KNDy neurone activation prior to the LH surge of the ewe is disrupted by LPS

in Reproduction
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C FerganiSchool of Veterinary Science, University of Liverpool, Liverpool, UK

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J E RoutlySchool of Veterinary Science, University of Liverpool, Liverpool, UK

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D N JonesSchool of Veterinary Science, University of Liverpool, Liverpool, UK

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L C PickavanceSchool of Veterinary Science, University of Liverpool, Liverpool, UK

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R F SmithSchool of Veterinary Science, University of Liverpool, Liverpool, UK

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H DobsonSchool of Veterinary Science, University of Liverpool, Liverpool, UK

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Correspondence should be addressed to C Fergani; Email: cfergani@bwh.harvard.edu

(C Fergani is now at Division of Endocrinology, Diabetes and Hypertension, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA)

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In the ewe, steroid hormones act on the hypothalamic arcuate nucleus (ARC) to initiate the GnRH/LH surge. Within the ARC, steroid signal transduction may be mediated by estrogen receptive dopamine-, β-endorphin- or neuropeptide Y (NPY)-expressing cells, as well as those co-localising kisspeptin, neurokinin B (NKB) and dynorphin (termed KNDy). We investigated the time during the follicular phase when these cells become activated (i.e., co-localise c-Fos) relative to the timing of the LH surge onset and may therefore be involved in the surge generating mechanism. Furthermore, we aimed to elucidate whether these activation patterns are altered after lipopolysaccharide (LPS) administration, which is known to inhibit the LH surge. Follicular phases of ewes were synchronised by progesterone withdrawal and blood samples were collected every 2 h. Hypothalamic tissue was retrieved at various times during the follicular phase with or without the administration of LPS (100 ng/kg). The percentage of activated dopamine cells decreased before the onset of sexual behaviour, whereas activation of β-endorphin decreased and NPY activation tended to increase during the LH surge. These patterns were not disturbed by LPS administration. Maximal co-expression of c-Fos in dynorphin immunoreactive neurons was observed earlier during the follicular phase, compared to kisspeptin and NKB, which were maximally activated during the surge. This indicates a distinct role for ARC dynorphin in the LH surge generation mechanism. Acute LPS decreased the percentage of activated dynorphin and kisspeptin immunoreactive cells. Thus, in the ovary-intact ewe, KNDy neurones are activated prior to the LH surge onset and this pattern is inhibited by the administration of LPS.

Abstract

In the ewe, steroid hormones act on the hypothalamic arcuate nucleus (ARC) to initiate the GnRH/LH surge. Within the ARC, steroid signal transduction may be mediated by estrogen receptive dopamine-, β-endorphin- or neuropeptide Y (NPY)-expressing cells, as well as those co-localising kisspeptin, neurokinin B (NKB) and dynorphin (termed KNDy). We investigated the time during the follicular phase when these cells become activated (i.e., co-localise c-Fos) relative to the timing of the LH surge onset and may therefore be involved in the surge generating mechanism. Furthermore, we aimed to elucidate whether these activation patterns are altered after lipopolysaccharide (LPS) administration, which is known to inhibit the LH surge. Follicular phases of ewes were synchronised by progesterone withdrawal and blood samples were collected every 2 h. Hypothalamic tissue was retrieved at various times during the follicular phase with or without the administration of LPS (100 ng/kg). The percentage of activated dopamine cells decreased before the onset of sexual behaviour, whereas activation of β-endorphin decreased and NPY activation tended to increase during the LH surge. These patterns were not disturbed by LPS administration. Maximal co-expression of c-Fos in dynorphin immunoreactive neurons was observed earlier during the follicular phase, compared to kisspeptin and NKB, which were maximally activated during the surge. This indicates a distinct role for ARC dynorphin in the LH surge generation mechanism. Acute LPS decreased the percentage of activated dynorphin and kisspeptin immunoreactive cells. Thus, in the ovary-intact ewe, KNDy neurones are activated prior to the LH surge onset and this pattern is inhibited by the administration of LPS.

Introduction

During the late follicular phase of the ewe, the decrease in plasma progesterone concentrations after luteolysis, along with an increase of oestradiol from the dominant follicle(s), triggers the onset of sexual behaviour, closely followed by a sudden and massive release of gonadotrophin-releasing hormone (GnRH) and therefore, luteinising hormone (LH), leading to ovulation. By contrast, various types of stressors, such as an acute bolus injection of the E. coli endotoxin (lipopolysaccharide (LPS)) during the late follicular phase decreases plasma oestradiol concentrations and abolishes both sexual behaviour and the LH surge (Fergani et al. 2012) via mechanisms that remain largely unknown.

In the ewe, oestradiol acts, at least in part, in the vicinity of the arcuate nucleus (ARC) to initiate positive feedback mechanisms (Blache et al. 1991, Caraty et al. 1998). In line with this hypothesis, we have recently shown that the number of ERα-containing cells that are activated in the ARC (as measured by co-localisation with c-Fos) increases dramatically at least 6–7 h prior to the surge onset and remains elevated throughout the LH surge (Fergani et al. 2014). Furthermore, this pattern of activation is attenuated, if preceded by acute administration of LPS (Fergani et al. 2014). Undoubtedly, kisspeptin signalling is the key pathway in mediating oestradiol-positive feedback on GnRH neurones in all species studied to date (Smith 2009, Lehman et al. 2010, Fergani et al. 2013) and is therefore a primary candidate for initiating the GnRH/LH surge. However, ARC kisspeptin cells are activated during the LH surge but not at other times in the follicular phase (Merkley et al. 2012, Fergani et al. 2013). Thus, there are other cell types activated in the ARC before the expected surge onset that are not kisspeptin immunoreactive cells, but contain ERα.

In this context, the ARC contains tyrosine hydroxylase (TH; a biosynthetic enzyme marker for dopamine), β-endorphin and neuropeptide Y (NPY) cells, sub-populations of which contain ERα (Antonopoulos et al. 1989, Lehman & Karsch 1993, Lehman et al. 1993) and have been implicated in reproductive neuroendocrine mechanisms, and also in the pathophysiology of stress-induced reproductive disruptions (Melis & Argiolas 1995, Fabre-Nys et al. 2003, Taylor et al. 2007). These neuropeptides are, therefore, potential candidates for contributing to the generation of sexual behaviour and/or GnRH surge secretion. More importantly, however, nearly all kisspeptin cells in the ARC co-localise two other neuropeptides that are key in the control of GnRH secretion: neurokinin B (NKB) and dynorphin (Goodman et al. 2007, Navarro et al. 2011) and thus are termed KNDy cells (kisspeptin, neurokinin B and dynorphin; (Cheng et al. 2010, Navarro et al. 2011). As 94% of kisspeptin cells co-localise dynorphin and 80% co-localise NKB, with an equally high reciprocal co-localisation (Goodman et al. 2007), immunohistochemical detection of kisspeptin protein would potentially reflect presence of all three neuropeptides. However, in the ewe, KNDy peptide immunoreactivity and/or gene expression fluctuates depending on hormonal and gonadal status (Foradori et al. 2006, Smith 2009). Thus, it is plausible to speculate that in ovary-intact ewes, endogenous fluctuation of the ovarian steroid hormone milieu during the follicular phase may be associated with differential protein expression within KNDy cells and, therefore, different activation patterns for each neuropeptide.

Lastly, there is evidence that kisspeptin neurones mediate the effects of stressors on the reproductive neuroendocrine axis. For example, there is downregulation of the hypothalamic kisspeptin system in rats after administration of LPS (Iwasa et al. 2008, Kinsey-Jones et al. 2009). Furthermore, immunohistochemical analysis of kisspeptin combined with c-Fos, revealed that LPS administration is accompanied by reduced activation of kisspeptin cells in the ARC of the ewe (Fergani et al. 2013, 2014). However, the effects of stressors on the levels dynorphin and NKB immunoreactivity and activation in the ARC have not received as much attention.

We have shown that activation of ERα-containing cells in the ARC dramatically increases at least 6–7 h prior to the LH surge onset and this pattern is attenuated if preceded by a bolus injection of LPS (Fergani et al. 2014). Furthermore, at least some of these ERα-containing cells are not kisspeptin cells and therefore, their phenotype remains to be elucidated (Fergani et al. 2013, 2014). For the present study, we collected brain tissue from ewes at various times in the follicular phase and used immunohistochemistry to pinpoint the time when dopamine, β-endorphin, NPY and kisspeptin-NKB-dynorphin (KNDy)-expressing cells become activated (i.e., co-express c-Fos). Furthermore, these activation patterns were correlated to peripheral plasma oestradiol and progesterone concentrations as well as the timing of different sexual behaviours and the LH surge onset. Lastly, we sought to determine whether the disruption of sexual behaviour and/or the surge after LPS administration is associated with altered activation of any or all of these cell types, adding to the evidence of their involvement in the physiological oestrus or surge generating mechanism.

Materials and methods

Animals, study design, tissue collection, blood collection and hormone assays

All procedures were conducted within requirements of the UK Animal (Scientific Procedures) Act 1986 and approved by the University of Liverpool Animal Welfare Committee. Experiments were carried out on adult, ovary-intact Lleyn crossbred ewes (6 groups of 4–6 ewes per group) during the mid-breeding season. After follicular phase synchronisation, ewe and ram oestrus behaviour was monitored during 30-min observation periods before each blood sample collection at 0, 16 and 24 h and subsequently at 2-h intervals till 40 h after PW (PW; progesterone intravaginal device withdrawal). The following behaviours of oestrus were recorded: (1) ewe is within one metre of a ram (behavioural scan sampling; (Martin 1986)), (2) ram nosing perineal region of ewe, (3) ewe being nudged by ram without moving away and (4) ewe mounted by ram without moving away. Frequent blood sampling, as well as the administration of all substances, was facilitated by insertion of a silastic catheter (Dow Corning, Reading, UK). Duplicate blood samples were analysed by enzyme-linked immunosorbent assays (ELISAs) for LH, pregnane metabolites (equivalent to, and hereafter referred to as, progesterone) or cortisol. LH results were expressed as ng equivalent of NIAMDD ovine LH 21 per mL plasma. Oestradiol was measured in duplicate by radioimmunoassay (RIA) using 0.5 mL plasma extracted with 3 mL diethyl ether followed by evaporation to dryness. Contemporary inter-assay and intra-assay coefficients of variation for LH, progesterone, cortisol and oestradiol were all less than 12%. The minimum detectable amounts were 0.02, 0.16, 0.8 ng/mL and 0.2 pg/mL and assay precisions (in the mid-range of the standard curve) were 0.1, 0.01, 0.2 ng/mL and 0.2 pg/mL respectively. All samples from individual animals were measured in the same assay for each hormone. One group of ewes was killed at 0 h (0 h control group; n = 5) and another group at 16 h after PW (16 h control group; n = 4). At 28 h, the remaining animals received 2 mL saline vehicle or endotoxin (lipopolysaccharides from E. coli 055:B5, LPS, Sigma-Aldrich; i.v. dose of 100 ng/kg body weight). The dose of LPS had been determined previously to evoke a robust increase in plasma cortisol followed by a delay in the LH surge onset (Fergani et al. 2012). The timing of the treatments was chosen in order to precede all sexual behaviours and not just mounting. Two groups were killed at 31 h (31 h control, n = 6 and 31 h LPS group, n = 5) and two groups at 40 h after progesterone withdrawal (40 h control, n = 5 and 40 h LPS group, n = 5). Ewes were killed with pentobarbitone and perfused with: 2 L 0.1 M phosphate buffer (PB; pH 7.4) containing 25,000 IU per litre of heparin and 1% sodium nitrate; then 2 L Zamboni fixative (4% paraformaldehyde) and 7.5% saturated picric acid in 0.1 M PB, pH 7.4); followed by 500 mL of the same fixative containing 30% sucrose. Hypothalamic blocks (17 mm in width) were obtained (extending from the optic chiasma to the mammillary bodies). Free-floating sections were stored in cryoprotectant solution and stored at −20°C until processed for immunohistochemistry.

Dual-labelled immunofluorescence for c-Fos and DA, β-endorphin, NPY, dynorphin or NKB

Dual-label immunofluorescence was carried out on 40 μm sections containing ARC. All steps were followed by washes in 0.1 M phosphate buffer saline, pH 7.2 (PBS) and performed at room temperature unless otherwise stated. Antibodies were diluted with 2.5% normal donkey serum (catalogue item S2170, Biosera, UK), 1% Triton X-100 (T9284, Sigma-Aldrich) and 0.25% sodium azide (Sigma) in 0.1 M PBS. Free-floating sections were washed thoroughly in PBS for 2 h to remove the cryoprotectant solution followed by 1-h incubation in blocking solution (10% donkey serum in PBS) and a 72-h incubation at 4°C with polyclonal rabbit anti-c-Fos antibody (AB-5, PC38, Calbiochem; 1:5000). Next, sections were incubated with donkey anti-rabbit Cy3 (711-165-152, Jackson Immunoresearch, West Grove, PA; 1:500) for 2 h. A second immunofluorescent procedure was then performed, as described previously, to localise the second primary antibodies: mouse anti-tyrosine hydroxylase serum (MAB318, Millipore; 1:20,000) or rabbit anti-β-endorphin serum (T-4041, Peninsula Laboratories, San Carlos, CA, USA; 1:500) or rabbit anti-neuropeptide Y serum (N9528, Sigma-Aldrich; 1:5000), or rabbit anti-dynorphin serum (T-4268, Peninsula Laboratories, LLC; 1:10,000) or rabbit anti-NKB serum (T-4450, Peninsula Laboratories, LLC; 1:1000); each incubated for 72 h at 4°C and then visualised using donkey-anti-rabbit Dylight 488 (715-485-152, Jackson Immunoresearch; 1:500) or donkey-anti-mouse Dylight 488 (715-485-151, Jackson Immunoresearch; 1:500), accordingly. Thereafter, sections were mounted on chrome alum gelatin-coated slides and cover-slipped with Vectashield anti-fading mounting medium (H-1000, Vector Laboratories, UK). The c-Fos (Ghuman et al. 2011), tyrosine hydroxylase (Robinson et al. 2010), β-endorphin (Ghuman et al. 2011), neuropeptide Y (Skinner & Herbison 1997), kisspeptin (Franceschini et al. 2006), dynorphin (Foradori et al. 2006) and NKB (Goodman et al. 2007) antibodies have been validated previously for use in ovine neural tissue.

Triple-labelled immunohistochemistry for c-Fos, kisspeptin and dynorphin

Interestingly, we observed a different dynorphin cell activation pattern compared to what we had previously reported for kisspeptin (Fergani et al. 2013). In order to confirm this discrepancy, we performed triple-label immunohistochemistry for c-fos, kisspeptin and dynorphin. This consisted of an immunoperoxidase protocol in which nuclear c-Fos was detected first with diaminobenzidine as chromogen (DAB; brown reaction product) followed by visualisation of kisspeptin and dynorphin with immunofluorescence. As kisspeptin and dynorphin antibodies were both derived in the rabbit, we used a previously described modified protocol (Hunyady et al. 1996, Cheng et al. 2010). Free-floating sections were washed thoroughly in PBS for 2 h to remove the cryoprotectant solution followed by a 15-min incubation in 40% methanol and 1% hydrogen peroxide (H2O2; 316989, Sigma-Aldrich) in PBS to inactivate endogenous peroxidases. Sections were then incubated for 1 h in blocking solution (10% donkey serum in PBS), followed by a 72-h incubation in rabbit anti-c-Fos antibody (1:5000) at 4°C. After, sections were labelled with biotinylated donkey anti-rabbit IgG (711-065-152, Jackson Immunoresearch; 1:500) for 2 h, followed by 90 min in Vectastain Elite ABC kit (1:250 in PBS; PK6100, Vector Laboratories Ltd). Nuclear c-Fos was visualised by 5-min incubation in DAB (SK-4100, Vector Laboratories). The second immunohistochemical procedure consisted of incubation for 72 h in rabbit anti- kisspeptin (lot #564; gift from Prof. Alain Caraty, INRA Nouzilly, France; 1:150,000) at 4°C. Following incubation, sections were labelled with biotinylated donkey anti-rabbit IgG (1:500; for 2 h) and then incubated in Vectastain Elite ABC kit (1:250; for 90 min). The signal was amplified in TSA for 10 min (1:200; New England Nuclear Life Science Products Life Sciences, Boston, MA, USA) and diluted in PBS with 0.003% H2O2 as substrate (Cheng et al. 2010) and then labelled with streptavidin-conjugated Alexa Fluor 488 (S11223, Molecular Probesy; 1:100) for 2 h. A third immunohistochemical procedure was then performed with a 72-h incubation with rabbit anti-Dynorphin (T-4268, Peninsula Laboratories, LLC; 1:10,000) and subsequent labelling with donkey anti-rabbit Cy3 (711-165-152, Jackson Immunoresearch; 1:500) for 2 h. Finally, sections were washed, mounted on chrome alum gelatin-coated slides, dried and cover-slipped with Vectashield anti-fading mounting medium.

Data collection and analysis

Hormone and immunohistochemistry data were analysed with Minitab 15 statistical package (MINITAB Inc, Pennsylvania, USA). Statistical significance was accepted when P < 0.05.

Quantitative analysis was carried out on three sections from each of the middle and caudal divisions of the ARC from each animal, where the largest numbers of cells are located (Lehman et al. 2010). Sections were examined under an epi-fluorescent/brightfield microscope (Zeiss Axio Imager. M1) and photographed by digital microphotography (Hamamatsu ORCA I-ER digital camera, Hamamatsu Photonics, Welwyn Garden City, Herts) using a 20× objective. Photographs (three per section) were acquired with an image analysis program AxioVision (Zeiss Imaging Systems) and consisted of single c-Fos staining, single dopamine, β-endorphin, neuropeptide Y, kisspeptin, dynorphin or NKB staining as well as merged fluorescent images to produce a spectral combination of green (fluorescein) and red (rhodamine). All photographs were imported into ImageJ, version 1.42q, and counts performed using the cell count plug-in. Triple co-localisation was determined by switching through the single-labelled brightfield/fluorescent photographs. The observer was unaware of the animal identity and group. The mean total number and percentage of single-, dual- or triple-labelled cells was summed from the photographs of each section and then averaged for each ewe and compared using GLM ANOVA, followed, when appropriate, by Tukey’s multiple comparisons post hoc test. Mean data (±s.e.m.), as presented in figures and results, were calculated by averaging values for each group.

The data were analysed in two ways: the first consisted of data derived from control ewes, grouped according to time as well as hormonal and sexual behaviour status; i.e., those killed at 0 or 16 h after PW, those killed at 31 h after PW but before the onset of sexual behaviour (Before sexual behaviour, n = 3), those killed at 31 or 40 h after PW but after the onset of sexual behaviour and before exhibiting an LH surge (during sexual behaviour, n = 5) and those killed after the onset of both sexual behaviour and during the LH surge (Surge, n = 3). This grouping was used to describe the timing of each neuropeptide cell activation relative to the LH surge onset. Secondly, control and treated ewes were grouped according to time of killing after PW, and these data were used to compare LPS treatment effects.

Lastly, regression analysis was used to correlate plasma oestradiol and progesterone concentrations (percentage of change from 0 h) to the percentage of DA, β-endorphin, NPY, kisspeptin, dynorphin or NKB cells that co-localised c-Fos (i.e., were activated) during various times in the follicular phase of control ewes.

Results

Animals treated with LPS did not show any signs of illness, with very few exceptions of mild coughing and increased respiration rate.

Luteinising hormone (LH), sexual behaviour and plasma hormone profiles

Detailed LH, sexual behaviour and plasma hormone profiles have been published previously (Fergani et al. 2013). In brief, there was no sexual behaviour or LH surge recorded in control ewes killed at 0 or 16 h. Eight of eleven control animals, killed at 31 or 40 h, began exhibiting sexual behaviour at 28.5 ± 2.4 h after PW, and three of five (from the 40 h control group) additionally had an LH surge with a mean onset at 36.7 ± 1.3 h after PW. None of the LPS-treated ewes exhibited an LH surge within the 40 h of study and only 3 out of 8 LPS-treated ewes displayed early signs of oestrus behaviour (near ram and being nosed), which ceased after treatment (Fergani et al. 2013).

In control ewes, plasma oestradiol concentrations began to increase at 28 h after PW and reached maximum values just before the LH surge onset (i.e., at 32 h after PW; 12.2 ± 1.8 pg/mL). However, treatment with LPS was followed by a decrease in oestradiol concentrations, which was evident 8 h after LPS administration (from 11.6 ± 1.6 pg/mL to 6.9 ± 1.8 pg/mL) and remained low until ewes were killed at 40 h. Plasma progesterone concentrations decreased from 0 to 28 h after PW in all groups (from 33.7 ± 2.0 ng/mL to 6.6 ± 0.4 ng/mL). However, LPS treatment increased progesterone concentrations from 6.9 ± 1.0 ng/mL before treatment to a mean maximum of 9.9 ± 1.6 ng/mL after treatment. In all control animals, mean plasma cortisol concentrations remained low throughout the study (10.5 ± 0.7 ng/mL). By contrast, LPS treatment increased cortisol concentrations to a mean maximum of 157 ± 19.8 ng/mL 2 h after treatment (Fergani et al. 2012).

Control ewes grouped according to sexual behaviour and the LH surge

c-Fos co-expression with dopamine or β-endorphin or NPY

The percentage of activated dopamine cells (% dopamine/c-Fos) decreased in the ‘before sexual behaviour’ group till the ‘surge’ (P < 0.05 for each comparison; Fig. 1A), whereas the % β-endorphin/c-Fos cells was greater in the 16 h and ‘during sexual behaviour’ groups compared to 0 h and ‘surge’ (P < 0.05 for both comparisons; Fig. 1B). In contrast, the % NPY/c-Fos cells did not fluctuate, but tended to be higher in the ‘surge’ group (P < 0.08; Fig. 1C). The % dopamine/c-Fos cells in the ARC was positively associated with progesterone concentrations (P = 0.001; Fig. 1D), whereas % NPY/c-Fos was positively associated with plasma oestradiol concentrations (P = 0.008; Fig. 1F).

Figure 1
Figure 1

(A, B and C) Mean % (±s.e.m.) dopamine, β-endorphin and neuropeptide Y cells respectively, that co-express c-Fos in the ARC at different stages during the follicular phase of control ewes as determined by dual-immunofluorescence. Animals were grouped according to time after PW as well as by hormonal and behavioural status, that is, grouped into those killed at 0 and 16 h after PW (n = 4–5), those killed before the onset of sexual behaviour (before sexual behaviour, n = 3), those killed after the onset of sexual behaviour but before exhibiting an LH surge (during sexual behaviour, n = 5) and those killed during sexual behaviour and an LH surge (surge, n = 3). Within each panel, differences between the percentages are indicated by different letters on top of each bar (P< 0.05) except * when P< 0.08. (D, E and F) Regression graphs showing the association between dopamine, β-endorphin and neuropeptide Y cells respectively, that co-express c-Fos in the ARC of control ewes against the % change in concentration from 0 h to the mean two consecutive highest or lowest concentrations for oestradiol (open circles, E; dashed line) or progesterone (filled, black squares, P; solid line) respectively.

Citation: Reproduction 154, 3; 10.1530/REP-17-0191

c-Fos co-expression with kisspeptin, dynorphin or NKB

The double-label immunofluorescence study (c-Fos and dynorphin) showed that the % dynorphin/c-Fos was greatest in the ‘before behaviour’ group compared to earlier stages in the follicular phase (P < 0.05; Fig. 2). Thereafter, there was a gradual decrease until the LH surge (Fig. 2). This was a surprising result, as we have previously shown that the greatest number of kisspeptin cells express c-Fos during the surge (Fergani et al. 2013). Therefore, we proceeded with co-staining hypothalamic sections with both proteins (kisspeptin and dynorphin) in addition to c-Fos, to confirm this novel finding.

Figure 2
Figure 2

Mean % (±s.e.m.) dynorphin cells that co-localise c-Fos in the ARC at various stages during the follicular phase of control ewes as determined by dual-labell immunohistochemistry (Dynorphin/c-Fos). Animals are grouped according to time as well as hormonal and behavioural status (for details, Fig. 1 legend). Within each panel, differences between the percentages are indicated by different letters on top of each bar (P< 0.05).

Citation: Reproduction 154, 3; 10.1530/REP-17-0191

The % kisspeptin/c-Fos sequentially increased during the follicular phase, with a two-fold increase during the ‘surge’ (P < 0.05; Fig. 3A). By contrast, a two-fold increase in the % dynorphin/c-Fos was observed earlier, in the ‘before sexual behaviour’ group, compared to 0 and 16 h (P < 0.05 for both; Fig. 3B). This increase was maintained to a lesser extent till the ‘Surge’ (Fig. 3B). The % NKB/c-Fos followed a similar pattern to that of kisspeptin and sequentially increased from 0 h till the ‘surge’ (P < 0.05 for ‘surge’ compared with other stages; Fig. 3C). Furthermore, the % kisspeptin/c-Fos and % NKB/c-Fos was positively associated with changes in oestradiol concentration (P = 0.005, RSq = 36% and P = 0.002, RSq = 41% for kisspeptin and NKB respectively; Fig. 3D and E), whereas the activation of all three neuropeptides was negatively associated with plasma progesterone values (P = 0.002, RSq = 41%, P = 0.001 RSq = 47% and P = 0.001, RSq = 50% for kisspeptin, dynorphin and NKB respectively; Fig. 3D, E and F).

Figure 3
Figure 3

Panels (A, B and C) Mean % (±s.e.m.) kisspeptin, dynorphin and NKB cells respectively, that co-localise c-Fos in the ARC at various stages during the follicular phase of control ewes as determined by triple- and dual-label immunohistochemistry (kisspeptin/dynorphin/c-Fos and NKB/c-Fos respectively). Animals are grouped according to time as well as hormonal and behavioural status (for details, Fig. 1 legend). Within each panel, differences between the percentages are indicated by different letters on top of each bar (P< 0.05). (D, E and F) Regression graphs showing the association between the % kisspeptin, % dynorphin and % NKB cells respectively, that co-localise c-Fos in the ARC against the % change in concentration from 0 h to the mean two consecutive highest or lowest concentrations for oestradiol (open circles, E; dashed line) or progesterone (filled, black squares, P; solid line) respectively.

Citation: Reproduction 154, 3; 10.1530/REP-17-0191

Kisspeptin, dynorphin and NKB cell numbers and percentage co-localisation

In order to address the differential activation of kisspeptin and dynorphin neurons we analysed the co-localisation between these two peptides throughout the follicular phase. Kisspeptin and dynorphin cell numbers were greater ‘during sexual behaviour’ (P< 0.05; Fig. 4A and B), whereas the number of NKB cells was greater during the ‘surge’ as well (P< 0.05 for all comparisons; Fig. 4C). However, the per cent co-localisation between kisspeptin and dynorphin immunoreactivity in the ARC varied during the follicular phase (Fig. 4D and E). The % of kisspeptin cells co-localising dynorphin was lower in the ‘surge’ group (that is, there were more single-labelled kisspeptin cells (P< 0.05 for all comparisons; Fig. 4D)). Furthermore, there were fewer dynorphin cells co-localising kisspeptin in the ‘before sexual behaviour’ group (that is, there were more single-labelled dynorphin cells at those times; P< 0.05 for all comparisons; Fig. 4A, B, C and D).

Figure 4
Figure 4

(A, B and C) Mean (±s.e.m.) number of kisspeptin, dynorphin and NKB cells respectively. (D) Mean % (±s.e.m.) kisspeptin cells co-localising dynorphin, and (E) mean % (±s.e.m.) dynorphin cells co-localising kisspeptin. (F) Mean (±s.e.m.) number of dynorphin (black bars) and kisspeptin (white bars) cells that co-localise c-Fos. Mean (±s.e.m.) numbers and percentages are per section from the ARC at various stages during the follicular phase of control ewes as determined by triple- and dual-label immunohistochemistry (kisspeptin/dynorphin/c-Fos and NKB/c-Fos respectively). Animals are grouped according to time as well as hormonal and behavioural status (for details, Fig. 1 legend). Within each panel and type of cell, differences between numbers and percentages are indicated by different letters on top of each bar (P< 0.05). (F) *P< 0.05 compared to activated dynorphin cells.

Citation: Reproduction 154, 3; 10.1530/REP-17-0191

Finally, the total number of activated dynorphin cells was greater in the ‘before sexual behaviour’ group compared to the activated kisspeptin cells (P < 0.05; Fig. 4F), whereas there were more kisspeptin activated cells during the ‘Surge’ compared to activated dynorphin cells (P < 0.05; Fig. 4F). Examples of photomicrographs of sections from the ARC that have been triple-labelled for c-Fos, kisspeptin and dynorphin as well as c-Fos and NKB are shown in Fig. 5.

Figure 5
Figure 5

(A, B and C) Sets of photomicrographs in the ARC that were triple-labelled for c-Fos, kisspeptin and dynorphin, as well as a merged image (D) in control animals at 31 h after progesterone withdrawal (that is, before the onset of sexual behaviour or the LH surge). Photomicrographs from the ARC nucleus that were dual-labelled for NKB cells and their co-localisation with c-Fos in control before the LH surge, but during sexual behaviour (E) as well as in control animals at 40 h and specifically during an LH surge (F). (A, B, C and D) Arrows indicate examples of single-labelled dynorphin cells co-localising c-Fos. (E and F) Arrows indicate examples of dual-labelled cells. Original magnification: ×20 (A, B, C, D and E), original magnification: ×10 (F). 3V = third ventricle.

Citation: Reproduction 154, 3; 10.1530/REP-17-0191

Comparison of control and LPS-treated ewes

To determine the effects of LPS treatment, data were analysed according to time of killing after PW (irrespective of sexual behaviour and the LH surge) and compared to control animals at each time point.

Dopamine or β-endorphin or NPY cell numbers and co-expression with c-Fos

The total number of immunoreactive dopamine cells increased in the 40-h controls compared to 0 h (P < 0.05; Table 1), whereas there were more β-endorphin cells in the 31- and 40-h control and LPS groups (compared to 0 h controls P < 0.05 for all; Table 1). The number of NPY cells did not vary across the follicular phase of controls and was not affected by treatment (Table 1). The % dopamine/c-Fos, % β-endorphin/c-Fos and % NPY/c-Fos was not affected by LPS treatment (Fig. 6A, B and C).

Figure 6
Figure 6

(A, B, C, D, E and F) Mean % (±s.e.m.) dopamine, β-endorphin, neuropeptide Y, kisspeptin, dynorphin and NKB cells respectively, that co-express c-Fos in the ARC across the follicular phase of control ewes as determined by immunofluorescence. Animals are grouped according to killing time after progesterone withdrawal (PW), that is, control ewes at 0, 16, 31 and 40 h (n = 4–6 per group; black bars) as well as after LPS at 31 and 40 h (n = 4 for both times; white bars). Treatment with LPS was at 28 h after PW. Within each panel, differences within controls are indicated by different letters on top of each bar (P< 0.05). Differences between control and LPS treated ewes, at each time point, are indicated with a star (P< 0.05).

Citation: Reproduction 154, 3; 10.1530/REP-17-0191

Table 1

 Mean (±s.e.m.) total number of dopamine, β-endorphin and neuropeptide Y immunoreactive cells in the middle and caudal ARC at various times after PW with or without liposaccharide (LPS).

Dopamine β-Endorphin Neuropeptide Y
0 h 28.0±6.2 47.5±11.3 22.6±9.1
16 h 42.3±10.5 56.5±15.4 38.2±9.5
31 h control 40.3±15.5 83.3±13.0* 22.6±3.6
31 h LPS 39.5±14.8 109.8±19.5* 26.4±7.6
40 h control 60.5±4.5* 92.0±9.4* 33.8±6.1
40 h LPS 53.1±10.8 93.9±6.6* 41.4±17.6

P< 0.05 within a column, greater than 0-h control group.

Kisspeptin, dynorphin and NKB cell numbers and co-expression with c-Fos

In controls, maximum kisspeptin and dynorphin cell numbers were recorded at 31 and 40 h after PW (Table 2). Of note, there where were more dynorphin cells than kisspeptin or NKB cells in the 31-h controls (Table 2). Treatment with LPS decreased the number of immunodetectable dynorphin cells compared to controls (Table 2). The number of NKB cells did not vary during the follicular phase or after LPS treatment (Table 2).

Table 2

 Mean (±s.e.m.) total number of kisspeptin, dynorphin or NKB immunoreactive cells in the middle and caudal ARC at various times after PW.

Kisspeptin Dynorphin Neurokinin B
0 h 22.6±6.8 28.9±9.5 19.1±4.0
16 h 29.8±12.1 35.1±11.9 23.3±9.1
31 h control 37.2±3.0* 59.9±6.2*# 30.0±7.5
31 h LPS 29.0±7.4 28.9±5.4 28.8±10.3
40 h control 42.1±11.9* 53.5±11.8** 39.2±6.9
40 h LPS 33.6±9.5 35.6±9.1 29.2±2.3

Kisspeptin: *P< 0.05 within a column, greater than 0 h control group; dynorphin: *P< 0.05 within a column, greater than 0 h, 16 h and LPS groups; **P< 0.05 within a column, greater than 0 h; #P< 0.001 within a row, greater than kisspeptin and NKB.

At 40 h after PW (i.e., 12 h after LPS administration), the % kisspeptin/c-Fos was markedly lower in LPS-treated animals compared to controls (P < 0.05; Fig. 6D). Interestingly, the % dynorphin/c-Fos decreased earlier than kisspeptin, i.e., at 31 and 40 h (3 and 12 h after LPS administration; P < 0.05; Fig. 6E). The % NKB/c-Fos was not affected by LPS administration within the 12 h post treatment (Fig. 6F).

Discussion

The present study demonstrates that various cell types within the ARC of the ovary-intact ewe are activated at different times during the follicular phase, leading up to the GnRH/LH surge. Specifically, activation of dopamine neurones was initially high, but decreased before the onset of sexual behaviour, whereas the activation of β-endorphin cells increased in the mid-follicular phase, decreasing a few hours later during the surge. The percentage of activated NPY cells tended to increase in animals undergoing an LH surge. Treatment with LPS had no effect on the activation of dopamine, β-endorphin or NPY cells raising the possibility that these cell types are only permissive in the surge induction process. Our observations are also consistent with a role for KNDy cells in the GnRH/LH surge mechanism as these cells became activated prior to the LH surge onset. Interestingly, in our ovary-intact ewe model, kisspeptin, NKB and dynorphin immunoreactivity and co-localisation vary throughout the follicular phase, leading to differential activation patterns for each individual KNDy peptide. Maximum kisspeptin and NKB immunoreactive cells were maximally activated during the GnRH/LH surge, whereas maximum activation of dynorphin positive cells occurred at least 6–7 h before that. Furthermore, LPS administration in the late follicular phase prevented kisspeptin and dynorphin-positive cell activation, and this was accompanied by a failure to exhibit an LH surge.

Dopamine has been implicated in the control of female sexual behaviour (Fabre-Nys & Gelez 2007). In the present study, dopamine neurons in the ARC were maximally activated in the early follicular phase but greatly decreased just before the ewes began exhibiting signs of estrous. These results are consistent with a biphasic role of dopamine as described by Fabre-Nys (Fabre-Nys et al. 1994, 2003), who showed that extra-cellular concentrations of dopamine in the mediobasal hypothalamus (MBH; containing the ARC and ventromedial nucleus; VMN) are initially high, followed by a sharp decrease preceding the onset of sexual behaviour (Fabre-Nys et al. 1994). The present data indicate that the source of dopaminergic input in the MBH could be derived, at least in part, from cells located in the ARC. Indeed, 20% of dopamine neurones in the ARC send projections towards the VMN (Qi et al. 2008) providing a possible signalling pathway involved in the initiation of sexual behaviours. However, in the present study, ewes treated with LPS did not exhibit signs of sexual behaviour but dopamine cell activation in the ARC was not affected, indicating that this pathway may be permissive but not indispensable for the initiation of oestrus.

In the ARC, 15–20% of β-endorphin cells contain ERα (Lehman & Karsch 1993), and β-endorphin or pro-opiomelanocortin (POMC) fibres directly innervate GnRH cells in the rat (Leranth et al. 1988) and monkey (Thind & Goldsmith 1988) or form close appositions in the ewe (Dufourny et al. 2005). In the present study, activation of β-endorphin cells slightly increased during the mid/late follicular phase, but not in animals exhibiting a GnRH/LH surge. These results are consistent with those of Domanski (Domanski et al. 1991) who demonstrated a decrease in β-endorphin concentrations in the ARC of ovary-intact ewes before the onset of the pre-ovulatory LH surge, but conflict with those of Taylor and coworkers (Taylor et al. 2007) who observed an increase in POMC mRNA at the time of the peak of the GnRH surge in OVX ewes. The reason for this divergence between studies is not known, although it may reflect differences in the timing of brain tissue sampling, as well methods of detection (i.e., protein vs gene expression). Furthermore, various POMC gene products other than β-endorphin, such as α-melanocyte-stimulating hormone may have differential effects on the reproductive axis compared to β-endorphin (Gonzalez et al. 1997, Scimonelli et al. 2000).

In the sheep, the role of NPY in the regulation of GnRH is not clear. NPY administered intracerebroventricularly (icv) suppressed release of LH in OVX and OVX oestradiol-treated sheep (Malven et al. 1992, Estrada et al. 2003), whereas in follicular phase ewes, icv administration of anti-NPY serum delayed the onset of the pre-ovulatory GnRH/LH surge, implying a stimulatory role in this process (Porter et al. 1993). In addition, a stimulatory effect on GnRH release by NPY infusion into the ME was observed in ovary-intact ewes, but only in the follicular, and not in the luteal, phase (Advis et al. 2003). In the present study, NPY activation tended to be higher in animals that were exhibiting an LH surge. Furthermore, this pattern of activation was positively correlated to plasma oestradiol concentrations. It is plausible to speculate that NPY is involved in the regulation of GnRH secretion (Kalra et al. 1991, Sahu et al. 1995) but specific actions depend on the prevailing endocrine status. For example, in rats, NPY stimulates GnRH release in the presence of oestradiol, but inhibits GnRH release during the absence of sex steroids (Kalra & Crowley 1992). Interestingly, LPS had no effect on β-endorphin or NPY cell activation. Therefore, it appears that neither of these phenotypes are essential in the surge induction process, nor to mediate the LPS-induced disruption of sexual behaviour or the GnRH/LH surge.

The immunoreactivity of all three KNDy peptides in the ARC was greater in the late, rather than early, follicular phase, adding to the increased evidence for the involvement of these cells in oestradiol positive feedback in the ewe. We have shown that kisspeptin cells are activated during the LH surge in ovary-intact ewes (Fergani et al. 2013) and a similar finding has been reported by Merkley and coworkers (Merkley et al. 2012) in OVX oestrogen-treated sheep undergoing an LH surge. More recently, an important role for NKB in oestradiol-positive feedback and the GnRH surge has emerged as local administration of an NKB receptor agonist (senktide) into the retrochiasmatic area stimulates surge-like LH secretion (Billings et al. 2010), whereas an NKB receptor antagonist (SB222200) administered in the same region decreased LH surge amplitude (Porter et al. 2014). NKB neurones located in the ARC are thought to be the source of input to this area (Grachev et al. 2016). In the present study, NKB cells in the ARC were gradually activated, with maximum activation during the LH surge (i.e., in a similar pattern to kisspeptin cells). Furthermore, there was a positive correlation of kisspeptin and NKB cell activation with plasma oestradiol concentrations and a negative correlation with progesterone. These data provide further evidence that kisspeptin and NKB neurones in the ARC are activated during, and may therefore be involved in, oestradiol-positive feedback and the surge phase of GnRH/LH secretion in the ewe.

To date, dynorphin neurones in the ARC of the ewe have been implicated in the negative feedback actions of progesterone to inhibit GnRH and LH pulse frequency (Goodman et al. 2011). Interestingly, we observed maximum activation of dynorphin immunoreactive cells occurred at least 6–7 h before the expected LH surge, at a time when activation of kisspeptin and NKB were comparatively lower, suggesting that dynorphin may play a distinct role in the GnRH surge induction process. The precise physiological role of an increase in dynorphin protein within KNDy cells prior to the LH surge is not known; however, these observations are consistent with the hypothesis that endogenous opioid systems in the hypothalamus are permissive of sexual behaviour and the GnRH/LH surge (Kalra 1993, Walsh & Clarke 1996) and may, therefore, be a critical part of the oestradiol-positive feedback mechanism (Smith & Gallo 1997, Zhang & Gallo 2003). Furthermore, recent evidence suggests that this dynorphin input originates from ARC KNDy cells, as ablation of these cells leads to an abnormal increase in the amplitude of the LH surge, whereas microinjections of dynorphin in to the POA of KNDy-ablated rats restored LH surge levels (Helena et al. 2015). We speculate that increased opioid influence during the mid-follicular phase plays a role in preventing premature activation of GnRH neurones, giving time for an increase in the releasable pool of GnRH, as well as an increase in GnRH receptor numbers in the pituitary (Clarke et al. 1988, Walsh & Clarke 1996).

Different activation patterns between kisspeptin/NKB and dynorphin is a novel finding in the present study, as all three neuropeptides co-localise in the same KNDy cell, as has been described in OVX ewes (Goodman et al. 2007). However, this can be explained by considering the expression of individual KNDy peptides as being differentially regulated by steroid hormones and gonadal status. For example, ovariectomy increases NKB and kisspeptin, but decreases dynorphin gene and protein expression in the sheep ARC (Pillon et al. 2003, Foradori et al. 2005, Navarro et al. 2009). Furthermore, recent findings in the rat, using immunoelectron microscopy, indicate that each neuropeptide is contained within separate neurosecretory vesicles, adding to the evidence that each KNDy peptide is differentially regulated within the KNDy neurone (Murakawa et al. 2016). Therefore, we hypothesise that fluctuating endogenous steroid concentrations in ovary-intact ewes result in differential peptide content and/or immunoreactivity within the KNDy neurone, leading to increased dynorphin immunoreactivity (and subsequently, activation), 6–7 h before the LH surge.

In the present study, acute LPS administration in the late follicular phase was accompanied by suppression of dynorphin and kisspeptin activation within the KNDy cell, a decrease in plasma oestradiol concentrations, and subsequent absence of a GnRH/LH surge. Several studies report downregulation of the ARC kisspeptin system in rats and male rhesus monkeys after metabolic or immune/inflammatory stressors, such as negative energy balance (Castellano et al. 2005), short-term fasting (Wahab et al. 2010) or administration of LPS (Iwasa et al. 2008, Kinsey-Jones et al. 2009). However, to the best of our knowledge, there are no equivalent data for the actions of stressors on dynorphin cells located in the ARC.

Regarding the potential mechanisms via which LPS inhibited the activation of kisspeptin and dynorphin within KNDy cells, it must be noted that plasma oestradiol concentrations decreased 8 h after administration of LPS (Fergani et al. 2013), presumably via inhibition of GnRH/LH pulses and deprivation of mature follicle(s) gonadotrophic drive. However, the decrease in the percentage of activated dynorphin neurones occurred sooner (within 3 h after LPS treatment) and therefore, lack of an efficient oestradiol signal cannot be the cause but could be the result of lack in KNDy cell activation.

Various other factors have been implicated in LPS-induced disruption of the oestrous cycle and at least some of those may be acting upon KNDy neurons. In our paradigm of an acute peripheral LPS administration, peripheral cortisol and progesterone concentrations increased within 2 h after the injection and are, therefore, potential candidates for the immediate inhibition of dynorphin neurone activity and the surge mechanism. In accordance, Pierce and coworkers (Pierce et al. 2008) and Wagenmaker et al. (Wagenmaker et al. 2009) report that cortisol disrupts the positive feedback effect of oestradiol to trigger an LH surge in the ewe, whereas progesterone has been implicated in both inhibition of GnRH pulses (Karsch et al. 1987) and of the surge mechanism (Kasa-Vubu et al. 1992, Richter et al. 2005). Glucocorticoid receptors (GR) co-localise with kisspeptin neurons in mice and rats (Takumi et al. 2012) and the tissue-specific deletion of GR in kisspeptin neurons eliminates cortisol-induced suppression of kisspeptin gene expression (Grachev et al. 2013). However, the absence of GR in kisspeptin neurones does not prevent the suppression of the reproductive axis following traumatic stress (Whirledge & Cidlowski 2013), and thus, GR signalling in KNDy neurons cannot fully account for LH surge disruption. Similarly, we have previously presented evidence that the progesterone/glucocorticoid receptor antagonist, RU486, was unable to reverse delays in the GnRH/LH surge induced by a metabolic stressor (Dobson & Smith 2000). Notably, corticotrophin-releasing hormone (CRH) has been demonstrated to be a powerful suppressor of the GnRH pulse generator in the rat (Li et al. 2010), whereas acute LPS administration increased the number of immunoreactive cells within the ARC/ME that contained CRH-type 2 receptors (Fergani et al. 2013). However, icv administration of CRF in the sheep either increases (Naylor et al. 1990, Caraty et al. 1997) or has no effect (Clarke et al. 1990) on LH pulse frequency. Clearly, this pathway requires further investigation.

Lastly, the action of interleukin- (IL-)1 β must also be taken into account, as this cytokine is secreted in response to LPS and is considered to be the most potent down regulator of reproductive processes during an immune/inflammatory challenge (Herman et al. 2012). IL-1 β has been described to act within the hypothalamus by inhibiting GnRH expression but also directly on pituitary gonadotropes to suppress GnRH receptor expression (Herman et al. 2012, Herman et al. 2013). Whether IL-1 β has any direct or indirect inhibitory effect on ARC KNDy neurones, merits further investigation.

Our results indicate that the activation patterns of ARC cells containing dopamine, β-endorphin and NPY differ throughout the follicular phase of ovary-intact ewes. However, a surge-inhibiting dose of LPS had no effect on the activation of these phenotypes, suggesting that they are not essential mediators of GnRH/LH surge release. More importantly, our results confirm a critical role for KNDy cells in the GnRH/LH surge mechanism in the ewe. Furthermore, cells immunoreactive for dynorphin were activated at least 6–7 h before the expected LH surge, at a time when activation of kisspeptin- and NKB-positive cells was comparatively lower, suggesting that dynorphin, possibly derived from KNDy cells, may play a distinct role in the GnRH surge induction process. The physiological relevance of this finding remains to be explored.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.

Acknowledgements

Thanks are due to Nigel Jones and the farm staff for care of the animals; Hilary Purcell, David Jones and Peter Taylor for technical assistance; and Prof. A Parlow and NIAMDD, USA for LH standard preparations. They are also grateful to Dr Richard Morris for his guidance in immunohistochemical techniques as well as Dr Michael Morris for help with animal observations.

References

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    Figure 1

    (A, B and C) Mean % (±s.e.m.) dopamine, β-endorphin and neuropeptide Y cells respectively, that co-express c-Fos in the ARC at different stages during the follicular phase of control ewes as determined by dual-immunofluorescence. Animals were grouped according to time after PW as well as by hormonal and behavioural status, that is, grouped into those killed at 0 and 16 h after PW (n = 4–5), those killed before the onset of sexual behaviour (before sexual behaviour, n = 3), those killed after the onset of sexual behaviour but before exhibiting an LH surge (during sexual behaviour, n = 5) and those killed during sexual behaviour and an LH surge (surge, n = 3). Within each panel, differences between the percentages are indicated by different letters on top of each bar (P< 0.05) except * when P< 0.08. (D, E and F) Regression graphs showing the association between dopamine, β-endorphin and neuropeptide Y cells respectively, that co-express c-Fos in the ARC of control ewes against the % change in concentration from 0 h to the mean two consecutive highest or lowest concentrations for oestradiol (open circles, E; dashed line) or progesterone (filled, black squares, P; solid line) respectively.

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    Figure 2

    Mean % (±s.e.m.) dynorphin cells that co-localise c-Fos in the ARC at various stages during the follicular phase of control ewes as determined by dual-labell immunohistochemistry (Dynorphin/c-Fos). Animals are grouped according to time as well as hormonal and behavioural status (for details, Fig. 1 legend). Within each panel, differences between the percentages are indicated by different letters on top of each bar (P< 0.05).

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    Figure 3

    Panels (A, B and C) Mean % (±s.e.m.) kisspeptin, dynorphin and NKB cells respectively, that co-localise c-Fos in the ARC at various stages during the follicular phase of control ewes as determined by triple- and dual-label immunohistochemistry (kisspeptin/dynorphin/c-Fos and NKB/c-Fos respectively). Animals are grouped according to time as well as hormonal and behavioural status (for details, Fig. 1 legend). Within each panel, differences between the percentages are indicated by different letters on top of each bar (P< 0.05). (D, E and F) Regression graphs showing the association between the % kisspeptin, % dynorphin and % NKB cells respectively, that co-localise c-Fos in the ARC against the % change in concentration from 0 h to the mean two consecutive highest or lowest concentrations for oestradiol (open circles, E; dashed line) or progesterone (filled, black squares, P; solid line) respectively.

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    Figure 4

    (A, B and C) Mean (±s.e.m.) number of kisspeptin, dynorphin and NKB cells respectively. (D) Mean % (±s.e.m.) kisspeptin cells co-localising dynorphin, and (E) mean % (±s.e.m.) dynorphin cells co-localising kisspeptin. (F) Mean (±s.e.m.) number of dynorphin (black bars) and kisspeptin (white bars) cells that co-localise c-Fos. Mean (±s.e.m.) numbers and percentages are per section from the ARC at various stages during the follicular phase of control ewes as determined by triple- and dual-label immunohistochemistry (kisspeptin/dynorphin/c-Fos and NKB/c-Fos respectively). Animals are grouped according to time as well as hormonal and behavioural status (for details, Fig. 1 legend). Within each panel and type of cell, differences between numbers and percentages are indicated by different letters on top of each bar (P< 0.05). (F) *P< 0.05 compared to activated dynorphin cells.

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    Figure 5

    (A, B and C) Sets of photomicrographs in the ARC that were triple-labelled for c-Fos, kisspeptin and dynorphin, as well as a merged image (D) in control animals at 31 h after progesterone withdrawal (that is, before the onset of sexual behaviour or the LH surge). Photomicrographs from the ARC nucleus that were dual-labelled for NKB cells and their co-localisation with c-Fos in control before the LH surge, but during sexual behaviour (E) as well as in control animals at 40 h and specifically during an LH surge (F). (A, B, C and D) Arrows indicate examples of single-labelled dynorphin cells co-localising c-Fos. (E and F) Arrows indicate examples of dual-labelled cells. Original magnification: ×20 (A, B, C, D and E), original magnification: ×10 (F). 3V = third ventricle.

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    Figure 6

    (A, B, C, D, E and F) Mean % (±s.e.m.) dopamine, β-endorphin, neuropeptide Y, kisspeptin, dynorphin and NKB cells respectively, that co-express c-Fos in the ARC across the follicular phase of control ewes as determined by immunofluorescence. Animals are grouped according to killing time after progesterone withdrawal (PW), that is, control ewes at 0, 16, 31 and 40 h (n = 4–6 per group; black bars) as well as after LPS at 31 and 40 h (n = 4 for both times; white bars). Treatment with LPS was at 28 h after PW. Within each panel, differences within controls are indicated by different letters on top of each bar (P< 0.05). Differences between control and LPS treated ewes, at each time point, are indicated with a star (P< 0.05).

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