temperature; TBS, Tris-buffered saline.

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